Selected Topics in the History of Biochemistry VII: Personal Recollections (Comprehensive Biochemistry, Vol 42)

COMPREHENSIVE BIOCHEMISTRY COMPREHENSIVE BIOCHEMISTRY SECTION I (VOLUMES 1^4) PHYSICO-CHEMICAL AND ORGANIC ASPECTS O...

Author: G Semenza | AJ Turner

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COMPREHENSIVE BIOCHEMISTRY

COMPREHENSIVE BIOCHEMISTRY

SECTION I (VOLUMES 1^4) PHYSICO-CHEMICAL AND ORGANIC ASPECTS OF BIOCHEMISTRY

SECTION II (VOLUMES 5^11) CHEMISTRY OF BIOLOGICAL COMPOUNDS

SECTION III (VOLUMES 12^16) BIOCHEMICAL REACTION MECHANISMS

SECTION IV (VOLUMES 17^21) METABOLISM

SECTION V (VOLUMES 22^29) CHEMICAL BIOLOGY

SECTION VI (VOLUMES 30^42) A HISTORY OF BIOCHEMISTRY

COMPREHENSIVE BIOCHEMISTRY Series Editor: GIORGIO SEMENZA Swiss Federal Institute of Technology, Department of Biochemistry, ETH-Zentrum, CH-8092 Zu«rich (Switzerland) and University of Milan, Department of Chemistry, Biochemistry, and Biotechnologies for Medicine, I-20133 Milan (Italy) VOLUME 42 SELECTED TOPICS IN THE HISTORY OF BIOCHEMISTRY PERSONAL RECOLLECTIONS. VII Volume Editors: GIORGIO SEMENZA Swiss Federal Institute of Technology, Department of Biochemistry, ETH-Zentrum, CH-8092 Zu«rich (Switzerland) and University of Milan, Department of Chemistry, Biochemistry, and Biotechnologies for Medicine I-20133 Milan (Italy)

A.J. TURNER School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT (UK)

AMSTERDAM BOSTON LONDON NEW YORK OXFORD PARIS SAN DIEGO SAN FRANCISCO SINGAPORE SYDNEY TOKYO 2003

ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 P.O. Box 211, 1000 AE Amsterdam, The Netherlands 2003 Elsevier Science B.V. All rights reserved. This work is protected under copyright by Elsevier Science, and the following terms and conditions apply to its use: Photocopying Single photocopies of single chapters may be made for personal use as allowed by national copyright laws. Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational institutions that wish to make photocopies for non-profit educational classroom use. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: [email protected]. You may also complete your request on-line via the Elsevier Science homepage (http://www.elsevier.com), by selecting ‘Customer Support’ and then ‘Obtaining Permissions’. In the USA, users may clear permissions and make payments through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA; phone: (978) 7508400, fax: (978) 7504744, and in the UK through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London WIP OLP, UK; phone: (þ44) 171 631 5555; fax: (þ44) 171 631 5500. Other countries may have a local reprographic rights agency for payments. Derivative Works Tables of contents may be reproduced for internal circulation, but permission of Elsevier Science is required for external resale or distribution of such material. Permission of the Publisher is required for all other derivative works, including compilations and translations. Electronic Storage or Usage Permission of the Publisher is required to store or use electronically any material contained in this work, including any chapter or part of a chapter. Except as outlined above, no part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Address permissions requests to: Elsevier’s Science & Technology Rights Department, at the phone, fax and e-mail addresses noted above. Notice No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. First edition 2003 Library of Congress Cataloging in Publication Data A catalog record from the Library of Congress has been applied for. British Library Cataloguing in Publication Data A catalogue record from the British Library has been applied for. ISBN: 0 444 50924 0 (Volume) ISBN: 0 444 80151 0 (Series) ISSN: 0069 8032 The paper used in this publication meets the requirements of ANSI/NISO Z39.48 -1992 (Permanence of paper) Printed in The Netherlands

v

PREFACE TO VOLUME 42

There is a history in all men’s lives. Shakespeare, Henry IV, Pt. 2 History is the Essence of Innumerable Biographies T. Carlyle, On History

Perhaps one of the most exciting events in science in our times has been the unprecedented development of biology, more exactly of molecular biological sciences ^ biochemistry, molecular biology, cell biology, biophysics, culminating in today’s functional genomics and proteomics. These developments have been so explosive in speed that they have produced the unique situation that these disciplines have come of age at a time when their founding fathers or their immediate scientific sons or daughters, are still alive and active. It seemed, therefore, self-evident to ask them to write, for the benefit of both students and senior scientists, about their scientific lives. With this idea in mind one of us (GS ^ who, incidentally, learnt for his biology exam, 1946, that genes are presumably proteins, the tetranucleotide structure of nucleic acids making them unlikely candidates as store houses of information) had already edited two volumes for John Wiley & Sons, which had, however, a different format. In Elsevier’s Comprehensive Biochemistry series, Vol. 35 and the following (with one exception) are composed of Personal Recollections. The editors hope that they convey to the reader lively, albeit occasionally subjective, views, or at least glimpses,

vi

PREFACE

of the environments in which the authors have operated and which have brought about new scientific concepts and significant advances in knowledge. The editors considered it presumptous to give the authors narrow guidelines: directness and straightforwardness should be given priority over uniformity. Indeed, most if not all chapters published in these volumes convey, alongside scientific information, the flavor of the authors’ personalities. Most scientists who have contributed to the explosive development of the biological molecular sciences in the twentieth century (although not necessarily all the authors in this volume) lived and operated during the so-called ‘‘Age of Extremes’’ ^ like other citizens they may have gone through one or more world wars, civil wars, political and other revolutions, their aftermath, including need and distress, lack of freedom and disrespect of human rights. It must have been very difficult in such circumstances always to put on the white lab coat, and even to find the straight and narrow path between the wolves and their prey. Worse, a few ^ very few ^ biologists chose a path within the wolves’ realm, or on its boundary. We have welcomed the possiblity of reproducing three texts written by Mu«ller-Hill (Chapter 10). The young ^ to whom the future belongs ^ have the right and duty to be told to remember: those peoples or persons who forget their past are condemned to experience it again. By now the reader knows what he will find in this volume: a series of chapters primarily devoted to molecular biological sciences (many of them happen to center on protein chemistry ^ a fitting subject for the year in which John Edsall would have celebrated his 100th birthday, and 100 years since Emil Fischer and Franz Hofmeister proposed that the amino acids in peptides and proteins are bound via the peptide bond), but also reminders of the tragedies of the twentieth century, in some of chapters, and particularly in one of them. The editors wish to express their gratitute to the authors for the beautiful work they have done and the fascinating stories

PREFACE

vii

they have to tell. Finally, we would like to thank all those concerned at Elsevier for managing the production of the volume so efficiently. Swiss Institute of Technology, Zu«rich, Switzerland, and University of Milan, Italy, 2002 School of Biochemistry and Molecular Biology, University of Leeds, England, 2002

Giorgio Semenza

A.J. Turner

This Page Intentionally Left Blank

ix

CONTRIBUTORS TO THIS VOLUME B. BLOMBA«CK Karolinska Institutet, Coagulation Laboratory, Nobels va«g 12a, Stockholm, SE-171 77, Sweden

LEOPOLDO DE MEIS Departamento de Bioqu|¤ mica Me¤dica, Instituto de Cie“ncias Biome¤dicas, Centro de Cie“ncias da Sau¤de, Universidade Federal do Rio de Janeiro, Cidade Universita¤ria, RJ, 21941 590, Brazil

HANS JO«RNVALL Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden

HANS KLENOW Department of Medical Biochemistry and Genetics, The Panum Institute, The University of Copenhagen, Copenhagen, Denmark

TORVARD C. LAURENT Department of Medical Biochemistry and Microbiology of the University of Uppsala. BMC, BOX 582, SE-751 23 Uppsala, Sweden

URIEL Z. LITTAUER Department of Neurobiology,Weizmann Institute of Science, Rehovot 76100, Israel

BENNO MU«LLER-HILL Institut fu«r Genetik der Universita«t zu Ko«lnWeyertal 121, D-50931 Ko«ln, Germany

x

CONTRIBUTORS

PHILIP J. RANDLE Nuffield Department of Clinical Biochemistry, Clinical Laboratory Sciences, Radcliffe Infirmary, Oxford OX2 6HE, UK

VLADIMIR P. SKULACHEV Department of Bioenergetics, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia

CHARLES TANFORD Emeritus Faculty, Duke University, Durham, N.C., USA

xi

CONTENTS VOLUME 42

A HISTORY OF BIOCHEMISTRY Selected Topics in the History of Biochemistry Personal Recollections.VII Preface to Volume 42................................................................. Contributors to this Volume .................................................... Contents ....................................................................................

v ix xi

Chapter 1. FiftyYears in theWorld of Proteins by C. TANFORD ............................................................................

1

Origins ......................................................................................... Cohn and Edsall ............................................................................ My First Project in Protein Science ................................................. The Invention of Polymer Science? ................................................... Macromolecules: Theoretical Research with J.G. Kirkwood ................ Antibodies .................................................................................... Genetic Basis for Antibody Diversity ............................................... Subunits and Allosterism ............................................................... Micelles and Membranes ................................................................ Membrane Proteins ....................................................................... Philip Handler and the Einstein Statue ............................................ Eastman Professor at Oxford .......................................................... Popular Writing: Travel Books ......................................................... Historical Perspective .................................................................... References ....................................................................................

1 6 11 12 14 19 23 24 28 33 36 39 42 46 49

Chapter 2. Proteins, Life and Evolution by H. JO«RNVALL ..........................................................................

53

xii

CONTENTS

Abstract ....................................................................................... Introduction ................................................................................. CVof the Guide Through the Text..................................................... Early Days, Scientific Fathers .......................................................... Cambridge, the Mecca at the Start ................................................... Development of Protein Sequence Analysis ....................................... Separation Techniques and Continued Progress ................................ Amino Acid Analysis, Complementary Approaches, and Further Development ................................................................................. Correlation with 3 -D Structures ...................................................... Developments in Molecular Genetics ............................................... Molecular Evolution: Development from Early ‘‘Diagonals,’’over the Atlas Issues, to Bioinformatics ........................................................ Mass Spectrometry ........................................................................ Present Situation, Including Comments on Karolinska, Nobel, and Funding ................................................................................. Summary of Revolutions ................................................................. Perspectives .................................................................................. Acknowledgments.......................................................................... References ....................................................................................

53 54 55 56 57 59 65 66 72 76 79 85 89 93 94 97 97

Chapter 3. PehrVictor Edman: The Solitary Genius by B. BLOMBA«CK .........................................................................

103

Nature is the Guide ........................................................................ Science Takes Hold......................................................................... Birth of an Idea ............................................................................. Idea Becomes True ......................................................................... Automation Must Come .................................................................. End of Journey .............................................................................. Edman, The Person ........................................................................ Acknowledgments.......................................................................... References .................................................................................... Selected References from Pehr Edman’s Biography ............................

105 106 109 113 120 125 127 131 132 133

Chapter 4. A Privileged Life by T.C. LAURENT .........................................................................

137

Introduction ................................................................................. Family Background ........................................................................ The Karolinska Institute ................................................................

137 138 140

xiii

CONTENTS Medical Studies ........................................................................... Experimental Histology 1949^1951 .................................................... Chemistry Department 1951^1953 ...................................................... Retina Foundation 1953^1954 ........................................................... Back in Stockholm 1954^1958 ...........................................................

140 141 143 147 149

Establishing a Research Career .......................................................

152

Retina Foundation 1959^1961 ........................................................... Uppsala 1961^1966 ........................................................................

152 156

.......................................................................

164

Preludes..................................................................................... New Duties ................................................................................. Research and Graduate Students 1966^1980 ......................................... The Turning Point: Australia 1979^1980 .............................................. Hyaluronan Research 1980^1996 ....................................................... New Developments ........................................................................ Interaction with Industry ...............................................................

164 165 172 179 181 187 190

The Royal Swedish Academy of Sciences ...........................................

192

The Academy ............................................................................... President 1991^1994 ...................................................................... The Nobel Prizes .......................................................................... Some Consequences of the Academy Work ...........................................

192 195 203 208

The Wenner^Gren Foundations ........................................................ Concluding Thoughts ..................................................................... References ....................................................................................

209 214 217

Permanent Position

Chapter 5. RNA Enzymology and Beyond by U. Z. LITTAUER ....................................................................... 221 Growing Up ..................................................................................

221

From Tel Aviv to Jerusalem ............................................................. Hebrew University and Hemed .........................................................

226 230

Weizmann Institute of Science ........................................................ St. Louis: Polynucleotide Phosphorylase ........................................... Isolation of High Molecular Weight Ribosomal RNA........................... The Single-Stranded Nature of rRNA ............................................... Early Studies on the Secondary Structure of RNA ............................. tRNA Regulation ...........................................................................

233 236 239 240 242 245

tRNA Nucleotidyl Transferase.......................................................... The IUB Congress in Moscow .......................................................... Purification of tRNA ..................................................................... The Function of Modified Nucleosides in tRNA .................................... Phage-induced tRNA ..................................................................... Isolation of tRNA Genes .................................................................

245 246 247 248 251 252

xiv

CONTENTS

PNPase Research Applications ........................................................

253

Monofunctional Substrates of PNPase................................................ The Tale of the Poly(A) Tail ............................................................. Plant Viral RNA ...........................................................................

253 254 256

Differentiation of Artemia salina Cysts ............................................. Establishment of the Department of Neurobiology .............................

257 259

Neuroblastoma as a Model System for Neuronal Differentiation ................ Surface Glycoproteins .................................................................... Plasminogen Activators .................................................................

260 261 263

Microtubule Proteins .....................................................................

264

Control of Tubulin Expression in the Developing Nervous System ........................................................................... Tubulin Microheterogeneity ............................................................ Microtubule-Associated Proteins ...................................................... A Unique MAP2 Species in Human Neuroblastoma Cells ......................... Neuroblastoma Laminin Binding Proteins ..........................................

264 265 268 269 271

Promoting International Links ....................................................... References ....................................................................................

272 280

Chapter 6. Some Selected Recollections from a Life with Biochemistry by H. KLENOW ............................................................................ 285 Introduction ................................................................................. Student years at the University of Copenhagen .................................. Experiments with Penicillin ........................................................... Pupil of Herman Kalckar and Experiments with Xanthine Oxidase ..... Excursion to Two European Strongholds in Biochemistry ................... Ribose 1,5 -Diphosphate .................................................................. National Institutes of Health 1952^1953 ............................................ Back in Copenhagen ...................................................................... Cancer Research ........................................................................... Experiments with 20 -Deoxyadenosine ............................................... Experiments with 30 -Deoxyadenosine ............................................... Back to the University of Copenhagen .............................................. Experiments with other Congeners of Adenosine ............................... Experiments with DNA Polymerase from E. coli................................. Ribonucleotides Again ................................................................... Regulation of Cellular Content of Nucleoside Triphosphates ............... Conclusion .................................................................................... References ....................................................................................

285 286 289 289 292 293 295 297 299 300 303 305 306 307 312 313 315 316

CONTENTS

xv

Chapter 7. A Risky Job: In Search of Noncanonical Pathways by V.P. SKULACHEV ......................................................................

319

Introduction ................................................................................. In the Beginning ...........................................................................

319 319

Short Excursion to CV ................................................................... Student Years: Junior Courses .......................................................... The Student Year: S.E. Severin and V.A. Engelhardt ...............................

319 320 321

Challenge 1: Nonphosphorylating Respiration in Thermoregulation .....

324

Attacking the Major Bioenergetic Paradigm ........................................ Thermoregulatory Uncoupling: History of Discovery. Sergey Maslov ............................................................................. Brown Fat and UCP1 ..................................................................... The ATP/ADP Antiporter ................................................................ UCPs in Tissues Other than Brown Fat ............................................... The Story is not Finished Yet ...........................................................

324 325 328 329 331 334

Challenge 2: Initially any Respiration is Nonphosphorylating; Chemiosmosis ...............................................................................

337

From Uncoupling to Coupling Mechanism: The Adenine Hypothesis ................................................................. Chemiosmotic Hypothesis: Peter Mitchell ........................................... A.N. Belozersky Bioenergetics, a Department in MSU and a Branch of Biological Sciences ................................................... Protonophores: Efim Liberman ........................................................ Membrane Potential Revealed by Penetrating Ions ................................ Proteoliposomes........................................................................... Bacteriorhodopsin: W. Stoeckenius and E. Racker .................................. Bacteriorhodopsin: L. Drachev, A. Kaulen, Yu. Ovchinnikov, and H. Khorana ..................................................... Protometer: A. Glagolev and S. Bibikov .............................................. Electric Motor Invented by Bacteria .................................................. Extended Mitochondria as Intracellular Electric Cables ......................... Hþ as a Convertible Energy Currency: Hþ Buffering by Naþ/Kþ Gradients ................................................

337 339 340 341 343 345 347 348 350 352 353 357

Challenge 4: Energy Coupling without Hþ: The Sodium World..........................................................................

359

Mystery of Alkaliphilism ................................................................ Three Laws of Bioenergetics ............................................................

359 363

Challenge 5: Respiration Which Kills Us...........................................

364

How to Prevent Formation of Reactive Oxygen Species ........................... Production of Poisons is the Biological Function of Some Respiratory Enzymes .................................................................................... From Weismann to Apoptosis ........................................................... Mitochondria and ROS in Apoptosis: Mitochondrial Suicide (Mitoptosis) ......................................................................

364 368 369 371

xvi

CONTENTS

Programmed Death at Supracellular Level: Bystanders; Organoptosis......... Phenoptosis, Programmed Death of Organism ...................................... Phenoptosis of Multicellular Organisms that Reproduce Only Once .................................................................... Phenoptosis of Repeatedly Reproducing Organisms: Why Aging is Slow? ....................................................................... How may Age-dependent Phenoptosis be Organized? Telomeres ................................................................................... Mutants Who Live Longer ............................................................... ‘‘Samurai’’ Law of Biology ...............................................................

380 381 383

Challenge 6: Life without Science ....................................................

386

Directorship in the Belozersky Institute ............................................. Election of the Rector of Moscow State University ................................. The Soros Foundation .................................................................... Some Other Duties ........................................................................

386 387 389 395

Acknowledgments.......................................................................... References ....................................................................................

401 401

Chapter 8. FiftyYears of Biochemistry as Enjoyed by a Medical Biochemist Motivated by an Interest in Diabetes by P.J. RANDLE ...........................................................................

411

Beginnings ................................................................................... Research ......................................................................................

411 420

Beginnings: Cambridge Biochemistry Department, 1952^1960 ................... Evolution and Revolution 1960^1964 .................................................. The Move to Bristol....................................................................... Biochemical Research at Bristol 1964^1975 .......................................... Acylglycerols in Muscle .................................................................. Fatty Acid Oxidation Inhibitors ........................................................ Pancreatic Islets in vitro ................................................................. Reversible Phosphorylation in the PDH Complex: 1971^1975 ......................

420 424 427 428 428 429 429 430

International Protein Phosphorylation Group ................................... Other Activities at Bristol ...............................................................

432 432

External Examining ...................................................................... Service on General Medical and Dental Councils .................................. Work for the Department of Health: Knighthood ...................................

432 433 434

Biochemistry Research at Oxford 1975^1993 ...................................... Mechanisms Mediating the Role of the PDH Complex in Regulating Glucose Oxidation with Special Reference to Inhibitory Effects of Starvation and Diabetes.................................... Regulation of the Mitochondrial Branched Chain 2 -Oxoacid Dehydrogenase Complex (BCDH Complex) by Reversible Phosphorylation ............................................................................

435

375 376 379 379

436

438

CONTENTS

xvii

Other Contributions and Services....................................................

441

Learned Societies ......................................................................... Retrospection ..............................................................................

441 441

References ....................................................................................

442

Chapter 9. My Happy Days with Lac Repressor ^ in a DarkWorld by B. MU«LLER-HILL ....................................................................

447

Family and Childhood .................................................................... Chemistry in Freiburg and Munich .................................................. With Howard Rickenberg in Bloomington, Indiana, 1963^1964 ............ In the Watson^Gilbert Group 1965^1968 ............................................ The First Ten Years in Cologne 1968^1977 .......................................... The Next Ten Years in Cologne 1978^1987 .......................................... The Last Ten Years Before my Retirement in Cologne 1988^1997 .......... The First Four Years after my Retirement 1998^2001........................... Outlook ........................................................................................ References ....................................................................................

448 453 458 461 468 477 483 490 492 494

Chapter 10. A Dark Side of Science in Difficult Times by B. MU«LLER-HILL ....................................................................

501

The Blood from Auschwitz and the Silence of the Scholars by B. MU«LLER-HILL ........................................................................... 502 Abstract ....................................................................................... Von Verschuer, Excellent Scientist and Outspoken Anti-Semite ........... Von Verschuer as a Practising Anti-semite and Racist ........................ Von Verschuer, Director, and Mengele, Former Post-doc, Now Guest and Collaborator, in the Berlin Kaiser Wilhelm Institute for Anthropology ................................................................................ The Documents of the Auschwitz Research Project ............................ Mengele in Auschwitz .................................................................... Von Verschuer’s Research Activities in the Records of the Deutsche Forschungsgemeinschaft ............................................ Project I: Genetic Defects in Families and Twins ............................... Project II: Genetic Differences among Jews, Gypsies and Others in Resistance to Infectious Diseases ....................................................

502 504 508

510 513 514 518 519 520

xviii

CONTENTS

1945^1951: Von Verschuer has to Wait for a Professorship ..................... Butenandt as Witness at the IG-Farben Trial in Nuremberg ................ Von Verschuer et al. Back to Normal Academic Life ............................ The Silence of Von Verschuer and his Colleagues, Scientists and Historians .................................................................................... Other Examples of the General Disinterest in the Inglorious Past ........ Is there a Bottom Line? .................................................................. References ....................................................................................

527 531 532 535 538 541 541

Selective Perception: The Letters of Adolf Butenandt Nobel PrizeWinner and President of The Max-Planck-Society by B. MU«LLER-HILL ........................................................................... 548 Abstract ....................................................................................... Introduction ................................................................................. Pre-History ................................................................................... Letters Exchanged between Butenandt and Von Verschuer from September 1944 to September 1945 ................................................... Letters Exchanged between Butenandt and Hillmann: the Destruction of the Top Secret Papers ................................................ War Important Projects of Butenandt .............................................. Butenandt and National Socialism .................................................. Butenandt: Master of Selective Perception........................................ Another View: Opportunisms and Power Hunger ............................... Acknowledgments.......................................................................... References ....................................................................................

548 548 549 554 557 559 564 572 575 577 577

The Fraud of Abderhalden’s Enzymes by B. MU«LLER-HILL ........................................................................... 580 Ute Delchmann and Benno Mu«ller-Hill ............................................ Abderhalden’s Fraud ...................................................................... Why No One Stopped Him .............................................................. Sinister Applications ...................................................................... Behind the Fraud........................................................................... Can it Happen again? ..................................................................... Acknowledgments.......................................................................... References ....................................................................................

580 581 582 584 586 588 589 589

CONTENTS

xix

Chapter 11. The Sarcoplasmic Reticulum Ca2þ -ATPase and the Processes of Energy Transduction in Biological Systems by L. DE MEIS ............................................................................

591

Introduction ................................................................................. Personal Memories ........................................................................

591 593

The Origins and the Choice of a Career .............................................. Tininho ..................................................................................... Postdoctoral Training in the USA ..................................................... The Military Regime and the Hard Years Back Home ............................. Heidelberg .................................................................................. The Bonanza and the Graduate Courses ............................................. The Department of Medical Biochemistry ........................................... Strategies and Great Friends ...........................................................

593 601 602 604 608 610 611 614

The Research Work ........................................................................

616

The First Steps ............................................................................ Phosphoenzyme of High and Low Energy ............................................ Binding Energy and Synthesis of ATP in the Absence of a Ca2þ Gradient ..... The Reaction Sequence .................................................................. Solvation Energy and Phosphate Compounds of ‘‘High’’and ‘‘Low’’ Energy .... Pyrophosphate of High and Low Energy ............................................. Role of Water Activity in the Process of Energy Transduction by Different Enzymes ................................................... The Thermogenic Function of the Ca2þ -ATPase: Uncoupled Ca2þ Efflux and Uncoupled ATP Hydrolysis ........................................................

616 618 622 625 626 630

Science and Education ................................................................... Science and Art .............................................................................

636 638

Illustrated Books ......................................................................... Theater...................................................................................... CD and DVD ...............................................................................

638 639 639

Final Statement............................................................................. References ....................................................................................

640 640

631 633

Name Index................................................................................ 643


G. Semenza and A.J. Turner (Eds.) Selected Topics in the History of Biochemistry: Personal RecollectionsVII (Comprehensive BiochemistryVol. 42) 2003 Elsevier Science B.V.

1

Chapter 1

FiftyYears in theWorld of Proteins CHARLES TANFORD Emeritus Faculty, Duke University, Durham, N.C., USA

Origins I was born in Saxony, in Halle-an-der-Saale, in 1921, although my father and mother actually lived in the big city of Leipzig, 45 km away. My mother was in fact a Leipzig girl; my father, though born in Poland (of Jewish parents), had lived in Leipzig for a decade or more and had a thriving business there. Our family moved to England in 1930 and my father lived in England until he died in 1971. Why was I born in Halle? It probably reflects nothing more than a shortage of maternity beds in Leipzig; 1921 was only three years after the end of a devastating war. A few years ago, I wrote to the British Home Office for information about my father’s background. They duly sent me the data in their records. My father was born in 1888, in the town of Brzesko, about 50 km east of Krakow. The Krakow area was part of Austria at that time and the Jewish community had been exceptionally well treated there. Since 1867 they had held full Austrian citizenship, which included voting rights and the obligation of military service. My father was in the Austrian army in the first world war and received shrapnel wounds that destroyed Mailing address: Tarlswood, Back Lane, Easingwold,York YO61 3BG, UK

2

C. TANFORD

his hearing in one ear. He took pride in being an Austrian (rather than a German) even after he lived in Leipzig. I remember when I was just a few years old that the first car he ever bought was Austrian-built (brand name Steyr, if I recall correctly). He was still an Austrian citizen when he moved to England and became naturalized there. One item in the Home Office report that came as a surprise to me was their reference to my father’s marriage in April 1922, in Leipzig. This was 5 months after I was born. I was thus illegitimate, a bastard, as they used to call it. Neither my father nor my mother ever discussed the matter with me, though my mother had plenty of opportunity to be frank after she was separated from my father around 1950, by which time births out of wedlock became almost the normal fashion. My mother moved to the United States with my much younger sister and eventually lived to the age of 99. Which prompts me to tell a lurid tale, dimly recollected as originating from gossipy cousins. The gist of it is that my mother was married to someone else when my father became infatuated with her; her husband was a gambler, who squandered his money at the gaming table; my father followed them to Baden-Baden and waited on the sidelines until the gambler had had a run of disasters at the Casino. He then offered to clear the man’s debts in exchange for ‘‘possession’’ of his wife. All parties seem to have agreed and he carried my mother off on the spot and set up a house with her in Leipzig. Divorce proceedings were slow in 1921 and the passage of more than a year between abduction and marriage does not seem unreasonable. In any case, there is no question about the identity of my biological father. Physical resemblance between us is unmistakable, increasingly so as I grow older. My father’s infatuation with my mother was short-lived. I never knew my parents other than as a bitter, loveless couple, who rarely spoke to each other. They still had sex (early Sunday mornings, I think), but that was their only communication. My father was away on business trips quite often and life at home

FIFTY YEARS IN THE WORLD OF PROTEINS

3

was free of overt tension then. But I shall never forget the chill that descended when he returned ^ my personal fear that I would somehow transgress and be punished by being made to share for some days the icy exclusion of my mother. One transgression would have been any contact with the opposite sex. I went to segregated schools, of course, which was the rule in England, but even close friendship with boys at school was likely to set off parental gloom; the grim spectre of ending up ‘‘in bad company’’ was ever kept before me. After I left home at age 17, I began to have normal opportunities for female companionship, but for many years I retained severe fears and inhibitions. My sex life was substandard, even by old-fashioned criteria, until well into my twenties. My father was clearly wealthy, had been so even before I was born, but I do not know how he made his money ^ he never talked about it; there was no fatherly boasting about how he had come from the back woods of Poland and made it in the commercial world; this was another subject that seemed to be taboo. We lived quite modestly in the context of the times. But poverty was never a threat: I lived through the great depression, but can honestly say that I was never made aware of it. Presumably there might have been money available for all sorts of extravagances, provided there was serious intent. But I never asked. In particular, I never took an interest in science, never longed for a chemistry set, never took clocks apart to see how they worked, never scooped tadpoles from ponds. In fact, I didn’t take much interest in any of my academic subjects. I loved cricket, that most numerical of sports, I collected stamps, I was a pretty good chess player, I read Dickens and Dorothy Sayers, but not much else. I left home (moving an ocean away to the US) when I was 17 and soon discovered a resonance with American society and a vocation for chemistry in particular. I obtained an excellent education and an undergraduate degree in chemistry at NYUs uptown branch in the Bronx; I went on to Princeton for a PhD in physical chemistry.

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My graduate studies were interrupted by a year spent at Oak Ridge, where I participated in a very small way in the creation of the first atomic bomb. I felt a sense of exhilaration when the news broke of its successful use, and my view has not changed in retrospect. Unlike many of my contemporaries, I feel no guilt. Had nuclear energy been first discovered and exploited in time of peace, we would all have celebrated. I am sure even now that humanity will eventually want to reap the as yet hardly imagined benefits that nuclear energy can provide. I went to Brzesko in May 1993 to get a feeling for place and time. I went to the office of registry of births and deaths in Brzesko: they pulled the appropriate volume off the shelf and the record for my father was there ^ handwritten ^ confirming exact date, names of parents, etc. They gave me a birth certificate, which I have kept as a memento. I also have a photo of the state office building in Brzesko, which can be seen to house every bureau under the sun. The office for births and deaths and marriages is called ‘‘Urzad Stanu Cywilnego’’. The birth record listed a house number (city-wide numbering system, presumably), but no street address. There is no Jewish community in Brzesko today and a couple of casual inquiries yielded no information about where such a community might have existed a century ago. Brzesko today is a prosperous-looking attractive town. The best way to try to visualize life there in my father’s time is probably to take Kazimierz (in Krakow) as a model ^ here the State of Poland is engaged in deliberate remembrance and restoration. I have found out since my visit to Brzesko that, although antisemitism has been a chronic ailment throughout Continental Europe ever since the Middle Ages, Galicia was relatively liberal and Jews lived here without interruption, reasonably well respected most of the time, from around 1300 until the 1940s holocaust. They were expelled from the city of Krakow itself in 1494 and resettled in neighboring Kazimierz, but this does not seem to have interfered with their banking and trading activities. The residential segregation was in any case abolished by


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the Austrians and in 1867 full citizenship was granted ^ my father served proudly in the Austrian army, as I have already mentioned. The more prosperous Jews returned to live in central Krakow in large numbers. This brief history of the Krakow area brings into sharp focus the unholy barbarism of the Germans in our own time. All the Jews were driven out of Krakow soon after the Germans occupied the area, first into a crowded ghetto in Podgorze (another district of Krakow), where they were enclosed within high walls and placed under constant police guard. Eventually they joined the masses of other Jews from all over Europe, marked for extermination. As it happens, the most notorious of extermination camps, Oswiecim and Brzezinka (Auschwitz and Birkenau), are in the Krakow area, about the same distance from the city as Brzesko, but in the opposite (westerly) direction. The camps have been preserved, a commemorative museum has been built, etc. They have become part of the normal tourist itinerary, with parking for tour buses, a cafeteria and souvenir shop. The horror is muted thereby, but the barracks and furnaces are still there; they and the photographs in the exhibits serve to preserve our feelings of revulsion ^ the naked women, for example, old and young, some clutching babies, being herded by SS men to the gas chambers. All of which led me to do some soul searching: had my family remained in Leipzig, I and my family would have been within the catchment area of the SS and would most likely not have survived. Have I been properly appreciative of my good fortune? The answer is negative. I was a callous youth, unaware of much of what was going around me. I did not exactly deny my background, but I did deliberately avoid talking about it. It was a decision I made while still a teenager; I did not want to be drawn into camaraderie with (or even identified with) a kind of people who occasionally showed up in our house, who seemed to have just a single topic of conversation, centered on their being Jewish, bemoaning persecution, longing for their

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distant Zion. Nobody at school talked like that, nor did my father’s more typical friends, those with whom he played golf or had lunch in the city. It was actually seeing Auschwitz, in the context of its proximity to benign Brzesko, that has made me fully aware of the horror of the holocaust. I could easily have been part of the gruesome procession. I have no knowledge of demography, but my untutored opinion is that the broad outline of my migration, from Germany to England to America, cannot have been unusual for middleclass people of my generation. I actually know a prominent American musicologist with whom I coincided every step of the way: same secondary school in London, NYU for undergraduate education, Princeton for graduate school. Such precise coincidence of actual institutions is statistically improbable and I take it as evidence that hundreds of us may exist altogether ^ former asylum seekers of sorts ^ who aspired to and attained careers in science and other academic professions far from their native lands. I have never talked to any of them, not even my musicologist ex-classmate, whose name appears occasionally in the press. We have exchanged letters, vaguely expressed an intent to get together, but never did.

Cohn and Edsall My undergraduate thesis in chemistry and my PhD thesis in physical chemistry both focussed on the mechanism of interaction of small molecules in the gaseous state ^ on combustion in a burner flame, in the case of my PhD dissertation. The ‘‘Tanford^ Pease theory of burning velocity’’ was briefly in the limelight, before more elegant theories took its place [1]. My first exposure to proteins and a huge transition from what I had been doing at Princeton came when I went to Harvard, to the medical school in Boston, to the Laboratory of Physical Chemistry related to Medicine, headed by Edwin Cohn and


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John Edsall, which was synonymous with the best in protein chemistry in those days. This career reorientation, which was not viewed with great fervor by most of the Princeton physical chemistry faculty, came about in large part through the arrival in Princeton, during my final year there, of Walter Kauzmann, a new assistant professor. Kauzmann had done his PhD at Princeton a few years earlier, with a theoretical dissertation on optical rotation. He spent the war years at Los Alamos and had then taken some time off to do some reading, to help him decide on his future research. In the course of this he had become enthralled by the treatise, Proteins, Amino Acids and Peptides [2], of which Edwin Cohn and John Edsall were co-authors, and that determined him to become a protein physical chemist. Kauzmann’s enthusiasm was infective. The work I had done on combustion was in a hot field, applicable to the development of jet fuels, and I had lucrative job offers from the industrial sector. But the prospects of novel and exciting research, which I naively equated in my mind with some romantic notions, as a sort of physico-chemical exploration of the vast mysteries of life, outweighed any question of salary and I decided to join Cohn and Edsall’s laboratories if they had a place for me ^ which they did, as a ‘‘post-doc’’, with a stipend less than half of what I could have had in industry. The Cohn and Edsall laboratory, established back in 1920, was a unique entity. A department of physical chemistry in the medical school, a juxtaposition of physics and chemistry with medicine, was unheard of in modern times, probably since the days of Islamic science in Spain in the middle ages. Equally unprecedented was the concept that the new department was to be entirely free of direct teaching duties, with no obligation to participate in the elementary instruction of medical students. Unlike many other academic science centers, the laboratory had flourished during the war years because it had had a vital military function ^ the mobilization of expertise in relation to blood transfusions, the need for which had been foreseen soon after the war began in Europe in 1939, before the US itself

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became a participant. The momentum acquired during the war persisted for a few years after the war and was still in evidence when I came there in 1947. Most importantly, no diversification had taken place. Cohn and Edsall and their associates were still working uniquely on proteins: their fractionation and purification; their individual sizes and shapes and other molecular properties. The department’s single-minded concentration on proteins was unparalleled, with no digression to metabolic pathways, genetics, enzyme kinetics, or other aspects of biochemistry that would have been considered essential concerns had they been part of a typical medical school Biochemistry Department. In this laboratory every experimental result, every day of the week, could hardly be described without reference to a mental picture of protein molecules. The upshot of the experience for me personally was to create in my mind a passion for proteins and an indelible picture of a globular protein ^ the category that includes most enzymes, antibodies, and binding proteins. Fibrous proteins were also being studied in the Cohn and Edsall laboratory (fibrinogen, for example, was of prime interest because of its central role in forming blood clots), but globular proteins interested me the most intensely and continued to do so in the years to come for a different reason, which was that they are the proteins that can be readily crystallized and subjected to X-ray analysis. They were thus capable of yielding ^ and as the years went by they did yield ^ increasingly precise details of molecular structure and organization. The salient features of my perceived molecular model may be summarized as follows: 1. Proteins are macromolecules, molecular weights ranging from around 10,000 to more than a million. 2. In contrast to fibrous proteins, the globular proteins were known to be compact particles of astonishingly small dimensions. Typical particle radii for proteins of moderate


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size were 20^30 —. In terms of a model, this means extremely tight packing, with virtual exclusion of solvent water from the interior. 3. An equally striking aspect of the molecular image was that all proteins are electrolytes, bristling with ionic charges (charges derived from the side chains of acidic and basic amino acids). In the neutral region positive and negative charges must co-exist: in the case of globular proteins, given the compact particle size, they must often be in close proximity, not more than 5 or 6 — apart. It was of course a fuzzy picture: even the -helix had not yet been proposed to lead us to think in terms of organized structural patterns. DNA still existed only in a chemical sense, its relation to proteins unknown. In addition, it must be appreciated that Cohn and Edsall did not create even this low-resolution model nor contribute compelling original work to its creation. It was the product of the efforts of many scientists: the high molecular weight was established by chemical means in the earliest days of protein studies and then confirmed by all conceivable physical methods; the compact folding was most convincingly demonstrated by Svedberg’s measurements of sedimentation rate with the ultracentrifuge; the ionic charges and their dependence on pH had been established both by electrophoresis and by more specific electrometric analysis. Details of the progress towards understanding and description of the critical experiments along the way (with pertinent original references) are given in the recent book on the subject by myself and Jacqueline Reynolds [3]. Edwin Cohn was director of the laboratory and held its purse strings. He was a ruthless manager, regarded by some of his underlings as an evil man without an ounce of human kindness. I did not see him as quite the ogre that some did, for I could see that, whatever his faults, he never compromised with scientific integrity. I remember one occasion when something dear to his

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heart had been demonstrated for the protein serum albumin. But while the work was in progress, a better way of preparing serum albumin had been devised. Cohn insisted that the work be repeated with the better preparation before he would allow publication ^ not very kind perhaps for the post-doc who had spent most of the year doing the experiments, but the sort of undisputable authenticity that one had come to expect was demonstrated. I was not surprised to learn around the time I left Harvard that Cohn needed (and habitually used) a daily injection of epinephrine to maintain the hectic pace of his professional life, of a dosage that would have killed most normal people. Cohn’s mind deteriorated in the last year or two ^ had perhaps begun to do so while I was still there ^ and when he died in 1953 he was found to have had a brain tumor. John Edsall, Cohn’s principal partner, was the opposite of Cohn, the epitome of a scholarly and sometimes absent-minded professor, with no desire to dominate anyone. He had joined the laboratory in 1926, while still a third year medical student at Harvard, under a program designed to give those who were so inclined a research experience to relieve the tedium of purely clinical studies. He never returned to medicine, but continued in basic science, specializing in the use of rather esoteric methods (light scattering, Raman spectra, flow birefringence) to investigate protein structure or molecular shape. Much later, he became editor of the Journal of Biological Chemistry ^ undoubtedly the best editor this journal has ever had ^ and he also started to take an interest in historical aspects of protein science and biochemistry. John and I have remained friends for life. The shining light for me during my Boston years was, however, neither Cohn nor Edsall, but George Scatchard, a professor at MIT without formal academic Harvard appointment, but despite that a vital member of the Harvard group. He was the most memorable among all my mentors, with an unmatched comprehension of solution thermodynamics ^ the modern


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inheritor of the depth of comprehension that J. Willard Gibbs had had 70 years earlier. He was a master of the use of partial derivatives. He made it absolutely clear that they were not merely mathematical symbols, but corresponded to precisely defined operations, observable changes in dependent variables that could be measured experimentally. Scatchard was also a potentially devastating presence at seminars, merciless when he detected confusion or misconceptions hiding behind a glib manner of presentation. I was asked to write his obituary for nature when he died in 1973 ^ anonymously, as was the custom then. ‘‘There was a perpetual frown on his face,’’ I said with reference to his severity, ‘‘deepening in intensity at each point where the speaker was glossing over theoretical or experimental difficulties. At the end of the seminar there was relief when he asked an innocuous question.’’ I remember one meeting, several years after I had left Harvard, when he said ‘‘Good paper, Tanford,’’ after a talk I had given. It was a great moment for me. Although the obituary in Nature was formally anonymous [4], it seemed to be common knowledge that I had written it. John Edsall and Walter Stockmayer, in their biographical memoir of Scatchard for the National Academy of Sciences [5], quote an entire paragraph from the obituary and attribute the authorship to me.

My First Project in Protein Science My own project in the laboratory (assigned by Cohn) was a study of acid^base equilibria in serum albumin: each of the more than 200 individual acidic and basic groups per molecule were titrated sequentially and the corresponding equilibria (apparent pKs) established [6]. But the underlying working model for the globular protein was an intrinsic part of the project because the equation for the overall titration, including effects of ionic strength and temperature, was dependent on it. The equation commonly used at the time had originally been

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derived by Kai Linderstrm-Lang [7] and it represented the protein particle as an equivalent sphere. Electrostatic interaction between different ionic groups was formally incorporated, but only crudely, in terms of the total charge, growing and waning as the pH was changed. These interactions (regardless of how they are cast into equations) depend inevitably on how close charges are to each other and they are thereby related to particle dimensions, the pertinent term in the Linderstrm-Lang equation being the so-called w factor. The interest of everyone in the laboratory was usually confined to the native state of any protein we studied. Cohn was very sensitive to the fragility of the native state (excessively so, it ultimately turned out) and the desire to keep all components in their native states was uppermost in his mind as fractionation and purification procedures were developed. The dimensional parameters that emerged from my titration were, therefore, those already known for the native state: the electrostatic equivalent radius for serum albumin was essentially the same as its hydrodynamic counterpart, determined by diffusion or sedimentation. A few years later, after I had left Harvard, I extended my acid^base titration study to denatured states of serum albumin. The w parameter used for the native protein, of course, no longer fitted the data; the new value that was needed required that charges be further apart, i.e., overall molecular dimensions had to be increased. On the basis of my results I was able to equate denaturation with molecular expansion ^ unfolding of the tight native structure. We went on to confirm our conclusion by use of more conventional hydrodynamic tools [8].

The Invention of Polymer Science? The most important feature of the working model for globular proteins that was imprinted on my mind was the fact that proteins are macromolecules ^ a fact as old as protein


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chemistry itself, generally accepted for 100 years or more. I was reminded of this just recently, when a contradictory historical opinion emerged [9]. On 19 April 1999 an ‘‘International Historic Chemical Landmark’’ was designated at the University of Freiburg in Germany, under the joint auspices of the Gesellschaft Deutscher Chemiker and the American Chemical Society. Its purpose was to recognize the ‘‘amazing accomplishment’’ of Hermann Staudinger, erstwhile member of the Freiburg faculty, who, according to legend, single-handedly ‘‘invented’’ the macromolecular concept in 1920 and fought for it against tenacious opposition. ‘‘Grudging acceptance of the idea came about only in the 1930s.’’ A symposium was held to mark the occasion; the ceremony began and ended with fanfares delivered by a brass ensemble, ‘‘a fitting acclamation for so great an achievement.’’ This legendary role played by Staudinger may accurately reflect historical opinion among many industrial organic chemists, but protein chemists, as I have said, have known that proteins are macromolecules for more than a century [3]. Edwin Cohn had certainly accepted it as established when the laboratory at the medical school was first created in 1920. In 1926, J.B. Conant, then professor of organic chemistry, soon to become Harvard’s president, was impressed by a German report that the freezing point depression of proteins dissolved in phenol was unexpectedly large, corresponding to molecular weights of only a few hundred. This suggested that proteins might be colloidal aggregates of intrinsically small molecules, phenol breaking up the larger particles normally seen in aqueous solution. Cohn felt that such a heretical idea needed to be challenged: Cohn and Conant worked on the problem together [10] and showed that the German result was an artefact, arising from failure to maintain constant thermodynamic water activity. When the measurement was repeated at controlled constant water activity, the result obtained was conclusive: proteins had the same high molecular weight in phenol as in aqueous solution.

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Macromolecules: Theoretical Research with J.G. Kirkwood I was still of course a physical chemist. My two-year stint at a medical school did nothing to alter my identity. My research interest in proteins was undoubtedly unusual for the typical physical chemist in a chemistry department, whose undergraduate classes held a mixture of aspiring industrial chemists and chemical engineers, but when it came to what the research was about, it was clearly the physical chemistry of proteins ^ the physical state of protein molecules, the thermodynamics of their behavior in solution ^ that fascinated me, much more than their biological function. When, at the end of my postdoctoral appointment, I looked for a permanent position, my search was confined to Chemistry Departments; I ended up at the University of Iowa, teaching undergraduate physical chemistry and graduate courses in thermodynamics and kinetics. Nothing better illustrates the uniqueness of the Cohn and Edsall group in the academic world ^ in 1949 ^ than the fact that it would never have occurred to me to consider a job in a Biochemistry Department or in any way connected to a medical school. (The academic world would change dramatically in this regard in the next 10 years, but I was of course unable to predict that.) Teaching at Iowa took up six lecture hours per week, plus laboratory supervision, which was less demanding. I took special pride in meeting my early morning physical chemistry class, which involved a one-mile walk from home, regardless of the weather, often with deep snow on the ground and temperatures down to 20 F. And the students, too, all took pride in missing no classes and in being there on time! (Do they still have classes meeting regularly at 8 o’clock in the morning?) (Figure 1). For me, despite the heavy teaching commitment, there was still time for research; and my research students always had a wealth of opportunities in chemical industry after they got their degrees ^ proteins are surely like polymers, the visiting talent recruiters


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Fig. 1. The author, explaining a fine point of physical chemistry. My ‘‘victim’’ here was Jean-Pierre Changeux.

would argue. And polymers were the big thing at Du Pont and Cyanamid and elsewhere in the industrial world. It was during my appointment at Iowa that I started writing my textbook, Physical Chemistry of Macromolecules [11]. I felt (correctly) that I lacked the background that I needed to expand my research on the physical chemistry of proteins beyond what I had done at Harvard. Existing books or reviews were inadequate, so I wrote my own ^ it seemed to me that I didn’t have much choice. My contacts with representatives of chemical industry were actually a factor in prompting me to undertake the task. The industrial recruiters were invariably impressed that viscosity measurements were an important tool in our research, because viscosity was also a vital tool in polymer chemistry, being used to measure molecular weights of polymers in solution. But the problem was that this was not an

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application which was compatible with what I knew about the viscosity of protein solutions. On the basis of Einstein’s equation and other fundamentals in the Cohn and Edsall doctrine, viscosity in protein solutions should be independent of molecular weight, but should instead be a measure of ‘‘shape’’and/or hydration ^ particularly dramatic in its ability to demonstrate the difference between fibrous and globular proteins of the same molecular weight [2,12]. The theoretical basis for this discrepancy between the polymer and protein chemist’s uses of the same experimental tool required a clear understanding of macromolecular dynamics, which, with no expert help at hand, I had to work out for myself from the available literature. It was difficult for me, and many other basic aspects of macromolecular physical chemistry generated equally challenging problems. The book eventually took eight years to write and ultimately sold more than 25,000 copies. It is likely that my struggles in working things out for myself (usually going back to original literature) contributed to the book’s ultimate readability, making it easier for the reader to appreciate the fundamentals from the standpoint of someone approaching the subject for the first time. The book almost did not come to be published, at least by John Wiley and Sons, the presumptive publisher during the years of writing; I have told that story in an earlier article [13]. For my sabbatical year (1956^1957) I enjoyed the benefits of a Guggenheim fellowship and spent the period at Yale University with J.G. Kirkwood, one of the foremost theoretical physical chemists of the day. My proposed project was to recast the calculation of the electrical work of charging a protein molecule as a function of pH: the equation known to me at the time was the one derived by Kai Linderstrm-Lang in 1924 ^ an extension of the Debye^Hu«ckel equation for simple ions ^ in which the total charge was treated as uniformly distributed over the surface of a sphere [7]. Actual protein charges, of course, have discrete locations: carboxyl groups derived from aspartic and glutamic acid side chains, amino groups derived from lysine side chains, etc., and to take this into account requires a much


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more sophisticated mathematical treatment than a theory based on uniform charge distribution. Kirkwood was an authority on the statistical mechanics of macromolecules and had actually had previous experiences with protein chemistry. He and George Scatchard had been contemporary postdoctoral fellows in Germany, partly in the laboratory of Peter Debye, and had published a paper together (in German) in the Physikalische Zeitschrift, which extended the Debye^Hu«ckel theory of ionic interactions to multipolar ions [14]. Kirkwood later became a research associate at MIT for a couple of years and further extended this work into a classic paper that dealt specifically with amino acids and proteins [15]. Through this connection Kirkwood had become, in effect, an adjunct member of the CohnÊdsall family, and he had contributed a brilliant chapter on dipolar ions to the CohnÊdsall treatise. Thus, when I came to Yale, Kirkwood immediately understood my problem and knew exactly how to deal with it. It took only a couple of hours of initial discussion to set me off on a whole year’s work. I had, of course, an ulterior motive for my undertaking. The forces that led to the folding of polypeptide chains into the compact particles of globular proteins had not yet been established. Intramolecular hydrogen bonds, first advocated by Pauling and Mirsky in 1936, were everybody’s favorite candidates and they took on the mantle of virtual certainties in people’s minds after the structures of the -helix and -sheet for the polypeptide backbone were brought forward in 1951. But it was generally recognized that backbone folding was not enough by itself to produce a globular particle and side-chain hydrogen bonds (e.g. carboxyl^tyrosyl) were invoked to provide the final tightening. This widely accepted mechanism was testable in a sense because it would be expected to modify the pK values of the affected acidic and basic groups and thereby change the simple model used for analysis of protein titration curves, which assumed that all groups with a common origin (e.g. all lysine amino groups) would have the same intrinsic pK ^ the pK value

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before electrostatic interactions were taken into account. Deviations of the right magnitude from the predictions of the model were indeed known from experimental data, but my point of contention was that they didn’t necessarily arise from variation in the intrinsic pK; they could just as well have their origin in variable electrostatic interactions due to irregular spacing of individual acidic and basic groups. I was able to demonstrate this possibility quantitatively in my calculations at Yale by varying the spacing of groups in a simple geometrical model and using the equations derived for discrete charges. The calculations were laborious. There were no computers then and Kirkwood didn’t even have a mechanical calculator ^ tables of logarithms were still used to multiply and divide, and the values of Legendre polynomials (which occurred as factors in the equations) were copied by hand from tabulations in heavy books that had been prepared by the WPA (works progress administration) as part of Franklin D. Roosevelt’s program to alleviate unemployment and lift America out of the great depression. It is no exaggeration to say that the calculations that took me months to carry out would today be done in minutes. But the labor was worthwhile: an important purpose was served. Other fundamental considerations showed, around that time, that the hypothesis of side chain hydrogen bonds as a force for intramolecular cohesion was unlikely to be valid. My calculations demonstrated that effects of the same magnitude could be explained on the basis of the discrete charge model [16]. Being a working member of a family of theoreticians was a revelation. Kirkwood was not the only senior professor at Yale who was a theoretician: Lars Onsager, who later (in 1968) won the Chemistry Nobel Prize for his work on the thermodynamics of irreversible processes, had the office next to mine and half a dozen students and postdoctoral associates made up a sizable and congenial group. I learned that experimental data alone were not the bedrock on which hypotheses succeeded or foundered. Mathematical equations encompassing the body of


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knowledge in theoretical physics, facts that had been previously established and had stood the test of time, were equally powerful criteria.

Antibodies As I have said, I was still a physical chemist. I was working with protein molecules that were constituents of living organisms ^ even essential catalysts of the whole process of life ^ but I was still looking at them as physical entities, composed of atoms, electrostatic charges, etc. I mentioned earlier in this account that I had been motivated to work in the Cohn and Edsall laboratory by romantic notions to the effect that physico-chemical understanding of proteins would provide useful clues for exploration of the vast mysteries of life, but I had never imagined that I would myself participate in such exploration. I had fondly imagined that if my work helped people to understand problems like that of the mechanism of folding of polypeptide chains, others would take over from me and put that understanding to good use. Nothing I did in my own laboratory had any direct connection to the mysteries of life. When I was invited in 1960 to become professor of physical biochemistry at Duke University, it was a challenge: I was expected to make my work directly relevant to the mechanism of enzyme action or other important life processes. Could I adapt my mindset to do so? Becoming part of a medical school faculty was itself a huge change. I found that even the basic scientists in a biomedical environment had a hunger for innovative answers to problems and less concern for critical assessment of the steps used to obtain such answers. When Harold Scheraga had lectured at Iowa, a few months earlier, on the subject of his hydrogen bond theories for explaining the specificities of protein structure^function relations; he was vigorously challenged in the

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postlecture discussion about the free energy of hydrogen bond formation which was a crucial factor. He used a value of about 6 kcal/mol per bond, which was within the accepted range for hydrogen bond formation in a vacuum, but surely could not apply to hydrogen bonds within proteins ^ at least not to globular proteins, which were normally studied in an aqueous environment. Here the unbonded reference state, with which the internally hydrogen-bonded state was to be compared, was not really ‘‘unbonded,’’ but would in fact be hydrogen-bonded to water molecules. Bond formation within the protein fabric would be an exchange of one hydrogen bond for another and this line of reasoning would lead theoretically to a much smaller free energy change; actually near zero as a first approximation. (Linus Pauling, incidentally, had made the same error when he first proposed the importance of intramolecular hydrogen bonds in 1936 [17].) When he lectured at Duke, Scheraga was viewed in a different light: in a sense he had preempted the entire field of the relationship between protein structure and biological function; he had concrete answers, all based on plausible patterns of specific hydrogen bonds, different patterns for each function. It was all respectable. If they worked, if predictions fit the data, why shouldn’t they be right? Questions asked after seminars tended to be guided by this principle. They often referred to the discussant’s own research ^ could tyrosine be hydrogen-bonded in this or that particular enzyme? Could that explain yesterday’s anomalous results in his laboratory? In 1963 Ed Buckley, a member of the Department of Medicine, a practising immunologist, asked to spend a year in my laboratory. We discussed various ongoing projects he could join, but finally settled on a new project, an investigation of immunoglobulins, the very proteins that were at the heart of Buckley’s own medical interests. A postdoctoral fellow, C.A. Nelson, and a graduate student, Phil Witney, joined the project and that is how I got involved for the first time in research which impinged directly on questions of biomedical importance.


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The first project ^ and this was in line with Buckley’s intent to learn how to do protein chemistry ^ was to characterize the immunoglobulin molecule (serum -globulin) in solution. This matter had puzzled me for some time, for all hydrodynamic measurements were consistent in indicating that the immunoglobulin molecule was very compact, but not quite as compact as the typical globular protein. The so-called frictional ratio ^ the ratio of the Stokes radius to that of a sphere of the same molecular weight ^ had the value of 1.45, compared to around 1.2 for most globular proteins. This anomaly was evident even in experiments that were not intended for study of globular proteins, but rather designed as controls in the characterization of fibrous proteins: fibrous proteins displayed prominent flow birefringence in aqueous solution, but globular proteins did not ^ highly viscous media were required. It was found that 90% glycerol had to be used for serum albumin and 70% glycerol for -globulin. No glycerol at all was needed for fibrinogen, so the difference here was not important, but the result did indicate again that whereas immunoglobulins were definitely not grossly extended, nothing like fibrous proteins, they were nevertheless not quite as tightly folded as egg albumin, hemoglobin, etc. [18]. On the other hand, the well-known molecular fragments that could be obtained from immunoglobulin by certain proteolytic enzymes did not have this anomaly: their frictional ratios were 1.21 and 1.24, respectively. These fragments were known to carry the antibody specificity of the parent molecule and there was much evidence to suggest that fragmentation occurs without significant local structural change. The available equations for interpretation of hydrodynamic data were limited. Commonly ^ when one was looking at hydrodynamic data without other considerations an ellipsoidal model was used, in which the deviation from spherical shape is described in terms of the ratio between major and minor ellipsoidal axes, but this was purely a formalism. It was generally understood, for example, that uniform expansion without

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Fig. 2. Immunoglobulin shape and domain structure [19]. The present conventional names are ‘Fab’ for the domains carrying the antibody site (I and II) and ‘Fc’ in place of III.

change in symmetry would produce a similar experimental effect. In the case of immunoglobulin, after much debate and taking functional needs into account, we finally agreed on the model shown in Figure 2 ^ which indeed ascribes increased friction to extension of overall radius without marked asymmetry [19]. The model has a central hinge to provide flexibility for antibodyântigen interaction. It was subsequently confirmed more directly by electron microscopy byValentine and Green [20]. I first presented these results at a symposium at which the principal speaker was Gerry Edelman, who was later (in 1972) to win a share of the Nobel Prize for his work on immunoglobulins. Edelman referred to molecular shape in his lecture, but did so in terms of the solid ellipsoidal model, apparently believing it was a reasonably close representation of the true shape [21]. Taking this model at face value meant that the immunoglobulin molecule was pictured as a long stiff cigar-shaped body; and Edelman had put the two binding sites for antigen at its distal ends, which made them about 240 — apart ^ not exactly suitable for the sort of adaptability in the formation of antibodyântigen


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precipitates that would be desirable. Besides, there was strong evidence against a rigid model. Rotational relaxation times of fluorescent markers attached to the binding sites had been measured in several laboratories and they were very fast, suggesting that the entity to which the marker was attached was relatively small, about the size of one of the antigen-binding domains ^ indicating that the domains were able to rotate independently of each other. I derived some satisfaction from being able to discuss the topic in more enlightened terms. By the time of his Nobel Award address, Edelman of course had the correct Y-shaped overall conformation, but he made no reference to our work, nor even to the electron microscopic picture of Valentine and Green, which was effectively a direct photograph, more easily appreciated than our conclusion that was reached by way of interpretive equations.

Genetic Basis for Antibody Diversity It was not possible in the 1960s to be involved with immunoglobulins without being aware that there was a more important unsolved problem than that of molecular shape. At that time it was still generally believed that the general axiom of protein chemistry, that amino sequence uniquely determines threedimensional structure and thereby function, did not apply to antibodies ^ it was thought implausible that the huge number of distinct immunological specificities that were known to exist could be matched by an equally huge number of distinct genetically inherited genes. Instead, the complementarity theory suggested by Linus Pauling in 1940 still held sway: antibody diversity was thought to be generated by an instructional mechanism, whereby the presence of an infecting agent (antigen) could influence a single amino acid sequence to take up any number of different structures uniquely complementary to the invading substance [22]. This was of course an example of pure speculation, without supporting evidence at all. But

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Pauling had captured public imagination with his -helix and tended to be believed no matter what he said. Coming upon the idea freshly by virtue of the foray into immunoglobulin structure just mentioned, I did not find Pauling’s idea plausible, given the increasingly firmly established doctrines about the genetic process and the mechanism of protein biosynthesis that went with it. These doctrines were still recent knowledge in the 1960s, perhaps not fully tested, but they carried conviction. Direct evidence related to antibody diversity was needed. A graduate student, Phil Whitney, took on the problem and was able to show unequivocally that the template theory of antibody synthesis was inconsistent with experimental evidence. He did this by fully denaturing and disrupting (breaking disulfide bonds) a specific antibody and showing that specific activity was recovered after renaturation. The conclusion was that sequence determines three-dimensional structure, as it does for all proteins [23]. The same result had actually been obtained a little earlier by Ed Haber, an immunologist at MIT, while Whitney’s work was still in progress [24]. Haber had used an antibody against the enzyme ribonuclease; Whitney had used an antibody against the haptenic dinitrophenyl group. Control experiments in both cases showed that the presence of antigen during renaturation had no effect. The bottom line overall is that we had accomplished our goal. At the beginning of this section, I said that that when I first came to Duke, I was still looking at proteins as physical entities, composed of atoms, electrostatic charges, etc., with no direct connection to the biological function. This transitional hurdle had clearly been overcome.

Subunits and Allosterism And then quite by chance I even became involved with ‘‘molecular biology,’’ the pinnacle of the bright new look of biological research.


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After the DNA double helix ‘‘molecular biology’’ had moved to the top of the league in terms of relevance to the ultimate secrets of life. But proteins remained very much a part of the story. How many polypeptide chains? How big are they? Are they all alike? How are they linked? These questions were fundamental to an understanding of immunoglobulins, as seen in Figure 2, but they were also important for some proteins that were at the core of the genetic mechanism, part of the DNA ! messenger RNA ! protein story, and this made them part of the molecular biology agenda. This circumstance got me involved in a controversy, quite unintentionally; I was not even aware of it at first. The ultimate substantive matter in question was the subunit composition of an enzyme, aspartate transcarbamylase, a critical regulatory component in the biosynthesis of nucleotides ^ DNA and RNA. But the controversy itself centered on just one person, Jacques Monod, one of the half dozen people closest to the very heart of molecular biology. He shared the Nobel Prize for biology or medicine in 1965 with two of his colleagues from the Pasteur Institute, but has never been one of my favorite scientists. One historian describes him as having a zest for combat and a flair for performance, but I was only rarely impressed by the relevance of his battles to scientific priorities. The incident to which I am referring here has to do with allosterism, a well-defined concept in the chemistry of some enzymes and in the binding of oxygen to hemoglobin. In Monod’s perception it became much more; he saw allosterism as the fundamental element of all biological control; ‘‘the most elaborate product of molecular evolution.’’ As is well known, allosteric regulation in hemoglobin, is achieved by interaction between protein subunits ^ the constituent and polypeptide chains. Monod turned this into a generalization: protein subunits became part of the fundamental principle; although, actually there is no absolute mathematical necessity for multiple subunits to be involved. In any case, subunits and their molecular weights were at the heart of allosterism research and that is how my laboratory came into the picture: in a project dominated

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by two visiting polymer chemists from overseas, Savo Lapanje from Yugoslavia (now Slovenia) and Kazuo Kawahara from Japan, we had established the potency of concentrated guanidinium chloride as a protein denaturant. After disulfide bond disruption, all proteins in that solvent were found to be reduced to their constituent polypeptide chains, behaving physically as structureless ‘‘random coils.’’ We successively applied a series of discriminating physical methods to show rigorously that under these conditions the molecular behavior became essentially identical to that of a synthetic organic polymer dissolved in an indifferent organic solvent; indeed our project was not primarily directed at protein chemists or biochemists, but rather at polymer chemists, to convince them that it was often valid to apply the theories developed for polymer chemistry to the systems we were working on [25]. Use of concentrated guanidinium chloride as a solvent created a special problem for molecular weight measurements in the ultracentrifuge, where the buoyant mass of the protein particle is the determining factor. This factor includes bound solvent and requires knowledge of the proportions of water to guanidine, within the protein particles as well as in the bulk solvent: is one or the other solvent component preferentially bound? This problem was successfully investigated in my laboratory by Kirby Hade, a postdoctoral fellow who used to be a stockbroker and came to work with me to look for a less volatile career. Characteristically he chose the most esoteric and laborious method for determining preferential binding, by measurement of ‘‘isopiestic’’ compositions [26] ^ a method that was exact if you fully understood the partial differential equations that describe what you are doing. George Scatchard was the only chemist who had ever been known to actually use the isopiestic method in a biochemical context. Many years later S.N. Timasheff of Brandeis University became the unchallenged world expert on preferential binding by proteins in multicomponent systems ^ his work should be consulted for detailed analysis [27].


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The instigator of the rather acrimonious debate with Jacques Monod was the ‘‘molecular biology correspondent’’of the journal Nature, who, at that time, wrote unsigned weekly contributions related to current work in the field. He has since been identified as Walter Gratzer, well known today both as a molecular biologist and as a writer of semipopular works in general science. Gratzer expressed his dismay at the faddishness of the allosteric phenomenon, using the enzyme aspartate transcarbamylase as an example. He noted Monod’s enthusiastic ‘‘discovery’’ of the use of guanidine as an agent for denaturation (and polypeptide disruption) and as a medium for measurement of subunit molecular weights. Gratzer pointed out with some sarcasm (perhaps even jokingly) that it should really be called a ‘‘rediscovery,’’ citing the work from my laboratory and others in America as a precedent. Monod did not take kindly to criticism and attacked Gratzer’s judgment in a letter to the journal, which was not a very wise move, considering Monod’s own ignorance of physical chemistry. The importance of buoyant density in the interpretation of ultracentrifugal results, apparently just recognized by Monod, was at the core of the dispute. Gratzer appropriately referred to Kirby Hade’s earlier analysis of that problem as definitive. Another rather arrogant (as well as puzzling) view of Monod’s was that he thought he had free choice in what was a subunit and what was not ^ aspartate transcarbamylase should be called a tetramer, he asserted, not an octamer, which it seemed to be by actual count. As Gratzer put it, Monod’s intention appeared to be ‘‘a new definition of a monomer ^ or monodmer perhaps ^ elusive, but adaptable.’’ Such opportunities for a play on words are rare in serious scientific discourse. Monod did not seem to appreciate the play on words and was offended by being made a laughing stock. As for me, I could claim that work done in my laboratory had actually made an impact on the Olympian heights of molecular biology. (Or, as Gratzer put it in a comment on his own role: ‘‘It made me feel that I had not lived entirely in vain.’’)

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Micelles and Membranes From this point onwards, most of my work was done jointly with Jacqueline Reynolds, my right-hand associate in the laboratory, who quickly became my beloved partner in life as well. This kind of partnership has become much more common since my generation, but even now partners of this kind, though perhaps sharing the same employer, tend to retain separate identities and separate research interests. In our case, the research objectives were to a large extent shared and this creates obvious problems in the assignment of professional credit. In the text from here on, I shall use the pronouns ‘‘we’’ and ‘‘our’’ in referring to the scientific work published from our laboratory, regardless of whether one alone or both of us appear as authors. There were, of course, usually graduate students or postdoctoral fellows involved in any work that we did, as well as we, the nominal project directors. Moreover, they have all told me (in later years) that Jacqueline was a far more helpful research ‘‘advisor’’ than I had been. (Marc le Maire and Darrell McCaslin, for example, both superb graduate students, ostensibly working under my sole direction as thesis advisor.) In all of our work on molecular weights of membrane proteins, in particular, I know that my own contribution can only have been peripheral. For example, our innovative development of a method for using the ultracentrifuge to determine the molecular weight of the protein moiety in protein^detergent or protein^lipid complexes without actual knowledge of bound lipid or detergent is in this category [30]. Both the idea itself, which was an extension of the buoyant density principle, using D2O to adjust the solvent density so as to match the density of bound lipid or detergent ^ as well as the practical execution ^ were primarily Jacqueline’s work. An example I remember well is our collaborative efforts with Arthur Karlin on the acetylcholine receptor [31], where my job in the partnership was to make frequent trips to the airport (I had a fast car) to collect the samples that had been freshly


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isolated earlier that day in Karlin’s laboratory in New York, and were shipped to us via Eastern Airlines. Our transition from water-soluble proteins to membrane proteins may not seem to be a very profound change in direction today, but seemed to be at that time. It involved a much broader vista, which incorporated lipids (micelles, membranes) as well as proteins. Phil Handler, when he heard about it, thought I was out of my mind, sacrificing an assured status as a leading physical protein chemist by venturing into this new field with dubious prospects. In a sense Handler’s objections justified the change in direction, in that they illustrated the newness of the project, the magnitude of the leap into the unknown. Ventures that are dull and have predictable results do not carry much risk. To prepare myself for the change, sensing (correctly) that my physico-chemical background would be inadequate, I spent much of the early time in exploring old ground, to organize in my mind what surface chemists and surfactant chemists already knew: the thermodynamic extension of hydrophobicity to micelle formation, size, and shape of micelles, etc. This endeavor resulted in a new book, The Hydrophobic Effect [32]. Just as in the 1950s, when I ventured beyond the teachings of Cohn and Edsall and felt the need to write Physical Chemistry of Macromolecules, so now, again venturing beyond what I already knew, another textbook seemed the surest way to go. (On some later occasion, Walter Kauzmann and I argued about the philosophy of textbook writing. Kauzmann believed in the general rule that you only write a textbook after much experience, when you have become expert enough to be able to vouch personally for the truth of every word. My viewpoint was the opposite, that you use textbook writing to learn a subject, to express a consensus rather than a personal view.) In the laboratory, the catalyst for getting the new venture going was again the connection to the medical school. This time it was a medical student, John Gwynne, who had opted to spend his third year on a project in basic science and had dropped into my office, to see what might be on offer in our

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laboratory. Nothing we were doing was likely to be of immediate medical interest, I told him, but I did have a menial job for someone with a brain, which might eventually blossom into some research ^ and I told him about our desire to expand our work from focussing exclusively on proteins in aqueous solution to proteins which were in their native state in cell membranes and that it was an ambitious step for us for we knew nothing, whatever, about cells and membranes. Nor could I identify who among my colleagues might profitably be consulted on the subject: many of them could probably qualify as ‘‘cell biologists’’ of a sort, but surely the practical details of getting cells or membranes from living tissue into laboratory apparatus would be different for each of them: muscles, nerves, the eye, the blood stream, bacteria would all be expected to demand different techniques and specialized apparatus; and what you meant to do with the cells when you got them could make a difference, too ^ would an electron microscopist have different criteria from a physiologist studying transport? Red blood cells seemed particularly attractive to me (in my ignorance), being both abundant and easy to collect. Was this really true? And who in the diverse population of Duke Medical Center would have the answers? I told John Gwynne that if he could identify appropriate members of the medical faculty and find out how membranes could be prepared free from water-soluble cell contents, we could think of chemical approaches to begin to characterize the membrane proteins. I must admit that I expected a medical student to prefer more precisely defined projects, that did not begin in this way in uncharted waters. I was, therefore, surprised when Gwynne showed up again in a couple of weeks, mission accomplished. Red blood cells were indeed the most promising starting point, and Gwynne was ready to tell us just how red cell ‘‘ghosts’’ (as the membrane preparations were called) were to be made. We had during the preceding years established that most proteins, when dissolved in the potent denaturing agent


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guanidinium chloride and reduced to their constituent polypeptide chains by disulfide bond disruption, behave physically as structureless ‘‘random coils.’’ The constituent amino acids remain covalently linked by peptide bonds, but all effects of hydrogen bonds and other noncovalent structure-forming factors are lost: the physical behavior of the molecule becomes essentially identical to that of a synthetic organic polymer dissolved in an indifferent organic solvent. This means that physical parameters that depend on molecular weight become regular functions of molecular weight. One consequence was that it enabled us to adapt gel exclusion chromatography to molecular weight determination ^ strictly speaking, molecular length is the effective parameter in chromatographic sorting, but in most applications that will be proportional to molecular weight. The result was a method whereby polypeptides of a mixture could be separated on a column and the molecular weight of all separated fractions could be measured simultaneously [33] ^ a huge gain in convenience over the analytical ultracentrifuge. There was good evidence that guanidine hydrochloride would also disrupt noncovalent association between lipids and proteins, i.e., the method should be applicable to proteins from biological membranes (not ordinarily soluble in aqueous media) as well as to those that originate from a cell’s cytoplasm. Once John Gwynne could give us an accurate description of how to prepare red cell ghosts, separated from any other protein-containing matter, we had everything ready to go for molecular weight analysis. The most significant aspect of the result that emerged was that a major component of the mixture had an astonishingly high molecular weight, higher than 200,000 [34]. Apart from the heavy chain of myosin, no polypeptide of greater length was known. About this time, the detergent sodium dodecyl sulphate (SDS) surpassed guanidine hydrochloride as everyone’s favorite reagent for membrane disruption cum polypeptide chain segregation by molecular weight ^ using gel electrophoresis for the

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latter in place of the simpler gel filtration [35]. We knew that polypeptides in SDS, though thoroughly denatured, did not at all the resemble structureless random coils that they were in guanidine. The physical basis for sorting by molecular weight was, therefore, not immediately obvious, but we quickly worked it out [36], showing in the process that electrophoretic mobility on SDS gels was not as rigorously linked to molecular weights as was the case for gel filtration in guanidine, i.e., there were more likely to be discrepancies when molecular weights derived from SDS gels were compared precisely with values ultimately obtained by ultracentrifugation. (Molecular weights from SDS gels are in fact commonly called ‘‘apparent molecular weights.’’) We repeated the analysis of red blood cell membrane proteins in the new system and got similar results, but in SDS we clearly saw two distinct polypeptides around 200,000 molecular weight, which had merged into one broad band in gel filtration [37]. A year later we discovered an even larger new polypeptide, again lipid associated, though not in a membrane: this was the polypeptide constituent of low density lipoprotein (LDL) with a molecular weight of 250,000 [38]. Strangely, this proved to be controversial: the self-appointed authorities on molecular weight in the lipoprotein establishment (often referred to as the ‘‘Sardinian mafia’’) refused to recognize our measurement, though we repeated and extended it: they were advocating a vastly smaller figure, around 20,000. This controversy was one of very few truly unpleasant events in my experience; in retrospect probably best forgotten. Later genetic analysis showed that LDL can be separated into several different fractions ^ all of them have principal polypeptide chains with molecular weights above 250,000. In the process of this work, we became reasonably proficient in handling many practical aspects of amphiphile interactions and in understanding much of the underlying theory, which is of course governed by the quantitative canons of thermodynamics. Detergent micelles and phospholipid vesicles gradually became


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part of our everyday vocabulary; most of the proteins we handled needed their presence to remain in solution.

Membrane Proteins Guanidine and SDS solutions are denaturing solvents but we wanted, of course, to characterize the native states of membrane proteins as well. The difficulty here was that there was not yet a consensus on how to define or conceive the native state ^ which, to newcomers like ourselves, translated to uncertainty, because we did not have the kind of experience that would enable us to make a shrewd guess as to which of the contending ideas was likely to prove correct. Even the phospholipid bilayer, the one indispensible concept for the most minimal understanding of cell membranes, was still being disputed, despite the fact that it had been first demonstrated in red blood cells nearly 50 years before and that it was really the only conceivable arrangement on the basis of thermodynamics, a direct consequence of Irving Langmuir’s classic 1917 measurements with monolayers of amphiphilic substances. I have described the ludicrously slow acceptance of the bilayer concept in the intervening period in my book, Ben Franklin Stilled the Waves [39]. The point here is that skepticism still persisted when we became involved in membrane research. And among those who were convinced about the lipid bilayer, the manner of incorporation of proteins in membranes was still open to debate. Some, like our Duke colleague David Robertson, were reluctant to believe that proteins could actually penetrate or traverse a bilayer. Two quite disparate conceptual images, taken from a conference at the New York Academy of Sciences in 1972, are shown in Figure 3 [40]. One is Robertson’s ‘‘unit membrane,’’ with protein totally on the outside. The other gives Vanderkooi’s view of cytochrome oxidase complexes in mitochondrial membranes: the protein is positioned more realistically, but there is not yet any conception of distinct hydrophilic

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Fig. 3. Two disparate conceptual images of membrane proteins, both proposed at the same meeting in New York in 1972 [40]: (a) Robertson thought that proteins would be asymmetrically disposed, outside the two bilayer surfaces; (b) Vanderkooi’s view of cytochrome oxidase complexes in cross-section.

and hydrophobic domains and how they might dictate protein^ lipid interaction. Later in that same year of 1972, the now conventional idealized picture of phospholipid bilayers, with functioning proteins running all the way through them, finally became popularized by Singer and Nicolson’s ‘‘fluid mosaic’’ model [41]. In the laboratory these were exciting times, the most exciting I can remember. We learned how to use benign detergents, which, unlike SDS, could solubilize membrane proteins without gross denaturation, in an environment simulating the native state, but an environment where they would also be readily accessible for molecular characterization [42] ^ an advance in which we were in part anticipated by two likeable young men from Finland, Kai Simons and Ari Helenius [43].


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In practice we were still mostly confined to measuring molecular weight and asking how many polypeptide chains per molecule, but the proteins to which these questions were addressed had been separated by detergent treatment from other membrane components. Most important, many of the proteins had known cellular function and our measurements were relevant to these functions. I have already mentioned red blood cells, but we actually managed to cover a range of biologically relevant topics, spurred on in many cases by the missionary zeal of students. For example, a physiology graduate student, Stuart Grefrath, with an interest in neurophysiology, came to our laboratory to enumerate the polypeptide chains of an excitable membrane ^ in this case from garfish olfactory nerve ^ and this led us to subsequent involvement with several other active transport systems [44]. (Stuart himself sadly died as a result of a congenital cardiovascular disease before he could fulfil his early promise with an independent career.) Cerebral myelin was another membrane from the nervous system that we looked at [45]. Also worth singling out are two visitors to the laboratory who chose to work together: Neal Robinson, a postdoctoral fellow, and Leon Visser, who was on sabbatical leave from the CSIR in Pretoria, South Africa). They did a very neat job of quantitatively defining the domain structure of cytochrome b5, a sort of prototype for illustrating how membrane proteins are attached to membranes and still carry out their function in the adjacent cytoplasm [46]. In other projects we were responding to requests from distant colleagues. I have already mentioned the work with Arthur Karlin on the acetylcholine receptor from an electric fish. Another example was bacteriorhodopsin, for which we made measurements in detergent solution at the behest of Walter Stoeckenius from the University of California [47]. There was an important question here, related to the mechanism of this protein’s proton pumping activity: is the native protein functionally a monomer or (as some data seemed to suggest) is it a trimer? Our solubilized protein was unquestionably a monomer

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and spectral evidence indicated that it was going through the same cycle of molecular changes as in the native membrane. In most cases we ourselves didn’t become directly involved in trying to puzzle out how receptors or pumps or channels worked, but we inevitably became aware of what the problems were. It was a truly enriching experience, quite a difference from the days when serum albumin and -lactoglobulin (obtained from commercial suppliers) were the prime targets of our research. In the case of ion pumps we did actually enter into the physiology, mostly on the level of hypothesis or theory; we learned how to model kinetic schemes with a computer; we attended appropriate conferences; etc. Our final sabbatical leave ^ to the Max Planck Institute in Heidelberg ^ was important in this respect. The chairman of the physiology department at Duke, Ted Johnson, joined us on this occasion, so there were three of us in active collaboration [48,49]. Ted had been a computer enthusiast for a long time, and had connected every individual member of the Physiology faculty to the North Carolina Research Triangle central computer facility before it became fashionable to spend departmental funds in that way. In those days we had to write our own programs for the computer, which was difficult for me, but I believe we obtained some useful results, mostly on kinetic models for the pumping cycle of the ATP-driven Na,K pump.We worked unorthodox hours, including weekends and holidays, which sometimes discomfited our Heidelberg hosts, who were accustomed to turn off the central heating when the laboratory was supposed to be unoccupied. About every ten days we drove across the border into Strasbourg for a gourmet lunch ^ accompanied by Alsatian wines, the taste of which we have enjoyed ever since.

Philip Handler and the Einstein Statue Philip Handler was the chairman of the Biochemistry Department at Duke University Medical School in 1960, who


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was responsible for my being invited to join the department as professor of physical biochemistry. It was a position of distinction, first occupied by Hans Neurath, one of the only very few persons in America at the time who could honestly lay claim to the designation of ‘‘physical biochemist.’’ I certainly was not in that category, being a conventional physical chemist; only my choice of proteins as research material could be considered somewhat eccentric. Handler, in his letter of appointment to me, expressed the hope that I would change my colors, become involved in the marvels of intermediary metabolism and the enzymes that catalyze and regulate each step. Which I never did: I wanted to be ‘‘relevant,’’ but antibodies and cell membranes became my pathway to that goal. Philip Handler turned out to be unlike anyone I had encountered before. He was a masterful politician and relished political power, e.g., the power to make new faculty appointments without having to go through the formality of seeking the approval of existing faculty; or the power to determine which departments would go into a new building under construction. In Washington he sought and achieved the power to influence federal legislation ^ with persuasive arguments in favor of much more money for biochemistry. At the same time his grasp of scientific principles remained high. He himself had no direct interest in immunology, but he recognized immediately (without it being pointed out to him explicitly) that our work with antibodies challenged one of the sacred cows of immunochemistry: Pauling’s dictum of complementarity between antibody and antigen as an instructive factor in the generation of antibody diversity. One week after he first learned about it, I found that he had (without telling me in advance) invited a leading figure in the field of chemical immunology to come to Duke to give a seminar and to educate Handler himself to the point where he could make his own judgement about our work. Handler was also a man with an acute sense of values that I could not help but admire, a sense of duty, a sense of right

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and wrong ^ I don’t know exactly how to express it in a single word. Handler would never have made his departmental power play, preempting new laboratory space for biochemistry that had been initially planned for the department of anatomy, if he had not been convinced that it was in the best interest of Duke University to do so. And he was right, of course: Duke’s biochemistry was far better than anatomy. Handler was elected president of the National Academy of Sciences in 1970, a post for which he was obviously eminently qualified. His most effective achievement in the public service in that position was to my mind the commissioning and subsequent seeing through to fruition of the statue of Albert Einstein in front of the Academy building, on Constitution Avenue, in the heart of Washington. There was no conceivable political gain in the project. On the contrary, the statue’s erection faced huge opposition; Handler alone seemed to fight for its existence. In the words of his biographers [50]: ‘‘He conceived the idea for the statue, raised funds for it, commissioned the sculptor, and followed in great detail the sculpture itself before and during its construction.’’ The statue is testimony to Handler’s unwavering faith in his sense of values, including his patriotism. ‘‘To Phil, Einstein symbolized the best in science and humanity of our century.’’ What seemed initially, especially, remarkable in view of Phil’s usually evident partisanship is that Einstein wasn’t even a member of Handler’s own constituency within science, not a biochemist, but a physicist. He was an American and a member of the National Academy, but he was not born in America, nor was his great scientific work done in America. It was done in Switzerland, the country of the cuckoo clock (in the famous words of Orson Welles). The statue’s presence here proclaims our faith in all of science, our regard for it as a beacon to guide the future. We can all see the statue now, just a few steps from the Lincoln Memorial, the Washington monument, the Vietnam Veterans’ memorial, the statue of John Paul Jones. Heroes of war and revolution have their merited prominence


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here, as they do in most countries, but in America, the statue seems to say, a hero of science has equal stature. The opposition to the Einstein statue came from both within the National Academy and from the public at large. The Washington Post opposed it; so did the weekly journal Science. Physicists were appalled at the expense: you could fund ten fellowships a year for the sum involved and wouldn’t that be a better way to celebrate science? Artists were upset by what they saw as the statue’s ugliness ^ ‘‘gross as well as trite,’’ in the words of one critic. Curiously, the statue itself has come to answer the critics, no words are needed. On a warm day you will find not only Academy workers, but other Washingtonians as well, having their lunch, sitting on its pedestal. And children play around the statue, often climbing on Einstein’s lap, perhaps absorbing some reverence (one hopes) for Einstein’s intellectual and humanitarian values.

Eastman Professor at Oxford For the year 1977^1978, I accepted an invitation to be the George Eastman visiting professor at Oxford. This position was established in 1930 through the philanthropy of the American manufacturer, George Eastman. It was limited to American citizens, creating a kind of inverse of Rhodes scholarships. Rhodes scholars have often been viewed in England as visitors from a relatively backward former British colony, coming to Oxford for a taste of culture and scholarship; the Eastman professorship represents the other side of the coin ^ as if to say that we have scholars in America, too, whose teaching can benefit Oxonian students. Oxford freely selects the recipient of this award. There is no influence from the American side. About half the appointees have been scientists, including Linus Pauling in 1948 and George Beadle in 1958. Beadle became a Nobel laureate while in residence at Oxford. Beadle’s wife,

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Muriel Beadle, wrote an amusing account of their year in Oxford in a book, entitled These Ruins are Inhabited. The best known of all the holders of the Eastman chair is Felix Frankfurter, who was a professor of law at Harvard University at that time (1933), and later became a memorable justice of the American Supreme Court. He has left behind an extended description of his Oxford year, which he characterized as ‘‘rich, stimulating, exciting, affectionate.’’ He also pointed out the many anomalies ingrained in Oxford tradition, small obstacles that nobody warns you about and that sometimes seem deliberately designed to prevent the visitor from ever feeling entirely at home. Though unable to arrange for American choice of who the Eastman professor was to be, George Eastman did influence what he would do. Suspicious that the recipient might take advantage of the traditional Oxford liberal definition of professorial duties and spend his year gallivanting around Europe, Eastman specified that the Eastman professor must give 24 lectures in the course of his year. This was, of course, virtually impossible. Oxford operates on the tutorial system: students ‘‘read for a degree’’ under supervision of a tutor, who must be a fellow of the college in which they are enrolled. Students may attend lectures by professors (who are appointed for the university as a whole, not limited to a single college), but there is no registration for the lectures, no record of who has attended, no examination for the course, no examination grades. No Oxford professor has ever been known to give as many as 24 lectures in one year. With respect to the Eastman professor, I found that the Oxford authorities interpreted the statutory requirement with typical liberality: many possible circumventions had acquired validity through precedence. I personally thought that Eastman’s rules should be honored, at least in spirit, and I gave 12 carefully prepared formal lectures. They were well received and I held on to my audience (which was never under any compulsion to return) better than most of my predecessors. The lectures were, of course, based on my own interests and


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experience ^ proteins, membranes, etc. ^ and were directed at students. I don’t recall that they led to any inspired discussions with my professional peers, nor to any exciting collaborative research related to these topics. There was no resemblance to my year at Yale, where I had acquired at least a glimmer of special erudition ^ theoretical proficiency in this case ^ from my hosts and companion scholars. In the absence of strong professional stimulation, my most vivid memories of the year are of Balliol College and the collegiate life. Not only students, but also professors, must be collegiate fellows. The Eastman professorship is attached to Balliol College, and the Eastman professor is a member of Balliol for his year of residence. He can attend college meetings, have lunch at high table, and can become as involved in collegiate affairs as he wishes. One is thereby constantly exposed to a system of education, stressing individual initiative, intrinsically elitist, which has no parallel in America. I am not even sure how closely my memories correspond to Oxford as it is today. Since coming to England as a permanent resident, I have been back to Balliol only casually: one Gaudy, one meeting that happened to be sited there. I know from reading the newspapers that changes have taken place, that there is a need to survive in what has become an increasingly egalitarian society. Balliol College has had an American master since my time (originally one of the Eastman professors), so anything is possible. I should not fail to mention the innumerable dinner parties ^ a perennial feature of Oxford life ^ where the food and wine were superb and the seating arrangement was invariably determined by the hosts in advance, indicated by a seating chart posted on a bulletin board and by formal place cards at the table. Felix Frankfurter found that his card was always labelled ‘‘The Eastman Professor,’’ and he thought it likely that most of his dinner partners never did discover his name [51]: It was a beautiful dinner. Those were still the days when there were four lovely glasses at each place, four courses of wine. It was

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a big party. The invitation was addressed as I was addressed the rest of the year, ‘‘The Eastman Professor.’’ I had no name, I wasn’t a person.You were known by your title.

His description remained true in my time, almost fifty years later. Felix Frankfurter was not alone in his admiration of Oxford. Most Eastman professors were enthusiastic about the experience and marvel at the intellectual freedom that the Oxford system affords, as indeed I did myself in an article entitled ‘‘A clerk at Oxenford,’’ published in a faculty newsletter that Duke University used to put out for its staff. In retrospect, I am not sure whether my admiration was fully justified; is the system really still viable from an educational standpoint? Freedom to excel also allows for freedom to fail and I suspect that I saw many failures. Today we are faced with an increasingly egalitarian world ^ a society that is more regulated, with rules spelt out to give everyone equal access. Somewhere along the line, modern teachers are supposed to tell the student exactly what he needs to do to pass exams and to fill employers’ expectations. The American system and the bulk of Britain’s less prestigious universities may be better suited for this task than the fabled ‘‘Oxbridge’’ of Eastman memories and English novels.

Popular Writing: Travel Books I retired from Duke University in 1988 and so did Jacqueline, who was by then an independent full professor and a powerful force in university affairs, something that I had never been. Our retirement was a complete break: we burned our bridges behind us, both geographically and professionally. We sold our home, packed our bags and sailed off into the unknown ^ quite literally, for we did not know where in England we would eventually settle.We made no new academic connections, though there would have been opportunities aplenty, had we so desired. We


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have never again worked in a laboratory or developed new theories or wrote reviews at the cutting edge of current research. Which does not mean we were idle. On the contrary, we made plans for two major projects: 1. A travel guide to places in Europe that were associated with the lives of great scientists of the past or that were sites of special scientific interest in a more general way ^ institutions, archaeological or geological features, etc. We had always enjoyed traveling, especially in Europe, and had been annoyed by the one-sidedness of most travel guides, which tend to extol the virtues of liberating revolutionaries, battle heroes, architects, painters, and politicians, but almost invariably ignore scientists and scientific discoveries that merit equal prominence in popular culture. We wanted to remedy this by providing appropriate raw material for writers of guide books to enable them to appreciate the rich heritage of science and hopefully to transmit some of it to their readers. 2. In a more scholarly vein, we wanted to write a history of the subject that we had spent most of our lives working on, namely proteins: their chemistry, structure, function, and genetics. By the time we actually got going on this project, proteins had catapulted to a position of stupendous prominence on the world scene, and we were urged to modify our goal, to write about the future: designer proteins, the marvelous feats which proteins could hope to accomplish with the aid of genetic manipulation. But we stuck to original intent. We wanted to write the history. Proteins had been perceived to be capable of marvelous feats long before what we now achieve by purposeful engineering ^ we wanted to put that past into perspective for today’s reader. Writing the travel book [52] took us all over Europe, for we found that the roots of science are not confined to places that

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we now see as its most fertile ground. Moreover, we absorbed a great deal of general European history in the process, both old and new. We were in Germany when East and West were still divided, before the Berlin wall came down.We managed to get to Greece before too many sun-seeking tourists arrived to spoil the view. Poland gave us Copernicus, nearly 500 years ago, and the country today takes pride in his pioneering achievement with well-planned memorials in Frombork, Lidzbark Warminski, and Torun. (The official guidebook to the area existed only in the Polish language at the time we visited there, and we had to translate it laboriously with the aid of a dictionary from our local library.) Poland also brought us face to face with Auschwitz, as I mentioned at the beginning of this article ^ an unsavory truth with scientific overtones that it would be folly to ignore. In Greece, of course, we were able to go much further back in time than Copernicus. To the imposing statue of Aristotle, erected close to where he was born in Stagira in 384 B.C., to the first profit-making health center founded by Hippocrates on the island of Kos in about 360 B.C., and to the island of Samos, which was the home of Pythogaras (where presumably, among other achievements, he first squared the hypotenuse) and the home of the astronomer Aristarchus, who had proposed a heliocentric solar system 1500 years before Copernicus. Cemeteries were a rich source of material. In Vienna’s Zentralfriedhof we found Ludwig Boltzmann, one of our personal heroes in the struggle for recognition of the atomic^molecular picture of matter, interred here alongside some of Vienna’s famous musicians: Beethoven, Schubert, Brahms, and Strauss. Boltzmann’s tombstone bears the famous legend ‘‘S ¼ k log W,’’ the equation that links atoms and molecules to undifferentiated ‘‘matter.’’ Go«ttingen’s town cemetery is another prestigious resting place. It contains the tombs of many famous physicists and physical chemists, one of whom (Walther Nernst) required much family pressure and two less prestigious interments before he made it to this place of honor.


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And there’s Neanderthal, close to Du«sseldorf, where the remains of our beetle-browed eponymous ancestor were discovered during quarrying operations in 1830. Cave paintings and relics of a somewhat more modern ancestor, Cro-Magnon man, are celebrated in the Dordogne in France. Stonehenge man was even more modern than that (his skeletons would be indistinguishable from ours), but here we have an enigma regarding the provenance and the social significance of the familiar stone circles. There are similar stone circles further north in the British Isles, e.g. on Orkney Island. Places of unforgettable natural beauty and inviting deep questions about prehistoric science ^ prehistoric in the sense that we have no written history, only the stones themselves. These notes give just a tiny fraction of the places we visited, but should suffice to convey that we ranged far and wide and that we had an enjoyable time in doing so. The book was quite successful and Geoff Farrell, the editor at Wiley’s Chichester branch, asked us to write a follow-up limited to Britain and Ireland alone. His instructions were to see if we could find a scientifically interesting site within walking distance of every Marks and Spencer shop, and we had come reasonably close to that when the book was finished in 1994 [53]. In Britain we visited John Dalton’s birthplace in Cumbria and Isaac Newton’s in Lincolnshire; Charles Darwin’s house in Kent and his grandfather Erasmus’s in Staffordshire; Jenner’s Temple of Vaccinia in Gloucestershire; the Greenwich Observatory and Michael Faraday’s magnetic laboratory in London; James Clerk Maxwell’s school in Edinburgh. We became aware of many less famous scholars and of scientific endeavor in more obscure places: Jeremiah Horrocks, curate of St. Michael’s Church in Much Hoole, Lancashire, who correctly predicted the transit of Venus across the face of the sun in 1639 and confirmed his prediction with home-made equipment; Mary Anning, teenage beachcomber, who discovered the first complete skeleton of a giant marine lizard at Lyme Regis in Dorset in 1811 and sold it to the British Museum for »25; John Napier,

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Scottish laird, who discovered the utility of logarithms, but even before that had invented a simple multiplication aid, called ‘Napier’s bones.’ Britain’s public houses proved useful to us in writing this second guide, for we often didn’t know whether there would be a monument or even a memory where one might logically be expected. The publican usually knew: ‘‘There’s a pile of stones up on the village green ^ I think that’s the man you want.’’And it usually was, though sometimes we were directed to seek out soand-so down the road and got the desired information from that source. Only once do I remember this approach to fail altogether, at the Eagle pub in Cambridge, frequented by Watson and Crick, where Francis Crick reports he went on the day in 1953 when the DNA structure was solved and announced it to the customers with the words ‘‘We have just solved the secret of life.’’ When we were there in 1994, the pub had just reopened after being been closed for renovation ^ surely the right time for installation of a memorial plaque. We never did find out whether one has been or was going to be put up. When we asked the barman, his reply was: ‘‘Who did you say? Watson and Crick? Never heard of them.’’

Historical Perspective I turn now very briefly to our more serious project of writing a history of proteins: the book was completed early in 2001 and published later that year. I should acknowledge at the start our indebtedness to Walter Gratzer’s active promotion of the project. Walter recommended it to Oxford University Press; he tried valiantly to get us to appeal to the ‘‘popular science’’ market ^ a genre where he himself has been so skilful. He is even the person who proposed the essence of the book’s title: he judged our original title,‘‘A History of Proteins,’’ to be too academic and suggested the analogy to robots as part of his popularization


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efforts. After publication, when reviews seemed to be slow in coming, he used his journalistic connections to assure a competent review. ‘‘Nature’s Robots’’ was the book’s title eventually agreed with the publisher, with ‘‘A History of Proteins’’ as subtitle. It turns out to be a reasonably realistic title, not just a sales gimmick. The final text of the book is divided into four parts: two of them deal with chemistry and molecular structure, the other two address physiological function and how it is dictated by the genes ^ which is where the robotic analogy come in [3]. Two motivating factors spurred us on. One is the fact that proteins have leapt into new and disproportionate prominence in the last few years. ‘‘Proteomics’’ is the new buzzword for the first decade of the 21st century, displacing ‘‘genome.’’ A concise history of how this happened is clearly necessary to put the present status of proteins into proper perspective. Where did it start? Who discovered the peptide bond? Questions of this sort needed to be addressed. (To put it more precisely, we ourselves wanted to know the answers. The level of curiosity among presently practising protein scientists is a matter for conjecture.) The second motivating factor was that such history, as exists, is inadequate and often needs correcting. I mentioned earlier (under the heading ‘‘The invention of polymer science?’’) the chemistry establishment’s ludicrous promotion of Hermann Staudinger to the status of a hero, who in 1920 is said to have ‘‘invented’’ the concept of macromolecules and then to have done battle for it against tenacious opposition. This is only one example out of many historical distortions that need to be put right; on the other side of the coin we find dedicated figures who have been unjustly assigned to oblivion and merit a more honored place in history. We based our history as much as we could on original papers in professional journals or on similar material in books. We wanted to use our own judgement to identify the key moves in that history, which were often the perception of new ideas, but

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could sometimes be just new experimental data, not necessarily linked to any particular hypothesis or theory. We had been in the game ourselves, through a good part of the period, when this history unfolded, so that most of it was familiar territory and we could recognize experimental failings or intellectual brilliance, even long after the fact. We thought we could identify imposters as well ^ incompetent and/or stupid and sometimes possibly even guilty of foul play. One glaring example of the negative side of things was the notorious Dorothy Wrinch. She is a favorite martyr of the current feminist movement, supposedly the victim of discrimination because of her sex [54,55]. She was actually grossly incompetent and as close to being a fraud as anyone you can find in the scientific record, roundly denounced in letters to Nature by illustrious contemporaries from both sides of the Atlantic. Another and better known example is provided by the colloid chemists who stubbornly refused to believe that enzymes were proteins ^ one has to be charitable to regard them as ‘‘misguided.’’ An example of a good guy, meriting more credit than he usually gets, is Gerrit Mulder, the very first person to use the word ‘‘protein’’ in 1838. He was an accurate analyst who recognized from the start that sulfur was normally present in only small amounts, and therefore, that proteins had to be large molecules unless we allow the existence of fractions of a sulfur atom per molecule [3: pp. 11^20]. Moreover, the list of plaudits continues with Emil Fischer and Franz Hofmeister, who independently discovered the peptide bond in 1902 [56], and with The Svedberg and Arne Tiselius, who taught us how to measure molecular size and electrical charge. A less familiar name is that of J.C. Jacobsen, founder of the Carlsberg Laboratory in Denmark, which generated not only oceans of good beer, but also some of the outstanding protein science and scientists of the first half of the twentieth century [57]. John and Charlotte Schellman, who spent a year there almost 50 years


49

ago, characterize it as having been ‘‘a Mecca for quantitative biochemists.’’ [58] Mostly, of course, it is the successes that one writes about and, inevitably, our protein history comes to a climax with the famous names and achievements of the Cambridge MRC, where molecular biology, the great scientific revolution of our times, was born and brought to maturity.

REFERENCES [1] Tanford, C. and Pease, R.N. (1947) Theory of burning velocity. J. Chem. Phys. 15, 433^439, 861^865. [2] Cohn, E.J. and Edsall, J.T. (1943) Proteins, Amino Acids and Peptides, as Ions and Dipolar Ions. New York, Reinhold. [3] Tanford, C. and Reynolds, J. (2001) Nature’s Robots: A History of Proteins. Oxford University Press. [4] Obituary of George Scatchard (unsigned) (1974) Nature 248, 367. [5] Edsall, J.T. and Stockmayer, W.H. (1980) George Scatchard (1892^1973) Biog. Mem. Natl. Acad. Sci. 52, 335^377. [6] Tanford, C. (1950) Hydrogen ion equilibria in native and modified human serum albumin. J. Amer. Chem. Soc. 72, 441^451. [7] Linderstrm-Lang, K. (1924) On the ionisation of proteins. Compt. rend. trav. Lab. Carlsberg 15(7), 1^29. [8] Tanford, C., Buzzell, J.G., Rands, D.G. and Swanson, S.A. (1955) The reversible expansion of bovine serum albumin in acid solutions. J. Amer. Chem. Soc. 77, 6421^6428. [9] Editorial (1999) Inventing polymer science. Chem. Heritage 17(3), 5^6. [10] Cohn, E.J. and Conant, J.B. (1926) The molecular weight of proteins in phenol. Proc. Natl. Acad. Sci. 12, 359^362. [11] Tanford, C. (1961) Physical Chemistry of Macromolecules. New York,Wiley. [12] Einstein, A. (1906) Eine neue bestimmung der moleku«ldimensionen. Ann. Physik 19, 289^306; (1911) 34, 591^592. [13] Tanford, C. (1994) Recollections. Protein Sci. 3, 857^861. [14] Scatchard, G. and Kirkwood, J.G. (1932) Das Verhalten von Zwitterionen und von mehrwertigen Ionen mit weit entfernten Ladungen in Elektrolytlo«sungen. Physik. Z. 33, 297^300. [15] Kirkwood, J.G. (1934) Theory of solutions of molecules containing widely separated charges with special application to zwitterions. J. Chem. Phys. 2, 351^361.

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[16] Tanford, C. and Kirkwood, J.G. (1957) Theory of protein titration curves. J. Amer. Chem. Soc. 79, 5333^5339, 5340^5347. [17] Mirsky, A.E. and Pauling, L. (1936) On the structure of native, denatured, and coagulated proteins. Proc. Natl. Acad. Sci. 22, 439^447. [18] Edsall, J.T. and Foster, J.F. (1948) Double refraction of flow: human serum_ globulin and crystallized serum albumin. J. Amer. Chem. Soc. 70, 1860^1866. [19] Noelken, M.E., Nelson, C.A., Buckley, C.E. and Tanford, C. (1965) Gross conformation of rabbit 7S -immunoglobulin and its papain-cleaved fragments. J. Biol. Chem. 240, 218^224. [20] Valentine, R.C. and Green, N.M. (1967) Electron microscopy of an antibody-hapten complex. J. Mol. Biol. 27, 615^617. [21] Edelman, G.M. and Galley, J.A. (1964) A model for the 7S antibody molecule. Proc. Natl. Acad. Sci. 51, 846^853. [22] Pauling, L. (1940) A theory of the structure and process of formation of antibodies. J. Amer. Chem. Soc. 62, 2643^57. [23] Whitney, P.L. and Tanford, C. (1965) Recovery of specific activity after complete unfolding and reduction of an antibody fragment. Proc. Natl. Acad. Sci. 53, 524^532. [24] Haber, E. (1964) Recovery of antigenic specificity after denaturation and complete reduction of disulfides in a papain fragment of antibody. Proc. Natl. Acad. Sci. 52, 1099^1106. [25] Tanford, C. Kawahara, K. and Lapanje, S. (1966) Proteins in 6M guanidine hydrochloride. Demonstration of random coil behavior. J. Biol. Chem. 241, 1921^1922. [26] Hade, E.P.K. and Tanford, C. (1967) Isopiestic compositions as a measure of preferential interactions of macromolecules in two-component solvents. Application to proteins in concentrated aqueous cesium chloride and guanidine hydrochloride. J. Amer. Chem. Soc. 89, 5034^5040. [27] Timasheff, S.N. (1998) Control of protein stability and reactions by weakly interacting cosolvents: the simplicity of the complicated. Adv. Protein Chem. 51, 356^432. [28] Gratzer,W.G. (1968) ‘‘Molecular biology correspondent’’, more allosterism. Nature 217, 803^804, 902^903. [29] Monod, J. (1968) Correspondence with reply by ‘‘Molecular Biology Correspondent’’. Nature 218, 106. [30] Reynolds, J.A. and Tanford, C. (1976) Determination of molecular weight of the protein moiety in protein-detergent complexes without direct knowledge of detergent binding. Proc. Natl. Acad. Sci. 73, 4467^4470. [31] Reynolds, J.A. and Karlin, A. (1978) Molecular weight in detergent solution of acetylcholine receptor from Torpedo californica. Biochemistry 27, 2035^2038.


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[32] Tanford, C. (1973) The Hydrophobic Effect: Formation of Micelles and Biological Membranes. New York,Wiley; 2nd edn. 1980. [33] Fish, W.W., Mann, K.G. and Tanford, C. (1969) The estimation of polypeptide chain molecular weight by gel filtration in 6M guanidine hydrochloride. J. Biol. Chem. 244, 4989^4994. [34] Gwynne, J.T. and Tanford, C. (1970) A polypeptide chain of very high molecular weight from red blood cell membranes. J. Biol. Chem. 245, 3269^3273. [35] Weber, K. and Osborn, M. (1969) The reliability of molecular weight determinations in dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406^4412. [36] Reynolds, J.A. and Tanford, C. (1970) The gross conformation of proteinsodium dodecyl sulfate complexes. J. Biol. Chem. 245, 5161^5165. [37] Trayer, H.R., Nozaki,Y., Reynolds, J.A. and Tanford, C. (1971) Polypeptide chains from human red blood cell membranes. J. Biol. Chem. 246, 4485^4488. [38] Smith, R., Dawson, J.R. and Tanford, C. (1972) The size and number of polypeptide chains in human serum low density lipoprotein. J. Biol. Chem. 247, 3376^3381. [39] Tanford, C. (1989) Ben Franklin Stilled the Waves. Durham, N.C. Duke University Press. [40] Green, D.E. (ed.) (1972) Membrane structure and its biological applications. Ann. N.Y. Acad. Sci. 195, 1^510. [41] Singer, S.J. and Nicolson, G.L. (1972) The fluid mosaic membrane. Science 175, 720^731. [42] Tanford, C. and Reynolds, J.A. (1976) Characterization of membrane proteins in detergent solutions. Biochim. Biophys. Acta 457, 133^170. [43] Helenius, A. and Simons, K. (1975) Solubilization of membranes by detergents. Biochim. Biophys. Acta 415, 29^79. [44] Grefrath, S.P. and Reynolds, J.A. (1973) Polypeptide components of an excitable plasma membrane. J. Biol. Chem. 248, 6091^6094. [45] Reynolds, J.A. and Green, H.O. (1973) Polypeptide chains from porcine cerebral myelin. J. Biol. Chem. 248, 1207^1210. [46] Visser, L., Robinson, N.C. and Tanford, C. (1975) The two-domain structure of cytochrome b5 in deoxycholate solution. Biochemistry 14, 1194^1199. [47] Reynolds, J.A. and Stoeckenius, W. (1977) Molecular weight of bacteriorhodopsin solubilized in Triton X-100. Proc. Natl. Acad. Sci. 74, 2803^2804. [48] Reynolds, J.A., Johnson, E.A. and Tanford, C. (1985) Incorporation of membrane potential into theoretical analysis of electrogenic ion pumps. Proc. Natl. Acad. Sci. 82, 6869^6873.

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[49] Tanford, C., Reynolds, J.A. and Johnson, E.A. (1987) Sarcoplasmic reticulum calcium pump: a model for Ca2þ binding and Ca2þ -coupled phosphorylation. Proc. Natl. Acad. Sci. 84, 7094^7098. [50] Smith, E.L. and Hill, R.L. (1985) Biography of Philip Handler. Biog. Mem. Natl. Acad. Sci. 55, 305^353. [51] Phillips, H.B. (1960) Felix Frankfurter Reminisces. London, Secker & Warburg. [52] Tanford, C. and Reynolds, J. (1992) The ScientificTraveler. NewYork,Wiley. [53] Tanford, C. and Reynolds, J. (1995) A Travel Guide to Scientific Sites of the British Isles. Chichester,Wiley. [54] Julian, M.M. (1984) Dorothy Wrinch and a search for the structure of proteins. J. Chem. Ed. 61, 890^892. [55] Abir-am, P.G. (1987) Synergy or clash: disciplinary and marital strategies in the career of mathematical biologist Dorothy Wrinch. In Uneasy Careers and Intimate Lives (Abir-am, P.G. and Outram, D., eds.), pp. 239^280. New Brunswick, N.J., Rutgers Press. [56] Fruton, J.S. (1985) Contrasts in scientific style. Emil Fischer and Franz Hofmeister: their research groups and their theory of protein structure. Proc. Amer. Phil. Soc. 129, 313^370. [57] Holter, H. and Mller, M. (eds.), (1976) The Carlsberg Laboratories 1876/1976. Copenhagen, Rhodos. [58] Schellman, J. and Schellman, C. (2000) Kaj Linderstrm-Lang (1896^1959) In Selected Topics in the History of Biochemistry, Comprehensive Biochemistry (Semenza, G. and Jaenicke, R., eds.), Vol. 41. Elsevier, Amsterdam.


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Chapter 2

Proteins, Life and Evolution HANS JO«RNVALL Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden

Abstract The development of protein analyses and interpretations is being monitored through my life in science. It started with manual stages in biochemistry. Then came separate stages of automations, integrations with three-dimensional analyses, combinations with DNA technology into molecular biology at large, development of bioinformatics, integration with mass spectrometry, and all now combining into the present stage of proteomics. Personal encounters with leading scientists and laboratories are given, stressing trends and developments. Increases in sensitivity with at least nine orders and in speed with at least five orders are seen during my scientific life, making this generation unique in both the history and the future regarding sensitivity increases in protein analysis. Current trends promise further developments in miniaturizations, microfluidics, biophysical methods, computerizations and data banks, ensuring important eras for protein science also in the future.

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Introduction Asked to write an account of my scientific life, I am happy to do so, but have two clarifications: First, although I have had close to 40 years of experience in protein chemistry, I write this account while still living an active life in science. Thus, this is not to be understood as a life account at the end of a career, but rather as a progress report while I am still working. Second, this account is written by a happy person who is grateful to fate for having experienced exactly these decades of science, first at formal schools and then at the school of life. When I was born, proteins were unknown as discrete molecules, were not understood in relation to their genetic representation, could not be easily purified, and could not be analyzed at any level of structure. Today, all this has changed. We now take for granted almost all those analyses and relationships of proteins that were considered impossible, or ‘‘science fiction,’’ at the start of my career. I am very lucky to have lived at this period, experienced the progress, taken part in the steps, and followed the complete revolution. I feel privileged and honored to reflect on some parts of the history of science through this account. Although the text centers around me, the relative lengths of the sections do not parallel their lengths in my life. Instead, I have tried to concentrate on periods, laboratories, people, inventions and meetings of major interest to best reflect important methodologies and breakthroughs. Perhaps my early period in Cambridge may appear overemphasized, but it was an interesting time, both to me and to science. Hopefully, a balance emerges at the end, to develop the points I consider relevant. Originally, the passage above was all words that I had intended to include before starting my voyage through this generation’s developments in protein chemistry. However, an editorial advice to me was to also give some conventional biography data up front, for the reader to get to know the author/guide before entering the adventure, and I agree. Basic CV data are, therefore, now given

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first to introduce the present guide for the travel through protein science from about the early 1960s up to now.

CVof the Guide through the Text Born in 1942 in a southern suburb of Stockholm, Sweden, I became interested in chemistry early, and soon, especially, in proteins. After a local school, I came to a gymnasium in the capital, and graduated from there after everyday commuting by train. I immediately continued at the university level, and I directly knew it would be in chemistry, but I did it at first not know exactly where: through the medical, technical, or natural science entrance. I chose the medical approach to chemistry, and started at Karolinska Institutet ^ a choice that I now know was good, and one that I have never regretted. After a stay in Cambridge, UK (below), I returned to Karolinska and there I passed my PhD and MD in 1970, at the same time getting the Docent title, which is equivalent to Assistant professor, but coming without position or salary. I got the latter two through various teacher/research/postdoc positions, and from 1974, a personal research position in ‘‘Medical Protein Chemistry,’’ at the Swedish MRC but localized to Karolinska. I also spent much time at laboratories abroad, mostly US, but also some in Canada and Russia (then USSR), in total over four years. In 1982, I became Professor of Physiological Chemistry and head of the Department of Chemistry I. In 1987, I became the chairman of the Karolinska chemistry departments, and in 1993 the chairman of the then further combined Department of Medical Biochemistry and Biophysics, MBB. In 1983, I became an EMBO member, and in 1990 an honorary member of the American Society for Biochemistry and Molecular Biology. In 1989, I was elected into the Karolinska Nobel Assembly, and in 2000 I became its scientific Secretary. I have also had positions at the Faculty Board of Karolinska, the Board of the Swedish MRC, as Editor of the

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European Journal of Biochemistry (for 10 years, handling close to 3000 manuscripts), and as Advisory Board member of journals (including Biochemistry for over 15 years). I have received national and international awards, some making me especially happy, like for research from both the Slovak Academy of Sciences and the Royal Swedish Academy of Sciences, and one (‘‘Ma«ster’’) for education from the Karolinska medical students. I have had 25 graduate students passing their theses with me, and about equally many more doing parts of their thesis work with me. In total, I have published over 700 reports. After these CV basics, let us start the trip through this generation’s developments in protein chemistry. Early Days, Scientific Fathers With increasing clarity, I see how scientific progress is a consequence of previous research, and that we follow in the footsteps of our scientific predecessors. Also in the field of protein chemistry this is true. I was fortunate in having three ‘‘scientific fathers’’: Hugo Theorell (Theo), Ieuan Harris (in Fred Sanger’s department), and Viktor Mutt. Although separated in time, space, and type of interactions with me, they all were true supporters and gave different kinds of help, experience, and knowledge. I started with Theo, at Karolinska Institutet, and I was asked as a fresh medical student to follow him in a long research chain on enzymes, in particular alcohol dehydrogenase, ADH. This involved, among other things, going to Cambridge, coming under the wings of Ieuan Harris, and picking up the many Cambridge methods of those days. Coming back home, I got to know Viktor Mutt. But with Viktor it was different: he was a true collaborator at all levels, with help through instruments and economy at my early postdoc years, when it was grant-wise difficult for me. In this regard, Erik Jorpes, previous head of Viktor, should be mentioned too. He was also devoted, wise, and warm. Although I met him largely after his career, he was an appreciative mental support. His life


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constitutes another chapter in this series (Vol. 41), contributed by Viktor Mutt and Margareta Blomba«ck. Coming back to Viktor, my initial close contact with him was when he was the external examiner of my thesis.We liked each other immediately, and soon started a longtime collaboration. It lasted for well over 20 years, until his death in 1998, and we produced 52 joint research reports giving the characterization of about equally many novel, bioactive peptides in the gastrointestinal tract and the central nervous system. Our last joint student, Zheng-wang Chen, passed his thesis in 1997 with characterization of four novel bioactive peptides, two of which he gave fancy names: dopuin [1] and daintain [2], alluding to Chinese words for their properties. But before continuing the story at Karolinska, we revert back to my early days, and follow the separate stages of development of protein chemistry.

Cambridge, the Mecca at the Start My PhD task, with Theo on ADH, was to characterize the subunit size, composition, and primary structure of the enzyme. A big challenge at the time when no enzyme that size had been characterized, isozyme complexity was far from realized, and the subunit size was not unanimously agreed upon. I started with this at Karolinska Institutet in the fall of 1963, as Theo’s last PhD student. I did my PhD studies part-time, at the same time as I continued my medical studies, following the general practice of the students at the department. To solve the structural tasks, I had to learn many methods (very good, many thanks to Theo and to all my subsequent teachers!). I ended up with good results which have by now through much further work transformed ADH to a model system, illustrating molecular evolution and many aspects of structure^function relationships. This story parallels that on several other enzymes by other groups and was always in the scientific forefront. Great fun and much joy to me!

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Very soon in this chain of events, it became clear that novel techniques were required, and that they did not exist at Karolinska. Ieuan Harris from Cambridge, UK, and Bert Vallee from Harvard published short sequence studies of the active site of ADH. I saw the publications [3,4] and was thrilled. So did Theo, and soon he arranged for me to go to Fred Sanger’s department at the MRC Laboratory of Molecular Biology in Cambridge, UK, to study further with Ieuan Harris. (I later collaborated much also with Bert Vallee). I accepted Theo’s suggestion immediately, and so I was off to Cambridge in early 1967 for well over a year’s stay. Great luck! I arrived there at the peak period. A fantastic time! Much of the leading protein and DNA science of the world in those days centered around that whole department. Perutz’s wife was head of the canteen, knowing everyone and taking care of all, including me. I was taught to punt on the river Cam by him and his family. I was taught science by Ieuan and his students, in particular three other dehydrogenase students: Barrie Davidson with lobster tail glyceraldehyde 3-phosphate dehydrogenase, a work that was completed [5], Graham Jones with yeast glyceraldehyde 3-phosphate dehydrogenase, and Jo Butler with yeast alcohol dehydrogenase. In the lab opposite the corridor, Cesar Milstein taught me how to carboxymethylate a protein and gave practical hints. In the other lab of that wing, Brian Hartley and my fellow-Swedish visitor, Staffan Magnusson, taught me further methods. Jointly, the three labs of Ieuan, Cesar, and Brian were a true heaven for a young fresh protein student like me. The atmosphere was exciting: we felt we could analyze almost anything. We would sit daily in the canteen including Francis Crick and Sidney Brenner, for all meals, discussing everything from DNA to primordial soups that created life! The lab had parties with ‘‘plays’’ ending with a person coming in and imitating Max Perutz by saying something like ‘‘we had the same experience with hemoglobin.’’ In all labs I have worked at, the atmosphere has been rewarding and lab parties a natural phenomenon. Cambridge was fantastic, a true ‘‘think tank’’ of early molecular


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biology, and a real explosion of science! I was soon a genuine and accepted member of this scientific revolution. In my limited spare time, I experienced British nature. Staffan Magnusson, in the lab alongside, and I spent ornithological sessions in adjacent marshes and forests. I have later found that many biochemists are interested in birds! I did much work in England, utilizing the advantages of the Cambridge methods.They were genuinely manual, semiquantitative (at most!), and truly British! In retrospect, and knowing all later automations and quantifications, it appears amazing that the ‘‘dansyl-Edman’’ sequencing technique of those days could get so popular and reliable. But it brought great prospects with only cheap investments and was a necessity to protein science at that time. I came home from Cambridge ^ to set up a similar lab in Sweden, with the support of Theo. I published two papers with Ieuan in Cambridge and soon afterwards many more papers from my Swedish lab ^ and suddenlyADH was known in primary structure and in many of its properties! In the process, I got my PhD, and also my MD (always keeping my medical studies in parallel until then). What a time and what an explosion, and many thanks to all involved: Theo, —ke —keson (Theo’s ‘‘right hand’’ in biochemistry for decades and my real teacher at the start), Anders Ehrenberg (then biophysicist with Theo, and my formal employer at the start), Ieuan Harris, and many others. After this, in 1970, I was ready for the real life and had learnt the current protein methods at the Cambridge ‘‘Mecca’’ of protein chemistry of those years, had got equipment, thanks to Theo, and was set to apply all methods further. Before continuing this story, now a section on the methods of the to show both the breakthroughs and the hardships.

Development of Protein Sequence Analysis The general breakthroughs of those days in the 1960s were structural analysis ^ by X-ray crystallography centering on Perutz’s

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and Kendrew’s work on globins ^ and by chemical sequence analysis centering on Sanger’s and collaborator’s start with peptide hormones and enzymes. All these authorities’work is well-known. Sanger’s sequencing techniques, first in protein chemistry and then in DNA sequencing, gave him two Nobel Prizes, the only one to get the Nobel Prize in the same subject twice (Chemistry 1958, 1980) and similarly Kendrew and Perutz got the Chemistry prize (1962) for their studies of the structure of globular proteins! What were the secrets of Cambridge at that time and how did its rapid progress come about? All sequencing ‘‘secrets’’ were because of methodological progress, and in my view there were two particular ‘‘secrets’’ in the chemical revolution, both very elegant and very ‘‘British,’’ if I may say so, as one of the supporters of that era in British science: one secret or progress was the development of many smart and manual methods! The other was their use in smallscale lab facilities! Of course, both pieces of progress were dependent on Pehr Edman and his method of sequence degradation. More about that later. These British ‘‘secrets’’ were the two conditions that made it possible to characterize proteins in single-person, low-economy, PhD-type projects! To a large extent, it was a transient period, those methods were semiquantitative, relying on manual solutions rather than present-day high-tech sequencing equipment. But it worked and gave rapid progress. In particular, Barrie Davidson and Richard Perham, with Ieuan Harris, solved the primary structures of glyceraldehyde 3-phosphate dehydrogenase from lobster [5] and pig [6], and I that of alcohol dehydrogenase [7] ^ the horse liver ADH EE isozyme, at that time the longest enzyme protein chain sequenced and the one also including characterized isozyme substitutions [8]. We each did these determinations more or less in single-person work, far from large labs, which centered on column methods and later sequencer analysis, while we were truly manual and extremely low-scale in budget. My determination involved a 374-residue subunit. It was at a time when Edelman and Porter got the


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Nobel Prize for characterizations of other primary structures (1972, in Physiology or Medicine, ‘‘for their discoveries concerning the chemical structure of antibodies’’). This is not said to boost us PhD students ^ but to illustrate that this was a time (late 1960s) in protein science ^ when the step from great Nobel laureate to ordinary, small-scale PhD student was minimal, and the intervening bridge really made possible by the many elegant British inventions in low-budget, chemically smart, and manualsequencing techniques. One such invention was the creation of an excellent N-terminal labeling group: the dansyl chloride and its subsequent use in subtractive Edman degradation [9,10], allowing fast and efficient sequence analysis. The heart of this success was from Sanger’s earlier key invention of a labeling method (then using fluorodinitrobenzene [11]). This was now combined with manual Edman degradation, but using the Edman technique in the theoretically wrong mode of ‘‘subtractive Edman’’ rather than the theoretically correct mode of ‘‘direct Edman’’ degradation. This allowed efficient chromatography for resolution of all resulting dansyl amino acid derivatives. Those early days of protein chemistry were not limited by the chemistry, since both PITC and dansyl-Cl were available, thanks to Edman, and Gray and Hartley, respectively, but by lack of fast detection methods. Two-dimensional separations, first by paper electrophoresis in the tradition of the Cambridge era, and then by chromatography on polyamide sheets [12], changed that and made the Edman method available in the dansyl-subtractive mode to the general public of many small labs. Combined with efficient peptide purifications (below), using multidimensional paper electrophoresis, again along the Cambridge tradition, all tools for protein analysis were available. True, separations were still troublesome, many papers burnt in electrophoretic accidents, and all nonquantitative identifications at first made the sequences difficult to publish, since journals wanted quantitative approaches and more ‘‘solid’’ data than those available from these practical methods. But times changed rapidly, journals learnt that the

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dansyl-Edman method was reliable, and the papers were accepted. From then on, this paved the road to the now routine reports of sequencer-based primary structures of proteins. Such reports are often not any longer considered ‘‘science,’’ but just routine methods, left to be determined at core facilities! Three basic sets of pioneers with discoveries/inventions paved this progress from ‘‘the 1960s’’ to the ‘‘post-1980s’’ of protein science, by showing that all analyses are possible, and after the 1980s just routine. The three pioneers were: 1. Pehr Edman: His method of degradation [13, 14] made the concept of sequence analysis efficient and paved the road into protein primary structures. The method is still used and one of the few ‘‘old chemical’’ methods that even today is great science and much used in thousands of instruments worldwide, after over 50 years of service to the scientific community. Pehr Edman is the subject of another chapter in this volume, contributed by Birger Blomba«ck. 2. The Cambridge teams: Many individuals, starting with Sanger and continuing with Harris^Milstein^Hartley and their groups, converted scientific methods to every laboratory’s reach and added to the field a number of manual steps, shortcuts, and electrophoretic separations, making progress in the 1960^ 1980 transition period that opened the field to automation from others. 3. The Edman^Begg [15] and Hewick^Hunkapiller^Hood^ Dreyer [16] era: The Edman sequencer methodology, which was published in 1967 in the first issue of EJB [15], is a true classic and coined the concept of sequenator or sequencer, starting a new industry era. Subsequently, sequencers have automated both protein and DNA analysis, now making also genome projects just routine! Similarly, the ‘‘gas-phase’’ sequencer [16] is also classic, and brought protein science into molecular biology by supplying a step in literally many thousands of clonings worldwide. I will never forget the Edman^Begg EJB paper and that period. I read it in the library


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of the Cambridge lab and was amazed. Not only that report, but also that journal, EJB, and the persons behind its scene, deserve some comments: at that time, Ieuan Harris, my Cambridge mentor, was the leading Protein Editor of EJB and little could I then imagine that I was to get that same role over 20 years later, in the chain of Protein Editors from Ieuan Harris, via Pierre Jolle's, to myself, where I served for 10 years, 1990^1999. A nice experience and an excellent insight into the development of protein chemistry! At my resignation, Jan Johansson, one of my leading collaborators, became the next link in the chain and is now a Protein Editor with EJB since year 2000. Great, and fun to experience this continuity, with a tradition from previous leaders to coming heads! I feel the responsibility both backwards to the great deeds of Ieuan Harris, to the similarly fantastic deeds and amazing impartiality of Pierre Jolle's, and forwards to the young and promising scientist in Jan Johansson. Who could have dreamt this in 1967? Not me, and not Ieuan, but really showing the importance of tradition, knowledge, and continuity in scientific research. In addition, another of Ieuan’s early students and collaborators, Richard Perham, is now the Managing Editor of the same journal. To me, all these successions serve to show the importance also of editorship ^ and scientific administration ^ not only technology, but also in protein chemistry! Probably, the interconnecting chain of successors illustrate one more fact: the field was after all perhaps not that large, and the most active branches, therefore, often knew of each other or had worked together at one stage or another. Combined, the three legs of the Edman reagent, the Cambridge grouping, and the development of ‘‘gas phase’’sequencers through Edman^Dreyer^Hood^Hunkapiller, in my view explain the scientific transition from the 1950 realization that proteins are possible to analyze (thanks to Sanger and Edman), to the 1980 realization that sequence analysis is just a routine method (thanks to Hunkapiller and Hood). In this chain of events, it was a great pleasure for me to see that Hood and Hunkapiller were the recipients of the year 2000 Edman award at the MPSA (Methods

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in Protein Structure Analysis) series of meetings.Their ABI ‘‘gasphase’’ sequencer stands behind thousands of molecular biology protein characterizations, leading to the cloning and identification of innumerable metabolic and regulatory structural systems in humans, animals, plants, and lower organisms. Furthermore, equally rewarding was the choice of Geoffrey Begg and Brigitte Wittmann-Liebold as the 1990 Edman awardees, and Richard Laursen as the 1988 Edman awardee. Begg was the coworker with Edman himself in the development of the spinning cup sequencer. Wittmann-Liebold was the real constructor of many sequencer refinements, including valves, cartridges, and cold traps [17], and Laursen was the father of the solid-phase sequencer launches [18]. Hence, scientists behind all major branches of protein sequencers have been recognized with the Edman award. Equally satisfying for me to experience is that also subsequently (below), major developers of mass spectrometric protein analysis have been similarly recognized with the Edman award. Finally, in relation to Pehr Edman: I was fortunate to meet him closely a few times, and he was a great man. Interestingly, he also gave me good hints in my sparetime hobby of ornithology.Thanks to hints from him, brought to me through his wife, Agnes, at a lecture when we met at Karolinska in Stockholm, I saw my first special Australian endemic birds. Over and over again, I have noticed that ornithology attracts many biochemists, and Pehr Edman was no exception! Coincidentally, a chapter on him appears in this series (even the same volume), contributed by Birger Blomba«ck, with whom I have also been fortunate to collaborate at a few occasions. Before leaving the subject of analytical breakthroughs, one further detail should be mentioned: the development of separation techniques. The analytical progress, reflected by my lumping of ‘‘Edman ^ Cambridge ^ Sequencer’’ into a triad, was only one leg that transformed ‘‘chemical protein science’’ into ‘‘modern, present-day proteomics.’’ Two other legs were equally important in the final transition, and two where I too had the privilege to follow the progress: separation techniques (first) and protein mass spectrometry (later).


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Separation Techniques and Continued Progress Peptide (and protein) separations are crucial in all present proteomics approaches. There were many steps involved in the progress, and again, the Cambridge methodology gave one leap, by development of multidimensional paper electrophoretic methods [19,20]. Apart from all the chemistry at Cambridge, the central ‘‘hightech machine’’ in the Sanger era was the sewing machine for attaching papers to each other in between the different electrophoretic steps. Notably, a used sewing machine was one of the initial pieces of equipment that I bought for my first scientific grant in the late 1960s, and that sewing machine subsequently contributed to over 10,000 multidimensional paper electrophoretic separations, making all separations possible. A great fun and an early help to me! In between, it also repaired our lab-coats! Of course, many other groups and laboratories contributed to the further developments of separation techniques. This was true also for Theo’s lab, and one of the early scientists there (Sven Pale¤us), whom I met, but barely, was one of the first to apply ion-exchange chromatography to protein purification. So did Theo, and Keith Dalziel in his laboratory early developed a multistep purification of ADH [21]. More steps of separation progress followed and from many different origins, and increasingly involving industrial instrument manufacturers. We all remember the great impacts of Sephadex, further extensive miniaturizations and corresponding pump technologies with HPLC and its reverse-phase applications [22]. This continued with FPLC and its ion exchangers, Microblotters for sequencer applications, and further microbore uses. And then, of course, SDS/polyacrylamide gel electrophoresis [23], becoming a standard control and molecular weight evaluator for all protein subunits worldwide, and leading up to presentday fantastic separations on 2-D gels. Again, I have followed these methods and bought many of the instruments. Indeed, industrial manufacture and technical developments have brought much to our separation possibilities. However, apart

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from Jerker Porath (Edman prize 1996), these methods are not much person-fixed or place-fixed in my memories and, therefore, now left from further description here, except just mentioned because they have been essential for the development of protein chemistry at large. So has mass spectrometry, and it deserves a special section (below), but first yet other developments.

Amino Acid Analysis, Complementary Approaches, and Further Development Perhaps, this is the place also to correct and complement my presentation somewhat. Up to here, my description may sound as if much of the early events happened in Britain, or possibly around a few other scientists, like Edman and those behind the California sequencer. And much did happen in Britain (!), but not all. US was also a biochemical superpower, important since long, and had many further contributions. To give a balanced account, at least two further background advancements from there should be mentioned. They really set equally much of the conditions for protein chemistry and its future, and they meant much for me personally already then (but not initially noticed by me) and especially in the future (noticed!). The reason they, anyway, come late now is that they, to a large extent, happened before or after my scientific start with Theo and at Cambridge. One of these background advances is the art of amino acid analysis, the other the impact of large groups with early sequence studies independent of the Cambridge grouping. Regarding amino acid analysis, this breakthrough in the early 1950s was crucial. Ninhydrin reagents paved the way [24], ionexchangers for fractionation of amino acids gave the tools [25], and automated amino acid analyzers [26,27] made it all usable to everyone. Much of this centered around Moore and Stein with coworkers at the Rockefeller University in NewYork. It set a new era. This whole development made amino acids, and thereby peptides and proteins, amenable to analysis. It gave quantification


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to all analyses and purifications. Although not directly giving sequence information (but possible to use to this end, too, via the ‘‘subtractive Edman’’ approach, cf. above), it gave solid data on all compositions and hence promoted the whole subsequent explosion in protein sequence analysis. When I started with Theo, he had just built an analyzer, following descriptions from the literature. The machine was quite enormous, filling a room, and his technician was constantly climbing about, loading bottles, columns, and checking it all. My first project was to hydrolyse ADH, at that time in large volumes (milliliters) of 5.7 M HCl in very thick tubes. What a change later, in Cambridge, with volumes in microlitres and thin, minute glass tubes! But from now on, at the end of the 1950s, hydrolysis was the check-up and secured all subsequent sequence analyses! Although amino acid analyzers are nowadays sometimes considered a little out-offashion in the world of mass spectrometers and core facilities, they are still reliable and constitute a quantitative method, not to be forgotten. Last year, I bought a new analyzer, again based on ninhydrin, and acquired 38 years after I first used the homebuilt prototype instrument in Theo’s lab. I was also lucky to meet both Stein and Moore when they visited Theo. Later on, I met Stanford Moore quite much. Many scientists from those days knew of Stanford Moore and his care of us all! He was a genuine gentleman, wishing always to promote science, and making scientists to meet in small groups with him for breakfast, lunch, or dinner at all scientific meetings where he attended. A true genius in associating people, a promoter of science, and above all, a fantastic person! He was the one, who at a breakfast meeting (at the IUB conference in Hamburg, 1976), introduced and associated me with Ettore Appella of NIH, with whom I from that time started to collaborate for decades on methodological aspects of protein sequence analysis. I also visited Stanford Moore at his Rockefeller site. To a considerable extent, he and his social meetings gave me contacts and formed my future. He did the same with many others; for example, at that same Hamburg meeting,

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Ieuan Harris was also invited and introduced to new friends, but at a higher level: since he was my former teacher, he was already at the lunch club with Stanford Moore, while I was at the breakfast club. In short, devoted protein scientists were to a large part a family, thanks to Stanford Moore. He was a true and benevolent gentleman, in addition to being a great scientist and one who, together with his collaborators, permanently brought reliable total compositions into protein chemistry. A greater revolution than what we may nowadays realize! The other background development contributing to protein science that should perhaps be mentioned here is the influence of some largescale early sequencing projects. The corresponding laboratories did not have the same type of impacts as those behind the Edman reagent, the dansyl technique, the amino acid analyzer, and the sequencer automations, but they gave scientific possibilities, brought much activity into protein science, and also came into my life. One such group was the laboratory of Emil Smith and associates at UCLA. Irving Zabin’s laboratory at UCLA (see later), was where I was situated when the molecular biology revolution hit protein science. All those sequencing projects have meant much to protein science and knowledge! Another such laboratory was the Seattle grouping, started by Hans Neurath, and continued by many of his collaborators and successors. They all meant much. I was fortunate in coming into close contact with this grouping, also. I met Hans Neurath at a meeting in Germany in 1979, came into the Advisory Board of Biochemistry through suggestions by him and Bert Vallee, and then initially joined also when he founded the next journal, Protein Science. Combined, these large scientists and great US groups have meant much to the whole subject of protein science. It is sad, now to realize that those early eras have gone. That generation is now disappearing. My early mentors have already died, Theo, Ieuan Harris, and Viktor Mutt, and during the time that I write and polish this text, some of my subsequent supporters just died: Max Perutz and now Hans Neurath. In this context, I will never forget one of my absolute highlights regarding scientific lectures: the protein


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science meeting in Seattle, 1989, with Linus Pauling giving an enormous lecture, and Emil Smith and Hans Neurath being chairmen and getting awards. Close to a thousand scientists, including me, were fascinated by that Pauling talk and that whole evening of celebration with all these three protein scientists on the scene. Ovations were long, and I still have the menu with dedicated signatures of all these three great men. With this section on amino acid analysis and emphasis on research in the US, I hope to have balanced the previous sections, where much or nearly all progress was ascribed to Cambridge at one stage, and to three sequence developments at separate stages. All this is still true, of course, centering on sequence analysis per se and on the inventions. But when it comes to all of chemical protein analysis in the world, the fields are larger and more involved as shown by this section. The automation in chemical sequence analysis and the general complements above, constitute my recollections on the further development of chemical protein sequence analysis. I continued for decades to work in this field. From 1978, when I got my first spinning cup Beckman sequencer, I turned into a ‘‘machine biochemist.’’ Over the years, I have bought many sequencers. With the help of Brigitte Wittmann-Liebold, I got one rebuilt to include automatic PTH conversion in a separate cell, more efficient vacuum by a liquid-nitrogen cooling trap, and a more permanent pick up line in gold, all as invented and described by her [17]. Soon after these big changes were achieved, progress continued, and I bought further sequencers in rapid succession, and more recently, three further latest version ABI models, including also an instrument for C-terminal degradation [28]. The latter has been very good, and thanks to Ella Cederlund and Tomas Bergman with me, we have upgraded and modified that technology to get an excellently working sequencer for quite extraordinary C-terminal degradations [29]. All these instruments lasted for about a decade each, meaning that I constantly had two or three instruments which were running year-round, day and night, making it possible for my

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group and me to determine hundreds of protein and polypeptide primary structures and to use them in a large number of structural, functional, and evolutionary interpretations. This was a very productive time in my career, I became known for work on dehydrogenases, bioactive peptides, protein structure analysis, and molecular evolution. I participated in nearly all the MPSA conferences, from the first one in Boston in 1975 [18], and other early ‘‘preseries’’ meetings (like the one in Montpellier in 1971) and I was the organizer of the MPSA meeting in 1990 [30]. I decided to hold it in the northernmost Swedish town of Kiruna, above the Arctic Circle. I occupied much of four jets flying all participants north. Much organization and a hectic time, but great fun, and I got help from my staff, in particular Jan-Olov Ho«o«g (close collaborator on ADH enzymes) and Ann-Margreth Gustavsson (now my wife). Many participants remember this meeting and still tell me that it is the northernmost scientific meeting that they have ever been to. It was a success and a great time in my life. However, fate also made that meeting very bad in my mind for other reasons: at that time, we were three close friends of Swedish protein/peptide chemists:Viktor Mutt, Staffan Magnusson, and I.Viktor and Staffan were the first ones I invited to the Kiruna meeting, and we were going to celebrate methodological protein analysis and have a nice meeting. But none of us made it intact through that meeting: Viktor got ill in the spring before the meeting, in the disease that finally took his life years later. I myself, got a retinal rupture about a week before the meeting, when I was standing by the fax more or less all days and nights in final arrangements with all participants, and I could thus also not participate, but was hospitalized without vision during the meeting. And Staffan participated, but died on his return travel when he stopped by in Stockholm and we met. On his last evening, we went out for an ornithological session ^ as already mentioned, Staffan was another biochemist with an ornithological interest ^ both of us heavily crippled, I not seeing and he not easily walking. Strange MPSA meeting and serious set-backs!


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At the Kiruna meeting, the main role of substituting me was kindly handled by my friend and ADH collaborator, Bert Vallee from Harvard. I phoned him just a few days before, when I was lying without vision. He flew over immediately, visited me at the hospital, went to Kiruna, and ran it all excellently. Thanks! Overall, Bert and Kuggie are great friends and were collaborators for many years, giving wisdom and life experience! My lack of vision was a difficult problem and was critical, but after six surgeries my ophthalmologist surgeons finally managed to fix my eyes, and after about a year of little or no vision, I saw again. A fantastic feeling! Since then, I have had no eye problems and see better than ever before, but much inside my eyes has ever since been only plastics. It is not only protein chemistry, but also clinical research and ophthalmologic surgery that has made great progress during the 1980s and 1990s! During these decades, I also made much of my formal, scientific career, as summarized by years in the initial passage on my CV. I wrote hundreds of papers and had a maximum of 11 simultaneous PhD students. My career was a consequence of my longtime work and early devotion to chemical protein analysis. However, my scientific methodology and my life did not stand with just analyses, and was for much of my leaps equally dependent on other methods and interests. Thus, for me, like for most protein chemists in progress, chemical sequence analysis was only one leg, and perhaps just the entrance. To get further, to understand Life and Biochemistry, much more was needed, in particular: 1. 3-D structure analysis with X-ray crystallography, or for small proteins, NMR 2. from the late 1970s, transition to molecular biology and acquisition of sequence data by cloning and cDNA sequencing, and 3. from the late 1980s transition to mass spectrometry, which may soon be the method that finally puts an end to Edman chemistry after its about five decades of reign in protein sequence analysis, and

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4. informational studies, using bioinformatics and including studies in molecular evolution. I was fortunate also in these applications and transitions. Although I am not a crystallographer, molecular geneticist, mass spectrometrist, or mathematical evolutionary biologist, I came close to all these fields, with collaborations with corresponding scientists, and got these fields into my lab; thanks to excellent coworkers, students, visitors, and postdocs. Especially, in mass spectrometry, my transition of emphasis is profound, and during the last decade, I have acquired mass spectrometers at about the same speed as I previously bought and used chemical sequencers. I recently bought a novel ES-Q-TOF Ultima with promising possibilities! Of course, I realize that these transitions should not be summarized as ‘‘follow-up’’ methods to chemical protein analysis. Many more scientists have entered ‘‘Protein Science’’ via X-ray crystallography, NMR, molecular genetics, mass spectrometry, or evolutionary biology, than via chemical protein analysis, as I did. All these approaches are equally important, and neither is now a ‘‘follow-up’’ to the others. It is only in the chronology of my life that all beyond sequence degradation got the ‘‘follow-up’’ timing, and I, therefore, hope the reader excuses my order of presentation, with my initial emphasis on the biochemical protein analysis. Now, thus my recollections on these other approaches.

Correlation with 3 -D Structures I have collaborated with excellent crystallographers. Indeed, this is a necessity for meaningful interpretations of biochemical analyses and for the real understanding of protein structures. Theo saw this connection, too. Much like he initially sent me to Cambridge to pick up the biochemical methods, he made the then emerging crystallographic star in Sweden, Carl-Ivar Bra«nde¤n, interested in ADH. Carl had already been on a


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postdoc to Cambridge and started at his home lab in Uppsala to embark on the ADH structure. Thanks to him and his excellent collaborators, I got early access to the 3-D structure, I got to participate in many of the initial functional enzyme interpretations, and we had a joyful and productive collaboration. We adjusted ‘‘my’’ sequence to the, at that time, emerging diffraction pattern and had lots of joint meetings (including with Theo at a press conference in 1970, Figure 1). I was close to start working with

Fig. 1. Press conferences and celebrations are promoting joy and scientific collaboration within the groups. Here,Theo (furthest right), Carl-Ivar Bra«nde¤n (furthest left), —ke —keson (right center), and myself (left center) at a press conference, celebrating the 6 — 3-D model of ADH, directly after my dissertation on that primary structure in 1970. The photo also illustrates the shifting clothing modes. At this time, glasses were heavy and dark, showing essentially four similar glasses enjoying the structural model. Photo from the Swedish newspaper Dagens Nyheter.

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Carl in Uppsala, and had not fate given me other positions, I might have continued my career close to him. Carl also shared an ornithological interest, and at one of our joint conferences in England, we were invited by Sir David Phillips. Unfortunately, we came a bit late. Sir David seemed to have little understanding that we had stopped for a Gadwall at a pond on the way to him. Luckily enough, myADH amino acid sequence after many years of refinements finally fit into the diffraction pattern [31] without corrections, and although Carl and coworkers initially were questioning an internal charge at position 68, that Glu also turned out to be correct. It may even have a special function as a potential, additional or alternative ligand to the zinc ion at the active site as later also pointed out by others [32]. Carl, his collaborators, and I wrote the ADH review in‘‘The Enzymes’’ [33], made joint and early adjustments of also my next primary structure, that of the yeast ADH [34], and this was then followed by long sessions of modeling with sorbitol dehydrogenase including one of my longtime postdoc visitors, Jonathan Jeffery from Aberdeen [35]. In all, my time and experience with Carl and his group in Uppsala was great and productive, much like my earlier stay in Cambridge. A chapter by him will also appear in this series. When Carl moved to further projects and positions in Europe, Hans Eklund in Uppsala and I continued the cooperation. With the help of our coworkers, we have now finished characterizations of quite many enzymes, the hitherto latest being a plant pectin methylesterase [36], and with a bacterial ADH structure still in the pipeline. All my contacts with Hans and his coworkers have been a great pleasure, have taught me much on protein structures, and have been part of my own progress. Of course, at the same time, the early supply of primary structures have been useful also to the crystallographic group, and I think a joint cooperation is to be recommended to all protein biochemists and X-ray crystallographers alike. I have benefited greatly from this. Gradually, I came into similar cooperations also with other crystallographers.This diversification was largely due to the fact that we got more proteins than could be handled together with


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the Uppsala group, but was also due to the fact that we happened to enter a new protein family, when one of our ADH proteins turned out to be an SDR enzyme (more below). And since that enzyme was initially crystallized by Debashis Ghosh in Buffalo [37], I then came into natural contact with him, and later for similar reasons with Rudolf Ladenstein in Huddinge (also at Karolinska). Together the longtime contacts, studies, and collaborations with Hans, Deb, and Rudolf have been a true grace, joy, and humble study of nature and its evolution and mechanisms. It has also been very interesting for me to follow the progress in Xray crystallography so closely. Initially, interpretations were taking years, models were hand-built (cf. Figure 1), and computer power was low. Now, everything is rapid, computerized, and sophisticated. A development I see paralleled by the similarly computerized progress in both mass spectrometry and bioinformatics (below). I am happy to have seen the enormous progress in all these fields, which, together with molecular genetics, have revolutionized protein science! Of course, NMR is also part of the protein science family, and equally important as the other members. In my case, however, contacts with NMR have been limited, mainly because my enzyme side has involved larger proteins than those early handled by NMR. Hence, my personal entrance into the 3Dworld was largely via X-ray crystallographers rather than via NMR spectrometrists. However, my peptide side experienced NMR, and one of my close coworkers, Jan Johansson, went for a postdoc to Kurt Wu«thrich, determined the tertiary structure of the lung surfactant protein C [38], a protein from a source we only some years earlier had distinguished [39] from a blurry lipid mixture. Jan has since continued in our division with peptide conformations of importance to Alzheimer’s and the other aggregation diseases [40], where NMR is a natural must. In this regard, I also had the pleasure of inviting Kurt Wu«thrich to several of our conferences on evolution and life, and during my chairmanship at our department, we secured recruiting Gottfried Otting from Zu«rich for the first NMR professorship

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Fig. 2. Establishment of the sorbitol dehydrogenase (SDH) relationship to ADH in 1981 defined two major protein super-families, now called SDR (short-chain dehydrogenases/reductases) and MDR (medium-chain dehydrogenases/reductases). Today both have thousands of known members in data banks (presently 1000 MDR forms and > 3000 SDR forms; with at least 63 SDR and 23 MDR genes in the human genome), but the start was the ADH/SDH structures, which showed both splits and links in the complex evolution of large super-families. Splits from the fact that the same type of pattern (then ADH and PDH [polyol dehydrogenase]) occurs in two separate protein types (SDR and MDR), establishing the two super-families from an early start point (top); and links from the fact that each super-family includes many enzyme families (ADH and PDH first defined), establishing parallel evolutionary pathways in different lines. Modified from figure in Ref. [54].

at Karolinska Institutet. In short, NMR is a method for the future, like also X-ray crystallography!

Developments in Molecular Genetics The addition of Molecular Genetics in the 1970s had a profound impact on protein science. In a few leaps it became possible to


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get protein primary structures via DNA sequencing [41,42] and to produce any protein or site-directed mutant via DNA technology from bioexpression of corresponding DNA structures. A true revolution in protein science! By this, it has become possible to construct and produce proteins at will, plus rapidly and reliably to analyze them independently of solubility, purity, amount, or activity. The journal Nature, at the start of DNA technology, saw the demise of protein chemistry, drew the parallel to the revolution of protein chemistry that had been brought about by X-ray crystallography and had a commentary called ‘‘Rise and fall of protein chemistry’’ to allude to the, at that time, previous and present situation. Sanger got his second sequencing Nobel Prize, and protein chemistry became a branch of molecular biology at large. Of course, we now know that this development was not the demise of protein science, rather a rebirth. It was a revolutionary methodology, and above all, it brought possibilities. Suddenly, membrane proteins, adhesion molecules, gigantic proteins, and trace-occurring receptors became amenable to structural analysis. This opened new fields and meant an enormous leap forward. After about two decades of this progress, and subsequent completion of a number of genomesincluding the human genomewe now know that science is coming back to protein chemistry. With the present era of proteomics, protein analysis is revived at a ‘‘global’’characterization level in the cell, and is coupled with mass spectrometry [43]. Protein chemistry is back and modern again, but now forever combined with molecular genetics, crystallography, and mass spectrometry. Great, and I am happy to have experienced this transition, too. To me personally, this mid-1970s revolution came at a nonoptimal timing. I had just started a gigantic ‘‘traditional’’ protein sequencing project, to characterize the adenovirus hexon protein with about 1000 residues and to do that by chemical methods! For this purpose, in collaboration with Lennart Philipson, later being head of EMBL and later still of Skirball Institute for Biomolecular Medicine in New York, I had gone to UCLA for a

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sabbatical, studying large protein sequencing projects with Irving Zabin and Audre¤e Fowler (who had just completed the 1021-residue -galactosidase [44]), neighboring the laboratory of Emil Smith (who had completed much of the 1030-residue [45] NAD-specific Neurospora crassa glutamate dehydrogenase [46]). I was at the center of such a hopelessly large protein project, using ‘‘traditional’’ methods, and I was spatially divided, with me in US using UCLA sequencers and my collaborators (in particular Hedvig von Bahr-Lindstro«m) back in Sweden still pushing dansyl-Edman sequencing, coordinated in the same project. And just when we were seeing the end of our large project, the new era appearedin addition, the recombinant techniques were early applied to just the same virusadenovirus, as we were studying. What to do? Lennart visited me in Los Angeles and said: attack! That was correct, and I did so. Essentially, we solved it mostly by the traditional methods, got 966 residues [47] in sequence, and received only some help from the new methodology of DNA sequence analysis (Ulf Pettersson and Go«ran Akusja«rvi) in Lennart’s laboratory in Uppsala. The sequence, although pushed, was largely correct, and we only missed a tryptophan position (Trp-463) and a few details, to later come out with the final solution [48]. A thrilling time and an experience! But Lennart taught me a good lesson: when all looks difficult and hopeless, attack and push even more! That was a time when I got further sequencers and we got the end of our project, a new structurethe adenovirus hexon protein. But the new era was here to stay, and I switched, too. Keeping the protein sequencing methods, but adding the DNA sequencing and molecular genetics. Again, this started for me with my ADH projects. I went to the Memorial University, Newfoundland, Canada, to stay with Bill Davidson to get hands-on personally, and started cooperation with Gregg Duester and Moyra Smith [49] and with Bert Vallee’s molecular biology unit [50], and soon we had the molecular genetics techniques in our lab, too, with talented scientistsHedvig von


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Bahr-Lindstro«m, Jan-Olov Ho«o«g, and later Udo Oppermann. And now this combined approach, having access to both protein chemistry and DNA biotechnology, gives usas well as all versatile laboratoriesmaximal use of both methods. Presently, we keep protein sequencing, keep the DNA sequencing and mutagenesis, keep the crystallographic cooperations, plus add protein mass spectrometry. In all, this gives increased versatility and speed. Instead, I now sometimes wonder on my real expertise, as to which subfield all of us belongbut I guess at least some are still in protein chemistrybut now with reemphasis on proteomics. In innumerous cooperations and core facility studies, we have promoted molecular biology at large. It has been a great time and it constitutes a nice memory to have experienced this transition. After this description, now a switch again to recollections in molecular interpretation, molecular evolution, and bioinformatics.

Molecular Evolution: Development from Early ‘‘Diagonals,’’ over the Atlas Issues, to Bioinformatics With the availability of primary structures in the 1960s, I, like many others, started to do comparisons. Gradually, they grew more and more sophisticated, and these developments have formed part of my life, much like the chemical protein analyses have. Once I had the first ADH primary structure (horse EE form), and small parts of some others (yeast, the horse ES form and a human and a rat form), two things became apparent: (1) the isoforms were closely related and constituted an isozyme family, and (2) the yeast and mammalian forms were highly dissimilar, the initially detected and reactive cysteine residues were not even orthologous, although initially compared [3]. The isozyme/class relationships turned out to be highly complex, representing successive and repeated duplications in early vertebrate lines to form separate classes, and then

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different further duplications in the separate lines to form nonidentical isozyme sets in separate species. This whole system, with the successive clarification of the orders of the duplications, and the separate characteristics of each form became my next 20-year project after my initial isozyme report [8]. Gradually, my group has characterized much of this system, ending up with a summary of the evolution of this molecular system [51]. In this investigation, we studied in total 50-odd different ADHs, selecting the classes of the human enzyme, as well as highly different life forms from plants, through prokaryotes and nonvertebrates to a number of vertebrates, all in collaborations with many students passing through, and with separate foreign collaborating groups. After myself, PhD students on ADH from my group or from separate subgroups in my department have already passed eleven theses on ADH and related enzymes, with soon five more coming for 2002 and 2003, all with me, my ADH-collaborators, Jan-Olov Ho«o«g, Bengt Persson, and Udo Oppermann, or we in different combinations, as mentors. In addition, I have had close collaborations in these studies with first US scientists (initially Bert Vallee and Gregg Duester), then much with two groups around excellent Spanish scientists (Xavier Pare¤s and Roser Gonza'lez Duarte) and all the time with crystallographic collaborators (Carl-Ivar Bra«nde¤n, Hans Eklund, Debashis Ghosh, Rudolf Ladenstein in that order), from whose laboratories further PhD theses have also arisen. In total, this large set of characterizations has made the whole MDR and SDR dehydrogenase/reductase system into a model system with many known relationships. This family system could itself be the subject for a review, but some of my own highlights in this chain of evolutionary discoveries are just mentioned here: 1970: isozyme characterization showed close relationships [8]. 1976: initial characterization of Drosophila ADH showed this form to be completely different [52], later leading to the distinction of two large enzyme families (SDR and MDR, below).


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1977: comparisons of completed primary structures (one yeast and one horse form) showed distant relationships and established the different characteristics of dimeric and tetrameric ADHs [53]. 1981: characterizations of sorbitol DH established the ADH family to involve also other enzymes, and defined the two separate super-families, later called medium-chain dehydrogenases/ reductases, MDR, and short-chain dehydrogenases/reductases, SDR (Fig. 2) [54]. 1988: characterization of the separate evolutionary properties of the classes of the vertebrate ADH [55] 1992: definition of the early duplication forming the vertebrate ADH system, with separate classes, tracing enzymogenesis [56] and eventually characterization of the separate evolutionary properties of the classes [51]. (Fig. 3) 1995: definition of the SDR family relationships [57]. This formed the start of an ‘‘explosion’’ of SDR discoveries. SDR is now one of the large gene/protein families in the human: with minimally 63 [58] genes involved in many regulatory steps of which we have studied in particular glucocorticoid activation/ inactivation through separate forms of the 11-hydroxysteroid DH in humans [59]. Apart from the mere fact of this chain of events, it is of special interest to study the methodological development of comparisons. Thus, evolutionary studies with initially manual comparisons have, like all data handling, been explosively automated and now form a separate subfield of biochemistry/molecular biology, in the name of Bioinformatics. My initial sequence comparisons were very unsophisticated, involving mainly ‘‘diagonal comparisons’’ [60], initially assembled manually with a mm-paper! A great push for the early development of the science of molecular evolution came with Margaret Dayhoff, who founded the Atlas summaries, leading to the National Biomedical Research Foundation, then at Silver Spring in Maryland, US, and later to the PIR (Protein

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Fig. 3. Characterization of the separate evolutionary properties of the classes of human ADH. Top and bottom folds show the enzyme in stereo, with the most variable regions (thick-line tracing) at different places in class III (top) versus class I (bottom). In III, these regions are at nonfunctional segments of ancestrally locked enzyme forms, like for conserved proteins with constant functions. In I (and all other non-III classes), these regions are at functional segments of evolving enzymes, directly illustrating ‘‘enzymogenesis’’ [56]. The two middle boxes represent the corresponding primary structures (again III at top, and I at bottom) with a vertical black bar at each position where five divergent species (given left of the boxes) have identical residues, thus showing the variable regions as two white segments (indicated and numbered below the top box) and three white segments (indicated and numbered above the bottom box). These segments are identically denoted also in the top and bottom folds. The boxes further show (by similar amounts of black in both boxes) that similar variability requires more divergent species in III (human ^ yeast) than I (just vertebrates), thus illustrating the parallelism between greater overall and greater segmental (at functional sites) evolutionary speeds for class I versus the in both aspects more constant class III. Figure from Ref. [51].


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Identification Resource) data base. The first issues of the Atlas are now classic and define much of the principles for tracing molecular evolution. I still remember with great feelings the enthusiasm I felt for the knowledge that came from each Atlas issue. My reading of the early Atlas issue 1966 [61] was scientifically a high moment for me, much like the initial reports on the Edman [15] and gas-phase [16] sequencers. When computers became available, I first got help from a young computer scientist in my immediate surroundings: Magnus Persson, but soon centered on the development of an in-group specialization around one of my PhD students, Bengt Persson. Together Bengt and I characterized the functional assignments and ancestral relationships of the peptides and proteins which were determined within my groupa very successful collaboration which has continued throughout the years. After Bengt’s dissertation (1991), he went for a postdoc at the EMBL computer center, then came back to move on to a position at the Stockholm Bioinformatics Center, and has just been appointed Professor in Bioinformatics at Linko«ping University in Sweden. It has been a great privilege for me to follow the progress in Bioinformatics, growing from manual unsophistication to now great data banks and enormous computer powers. And to closely see this, with one of my students, participating in forming a new scientific field, where Bioinformatics is now a science by itself. It has been equally great to follow this methodological development as it has been on the analytical side. However, with the diversification, I got less time myself for all stages from analyses to interpretations (Figure 4). Finally, regarding this passage, two further personal notes are appropriate. The first is that at the end of this writing two more of my students got nominated for Professorship discussion. Thus, not only Bengt Persson in Bioinformatics, but also Jan Johansson in Physiological Chemistry and Tomas Bergman in Protein Analysis and Proteomics. This trebles my joy. It is a special feeling to see students growing into professors and centers, themselves! It also illustrates the speed

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Fig. 4. John Hempel, an excellent American scientist and friend, stayed with me for over four postdoc years. We almost made him into a Swede, and his children, born here, got Swedish names.When I later visited his Pittsburgh lab, he had put a Swedish flag on the lab walls. During his stay, my projects started to increase in number, and hence I was often elsewhere or in other projects when he searched for me at my office. He put up this sign on the door to my office (around 1984), in all friendliness, suggesting that those who wish to see me could do so by looking at this photo. The photo with its text is still on my office door, although the office has moved several times since then.

of protein science today: this subject covers all their subfields and includes many branches. A nice spread and much versatility! The second personal point in this field is my son Henrik. He early became ‘‘computerized’’ and he completely independently, with me initially not even knowing of his starting that work, developed a program, Motifer, for powerful detection of distant relationships, and for doing that directly from DNA sequences [62]. Good!


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Mass Spectrometry As obvious from my recollections above, I have followed two separate, long-term developments in protein chemistry. One is the ‘‘wet’’ chemistry part, with protein chemistry and analysis, ranging from early manual methods, via sequencers, to cloning and mutagenesis, separations, X-ray crystallography/NMR, and into the present proteomics. The other is the ‘‘in silico’’ branch, with interpretations, functional assignments, comparisons, molecular modeling, and bioinformatics. Both these fields continue to develop and to subdivide further. One of these subdivisions, which forms a part of the analytical leg, is so strongly emerging that I think it should get a special chapter by itself. It is protein mass spectrometry. Of course, protein mass spectrometry is analytical, and in that sense ‘‘wet chemistry,’’ in contrast to ‘‘in silico’’ chemistry, but it concerns minute amounts, and largely physical rather than chemical methods. It appears pertinent to have this section at the end, both because it is the hitherto last scientific leg of my scientific life, and because it is still emerging, with great developments now changing the field of protein analysis at large. A description of my protein life in analysis would not be complete without some recollections on the development of mass spectrometry (MS). Although I started in enzymology with Theo, being theoretically far from mass spectrometry of those days, I was physically close: Theo was in the Department of Biochemistry at Karolinska, and just opposite the local street was the then much bigger ‘‘mother’’ department of Medical Chemistry. It was at that time an analytical center for lipid biochemistry, where mass spectrometry already constituted a central method. Centered around Sune Bergstro«m (then the head), Bengt Samuelsson, and Jan Sjo«vall, with their early research on steroids and prostaglandins, novel mass spectrometers were born.Their leading mass spectrometrist was Ragnar Ryhage. Around him and Jan Sjo«vall, the first commercially available gas-chromatography/mass spectrometry instrument was developed in collaboration with the Swedish

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instrument firm LKB, making the LKB 9000 mass spectrometer in 1965, and developing the field for biochemical analysis, mainly on lipids. Until this day, Jan Sjo«vall is a leading mass spectrometrist, now as emeritus at our department. Jan, together with Sune Bergstro«m, was also the one who originally recruited me into the Department of Chemistry after my dissertation and Theo’s retirement in 1970. Over the years, both Sune and Jan have been devoted chemists. Sune sometimes phoned me, at any time, and is the only colleague who has ever phoned me late New Year’s eve with a scientific question. Later, I succeeded Sune on his chair, a result that I could not even have dreamt of at the time when he first saved me to the Chemistry department. Jan retired later, and then joined my grouping to promote protein mass spectrometry. Again, these transfers illustrate the adjustments of science, and the good results of combinations of methodology and project-driven science. During the early days, protein chemistry had little use of mass spectrometry, because of lack of volatility of peptides, and even worse, of proteins! True, all protein chemists, at meetings, in particular the MPSA series, always had sessions on mass spectrometry, but they were at that time for special problems, modified residues, and small compounds. Early on, we also all heard of and experienced the progress of tandem mass spectrometry for sequence analysis, and I several times heard Klaus Biemann give his impressive lectures on the successful progress, but mass spectrometry did at that time not make it to every-day protein analysis. This changed drastically with the development of matrix assisted laser desorption [63] and of electrospray [64] ionization techniques in the late 1980s. Suddenly, mass spectrometry became available for protein chemists. Peptides and proteins were flying, independently of their large size! After that, the mass spectrometry explosion in protein science has continued. Mass spectrometry is not any longer just a tool or possibility, but a necessity in protein chemistry, and a method soon eliminating many other analytical techniques, including sequencers.


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If the trend continues, it may be the method that finally buries, as mentioned above, the Edman phenylisothiocyanate method after its service to protein chemistry for over half a century. The development of mass spectrometry, like that of other analyses, has had a twofold basis of progress: first in instruments/methodology, and then equally much in computerization/data processing. What used to be time-consuming mass spectral outprints on special paper and slow scannings, is now real time data searches and ion interpretations to give on-line identifications toward databanks of genome sequences. In this manner, enormous numbers of mass spectra constitute no hinder, at the same time as matrix-assisted laser desorption and electrospray ionization have eliminated the protein volatilization problems. Ever-increasing sensitivity is now achievable, continuous nanospray flows [65] are routine applications, and correct mass picking in ion-traps plus on-line LC applications [66] make it possible to combine the separation/purification presteps with the analytical steps. Further developments in Fourier transform applications, or in path lengths, reflectors, and delayed extractions in MALDI applications, continuously increase resolution, availability, and sensitivity. Perhaps, one day, the old dream of close to one-molecule analysis is possible, not only with fluorescence correlation spectroscopy [67], as it already is, but also with mass spectrometry, showing the power of biophysical methods in present-day protein science. Like in the other analyses (chemical, molecular-biology, and X-ray crystallography) and in interpretations (bioinformatics), fate was kind to me in mass spectrometry, by bringing me correct constellations. When Jan Sjo«vall and his group joined me, we started an integration of our protein and mass spectrometry resources. At the same time, I got the responsibility to start a Protein Analysis Center (PAC) facility at Karolinska, 1997. I equipped this with new sequencers and new mass spectrometers, including in the unit also Bill Griffiths, the excellent

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mass spectrometrist in Jan Sjo«vall’s group, and Tomas Bergman from my group. And so it is, that I have come to witness also the mass spectrometry era in my life of proteins. It is clear that mass spectrometry is inside protein science to stay, and to become a leading or primary tool! But it is not yet the only tool. Even some sequencers, like especially C-terminal sequencers for degradation of large proteins, may still compete at practical characterizations of large recombinant protein products. But mass spectrometry is here. Last year, we had an international evaluation of our PAC facility, and we were asked to increase emphasis on mass spectrometry still further. We did so, and have just bought the new Q-TOF Ultima ES instrument, plus supported the present chairmanKarl Tryggvasonto buy also such a Q-TOF MALDI instrument. In the same manner, as I during the 1980s and 1990s bought nine chemical sequencers, my extended group and I have now during the 1990s and 2000s bought six mass spectrometers. What a transition and what a rapid start to my now next era of protein analysis. The last two years, two students of mass spectrometry have passed their PhDs in my group, including a versatile researcher (Andreas Jonsson) with an excellent thesis of seven papers [68]. He was immediately employed by Micromass Inc. True, we still keep also the sequencers and an amino acid analyzer, but I see the continuous development of mass spectrometry. This is the present situation and where my research is now positioned. I look forward to a promising future and further analyses at a still increasing speed and sensitivity. It should be added that mass spectrometry is not only primary structure analysis, but also allows analyses of complex interactions in biological systems. Associated proteins or proteins with cofactors and ligands, can fly together. Mass spectrometry is increasingly useful in establishing protein^protein interactions, metal binding, quaternary structure transitions, and ligand^ receptor interactions in all sorts of present and future projects in complex cell biology. A powerful technique with more to give!


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Present Situation, Including Comments on Karolinska, Nobel, and Funding This account has illustrated the multiple transitions in Protein Chemistry that have occurred during the last 40 years or so. I have been lucky to participate in a nice saga towards our further understanding of nature. And one issue has convinced me: the power and individuality of proteins will ensure that protein science will continue to flourish for a long time to come. With the postgenomic era of proteomics ahead, with all previous methodological eras of Protein Chemistry behind, and with Bioinformatics and Mass Spectrometry rapidly growing, protein science will be at the front during the next stages of development, interacting with other fields, such as neurobiology and cell biology. Before summarizing and trying to look at the future, let us start with the present situation. Perhaps, my description sounds as if protein chemists, in general, now are well integrated and have carried through all transitions completely at each stage of development. This is not the full truth, and probably we are all still biased towards or feel most at home with some fields. Similarly, transitions have in parts been integrations as a result of new or additional scientists, students, and technicians, rather than genuine alterations. Nevertheless, some transitions are indeed genuine: we and many others do have all these interests, we crystallize our proteins, we prepare our clones of cDNA, we are versatile in mass spectrometry, and we are fully computerized with all individuals independently drawing functional conclusions. However, several of our thoughts and hands still center on chemistry and biochemistry. Similarly, in project spread, subjects get deepened and enlarged. Thus, early on, much of me was dehydrogenases/reductases. While always keeping some of that, I then turned to viral proteins, bioactive peptides/hormones, lung surfactants (lipophilic peptides), methodology, core facility set up, and most recently, together with John Wahren (Karolinska Hospital), further peptide

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hormones, to the role of proinsulin C-peptide (Figure 5, [69,70]) and the possible pharmaceutical use of human C-peptide and its derivatives/analogs. Hence, not only changing methods, but also extended projects have governed my life, as that of others, and have developed over the years to give the present picture. During the last few years, I have got one further scientific thrill and considerable widening of interest, since I became the Secretary of the Karolinska Nobel Assembly and Committee from the year 2000. The Nobel work means much studies including monitoring of up-to-date fields. Interesting, and a ‘‘gift from heaven!’’ In fact, I am impressed by the dedication paid by colleagues in the whole Nobel system. We are lucky in Sweden to have this system, giving us both internal education in monitoring of science, and constant attention

Fig. 5. My latest major project, C-peptide and its mechanisms as a novel bioactive peptide, of great interest for pharmacological prevention of diabetic long-term complications. Left: Our current model of the tripartite nature of proinsulin C-peptide with a potentially structured N-terminal part, an unstructured central loop, and a bioactive C-terminal fragment. Right: Apparent biosignaling pathways for C-peptide. The arched top line represents the cell surface, with a G-protein coupled receptor (GPCR) as the most interesting C-peptide receptor candidate, but with some effects also via ligand-gated ion-channel receptors coupled to a glutamate receptor (Glu) and the insulin receptor (Ins), all working via signaling pathways involving intracellular calcium, protein phosphorylation (protein kinase C, PKC; mitogenactivated protein kinase, MAPK; phosphatidyl inositol 3-kinase, PI3-K; endothelial NO synthase, eNOS; and Naþ,Kþ-ATPase). Originals from [69,70].


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Fig. 6. Swedish postal stamp issue from 1996, showing some Swedish Nobel Laureates in Physiology or Medicine. Two earlier Laureates (Allvar Gullstrand and Ulf von Euler) and one later (Arvid Carlsson) also exist. The stamp set is rare for me: not only did I write the short captions on the set for three of the persons, but I also had close personal contacts with all four of these scientists: one (Sune Bergstro«m) was my predecessor on my chair in medical chemistry (top), one (Bengt Samuelsson) my previous head and in some cases collaborator (topmiddle), one (Theo) my initial mentor (bottom-middle), and one (Ragnar Granit) helping me during my course in medicine (bottom). A rare coincidence, and also part of my philatelic interests.

from the scientific (and society!) eyes of outside. Many have praised the carefulness of the Nobel evaluation system, and I share this admiration. It is a rewarding work. In addition, it is a great fun, uniting different subjects at the faculty level, learning to know fields and distant colleagues. In this connection, I have also been involved in a Post-Office philatelic stamp issue (Figure 6). Above all, Nobel work has a positive attitude, always trying to find the best, and at the end participating

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in celebrations, with the progress of science as a goal. I, we, and Swedish science, are grateful to the big donator starting it all! In retrospect, I now realize how foresighted Alfred Nobel was: Karolinska was founded by a Royal decree in 1810, but had to defend its position against the then existing universities, and did not get rights to examine its own medical students until 1874. It was this institution, already then important and powerful, but only 21 years old as a student examiner (!), that Alfred Nobel in his foresight (will of 1895) bestowed with the international responsibility of awarding his Prize in Physiology or Medicine for the ‘‘greatest benefit on mankind’’ to ‘‘the most important discovery within the domain of Physiology or Medicine!’’ Only recently was Karolinska formally named a Medical University. But it has always stressed research, and jointly the Karolinska and the Nobel system have grown in size, importance, and esteem. Karolinska was also one of the first, if not the very first Medical Faculty worldwide, to have a Professorship in chemistry. Already one of the four initial professors was a chemist, and this was Jo«ns Jacob Berzelius, one of the founders of modern Biochemistry. When we now start planning for the 200-year centennial of Karolinska, therefore studying its history, I learn from the annals that I am number nine on the chair from Berzelius. A great responsibility, but also another sign of the importance of tradition in academic research! With a long record, and a sufficient scientific body, basic research has flourished, giving the necessary progress, multiplicity, and freedom. Recently, Sweden and many countries have seen a relative decline in the state resources to basic research, which have been surpassed by external fundings and more applied research. EMBO in a recent survey and a site visit saw a possible decline in Swedish molecular biological production. Of course, I and many scientists are concerned. Let us hope that we can all revert this trend. In this regard, proteins and proteomics, with their flashy subjects, seem to offer great hopes, and may be a strong base for emphasis on research


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worldwide. Now, after this digression on Karolinska, Nobel and funding-policy, let us summarize the progress and try to look toward the future.

Summary of Revolutions My scientific life started at a time when only few peptides were known in structure, and when manual methods were the rule. Since then, I have seen at least five revolutions in protein science: 1. Instrumental automation, with sequencers (for both protein and DNA analysis) making much of the old science of Protein Structure Analysis to become routine, for core facilities. 2. Correlation with X-ray crystallography and NMR making molecular mechanisms and functional assignments understandable. 3. Development of molecular genetics in protein chemistry, making proteins of any size, complexity, and insolubility amenable to analysis, and allowing direct construction of new and mutant forms to test functional hypotheses and conclusions. 4. Computerization at all interpretational levels, allowing not only data acquisitions (as in mass spectrometers) but also the build up and on-line screening of data banks, leading to the new branch of bioinformatics with molecular graphics and ‘‘in silico’’ mimicking of all analyses, interactions, reactions, and mechanisms. 5. Ionization methods making mass spectrometry applicable to proteins, and leading to the explosion and take over of mass spectrometry in most protein analyses, now reaching already the attomol or so analysis level. Together with other scientific advances, this means that all that we know today, take for granted, and can analyze, could not even be dreamt of, visualized, or formulated when I started

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in protein chemistry. My generation has been fortunate in living through these revolutions. No generation before have had such a development, and although one should not exaggerate, at least some of these steps cannot happen to a similar extent to any other generation in the future either. One such unique leap is sensitivity. I started at about the micromole level of sequence analysis and have now experienced sub-femtomol sequencing, thus a leap of over nine orders of magnitude in sensitivity. Since we cannot possibly analyze below the 1-molecule level, no other generation can see the same great sensitivity increase (over nine orders) as we have seen. We have already reached more than halfway down to what remained to the 1-molecule level and we have consumed much of Avogadro’s number in sensitivity cuts. In fact, this sensitivity increase, and the data handling, are probably the two most impressive advances. Cuts in necessary time for analysis have also been impressive, but not equally so as sensitivity. Time is down about five orders of magnitude (from half week to seconds for an identification).

Perspectives For the perspectives, how do I expect the development of protein chemistry for the next generation? Of course, this is hopeless to predict, much like the present stage was outside all our science fiction grasps when I started. But it does perhaps not take too much imagination now to see at least some of the start of the future developments. Thus, for the future, I believe very much in: 1. 1-molecule analysis: sensitivity at this level or there about is expected. Fluorescence correlation spectroscopy [67], as already mentioned, is almost there, and the atomic force technique [71], further mentioned below, can observe single molecules. Some other spectroscopic and immunological reagents are also closing in. And with just a few pushes more in ion


2.

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handling, improved configurations, or transformations, mass spectrometry may soon approach this stage, too. Thus, the 1-molecule goal may be around the corner. Biophysics: Electromagnetically handled tools beat in speed any type of mixing, chromatography, or instrument moving part. Spectroscopies and activators/sensors are rapid tools already now, and Biophysics is for the future! 3 -D analysis:. Beams of increased intensity already push X-ray crystallography further in speed and sensitivity. This has helped to bring 3-D determinations into the proteomics concept of analyses of all components in a system. Standardized purification schemes will increase throughput further and promote also NMR analyses. Complementary methods may come. The old ‘‘science fiction’’of having a series of ever smaller, resistance-feeling, miniaturized ‘‘hands or gloves’’ with which one could directly feel the shape of a molecule, much like you can feel any surface by touching/ caressing it, and as proposed by the Nobel laureate Archer Martin long ago when he visited Karolinska with a lecture, this series of successively smaller hands is still science fiction, but the atomic force technique [71] appears to be a step into that direction and already gives rapid information on 3-D surface relationships. Bioinformatics: continues, including enormous databanks, on-line access during any analysis, and fantastic predictions, comparisons, and calculations. Of course! Miniaturization: also continues. This is dramatic already now. By decreasing volume, we can keep concentration constant when going down in amount, thus avoiding background blurring, need for purer samples than now, or disturbance from external factors. Some examples already achieved are: micro-cuvettes giving almost 1 cm light path, as in old cuvettes, but now with a total volume of about 100 nL; pumps, with stable flows down to 1 mL/min, allowing direct visualization and collection of eluted material in nanoliter volumes; chromatographies on multiple column

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lanes (10 nL) in CDs, giving elution flows down to 1 nL/sec for subsequent MALDI-MS [studies in progress with the Gyros company]; sequencer chips already constructed, degrading protein amounts so small that liberated amino acid derivatives cannot yet be detected, but will soon become detectable via mass spectrometry and novel processes [72]; microfluidics, already depending on new science, promises to come with further instruments, like concentrators [work in progress with Astorga-Wells et al.]; micro-dialysis equipment for in vivo measurements is also already existing and well-known, although not available to proteins but to peptides and other small molecules for continuous and direct monitoring [73]. 6. Integration: also continues, combining detection methods with microfluidics, computerization control, and data-bank screenings. Some of this already exists, with mass spectrometry as detection method (e.g. in Ciphergene and Gyros instruments), but additional integrations and cross compatibilities may produce further breakthroughs. 7. Complex cellular analysis: is also coming. Much of previous analyses have been performed on pure or purified samples. But the future mode is direct analysis in complex systems, allowing evaluation of the interplay between regulatory proteins in cellular processes, such as systems in neurobiology or cell biology at large. Again, mass spectrometry may prove powerful also here, with its possibilities to study also protein^protein and protein^ligand interactions. Further complexity is to come in all analyses! Perhaps these examples are sufficient to hint at some promising developments.The next generation of scientists will see much further miniaturizationmicrofluidics, mass spectrometry, biophysics, and continued computerization! I believe in those methods, and to balance it all, I also believe in much promise for two further fields, complementing protein chemistry: one is the study of transgenic animals to functionally assign all gene products and expression localities; this revives


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old-time physiology! The other is revival also of chemistry to functionally understand the interactions; this means coming back to where my dream was when I started, in other words, revival of biochemistry, too! ACKNOWLEDGMENTS

I wish to express my sincere gratitude to all my collaborators, supporters, students, and technicians. In spite of knowledge and methods, we all depend on personnel resources, and I have been fortunate in always having many scientists, students, and technicians surrounding me. Apart from those mentioned in the text, I have large numbers of additional collaborators. You are remembered and thanked! If, in addition, I should highlight anyone further, it would be my scientific fathers as mentioned here, my excellent students throughout my career, and two of my granitestrong technical supportersElla Cederlund and Carina Palmberg. Ella started with me at the age of 19, directly from her engineer exam, and last month we celebrated her 50th birthday! Similar with Carina! Similar also with my family. In particular my son Henrik, with whom I did my evening socializations by seeing him doing the pipetting extractions from the sequencers already at the age of three; And Ann-Margreth, keeping much of the lab order for almost two decades, and lately also me. Finally, I wish to acknowledge all research grants received, in particular from the Swedish Medical Research Council (now Swedish Research Council), the Swedish Cancer Society, and the Knut and Alice Wallenberg Foundation. These bodies have long been very beneficial for much of Swedish science!

REFERENCES [1] Chen, Z.-W., Bergman,T., O«stenson, C.-G., Efendic, S., Mutt,V. and Jo«rnvall, H. (1997) Characterization of dopuin, a polypeptide with special residue distributions. EJB 249, 518^522.

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[2] Chen, Z.-W., Ahren, B., O«stenson, C.-G., Cintra, A., Bergman,T., Mo«ller, C., Fuxe, K., Mutt,V., Jo«rnvall, H. and Efendic, S. (1997) Identification, isolation and characterization of daintain (allograft inflammatory factor 1), a macrophage polypeptide with effects on insulin secretion and abundantly present in the pancreas of prediabetic BB rats. Proc. Natl. Acad. Sci. USA. 94, 13879^13884. [3] Harris, I. (1964) Structure and catalytic activity of alcohol dehydrogenases. Nature 203, 30^34. [4] Li, T.-K. and Vallee, B.L. (1964) Active-center peptides of liver-alcohol dehydrogenase. I.The sequence surrounding the active cysteinyl residues. Biochemistry 3, 869^873. [5] Davidson, B.E., Sajgo, M., Noller, H.F. and Harris, J.I. (1967) Amino acid sequence of glyceraldehyde 3 -phosphate dehydrogenase from lobster muscle. Nature 216, 1181^1185. [6] Harris, J.I. and Perham, R.N. (1968) Glyceraldehyde 3 -phosphate dehydrogenase from pig muscle. Nature 219, 1025^1028. [7] Jo«rnvall, H. (1970) Horse liver alcohol dehydrogenase. The primary structure of the protein chain of the ethanol-active isoenzyme. EJB 16, 25^40. [8] Jo«rnvall, H. (1970) Differences in E and S chains from isoenzymes of horse liver alcohol dehydrogenase. Nature 225, 1133^1134. [9] Gray, W.R. and Hartley, B.S. (1963) A fluorescent end-group reagent for proteins and peptides Biochem. J. 89, 59P and 379^380. [10] Gray, W.R. (1967) Sequential degradation plus dansylation. Methods Enzymol. 11, 469^475. [11] Sanger, F. (1945) The free amino groups of insulin. Biochem. J. 39, 507^515. [12] Woods, K.R. and Wang, K.-T. (1967) Separation of dansyl-aminoacids by polyamide layer chromatography. Biochim. Biophys. Acta 133, 369^370. [13] Edman, P. (1950) Method for determination of the amino acid sequence in peptides Acta Chem. Scand. 4, 283^293. [14] Edman, P. (1953) Note on the stepwise degradation of peptides via phenyl thiohydantoins. Acta Chem. Scand. 7, 700^701. [15] Edman, P. and Begg, G. (1967) A protein sequenator. EJB 1, 80^91. [16] Hewick, R.M., Hunkapiller, M.W., Hood, L.E. and Dreyer,W.J. (1981) A gasliquid solid phase peptide and protein sequenator. J. Biol. Chem. 256, 7990^7997. [17] Wittmann-Liebold, B. (1981) Chemical Synthesis and Sequencing of Peptides and Proteins. (Liu, T.Y., Schechter, A.N., Heinrikson, R. L. and Condliffe, P.G. eds.). pp. 75^110. Amsterdam, Elsevier/North-Holland. [18] Laursen, R.A. (ed.) (1975) Solid-Phase Methods in Protein Sequence Analysis, pp 1^286. IL, USA, Pierce Chemical Co. Rockford.


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[19] Ryle, A.P., Sanger, F., Smith, L.F. and Kitai, R. (1955) The disulphide bonds of insulin. Biochem. J. 60, 541^556. [20] Ambler, R.P. (1963) The amino acid sequence of Pseudomonas cytochrome c-551. Biochem. J. 89, 349^378. [21] Dalziel, K. (1958) On the purification of liver alcohol dehydrogenase. Acta Chem. Scand. 12, 459^464. [22] Zimmerman, C.L., Pisano, J.J. and Appella, E. (1973) Analysis of amino acid phenylthiohydantoins by high speed liquid chromatography. Biochem. Biophys. Res. Commun. 55, 1220^1224. [23] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680^685. [24] Moore, S. and Stein,W.H. (1948) Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem. 176, 367^388. [25] Moore, S. and Stein,W.H. (1951) Chromatography of amino acids on sulfonated polystyrene resins. J. Biol. Chem. 192, 663^681. [26] Moore, S., Spackman, D.H. and Stein, W.H. (1958) Chromatography of amino acids on sulfonated polystyrene resins. An improved system. Anal. Chem. 30, 1185^1190. [27] Spackman, D.H., Stein, W.H. and Moore, S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30, 1190^1206. [28] Boyd, V.L., Bozzini, M., Guga, P.J., DeFranco, T.J. and Yuan, P.-M. (1995) Activation of the carboxy terminus of a peptide for carboxy-terminal sequencing. J. Org. Chem. 60, 2581^2587. [29] Bergman, T., Cederlund, E. and Jo«rnvall, H. (2001) Chemical C-terminal protein sequence analysis: improved sensitivity, length of degradation, proline passage, and combination with Edman degradation. Anal. Biochem. 290, 74^82. [30] Jo«rnvall, H., Ho«o«g, J.-O. and Gustavsson, A.-M. (eds.) (1991) Methods in Protein Sequence Analysis. pp. 1^398. Basel Birkha«user. [31] Eklund, H., Nordstro«m, B., Zeppezauer, E., So«derlund, G., Ohlsson, I., Boiwe, T., So«derberg, B.-O., Tapia, O., Bra«nde¤n, C.-I. and —keson, —. (1976) J. Mol. Biol. 102, 27^59. [32] Ryde, U. (1995) On the role of Glu- 68 in alcohol dehydrogenase. Protein Sci. 4, 1124^1132. [33] Bra«nde¤n, C.-I., Jo«rnvall, H., Eklund, H. and Furugren, B. (1975) Alcohol dehydrogenases. The Enzymes, 3rd Ed.,Vol. 11, pp. 103^190. [34] Jo«rnvall, H., Eklund, H. and Bra«nde¤n, C.-I. (1978) Subunit conformation of yeast alcohol dehydrogenase. J. Biol. Chem. 253, 8414^8419. [35] Eklund, H., Horjales, E., Jo«rnvall, H., Bra«nde¤n, C.-I. and Jeffery, J. (1985) Molecular aspects of functional differences between alcohol and sorbitol dehydrogenases. Biochemistry 24, 8005^8012.

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[36] Johansson, K., El-Ahmad, M., Friemann, T., Jo«rnvall, H., Markovic, O. and Eklund, H. (2002) Crystal structure of plant pectin methylesterase. FEBS Lett. 514, 243^249. [37] Ghosh, D., Weeks, C.M., Grochulski, P., Duax, W.L., Erman, M., Rimsay, R.L. and Orr, J.C. (1991) Proc. Natl. Acad. Sci. USA 88, 10064^10068. [38] Johansson, J., Szyperski, T., Curstedt, T. and Wu«thrich, K. (1994) The NMR structure of the pulmonary surfactant-associated polypeptide SPC in an apolar solvent contains a valyl-rich a-helix. Biochemistry 33, 6015^6023. [39] Curstedt, T., Jo«rnvall, H., Robertson, B., Bergman, T. and Berggren, P. (1987) Two hydrophobic low-molecular mass protein fractions of pulmonary surfactant. Characterization and biophysical activity. EJB 168, 255^262. [40] Kallberg, Y., Gustafsson, M., Persson, B., Thyberg, J. and Johansson, J.(2001) Prediction of amyloid fibril-forming proteins. J. Biol. Chem. 276, 12945^12950. [41] Maxam, A.M. and Gilbert,W.A. (1977) A new method for sequencing DNA. Proc. Natl. Acad. Sci. USA 74, 560^564. [42] Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463^5467. [43] Jolle's, P. and Jo«rnvall, H. (2000) Proteomics in Functional Genomics. pp 1^236. Basel, Birkha«user. [44] Fowler, A.V. and Zabin, I. (1978) Aminoacid sequencing of -galactosidase. J. Biol. Chem. 253, 5521^5525. [45] Haberland, M.E. and Smith, E.L. (1977) Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neuospora crassa. J. Biol. Chem. 252, 8196^8205. [46] Austen, B.M., Haberland, M.E., Nyc, J.F. and Smith, E.L (1977) Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neuospora. J. Biol. Chem. 252, 8142^8149. [47] Jo«rnvall, H., Akusja«rvi, G., Alestro«m, P., von Bahr-Lindstro«m, H., Pettersson, U., Appella, E., Fowler, A.V. and Philipson, L. (1981) The adenovirus hexon protein: The primary structure of the polypeptide and its correlation with the hexon gene. J. Biol. Chem. 256, 6181^6186. [48] Akusja«rvi, G., Alestro«m, P., Pettersson, M., Lager, M., Jo«rnvall, H. and Pettersson, U. (1984) The gene for the adenovirus 2 hexon polypeptide. J. Biol. Chem. 259, 13976^13979. [49] Duester, G., Hatfield, G.W., Bu«hler, R., Hempel, J., Jo«rnvall, H. and Smith, M. (1984) Molecular cloning and characterization of a cDNA for the subunit of human alcohol dehydrogenase. Proc. Natl. Acad. Sci. USA 81, 4055^4059.


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[50] Sharma, C.P., Fox E.A., Holmquist, B., Jo«rnvall, H. and Vallee, B.L. (1989) cDNA sequence of human class III alcohol dehydrogenase. Biochem. Biophys. Res. Commun. 164, 631^637. [51] Danielsson, O., Atrian, S., Luque, T., Hjelmqvist, L., Gonza'lez-Duarte, R. and Jo«rnvall, H. (1994) Fundamental molecular differences between alcohol dehydrogenase classes. Proc. Natl. Acad. Sci. USA 91, 4980^4984. [52] Schwartz, M.F. and Jo«rnvall, H. (1976) Structural analysis of mutant and wild-type alcohol dehydrogenases from Drosophila melanogaster. EJB 68, 159^168. [53] Jo«rnvall, H. (1977) Differences between alcohol dehydrogenases. Structural properties and evolutionary aspects. EJB 72, 443^452. [54] Jo«rnvall, H., Persson, M. and Jeffery, J. (1981) Alcohol and polyol dehydrogenases are both divided into two protein types, and structural properties cross-relate the different enzyme activities within each type. Proc. Natl. Acad. Sci. USA 78, 4226^4230. [55] Kaiser, R., Holmquist, B., Hempel, J., Vallee, B.L. and Jo«rnvall, H. (1988) Class III human liver alcohol dehydrogenase: A novel structural type equidistantly related to the class I and II enzymes. Biochemistry 27, 1132^1140. [56] Danielsson, O. and Jo«rnvall, H. (1992) ‘Enzymogenesis’: Classical liver alcohol dehydrogenase origin from the glutathione-dependent formaldehyde dehydrogenase line. Proc. Natl. Acad. Sci. USA 890, 9247^9251. [57] Jo«rnvall, H., Persson, B., Krook, M., Atrian, S., Gonza'lez-Duarte, R., Jeffery, J. and Ghosh, D. (1995) Short-chain dehydrogenases/reductases (SDR). Biochemistry 34, 6003^6013. [58] Kallberg, Y., Oppermann, U., Jo«rnvall, H. and Persson, B. (2002) Shortchain dehydrogenase/reductase (SDR) relationships: a large family with eight clusters common to human, animal and plant genomes. Protein Sci. 11, 636^641. [59] Oppermann, U.C.T., Persson, B. and Jo«rnvall, H. (1997) The 11-hydroxysteroid dehydrogenase system, a determinant of glucocorticoid and mineral ocorticoid action. Function, gene organization and protein structures of 11-hydroxysteroid dehydrogenase isoforms. EJB 249, 355^360. [60] Jo«rnvall, H. (1973) Partial similarities between yeast and liver alcohol dehydrogenases. Proc. Natl. Acad. Sci. USA 70, 2295^2298. [61] Eck, R.V. and Dayhoff, M.O. (1966) Atlas of Protein Sequence and Structure 1966, pp 1^215. Silver Spring, MD, USA, National Biomedical Research Foundation. [62] Jo«rnvall, H. (1999) Motifer, a search tool for finding amino acid sequence patterns from nucleotide sequence databases. FEBS Lett. 456, 85^88.

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[63] Karas, M. and Hillenkamp, F. (1988) Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal. Chem. 60, 2299^2301. [64] Fenn, J.B., Mann, M., Meng, C.K.,Wong, S.F. and Whitehouse, C.M. (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246, 64^71. [65] Wilm, M., Shevchenco, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T. and Mann, M. (1996) Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379, 466^469. [66] Martin, S.E., Shabanowitz, J., Hunt, D.F. and Marto J.A. (2000) Subfemtomole MS and MS/MS peptide sequence analysis using nanoHPLC micro-ESI Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 72, 4266^4274. [67] Eigen, M. and Rigler, R. (1994) Sorting single molecules: Application to diagnostics and evolutionary biotechnology. Proc. Natl. Acad. Sci. USA 91, 5740^5747. [68] Jonsson, A. (2001) Mass Spectrometry in Protein Structure Analysis. Diss., Karolinska Institutet. [69] Jo«rnvall, H., Nordling, E., Persson, B., Shafqat, J., Ekberg, K., Wahren, J. and Johansson, J. (2002) A structural basis for proinsulin C-peptide activity, in Nordling, E. (2002) Biocomputational studies on protein structures. Diss., Karolinska Institutet. [70] Johansson, J., Ekberg, K., Shafqat, J., Henriksson, M., Chibalin, A., Wahren, J. and Jo«rnvall, H. (2002) Molecular effects of proinsulin C-peptide. Biochem. Biophys. Res. Commun. 295, 1035^1040. [71] Viani, M.B., Pietrasanta, L.I.,Thompson, J.B., Chand, A., Gebeshuber, I.C., Kindt, J.H., Richter, M., Hansma, H.G. and Hansma, P.K. (2000) Probing protein^protein interactions in real time. Nature Struct. Biol.7, 644^647. [72] Wurzel, C. and Wittmann-Liebold, B. (2000) New approaches for innovations in sensitive Edman sequence analysis by design of a wafer-based chip sequencer. In: [43], pp. 145^157. [73] Ungerstedt, U. (1991) Microdialysis ^ principles and applications for studies in animals and man. J. Intern. Med. 230, 365^373.


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Chapter 3

PehrVictor Edman: The Solitary Genius BIRGER BLOMBA«CK Karolinska Institutet, Fogdevreten 2A, Stockholm, SE-171 77, Sweden

One of the main problems in protein chemistry half a century ago was how to efficiently elucidate the sequence of amino acids in proteins. With laborious methods, only small protein structures had so far been resolved. For insulin, the most famous example, the sequence of its 51 amino acid residues took a decade to unravel. Entertaining the thought of establishing the amino acid sequences for protein molecules with hundreds or even thousands of amino acids was, in general, considered impossible, unrealistic, or meant for a far distant future. Still, there was a general agreement in the biosciences that this information was fundamental to the understanding of various life processes and evolutionary aspects. Then came, what I would call, the Edman revolution.1

1 In the 1960s I applied for an appointment as professor at a University in Sweden. Among the credentials I mentioned was my intention to determine the primary structure of fibrinogen, although at the time I only knew short sequences of the N-terminal ends of the three chains, determined by Edman’s method, but my belief in the method was steadfast. One of the reviewers admired my resolution but ended with a warning, saying poetically, that the endeavor was like a promise to reveal the ice-hidden land of Antarctica, when only pieces of coastline geography was known. This was how biochemists in those days looked at the prospect of elucidating the primary structure of proteins.

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Pehr Edman in 1960s. To the right is a photo of Pehr at the age 4^5.

In the history of protein chemistry, Pehr Edman will be remembered for his outstanding, painstaking, and, not the least, brilliant work that gave us a novel tool to establish amino acid sequences in proteins; a technique by which amino acids in the protein molecule can be removed and identified ^ one after the other in a stepwise fashion. The rapid developments in molecular biology during the past 30^40 years would not have been possible without Edman’s amino acid sequence method. It is true that today, amino acid sequences of even large proteins are rapidly deduced from the nucleotide sequences of their genes. Still, in order to catch the complementary DNA (cDNA) representing the protein, one needs a partial amino acid sequence, albeit short, and to establish that sequence, one is in need of what is generally known by the

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eponym Edman degradation. In addition, only direct protein sequencing can provide hard information about the final primary structure of the functioning protein. The chemistry discovered by Edman half a century ago is virtually the only tool we have for this purpose. Already in the 1940s Pehr Edman set out to solve the problem of rapid and accurate stepwise degradation of protein chains. Throughout his research career, he resisted all temptations to deviate from his set course until he considered the task completed and well done. Rewards for early application of his method to interesting biological and medical problems went to others. At the time of his death he was still engaged in optimizing the method.2

Nature is the Guide Pehr Victor Edman was born in Stockholm, Sweden, on April 14, 1916. His family lived in the O«stermalm area of Stockholm. His father, Victor Edman, was a judge. On the paternal side, male members of the family had, for generations, served as public officers in state agencies and the armed forces. His father appears to have been a rather serious man and was a devoted Christian. The mother, Alba Edman, was of a more joyous and lively nature. As a boy, Pehr Edman first attended the elementary public school in Stockholm and later high school in ‘‘Norra Latin,’’ a school with special emphasis on the humanities. For some reasons, among them a sadistic teacher, he was unhappy there but got a new start at the ‘‘Norra Realskolan,’’ where mathematics and the natural sciences were in focus, and luck turned. When discussing this period in his life, Pehr Edman often mentioned a biology teacher, Mr. Ringselle, who seemed to have been instrumental in awakening his interest in nature 2 Over the years since his death, a few monographs on Pehr Edman have appeared [1^3]. The present one is partly based on the articles in References 1 and 2.

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and in the biological sciences. Edman would often talk with warmth and appreciation about Mr. Ringselle and this time of his life and gave this short description of the man: stout, jovial, and warm. He spent several summers with Mr. Ringselle out on the island of Singo« in the northern archipelago of Stockholm, where Mr. Ringselle had a house. Under his expert guidance, Pehr went out for botanical studies, bird watching, or fishing. Pehr Edman’s brother tells that he sometimes used to join Pehr on these excursions. He recalls how Pehr could sit for hours watching some natural phenomenon ^ be it live or dead, organic or inorganic. In the same way Pehr Edman would, later on in life, at times sit for hours at his laboratory bench meticulously watching chemical reactions or physical phenomena. His natural gift of observation and deduction, together with his logical mind and stringency of expression, were apparent already as a boy. In 1935 Pehr Edman passed his matriculation examination with excellent records. This examination in Sweden is a great step in the career of a young man. On the day of the examination, family and friends would come to school with flowers and gifts. They would wait outside the school for the students to come out after being through with their exams. Pehr Edman’s family had to wait in vain. He had left the school through the back door and gone straight home. A tendency towards shyness was another of his qualities, and he certainly disliked pompous performances especially when he himself was at the center of attention.

Science Takes Hold After the matriculation examination, Pehr Edman decided to study medicine and therefore applied to the Karolinska Institutet in Stockholm, a medical school. He started his medical studies in 1935 and received a bachelor of medicine degree in 1938. He graduated as a physician in 1946. During his studies at the Karolinska Institutet, he joined a socialist political organization,


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Clarte¤.3 Several Swedish intellectuals, politicians, and artists were its members. Tage Erlander, who later became Prime Minister of the Swedish government, was one of them. Among the artists was a famous poet, Nils Ferlin, whose anarchistic ideas and whims quite often turned the meetings to pandemonium, as Pehr Edman recalls. During his studies Pehr Edman met his first wife, Barbro Bergstro«m, whom he married in the early 1940s. Concurrently with his studies in medicine, he started his training in biochemistry under the guidance of Prof. Erik Jorpes and for a short period he also studied in Prof. Hugo Theorell’s department. It was in Prof. Jorpes’s department that he first became interested in protein chemistry. Prof. Jorpes’s main interest at the time was in the biochemistry of mucopolysaccharides, especially heparin. Erik Jorpes had also an interest in secretin and insulin. Pehr Edman took some part in the work on secretin but soon started on a project of his own; isolation and characterization of angiotensin (at the time called hypertensin or angiotonin). He had to deal with a compound that was present only in trace amounts. As starting material he used horse blood, obtained in early morning hours from a nearby slaughterhouse. Out of one ton of blood, he obtained 30 mg of purified angiotensin, having 700 times higher specific activity than the starting material. During this work, he became acquainted with the preparatory and analytical tools used in those days; one of which was column chromatography. The innovative trait in him was apparent both in the preparative toil and in the various analyses. For example, he devised new solvent systems for two-dimensional paper chromatography, useful in analyses of amino acids. His work on angiotensin resulted in a preparation that he considered chemically pure. He determined the amino acid composition and other chemical characteristics including molecular mass. This work resulted in a thesis that was presented at the Karolinska Institutet in 1945, 3 In a previous review [2] this chapter was erroneously stated to be a Marxist organization.

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receiving the mark of Summa cum Laude. Pehr Edman’s thesis work took place during World War II, and for a period he was drafted to serve as a physician in the armed forces. Knowing Pehr Edman’s dislike for the military establishment, this was surely a rather dull time for him. However, he got some relief by being offered a horse for transportation between the military units; he came to enjoy horse riding. After his dissertation, Pehr Edman applied for a position as docent at the Karolinska Institutet, which was granted. However, Pehr Edman now wanted to widen his experience and perspectives in protein chemistry and therefore applied for, and shortly thereafter received, a Rockefeller fellowship. This was for a one-year stay (1946^1947) at the Rockefeller Institute in Princeton (a division of the Rockefeller Institute in New York), in the laboratory of

Pehr Edman at Rockefeller Institute. From left to right: Moses Kunitz, Roger Herriott,Winston Price, John Northrop, Pehr Edman.


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Dr Northrop and Dr Kunitz: at that time one of the most prestigious places for protein chemists ^ a laboratory where many enzymes had been isolated in crystalline form.

Birth of an Idea Let us start with some historical remarks. Traditionally, proteins were regarded as biocolloids in the 19th century and few people supposed that any protein preparation contained identical molecules. At the end of the 19th century, Olof Hammarsten in Uppsala isolated fibrinogen out of horse4 plasma [4]. This clotting protein was one of the first proteins to be isolated in pure form; it appeared to be a homogeneous component on the basis of being almost completely transformed into fibrin by thrombin. The idea that there were distinct species of proteins became more and more in fashion as other proteins and peptides were isolated in what appeared to be unique forms. Crystallization of enzymes in the first half of the 20th century, and the demonstration by Sumner that the enzyme activity was an inherent property of the protein, added credibility to this concept [5].5 So did also the demonstrations by Svedberg [6] and Tiselius [7] that purified proteins migrated as single boundaries in a centrifugal or electrical field. The early X-ray diffraction patterns of various proteins pointed in the same direction [8]. Interwoven was the technical upsurge in new capabilities in preparation of proteins in pure form and in quantitative analysis of amino acids. As to the latter, Martin and Synge [9] demonstrated that solutes, such as amino acids, are distributed between two liquid phases, one of which could be immobilized 4 Hammarsten choose horse blood since the erythrocyte sedimentation rate in this blood is high; thus making centrifugation, not available to him, superfluous. Also, for Pehr Edman centrifuges were not available and he, therefore, also used horse blood in his studies on angiotensin. 5 James B. Sumner, John H. Northrop, and Wendell M. Stanley received the Nobel Prize in 1946 for their work on enzymes and virus proteins.

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by mixing it with an absorbent powder. This was followed by the introduction of paper chromatography by Consden et al. in 1944 [10]. A further development in quantitative analysis came with the use of synthetic ion exchange resins as reversible adsorbents of amino acids by Moore and Stein [11]. All this brought about a changing view about proteins that was eventuallyaccepted bychemists and physicists alike.Thus, by 1950 the realization had dawned that individual proteins contained molecules which were identical replicas, had exact molecular masses and amino acid compositions, and identical packing of the polypeptide chains. This view gained final acceptance when crystalline preparations of proteins became available for analysis of their X-ray diffraction pattern; it was found that crystals gave rise to diffraction patterns that could be interpreted on the same basis as the crystals of simple inorganic salt compounds, although requiring tremendous computer capabilities. Although isolation of proteins and the analysis of amino acid composition had become little more than a matter of routine, an important feature, the sequence of the amino acids in the polypeptide chain, was still unknown. The first successful attack on the problem of sequence determination in proteins was that mounted by Frederick Sanger and published during the period 1945^51. Sanger used insulin as model protein. Sanger’s idea was simple. All that is necessary for the determination of the sequence of a dipeptide is amino acid analysis and a method of determining which amino acid is N-terminal. Sanger was the first to realize that even with longer peptides, the unique sequence could be established by locating the N-terminal in the starting molecule and in each of the smaller peptides derived from it by random partial acid hydrolysis or by digestion with proteolytic enzymes. He used column chromatography on silica gel for separation of peptides and paper chromatography for determining amino acid composition. The N-terminal amino acid (vide infra) was recognized in any peptide and, if longer than a dipeptide, its sequence could be worked out by further partial hydrolysis and by fitting overlapping sequences. He devised a method of labeling the


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N-terminal end of the polypeptide chain by reacting it with 1-fluoro-2,4-dinitrobenzene (FDNB) to form the dinitrophenyl derivative of the N-terminal amino group. These dinitrophenyl (DNP) derivatives were substantially stable to acid hydrolysis and their bright yellow color facilitated both qualitative and quantitative analysis. Using this technique, Sanger and coworkers were able to elucidate the order of the amino acid residues in the A- and B-chains derived from insulin after reduction and subsequent oxidation with performic acid. In 1951 the complete sequence of the 30 amino acid residues in the B-chain of insulin was published [12,13]. Moreover, it was shown that the B-chain were identical in beef, pig, and sheep insulin; whereas, in the A-chain a few substitutions were noticed in some species. These achievements certainly marked a milestone in protein chemistry and demonstrated unequivocally that a protein in a given species was carrying a unique primary sequence. The time Pehr Edman spent at the Rockefeller Institute in 1946 and 1947 was in a way crucial to his scientific career since it was here that he made the first attempts toward stepwise degradation of proteins. However, the turning point in his thinking had occurred before that. During his work on angiotensin, it became clear to him that molecular mass and amino acid compositions were not parameters that would give information explaining the biological activity of the protein. Most likely, this must somehow reside in the amino acid sequence of the protein.6 Edman was well read in biochemistry, so he certainly 6 Many years later, in the1960s, this prediction was boosted when Chinese scientists at Academia Sinica in Shanghai synthesized insulin de novo from synthesized amino acids and showed that the compound had almost full biological activity and otherwise resembled native insulin in physical and chemical characteristics [14,15]. Although the authors saw the achievement as a confirmation of the materialistic theory of Marx and Engels, it was nevertheless a major breakthrough in our view of the structure^function relationship in proteins, ‘‘was not this what I thought’’ Edman said to me in 1962. It is true that for the function of most proteins, tertiary structure and conformation are the final determinants. Still, the information necessary to give a protein, its active form, is inherent in its primary structure and this was what the Chinese scientists had demonstrated for the first time. The truth of this was further confirmed by Christian B. Anfinsen (Nobel Prize 1972) in his studies on the folding of ribonuclease, albeit in a more indirect way.

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was, at that time, aware of the work on the amino acid sequence of insulin by Sanger’s group. Nevertheless, he was convinced that the method used by them would have severe limitations when it came to sequencing large protein molecules. At the Rockefeller Institute, Pehr Edman was, for a while, considering using enzymes for sequence purposes but soon considered it impractical. He turned his attention to chemical reagents that would allow both carbamylation of the -amino acid residue in proteins and subsequent rearrangement and release of the N-terminal amino acid under mild conditions. Sanger, on the other hand, had just wanted a stable label on the N-terminal that could survive acid hydrolysis of all peptide bonds. Edman’s first approach to use phenylisothiocyanate for carbamylation of peptides was influenced by the work of previous investigators [16^18]. Bergman and coworkers [16] had already, in 1927, used phenylisocyanate to label the N-terminal amino acid in dipeptides and shown that on subsequent acid hydrolysis, the phenylhydantoin of the N-terminal was formed. Later Jensen and Evans [17] used the method on insulin and were able to show the presence of the hydantoin of phenylalanine, one of the N-terminals, after acid hydrolysis. Abderhalden and Brockmann were the first to use phenylisocyanate for stepwise degradation of polypeptides [18]. The N-terminal amino acid was released as a hydantoin from the carbamylated peptide in methanol saturated with HCl. Edman at first thought that this was due to a nucleophilic attack by phenylisocyanate on the first CO^NH bond of the peptide.7 However, whatever the reaction mechanism involving phenylisocyanate, it was inconvenient for sequence purposes since the labeled N-terminal amino acid could not be released without breaking other peptide bonds. What Edman was pondering over was finding another, more efficient, nucleophile, i.e. a label on the N-terminal, that would 7 Much later, in 1970, Edman realized that his assumption in regard to phenylisocyanate had not been correct, since cleavage of the peptide bond precedes formation of hydantoin [19]. Nevertheless, it put Edman on the track to find a nucleophile and that was worthwhile.


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present the CO^ group of the linkage to the next amino acid with an energetically more favorable reaction partner (than the NH^group) and thus cause fission of the bond without breaking other peptide bonds. Edman considered phenylisothiocyanate to be such a nucleophile. This was a new daring idea. It may appear to us that Edman’s idea to use phenylisothiocyanate as a nucleophile came out of the blue. Or some may hint that someone else gave him the idea. Neither of these inklings, I believe, is correct. Edman knew his organic chemistry and the order of nucleophilicity among elements. Certainly, he knew that a ketonic sulfur would be expected to be a good supplier of electrons and the sulfur in phenylisothiocyanate seemed to offer a good prospect.8 Toward the end of his stay at the Rockefeller Institute, he had tried phenylisothiocyanate on model peptides and obtained evidence for the soundness of his unique idea.

Idea Becomes True After his return to Sweden, Pehr Edman applied for and was awarded a vacant associate professorship at the University of Lund. He continued to work there on the use of phenylisothiocyanate for sequence analysis. For a while, we find him working also on other problems in protein chemistry. So, in 1948 he designed the first clock operated chromatographic fraction collector. He also undertook some studies on separation of chymotryptic peptides by partition chromatography on starch, 8 To support the above statements, I quote from Edman’s lecture at the meeting of the American Chemical Society in 1951 as recorded by J. S. Fruton [20]: ‘‘In their method (Abderhalden and Brockmann’s, author’s remark) the phenylcarbamyl peptide was exposed to prolonged heating at 60 C in methanol saturated with HCl in order to bring about the desired splitting off of the hydantoin. Concomitantly, however, to the desired cleavage other peptide bonds in the chain were also attacked to some extent, a fact that made the method less satisfactory.The present attempt to employ the same reaction for the determination of amino acid sequences in peptides was guided by several considerations. First, it was postulated that the ease of the reaction parallels the ease of ring closure and from that point of view the phenylthiocarbamyl derivative should be preferable . . .’’

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evidently inspired by Martin and Synge. Nevertheless, sequence analysis was his major scientific interest. In 1949 the first version of Edman’s method for determination of the amino acid sequence of peptides was ready for publication. It featured a concept of sequencing which was novel and which, as time was to show, had ultimately a far greater potential than Sanger’s method for development and also for automation. In this first method, he showed that coupling of phenylisothiocyanate to amino groups of peptides and proteins occurred easily. Furthermore, the carbamylated N-terminal amino acid was swiftly rearranged and released as ‘‘thiohydantoin’’ in acid media; and the ring closure appeared to occur much easier with phenylisothiocyanate than with phenylisocyanate used earlier by Abderhalden and Brockmann.9 He showed that by carrying out the reaction in acid anhydrous nitromethane, secondary hydrolysis of peptide bonds did not occur to any appreciable extent. Edman suggested that this was because ring closure to form the supposed ‘‘thiohydantoin’’ does not require water, whereas breaking of peptide bonds does. In this work, the released ‘‘thiohydantoin’’ derivative was identified as amino acid by paper chromatography after hydrolysis of the hydantoin in alkali solution. In this study, Edman used di- and tripeptides. In order to make the method more generally applicable to sequence analysis, Pehr Edman now performed thorough investigations on the reaction mechanisms involved, as well as procedures for characterization of the ‘‘thiohydantoins’’ formed during the reaction. The second version of Edman’s method was published in 1950. He now finds the following optimal conditions for sequencing: the peptide is dissolved in pyridine, which is a good solvent both for phenylisothiocyanate as for peptides. After addition of phenylisothiocyanate, pH was adjusted to about 9. Following carbamylation, the excess of phenylisothiocyanate was removed by extraction with benzene and the 9 Edman was not aware of the intermediate thiazolinone at this time. It was, in fact, the formation of the latter that he was observing, and this is a very fast reaction as compared to formation of thiohydantoin.


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phenylthiocarbamyl derivative of the peptide dissolved in acid anhydrous nitromethane. Ring closure of the N-terminal amino acid to what Edman at the time still believed was a phenylthiohydantoin derivative now occurred. Following its release from the rest of the peptide, the latter spontaneously precipitated in the water-free medium. Eventually, the ‘‘thiohydantoin’’ in the organic phase was hydrolyzed in alkali and the amino acid identified by paper chromatography. A drawback of this method, Edman said, was the rather limited solubility of the peptides in nitromethane leading to sluggish cleavage. A further disadvantage was that ‘‘thiohydantoin’’ derivatives of asparagine and glutamine were hydrolyzed during the hydrolysis in alkali preventing their positive identification. Dr Ikuo Yamashina was, very likely, the first, outside Edman’s group in Lund, to learn this second version of the phenylisothiocyanate technique in 1955. Yamashina, at the time a visiting scientist from Prof. Jorpes’s laboratory in Stockholm, applied the method for N-terminal analysis of enterokinase. On his return to Stockholm, he told how N-terminals in proteins could be easily determined in a quantitative fashion with Edman’s method. Therefore, Ikuo Yamashina and I tried the method in 1957 for determination of the N-terminal amino acids in fibrinogen and fibrin. This turned out to be worthwhile because it showed for the first time that the molecule from several animal species was composed of three pairs of polypeptide chains indicating a dimeric structure of the molecule [21]. Monomeric fibrin had the same basic structure although small peptides had been released by thrombin from the N-terminal end of two of the three chains. For our purpose, we were using a modified procedure for obtaining the phenylthiohydantoins and also for their analysis.10

10 Since neither fibrinogen nor fibrin are soluble in acid nitromethane, we chose to perform the cyclization step in 1 N HCl at 100 C. Furthermore, we identified the thiohydantoins directly by paper chromatography using the solvents developed by Sjo«quist and Edman at that time.

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During the following years, Pehr Edman introduced a number of improvements to his method. Anhydrous acetic acid was introduced instead of nitromethane, since cyclization appeared to occur as fast in this solvent and furthermore this is a perfect solvent for peptides. Edman also engaged himself in a thorough study on the reaction mechanism of the thiohydantoin formation. It resulted in a publication in 1956 that came to be of utmost importance for the practical execution of stepwise amino acid sequencing. It now became evident that the product formed in anhydrous media was not a phenylthiohydantoin derivative, as Edman had assumed before, but rather a novel compound, the isomeric anilinothiazolinone (2-anilino-5-thiazolinone) (Figure 1). This derivative has a different absorption spectrum in ultraviolet light and different migration during paper chromatography. Presumably, it was the latter observations that initially led Edman to make a thorough chemical characterization of the compound. Its formation following the nucleophilic attack by the thioketonic sulfur in phenylisothiocyanate was extremely fast. Its importance for the success of stepwise degradation lies in its speed of formation and in the fact that its formation is not due to a hydrolytic process but occurs under water-free acid conditions. The risk for cleavage of other peptide bonds than that involving the N-terminal amino acid is negligible. After its release, it can be removed from the residual peptide by extraction and subsequently under hydrolytic acid conditions transformed to the thiohydantoin. The latter occurs in two steps. First, the thiazolinone undergoes rearrangement in water to the corresponding thiocarbamyl derivative of the amino acid, and then under hydrous acid conditions, cyclization to the thiohydantoin takes place (Figure 1). The latter reaction is catalyzed by acid but it is slower than the thiazolinone formation. All phenylthiohydantoin derivatives of amino acids show strong absorption in the ultraviolet (with a maximum around 268 nm) useful for their quantification. It was now clear that three discrete reaction steps were involved: (1) coupling with phenylisothiocyanate, (2) cleavage to


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Fig. 1. Edman’s final reaction scheme with intermediary thiazolinone formation. (1) Coupling with phenylisothiocyanate. (2) Carbamylated peptide. (3) Release of Nterminal as thiazolinone. (4) Hydrolysis of thiazolinone to carbamylated amino acid. (5) Ring closure in acid to thiohydantoin. (Adapted from Partridge and Blomba«ck (1979))

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form anilinothiazolinone under anhydrous acid conditions, and (3) conversion to phenylthiohydantoins under hydrous acid conditions. Pehr Edman was quite satisfied with the development so far.11 As discussed by Edman and Henschen in 1975, the generality of thiazolinone formation in stepwise amino acid sequencing has been demonstrated in many other stepwise reactions using other reagents, but the key reaction is always the formation of a thiazolinone. Pehr Edman now also describes the synthesis of phenylthiohydantoin (PTH) derivatives of most of the naturally occurring amino acids;12 characterizes them by melting point and elementary analysis. With a few exceptions, the chemical stability of PTH-amino acids were excellent. In general, they are easily obtained as crystalline compounds with high melting points. The less stable compounds are those with an ÔH or ^SH group on the -carbon of the amino acid and those show in varying degrees a tendency for -elimination. In PTH-cystine and PTH-cysteine, the tendency for -elimination make them not useful for identification purposes. On the other hand, the PTH derivatives of S-alkylated cysteine are more stable as is PTH-cysteic acid. The PTH-derivatives of asparagine and glutamine tend, to some extent, to lose the amide group in acid. Therefore, in sequence determination, these PTH-amino acids are found contaminated by the corresponding acids. Solutions of PTH-amino acids show a tendency for photodecomposition, which becomes apparent after prolonged exposure to daylight. Pehr Edman’s coworker, John Sjo«quist, developed paper chromatography systems for resolution of PTH-amino acids [23^25]. These systems permitted a direct identification and 11 The following quotation reflects on the importance Pehr Edman placed on the discovery of his sequence method. Pehr Edman’s mother, in a discussion with the author in 1962, tells the following: ‘‘One day Pehr came home to me and asked me to sit down with him because he had something interesting to tell me. He then told me that he had discovered a way to analyze proteins which had not been possible before and that this discovery would certainly be of great importance for biochemistry in the future.’’ 12 PTH threonine and PTH serine were synthesized by Ingram in 1953 [22].


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quantification of all amino acids as PTH derivatives instead of, as previously, indirectly by amino acid analysis after alkaline hydrolysis of the PTH derivatives. Several investigators have used indirect methods for the identification of N-terminal amino acids released during a degradation cycle. Hirs et al. [26], in their classical elucidation of the first enzyme structure, compared the amino acid composition of the peptide before and after each degradation cycle. The indirect method has also been used in a different way by Gray and Hartley [27], who determined the new N-terminal amino acid using the sensitive dansyl technique after each degradation cycle. This requires complete hydrolysis of a small portion of the peptide after each cycle. This method permits sequence determination on small amounts of peptide because of the great sensitivity inherent in fluorescence measurements. Edman, however, consistently favored the direct identification of PTH-amino acids for the following reasons: 1. His chief criticism of the indirect method relates to the fact that it requires complete hydrolysis of the peptide. Therefore, the difference in composition before and after hydrolysis relates only to the hydrolysate of the peptide. Thus, amino acid residues like asparagine, glutamine, and tryptophan cannot be identified. 2. Many proteins contain modified amino acid residues, for example, by a covalently bound carbohydrate moiety. If these groups are removed by hydrolysis in acid, they cannot be located by the indirect method. Edman, therefore, concentrated his efforts on improvements of the direct method, which led to a whole arsenal of procedures for rapid identification of the PTH-amino acids, including thinlayer and gas chromatography, mass spectroscopy, and, later, high performance liquid chromatography.

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Since the value of a sequencing technique depends largely on the length of the amino acid sequence that may be determined, high repetitive yield is an important factor, i.e. the yield of amino acid calculated from one degradation cycle to the next. Losses of even a small percentage severely limit the practical length of the degradation. Edman emphasized the importance of the repetitive yield by illustrating the point with a simple calculation which showed that repetitive yields of 97, 98, and 99% make possible 30, 60, and 120 degradation cycles, respectively.

Automation Must Come In 1957 Pehr Edman accepted an offer as Director of Research at the newly established St. Vincent’s School of Medical Research in Melbourne where he would remain for 15 years and became an Australian citizen. The reasons for his leaving Sweden were rather complex. Pehr Edman’s time at the biochemistry department in Lund had not been altogether happy, possibly because of personal frictions. Also, his scientific endeavor was apart from the mainstream scientific aims of the department. Few were the persons he would enjoy discussing scientific matters with; one of those was Dr John Sjo«quist, his close associate, another was Dr Arvid Carlsson (Nobel Prize Laureate in year 2000). Pehr Edman also emphasized the circumstance that he did not get enough scientific support in Sweden, supposedly because of a lack of understanding for the work he was engaged in. The impending breakdown of his first marriage may have been another factor playing a role in his decision to leave. It was in Australia that Pehr Edman brought to perfection the manual three-stage degradation technique. With the knowledge of the reaction mechanism, it was now possible for Edman to work out a practical scheme, i.e. the three-stage procedure, for sequence analysis of peptides and proteins. Briefly, the


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manual three-stage method was performed as this: protein or peptide (0.25^2 mmol) is coupled with phenylisothiocyanate in buffer at pH 9. After coupling, excess phenylisothiocyanate and byproducts are removed by extraction with benzene and the water phase freeze-dried. The residue is extracted with ethyl acetate and dissolved in water-free trifluoroacetic acid that leads to the release of the amino terminal amino acid as a anilinothiazolinone derivative. Trifluoroacetic acid has the advantage of being both a good catalyst for cyclization and a good solvent for proteins. From this solution, the residual protein or peptide is precipitated with ethylene chloride and is now ready for the next degradation cycle. The ethylene chloride phase containing the thiazolinone is evaporated and the residue taken up in diluted hydrochloric acid for conversion of the thiazolinone to thiohydantoin at elevated temperature. Margareta Blomba«ck and I came to Edman’s laboratory as visiting scientists in 1961 and tried the three-stage technique on fibrinopeptide A, which consists of 16 amino acid residues. Starting with a few micromoles of peptides, we were able to make a complete stepwise degradation with good yields up to the very last residues.13 These results impressed upon Pehr Edman and certainly strengthened his already strong conviction that long peptides could be degraded in even better repetitive yields. The need for automation was evident, and by 1962 the stage was set for automation of the procedure. Standard reaction conditions applicable to all amino acids were already known, and side reactions had largely been eliminated. At the end of 1961, Pehr Edman and his laboratory assistant Geoffrey Begg 13 Back in Stockholm in 1962, we successfully used the three-stage technique on a variety of proteins and protein fragments, mainly fibrinopeptides. I was also passing details of this fantastic method to other scientists. The trouble was that the three-stage technique had not been published by then. I warned my informants of that fact. When, nevertheless, other workers released details of the procedure to the press, it caused me considerable consternation and anger. After all, I was the leak and the publications were a possible threat to the automation work Edman and Begg were performing at that time.

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Fig. 2. Spinning cup. An aqueous solution on the surface (white) containing the protein is being extracted by an organic solvent (black). (From Partridge and Blomba«ck (1979))

started to explore different possibilities to solve the automation problem.14 The problem was first to find a single physical process that could accommodate the various operations in the manual procedure. Guided by Edman’s great knowledge, imagination, and intuition, they, almost from the very start of this endeavor, conceived the idea of the spinning cylindrical cup, in which all reaction media were spread out as thin films 14 Geoffrey Begg in an open letter to Citation Classic (CC/number 9, February 27, 1984) tells how the idea of automation was conceived: ‘‘After observing the sequencing of proteins, I soon realized how repetitive it was. At a morning tea break one day, I suggested that a machine could be made to do this work, but because of my junior status, and the fact that Edman degradation was a highly skilled technique, my comments met much derision from the other staff. About a week later, Edman (who, unknown to me, was also considering such a machine) took up the suggestion and after a day of intense discussion we had a clear idea of how a protein sequencing machine would work. . . . A vertical rotating column with a base would allow extracting liquids introduced to the bottom to rise up over protein on the wall and be scooped off at the top. Placed into a chamber that could be evacuated, we had the ‘machine.’ . . .Continually referring to the ‘machine’ soon became tiring so we affectionately named it ‘Mathilda’ (Waltzing Mathilda), but when it came to publish, we renamed it ‘sequenator.’ ’’


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Prototype of sequenator. Pehr Edman explains the automation process to Agnes Henschen.

on the vessel wall (Fig. 2). This established a large surface and accomplished the equivalent of rapid stirring. The rotating film containing the protein would be well suited for extraction with a solvent, since the extraction fluid is continuously fed at the bottom of the cup, glides over the surface of the protein film, and is subsequently removed in the upper part of the cup. The surface film is also well suited for carrying out drying and other procedures and furthermore, the whole degradation cycle could be programmed. Edman and Begg started the experimental work with rotating glass cups in early 1962. I recall Pehr in his laboratory, day after day sitting in front of the spinning cup critically watching in strobe light the liquid phase passing over the surface of the spinning cup. The automated instrument was called a sequenator or in laboratory slang ‘‘Waltzing Mathilda.’’

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This instrument was designed to contain reservoirs to hold all the reagents required for the reaction cycles together with receivers for effluents and means for controlling reaction temperature. A system of feed tubes and automated valves was provided and programmed to supply the reagents and extraction solvents to the spinning cup in the correct order at preset time intervals. The process embraced the coupling step with formation of the phenylthiocarbamyl derivation of the protein and release of the N-terminal amino acid as anilinothiazolinone. The thiazolinones of each N-terminal amino acid were automatically transferred to tubes in a fraction collector and were then, in a separate operation, converted to the corresponding phenylthiohydantoins for identification by thin layer chromatography or other suitable procedures. The steps in the sequenator were largely copies of the manual three-stage method, but for logistic or other reasons certain changes had to be made. For example, trifluoroacetic acid in the cleavage step was changed to heptafluorobutyric acid to avoid excessive evaporation. The instrument allowed the degradation of even large polypeptides to an extent that had never before been possible. The degradation cycle proceeded at a rate of about 15 cycles in 24 h and with a repetitive yield of about 97%. By comparison, the rate using the manual technique is one or two amino acids per day. The three-stage sequence method had changed the strategy of sequence determination and automation made it widely available. It was no longer necessary to begin by cleaving the protein backbone into many small peptides since long direct sequences were possible. In 1963 Edman reported for the first time (at a meeting of the International Committee on Thrombosis and Hemostasis at Gleneagles in Scotland) on the progress in automatic stepwise degradation of proteins. Pehr Edman concluded his presentation with the following: ‘‘Our experience of the performance of this apparatus is as yet limited. However, trial runs have shown that it is capable of an output of at least 15 amino acids in 24 h. Furthermore, the high repetitive yield of 97% indicates that the


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determination of extended sequences will be possible. These expectations are supported by the results of sequence determinations on intact proteins currently being made in our laboratory. However, more work is required before a full assessment is possible.’’ Later on, Edman applied the process to apomyoglobin from the humpback whale and showed that it was possible to establish the sequence of the first 60 amino acids from the N-terminal end with a repetitive yield of 98%. About 0.25 mmol of the protein was required for this operation. In 1967 the work on the automated sequence analysis was finished and published. Edman and Begg did not seek to patent the sequenator technology as they believed that this would slow down the industrial development of the instrument and increase the cost for the scientific community. An important part of Edman’s work since the original description of the method consisted in optimizing the reaction and identifying and eliminating side reactions, and this search continued up to the time of his death. At one point he identified side reactions caused by impure reagents, in another instance it was found that the thiocarbamyl group easily lost sulfur by oxidation with the result that the carbamyl group was not reactive and the degradation came to an end. This prompted the reaction to be carried out in a nitrogen atmosphere. Further, although reaction conditions for coupling, cleavage, and conversion were equally suitable for all amino acids, it was discovered that proline, depending on its position in the sequence, sometimes was much more slowly released than other amino acids during the cleavage reaction.

End of Journey In the early 1970s, Pehr Edman accepted an offer to be Director of the Department of Protein Chemistry I of the Max Planck Institut fu«r Biochemie in Martinsried, near Munich. He and his wife Agnes Henschen moved there in 1972.

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During the last years of his life, he continued his work on the sequenator with the main objective of improving the yields. He was of the opinion that high repetitive yields are crucial in stepwise sequencing and that all efforts must be directed toward this goal. The actual yield of 98% had made possible 60 sequence steps but he believed 99% repetitive yield was possible, allowing, as mentioned earlier, 120 sequence steps to be performed. The urgency he felt for obtaining high yields was absolutely sound in those early days in the history of sequencing; remember the prospect of having millions of proteins being sequenced. Nowadays, although still desirable, with the advance of molecular genetics in the last decades, high repetitive yields may have lost some of its former imperative. Relatively short amino acid sequences of a protein are now required for identifying the cDNA sequence corresponding to the protein. The cDNA is subsequently decoded in terms of a protein sequence. It is usually taken for granted that this virtual sequence represents the true amino acid sequence of the protein. I doubt that Edman would have altogether agreed with this concept. Another concern, in addition to refinement of the sequence methodology, Edman had, in those final years of his life, was the necessity of establishing adequate arrangements for data storage, data retrieval, and data processing. He was of the opinion that one of the most potentially powerful applications of sequence studies lies in the search for evolutionary relationships between different proteins; but this becomes valuable only in proportion to the number of sequences available for comparison. The opportunity to set up adequate computer storage facilities should not be lost in view of the rapid rate at which sequence data became available. In his own words ‘‘we may in time expect the unraveling of a new systema naturalis among the biomolecules. It would be tragic if this development would be endangered through our own negligence.’’ His laboratory at Martinsried in Munich was endowed with a wealth of hard-won experience in sequencing and was engaged


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in a number of sequence projects. Of special importance to Pehr Edman, I believe, was the work that Agnes Henschen and her coworkers did on the elucidation of the primary structure of fibrinogen. This endeavor brought to light the complete primary structure of all three chains of fibrinogen and eventually of the whole disulfide bonded molecule [28]. It should be mentioned that fibrinogen is one of the few large proteins in which all amino acids in the sequence were directly identified by protein sequencing. In February 1977 when leaving a scientific lecture in Martinsried, Pehr Edman suddenly fell down unconscious. After a few weeks of illness he died on March 19. A tumor of the brain was diagnosed; there had been no symptoms before he was struck by unconsciousness. Pehr Edman received several honors for his achievements in science: the Britannica Australia Award, the Berzelius Gold Medal, the Gold Medal of the Swedish Academy of Engineering, the Linderstro«m-Lang Medal. Pehr Edman was a Fellow of the Australian Academy of Science, Fellow of the Royal Society of London, and a scientific Member of the Max Planck Society.15

Edman, The Person I believe that most people who met Pehr Edman for the first time got the impression of a courteous, kind but also reclusive man with a hint of shyness. There was, in his personality, a certain aloofness, which people who did not know him may mistakenly have taken for snobbishness. People who came closer to him could fully appreciate other qualities: generosity, warmth, humor, and sympathy. 15 Many scientists have asked me why Pehr Edman never got the Nobel Prize. Considering the importance of his work, as we see it today, the question is relevant but I do not have any definite answer. Perhaps, the scientific community did not, during his lifetime, fully appreciate the width of Pehr Edman’s contribution to protein chemistry and molecular biology.

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Pehr Edman had a vast knowledge in many areas. His mind was logical, he was stringent in expression, and, most of all, had an admirable and respected integrity. In his opinions he was rock-firm, almost to the extent of stubbornness; but he could change views if well-founded reasons were presented. Pehr Edman was not an individual who thoughtlessly followed fashions in his thoughts; he was too analytical and had too strong desire to reach for causes at fundamental levels. In trying to describe Pehr Edman’s character, words like incorruptible and uncompromising come to mind, as do sincerity and loyalty to his friends. In Pehr Edman’s language ‘‘yes’’ and ‘‘no’’ stood for fundamentally opposite meanings. He had courage and dared to express opinions, which he felt were morally right; without hesitation and without political or opportunistic consideration.16 The uncompromising quality of his personality made him distrust politicians with few exceptions. At the core of his personality was a sincere humanism. Therefore, he was, on the whole, against violence and oppression and he was a sworn enemy of militarism in the world. His expression of intense dislike for the oppression of black people he had witnessed in Africa in 1957, on his journey by ship to Australia, is an example of this. There was in Pehr Edman’s personality an encompassing trait of purism that may be common to many scientists of his caliber; his teacher Erik Jorpes had it, and so had Jo«ns Jakob Berzelius, the first professor of chemistry at the Karolinska Institutet. 16 This brings to mind a letter written by Pehr Edman to Joseph Fruton in connection with the failure to obtain a visa for travel to USA to attend the meeting of the American Chemical Society in 1951 (20). I quote from the letter: ‘‘This is to inform you that I failed to obtain a visa for the United States. On my visit to the American Consulate in Gothenburg it was made clear to me that I was suspected of holding views dangerous to the security of the United States. The reason for this suspicion was the fact that I had signed the Peace Appeal (the so called Stockholm Appeal). In spite of the fact that this appeal contains no one-sided or ideological statement a signer is apparently considered to be a Communist. My declaration that I was not and had never been a member of the Communist Party was not satisfying since I was called on to state my political views. This I refused to do. As a consequence my application for a visa was declined.The reason for my refusal should be obvious to anybody to whom freedom of thought is an indefeasible right.’’


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Pehr Edman’s urge for purity and perfection in life may, therefore, partly have been a cultural heritage. Whatever its origin, inborn or acquired, this quality was very likely in play when he joined the socialists as a young man in the thirties, when he chose the self-imposed expatriation in the fifties, and it was probably a strong driving force in his scientific accomplishments. For a purist nothing except the whole is good enough. He is beset by one idea: to reach perfection and impeccability. This quality in Pehr Edman’s personality was most likely a prerequisite for his motivation to spend so much time and effort on perfection of the phenylisothiocyanate method. Pehr Edman had broad interests outside science. He loved nature and this goes all the way back to his childhood. Birds were of special interest to him. I believe he was almost as knowledgeable in ornithology as he was in chemistry. Music was another of his preoccupations. He could sit for hours listening to his favorite records in classical or contemporary music. His thoughts also turned to literature, apart from science, where he was also well read. Pehr Edman had many friends, but most of them were from circles outside his scientific field. There were a number of Swedes in Melbourne during his years in Australia and many of them became his closest friends. Among them his shyness seemed to disappear and he would allow his distinct and clear Swedish to flourish and there was his wonderful humor, sometimes drastic, but to the point. I believe that he felt most at home in this company. At this point it should be mentioned that Pehr was a gourmet, a fine wine connoisseur as well as an exceedingly fine cook. Of course, we cherished his friendship on its own merits, but animated discussions gave extra pleasure when enjoined with dinner, prepared by Pehr. It may have been dishes with crispy duck or simply with Swedish meatballs. The dishes prepared by Pehr Edman were always cooked in a masterly way ^ thoughtfully and meticulously. Margareta Blomba«ck and I met Pehr Edman for the first time in 1957, shortly before he left Sweden for Australia. We had

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tried the phenylisothiocyanate method to determine end groups in fibrinogen. We discussed our results with Pehr and he gave us suggestions on how to proceed. Pehr apparently liked our work and a few years later we were invited to come to the St.Vincent’s School of Medical Research in Melbourne, for a one-year stay as visiting scientists. Before we left Sweden, some people hinted that Pehr Edman was not the easiest to work with, unsociable, and difficult in general. The man we met was nothing of that kind. Toward us he was warm, friendly, and considerate and we became close friends. I recall our stay in Melbourne with happiness. Not only did I learn a lot about protein chemistry and how to think scientifically, but also had the rare opportunity to come close to him as a person. I remember the evenings at our favorite restaurant with enjoyable discussions for hours and the fact that Pehr was so widely read surely increased the pleasure of those occasions. Other memories pop up; the talks in his

Pehr Edman with family. From left to right: Helena, Pehr, Carl, Agnes.


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home in front of the fireplace after wonderful meals prepared by Pehr, or the long excursions into the wilderness of Australia to Wilson’s Promontory for hiking, fishing, or bird-watching. Or, on Sundays in winter time the shorter trips to Sherbrook forest to listen to the lyre-birds in the damp woodland; and we grilled lamb chops over an open fire and ate them with potato salad and drank red wine. It was not without sadness that we left Australia for Sweden in 1962. But Pehr made several visits to Sweden during the following years. We happened to have bought a country house on a small island off the island of Singo«, in fact quite near, separated only by an inlet from the place where Pehr used to spend his summers during boyhood. Pehr visited us there several times. He was delighted to come back to the nature of his boyhood ^ there were his flowers, pike inlet, birds. A circle was somehow closed. In 1966 we returned to Australia to attend a conference. With us on the travel was Agnes Henschen. Pehr met Agnes and what follows is a love story that would last until the end of his life. They married in 1968. The encounter with Agnes changed Pehr Edman’s personal life to a happier direction. He now appeared relaxed and at peace with himself and the years to follow were certainly among the happiest in his life. They had two children, Carl and Helena. In his first marriage Pehr Edman had two children, Martin and Gudrun.

ACKNOWLEDGMENTS

The personal photos in the present communication were kindly provided by Agnes Henschen-Edman. She and Pehr Edman’s son Carl furthermore gave much helpful commentaries and corrections of the manuscript during its preparation. All of this is gratefully acknowledged.

B. BLOMBA«CK

132 REFERENCES

[1] Blomba«ck, B. (1977) Thrombosis Research, 11, 695. [2] Partridge, S.M. and Blomba«ck, B. (1979) Biographical Memoirs of Fellows of the Royal Society, 25, 241. [3] Morgan, F.J. (1990) Historical Records of Australian Science, 8(2). [4] Hammarsten, O. (1876) Nova Acta Reg. Soc. Scient. Ups. Ser. III, 10(1). [5] Sumner, J.B. (1926) J. Biol. Chem., 69, 435. [6] Svedberg, T. (1926) Z. Phys. Chem., 121, 65. [7] Tiselius, A. (1937) Trans. Faraday Soc., 33, 524. [8] Bailey, K., Astbury,W.T. and Rudall, K.M. (1943) Nature, 151, 716. [9] Martin, A.J.P. and Synge, R.L.M. (1941) Biochem. J., 35, 1358. [10] Consden, R., Gordon, A.H. and Martin, A.J.P. (1944) Biochem. J., 38, 224. [11] Moore, S. and Stein,W.H. (1951) J. Biol. Chem., 192, 663. [12] Sanger, F. and Tuppy, H. (1951) Biochem. J., 49, 463. [13] Sanger, F. and Tuppy, H. (1951) Biochem. J., 49, 481. [14] DuYu-Cang, ZhangYu-Shang, Lu Zi-Xian and Tsou Chen-Lu (1961) Scientia Sinica, 10, 84. [15] Kung Yueh-Ting, Du Yu-Cang, Huang Wei-Teh, Chen Chan-Chin, Ke LinTsung, Hu Shih-Chuan, Jiang Rong-Qing, Chu Shang-Quan, Niu Ching-I, Hsu Je-Zen, Chang Wei-Chun, Chen Ling-Ling, Li Hong-Shueh, Wang Yu, Loh Teh-Pei, Chi Ai-Hsech, Li Chung-Hsi, Shi Pu-Tao, Yieh Yuen-Hwa, Tang Kar-Lo, Hsing Chi-Yi (1966) Scientia Sinica, 15, 544. [16] Bergman, M., Kann, E. and Mieckely, A. (1927) Ann., 458, 56. [17] Jensen, H. and Evans, F.A. (1935) J. Biol. Chem., 108, 1. [18] Abderhalden, E. and Brockmann, H. (1930). Biochem. Z., 225, 386. [19] Hagel, P. (1970) N-terminal amino acid determination. Ph.D. Thesis. University of Amsterdam. [20] Fruton, J.S. (1992) Int. J. Peptide Protein Res., 39, 189. [21] Blomback, B. and Yamashina, I. (1958) Ark. Kemi., 12, 299. [22] Ingram,V.M. (1953) J. Chem. Soc., 3717. [23] Sjo«quist, J. (1953) Acta Chem. Scand., 7, 447. [24] Sjo«quist, J. (1959) Arkiv. Kemi., 14, 291. [25] Sjo«quist, J. (1960) Biochem. Biophys. Acta., 41, 20. [26] Hirs, C.H.W., Moore, S. and Stein,W.H. (1960) J. Biol. Chem., 235, 633. [27] Gray,W.R. and Hartley, B.S. (1963) Biochem. J., 89, 379. [28] Henschen, A., Lottspeich, F. Kehl, M. and Southan, C. (1983) Ann. N.Y. Acad. Sci., 408, 28.


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SELECTED REFERENCES FROM PEHR EDMAN’ S BIOGRAPHY (1944) (1945) (1945) (1947) (1947) (1947) (1948) (1949) (1950) (1950) (1953) (1953) (1956)

(1956) (1956) (1956) (1957) (1957)

(1957)

On the purification of hypertensin (Angiotonin). Ark. Kemi Miner. Geol. 18B(2). Preliminary report on the purification and the molecular weight of hypertensin. Nature (London.) 155, 756. On the purification and chemical composition of hypertensin (Angiotonin). Ark. Kemi Miner. Geol. 22A(3). A note on the action of tyrosinase on pepsin, trypsin, and chymotrypsin. J. Biol. Chem. 167, 301. The action of tyrosinase on chymotrypsin, trypsin, and pepsin. J. Biol. Chem. 168, 367. Note on the cleavage of insulin by chymotrypsin. Arta Chem. Scand. 1, 684. A technique for partition chromatography on starch. Acta Chem. Scand. 2, 592. A method for the determination of the amino acid sequences in peptides. Arch. Biochem. 22, 475. Preparation of phenylthiohydantoin from some natural amino acids. Acta Chem. Scand. 4, 277. Method for determination of the amino acid sequence in peptides. Acta Chem. Scand. 4, 283. Note on the stepwise degradation of peptides via phenyl thiohydantoin. Acta Chem. Scand. 7, 700. Selective cleavage of peptides In: The Chemical Structure of Proteins (Wolstenholme, G.E.W. ed.), pp. 98. J. & A. Churchill Ltd., London. (With K. Lauber) Note on the preparation of phenyl thiohydantoins from glutamine, S-carboxymethyl cysteine, and cysteic acid. Acta Chem. Scand. 10, 466. Mechanism of the phenyl isothiocyanate degradation of peptides. Nature (London) 177, 667. On the mechanism of the phenyl isothiocyanate degradation of peptides. Acta Chem. Scand. 10, 761. (With J. Sjo«quist) Identification and semiquantitative determination of phenyl thiohydantoins. Acta Chem. Scand. 10, 1507. (With K. Heirweg) Purification and N-terminal determination of crystalline pepsin. Biochim. Biophys. Acta. 24, 219. (With others) Isolation of the red pigment concentrating hormone of the crustacean eyestalk. Trans. 2nd Int. Symp. Neurosecretion, pp. 119. Berlin. Springer-Verlag. (With L. Josefsson) Reversible enzyme inactivation due to N,O-Peptidyl Shift. Nature (London) 179, 1189.

134 (1957) (1957) (1959) (1960) (1962) (1962) (1962)

(1963) (1963) (1963)

(1963) (1966)

(1967) (1967) (1968) (1969) (1969) (1970) (1970) (1970) (1970)

B. BLOMBA«CK Phenylthiohydantoins in protein analysis. Proc. R. Aust Chem. Inst. pp. 434. (With L. Josefsson) Reversible inactivation of lysozyme due to N,O-peptidyl shift. Biochim. Biophys. Acta. 25, 614. Chemistry of amino acids and peptides. Ann. Rev. Biochem. 28, 69. Phenylthiohydantoins in protein analysis. Ann. N.Y. Acad. Sci. 88, 602. (With H. Smith & J.A. Owen) N-terminal amino acids of human haptoglobins. Nature (London) 193, 286. (With H. Niall) The N-terminal amino acids of human plasma proteins. J. Gen. Physiol. Suppl. 45, 185. (With B. Blomba«ck, M. Blomba«ck & B. Hessel) Amino-acid sequence and the occurrence of phosphorus in human fibrinopeptides. Nature (London) 193, 883. (With D. Ilse) The formation of 3 -phenyl-2 -thiohydantoins from phenylthiocarbamyl amino acids. Aust. J. Chem. 16, 411. (With B. Blomba«ck & M. Blomba«ck) On the structure of human fibrinopeptides. Acta Chem. Scand. 17, 1184. (With B. Blomba«ck, M. Blomba«ck, R.F. Doolittle & B. Hessel) Properties of a new human fibrinopeptide. Biochim. Biophys. Acta 78, 566. Determination of amino acid sequences in proteins. Thromb. Diath. Haemorrh. Suppl. 13, 17. (With B. Blomba«ck, M. Blomba«ck & B. Hessel) Human fibrinopeptides; isolation, characterization and structure. Biochim. Biophys. Acta 115, 371. (With G. Begg) A protein sequenator. Eur. J. Biochem. 1, 80. (With H.D. Niall) Two structurally distinct classes of kappa chains in human immuno-globulins. Nature (London) 216, 261. (With A.G. Cooper) Amino acid sequence at the N-terminal end of a cold agglutinin kappa chain. FEBS Lett. 2, 33. (With K.J. Fraser) On the amino acid sequence of light chains from arsonic antibody. Proc. Aust. Biochem. Soc. 2, 37. (With G. Mamiya & A. Henschen) Structure of jack bean urease. Proc. Aust. Biochem. Soc. 2, 26. Sequence Determination. In: Protein Sequence Determination (S.B. Needleman ed.), pp. 211. Berlin: Springer Verlag. Sequence determination. Mol. Biol. Biochem. and Biophys. 8, 211. (With A.S. Inglis) Mechanism of cyanogen bromide reaction with methionine in peptides and proteins I. Analyt. Biochem. 37, 73. (With A. Henschen) Variants in fibrinogen. Proc. Aust. Biochem. Soc. 3, 26.

PEHR VICTOR EDMAN: THE SOLITARY GENIUS (1970)

(1970)

(1970)

(1970) (1972)

(1975) (1976)

(1977)

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(With C. Rochat & H. Rochat) Some S-alkyl derivatives of cysteine suitable for sequence determination by the phenylisothiocyanate technique. Analyt. Biochem. 37, 259. (With H. Rochat, C. Rochat, F. Miranda & S. Lissitzky) The amino acid sequence of neurotoxin I of Androctonus australis Hector. Eur. J. Biochem. 17, 262. (With H. Rochat, C. Rochat, C. Kupeyan, F. Miranda, S. Lissitzky) Scorpion neurotoxins: a family of homologous proteins. FEBS Lett. 10, 349. (With K.J. Fraser) The N-terminus of light chains from rabbit arsonic antibody. FEBS Lett. 7, 99. (With A. Henschen) Large scale preparation of S-carboxymethylated chains of human fibrin and fibrinogen and the occurrence of y-chain variants. Biochim. Biophys. Acta 263, 351. (With A. Henschen) In: Protein Sequence Detemination (S.B. Needleman ed. ) (2nd edn.), pp. 232. Berlin: Springer-Verlag. (WithW.F. Brandt, A. Henschen & C. von Holt) Abnormal behaviour of proline in the isothiocyanate degradation. Hoppe-Seyler’s Z. Physiol. Chem. 357, 1505. Unwinding the protein. Carlsberg Res. Comm. 42, 1.



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Chapter 4

A Privileged Life TORVARD C. LAURENT Department of Medical Biochemistry and Microbiology of the University of Uppsala, BMC, Box 582, SE-751 23 Uppsala, Sweden

Introduction When asked to publish my ‘‘Personal recollections’’ I hesitated for two reasons; I have not lived an especially adventurous life that would interest people, and I have no literary qualifications. However, I had already considered writing my life history, just privately, for my children and grandchildren inspired by my great-grandfather, Carl-Erik Bergstrand, who wrote his unpublished memoirs in 1907^1910. I found his lifestory in a drawer in my parent’s home when my mother had died, and I have been fascinated by the description of his family background, of his work and of life in Sweden in the 19th century. Getting older I have realized that I owe my own happy life to a great extent to previous generations, not only the genetic inheritance but also the traditions in which I grew up. I would very much like to convey this to future generations of my own family. To simplify my task I have, in the present chapter, selected the part that essentially concerns my professional activity as a biochemist.

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Fig. 1. Torvard C. Laurent.

Family Background I was born in Stockholm in 1930 as the second son of Torbern and Bertha Laurent. My parents had very different backgrounds. My mother’s relatives had been farmers although her father was manager, first of a brewery and then of a local bank. As can be deduced from my name, I have a French origin on my father’s side. My ancestors were Huguenots who fled from Montpellier in 1695. After some time in Switzerland they were enrolled at the Prussian court in Berlin as skilled craftsmen. When Lovisa Ulrika, the sister of the Prussian king, married the Swedish crownprince Adolf Fredrik in 1744, she brought her perfume and glove maker to the Swedish court. I am the 8th generation descendant of him.

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My great-grandfather, Carl-Erik Bergstrand, was the man who started the academic tradition of our family. He was born in 1830, grew up in a poor peasant family 90 km from Uppsala and was able ^ against all odds ^ to get an education and to study chemistry at the university. Bergstrand defended the first doctoral thesis of nutrition in Sweden in 1857 and became an agricultural chemist in Uppsala. He, subsequently, was appointed professor, became head of the experimental station of the Academy of Agriculture in Stockholm and a member of the Royal Swedish Academy of Sciences. In his memoirs he describes the development of agriculture in Sweden but also depicts many other aspects of the Swedish society in the 19th century from the poor farming conditions, the educational system, the industrial revolution, the growth of Stockholm, the scientific development, artists, international connections, the first Nobel prize awards, and his meetings with royalty. Carl-Erik had a large family. His eldest son became professor of astronomy in Uppsala. In the next generation my father ended up as professor of telecommunications at the Royal Institute of Technology in Stockholm, and his sister became a professor of botany at Cairo University. In my generation, my older brother held a chair of theoretical physics at Stockholm University and I ended up as professor of medical and physiological chemistry at the University of Uppsala. Of these persons, my aunt Vivi Ta«ckholm is probably the most well-known. After having received a BSc degree with botany as main subject, she became a journalist and a writer of children’s books. Then she married Gunnar Ta«ckholm, a botanist who became the first head of the Department of Botany at Cairo University. Gunnar and Vivi started the herbarium collection at the university but Gunnar died after a few years. Vivi formally took over the chair when she had received a PhD h.c. for her work on the Egyptian flora. She became a legend in Cairo where she stayed for about 50 years. Vivi lived in Sweden during World War II and in the summers

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I made botanical excursions with her. All the plants that I collected are now part of the herbarium in Cairo. Although I was born in Stockholm the family moved after a couple of years to Lidingo«, a suburb situated on an island in the inner archipelago of Stockholm. We lived a small-town life even if we were only twenty minutes from the center of Stockholm. I went to school during the war time years and finished highschool at the age of 17.

The Karolinska Institute Medical Studies I was admitted to medical school in 1948 and the term started in September at the recently built Anatomy Department at the new campus of the Karolinska Institute in Solna outside Stockholm. For the first four and a half years, I was strictly going to follow the curriculum, which meant two and a half years of preclinical subjects and then clinical work including internal medicine and surgery. The whole of the first year was devoted to morphology. The second year was considerably more interesting when we studied chemistry and physiology. The Chemistry Department had not yet moved to the new campus but was situated in a very old building close to the city hall in Stockholm and close to the old university hospital ^ The Serafimer Hospital. It was here that Jo«ns Jacob Berzelius had moved together with the Karolinska Institute at the beginning of the 19th century. A large part of the chemistry course was taught by a young assistant professor, Peter Reichard, who later became a colleague and friend when we moved to Uppsala. We also attended lectures by the two professors, Einar Hammarsten and Erik Jorpes. Jorpes examined us in general chemistry and Hammarsten in medical chemistry. Jorpes never asked me a question during the exam. Hammarsten asked us to write an essay about one of ten topics given.

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It turned out that one of the topics was ‘‘mucopolysaccharides’’of which I knew quite a lot (see below) and my exam in medical chemistry was quite successful. Chemistry was followed by physiology, pharmacology, and general pathology before we got into contact with patients. My experience of clinical work was positive but at that time I had other interests that occupied my mind and I probably did not engage as much in my medical studies as I should have done.

Experimental Histology 1949^1951 After my first year at the Karolinska Institute, I was asked by Professor Hjalmar Holmgren to be an unpaid instructor of histology. This was a common way at the time to recruit graduate students. Hjalmar Holmgren held a personal chair of experimental histology. His main contribution had been the identification of heparin in mast cells, but he was also very interested in the regulation of the daily rhythm as mirrored in the biochemical processes in the liver. He was a young energetic man, coming from a well-known academic family, and he had a great sense of humor. Hjalmar Holmgren surrounded himself with a group of very active researchers. Two associates were Hungarian and one of them, Endre Balazs became my teacher. His nickname used by everybody is Bandi. Balazs had escaped from Hungary in 1947 before the communists took over. His main interest was connective tissue and the interplay between fibroblasts and extracellular matrix and he was especially interested in the mucopolysaccharide hyaluronic acid (later renamed glycosaminoglycan and hyaluronan, respectively). We were two young students engaged by Balazs, Jan von Euler and myself. Jan, who was a son of Hans von Euler and a brother of Ulf von Euler (both Nobel laureates), was a highly intelligent boy and very much appreciated by everybody. He was put on a project to determine the presence of hyaluronidases in serum of cancer patients, but I do not think that any results came out of

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it at that time. Jan ^ still young ^ succumbed to depression, which shocked us all. I myself was asked to prepare hyaluronan from umbilical cords. Balazs wanted to test its effect on cell growth in tissue culture. In order to obtain fresh umbilical cords, I had to bribe the midwives at the Karolinska Hospital with cakes. Then I followed well-published procedures to prepare the polysaccharide, which included extraction with a salt solution, removal of proteins by shaking with chloroform and isoamyl alcohol (Sewag technique), and repeated precipitations with ethanol. When the polysaccharide was sufficiently pure it had to be sterilized. Due to the high viscosity of the solutions, we could not use ultrafiltration and finally we had to autoclave them. Tissue culture at this time had to be performed under strictly sterile conditions as we had no antibiotics. Small pieces of chicken hearts were placed in a hanging drop of fibrin, and the migration of fibroblasts out of the tissue was followed microscopically. The microscopic picture was enlarged and the area that was covered by cells was measured by planimetry. During this work we made several interesting observations. In the extraction of hyaluronan from the umbilical cords at different salt concentrations and at varying pH, I found highly different viscosities of the extracts and thought that the extraction procedures were not equally effective. However, it turned out that the viscosity of hyaluronan was highly dependent on pH and ionic strength. Today, this is trivial but at that time it had only been shown for some synthetic polymers by Raymond Fuoss. The observation resulted in a publication in Journal of Polymer Science: ‘‘Viscosity function of hyaluronic acid as a polyelectrolyte.’’ In our search for techniques to sterilize hyaluronan, we also used ultraviolet irradiation. To our surprise the viscosity disappeared. Later it was also shown that irradiation with electrons degraded hyaluronan. What we had observed was probably the first example of free radical degradation of hyaluronan. Finally, we could show that hyaluronan promoted fibroblast growth while sulphated polysaccharides were

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inhibitory. This was the first example of how cellular functions can be regulated via an interaction with hyaluronan. In the resulting publication we also showed that heparin could not be a sulphated hyaluronan and we also, probably for the first time, described biological effects of heparan sulphate [1]. As a side line, I also published a paper on the effect of ultraviolet irradiation of glucose. My initial collaboration with Bandi Balazs lasted for one and a half years.When the Korean war broke out he decided to move to Boston, where he was employed by an ophthalmic surgeon, Charles Schepens, to do research on the eye at a laboratory named Retina Foundation. In a very short time, Bandi developed this laboratory into a major eye research institute. Endre Balazs has continued to be an important person in my life and it is appropriate at this stage to tell the reader why he always has fascinated me. It is only a ten-year age difference between us but with time we almost developed a father^son relationship. The characteristics of Bandi could be summarized in the words: warmth and generosity, scientific imagination, hard working, unyielding, and entrepreneurial. He has always had numerous ideas, has never been afraid of starting large-scale projects, has been able to convince granting agencies and others to finance them, and his success rate has been high. It is not surprising that I found it stimulating to work with him but then ^ in the end ^ I realized that we had to go separate paths if I should be able to carry out my own ideas.

Chemistry Department 1951^1953 Hjalmar Holmgren was seriously ill with cancer at the time when Bandi left Sweden, and he died a few months later. His research group dissolved. A virologist working in Holmgren’s laboratory had a friend in the Chemistry Department, Bertil Jacobson, and suggested that I should move to him. At this

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time the Chemistry Department had moved to the Solna campus and to a completely new building. I started there in the fall of 1951. The Chemistry Department was divided into two sections headed by Einar Hammarsten and Erik Jorpes, respectively. There was a large open hall in the center of the building surrounded by laboratories on two floors. Section I, to which I belonged, was housed on the first floor and Section II on the second floor. In addition there was a connected building housing the teaching facilities. Further research facilities occupied the basement of the main building and a lower floor of the teaching unit. There was an open animosity between Hammarsten and Jorpes. They had very different personalities. Einar Hammarsten came from an academic family ^ his uncle, Olof Hammarsten, had been professor of medical and physiological chemistry in Uppsala and a very prominent scientist. Einar had made pioneering work on nucleic acids and had surrounded himself with an outstanding group of students, which was going to dominate Swedish medical biochemistry for a long time. Persons who came from his laboratory included Torsten Teorell, Hugo Theorell, Torbjo«rn Caspersson, Erik Jorpes, Peter Reichard, and Ulf Lagerkvist. He also collaborated with others, e.g., with Einar Stenhagen to develop mass spectrometry for metabolic studies and this work was in the hands of Ragnar Ryhage. During my time in the department his closest student was Hans Palmstierna, who later became an important voice in Swedish environmental debate. Palmstierna later disappeared mysteriously during a boat trip. Einar Hammarsten was one of the most informal professors I had met at the time. Everybody addressed him by first name that made it difficult for me, due to my upbringing, to talk to him. It was not until I had finished my doctorate and Einar had retired from his chair that we got a closer personal relationship; and I have a fond memory of the day he came home to us for dinner when our first child was born.

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Einar had no office ^ it had been turned into a coffee room. Instead he had a very small desk in the laboratory and he was completely engaged by his research and a pipe, which always was in his mouth. In spite of his informality, Einar Hammarsten was one of the most influential persons at the Karolinska Institute and in the medical Nobel committee. In contrast, Jorpes grew up in a fishing village on the island of Ko«kar in the —land archipelago under poor circumstances. He was able to go to school in Turku, Finland. During the Russian revolution, he joined the red forces and he was one of the founders of the Finnish communist party, which was constituted outside Moscow in 1918. During the civil war in Finland, the communists lost and Jorpes had to flee to Sweden. In Stockholm, he was able to continue his medical education and do research in biochemistry. He made important contributions for the production of insulin in Sweden and to solve the structure of heparin. He and his coworkers were engaged in research on the mechanism of blood clotting, on mucopolysaccharide chemistry, and on the isolation of peptide hormones. Jorpes had a very complex personality. He was very much feared. He could not be relied upon and he persecuted people, whom he for some reason did not like. He disliked physical chemistry and therefore I was not one of his favorites. However, he supported some of his students, who were in need, with personal funds and he made large donations. He could do this due to his income from insulin and heparin production. It was very difficult to come close to Jorpes as a person. My new teacher, Bertil Jacobson, was a complete contrast to Bandi Balazs. He was a physical biochemist and his great strength was his theoretical mind and his ability to build refined instruments. If Bandi had been an extrovert, Bertil was an introvert. Bertil was, for example, at that time greatly influenced by psychoanalysis. However, also Bertil was a warm and generous person.

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My first assignment was to calculate intermolecular distances in organic liquids and correlate them to viscosities. However, Bertil was, at that time, also very interested in the macromolecular properties of DNA and this overlapped with my interest in hyaluronan. A group in the Chemistry Department had measured the dielectric properties of DNA at various frequencies in an alternating field and interpreted the variation in dielectric constant in similar terms as for proteins, i.e., that the nucleic acid rotated in the oscillating field. It was concluded that the only possible rotation would be around the long axis of the molecule. To test if this conclusion was correct, Bertil built an instrument similar to a rotating viscometer in which the DNA molecules were lined up by the stream lines and measured the dielectric dispersion both parallel and perpendicular to the stream lines. Both for DNA and hyaluronan, we got similar dielectric dispersions in the two measurements, and the theory of rotation had to be abandoned. Bertil instead proposed that both polymers surrounded themselves with ice-like hydration shells and that we measured the dielectric dispersion of ice. It was quite an ingenious theory, although we now know that the effect is due to oscillations of counter ions within the polyelectrolyte domain. Even if there existed an animosity between the two professors in the department, there was no barrier between the younger scientists in the two sections. This was important for me because it was in Jorpes’ section that I found the expertise in polysaccharide chemistry. Sven Gardell, later professor at the University of Lund, could separate and analyze connective tissue polysaccharides and Harry Bostro«m, later professor of internal medicine in Uppsala, studied their metabolism. My friend Lennart Rode¤n did his graduate work under the guidance of Harry Bostro«m. He isolated the factor in serum, which enhances sulphate incorporation into cartilage and showed it to be glutamine. Others of my contemporaries in Jorpes’ section were Birger and Margareta Blomba«ck, who did pioneering work on blood coagulation, and Viktor Mutt, who

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became the major force in isolating biologically active intestinal peptides.1 Retina Foundation 1953^1954 When Bandi had established himself in Boston, he wrote and asked me if I would like to work in his laboratory. The offer was very tempting and the best time would be immediately after the course of surgery. An interruption of my medical studies at this point of time would not be deleterious as the remainder could be carried out in a less rigid time sequence. Thus, I accepted to come in the beginning of 1953. The only problem at that time was that by then I had a girl friend; one of my class mates in medical school, Ulla Hellsing. We got engaged in February 1953. A few days later, I left for USA on a cargo ship from Stockholm harbor only 2 km from my parents’ home. After a rough crossing of the Atlantic I ended up in Portland Maine, where Bandi and two of his laboratory assistants picked me up. Half a year later, Ulla followed me on a regular passenger ship to New York. We got married on her arrival, and after a short visit to Boston we made our honeymoon trip in a Greyhound bus over the American continent. Retina Foundation, founded by Charles Schepens, was housed in an old apartment building in the Italian district of North Boston not far from Massachusetts General Hospital and Massachusetts Eye and Ear Infirmary. The building was rather dilapidated and the whole district was torn down a few years later. The value of the equipment in the laboratory exceeded many times the value of the building itself. Although Schepens had employed people and equipped a machine shop for construction of optical instruments and also employed a skilled electron microscopist, it was Bandi who had taken over most of the enterprise and occupied the main part of 1 See also chapter on E. Jorpes by V. Mutt and M. Blomba«ck in Volume 41 of this series and that on P. Edman by B. Blomba«ck in this volume.

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the house. He had started a large program to study the composition and turnover of the vitreous body of the eye by various techniques. The rationale for this topic was the presumed importance of the vitreous body in the formation of retinal detachments, the speciality of Schepens. In recruiting staff to the laboratory, Bandi had a preference for Hungarians and Swedes. John Gergely was one of Bandi’s Hungarian friends. He was a biochemist at Massachusetts General Hospital and was studying muscular contraction. Gergely had equipment for measuring light-scattering from macromolecules, a technique which had gained interest in USA but not yet reached Sweden. I learnt the technique from Gergely and we characterized hyaluronan from umbilical cord and showed that it had a molecular weight of several millions and behaved as a somewhat stiff random coil in solution. The resulting paper became a corner stone of my doctoral thesis [2]. In a subsequent paper I isolated hyaluronan from the bovine vitreous body and showed that it had a much lower molecular weight and was highly polydisperse. When Ulla came we both became engaged in two aspects of Bandi’s characterization of the vitreous body, the variation in composition between different species and the development of the vitreous in the cattle eye. For the first project we had to collect eyes from many animals including squid, turtles, frogs, various birds, and various mammals. I remember especially one Saturday when we were alone in the lab and there came a shipment of bullfrogs from Wisconsin. We did not know what to do with them so we put them in the tub in one of the bath rooms in the house. On Monday morning when the laboratory technicians opened the door to the bath room all the frogs jumped out to their horror. In collecting cattle fetuses, I had to stand at the slaughter house and wait for pregnant cows on the line to catch their wombs. This was disapproved of by the butchers as it interfered with their work and they threw hooves at me to keep me away. It was time for us to break up from Boston in the summer of 1954 and go back to Stockholm and medical school.

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Back in Stockholm 1954^1958 From now on Ulla concentrated on finishing her MD while I went back to the Chemistry Department to continue my graduate studies interwoven with some medical courses. My further work took two directions. First, Bertil Jacobson wanted to pursue his idea that DNA and hyaluronan surrounded themselves with large icelike hydration shells and to prove this by X-ray diffraction. He built X-ray cameras to record the amorphous diffractograms from water solutions of these polymers and I did the experiments and calculations. For this work I used BESK ^ the first electronic binary computer in Stockholm ^ to do one-dimensional Fourier analyses. I spent two years on these experiments but could never record any changes in the water structure, which of course was especially negative for Bertil Jacobson as this was the main theme of his research. His work from then on became directed essentially toward development of instruments and he later became professor of medical technology. My second research line was a continuation of the lightscattering work I had started in Boston. Einar Hammarsten had realized the importance of light-scattering for studies of DNA and he helped me to get a grant for a light-scattering photometer. He also arranged a position for me as teaching assistant in medical physical chemistry, which helped our economical situation but also meant more teaching obligations to medical students. With the new light-scattering equipment, I made a more thorough study of the molecular properties of hyaluronans isolated from different tissues. At this time a new technique to isolate and fractionate polyanions had been published by John Scott in Manchester. He used detergents, especially cetylpyridinium chloride (CPC), to precipitate the polymers. Differently charged polymers precipitated at different ionic strengths. The technique suited me well for the isolation of hyaluronan free of sulphated polysaccharides from the tissues. My new results fitted my previous description of hyaluronan as a random coil structure (Figure 2). I also found

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Fig. 2. A slide frequently used 40 years ago to compare the size of a hyaluronan molecule with that of other macromolecules.

out that I could dissolve a cetylpyridinium^hyaluronate complex in methanol and for the first time study the polysaccharide in an organic solvent. My doctoral thesis [3] was defended in public on October 12, 1957. At that time the defence was held in formal dress, i.e., I had to wear tails. The thesis was a summary of eight publications originating from Experimental Histology, Retina Foundation, and the Chemistry Department. My first examiner appointed by the faculty was Lars-Go«ran Allge¤n, who previously had done work on dielectric properties of DNA, and as second examiner I had asked John Scott to come from Manchester. John Scott was wearing ^ on top of the tails ^ an English academic robe and felt very uncomfortable and his performance was therefore short. Otherwise everything went

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well except at the dinner in the evening. I had spent all my time in preparing my defence and completely disregarded the fact that at Swedish formal dissertation dinners there should also be well-prepared dinner speeches. Having finished my thesis, it was high time to think of how I should earn a living in the future. An academic career in Sweden was highly uncertain especially since I did not belong to ‘‘an academic school.’’ The most natural would be a clinical career and my first objective was to finish my MD, which I did in the spring of 1958. I had also applied to become a ‘‘docent’’ (assistant professor title) in medical physical chemistry and this was granted. At the end of May on a memorable day, when all the academic schools in Stockholm had a graduation ceremony in common in the Stockholm City Hall, I officially received my doctorate. At this occasion my father gave the official graduation lecture and was also ‘‘promotor,’’ i.e., he awarded the doctorates to graduates from the Royal Institute of Technology. In the meantime during my graduate work Ulla had started a career in ophthalmology. We had got our first child, Birgitta, in April 1957 and we expected our second child late in the fall of 1958. We had come to a turning point in life. What were we going to do next? Bandi had offered us to come back to the Retina Foundation and we decided to accept, well aware that, in reality, we might emigrate to the USA. I had no department in Sweden to return to. Einar Hammarsten had retired and his successor, Sune Bergstro«m, came from Lund and filled all the positions in the Chemistry Department with his disciples. My temporary appointment ended in 1958. We decided to move in January 1959 when our second child was born. It was a boy, Claes. Before we left I also finished some collaboration with Birger Blomba«ck on the formation of fibrin from fibrinogen and with Hans Palmstierna and Tord Holme on the characterization of glycogen in Escherichia coli. Most importantly, I got my first graduate student. One of the medical students I had taught, Ingemar Bjo«rk, asked if he could work with me. He followed me to Boston and then to Uppsala.

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Establishing a Research Career Retina Foundation 1959^1961 Equipped with immigration visa, we flew in the beginning of January in a slow propeller plane to New York. The Retina Foundation had grown since our last stay. Claes-Henrik Dohlman, a Swedish corneal surgeon whom we knew since our last visit in Boston, had brought two of his colleagues from Sweden, Arvid Anseth and Bengt Hedbys. Claes-Henrik shared his time between clinical work at Massachusetts Eye and Ear Infirmary and basic research on the cornea at the Retina Foundation. As mentioned Ingemar Bjo«rk joined me as my graduate student and started his thesis work on lens proteins. Ulla, when not caring for the children (the third one, Agneta, was born in Boston) worked part time in the laboratory on projects concerning the vitreous body.There was also a stream of other scientists working in the laboratory, e.g., David Maurice, a well-known corneal physiologist from England, and Saichi Mishima, who later became head of the Department of Ophthalmology at Tokyo University. Also the instrument park had grown and I was in charge of the physicochemical equipment, which now also included an analytical ultracentrifuge placed in the basement. To protect this instrument against floods from broken sewers we had to build a plastic tent over it. I also took over a laboratory technician, Adolph ‘‘Pete’’ Pietruszkiewicz, who was running the centrifuge and who became a most valuable coworker. When Bandi left for a year in Sweden, I was asked to take charge of the administration of the laboratory, which was both a good experience and a challenge. While I developed my own research program, I had to help Bandi’s collaborators; and furthermore, I became partly involved in advising ClaesHenrik’s coworkers. As a continuation of my thesis work, I decided to use the CPC-fractionation technique developed by John Scott to fractionate hyaluronan from bovine vitreous body in order to check if

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it contained more than one component. The polysaccharide was dissolved at a high ionic strength together with CPC and the solution was diluted with water in defined steps. At each step a precipitate was formed, which I analyzed chemically and physicochemically. It turned out that I had obtained a molecular weight fractionationwith fractionsbetween100,000 and1.5million Da [4]. We suddenly had a versatile tool for studying molecular weight polydispersity of charged polysaccharides. The next step was obvious. Heparin had been fractionated by CPC precipitation by John Scott, and fractions of varying biological activities had been obtained. It had been assumed that this was due to a fractionation according to chemical composition especially sulphate content, which would give variation in charge density. When I fractionated heparin with the CPCtechnique, I found a molecular weight fractionation rather than charge fractionation and the conclusion was that the anticoagulant activity of heparin was molecular weight dependent. My conclusion was heavily criticized by the guru of heparin research, Erik Jorpes, and he even promised that he would stop me from getting a permanent position in Sweden. However, many years later it was possible for me to confirm the results by independent techniques and propose the mechanism for the molecular weight dependence [5]. When heparin acts as an anticoagulant it catalyzes a reaction between thrombin and antithrombin, which both bind to the polymer during the process. Antithrombin requires a very specific binding site and the statistical probability that this binding site is present next to a site, where thrombin can bind, increases with increasing molecular weight. I regard this paper as one of my best intellectual contributions. Arvid Anseth’s thesis project was a study of the composition of corneal polysaccharides at normal and pathological conditions and we developed together a technique to fractionate them by ion exchange chromatography. In this case we found that the chondroitin/chondroitin sulphate was fractionated essentially according to sulphate content, while keratan

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sulphate was fractionated according to molecular weight. The precipitation of the keratan sulphate fractions with CPC showed an ionic strength-dependent pattern similar to that of hyaluronan and heparin. The theory for the molecular weight fractionation with CPC was worked out a few years later by John Scott and we published a note together in Nature [6]. In our analyses of the hyaluronan fractions from the bovine vitreous body, we had determined the sedimentation coefficients at infinite dilutions for each fraction. This could only be made by extrapolations from measurements made at several concentrations. We then found that the sedimentation coefficient is strongly concentration-dependent and the larger the molecule the stronger the dependence at high dilutions. In contrast, at concentrations above 0.2% the sedimentation coefficient was slow and essentially independent of molecular weight [4]. When searching the literature, I found that Ogston and coworkers had published a similar pattern for other polymers and they had proposed that the polymers at high concentrations form a continuous chain network with a high resistance toward water flow, which inhibited the sedimentation. It was only at very high dilution that the molecules sedimented freely. This observation became the starting point of twenty years of research on the physiological function of hyaluronan and other connective tissue polysaccharides. If hyaluronan forms a network at physiological concentrations in the ultracentrifuge it must also do so in the extracellular matrix in between the cells. The properties of these polymer networks should determine physiological events in connective tissue. For example, the holes in the meshwork should regulate the transport of other molecules. This was possible to check in the analytical ultracentrifuge and we determined the sedimentation rate of various globular compounds through hyaluronan solutions of different concentrations [7]. It became quite apparent that hyaluronan acted as a sieve, which retarded large molecules much more than small molecules. The phenomenon was also confirmed by studies of the diffusion of various molecules

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through the network. It became possible to formulate an empirical equation that described the retardation as a function of mesh work density and diameter of the penetrating molecule. My personal economic support during this time was a postdoctoral fellowship from the Helen Hay Whitney Foundation, which supported research on arthritis. I was very fortunate to obtain one of these fellowships after having been interviewed by Jerome Gross, one of the pioneers in collagen research, and Walter Bauer, professor of internal medicine at Harvard. I have, later in life, used my experience from these interviews when I myself have been in charge of fellowship programs. The Helen Hay Whitney fellows met once a year in Princeton together with the scientific board, of which Walter Bauer was chairman. We had to present our research to a qualified audience, which made me somewhat uneasy, but it was a very good school. Bauer was an impressive person. For Swedes he was mainly known as the man, who had come to Sweden after the war and introduced us to penicillin, and he was thus familiar with Swedish medicine. When we were travelling back in the same car from Princeton to Boston, he asked me of my opinion of American medical practice in contrast to Swedish socialized medicine. In USA socialized medicine had a very bad reputation and I must say that I hesitated to tell the foremost representative of American medicine that I preferred the Swedish version ^ but I did. To my surprise he said that so did he. It is difficult not to mention the exciting political situation during our stay in Boston. In 1960 John Kennedy and Richard Nixon competed for the presidency. Kennedy lived at Beacon Hill close to our laboratory. We were all for Kennedy, who had the youth, vigour, and academic contacts that appealed to us. I carried a straw hat with Kennedy’s picture on top and my neighbors in Arlington, who were republicans, called me a communist. I also took part in a big rally in Boston Garden the night before the election and on election day Kennedy himself voted in the building next to our laboratory. I have never before or after felt so personally engaged in politics.

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It was a golden time for research in USA after the war and there was a strong temptation to stay in Boston. With time I realized that I ought to break the ties with Bandi. I had an offer to go to Karl Meyer at Columbia University in New York but that would mean to leave all our friends in Boston and we knew nobody in New York. The solution came when I received an offer to come to Uppsala.

Uppsala 1961^1966 The new environment The professor of medical and physiological chemistry in Uppsala, Gunnar Blix, was due to retire in 1961 and there was a lack of senior investigators in the department. Therefore, my friend Lennart Rode¤n, who lived in Stockholm, held the position as docent (associate professor) and commuted to Uppsala. Lennart saw a possibility of getting me to Uppsala and convinced Gunnar Blix that he should propose an established investigatorship of ophthalmic biochemistry in the Swedish Medical Research Council and mention me as a possible applicant. The council accepted the idea and created such a position. Thus, I came to Uppsala in September of 1961. As Lennart Rode¤n and I had closely related research interests, I had looked forward to interact with him. However, Lennart at this time left Uppsala and moved to Albert Dorfman in Chicago and later he became professor at the University of Birmingham in Alabama. Lennart did fundamental work on the structure of cartilage proteoglycans and he was the one, who established the structure of the polysaccharide^protein linkage region, a project in which I became partly involved as physical chemist [8]. Although Lennart left Uppsala, another colleague from Stockholm instead appeared. Peter Reichard succeeded Gunnar Blix as professor at the same time as we arrived. Peter

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became a great support to me. Gunnar Blix was still active as emeritus and had a few graduate students. The Departments of Medical Chemistry and Pharmacology were housed in a building from 1945, which had a considerable charm but numerous defects due to its war time quality. I got a laboratory in the basement and my first task was to apply for financial support as I arrived with two empty hands. However, I could recover some of my equipment from Stockholm, and with the help of Peter Reichard I was able to get a grant from the Wallenberg Foundation for an analytical ultracentrifuge. Looking back it was at that time probably easier to establish yourself from a ‘‘zero’’ position than it would be today. In the meantime, when equipping the laboratory, I had plenty of time to establish contacts with other scientists in Uppsala. Peter Reichard studied the synthesis of deoxyribonucleotides, which was far from my interest, but at least I helped him to determine the molecular weight of thioredoxin, which he had discovered. He surrounded himself with a number of very talented graduate students, who are now filling chairs in various parts of Sweden. The second professor in the department, Gunnar —gren, worked on nutrition and protein phosphorylation, but when I came his research was no longer at its peak. However, his students Lorentz Engstro«m and O«rjan Zetterqvist continued the tradition of protein phosphorylation. Ernst Ba¤ra¤ny was professor of pharmacology in the department next door and his special interest was eye physiology and pathophysiology, especially glaucoma, i.e., close to my interest. He was a charismatic person with numerous ideas; he was liked by everybody and he had a strong position in the Faculty of Medicine. He often came over to me to test his ideas and I went to him and asked for advice. A society for experimental biology was very active to organize seminars. Biologists from various parts of the university met regularly in this forum. To the meetings came biochemists from the department of Arne Tiselius, zoophysiologists, researchers from the pharmaceutical company Pharmacia,

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clinical scientists from the university hospital and researchers from the preclinical departments. At that time it was easier to assemble scientists for interdisciplinary discussions than it is today. I became engaged in this society first as secretary and later as chairman and could invite lecturers from other universities. It was thus easy to establish contacts with other scientists. Furthermore, there was a very strong tradition of polysaccharide research in Uppsala. The use of dextran as a plasma substitute had been developed by Bjo«rn Ingelman and Anders Gro«nvall in Tiselius’ department, and dextran had become the main product of Pharmacia. Many research groups studied the biological effects of dextran as well as the use of dextran in separation techniques. It was easy to find points in common with hyaluronan research.

Properties of polysaccharide networks As mentioned previously, my main idea at this time was to understand the physiological functions of hyaluronan in terms of physical^chemical properties of three-dimensional continuous networks. During our studies of the sieve effect of hyaluronan, an interesting paper was published by Ogston and Phelps in which they described equilibrium dialysis experiments indicating that hyaluronan excluded space for other macromolecules and they assumed that this was a sterical phenomenon. Ogston had previously calculated the available space for spheres in a network of randomly distributed rods but unfortunately the experiments showed a much larger effect than theoretically predicted. The concept of mutual exclusion of spherical molecules had previously been used to explain the concentration dependence of various properties of protein solutions. The effects should be larger if asymmetric polysaccharide chains exclude globular proteins and therefore such sterical interaction could be physiologically relevant. I decided to repeat the equilibrium dialysis

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experiments of Ogston and Phelps but also to study possible exclusion effects by osmometry and solubility experiments. These experiments did not require extensive resources. I built simple equilibrium dialysis cells in the machine shop of the department, and I used some ordinary capillary osmometers for studies of the interaction between serum albumin and hyaluronan. As the solubility studies required excessive amounts of polysaccharide, these experiments were made with dextran instead of hyaluronan. The results were striking. The equilibrium dialysis experiments were at variance with the data obtained by Ogston and Phelps but agreed fairly well with what was to be expected from the theoretical calculations by Ogston [9]. I started a correspondence with Ogston and also sent him my osmometry data on which I needed expert help to interpret. He became very enthusiastic and we published the osmometry data, which agreed with the exclusion theory, in a joint paper [10]. Although I had met Alexander ‘‘Sandy’’ Ogston already a few years earlier, this collaboration started a life-long correspondence between us and exchange of ideas. Also the solubility experiments confirmed that polysaccharides exercise a dramatic exclusion effect on proteins. The solubility decreases in the presence of the polymer and the effect is larger for large proteins [11]. Two years before we arrived in Uppsala, Jerker Porath and Per Flodin had published the technique of gel filtration. When proteins are chromatographed on a granulated cross-linked dextran gel (‘‘Sephadex’’), large molecules are eluted before small ones. The name ‘‘gel filtration’’ had been coined by Tiselius. Others had introduced the term ‘‘molecular sieve chromatography’’ implying that the effect was due to some kind of filter effect. The process had also been described as an exclusion effect although there were no quantitative data which proved the case. With my new knowledge of exclusion phenomena, it became quite obvious to try to explain the data by the Ogston model. Together with Johan Killander, I collected gel filtration data for a large number of proteins and several different

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Sephadex gels and showed that the data were completely compatible with the exclusion of the proteins from a random network of dextran chains. Furthermore, the exclusion from the dextran gels agreed with the protein solubility in dextran solutions confirming the theory. The resulting paper [12] became my most cited publication. As we now knew the mechanism, I decided to use the term ‘‘gel chromatography’’ instead of gel filtration. As an extension of this work we cross-linked hyaluronan to a gel and used it for gel chromatography [9]. The results confirmed beautifully the equilibrium dialysis experiments we had made previously. Later I also characterized agarose gels by gel chromatography (Figure 3). As a side-line my father, my brother Estan, and I constructed an electrical analog computer to describe the gel chromatographic process [13]. It was quite ingenious and could describe the effects of column lengths and flow rate on elution pattern and resolution (Figure 3). The computer was never of any practical use but I value this work very much because it gave me an insight into my father’s creativity and engineering skill, which I had not been able to observe so closely previously.

Co-workers The move to Uppsala also made it possible to recruit students. Arvid Anseth had returned to Lund and defended his thesis on corneal polysaccharides in 1961. He later became professor of ophthalmology first in Tromso« and finally in Oslo, Norway. Ingemar Bjo«rk came to Uppsala when I had established myself. He got his doctorate in 1964 and did post-doc work with Charles Tanford. He continued work on proteins, e.g., immunoglobulins, coagulation factors, and proteolytic enzymes, which earned him a chair of medical chemistry at the Veterinary Faculty of the Agricultural University. Lennart Rode¤n had recruited two young Uppsala students to Chicago, Birgitta and Ulf Lindahl. When they returned to Uppsala in 1964, Ulf joined my group

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Fig. 3. Demonstration of the principle of gel chromatography. A. The density of a three-dimensional random network of rod-like molecules determines the available space for different sized spherical molecules. B.When macromolecules are chromatographed on a gel column, those with less available space in the gel will partition to the liquid phase and move fast. C. Chromatography of three substances of varying molecular size on a hyaluronan gel. D.The available space calculated from the chromatogram is the same as calculated theoretically. E. Analog computer, that describes the gel chromatographic process.

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and finished his thesis in 1966 on the structure of the heparin^ protein linkage region. My first student recruited in Uppsala was my brother-in-law Krister Hellsing. He started a project dealing with immune reactions in polymer media. As polysaccharides decreased the solubility of large proteins, the effect should be especially pronounced for large immune complexes and could be the reason for immune precipitates in connective tissues in immune diseases. Krister could show a rather dramatic effect of dextran and hyaluronan on the precipitin reaction and analyzed various parameters, which regulated the effect and which all indicated that it was an exclusion effect. He moved to clinical chemistry, where he utilized his new knowledge to increase the sensitivity of clinical immune analyses. In 1962 I got my analytical ultracentrifuge and employed a young engineer, Hkan Persson (later Pertoft), straight from a technical high school, to run it for me. His salary was financed by grants from the Medical Research Council. Hkan stayed with me for 35 years, until I retired and lost my research grant. He has been very important for my research program and I will return to his contributions below. During the first years I was also helped by some talented students, who were trained to become laboratory technicians, and I would like to gratefully acknowledge their contributions.

Spreading the message An important aspect of all scientific activities has been the presentation of data at conferences. My first lecture was held at an American Chemical Society Meeting in 1953 and dealt with ultraviolet irradiation of glucose. The first international meeting was the 4th international congress of biochemistry in Vienna, which Ulla and I and many other Swedes attended in 1958. The Austrians had, despite meagre material resources after a long communist occupation, done the utmost to be good

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hosts and they succeeded. It was a relaxed atmosphere, it was easy to meet and talk even to the famous men you only knew from the text books. You met an old and refined culture in the city. The next international congress was held in Moscow in 1961, just after our return to Sweden. This conference was a direct contrast to the one in Vienna ^ travel was complicated, the organization was bureaucratic, the conference was spread over a large area at the University of Moscow so that it was difficult to move from one section to another, the program was not followed and many speakers, especially Russians, never turned up. We were prevented from speaking to Russian delegates. One symposium was canceled so that Titov, who had just returned from space, could give a press conference. No information reached us from outside Russia and only when we came home we heard that the communists in the meantime had built the wall in Berlin. For a Swede it was an important experience forty years ago to have seen both USA and Russia. It was at two conferences in 1964 (in St. Andrews, Scotland, and Swampscott, MA) that I could summarize for the first time to a larger audience my new ideas of the physiological function of polysaccharide networks (see Table 1). Although I had lectured on various parts of this topic previously, I realized afterwards that it was at these occasions that the new concepts TABLE 1 Physical chemical properties of polysaccharide networks connected with physiological and pathological functions in tissues Anomalous osmotic pressure Flow resistance Exclusion of macromolecules Decrease in solubility of macromolecules Retardation of macromolecular diffusion Viscoelasticity

Regulation of hydration of tissues Regulation of flow through tissues Regulation of macromolecular content in the matrix Physiological and pathological precipitations in the tissues, e.g. amyloid, lipids, immune complexes Regulation of macromolecular transport in tissues Lubrication

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of the physiological role of polysaccharide networks was accepted. In the following two years I was invited to give about twenty lectures on the subject, especially in USA, but also in Germany, Italy, and France.

Permanent Position Preludes Already in 1963 Peter Reichard returned to Stockholm when he was appointed successor of Erik Jorpes at the Karolinska Institute. It was unfortunate for our department to lose not only Peter but also his very talented graduate students. When looking back to the 1960s, I guess that I never worked in a place with such a high concentration of brilliant students. As a result the chair of medical and physiological chemistry in Uppsala was again vacant and we were seventeen applicants at the deadline. When looking back on the list it included a large number of highly qualified persons and at that time I took for granted that my chances in this group were rather slim. There were few openings in the academic hierarchy and the competition was fierce. The Nobel laureate Hugo Theorell wrote an article in one of the daily newspapers entitled ‘‘What will happen to the sixteen?’’ In retrospect we now know that nearly all of them became professors. The selection process became complicated and the whole procedure took more than two years. Finally, I was appointed professor of medical and physiological chemistry from February 1, 1966. It was an historical chair, which now was mine. The first professor in medicine in Uppsala had been Johannes Chesnecopherus, appointed in 1613. Fourteen years later the position was divided into two and Chesnecopherus continued on the chair dealing with theoretical medicine. In the end of the 17th century, this position was held by Olaus Rudbeckius, the universal genius who discovered the lymph vessels, and in

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the 18th century by Linnaeus, the botanist. The name of the chair was then ‘‘medicine and botany.’’ Linnaeus also lectured in chemistry to medical students. In 1852 botany was moved to the Faculty of Science and the chair was turned into medical and physiological chemistry. Before me, there had been eight professors of the subject of whom Olof Hammarsten (1873^1906) probably was the most revered. My official inauguration took place on October 1, 1966, in the main lecture hall of the university. At that time it was a public, very solemn ceremony conducted by the vice-chancellor, who then was the legendary Torgny Segerstedt. It included an academic procession in formal dress, music by the academic orchestra and a lecture, for which I had chosen the subject ‘‘The physiological chemistry of connective tissue.’’ Afterwards, as was the custom, we had invited 150 persons for dinner. To our joy many of our friends were there, also from other universities, and I especially remember with affection that Einar Hammarsten came ^ it was the last time I saw him.

New Duties Although I had been teaching and administrating earlier, this had been far from a major occupation. Suddenly, one duty after the other fell upon my shoulders and it is unavoidable to mention them in a chapter of personal recollections. For the period 1966^1979 the main part of my time was occupied by administration and scientific evaluation. The fact that our research still was alive must be attributed to my gifted co-workers and students.

The Department Even if we were several persons, who shared the teaching load, I had to prepare lectures covering large parts of my subject and

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this was not the least important for my own education. With time, a certain subdivision of the responsibilities developed and I lectured for many years on tissue and organ chemistry (connective tissue, mineralization, muscular contraction, neurochemistry, etc.), which I very much liked. However, I also had to lecture on those parts, which nobody liked and to them belonged amino acid metabolism. Later the department got special positions for university lecturers who took over much of the responsibility of the undergraduate teaching. An expansion period at the Swedish universities without any previous counterpart came in the 1960s. The number of students tripled in our department, which also meant more teachers and new laboratories. A completely new site for the preclinical departments, the Biomedical Center (BMC), was planned and the driving force behind the construction was the dynamic professor of physiology, Karl-Johan O«brink. We engaged ourselves in planning our new department and the teaching facilities were built in 1968. The research remained in the old building until 1974. Even if BMC from the beginning was planned essentially for the Faculty of Medicine it soon changed character. It was decided that the Veterinary School and the School of Pharmacy in Stockholm should move to Uppsala and be placed at BMC (at least partly in case of the veterinarians). Arne Tiselius declared that he was interested in moving the Department of Biochemistry in the Faculty of Science close to the medical school and therefore also biochemistry, microbiology, and molecular biology were included. Other facilities have later been incorporated and when the BMC was completed it housed 1500 employed persons and numerous students on 100, 000 m2 area. It is one of the largest centers for life sciences in Europe. For extended periods I served as deputy director of BMC. In our department we shared the administrative responsibilities and the chairmanship rotated. I myself served for two periods. As our department was the largest in the faculty, there was quite a lot of routine work but there was never any

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great problem. We had competent staff and surprisingly few conflicts.

The Faculty of Medicine After only a year, I became involved in the administration of the faculty as a member of the faculty board and soon as deputy dean and treasurer of the faculty. Other assignments were on the faculty teaching board, as chairman of the committee for graduate education, as member of the board of the University Hospital, and more recently member of the board of the University. Of these jobs the one concerning graduate education was probably the most important. The government introduced in the end of the 1960s, a formal curriculum for research training. Previously, the only requirement for a PhD was a thesis describing research, which the candidate had independently carried out and defended in public. Such a thesis often summarized a number of articles in scientific journals with the student as the sole or main author. Often the thesis was a lifetime’s work. According to the new system, a PhD degree should only take four years, it should be carried out within a research group with less degree of independence and the candidate should get a formal education including more or less compulsory courses. It fell upon me to organize the new PhD curriculum. It was a challenge as it was not easy to request more formal teaching from the professors. One somewhat odd assignment for a biochemist was on the board of a spa, Sa«tra Brunn, owned by the faculty and run as a rehabilitation center for rheumatic patients. I had heard about the glorious crayfish parties held in connection with the board meetings at the spa; and when there was a need for a substitute on the board in 1972, I volunteered. At the first board meeting the crayfish had been infested by disease and I never saw a crayfish at Sa«tra Brunn. I stayed on the board for nearly 20 years ^ the last 12 years as chairman. The director and

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chief physician of the spa, Nils-Johan Ho«glund, was not only a very good doctor but also a man who cared for the traditions of this 300 year old institution. Even if we always had financial problems, it was a privilege to serve this humane establishment.

The University and the Academic Life My contacts with the central management of the university were for a long time only informal but still rather interesting. Our vice-chancellor, Torgny Segerstedt, had a special way of getting feedback from the grass-roots. There was a small society housed in a building close to the university. Segerstedt was its president and young professors were the active members.We met every month, sometimes even more often, and had interdisciplinary discussions on topics of current interest. Segerstedt used the society as a sounding board for new ideas. I was active for many years in this group. I became a member of the The Royal Society of Sciences in 1974. This is the oldest of the scientific societies in Uppsala. As a matter of fact, it is the oldest academy in Sweden. It was founded in 1711 when, during an epidemic, the university had to be closed temporarily and the professors needed an active forum to meet. It has since served as an interdisciplinary meeting place in Uppsala. For a long time my friend and colleague Lars-Olof Sundelo«f has been secretary. A very positive memory was the 500th anniversary of the university. It was founded by Jacob Ulvsson in 1477, by permission of the Pope and a charter from the Swedish government, and became the oldest university in northern Europe. The quincentenary celebration started with a concert for all university employees on New Year’s eve. Forty international conferences and many concerts, exhibitions and other events were held throughout the year and in the end of September at the ‘birthday’ there were three days of festivities. I had got the

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responsibility and financial support to organize a conference on ‘‘Biology of Connective Tissue’’ on September 4^9. The planning took me two years and I engaged all my staff and students. Three hundred persons participated and we had the world elite as speakers. In retrospect, I do not think that we could have organized a better conference. At the jubilee festivities, one hundred distinguished foreign scientists were made honorary doctors. On September 28 these scientists gave lectures in the various departments; on Jubilee day, September 29, there were ceremonies and congratulations in the main hall including a lecture by the Nobel laureate Glenn Seaborg, who had Swedish ancestors, the first performance of a symphony by the Swedish composer Allan Pettersson and a banquet. The last day was the day of conferment. I was confering the degree to twenty honorary medical doctors, two of which were pioneers in hyaluronan research, Karl Meyer and Sandy Ogston. This conferment protocol should be in Latin but I had translated it into English, which even the critics eventually accepted. I also gave the official conferment lecture in the aula. It had the title ‘‘Modern medicine is founded on basic research’’ and contained examples from medicine in Uppsala. The evening ended by a number of balls at student clubs.

Duties on the national scene Although Swedish biochemistry held an internationally prominent position, the Swedish Biochemical Society led a languishing life. There were forces to activate the society and Peter Reichard convinced me to take over as secretary for the period 1967^70. Bo Malmstro«m was then chairman. I did myself serve as chairman in 1973^76. We organized national meetings and did also represent Sweden in the Federation of European Biochemical Societies (FEBS), where I served a few times on the FEBS-council. A memorable meeting was held in Prague in 1968 to which I travelled with my whole family. The Czechs were

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trying to liberate themselves from the Russians and there was a nervous atmosphere as everybody knew that Russian troops were assembled at the borders. After the meeting we travelled for a few days in the country and left on the eve of the Russian entry. Back in Uppsala we could see on TV the Russian tanks on the streets of Prague where we had walked two days earlier. Without anyone asking me I was appointed a member of the Swedish Natural Science Research Council in 1968. In the council I suddenly had to take major science policy decisions on matters of which I had no background knowledge. I remember especially when I was placed on a committee, which should deal with an application for a radiotelescope. The amount requested corresponded to more or less the total budget of the council. We had hearings with the applicant, who was a colleague of my father, and I wondered what he would think of a young whipper snapper like me. I was very much against the whole project because it would impoverish Swedish science in general. However, the Swedish government eventually financed the telescope and it became a very successful investment. My main duty in the council was evaluation of grant applications in chemistry and I was the only biochemist on the evaluation committee. Although biochemistry was not such a large field as it is today, it was hard work for an inexperienced young professor. In 1970 the Medical Faculty in Uppsala appointed me as its representative in the Medical Research Council and I resigned from the Natural Science Council. The chairman of the Medical Research Council was a county governor without any experience of research and therefore the secretary of the council, first Bengt Gustafsson ^ a microbiologist ^ and then Henry Danielsson ^ a medical biochemist like myself ^ became the influential persons. Due to Bengt Gustafsson and Henry Danielsson, the Medical Research Council had a very small but superefficient administration. Being a member of the council, I automatically became chairman of one of the two evaluation committees dealing with biochemistry. My committee processed essentially applications

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on macromolecular chemistry while the other dealt with metabolism and clinical chemistry. The suggestions from the various evaluation committees were adjusted to a final granting decision by the executive committee. As the chairman of the council was a layman, the executive committee was headed by the deputy chairman and I served in this position during the period 1973^77. I remember the work on the councils as inspiring and it gave a lot of insight into Swedish science policy. Looking back on the work in the Medical Research Council, there is one thing that I am especially happy about. At the time there was a political pressure to increase support to rheumatic diseases and a number of chairs in rheumatology had been established at the Swedish universities. However, there were few competent persons, who could fill them. Together with Bo«rje Olhagen, a rheumatologist in Stockholm, I made a survey of research in rheumatology and suggested a program of how the research council could build competence in the field. This program was accepted and turned out to be very successful. Sweden now plays a leading role in rheumatology. I also worked together with Bo«rje Olhagen to distribute grants for research in rheumatology from the King Gustaf V 80-year Birthday Fund and I have been associated in various capacities with this fund for about 40 years. Another rather burdensome type of evaluation in the Swedish academic life was ^ at least 30^40 years ago ^ the appointment of professors. Each time a committee of three specialists were asked to read the complete production of all applicants and to write individual statements, which easily could be 40^50 pages long. During the expansion of the universities during the 1960s and 70s, numerous new professors were appointed and a lot of our time was used to evaluate our colleagues. I got my full share of this work and according to my records I have been on evaluation committees twenty-six times; to this should be added evaluations of candidates of a large number of other academic positions, of dissertations and of various publications.

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Research and Graduate Students 1966^1980 The biological role of connective tissue polysaccharide networks continued to be my own main interest until 1980. However, as should be clear from the previous chapter, there was little time left for me to be at the lab bench even if I was assisted by very efficient technicians. Much of the work was carried out together with guest scientists and many of the concepts had already been formulated. Instead, the new ideas and research lines came from my students and successively from their students. Furthermore, several other active research groups developed in the department, which created a fertile environment. Below I will try to describe the research activities in my group.

Characterization and biology of polysaccharide networks My contacts with Sandy Ogston continued to be close. He moved from Oxford to the National University of Australia in Canberra and I planned about 1965, when I had my doubts of getting the chair in Uppsala, to go for a sabbatical to him. For obvious reasons this could not be realized but instead a successful collaboration developed with a couple of his younger collaborators and their coworkers. Barry N. Preston, who had done a lot of physical chemical characterization of hyaluronan with Sandy, came to Uppsala in 1972 and since then there has been an almost continuous presence of Australians in our laboratory until I retired (all in all eleven Australians and many have returned several times). Furthermore, Ulla and I and our daughter Agneta stayed for a year (1979^1980) in Preston’s lab at Monash University in Melbourne. Two other colleagues, who have been central in the polysaccharide studies, were Robert L. Cleland and Lars-Olof Sundelo«f. I met Bob Cleland, a very competent polymer physical chemist, already in 1960 when he worked at the Retina Foundation on hyaluronan. He became professor at Dartmouth College in New Hampshire, but had

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a sabbatical in Uppsala in 1968, and from 1977 he came to Uppsala regularly for extended research until his untimely death in Uppsala in 1993. Lars-Olof Sundelo«f, a specialist on diffusion, came from the Department of Physical Chemistry, a classical place for polymer chemistry, and he took over the chair in physical and inorganic chemistry in the Pharmaceutical Faculty at BMC. Our scientific contacts started soon after I came to Uppsala when we were introduced to each other by Kai Pedersen, the long-time collaborator of The Svedberg. We found out that we had transport processes as a common interest. Many of the studies on polysaccharide^protein interactions during the 1970s were directed to a refinement and verification of previous ideas and to find biologically relevant systems. We studied not only the translational transport of globular particles through polysaccharide networks but also the retardation of elongated molecules like nucleic acids and found that they more easily penetrated the meshwork. Even if translational diffusion was hindered for a spherical protein, its rotational diffusion was essentially unhindered as expected if it was placed in a cavity formed by polysaccharide chains. We tried to differentiate between charge interactions and steric interactions, we studied the effect of temperature on the interactions and we measured the effect of exclusion on denaturation of polynucleotides and on enzyme reactions. Five new graduate students joined me during the 1960s after I had become professor and they were given topics for their theses in line with our general interests. Torsten Helting came from Lennart Rode¤n in Chicago with a half-completed thesis on the synthesis of the protein^polysaccharide linkage region, which he finished in 1970. —ke Wasteson, who also graduated in 1970, developed techniques to fractionate and determine molecular weight of chondroitin sulphate and he applied his methods in studies of metabolic problems. Bjo«rn O«brink (1971) analyzed the steric and charge interaction between collagen and connective tissue polysaccharides and the importance of these interactions in collagen fiber formation.

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Per-Henrik Iverius (1971) described the interaction between polysaccharides and lipoproteins and lipoprotein lipase. Anund Halle¤n (1974), a specialist in geriatrics, whom I knew from my graduate studies in Stockholm, fractionated polysaccharides by ion exchange chromatography. He was especially interested in the age changes in the nucleus pulposus of the spine. Wasteson, O«brink, and Iverius were all going to start their own research groups, although Iverius later moved to USA. Torsten Helting went to the pharmaceutical industry, but came back to us as adjunct professor many years later, and Anund Halle¤n returned to clinical work. Several foreign guest scientists also enlivened the team when they worked on various biological aspects of polysaccharides. The interest in the physiological role of connective tissue polysaccharides increased dramatically not only with matrix biologists but also capillary physiologists and I got many invitations to lecture and to write review articles. The most comprehensive overview on the subject was written in 1978 in collaboration with Wayne Comper, a student of Barry Preston [14].

Blue fingers When Barry Preston came to Uppsala in 1978, he brought some experimental data on transport in concentrated polymer systems, which were hard to explain. He had studied the diffusion of polyvinylpyrrolidone (PVP) with a molecular weight of 360, 000 in dextran solutions and found that it diffused with paradoxic speed.We tried to interpret the data in terms of exclusion and sent a note to Nature. However, the phenomenon demanded a closer analysis and Lars-Olof Sundelo«f built a simple and ingenious diffusion cell, which allowed a series of parallel experiments in a short time. By a shearing mechanism a horizontal boundary could be formed between an upper and a lower chamber, which both could be filled with dextran. Labeled PVP was placed in the lower chamber and the transfer of label

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to the upper chamber was recorded at different times. Supplied with this instrument I went on a sabbatical year to Barry at Monash University in 1979. In collaboration with Barry, Wayne Comper, and Greg Checkley, I could rapidly verify the previous experiments and furthermore, if we determined the transport of PVP and a low-molecular weight compound, sorbitol, at the same time in the same cell, sorbitol diffused with normal rate while PVP was transported much faster with a different kind of kinetics than diffusion. In one of our discussions, I said that I would like to see with my eyes what is happening and then Barry remembered that he could stain PVP blue. When the experiment was repeated with blue PVP we could see blue, ordered, finger-like structures moving with a constant rate from the lower compartment into the upper one and colorless fingers moving in the opposite direction (Figure 4). We had discovered a system of transport by ordered convection. The

Fig. 4. Blue fingers.

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fingers were on the cover page of Nature [15] and in a series of papers we could characterize the phenomenon and show that the driving force was an osmotically driven redistribution of water in the boundary layer leading to a gradual density inversion. In the continuation, Wayne Comper took an especially active part and later also John Wells, another pupil of Sandy Ogston. Although we speculated a lot about the applicability of the phenomenon for biology, we never were able to show it.

Cell separations in Percoll In our early studies on the transport of globular particles through polysaccharide networks, we received samples of viruses from my colleague Lennart Philipson. The concentrations of these viruses were too low for detection in the analytical centrifuge and we had to do the experiments in a preparative centrifuge, where we could fractionate the content and determine the biological activity in the fractions. To avoid convection in the centrifuge tube, we needed a density gradient. The traditional gradient materials would be sucrose or CsCl but I had a bottle of colloidal silica on the shelf, which was used for calibration of my light-scattering instrument, and I suggested that we used it. To our surprise we did not have to preform a gradient ^ it was automatically formed by centrifugation at high speed due to the polydispersity of the colloid. Furthermore, the gradients that formed were within the physiological density interval of living cells, had low osmotic pressures and the salt conditions could be adjusted to the physiological. Hkan Pertoft, who did the experiments, took up this lead and showed that the gradients could be used routinely for cell and particle separations (Figure 5). However, colloidal silica does lyse red blood cells and in order that the silica particles should not interact with the cells they had to be coated with a polymer. Hkan wrote a doctoral thesis on the technique and received permission from the government to defend it for the title

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Fig. 5. Separation of parathyroid cells after centrifugation in a Percoll gradient. To the left: marker beads of known densities. To the right: a sample of parathyroid cells separated into a nonviable cell fraction, two populations of viable glandular cells and a band of contaminating erythrocytes. (Courtesy of H. Pertoft.)

‘‘docent’’even if he did not have the proper academic background to obtain a PhD. This cell separation medium is now sold by Amersham-Biotech under the trade name Percoll (Pertoft’s colloid) [16]. Percoll is extensively used today in cell biology but it has also been the basis for several new research lines in our own laboratory. A surgeon, Go«ran —kerstro«m, used it for density determinations of parathyroid glands and related the density to cellular composition. This made it possible already in the operating theatre to determine quantitatively how much active endocrine tissue had been removed during parathyroidectomy. Go«ran is now a professor of surgery in Uppsala. Another important use was the fractionation of liver cells by Brd Smedsrd to be described later. Hyaluronan and the Pharmacia connection When I came to Uppsala it was easy to get in contact with polymer chemists at the drug company Pharmacia. As Pharmacia

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had developed from collaboration with the university, there was a very open attitude for discussions with basic scientists. At an early stage I started to work with Kirsti Granath, who was in charge of polymer characterization at Pharmacia. The easiest way to get to her lab was to drive to the backside of the company and climb through her window. She supplied me with large amounts of well-characterized dextrans and she had gel columns on which one could determine molecular weight distributions of both dextran and hyaluronan. Bjo«rn Ingelman was the living encyclopedia of dextran. Pharmacia also had competent capillary physiologists who could act as a sounding-board in discussions on the biological role of polysaccharides. A new turn on the hyaluronan story came in 1971. Endre Balazs was in Uppsala and we discussed his work on the medical use of hyaluronan. He had developed a technique to prepare pyrogen-free material from umbilical cord and together with a veterinarian he had shown that this could be used for treatment of traumatic joints in race horses [17]. Bandi was now looking for a partner to commercialize the material. Due to my good connections, I suggested Pharmacia and wrote in August 1971 a letter to the research director in which I proposed a collaboration with Balazs. After long discussions, he convinced the management to accept the proposal. Pharmacia set up production of hyaluronan from rooster combs and produced it for veterinary use while clinical tests on humans were started. However, the effect on human joints was doubtful. At the end of the 1970s it was shown that hyaluronan could be used as a protective agent in eye surgery. When cataractous lenses are removed from the eye and replaced by intraocular plastic lenses an elasto-viscous solution of hyaluronan in the anterior chamber protects the tissues from damage [18]. From 1980 hyaluronan was sold for eye surgery under the trade name Healon and it became one of the major products of Pharmacia. Since then hyaluronan has been manufactured in many places of the world and used for an increasing number of applications. A recent account can be found in the published

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contributions from a symposium that I organized in 1996 to honor Bandi [19]. The interest of Pharmacia in hyaluronan led to new collaborations. Ove Wik came over from Pharmacia and made his doctorate on characterization of hyaluronan and hyaluronan solutions, which he defended in 1979. There had also been another turning point in hyaluronan research in the 70s. It was discovered that cartilage proteoglycans recognize and bind noncovalently to hyaluronan. My graduate student, Anders Tengblad, was studying this interaction and he used hyaluronan binding proteins to develop a very sensitive assay for the polysaccharide [20]. It became possible to analyze for nanograms of hyaluronan in tissue samples. Anders came to be employed by Pharmacia and was central in their research until his sad death in connection with a heart operation a few years later.

The Turning Point: Australia 1979^1980 In 1979 our youngest daughter would finish high-school and presumably leave home. The two eldest children were already at the university studying law and engineering. We were at a turning point of our lives when we were no longer bound to home out of responsibility to the children. If we should try something new ^ this was the time. The answer was to go for a sabbatical year to find new ideas and time to do bench work. A natural place would be Monash University in Melbourne where Barry Preston was active. We faced one obstacle. Ulla had got a position in the eye clinic and if she took a year off for unspecified reasons she was certain to lose her job. However, there was a possible solution; if she did something of value for the eye clinic, when being away, she could get an unpaid leave. I succeeded in obtaining a grant from Pharmacia so that she could work in the laboratory on an ophthalmological project. When this problem was solved our daughter Agneta declared that she would join us on the trip.

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We employed her as a dish-washer and lab technician so that she had something to do. The sabbatical year (1979^1980) became the turning point, which I had hoped for. I have already described the discovery of the blue fingers but there was so much more to be grateful for. We rented a small house on the campus of Monash University outside Melbourne and we bought an old car. The telephone exchange on the campus was closed during Swedish working hours so we were seldom reached by telephone and the first months there was a mail strike which further isolated us. Instead, we made numerous new friends and saw new places. Usually, we worked hard a couple of weeks and then took off in the car for sight-seeing in Victoria and neighboring states. We flew to Townsville in Queensland to visit one of our previous guest scientists and we were swimming at that time at the barrier reef. We were in Sydney, Canberra, Adelaide, and later in Perth for scientific contacts and leisure. But foremost we worked hard in the laboratory. Ulla took up research where she had left it twenty years earlier. She had studied the composition, especially hyaluronan, of the vitreous body and aqueous humor. Now, thanks to Anders Tengblad, we had a new principle for determination of hyaluronan in nanogram amounts. Ulla made a thorough study of the applicability of the technique for various biological samples. Anders came down from Uppsala to Melbourne to discuss his thesis and Ulla and Anders then coauthored a paper on the practical aspects. Ulla could now determine the hyaluronan content of single samples of aqueous humor and the cornea. She used this technique also for various clinical applications. Especially interesting was the finding of hyaluronan in blood serum and its origin in lymph [21]. The Australian visit thus became highly rewarding also for Ulla. Two years later she defended her doctoral thesis in Uppsala [22]. At the end of our visit our two elder children came to visit us after a trip through India and Southeast Asia. We all returned

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to Sweden via New Zealand, the Pacific Islands, USA, and Canada full of energy and with new ideas. The most important new contact of all in Melbourne was Robert Fraser at the Department of Medicine of the University of Melbourne. Before we were leaving he approached me and asked if he could come for a sabbatical to Uppsala. He came only two months after we had returned to Uppsala and he became my closest collaborator for the next fifteen years.

Hyaluronan Research 1980^1996 The discovery of the blue fingers more or less closed my own work on the physical chemistry of polymers even if some of my collaborators and foreign guests continued. Instead, the polymer work became concentrated to the department of Lars-Olof Sundelo«f.

Turnover of hyaluronan in blood When coming back to Uppsala in 1980, our interest was focused on the new discovery that we could determine hyaluronan in blood and that it came from lymph. Bob Fraser arrived in the fall and brought a sample of hyaluronan labeled with tritium in the acetyl moiety. We decided to look for its fate after intravenous administration. When injected into rabbits, it had a halflife of only 2^5 minutes and almost all radioactivity went to the liver. Smaller amounts were found in the spleen. Tritiated water appeared in blood already after 20 minutes. Apparently, there existed a highly efficient scavenger system for hyaluronan in the liver. When we roughly fractionated the cells of this organ, we found that it was not the hepatocytes but the nonparenchymal cells, which were responsible for the uptake [23]. Bob returned to Australia and synthesized more labeled polysaccharide. This was then used for experiments on ourselves

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(Bob, Ulla, myself, and a graduate student, Anna Engstro«m). We exhibited the same rapid uptake and degradation of hyaluronan as the rabbits. In publishing these results we encountered some problems because we had not asked for permission to do human experiments. Also when lecturing about them, I have frequently been asked about the ethics of injecting radioactive hyaluronan into your wife. As a matter of fact, the experiments were done in a very safe way due to the experience of Bob Fraser and the results were important. When labeled hyaluronan was injected in rats used for wholebody autoradiography, the heavy uptake in the liver was verified but we could also see radioactivity in spleen, lymph nodes and bone marrow. Bob later showed that lymph nodes were important catabolizing sites of hyaluronan that had been carried from the tissues by the lymph. Liver endothelial cells. A Norwegian cell biologist and graduate student, Brd Smedsrd, had come to the laboratory at the time when we returned from Australia. He had planned studies on cell adhesion but his proposed supervisor had moved to USA and Brd instead joined my group. Brd together with Hkan Pertoft started to look for the cells in the liver that were responsible for the uptake of hyaluronan. They invented a separation technique based on Percoll centrifugation and matrix adhesion and separated hepatocytes, Kupffer cells, and sinusoidal endothelial cells from each other and could keep them in culture. These cell preparations were used for our further studies. To our surprise it was not the Kupffer cells but the endothelial cells, which were the scavengers endocytosing hyaluronan [24] (Figure 6). We could show that they carried receptors for hyaluronan, which internalized the polysaccharide with great efficiency for degradation in the lysosomes. Brd subsequently showed that the liver endothelial cells also had important scavenger functions in general, a knowledge that had been forgotten when the concept of the ‘‘reticuloendothelial system’’ had been scrapped in the belief that it was macrophages that were the real scavengers [25]. That

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Fig. 6. Liver endothelial cells in culture: (a) morphology; (b) uptake of fluorescent hyaluronan.

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liver endothelial cells are responsible also in vivo was shown by Bob Fraser by use of electron microscopic autoradiography. Later it became clear that also chondroitin sulphate could bind to the receptor and that high molecular weight hyaluronan was more efficiently taken up than smaller molecules. Various attempts to isolate and characterize the hyaluronan receptor on the liver endothelial cells have been made both in our laboratory and elsewhere. Recent work by Peter McCourt in Staffan Johansson’s group in our department seems to have been successful. Peter has now joined Brd Smedsrd at the University of Troms, Norway, where Brd is professor of cell biology.

Concentration of hyaluronan in serum and clinical research Already at Monash University we had observed that a patient with alcoholic problems had a high hyaluronan level in serum. Could it be due to liver cirrhosis? Could determination of serum hyaluronan be of clinical interest? A rheumatologist, Anna Engstro«m-Laurent, married to a distant relative of mine, had contacted me for graduate studies. She was asked to make a thorough analysis of the normal variation of serum hyaluronan and she found a clear increase of the level with age. Furthermore, after physical activity it increased, presumably due to a pumping of lymph from the periphery to the blood. However, there were also pathological conditions in which very high levels were found, e.g., in patients with liver cirrhosis, when the liver endothelium did not function, and rheumatoid arthritis. In the latter case Anna could show that serum hyaluronan increased dramatically when the patients started to walk in the morning and when hyaluronan from their swollen joints was carried to the blood stream. This explained for the first time the cause of a cardinal symptom in rheumatoid arthritis, i.e., morning stiffness. Hyaluronan is accumulated in the arthritic joints

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during night and causes an oedema, which makes the tissues less mobile [26]. Anders Tengblad, when employed by Pharmacia, developed a commercial kit for hyaluronan determinations. Several other techniques based on similar principles were described in other countries. A clinical chemist was asked to make a comparison between the different techniques, in order to standardize the results, and this led to a rapid expansion of clinical studies. Another useful technique in this connection, which has a background in several laboratories, was the specific histological staining of hyaluronan by which pathological accumulations in various tissues could be visualized. During a decade about 15 graduate students worked in Uppsala or at other universities in collaboration with us on various clinical problems. There were also several guest scientists joining the group in these efforts. As hyaluronan commonly is increased in inflammatory foci, can be produced by tumors, is part of repair processes and is increased in some hereditary diseases, numerous diseases exhibit anomalous hyaluronan concentrations in tissues and body fluids. Valuable observations were made pertaining both to the use of hyaluronan for diagnostic purposes and for the understanding of the pathophysiological role of hyaluronan in disease. Much of this work has been summarized in reviews [27,28].

Basic studies on hyaluronan turnover All the clinical data necessitated a deeper understanding of factors, which regulate the synthesis of hyaluronan, its turnover and degradation. For this reason we needed hyaluronan with new kinds of label, physiological expertise, and more advanced experiments in animals. Bob Fraser supplied us with tritiumlabeled hyaluronan, Ove Wik gave us fluorescently labeled material and Lauritz Dahl synthesized hyaluronan substituted with 125 I-labeled tyramine cellobiose. The tyramine cellobiose has the

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property of remaining intracellularly and could therefore be used to trace the cells and organs where hyaluronan had been internalized and catabolized. Colleagues both in Australia and Sweden helped us with animal experiments. Rolf Reed came from Bergen in Norway and took a very active part and so did Ulla. In summary, we found that turnover of hyaluronan is unexpectedly rapid. If we extrapolate from a rat, a full-grown man should contain about 15 grams of hyaluronan of which about a third turns over each day. This is not what one expects from an extracellular matrix component. The major uptake and degradation takes place locally but part is carried by lymph from the tissues to lymph nodes and the amount is a function of lymph flow. A large amount is metabolized in the nodes and only minor amounts are entering the general circulation and are degraded mainly in the liver. However, there must also be a degrading activity in the circulatory tree as it is possible to demonstrate a depolymerization of hyaluronan during the circulation.

Hyaluronan biosynthesis The other side of the coin concerns the regulation of biosynthesis of hyaluronan. In 1987 Paraskevi (Evi) Heldin joined my group. She came from Lorentz Engstro«m, where she had worked on protein phosphorylation. As we knew that there exists a connection between cell proliferation and hyaluronan synthesis she started to investigate the effect of growth factors on hyaluronan production in vitro. Especially platelet-derivedgrowth-factor (PDGF) induced hyaluronan production by fibroblasts [29]. With this observation as a basis, she has continued to study signal transduction by which growth factors transmit their message. In the meantime the hyaluronan synthases were discovered and characterized and she has been able to go deeper into the molecular mechanism by which the synthesis of

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hyaluronan is being regulated at the same time as she has taken up biological problems in which hyaluronan production is central. It has been an inspiration to follow her work.

The international hyaluronan community The growing interest in hyaluronan during the 1980s, especially in clinical work, was mirrored by an increasing number of invitations to lecture and write review articles on the subject. In many of these reviews, I collaborated with Bob Fraser. The time also came when international symposia could be organized entirely on hyaluronan. The first was arranged by Endre Balazs in St. Tropez, France, in 1985. The second was a CIBA Foundation symposium in 1988 ‘‘The Biology of Hyaluronan’’ of which I was responsible myself. It was subsequently published [27]. Also the third meeting ‘‘The Chemistry, Biology and Medical Applications of Hyaluronan and its Derivatives’’ was my initiative and held in Stockholm in 1996 and published in 1998 [19]. It was held in honor of Endre Balazs, who had reached his 75th birthday the year before. His 80th anniversary was celebrated at a symposium ‘‘Hyaluronan 2000’’ in Wrexham in Wales and at that time I gave the introductory plenary lecture, which had the title ‘‘Hyaluronan before 2000.’’ The attendance at this symposium was about 300, which gives an idea of the growth of the field. A new symposium is planned for Cleveland, Ohio, in 2003.

New Developments Many of my younger colleagues have continued an academic career and very successfully broken new ground within our research group. It is appropriate to mention a few of these projects, which have been very successful.

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Heparin Ulf Lindahl already from the beginning showed great independence. After his thesis on the heparin^protein linkage he went over to study the structure of heparin and its proteoglycan and the biosynthesis and degradation of heparin in mastocytomas. He inspired many students in this work. Heparin inhibits blood coagulation by catalyzing the binding of thrombin and Factor Xa to antithrombin. Lindahl and collaborators made a key discovery in 1976 when they showed that only a fraction of the heparin molecules could act in this process [30]. This led to the elucidation of the antithrombin binding region in heparin and the production of low-molecular weight heparin (Fragmin) for clinical use. Fragmin is now an important product sold by Pharmacia-Upjohn. Ulf became professor at the Veterinary Faculty already in 1973 at the age of 33 and continued in this position until 1991 when he moved back to our department and became my closest colleague. He has made Uppsala a world center for research on heparin and heparan sulphate. Ulf’s first student, Magnus Ho«o«k, who wrote his thesis on heparin biosynthesis, was very active in the group during the 70s but moved to the USA in 1980. Magnus was accompanied by two young graduate students, Lena Kjelle¤n and Staffan Johansson. When back in Uppsala, Lena graduated on heparan sulphate and Staffan on cell^matrix adhesion. Both have continued these research lines and both are now professors in our department.

Cell^cell and cell^matrix adhesion In the summer of 1971 Ulf Lindahl and I attended a Gordon Conference in New Hampshire, where we heard a lecture on cell^cell adhesion in colonies of marine sponges. Macromolecular compounds containing carbohydrate aggregated sponge cells but the compounds were very specific for

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each type of sponge. On the plane trip back, we came to the conclusion that there must be similar compounds in higher organisms, which are responsible for the aggregation of specific cells to form organs. We decided to suggest to —ke Wasteson and Bjo«rn O«brink that they should search for such substances and they were joined by a pathologist, Bengt Westermark. Ater a while Wasteson and Westermark left the project but O«brink continued. The problem was not simple. After much work O«brink established a preparation of dispersed hepatocytes, which could be used to test compounds both for cell^cell aggregating activity and cell^substrate adhesion. O«brink and collaborators could define a glycoprotein responsible for intercellular adhesion of hepatocytes and named it Cell-CAM 105 [31]. Much of O«brink’s work has since then focused on this component. O«brink’s pioneering work on cell^cell adhesion gave him a chair of cell biology at the Karolinska Institute in 1987. The attachment of liver cells to collagen and fibronectin became the theme of Kristofer Rubin. He was the first of O«brink’s pupils to graduate in 1980. Rubin and his collaborators isolated integrins from the cell surface of hepatocytes and fibroblasts, which recognize collagen, and antibodies directed toward the integrins prevented attachment [32]. Rubin also made the interesting observation, when fibroblasts were growing in a collagen gel, that the gel contracted but that this contraction could be inhibited by the antibody. The capillary physiologist Rolf Reed from Bergen, Norway, collaborated with me on hyaluronan at that time and when he got in contact with Rubin he immediately realized the physiological importance of gel contraction. Reed and Rubin set up in vivo experiments and measured the interstitial pressure before and after injection of antibodies directed toward the collagen-binding protein [33]. The experiment showed that the fibroblasts played an important role in determining the interstitial fluid pressure. This was a revolutionary discovery for capillary physiologists, who

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until then only had played with osmotic and hydrostatic pressures. Kristofer Rubin is now professor of biochemistry of connective tissue in Uppsala, and Rolf Reed professor of physiology and dean of the Medical Faculty in Bergen.

Platelet-derived growth factor While Bjo«rn O«brink pursued the cell^cell adhesion problem, —ke Wasteson and Bengt Westermark got engaged in the problem of growth promotion. They were joined by a young graduate student, Carl-Henrik Heldin. It was known since a long time that serum contained a factor, which increases growth rate of fibroblasts and which comes from platelets (platelet-derivedgrowth-factor; PDGF). The group purified PDGF and characterized the molecule [34]. It was a protein with a molecular weight of 33,000 containing two chains. Heldin defended his thesis on PDGF in 1980. Soon the group had determined the amino acid sequence of the molecule and found sensationally that PDGF was identical to an already known tumor promoting factor [35]. For the first time a tumor promoter was shown to be a normal growth factor. This started a new era in cancer research. The Ludwig Foundation placed an institute for cancer research at BMC in Uppsala and Heldin became its director. He has continued high-level work on growth factors and is today the most cited scientist in Sweden. Wasteson moved to the University of Linko«ping and introduced preclinical teaching and research at its new medical school. He later became dean while Bengt Westermark today is dean in Uppsala.

Interaction with Industry Although as a principle I have tried to keep my activities within the academic world, it has sometimes been necessary to act also in the commercial area.

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As mentioned above I had a lot of experience from scientific collaboration with colleagues employed at the drug company Pharmacia. The introduction of hyaluronan in eye surgery around 1980 created a dramatic boost for Pharmacia, which was both positive and negative. On the positive side was the increase in research and development, on the negative side was the growth from a small company with informal ties to the university to a large company that became a game on the stock market. Pharmacia was mainly owned by the Lundberg family in Gothenburg. They wanted to sell their shares for tax reasons and sold them to Volvo, the car manufacturer. The executive director of Volvo, Pehr Gyllenhammar, promised that Volvo was making a long-term commitment. However, only a year later (1986) it was announced at a press conference that Volvo would merge Pharmacia with Fermenta, owned by an Egyptian, Refat El-sayed, who had made money on large scale production of antibiotics for veterinary purposes. Gyllenhammar expressed his great confidence in El-sayed. Fortunately, it became known in time that El-sayed had lied about having a PhD-degree and Gyllenhammar was forced to withdraw from the deal. During this process I took an active part trying to stop the affair. I wrote a personal letter to Gyllenhammar informing him about the risks of destroying the informal collaboration between the university and Pharmacia. I also took part in the public debate. A merge with Fermenta would have been a catastrophe. Later that year I received ‘‘The Pharmacia Award’’at the share holders meeting for my effort. Unfortunately, this blow to the prestige of Gyllenhammar demanded some kind of revenge. He declared that he wanted to structure and rationalize the Swedish drug industry and started to fuse a number of small drug companies with Pharmacia even if the companies had very different products and internal cultures. This started a turmoil in the company with internal feuds going on for many years. In the end Pharmacia was merged with international companies and

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today it is based in the USA. What is left in Uppsala are some production units while most research is gone. It has been a tragedy to follow the development of Pharmacia in Uppsala. The disintegration of Pharmacia has forced a large number of former employees to start small biotechnology companies. Uppsala has become a center for new developments. For some years I was on the board of one of these companies, Medisan (later named Biophausia), which had taken over the sale of dextran from Pharmacia. The basic idea of the company was good; income from the commercial part should finance development of new products of which some also were connected to hyaluronan. Unfortunately, the market for dextran went down and the company ran into financial problems. I also became involved as a consultant in a Canadian company, Hyal, which tried to introduce hyaluronan products for clinical use without success. In contrast my former neighbor, Bengt —gerup, has been very successful in commercializing intra-dermally injected cross-linked hyaluronan for cosmetic purposes and his company, Q-med, has been one of the fastest growing biotechnology companies in Uppsala.

The Royal Swedish Academy of Sciences The Academy The Royal Swedish Academy of Sciences is, like most academies of the western world, a private organization. In 1982 I was elected a member of its class of medicine. It was certainly very flattering to become part of this illustrious club. I attended meetings when I was in Stockholm but did not have any special assignment in the academy until to my great surprise I was nominated to become president in 1991 (Figure 7). The academy was founded in 1739 and modeled on The Royal Society in London. Linnaeus, the botanist, was the most well

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Fig.7. Ingvar Lindqvist turns over the presidency of the Royal Swedish Academy of Sciences to the author on April 10 1991.

known of the five founding fathers. Due to good contacts with the royal court the academy soon received royal status. The purpose of the new society was primarily very applied, to aid Swedish farming and manufactories, and its motto was ‘‘for our descendants.’’ The King decided that this was an important aim and that therefore the academy should be supported by getting all the revenues from selling calendars (the almanacprivilege). This secured the economy of the academy for more than 200 years. Swedish science has never experienced such a time of glory as in the middle of the 18th century with names like Celsius, Linnaeus, Klingenstierna, Polhem, Scheele, Rose¤n, Swedenborg, and Wargentin, and it was a very active society which met in Stockholm; in the beginning the meeting place

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was the House of Nobilities. A new glorious period came in the first half of the 19th century when the chemist Jo«ns Jacob Berzelius was secretary. However, at that time basic rather than applied science was prevailing in the society. At the beginning of the 20th century, the Nobel prizes were introduced and increased the international fame of the academy. In the middle of the 20th century, the reputation of the academy was at a low level after an internal scandal. Olof Palme decided to withdraw the almanac-privilege, which added to the problems. The academy was saved by a new secretary, CarlGustaf Bernhard, a physiologist, who realized the strength of studying environmental problems and put the academy in the international frontline in this field. When I became a member, Bernhard had left as secretary but he was still very much present. The academy has 161 Swedish members below 65 years of age and, in total, about 340 members. When someone turns 65 a new member can be elected. There are also 161 foreign members. The members are divided into ten classes according to scientific fields. The class of medicine has 25 members below 65 and the majority of them come from the Karolinska Institute. When I was elected we were only two from Uppsala. The class of medicine is therefore not at all representative of the Swedish medical establishment and has for that reason only minor influence on Swedish medicine. It is a very different situation in other classes and especially the classes of physics and chemistry play important roles as they are in charge of the selection of Nobel laureates in these fields. The academy has its localities close to the university campus in Northern Stockholm. It is run on capital income and various grants but has very little support from the state; the total yearly budget is about 10 million dollars. It operates several research institutes but the main activities are meetings, international contacts, scientific evaluations, and formulation of policies for science. The academy also hands out a number of awards including the Nobel prizes.

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President 1991^1994 According to the by-laws the president is the foremost advocate of the academy. However, the permanent secretary serves as acting director and is head of the central secretariat of about thirty persons. There is a board of the academy meeting every month and an inner group consisting of the president, three deputy presidents, the permanent secretary and his deputy, which meets more frequently. Embedded in the constitution of the academy, there is an apparent risk for the creation of a conflict between the president and the secretary. I was lucky to avoid this situation. The permanent secretary during my term was Carl-Olof Jacobson, professor of zoology from Uppsala, and we worked well as a team. After academy sessions, we often kept company back to Uppsala in my car and had a chance to discuss common problems.

Regular duties The work in the academy took far more time than I had anticipated but it also became a very interesting experience. My colleagues in the department in Uppsala kindly relieved me from teaching duties as it became impossible to make long term planning of my program. Many times I had to be at the academy at short notice. The academy meets in the afternoon of the second and fourth Wednesday every month. Except for business matters members often give short reports on scientific progress in different fields. After the regular business meeting there is a lecture ^ also open to the public ^ by an invited speaker. The evening ends with a supper in the club house where the president is supposed to give a speech and thank the invited lecturer. These dinner speeches belonged to the most stressful of duties. After a while it was difficult to find anything new and original to say. A secretary at the information department was one of my best sources for

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topics. She supplied me with old documents from the archives. Another source was the previously mentioned memoirs of CarlErik Bergstrand; it was many times possible to find some historical note, which could be connected with the lecturer and the theme of the evening. At one supper speech that I especially remember, I should address Carlo Djerassi, well-known chemist, the father of the contraceptive pill and also author of novels. It was the same day that we had voted for the Nobel prizes in physics and chemistry and the prize in medicine had been decided a few days earlier. Djerassi, I am sure, was aware that his contributions could have merited him both for the prizes in chemistry and medicine and furthermore, he had just written a novel ‘‘Cantor’s dilemma’’ dealing with the problems of Nobel awards. In my speech I regretted that he had not received a prize in medicine or chemistry this year either and that there was now only one prize left and that was the peas-prize. I awarded him a can of green peas. A few years later we had invited Istvan Hargittai from Budapest to lecture on the same day as we announced the Nobel prize in chemistry. He was speaking about symmetry ^ his favorite topic. What he did not know was that his lecture to a large extent was going to deal with the prize of that year ^ the discovery of fullerenes. He had interviewed a number of people involved in fullerene research for his journal ‘‘The Chemical Intelligencer’’ and had explicitly asked them whom they thought should get the prize. Except for the regular bimonthly meetings, the academy also has a formal yearly celebration on March 31 in the presence of the King and Queen. At this occasion we also invite guests from other academies, prize winners, politicians, and other Swedish and foreign contacts. The formal business meeting is followed by a banquet hosted by the president. As there are eight other national academies in Stockholm, which have similar yearly celebrations, and also a number of other occasions when formal dress is required, long skirt and tails became somewhat the working attire for Ulla and myself.

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My experience of royalty was nonexistent before my turn at the academy. However, it was not only at formal occasions that we met them. The King is the patron of the academy and he did also come to some of the ordinary meetings. I completely lacked knowledge of the customs and made a number of mistakes when addressing the King and Queen. A neighbor, who got embarrassed, bought me a book about etiquette. However, Swedish royalties are easy to socialize with and the Queen is absolutely charming. Having earlier been a stern republican I became a practising monarchist.

International contacts The academy serves as an umbrella organization for a lot of Swedish international scientific collaborations. Therefore, much of my time was spent on international contacts. For example, the academy has established a number of national committees on various subjects, which are the main contact agencies to the international scientific unions. The activity of these committees varied. For example, when the international congress of biochemistry was organized in Stockholm in 1973, the national committee of biochemistry was heavily engaged, but later when I served on the committee myself, also as chairman, the activity was very low. As president I tried to activate these committees to work also on the national scene and I am especially pleased that the national committee of chemistry started a lot of interesting activities in Sweden. During the cold war the academy served as a very important bridge over the iron curtain. The academy in Sweden, a neutral country, was able to write agreements on exchange of scientists with the academies of the eastern countries. Although the Soviet Union had already disintegrated at the time of my presidency these ties were still very important. I travelled to Moscow and other capitals in the former eastern block and signed treaties. In Estonia our academy helped to organize an

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evaluation of the whole research organization and suggested drastic changes. We had frequent visits from the Baltic academies and I made two trips to Riga and Tallin. Important companions on these visits were the foreign secretary of the academy, Olof G. Tandberg, and Sascha Edblad, who spoke Russian. At a visit of the Swedish minister of finance to Moscow he had promised 10 MSEK for Swedish^Russian scientific collaboration. The academy was asked to manage the program and one of my first tasks was to chair the first meeting when the money should be allocated. Another international collaboration, in which I rapidly became involved, was the All-European Academy Meetings (ALLEA). The Dutch and the Swedish academies had started this organization and the first meeting was held in The Netherlands. I became host of the second meeting in Stockholm in March 1992, was on the organizing committee of the third meeting in Paris in 1994 and was personally invited to the fourth in Budapest in 1996. Each meeting required a lot of preparations and the Stockholm meeting was complicated as the Dutch and Swedish delegates envisaged a different purpose for the organization. The Swedes wanted to keep ALLEA as an informal forum for information and discussions while the Dutch wanted a strong organization, which could act as a pressure group for science in the European Union. One evening there was an open debate on the subject between me and Peter Drenth, the president of the Dutch Academy. It was an irony that the debate was held in the museum of the Wasa-ship, which sank in the harbor of Stockholm in 1628 because of faulty construction. The ship was built in Stockholm but the constructor was Dutch. Anyhow, the meeting in Stockholm was quite successful and both views were respected. The organization survived, which was amply demonstrated at the meetings in Paris, organized by Paul Germaine, and in Budapest, chaired by Domokos Ko¤sa¤ry. Some of the foreign travel was solely representation and quite enjoyable, especially when Ulla could join me. I made a courtesy

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visit to l’Academie des Sciences in Paris and another time we were invited to the Hungarian Academy of Sciences. We took part in the 250th anniversaries of the Danish Academy and the American Philosophical Society and I was also going to the 50th anniversary of the Russian Medical Academy. In other instances, I was asked to take part in conferences even if the topics were outside my expertise. The first time it was a request from the World Economic Forum in Geneva that I should be their advisor. I hesitated but finally accepted and was invited to a meeting in Davos in 1992 together with a large number of heads of states and ministers. It was a memorable week. My duties consisted in taking part in a discussion on economy, education, and environment led by Maurice Strong. This was supposed to be a preliminary to the UN conference on environment in Rio de Janeiro. The economists were arguing that all educational and environmental problems should be solved by market economy and I reacted violently. According to Domokos Ko¤sa¤ry, the president of the Hungarian academy, who had been in jail during the Stalin era, I expressed the most socialistic views of all. Another duty at this meeting was to act as an oracle together with some very prominent scientists, like James Watson, and tell journalists where I believed that the great commercial breakthroughs through research would come in the future. In September the same year, I was invited to give the Berzelius lecture at the Academia Nazionale Delle Scienze in Rome and made at the same time a courtesy visit to the Third World Academies in Trieste where Abdu Salam was president. I was asked by The Third World Academies to go to their meeting in Kuwait in November 1992 and chair a discussion on how to promote research in the Third World. They chose a Swede for this task because Sweden did not belong to any power block and had not been a colonial power. This was one year after the Gulf war and the meeting became an interesting experience. We stayed at a luxury hotel in the desert and noticed very little of the effects of the war. Most of the discussions were centered on

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the endowment of large sums of money to build universities and I had to tell the participants that without the help of charismatic leading scientists, who developed research, there was no use for the money. I took as an example from the Arab world my own aunt,Vivi Ta«ckholm, who through her enthusiasm had made the Botany Department at Cairo University a leading institute. To my delight she had been teacher to many of the Arabic delegates present. Another example of my ‘‘pseudoscientific’’ engagements was the workshop ‘‘An integrated approach to Science & Technology Policy’’ organized by Academia Europea in Portugal in 1994. I spoke about ‘‘Where does medical research take us?’’ and raised the controversial question whether medical research, through its effect of increasing the human population, was negative for mankind. The same year I was also asked to write an article in the Turkish daily newspaper ‘‘Cumhuriyet’’ about ‘‘Third millenium ^ fears and hopes’’ (in Turkish). Part of my duties in Stockholm included the opening of various international conferences both at the academy and elsewhere. Especially, I remember the opening of the first Stockholm Water Symposium in connection with the award of the first Stockholm Water Prize. I have been fascinated by water ever since I worked on the hydration of hyaluronan.

Research stations The academy has throughout its history started a large number of research institutes or other organizations, which later have been transferred to other principals, usually the government, when they had proved themselves viable but too expensive for the academy. Examples of such units are the bureau of statistics, the bureau of meteorology, the museum of natural sciences, the Stockholm observatory, the academy library, the Nobel institutes etc. Still the academy runs at present seven very active institutes and I made a point of visiting them. The only one that I

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never saw was the Research Station of Astrophysics at La Palma. The visit to our Arctic Research Station in Abisko was made in connection with a vacation tour to the North Cape and it was a delight to see its activity. In contrast I had to go to our Marine Biological Station at Kristineberg several times as we had organizational problems, which were difficult to solve and caused some turbulence. The other research institutes, The Beijer Institute of Ecological Economics, The Bergius Foundation (Botany), The Center of the History of Science and the Mittag-Leffler Institute (Mathematics) are all in Stockholm and playing important roles in international science. It may seem out of date that a private organization like the academy runs research institutes separate from the government. However, experience has taught us that it is much easier to start something new without the governmental bureaucracy and also to put an end to it if it is not interesting. The academy has proven that it can act as fertile soil for important new public activities.

Interaction with the government At the same time as I became president of the academy, Sweden got a non-socialistic government. The new minister of education, Per Unckel, decided to dramatically liberate the universities from government regulations. One day I could read in the newspapers that he had proposed that the Royal Swedish Academy of Sciences should be the authority supervising the universities. Some days later he came to the academy for a discussion. I mentioned what I had read in the newspaper and told him that the academy was a private organization, which did not take orders from the minister. However, we were generally interested in discussing university matters and could give advice, but we were not accepting directives from the government. To my surprise and delight he appreciated our standpoint and said that he

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wanted a completely independent advisor. This started an interesting dialogue between us. Per Unckel proposed a doubling of PhD exams in Sweden before the year 2000 in order to secure Swedish competence in the growing high-technology industry. To reach this goal he asked a number of authorities, including the academy, for evaluation of his program, which he had named ‘‘Agenda 2000.’’ The academy appointed a committee, which I chaired, to write the report and we worked with enthusiasm, and fast. We got input from all parts of the academy and it became quite clear that it would not be possible to reach the goal due to the low number of high-school children, who had chosen science subjects in their curriculum. There were too few students recruited to science at the universities. The problem could be traced back to a school reform in 1968 when primary school teachers started to be recruited from students without knowledge of science. These teachers therefore avoided to take up scientific topics with their adepts. A reform of the Swedish university system had to start in primary school. The report ‘‘The view of the Academy of Sciences on knowledge and competence for the 21st century’’ was the first report published in the series Agenda 2000. It became an important contribution to Swedish public debate. During the reign of the social democrats and under pressure from the trade unions a scheme had been developed to socialize Swedish industry. Companies had to pay a special tax, which was collected in special funds controlled by the trade unions. These funds were gradually going to invest in Swedish industry until they owned it. Naturally, the new conservative government wanted to dispose of these funds and it was suggested that the money should be used for research. The academy and other organizations were asked for advice and I was chairing a committee, which proposed that the money should be placed in an independent research foundation, which should be responsible for the search of frontline projects of special importance for Sweden. I also wrote an article in a

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daily newspaper about our proposal. We got a positive response from the Swedish academic community and in the end the result came close to our suggestion. The Swedish Medical Research Council was also asked for advice and it proposed that a large share should be given for specific projects in which ^ it turned out ^ the council members were personally involved. The medical establishment outside the council got outrageous and demanded publicly that the council, including the secretary, should resign. In the end the council also had to go. In this debate it was pointed out that the proposal by the academy was the most appropriate. During this turmoil I was informally asked if I could take over as secretary of the Medical Research Council but I felt that I was too old for such a demanding job. In the end the conservatives instituted several research funds completely independent of the government. When the social democrats came back in power, we got a new minister of education, Carl Tham, who convinced the parliament to introduce a new law, which made it possible for the minister to appoint the boards of these funds and again take charge of the money. Carl Tham antagonized the Swedish academic community by the introduction of a number of badly prepared reforms and an arrogant attitude in the debate. In 1996 I wrote a newspaper article with the title: ‘‘A deep gap in confidence between Tham and the research community. It can be difficult for Tham to continue governing ^ which respected researcher will take commission from him?’’ I have never received such a positive response on an article as this one. It was no surprise that Carl Tham had to leave as minister after the next election.

The Nobel Prizes The great prestige of the academy is to a large extent due to its role in selecting Nobel laureates. The Nobel Foundation is taking care of the endowment and organizes the awarding

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ceremony but independent institutions select the laureates. The prizes in physics and chemistry are decided by the academy after considerations in the Nobel committees. The academy also selects the prize winner in economics to the memory of Alfred Nobel. The Nobel Committee of Chemistry. The members of the Nobel committee of chemistry are appointed by the academy after recommendations from its class of chemistry. Traditionally, the five permanent members and a few adjunct members have been selected from the chemistry class. About 1990 there was a move in the academy that the committee should include chemistry specialists, who belonged to other classes. Not surprisingly by the chemists resisted but the pressure became too large. As a consequence I was chosen as a member of the Nobel committee of chemistry in 1992. I remained on the committee for 9 years. The records of the committee will be secret for 50 years and it is therefore not possible to describe the work except from a formal point of view. However, the procedures to select the laureates are impressive. Personal invitations to nominate candidates are distributed to about 3000 chemists all over the world and about 400 nominations are received each year. The first evaluations are made by committee members, but when a special field or a special candidate becomes hot, one or several evaluations by experts outside the committee are often also requested. During my time in the committee, we also started hearings with external experts. When I joined the committee, an organic chemist from Lund, Salo Gronowitz, was chairman. The biochemist from Stockholm, Bertil Andersson, took over for a year, before his time on the committee was out, and he was followed by Lennart Eberson from Lund. Eberson died the day before the first committee meeting of year 2000 and I, being the oldest member, had to substitute for a month before Bengt Norde¤n, physical chemist from Gothenburg, could be elected by the academy. I thus served four chairmen. Being a biochemist, I had to share

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evaluation work with Bertil Andersson and later his successor on the committee Gunnar von Heijne. I have the highest respect for their knowledge and analytical stringency. The secretary is an important person on the committee. My first secretary was an inorganic chemist, Peder Kierkegaard and, after his untimely death, a biophysicist, Astrid Gra«slund, both from the University of Stockholm and both positive and effective persons who were ideal for the job. The secretariat of the committee occupies ^ together with the physics secretariat ^ a special inaccessible floor in the academy and new and safe archives were being built during my term as president. The working year for the committee starts a few days after the nominations have arrived on February 1. Major evaluations have to be decided during the first meeting. After sessions during the spring, which also include consultations with the committees of medicine and physics, a probable outcome of the selection process is known in the beginning of June. The summer is used by the chairman to collect material and write the report. In the end of August the committee meets again for the final decision. If the members are unanimous in the decision they are allowed to eat a gourmet dinner after the meeting. It is not surprising that the committee always seems to be unanimous. The committee report is submitted to the class of chemistry in September. The class adds a written opinion before the whole academy meets in the middle of October. At this meeting the committee chairman gives a review of the various nominations and a specialist on the committee describes the proposed candidate. After an open discussion the academy makes the official decision by a closed vote. Immediately after the decision the secretary of the academy tries to phone and inform the new laureate(s) before the official announcement is made at a press conference. The prize is then awarded on December 10 but celebrations and lectures actually occupy a full week in December. The laureates give their Nobel

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lectures; each Nobel awarding organization has a reception; the embassies of the countries from which the laureates originate give lunches or dinners. On the Nobel day there is a banquet in the City Hall and the next day The King hosts a dinner in the palace. The laureates also often lecture at Swedish Universities outside Stockholm. During my term on the committee, the prize was awarded twice to biochemists. In 1993 Kary Mullis and Michael Smith shared the prize for the PCR technique and genetic engineering, respectively, and in 1997 Paul Boyer and John Walker received half the prize for the structure of ATP synthase and Jens Skou the other half for the discovery of Naþ, Kþ ATPase. However, I felt as much involved when the laureates came from other fields. It was remarkable from a psychological point of view that after having tried to weigh their contributions you usually felt a personal engagement and enthusiasm even if you were not a specialist. To the prizes that caught my imagination belonged the ones given for discoveries in atmospheric chemistry in 1995, of the fullerenes in 1996, and on femtochemistry in 1999. It was also an experience to meet the persons behind the discoveries. Most of them I had not met before the award but I have seen several of them on later occasions.

The Board of Trustees of the Nobel Foundation The academy and the other institutions, which select laureates also appoint members of the board of trustees of the foundation. I became a member of this board in 1992 and was chairman in 1994^2001. The duties of the trustees are to appoint the board of the Nobel Foundation and to approve its actions after audit. The trustees only have one regular meeting per year and the meeting is usually short. In the evening The Nobel Foundation invites the trustees to a very nice dinner and it has been one of my duties to give a dinner speech at these occasions. The host and the chairman of the Nobel Foundation during most of my

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time as trustee has been Bengt Samuelsson, Nobel laureate and my colleague as medical biochemist. There has never been any reason to give anything but praise to the board and to the director of the Nobel Foundation at these occasions. The foundation has always been exemplarily managed first by Stig Ramel and then by Michael Sohlman as directors. I spent most of the time of my dinner speeches on science history and pseudophilosophical matters. During the last decade we have had a number of jubilees. Stig Ramel organized in 1991 a 90-year jubilee of the first Nobel prizes. There was a memorial occasion in Paris at the centennial of Nobel’s signing of his will in 1995, and in December of 2001 we had a glorious celebration of the centennial of the first Nobel prizes. More than 160 previous laureates attended the award ceremony at this occasion.

Lindau In 1998 the Nobel Foundation asked me to be its representative at the Week of Nobel Laureates in Lindau, Germany. After World War II Count Lennart Bernadotte, a relative of the Swedish king, initiated a ‘‘summer school’’ for German graduate students every year in Lindau and invited Nobel laureates to lecture. Lennart Bernadotte lives on the island Mainau in the Boden lake not far from Lindau and the program was actually started to promote the region. The initiative was in the beginning very much criticized by the Nobel Foundation, which did not like the official Nobel character of this course. During the term of Stig Ramel as director, there was a reconciliation and the Nobel Foundation now sends an official representative. The meeting has increased in size during the years and the graduate students now also come from abroad. In 1998 the subject of the Nobel week was chemistry and I was told that the only thing I had to do was to speak a few minutes at the opening session. Otherwise Ulla and I could regard the

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trip as a week of vacation. It was not difficult to accept the assignment. Next, I was asked also to host a dinner for Swedish graduate students. Then it was realized that the man, who had organized the program, could not come and I was asked to co-chair the conference. Although it meant a lot of preparations it was really enjoyable. In Lindau we again saw, under much more relaxed conditions, about twenty of the laureates that we had met in Stockholm in previous years. They lectured, took part in round table discussions and talked with the students. Nowadays Lennart Bernadotte’s charming wife Sonja is the driving force behind the Lindau meetings and it is really a very successful undertaking. The last day we were all invited to a boat trip to Mainau and lunch at the castle. When Sonja Bernadotte later contacted me and asked me to help with the 50-year jubilee of the Lindau meetings, I was very sorry that I had no opportunity to help her.

Some Consequences of the Academy Work ICSU The International Council of Scientific Unions (ICSU) is the umbrella organization of all the major scientific unions and has its headquarters in Paris. The director of ICSU at the time I was engaged at the academy was Julia Marton-Lef e'vre and I met her at several conferences in Europe. She is a very competent and engaging person and therefore, when I was asked in 1995 to serve on the finance committee of ICSU, I accepted and stayed for five years. It was not a heavy load as we met in Paris only once or twice a year. When I started, I knew very little about ICSU but I found that it was very efficiently run by Julia. However, there were reasons to criticize the man responsible for investing the funds of ICSU in shaky Mexican bonds and shares in a tobacco company, Philip Morris. Furthermore, it was surprising to find how little

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cooperation there actually was between the different unions, which induced us to an interesting Wenner-Gren symposium. The ICSU-work created useful contacts and it was always nice to go to Paris in the spring.

Auditing of research funds As mentioned above, the conservative government started several independent research foundations from the money collected to socialize Swedish industry. The largest was named ‘‘The Fund for Strategic Research.’’ On the advice of the academy I was asked to be an auditor of the fund to scrutinize if the money was used according to the intentions, i.e., to promote research of importance to strengthen Swedish industry. The Foundation started up in a serious way but when the socialists took over the reign and wanted to use the money as substitute for a reduced budget to the research councils I resigned. Instead, I soon became auditor of another of these funds named STINT, which supports scientific international exchange.

The Wenner-Gren Foundations In the fall of 1992 I received a phone call from a colleague in Stockholm, who unofficially asked if I would consider to become Science Secretary of the Wenner-Gren Foundations (WGS). It was in the middle of my term as president of the academy and I could hardly think of any further duties. I would only accept if the assignment was postponed until 1994. I then got an official invitation to talk to the chairman of the board, Jan Wallander, a former director of one of the major banks in Sweden. We agreed that I should become a member of the board of WGS immediately, and that I should be responsible secretary from September 1993, but that the retiring secretary, David Ottoson, should help me the first year.

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Background history The Wenner-Gren Foundations originated from a donation in 1955 by the Swedish financier Axel Wenner-Gren, the founder of the company Electrolux. The aim of WGS is, according to the statutes, to promote international scientific exchange. The Swedish government supplied a piece of property in the northern part of Stockholm on which the Wenner-Gren Center was completed in 1962. It consists of a 24-floor high building, a lower ordinary building and a large semicircular complex containing 155 guest apartments for visiting scientists. The highrise building was intended to be an administrative center of Swedish science but in reality it became with time occupied by private business. For a long time WGS was plagued by a weak economy and the main activity was to maintain the guest apartments. However, the first science secretary, the physiologist Yngve Zotterman, who was very energetic and imaginative, raised funds from outside sources to organize a large number of international scientific symposia. His successor, David Ottoson, concentrated essentially on symposia within neurobiology. The chronically bad finances of WGS had to be put on a sounder basis.When Jan Wallander became chairman he sold the two central buildings in 1990 and invested the capital to yield a good return. The WGS kept the guest apartments and continued to subsidize the living costs of foreign scientists. But on top of that there were now funds to give scholarships. This possibility emerged in a small scale during the time of David Ottoson but it was not until I became secretary that the program got into full operation. The budget for grants for scientific exchange from WGS is today in the order of 5 million euro. The science secretary is in charge of the scientific program of WGS while a director is in charge of economic matters and the administration of our guest apartments. The most important person has been my secretary, Gun Lennerstrand, who was working at WGS already 40 years ago. She has an enormous routine and is very efficient.

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International symposia My time at the WGS has been shared about equally by the organization of international scientific symposia and the granting program. We have hosted about five symposia per year. A Wenner-Gren symposium usually lasts for three days and allows time for about 25 invited speakers. Proposals for symposia from interested scientists are reviewed by our scientific board, which selects suitable topics and organizers. Most conferences have dealt with topics in biomedicine but we have also had conferences in such diverse fields as history, economics, and military and political strategy. Many symposia have dealt with frontline research, e.g., ‘‘Chemical and Biological Combinatorial Libraries for Ligand Design’’ in 1997 and ‘‘Novel Aspects of Structural Biology’’ in 2001; others have had more of an overview character, e.g., ‘‘ReninÂngiotensin’’ in 1997 and ‘‘Plant Systematics for the 21st Century’’ in 1998. These two symposia honored a discovery and two scientists, respectively, who were a hundred years old. Many times we have collaborated with international organizations, e.g., Academia Europaea on topics like ‘‘The Impact of Electronic Publishing on the Academic Community’’ in 1997. We have also recently started a series together with European Science Foundation and the first symposium was held in 2001 on ‘‘Perils and Prospects of the New Brain Sciences.’’ I was personally responsible for the symposium ‘‘The Chemistry, Biology and Medical Applications of Hyaluronan’’ in 1996, which was held to honor Endre Balazs [19]. Moreover, I organized together with the Canadian Research Council a most interesting conference on ‘‘Nature and Dynamics of Interdisciplinary Research’’ in 1998. The idea of a symposium grew out of discussions at ICSU with the Canadian geologist Hugh Morris on how to promote collaborations between the international unions. A central person in the organizing committee was Richard Zare at Stanford. The conference took up four case studies, which could only be solved by expertise from

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many fields, and we then discussed the obstacles for the collaboration. I believe that our efforts made some impression on US science politics. In the year 2000, I organized a symposium together with the Hungarian chemist Istvan Hargittai on ‘‘Symmetry.’’ The subject was discussed from all possible angles from mathematics and physics to art and poetry. From the beginning, the Wenner-Gren symposia were all published in ‘‘Wenner-Gren International Series,’’ published by Pergamon Press.With time it became clear that in a fast-moving field it was meaningless to publish a symposium volume, which would be out of date when printed. Therefore, only symposia of review character or of more general interest are now printed.We have also changed publisher a couple of times and today Portland Press in London (the publishing arm of the U.K. Biochemical Society) is responsible. The latest volume of the series is number 80, which is the‘‘Symmetry 2000’’symposium. The symposia have no doubt contributed to the purpose of WGS, i.e., to promote international scientific exchange. Apparently, the Wenner-Gren symposia are so attractive to attend that most top scientists approached have accepted to participate. It has given the Swedish academic community all possibilities to interact with them. It is interesting that many young students, whom WGS have supplied with postdoctoral fellowships, have met their future hosts at Wenner-Gren symposia.

Fellowship program The larger share of the WGS budget is used for fellowships. On the basis of applications, we select young Swedish PhDs for postdoctoral work abroad and similarly we support young foreign scientists, who would like to come to Sweden. The fellowships can be extended for up to two years. We also support foreign professors who would like to spend sabbatical years in Sweden.

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Most of our Swedish and other European postdocs go to the US for training and many of the best are recruited to American laboratories and do not return. There is no reciprocal exchange. Therefore, other countries support American science both economically and with their best young brains. For this reason I proposed in 1994 a new Wenner-Gren program in which we support post-docs for five years ^ up to three years abroad as fellows and two years after return as assistant professors. As this is a very expensive program, we can at present only afford five such fellowships per year. However, the new program also necessitated a more scrutinizing selection system and the scientific board has to reserve a week in March for personal interviews with the most merited candidates. The first batches of these special postdocs have now passed their five years and they generally show very impressive academic records. Other items that we support are travel to conferences for young scientists, guest lecturers, who come to Sweden, and various minor international symposia. However, we also have a special program for distinguished lecturers, who are invited to speak at several universities. They often tour Sweden for a week. So far we have had 25 such distinguished guests. Traditionally about half have come from France and both Pierre-Gilles de Gennes and Claude Cohen-Tannoudji were WGS lecturers the same year that they got their Nobel prizes. Finally, WGS has the possibility to support a few special projects outside the general program and this may be to publish a book, to finance a scientific exhibition, to purchase historical documents for Swedish archives and to support international student activities. The year 2002 will be my last year at WGS ^ I have now already been involved in this organization for 10 years. It has been a positive experience. Late in the year we will celebrate that it was 40 years since the Wenner-Gren Center was opened. It is my firm opinion that the Foundation now has matured and found a niche in the Swedish and international scientific establishment, where it can work very fruitfully.

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Concluding Thoughts The 20th century has witnessed the most dramatic expansion of science in the history of mankind. World War II had just ended when I left school. The atomic bomb had been created after a concentrated effort of scientists and it was believed that research could solve all problems. I grew up in a country that had escaped the war, I came from a family with academic traditions, I entered academic life at a time when optimism and increased financial resources permeated the universities and I was lucky to have the right teachers. I got the best possible start in life and I have continued to be privileged. With one exception ^ when I applied for the chair in Uppsala ^ I have never been in serious competition with my colleagues, which has made my professional life uncomplicated and left time for productive work. I have also been especially lucky to have had so many excellent coworkers. Similarly, I have been lucky in my private life and there has been a close solidarity within the family. It was of course a delight when Ulla came back to research in 1980 and we could share work again. However, one event in 1983 changed the situation in the family dramatically. Our son Claes was hit by a car when he was 25 years old and just had started his career after having passed university. Since then he is bound to a wheel chair. It has been a great relief that his intellect and sense of humor have remained intact and that he has stoically adapted to his difficult situation.

Some thoughts on leading a research group Looking back on my experiences in academic research, I feel that a positive interaction with colleagues, collaborators, and students has been the central part of all work. One of the most important activities has occurred during coffee breaks and we have consumed large amounts of coffee in the laboratory. Many

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new ideas develop through conversations and I am surprised how often a good idea first is formulated in spoken words before you have had any previous thoughts on the matter. Presumably, thinking is taking place through a series of associations and, by connecting a number of brains into a network, the number of associations increase. To let young people feel that they work independently and that they handle their own research projects is another important factor. This is something I found out by necessity. As a young professor I was heavily involved in administration and my time with the graduate students became limited. The research group had evening seminars once a week, often in the basement of our home, and the door to my office was always open when I was in the laboratory. However, many times I was somewhere else. Therefore, the students had to take a great responsibility for their own projects and were often sole authors on the publications. This promoted creativity and led to new original research directions. My main role became to encourage students to take their own initiatives and give them whatever support I could and also to try to create as harmonious an atmosphere as possible in the research group.

On administration It must be clear from my recollections that a considerable amount of my life has been spent on administrative work. Would it not have been better to concentrate on research? I have many times asked myself this question, especially during my first 15 years as professor. The answer has always been that one cannot decline to take part in organizational matters; it is a responsibility. If the scientists do not do it, professional administrators and politicians without practical knowledge will decide, which could be disastrous.

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Women in science In view of the present debate regarding the low representation of women in science, I cannot resist making a remark. When Ulla and I went for our sabbatical year at Monash University the university administration first did not allow us to work in the same department as we were married. It was against the university regulations. In Uppsala it never bothered me if husbands and wives worked together ^ just the opposite. Nothing can be more stimulating for a couple than having a common goal. The number of current and emeritus professors in our department working in biochemistry is presently nine and six of them have been or are scientifically working with their spouses. I am proud that I have had three female graduate students, who, when they had graduated, convinced their husbands to join my group and also get doctorates. The first woman to get her PhD in our department during my time graduated in 1978. About twenty years later we got our first two female professors. This is about the time it takes to reach the top of a research career. In my opinion it is better for everyone if men and women are treated equally and judged only on their scientific merits. This is much more healthy than to preferentially promote the under-represented sex. I have no doubt, also after having judged postdoc applicants at WGS, that women can compete on equal basis and with great success. Furthermore, looking back on my own family history there is no lack of competent women.

Growth of science One very marked change during my more than 50 years in research is the development of biomedicine from a small science into a big science. This has been especially marked in the last years in genomic research. One example is the determination of the base sequence in the human genome. Recently, I served as chairman of a consortium of seven universities, which had got a

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500 million SEK grant from a private foundation for collaboration within genomics. The technology is now so expensive that it must be utilized maximally and be shared by as many as possible.This type of research requires organization on a level, which previously only was known from, e.g., physics and astronomy. There is a clear risk that big science leads to an increasing specialization, while many problems rather need interdisciplinarity to be solved. I am convinced that small research groups, working on original ideas, still have a place in the organization if they are placed in departments, where they can interact with other scientists working on other problems. A high intellectual density is the most fertile environment. Looking in the rear view mirror Already the title of these personal recollections tells the reader that I have been very fortunate in life. But what have I accomplished? The answer has to be given by others. Many times I have felt satisfaction and I have received both official and private signs of appreciation. Still I know that my contributions are tiny drops in the ocean of knowledge. I do see myself essentially as a link between a previous generation and the succeeding one. During my active time seventy-five young scientists trained by me or my younger collaborators in the department have received their doctorates. Of these half a dozen of my scientific children, an equal amount of my scientific grandchildren and soon three or perhaps four of my scientific great-grandchildren have been appointed professors. I am most grateful to my teachers and I feel delighted when I see the success of the next generations. REFERENCES [1] Balazs, E.A., Ho«gberg, B. and Laurent, T.C. (1951) The biological activity of hyaluron sulfuric acid. Acta Physiol. Scand. 23, 168^178.

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[2] Laurent,T.C. and Gergely, J. (1955) Light scattering studies on hyaluronic acid. J. Biol. Chem. 212, 325^333. [3] Laurent, T.C. (1957) Physico-chemical Studies on Hyaluronic Acid, pp. 1^28. Uppsala, Almqvist & Wiksells Boktryckeri AB. [4] Laurent,T.C., Ryan, M. and Pietruszkiewicz, A. (1960) Fractionation of the hyaluronic acid. The polydispersity of hyaluronic acid from the bovine vitreous body. Biochim. Biophys. Acta 42, 476^485. [5] Laurent,T.C.,Tengblad, A.,Thunberg, L., Ho«o«k, M., and Lindahl, U. (1978) The molecular-weight-dependence of the anti-coagulant activity of heparin. Biochem. J. 175, 691^701. [6] Laurent,T.C. and Scott, J.E. (1964) Molecular weight fractionation of polyanions by cetyl pyridinium chloride in salt solutions. Nature 202, 661^662. [7] Laurent, T.C. and Pietruszkiewicz, A. (1961) The effect of hyaluronic acid on the sedimentation rate of other substances. Biochim. Biophys. Acta 49, 258^264. [8] Gregory, J.D., Laurent, T.C. and Rode¤n, L. (1964) Enzymatic degradation of chondromucoprotein. J. Biol. Chem. 239, 3312^3320. [9] Laurent, T.C. (1964) The interaction between polysaccharides and other macromolecules. 9. The exclusion of molecules from hyaluronic acid gels and solutions. Biochem. J. 93, 106^112. [10] Laurent, T.C. and Ogston, A.G. (1963) The interaction between polysaccharides and other macromolecules. 4. The osmotic pressure of mixtures of serum albumin and hyaluronic acid. Biochem. J. 89, 249^253. [11] Laurent, T.C. (1963) The interaction between polysaccharides and other macromolecules. 5. The solubility of proteins in the presence of dextran. Biochem. J. 89, 253^257. [12] Laurent, T.C. and Killander, J. (1964) A theory of gel filtration and its experimental verification. J. Chromatog. 14, 317^330. [13] Laurent, T.C. and Laurent, E.P. (1964) An electrical analogy to the gel filtration process. J. Chromatog. 16, 89^98. [14] Comper, W.D. and Laurent, T.C. (1978) Physiological functions of connective tissue polysaccharides. Physiol. Rev. 58, 255^315. [15] Preston, B.N., Laurent,T.C., Comper,W.D. and Checkley, G.J. (1980) Rapid polymer transport in concentrated solutions through the formation of ordered structures. Nature 287, 499^503. [16] Pertoft, H. and Laurent, T.C. (1982) Sedimentation of cells in colloidal silica (Percoll). In Cell Separation: Methods and Selected Applications. (Pretlow, T.G. and Pretlow, T.P. Eds.) Vol. 1, pp. 115^152. Academic Press, New York. [17] Rydell, N.W., Butler, J. and Balazs, E.A. (1970) Hyaluronic acid in synovial fluid. VI. Effect of intra-articular injection of hyaluronic acid on the clinical symptoms of arthritis in track horses. ActaVet. Scand. 11, 139^155.

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[18] Miller, D. and Stegmann, R. (1980) Use of Na-hyaluronate in anterior segment eye surgery. Am. Intraocular Implant. Soc. J. 6, 13^15. [19] Laurent, T.C. (Ed.) (1998) The Chemistry, Biology and Medical Applications of Hyaluronan and its Derivatives. Wenner-Gren International Series 72, pp. 1^339. Portland Press, London. [20] Tengblad, A. (1980) Quantitative analysis of hyaluronate in nanogram amounts. Biochem. J. 185, 101^105. [21] Laurent, U.B.G. and Laurent, T.C. (1981) On the origin of hyaluronate in blood. Biochem. Int. 2, 195^199. [22] Laurent, U.B.G. (1982) Studies on endogenous sodium hyaluronate in the eye. Acta Universitatis Upsaliensis. Abstracts of Uppsala Dissertations from the Faculty of Medicine. 428. pp. 1^40. [23] Fraser, J.R.E., Laurent,T.C., Pertoft, H. and Baxter, E. (1981) Plasma clearance, distribution and metabolism of hyaluronic acid injected intravenously in the rabbit. Biochem. J. 200, 415^424. [24] Smedsrd, B., Pertoft, H., Eriksson, S., Fraser, J.R.E. and Laurent, T.C. (1984) Studies in vitro on the uptake and degradation of sodium hyaluronate in rat liver endothelial cells. Biochem. J. 223, 617^626. [25] Smedsrd, B., Pertoft, H., Gustafsson, S. and Laurent, T.C. (1990) Scavenger functions of the liver endothelial cell. Biochem. J. 266, 313^327. [26] Engstro«m-Laurent, A. (1985) Circulating sodium hyaluronate, a study of its concentration and turnover in serum with special regards to liver disorders and inflammatory connective tissue diseases. Acta Universitatis Upsaliensis. Abstracts of Uppsala Dissertations from the Faculty of Medicine, 525, 1^41. [27] Evered, D. and Whelan, J. (eds.) (1989) The Biology of Hyaluronan. Ciba Found. Symp. 143 pp. 1^298. Chichester, John Wiley & Sons. [28] Laurent,T.C., Laurent, U.B.G. and Fraser, J.R.E. (1996) Serum hyaluronan as a disease marker. Ann. Med. 28, 241^253. [29] Heldin, P., Laurent, T.C. and Heldin, C.-H.(1989) Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts. Biochem. J. 258, 919^922. [30] Ho«o«k, M., Bjo«rk, I., Hopwood, J. and Lindahl, U. (1976) Anticoagulant activity of heparin: separation of high-activity and low-activity heparin species by affinity chromatography on immobilized antithrombin. FEBS Lett. 66, 90^93. [31] Ocklind, C. and O«brink, B (1982) Intercellular adhesion of rat hepatocytes. Identification of a cell surface glycoprotein involved in the initial adhesion process. J. Biol. Chem. 257, 6788^6795. [32] Rubin, K., Gullberg, D., Borg, T.K. and O«brink, B. (1986) Hepatocyte adhesion to collagen. Isolation of membrane glycoproteins involved in adhesion to collagen. Exp. Cell Res. 164, 127^138.

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[33] Reed, R.K., Rubin, K.,Wiig, H. and Rodt, S.—. (1992) Blockade of 1-integrins in skin causes edema through lowering of the interstitial fluid pressure. Circ. Res. 71, 978^983. [34] Heldin, C.-H., Westermark, B. and Wasteson, —. (1979) Platelet-derived growth factor: purification and partial characterization. Proc. Natl. Acad. Sci. USA 76, 3722^3726. [35] Waterfield, M.D. et al. (1983) Platelet-derived growth factor is structurally related to the putative transforming protein p28sis of simian sarcoma virus. Nature 304, 35^39.


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Chapter 5

RNA Enzymology and Beyond URIEL Z. LITTAUER Department of Neurobiology,Weizmann Institute of Science, Rehovot 76100, Israel

Young man, why not study the human brain? (David Ben-Gurion)

Growing Up At home, in Palestine, there were only three of us: father, mother, and myself. I always envied my schoolmates who were surrounded by large families. During their youth, my parents joined the Zionist Blau-Weiss organization, which was motivated by an idealistic desire to build a new-old homeland for the Jewish people. In 1923, they decided to fulfil their dream and left Germany to settle in EretzIsrael (Land of Israel, as the Jewish population called Palestine). The idea of moving to an Asiatic, desolate country did not appeal to their families, who preferred to continue their comfortable lifestyle in Germany.

e-mail: [email protected]

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Fig. 1 Uriel Z. Littauer. Photographed by the Photography Department of the Weizmann Institute.

Upon my parents’ arrival in Palestine, long before the establishment of the State of Israel, they found a sparsely populated country, governed by the British under a mandate from the League of Nations. They settled in Tel Aviv (in Hebrew: Spring Hill), where I was born on February 24, 1924. Tel Aviv was a small city at that time, with a population of about 22,000 inhabitants, built on barren sand dunes along the Mediterranean Sea, with many unpaved roads and few cars. Electricity was introduced in 1923 and was not common in all households. I can still remember the horse-drawn carriages and camel convoys passing through the city with their jingling bells. The city consisted mainly of ordinary, whitewashed, cement brick apartment buildings, and a few houses belonging to the wealthy,

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built in an eclectic style. Later, with the new wave of immigration, architects trained at the German Bauhaus Institute introduced their modern concepts of streamlined functional buildings. On Saturdays, the beach attracted crowds of people who would walk in the streets dressed in their swimming suits. When my father bought a motorcycle, the neighbors complained to the Mayor, Meir Dizengoff, about its noise. His answer was ‘‘I wish Tel Aviv would have more traffic and grow to become a modern and prosperous city.’’ Too soon, too noisy, and probably not to his liking, his wishes materialized. I was curious to learn about the origin of my family. From time to time, I heard some stories from my parents, but my knowledge of my roots was (and remains) rather fragmentary. From the little I was able to gather, it appears that my paternal grandfather, Saul Littauer, was born in Rawitsch, a small town in Poznan. He lived most of his life in Berlin and owned a textile factory in Leipzig. His wife, Caecilia Sara ne¤e Bernstein, a distinguished lady, came from a family of intellectuals and politicians. After the death of her husband, she continued to live in Germany and was later deported by the Nazis to Theresienstadt concentration camp, where she perished. Years later, in October 1984, I tried to find the house of my paternal grandparents in Berlin. At that time, I was involved in organizing an International Symposium on the Molecular Basis of Nerve Activity, which was being held in Berlin in memory of Professor David Nachmansohn, a distinguished biochemist from Columbia University College of Physicians and Surgeons, New York. Nachmansohn, a close friend of the Weizmann Institute, had spent, during the twenties, several years at the famous laboratory of Otto Meyerhof at the historical research center of the KaiserWilhelm-Gesellschaft fu«r Biologie [1]. The symposium was initiated jointly by the Department of Neurobiology of the Weizmann Institute of Science, the Socie¤te¤ Francaise de Chimie Biologique, the Free University of Berlin, and the MaxPlanck-Gesellschaft [2]. The late Professor Heinz Gu«nter Wittmann, then the director of the Max-Planck-Institute for

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Molecular Genetics, and his wife Brigitte were kind enough to drive me in their car to the address of my paternal grandparents. However, all I could find was a small garden at the location of their house, which had apparently been destroyed during the Second World War. My attempts to locate the house of my maternal grandparents in Leipzig were more successful. In 1998, the city of Leipzig officially invited me to visit as their guest. This gave me the opportunity to gather some information on my ancestors. My maternal grandfather, Benjamin Wolf Lehrfreund, a prosperous grain merchant, was born in Krakow, Poland, and moved in 1902 to Leipzig, which was known as a commercial business center. After the death of his first wife, Salomea, he married Rive Regine Stern, who raised his large family of eight children. Walking through the streets of Leipzig and visiting the former Jewish schools, modern hospitals, and old age homes, I was overcome by the terrible tragedy suffered by this once active and lively Jewish community. It vanished completely. It took me some time to locate the house of my grandparents, as the layout of Leipzig has changed over the years. Some of the street names had been changed twice, once during the Nazi period and later by the East German authorities, while others do not exist any more. With the aid of a 1938 street map I obtained from my hosts, I found my grandparents’ home. The still elegant house is close to a large park and is well kept, except that their large nine-room apartment has been divided into three small flats. My hosts arranged for me to visit the public library and trace the address of my grandfather’s factory with the aid of a 1918 telephone book. But neither his house, nor the factory, exists any more. My father, Franz Shimon Littauer, was born in 1893 in Berlin. In 1914, while still a student, he visited Palestine. Impressed by the country, he decided to settle there after completing his studies in engineering. Being an avid photographer, he took some unique pictures that documented this early pioneering period. During the First World War, he served in the Imperial


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German Army as an officer, quite a distinction for a Jew. In 1921, he graduated in chemistry from Leipzig University. To prepare himself for his move to Palestine, he also studied botany and later received his PhD degree from the Faculty of Agriculture of the University of Breslau. My mother, Regina (Gina) ne¤e Lehrfreund, was born in 1900 in Krakow, Poland. At the age of two, she moved with her family to Leipzig, where she graduated from a teacher’s college. She later specialized in the Montessori method of teaching that aims to develop a child’s creative potential. It was considered at that time a modern and even vanguard school of education. After her arrival to Britishruled Palestine, my mother established the first kindergarten that employed the Montessori method (probably the only one in the Middle East) and many generations of children graduated from her institution. In the early 1920s, few professional positions were available in Palestine. My father applied to the ‘‘Agricultural Experiment Station’’ of the Jewish Agency. The Station, which eventually became known as the Volcani Center of the Agricultural Research Organization, was poorly equipped. However, when my father mentioned that he brought a microscope with him from Germany, he was hired on the spot and appointed as a senior plant pathologist. After his retirement, it took me some time to retrieve the microscope, which may be considered now as an antique piece. During the time of the British Mandate, the major source of income in Palestine came from exporting citrus fruit to Britain. The Jaffa oranges, as they are called, were then handled by primitive methods and the rate of decay was high, more than 20%. In 1937, my father founded and headed the Division of Fruit and Vegetable Storage, which played an important role in strengthening the economy of the country. Thanks to his research on the properties of various fungistatic agents, my father was able to introduce methods, still in use today, that reduced the incidence of rotting in the exported fruit to less than 2%.

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From Tel Aviv to Jerusalem Shalva, the private elementary school I attended in Tel Aviv, had an informal and friendly atmosphere. Teachers were addressed by their first name and were not very demanding. One of my classmates, Ted Arison, a good natured and rather quiet boy, used to invite us to the terrace of his flat to watch a movie projected in the nearby open-roof movie theatre. In due time, Arison accumulated a fortune in the pleasure boat business in the US. Sixty years later, I met him again when he generously donated funds towards the construction of a new Neurobiology building at the Weizmann Institute that was to eventually bear his name (Fig. 2). In 1936, we left Tel Aviv and moved to Rehovot (‘‘Hamoshava’’ ^ the colony, as it is still referred to by old-timers), a small village with about 7000 inhabitants, surrounded by orange groves,

Fig. 2 School children celebrating the wheat festival, Shavuot, on a street in Tel Aviv. Photographed by Dr. F.S. Littauer in 1930.


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21 km south of Tel Aviv. My parents built their house within walking distance of the Agricultural Experiment Station and the nearby Daniel Sieff Research Institute, which was the first building to be established in what was to become known, some twelve years later, as The Weizmann Institute of Science campus. At that time, Rehovot did not have a high school, and I had to commute to school by bus through a narrow winding road to Tel Aviv. To protect the passengers from stone throwing and occasional shooting by Arab terrorists, the bus was armored with steel plates and fenced windows.When the bus ride became too dangerous, I used to stay with my uncle in Tel Aviv. In contrast to the liberal elementary school, the ‘‘Herzlyyia’’ Gymnasium I attended was formal and demanding. Teachers had to be addressed by the students standing up, in the third person, and discipline was strictly enforced. It took me some time to adapt to the change in atmosphere. However, our teachers were among the best intellectuals, writers, and scientists who had immigrated to the country, often with PhD degrees. In particular, I enjoyed the lessons in biology, botany, physics, and soil chemistry. Many friends used to visit my parents’ home, and their learned discussions influenced my interest in science. I still remember their criticism of the theories advanced by Trofin D. Lysenko, the controversial Soviet scientist and politician who denied, among other things, the existence of genes. His vague ideas about heredity induced me to perform my first experiments in genetics. In an attempt to determine the function of chromosomes, I grew in our garden various plants belonging to the cucumber family and tried to crossbreed them. The success of these breeding attempts was then correlated with the number of chromosomes from each of the plants. In spite of my enthusiastic efforts, I failed to reach a definite conclusion. At the age of fifteen, I joined the ‘‘Haganah,’’ the self-defense organization of the Jewish community in Palestine (the ‘‘Yishuv’’). There, my friends and I received paramilitary training. We were taught to communicate with wireless instruments

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and handle a heliograph, a signaling apparatus that sends flashes of reflected sunlight from a moving mirror. On sunny days, we were very proud to establish communications with settlements as far as 30 kilometers away, by means of Morse code. These simple devices were used to transmit messages to neighboring towns without being intercepted by Arab terrorists or the British authorities. After my high-school graduation, I joined a youth movement whose members went to various kibbutzim for agricultural training. Our group of about 20 girls and boys spent a year working in Kibbutz ‘‘Ramat Yohanan,’’ near the city of Haifa. We planned to settle later in the Jordan valley and form a kibbutz of our own. The widespread feeling that cultivation of the arid land was an important pioneering mission to the fledgling country influenced the choice of my move. In 1942, the German armies, headed by Field Marshal Erwin Rommel, were advancing in North Africa eastward. It was feared that that they might soon occupy Egypt and then reach Palestine. Together with three fellows of our group, I volunteered for the ‘‘Palmach’’ (short for Peluggot Machatz, i.e. crack units), the elite fighting force of the Haganah. Their units were stationed around the country disguised as Kibbutz members and combined farming with extensive military training that included the use of weapons, explosives, and field maneuvers. Although the Palmach was an illegal military organization, the British considered it as a potential anti-German fighting force. Indeed, there was some measure of cooperation with the British army, at least as long as the Germans were advancing towards Egypt. My Palmach unit was moved to Kibbutz ‘‘Tel Yoseph,’’ in the Jezreel Valley. Instead of the usual tents, my comrades and I were housed under primitive conditions in the attic of a large cowshed. My company commander was Yitzhak Rabin, a reserved, somewhat shy person, who could on occasions become quite blunt, yet we got along very well. Years later, he was to distinguish himself during the Six-Day War as a successful Chief-of-Staff of the Israeli Defense Forces, and he eventually


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became the Prime Minister of the State of Israel. Toward the end of my service, I was assigned to Kibbutz Caesaria, where I joined the marine unit of the Palmach (the ‘‘Paliam’’). Although I had no sailing experience, I was selected for this unit because of my swimming skills. In the Paliam, we learned to navigate boats and sail in the Mediterranean Sea. The training was to prepare us to smuggle Jewish holocaust survivors from Europe. Some time later, the Paliam managed to bring about 65 boats carrying 75, 000 refugees to Palestine. The British Navy intercepted most of the boats and the refugees were deported to camps in Cyprus. They were only released in 1948 after the establishment of the State of Israel. In my last year of service, I suffered severe attacks of malaria. The doctors advised me to move to Jerusalem in the hope that the change of climate would help my recovery. I was, therefore, transferred in the fall of 1944 to the reserve unit of the Palmach in Jerusalem, a fortunate move that allowed me to study at the Hebrew University of Jerusalem. During the few vacations I had from my Palmach unit, I met and fell in love with my wife-to-be, Atida, a beautiful blue-eyed student, at a teacher’s seminar, and a native of Rehovot. Six generations ago, in 1830, Atida’s paternal forefather, Israel Buck, left Berdichev (at present a town in the Ukraine) to settle in Safad, a sacred city in the hilly region of northern Palestine. A printer by trade, he brought with him his printing machines and a group of his skilled workers. He planned to publish prayer books, which he hoped would have a special appeal as they were printed in the Holy Land. However, a severe earthquake on January 1, 1837 destroyed Safad together with his printing machines, and he was forced to move to Jerusalem, where he became one of the distinguished leaders of the Jewish community. With his son, Nissan Buck, he published a newspaper named ‘‘Havazelet’’ (The Lilly) which was the first to be printed in Modern Hebrew. Atida’s father, Achisamach, was a farmer who owned an orange grove in Rehovot. Her mother, Hanna, came to

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Palestine in 1924 as a young woman from L’vov, which was part of the Austrian^Hungarian Empire, later Poland and now in the Ukraine. We were married in Rehovot on July 12, 1946 and on March 13, 1949, we were thrilled with the birth of our first daughter, Tahel. Our second daughter, Michal, and our son, Dan, were born years later, on June 2, 1957 and March 31, 1969. We returned to Jerusalem to complete our studies, and rented a room in a house that served during the nineteenth century as a caravan-inn. Its courtyard contained a huge water-well equipped with an old, hand-operated pump. During the War of Independence, when the water supply was cut from the besieged Jerusalem, we were lucky to have the well that provided water to the whole neighborhood and ourselves.

Hebrew University and Hemed The Hebrew University of Jerusalem, the only university that existed then in the country, was founded in 1925. The campus was built on Mount Scopus overlooking the Old City of Jerusalem with its beautiful surrounding stone wall, that was erected between 1537 and 1540 by the Turkish Sultan, ‘‘Su«liman the Magnificent’’ and his famous architect, Sinan. The University’s amphitheater was a favorite meeting place for its students and on clear days, one could observe a grand view of the Judean Desert, the Jordan valley, and the Dead Sea. The student body numbered about 700 and the choice of faculties was somewhat limited. My hope was to study medicine, but the plans to open a medical school were still at the drawing board stage. I, therefore, chose to study chemistry with biochemistry and bacteriology as minor subjects, which I thought I would need later if I went into medicine. About that time, there was a determined attempt by the mandatory power to crush any action that could aid in the establishment of a Jewish State; the gates of Palestine were virtually


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closed to the immigration of Jewish holocaust survivors. This, in turn, intensified the conflict between the British and the various Jewish underground organizations. On several occasions, the Palmach reserve unit I belonged to was called upon to perform sabotage acts. One that stands out in my memory was the blowing up, between 16^17, June 1946, of the Allenby Bridge that spanned the Jordan River, connecting Palestine with Jordan. At the University, I met two remarkable scientists, Aharon Katchalsky and his brother Ephraim Katchalski (Katzir). Both brothers, then young lecturers at the Hebrew University, were known for their modern research and wide interest in biological problems, in culture, and in science policy. Their lectures were popular among the students and attracted huge crowds. Aharon and Ephraim suggested that I join a group of students that was involved in clandestine scientific work for the Haganah. They were making defense tools for impending emergencies and thought that my previous Palmach experience would be of use to them. I was also asked to give this group of students some basic military training. Later, in 1948, both brothers joined the Weizmann Institute. Aharon distinguished himself as a polymer chemist and was one of the Institute’s most eminent scientists, where he founded the Department of Polymer Chemistry. His brilliant and dynamic career ended tragically when, in 1972, he was murdered by Japanese Red Army terrorists at Ben Gurion Airport. Ephraim founded the Institute’s Department of Biophysics, and carried out outstanding work on synthetic polyamino acids as protein models. His Department was internationally acclaimed for its excellent achievements and attracted many foreign scholars. In 1973, he was elected to be the fourth President of the State of Israel, and upon the completion of his five-year presidential term he returned to research. The mounting international pressure on the British to allow the holocaust refugees to enter the country led the British to hand over the Palestine case to the United Nations. Some time later, the UN General Assembly voted on November 29, 1947, for the establishment of two separate states in Palestine, one

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Jewish and one Arab. The atmosphere in the center of Jerusalem was electric, with people listening anxiously to the results of the UN votes that were broadcast on the radios of every coffee shop along its main streets. After the results were announced, people sang and danced in the streets and the celebrations continued throughout the night. The Arabs, however, rejected the UN resolution and reacted with violent murderous attacks on the Jewish population, which soon escalated to a full-scale war. The armies of seven Arab states invaded the country, and the old city of Jerusalem was conquered by the Jordanian Arab Legion. Most of the old Jewish quarter was destroyed, including ‘‘Tiferet Israel’’ ^ the synagogue that was built in 1872 by Israel and Nissan Buck, Atida’s ancestors. The western part of Jerusalem was under siege and its population suffered from a severe shortage of water and food supply. The University was forced to close its gates and most of us volunteered for military service. On May 15, 1948, David Ben-Gurion declared the establishment of the State of Israel in the old Tel Aviv Museum. Although fighting was going on, everyone recognized the historic importance of this event. I was transferred from the Palmach reserve to ‘‘Hemed,’’ the scientific corps of the Israeli Defense Force, and appointed as the officer in charge of its chemical unit. The leading force, and one of Hemed’s commanding officers, was Aharon Katchalsky. He moved swiftly to recruit students and young university instructors to Hemed. Based on his earlier Haganah military activities, Aharon was able to assign them to a variety of projects that contributed to the defense of Jerusalem. Since the University was closed and the only road leading to Mount Scopus from the center of the city was cut off, we had to find alternative laboratory space for Hemed. I suggested contacting Professor Ernst David Bergmann, the Scientific Director of the Daniel Sieff Research Institute in Rehovot, whom I had met on previous occasions. Ernst Bergmann received me at the clubhouse of the Institute and was very sympathetic to our request. At that time, a new building adjoining the Sieff Research


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Institute had almost been completed, and Bergmann allocated its space to Hemed activities. In August 1948, during a temporary truce period, a large group of young Hemed members left Jerusalem and settled in a camp near the Weizmann Institute campus. Recruits from Tel Aviv joined them and eventually the whole building was bustling with activity. Bergmann soon became the leading figure of the scientific establishment of the Israel Defense Force, a position he held for many years. In Rehovot, under the guidance of Aharon Katchalsky, I was involved in developing solid composite rocket propellants that were based on mixtures of polymethyl methacrylate and potassium perchlorate. Ten years later, we worked out a method for calculating the specific impulses of composite propellants, which accounted well for our previous experimental data [3].

Weizmann Institute of Science Early in 1949, Hemed members were gradually transferred from Rehovot to other camps, and the scientific activities at the Weizmann Institute returned to normal. I received an extended leave of absence from the Army, which enabled me to complete my master’s thesis. In June 1949, Bergmann invited me to join him in the Weizmann Institute as his PhD student. I gladly accepted. Professor Chaim Weizmann, the first President of the State of Israel and the Zionist statesman, championed the idea of creating modern scientific centers that could help build a nation lacking natural assets. In discussions with Albert Einstein, Carl Neuberg, Richard Wilsta«tter, and other leading scientists, Weizmann developed the plans for the Daniel Sieff Research Institute in the early 1920s. The foundation of the KaiserWilhelm-Gesellschaft in Germany served as his model. The Daniel Sieff Research Institute was opened in Rehovot in 1934 and was devoted to chemical and pharmaceutical research. Weizmann’s dream of expanding the campus and building

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a modern scientific center was realized on November 2, 1949 with the inauguration of the Weizmann Institute of Science. The official opening of the Institute is still vivid in my memory. It was celebrated in a grand style typical of Meyer Weisgal, the energetic Chairman of the Board, who was the moving spirit behind the establishment of the Institute. A large crowd gathered on the lawn facing the first new building. On the stage beside Chaim Weizmann sat David Ben-Gurion, the Prime Minister, and members of the Israeli government, as well as famous scientists, donors, and other dignitaries. The ceremony was followed by a symposium that was attended by the entire scientific staff. Because there was no suitable lecture hall at the Institute, it was held at the ‘‘Beth Haam’’ hall, the cultural center of the small town of Rehovot. In the evening, the Israel Philharmonic Orchestra ended the festivities with a special concert conducted by Paul Paray and with the violinist Zino Francescatti. At the time of its inauguration, the Institute consisted of eight departments with sixty laboratories. Since then, it has expanded considerably and is a monument to Chaim Weizmann, who served as its President until his death in 1952. Ernst Bergmann served as the Institute’s Scientific Director. He was an organic chemist, a former student of Wilhelm Schlenk and coworker of Chaim Weizmann for many years [4]. Bergmann had a dynamic and brilliant personality with an encyclopedic knowledge of chemistry and a broad interest in science. He used to spend long hours in the library and distributing notes with instructions, ideas, and lists of literature references each morning. Impressed by the concept of the ‘‘energy-rich phosphate bond’’ introduced by Fritz Lipmann [5], Bergmann suggested that I try to synthesize acetyl-pyrophosphate and examine its biochemical properties. It took me several weeks until I succeeded in the synthesis; however, acetylpyrophosphate turned out to be too labile to serve as a metabolic intermediate. The only result of this study was the determination of the infrared absorption spectra of a number of inorganic and organic phosphates and pyrophosphates [6]. About ten years


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later, on December 1, 1959, a German delegation from the MaxPlanck-Gesellschaft consisting of Otto Hahn, Wolfgang Gentner, and Feodor Lynen arrived at the Weizmann Institute to explore the possibility of scientific collaboration between the two institutions. Feodor Lynen was known for his studies on the mechanism and regulation of cholesterol and fatty acid metabolism, and shared the 1964 Nobel Prize for Medicine with Konrad Bloch. During his visit to the Institute, I learned from Lynen that I was not the only one to synthesize acetyl-pyrophosphate in vain. For my doctoral dissertation, Bergmann recommended that I choose a subject close to Professor Chaim Weizmann’s scientific interests, namely the mechanism of pentose fermentation in bacteria. I conducted my research in the laboratory of Benjamin Volcani, who is known for isolating halophilic microorganisms from the Dead Sea. Until Volcani’s discovery, the Dead Sea was considered devoid of any living organism, as its name implies. In my studies, I found that the oxidation of D-xylose and L -arabinose in Escherichia coli cells are adaptive processes. Cell-free extracts obtained from D-xylose grown cells contained a D-xylose isomerase, catalyzing the reversible isomerization of D-xylose to D-xylulose, thus indicating that the first step in xylose degradation is the isomerization to D-xylulose, which is then phosphorylated. In the case of L-arabinose, a similar isomerase was present in extracts of L-arabinose grown cells, while in the case of D-ribose, no isomerization occurs, but a direct phosphorylation to ribose-5-phosphate takes place. I further showed the participation of cocarboxylase in ribose-5phosphate breakdown. I was also able to demonstrate the role of sedoheptulose-7-phosphate in the metabolism of pentoses and glucose in these cells. The results of these experiments suggested that the pentose phosphate molecule is split by transketolase into triose-phosphate and an activated C2 compound, which is presumably bound to thiamine-pyrophosphate [7,8]. While I was in the advanced stages of my doctoral work, Sol Spiegelman came to Rehovot to consider an offer to join the Weizmann Institute. He suggested that for my postdoctoral

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studies I contact Arthur Kornberg, Head of the Department of Microbiology of the Washington University School of Medicine, in St. Louis, Missouri. Kornberg had just moved from the National Institutes of Health (NIH) and was recruiting people for the new Department. I was greatly impressed by his recent publications on coenzyme and nucleotide synthesis, and decided to write and ask if he would accept me as a postdoctoral fellow. Spiegelman offered to talk to Kornberg on my behalf, and on the strength of his recommendation, I was fortunate to be accepted and to receive a fellowship from the Dazian Foundation. The time I spent in association with Arthur Kornberg, I consider as a most influential, unusually rich, and satisfying experience.

St. Louis: Polynucleotide Phosphorylase In March 1955, we settled in St. Louis, Missouri. The summers were too hot even for us Israelis. In our small apartment, we installed a window fan that blew out the hot air to blow in hot air. When it became unbearable, we found refuge in an airconditioned movie theatre that was close-by. Occasionally, Robert Lehman, a friend and postdoctoral fellow with Arthur Kornberg, would join us there. Compared to the sparsely supplied Weizmann Institute, the Department of Microbiology in St. Louis was well equipped and efficiently run. Arthur Kornberg attracted a number of excellent, enthusiastic young scientists to the Department, including Paul Berg, Maurice Bessman, Melvin Cohn, Irving Craword, Robert De Mars, Jose Fernandez, David Hogness, Jerard Hurwitz, Sylvy Kornberg, Robert Lehman, Irving Lieberman, Jim Ofengand, Ernie Simms, Herbert Wiesmeyer, and Philip Varney. In keeping with a tradition established at the NIH, there was a daily lunchtime journal club. Its informal discussions were critical and lively and often continued in the corridor and laboratories. A short time after my arrival in St. Louis, during a party at Kornberg’s house, I was introduced to Carl Cori and Gerty Cori,


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the 1947 Nobel Laureates for Physiology or Medicine, known for their work on the catalytic conversion of glycogen. When Gerty Cori learned that I was an Israeli, she started a conversation on the political problems of the Middle East, and I was greatly impressed by her knowledge of the history of the region. One of her favorite subjects was the Hittite culture, whose empire in the 2nd millennium B.C. rivaled that of the Babylonian and Egyptian. Carl Cori invited me to attend the Department of Biochemistry weekly seminar that attracted considerable attention among the St. Louis scientific community. The atmosphere of the seminars was formal and the lectures ended with everyone waiting for Gerty Cori to start the discussion. At Kornberg’s house, I also met Rita Levi-Montalcini, the 1986 Nobel Laureate for Physiology or Medicine.We had long discussions on the nature of the nerve growth factor she had just discovered. Years later, in 1978, we renewed our acquaintance when she came to the Weizmann Institute to receive an Honorary Doctorate. Arthur Kornberg’s interests centered on certain aspects of nucleotide metabolism leading to nucleic acid synthesis. He suggested that I try to construct a cell-free system that would catalyze the synthesis of RNA. As substrate, I used 14C-labeled ATP, which I had to synthesize myself from 14C-labeled adenine by a series of enzymatic reactions, since commercially labeled nucleotides were not available then. Within a short time, I was able to show that cell-free extracts from E. coli cells can convert 14 C-labeled ATP to an acid-insoluble product, which I presumed to be a polyribonucleotide. Moreover, the addition of adenylate kinase (myokinase) to the system increased the rate of the reaction [9]. Although the activity was barely detectable, Kornberg suggested that I attempt to characterize the polynucleotidesynthesizing system. While making rapid progress in purifying the E. coli enzyme, we learned from Herman Kalckar, who came for a visit, that Marianne Grunberg-Manago and Severo Ochoa at New York University had independently discovered an activity similar to ours in extracts of Azotobacter vinelandii (A. agilis). The enzyme was named polynucleotide phosphorylase

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(PNPase), and was shown to catalyse the conversion of nucleoside diphosphates into long polyribonucleotides, with the release of free phosphate. Although we were disappointed by the news of their findings, we decided to continue with our studies with the purified E. coli enzyme. Acting on this new information, we shifted to using ADP rather than ATP and found it to be the preferred substrate in our system [10]. PNPase was the first enzyme to be discovered that catalyzes the synthesis of polyribonucleotides with a 30, 50 -phosphodiester bond. The reaction is reversible and, in the presence of phosphate ions, PNPase is a processive 30 !50 exoribonuclease that catalyzes the phosphorolysis of polyribonucleotides, liberating nucleoside diphosphates. At that early stage, we pointed out that it is not clear how the specific nucleotide composition of the RNA of a given species is produced by an enzyme that appears to polymerize the available nucleoside diphosphates in a random fashion [10]. Subsequent research in other laboratories showed that the cellular function of the enzyme is to degrade RNA and that RNA synthesis is catalyzed by DNA-directed RNA polymerase. PNPase has been the subject of numerous studies; it was employed as a tool for producing model nucleic acids and solving many important biological problems. Thus, establishing the genetic code was facilitated by the ability of PNPase to synthesize heteropolymers and triplet nucleotides. The advances made in the understanding of the physicochemical properties of polynucleotide chains and their hybridization reactions, as well as the synthesis of polynucleotide inducers of interferon, are further examples of the role played by the enzyme. Interest in PNPase was renewed by the finding that mRNA degradation in E. coli cells involves a multiprotein complex (named the RNA degradosome), that consists among others, of PNPase, RNase E, and DEAD-box RNA helicase. Recently, I was glad to have the opportunity to publish a review, together with Marianne Grunberg-Manago, that summarizes the voluminous studies on this enzyme that have accumulated throughout the years [11].


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During our stay in St. Louis, our daughter, Tali, developed a serious kidney disease and had to be hospitalized. At that time, St. Louis Children’s Hospital pioneered the use of steroids; a treatment that we believe saved her life. The loving care of the attending physicians and the sympathy and help of Sylvy and Arthur Kornberg were a great comfort to us. Arthur Kornberg continued his brilliant research on the enzymatic synthesis of DNA and shared with Severo Ochoa the 1959 Nobel Prize for Physiology or Medicine.

Isolation of High Molecular Weight Ribosomal RNA Toward the end of my stay in St. Louis, Alex Rich and Leon Heppel invited me to the NIH to acquaint them with PNPase. Heppel was very generous in providing me with samples of his RNA collection, which I intended to use upon my return to the Weizmann Institute. Back in Rehovot, I was faced with a problem: although the enzyme readily catalyzed the phosphorolysis of synthetic oligoribonucleotides and TMV RNA, all the other RNA samples had not been affected by the enzyme. I suspected that the RNA samples were degraded products that were resistant to phosphorolysis. I decided to try to improve on the existing methods for RNA isolation. My attempts to extract intact RNA from ribosomes yielded somewhat degraded preparations, while direct extraction of E. coli cells with a phenol^water mixture resulted in RNA preparations that were heavily contaminated with polysaccharides. Eventually, I devised a method for isolating undegraded RNA from E. coli protoplasts [12].This method yielded excellent RNA preparations, with sedimentation constants of 4.1S, 16.5S, and 23.7S. The two high-molecular-weight RNA components were separated from the low-molecular-weight fraction by ammonium sulphate precipitation, and later turned out to be derived from ribosomes. This was not a trivial finding as a number of investigators considered ribosomal RNA (rRNA) as an aggregate of short

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polyribonucleotides (see below). My method proved of general use for the preparation of undegraded rRNA from bacteria. Encouraged by the success with bacterial rRNA, we demonstrated that rRNA from animal ribosomes consisted of two high-molecular-weight species with sedimentation values of 18S and 28S. Together with one of my PhD students, Yosef Kimhi (now, Vice President of Scientific Affairs, Yeda Co.), we found, as expected, that all the high-molecular-weight rRNA preparations were readily phosphorolyzed by PNPase. In contrast, transfer RNA (tRNA) preparations were attacked more slowly and to a limited extent (20^30%). Urea was found to increase the degree of breakdown of tRNA, indicating that the secondary structure of tRNA hinders the enzymatic attack. PNPase was also employed for the preparation of a variety of labeled nucleoside di- and triphosphates [13^15]. In May 1957, the Section of Biochemistry was founded at the Weizmann Institute. It consisted originally of four groups, with Theodore (Ted) Winnick, formerly of Iowa State College, as the head. His group studied protein and peptide biosynthesis. Another group, led by the late David Elson (a former student of Erwin Chargaff ), dealt with the structure and function of ribonucleoproteins. My own group examined the enzymatic synthesis of RNA, its physicochemical characterization, and its relation to protein synthesis. A year later, we were joined by the late Mordhay Avron, whose interests centered on the mechanism of photosynthesis. In 1961, Ted returned to the USA. Soon after his departure, the three of us were the first to introduce at the Institute, a system of rotating Department Chairmanship.

The Single-Stranded Nature of rRNA Having at hand high-molecular-weight rRNA preparations, it was only logical to approach my good friend and colleague, Heini Eisenberg from the Polymer Department, and suggest that we collaborate in an attempt to determine their physical


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properties. Heini had already distinguished himself in the study of solution properties of DNA and agreed immediately to my proposal. It should be noted that at that time, the structure of RNA in solution, unlike that of DNA, was unknown, mainly because of the lack of undegraded RNA preparations. We were surprised to find that on increasing the ionic strength of the high-molecular-weight E. coli rRNA solution, the limiting viscosity decreased about 100-fold. Similar results were obtained with rat liver RNA. Thus, rRNA behaves quite unlike DNA, where the limiting viscosity shows a much smaller dependence upon ionic strength. We proposed, therefore, that rRNA behaves as a flexible, contractile single stranded coil [12]. In support of this assumption, birefringence of flow measurements showed that rRNA in aqueous solution gave moderate positive values, which disappeared upon addition of salt. This is in contrast with DNA solutions, where considerable negative birefringence persists even in the presence of 2 M NaCl. The positive birefringence observed for rRNA could be compared with the measurements of Rosalind Franklin, who found a positive contribution to birefringence of flow in TMV suspension, which disappeared upon removal of the RNA constituents from the virus particle. Thus, our observations gave a clear indication of the considerable difference between DNA and RNA structure [16]. In September 1958, I presented our results at the Fourth International Congress of Biochemistry in Vienna [17]. Our conclusion that rRNA is a contractile single-stranded coil was well received. After the lecture, I met with Francis Crick, Paul Doty, Alex Rich, and Vittorio Luzzati for lunch. Doty told me that they also had evidence that RNA is made of single-stranded chains. He believed that rather than being a continuous chain, rRNA is an aggregate formed by a number of short fibers that are dissociated upon heating or urea treatment [18,19]. I felt, however, that the rRNA preparations Doty used were degraded. The correlation between the sedimentation velocity and viscosity data that we had found for rRNA at different ionic strength concentrations supported the notion that each ribosomal

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subunit contained a single continuous uninterrupted RNA strand. At our lunch meeting, Crick made the interesting suggestion that the RNA fiber can, under certain circumstances, fold onto itself and give secondary structure characteristics, which might explain the reversible curve obtained in our potentiometric titration measurements. By 1959, our work had been published in detail, as had that of Doty and his collaborators, and the single-stranded nature of RNA in solution was established. After the Congress, I visited Martin R. Pollock at the National Institute for Medical Research, Mill Hill.We discussed the claim of M. Kramer of the University of Budapest that penicillinase could be formed de novo in Bacillus cereus cells, following a suitable treatment with an RNA fraction isolated from constitutive penicillinase-producing cells. On my return to Rehovot, I extracted RNA from the B. cereus cells and mailed the preparation to Pollock. However, our hope to find a biological assay for RNA did not materialize. At Mill Hill I also met Alick Isaacs and was greatly impressed by his discovery of interferon. In April 1959, I was the first scientist at the Institute to receive an NIH grant. The NIH kept its support in various forms for a period of fourteen years and was a tremendous help to our studies.

Early Studies on the Secondary Structure of RNA In collaboration with Robert Cox (now at the National Institute for Medical Research, Mill Hill), the changes in rRNA configuration brought about by the addition of electrolytes were examined further. Robert, then a visiting scientist, arrived in 1958 from Arthur Peacocke’s laboratory at Birmingham; his previous experience with potentiometric titration measurements of DNA was of great help to our RNA studies. We noted that the abrupt fall in viscosity upon increasing the concentration of sodium chloride was greater than that found for simple polyelectrolytes [20]. Electron microscopical studies in collaboration


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with David Danon, from the Institute’s Section of Biological Ultrastructure, also supported the notion that rRNA is a molecule capable of a great degree of coiling [21,22]. The close correlation of the ionic strength dependencies of optical rotation, optical density, and hydrodynamic properties, indicated that rRNA as well as tRNA possess a significant secondary structure [14,15,20,23,24]. Similar evidence that TMV RNA and rRNA are constrained by intramolecular bonds was obtained in the laboratories of Alfred Gierer, Paul Doty, Helga Boedtker, Aleksandr Sergeevich Spirin, and their colleagues. From all of these observations it was estimated that, in dilute salt solutions, RNA has a partial (40^60%) helical structure. Furthermore, the RNA molecule was considered as a composite of short DNA-like rigid helical regions, connected by flexible single-stranded sections. To explain how half the bases in RNA could fit into helical regions, Jacques Fresco, Bruce Alberts, and Paul Doty proposed that the double helical regions are not perfect helices, but contain looped out residues. The predominant element of RNA secondary structure is the hairpin that is formed when a local region of polynucleotide chain folds back on itself to form a short, intramolecular base-paired helix called the stem. This model is still the basis of our understanding of the secondary structure of RNA (for reviews see Refs. 19, 25, 26). We have extended our studies of the structure of rRNA and tRNA from various sources with similar results. Together with Mordhay Avron, we noticed that RNA preparations from Swiss chard chloroplasts contain some DNA (cf. [14]). Regrettably, we did not follow up this lead to establish the presence of a unique chloroplast DNA. Several years later we demonstrated, together with my graduate student, Inder M. Verma (now a Professor and leading molecular biologist at the Salk Institute) and Marvin Edelman (then a visiting scientist from Harvard Medical School), the presence of rRNA in mitochondria from several fungal species. We also showed that the mitochondrial rRNA possesses a unique ordered structure that differs from that of the homologous cytoplasmic rRNA. At this stage,

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Cyril Kay, a visiting scientist from the University of Alberta, Edmonton joined us in our efforts. Circular dichroism measurements and optical absorbency properties suggested that both base pairing in helical regions, as well as base stacking, play a role in stabilizing mitochondrial rRNA and cytoplasmic rRNA structure, and indeed suggested that the two forces are not independent of one another. Our results indicated that the forces which contribute to the ordered structure of mitochondrial rRNA resemble those operative in other ribosomal RNAs. In the absolute conformation, however, the unique nature of mitochondrial rRNA becomes apparent [27^30]. These were the last physicochemical experiments that we carried out with RNA. Inder finished his PhD thesis in record time and left to join David Baltimore’s laboratory at MIT, and Marvin joined the Weizmann Institute (where he is now Professor of Agricultural Molecular Biology). The elucidation of the primary structure of tRNAAla by Robert Holley and his colleagues, and of tRNASer by Hans Zachau and his colleagues, marked the rapid progress made in the understanding of tRNA structure. Their studies provided the basis for the ‘‘cloverleaf’’ model in which about half the residues are base-paired. By 1969, it became clear that the ‘‘cloverleaf’’ pattern could be fitted to several other tRNA sequences. In the late 1970s, comparative sequence analysis of rRNA led to its secondary-structure models as well. It was obvious at that time that, in addition to the secondary structure of RNA, tertiary interactions must also take place. Current understanding of tertiary RNA folding is based on X-ray analysis of several tRNA species and ribosomes. These studies showed that many interactions which maintain RNA tertiary structure are of a novel type, such as base triples and interactions between bases and the ribose-phosphate backbone itself, as well as the ability to form pseudoknots. In addition, most of the bases are stacked playing a major role in stabilizing the architecture of the molecule (for a recent review see Ref. [26]).


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tRNA Regulation The lack of functional assays for rRNA stimulated our interest in mechanisms that govern the regulation of tRNA and mRNA activity. We, therefore, devised methods for the purification of specific tRNA species, as well as examined the in vitro transcription of tRNA, the processing of precursor molecules, and posttranscriptional modification of tRNA chains [31].

tRNA Nucleotidyl Transferase The late Violet Daniel, my first graduate student (who later went on to become a Weizmann Institute Professor of Biochemistry), joined my laboratory in 1957. She isolated and purified tRNA nucleotidyltransferase, an enzyme that incorporates AMP and CMP residues into tRNA molecules lacking all or part of the 30 -CCA terminus [32]. The enzyme has an important role in the synthesis, repair, and proofreading of tRNA. Hans Bloemendal, a research scientist at the Department of Biochemistry, Netherlands Cancer Institute (now a Professor of Biochemistry at the University of Nijmegen), came to my laboratory in 1960 for a short visit and collaborated with Violet in the initial phase of these studies. In addition to being an excellent scientist, it turned out that Bloemendal’s visit caused quite a stir in the press, as he was a famous cantor in the Jewish synagogue of Amsterdam and his recordings had a wide circulation. Ten years later, Jacov Tal, a graduate student (now Head of the Virology Department at Ben Gurion University) and Murray P. Deutscher, a visiting scientist from the University of Connecticut, demonstrated that tRNA nucleotidyltransferase adds CMP to tRNA N by a nonprocessive mechanism. They improved the conditions for the stepwise degradation of 30 -terminal nucleotides and were able to show that, contrary to our earlier observations, tRNA NCA had essentially no amino acid acceptor activity. [33].

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In August 1960, Leendert Bosch and Hans Bloemendal invited me to participate in a symposium on Protein Biosynthesis held in ‘‘De Pauwhof,’’ a recreation center for artists and intellectuals in Wassenaar, The Netherlands [14]. The quiet atmosphere of the estate created a favorable climate for personal contacts and informal discussions. Nobel laureate Fritz Lipmann from the Rockefeller Institute, New York, opened the meeting. To the astonishment of the organizers, he suggested, with a smile, that we might save time by skipping the greetings of all the dignitaries and start with the lectures directly. The meeting marked the rapid progress that had been made in understanding the mechanism of protein synthesis and the involvement of tRNA and ribosomes in this process. Lipmann reported on the work of Harold Bates, who claimed to synthesize glutathione from glutamylcysteine and glycine in pigeon liver extracts with the participation of a soluble RNA fraction. Furthermore, oxidation of a solution containing GluCySH-RNA resulted in its precipitation in a pure form. The results caused quite a sensation among all of us. Unfortunately, all these claims were subsequently withdrawn. Surprisingly, the most exciting news, the possible existence of mRNA, or ‘‘tape RNA’’ as it was then called, arose only in informal private discussions.

The IUB Congress in Moscow The fifth IUB Congress of Biochemistry, held in Moscow on 10^16 August 1961, was exceptional in several respects. The Soviet Union was celebrating the successful orbit in space of its second astronaut, Gherman Titov, with a grandeur parade in the Red Square. I was invited to a lavish reception in his honor and to a press conference, that emphasized the superiority of the Soviet space technology. Who could predict then the disintegration of this superpower several decades later? The scientific highlight of the meeting was the report by Marshall W. Nirenberg and Johan Matthaei, that poly(U) served


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as a template for the synthesis of polyphenylalanine in a cellfree protein synthesizing system. Nirenberg’s unassuming lecture was given in a small hall and for the few of us who attended his talk, it was a complete surprise. The importance of his discovery, which led to the elucidation of the genetic code, was apparent. The news that UUU, the first word of the genetic code had been cracked was immediately related to Francis Crick. In a dramatic move, he invited Nirenberg to repeat his lecture the next morning in one of the main symposia before a packed audience. Some of the Russian colleagues I met remarked that this was the first time that genes were allowed to be mentioned in public in the USSR. By 1965, the elucidation of the genetic code was completed and a new era in biology had begun.

Purification of tRNA A related topic, with which I was occupied at the time, was the purification of amino acid-specific tRNA species. In collaboration with Ephraim Katchalski (Katzir), we developed a general chemical method for the purification of tRNA species, based on the ability of the primary amine group of aminoacyl-tRNA to initiate N-carboxy--amino acid anhydride polymerization. Together with Saul Yankofsky, Abraham Novogrodsky, and my graduate student Shulamith Simon, we invested considerable efforts in these studies. In brief, the method involved the enzymatic esterification of tRNA with a desired amino acid. The resulting aminoacyl-tRNA was then separated from the bulk of unesterified tRNA by reacting this mixture with N-carboxy-benzyl-L-aspartate anhydride to yield the corresponding (-benzyl-L-aspartyl)n-aminoacyl-tRNA. The polypeptidyl-tRNA precipitates out from the reaction mixture, whereas uncharged tRNA chains, which do not react with N-carboxy--benzyl-Laspartate anhydride, remain in solution. The polypeptidyl ester was then hydrolyzed by pronase to liberate a purified tRNA

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preparation. In the case of tRNAVal about 70% of the expected amino acid acceptor function was obtained [34^36]. Robert Stern, who came to my laboratory from the NIH (now Professor at the Department of Pathology, University of California, San Francisco), developed an alternative method for the purification of amino acid specific tRNA species. It involved the attachment of an aromatic substituent onto the amino group of aminoacyl-tRNA. The substituted aminoacyltRNA shows increased affinity for methylated albumin-silicic acid (MASA) columns, thus allowing it to be separated from the unsubstituted tRNA. However, other and more efficient methods for purification of tRNA were soon developed in the laboratories of A. D. Kelmers and I. Gillam and their colleagues. They employed chromatography on reversed-phase columns or benzoylated DEAE-cellulose columns. For analytical purposes, Robert used MASA columns, as they provided better resolution of aminoacyl-tRNAs than chromatography on methylated albumin kieselguhr columns, which were in common use at that time. The MASA column also allowed us to distinguish between uncharged, aminoacylated and N-blocked-aminoacyl-tRNAs, probably based on differences in their conformation [37^39]. The MASA column was also used by Phil Leder (a visiting scientist from the NIH) to separate N-formyl-met-tRNA from met-tRNA [40].

The Function of Modified Nucleosides in tRNA In the spring of 1962, I took off for a sabbatical in the Department of Biochemistry at Stanford School of Medicine. My host was Nobel Laureate Paul Berg, a friend since our stay with Arthur Kornberg in St. Louis. Together with Karl Muench, we decided to study the function of methylated nucleosides in tRNA, as a striking feature of tRNA is its high content of modified nucleosides. Many of the unusual bases are methylated derivatives of the four common bases, and the methyl group of


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methionine is the precursor of these methyl groups. An experimental approach to the function of methylated bases in tRNA was provided by the finding of Ernest Borek from the College of Physicians and Surgeons, Columbia University, that methyldeficient tRNA is synthesized by a‘‘relaxed’’ methionine requiring mutant of E.coli grown in the absence of methionine. We demonstrated that the methyl groups are unessential for amino acid acceptor activity of tRNA. In the winter of 1962, I moved to the laboratory of Nobel Laureate James Watson in the Biological Laboratories of Harvard University, to examine the participation of methyl-deficient tRNA in the transfer reaction using polyuridylate-directed synthesis of polyphenylalanine. Both methylated and methyl-deficient tRNA were found to participate in the transfer reaction. In my laboratory, Michel Revel (now Professor of Virology at the Weizmann Institute) used methylated albumin-kieselguhr (MAK) column chromatography to separate methyl-deficient from normal tRNAPhe. However, the tRNA recovered from the column fractions contained traces of MAK as a contaminant that interfered with the assays of biological activity of tRNA. Later, Robert Stern continued these studies in collaboration with Fabio Gonano (Naples) and Erwin Fleissner (New York). To separate the methyl-deficient from the normal tRNAPhe species, they used MASA columns or fractionation by countercurrent distribution. The recovered tRNA from the separated fractions was found free of interfering contamination. Ribosome binding assays showed that the methyl-deficient phenylalanyl-tRNA was less efficient than normal phenylalanyltRNA in binding to poly U or poly UC. On the other hand, in the in vitro hemoglobin-synthesizing system, the tryptic peptides derived from normal and methyl-deficient phenylalanyl-tRNA were indistinguishable [41]. In addition, Ruth Milbauer (a Research Assistant) found that reduced transfer activity is not confined to tRNA synthesized in a ‘‘relaxed’’ strain during amino acid starvation, but can also be produced in cells, where no new tRNA is synthesized during amino acid starvation. It was concluded that most of the methylated bases in tRNA are not likely

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to be essential for viability but, depending on the type of base modification and position along the tRNA chain, may play a role in the fine-tuning of tRNA activity. Together with the late Hiroshi Inouye, and with Sara Fuchs and Michael Sela from the Department of Chemical Immunology, we developed specific immunological methods for detecting tRNA species containing inosine [42]. In other studies, affinity chromatography on columns of anti-Y antibodies allowed Raphael Salomon (a Senior Scientist), Sara Fuchs, and the late Aharon Aharonov to identify and purify isoaccepting tRNAPhe species containing the highly modified tricyclic imidazopurine (Y-base) [43]. A similar immunological method was put to use by Salomon and Kimhi to show that tRNAPhe from mouse neuroblastoma cells lacks the peroxy Y-base that is present in normal mammalian tRNAPhe. Thus, in this respect, mouse neuroblastoma cells differ markedly from normal brain cells [44]. David Shugar from the Polish Academy of Sciences Institute of Biochemistry and Biophysics, Warsaw, visited our laboratory in the summer of 1965. He was interested in the possible polymerization of Et2UDP and dihyroUDP by PNPase. I promised to try to examine whether they could serve as substrates for the enzyme. Later, he asked me to participate in a symposium on Properties and Function of Genetic Elements at the 3rd FEBS meeting held in April 1966 in Warsaw. The meeting took place at the Palace of Culture and Science, a huge ugly, high-rise building, typical of neoclassical Stalinist architecture. I took some purified PNPase with me as well as a collection of nucleotides that were not available in Shugar’s laboratory. At the airport, I was met and whisked through the lengthy line of people waiting for passport control and spent a pleasant evening at the home of Grace and David Shugar together with Severo Ochoa and Beverly Griffin. I was amazed to find a person of Shugar’s stature, highly regarded by authorities and the head of a scientific institute, living in a humble and sparsely furnished apartment. On the eve of Passover, the Israeli Ambassador to Poland, Mr. Dov Sattath invited me to a ‘‘Seder’’at his home. He told me that


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he had only few contacts with the Polish government and felt isolated in Warsaw. Later at a cocktail party given by the Polish Vice Prime Minister, Eugenius Szer, I tried to convey the Ambassador’s feeling of isolation and was assured that a meeting would be arranged. At the ‘‘Seder,’’ I met the famous Yiddish actress Ida Kaminska and went later to her performance at the Yiddish Theatre. Most of the actors were not Jewish and memorized the Yiddish text by heart. The theatre was the only remains of the rich and abundant Jewish cultural life in Poland. On my return trip from Warsaw, the office of Austrian Airlines informed Phil Leder and me that we had been moved to a waiting list, as the plane was overbooked with an unknown number of passengers arriving from Moscow.We were somewhat distressed, as the next flight was scheduled to leave two days later. We finally managed to board the plane, thanks to a highranking officer we had met two days earlier at a party in Warsaw.

Phage-induced tRNA Early studies of RNA synthesis in E. coli cells showed that after infection with a T-even bacteriophage, there was a rapid incorporation of 32P-orthophosphate into RNA. While most of the labeled RNA synthesized after infection was phage-specific mRNA, it was noticed by Sol Spiegelman and his colleagues that some eight percent were found as a low molecular weight RNA fraction. Because of this finding, we attempted to characterize this 4 S RNA fraction and determine whether it has amino acid acceptance activity. Together with Violet Daniel and Sara Sarid (from the Department of Biophysics), we isolated the 4S RNA from T4 phage-infected E. coli cells. The presence of pseudouridylic acid in the T4 phage-coded 4S RNA fraction led us to suggest that it contained tRNA [45]. Later, we developed two methods that enabled us to reveal the presence of tRNA specified by the T4 genome. In the first procedure, biologically

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active tRNA was isolated from its hybrid with T4 DNA [46]. In the second, the tRNA isolated from the T4-infected cells was charged with a given-labeled amino acid, then hybridized with T4 DNA under conditions that kept the aminoacyl-tRNA ester bond intact. This novel method that we developed allowed us to monitor the hybridization of individually labeled aminoacylated tRNA species with DNA. To preserve the aminoacyl-tRNA ester bond, the aminoacyl-tRNA was acylated with N-hydroxysuccinimide acetyl ester to yield N-acetylaminoacyl-tRNA. We synthesized the latter, since we have previously shown that N-substituted aminoacyl-tRNA derivatives are considerably more resistant to hydrolysis than the corresponding unblocked aminoacyl-tRNA derivatives. Using N-acetyl[3H]aminoacyltRNA molecules, we were able to reveal the presence of several T4phage-codedtRNA species [47]. Our discovery was well received during an EMBO workshop on tRNA that was organized by Sydney Brenner in Cambridge in March 1969. During the meeting, I was particularly impressed by the work of John Smith and his colleagues who had isolated a series of mutants of the su3 locus. These included mutants with reduced suppressor activity, some of which appeared to affect the rate of maturation of tRNATyr, resulting in the accumulation of precursor molecules.

Isolation of tRNA Genes In another project that involved Violet Daniel, Jacques S. Beckmann, Sara Sarid, Jacob I. Grimberg, and Max Herzberg, we were the first to isolate a tRNA gene. We used the phage Tyr gene with its promoter^ 80suþ 3 for the isolation of a tRNA operator region extant. The purification procedure made use of two specialized su3 transducing phages carrying an E. coli tRNATyr gene inserted into their DNA in opposite orientation. The separated heavy strands of the two phages were annealed and the single-stranded tails of the resulting hybrid were removed by digestion with Neurospora endonuclease. The


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isolated duplex then served as a template for the in vitro transcription of tRNATyr-like molecules [48]. With present-day techniques, isolation of a tRNA gene is a relatively simple project; at the time, however, it required considerable efforts on our part. After completing his graduate studies, Jacques Beckmann headed off to France, where he became Associate Director of the French National Genotyping Center. He is now a Professor at the Weizmann Institute Human Genome Center. During these early days, the knowledge of nucleic acids was quite rudimentary among Israeli scientists, as it was hardly taught at the Universities. I decided, therefore, to introduce the subject to our graduate students by giving an extensive lecture course on the chemistry of DNA and RNA, and a more advanced one on molecular biology. In addition, together with Yosef Kimhi, we wrote two detailed articles in Hebrew on nucleic acids and the genetic code in Mada, a popular science magazine that was edited by my good friend Nathan Sharon [49]. The Mada articles became quite popular and were put to use as mandatory reading material by university students.

PNPase Research Applications Monofunctional Substrates of PNPase Gabriel Kaufmann, then a graduate student (now Professor and Head of the Department of Biochemistry at Tel Aviv University), studied the substrate specificity of PNPase. The enzyme was found to direct the reversible addition of a single deoxynucleotidyl residue to ribooligonucleotide primers, while further addition of oligodeoxynucleotide residues to the resulting product was very sluggish [50]. The enzyme was also found to phosphorolyze aminoacyl-tRNAs, thereby yielding aminoacyl-ADP and nucleoside diphosphates [51]. These findings prompted us to investigate the properties of ribonucleoside diphosphate analogs modified in their sugar moiety as substrates for this enzyme. We suggested

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that blocking of NDPs at the 30 -hydroxyl-function would yield ‘‘monofunctional’’ substrates, of which only one residue may be added to an oligonucleotide primer, thus serving as chain terminators.Together with Matityahu Fridkin from the Department of Organic Chemistry, we synthesized a series of 20 (30)-O-isovalerylesters of NDPs that fulfilled these properties.The blocking group was subsequently removed by mild alkaline hydrolysis from the oligonucleotide products, permitting a succession of single addition reactions. This procedure was employed for the stepwise synthesis of polyribonucleotides of defined sequence [52,53]. Combinations of these reactions and using T4 RNA ligase to ligate the synthesized oligonucleotides allowed the synthesis of appreciable long oligonucleotides. The use of T4 RNA ligase was further adapted by Kaufmann for unique sequence insertions and alterations in tRNA anticodon loops [54].

The Tale of the Poly(A) Tail The discovery that the majority of eukaryotic mRNA species terminate in a poly(A) tract at their 30 -end, has stimulated many speculations as to its function. The search for the role of the poly(A) tail of eukaryotic mRNA started as a straightforward project, and as it continued in many laboratories, unraveled a multitude of mechanisms that control the functional stability of individual mRNA species, both in the nucleus and in the cytoplasm. We were in a good position to study the cytoplasmic role of this poly(A) sequence. At about that time, Hermona (Mona) Soreq, then a graduate student (now Professor and Head of the Institute of Life Sciences at the Hebrew University of Jerusalem) succeeded in purifying E. coli PNPase to homogeneity. The purified enzyme was virtually free of contaminating nucleases, which allowed us to develop a specific method for the removal of the poly(A) tail from mRNAs. The method is based on the processive phosphorolysis of the poly(A) tail using molar excess of PNPase at 0 C, conditions


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that leave the rest of the mRNA molecule intact. Together with Uri Nudel, Raphael Salomon, and Michel Revel, we found that globin poly(A)-free mRNA can still be translated in a Krebs ascites tumor cell-free extract (the nuclease-treated reticulocyte lysate cell-free system was not yet developed). We also observed that at longer periods of incubation, the rate of globin synthesis appeared to level off more sharply with deadenylated mRNA than with native mRNA [55,56]. The in vitro systems survive no longer than 2 h and are inadequate for detection of long-term effects of the poly(A) tail on mRNA stability. We, therefore, turned our attention to the use of Xenopus laevis oocytes, and were fortunate to collaborate with Georges Huez and Ge¤rard Marbaix from the University of Brussels, who were experts in the use of this relatively new system. To examine the functional stability (ability to be translated) of the mRNA, Mona took the deadenylated mRNA samples to Brussels and microinjected them into the oocytes. The results showed that the rate of globin synthesis with poly(A)-free mRNA is considerably lower than with native mRNA, and this difference became more pronounced at longer periods of incubation [57]. In subsequent experiments, we were able to show that readenylation of poly(A)-free globin mRNA restores its functional stability [58]. In retrospect, we were more than fortunate in choosing globin mRNA for our studies, as there are examples where the removal of poly(A) tracts from some other mRNA species does not affect their stability (reviewed in [19,59]). The functional stability of globin mRNA with poly(A) tracts of varying size was examined by Uri Nudel and Mona Soreq in Xenopus oocytes. Molecules with 32 or more adenylate residues had equivalent functional stability, whereas those with less than 32 adenylate residues were tenfold less stable. We suggested that a minimal size of poly(A) segment is essential for binding to poly(A) binding proteins (PABP), thereby protecting the mRNA from nucleolytic degradation [60].This suggestion correlates well with the observation of Roger Kornberg that the minimal length of the poly(A) tail necessary for PABP binding is 27^30 residues

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[61]. In order to account for the great variability among mRNA species, we proposed that the 30 -untranslated region (30 -UTR) can modulate the affinity of PABP for the poly(A) segment, thus permitting control of the poly(A) stability in individual mRNA species [19]. The injected oocyte system was used several years later by Irith Ginzburg and Pierre Cornelis (an EMBO Fellow from the Catholic University of Louvain) to show that treatment of rats with ethionine, a hepatocarcinogen, causes severe impairment in the aminoacylation capacity of tRNA and its participation in protein synthesis [62]. Renewed interest in the regulation of mRNA stability came about with the observation that c-fos and c-myc mRNAs turn over rapidly in cells. Thus, it has been suggested that the AUrich sequence contained in the 30 -UTR of c-fos mRNA, accelerates deadenylation of the poly(A) tail and mRNA degradation (reviewed in [63]). It also appears that the interaction between the poly(A) tail^PABP complex and cap-associated initiation factors may be important in maintaining the physical integrity of an mRNA, as it is in promoting efficient translation initiation (reviewed in [64,65]). Thus, there is a multitude of systems that use the poly(A) tract to control the expressions of specific mRNA species. The number of cis-acting elements and transacting factors regulating turnover of mRNA is increasing rapidly, and the complexity of these processes grows in parallel. We also used the 30 -exonucleolytic activity of PNPase to analyze the size of the poly(A) tails from various mRNA and viral RNA species [55,60,66,67] as well as for the sequence analysis of short oligoribonucleotides [53]. Additional applications of the enzyme developed by Yosef Kimhi and Mordhay Avron were for the radiolabeling of NDPs and NTPs at their -position [68]. Plant Viral RNA RNA obtained from various plant viruses such as TYMV, TMV, and BMV, are terminated with tRNA-like structures, which are


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recognized by tRNA-specific enzymes such as aminoacyl-tRNA synthetases. Sequence analysis of the 30 -terminal region of the viral RNAs showed that the cloverleaf structure, as found in tRNA, appeared to be absent. However, it was shown by C. W. Pleij from Leiden University that three-dimensional folding of the 30 -domain based on pseudoknotting, imposes tRNA-like mimicry that shows a great resemblance to the L-shape configuration of tRNA, and thus could explain the amino acid acceptance capacity of these plant RNA viruses [26,69]. In support of this theory, Raphael Salomon, Hermona Soreq, and colleagues utilized a highly purified nuclease-free histidyl-tRNA synthetase to determine the precise location of the aminoacylation site of tobacco mosaic virus (TMV) RNA. In order to stabilize the TMV histidyl-RNA ester bond, TMV N-acetyl [3H]histidylRNA was prepared. Upon electrophoresis in polyacrylamideagarose gels, the TMV N-acetyl [3H]histidyl-RNA showed a migration identical to that of unacylated TMV RNA. These results indicated that the histidine-acceptor site is located at or near the 30 -terminus. It also suggested that no new sites for aminoacylation are exposed during the incubation period. Removal of 5 to 10 nucleoside residues from the 30 -terminus by limited phosphorolysis with PNPase, or oxidation of the 30 -terminal ribose with periodate eliminated the aminoacylation capacity of TMV RNA as well as its infectivity in tobacco leaves. It was, therefore, concluded that the histidine aminoacylation site is located at the 30 -terminal adenosine of TMV RNA [66]. Differentiation of Artemia salina Cysts During a Gordon Conference, I met A. K. Kleinschmidt, who introduced me to the strange biology of the brine shrimp. A subsequent visit to the laboratory of Severo Ochoa, who was studying this system, persuaded me that the developing cysts might serve as a unique model for studying differentiation and protein synthesis regulation. The crustacean Artemia salina

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(brine shrimp) can reproduce in two ways: either the fertilized egg in the female ovisac directly evolves into nauplius larvae, or, during the dry season, it stops development at the stage of the gastrula, encysts, and is released in this form. These cysts may be desiccated by natural drying or osmotically by high environmental salinity, after which they enter a state of dormancy called cryptobiosis. It seems that desiccation of the cysts activates the gastrula which, when rehydrated, will undergo further differentiation to the prenauplius stage; this latter process taking place without cell division in about 15 h. Further incubation by the encysted prenauplii induces hatching and gives rise to a free-swimming nauplius larva. At this stage, cell division is resumed. The dehydrated cysts are sold as tropical fish food, and I decided to buy a few cans in New York City and take them with me to Rehovot. About that time, Henry Schmitt, a visiting scientist from the University of Brussels, joined the lab, and I suggested to him that we study together with Haim Grosfeld, then a graduate student, the biogenesis of the developing brine shrimp mitochondria. They soon found that when isolated from the cysts, the mitochondria were devoid of cristae and possess low respiratory capabilities. Hydration of the cysts induced marked biochemical and morphological changes in the mitochondria, and their biogenesis proceeds in two stages. The first stage is completed within one hour and is characterized by a rapid increase in the respiratory capability of the mitochondria, their cytochrome oxidase, cytochrome b, cytochrome c, and by a few morphological changes. In the second stage, there is an increase in the protein-synthesizing capacity of the mitochondria, as well as striking changes in mitochondrial morphology, leading to formation of cristae [70]. Further studies by Haim Grosfeld revealed marked differences in the protein composition of the cysts and nauplii. Thus, low molecular weight protein species, characteristic of the undeveloped embryos, give way to those of higher molecular weight [71]. He also demonstrated that dormant A. salina cysts are not devoid of mRNA, but that it is


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present mainly as a masked mRNA ribonucleoprotein complex, sedimenting at 40S, which disappears following rehydration. In addition, a small fraction of mRNA is present in polysomes, whose amount increases dramatically with development [72].

Establishment of the Department of Neurobiology Several years after returning from my postdoctoral studies in St. Louis, David Ben-Gurion, the first Prime Minister of the State of Israel, visited the Weizmann Institute of Science. He inquired about the nature of my scientific work and listened intently to my explanations of the genetic code. ‘‘I have to confess,’’ he remarked,‘‘that if I could have started life all over again I would have dedicated myself to research in biology.’’ He then asked ‘‘young man, why not study the human brain? After all, the superiority of all humans are their minds.’’ With this remark, he left my laboratory. A recollection of our conversation came back to me in 1968, when I proposed to the management of the Weizmann Institute that they should initiate research in neurobiology and open a department devoted to this field. My interest in neuroscience developed slowly, coinciding with the advances made in molecular biology and genetics. It became obvious that the time was ripe to apply similar interdisciplinary approaches to neurobiology. In October 1969, I was invited by the NIH as their first Fogarty Scholar-in-Residence. This was an opportunity to learn more about neurobiology. I chose to join Marshall Nirenberg, Chief of the Laboratory of Biochemical Genetics at NHLBI, as his interests had also shifted from molecular biology to neurobiology. My stay was immensely profitable; I worked all day in the lab, and at night, managing to complete a backlog of 14 papers. I learned a great deal during my stay in Marshall’s lab, and out of that I found, upon my return to Israel, the Department of Neurobiology at the Weizmann Institute and served as its Chairman until 1988. Throughout the years,

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I maintained a close connection with Marshall and visited him on several occasions. In 1978, we were particularly pleased when he came to the Weizmann Institute to receive an Honorary Doctorate.

Neuroblastoma as a Model System for Neuronal Differentiation Marshall very generously allowed us to use the cloned cell lines he had isolated from a spontaneous mouse neuroblastoma tumor, C-1300. The cell lines serve as a useful model system for exploring certain aspects of nerve cell differentiation, especially since they differ in their morphology, ability to extend neurites, neurotransmitter metabolism, and capacity to generate an electrically active membrane. Together with Yosef Kimhi, Clive Palfrey (a graduate student), and Ilan Spector (a neurophysiologist who joined us from the University of Paris), we examined the effect of dimethylsulfoxide (DMSO) on mouse neuroblastoma cell lines. We were influenced by Charlotte Friend’s discovery in 1971 that DMSO is a potent inducer of differentiation of Friend virus-infected murine erythroleukemic cells. We, therefore, examined its effect on neuroblastoma cells. Addition of DMSO, at a concentration of 2%, to several of the mouse cell lines induced differentiation, as judged by extension of neurites, development of highly excitable membranes, and the ability of most of the treated cells, to fire repetitively in response to prolonged depolarizing stimuli. Our results also suggested that the development of the excitable membrane could have taken place independently of the induction of neurospecific enzymes and did not require a sustained elevation of cyclic AMP levels [73,74]. In addition, Clive Palfrey demonstrated that a convenient way to monitor the development of the density of sodium channels was to measure the increase in the rate of efflux of 86Rbþ from preloaded cells on application of the neurotoxins, veratridine, and scorpion venom [75]. This


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method was subsequently used as an indicator to probe the maturation of excitable membranes in cells treated with various inducing agents. Thus, in collaboration with Paul Marks (then at Columbia University) and colleagues, we demonstrated that hexamethylene bisacetamide (HMBA) is effective in inducing differentiation of mouse neuroblastoma cells at concentrations 50-fold lower than that of DMSO [76], an observation that has clinical implications.

Surface Glycoproteins Mary Catherine (Susy) Glick, a sabbatical visitor from The Children’s Hospital of Philadelphia (CHOP), had begun, in 1971, a completely new line of research on the glycoproteins from surface membranes of neuroblastoma cells. Together with Yosef Kimhi, we examined the composition of surface membrane glycopeptides isolated from different clones of mouse neuroblastoma cells. Our results demonstrated that there appeared to be a positive correlation between the capacity to differentiate morphologically, as well as electrically, and the proportion of a particular group of glycopeptides present on the cell surface [77,78]. In another study, Nava Zisapel (a visiting scientist and currently Professor at the Department of Neurobiochemistry of Tel Aviv University) synthesized a new cationic hydroxysuccinimide ester that served as a reagent for labeling exterior membrane proteins [79]. I have continued collaborating with Susy Glick and have visited her lab at CHOP for extended periods. Together with Maria Giovanni, then a graduate student at Susy’s laboratory, we examined the neuronal properties of human neuroblastoma cells. The human neuroblastoma cell lines were obtained from different tumors, and have preserved a near diploid chromosomal number when maintained in culture. These human cells circumvent the disadvantages of the mouse neuroblastoma cell lines which are all derived from a single tumor, C-1300,

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and which show anaploidy constantly changing in culture. In contrast to the mouse cells, some of the human neuroblastoma cloned cells possess NGF receptors, while other cell lines display primitive neuroblastic properties that can be induced to express genes characteristic of neuronal, Schwanian, or melanocytic cells. As with mouse neuroblastoma, we have found that several human cell lines in culture display differentiated neuronal properties, such as neurotransmitter synthesizing enzymes and active Naþ channels. They could also be induced to differentiate with different external biological response modifiers [80]. The ability of some of the human neuroblastoma tumor cell lines to differentiate in vitro provides a model system to study the molecular events regulating cell growth and differentiation of these neural crest derivatives and may provide insight into the molecular mechanisms contributing to their tumorigenic conversion. We refined our experimental procedures to allow us to detect minor changes in the surface glycoproteins. This was done by developing a shearing method for the isolation of neurites and metabolically labeling of the cells with the specific glycoprotein precursors, L-[3H]fucose or D-[3H]glucosamine. With these more sensitive methods, we detected a 200-kDa glycoprotein, which was associated with mouse, and human neuroblastoma cells displaying active Naþ channels. In contrast, the level of this glycoprotein was significantly reduced in surface membranes from nondifferentiated cells, and a radioactive glycoprotein of similar molecular weight was found in the growth medium. Finding the 200-kDa glycoprotein in mouse cells as well the human neuroblastoma cells indicated that it might be neuronal-specific, and thus of importance in the expression of the differentiated functions. Moreover, the appearance of this 200kDa glycoprotein in concert with the active ion channels of these cells suggested that it could be directly involved with the ion flux itself [80^82]. During one of my visits to Philadelphia, Professor Klaus Hummeler, then the Director of The Joseph Stokes, Jr.


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Research Institute at CHOP, and I were instrumental in establishing a collaborative agreement between the Weizmann Institute and CHOP that supported complementary aspects of basic and clinical research projects of common interest.

Plasminogen Activators Another project we studied at the Weizmann Institute was the expression of plasminogen activators (PAs) in cultured neuroblastoma cells. The PAs, tissue-type (tPA) or urokinase-type (uPA), are serine proteases, that function in the generation and control of extracellular proteolysis by converting the inactive zymogen plasminogen into the active protease plasmin. The plasminogen system has been linked to biochemical changes in the matrix that surrounds nerve cells, thus influencing neuron growth and the establishment of synaptic connections that permit brain cells to communicate with each other. In our studies Mona Soreq and Aliza Zutra, my technician, collaborated with Ruth Miskin, an expert on PA from the Institute’s Department of Biochemistry. Cultured mouse neuroblastoma cells were found to display significant intracellular and secreted tPA activities, and upon differentiation with dibutyryl cyclic AMP, the level of tPA increased about 20-fold. Plasminogen-dependent proteolysis did not seem to influence the initiation of neurite extension, the appearance of toxinactivated Rbþ-efflux, or acquisition of electrical properties. However, significant spatial modulation of tPA activity was found to accompany the differentiation process and reflected selective differences in the properties and the function of individual neuronal processes and their growth cones [83]. The high level of secreted tPA in neuroblastoma cells could be linked to recent results suggesting the involvement of plasmin in the processing of human amyloid precursor protein (reviewed in Ref. 84).

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Microtubule Proteins Microtubules are a ubiquitous class of cytoskeletal organelles that are found in all eukaryotic cells and are dynamically involved in numerous cellular processes. In mature neurons, axon outgrowth, synapse formation, and axoplasmic transport depend on microtubule integrity. The diversity of microtubule functions has led to the idea that not all microtubules are alike and that different microtubules with distinct functions are present in different cells and even within single cells (reviewed by Ref. 85). The major structural subunit protein of microtubules is tubulin, a heterodimer composed of two related - and -tubulins. The tubulin hetrodimers polymerize together with microtubule-associated proteins (MAPs) to form the cylindrical wall of hollow microtubule filaments.

Control of Tubulin Expression in the Developing Nervous System Because of its universal occurrence and importance for neurite outgrowth, we decided to investigate the regulation of tubulin synthesis at both the transcriptional and translational levels. As a first step, Henri Schmitt and Illana Gozes (then a graduate student, currently a Professor and Head of the Department of Clinical Biochemistry at Tel Aviv University) studied the synthesis of tubulin in rat brain. They showed that during the first ten days after birth, the percentage of cytoplasmic rat brain tubulin increases moderately or remains almost constant. Thereafter, a progressive decrease in the percentage of cytoplasmic brain tubulin occurs until the rat reaches adulthood [86]. The postnatal decline in tubulin level occurs in the soluble fraction and is accompanied by a decrease in the relative amounts of its mRNA chains [87]. This decrease may be characteristic of all cell types of the brain and results from an age-dependent


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reduction in the percentage of the mRNA molecules coding for tubulin. Alternatively, since glial cell division mainly occurs postnatally, whereas neuronal mitosis ceases almost completely at birth in rat brain, the observed decrease may be a manifestation of lower synthesis of tubulin in glial cells. Neurons and glia are intimately interwoven in the brain and, therefore, separation of these cells is extremely difficult. However, an intensive study by Illana Gozes, together with Michael Walker and Alvin Kaye (then at the Department of Hormone Research), showed that neuronal and glial nuclei can be separated using sucrose gradients. Moreover, under certain conditions, the nuclear fraction can serve as an in vitro model system for protein synthesis that occurs in intact cells. Using neuronal- and glial-enriched nuclear fractions, it was concluded that the reduction in tubulin content during brain development occurs in parallel for both neurons and glia, and results from changes in the proportion of the corresponding mRNA [87,88]. It is gratifying to learn that the concept of nuclear protein synthesis was recently confirmed [89]. It should also be noted that tubulin is not only confined to the cytoplasm but is also found to be associated with various membranes. A striking feature is the observation by Gozes that the - and -tubulin subunits differ in their association with brain presynaptic membranes [90]. It appears that the -tubulin subunit is an integral synaptic membrane protein, whereas most of the -tubulin subunit can be easily dissociated from these membranes. Whether the membrane-associated tubulin functions by a mechanism other than polymerization into microtubules remains to be determined.

Tubulin Microheterogeneity Further studies by Illana Gozes led to the exciting discovery that brain -tubulin and -tubulin display extensive microheterogeneity, and several nonidentical isotubulins, that differ in isoelectric points but have similar molecular weights, had been

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identified. The extensive microheterogeneity of tubulin appeared to be more prominent in the brain than in other tissues such as spleen or liver. Moreover, brain tubulin microheterogeneity was found to be developmentally determined, increasing in the mature brain [91]. My tubulin group, which initially consisted of Schmitt and Gozes, quickly grew to include the visiting Scientists: Annie de Baetselier (Universitaire Instelling Antwerpen), Arlette Fellous (INSERM, Bicetre, France), and Susanna Rybak (Stanford University), as well as Huub J. Dodemont (a Postdoctoral Fellow from the University of Nijmegen) and Talma Scherson (a Graduate Student). Subsequent studies by Gozes and de Baetselier showed that some of the age-dependent variations in tubulin microheterogeneity are controlled at the mRNA level and that there is dissimilarity in the distribution of isotubulins isolated from various regions of rat brain. These observations may reflect, to some extent, differences in tubulin microheterogeneity within the different brain-cell types [92,93]. However, multiple tubulin forms were found to be expressed by a single neuron, suggesting an additional hypothesis, namely, that the various isoforms may be differentially utilized in different subcellular structures [94]. At this stage of our work, I was fortunate to have been able to recruit Irith Ginzburg (now a Professor at the Department of Neurobiology of the Weizmann Institute) and establish with her a collaboration that lasted for ten years. It became clear that further progress in our studies required cloning of the tubulin genes and their sequence determination. What is now almost a routine exercise was in those early days (end of the 70s) a difficult and demanding task. The goal was achieved by Irith Ginzburg and her colleagues, who were able to isolate several cDNA clones bearing sequences coding for rat brain -tubulin and -tubulin, as well as actin. In addition, several rat -tubulin pseudogenes were identified and sequenced [95^97]. We were not alone in this endeavor. Simultaneous studies in many other


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laboratories established that - and -tubulins are encoded by dispersed multigene families that are subject to differential regulation during development and differentiation. It was also observed that the large number of tubulin isotypes is due not only to expression of multiple tubulin genes, but also to various posttranslational modifications. However, the possible functions of the tubulin isotypes remains elusive (reviewed by Refs. 98,99). We used the tubulin and actin cDNA clones to study the control of mRNA expression in a number of cell systems. Some insight into the regulation of tubulin genes was obtained from our studies with nonneuronal cells. Thus, Arlette Fellous and Irith Ginzburg showed that treatment of human lymphoblastoid cells with either - or -interferon (IFN) induced a marked increase in the amounts of tubulin mRNA sequences. The specific induction of tubulin mRNA in IFN-treated cells was abolished when these cells were pretreated with colchicine, suggesting two independent regulatory sites for tubulin mRNA expression. The expression of - and -tubulin did not seem to be coordinately controlled, since the treatment with IFN- induced a more pronounced increase in the level of -tubulin than of -tubulin mRNA. It is interesting to note that the cytoplasmic actin mRNA levels were not affected following IFN treatment of these cells, indicating that the two cytoskeletal elements, tubulin and actin, are differentially regulated [85,100]. In another study, Irith Ginzburg, Susanna Rybak, and Yosef Kimhi used cDNA probes to reveal a biphasic regulation by dibutyryl cyclic AMP of tubulin and actin levels in neuroblastoma cells. The expression of actin mRNA during the differentiation of these cells differed from that of tubulin mRNA, suggesting again that the mRNAs of these two cytoskeletal proteins are independently regulated [101]. Irith also pioneered in the use of tubulin antisense oligodeoxyribonucleotides to prevent neurite outgrowth in nerve growth factor induced PC12 cells. The results showed that at least two tubulin isoforms are involved in neurite outgrowth [102].

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Microtubule-Associated Proteins In addition to tubulin, microtubules contain a heterogeneous class of proteins, known as microtubule-associated proteins (MAPs), which promote tubulin assembly and affect the stability of polymerized microtubules. Brain microtubules contain a variety of MAPs, and over 20 distinct species have been identified. MAP1, MAP2, and tau are the most prominent MAPs. It appears that distinct microtubules are present in different cell types, and even within a single cell like the neuron, microtubule heterogeneity is involved in the formation of microdomains that may be the basis for neuronal polarity. Thus, MAP2 is predominantly localized in dendrites, whereas tau is targeted to the axons. Additional interest in tau proteins has recently stemmed from its identification as a major component of paired helical filaments, the abnormal structures characteristic of Alzheimer’s disease. Several groups found that during brain development, there is a marked increase in the diversity of tau isotypes and an enhancement of their ability to induce microtubule assembly. In our laboratory, Irith Ginzburg and Talma Scherson showed that some of the age-dependent differences in tau proteins are controlled at the RNA level [103,104]. Thus, the expression of microtubule proteins in the brain is characterized by a wider variety of isoforms than in other tissues, as well as by a specific time of their activation during brain differentiation. Moreover, Drorit Neuman (then a graduate student), Michal Schwartz (currently a Professor at the Department of Neurobiology), and colleagues observed enhanced synthesis of 2-isotubulin species and two tau isoforms in the goldfish retina during optic nerve regeneration. It was, therefore, suggested that these isoforms are involved in the formation of the new axonal matrix [105]. With the help of my devoted technician, David Giveon, we were able to develop a specific binding assay that monitors the interaction of labeled MAPs with tubulin cleavage peptides fixed to nitrocellulose membranes. To identify


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the tubulin-binding domains for MAPs, we examined, in collaboration with Herwig Ponstingl (from the German Cancer Research Center, Heidelberg), the binding of rat brain MAP2 and tau factors to sixty cleavage peptides derived from pig - and -tubulin. MAP2 and tau factors were found to specifically interact with peptides derived from the carboxyl-terminal region of -tubulin. To narrow down the location of the -tubulin binding site that is common to MAP2 and tau factors, we synthesized five peptides that are homologous to overlapping sequences from the porcine or rat carboxyl-terminal region. Binding studies with the synthetic peptides suggested that amino acid residues 434^440 of -tubulin are essential for the interaction of MAP2 and tau factors [106]. Binding to -tubulin cleavage peptides yielded less clear results, but evidence from other groups indicated that the carboxyl-terminal regions of both - and -tubulin are involved in the binding of MAPs. It is interesting to note that the main amino acid sequence divergence of the various isotubulins and most of the post-translational modifications take place within their C-terminal regions. Moreover, the same region was found to include, at least in part, the binding site to MAP2 and tau factors. This led to the hypothesis that the variability in the C-terminal region will determine the strength and specificity of interaction of isotubulins with the various MAPs, thus generating functionally different microtubules. This hypothesis was supported by Irith Ginzburg and Sue Griffin (a visiting Professor from the University of Arkansas) who found that antibodies elicited against peptides spanning the Cterminal region of -tubulin allowed the identification of unique microtubule structures in different cerebellar cells [107].

A Unique MAP2 Species in Human Neuroblastoma Cells While this work on the tubulin binding sites for MAPs was in progress, we had begun a new line of investigation on the role of

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MAPs and laminin in the differentiation of human neuroblastoma cells. My interest in human neuroblastoma was stimulated during my frequent visits to The Children’s Hospital of Philadelphia. Human neuroblastoma is one of the most common solid tumors in children. It is of presumed neuronal crest origin and can occasionally undergo maturation into a more differentiated benign tumor called ganglioneuroma. The in vivo mechanisms responsible for the maturation of neuroblastoma are not known. However, the similarity of neuroblastoma cells to neuroblasts and their ability to mature spontaneously to a more benign tumor form, suggest that the origin of the disease is a block in the differentiation of a precursor cell. About that time, Joachim Kirsch, a visiting scientist, and currently Professor at the faculty of Medicine, University of Ulm, joined the lab. Together with Aliza Zutra, he was able to identify three MAP2 species (270, 250, and 70 kDa) in cultured human neuroblastoma cells. The expression of the 250-kDa isoform, termed MAP2d, appeared to be unique for cultured human neuroblastoma cells and fetal human brain. On the other hand, in adult human cerebellum, the same antibody detected only the 270-kDa MAP2 species. In addition, it was observed that human neuroblastoma cells show a shift from the embryonic 250-kDa MAP2d isoform to the more mature 270-kDa species when induced to differentiate with dibutyryl cAMP. Thus, the level of MAP2d may serve as a differentiation marker for neuroblastoma and nerve cells [108]. Analysis of neural-derived tumors showed that MAP2d is found in solid tumors diagnosed as neuroblastoma, but hardly in ganglioneuroma tumors. In contrast, both neuroblastoma and ganglioneuroma tumors contained significant levels of MAP1B (MAP5). These studies, carried out in collaboration with Lynn Meister and David Pleasure from The Children’s Hospital of Philadelphia, indicated that MAP2d is associated with human embryonic nerve cells and immature undifferentiated neuroblastoma tumors and, thus, may lead to a new prognostic tool for neuroblastoma tumors.


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Further investigation by Kirsch revealed that neurite outgrowth is accompanied by reorganization of the microtubular cytoskeleton. Thus, upon neurite extension in neuroblastoma cells, microtubule bundles are formed in which MAP2 is found in the proximal part and in branching points. In contrast, MAP1B (MAP5) is distributed along the entire length of the neurite. The differences in spatial distribution between MAP1B and MAP2 along the neurites and its identity with neuraxin illustrate the heterogeneity in microtubule composition in various parts of the cell process, and may suggest a different function for MAP1B [109,110]. A completely distinct pattern of reorganization was observed for the intermediate filament proteins ^ peripherin and vimentin. In undifferentiated cells, these two intermediate filament systems were organized as perinuclear whorls as in other cell systems. However, upon induction of differentiation, both these intermediate filaments were found in the growth cones, which represent the most dynamic part of the growing neurite. These observations suggest that the intermediate filament proteins are translocated toward the growth cones and are involved in neurite elongation [108,111]. Other studies by Joachim Kirsch in collaboration with Heinrich Betz at the Max-Planck-Institute for Brain Research, Frankfurt, have demonstrated that a 93-kDa glycine receptorassociated protein binds to tubulin. This 92-kDa protein (termed Gephyrin) appears to anchor the glycine receptor at postsynaptic sites via binding to subsynaptic tubulin and thus serves an important role in the topological organization of the postsynaptic membrane [112].

Neuroblastoma Laminin Binding Proteins Laminin, the major glycoprotein of basement membrane, is known to promote cell adhesion, growth, migration, and neurite outgrowth. I have, therefore, examined the involvement of

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laminin in the differentiation of cultured neuroblastoma cells. Together with Nira Garty and Ilana Bushkin-Harav, we have noticed that the differentiation of human neuroblastoma cells is accompanied by increased adhesion to laminin. The major binding site in laminin, mediating cell attachment, was then identified as containing the YIGSR peptide sequence on the 1 chain. Furthermore, affinity chromatography with immobilized C(YIGSR)3^NH2 peptide amide revealed one major YIGSR-binding protein with an apparent molecular weight of 67 kDa. The 67-kDa laminin-binding protein (LBP) appeared to be downregulated upon differentiation of human neuroblastoma cells. The decline in the levels of the 67-kDa LBP was accompanied by a decrease in the corresponding mRNA levels. It appears that in neuroblastoma cells, the 67-kDa LBP binds to the YIGSR sequence in a cooperative manner through an association with another protein. Thus, cross-linking experiments with SulfoMBS detected an additional protein with a molecular mass of 116 kDa that binds with biotin labeled C(YIGSR)3^NH2. Incubation of the cells with C(YIGSR)3^NH2 peptide amide or antibody directed against the 67-kDa LBP, induces tyrosine phosphorylation of proteins with a molecular mass ranging from 115 to 130 kDa and another heterogeneous protein group of 32 kDa. These results suggest the possibility that the 67-kDa LBP may mediate signaling events in neuroblastoma cells [113].

Promoting International Links In the early days, after the establishment of the State of Israel, scientists in Israel felt isolated from the international scientific community. Efforts were made to change the situation by joining several international scientific societies, host meetings in Israel, and support collaborative exchange programs with foreign organizations. One of the few organizations created in a politically divided Europe, the Federation of the European Biochemical Societies


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(FEBS), allowed and encouraged scientific cooperation among members of its constituent societies. It sponsored annual scientific congresses alternating between Western and Eastern Europe, and was the only means, at the time of the cold war, which offered an international forum for interaction among its members. The first FEBS Council Meeting took place in London on March 22, 1964. Nobel Laureate Edmond H. Fischer from the University of Washington, Seattle, who had just returned from a visit to the Weizmann Institute, was delegated by the Israel Society for Biochemistry to present its application to join the Federation. However, several Council members argued that geographically Israel was not part of Europe, and Fischer’s efforts failed. A second opportunity presented itself during the 2nd FEBS Congress, which took place in Vienna on 21^24 April, 1965. I was invited by Otto Hoffmann-Ostenhof, President of the meeting, to deliver a lecture and to serve as a chairman in one of the symposia. Prior to the meeting, I suggested to Hoffmann-Ostenhof that the membership of the Israel Society for Biochemistry should be reconsidered at the forthcoming FEBS Council Meeting, to which he agreed. Upon arriving in Vienna, I lobbied our case among several delegates. Hoffmann-Ostenhof, who was eager to help, told me that the Biochemical Society of Yugoslavia had applied to join FEBS. He suggested that, as the Chairman of the FEBS Council, he would bring up both, our application and that of Yugoslavia, for consideration at the same time, thereby avoiding a negative vote by the delegates of the Eastern European biochemical societies. To block the geographical argument, I proposed that the name of FEBS be changed to the ‘‘Federation of Biochemical Societies in the European Area.’’ The Council met in the famous Hotel Sacher and was chaired by Hoffmann-Ostenhof who welcomed the seventeen delegates of the adhering Societies. Nobel Laureate Severo Ochoa, the President of the International Union of Biochemistry (IUB), attended the meeting and took an active

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part in the discussions. I described our situation that Israeli biochemists, which were scientifically active, felt isolated from their colleagues in Europe. I also mentioned a number of analogous cases in which nonscientific European Federations had accepted an Israeli society as a full member. Otto HoffmannOstenhof, and Ernst Auhagen (representing West Germany) endorsed our application. Finally, at the suggestion of Bill Whelan (UK), the Israel Society for Biochemistry was accepted as a full member nem con with five abstentions. At the Council meeting, the dynamic Bill Whelan was appointed as Secretary-General. He suggested that FEBS might venture into the field of publication, which aroused prolonged discussions. A subcommittee was set up to examine the feasibility of launching a biochemical journal. Its members consisted of Jean Courtois, Otto Hoffmann-Ostenhof, Claude Lie¤becq, Pavao Mildner, Peter Reichard, Bill Whelan, and myself. On 25^26 October 1965, we met again in Courtois’s office in Paris. FEBS did not have the funds to defray the traveling expenses of the subcommittee, and I had to shift a lecture engagement to be able to participate at the meeting. The majority opinion favored a conventional type of journal rather than one devoted to publishing rapid short communications, which was favored by some members of our Committee. During this meeting, Claude Lie¤becq was nominated as Editor-in-Chief. I imagine he was not totally surprised as, several months earlier at the final dinner in Schloss Laudon near Vienna, I hinted to Suzanne Lie¤becq that her husband was being considered for that position. The subcommittee outlined the specification for the journal and a number of publishers were approached. In the meantime, Theodor Bu«cher, the President of the German Gesellschaft fu«r Biologische Chemie, had suggested to Bill Whelan that instead of establishing a new journal, the Biochemische Zeitschrift be converted into the European Journal of Biochemistry. At its meeting in Warsaw in March 1966, the FEBS Council enthusiastically adopted this suggestion [114,115]. A short time later, I accepted an invitation to join the


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Editorial Board of the journal, which started to appear in March 1967. During the first Editorial meeting that took place in Heidelberg in July 1966, I had long discussions with the late Lars Ernster about the possibility of his joining our Department of Biochemistry at the Weizmann Institute. Although he decided to remain in Stockholm, we stayed in touch and he visited us in Rehovot on several occasions. The Sixth FEBS Council Meeting was held in Oslo on July 3, 1967. The Council decided to start publishing an additional journal, FEBS Letters, devoted to short papers. It also elected Peter Campbell as Chairman of the FEBS Summer School Subcommittee, and Henry Arnstein as Secretary-General. Both Peter and Henry approved of my suggestion to organize a FEBS Summer School devoted to nucleic acids at the Weizmann Institute. In September 1968, we hosted the Summer School and were very pleased to secure the participation of a distinguished group of outstanding biochemists (Figure 3). The School attracted considerable attention, and about 190 scientists from seventeen countries attended. To house the participants, I reserved all the rooms in a nearby dormitory and persuaded the management of the Weizmann Institute to build an additional floor to the Institute’s guesthouse. I maintained close contact with FEBS and represented the Israel Society for Biochemistry at its Annual Council Meetings for many years. In July 1975 in Paris, the Council accepted my offer to hold the 13th FEBS Congress in Jerusalem in 1980, which turned out to be a successful event. Between the years 1984^1989, I joined the Executive Committee, and was elected Chairman of the FEBS Publication Committee, and in 1999 I was awarded, in Nice, the FEBS Diplo“me d’honneur for my efforts on behalf of the Federation over the years. In March 1964, I received a letter from Max Perutz, the 1962 Nobel Laureate in Chemistry, asking me to become one of the founding members of EMBO, the European Molecular Biology Organization. The aim of the Organization was, and continues to be, to promote molecular biology studies in Europe. EMBO

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Fig. 3 Although at a casual glance, it may appear that Fred Sanger (right) is presenting Francis Crick with some special FEBS award commemorating the Summer School on Nucleic Acids, in actual fact, the fraternal embrace is merely the transfer of a microphone from one speaker to another. Rehovot 1^6, September 1968. Photographed by the Photography Department of theWeizmann Institute.

managed to raise funds for research and overcome the rigid division of science into classical disciplines that existed in many European universities. Through its fellowship program, it was the only organization in those early years in Europe to support interchanges of research workers and ideas between European laboratories. I familiarized myself with the operation of the Organization when I served, from 1980 through 1983, on the EMBO Course Committee. Several years earlier, from February 24 to 28, 1975, I organized a successful EMBO Workshop on tRNA Structure and Function. It was held in the guesthouse of Kibbutz Ginossar on the shore of the Sea of


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Galilee, 200 meters below sea level. For many of the 90 participants, it was their first visit to Israel and gave them an opportunity to acquaint themselves with the communal life of the Kibbutz. The meeting served to highlight the turning point of the biochemical investigations of tRNA and emphasized the importance of the new X-ray crystallographic studies in elucidating the three-dimensional structure of tRNA. The International Union of Biochemistry and Molecular Biology, IUBMB (formerly IUB), is another organization I was associated with for many years. IUBMB unites biochemists and molecular biologists in 66 countries. It is devoted to promoting research and education in biochemistry and molecular biology throughout the world, and gives particular attention to areas where the subject is still in its early development. Every three years, the Union sponsors International Congresses of Biochemistry, which are major meetings where current research is considered. Among other activities, IUBMB sponsors international conferences, symposia, workshops, and training sessions on biochemical education. In 1970, I was invited to join the newly formed IUB Advisory Symposium Committee, which enabled me to learn about the operation and organization of the Union. From 1983 through 1988, I was elected as its chairman (renamed IUBMB Committee on Symposia, since 1983). During this period, we initiated 54 symposia worldwide. In this capacity, I was a member of the IUBMB Executive Committee. I devoted considerable time organizing the 15th International Congress of Biochemistry, which was scheduled to take place in Jerusalem in August 1991. The outbreak of hostilities in the Gulf region on January 17, 1991, and the missile attack by Iraqi forces on Israel made it uncertain whether the meeting would take place. As President of the meeting, I was under considerable pressure from the IUB Executive Committee to move the meeting to another location. I remember the urgent discussions I had with E. C. (Bill) Slater, the President of IUB. One telephone call took place, at night, during a SCUD missile attack on Tel Aviv.

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Through my window in Rehovot, I was watching two bright mushrooms of exploding missiles when I answered the telephone. However, I was determined to wait as long as possible before making the final decisions. During this period, Bill Slater was very helpful and supportive of our efforts to draw up an emergency plan for the meeting. The fighting had stopped by the end of March, and we were in a position to continue with our preparations in a more normal atmosphere and conduct a most successful Congress as scheduled. The Congress was held in the Jerusalem Convention Center and the adjoining Jerusalem Hilton with 2520 participants from 45 countries. It contained 68 symposia with 440 lecturers and 1068 poster presentations. The six Plenary Lectures were given by Paul Berg (USA), Tom Blundell (UK), Adrienne E. Clarke (Australia), Yasutomi Nishizuka (Japan), Bert Sakmann (Germany), and Robert Tjian (USA). Seven satellite meetings were held before and after the Congress, one in Gothenburg, Sweden, one in Moscow, USSR, and five in Israel. Before the Congress, we also organized the IUB Fellows Course in the guesthouse of Kibbutz Ramat Rachel with over 100 enthusiastic young participants. The 5th IUBMB Conference on Biochemistry of Health and Diseases was another successful and well attended meeting that I hosted in Jerusalem, in 1998. My activities with IUBMB were concluded in July 2000, at the Executive Committee meeting in Birmingham UK, when I was honored to receive the IUBMB Distinguished Service Award. Another aspect of my efforts to create international scientific links was my association with the Aharon KatzirKatchalsky Center of the Weizmann Institute. The Center was established in 1972, following the tragic death of Professor Aharon Katzir-Katchalsky at the hands of terrorists. It is chaired by Professor Ephraim Katzir and promotes various activities in the Life Sciences. Of particular concern to the Center is the impact of scientific and technological advances on human society. During 1990^1996, I became the Director of


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the Center and was closely involved with its activities including the Student Travel Fellowship Program, the Annual KatzirKatchalsky Lecture and the Annual Katzir-Katchalsky Conferences, both in Israel and abroad. A particularly successful meeting that brought forward the most recent advances in molecular neurobiology was the Fifteenth Aharon Katzir-Katchalsky Conference, held in Leeds Castle Maidstone, Kent, UK from June 1 to 5, 1986. Eric A. Barnard (Cambridge), Yadin Dudai (Rehovot), Israel Silman (Rehovot), and myself served on the organizing committee. There were 90 participants from 15 countries, with 43 lectures and 28 poster presentations. The first session was devoted to neuronal recognition with contributions from S. Benzer (Pasadena), M. C. Raff (London), G. Fishbach (St. Louis), Z. Vogel (Rehovot), M. Israel (Gif-sur-Yvette), and C. Goridis (Marseille). The second session was devoted to the molecular biology of GABA, and ACh receptors. It included presentations by E. A. Barnard (Cambridge), A. Karlin (New York), and A. Borsodi (Szeged). In another session,W. A. Catterall (Seattle), M. Lazdunski (Nice), B. Sakmann (Go«ttingen), and I. Steinberg (Rehovot) described the molecular properties of ion channels, whilst the structure of AChE was illustrated by I. Silman (Rehovot) and W. H. Randall (Cambridge). In another session, M. Hanley (Cambridge), S. K. Fisher (Ann Arbor), and D. Lancet (Rehovot) examined signal transduction pathways in nerve cells. F. H. Crick (La Jolla), Y. Dudai (Rehovot), and P. Goelet (New York) devoted their lectures to the molecular basis of learning and memory. A session dealing with the various components of neuronal cytoskeleton involved presentations by E. Lazarides (Pasadena), K. Weber (Go«ttingen), F. Gros (Paris), J. Nunez (Creteil), and myself. The last session dealt with regulatory mechanisms and trophic factors with lectures by M. Nirenberg (Bethesda), E. Shooter (Stanford), H. Thoenen (Planegg-Martinsreid), and D. Monard (Basel). Many more presentations added to the interesting and lively discussions during this meeting.

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In closing, I hope I have conveyed the satisfaction and pleasure that I have had in pursuing scientific research. I was also privileged to be the founder of two flourishing departments at the Weizmann Institute. Throughout the years, my efforts to secure international collaboration brought me in contact with colleagues worldwide. I feel fortunate to have developed longlasting friendships with many of them.

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[42] Inouye, H., Fuchs, S., Sela, M. and Littauer, U.Z. (1973) J. Biol. Chem. 248, 8125^8129. [43] Salomon, R., Fuchs, S., Aharonov, A., Giveon, D. and Littauer, U.Z. (1975) Biochemistry 14, 4046^4050. [44] Salomon, R., Giveon, D., Kimhi, H. and Littauer, U.Z. (1976) Bichemistry 15, 5258^5262. [45] Daniel,V., Sarid, S. and Littauer, U.Z. (1968) Proc. Natl. Acad. Sci. USA 60, 1403^1409. [46] Daniel,V., Sarid, S. and Littauer, U.Z. (1968) FEBS Lett. 2, 39^41. [47] Daniel,V., Sarid, S. and Littauer, U.Z. (1970) Science 167, 1682^1688. [48] Daniel, V., Beckmann, J.S., Sarid, S., Grimberg, J.I., Herzberg, M. and Littauer, U.Z. (1971) Proc. Natl. Acad. Sci. USA 68, 2268^2272. [49] Sharon, N. (2000) Selected Topics in the History of Biochemistry: Personal Recollections VI (Comprehensive Biochemistry Vol. 41), (Semenza, G. and Jaenicke, R. eds.), pp. 707^748. Amsterdam, Elsevier. [50] Kaufmann, G. and Littauer, U.Z. (1969) FEBS Lett. 4, 79^83. [51] Kaufmann, G. and Littauer, U.Z. (1970) Eur. J. Biochem. 12, 85^92. [52] Kaufmann, G., Fridkin, M., Zutra, A. and Littauer, U.Z. (1971) Eur. J. Biochem. 24, 4^11. [53] Kaufmann, G., Grosfeld, H. and Littauer, U.Z. (1973) FEBS Lett. 31, 47^52. [54] Kaufmann, G. and Littauer, U.Z. (1974) Proc. Natl. Acad. Sci. USA 71, 3741^3745. [55] Soreq, H., Nudel, U., Salomon, R., Revel, M. and Littauer, U.Z. (1974) J. Mol. Biol. 88, 233^245. [56] Soreq, H. and Littauer, U.Z. (1977) J. Biol. Chem. 252, 6885^6888. [57] Huez, G. et al. (1974) Proc. Natl. Acad. Sci. USA 71, 3143^3146. [58] Huez, G. et al. (1975) Eur. J. Biochem. 59, 589^592. [59] Littauer, U.Z. and Soreq, H. (1982) Prog. Nucleic Acid Res. Mol. Biol. 27, 53^83. [60] Nudel, U., Soreq, H. and Littauer, U.Z. (1976) Eur. J. Biochem. 64, 115^121. [61] Baer, B.W. and Kornberg, R.D. (1983) J. Cell. Biol. 96, 717^721. [62] Ginzburg, I., Cornelis, P., Giveon, D. and Littauer, U.Z. (1970) Nucleic Acids Res. 6, 657^672. [63] Atwater, J.A., Wisdom, R. and Verma, I.M. (1990) Ann. Rev. Genet. 24, 519^541. [64] Gallie, D.R. (1988) Gene 216, 1^11. [65] Deo, R.C., Bonanno, J.B., Sonenberg, N. and Burley, S.K. (1999) Cell 98, 835^845. [66] Salomon, R., Sela, I., Soreq, H., Giveon, D. and Littauer, U.Z. (1976) Virology 71, 74^84. [67] Grosfeld, H., Soreq, H. and Littauer, U.Z. (1977) Nucleic Acids Res. 4, 2109^2121.


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[93] Gozes, I., de Baetselier, A. and Littauer, U.Z. (1980) Eur. J. Biochem. 103, 13^20. [94] Gozes, I. and Sweadner, K.J. (1981) Nature 294, 477^480. [95] Ginzburg, I., Behar, L., Givol, D. and Littauer, U.Z. (1981) Nucleic Acids Res. 9, 2691^2697. [96] Ginzburg, I., Teichman, A., Dodemont, H.J., Behar, L. and Littauer, U.Z. (1985) EMBO J. 4, 3667^3673. [97] Ginzburg, I., Teichman, I. and Littauer, U.Z. (1986) Ann. N.Y. Acad. Sci. 466, 31^40. [98] Sullivan, K.F. (1988) Annu. Rev. Cell Biol. 4, 687^716. [99] Boucher, D., Larcher, J.C., Gros, F. and Denoulet, P. (1994) Biochemistry 33, 12471^12477. [100] Fellous, A., Ginzburg, I. and Littauer, U.Z. (1982) EMBO J. 1, 835^839. [101] Ginzburg, I., Rybak, S., Kimhi, Y. and Littauer, U.Z. (1983) Proc. Natl. Acad. Sci. USA 80, 4243^4247. [102] Teichman-Weinberg, A., Littauer, U.Z. and Ginzburg, I. (1988) Gene 72, 297^307. [103] Ginzburg, I., Scherson, T., Giveon, D., Behar, L. and Littauer, U.Z. (1982) Proc. Natl. Acad. Sci. USA 79, 4892^4896. [104] Ginzburg, I., Scherson,T., Rybak, S., Kimhi,Y., Neuman, D., Schwartz, M. and Littauer, U.Z. (1983) Cold Spring Harbor Symposia on Quantitative Biology,Volume XLVIII, 783^790. [105] Neuman, D., Scherson, T., Ginzburg, I., Littauer, U.Z. and Schwartz, M. (1983) FEBS Lett. 162, 270^276. [106] Littauer, U.Z., Giveon, D., Thierauf, M., Ginzburg, I. and Ponstingl, H. (1986) Proc. Natl. Acad. Sci. USA 83, 7162^7166. [107] Ginzburg, I., Behar, L., Littauer, U.Z. and Griffin, W.S.T. (1989) J. Neurosci. 9, 1961^1967. [108] Kirsch, J., Zutra, A. and Littauer, U.Z. (1990) J. Neurochem. 55, 1031^1041. [109] Rienitz, A. et al. (1989) EMBO J. 8, 2879^2888. [110] Kirsch, J., Littauer, U.Z., Schmitt, B., Prior, P., Thomas, L. and Betz, H. (1990) FEBS Lett. 262, 259^262. [111] Kirsch, J., Zutra, A. and Littauer, U.Z. (1991) In Advances in Neuroblastoma Research (Evans, A.E., D’Angio, G.J., Knudsen A.G. Jr. and Seeger, R.C. eds.),Vol. 3. pp. 327^333. New York,Wiley-Liss. [112] Kirsh, J., Langosch, D., Prior, P., Littauer, U.Z., Schmitt, B. and Betz, H. (1991) J. Biol. Chem. 266, 22242^22245. [113] Bushkin-Harav, I., Garty, N. and Littauer, U.Z. (1995) J. Biol. Chem. 270, 13422^13428. [114] Whelan,W.J. (1974) FEBS Lett. 40 (Suppl.), S154^S159. [115] Lie¤becq, C. (1992) Eur. J. Biochem. 204, 421^432.


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Chapter 6

Some Selected Recollections from a Life with Biochemistry HANS KLENOW Department of Medical Biochemistry and Genetics, The Panum Institute, The University of Copenhagen, Copenhagen, Denmark

Introduction I grew up in a typical Danish middle-class family. We were three children. I have an elder and a younger sister. We lived in one of the northern suburbs of Copenhagen in a beautiful area with lakes and forests. Our parents liked to take us children on long walks on Sundays into the wonderful North Zealand. So we learned early to love outdoor life. We were spared for major worries and we lived a happy life. My father was a civil engineer, and his father and his father again were tanners. My great grandfather emigrated in the middle of the 19th century to Denmark from Mecklenburg, the German province close to the Baltic Sea. My mother’s father was a butcher, but my mother told me that he much preferred to stand behind the easel making oil paintings. Both of my grandfathers died before I was born in 1923. I remember, however, vividly and with much devotion my two grandmothers.

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Student Years at the University of Copenhagen I liked very much my time as a student at the University of Copenhagen. There was a good and relaxed atmosphere. We were very few (less than 10) biochemistry students all together at all levels. We had good teachers both at the practical courses and at the lectures, and they were excellent scientists too. One of our teachers in plant physiology (Poul Larsen) had just found a plant hormone that stimulated the growth of dicotyledones. The compound was isolated and given to our teacher in organic chemistry, K.A. Jensen, and he soon identified it as indolyl acetic acid. K.A. Jensen gave fine lectures that often ended in a quite dramatic way. One day he demonstrated the effect of

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photochemistry. He produced a mixture of hydrogen gas and chlorine gas in a big glass container and as he left the lecture room he lit a lamp that irradiated the gas mixture leading to formation of HCl through an impressive explosive reaction. K. A. Jensen later wrote an excellent textbook in chemistry. He was my teacher in the laboratory course in chemical synthesis in the spring of 1945. The chemistry building was situated at one end of the Botanical Gardens, which formed a depression in the landscape. On March 21st we were highly excited to see a number of small aircrafts coming at a terrible speed virtually through the depression almost below us. We rushed to the windows and saw that it was R.A.F. Mosquito airplanes which seconds later dropped their bombs at the headquarter of the Gestapo near the center of Copenhagen. The Gestapo had placed their most prominent prisoners from the resistance movement on the top floor as a kind of shielding to protect the lower floors of the building where they had their files including those on the members of the resistance movement. The R.A.F. knew this and managed to hit the lower floors of the building with their bombs. This enabled most of the Danish patriots to escape in the following turmoil among them our teacher in zoophysiology, P. Brandt Rehberg, known for his fundamental work on renal physiology. He managed to hide from the Germans until the liberation of the country by the Allied Armed Forces on May 5th. He soon after resumed his lecturing and I remember that at one occasion he talked about the senses and emphasized that in many cases it was possible to adapt to sense impressions. However, there was one exception he said, and that was the sense of pain. When he said that he turned pale and he had to leave the lecture room. He had experienced himself that one cannot adapt to the impression of pain. The Gestapo had tortured him while he was their prisoner. Hans H. Ussing, who is famous for his monumental work on transport of ions across the cell membrane, taught us basic biochemistry. The textbooks available were in general not very

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exciting and that held not least in biochemistry. Of course, biochemistry was at a quite primitive level compared to what we know today. I remember that the author of one of the textbooks stressed that the nature of enzymes was quite uncertain. Only in few cases was it known for sure to be a protein. Until a few years ago, it seemed odd to think that an enzyme could be anything but a protein. Yet, Tom Cech taught us that some RNA molecules have catalytic activity, they may function as enzymes. Shortly before this discovery in 1983, Tom Cech worked in our institute as a guest scientist. He came primarily to do experimental work with my young colleague Jan Engberg. It was soon after that Jan together with others in elegant experiments had shown that in the protozoon Tetrahymena the gene for ribosomal RNA had palindromic structure [1]. Vagn Leick,

Institute for Cytophysiology. The University of Copenhagen, 1950. Standing: Gu«nter Stent, Niels Ole Kjeldgaard, Hans Klenow, James D. Watson, Vincent Price. Sitting: Herman M. Kalckar, Audrey Roschou Nielsen, Jytte Heisel, Eugene Goldwasser,Walter Scott MacNutt, E. Hoff-Jrgensen.


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another of my colleagues at the institute, had already made thorough studies on the formation of ribosomes in Tetrahymena.

Experiments with Penicillin In a period during the German occupation of Denmark, all lectures were abandoned at the University of Copenhagen. A close friend of mine, Rolf Brodersen, suggested therefore that I in this period performed experimental work together with him at the University Institute of General Pathology where he had a job. At the institute the professor (K. A. Jensen, but not the chemist of the same name) had managed to isolate a sample of a mould that apparently produced penicillin. He and his assistant worked eagerly in the laboratory to isolate the active compound. A simple and convenient bio-assay based on the inhibitory effect of the presumed penicillin on the growth of Staphylococcus aureus was used. At that time nothing at all was known about the chemistry of penicillin. Rolf, who was an excellent biochemist and physical chemist, planned experiments intended to determine the molecular weight of the substance based on its diffusion constant. We first made model experiments with known compounds measuring their free diffusion constants in a simple setup and the relationship between these constants and the respective molecular weights were obtained. Consequently, it was my task to determine the diffusion constant of the so-called Danish penicillin. The results came out quite well and the molecular weight computed was in fair agreement with the later on published value based on the constitutional formula of penicillin [2].

Pupil of Herman Kalckar and Experiments with Xanthine Oxidase In the spring of 1947, it was time for me to find a laboratory where I could do biochemical graduate laboratory work. I had

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heard a lecture by a marine biologist who described his work on the determination of the formation of dry matter in seawater based on an isotope method. I talked to him afterwards and he kindly agreed to accept me in his laboratory in the following fall. However, as time went by, I felt that marine biology might not be the right field for me. One day when I had lunch with my fellow students, one of them mentioned that a certain Dr. Kalckar had returned to Denmark after having spent the time during the war in the USA doing biochemical research there. It sounded very interesting to me and I decided to approach Kalckar. His personality and his charisma immediately fascinated me. In addition, his work on nucleosides and pteridines sounded so exciting that I asked him right away if he was willing to accept me as his student. I was very happy that he did so but I felt uneasy about asking the marine biologist to release me from our agreement. But he generously did so and it was thus my great fortune that Herman Kalckar, this unique character and great scientist, became my teacher and mentor and later my close friend. I soon became aware that Kalckar already before the war had discovered the phenomenon fundamental to all aerobic organisms: oxidative phosphorylation; i.e. that cellular oxidation of metabolites is coupled to phosphorylation of ADP to ATP. Among his many activities in USA were his discoveries of several enzymes that catalyze the metabolism of nucleosides and nucleotides. He had developed the very sensitive and rapid differential spectrophotometric method for analysis of the enzymatic interconversion of purine compounds based on their different ultraviolet absorption spectra. At the time when I began my work in his laboratory, he was particularly interested in the very reactive pteridines, and among them especially in folic acid. Others had shown that pteridine-6 -aldehyde was a contaminant of folic acid that inhibited the activity of xanthine oxidase from milk. Together with Niels Ole Kjeldgaard we found that the 6 -aldehyde was


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a substrate for this enzyme and that preincubation of preparations of folic acid with xanthine oxidase released their inhibitory effect on the oxidation of hypoxanthine. These studies led to characterization of the broad specificity of xanthine oxidase [3]. I managed to show that xanthine oxidase catalyzes the oxidation of adenine to the highly insoluble 2,8-dioxyadenine [4]. This compound is usually not present in the human organism but it has some years ago been found that it accumulates in the blood and precipitates in the kidneys in persons who lack the enzyme adenine phosphoribosyltransferase. They suffer from a seldom genetic disease, and they are not able to reutilize free adenine through this enzyme reaction, and adenine is instead being converted to 2,8-dioxyadenine probably catalyzed by xanthine oxidase [5]. In postwar Denmark the housing problem was considerable. But in the summer of 1949, my fiance¤ Margrethe Felbo and I were lucky to obtain access to a small apartment in a newly built apartment house. This was in a village close to where we both had lived as children. We had fallen in love with each other already almost 7 years before but now we could finally begin to live our lives together and my wife’s salary as a trained nurse at a hospital in Copenhagen supported our daily life. I hoped to obtain my university degree soon after, and in November, the final and oral examination for the degree of magisterium scientiarum took place. There were three professors: one in organic chemistry, one in physical chemistry, and one in biochemistry. They examined in turn: when one was examining the two others were censors. The verdict after the examination would be either ‘‘passed’’ or ‘‘not passed’’. I was lucky to belong to those of first category. Kalckar’s research was supported by several American funds including the Rockefeller Foundation and by the Carlsberg Foundation in Denmark. It was my great fortune that he, generously out of this money, offered to have me as his assistant at his Institute of Cytophysiology under the Medical Faculty of the University of Copenhagen.

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Excursion to Two European Strongholds in Biochemistry In the late 1940s, filterpaper chromatographic methods were developed and they were soon recognized as strong analytical tools not least in biochemical laboratories. Kalckar wanted the method introduced in his laboratory and arranged for me to go to Cambridge to pick it up where Haines and Isherwood had developed it to perfection. Kalckar had close connections to Otto Warburg in Berlin whom he had visited in the spring of 1950. Warburg was also interested in the new method and it was suggested that I, on my way home from Cambridge, should make a stop in Berlin and pass on my newly acquired knowledge to Warburg’s institute. I was very kindly received in Cambridge in November 1950 where, especially, Charles Haines took my hand and taught me many small tricks of the method. He supplied me with a set of small tools and gadgets that made it possible for me to demonstrate the method in Berlin and at home. It was a great experience for me to be exposed to the special atmosphere that prevailed in the biochemical laboratories in Cambridge and thanks to Kalckar. I had even the opportunity to visit several leaders in the fields including Dorothy and Joseph Needham in their home for a nice 5 o’clock cup of tea. I had a rough passage to Berlin flying at low altitude in a windy weather over the waves of the North Sea in an old aircraft. I felt a bit miserable when we landed at the Tempelhof Airport in Berlin. But there I was received by a chauffeur who drove me in one of the very few privately owned cars to the Kaiser Wilhelm Institut fu«r Zellphysiologie. On the way, I saw that the streets ran between large heaps of bricks and all over people were sitting cleaning bricks for plaster and there were many begging children. Very few houses were undamaged, one of them being the building of Warburg’s institute. It had been built in 1931 in a beautiful style, but in 1943, the air attack on Berlin forced Warburg to move his institute to a mansion north of Berlin. In the spring of 1950,


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the American commander-in-chief who had used the institute for his headquarters returned it in restored state toWarburg. Warburg was highly esteemed as a scientist for his monumental contributions to biochemical research, but he was also known for his angry remarks to those of his fellow scientists who were his opponents or who did not meet his requirements on high quality science. I was quite scared to meet this scientific giant, but it was probably my advantage that I was just a completely unknown young guy who hoped to become a scientist. I was kindly received and spent two interesting weeks at this famous place. The demonstration of my newly acquired knowledge seemed to be well received and I had the opportunity to follow experiments going on concerning the quantum yield of photosynthesis as studied in green algae. It was a great help to me that the American biochemist Dean Burk (known especially from the so-called Lineweaver-Burk plot in enzyme kinetics) was a visiting scientist at the institute at the same time. He was extremely kind and helpful to me and in the weekend he took me on a guided tour from the American sector of Berlin to the French sector, the British sector, and the Russian sector. Warburg did not want to spend much time on administration and he did not have a secretary. He personally wrote his letters in a short and concise style on a typewriter placed in a corner of the big library. One day when I was sitting reading in the library, Warburg came in and sat down at the typewriter and began to write a letter. Suddenly he stopped writing, turned to me and said: You may wonder why I do not take copies of my letters ‘‘es ist aber gar nicht notwendig, weil ich immer nur die Warheit schreibe.’’

Ribose 1,5-Diphosphate In 1947 Kalckar found an enzyme that catalyzed the phosphorolytic cleavage of inosine or guanosine leading to formation of ribose-1-phosphate (R-1-P) and hypoxanthine or guanine,

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respectively. This reaction resembled the one catalyzed by glycogen phosphorylase giving rise to glucose-1-phosphate (G-1-P). Victor A. Najjar had shown that G-1-P could be converted to glucose-6 -phosphate (G-6 -P) catalyzed by phosphoglucomutase from muscle, an enzyme that he obtained in pure crystalline form. It was later found in Leloir’s laboratory in Argentina that this enzyme reaction requires a coenzyme identified as glucose-1,6 -diphosphate (G-1,6 -P2). The mechanism of the reaction was a transfer of the 1-phosphate of a coenzyme molecule to the 6 -position of G-1-P whereby a new coenzyme molecule and the reaction product G-6 -P were formed. R-1-P was known similarly to be enzymatic converted to ribose -5-phosphate. I studied this reaction in extracts from muscle and found that it resembled the phosphoglucomutase reaction and when the procedure of Najjar for the isolation of the crystalline enzyme was followed, the ratio between the two phosphomutase activities was almost unchanged at the different purification steps. In addition, G-1,6 -P2 served as a coenzyme also for the reaction with R-1-P as a substrate. When substrate amounts of G-1,6 -P2 were incubated with enzyme and excess of R-1-P, G-6 -P accumulated together with equivalent amounts of a compound which was isolated in pure form. The chemical and chromatographic properties of the compound suggested that it was ribose-1,5-diphosphate [6]. This structure was soon confirmed when H. Gobind Khorana kindly sent me a sample of -D-ribofuranosyl-1,5-diphosphate, which he had prepared by chemical synthesis. It appeared to have the same coenzyme properties in the phosphoribomutase reaction as the compound, which I had isolated. At that time it was known that adenine may serve as a precursor for the formation of adenosine monophosphate in a number of mammalian tissues. Several leading scientists in the field suggested that a reaction might take place between adenine and ribose-1,5-diphosphate leading to formation of AMP and orthophosphate.This would be similar to the nucleoside phosphorylase reaction. Two guest scientists (Saffran and Scarano) in the


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Copenhagen laboratory seemed to have obtained good evidence with extracts from pigeon liver that this was actually the case [7,8]. However, soon afterwards, Arthur Kornberg found an enzyme that catalyzed the formation of 5-phosphoribosyl1-pyrophosphate from ATP and ribose-5-phosphate. This compound was soon shown to react through another enzyme reaction with adenine to form AMP and pyrophosphate. A full explanation of the findings in Copenhagen was never obtained.

National Institutes of Health 1952^1953 In the month of March 1952, my wife, our 21 months old son (Steen Klenow), and I were on our way from Copenhagen to NewYork on board a small Danish freighter. After a rough voyage we spent a few days in NewYork and went by train toWashington D.C. where Bernie Horecker picked us up. It was my good fortune that Arthur Kornberg had accepted me as a member of his group at The Enzyme Section of the National Institutes of Health in Bethesda, Maryland. I had obtained a fellowship from the Danish Egmont H. Petersen Foundation, which made it possible for us to spend a year abroad. I became directly affiliated with Bernie and his work on the oxidative pathway of glucose metabolism. I had a most fruitful and rewarding time at this very well organized laboratory where certain good rules were to be followed. At noon every day there was a journal club session from 12:00 to 12:30 for those who were invited to participate. I was lucky to be among them. Other participants were, in addition to Horecker, Kornberg, and Leon Heppel, also as guests Osamu Hayaishi and H. A. Barker. From other laboratories at NIH came Herbert Tabor and Alan Mehler. If a member of The Enzyme Section was invited to give a seminar on his work at another laboratory or at a meeting like the Federation Meeting he first had to give a rehearsal for the group. If there was too much criticism from the audience he had to repeat it until it was in an acceptable form. Likewise, written publications were not to be submitted before the draft

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had circulated among the members of the staff and corrected according to the comments and the criticism. At that time the metabolic pentose phosphate pathway was not known. It was known, however, from the work of Otto Warburg with Zwischenferment (glucose-6 -phosphate dehydrogenase) that glucose-6 -phosphate may be oxidized to 6-phosphogluconic acid with NADP as a coenzyme. Bernie had studied the further enzyme-catalyzed oxidation of 6 -phosphogluconate and found the products besides CO2 to be ribulose-5-phosphate and again NADP served as a coenzyme. In most enzyme preparations, ribulose-5-phosphate was in enzyme catalyzed equilibrium with ribose-5-phoshate. When I joined Bernie, he had just found that extracts of liver and spinach contained an enzyme that catalyzed a reaction between two pentose-5-phosphate molecules giving rise to the formation sedoheptulose-7phosphate and triose phosphate. We now purified the enzyme and it was soon found that it catalyzed the formation or the breakdown of keto linkages and it was, therefore, referred to as transketolase. The substrates for the enzyme reaction was later found to be xylulose-5-phosphate and ribose-5-phosphate. It soon appeared that the transketolase reaction required thiamine pyrophosphate as a coenzyme. Since the reaction consisted in the transfer of a glycolaldehyde group from a ketose compound to an aldehyde compound it was suggested that ‘‘active’’glycolaldehyde was formed as a complex with thiamine pyrophosphate. Free glycolaldehyde could not be detected during the enzyme reaction. A number of compounds were found to act as donors, respectively as acceptors of glycolaldehyde in the transketolase reaction. This gave rise to quite a puzzle to account for the possible metabolic pathways of trioses, tetroses, pentoses, hexoses, and heptoses. Experiments with ribose 14C labeled at specific carbon atoms were very helpful in further identification of the metabolic conversions of ribose 5-phosphate [9,10]. Before I left, the transaldolase was isolated and now all of the steps of the pentose phosphate pathway could be written down for the first time.


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In the spring of 1953, my Danish fellowship expired so my wife and I had to decide what to do next. I was, of course, very tempted to accept a generous invitation from Jack Buchanan to come and work with him at MIT on the biosynthesis of purine compounds. However, major changes at home in Kalckar’s institute seemed to occur. Since it might affect my future there, I felt that it was important for me to go back. In addition, my wife was not too keen to spend another year abroad not least because she was expecting our second child. The final result was that my stay at The Enzyme Section was extended for 3 months supported by the NIH. I had had a most exciting and rewarding time there. The experimental work had been a major exercise in enzymology and carbohydrate chemistry and I had not only learned a lot, I had also obtained a good acquaintance with American biochemistry not least by participating in a Federation Meeting in New York and in another one in Chicago. We left Bethesda in the month of May 1953. We went by train from Washington D.C. to New York where the parents of my friend Jonathan Wittenberg generously had invited us to stay for some days in the top flat of their charming house in Greenwich Village, downtown New York. We enjoyed being tourists in this metropolis and it was nice having some time off together. Vacation was not at the agenda of The Enzyme Section. We had a pleasant voyage to Gothenburg, Sweden, on board the liner ‘‘Gripsholm’’of the Svenska Amerikalinien. After a short journey by train to Copenhagen we had a happy reunion with our families. A few weeks later, my wife gave birth on Midsummer Day to our second son (Lars Klenow).

Back in Copenhagen In those years there was a growing excitement about enzyme reactions involving DNA and other 2-deoxyribose compounds. In 1950 Herman Kalckar together with E. Hoff-Jrgensen and

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Walter MacNutt, an American research fellow, had found that extracts of Lactobacillus helveticus contained an enzyme that catalyzed the exchange of purines and pyrimidines between 20 -deoxyribosides without the intervention of phosphorolysis, i.e. by a trans-N-glycosidase reaction. Kalckar and Morris Friedkin had also shown that nucleoside phosphorylase was active even with 20 -deoxyinosine or 20 -deoxyguanosine as substrates giving rise to formation of hypoxanthine and guanine, respectively together with the highly acid labile 2-deoxyribose1-phosphate. In the year after his time in Copenhagen with Ole Maale and Kalckar, Jim Watson together with Francis Crick published in 1953 the startling discovery of the structure of DNA. J. R. Totter had shown that the carbon atom of formate served as a precursor for the carbon of the methyl group in thymine in DNA both in vivo and in vitro. In 1956 Arthur Kornberg published the first of a number of fundamental papers on the enzymatic synthesis of DNA. I became interested in the myokinase reaction and found together with Eleanor Lichtler from USA that it could take place albeit at lower rate also with 20 -deoxyAMP as a phosphate acceptor instead of AMP and with ATP as a phosphate donor. Both 20 -deoxyADP and 20 -deoxyATP were isolated in preparative amounts. They were easily separated from the respective ribose moieties on columns of cellulose powder in the presence of borate, a compound that make complexes with ribose residues but not with 20 -deoxyribose residues [11]. Together with a biochemistry student (Bjrn Andersen), I found that 20 -deoxyATP could substitute for ATP in the enzymatic reaction that led to the formation of the coenzyme NAD from ATP and nicotinamide mononucleotide. This 20 -deoxyribose analog of NAD was isolated and it was shown to serve as a hydrogen acceptor in several dehydrogenase reactions although less efficiently than if NAD was a hydrogen acceptor [12]. Kalckar had in a relatively short span of years after his return to Denmark attracted a large number of highly gifted foreign scientists to his institute in Copenhagen. They came


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from several European countries and from the USA. Among these were two later Nobel Prize winners, Jim Watson and Paul Berg. However, by 1954 Kalckar accepted an invitation from NIH to conduct basic medical research in a field of biochemistry in Bethesda. During the following year it became clear that he probably would stay in the USA for good. This meant that the institute in Copenhagen that was established for him sooner or later would be closed down. We were three Danish pupils of Herman’s, Agnete Munch-Petersen, Paul Plesner, and myself who had to look for other possibilities if we wanted to continue in the field of research that Herman had introduced to us in such a fascinating way. The outlook was not promising, indeed.

Cancer Research In 1956 I was lucky to be approached by leaders from the Danish Cancer Society. Among their activities they were running a laboratory (The Fibiger Laboratory). It had a biological section and they now wanted to extend it with a biochemical section, and they generously offered me to act as the leader of this new laboratory. This was, of course, a most welcome and appealing opening and it was even arranged that Agnete and Paul could join me for a period until they found more permanent affiliations. In addition, I was fortunate to get permission from both Herman Kalckar and from the university to transfer the scientific equipment that Kalckar had acquired for his institute to the premises of The Fibiger Laboratory. This meant that our experimental work could continue almost without interruption. In this period our third son (Anders Klenow) was born (on the first day of 1955). In 1956 I moved to The Fibiger Laboratory and two years later (on the longest day of the year) our fourth son (Niels Klenow) was born.

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Experiments with 20 -Deoxyadenosine As a researcher in cancer, I found it relevant to study DNA synthesis in tumor cells. I chose Ehrlich ascites tumor cells. They were easy to obtain in relatively large amounts at low cost from the peritoneum of mice as suspensions of single cells of which many were in the S-phase of the cell cycle. I first asked if adenosine or guanosine might serve as precursors for the synthesis of DNA. Ascites tumor cells were incubated with either of the two purine ribosides labeled uniformly with 14C. Afterwards the DNA of the cells was isolated and enzymatically degraded to 20deoxyribonucleotides. Together with Eleanor Lichtler I found that 20 -deoxyAMP from DNA was 14 C labeled both in the 20 -deoxyribose part and in the adenine part of the molecule in a way that suggested that most if not all of 20 -deoxyadenosine from DNA had been formed from adenosine without cleavage of the N-glycosidic linkage. Similar conclusions could be drawn from the experiment with uniformly labeled guanosine. Also in this case the labeling pattern suggested that the purine ribonucleoside might be converted to the deoxyribose moiety without cleavage of the N-glycosidic linkage [13]. I next asked if precursors of DNA like adenine, adenosine or 20 -deoxyadenosine had any effect on DNA synthesis. Cell suspensions were incubated with 14C formate in the absence or in the presence of either of the mentioned compounds. DNA was then isolated and enzymatically degraded to 20 -deoxyribonucleotides. TMP was heavily labeled in all cases except when the cells had been incubated with 20 -deoxyadenosine. In this case the labeling was only 5^10% of that in the control cells. Since it was found that the specific activity of the acid soluble thymine precursor compounds were almost the same in control cells and in the experimental cells it was concluded that 20 -deoxyadenosine inhibited DNA synthesis. When the experiments were repeated with 32P orthophosphate as tracer instead of 14C formate results that led to the same conclusion were obtained [14].


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It soon became clear that not 20 -deoxyadenosine itself but a labile intracellular metabolite thereof was the inhibitor of DNA synthesis. From experiments by Agnete Munch-Petersen and with Kay Overgaard-Hansen it was suggested that 20 -deoxyATP which accumulated in the cell was the active compound [15,16]. This was further supported by experiments performed by Alan Coddington whom we were fortunate to have with us from the UK. He and a biochemistry student of ours (Margit BaggerSrensen) showed that there was a close correlation between the cellular concentration of 20 -deoxyATP and the degree of inhibition of DNA synthesis [17]. It was further found that the inhibition of DNA synthesis caused by 20 -deoxyadenosine was immediately and almost completely lifted by the addition of both 20 -deoxycytidine and 20 -deoxyguanosine to the cell suspension while either of the two compounds alone had no effect. This indicated that 20 -deoxyATP inhibited DNA synthesis by preventing the reduction of guanosine and cytidine phosphates to the corresponding 20 -deoxy-derivatives. The antagonistic effect of 20 -deoxyguanosine and 20 -deoxycytidine might then be ascribed to their direct phosphorylation. The enzymatic reduction of guanosine compounds and of cytidine compounds to the respective 20 -deoxy-derivatives would consequently be bypassed [18]. Experiments by Peter Reichard in Sweden supported this possibility. He was studying an enzyme from chicken embryos that catalyzed the reduction of all four ribonucleotides to 20 -deoxyribnucleotides. He now found that micromolar concentrations of 20 -deoxyATP inhibited the reduction step. This key enzyme (nucleoside diphosphate reductase) in DNA synthesis has been studied in all details in Peter Reichard’s laboratory. It appeared to be an enzyme, the activity of which is highly regulated and subject to complicated allosteric control. At this point it was clear to us that even if the principle of inhibition of DNA replication as we had observed should be applicable in treatment of some sort of cancer the use of 20 -deoxyadenosine as such or alone would probably be out of

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question. This was due to the ubiquity of adenosine deaminase in human tissues. This enzyme is highly active not only with adenosine but also with 20 -deoxyadenosine as a substrate. The rate of deamination of 20 -deoxyadenosine to the inactive 20 -deoxyinosine derivative would, not least in the blood stream, prevent the accumulation of 20 -deoxyATP in cells in vivo from added 20 -deoxyadenosine. The problem might be solved in one of the following two ways. It might be possible to obtain a compound that efficiently inhibited adenosine deaminase. Alternatively it might be possible to develop a structural analog or derivative of 20 -deoxyadenosine that was resistant to adenosine deaminase but retained the other properties necessary for inhibition of DNA replication. We made some attempts in both directions. A biochemistry student of ours (Sune Frederiksen) prepared 2-aminopurine-20 -deoxyribonucleoside from 2-aminopurine and thymidine in a reaction catalyzed by deoxyribosyltransferase from L. helveticus. This compound appeared to be an excellent inhibitor of adenosine deaminase [19]. Likewise, Alan Coddington prepared N-6 -methyl-20 -deoxyadenosine by chemical synthesis [20]. This compound was also a potent inhibitor of adenosine deaminase. When it was present in a cell suspension together with 20 -deoxyadenosine the endogenous adenosine deaminase of the cells was inhibited. This resulted in extension of the duration of the period in which both 20 -deoxyadenosine and 20 -deoxyATP was present in the cells and also of the period in which DNA synthesis was inhibited [17]. Later on, even much more potent inhibitors of adenosine deaminase were developed in other laboratories. Of special interest was the appearance in the early 1980s of the highaffinity adenosine deaminase inhibitor 20 -deoxycoformycin. This compound may give rise to accumulation in the blood cells of 20 -deoxyadenosine and of 20 -deoxyATP, both derived from DNA under erythrocyte formation. The compound had a pronounced antileukemic effect. 20 -Deoxycoformycin is in recent years being used with considerable success in many


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oncological wards in the treatment of chronic lymphocytic leukemia and hairy-cell leukemia. Of a number structural analogs and derivatives of 20 -deoxyadenosine that we tested, the only one which apparently mimicked the effect of 20 -deoxyadenosine on DNA synthesis was 20 -deoxyadenosine-1-N- oxide. Sune Frederiksen and I had prepared it by treatment of 20 -deoxyadenosine with monoperphthalic acid [21]. This compound was not a substrate for adenosine deaminase and was not converted to the corresponding triphosphate in ascites tumor cells. It did, however, give rise to accumulation of 20 -deoxyATP and to inhibition of DNA synthesis in the tumor cells. What happened was that an enzymatic process slowly reduced the 1-N-oxide to the parent compound, which was then phosphorylated to the triphosphate [22]. The search for congeners of 20 -deoxyadenosine resistant to adenosine deaminase and with properties that resulted in inhibition of DNA synthesis was in the following years pursued in several laboratories. Halogenated derivatives of 20 -deoxyadenosine and other adenine nucleosides were developed in the 1980s. Of these the adenosine deaminase resistant compounds 2-chloro-20 -deoxyadenosine and arabinosyl-2-fluoroadenine were of special importance since they, after intracellular phosphorylation, inhibited DNA synthesis at various levels. In the late 1980s, they were found to have antileukemic effect and in recent years they are being used in many oncological wards with excellent results in the treatment of chronic lymphocytic leukemia and hairy-cell leukemia. Experiments with 30 -Deoxyadenosine In 1961 A. J. Guarino of Michigan generously provided me with a sample of cordycepin. It had been isolated from the liquid growth medium of the mould Cordyceps militaris and it had been shown to inhibit the growth of several strains of bacteria and to prolong the life of mice bearing Ehrlich ascites tumor

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cells. Cordycepin was believed to be an adenine nucleoside with a branched carbon chain of the glycosidic part of the molecule. However, in 1964 it was definitely shown that the chemical structure was that of 30 -deoxyadenosine, a hitherto unknown naturally occurring substance. In our laboratory the 30 -deoxyadenosine was subject to several studies on its effect on the metabolism of Ehrlich cells. When we later on ran out of the supply, we developed a procedure for the isolation in few steps of the pure crystalline compound in high yield. An amateur gather of mushrooms had guided us to a place in a park near Copenhagen where characteristic sporophores of Cordyceps militaris were growing on insect pupae. Several monospore cultures were isolated and the best producer of 30 -deoxyadenosine was grown in large scale for isolation of the compound [23]. We found that at relatively low concentrations of 30 -deoxyadenosine, the compound was efficiently taken up by Ehrlich cells and converted to the corresponding triphosphate while only a minor part was deaminated to the inactive 30 -deoxyinosine. This gave rise to inhibition after an induction period of the synthesis of RNA but not of DNA [24,25]. With Kay OvergaardHansen it was found that under such conditions the incorporation of adenine into AMP was prevented. Experiments with isolated enzymes showed that it could be explained by the finding that 30 -deoxyATP inhibited the enzyme that catalyzed the formation of 5-phosphoribosyl-1-pyrophosphate [26,27]. This was in agreement with the finding of others that 30 -deoxyadenosine may inhibit the de novo synthesis of ribonucleotides in cells. RNA synthesis was also studied in a cell-free system obtained from isolated nuclei. It consisted of a DNA^protein complex and catalyzed in the presence of all four ribonucleoside triphosphates, the synthesis of RNA as assayed by the incorporation of 32P-labeled AMP into the acid-insoluble fraction. In the presence of 30 -deoxyATP, the kinetics showed a complete blockade of the reaction even when the ratio of ATP to 30 -deoxyATP was high (i.e. about 50). However, there appeared to be an induction period of continued RNA synthesis before the blockade. This


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period varied directly with the ratio of ATP to 30 -deoxyATP in the reaction mixture. This type of kinetics was to be expected to result from the irreversible incorporation of a nucleoside monophosphate lacking the 30 -OH group into the 30 -end of a polyribonucleotide chain. The compound thus appears to stop the growth of RNA chains [28]. When we treated Ehrlich cells with relative high concentrations of 30 -deoxyadenosine, also the corresponding diphosphate and eventually the monophosphate accumulated in the cells and now also the synthesis of DNA was inhibited [25]. Since 30 -deoxyadenosine was found to be an excellent substrate for adenosine deaminase the 1-N-oxide also of this compound was prepared. Again in this case the mono-, di- and the triphosphate of the parent compound accumulated in cells when they were treated with the adenosine deaminase resistant 1-Noxide [29]. Thus a slow continuous administration of 30 -deoxyadenosine took place and inhibition of the synthesis of the nucleic acids resulted. Later on, Sune Frederiksen and Albert H. Rasmussen showed that the 1-N-oxide was much more efficient than 30 -deoxyadenosine itself both with regard to causing significant increase in the survival time of mice bearing Ehrlich ascites cells and with regard to inhibiting the growth of these cells in vivo [30].

Back to the University of Copenhagen In 1963 a professorship in biochemistry under the Medical Faculty at the University of Copenhagen became vacant. For me it was not immediately an appealing possibility. We had a good group of devoted young people and in addition to our studies on the effect of various adenine nucleosides on tumor cell metabolism, also the problem of cellular uptake of purine compounds was dealt with. Ulrik V. Lassen studied the mechanism of uptake of uric acid and related compounds. He published several papers partly together with Overgaard-Hansen. Our

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working conditions were in many ways excellent and we had a minimum of administrative obligations. Also the financing of our work was no big problem. In addition, I could hardly see myself as one of these wise and influential professors with heavy responsibilities. On the other hand, there were within the Danish Cancer Society diverging viewpoints with regard to which direction cancer research ought to follow. I had found that it had been a problem that the direction that I had chosen was not among the popular ones and that it probably would not be so in the future either. After many considerations and with reluctance I finally applied for the position. The scientific committee expressed itself positively about my research and the faculty approved. I obtained my appointment on June 1, 1964. In the time to follow, I was kept busy establishing a new biochemistry department. I was happy to be able to employ several of the people who had worked at the biochemistry section of The Fibiger Laboratory. This was 4 years before the turmoil of the world wide students’ revolt in 1968, which also hit Copenhagen. It later led to fundamental changes of the statute of the university.

Experiments with other Congeners of Adenosine In 1966 we were fortunate that John T. Truman from Harvard Medical School joined us for a longer period of time. He first studied the effect of 30 -amino-30 -deoxyadenosine on the metabolism of Ehrlich cells. The compound (a gift from H. A. Lechevalier) had been isolated from culture filtrates of the fungus Helminthosporium. The effect of this compound resembled very much that of 30 -deoxyadenosine. Again, incorporation of various radioactively labeled compounds into both RNA and DNA was inhibited and the di- and the triphosphate of 30 -amino-30 -deoxyadenosine accumulated in the cells. The triphosphate was isolated and it was found to function as a chain stopper in a DNA dependent RNA polymerase reaction with


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almost the same efficiency as that of 30 -deoxyATP [31]. John Truman and Sune Frederiksen showed that treatment of cells with 30 -deoxyadenosine had a differential inhibitory effect on the synthesis of various types of RNA. Again almost the same results were obtained in cells treated with 30 -amino 30 -deoxyadenosine [32]. So, the presence in the 30 -position of adenosine of an amino group instead of a hydrogen atom seemed offhand, not to make much difference. Yet, some years ago it was found that 30 -amino-30 -deoxyadenosine in contrast to other adenosine congeners including 30 -deoxyadenosine inhibited the replication of HIV virus in acutely infected cells [33]. In those days there was much interest (not least in the USA), in congeners and derivatives of adenosine compounds that were either naturally occurring or made by chemical synthesis. People were very generous in sending samples on request and we thought that it would be worthwhile to see if they were substratesfor adenosine kinase. Birte Lindberg partially purified the enzyme from rabbit liver and ascites tumor cells. Of the 38 compounds tested 20 were substrates and showed Michaelis^Menten kinetics with highly different KM-values [34]. These findings opened up for an almost embarrassing number of interesting possible further experiments with these compounds. Not knowing where to start or where to end I decided to look for other pastures. Experiments with DNA Polymerase from E. coli In the late 1960s a detailed knowledge of the mode of action of DNA polymerase from E. coli (later known as DNA polymerase 1) was available. This was almost entirely due to the monumental work carried out in Arthur Kornberg’s laboratory at Stanford. A number of properties of the enzyme made it, however, unlikely that this enzyme alone could account for the replication of DNA in vivo. Among the problems was that the enzyme could not initiate the synthesis of new DNA chains but only

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extend chains in the 50^30 direction. Another problem was the exonuclease activity associated with the enzyme. With nicked double-stranded DNA as a substrate, this 50^30exonuclease specific for double-stranded DNA would catalyze degradation of DNA ahead of the nick at the same rate as the chain behind the nick was extended. It was, therefore, believed that other enzymes with additional properties might exist. I had some faint ideas about these problems that I wanted to test. But I thought that I first should make myself familiar with the well-described enzyme from coli bacteria. So, my technical assistant Ida Henningsen and I set out to prepare the enzyme according to the procedure from Arthur Kornberg’s laboratory; Kay Overgaard later on joined us. At one instance we thought that we had lost a considerable amount of enzyme activity until we became aware that we, by a mistake in the reaction mixture, had used sodium phosphate buffer instead of the recommended potassium phosphate buffer for the assay of the enzyme activity. There had been a few reports in the literature that indicated that sodium ions might be inhibitory to the enzyme activity. We now decided to examine the effect of monovalent cations in the presence of buffers made from HCl and amines like collidin. It appeared that increasing concentrations of KCl had a pronounced (up to 15 fold) stimulating effect on the enzyme activity. Beyond the optimum concentration KCl became inhibitory. With the chlorides of rubidium, cesium, and ammonium similar but not quite so pronounced stimulation was observed. The activation constant for KCl was determined and it appeared to decrease drastically when the concentration of the amine buffer was increased. Lithium chloride and sodium chloride had no stimulating effect on the polymerase activity; in fact both salts counteracted the stimulating effect of the four above-mentioned salts. By manipulating the concentration of amine-HCl buffers and that of KCl, the polymerase showed full activity in the pH range from 7.4 to as high a value as 10.5 [35]. I gave a preliminary account of these findings at the FEBS meeting in 1968 in Prague. It was in those days when the


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atmosphere in that capital was very special and very optimistic; but few weeks later the Soviet army invaded Czechoslovakia. After my talk I was approached by a young American biochemist Tom Jovin. He came from Arthur Kornberg’s laboratory at Stanford and was going to spend some time at the University of Gottingen. He told me that he and his group at Stanford had prepared the coli DNA polymerase as a homogeneous protein and shown it to consist of a single polypeptide chain with a molecular weight of 109,000. He, kindly made the as yet unpublished procedure available to me. Back in Copenhagen I was busy finishing the experiments on the effect of salts on the polymerase activity. I afterwards wanted to make similar experiments on the exonuclease activity that was known to be a part of the same enzyme. For this purpose we made another preparation of the enzyme, this time according to Jovin’s procedure. A crucial step in this procedure was chromatography on a column of phosphocellulose. However, to our surprise we found that this step in our hands gave rise not to one but to two polymerase activity peaks of almost the same size.When the preparation was repeated the same phenomenon showed up again. At that time Tom Jovin visited us in Copenhagen. I had invited him to give a talk at a meeting of the Danish Biochemical Society. We discussed our findings of the two polymerase activity peaks but he too could not give a good explanation. He was, however, going on a short trip back to Stanford and would bring a sample of the authentic enzyme with him when he returned so that could compare it to the two activities of our preparations. Meanwhile, still another batch of enzyme was prepared and this time only one polymerase activity peak appeared as a result of the phosphocellulose chromatography. In addition, we were not able to detect any exonuclease activity towards nicked double-stranded DNA, the activity known to be associated with DNA polymerase from E. coli. At that time you had to prepare your own radioactively labeled double-stranded DNA as substrate for the exonuclease. So we thought that we had not made

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the right substrate. But when even poly d(A^T) (a molecule that inevitably would be double-stranded) did not serve as an exonuclease substrate we had to look for another explanation. It then occurred to me that this enzyme preparation had been interrupted at an early step in the procedure and that it had been left in the fridge for some days. All of the polymerase activity was, however, still present so we brought the preparation to the final step with the expected yield of polymerase activity. I then remembered that Linderstrm-Lang and Martin Ottesen at the Carlsberg Laboratory had experienced that a sample of crystalline ovalbumin after it had been left in the fridge for quite some time was converted to the so-called plaque albumin. It appeared that the fridge had been contaminated with B. subtilis, a bacterium that produced the highly active proteolytic enzyme subtilisin, which apparently had catalyzed limited proteolytic degradation of ovalbumin. In our case proteolytic enzymes might have been present in an early step of the preparation and could have caused a selective elimination of the exonuclease activity by limited proteolysis. In addition, when we analyzed the exonuclease free preparation on a gel filtration column we found it to have an apparent molecular weight of about 70,000. When the two polymerase activity peaks first obtained from the phosphocellulose chromatography were analyzed by gel filtration chromatography their apparent molecular weights were about 70,000 and 150,000, respectively, and only the latter enzyme was associated with exonuclease activity. Martin Ottesen encouraged me to treat this same enzyme with subtilisin. The result came out nicely: under proper conditions the exonuclease activity was selectively eliminated, and the apparent molecular weight of 150,000 was reduced to about 70,000. When Tom Jovin presented us with a sample of the authentic DNA polymerase from Arthur Kornberg’s laboratory, the same type of results were obtained when the enzyme was treated with subtilisin [36]. We observed that the action of subtilisin on the native enzyme under certain conditions could be monitored by a concomitant,


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at least, 2-fold increase in polymerase activity. The kinetics of the simultaneous decrease in exonuclease activity suggested that possibly a fragment of the enzyme harboring exonuclease activity was first formed followed by its deterioration. Enzymes some times have a more tight conformation and are less susceptible to proteolysis when bound to their substrate. Consequently, the subtilisin treatment of the native enzyme was repeated in the presence of the substrate for the exonuclease, nicked doublestranded DNA. Now there was an initial increase also in the exonuclease activity and when the reaction mixture was analyzed on a gel filtration column, the polymerase activity peak with an apparent molecular weight of 70,000 was followed by an exonuclease peak with an apparent molecular weight of about 35,000. These two molecular weights added fairly nicely up to the molecular weight of 109,000 found by Tom Jovin. Our finding that the apparent molecular weight of the intact enzyme was about 150,000 as determined by gel filtration was, of course, a problem. But the method for determination of molecular weight used by Jovin (sedimentation equilibrium analysis) was independent of the shape of the molecule while the results from the gel filtration method holds only for spherical molecules.We concluded, therefore, that the native enzyme had a shape that deviated considerably from that of a sphere. This enzyme might then consist of two separate folded structures connected by a short polypeptide chain particularly susceptible to proteolytic attack [37]. The determination of the N-terminal amino-acid sequences of the native enzyme and that of the two proteolytic fragments showed unambiguously that the polypeptide corresponding to the small fragment was placed in the N-terminal end of the native enzyme. We suggested that this multifunctional enzyme might have evolved by the fusion of two separate genes for the two functions. Before fusion the two separate genes may have been in a neighboring position [38]. As far as I remember, it was in 1975 at a FEBS meeting that Fred Sanger contacted me and said that he thought that the large fragment probably could be a useful tool in connection

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with development of a method for sequencing of DNA. Soon after he published his elegant sequencing procedure based on the synthesis of DNA chains of varying lengths catalyzed by the large fragment of DNA polymerase 1 and with 20,30 -dideoxyribonucleoside triphosphates as chain stoppers, followed by the separation of DNA chains according to their lengths by gel electrophoresis. In 1968 it had been found in Ed. Reich’s laboratory that the intact DNA polymerase contained yet another enzyme activity, a 30^50exonuclease specific for single-stranded DNA. This activity was in Arthur Kornberg’s laboratory shown to have a proof reading function for the DNA polymerase reaction. We found that this activity was located to the large proteolytic fragment of the enzyme.

Ribonucleotides Again Hans Flodgaard introduced me to automated HPLC technique. We were interested in the nucleotide content of human leukocytes. They were isolated by centrifugation of whole blood and the acid-soluble fraction was analyzed by HPLC on a column of an anion exchanger. However, in addition to the four expected UV-absorbing peaks, there was an unexpected fifth one in the triphosphate region. According to its position, it could be diadenosine 50,5000 -P1,P4 -tetraphosphate (Ap4A). When we examined the cell preparation under the microscope, it appeared that the cells inevitably were contaminated with blood platelets. To each cell was attached one or more platelets. Platelet preparations were easy to obtain in pure state and analyses showed now a ratio of putative Ap4A to ATP that was much higher than that obtained from the cell preparation. By enzymatic degradation it was unambiguously shown that human platelets contain relatively abundant amounts of Ap4A. Platelets are known to contain so-called dense granules that are characterized by the presence of very high concentrations of nucleotides like


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ATP, ADP, GTP, and GDP. These compounds are metabolically inactive in the dense granules; i.e. they are in contrast to the cytoplasmic nucleotides not labeled when platelets are treated with compounds like 3H adenosine. They are, however, released when platelets are induced to aggregate by treatment with e.g. thrombin. By these criteria we found Ap4A to be localized to the dense granula of the platelets of both man, rabbit, and rat [39]. The content of Ap4A of whole blood was found to correspond to the contribution from the platelets. On this basis we developed together with Paul Zamecnik a diagnostic test of a storage pool deficiency the so-called Chediak-Higashi disease. This sickness is a rare genetic disorder, which occurs in man and several other species, and it is characterized by the virtual lack of dense granules in the blood platelets. It appeared that this storage pool deficiency could be diagnosed by rapid and simple measurement of Ap4A in trichloroacetic acid extract of whole blood [40]. Paul Zamecnik had previously shown that Ap4A might be formed in the back-reaction of the amino acid-activation reaction. It was found to be present in minute amounts in several types of eukaryotic cells and the level varied widely with the proliferative activity of the cell. Based on a number of observations, it has been suggested that there might be a link between the amino acid-activation process and DNA replication in mammalian cells. It seems not to be quite clear what the function of abundant amounts in Ap4A in platelets is, but it has been found that it causes a dispersal of ADP-induced platelet aggregation.

Regulation of Cellular Content of Nucleoside Triphosphates In the early experiments with 20 -deoxyadenosine, I had observed that concomitant with the accumulation of 20 -deoxyATP in the cells their content of ribonucleotides (i.e. primarily ATP) declined to very low values. Later on others found similar

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phenomena in patients treated with potent inhibitors of adenosine deaminase and also in patients suffering from the lack of adenosine deaminase activity. In both cases 20 -deoxyATP (derived from DNA during erythrocyte formation) accumulated in the erythrocytes and their content of ATP was greatly reduced [41]. With the availability of the HPLC technique I wanted to return to these problems. Meanwhile, we had noticed that also with other nucleosides like 30 -deoxyadenosine similar effects were obtained, i.e. the cellular content of ATP was depleted as the ATP analog accumulated. We now looked at the effect of adenosine on the cellular content of ATP and on the metabolism of ATP. The latter was made possible by using cells in which the adenine ribonucleotides were labeled by pretreatment with radioactive adenine. By determination of the change of the specific radioactivity of ATP and of the increase in the total radioactivity of the growth medium the rate of turnover of ATP could be determined. Adenosine had two effects on the cells. It induced the catabolism of adenine ribonucleotides to inosine plus hypoxanthine and it could lead to expansion of the cellular pool of ATP. These two events appeared to be separate phenomena. Under certain conditions (low concentration or no content of orthophosphate in the growth medium) adenosine did not lead to any change of the ATP pool but it gave rise to accumulation at constant rate of inosine and hypoxanthine in the growth medium. There was, therefore, a complete coupling between the induced catabolism of adenine ribonucleotides and the counter balancing phosphorylation of adenosine. This may best be explained by assuming that adenosine is converted to AMP by a transphosphorylase reaction with inosine monophosphate (IMP) as phosphate donor, a reaction that may be catalyzed by 50 -nucleotidase. At the same time equivalent amounts of AMP is deaminated to IMP catalyzed by AMP deaminase. This reaction seems to be activated by the simultaneous decrease in the intracellular concentration of orthophosphate. If, instead of adenosine, an adenosine congener that is subject to intracellular


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phosphorylation is used in such experiments similar reactions may take place. The congener may be phosphorylated by a transphosphorylase reaction with IMP as phosphate donor. However, the monophosphates of many adenosine congeners (such as those of compounds like 30 -deoxyadenosine, purine riboside or even 20 -deoxyadenosine) are not substrates for AMP deaminase and therefore not subject to catabolism. A transphosphorylase reaction between IMP and e.g. 20 -deoxyadenosine leads then to depletion of the adenine ribonucleotide pool concomitant with formation of an equivalent pool of 20 -deoxyadenosine phosphates. If orthophosphate is available to cells, adenosine gives rise not only to turnover of ATP but also to expansion of the pool of ATP at a constant rate for more than two hours. This may lead to an increase of the cellular content by several folds. The latter phenomenon has a number of consequenses for the volume of the cells and for their content of inorganic ions [42]. The two effects of adenosine are separate and not interdependent. Inhibitors of either AMP deaminase or 50 -nucleotidase may prevent ATP turnover without affecting the rate of expansion of the pool of ATP. In addition, the activation constant for adenosine in the two reactions differ by a factor of about 6. The expansion of the ATP pool has the smaller activation constant for adenosine, and its value is of the same magnitude as the KM-value for adenosine kinase [43,44].

Conclusion Half a century ago, biochemistry was regarded as being a bit esoteric and of limited interest for the society. The family of biochemists was so relatively small that almost all in the field knew each other, or at least knew of each other. Looking back over the many years, it is overwhelming to recognize the enormous development of biochemistry in this period. It has been a greatly awarding intellectual experience and a great privilege to

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have had the opportunity to try to follow this progress. This holds especially for the events that led to the discovery of the structure of the most fundamental biological macromolecules and to the unraveling of the mechanism of their biosynthesis. I have been very privileged to have had great scientists as my mentors and to have had many gifted and devoted colleagues of whom several have become my close friends.

REFERENCES [1] Engberg, J., Andersson, P., Leick,V. and Collins, J. (1965) J. Mol. Biol. 104, 455^470. [2] Klenow, H. (1947) Acta Chem. Scand. 1, 328^334. [3] Kalckar, H.M., Kjeldgaard, N.O. and Klenow, H (1950) Biochim. Biophys. Acta 5, 575^585. [4] Klenow, H. (1952) Biochem. J. 50, 404^407. [5] Simmonds, H.A., Sahota, A. and Van Acker, J.K. (1995) In The Metabolic and Molecular Basis of Inherited Diseases, 7th Ed. (Stanbury, J.B., Wyngarden, J.B. and Frederickson, D.G. eds.), pp. 322^348. New York, McGraw-Hill. [6] Klenow, H. (1953) Arch. Biochem. Biophys. 46, 186^200. [7] Saffran, M. and Scarano, E. (1953) Nature 172, 949^951. [8] Scarano, E. (1953) Nature 172, 951^953. [9] Horecker, B.L., Smyrniotis, P.Z. and Klenow, H. (1954) J. Biol. Chem. 207, 393^403. [10] Horecker, B.L., Gibbs, M., Klenow, H. and Smyrniotis, P.Z. (1954) J. Biol. Chem. 207, 393^403. [11] Klenow, H. and Lichtler, E. (1957) Biochim Biophys. Acta 23, 6^12. [12] Klenow, H. and Andersen, B. (1957) Biochim. Biophys. Acta 23, 92^97. [13] Klenow, H. and Lichtler, E. (1957) Acta Chem. Scand. 11, 1080. [14] Klenow, H. (1959) Biochim. Biophys. Acta 35, 412^421. [15] Munch-Petersen, A. (1960) Biochem. Biophys. Res. Commun. 3, 392^396. [16] Overgaard-Hansen, K. and Klenow, H. (1961) Proc. Natl. Acad. Sci. USA 47, 680^686. [17] Coddington, A. and Bagger-Srensen, M. (1963) Biochim. Biophys. Acta 72, 598^607. [18] Klenow, H. (1962) Biochim. Biophys. Acta 61, 885^896. [19] Frederiksen, S. (1965) Biochem. Pharmacol. 14, 651^660. [20] Coddington, A. (1965) Biochim. Biophys. Acta 99, 442^451.


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[21] Klenow, H. and Frederiksen, S. (1961) Biochim. Biophys. Acta 52, 384^386. [22] Frederiksen, S. and Klenow, H. (1962) Cancer Res. 22, 125^130. [23] Frederiksen, S., Malling, H. and Klenow, H. (1956) Biochim. Biophys. Acta 95, 189^193. [24] Klenow, H. (1963) Biochim. Biophys. Acta 76, 347^353. [25] Klenow, H. (1963) Biochim. Biophys. Acta 76, 354^365. [26] Klenow, H. and Overgaard-Hansen, K. (1964) Biochim. Biophys. Acta 80, 500^504. [27] Overgaard-Hansen, K. (1964) Biochim. Biophys. Acta 80, 504^507. [28] Klenow, H. and Frederiksen, S. (1964) Biochim. Biophys. Acta 87, 495^498. [29] Frederiksen, S. (1963) Biochim. Biophys. Acta 76, 366^371. [30] Frederiksen, S. and Rasmussen, A.H. (1967) Cancer Res. 27, 385^391. [31] Truman, J.T. and Klenow, H. (1968) Mol. Pharmacol. 4, 77^86. [32] Truman, J.T. and Frederiksen, S. (1969) Biochim. Biophys. Acta 184, 36^45. [33] Vander Heyden, N., Rodi, C. and Rarner, L. (1989) AIDS Res. and Human Retroviruses 5(6), 647^653. [34] Lindberg, B., Klenow, H. and Hansen, K. (1967) J. Biol. Chem. 242, 350^356. [35] Klenow, H. and Henningsen, I. (1969) Eur. J. Biochem. 9, 133^141. [36] Klenow, H. and Henningsen, I. (1970) Proc. Natl. Acad. Sci. USA 65, 168^175. [37] Klenow, H. and Overgaard-Hansen, K. (1970) FEBS Letters 6, 25^27. [38] Jacobsen, H., Klenow, H. and Overgaard-Hansen, K. (1974) Eur. J. Biochem. 45, 623^627. [39] Flodgaard, H. and Klenow, H. (1982) Biochem. J. 208, 737^742. [40] Flodgaard, H., Zamecnik, P.C., Meyers, K. and Klenow, H. (1986) Thrombosis Res. 37, 345^351. [41] Siaw, M.F.E., Mitchel, B.S., Koller, C.A., Coleman, M.S. and Hutton, J.J. (1980) Proc. Natl. Acad. Sci. USA 7, 6157^6161. [42] Marcussen, M., Overgaard-Hansen, K. and Klenow, H. (1997) Biochim. Biophys. Acta 1358, 240^248. [43] Klenow, H. and stergaard, E. (1988) J. Cell. Physiol. 137, 565^579. [44] Overgaard-Hansen, K. and Klenow, H. (1993) J. Cell. Physiol. 154, 71^79.

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Chapter 7

A Risky Job: In Search of Noncanonical Pathways VLADIMIR P. SKULACHEV Department of Bioenergetics, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia

Introduction When my old friend Giorgio Semenza asked me to write this chapter, I immediately accepted his kind invitation. I did this not only because it is great honor to write for this prominent Comprehensive Biochemistry collection but also because I am now 67, an age to turn back and see what was already done and to avoid mistakes in the future. With age, mistakes become more and more expensive...

In the Beginning Short Excursion to the CV I was born in Moscow on February 21, 1935. My father Petr Stepanovich Skulachev (1907^1961) was an architect. He originated from a family of Russian peasants living in the Bryansk region (to the south-west of Moscow). My mother Nadezhda Aronovna Skulacheva (born 1910, nee Levitan) is also an architect. She is from a family of Ukrainian Jews who emigrated to

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Europe after the first Russian revolution of 1905 and returned to Russia after the second revolution (1917).

Student Years: Junior Courses In 1952, just after secondary school, I entered the Faculty of Biology, Moscow State University (MSU). Traditionally, biological education at MSU starts with zoology, botany, physics, chemistry, and mathematics whereas experimental branches of biology occupy senior courses. This is why my first small scientific work performed in the first year was related to zoology and botany being called ‘‘How flowers defend their nectar from ants.’’ This was a tribute to my interest in ants, which arose when I was in primary school to be with me all my life. Nevertheless, I did not regard myrmecology as my future occupation.

A RISKY JOB: IN SEARCH OF NONCANONICAL PATHWAYS

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When entering MSU, I wrote ‘‘Biochemistry’’ answering the question concerning a department of the Faculty of Biology which I prefer.

The Student Year: S.E. Severin and V.A. Engelhardt In the second year, I joined the famous seminar at the Department of Biochemistry chaired by the Head of the Department Professor Sergey Eugenyevich Severin and Professor Vladimir Alexandrovich Engelhardt. The latter name is well known in the West because of his two most famous discoveries, namely, oxidative phosphorylation (early 1930s) and ATPase activity of myosin (middle 1940s). On the other hand, S.E. Severin was less popular abroad although in the USSR his contribution was well recognized: he was founder (1939) of the MSU Department of Biochemistry and the head of this department during half a century (!), retiring in 1989 at age 88. In this period of time, the majority of the Russian biochemists graduated from his department. For many years, he was President of the Biochemical Society of the USSR and the Editor-in-Chief of Biochemistry (Moscow). Severin’s scientific career was unusual. He was a beloved pupil of Professor V. S. Gulevich, who discovered carnosine and carnitine in the very beginning of the twentieth century. Carnosine (-alanyl histidine) was, in fact, the first low molecular mass peptide described in living organisms. Now peptide biochemistry is a very important branch of the life sciences, but a long time proved to be necessary to recognize how important peptides are. In the 1930s, Gulevich asked Severin to identify the biological function of carnosine. Severin, in fact, solved the problem in 1953 when he found that addition of carnosine strongly increased the ability of isolated muscle to contract in response to an electric stimulus. The only change revealed in the carnosine-treated muscle was shown to be the accumulation of a huge amount of lactate. The explanation was

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obvious: the pK of carnosine is about 7, so carnosine neutralized Hþ produced by glycolysis and allowed the muscle to accumulate lactate without risk of acidification of the tissue. Paradoxically, Severin did not accept this explanation, deciding that pH buffer function is too primitive for a compound which was the main subject of his studies for so many years. He continued his search for carnosine function even after 1978 when Effron and coauthors reproduced ‘‘Severin’s phenomenon,’’ replacing carnosine with Tris buffer. In his studies, Severin addressed himself to quite different aspects of biochemistry, trying to find a place for carnosine in various metabolic processes or mechanisms of their regulation. After all, Severin’s pupils described an antioxidant effect of carnosine. In certain rare cases, it proved to be a neurotransmitter. Some other functions of this dipeptide were found by Russian and foreign biochemists, which confirm once more the statement that the majority of cell substances are polyfunctional. Such a broad field of interests resulted in Severin becoming a researcher of really encyclopedic learning in biochemistry, which accounts for his enormous success as university teacher and supervisor of several generations of young researchers graduated from the Department of Biochemistry, MSU. Engelhardt’s interests were always much more focused: he moved as deep as possible into the problem. I attended his course called ‘‘Enzymology.’’ He started with glycolysis but in the end of the course he failed to complete consideration of this chain of enzymic reactions. As to other numerous enzymes, they were not even mentioned. However, those glycolytic enzymes that were the subjects of the lecturer proved to be comprehensively analyzed. Thus, Engelhardt’s and Severin’s approaches were quite complementary, a fact explaining the great success of their seminar among the MSU students. Moreover, both of them were brilliant lecturers who liked teaching and students, one more attractive feature for those who decided to enter science.


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The seminars reinforced my decision to be a biochemist, and I entered Severin’s department in 1954. Here Professor N.P. Meshkova was my first supervisor. She asked me to isolate mitochondria from liver and skeletal muscle and compare their properties keeping in mind that muscle, but not liver, contains high concentration of carnosine. Later, Severin became my supervisor. He decided to reproduce, in the muscle mitochondria, the process of oxidative phosphorylation coupled to the cytochrome oxidase span of the respiratory chain, described in 1954 in liver mitochondria by Lardy’s [1] and Lehninger’s [2] groups. The goal of this study (in fact, my degree work) was quite traditional for Severin’s group; namely, to test the effect of carnosine on the studied biochemical reaction. Unfortunately, I failed to obtain ATP synthesis when ascorbate-reduced cytochrome c was oxidized by skeletal muscle mitochondria. Under the same conditions, succinate, pyruvate, and malate supported some oxidative phosphorylation. On the other hand, in liver mitochondria oxidative phosphorylation could be shown also with the ascorbate^cytochrome c system, just as in experiments of Lardy and Lehninger. I was quite disappointed by such a failure of my first attempt to find something new. I could not believe that in muscle, in contrast to liver, cytochrome oxidase is not coupled to energy conservation since substrates other than ascorbate þ cytochrome c were oxidized in the two tissues with the same P/O ratio. Apparently, muscle mitochondria proved to be less resistant than liver mitochondria to a hypotonic treatment required to allow added cytochrome c to reach the mitochondrial cytochrome oxidase. In other words, it was easier to uncouple mitochondria from muscle than from liver. However, what does uncoupling of respiration and phosphorylation mean? Why cannot glycolysis be uncoupled? Is uncoupling an in vitro artifact, or is there some biological function behind this strange phenomenon? I put all these questions but could not find answers. This is why there were more problems than solutions in my degree work. The great Engelhardt came to the department session

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when I defended this work. I was afraid that the founder of the phosphorylating respiration paradigm would attack and ruin my speculation that uncoupled respiration can also be of some physiological importance. However, Engelhardt did not criticize but instead even encouraged me saying that a few weeks earlier he met Lehninger who mentioned a surprising result of his group, pointing to existence of separate pathway of ‘‘nonphosphorylating respiration’’ in mitochondria in vitro. This was a happy end to my undergraduate student work.

Challenge 1: Nonphosphorylating Respiration in Thermoregulation Attacking the Major Bioenergetic Paradigm As already noted, studies of energetics of living systems resulted, in the 1950s, in a paradigm assuming that ATP synthesis is the only physiological function of respiration. This was a consequence of the following findings: 1. ubiquitous distribution of phosphorylating respiration among aerobic organisms, 2. role of ATP as convertible energy currency, and 3. identification of respiratory ATP synthesis as the major lightindependent energy-conserving mechanism of aerobes. As to respiration without phosphorylation, it was regarded as an in vitro artifact. This point of view was especially popular in Russia since the leading Russian biochemist V.A. Engelhardt made crucial contribution to all the three findings above. When a student came saying that nonphosphorylating respiration may also be of some physiological importance, the chance to succeed with such a nonconformist idea was close to zero.


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This is why Engelhardt’s public support at the defense of my degree work was so important for all the future studies that I performed within the framework of the nonphosphorylating concept. Severin, who became supervisor of my PhD thesis, also encouraged this kind of thinking because of his intuition as a broad biochemist and a hope to oppose something to triumphal Engelhardt’s success which always shifted to shadow Severin’s findings. When I became a postgraduate at Severin’s Department of Biochemistry in 1957, I started with summarizing rare observations found in the literature concerning nonphosphorylating respiration in vitro [3]. Together with Lev Kisselev, my first undergraduate student, I performed some in vitro experiments with mitochondria trying to demonstrate that uncoupling of respiration and phosphorylation can sometimes be reversible [4]. This was a piece of indirect evidence that uncoupling may be regulated, a feature surprising for an artifact. However, direct proof for the in vivo existence of nonphosphorylating respiration was needed. This was the goal of a series of experiments on the mechanism of so-called chemical thermoregulation, started in 1959.

Thermoregulatory Uncoupling: History of Discovery. Sergey Maslov For me, it seemed obvious that uncoupling of respiration and phosphorylation, if really exists in vivo, should take place in the warm-blooded animals under cold stress conditions when it is heat rather than ATP that is urgently needed. For a long time, it was known that cold exposure caused a strong increase in the oxygen consumption of mammals and birds. The question was: is this additional respiration coupled to ATP synthesis or is it uncoupled? In 1958, just before I started with this study, it was found in laboratories of Beyer [5], Hannon [6], and Smith [7] that a cold exposure results in some decrease

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in P/O ratio in rat liver mitochondria. However, the effect was rather small (no more than 30%) and required several weeks of the cold exposure, whereas stimulation of the in vivo oxygen consumption was much stronger and took minutes, not weeks. Therefore, it was clear that liver mitochondria of rats acclimated to cold are hardly a good experimental model to study in vivo uncoupling. One day my school-fellow physiologist D. Afanasiev, also an MSU postgraduate, asked me how to explain striking fast adaptation of pigeons to severe cooling, a phenomenon which he disclosed when searching for a hypothermia model. A pigeon was shorn to avoid physical thermoregulation and put into a refrigerator at 15 C with a strong wind produced by a ventilator. Under such terrible conditions, the pigeon died in less than 1 h if treated for the first time. However, the bird survived for several hours and maintained almost normal body temperature if put into the refrigerator for the second time on the next day after a 20^40 min first cold exposure. I immediately recognized that the Afanasiev’s pigeons may be an excellent model for my study. I suggested that at first cooling hypothermia develops so fast that an uncoupling mechanism fails to switch on, whereas at the second cooling something happened to prepare the animal to cold stress, uncoupling occurs in time, and saves the animal. Experiments I performed together with my friend Sergey Maslov, a very talented zoologist, confirmed the above reasoning. However, success did not come immediately. We started, like Beyer, Hannon, and Smith, with liver mitochondria and absolutely failed. No change in energy coupling was observed. On the other hand, breast muscle mitochondria proved to be almost completely uncoupled when isolated from a pigeon cooled for the second time. This uncoupling was accompanied by more than twofold increase in the oxygen consumption measured in vivo [8,9] (for review, see Ref. [10]). As to the first cold treatment, it also lowered P/O ratio in the muscle mitochondria but the effect was much smaller than after


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the second exposure to cold. The in vivo O2 consumption was unstable: initial stimulation changed to inhibition when the body temperature dropped drastically. In the same series of experiments, it was found that the P/O ratio of muscle mitochondria before the second cooling was almost as high as that of the nontreated control. This means that uncoupling upon the second cold exposure developed on the minute time scale, just as stimulation of the in vivo respiration [8,9,11]. Similar relationships were revealed in experiments on mice [9]. Here a very important prediction was verified by S. Maslov, namely, that in the first cold stress an animal fails to switch on the uncoupling mechanism and therefore dies. It was found that injection to mice of an artificial uncoupler, 2,4-p-dinitrophenol (which under normal conditions is a poison), prolonged the survival time at the first cold exposure [8]. These data just as the measurements of oxygen consumption by living animals, also performed by Maslov, were done to exclude explanation of the cold stress-induced uncoupling in isolated mitochondria as an in vitro artifact, like lower stability of mitochondria from the cold-treated birds or mammals. Thus, the cooperation of biochemist and zoologist proved to be fruitful. We coined the new phenomenon ‘‘thermoregulatory uncoupling,’’ being sure that the main part of the work was over. However, this was not the case. In fact, the topic of my first successful experiment followed me through all my life in science. It never became the major subject of my interest but was always present among other items in the research plans, just as my teacher in biochemistry, S.E. Severin, all his long life was searching for the function of carnosine. The next problem was to find out what the mechanism of thermoregulatory uncoupling was. The first observation concerning the mechanism was done in my experiments with pigeon and mouse muscle mitochondria. It was found that uncoupling caused by in vivo cooling could be in vitro abolished by serum albumin [9,11]. When I reported this observation at my first international meeting, 5th IUB Congress in Moscow (1961),

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Efraim Racker suggested in the discussion that the albumin effect may be an indication of the involvement of fatty acids as thermoregulatory uncouplers (uncoupling by fatty acids was already described by Pressman and Lardy [12] and Scholefield [13]). Our future studies confirmed this suggestion. We found that repeated short-term cold exposures resulted in an increase in concentration of free (nonesterified) fatty acids both in skeletal muscles and in isolated mitochondria. Fatty acids extracted from cold-treated pigeons caused uncoupling when added to the control mitochondria [14]. A symposium of the Moscow Biochemical Congress devoted to intracellular respiration was organized by S.E. Severin. I suggested calling it ‘‘Phosphorylating and nonphosphorylating oxidative reactions’’ and my supervisor accepted this title and invited me as a symposium speaker. Thus, I became the youngest speaker of the Congress. Unfortunately, just after the Congress a tragic event occurred: my father died of a heart attack... The role of fatty acids was later confirmed when Grav and Blix observed some uncoupling in skeletal muscle mitochondria from fur seals acclimated to cold under natural conditions, and this uncoupling could also be abolished by serum albumin [15]. More recently, Barre et al. identified skeletal muscles as the major site of nonshivering thermogenesis in ducklings under cold exposure [16] and revealed a fatty acid-mediated thermoregulatory uncoupling in skeletal muscle mitochondria of these birds [17,18]. However, real progress in understanding the mechanism of the phenomenon was made when another tissue was studied i.e., brown fat.

Brown Fat and UCP1 In late sixties, it became clear that in some mammals there is a tissue specialized in thermoregulatory heat production. This is brown fat [19].


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Brown fat mitochondria proved to be well equipped to form heat rather than ATP. They have a high level of the respiratory chain enzymes, low level of Hþ-ATP-synthase, and possess so-called uncoupling protein 1 (UCP1) discovered by Ricquier and Kader [20]. UCP1 composes 10^15% of the total protein content of the inner membrane of the brown fat mitochondria. No UCP1 was found in other tissues (for reviews, see Ref. [10,21^23]). The following chain of events resulting in uncoupling was identified: 1. Cold receptors in the skin send signals to hypothalamus and then via sympathetic neurons to the brown fat where noradrenaline is released. 2. Noradrenaline via -adrenalgenic receptors in the outer membrane of the brown fat cells activates adenylate cyclase in cytosol of these cells to form cAMP. 3. cAMP via a protein kinase cascade activates a triglyceride lipase so that free fatty acids are formed. 4. Fatty acids (i) come to mitochondrial matrix via fatty acyl CoA^fatty acyl carnitine system to be oxidized by the -oxidation and respiratory chain enzymes; (ii) uncouple their own oxidation by means of UCP1 already present in the brown fat mitochondria; (iii) induce de novo synthesis of UCP1 by stimulating activity of the nuclear gene encoding this protein. All these steps have been proved by direct experiments performed in several laboratories (reviewed in Refs. [10,21,22]).

The ATP/ADP Antiporter The brown fat studies, in spite of obvious progress in solution of the problem of the thermoregulatory uncoupling mechanism, failed to answer the question of what happens in other tissues

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where an additional amount of oxygen is consumed in response to cold. In mammals, brown fat amounts to too small portion of the total body weight to be responsible for all the cold-induced increase in the O2 consumption. In birds, where thermoregulatory uncoupling was discovered, there is no brown fat at all. Nevertheless, this uncoupling was fatty acid-dependent like that in brown fat. This forced me to search for protein(s) that are similar but not identical to UCP1 and can catalyze uncoupling by fatty acids in skeletal muscles. Structurally, UCP1 belongs to the family of mitochondrial anion carriers. Like these carriers, it is of about 30 kDa molecular mass, composed of three domains, each domain containing two transmembrane -helices [21]. Especially impressive similarity was revealed between UCP1 and the ATP/ADP antiporter. These proteins were found to have homology in primary, secondary, and tertiary structures. They form dimers and possess a purine nucleotide-binding site (one per dimer). Both of them contain no special signal sequence at the N-terminus of the polypeptide chain (reviewed in Refs. [21,22]). This is why I suggested that the ATP/ADP antiporter might be responsible for the fatty acid-induced uncoupling in skeletal muscle mitochondria. In 1987, my coworkers ^ E. Mokhova, A. Andreyev and some others ^ started with studies on the possible role of mitochondrial anion carriers in the fatty acid uncoupling. It was found that carboxyatractylate, the most potent and specific inhibitor of the ATP/ADP antiporter, recouples rat skeletal muscle mitochondria uncoupled by added fattyacids [24^27]. Later, the effect was reproduced in ATP/ADP antiporter proteoliposomes [28,29]. In yeast mitochondria, it was found that a mutation in the ATP/ADP antiporter strongly lowers uncoupling efficiency of fatty acids [30], and deletion in the antiporter gene abolishes the recoupling action of carboxyatractylate on the fatty acid uncoupling [31]. Further studies carried out in my group by Samartsev et al. [32,33] revealed that aspartate/glutamate antiporter can also be involved in the fatty acid uncoupling. This is apparently the case


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for the phosphate, dicarboxylate, and some other anion carriers (for review, see Ref. [10]). To explain these relationships, I put forward a suggestion that UCP1 and the anion carriers mentioned facilitate electrophoretic export of fatty acid anions from the internal to external leaflet of the inner mitochondrial membrane. On the external membrane surface, the fatty acid anions are assumed to be protonated to return with proton to the internal leaflet by a flip^flop mechanism. On the internal surface, protons release to the mitochondrial matrix, the fatty acid anions being regenerated [25,34]. This concept called ‘‘fatty acid circuit’’ was later supported by Garlid, Jezek, and coworkers [35], who obtained several pieces of evidence confirming separate steps of the above scheme. On the other hand, Winkler and Klingenberg proposed another mechanism assuming that fatty acid occupies a fixed position inside the hydrophobic core of UCP1 or of an anion carrier, thereby facilitating the Hþ trafficking [36]. Unfortunately, this hypothesis does not explain why all the mitochondrial proteins mediating fatty acid uncoupling are anion carriers. Even UCP1 can translocate wide range monoanions (Cl, hexane sulfonate, etc.), the translocation efficiency increasing with increase in the anion hydrophobicity. Garlid’s group found that undecanesulfonate is not only transported by UCP1 but also competes with laurate, inhibiting the laurate-mediated Hþ transport [35]. Moreover, only those fatty acid derivatives that easily penetrate a phospholipid bilayer in their protonated form can uncouple at low concentrations [37].

UCPs in Tissues Other than Brown Fat In the mid-1990s, state of the art in the studies on thermoregulatory uncoupling could be summarized as follows: 1. In brown fat, the mammalian tissue specialized in thermogenesis, there is a special protein, UCP1, which carries out

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the fatty acid-mediated uncoupling by facilitating the fatty acid anion trafficking through the inner mitochondrial membrane. 2. In birds and as well as in mammalian tissues other than brown fat, the same function is performed by the ATP/ADP antiporter and other anion carriers. This picture was supplemented with a quite new component when Ricquier, Fleury, and their colleagues reported in 1997 that there is a gene in the human genome that encodes a protein of 59% amino acid identical to UCP1. A very similar gene was also found in mice. In contrast to the UCP1 genes, the gene in question was found to be expressed in all the tissues but liver. It was ectopically expressed in yeast, and this resulted in uncoupling and inhibition of growth. The protein encoded by this gene was called UCP2 [38]. Also in 1997, Giacobino and coworkers identified one more UCP1-like gene inherent in skeletal muscles and brown fat, which has 57 and 73% identity to UCP1 and UCP2, respectively [39,40]. Later UCP4 [41] and Brain Mitochondrial Carrier Protein 1 (BMCP1, or UCP5) [42], also clearly belonging to the UCP family, were described. In 2001, a UCP was found specifically expressed in skeletal muscles of chicken, ducks [43], and hummingbirds [44]. Its sequence proved to be intermediate between mammalian UCP2 and UCP3. A family of UCP genes were found in plants [45,46] as was initially suggested by Vercesi [47]. Attempts to verify the participation of the new UCPs in thermoregulatory uncoupling were recently made in several laboratories. In Giacobino’s [39] and Ricquier’s [48] groups, it was found that the UCP2 mRNA level increases when an animal is exposed to cold. A similar cold effect was observed when plant UCPs were studied [45,49]. This was also confirmed by Lin et al. [50], who showed that in skeletal muscles the UCP2 mRNA doubles on the 3rd day of cold exposure of rat to be normalized on the 6th day. As to the UCP3 mRNA, it increases by factor 3 on


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the first day of cooling and decreases below the control level on the 3rd^6th days. Three hour cold exposure is without effect on both UCP2 and UCP3 mRNAs but already increases the mRNA of the plasma membrane glucose transporter 4, a protein responsible for glucose import by the muscle cells. The above dynamics points to existence of several lines of adaptive responses to cold at the level of the muscle cell gene expression: (i) greater supply of the muscle with the fuel (glucose) (takes hours); (ii) the UCP3-mediated uncoupling (1 day); (iii) the UCP2-mediated uncoupling (several days). Such a scheme does not exclude a contribution of the ATP/ADP antiporter and other anion carriers to thermoregulatory uncoupling. Just such a contribution seems to explain some data that the UCP3 gene knockout mice can still maintain almost normal body temperature during cold exposure [51] although knockout resulted in some increase in the energy coupling in the skeletal muscle mitochondria [51,52]. In the same way, one can account for the fact that knockout of the UCP1 gene proved to be critical for thermoregulation in some mouse strains [53] but not in others [54] (for discussion, see Ref. [55]). To elucidate the contribution of various fatty acid-mediated mechanisms to the thermoregulatory uncoupling, we recently studied effects of two- and one-day cold exposures of rat on the heart and skeletal muscle mitochondria, respectively. The treatments were shown to cause maximal increase in the UCP2 and UCP3 mRNA levels in the corresponding tissues. To be sure that not only the UCP mRNA but also the UCP protein levels are increased by cold, I asked Professor J.-P. Giacobino and his colleagues in Geneva to measure the amount of UCP3 in muscles before and after cold exposure. Experiments performed in my group by Dr. R. Simonyan [56,57] clearly showed that the cold exposure of an animal (i) decreases energy coupling in isolated muscle mitochondria and (ii) increases sensitivity of these mitochondria to uncoupling by the in vitro added fatty acids. Parallel measurement of the amount of UCP3 in the rat skeletal muscle mitochondria

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showed threefold increase in [UCP3] after 1 day cold exposure, which is in good agreement with data of Lin et al. [50] on UCP3 mRNA. These results seem to exclude explanation of uncoupling in mitochondria from cold-treated animals by an in vitro artifact such as absorption by mitochondria of extramitochondrial fatty acids during tissue homogenization or changes in sensitivity of mitochondria to the isolation and incubation procedure (we should always keep in mind explanations of this kind when trying to follow some in vivo effect by means of in vitro energy coupling measurements). A strong cold-induced increase in the amount of UCP3 cannot be accounted for by any in vitro artifacts. It seems quite reasonable to expect that mitochondria having higher concentration of an uncoupling protein have lower energy coupling.

The Story is not Finished Yet On the face of it, our and Giacobino’s data allow us to conclude that thermoregulatory uncoupling in skeletal muscles is directly proved and my postgraduate work is, after all, completed. However, a feature of biology, which attracts young students to this branch of science and simultaneously disappoints researchers of older ages, consists in that the system under study is so complex that it is rather easy to find a piece of evidence of something quite new but, on the other hand, it is terribly difficult to directly prove the finding in question. In other words, in biology it is very easy to start but it is very difficult to finish. Thermoregulatory uncoupling is not an exclusion from the above rule. Here, there are still some problems which remain obscure. One of them is why the concentration of UCP3 in either skeletal muscle or brown fat was very much lower than that of UCP1 in brown fat. [UCP1] in the brown fat mitochondria from hamster is about 500-fold higher than [UCP3] in the skeletal muscle mitochondria from warm-adapted rat [58]. Certainly, we should take into account the fact that brown fat


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of hamster, a hibernating animal, must be always ready for huge heat production, the only function of this tissue, whereas for skeletal muscle of rat, it is the ATP-dependent mechanical work that is the major function. Thus, it would be dangerous for muscle mitochondria to have a large level of an uncoupling protein. Moreover, the cold increases [UCP3] threefold, which decreases the difference between [UCP1] in brown fat and [UCP3] in muscle. According to Boss et al. [40], the level of UCP3 mRNA is much higher in the fast-twitch than in slowtwitch muscles. This seems to indicate that the UCP3-linked uncoupling is inherent in fast-twitch muscles, whereas in slowtwitch ones uncoupling is mediated by other mechanisms, e.g., the ATP/ADP and the aspartate/glutamate antiporters. In such a case, the UCP3 amount per mg protein of mitochondria isolated from total muscle tissue, measured by Brand and coworkers [58], should be lowered compared with that from fast-twitch muscles. As to the antiporters’ contribution to the uncoupling, it decreases, according to Simonyan’s data [57], from 59 to 47% after one-day exposure of the rat to the cold. It seems reasonable to assume that this is due to an increase in contribution of UCP3 whose concentration rises. We tried to verify this suggestion by measuring recoupling effect of GDP, a nucleotide which is assumed to specifically arrest the UCP activity. Surprisingly, the GDP recoupling is lowered, instead of being increased, by the cold [57]. This paradoxical observation is one more problem to be solved. A tentative explanation might be that cold exposure not only induces the UCP3 synthesis but also, in some way, desensitizes it to the GDP inhibition. As a result, both amount and activity of UCP3 rises [57]. If this is the case, one can expect that such an effect should disappear in UCP3/ animals. Now we are planning such an experiment. At any rate, it seems obscure how UCPs uncouple if there are always millimolar concentrations of purine nucleotides in the cell that should completely arrest their activity. This question is one more topic for future investigation in the thermoregulatory uncoupling.

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Among unsolved problems, I may mention the question of how an organism cancels thermoregulatory uncoupling in muscle, and what alternative mechanisms are activated to maintain the body temperature during long-term cold adaptation. Apparently, a decrease in energy coupling in tissues other than skeletal muscle is one of these mechanisms. Quite recently, Moreno-Sanchez and his colleagues [59] revealed UCP2 induction in liver at cold acclimation. Such an induction was found after 15 day cold exposure of rat, which was accompanied by some decrease in the ADP/O ratio, the short-term cooling being without effect. These observations are in excellent agreement with data obtained more than 40 years ago by the pioneering works on thermoregulatory uncoupling [5^7]. Finally, when speaking about perspectives, I should point to the molecular mechanism of transport of fatty acid anions by UCPs, the ATP/ADP antiporter, and other mitochondrial anion carriers. Here, crystallization and X-ray analysis of these proteins and their complexes with fatty acids are badly needed. In any case, there is still a lot to do when studying the thermoregulatory uncoupling, a phenomenon with which I started my scientific carrier so many years ago. Even more, analysis of current literature clearly shows that problem of physiological role of nonphosphorylating respiration is very much more popular now than previously. Discoveries of new UCPs by Ricquier’s and Giacobino’s groups in tissues other than brown fat strongly stimulated interest not only to the role of uncoupling in heat production but also in the functions of respiration alternative to the ATP production. In my first book published in 1962, I wrote that respiration is involved in four alternative functions; namely, energy conservation, energy dissipation, formation of useful substances, and removal of harmful substances [11]. The last function has now become especially popular in relation to defense against oxygen. High concentration of O2 is harmful, being favorable to generation of toxic reactive oxygen species. It is obvious that uncoupling mechanisms and, in particular,


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UCPs can facilitate performance of this and other alternative functions of respiration.

Challenge 2: Initially any Respiration is Nonphosphorylating; Chemiosmosis From Uncoupling to Coupling Mechanism: The Adenine Hypothesis After discovery of the phenomenon of thermoregulatory uncoupling, I faced the problem of what its molecular mechanism was. However, I recognized very soon that there is no chance to solve the problem of uncoupling before the mechanism of coupling of respiration and phosphorylation becomes clear. In the early 1960s, the traditional point of view was that respiration is coupled to phosphorylation in the same way as for already well-studied glycolysis. In glycolytic phosphorylation, substrates are oxidized in such a manner that a phosphorylated product is formed. Then, high-energy phosphoryl is transferred to ADP to produce ATP. There were many hypotheses trying to explain how electron carriers of the respiratory chain might produce a phosphorylated intermediate. Among them, one of the most extravagant was formulated by Professor L.A. Blumenfeld, Head of Department of Biophysics, Physical Faculty, MSU, and his colleague, Dr. M.I. Temkin [60]. They calculated that the free energy difference for protonation of an aromatic amine (aniline) and aliphatic methyl amine is equal to 8.4 kcal mol1, which is of the same order of magnitude as the energy price for ATP synthesis from ADP and phosphate. This proved to be a basis for speculation that the adenine part of ADP forms complexes with respiratory chain carriers which reduce adenine, an event resulting in conversion of aromatic amine of adenine to an aliphatic one. Such an event was assumed to be followed by phosphorylation of reduced adenine. Subsequent oxidation of reduced adenine by the next

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respiratory chain carrier should energize the phosphoryl residue which is then transferred to the pyrophosphate tail of ADP to form ATP. When I decided to switch from uncoupling to coupling, I badly needed some attractive new idea since my feeling was that the popular hypotheses assuming phosphorylated respiratory chain carriers are fundamentally wrong. The Blumenfeld and Temkin’s scheme did not require such an assumption. Moreover, it seemed to account for some effects of adenine nucleotides on mitochondrial respiration described in my group which I organized at the Department of Biochemistry, MSU. The group consisted of several under- and postgraduate students including Inna Severina, my future wife. It was Inna who studied redox properties of adenine and related compounds and their low temperature fluorescence which, we hoped, would be used to monitor postulated interaction of ADP with respiratory chain. This work resulted in some new physicochemical observations concerning adenine. However, all attempts to prove the speculation about participation of adenine nucleotides in the respiratory chain electron transport failed. Nevertheless, I am grateful to the Blumenfeld and Temkin’s scheme and to my old friend biophysicist Simon Shnol who attracted my attention to this scheme. It not only helped me to enter the energy coupling field but also allowed me to imagine how bioenergetic mechanisms could originate. I suggested that, when first living cells appeared, it was a photon of ultraviolet light, rather than reduction, that facilitated phosphorylation of the adenine moiety of ADP with subsequent ATP formation [61,62]. This simple mechanism, I think, was replaced by more complicated devices when UV became unavailable due to formation of the ozone layer in the atmosphere. As to ATP, it remained a convertible energy currency since too many energyconsuming processes were already supported by energy of ATP hydrolysis. The adenine mechanism of energy conservation, which is impossible on the modern Earth seems to be possible on Mars


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where ozone is absent. This is why I am impatiently waiting for results of the search for life on that planet.

Chemiosmotic Hypothesis: Peter Mitchell One of the attractive features of the adenine hypothesis of energy coupling was that it did not require any high energy intermediates of respiratory chain electron carriers since ATP was presumed to be immediately formed from ADP and phosphate. A search for such intermediates was performed in many laboratories in the late 1950s and 60s. Results proved to be quite negative and initiated numerous confusions. Independently from Blumenfeld and Temkin, a quite new hypothesis, called ‘‘chemiosmotic,’’ was introduced by Peter Mitchell. In 1961, he published a paper in Nature [63] postulating that electrochemical Hþ potential difference ð Hþ Þ exists on inner mitochondrial membrane and that it is Hþ that plays the role of a mysterious high energy intermediate of respiratory phosphorylation. Hþ was hypothesized to be generated by the respiratory chain. When formed, Hþ is consumed by Hþ-ATPsynthase, a hypothetic Hþ -utilizing enzyme immediately producing ATP from ADP and phosphate. According to the scheme, the function of respiration consists in an uphill Hþ transport through the mitochondrial membrane, the ATP formation being coupled to the oppositely directed downhill Hþ transport. If this is the case, respiration is always ‘‘nonphosphorylating.’’ As to phosphorylation, it is a further event occurring when the respiration-generated Hþ is used to actuate Hþ-ATPsynthase. Obviously, such coupling is very easy to destroy. To this end, it is enough to allow a downhill Hþ transport bypassing Hþ-ATP-synthase. Just in this way Mitchell explained the action of 2,4-dinitrophenol and other uncouplers that, as he supposed, facilitate the Hþ leakage through the membrane.

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A.N. Belozersky; Bioenergetics, a Department in MSU and a Branch of Biological Sciences I liked Mitchell’s hypothesis since it gave a chance to explain the mechanism of the thermoregulatory uncoupling as well as other numerous cases when respiration occurs without phosphorylation. However, I proceeded to its verification only five years after its publication. Such a delay was due, first of all, to that I tried to exhaust possibilities of an alternative (adenine) hypothesis. Moreover, the period between 1960 and 1965 was overcrowded by events only indirectly related to science or unrelated to it at all. In 1960, I married my first wife, Ksenia Myasoedova. In 1961, my father died and suddenly I found myself as the head of family. In the same year, I defended my candidate (PhD) thesis. In the next year I wrote my first book, ‘‘Interrelation of oxidation and phosphorylation in the respiratory chain’’ [11]. In 1964, my first beloved child appeared, daughter Tatiana. In 1965, I took part in organization of an Interfaculty Laboratory of Bioorganic Chemistry, which later was transformed to the Belozersky Institute of Physico-Chemical Biology, MSU. Professor Andrey Nikolaevich Belozersky, the Head of Department of Plant Biochemistry, MSU and Vice President of the USSR Academy of Science, was the founder of the Laboratory. This great man discovered, in the 1930s, DNA in plants and postulated that DNA is a compound of ubiquitous distribution in living organisms. In the 1950s, together with his brilliant student Alexander Spirin, he predicted the existence of mRNA. In the 1960s, Belozersky decided to perform an experiment on reintroduction of science into universities. Before the Second World War, science was expelled from universities in the USSR to be concentrated exclusively in specialized research institutes. Belozersky understood how bad this was for both science and education and convinced his friend and great mathematician I.G. Petrovsky, the Rector of MSU, to construct a new building, buy the best equipment, and organize a new quite large research


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laboratory to study various topics on frontiers of modern biology, chemistry, physics, and mathematics. The laboratory was composed of several small departments covering, in fact, all fields of biochemistry, biophysics, and molecular and cell biology. The average age of the heads of these departments was about 33, which was more than 20 years younger than that of heads of traditional education departments in MSU and other USSR universities. The laboratory was founded in 1965 when I was 30 and occupied the position of junior researcher at Severin’s Department of Animal Biochemistry. Belozersky invited me to organize a department in his laboratory saying that he is too old (at that time he was 60) to discover something and, therefore, he guaranteed me complete freedom in deciding how to call the future department and what topics I shall study. He kept this word and during the next seven years when he was Director of the Laboratory he never tried to interfere with any decisions concerning work of my department. When Belozersky was my Director, he did a lot to support my work. He always was for me a wise older friend rather than a boss or supervisor. This is why I am very very grateful to Andrey Nikolaevich Belozersky. I dubbed the new department ‘‘Bioenergetics’’ (this word was coined by A. Szent-Gyorgyi on the title page of his book published in the 1950s). In 1968, I took part in a conference on oxidative phosphorylation in Polignano-a-Mare, near Bari. At this conference, a special session was devoted to the question on how to call a new branch of science dealing with mechanisms of biological energy transductions. I suggested the name Bioenergetics. Being asked what the reason for such a term was, I explained that this is the name of my new department in Moscow. Surprisingly, the word was accepted.

Protonophores: Efim Liberman Keeping in mind the mechanism of thermoregulatory uncoupling, I started with a program of verification of that part

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of the chemiosmotic theory that deals with uncouplers. The program in question was initiated by my visit to a workshop on oxidative phosphorylation organized by L. Wojtczak just after the Warsaw FEBS Meeting in April, 1966. Here, for the first time, I met Peter Mitchell. I was very much impressed by his personality in spite of the fact that he absolutely failed to Britton Chance in discussing Methylene Blue responses of mitochondria. At the same workshop, Chappell presented his data showing that an artificial uncoupler is operative on liposomes, accelerating transmembrane equilibration of Kþ in the presence of valinomycin [64]. A similar effect was previously reported in experiments with mitochondria [65]. Mitchell explained it assuming that valinomycin allows Kþ to electrogenically cross the membrane, whereas uncoupler discharges the formed membrane potential by the Hþ flux in the direction opposite to that of the Kþ flux [66]. Chappell’s experiment clearly showed that the lipid, rather than the protein, component of mitochondrial membrane is responsible for effects of both valinomycin and uncoupler. In the same year, 1966, Lehninger’s group published, in Biochem. Biophys. Res. Commun. [67], a short note that a classical uncoupler 2,4-dinitrophenol (DNP) decreases electric resistance of a bilayer planar phospholipid membrane (BLM), one more effect predicted by Mitchell’s scheme of uncoupling. When I returned from Warsaw, I met a Moscow biophysicist, Efim Liberman, who observed an effect similar to Lehninger’s but with a fatty acid instead of DNP. Once after the famous Gelfand’s biological seminar, Efim kindly offered a lift in his car to my home. All the journey took less than ten minutes, but this time proved to be sufficient for us to come to an agreement to perform joint experiments which resulted in cooperation for many years. Liberman was one of the best specialists in BLM in the world at that time. I have never met in my life a scientist who was thinking so fast and sharp. The idea of our experiment was very simple. Both Lehninger’s and Liberman’s data could be explained by some occasional damage to BLM, caused by DNP or fatty acid, since resistance


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of the BLM, initially extremely high, was known to be lowered by quite different factors disturbing the regular lipid bilayer structure. On the other hand, if the Mitchell scheme was right, we may hope to find that (i) not only the two above mentioned uncouplers but all of them should decrease the resistance, (ii) efficiencies of uncouplers in mitochondria and BLM should correlate with each other, and (iii) the resistance decrease in BLM should be due to specific Hþ conductance. All these predictions proved to be right. Sixteen different synthetic uncouplers strongly decreased the BLM resistance. The C1/2 values in BLM and mitochondria were found to correlate. It was Hþ conductance that was responsible for the effect on the BLM. Experiments with tetrachlorotrifluoromethyl benzimidazole (TTFB) were especially demonstrative. In this compound, there is only one H atom and just it must be responsible for Hþ conductance due to dissociation of TTFBH to TTFB. It was shown that substitution of this H by ^CH3 completely abolishes activity of this very potent uncoupler in both mitochondria and BLM. I dubbed uncouplers operating in such a way as ‘‘protonophores.’’ This word proved to be my first contribution to the scientific English language [68].

Membrane Potential Revealed by Penetrating Ions Elucidation of the protonophorous mechanism of uncoupling directly confirmed one of the crucial postulates of the chemiosmotic hypothesis. The next piece of evidence in favor of this concept was obtained when we tested Mitchell’s assumption that respiratory and photosynthetic electron carriers, as well as membranous ATPases, generate transmembrane difference in electric potentials () [63,66]. To this end, we decided to find artificial ions penetrating through biomembranes. By definition, biomembranes should be impermeable for hydrophilic substances. Ions are hydrophilic due to dipoles of water molecules surrounding ionized atoms. Hydrophobic

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residues fail to make an ion penetrating since the resulting molecule tends to localize in the membrane^water interface, ionized group facing water and hydrophobic part immersed into the lipid layer of a membrane. To solve the problem, we tried ions where the charge of the ionized atom is delocalized over residues connected with this atom. I had a plan to recruit chemists to synthesize such molecules. However, Liberman could not wait and revised collections of organic compounds in MSU and Moscow research institutes, being sure that substances we badly need are getting dusty on a shelf of a chemical laboratory. And he proved to be right. Soon, we already had at our disposal several synthetic organic ions of the required structure. At first, they were tested in BLM. The prediction was that a trans-BLM concentration gradient of a penetrating ion should generate the Nernst diffusion potential of 60 mV per tenfold concentration difference. For cations and anions, the compartment with higher ion concentration should be charged negatively and positively, respectively. Such ions were really found. The most demonstrative proved to be tetraphenyl phosphonium cation (TPPþ) and tetraphenyl borate anion (TPB). These two ions differ by only a single central atom which was charged positively in TPPþ and negatively in TPB. Then the same ions were tested in mitochondria. This was done by my Lithuanian coworker and friend Antanas Jasaitis, who worked at that time in my department. It was shown that energization of mitochondria by respiration or ATP hydrolysis initiates fluxes of penetrating ions, cations, and anions moving into and out of mitochondria, respectively. The ion fluxes were completely abolished by protonophores [69]. This observation was later extended to inside-out submitochondrial particles and bacterial chromatophores where cations moved out of the vesicle whereas anions were taken up. In the case of chromatophores, not only respiration and ATP hydrolysis but also light could be the energy source [70^73]. These data proved convincing for the bioenergetic community who accepted the idea of electric current generators in the


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coupling membranes. It was important that we played with artificial ions. For such ions (in contrast to natural ions like Kþ), it would be hardly realistic to expect existence of any carriers in the mitochondrial membrane. Our observation was favorably accepted by the audience. At the FEBS Meeting in Madrid in 1969, my seven-minute talk was appreciated as a sensation. The data were immediately published in Nature. A series of four papers were accepted by Biochim. Biophys. Acta. In the latter case, I received from the editor a message written in Russian: ‘‘We are proud to publish your works.’’ David Green entitled one of the sections of his review ‘‘Skulachev ions’’ (Skþ and Sk for penetrating cations and anions, respectively) [74].

Proteoliposomes The main difficulty in interpreting and Hþ measurements on natural membranes is due to their complicated enzymatic and ion-carrier patterns. For instance, one cannot exclude that instead of being an intermediate between respiration and ATP synthesis, Hþ is a side product of one of them required for energy coupling as an allosteric control mechanism. To simplify the situation, one had to reconstitute a model membrane containing only one type of Hþ generators. This was first done in the early 1970s by Kagawa and Racker [75], who reconstituted vesicles from mitochondrial Hþ-ATPsynthase and phospholipids. An indication of ATP-dependent Hþ formation was obtained by means of the valinomycin and uncoupler probes. A similar approach was applied by Hinkle et al. [76] in Racker’s group and by Jasaitis et al. [77] in my group where the cytochrome oxidase Hþ generator was reconstituted. Curiously, I made a mistake in the title of the paper in Biochim. Biophys. Acta, reporting about these data. I wrote ‘‘Liposomes inlayed with cytochrome oxidase.’’ Later, I found the same spelling in the title of a paper by an Indian bioenergeticist who

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apparently decided, like the BBA editors, that incorporation of a protein into the liposome membrane should be written ‘‘inlayed’’ rather than ‘‘inlaid.’’ Fortunately, such a perversion of English was not accepted by the membranologists. On the other hand, the word ‘‘proteoliposome’’ which I introduced for reconstituted protein^lipid vesicles is now widely used ^ my second contribution to English... In 1973, Racker and Kandrach [78] obtained proteoliposomes inlaid with both cytochrome oxidase and Hþ-ATP-synthase. The vesicles proved to be competent in respiratory phosphorylation when those cytochrome oxidase molecules were actuated that were reduced by intraproteoliposomal cytochrome c. If electrons were supplied by both internal and external cytochromes c, respiration proved to be nonphosphorylating. This result was in line with our observation made by means of penetrating ions.We found that direction of in the case of internal cytochrome c was the same as that in the case of ATP hydrolysis, whereas external cytochrome c mediated generation of of the opposite direction [77]. Years 1972^1973 proved to be overcrowded with events of personal importance for me. A day before the New Year 1973 Belozersky died because of cancer of a very aggressive type. I was appointed as his successor at the position of Director of Laboratory of Bioorganic Chemistry. The appointment was quite unusual since, according to Soviet tradition, such a large institution (the staff, about 250 people) had to be headed by a communist party member. I have never been a communist and the reason why I was appointed was that Rector of University, Professor I.G. Petrovsky was also a nonparty man. For many years, he was like a symbol illustrating that nonparty people can also occupy top-level positions in the USSR. In fact, this was an absolute exclusion approved personally by Stalin who mentioned when appointing Petrovsky: ‘‘I know him. He is a nonparty man but (!) an honest person.’’ Petrovsky died the next day after he appointed me as the Laboratory Director.


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In 1973, I married for the second time, to Inna Severina who bore me three sons: Maxim (1973) and twins Konstantin and Innokenty (1976), making me quite happy...

Bacteriorhodopsin: W. Stoeckenius and E. Racker In January of 1973, I had a talk at a Bioenergetics conference organized by David Green in the New York Academy of Sciences. After the talk, somebody approached me saying that he has interesting information. This was Walter Stoeckenius, an electron microscopist involved in studies on membrane ultrastructure. We came to his room in the Americana Hotel where Stoeckenius put many figures on the table and bed. After an hour of conversation, it became clear for me that my new acquaintance had discovered a novel type of photosynthesis where the role of chlorophyll-containing electron carriers is performed by a retinal-containing protein, bacteriorhodopsin. As was shown by W. Stoeckenius and his younger colleague D. Oesterhelt, illumination of Halobacterium salinarium cells resulted in acidification of the medium, which was completely abolished by a protonophore. From New York, I came to Ithaca, being invited by E. Racker for a lecture. Here I told to Racker about Stoeckenius and Oesterhelt’s observation. He did not know about this and on the next day called Stoeckenius asking for a sample of bacteriorhodopsin. Soon Stoeckenius came to Ithaca with this protein and an experiment was performed which proved to be the last piece of evidence needed in favor of the chemiosmotic hypothesis. Racker and Stoeckenius [79] reconstituted proteoliposomes from a mixture of bacteriorhodopsin, beef-heart Hþ-ATPsynthase, and soybean phospholipids. The vesicles composed of constituents from the three kingdoms of living organisms (bacteria, animals, and plants) were shown to be competent in ADP photophosphorylation. In this system, mitochondrial Hþ-ATPsynthase utilized a Hþ that was produced in a manner quite

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different from the natural one (bacteriorhodopsin instead of respiratory chain enzymes). The results of bacteriorhodopsin studies shattered the ‘‘antiMitchellian’’concepts, bringing about a drastic change in public opinion; thus, the proton cycle scheme was accepted by the bioenergetic community as an experimentally proved theory. Four years later, in 1978, Mitchell received the Nobel Price in chemistry. As for me, I was invited as a plenary lecturer to the 11th International Congress of Biochemistry in Toronto, 1979 (The lecture was published in Can. J. Biochem. [95]).

Bacteriorhodopsin: L. Drachev, A. Kaulen, Yu. Ovchinnikov, and H. Khorana When I returned to Moscow from visits to New York and Ithaca in January, 1973, I told about Stoeckenius’ discovery to Yuri Ovchinnikov, a known Moscow bioorganic chemist and membranologist, Vice President of the Russian Academy of Sciences. We decided to organize a project called ‘‘Rhodopsin’’ to compare bacterial and animal retinal proteins trying to elucidate mechanisms of their functioning. The project proved to be successful. The structural part of the work done in Ovchinnikov’s Institute resulted in elucidation of the primary structure of bacteriorhodopsin, which proved to be the first membrane protein whose sequence was determined. The structure was published in 1978 by Yu. Ovchinnikov, N. Abdullaev et al. [80]. In 1979, the same work was completed by H. Khorana and coworkers [81]. As to my role in the project, I was responsible for the functional studies. To this end, I invited Dr. Lel Alexandrovich Drachev, a brilliant physicist who never studied biology previously and Dr. Andrey Kaulen, my pupil, young, and very talented bioenergeticist. The tandem proved to be extremely productive. In fact, L. Drachev and A. Kaulen invented a quite new method allowing to directly follow charge translocation not only across a membrane but also inside a membrane protein


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[82,83]. The method consisted in that proteoliposomes or natural membrane particles were adsorbed on the surface of a phospholipid-impregnated collodion film. Two electrodes are immersed to the salt solutions on both sides of the film. Actuating an electric current-generating protein by adding the light or a substrate resulted in charge displacement, which could be measured by a sensitive electrometer. The method was especially effective if the light can be used as an energy source. In such cases, short laser flash initiated single turnover of the studied protein, and electrogenic steps of the overall charge translocation process could be resolved. Later, A. Kaulen and A. Konstantinov succeeded in application of the method to such a ‘‘dark’’ Hþ generator as cytochrome oxidase. A lightsensitive electron donor was employed [84]. Originally, the direct method of measurement was developed by L. Drachev and A. Kaulen for bacteriorhodopsin [85]. The value measured under continuous illumination reached 200 mV, whereas the single turnover-inducing flash resulted in up to 60 mV [85,86]. The method revealed correlation of main steps of the bacteriorhodopsin photocycle and generation. Playing with mutants obtained by Khorana’s group, we identified some crucial amino acid residues involved in the Hþ translocation inside the bacteriorhodopsin molecule [87]. The same was done by L. Drachev, A. Semenov, A. Konstantinov, and coworkers for photosynthetic reaction centers from Rhodopseudomonas viridis [88]. These data combined with X-ray data ofH.Michelandcoworkers [89]allowedusto explainthe molecular mechanism of generation by this complex. It proved to be the first Hþ generator for which the principle of energy conservation was elucidated [25]. In the 1970s, the same approach was used in my laboratory to study cytochrome oxidase [84,90], Hþ-ATP-synthase [91], transhydrogenase [92], and some other Hþ generators [93]. These and many other aspects of biological membrane-linked energy transduction were summarized in my book ‘‘Membrane Bioenergetics,’’ published by Springer-Verlag in 1988.

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Protometer: A. Glagolev and S. Bibikov Photoreception was the first biological function postulated for bacteriorhodopsin by Oesterhelt and Stoeckenius [94]. Such a suggestion was based upon the facts that (i) Halobacteria, where bacteriorhodopsin was discovered, possess a phototaxis, and (ii) visual rhodopsin is a key component of the animal photoreceptor system. However, later the authors changed their mind assuming that it is the light-dependent Hþ generation that is the function of bacteriorhodopsin. Works of mine and some other laboratories’ directly proved that the Hþ generation is really inherent in bacteriorhodopsin. Nevertheless, some explanation for the halobacterial phototaxis mechanism was still required. In this connection, we hypothesized that bacteriorhodopsin is involved in photoreception but indirectly. It was assumed that the bacterium monitors Hþ by a hypothetical device which I called a ‘‘protometer’’ (a mechanism to measure the protonmotive force). According to the scheme postulated, a decrease or an increase in Hþ are the repellent or attractant signals for bacteria. If it is the case, (i) the light should result in an attractant photoeffect by inducing a bacteriorhodopsin-mediated Hþ increase, and (ii) bacteria should be more sensitive to the light if the light-independent Hþ generators are inhibited and bacteriorhodopsin becomes the only Hþ -generating mechanism. This prediction was verified by my young coworker Alexey Glagolev1 and his postgraduate V. Baryshev. It was found that inhibition of respiration and utilization of glycolytic ATP by cyanide and dicyclohexyl carbodiimide (DCCD), respectively, resulted in that much smaller decrease in the light intensity was perceived by the bacterium as a repellent signal [96]. The paper was published in Nature and 1

The Glagolev life story is rather complicated. He was born in USA as Kim Lewis. Then he was moved to USSR when he was a child. Here his mother changed his name to Alexey Glagolev. In late 1980s, during Gorbachev’s ‘‘perestroika,’’ Glagolev returned to his homeland. Now he successfully studies microbial bioenergetics as Professor Kim Lewis of the North-East University, Boston.


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the situation seemed to be clear until a family of rhodopsins were discovered in H. salinarium. It was found that this bacterium possesses, besides bacteriorhodopsin, three more retinal proteins: halorhodopsin which pumps Cl instead of Hþ and two sensory rhodopsins taking part in attractant and repellent effects of the light (for review, see Ref. [25]). Sensory rhodopsins, like animal rhodopsin, can operate as counters of photons making a bacterial cell sensitive to the minimal portion of light, a single photon. Thus, it was assumed that sensory rhodopsins, rather than bacteriorhodopsin, are responsible for phototaxis in H. salinarium. However, this assumption failed to explain our data on sensitization of bacteria to the light by adding cyanide and DCCD. To clarify the situation, I decided to create such a H. salinarium mutant that would correspond to the level of our knowledge concerning these bacteria at the time when we believed that bacteriorhodopsin is the single retinal protein of H. salinarium. To this end, I sent my postgraduate S. Bibikov to D. Oesterhelt’s lab in Germany to play with H. salinarium mutants. At first, a mutant was constructed which contained no retinal proteins at all. Then gene of bacteriorhodopsin was reintroduced into this mutant. When he returned to Moscow, Bibikov measured phototaxis of the mutants obtained. It was found that the mutant lacking all the rhodopsins is absolutely blind, whereas a mutant containing bacteriorhodopsin as a sole retinal protein has a phototaxis. However, its sensitivity to the light proved to be much lower than in the wild type. As to effects of cyanide and DCCD, they were well reproduced in the bacteriorhodopsinonly mutant [97,98]. From these data, I concluded that sensory rhodopsins and bacteriorhodopsin are responsible for sensing of dim and bright light, respectively (just as rods and cones in our eyes). Sensory rhodopsins, being counters of single photons, are extremely sensitive but, just for these reasons, they are saturated very soon when the light intensity increases. Bacteriorhodopsin is much less sensitive since it is involved in the light sensing in an indirect way ^ as a Hþ generator. One needs

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very many photons to be consumed by bacteriorhodopsin to obtain such a Hþ change which is measurable for the hypothetic protometer. It remained obscure what the protometer is in H. salinarium. There are some indications that in E. coli this role is performed by the so-called Arc system. We tried to attack this problem but the project was unhappy because of some objective and subjective reasons, namely, economic crises in Russia and my new love ^ sodium energetics. However, before switching from Hþ to Naþ, I was involved in studies on some consequences of the Hþ cycle theory.

Electric Motor Invented by Bacteria In 1974, Adler and his colleagues [99] observed that an E. coli mutant, competent in respiration but lacking oxidative phosphorylation, required oxygen for motility although the ATP level was O2 -independent. Moreover, strong lowering in the ATP level did not stop the oxygen-supported motility, while the addition of a protonophore did. The authors concluded that ‘‘a nonphosphorylating intermediate of oxidative phosphorylation,’’ rather than ATP, is the driving force for bacterial motility. By that time, it was already clear for us that such an intermediate is identical to Hþ . Therefore, we undertook a study to directly demonstrate this new function of Hþ . Such an investigation seemed very risky since it was based on a single observation of Adler’s group on a specific E. coli mutant. On the other hand, for a long time the mechanism of mechanical work in biological system was believed to be associated exclusively with the Engelhardt’s ATPase of the actomyosin type. ATPases were shown to participate not only in muscle contraction in animals but also in motility of spermatozoa, some plants, fungi, and protozoa, as well as in the movement of intracellular structures in eucaryotes. In particular, an ATP-dependent mechanism was shown to be operative in eucaryotic flagella.


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When it was found that bacterial motility is also flagellumlinked, the simplest idea was that it is ATP-driven, too. In 1975, Glagolev in my group confirmed Adler’s observation for quite another bacterium, Rhodospirillum rubrum. It was shown that also in this case bacterial motility did not correlate with the ATP level. At the same time, there was a good correlation between the motility rate and the level of membrane potential produced by the photosynthetic redox chain [100,101]. Later, our group [102,103] and two other laboratories [104,105] found that Rh. rubrum, Streptococcus, and Bacillus subtilis, paralyzed by exhaustion of the endogenous sources of Hþ generation, become motile for several minutes when artificial or pH of proper directions were imposed. In 1978, Glagolev and I published a paper in Nature summarizing our studies on this subject [103]. It was assumed that bacteria possess a molecular motor rotated by a transmembrane electric field or a pH gradient. A mechanism of rotation was postulated, namely, proton movement via a Hþ half channel leading (i) from outside to a single proton-acceptor group of the stator and (ii) from one of numerous proton-acceptor groups localized on the rotor to the bacterial cytoplasm. Now, this scheme is widely applied to explain rotation of core subunits of Hþ-ATP-synthase.

Extended Mitochondria as Intracellular Electric Cables The electric conductance of the media on both sides of a biological membrane is always very high whereas the transmembrane conductance can be extremely low. This means that , if produced by a Hþ generator molecule inlaid in this membrane, cannot avoid fast irradiation over the membrane surface. As to pH, it also must irradiate quickly due to the high rate of Hþ diffusion in water and the high concentration of mobile pH buffers which cause a further increase in the rate of Hþ movement. Thus, Hþ produced by a Hþ generator in a certain

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area of the membrane can be transmitted as such along the membrane and transduced to work when used in another region of the same membrane. In 1969^1971, I extended this line of reasoning to the hypothesis that coupling membranes act as power-transmitting cables at the cellular level [61,107]. Initially, this idea was confirmed byA. Glagolev and I. Severina in experiments on cyanobacterial trichomes [108,109]. Later, we came to mitochondria. Here the main obstacle was that it was generally believed that mitochondria are numerous small spherical bodies suspended in the cytosol. By definition, such structures cannot be used to transport power for a distance comparable to the size of a eucaryotic cell. However, the first students of mitochondria, who employed the light microscope, always indicated that mitochondria may exist in two basis forms: (i) filamentous and (ii) spherical or ellipsoid. The very term ‘‘mitochondrion’’ translated from Greek means ‘‘thread-grain.’’ The development of the electron microscope and thin section techniques changed the original opinion in such a way that filamentous mitochondria came to be regarded as a very rare exception, while the spherical shape was assumed to be canonical.This change of view stemmed from the fact that single section electron microscopy deals with a two-dimensional, rather than threedimensional, picture of the cell. A clew considered in this way may be erroneously interpreted as a number of small grains if its reconstitution with the aid of many parallel sections is not carrier out. Three approaches have shaken the dogma of spherical mitochondria, namely: (i) reconstitution of three-dimensionalelectron micrographs of the whole cell by means of serial thin sections, (ii) high-voltage electron microscopy allowing one to increase the thickness of the studied preparation, and (iii) staining of mitochondria with fluorescent penetrating cations. It seemed most interesting to verify the cable hypothesis in muscles since here the cell size is very large and intracellular energy gradients can limit the performance of work. Dr. L. Bakeeva and Professor Yu. Chentsov in our Institute kindly agreed to investigate serial sections of the diaphragm muscle


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and found that mitochondria in this tissue are united in a very extended three-dimensional framework composed of branched filamentous mitochondria which I called ‘‘mitochondrial reticulum’’ [106,110]. In heart muscle, numerous spherical mitochondria proved to be attached to each other with special structures (mitochondrial junctions) composed of four membranes of two adjusted mitochondria with some electron-dense material in the intermembrane spaces. The system was dubbed ‘‘Streptio mitochondriale’’ [111]. The next question was whether all those complicated structures were equipotential or they were composed of many electrically isolated small mitochondria. To solve this problem, we needed to return to the light microscopy and living cells instead of electron microscopy that always deals with dead (fixed) material.With the light microscopy, functional analysis is possible; but the great disadvantage is low resolution, which is insufficient to see mitochondrial filaments. However, such a limitation is absent if we deal with the light emission instead of the light absorption or scattering. The stars are seen in the night as light sources although they are not seen in the daytime as the light-absorbing bodies. Thus, the problem was how to obtain the light emission from mitochondria. An obvious approach consisted in that the cell should be treated with penetrating fluorescing cations which can electrophoretically accumulate in the mitochondrial matrix. In this connection, we turned our attention to rhodamine. It has been known since 1941 that rhodamines specifically stain mitochondria [112]. Ethyl-substituted rhodamine was found by I. Severina [108,113] to be a cation penetrating BLM. Then D. Zorov tried to employ ethylrhodamine for detecting a mitochondrial reticulum in the diaphragm muscle, where it was previously described using the electron microscope. Unfortunately, this tissue was hardly the best one for the new method because of high light scattering of the actomyosin fibrils and very complicated pattern of mitochondria profiles.

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Americans L.B. Chen and colleagues had better luck. They found that the treatment of fibroblast culture with a commercially available sample of rhodamine B-conjugated IgG antibodies resulted in the staining of some ‘‘snakelike structures’’ inside cultured cells, which were later identified as mitochondria [114]. Further study revealed that the staining was caused by contaminations of free rhodamine B in this sample [115]. After these publications, we turned from muscle to fibroblasts. Combining a laser and a fluorescent microscope,V. Drachev and D. Zorov in my group [116] succeeded in illuminating a small part of a single mitochondrial filament in a human fibroblast cell stained with a fluorescing cation, ethylrhodamine. A very narrow laser beam (the diameter of the light spot was commensurable with the thickness of the filament) was used to cause local damage to the filament. The laser treatment resulted in disappearance of the rhodamine fluorescence in the entire 50 mM filament. The illuminated filament retained its continuity when scrutinized under a phase-contrast or electron microscope. Other filaments remained fluorescent [25,117,118]. Further experiments on a culture of cardiomyocytes revealed that illumination of a single spherical mitochondrion gives rise to quenching of the ethylrhodamine fluorescence not only in the illuminated organelle but also in large cluster of mitochondria connected by mitochondrial junctions to the illuminated one. Some of the quenched mitochondria were localized as far as 40 mM from the site of illumination. On the other hand, mitochondria localized near the illuminated organelle but not connected to it by the junctions proved to be unaffected [25,117,118]. The above data clearly showed that the mitochondrial filament in fibroblast, as well as junction-connected Streptio mitochondriale in cardiomyocyte, represent electric continua. A hypothesis has been put forward that mitochondria localized close to the outer cell membrane consume oxygen and form Hþ which is then transmitted via the Reticulum (or Streptio) mitochondriale to the cell core mitochondria when Hþ is converted to ATP. Such a power-transmitting mechanism should


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facilitate energy supply of the cell core since very fast Hþ transmission substitutes for slow diffusion of respiratory substrate and O2 (or ADP, phosphate, and ATP) in cytosol crossed by intracellular membranes and cytoskeleton structures. Moreover, it allows a very large cell like a myocyte to operate in such a fashion that oxygen is present only in a narrow peripheral zone, the major part of the cell being anaerobic. This might create a line of antioxidant defense for the cell [119]. Dl Hþ as a Convertible Energy Currency: Dl Hþ Buffering by Naþ/Kþ Gradients In Mitchell’s chemiosmotic hypothesis, the role of Hþ was confined to be a transient on the way from electron transfer to ATP synthesis [63]. However, later in many laboratories, including ours, some pieces of evidence were accumulated indicating that Hþ is a polyfunctional component resembling, in this respect, to ATP. Like ATP, Hþ can be formed by several energy-conserving systems [complexes I, III, and IV of the respiratory chain, and (in photosynthetic cells) complexes of the reaction centers of photosystems I and II]. When formed, Hþ can be utilized to support all the types of work, namely chemical (synthesis of ATP and pyrophosphate, reverse electron transfer via complexes I and III, NADH ! NADPþ transhydrogenation), osmotic (uphill transport of metabolites through coupling membranes), mechanical (rotation of bacterial flagella), and heating (thermoregulatory uncoupling). Moreover, again like ATP, Hþ proved to be a transportable form of energy (power transmission along extended mitochondrial profiles or mitochondrial clusters). This allowed me to conclude in 1977 that ATP and Hþ are two convertible forms of energy employed in the water and membrane fractions of the cell, respectively [102]. To perform such a role, the equivalents of Hþ [or component(s) equilibrated with Hþ ] must be present in an amount

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sufficiently large to buffer the rate of fluctuations of the Hþ producing and Hþ -consuming processes. Calculations showed that to form Hþ equal to 0.25 V, a bacterial cell should extrude amounts of Hþ commensurable with that of enzymes in its membrane [25]. To store membrane-linked energy in a ‘‘substrate’’ (rather than in a ‘‘catalytic’’) quantity, one must discharge the membrane by a flux of ion(s) other than Hþ. A flow of penetrating ions across the membrane discharges and, hence, allow an additional portion of Hþ to be extruded from the bacterium by Hþ generators. Now pH, rather than , appears to be a factor limiting the activity of Hþ generators. The pH-buffering capacity of the cytoplasm is by two orders of magnitude higher than the electric capacity of the cytoplasmic membrane, so that the amount of energy stored in pH appears to be much larger than that in . A further increase in the stored energy can be achieved if the formed pH is utilized by a cation/Hþ antiporter to substitute p(cation) for pH. Involvement of such an antiporter exchanging, e.g., inner Naþ for outer Hþ, is equivalent to an increase in the concentration of pH buffer as was recognized already in 1968 by Mitchell [120]. As to Kþ, Mitchell ignored its possible role. Accumulation of Kþ down is, according to him, an unfavorable, but inevitable, consequence of negative charging of the bacterial interior [120]. In 1978, I suggested [121] that influx of Kþ is used by the bacterial cell to induce ! (pH, pK) transition. The formed pK was postulated to buffer the component of Hþ . As to pH, it is utilized by a Naþ/Hþ antiporter so that pNa buffers the pH component of Hþ . According to this hypothesis, Kþ is accumulated in, and Naþ extruded from, the bacterial cell when there is an excess of energy sources. If, then, energy sources are exhausted, and pH tend to lower. Such a lowering should reverse the Kþ and Naþ fluxes: Kþ goes out, while Naþ goes in, the cell. This results in generation of and pH, respectively. Thus, cooperation of the -driven Kþ influx and pH-driven Naþ-efflux seems to meet the


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requirements for the Hþ buffer. Naþ is the most common cation of the outer medium. Therefore, a cell pumping out Naþ can easily create a large pNa. [Kþ]out is usually much lower than [Naþ]out, so a large pK can be obtained via Kþ import. On the other hand, [Kþ]out is not so low as to hinder a search for this cation in the medium. Suggesting the scheme, I based upon the observations that bacteria possess carriers competent in electrophoretic Kþ accumulation [122], a fact that can hardly be explained by Mitchell’s assumption that Kþ influx is an unfavorable side effect. As to Naþ/Hþ antiport, it was described as early as in 1974 by West and Mitchell [123]. To verify the Naþ/Kþ hypothesis, we used motility as a probe for the membrane energization. I. Brown and others in my group [124] found that pK and pNa of proper direction can really support motility of various bacteria for some time after the depletion of the energy sources. The capacity of the Naþ/Kþ energy buffer was shown to be directly proportional to the ambient salt concentration in the environment. It was maximal in halophilic H. salinarium and minimal in the fresh-water Phormidium uncinatum where only pK, but not pNa, proved to be effective. In H. salinarium preincubated in the light, Naþ and Kþ gradients were still competent in supporting measurable motility more than 8 h after the cessation of illumination and exhaustion of O2. This means that halobacteria invest a very large portion of energy into pK and pNa during daytime when solar energy is available, to regain it throughout the night. Challenge 4: Energy Coupling without Hþ: The Sodium World Mystery of Alkaliphilism The studies described in the preceding section contributed to verification of the chemiosmotic hypothesis and extended it in

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such a way that Hþ proved to be not only a transient between oxidation and phosphorylation but also convertible and transportable energy currency buffered in bacteria by the Naþ/Kþ gradients. However, an exception clearly inconsistent with the chemiosmotic scheme was still waiting for explanation. I mean the life under alkaline conditions. Some bacteria can survive at pH as high as 11 when extrusion of Hþ by a redox chain can hardly produce sufficient Hþ since there is a large pH of wrong direction (the cell interior much more acidic than the medium). Several solutions for the above problem were suggested by bioenergeticists. One of them consists in assumption that at high pH, some other ion substitutes for Hþ as the coupling substance. In this connection, A. Glagolev attracted my attention to Naþ. Really, in 1980 Dimroth reported on discovery of a procaryotic, primary Hþ generator creating a Naþ potential with no Hþ involved [125]. This was membrane-linked oxaloacetate decarboxylase of Klebsiella aerogenes. Such a nonoxidative, energy-releasing enzyme was found to expel Naþ from the bacterium so that (inside negative) and pNa (inside low [Naþ]) were formed. However, it was not clear (i) how the Naþ formed can then be utilized and (ii) whether this mechanism is related to alkalophilism (K. aerogenes is a neutrophil). The situation was exacerbated in the next year when Tokuda and Unemoto published a short communication in BBRC that alkali-tolerant (!) Vibrio alginolyticus possesses a Naþ-motive NADH-CoQ reductase [126]. The observation was so strange that, in fact, almost nobody paid any attention to this discovery although in 1982 the authors published a full-size report in J. Biol. Chem. [127]. In that time, four years after Mitchell’s Nobel Prize, the community was still under the influence of the original version of his scheme where Hþ was generated due to separation of H atom to Hþ and e^, and ATP was synthesized by means of a process when the Hþ formed is used to protonate the ADP or phosphate anion [63]. It is quite obvious that Naþ


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cannot substitute for Hþ as the coupling ion if the mechanism of coupling is organized in such a fashion. However, we were already prepared to modify the above scheme since our studies clearly showed that mitochondrial transhydrogenase is a reversible Hþ generator operating without release to water of the H atom which is involved in oxidoreduction [70,128]. This is why we started with studies on a possible role of Naþ as a coupling ion. Soon it became clear that at least one energy-linked function, motility, can be supported by Naþ [129]. P. Dibrov in my group revealed that motility of V. alginolyticus (i) occurs only if Naþ is present in the medium, (ii) can be supported by an artificially-imposed pNa in a manner sensitive to monensin, an Naþ/Hþ antiporter, (iii) is carried out in the presence of high concentration of a protonophore, and (iv) 100-fold lower protonophore concentration completely arrested the respiration-supported motility if monensin is present [129]. It should be mentioned that previously the Naþdependent motility was observed in Pseudomonas stutzeri [130] and in some bacilli [131]. However, V. alginolyticus proved to be the first species which was alkali-tolerant and possessed a primary Naþ generator. In further experiments, we found that addition of 250 mM NaCl to V. alginolyticus cells incubated without Naþ results in transient manifold increase in the ATP level which was protonophore-resistant but sensitive to monensin. Addition of 50 mM NaCl was ineffective whereas addition of a respiratory substrate after 50 mM NaCl gave rise to steady increase in the ATP level in a monensin-sensitive, protonophore-resistant fashion [132]. It was concluded that artificially-imposed or respiration-produced Naþ can be utilized by V. alginolyticus to synthesize ATP. As was independently shown in the Y. Unemoto lab, Hþ is the driving force to import various amino acids and sugars by the V. alginolyticus cells [133]. In a plenary lecture to the FEBS Meeting in Algarve (1985), I summarized these observations saying that for V. alginolyticus, Naþ is a convertible energy currency formed by respiration

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and utilized to support chemical (ATP synthesis), osmotic (uphill transport of metabolites), and mechanical (motility) types of useful work. I called this novel bioenergetic pattern the ‘‘Sodium Cycle’’ [134, 135]. Further studies revealed that such a cycle substituting for the Mitchellian proton cycle is inherent in very many marine aerobic and anaerobic bacteria. In particular, a unique sequence corresponding to the Naþ-motive-CoQ reductase has been found in aerobes whereas Naþ-motive decarboxylases proved to be typical for several anaerobes. Among them, there are the most terrible pathogens, such as Yersinia pestis, Vibrio cholerae, V. parahaemolyticus, Treponema pallidum, T. denticola, Chlamidia, Streptococcus pyogenes, Clostridium defficala, Neisseria meningitis, N. gonorrhoeae, Haemophilus influenzae, Pseudomonas aeruginosa, Salmonella enterica, and Klebsiella pneumoniae [136,137]. In some cases, it proved possible to identify a special Naþ-ATPase responsible for the Naþ ! ATP energy transduction [25]. In anaerobic Enterococcus hirae, Kakinuma and Hiragashi showed that Naþ-ATPase is induced by alkaline pH, addition of a protonophore, or mutation in the Hþ-ATPase which is also present in this microbe, i.e. under conditions when Hþ could not be used as the energy currency [138,139]. Naþ-ATP-synthase from Propionigenium modestum was studied in detail by Dimroth [140, 142]. A. Bogachev and coworkers in my group [141] studied Naþmotive NADH-CoQ reductase in Vibrio alginolyticus cells and found that the Naþ/e^ stoichiometry is about 1, which is lower than Hþ/e^ stoichiometry of E. coli Hþ-motive NADH-CoQ reductase (complex I). A further difference between the two enzymes was revealed when their redox centers were identified. Instead of FMN and six FeS clusters in complex I, the Naþmotive reductase was shown to contain one FAD, two FMN, and one FeS cluster [143,144]. Subunit composition of the Naþmotive enzyme was simpler (six) than that of the Hþ-motive (fourteen). Mechanisms of Naþ and Hþ pumping by both systems are still obscure. This problem is now actively studied


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in my lab by Bogachev cooperating with R.B. Gennis’ group in Urbana [143]. One more intriguing question is whether Naþ is pumped also in the terminal segment of the respiratory chain. Some indications that such a mechanism really exists were obtained in our group in the 1990s. This problem is now investigated in my lab by Dr. M. Muntyan. It should be noted in this context that the outer membrane of the animal cell is a ‘‘sodium membrane.’’ It is energized by Naþ/ Kþ-ATPase which forms Hþ which is then utilized by numerous Naþ, metabolite symporters [25]. This observation may be regarded as one more piece of evidence that high Naþ-containing blood and intercellular liquid represent a small part of the ocean from which multicellular animals are assumed to have originated.

Three Laws of Bioenergetics Discovery of the Sodium World allowed the filling of a significant gap in our knowledge concerning cellular energetics based on the Mitchellian Hþ cycle scheme. This is why I have decided to formulate some general bioenergetic principles of universal applicability to living cells. In 1992, I published a paper in Eur. J. Biochem. called ‘‘The laws of cell energetics’’ [145]. The first law was presented as follows: ‘‘The living cell avoids direct utilization of external energy sources in the performance of useful work. It transforms energy of these sources to a convertible currency, i.e. ATP, Hþ , or Hþ, which is then spent to support various types of energy-consuming processes.’’ In other words, the cell prefers to deal with energy in a money-type circulation rather than with barter. In fact, this law extended a principle put forward in 1941 by F. Lipman when he assumed that ATP is the biological energy currency [146]. It was now postulated that similar role can be performed in some cases also by protonic or sodium potentials. The validity of the postulate was proved by numerous

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cases when Hþ or Naþ were generated by some energyreleasing enzymes to be consumed by other proteins to perform all types of the useful work in the cell with no ATP involved. The second law proved to be shorter: ‘‘Any living cell always possesses at least two energy currencies, one water-soluble (ATP) and the other membrane-linked ( Hþ and/or Naþ ).’’ Continuing with the analogy between cell bioenergetics and everyday life, this law states that the cell always has some currency in cash and some in checks. Really, there is not a single well documented and reproducible observation that a living cell uses ATP but not Hþ or Naþ and vice versa. The third law was also rather short: ‘‘All the energy requirements of the living cell can be satisfied if at least one of three energy currencies is produced at the expense of external energy sources.’’ This law can be paraphrased as it does not matter how an income is received, in cash or in checks, as long as they are interconvertible. Frequently, there are both ATP- and Hþ ( Naþ )-producing mechanisms coexisting in one and the same cell. However, some bacteria produce only one type of the currency by primary energy-releasing mechanisms. There are anaerobes using ATP-producing fermentation as the primary energy source. In this case, Hþ and/or Naþ are generated by Hþ-(Naþ)-ATPases. In certain respiring bacteria, respiratory substrates and O2 are the primary energy source, and Hþ is formed to be converted to ATP by Hþ-ATP-synthase and to Naþ by the Naþ/Hþ-antiporter. In other bacteria, like Propionigenium modestum, Naþ is primarily formed by, say, Naþ-motive decarboxylase. As to ATP, it is produced by Naþ-ATP-synthase.

Challenge 5: Respiration Which Kills Us How to Prevent Formation of Reactive Oxygen Species For me, formulation of bioenergetic laws proved to be an important milestone and a signal that this is the right moment to


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change field of main interest. The cause proved to be an answer of my old friend Sergio Papa to my question concerning possible functions of nonphosphorylating respiration, which I put to him at a conference in Prague. Listing such functions, Sergio mentioned decrease in intracellular [O2] to minimize harmful effects of oxygen. It is generally accepted that at least 98% of cellular respiration produces inoffensive H2O whereas no more than 2% entails formation of superoxide anion (O2. ) which can then be converted to H2O2 and very aggressive oxidant OH.. All these compounds are called reactive oxygen species (ROS). The redox potential of the O2. /O2 pair is 0.16 V. This means that complexes I, II, and III can be competent in the one-electron reduction of O2 to O.2. In contrast to H2O-forming cytochrome oxidase which is operative at maximal rate down to very low [O2], production of O.2 by these complexes is strongly inhibited when [O2] decreases below the level found in water under normal atmospheric pressure. This is why a decrease in the intracellular [O2] might be simple but effective mechanism preventing O2. generation by the respiratory chain. To some degree, respiration, if it were competent in lowering of oxygen level inside the cell, could be regarded as an antioxidant mechanism and oxygen consumption to decrease this level could represent one more detoxifying function of respiration. On the face of it, lowering of [O2] should be carried out by phosphorylating respiration. However, this may not be the case always. For example, the above mechanism is quite good during active work when formed ATP is decomposed to ADP and Pi. On the other hand, in the resting state when the rate of respiration strongly lowers, some problems may arise for such respiratory function as [O2] lowering. In 1994, I suggested that uncoupling might be a mechanism facilitating [O2]-lowering function of mitochondrial respiration [147] (for details, see Ref. [148]). In fact, uncoupling not only allows making respiration independent of ATP turnover but also should decrease the steady-state

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[Q. ] level in complex III. This may be regarded as one more antioxidant effect since Q. is an excellent one-electron O2 reductant. The third antioxidant function of uncoupling was revealed when the rate of H2O2 and O2. production by mitochondria was measured as a function of membrane potential. Our experiments [148^150] revealed very steep dependence of ROS generation upon , indicating that even ‘‘mild’’ uncoupling, i.e. small Hþ decrease, is sufficient to prevent ROS formation. Such a ‘‘mild’’ uncoupling was shown to be obtained by low concentrations of free fatty acids. In the same experiments carried out by A. Starkov and S. Korshunov, it was shown that (i) the contribution of complexes I and III to ROS formation in the resting (State 4) mitochondria are 80 and 20%, respectively, and (ii) the level of ROS production by mitochondria is directly proportional to that of reverse electron transfer from succinate to NADþ. In fact, mitochondria oxidizing succinate produce ROS mainly in a rotenone- and uncoupler-sensitive fashion. Therefore, I concluded that we deal here with the Hþ -consuming reverse electron transfer from succinate (redox potential of succinate/fumarate is þ 0.03 V) to O2 (redox potential of O.2 /O2 is 0.16 V). This process proved to be only slightly slower than the maximal rate of ROS production in mitochondria observed when antimycin A was added [149]. However, the antimycin Atreated mitochondria are clearly pathological whereas the State 4 mitochondria are normally functioning organelles. The only problem is whether such a process is an inevitable consequence of aerobiosis when small penetrating molecules of O2 occasionally attacks respiratory chain electron carriers of negative redox potential, or alternatively there is a biological function behind O2 consumption by complex I. This exciting question will be considered in the next section. To conclude the above discussion concerning mechanisms preventing ROS formation, I would like to stress that the great majority of studies on antioxidant systems deal with ROS scavengers purifying the cell from already formed ROS. Such


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a tradition is rather strange since obviously it is much better to prevent the evil than to fight its consequences. The first work on preventing ROS formation by respiratory O2 conversions to H2O was performed as early as in 1969 by Dalton and Postgate who studied Azotobacter vinelandii [151,152]. They suggested that the very high rate of the A. vinelandii respiration, which is higher than in any other bacterium known, is required to lower intracellular [O2] and, hence, to permit N2 reduction of nitrogenase, an enzyme extremely sensitive to oxygen. They called this phenomenon ‘‘respiratory protection’’ assuming that there is special respiratory chain responsible for fast conversion of O2 to H2O to lower [O2] inside the bacterium. Later, it was really shown that A. vinelandii possesses two respiratory chains: one very similar to mitochondrial (complexes I and III and cytochrome o, which closely resembles complex IV) and another one terminated by cytochrome bd. The cytochrome bd chain is induced under conditions when N2 becomes the only nitrogen source. It was assumed that this chain is nonphosphorylating in order to avoid any restrictions of the respiration rate by the ADP availability. A. Bogachev in my group reinvestigated this problem. It was found that the initial step of the respiratory protection chain is really noncoupled. A. Bogachev and his colleagues sequenced the gene coding for the corresponding enzyme. The protein proved to be a typical representative of the so-called NADH dehydrogenase II (NDHII) family, a simple enzyme reducing CoQ by NADH with no Hþ or Naþ formed [153]. As to cytochrome bd, it was found to be Hþ -generating but Hþ/e^ stoichiometry was twofold lower than that of cytochrome o [154]. We concluded that the efficiency of the respiratory protection chain (NADH ! NDHII ! CoQ ! cytochrome bd ! O2 ) is fivefold lower than the cytochrome o chain (NADH ! complex I ! CoQ ! complex III ! cytochrome c ! cytochrome o ! O2 ), i.e. 2Hþ instead of 10Hþ per NADH oxidized. This means that for the respiratory protection chain, it is needed to consume

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fivefold more oxygen to produce the same amount of ATP than for the ‘‘canonical’’ chain.

Production of Poisons is the Biological Function of Some Respiratory Enzymes When I started with study of reactive oxygen species, my initial interest was concentrated on the possible role of uncoupling in respiratory protection. It seemed reasonable to assume that respiratory protection carried out by a special electron transport chain in A. vinelandii might also be organized in mitochondria by their sole respiratory chain if it is slightly uncoupled [147,148]. However, later I came to another, and more general, intriguing problem, namely why the respiratory chain, besides its protective function, generates ROS, i.e. compounds against which it should protect the cell. Sometimes, such a ROS production can be explained by damage to respiratory enzyme complexes (e.g., antimycin A-induced ROS formation). On the other hand, we clearly showed that the O.2 -producing reverse electron transfer from succinate to O2 is maximal only in tightly coupled mitochondria, which are usually regarded as the most intact organelles. It seems even more puzzling that there are respiratory enzymes always converting O2 to O2. or H2O2, not to H2O. At least in one special case enzymatic ROS formation has quite clear explanation. I mean production of O2. by a NADPH-oxidizing respiratory chain localized in the outer membrane of phagocytes. FAD and autoxidable cytochrome b serve as electron carriers. The chain is oriented across the membrane so that NADPH is oxidized and O.2 is formed on the inner and outer membrane surfaces, respectively. Extracellular ROS generated by this mechanism are used by a phagocyte to kill bacteria. Thus, ROS were selected during evolution as a biological weapon applied by macroorganisms as an


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antibacterial tool [155,156]. However, this is hardly an invention of metazoa. As Malatesta and coworkers reported, terminal oxidase of the bacterium Pseudomonas nautica reduces O2 to H2O2. If H2O2 is produced on the outer surface of the bacterial membrane, it could be employed to fight against cells of other species. Catalase sequestered in the P. nautica cytosol may protect this bacterium from the harmful effects of H2O2 generated by its respiration [157]. The above reasoning cannot be applied to cases when the ROS-producing enzyme is localized inside the cell so that ROS formed are released to cytosol or intracellular organelles. This is the case for monoamine oxidase of the outer mitochondrial membrane, cytochrome P450 of the endoplasmic reticulum, some respiratory enzymes localized in peroxisomes, etc. Cytosolic xanthine oxidase belongs to the same group. This is an enzyme oxidizing hypoxanthine and xanthine by O2 to uric acid, O2., and H2O2. Xanthine oxidase is formed from xanthine dehydrogenase which employs NADþ as oxidant. To acquire the oxidase activity (i.e. to reduce O2 instead of NADþ), xanthine dehydrogenase should undergo either limited proteolysis or oxidation of its sulfhydryl groups [158]. The latter may be done by ROS. Thus, xanthine oxidase forms ROS which can, in turn, cause formation of new portions of xanthine oxidase from xanthine dehydrogenase. In other words, a poison stimulates its own formation. Such an autocatalysis should result in a burst of ROS generation inside the cell. To understand such paradoxical relationships, I addressed myself to the phenomenon of programmed cell death. Really, burst in ROS formation might be accounted for assuming that this is a mechanism of cell suicide.

From Weismann to Apoptosis More than 120 years ago, the great German biologist August Weismann put forward a paradoxical idea that death due to

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aging was invented by evolution as an adaptive mechanism. For the first time, this concept was presented in his lecture to a meeting of the Association of German Naturalists in September, 1881. The hypothesis was published in German (1882) and then in English (1889). Weismann wrote: There cannot be the least doubt, that the higher organisms, as they are now constructed, contain within themselves the germs of death. . . The question arises as to how this has come to pass. . . Worn-out individuals are not only valueless to the species, but they are even harmful, for they take the place of those which are sound. . . I consider that death is not a primary necessity, but that it has been secondarily acquired as an adaptation. I believe that life is endowed with a fixed duration, not because it is contrary to its nature to be unlimited, but because the unlimited existence of individuals would be a luxury without any corresponding advantage [159].

In the 1950s, Weismann’s suggestion was strongly criticized by Medawar [160], who assumed that death because of aging is a laboratory artifact whereas under natural conditions the great majority of animals die before they become old. This assumption, however, cannot be applied at least to some periods of evolution of many species as was recently indicated by Bowles [161]. Moreover, many pieces of evidence were obtained that individuals with modified genomes can dramatically affect the very fate of a population even if they amount to only a quite small part of the population (see, e.g., Ref. [162]). In 1990s, the Weismann concept was revisited [161, 163^165] due to discovery that the programmed death mechanism (apoptosis) is inherent in cells of multicellular organisms. It was found that it is apoptosis that is responsible for purification of tissues from defective or infected cells, for cell elimination during ontogenesis, for suicide of homeless cells, or cells of the


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immune system producing antibodies to their own proteins, etc. (for review, see Ref. [164,165]). As was mentioned by Savill and Fadok, ‘‘while philosophers seek the meaning of life, cell biologists are becoming even more interested in the meaning of death’’ [166].

Mitochondria and ROS in Apoptosis: Mitochondrial Suicide (Mitoptosis) In 1996, an event occurred which initiated quite a new wave of mitochondrial research. Kroemer and coworkers in France reported [167] that in the mitochondrial intermembrane space a protein is hidden which causes apoptosis when released from mitochondria to the cytosol. The novel protein was called Apoptosis-Inducing Factor (AIF). It proved to be a FAD-containing enzyme resembling plant dehydroascorbate reductase also localized in the intermembrane space [168]. AIF was found to activate a DNAse cleaving nuclear DNA. This apoptotic pathway is required at least for the first wave of apoptosis during embryogenesis [169]. In 1996^1997, laboratories of Wang and Newmeyer in the USA reported that cytochrome c is also a pro-apoptotic protein [170,171]. It was found that many apoptotic stimuli cause cytochrome c release from the mitochondrial intermembrane space to the cytosol where Apoptotic Protease-Activating Factor 1 (Apaf-1) is present. The cytochrome cÂpaf-1 complex combines with several molecules of procaspase 9 in a dATP-dependent fashion.This results in cleavage of procaspase 9 to active caspase 9, a protease, which, when formed, attacks procaspase 3 so that active caspase 3 appears.The latter hydrolyzes a group of proteins occupying key positions on the metabolic map or responsible for structural organization of the cell.The final of all these events consists in hydrolysis of DNA and other biopolymers to monomers. In this way, the apoptotic cell, in fact, decomposes itself to simple constituents that can be utilized by other cells [172].

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The question arises why the mitochondrial intermembrane space is used by the cell to hide the suicide proteins like AIF or cytochrome c. In 1996, I suggested that the answer may be found if we take into account ROS [173]. To form a very large amount of ATP (in a 70 kg man, 40 kg ATP is produced per day), mitochondria convert to H2O about 0.4 kg O2 per day. If the O.2 production is equal to 1% of the O2 consumed, this gives 4 g O.2 per day. For sure, this amount is quite sufficient to damage all our DNA and, hence, to kill us. To avoid such a danger, mitochondria must form much lower O2. amounts and antioxidant systems of organisms must effectively detoxify the O2. formed. Really, mitochondria are equipped with a multifaceted mechanism minimizing the ROS danger. According to our data [174], it includes (i) lowering of [O2] inside, and in vicinity of, mitochondria; (ii) a Hþ decrease below the level critical for reverse electron transfer from CoQH2 to O2 in Complex I as well as for inhibition of cytochrome bl oxidation in Complex III; (iii) oxidation of O.2 back to O2 by cytochrome c when this protein is desorbed from the inner membrane surface to the intermembrane space. If these measures preventing the O2. formation or inactivating O2. formed fail, the next lines of defense become operative. This is (a) superoxide dismutase converting O.2 to H2O2 and H2O, (b) catalase and glutathione peroxidase decomposing H2O2, and (c) lipid-soluble (tocopherol) and watersoluble (ascorbate) antioxidants inactivating various forms of ROS [148,174]. In spite of all the systems listed, mitochondria can produce large amounts of ROS at least under certain in vitro conditions. This happens, e.g., when complex III is inhibited at the stage of cytochrome bl oxidation, i.e. in the antimycin A-sensitive site. Such an inhibition is apparently not so infrequent event since it is inherent in many hydrophobic xenobiotics. It is even more important that just this site, according to Boveris and his colleagues [174a], is inhibited by NO., a key metabolic regulator, resulting in huge increase in O.2 production. For cases of this type, I have postulated one more, and the last, line of mitochondrial antioxidant defense


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i.e. self-elimination (suicide) of the ROS-overproducing mitochondria, coined mitoptosis [23]. In 1992, my coworker Dr. D.B. Zorov mentioned a possibility that the mitochondrion might possess a mechanism of its programmed death, namely the so-called permeability transition pore (PTP) [175]. The PTP is formed in the inner mitochondrial membrane due to oxidation (or any other modification) of the cysteine 56 SH-group in the ATP/ADP antiporter. Oxidation results in conversion of the antiporter to a nonspecific high conductance channel permeable to substances of molecular mass 51.5 kDa. This conversion is catalyzed by a special protein, mitochondrial cyclophilin [176]. If a mitochondrion fails to prevent a [ROS] increase, the SH-group is oxidized, the PTP opens, and all the electrical and ion gradients between the matrix and the intermembrane space disappear. In fact, the mitochondrion with open PTP will perish just as a ship with open Kingstons. Several processes that are of vital importance for the mitochondrion require and ion gradients across the inner membrane to be maintained. In particular, collapse of makes impossible electrophoretic import of precursors of mitochondrial proteins and their proper arrangement in the inner membrane. Thus, the repair processes cease in the PTP-bearing mitochondrion. It is noteworthy that the mitochondrion does not require any extramitochondrial proteins to open the PTP when [ROS] rises. This means that a death of the mitochondrion, induced by its own ROS, can be regarded as a mitochondrial suicide. I proposed that such an event is used by the cell to purify its mitochondrial population from ROS-overproducing organelles [147,148,165]. PTP opening, besides initiating mitoptosis, has one more consequence, namely, swelling of the matrix because of the appearance of an osmotic disbalance between matrix and intermembrane space. Swelling, in turn, causes disruption of the outer mitochondrial membrane (for details, see Ref. [173]). As a result, cytochrome c, AIF, and some other pro-apoptotic

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proteins hidden in the intermembrane space are released to the cytosol. If it happens in a single mitochondrion, concentrations of pro-apoptotic proteins appear to be too small to initiate apoptosis. However, apoptosis is initiated if many mitochondria in the same cell commit suicide. It is important that PTP can be opened by any ROS independently of whether it is generated inside or outside the mitochondrion. Therefore, a cell overproducing ROS by an extramitochondrial mechanism will be killed by its own mitochondria. Thus, ROS-induced, mitochondria-mediated apoptosis may be regarded as a way to purify a tissue from ROS-overproducing cells (situation symmetric to purification of mitochondrial population from ROS-overproducing mitochondria). In 1994, I postulated that mitoptosis is an event preceding apoptosis [147]. In the same year, Newmeyer and coauthors published the first indication of a requirement of mitochondria for apoptosis [177]. Quite recently, Tolkovsky and her colleagues presented direct proof of the mitoptosis concept [178,179]. In a cell culture apoptosis was initiated by deprivation of a growth factor. A day later, processing of the apoptotic signal was interrupted downstream of mitochondria by a pan-caspase inhibitor Boc-Asp(O-methyl)-CH2F (BAF). As a result, the cells survive for some period of time but in the majority of them all mitochondria disappear within three days after the BAF addition. This was shown to be accompanied by disappearance of mitochondrial DNA and the cytochrome oxidase subunit IV encoded by nuclear DNA (a marker of the inner mitochondrial membrane). On the other hand, nuclear DNA, Golgi apparatus, endoplasmic reticulum, centrioles, microtubules, and plasma membrane remained undamaged. Overexpression of antiapoptotic protein Bcl-2 preventing ROS-induced changes in mitochondria was found to arrest mitoptosis. According to data recently obtained in our group by O. Pletyushkina, mitoptosis can also be observed under conditions when the apoptotic cascade is not inhibited. In certain apoptotic cells, the entire cytochrome c pool appears in the cytosol,


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whereas subunit IV of cytochrome oxidase practically disappears. Electron microscopy revealed that apoptotic stimuli result in swelling of mitochondria. Then, swollen mitochondria are transformed to small and ultracondensed, most probably, dead mitochondria. At a late stage of apoptosis, such mitochondrial corpses are engulfed by autophagosomes (Bakeeva et al., in preparation).

Programmed Death at Supracellular Level: Bystanders; Organoptosis There are some indications that apoptotic signal(s) can be transmitted from an apoptotic cell to neighbor cells (so-called ‘‘bystander effect’’) (for reviews, see Refs. [180,181]). This may be used as a line of antiviral defense when an infected cell organize a ‘‘death area’’ around itself inducing suicide of nearby cells that are the most probable targets for the virus during expansion of the infection. I have suggested that H2O2 produced by the apoptotic cell and released to the intercellular space may be a signal for bystanders to commit suicide [180]. The suggestion was recently confirmed by G. Bakalkin and coworkers [181] and then by O. Pletyushkina (in preparation) in my group. In the latter case, HeLa cells were growing on the surface of a cover glass. When a cell monolayer was formed, the glass was broken into two halves and one half was treated with tumor necrosis factor (TNF) to induce apoptosis. Later, TNF was washed out and the TNF-treated part of the glass was placed side by side with the nontreated one. It was found that cells on the nontreated part of the glass commit suicide, the effect being sensitive to catalase. It is obvious that massive apoptosis of cells composing an organ should eliminate the organ. Such a process can be defined as ‘‘organoptosis.’’ In fact, the autumn shedding of the leaves, preventing a tree from being broken by snow, is due to apoptosis of some cells in grafts and should be regarded to such kind of

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phenomena. It is remarkable that the term ‘‘apoptosis’’ was introduced by the Roman physician Galen who concluded that shedding of the leaves is an active process which does not occur in a broken branch. For sure, organoptosis is a mechanism of ontogenesis and can be mediated by H2O2. As was recently reported by Kashiwagi et al. [182], addition of thyroxine to severed tails of tadpoles, surviving in a special medium for days, caused shortening of the tails that occurred within hours. The effect proved to be a consequence of an induction of NO. synthase in cells of the tail by thyroxine, inhibition of catalase and glutathione peroxidase by NO., which in turn resulted in strong increase in the H2O2 level. Moreover, it is quite probable that not only H2O2 decomposition but also H2O2 production was increased by NO. due to inhibition of the cytochrome bl oxidation [174a].

Phenoptosis, Programmed Death of Organism On the face of it, death of an organism should be considered to be a result of a lethal pathology of no biological sense. However, this may not be the case if the organism in question is a member of a kin, community, or population of other individuals. Here, altruistic death of individuals may appear to be useful for a superorganismal unit, being a mechanism of adaptation of the group of organisms to a changing environment, just as was suggested by Weismann (see above). From this point of view, it seems reasonable to supplement the chain of events: ‘‘mitoptosis ! apoptosis ! organoptosis’’ with more step, that is, the programmed death of an organism. Let us call this event ‘‘phenoptosis.’’ Phenoptosis can be defined as a mechanism purifying kin or community from individuals that are no longer wanted [163^165]. For unicellular organisms, phenoptosis represents programmed death of the cell. The simplest examples of phenoptosis may be found in bacteria. Here altruistic programmed death


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was shown to occur (i) to prevent expansion of some phage infections in a bacterial population, (ii) to purify this population from those cells whose genome or some other key systems are damaged, (iii) to optimize the number of bacterial cells in the medium (‘‘quorum sensing’’), etc. [165, 183^186]. Quite recently, phenoptosis was demonstrated in a unicellular eucaryote. My son Fedor Severin and Dr. Antony Hyman [187] working in Dresden studied vegetative-to-sexual reproduction switch, a typical response of yeast to deterioration of ambient conditions. This process is known to be mediated by production of pheromones that stimulate agglutination of haploid cells of opposite mating types. However, higher pheromone concentrations kill these cells. Severin and Hyman have found that the pheromone-induced death is programmed, showing typical markers of animal apoptosis. The death was prevented by inhibitors of protein synthesis (cycloheximide) and of the permeability transition pore (cyclosporin A). The mitochondrial DNA and cytochrome c were shown to be required for the programmed death. ROS production preceded the death, occurring upstream of cytochrome c. It was mediated by the pheromone-induced protein kinase cascade. When haploids of the opposite mating types were mixed, some cells died, the inhibitory pattern being the same as in the case of the killing by pheromone. The percentage of dead cells strongly increased when mating was arrested. This study seemed to finalize verification of the programmed death in yeast, being in line with results of several previous investigations. Since 1994, some pieces of evidence were published indicating that (i) certain mammalian pro- and antiapoptotic proteins (e.g., Bax and Bcl-2) affect ROS formation and the very fate of yeast cells [188^194], (ii) some mutations [195] or toxic agents (H2O2, acetic acid, plant antibiotic osmotin) [196^198] kill yeast in a way similar to apoptosis in animals. Quite recently Madeo et al. [199] reported that death caused by H2O2 is mediated by a protein whose sequence and properties resemble those of the mammalian caspases. Inactivation of the corresponding gene increased the lethal dose of H2O2 required

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to kill yeast and prevented the age-dependent death in a small portion (less than 10%) of the yeast population. Superexpression of the protein called yeast caspase 1 (YCA1) decreased the H2O2 lethal dose. It is remarkable that a programmed death is also inherent in the vegetative reproduction of yeast. In fact, the life span of such a yeast is about 3 days. During this period of time, a mother cell forms about 30 buds and then dies. It was quite recently found that certain apoptotic markers, including the caspase induction, appear in old mother cells [199,200]. Summarizing the above data, I have concluded [201] that a response of the yeast cell to a change in ambient conditions for the worse results in the following. (i) Transformation of diploid cells to spores, a latent form of life, (ii) formation of haploid cells from spores, (iii) substitution of sexual for vegetative reproduction, and (iv) initiation of pheromone-induced programmed death resulting in strong shortening of the life span and, hence, in acceleration of changes of generations. The latter, like sexual reproduction, might be favorable for progressive evolution. Moreover, the pheromone-induced programmed death may be regarded as a mechanism purifying yeast population from haploid cells, which for some reason fail to mate. Such an effect may eliminate nonproductive haploid cells from cell agglutinates arising when haploid cells of opposite sex stick to each other and high pheromone concentrations appear in clefts between adhered cells. This is why chloroquine, an inhibitor preventing zygote formation but not agglutination, strongly stimulated death when cells of opposite sex were mixed [187]. If diploids formed as a result of mating of haploid cells are not adapted yet to new conditions, the above chain of events is repeated until a novel trait appears, which helps to overcome disadvantages of environmental changes that have occurred. Thus, yeast seems to be a form of life which oscillates between short-lived and long-lived modes, the oscillation being a mechanism of adaptation to changing conditions. In this sense, Weismann’s paradoxical hypothesis that death can be a kind of


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adaptation is proved by the above experiments. The next (and the most important!) problem is: ‘‘to what degree the principle of phenoptosis can be applied to higher organisms?’’

Phenoptosis of Multicellular Organisms that Reproduce Only Once Considering possible cases of phenoptosis in multicellular organisms, I addressed myself, first of all, to this group of living creatures. Obviously, some of them are constructed in a way predetermining death shortly after reproduction. Remember mayflies, their imagoes die in a few days since they cannot eat, due to lack of a functional mouth, and their intestines are filled with air. In the mite Adactylidium, the young hatches inside the mother’s body and eat their way out. However, more often a special phenoptotic program is switched on immediately after the act of reproduction. The male of some squids dies just after transferring his spermatophore to a female. The female of some spiders eats the male after copulation. Bamboo can live for 15^20 years reproducing vegetatively but then, in the year of flowering, dies immediately after the ripening of the seeds. The pacific salmon dies soon after spawning, and this happens due to actuation of a specific corticosteroid-mediated program [165].

Phenoptosis of Repeatedly Reproducing Organisms: Why Aging is Slow? On the face of it, also in these organisms, age-dependent phenoptosis, if it exists, might be organized as a sudden death. Bowles mentioned that a marine bird suddenly dies at about age 50, without any indications of aging [202]. However, this is certainly an exception rather than a rule.

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For evolution of repeatedly reproducing organisms, slow aging should be expected to have an advantage over acute phenoptosis. The function of an age-dependent phenoptosis is to reduce the pollution of the population by long-lived ancestors, thereby stimulating evolution. Slow phenoptosis facilitates performance of this function. In fact, the appearance of a useful trait may compensate for unfavorable effects of aging within certain time limits, thereby giving some reproductive advantages to an individual acquiring such a trait. A strong, largebodied deer, even after reaching a rather old age, still has a better chance than his younger but weaker rival to win a spring battle for a female or to escape from wolves [165].

How may Age-dependent Phenoptosis be Organized? Telomeres Bowles [202] suggested that, historically, the living cell invented the first specialized mechanism of aging when linear DNA was used instead of the circular DNA inherent in the majority of bacteria and Archae. This event immediately resulted in a specific kind of the DNA aging, a process consisting of replication-linked shortening of DNA. Such shortening inevitably accompanies replication of linear DNA since the replicative complex operates with linear DNA in the same way as it does with circular DNA. To produce a copy of a template, this complex should have some nucleotide residues to the left and to the right from the place where it combines with DNA. This is always the case if it deals with a circular DNA. However, with a linear DNA, the operation of such a mechanism results in underreplication of the ends of the DNA molecule, as was first indicated by my old friend Alexey Olovnikov [203]. The question arises as to why eucaryotes, during many millions of years of their evolution, failed to improve such an important enzyme as that carrying out DNA replication, to adapt it to linear DNA, while at the same time solving much


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more difficult problems. A possible answer might be: it happened since unicellular eucaryotes used under-replication for some purpose. For instance, it might be a mechanism to count number of replications and to accelerate change of generations by shortening the life span. Evolution of the above mechanism resulted in appearance of special noncoding sequences (numerous hexanucleotide repeats called telomeres) added to the ends of linear DNA by a special enzyme, telomerase. In the majority of cell types composing our body, telomerase is operative only in the embryo, while during all postembryonal life the telomere shortens. In this way, it became possible to count cell divisions without damaging those DNA sequences that encode RNA. Thus, the old (genetic) DNA function was separated from the new one, phenoptotic. It is not yet clear whether telomere shortening determines the life span of modern multicellular organisms or only of some cells composing these organisms.

Mutants Who Live Longer The concept considering aging as a program predicts that mutations should exist which result in breaking this program and, as a consequence, prolongation of the life span. This effect is quite improbable within the framework of the alternative (traditional) paradigm assuming that aging occurs due to accumulation of occasional injuries in such a complicated system as a living organism. In this case, a mutation, as an additional injury, should shorten, rather than prolong, the life span. A demonstrative precedent that mutations can really be favorable for longevity has been furnished by Hekimi and coworkers [204,205], who studied the nematode Caenorhabditis elegans. A mutant worm with two damaged genes was obtained living 5.5 times longer than the wild type. It would be interesting to study mutations in homologous genes already found in other organisms (from yeast to man).

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In mice, Pelicci and his colleagues [206] have reported that (i) animals lacking a particular 66 -kDa protein (p66shc) lived 30% longer and (ii) fibroblasts derived from these mice did not respond to an H2O2 addition by initiating apoptosis. I suggested the following explanation of these data [165,207]. p66shc is involved in the ROS-induced apoptosis. Apparently, in young animals it helps to purify the tissues from ROS-overproducing cells. This is good for the organism and should prolong its life. In old animals, however, the same effect may be involved in phenoptosis (and, hence, shortens the life span) since an aging program is actuated resulting in an increase in the ROS production and/or decrease in the ROS neutralization. Phenoptosis could be a direct consequence of the fact that so many cells in organ(s) of living importance commit suicide that functioning of these organ(s) appear to be impossible and, hence, death comes. There are many indications that aging is accompanied (or perhaps even caused) by an increase in the oxygen danger. For instance, ischemia of isolated mouse heart damaged the cardiomyocite nuclear DNA much stronger if the heart was obtained from 22- to 24-month-old mice than from 6- to 7-month-old ones [208]. The traditional point of view is that the age-related sensitization to the oxidative stress is a consequence of occasional injuries resulting in disregulation in the antioxidant system in an old organism. The phenoptotic concept, however, takes into account an alternative possibility assuming that such a disregulation is specially induced to realize the suicide signal which comes from, e.g., intracellular sensors measuring the telomere length. If it were the case, the paradoxical observation of the Pelicci’s group would be explained. Quite recently, one more finding looking paradoxical from a common point of view was published by Donehower and coworkers [209]. They generated mice with enhanced activity of p53, the protein called ‘‘guard of genome.’’ Mutant mice proved to be resistant to spontaneous cancer appearing with age in the wild-type group. None of the thirty five mutant mice developed overt, life-threatening tumors, whereas over 45% of


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the wild-type animals developed such tumors. Surprisingly to the authors, the mutant mice lived shorter by about 20%. Shortening of the life span proved to be a result of the fact that in the mutants, aging started 20% earlier than in control. On the other hand, when started, aging developed in the same time scale in two groups of animals studied. Aging of the mutant mice showed many traits similar to those in the wild type, namely, reduction of body weight; loss of mass of liver, kidney, and spleen; lymphoid and muscle atrophy; osteoporosis; and hunchbacked spine. It proved to be difficult to diagnose the reason of death for the old mutants. An impression arose that total living strength of the organism was slowly decreased and, after all, becomes lower than some critical level required to maintain the life. Just these symptoms should accompany a death caused by massive apoptosis of cells in important organs. My tentative explanation of the p53 mutant story [210] consists in that this protein, performing its ‘‘guard-of-genome’’ function, recognizes two kinds of injuries in the nuclear DNA, i.e. (i) damages localized in its semantic part and (ii) shortening of telomeres. The former effect prevents cancer and prolongs the life while the latter promotes aging and shortens the life. Mechanistically, the two p53-linked effects seem to be different, the latter specifically including p66shc. Thus, one may hope to prolong the life span by a concerted activating p53 and inhibiting p66shc.

‘‘Samurai’’ Law of Biology Discoveries of programmed death mechanisms on subcellular (mitoptosis), cellular (apoptosis), supracellular (death of cells ^ bystanders, and organoptosis), and even organismal (phenoptosis) levels allowed me to put forward a general principle which I called the ‘‘Samurai law’’of biology. It can be formulated briefly as following: ‘‘It is better to die than to be wrong;’’ or, in more detail: ‘‘Any biological system from organelle to organism

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possesses a program of self-elimination. This suicide mechanism is actuated when the system in question appears to be dangerous for any other system of higher position in the biological hierarchy’’ [165,211]. An impression arises that living creatures always favoring controlled processes over spontaneous, try to operate in this way also when dealing with death, the final and most dramatic event of life. Controlled death allows the system to regulate and optimize the life span. Moreover, it may be regarded as a line of ‘antimonster’ defense. As was pointed out by my pupil K. Lewis, working now in the USA, ‘‘it is quite possible that the main danger unicellular organisms face are not competitions, pathogens, or lack of nutrients but their own kin turning into ‘‘unhopeful monsters’’ causing death of the population’’ [185]. There are some indications that this is also true for multicellular organisms where a change in activity of a single gene in a very small minority of organisms composing a population can be sufficient to kill this population (see, e.g., Ref. [162]). Paradoxically, the most dangerous in spontaneous death may be risk of recovery. When organism (or a suborganismal system) is so badly damaged that becomes close to a death, it cannot further guarantee preservation of its DNA against injuries that can lead to appearance of monsters among offspring. Perhaps, this is the reason why organelles, cells, and organisms follow the principle declared by a Moliere’s character who preferred to die according to all the rules than to recover against the rules. Weismann’s idea on death as adaptation, the Samurai law and Moliere’s principle... All of them, when applied to man, look like atavisms inherited from animals. They are pernicious for individual but good for evolution. In wild nature, phenoptosis is useful first of all for survival and evolution of populations in aggressive environments. Humans organize their life to minimize its dependence upon environmental conditions. As for evolution, we no longer rely upon its slow rate. If we need to fly, we construct a plane instead of waiting for wings to appear on our back. Even such a phenoptotic function as


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defense against asocial monsters that can appear in the progeny of old parents due to mutations accumulating with age may be replaced by, say, social regulations forbidding the bearing of children beyond some critical age. The Samurai principle that for a long time was dominant in Japanese society was, after all, replaced by more humanistic laws. The great Russian biologist Ilya Il’ich Metchnikoff, who discovered phagocytes, suggested the idea of immunity, and received one of the first Nobel Prizes, wrote in his Optimistic Studies which I like very much: ‘‘Traits of animal origin are inherent in humans, who appeared as a result of a long cycle of development. Achieving a level of mental development, which is unknown among animals, human retains many traits that are not only unnecessary for him, but even obviously harmful’’ [212]. Phenoptosis must be attributed to the most harmful among such traits. Attempt to arrest a phenoptotic program, if it is really the aging mechanism, might be a way to human longevity. To solve the problem, we should elucidate all the chain of events involved. This seems to include (i) an ‘‘age meter’’ (p53 or another system monitoring the telomere shortening?), (ii) a mechanism processing the ‘‘age meter’’ signal (a p53-induced burst of the ROS-producing respiration in mitochondria?), (iii) massive mitoptosis leading to apoptosis in organs of vital importance, (iv) slow development of dysfunction of these organs. An obvious complication on this way consists in that (a) several phenoptosis mechanisms operating in parallel may exist and (b) components of these mechanisms may be involved in functions other than phenoptosis. For example, p53 takes part not only in the age measuring but also in anticancer defense. Therefore, deletion of the p53 gene shortens the life span due to the great increase in cases of malignization [209]. In this fashion, nature excludes the possibility of appearance of immortal individuals. A low p53 level decelerates aging but stimulate cancerogenesis, whereas a high p53 level prevents cancer but accelerates aging [210].

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As to the number of phenoptotic mechanisms, it should be finite and hardly large. In this respect, the concept of phenoptosis has obvious advantage over traditional gerontology: it suggests to break a limited number of programmed death pathways rather than to repair numerous injuries in such a very complicated system as the human organism. Such an approach, I hope, has a chance to be promising. As we say in Russia, ‘‘it is easier to break than to repair.’’

Challenge 6: Life without Science Directorship in the Belozersky Institute When I made my first small research work as a first-year student, I felt happiness which I never had felt before. It was in 1953. For twenty years from that time, research was my sole occupation. In the very beginning of 1973, fate offered me for the first time a test: research vs. administrative carrier. Professor A.N. Belozersky, the founder and director of our Laboratory of Bioorganic Chemistry, suddenly died. I decided to try to replace him. In one day I became a formal boss of hundreds of people: researchers, engineers, technicians, under- and postgraduate students. A happy event in my personal life (I had just married Inna Severina) helped me to overcome initial difficulties in the quite new field of activity and to find the only possible style of behaviors: to reign but not to rule. It was especially reasonable since just this style was employed by my great predecessor, Belozersky. The problem, however, consisted in that Belozersky was Vice President of the Soviet Academy of Sciences, whereas I was a young Doctor of Science. Immediately after the death of Belozersky, the Laboratory was attacked by university authorities who decided to close it and distribute its researchers and equipment (that time, the best in MSU) between Biological and Chemical Faculties. Unfortunately, Professor I.G. Petrovsky,


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the MSU Rector, a personal friend of Belozersky and one of the founders of the Laboratory, died two weeks after Belozersky. Thus, my first goal was to save the Laboratory. After more than a year’s struggle, we won thanks to consolidation of the department leaders inside the Laboratory and its friends outside. Among the latter proved to be the new MSU Rector Professor R.V. Khokhlov and Professor Yu.A. Ovchinnikov who replaced Belozersky as Vice President of the Academy. After all, the Laboratory was not only preserved but even enlarged and transformed to the Belozersky Institute of Physico-Chemical Biology, MSU. Democratic style of administration, where all important problems are publicly discussed and solved by the Board of the Department Chairmen, was further developed in the beginning of the Gorbachev’s ‘‘perestroyka.’’ We proved to be the first in the USSR who started to elect the Director of institute by a secret ballot at a meeting of the institute researchers. Rather soon, when the situation around the Laboratory was improved, I succeeded in organizing my time in such a way that administrative duties took no more than one day per week. I have kept that regime during all the following years but with two important exceptions that I would like to describe below.

Election of the Rector of Moscow State University In the end of 1991, just after the Yeltsin peaceful revolution, a quite new and very democratic statutes of MSU were introduced. According to the statutes, the University Rector should be now elected by secret ballot of about 1000 MSU professors ^ members of Councils of the MSU faculties and institutes. The main candidates were Prof. V.A. Sadovnichii and a person who will be called below as Mr. X. Mr. X was a typical Soviet economist who tried all his life to convince the audience that the Kolkhoz system is the best one for agriculture.When Yeltsin won, Mr. X suddenly became one of

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the most active proponents of the new regime. He was actively supported by the authorities and mass media. In the evening before the election day, he was presented by the first TV channel as the new MSU Rector. Prof. Sadovnichii represented the previous MSU administration where he was the First Prorector and, in fact, the MSU leader since the Rector, Academician Logunov was also Vice President of the USSR Academy of Science and director of a huge physical institute in Protvino and thus had no time to be deeply involved in the university problems. In contrast to Mr. X, Sadovnichii was a good scientist and lecturer (mathematician, a pupil of famous I.M. Gelfand). During his work as Prorector, he had a chance to demonstrate his qualification as an excellent administrator. On the other hand, Sadovnichii was not involved in the new politics and was attacked by Mr. X as a proponent of the old communist regime, a point very impressive just after the revolution. I attended a meeting of Mr. X with the electors and realized that he is a Lysenko-style demagogue who may ruin MSU if being elected. This is why I agreed with a suggestion of Professor A.A. Bogdanov, Deputy Director of the Belozersky Institute and my old friend, to be also involved in the Rector elections as an alternative ‘‘noncommunist’’ candidate. In this case, it was a chance to take some votes of revolutionaryoriented electors supporting Mr. X. This was quite probable since, in fact, Mr. X, like Sadovnichii, was also a Communist Party member whereas I have never joined any party in my life. Certainly, there was some risk to be elected: the Belozersky Institute was very popular in MSU due to not only good science made here but also to its democratic traditions which sounded especially well in that time. For me, election as the Rector would mean the end of scientific carrier: it would be impossible to make research being simultaneously responsible for 60,000 people working and educating in MSU. Nevertheless, I liked the University too much to allow Mr. X to become Rector. In fact, all my life since age 17 was directly related to


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MSU where I was under- and postgraduate student, researcher, head of department, and director of institute. I cannot imagine any relations with Mr. X as the rector and understood quite well how dangerous for MSU his success would be. Moreover, in those days I remember my grandfather who was revolutionary and once, between revolutions of 1905 and 1917, even accepted a party fee from Lenin since for some time he was the head of organization of Russian communists emigrated to Belgium. At any rate, in March 1992, I joined the group of candidates to the Rector position. I wrote a program of development of MSU, presented dozens of speeches to electors at many MSU faculties and institutes and to those thousand people who had to elect the Rector. Fortunately, I was not elected; so all the story took only one month of my life. During this month, I always felt that I was doing something absolutely wrong and quite unnatural for me. On the other hand, our goal was achieved. Mr. X failed already in the first round of elections. According to the new MSU statutes, only two candidates should compete in the second round if nobody won more than 50% in the first one. This was the case. In the first round, Sadovnichii and I received more votes than other candidates. After all, Sadovnichii was elected so I luckily escaped the risk of finishing my scientific carrier in 1993. As to Sadovnichii, he proved to be really the excellent Rector, being re-elected in 1997 and 2001. In 2001, he earned 98% of the vote ^ the best indication of his obvious success during the preceding eight years.

The Soros Foundation In 1992^1993, economic crisis defeated Russia. In some months, inflation was as high as 300%. All the funds to buy scientific journals, chemicals, and equipment were exhausted. In many groups in my Institute, experiments ceased and in the evening the Institute building looked wistful, all its windows dark.

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The help came quite unexpectedly. In November, 1992, I visited Professor H. Michel in Frankfurt. A conversation after my talk was suddenly interrupted by an urgent call from the USA. It was Dr. A. Goldfarb who was speaking: ‘‘Yesterday Mr. George Soros decided to donate US $ 100 million for Russian science. You should come to Washington to attend his press conference and thank him for his generous gift.’’ In fact, a few months ago I had approached the famous American philanthropist asking for much smaller amount, $ 1 million, to complete construction of a new building of the Belozersky Institute but received no answer. Goldfarb, a Russian molecular biologist emigrated to the USA, was one of Soros’ friends and promised me to assist in my contact with the philanthropist. This is apparently why Goldfarb had chosen me as a person who should attend the Washington event. In the USA, I met Soros for the first time and had a very interesting discussion with him.When I returned to Moscow, I visited Mr. B.G. Saltykov, Russian Vice-Prime Minister and Minister of Science, to report concerning the Soros initiative. A few days later, Soros appointed me and several other Russian scientists as members of the Russian Board of the International Science Foundation established to distribute his money in republics of the former Soviet Union. The first meeting of this Board occurred in the Kremlin office of Dr. A.V. Yablokov, a Board member and one of the advisors of President Yeltsin. From the American side, Dr. A. Goldfarb and Mr. G. Johnson, a Soros adviser, were invited. Curiously, Mr. Johnson forgot his passport in the hotel and the driving license proved the only document he had with him. This was sufficient to enter the Kremlin, a feature typical for that time. At this meeting, my colleagues asked me to be the Board Chairman and I failed to avoid this appointment since I realized how important this money may be for Russian science in such a difficult time. I have never felt such a great responsibility for my actions as in next several months. The goal was not only to distribute money as fast as possible and in the best manner but also to


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introduce to Russia the grant system absolutely unknown here previously. Dangers came from absolutely unpredictable sites. In January, 1993, George Soros was invited to the Kremlin to meet President Yeltsin who wished to personally thank the philanthropist for his enormous donation. In the Kremlin, Soros was accepted by Yeltsin as a head of the state. The two men simultaneously entered a huge hall in the Kremlin Palace to meet in its center for greetings. Yeltsin was accompanied by Vice-Prime Minister Saltykov and me, Soros, by Goldfarb, and Dr. Nina Fedoroff, a member of the ISF International Board. Then Mr. President invited all of us to a much smaller room for an informal conversation. After short exchange with compliments, bosses came to the question how to distribute $ 100 million among Russian scientists. Yeltsin started with very general statements but then mentioned that $ 100 million will be sufficient to support, say, 10 research projects. Each word was fixed by the President’s secretary and I suddenly realized that any statement put forward or approved by Yeltsin will be the final decision and, to change something, it will be necessary to meet the President once more, which was hardly probable in the near future. I was quite impolite but interrupted Yeltsin, saying the Russian ISF Board already elaborated a detailed scheme which is in line with general principles just formulated by the President. Yeltsin and Soros were a little bit surprised, the Vice-Prime Minister was very much frightened. The President relieved tension and spoke: ‘‘Genius thoughts may simultaneous come to the heads of two genii!’’ Thus, our scheme was rescued. The scheme in question cost me much ATP or Hþ and many sleepless nights. In the International ISF Board, an opinion dominated that just this Board, rather than the Russian one, should be responsible for elaboration of the ISF principles and regulations since Russians are not familiar with such kind of operations. As to the Russian ISF Board, it was not unanimous. One of the Board leaders, the great Russian physicist, Professor Lev Borisovich Okun already had an experience of how to

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distribute American money among Russian scientists. In fact, the American Physical Society allocated some amount for this purpose a year before the Soros initiative. This money was distributed by personal decisions of a small group including Okun and several other leading Russian physicists. Okun has proposed a similar scheme for ISF. Goldfarb and I had suggested an alternative plan which was, after long and exhaustive discussions, accepted by both the International and Russian boards. It consisted of two steps. 1. Distribution, as soon as possible, of a smaller part of $100 million as urgent help to all the Russian scientists whose publications are above some critical level. Each could apply for the help if he or she published during the last five years at least three papers in journals of impact factor higher than a certain value. This value corresponded to the medium impact factor of the reviewed Russian journals. As a result, more than 24,000 people applied for the help and obtained $500 each so we spent more than $12 million for this purpose. Due to the crazy dollar^rouble exchange rate, the $500 sum was equivalent to salary of an ordinary researcher for about one year. The action was completed in four months. 2. The larger part of the donation was distributed as ISF grants to those who won a contest organized on the basis of peer review. About 50,000 reviewers (88% from abroad) were involved. More than 16, 000 grant proposals were submitted, and $80 million were spent to support about 3000 grants. Moreover, some money was used to buy journals for scientific libraries ($4.8 million) and to pay for expenses of our scientists attending international meetings ($14 million). In the latter case, to obtain support it was sufficient to present a letter from the meeting organizers that at least a poster of the applicant had been accepted. Almost 10,000 trips were paid. $4 million were spent for telecommunication programs allowing


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Russian researchers to have e-mail and access to Internet. All the programs were operative for three years (1993^1995). Then during an additional year (1996), they were supported by extra money ($30 million) allocated by Soros and the Russian Government in proportion 1 :1. After this, ISF was transformed to CRDF supported by the US Government and ruled by other people. As to me, I was involved in the ISF activity during five years. During the first three months, ISF took so much time that my principle: ‘‘No more than one day per week for nonscientific purposes’’ was inverted and I spent only one day with my science. This was usually Thursday when I gave seminars. Such a regime resulted in severe hypertension which never troubled me before and after these three months. Apparently, research is the only possible modus vivendi for me... The most difficult was to develop regulations optimal for fair distribution of the support. After some unsuccessful attempts to solve this problem, I discovered a simple approach which helped me a lot. I have a younger brother Dmitry, a talented physicist working in a field quite far from mine (space physics). So I tried to create ISF regulations, keeping in mind Dmitry as a recipient of the help. For example, I learned that an old refrigerator in the brother’s apartment was broken and he has no possibility to buy a new one because of catastrophic inflation. This was a personal reason for me to fight for $500 urgent help as the first ISF action. When personal checks for the ISF applicants were prepared in New York, I agreed to transport the first portion of these checks in my bag when I returned from New York to Moscow. This was the only chance to do this fast since the international bank transfer system was not operative yet in that time in Russia. The total amount was equal to several hundred thousand dollars. I was a little bit nervous in New York, receiving such a huge amount of money and I counted the checks only when I came to Moscow. Here I found that the total amount was more than $1 00,000 less than it should be. It was Sunday. Because of the time difference, I ought to wait till Monday

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evening to call to the New York ISF office to learn that it was an ISF cashier who made a mistake and indicated the wrong amount in a form enclosed. For sure, this mistake added some grey hairs on my head. On the other hand, I thought that perhaps I was transporting the brother’s check so I gave him a chance to buy a refrigerator before the beginning of hot Moscow summer. This is why I decided to take this risky job. Fortunately, later the ISF mechanism worked much better thanks to enormous efforts of the ISF Executive Director A. Goldfarb and Chief of the Moscow office P. Arseniev. Any tensions in the relations of the International and Russian Boards disappeared when the former was headed by the famous James Watson. Inside the Russian Board, I had no problem since, after all, Professor L. Okun and I became close friends. As a result, I returned to science and my blood pressure was normalized. The ISF actions were greatly appreciated by scientists of the former Soviet Union. Dealing with thousands of people, it was operating in such a way that a conflict commission which we planned to establish did not have any job. Not a single complaint was received by the Moscow ISF office although the majority (80%) of applicants to the second round grants failed as a result of the peer review procedure. Partially, this was a consequence of that all of them already obtained the first round $500 help. The obvious success of ISF was to open for me a way to a career of politician, but I had a narrow escape of this fate in spite of numerous attempts of some members of Yeltsin’s team to recruit me. My further activity in this field was restricted by membership in the boards of the Russian Foundation for Basic Research (a Russian ISF analog completely supported by the government) and of the Program Supporting Russian Scientific Schools. The latter initiative was independently put forward by me and Professor V.E. Zakharov, and the Russian parliament approved it. Its goal is to support leading Russian scientists and their young students on the basis of their contributions already made in science. Professor M.P. Kirpichnikov, Minister


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of Science, asked me to head this Program but I rejected his request.

Some Other Duties In this context, I should mention some other activities which also consumed my energy for a long period of time. This is, first of all my editorial activity. For 14 years I was a member of the FEBS Letters editorial board. During this time, I handled 2235 papers. I calculated that this work took almost one year of my life. I am very proud that since 1988, when I joined the FEBS Letters Editorial Board, the percentage of the Russian papers strongly increased and this journal is now the most ‘‘Russian’’ among international scientific magazines. This parameter paralleled the impact factor increase, a fact indicating that Russians at least did not reduce the quality of the journal. Before Gorbachev, the Russian scientific community, like other parts of the Russian society, was isolated from the international one, and integration of our researchers to the ‘‘Scientific International’’ was an important goal for Russian scientists. For many (perhaps even for a majority!) Russian biochemists, a FEBS Letters paper proved to be the first publication in English. Some of their manuscripts I rewrote myself since certain quite brilliant Russian authors failed to write a paper in English. Fortunately, the situation dramatically improved during last decade. Moreover, I suggested to the Editorial Board to strongly increase number of invited mini-reviews and for some years the majority of their authors were recruited by me. When dealing with authors submitting an interesting observation which could not be published due to absence of, say, some controls, I returned the manuscript inviting them to resubmit the paper when the work is completed. Some times I returned a manuscript for two or even three times and after all accepted it. My personal record proved to be a paper from a bioenergeticist from Tenerife, who worked in Laguna University, being in fact

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isolated from the international bioenergetic community. He submitted a potentially interesting manuscript which was accepted after four versions. I am very grateful for support of my work in FEBS Letters to Professor Giorgio Semenza, the Editor-in-Chief. My work in the editorial boards of other international journals [Biochim. Biophys. Acta (Bioenergetics and Biomembranes), Europ. J. Biochem., J. Bioenerg. Biomembr., Intern. J. Biochem. Mol. Biol., IUBMB Life, Biosci. Rep.] took much less of my time than in FEBS Letters due to less important role played by the board members of these journals or to much smaller flow of manuscripts. My editorial work as well as many other kinds of my activity would be absolutely impossible without permanent and enormous help of my secretary. This position was subsequently occupied by Vera Dedukhova, Tatiana Konstantinova, and Oksana Malakhovskaya, clever, friendly and hardworking ladies who were always ready to come and help if it was really necessary. The role of the last one, Oksana, must be specially emphasized. She has performed her very difficult job already for 18 years. With years, Oksana became, in fact, my most important coworker, my memory and my ‘‘Internet liaison officer.’’ Her ability to discriminate between more and less important tasks, high intellectual level and fantastic accuracy made her irreplaceable in my university office, a single 20 m2 room occupied by me and her. I would like to give only one example of our cooperation, namely organization of a contest of Academia Europaea (AE) Prizes for young Russian scientists. I established these Prizes when I was elected to AE and became a member of its Council. The goal of the contest was to replace the Komsomol Prizes that disappeared together with the Komsomol after Yeltsin’s revolution. The Komsomol Prizes were the only nation-scale award for young scientists in the Soviet Union. The contest was organized by a staff of about 12 people occupying several rooms in the Komsomol Central Committee building near the Kremlin. A similar amount of work for the AE Prize contest was done for eight years by Oksana, who moreover was responsible for a lot


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of other duties. Oksana, the daughter of well-known chemist and MSU Professor O.M. Poltorak, was graduated from the Chemical Faculty, and started as a researcher. However, she switched to paper work because of an allergy to some chemicals. She lives, like me, in a professor’s apartment in the main MSU building, i.e. seven minutes by foot from my institute. For her, it is not a problem to come to the office at any time including holidays. Thus, it was my great good luck to invite Oksana Malakhovskaya to help me in my work. I was also lucky to work with two people who, in fact, helped in keeping Biochemistry (Moscow), the official journal of the Russian Biochemical Society. My supervisor, Professor S.E. Severin when he became 89 asked me to replace him as the President of this Society and Editor-in-Chief of the journal. I could not say ‘‘nyet’’ to him and agreed to be a candidate to these positions. Being elected by delegates of the Society Meeting, I decided to maintain the previous chiefs of both offices, Dr. M.B. Agalarova (the Society) and Dr. R.D. Ozrina (the journal). Fortunately for me, my duties in the Society proved to be rather formal because of uncertainties in the position of all the scientific societies during the Yeltsin’s times. Under conditions of permanent economic crisis, these organizations, previously supported almost exclusively by the state, proved to be without funding, and their activities were restricted to international connections and carrying out the national meetings once in five years. As to the journal, some radical improvements proved to be possible in a quite new political situation which arose in Russia since 1991. I succeeded in such a change of the agreement between the Editorial Board and Plenum Press publishing the English version of Biochemistry (Moscow) that the Board receives 50% income now instead of 11% in the Soviet times. The extra money was used to organize much better and faster English translation of the papers and to produce the electronic version of the journal. In this work, the contribution of Dr. R.D. Ozrina and Dr. R. Lozier proved absolutely crucial.

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Rada Ozrina, my old friend, was appointed as the Academic Secretary of the journal by Severin. She continued to perform this work when Severin was retired. For her, Biochemistry (Moscow) is like a beloved child. Rada spends almost all her time working for the journal and dealing with its numerous authors. Her contribution is impossible to overestimate. Dr. Richard Lozier is a rare example of the brain drain in the opposite direction, i.e. from the USA to Russia. I met him many years ago at one of the Retinal Protein Conferences. Richard was, in fact, one of the world leaders in this field. Later, he married a Russian girl and decided to come to Russia to occupy a permanent position here. I asked Richard to help me in reorganizing the English version of Biochemistry (Moscow). Soon he became the Editor of this version. Now he controls the quality not only of the language but also of the scientific level of the manuscripts. Very broad interests in biochemistry allowed Richard to perform his functions excellently. Having such a top level Western biochemist as Lozier at this position, Biochemistry (Moscow) moves, step by step, toward international standards. This is also facilitated by some reforms carried out in the journal, namely, simultaneous publication of the Russian and English version which now takes 4^6 months (previously, one and 2.5 years for the Russian and English versions, respectively), and publication of special issues composed of the mini-review series devoted to hot topics of biochemistry. These mini-reviews are written by the leading level specialists in the field (mainly from abroad). Already the first such issue organized by Dr. A.M. Olovnikov as the guest editor was of great success. Full texts of these special issues as well as 20% of the best regular papers are immediately available free of charge via Internet. It is not surprising that the impact factor of Biochemistry (Moscow) is growing. It has become fivefold higher than before the reorganization. More than 100, 000 people are visiting the journal website in Internet each month. To conclude this section, I would like to mention my education duties. Until recently, they were rather small. I am


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presenting one lecture per week during one semester at Biological Faculty and one seminar per week during all the year in my Institute. In both cases, the topic is the same (bioenergetics); so for me it is a pleasure rather than hard work. The bioenergetic seminar is now 35 years old. At the beginning, only a few people attended it ^ biochemist (M. Kondrashova), chemist (L. Yaguzhinsky), physicists (A. Borisov, E. Mokhova), and some undergraduate students, including I. Severina, who later became my wife. At first, we decided to use English at the seminar although none of us had systematically studied this language. We recognized that English is a tool of international communication between scientists and believed that through the seminar we would succeed in both discussing our science and improving our English. This caused problems: at the third seminar session, I forgot the English term ‘‘oscillations’’ and replaced it by ‘‘kolebations’’ (from ‘‘kolebaniya,’’ the Russian equivalent). This neologism caused great mirth, and since that time, we have used English at the seminar only if a speaker cannot speak in Russian. Later, the number of people attending the seminar increased. Now, we regularly get about 50 participants each week. They include researchers not only from the Department of Bioenergetics, MSU, but also from other places in Moscow and the Moscow region as well as students and postgraduates of biological, chemical, and physical faculties of the Moscow State University. The seminar starts with my talk on bioenergetics news (about 1^1.5 h). Usually, it is followed by talk on the same topic presented by one of the key participants.The talk(s) were interrupted many times by questions and short comments.Then, after 20 min break (tea with sandwiches), the second part comes. It includes a 30^60 min presentation from a seminar participant or an invited speaker, summarizing her/his own recent research, followed by about 30 min discussion. Uncompromising criticism and exhaustive discussion are two principles ^ and are applied to any speaker, from undergraduate students to Nobel Prize winners.

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Marten Wikstro«m, the famous Finnish bioenergeticist, told me that he has never been attacked so strongly as at my seminar. For my own bioenergetic news talk, I tried to focus not only on the most interesting publications and e-mails I have read during the last week, but also on the latest observations made by my research teams, or novel ideas that occurred to me. I am very grateful to my seminar. It has saved me from many mistakes at the outset of new projects. It has forced me to invent alternative approaches when old ones were exhausted. Nothing stimulates my thinking like this seminar. Apparently, public attack by young and aggressive people attending the seminar switches on those parts of my brain that cannot be actuated when I am sitting at the desk or staying on the balcony in my MSU apartment or my country house (I prefer to read or write at a desk on the balcony if the temperature is above 0 C). At the seminar, we try to follow the classical tradition of Russian science ^ to approach a problem as broadly as possible. This is why any new observation in bioenergetics as well as any biological discovery of principal importance may be the subject of our consideration. We are lucky to have in the audience many quite different specialists ^ from mathematicians to zoologists and archaeologists. One more feature of the seminar seems to be noteworthy ^ we try to maintain a merry mood even in difficult times. A joke or a funny story slotted into a talk is always welcome. Quite recently, an event happened which should be mentioned here. On February 15, 2002, I was appointed as the Dean of a new MSU faculty, School of Bioengineering and Bioinformatics (SBB). The idea is to establish a new type of MSU faculty able to help the most talented young people to become researchers of an international standard. The School will be small, 25^40 students will be graduated each year. The education will start before they enter MSU. Some senior pupils from the Moscow secondary schools will be attached to tutors, researchers of the Belozersky Institute, to study some basic aspects of the modern biology. For non-Muscovites, a biology class in the famous


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Kolmogorov Boarding School will be opened where youth will be educated during the last three years of the secondary school to be prepared to enter MSU. In MSU (five years), the SBB students will study more mathematics and English in addition to the best biological and chemical courses traditional for biochemical departments of the MSU biological and chemical faculties. The tutor system will also be operative for undergraduate students. After five years, the best people will continue education as postgraduates (three years more). Both underand postgraduate students will spend some time abroad for training at Western universities. Education in SBB will be accompanied by research in the Belozersky Institute under a tutor’s supervision. After receiving PhD, certain former postgraduates will continue research as the Belozersky postdocs. This will be the first postdoc in Russia ^ this institution is still unknown here. Thus, a youth entering the proposed education cycle at age about 15 will have a chance to spend as long as the next 13 years being included in the above system. My dream is that in the end of this way we shall have young researchers prepared to answer to challenges of the twenty first century. I am sure that for biologists, the main challenge will be to change the nature, including ourselves, rather than to explain it. One more challenge...

ACKNOWLEDGMENTS

I am very grateful to Ms. Oksana Malakhovskaya for the help in preparing the manuscript and Dr. Richard Lozier for its editing.

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[196] Madeo, F., Fro«hlich, E., Ligr, M., Grey, M., Sigrist, S.J., Wolf, D.H. and Fro«hlich, K.-U. (1999) J. Cell Biol. 145, 757^767. [197] Ludovico, P., Sousa, M.J., Silva, M.T., Leao, C. and Corte-Real, M. (2001) Microbiology. 147, 2409^2415. [198] Narasimhan, M.L., Damsz, B., Coca, M.A., Ibeas, J.I., Yun, D.J., Pardo, J.M., Hasegawa, P.M. and Bressan, R.A. (2001) Mol. Cell. 8, 921^930. [199] Madeo, F., Herker, E., Maldener, C., Wissing, S., La«chelt, Herlan, M., Fehr, M., Lauber, K., Sigrist, S.J., Wesselborg, S. and Fro«hlich, K.-U. (2002) Mol. Cell. 9, 911^917. [200] Laun, P., Pichova, A., Madeo, F., Fuchs, J., Ellinger, A., Kohlwein, S., Dawes, I., Fro«hlich, K.-U. and Bretenbach, M. (2001) Mol. Microbiol. 39, 1166^1173. [201] Skulachev,V.P. (2002) FEBS Zett. 528, 23^26. [202] Bowles, J.T. (1998) Med. Hypotheses 51, 179^221. [203] Olovnikov, A.M. (1971) Dokl. Akad. Nauk SSSR 201, 1496^1498 (in Russian). [204] Lakowski, B. and Hekimi, S. Science 272, 1010^1013. [205] Ewbank, J.J., Barnes, T.M., Lakowksi, B., Lussier, M., Bussey, H. and Hekimi, S. (1997) Science 275, 980^983. [206] Migliaccio, E., Giorgio, M., Mele, S., Pelicci, G., Revoldi, P., Pandolfi, P.P., Lanfrancone, L. and Pelicci, P.G. (1999) Nature 402, 309^313. [207] Skulachev,V.P. (2000) IUBMB-Life 49 177^180. [208] Azhar, G., Gao,W., Liu, L. and Wei, J.Y. (1999) Exp. Gerontol. 34, 699^714. [209] Tyner, S.D., Venkatachalam, S., Choi, J., Jones, S., Ghebranious, N., Igelmann, H., Lu, X., Soron, G., Cooper, B., Brayton, C., Hee Park, S., Thompson, T., Karsenty, G., Bradley, A. and Donehower, L.A. (2002) Nature 415, 45^53. [210] Skulachev,V.P. (2002) Ann. N.Y. Acad. Sci. 959, 214^237. [211] Skulachev,V.P. (2000) IUBMB Life 49, 365^372. [212] Metchnikoff, I. (1907) The Prolongation of Life: Optimistic Studies. London. Heinemann.


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Chapter 8

FiftyYears of Biochemistry as Enjoyed by a Medical Biochemist Motivated by an Interest in Diabetes PHILIP J. RANDLE Nuffield Department of Clinical Biochemistry, Clinical Laboratory Sciences, Radcliffe Infirmary, Oxford OX2 6HE, UK

Beginnings My interest in medicine was first aroused when, as a boy, I experienced surgery (tonsillectomy), and succumbed to most of the infectious diseases of childhood. I have recollections of being given a junior version of a stethoscope to play with at the age of six or seven or thereabouts but I have no recollection of how much of a nuisance it was or what happened to it! Presumably, I had expressed an interest which was to be replaced much later by the paraphernalia of organic chemistry. By the time I was ten, it was generally assumed in the family that I would take up medicine as a career. However, I was much more interested in outdoor activities (especially soccer and cricket) than in schoolwork, up to age fifteen. I was born and spent the first eighteen years of my life in Nuneaton which lies in the centre of England and conveniently located within easy reach of Birmingham, Leicester, and Coventry and on the main railway line between London and Scotland. My father had a bakery and grocery business founded

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by his father, and in those prewar and wartime years (1926^ 1945) bread was delivered mainly by horse-drawn vehicles. Up to the age of ten, we lived in a complex in the centre of town which contained the shop; a house behind where we lived; storage space for the carts which delivered bread; the bakehouse and associated storage space for flour, the other ingredients for the manufacture of bread and confectionery, the dough which was left overnight to ‘‘rise’’; and the coke for the ovens. The horses were kept in fields about two miles away in summer and in stables in a nearby part of town in winter. I gleaned something of the processes involved in the maturation of doughthe gas bubbles were obvious and I was told they were carbon dioxide (I had some very rudimentary chemical knowledge gleaned from my father). In 1936 when I was ten, we moved to a purpose built house on the outskirts of town, and located at the front of the field where the horses were kept. As a

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consequence, the horses were now available for riding some evenings and most Sundays. My father also harvested hay for winter feed from other fields which he rented. The fields behind our house also provided a venue for out of school sporting activities! In particular, it enabled me and my friends to play soccer (rugby football was the principal winter sport at the local Grammar School)and cricket in school vacations. My paternal grandparents had died before I was born so that on Sundays we invariably went with uncles, aunts, and cousins to my maternal grandmothers for tea. My maternal grandfather had also died before I was born. He was an undertaker (i.e. he arranged burial of the dead) and the transport this entailed was provided first by a collection of horse drawn carriages, then a fleet of Daimlers, and finally of Rolls Royces. These were located together with workshops in a very large garage accessible from my grandmother’s house. From time to time my cousins and I played in these cars before and after tea on Sundays until our parents decided enough was enough. The undertaking business was later taken over by two of my uncles; but in reality, their true interest was in farming ^ both acquired farms in due course which were managed for them on a day to day basis. As a consequence, I was able to add tractor driving and associated farming activities (e.g. ploughing, scuffling, and reaping) to horse riding, and hay cropping. As a result of these and other agreeable distractions, I was assigned to the B stream at school and did general science rather than physics and chemistry up until age 15. Schooling (1931^1944). My early education (1931^1936) at a state elementary school in Nuneaton was uneventful. Fortunately, from the point of view of my future career, my school from age 10 onwards (1936^1944) (King Edward VI Grammar School, Nuneaton) had an outstanding and inspiring chemistry master (Mr. C. Brown), who in the wartime also taught sixth form physics. He interested me in chemistry in particular, and as he thought I had some talent, he obtained a place for me in the science sixth form in 1941. [This was in spite of misgivings on

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the part of the headmaster who assured my father that I would not obtain scholarships to university; I obtained three in due course.] The school had no biology teacher or lab; but it was bombed in 1941 (Nuneaton was only eight miles from Coventry ^ a favourite target for bombers). At that point of time, I was due to join the sixth form (preparatory for university entry). As a result of the bomb damage, we were evacuated to the local girls’ high school which did have the necessary facilities for teaching sixth form biology. Brown’s teaching of chemistry in the sixth form was comprehensive and inspiring in all its branches. As a consequence, I went up to Cambridge with a knowledge of the subject as extensive as that of most chemistry undergraduates at the end of their first year. At that time medical students were required either to read organic chemistry at Cambridge in their first year, or to pass the equivalent of the first year examination from school. I elected to stay on in the sixth form for a third year to do the scholarship examinations at my chosen college (Sidney Sussex); the organic chemistry examination; and an entrance examination in Latin (Latin or Greek was then a compulsory requirement for entry). The whole of my summer vacation before going to Cambridge was overshadowed by the need to satisfy the entrance requirement in Latin. I was helped in this by an aunt who was a qualified teacher of classical languages. I only succeeded in satisfying the latter requirement one week before I went up. Failure would presumably have been meant being called up into the Air Force (I had been an air cadet in my wartime years at school); medical students were exempt from military service until qualified to practise. In the early 1960s I had the quiet satisfaction of voting with those who succeeded in abolishing the compulsory Cambridge entry requirement for a qualification in Latin or Greek! Undergraduate days and House Jobs (1944^1952). I went up to Cambridge in 1944. Cambridge in wartime was rather different from Cambridge in peacetime. The undergraduates were virtually all science and medical students, the few arts students being either medically unfit for military service, or from


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overseas. Teaching went on for six days a week and part of the college was occupied by civil servants evacuated from London. There was, of course, food rationing and in my third year, we were obliged to lunch at a nearby British restaurant (two courses then cost 5p in current UK currency). The town had sustained little, if any, bomb damage. At Cambridge medical students did the Natural Sciences Tripos with anatomy and physiology as compulsory two-year subjects. This left the choice of two one-year-half subjects. I chose biochemistry (and pathology). The biochemistry course gave me a lifelong interest in the subject; the Cambridge pathology course ^ principally morbid anatomy, histology, and bacteriology ^ was uninspiring for a preclinical student motivated primarily by chemistry and physiology and interested in mechanisms. In my first two years, I was tutored in biochemistry by Ernest Baldwin and in physiology by McCance and Neville Willmer. McCance, though interesting and well informed on medical matters, was not very helpful; his knowledge of general physiology was patchy and his areas of expertise did not cover the first year curriculum. He hated being asked questions to which he did not know the answer and in consequence, we were reduced oftimes to asking him questions that we knew he could answer. Fortunately, he was promoted to Professor at the end of my first year and I was supervised by Neville Willmer in my second year. He was not only well informed on most aspects of physiology, but he also taught me how to think about physiology and biological sciences in general and became a lifelong friend. I stayed on in Cambridge in 1946^1947 for the third year biochemistry degree course and as a consequence was hooked for life. The lecturers were distinguished, the course inspiring, and far sighted. That year they included Chibnall, Sanger (who had begun to sequence insulin with his fluorodinitrobenzene method), and Kenneth Bailey, on protein biochemistry; Max Perutz on X-ray crystallography (his visible computing facility at that time was a motor operated Brunswiga calculating machine); Keilin and Hartree on cell

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respiration; Malcolm Dixon and Edwin Webb on enzymes; Davy Bell on carbohydrate biochemistry; Robin Hill on photosynthesis and plant biochemistry; Guy Greville and T.R. Mann on metabolism, metabolic regulation, and hormones; and the staff of the Dunn Nutrition Unit on the biochemical basis of nutrition. Distinguished visiting lecturers included Martin and Synge (on their new technique of paper chromatography), Carl and Gerty Cori, Fritz Lipmann, and Albert Szent-Gyorgi. Our external examiner was Hans Krebs ^ awe inspiring but well suited from my point of view as my main interest ^ metabolism ^ was well represented in the examination papers! Although laboratory safety in those days was generally stringent, equipment was not as consistently safe as it is now. I recall a demonstration on X-ray crystallography in 1946 by the late Max Perutz in which students had to be advised rather exactly where to stand to avoid being radiated. I finished off my time in Cambridge by attending the 1947 meeting of the British Association for the Advancement of Science. Universities in the UK were each invited to nominate students (I think two) to attend this meeting with all expenses paid. I was lucky enough to be one of the Cambridge delegates and off I went to Dundee. There was much of interest for a medically inclined biochemist ^ most notably penicillin which had been isolated and put into medical practice by Florey and Chain and Fleming during the war (Chain spoke). That apart, it is always of interest to students to put faces to names so to speak and to begin the process of joining the community. I was to get to know Prof. (later Sir Ernst) Chain well in due course beginning with a memorable occasion in 1963 when he and Lady Chain came to Cambridge to play chamber music with Sir Rudolph and Lady Peters. [Chain was an accomplished musician. It was said that he had been a trainee conductor with the Berlin Philharmonic Orchestra in the days before Hitler.] The sitting room in his flat at Imperial College was dominated by two grand pianos [Lady Chain was also a pianist] though I never heard them played. Sir Rudolph, who was a good friend over


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many years, brought him down to my lab. Chain disagreed with some of my research findings, and in spite of his distinction I felt obliged to defend my work and did not hesitate to do so (politely and with tact or so I thought). The discussion became quite animated. After his departure, Ruby Leader, who had been in charge of laboratory animal care since the department opened under the chairmanship of Gowland Hopkins in the 1920s, came across from her room to enquire whether it was Prof. Chain’s voice that she had heard! She had first come to know him when he had worked in the department as a refugee from Nazi Germany before the 1939^1945 war. [Being the man he was, Chain subsequently took a helpful interest in my career behind the scenes though I did not know of this at that time.] In 1947 I went on (reluctantly) from Cambridge to do my clinical studies in London at UCH (University College Hospital). [Prof. Chibnall persuaded me to continue with my medical studies against my inclination at that time.] I soon came to be thankful for Chibnall’s persuasive influence. UCH was an exciting and agreeable clinical school and its situation in Gower Street close to University College was ideal from many points of view. For most of my time at UCH, I lived either in the student hostel in Gower Street or in the nearby quarters for resident hospital staff. Postwar London was a very different place from what it is now. There was virtually no traffic in Gower Street between 7pm and 7am and every evening sandwiches and other snacks could be obtained from a mobile snack bar which parked outside the college from 7 to 10 pm. The hostel, and the nearby student medical school common room, were equipped with snooker tables and we indulged ourselves before dinner or after an evening’s work. I had learned to play bridge in the wartime long vacation terms at Cambridge and there were many other students willing to make up a foursome. The college sports ground was not too distant and I arranged for Saturday cricket fixtures at Oxford and Cambridge in my spell as secretary. Lord’s cricket ground was

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nearby, and I can recall as a student being beguiled by friends to sneak away from ward rounds to spend sunny afternoons there during memorable visits from Australian and West Indian sides. Theatres (including the Covent Garden Opera House) sent free surplus tickets to the nearby nurses’ hostel and if the nurses did not want them they were available to students. That facility initiated my lifelong liking for opera (not very popular amongst nurses). I recall in particular a memorable season of Wagner operas with distinguished soloists (notably Hans Hotte and Kirsten Flagstadt) and also my first introduction to the haunting music of Der Rosenkavalier. There were concerts at the Royal Albert Hall and especially noteworthy were those given by Sir Thomas Beecham and the newly re-formed Royal Philharmonic Orchestra. [I first had the pleasure of listening to this orchestra under the baton of Sir Thomas in the Cambridge Guildhall in 1947.] HP (later Sir Harold) Himsworth was then Professor of Medicine at UCH. His major interests were diabetes and liver disease, but he was also promoting the biochemical approach more generally. His staff included CE Dent, a chemist who had turned to medicine and whom Himsworth had recruited to work on liver biochemistry. He chose to devote himself to the biochemistry of bone diseases when Himsworth departed and thereby achieved considerable distinction. Himsworth’s major contribution to diabetes in the 1930s had been his ‘‘insulinglucose tolerance test.’’ Through this he developed the concept of two types of diabetes characterised by insulin deficiency and insulin insensitivity, respectively. The chemical pathology department at UCH was of long-standing and its distinguished professoriate had included Sir Charles Harington, a discoverer of thyroxine. The head of department in my time at UCH was the porphyrin biochemist C. Rimington. I was familiar with his work from Part 2 biochemistry lectures at Cambridge. My interest in diabetes was initiated at UCH by Himsworth; and although he left to be Secretary of the Medical Research Council after a couple of years or so, this interest stayed with


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me and was to motivate my research throughout my subsequent career. All in all, the choice of UCH was most fortunate, another factor being the close proximity of University College which possessed a biochemistry department (then headed by Prof. Young ^ see later) which welcomed me to its seminars and to its lectures by visiting biochemists. I qualified in medicine in 1950, and embarked on a year of medical and surgical appointments at UCH followed by a further six months at St. Helier Hospital Carshalton. [There had been a six months’ delay in finals because of a shadow in a chest X-ray which might have been early tuberculosis ^ acquired from a highly infectious patient whom I had clerked. This delay turned out to be a blessing in disguise because, as a result, I was applying for a house physician job at the least competitive part of the year (December 1950).] I had no further trouble from this ‘‘X-ray illness.’’ It was my good fortune that my UCH medical house appointment was with Max Rosenheim and Charles Dent. Max (later Lord) Rosenheim, though mainly interested in renal disease and hypertension, remained a good friend and supporter until his untimely death in the early 1970s. My second house job was on a surgical firm which boasted both a neurosurgeon (Mr. Julian Taylor) and a genitourinary surgeon (Mr. ‘‘Tim’’ Merrington). My third house job (medical) was at St. Helier Hospital in Carshalton with a congenial and helpful physician from Guys Hospital (Dr. J.B. Harman) and a former medical registrar from UCH (Heinz Wolff). By the early summer of 1952, I had decided to pursue a career in biochemistry but wished to retain some interest in clinical medicine. In Cambridge Prof. Chibnall had by then retired and had been succeeded by Prof. F.G. Young, the leading UK researcher, into the biochemistry of diabetes at that time. I wrote to him and characteristically he invited me to meet him at the CIBA Foundation where he was attending a symposium related to diabetes. Our conversation was short and to the point and he advised me to apply for support for salary and research expenses from the Medical Research

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Council and how to proceed. It was characteristic of him that he then took me to the conference reception and introduced me to many of the leading figures in metabolic research at that time. He subsequently provided me with a lab in the Cambridge biochemistry department and a physician at Addenbrookes hospital (Dr A.P. Dick) employed me one day a week to look after his diabetic outpatients. The Medical Research Council provided a salary and research expenses and as a result of these arrangements I married and we bought a house in Cambridge. My wife (Elizabeth) and I celebrate our 50th wedding anniversary this year. After a couple of years, Sidney Sussex College appointed me to a research fellowship (1954^1957). My subsequent career was as a lecturer in biochemistry at Cambridge (1955^1964) with a fellowship as Director of Medical Studies at Trinity Hall (1957^1964); as first professor and head of the then new Department of Biochemistry at Bristol (1964^1975); and as Professor of Clinical Biochemistry at Oxford with a fellowship at Hertford College (1975^1993).

Research Beginnings: Cambridge Biochemistry Department, 1952^1960 My research career began with an attempt to bioassay insulin in blood plasma. Two possible assays had been described. Anderson and Long at the NIH had devised a bioassay which was sufficiently sensitive for the assay of insulin in pancreatic perfusate. This was based on the blood glucose lowering effect of insulin in alloxan-diabetic hypophysectomised adreno-demedullated rats but this was not sensitive enough for measurements in peripheral blood. Bornstein in Melbourne claimed greater sensitivity with alloxan-diabetic hypophysectomised adrenalectomised rats but no one else (including myself) succeeded


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with this assay (the animals failed to survive the last stage ^ adrenalectomy though they did survive resection of the adrenal medulla in confirmation of the work of Anderson and Long). I spent a year working on in vivo assays before abandoning this line. I then explored an in vitro bioassay based on the glucose uptake of rat diaphragm following an initial report by Groen in Amsterdam. This did yield results of interest, but the specificity of the method was clearly not straightforward ^ a problem more generally for bioassays for insulin in blood. It did, however generate results which attracted much interest at that time.The most interesting novel findings from my own studies in patients with this method were that plasma insulin activity is decreased in hypopituitarism and increased in acromegaly (excessive production of pituitary growth hormone) [1,2]. This was, perhaps, expected as it was known from the work of Houssay and others that sensitivity to the blood glucose lowering effect of insulin is enhanced in hypopituitarism and decreased by pituitary extracts in experimental animals and by pituitary hypersecretion of growth hormone in acromegaly in man. My mentor (Prof. F.G. Young) had achieved distinction for his work on the diabetes-inducing action of pituitary extracts and of purified growth hormone. He and others had established that growth hormone is not diabetogenic in rats but is diabetogenic in cats, dogs, ferrets, and man. He had discovered that prolongation of growth hormone treatment in cats and dogs could result in the development of a permanent form of diabetes which persisted when growth hormone injections were discontinued. The findings in acromegalics prompted a study of the effects of hypophysectomy (rats) and of treatment with growth hormone (rats and cats) on plasma insulin activity. These studies showed that hypophysectomy decreases plasma insulin activity in rats and that this is restored by growth hormone treatment. In cats plasma insulin activity was increased in response to treatment with growth hormone even when diabetes appeared but fell to abnormally low levels when the permanent form of diabetes developed [3].

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In 1954 Prof. Young was invited to give a paper at a Symposium on The Hypophyseal Growth Hormone, Nature, and Actions at the Henry Ford Hospital in Detroit. Other commitments made it impossible for him to attend. With characteristic generosity he asked if I might go in his stead. The committee agreed but stipulated that I must present the results of my own work. So, as a graduate student at the start of my third year, I flew off to Detroit somewhat nervously to read my first paper at a scientific meeting in the company of a host of distinguished speakers including Carl Cori and Bernardo Houssay. I then embarked on a tour of labs in Chicago, St. Louis, Nashville, Bethesda, Philadelphia, and Boston. Many of those that I met on this memorable first visit to the states were to become lifelong friends. Transatlantic flight (propeller driven) was very different in those days from what it is now. As a substitute for Prof. Young, I travelled first class with PanAmerican Airways. It is sad that what was then the most prestigious of American international airlines disappeared many years ago. The aeroplane was a Boeing stratocruiser. There were pull down, curtained bunks where overhead racks are located in present day passenger aircraft. The seats routinely had some five feet of leg room; and there was a downstairs bar with seating for ten. Flying time to New York was about 14^16 hours on a good day and usually involved a stop at Shannon on the west coast of Ireland to refuel both planes and passengers. In flight there was plenty of time both for talking in the downstairs bar and for sleep. There were separate men’s and ladies’ washrooms on either side of the aircraft. There the ladies (and some men) would change into night attire before tripping down the aisle, climbing ladders to the bunks, and drawing the curtains. [I did not bother with a bunk or night attire ^ the seats with extended leg rests were nearly as long as the bunks and as comfortable.] The changeover to jets at the end of the 1950s changed the whole character of long distance flying. It became much more routine. The bonhomie of the shared challenge and the varying flight plan


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largely disappeared. As a result of that visit, I got onto the ‘‘circuit’’ and in my Cambridge days was invited to further meetings in the USA in 1956, 1957, 1958, 1960, 1961, and 1963. I did not have what was then the customary year working in a lab in the USA, though I did spend a most enjoyable three-month period in 1960 working in Prof. C.R. Parks Physiology Department in Nashville (including one visit to the Nashville ‘‘Grand Old Opera’’ and the acquisition of a liking for baseball). The 1958 trip (to the Upjohn Conference facility at Brook Lodge in Michigan followed by trips to Toronto and Montreal) is remembered particularly because I missed the Cambridge biochemisty department’s celebration of Dr. Sanger’s first Nobel Prize. I was having dinner with Prof. John Beck and his wife in Montreal prior to the overnight flight home when the conversation turned to Fred Sanger and when he might get the Nobel prize. Mrs. Beck said there was something in the newspaper and there was the announcement. I got back to the lab the following day to find the untidy remains of the previous evening’s celebrations which I had missed! The problem of blood assays for insulin (and for other peptide hormones relevant to diabetes) was deemed of such importance that Dr. DeWitt Stetten of the NIH organised a meeting in Bethesda in 1957 on the assay of insulin (and other protein hormones) in blood. This was attended by bioassayists, and by those who were endeavouring to develop immunoassay methods. The latter were principally Yalow and Berson, who had successfully prepared radio-iodinated insulin and were in the process of developing radio-immunoassay (also G.M. Grodsky); and C.H. Reed who was attempting immunoassay of pituitary growth hormone with tannic-acid-coated red cells. It was obvious (to me at any rate) that radioimmunoassay could become the method of choice for assay of peptide hormones in blood. However, the Cambridge biochemistry department lacked the facilities for development of radioimmunoassay at that time. The necessary equipment became available in 1960 when H.G. Coore and I embarked on in vitro studies of the regulation of

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insulin secretion in rabbit pancreas pieces employing a double antibody method of radioimmunoassay developed in my lab by C.N. Hales (see later). The latter was subsequently marketed as a kit ^ by Wellcome and the Radiochemical Centre at Amersham. The demand for the kit was such that the paper on this method became a citation classic in Current Contents. Fortuitously, the work with rat diaphragm led to two observations on the side which proved to be important in other respects. In 1956 I discovered (initially by chance) that anoxia, inhibitors of respiration, and uncouplers of oxidative phosphorylation, stimulated in vitro glucose uptake by rat diaphragm [4,5]. By good fortune I had utilised a bicarbonate-buffered medium equilibrated with O2/CO2. This example of the Pasteur effect had been overlooked by earlier workers who had utilised phosphate-buffered media (phosphate buffering is inadequate for the high lactic acid production induced in diaphragm muscle by anoxia, uncouplers, and respiratory inhibitors). In collaboration with G.H. Smith (rat diaphragm) and H.E. Morgan (rat heart), it was shown in the late 1950s that activation of membrane transport and intracellular phosphorylation of glucose were the principal reactions accelerating glucose uptake [6^8]. Phosphofructokinase I (PFK1) was identified in later studies with E.A. Newsholme as the principal intracellular reaction mediating their effects on phosphorylation and glycolysis of glucose. The mechanism was identified as allosteric activation of PFK1 by altered concentrations of fructose 6 -phosphate, ATP , ADP, 50AMP, and Pi with secondary activation of hexokinase by decreased glucose 6 -phosphate. This general mechanism was first described by Passonneau and Lowry in their pioneering studies with the isolated enzyme [9].

Evolution and Revolution 1960^1964 In the mid 1950s I came across a paper given at the CIBA Foundation in 1952 by Drury and Wick in which they reported


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that injection of 3-hydroxybutyrate or acetate into eviscerated rabbits impaired glucose oxidation [10]. This prompted me to investigate and to show late in 1956 that 3-hydroxybutyrate inhibited glucose uptake by rat diaphragm in vitro in the presence of insulin (but not in its absence). This was the beginning of my career-long interest in regulatory interactions between glucose and lipid fuels and mechanisms of insulin resistance. I did not attempt to publish this finding at that time (a mistake that I was to regret) and was not able to pursue this line further until E.A. Newsholme joined me as a PhD student in 1959. By then the circulation of long chain nonesterified fatty acids (NEFA) in association with serum albumin had been discovered. Shipp et al. and Williamson and Krebs first reported inhibitory effects of albumin bound fatty acids and of ketone bodies respectively on glucose uptake in perfused rat heart in 1961 [11,12]. Newsholme and I began work in this field in 1960 and published our first results shortly thereafter [13]. In further studies with E.A. Newsholme and P.B. Garland and later with P.J. England, the pyruvate dehydrogenase (PDH), phosphofructokinase (PFK1), and hexokinase (HK) reactions were identified as the prime sites of inhibitory effects of fatty acid and ketone body oxidation on glucose utilisation in cardiac muscle. The mediators of these inhibitions were identifed as increased [acetylCoA]/[CoA] concentration ratios for PDH complex (and thereby glucose oxidation); and increased [citrate] (PFK1) and increased [glucose 6 phosphate](HK) for glucose uptake and glycolysis. The identity of these mediators has withstood the test of time notwithstanding the subsequent discovery of the role of reversible phosphorylation in the regulation of the PDH complex and of PFK1. The increase in citrate concentration was shown to be directly attributable to the increased [acetylCoA]/[CoA] ratio. Comparable increases in these ratios in cardiac and skeletal muscles of diabetic or starved rats were shown to be derived from accelerated oxidation of fatty acids. (In vitro this was shown to be the result of increased concentration and

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accelerated breakdown of muscle triacylglycerol.) We coined the term glucose fatty acid cycle to describe these regulatory interactions [13^15]. At much the same time (1961), C.N. Hales in my lab had successfully developed a double antibody radio-immunoassay for insulin in blood plasma [16] and this enabled H.G. Coore to embark on his studies in vitro of the regulation of insulin secretion. Whilst others skilfully employed perfused rat pancreas preparations for this purpose, Coore developed a much simpler and equally effective method employing pieces of rabbit pancreas incubated in vitro. Amongst other things, this allowed us to demonstrate for the first time the inhibitory effect of adrenaline on glucose-induced insulin secretion. (The vasoconstrictive effect of adrenaline which complicates perfusion did not interfere in experiments with pancreas pieces.) By a happy coincidence, Ernst Simon of Rehovoth visited our lab in Cambridge in 1961 in the course of returning to Israel from the USA. He discussed with us his work on the diabetogenic properties of the seven carbon sugar, D-mannoheptulose, which he and his colleagues purified in Israel from its abundant avocado pear crop. We agreed to collaborate and Coore was able to show that mannoheptulose, an inhibitor of hexokinases, specifically blocks stimulation of insulin secretion by sugars which are metabolised via hexokinases. These and contemporaneous studies by G.M. Grodsky in San Francisco with perfused rat pancreas provided the first convincing evidence that insulin secretory responses to sugars are mediated by their intracellular metabolism [17,18] and that the concentration dependence for glucose is determined primarily by the kinetics of glucose phosphorylation by glucokinase. Ultimately, the validity of this conclusion was to be substantiated by S.J.H. Ashcroft and others (see Ref. [19]). The mechanism ^ activation of ATP sensitive Kþ channels by glucose mediated ATP synthesis was established in the late 1980s/1990s by the work of the electrophysiologist F.M. Ashcroft with whom he collaborated [20].


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The Move to Bristol The 1960s ushered in a revolution in UK society as a whole, and especially in the attitudes and expectations of young people of university age whose numbers had expanded rapidly in the fifteen years since the end of the 1939^1945 war. One consequence of this was a nationwide expansion in student intake to universities. This necessitated the creation of new universities, the enlargement of existing ones, and the creation of new departments and disciplines. In 1963 Bristol University in common with a number of other universities, embarked on an expansion programme which included a new medical school building and the establishment of a Department of Biochemistry. I was fortunate to be appointed as the first professor and head of this new department. The university had a considerable and growing reputation and being situated in a very attractive city and part of the UK, it had few problems in attracting good staff. There was no shortage of applicants for lectureships and no shortage of new lectureships with the 1960s boom in university education and university-based laboratory research. Bristol had medical, dental, and veterinary schools and each required biochemistry teaching, which up until that time had been provided by the physiology department. There was also a degree course for scientists in biological chemistry which had been run jointly by the physiology, pharmacology, zoology, botany, and chemistry departments and which we inherited. As a consequence, the biochemistry department which acquired six members of academic staff and one technician on my appointment in 1964, had expanded to three professors and 28 members of academic staff on the university payroll and some 80 postdocs, research students, and technicians on outside grants, when I left in 1975. My contemporaries and I were fortunate to be born at exactly the right time to benefit from the explosive growth of universities and of funding for biomedical research in the 1960s and 1970s.

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Biochemical Research at Bristol 1964^1975 In the year following my move to Bristol, I was fortunate enough to receive recognition for our work on the Glucose Fatty Acid Cycle and on mechanisms regulating insulin secretion. This included invited lectures at Gordon and Laurentian Hormone Conferences, and the Banting Memorial Lecture of the British Diabetic Association (in 1965). This was followed in 1966 by award of the first Minkowski prize and lecture by the newly formed EASD (European Association for the Study of Diabetes).

Acylglycerols in Muscle My research in Bristol from 1964^1975 sought initially to expand on the two areas which I had developed in Cambridge in 1960^1964 with Newsholme and Garland, and Hales and Coore. R.M. Denton, a Cambridge graduate who had joined my lab in Cambridge in 1963 as a research student to work on the regulation of adipose tissue metabolism, moved to Bristol with me. In addition to his work on adipose tissue, he developed methods for the analysis of acylglycerols in muscles. In formulating the concept of a glucose^fatty acid cycle, we had proposed that accelerated breakdown of muscle acylglycerols was solely responsible for enhanced fatty acid oxidation in muscles of starved and diabetic rats in vitro, and hence for impaired glucose uptake and insulin sensitivity of cardiac and skeletal muscles from starved and alloxan diabetic rats. Denton and I were able to show by direct analysis that cardiac and gastrocnemius muscle triacylglycerol concentrations were increased by alloxan diabetes and by 24^72 h of starvation; and that lipolysis and release and oxidation of free fatty acids from endogenous triacylglycerol were accelerated in these muscles in vitro [22], (cf. [21]). These muscles were histologically free of adipose tissue and the triacylglycerol was visibly located within the muscle fibres. This provided further compelling evidence for


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the role of increased lipolysis and consequential free fatty acid oxidation in the disturbed carbohydrate metabolism in muscles in diabetes and starvation.

Fatty Acid Oxidation Inhibitors In 1967 Chase and Tubbs reported that carnitine acyl transferase and hence fatty acid oxidation are inhibited by 2-bromostearate (see Ref. [23] for later definitive reference). I was able to show in perfused rat heart that inhibition of fatty acid oxidation with 2-bromostearate reversed the insulin resistance in cardiac muscle in alloxan diabetes [24]. P.J. England, who graduated from the Bristol biochemistry department in 1965, provided sound quantitative evidence that increased glucose 6 -phosphate concentration mediates inhibitory effects of fatty acid oxidation on hexokinase and hence of intracellular glucose utilization [25]. This effectively completed my interests at the time in lipid^ glucose interrelations in the regulation of energy metabolism in muscles. My interest in this general area was to be reawakened in the 1970s by Lester Reed’s discovery that the pyruvate dehydrogenase (PDH) complex is regulated by reversible phosphorylation. Pancreatic Islets in vitro In the spring of 1965, I was joined in Bristol by S.J.H. (Steve) Ashcroft, who took over the programme of work on the mechanism of regulation of insulin secretion initiated in Cambridge by H.G. Coore. Ashcroft set out to separate pancreatic islets from acinar cells as a prerequisite to an investigation of our hypothesis that stimulation of insulin secretion by sugars is mediated by their metabolism within islet -cells. By happenstance, Moskalewski [26] and others had just described use of collagenase to release islets from within the exocrine pancreas. These could

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then be separated from acinar cells by hand picking with a wire loop under a dissecting microscope, or by differential centrifugation. (At that time hand picking provided the best functional islets.) Further development and refinement of this technique allowed Ashcroft to explore in detail the relationship between aspects of glucose metabolism and rates of insulin release in isolated islets.These studies in mouse (and also in human) islets led us to conclude in 1970 that glucose stimulation of insulin release was likely to be mediated by metabolism of the sugar [27,28]. The conclusions at the time were tentative (there is a phrase in the UK about ‘‘hedging ones bets’’!) but they turned out to be correct. Our hypothesis was that glucose-induced insulin secretion is effected by metabolism of the sugar and that concentration dependence is conferred predominantly by the glucose kinetic properties of islet glucokinase. Matschinsky who initially supported this hypothesis switched to expressing serious reservations [29]. Steve Ashcroft eventually took over this interest completely and his work [19] and further work by Matschinsky (after reconversion [30]) was to lead to acceptance of the hypothesis. Steve’s later collaboration with the electrophysiologist F.M. Ashcroft was to result in the discovery of the mechanism ^ i.e. the role of ATP-inhibited potassium channels as mediators of glucose stimulation of insulin release [20]. This discovery established finally that glucose-induced insulin secretion is indeed mediated by glucose metabolism (i.e. through the coupled synthesis of ATP and its effects on the ion channel).

Reversible Phosphorylation in the PDH Complex: 1971^1975 In 1969 Linn, Pettit, and Reed [31] showed that mammalian pyruvate dehydrogenase complexes (PDH) are regulated by reversible phosphorylation, phosphorylation being inactivating. This discovery, which was confirmed in due course by Wieland and Siess [32], and in my own laboratory in collaboration with Coore, Denton, and Martin [33], was clearly of substantial


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potential -importance in relation to the concept of a Glucose Fatty Acid Cycle and to Denton’s interest in the regulation of fatty acid biosynthesis. Our own immediate contributions included discovery of the role of Ca2þ as an activator of PDH phosphatase and thereby to increase PDH complex activity [34]; the ability of dichloroacetate and other halogenated carboxylates to activate PDH complex and promote glucose oxidation in tissues in vitro and in vivo through inhibition of PDH kinases [35]; evidence that the action of insulin to increase PDH complex activity in rat adipose tissue might involve activation of PDH phosphatase; and (contemporaneously with Reed and his colleagues and with Wieland) the activation of PDH kinase by increasing ratios of [ATP]/[ADP], [NADH]/[NADþ], and of [acetylCoA]/[CoA] [36,37,40,41]. The upshot of this was that increases in ratios of [NADH]/[NADþ] and [acetylCoA]/[CoA] were now considered to mediate effects of fatty acid oxidation to decrease rates of glucose oxidation and glucose utilisation in vivo by direct and indirect mechanisms (i.e. by direct inhibition and via protein phosphorylation respectively) [41]. The direct mechanism was reductive acetylation of lipoate; the indirect mechanism was the activation of PDH kinase which ensued. Dichloroacetate (as an inhibitor of PDH phosphorylation) and inhibitors of fatty acid oxidation were subsequently shown to produce a therapeutically useful lowering of blood glucose in Type 2 diabetics by promoting glucose oxidation. Unfortunately, the associated accumulation of NEFA in tissues and in the circulation constituted a potential hazard which eventually precluded their use in clinical practice ^ for more recent review see [42]. In 1973 Alan Kerbey joined my group in Bristol as a technician as his supervisor had gone to work in Geneva on sabbattical leave. Alan, who had been unable to take up a university place and who was to stay with me for twenty years, enrolled as a PhD student, moved with me to Oxford in 1975, and obtained his PhD in 1977.

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International Protein Phosphorylation Group In 1973 I had been invited to join the recently formed international protein phosphorylation group, an offer which I gladly accepted. I subsequently contributed to its meetings in Seattle (1973), Israel (1975), and Pitlochry (1977) [38,39]. These were memorable meetings ^ especially the Israel meeting at which visits to historic sites were incorporated into the programme (usually very early in the morning!). Especially noteworthy were the visits to Masada; and to the Israeli President’s house for afternoon tea. (The then President of Israel was Professor Ephraim Katchalski-Katzir, FRS of the Weizman Institute.)

Other Activities at Bristol External Examining One of the benefits (or disadvantages according to your point of view) of being a professor in the UK was the annual round of external examining in other universities. The first degree examinations for science students were generally held in June. Those for medical and veterinary students were held in spring or summer with re-examination of failed students in summer or autumn, respectively. Overall, this occupied about two weeks of my time in the summer and a few days in the spring and autumn. The advantage of being involved as an external examiner (for both medical/veterinary and science students) was that one rapidly acquired up-to-date knowledge of activities in different universities in respect of biochemistry teaching and research; and of problems being encountered elsewhere and possible solutions. In those days when the number of universities was much smaller than now, one rapidly got to know ones colleagues up and down the country, and some sort of consensus over common problems emerged via the periodic contact on the examination circuit.


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Service on General Medical and Dental Councils In the period from 1967 until my move to Oxford in 1975, I had represented the University of Bristol Medical School on the General Medical Council (GMC). [I also served on the General Dental Council but had relatively less to contribute as my knowledge of dentistry was minimal.] The GMC had two roles ^ it was responsible for maintaining standards in medical education and ensuring up-to-date curricula in medical schools; and it was responsible for maintaining standards in medical practice i.e. it had both educational and disciplinary roles. I took on the job as a favour to the Vice-Chancellor (others were unwilling!). I was always willing (within bounds) to try most things and I was almost certainly the first (and possibly the last) biochemist to sit on this august body. I enjoyed the work, mainly because I was interested in medical education, and by a mixture of good fortune and sympathetic treatment, I managed to keep my exposure to the Council’s disciplinary proceedings at a reasonable level. There was one unforgettable occasion when a train, which should have got me to the disciplinary committee with more than an hour to spare, broke down over the points outside Bristol station. As a result of this mishap, I was five minutes late arriving and as I had missed five minutes of the proceedings, I could no longer serve. The case went on daily for six weeks thereafter so that my mishap was rather fortunate in relation to my other commitments! The plans of medical schools in respect of curricula and staffing were reviewed by the GMC from time to time. In the early 1970s the Oxford Clinical School had, as a matter of course, furnished the GMC with a report on its plans for future developments in curriculum and staffing. The latter included proposals for a chair in clinical biochemistry. At that time the Oxford Medical School had a Reader in clinical chemistry (Dr. P.J. O’Brien) who was non-medical and nominally attached to the university biochemistry department. He was scheduled to retire in 1974. [The post was one of those

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funded by the benefaction of the Oxford motor car entrepreneur Lord Nuffield. In the early days of clinical biochemistry, it was common for laboratories in the UK to be directed by non-medical biochemists.] My work in establishing the Bristol biochemistry department was essentially complete; the Oxford clinical school (a postwar development) was growing and was acquiring a substantial reputation; and a change of scene seemed a good idea. An innocent comment of mine to the late Dr. D.H. Williamson about the planned chair, whilst examining a DPhil student with him in Oxford, was unexpectedly passed on by him to others in the school and I was encouraged to apply. The Oxford post was duly advertised in 1974 and I was fortunate enough to be appointed. After a year’s delay (to hand over responsibilities in Bristol), I moved to Oxford in 1975. Alan Kerbey moved with me to a research assistant post and completed his Bristol PhD shortly thereafter. He stayed with me with periodic promotions until the year before my retirement in 1993. Dr. S.J.H. Ashcroft, who had worked with me on biochemical mechanisms controlling insulin secretion at Bristol (as a research student and postdoctoral fellow), moved to Oxford as a lecturer and took over this line of research completely and with great effect.

Work for the Department of Health: Knighthood In my first four years at Oxford, the Department of Clinical Biochemistry was located at the Radcliffe Infirmary. I had a consultant contract within the National Health Service and I happily did whatever service work I was asked to undertake. The situation in Oxford was unusual for medical schools in that my university-based predecessor was a long-standing nonmedical biochemist. The appointment of a medically qualified NHS biochemist led inevitably to overall control of NHS work being assumed by the NHS consultant. Moreover, in the period following my appointment and before I took up my chair,


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the powers that be in the National Health Service (NHS) at Oxford had decided to appoint a second health service financed and medically qualified consultant in clinical biochemistry. This freed extra research time for me but it was desirable for me to find more health service work in what was a clinical chair. The problem was solved adventitiously by an appointment in the early 1980s by the Department of Health to chair a committee to advise the government on the vexed problem of diet and coronary heart disease. This was a contentious and time consuming subject in the 1980s and I deliberately suggested names for this committee which would represent varying points of view and be reflected in what I hoped would be seen as a very frank and balanced report. The enquiry itself was conducted over a threeyear period in the midst of a great deal of publicity and public debate. Many of my colleagues on the committee were frequently to be seen on television or heard on the radio (I limited myself to one radio appearance following publication of the report.). In consequence there was an air of expectation and we were fortunate that the report when published in 1985 burst the bubble of contention. It received a good press and the government gratefully accepted our findings and implemented our recommendations. The work entailed in producing the report, and a further six years of work in implementing its recommendations, provided me with more than sufficient NHS work to last me until my retirement in 1993. My good fortune in being knighted in 1985 was official recognition of these and other public services.

Biochemistry Research at Oxford 1975^1993 At the time of the move to Oxford, Dr Kerbey and I were heavily committed to elucidating the mechanisms, whereby diabetes and dietary carbohydrate deprivation inhibit glucose oxidation through phosphorylation and inactivation of the PDH complex. The regulation of glucose oxidation (and glucose utilisation

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more generally) was a problem which had interested me from the early 1960s and was to occupy me until my retirement in 1993 and beyond (I finally gave up laboratory research in about 1995 when my last graduate student completed her studies). This was an area of research which we pursued in parallel with the laboratories of L.J. Reed (Dallas) and O.H.Wieland (Munich).The other major area of interest was prompted by our discovery in the late 1970s/early 1980s that the branched chain 2-oxoacid dehydrogenase (BCDH) complex is also regulated by reversible phosphorylation (i.e. the enzyme complex which initiates oxidation of the 2-oxoacids formed by deamination of leucine, isoleucine, and valine). I first became interested in this enzyme complex in 1976 in collaboration with P.J.J. Parker (then a graduate student). I will consider our researches into these two topics separately starting with the PDH complex. These accounts are confined to the contributions in context which my laboratory made up until 1995 when I gave up research. I gave up my interest in the regulation of insulin secretion on moving to Oxford. This was taken over most productively by Dr. S.J.H. Ashcroft who moved to Oxford with me.

Mechanisms Mediating the Role of the PDH Complex in Regulating Glucose Oxidation with Special Reference to Inhibitory Effects of Starvation and Diabetes My move to Oxford coincided with a major development in research on PDH phosphorylation. This was the demonstration that increasing concentration ratios of [ATP]/[ADP], [NADH]/ [NADþ], and [acetylCoA]/[CoA] activate PDH kinase ^ both in rat heart mitochondria and in association with purified pig heart PDH complex [37,40,41]. Thus products of the PDH complex reaction and of -oxidation of fatty acids had been shown to inhibit the PDH complex reaction directly (end product inhibition) and indirectly (inactivation by protein phosphorylation).


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However, comparative mitochondrial studies showed that additional mechanism(s) were involved in the effects of diabetes or starvation. In studies with Drs. Kerbey Sugden and Hutson, three phosphorylation sites were identified in porcine and rat PDH complexes, and amino acid sequences around the sites were determined in porcine complex [44,45]. Inactivation was shown to require phosphorylation of only one of three sites (site 1) [43,44]. Later studies with ATPS showed that complexes thiophosphorylated in sites 2 plus 3 only are also inactive; i.e. inactivation is not site 1 specific [45].The eventual conclusion was that phosphorylation of sites 1 or 2, but not of site 3 are inactivating. Phosphorylation of sites 2 and 3 which occurs largely over the range of 70^100 percent phosphorylation of site 1was shown to inhibit reactivation by PDH phosphatase i.e. multisite phosphorylation provides a locking mechanism retarding reactivation [46,47]. This was shown to involve an increase in apparent Km for Ca2þ [48]. Multisite phosphorylation was shown to be ordered in pig and rat complexes. Relative rates were site 1 > site 2 > site 3 and inhibitors and activators of PDH kinase were effective with respect to phosphorylation at each of the sites; evidence for operation of this locking mechanism invivo was summarized [49]. It was considered to be of particular importance for glucose conservation in starvation and dietary carbohydrate deprivation; and for impaired glucose oxidation in diabetes. In these conditions the proportion of complex in the active dephosphorylated form fell by 75^85% in rat heart, kidney, adipose tissue, and liver (summarized in [50]). After some initial doubts these mechanisms were also shown to be involved in regulation of the skeletal muscle complex [51^53]. Longer term regulation of the PDH phosphorylation/dephosphorylation cycle was discovered when it was shown that the effect of starvation or alloxan diabetes to lower percent PDHa in rat tissues persists into isolated heart muscle mitochondria incubated with respiratory substrates in vitro. These and subsequent investigations with Denyer, Fatania, Stace, Jackson, Marchington, Jones, Priestman, Mistry, and Halsall led to the

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further discovery that 24^48 h of starvation or diabetes effects a stable 2-3-fold increase in the activity of PDH kinase in isolated rat heart, skeletal muscle, and liver mitochondria [54^56]. Complete reversal of the effect of starvation required 4 h of refeeding for liver and 24^48 h for muscle. In order to investigate these phenomena in more detail, studies were made in tissue culture of rat hepatocytes, cardiac myocytes, and soleus muscle strips [57^63]. These studies showed unequivocally that culture of these cell or tissue samples from fed rats with agents which increase cAMP (glucagon or dibutyrylcAMP) or with NEFA (n-octanoate or albumin bound palmitate) increased PDH kinase activity 2^3-fold within 24 h. and that the effect of glucagon was blocked by insulin. Culture of hepatocytes from starved rats reversed the effect of starvation by 60% in 24 h; this reversal was blocked by n-octanoate, dibutyrylcAMP, glucagon, or a combination. These studies thus provided strong evidence that the effects of diabetes and starvation to inhibit glucose oxidation are mediated by an increase in PDH kinase activity, thereby enhancing phosphorylation and inactivation of PDH complex. I gave up research in 1995 after two years of retirement (so-called). Since then, the further studies of R.A. Harris and of M.C. Sugden and their colleagues have identified four isozymes of PDH kinase and their respective roles in the regulation of PDH complex activity [64^70].

Regulation of the Mitochondrial Branched Chain 2-Oxoacid Dehydrogenase Complex (BCDH Complex) by Reversible Phosphorylation Earlier studies by Bowden, Connelly, Harper, Benevenga, and Wohlhueter had suggested the existence of enzyme complexes catalysing the oxidative decarboxylation of the branched chain 2-oxoacids formed by the deamination of leucine, isoleucine, and valine [71,72]. It seemed likely that the BCDH complex would be related in structure and operation to the PDH


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complex. Reed and colleagues first reported on the bovine kidney branched chain 2-oxoacid dehydrogenase complex in 1978. P.J. Parker (a new graduate student) and I began our studies on the bovine liver and rat liver and heart BCDH complexes in 1976. In our studies partial purification of the complex from ox liver mitochondria provided convincing evidence that a single complex, distinct from the pyruvate dehydrogenase complex, catalyses CoA and NADþ dependent oxidative decarboxylation of 4-methyl-2-oxopentanoate (ketoleucine), 3-methyl-2-oxobutyrate (ketovaline) and D- and L- 3-methyl-2-oxopentanoate (ketoisoleucine). We showed further that the reaction is inhibited by end productsisovalerylCoA (competitive with CoA)] and NADH (competitive with NADþ) [73]. We went on to show the presence of a comparable complex in rat liver and rat heart mitochondria [74]; inactivation of the rat heart complex by phosphorylation with ATP [75]; and interconvertible active (dephosphorylated) and inactive (phosphorylated) forms of the complex in rat heart and skeletal muscle [76]. Subsequent studies were with K.S. Lau and H.R. Fatania and with J. Espinal, M. Beggs, H. Patel, and J.M. Shaw. These demonstrated inactivation of rat liver and kidney BCDH complexes by ATP and the demonstration with purified ox kidney complex that this is effected by reversible phosphorylation [77,78]. Independent confirmation of regulation of BCDH complex by reversible phosphorylation was afforded by Hughes and Halestrap [79], Odyssey [80], and Paxton and Harris [81]. We went on to show with highly purified ox kidney complex inhibition of the BCDH kinase reaction by branched chain 2-oxoacids, ADP, and TPP [82] and reactivation by dephosphorylation with co-purified phosphatase [83]. Analysis of tryptic phosphopeptides from purified ox kidney complex revealed three phosphorylation sites; complete inactivation required phosphorylation of all three sites [84]. Purification of a protein activator of phosphorylated branched chain complex showed that it was the free branched-chain dehydrogenase component of the complex [85]. Further analysis revealed

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surplus free branched-chain dehydrogenase in rat liver and kidney but not muscle mitochondria. Analysis of rat tissues by immunoassay and bioassay showed relatively high total activities of the branched-chain complex in rat liver, kidney, and heart (57^82 units/g wet wt.) but lower activity in rat hind limb skeletal muscles [86]. In normal rats the percentage of complex in the active form was low in heart and skeletal muscles and higher in liver and kidney [87]. Low-protein diets decreased percentage of active complex and also the concentration of free E1 [88]. In tissues of rats on a normal diet, about 70% of whole body complex is in the liver and the major change effected by low-protein diets was in liver [89,90]. Re-evaluation of rat tissue concentrations by immunoassay and bioassay showed that the effects of diet and diabetes to decrease activity of branched-chain complex in liver is mediated almost wholly by inactivation of the complex by phosphorylation [91]. Activity of BCDH kinase was greater (3-fold) in heart than in liver mitochondria in rats on a normal diet. A low-protein diet (0% casein) for 10 days increased activity of the kinase in heart and in liver [89]. Further studies of temporal relationships showed that 0% casein diet decreased liver activity of BCDH complex within 4 days and increased activity of BCDH kinase within 9^19 days. Refeeding produced 50% reversal within 24 h and complete reversal after 20^30 days [92]. Further studies employing four mitochondrial marker enzymes showed that high-protein diet increased rat liver concentration and content of total BCDH complex by 31% by increasing mitochondrial specific activity [90]. My final paper on BCDH complex was devoted to the use of rat hepatocytes in tissue culture to study longer term regulation of the complex and of the role of BCDH kinase [91]. The main conclusions from the use of this technique were that branchedchain amino acids are involved directly in regulation of activities of BCDH complex and BCDH kinase; that mitochondrial uptake of branched chain 2-oxoacids is necessary for regulation of BCDH complex activity; and that the stable increase in BCDH kinase activity may function as a hysteresis mechanism.


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Other Contributions and Services Learned Societies As an MD and a PhD, I divided my activities between learned societies that were principally medical (the Royal College of Physicians (membership, then fellowship given on published work), those that were principally scientific (the Royal Society, the Biochemical Society, and the Society for Endocrinology), and those that were a combination of the two [the British Diabetic Association; the European Association for the Study of Diabetes (EASD); and the European Society for Clinical Investigation (ESCI)]. I was privileged to serve most of these organisations in various capacities: the Royal Society as a member of Council (1987^1989) and as a Vice-president (1988^ 1989); the Biochemical Society as a member of the Committee (1971^1974) and President (1995^2000); the Medical and Scientific Section of the British Diabetic Association as a member and later as its chairman (1971^1975); and the EASD as a founding member and President (1977^1980).

Retrospection I have been very fortunate in my career to have spent my time working for three outstanding UK universities (Cambridge, Bristol, and Oxford) liberal in their outlook and flexible in relations between clinical and scientific departments and for having had personal acquaintance with most of the outstanding biochemists and medical researchers in fields related to diabetes. I was fortunate that I embarked upon my career in 1952; the postwar development of universities in the UK was such that opportunities for promotion presented themselves at the most opportune moments for those who started their postgraduate careers in the early 1950s. Those whose careers were interrupted by the war had a more difficult time because

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opportunities for promotion at senior level in the UK were few and far between up until about 1960. The explosive growth in biochemistry and molecular biology starting in the 1950s has perhaps been much easier to comprehend and to put into perspective by those who lived through it than by those who had to comprehend it retrospectively. It is interesting to talk ^ as I do ^ to younger investigators who want to learn of the historical background to the current state of knowledge, and it is perhaps appropriate that these concluding remarks are written after one such session with a young graduate this morning!

REFERENCES [1] [2] [3] [4] [5] [6]

Randle, P.J. (1954) Lancet 1, 809^810. Randle, P.J. (1954) Lancet 1, 441^444. Randle, P.J. and Young, F.G. (1956) Endocrin. J. 13, 335^348. Randle, P.J. (1956) Nature (London), 178, 983. Randle, P.J. (1958) Biochem. J. 70, 490^500. Randle, P.J., Manchester, K.L. and Smith, G.H. (1958) Brit. Med. J. 1, 1028^1030. [7] Randle, P.J. and Smith, G.H. (1958) Biochem. J. 70, 501^508. [8] Morgan, H.E., Randle, P.J. and Regen, D.M. (1959) Biochem. J. 73, 573^579. [9] Passonneau, J.V. and Lowry, O.H. (1962) Biochem. Biophys. Res. Commun. 7, 10^15. [10] Drury, D.R. and Wick, A.N. (1953) Ciba Found. Coll. Endocrin. 6, 211^217. [11] Shipp, J.C., Opie, L.H. and Challoner, D.R. (1961) Nature 189, 1018^1019. [12] Williamson, J.R. and Krebs, H.A. (1961) Biochem. J. 80, 540^547. [13] Newsholme, E.A. and Randle, P.J. (1962) Biochem. J. 84, 79P. [14] Randle, P.J., Garland, P.B., Hales, C.N. and Newsholme, E.A. (1963) Lancet 1, 785^789. [15] Randle, P.J., Garland, P.B., Hales, C.N., Newsholme, E.A., Denton, R.M. and Pogson, C.I. (1966) Recent Progr. Horm. Res. 22, 1^44. [16] Hales, C.N. and Randle, P.J. (1963) Biochem. J. 88, 37^46. [17] Grodsky, G.M., Bennett, L.L.,Vcella, C., McWilliams, N.B. and Smith, D.F. (1963) Amer. J. Physiol. 205, 638^644. [18] Coore, H.G. and Randle, P.J. (1964) Biochem. J. 93, 66^78. [19] Ashcroft, S.J.H. (1980) Diabetologia 18, 5^15. [20] Ashcroft, F.M., Ashcroft, S.J. and Harrison, D.E. (1988). J. Physiol. 400, 501^527.


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[21] Garland, P.B. and Randle, P.J. (1964) Biochem. J. 92, 678^687. [22] Denton, R.M. and Randle, P.J. (1967) Biochem. J. 104, 416^422. [23] Chase, J.F. and Tubbs, P.K. (1972) Biochem. J. 129, 55^65. [24] Randle, P.J. (1969) Nature 221, 777. [25] England, P.J. and Randle, P.J. (1967) Biochem. J. 105, 907^920. [26] Moskalewski, S. (1965) Gen. Comp. Endocrinol. 5, 342^346. [27] Ashcroft, S.J.H., Hedeskov, C.J. and Randle, P.J. (1970) Biochem. J. 118, 143^154. [28] Ashcroft, S.J.H. and Randle, P.J. (1970) Biochem. J. 119, 5^15. [29] Matschinsky, F.M., Landgraf, R., Ellerman, J. and Kotler-Brajtburg, J. (1972) Diabetes. 21, (Suppl. 2): 555^569. [30] Matschinsky, F.M. (1996) Diabetes 45, 223^241. [31] Linn. T.C., Pettit, F.H. and Reed, L.J. (1969) Proc. Nat. Acad. Sci. U.S. 62, 234^241. [32] Wieland, O.H. and Siess, E.A. (1970) Proc. Nat. Acad. Sci. U.S. 65, 947^954. [33] Coore, H.G., Denton, R.M., Martin, B.R. and Randle, P.J. (1971) Biochem. J. 125, 115^127. [34] Severson, D.L., Denton, R.M., Pask, H.T. and Randle, P.J. (1973) Biochem. J. 140, 225^237. [35] Whitehouse, S., Cooper, R.H. and Randle, P.J. (1974) Biochem. J. 171, 761^774. [36] Pettit, F.H., Pelley, J.W. and Reed, L.J. (1975) Biochem. Biophys. Res. Commun. 65, 575^582. [37] Cooper, R.H., Randle, P.J. and Denton, R.M. (1975) Nature, 257, 808^809. [38] Randle, P.J., Cooper, R.H., Denton, R.M., Pask, H.T., Severson, D.L. and Whitehouse, S. (1994). In Metabolic Interconversion of Enzymes (Fischer E.H., Krebs, E.G. Neurath, H. and Stadtman, E.R. eds.), pp. 225^237. Berlin, Heidelberg, Netherlands, Springer-Verlag. [39] Randle, P.J. and Denton, R.M. (1996) In Metabolic Interconversion of Enzymes (Shaltiel, S. ed.), Berlin, Heidelberg, New York, Springer-Verlag. [40] Kerbey, A.L., Randle, P.J., Cooper, R.H., Whitehouse, S., Pask, H.T. and Denton, R.M. (1976) Biochem. J. 184, 327^348. [41] Portenhauser, R. and Wieland, O.H. (1977) Hoppe-Seyler’s Z. Physiol. Chem. 358, 647^658. [42] Randle, P.J. (1998) Diabetes Metab. Rev. 15, 263^283. [43] Kerbey, A.L., Radcliffe, P.M. and Randle, P.J. (1977) Biochem. J. 164, 509^519; 427^433. [44] Sugden, P.H. and Randle, P.J. (1978) Biochem. J. 173, 659^668. [45] Sugden, P.H., Kerbey, A.L., Randle, P.J., Waller C.A. and Reid, K.B.M. (1979) Biochem. J. 181, 419^426. [46] Radcliffe, P.M., Kerbey, A.L. and Randle, P.J. (1980) FEBS Lett. 111, 47^50.

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[47] Sugden, P.H., Hutson, N.J., Kerbey, A.L. and Randle, P.J. (1978) Biochem. J. 169, 433^435. [48] Kerbey, A.L., Randle, P.J. and Kearns, A. (1981) Biochem. J. 195, 51^59. [49] Kerbey, A.L. and Randle, P.J. (1979) FEBS Lett. 108, 485^488. [50] Hutson, N.J., Kerbey, A.L., Randle, P.J. and Sugden, P.H. (1978) Biochem. J. 173, 669^680. [51] Randle, P.J., Sugden, P.H., Kerbey, A.L., Radcliffe, P.M. and Hutson, N.J. (1978) Biochem. Soc. Symp. 43, 47^67. [52] Hagg, S.A.,Taylor, S.I. and Ruderman, N.B. (1976) Biochem. J. 158, 203^210. [53] Berger, M., Hagg, S.A., Goodman, M.N. and Ruderman, N.B. (1976) Biochem. J. 158, 191^202. [54] Ashour, B. and Hansford, R.G. (1983) Biochem. J. 214, 725^736. [55] Fuller, S.J. and Randle, P.J. (1984) Biochem. J. 219, 635^646. [56] Hutson, N.J. and Randle, P.J. (1978) FEBS Lett. 92, 73^76. [57] Denyer, G.S., Kerbey, A.L. and Randle, P.J. (1986) Biochem. J. 239, 347^354. [58] Fatania, H.R.,Vary, T.C. and Randle, P.J. (1986) Biochem. J. 234, 233^236. [59] Marchington, D.R., Kerbey, A.L., Giardina, M.G., Jones, E.A. and Randle, P.J. (1989) Biochem. J. 257, 487^491. [60] Marchington, D.R., Kerbey, A.L. and Randle, P.J. (1990) Biochem. J. 267, 245^247. [61] Stace, P.B., Fatania, H.R., Jackson, A., Kerbey, A.L. and Randle, P.J. (1992) Biochim. Biophys. Acta. 1135, 201^206. [62] Marchington, D.R., Kerbey, A.L. Jones, A.E. and Randle, P.J. (1987) Biochem. J. 246, 233^236. [63] Priestman, D.A., Mistry, S.C., Halsall, A. and Randle, P.J. (1994) Biochem. J. 300, 659^664. [64] Popov, K.M., Kedishvili, N.Y., Zhao, Y., Shimomura, Y., Crabb, D.W. and Harris, R.A. (1993) J. Biol. Chem. 268, 26602^26606. [65] Popov, K.M., Kedishvili, N.Y., Zhao, Y., Gudi, R. and Harris, R.A (1994) J. Biol. Chem. 269, 29720^29724. [66] Bowker-Kinley, M.M., Davis, W.I., Wu, P., Harris, R.A. and Popov, K.M. (1998) Biochem. J. 329, 191^196. [67] Huang, B.J., Gudi, R., Wu, P., Harris, R.A., Hamilton, J. and Popov, K.M. (1998) J. Biol. Chem. 273, 17680^17688. [68] Wu, P., Sato, j., Zhao,Y., Jaskiewicz, J., Popov, K.M. and Harris, R.A. (1998) Biochem. J. 329, 197^201. [69] Harris, R.A., Huang, B. and Wu, P. (2001) Adv. Enzyme. Regul. 41, 269^288. [70] Sugden, M.C., Bulmer, K. and Holness, M.J. (2001) Biochem.-Soc.-Trans. 29, 272^278. [71] Bowden, J.A. and Connelly, J.L. (1968) J. Biol. Chem. 243, 3526^3531. [72] Harper, A.E., Benevenga, N.J. and Wohlhueter, R.M. (1970) Physiol. Rev. 50, 428^558.


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[73] Parker, P.J. and Randle, P.J. (1978) Biochem. J. 171, 751^757. [74] Parker, P.J. and Randle, P.J. (1978) FEBS Lett. 90, 183^186. [75] Parker, P.J. and Randle, P.J. (1978) FEBS Lett. 95, 153^156. [76] Parker, P.J. and Randle, P.J. (1980) FEBS Lett. 112, 186^190. [77] Lau, K.S., Fatania, H.R. and Randle, P.J. (1981) FEBS Lett. 126, 66^70. [78] Fatania, H.R., Lau, K.S. and Randle, P.J. (1981) FEBS Lett. 132, 285^288. [79] Hughes,W.A. and Halestrap, A.P. (1981) Biochem. J. 196, 459^469. [80] Odessey, R. (1982) Biochem. J. 204, 353^356. [81] Paxton, R. and Harris, R.A. (1982) J. Biol. Chem. 257, 14433^14439. [82] Lau, K.S., Fatania, H.R. and Randle, P.J. (1982) FEBS Lett. 144, 57^62. [83] Fatania, H.R., Patston, P.A. and Randle, P.J. (1983) FEBS Lett. 158, 234^238. [84] Lau, K.S., Phillips, C.E. and Randle, P.J. (1983) FEBS Lett. 160, 149^152. [85] Patston, P.A., Espinal, J. and Randle, P.J. (1984) Biochem. J. 222, 711^719. [86] Espinal, J., Patston, P.A., Fatania, H.R., Lau, K.S. and Randle, P.J. (1985) Biochem. J. 225, 509^516. [87] Patston, P.A., Espinal, J., Shaw, J.M. and Randle, P.J. (1986) Biochem. J. 235, 429^434. [88] Espinal, J., Beggs, M., Patel, H. and Randle, P.J. (1986) Biochem. J. 237, 285^288. [89] Beggs, M., Patel, H., Espinal, J. and Randle, P.J. (1987) FEBS Lett. 215, 13^ 15. [90] Beggs, M. and Randle, P.J. (1988) Biochem. J. 256, 929^934. [91] Beggs, M., Shaw, J.M. and Randle, P.J. (1989) Biochem. J. 257, 271^275.

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Chapter 9

My Happy Days with Lac Repressor ^ in a DarkWorld BENNO MU«LLER-HILL Institut fu«r Genetik der Universita«t zu Ko«ln Weyertal 121, D-50931 Ko«ln, Germany

I never considered that I would be seventy years old, and that my laboratory would disappear. This is going to happen soon. So the moment was perfect when I was asked to write an autobiographical chapter for this series. Private life and science are intertwined and cannot be clearly separated as so many autobiographies of scientists demonstrate. This is also true in my case. In science one does not write about experiments which did not work. One does not speak about grant proposals which were rejected. In science only the positive counts. Why waste time on failures? Yet I have spent a considerable time on such a failure, human genetics in Nazi-Germany. I wrote a history about it. Thus, in the past seventeen years I have been asked again and again: ‘‘Why did you write this book ‘Murderous Science ^ Elimination by Scientific Selection of Jews, Gypsies, and Others in Germany, 1933^1945’?’’ [1]. In Germany a second question often followed: ‘‘Are you Jewish?’’ I denied the second question. For the first question I had a simple answer: ‘‘I discovered that almost nothing had been written on the topic, I was one of the first to enter this field. This is the main reason.’’And then I used to add that there were also other private reasons,

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but that this was more complicated. I will try to describe some of them here.

Family and Childhood I was born in 1933. I had no brother or sister. My father was a German lawyer. He never became a member of the Nazi party or any of its organizations. He was a liberal. His father was an engineer, one of his grandfathers a professor of physics. There were painters and art dealers in my father’s family. My mother was the daughter of a Swedish-British industrialist. Her mother was the daughter of a clergyman, one of her grandfathers had been mayor of Kalmar. There were apparently no workers or peasants in either family. However, if one went back long enough there they were. For example, my great-great-grandfather, Charles Hill, was the son of a British farmer, who died in 1826. His son Charles then,

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twelve years old, became a worker in a cotton factory in Bolton, a town close to Manchester. At the age of 20 he became an overseer. At the age of 27 he accepted the offer to organize a cotton factory in Sweden. A few years later he bought there his own factory. He died in Sweden as a millionaire. This provided my family with money. I enjoyed the presence of a most pleasant Dienstma«dchen who took care of me when my mother had no time, and this was rather often. His money also paid for part of my study-costs. My mother, Daisy, loved beautiful things, paintings, furniture. She cried, when forced to see ugly things. Her taste was impeccable. She forgot the real world. For example, the first day, when I went to school with her I had to wear red shoes and a red hat. So I had a hard time with my class mates. When my father left the house at the beginning of the war, she was crying. ‘‘Why do you cry, he is gone, we are now together,’’ I said to cheer her up. I remember the wonderful evenings we spent together on our balcony: swallows everywhere in the sky and the tower of the Mu«nster closeby. My mother read me poems. I recall just two: ‘‘Die Grenadiere’’ (the soldiers) and ‘‘Belsatzar.’’ Much later I learned that they were by Heinrich Heine. Then I made them part of my world. My father loved Goethe. I was five years old when he read Faust to me, in particular the student scene (Schu«ler-Szene) and the Walpurgisnacht. We played the student scene (Schu«lerSzene) at home. I played the student, my father Mephisto. I was told that one brother of my father was a professor of technical chemistry in Vienna and that the grandfather of my father had been a professor of physics at Freiburg University: science that was it. Then one day in 1938, Wolf, my father’s brother visited us. He had been just fired by the new Nazi government from his position as a professor at the University of Vienna. He was accused of having preferred Jewish collaborators over nonJewish ones. So there he was asking for legal advice from my father, the successful lawyer.

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Wolf stayed in the best hotel close to the train station. One afternoon, I was told, he was free between 3 and 5 p.m. I could visit him and ask all questions I wanted to ask. So we met in the salon of the hotel.We sat in deep leather arm chairs. He was smoking a cigar. I asked: uncleWolf, why can one look through glass but not through most stones? What is the difference between a metal and a stone? What is wood? What is electricity? The two hours came to an end, and I had come to the conclusion that chemistry was straightforward, I would understand it, but that physics was too complicated. Wolf then left. I never saw him again. He was reinstalled in his position in 1939, but died in 1941. In 1943 my parents sent me to a Gymnasium (grammar school) where I should learn Latin and Greek. At the same time I entered the compulsory youth organization of the Nazis, the Jungvolk. Membership and presence were obligatory. One afternoon we stood in uniforms orderly in groups when we saw one of our group, dressed in normal cloth, with his mother passing by. How dare he do so? This was too much courage. We were told that his father, a social democrat, was in a concentration camp. The next time we were ordered to march to the house where his family lived on the top floor. We went there, banged at the door, and screamed in the staircase to terrorize the boy and his mother. When we left I had a bad feeling. When the war was over, I saw him again in my class in the Gymnasium. I felt uneasy. He looked sick, I knew I should apologize, but I did not. And then one day he was absent and after some weeks our teacher told us that he had died of diabetes. I still feel uneasy not to have talked to him. My group became part of a special unit, organized by an SS-veteran. Life became unbearable. We were prepared for the war. On command we had to crawl rapidly through dirt and thorny bushes. We had to beat up each other. It was too much for me. I could no longer speak. My mother went with me to our Latin teacher. He seemed to understand. Large formations of allied bombers flew over Freiburg during daytime. Defence was nonexistent. One day, several units of


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concentration camp inmates were driven by SS men through the street where we lived. I stared at them. They were in terrible shape. I should have given them some bread. They had disappeared before I could do something. One day my friends and I decided not to go to the Jungvolk meetings anymore. Our commander came to the backyard, where we assembled, to bring us back to order. He was several years older and much stronger than we. We had asked some older boys for help. They and we beat him up. Blood everywhere. He finally fled. We expected the police to come. But one night later, November 27, 1944 the Allies bombed Freiburg. More than half of the city was destroyed. We were in the midst of it. About 2780 people were killed that night. About 2000 were severely wounded; many of them died in the following weeks. We walked through the fire storm and barely survived. I have never again seen so many corpses, heaps of them in many places. Women, men, and children. But I was free. No more special unit of the Jungvolk. It had all disappeared in the fire storm. Some friends gave us a room. My father joined us in July 1945. He had been an army judge. He had not been in the Nazi party. So he became a state prosecutor and director of the local prison. When years later I asked my colleagues or others of about the same age, how they had liked the Jungvolk or the Hitler-Jugend, almost all of them said that it had been wonderful. Then and later they all had made their careers. My experience was apparently unique. And I began to distrust them. In the Gymnasium I had one experience with profound impact. In 1950 or around that time, we had to see a particular planet through a telescope. We were standing in the court in a row, the largest students first, the smaller ones at the end. I was almost at the end. ‘‘Do you see the star?,’’ asked our teacher every student. Some had difficulties but then the teacher told them, how to sharpen the sight. Finally came the student in front of me, Harter.‘‘I do not see anything,’’ he said. The teacher showed him how to sharpen the view. ‘‘No, it is completely dark, I do not see anything.’’ The teacher yelled at him ^ and looked at the front of

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the telescope. There he and we saw that the telescope had been closed. Nobody could have seen a thing. Three of those who have seen the planet, where there was complete darkness, have become professors of physics or biology. Another one has become professor of philosophy and chief of a TV station, that I understand. The student who honestly said that he saw nothing had to leave the Gymnasium. He was insolent. His father was a worker, and thus he did not fit our institution.What would I have said? My parents gave me a large chemistry kit. I bought myself some chemicals and started to do experiments in inorganic chemistry. There I almost killed myself with H2S: I inhaled too much and almost fainted. After this experience I became more prudent. The main things we learned in the Gymnasium were languages and literature but not science. The ancient Greek allowed me to read the original poems of Sappho. The Latin invited me to read the ‘‘Nature of Things’’ by Lukretius. The French made me read poems of Villon, essays of Montaigne and novels of Sartre. The Swedish I had learnt from my mother made me read Strindberg: I fell in love with the French movies ‘‘Les jeux sont faits,’’ ‘‘Orphe¤e,’’ and ‘‘Les enfants du paradis.’’ I read Faulkner’s ‘‘Absalom,’’ and Sartre’s ‘‘Nause¤e.’’ I discovered poems by Brecht and the paintings of Picasso. I saw in Freiburg an exhibition of Wols, which touched my heart. I did not believe in God. I believed in molecules. I felt outside the normal German world. It was the idea of Sartre, that we decide what we do, which influenced me most. It confirmed my own thinking. Neither the state nor the family could determine what I wanted to do. I have kept this idea to the present day. Thus, I do not believe that our fate lies in our genes. While in the Gymnasium I took a special course in chemistry. I finished the Abitur scoring 17 points in 20. Chemistry seemed to be my subject. Then an expert came and gave us advice about what we should study. ‘‘You are too intellectual and too nervous to study chemistry,’’ he told me. I still do not understand how


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he came to that conclusion. So when my father asked me what I would study I said ‘‘law.’’ My father told me that he would not finance law. I would be a disaster as a prosecutor, I should study chemistry like his brother Wolf. So I studied chemistry.

Chemistry in Freiburg and Munich I began to study chemistry at the University of Freiburg. Here everything was solid and slow. I heard that Munich was much better. My parents financed three half-years there with the money inherited from Charles Hill. There I took first the lab course of quantitative inorganic analytical chemistry. My first experiment was to determine Mgþþ as MgNH4PO4. It took a full day to do so. This I did and in the late afternoon I presented my results to the postdoc running the lab.‘‘Wrong,’’ he said. I did this four more times, and every time it was ‘‘wrong.’’ Then I told the story to my neighbor in the lab, a more advanced student. He asked me ‘‘Did you use the factor?’’ I had no idea. So I was informed that the concentration of Mgþþ one had determined had to be multiplied by an empirical factor of let us say, 1.37. The concentration the course-organizer used was simply wrong. So I used the factor and my result was OK. Chemistry in Munich seemed full of such tricks. The students had collections and lists of all the metals we had to analyze. When we had to determine a certain cation it was easier to determine its anion, and so on and on. When I went to criticize this system of quantitative inorganic chemistry, I was told, to go to physical chemistry then you will see what is worse. I was told for example that there existed a machine where nothing was inside but where everybody in the past few years had gotten excellent results. One has to learn to make the calculations backwards, I was told. In Munich I lost my innocence. I had admired various female students, but I had never got close to any of them. So it came as

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a complete surprise when the girlfriend of a student I knew in Freiburg came to visit me in my room. To my great astonishment she undressed herself and me, we did it, and then she disappeared. In Freiburg the carnival of the Musikhochschule was famous. It was held in the baroque Wenzinger-Haus in front of the cathedral. Excellent live dance music and women in beautiful costumes. I met there Marianne Kesting, a Brecht specialist, and Dorothe Spatz, a flutist, who later married the biophysicist Friedrich Bonhoeffer. It was wonderful, no kisses, nothing of this type. Marianne Kesting had started to write her biography of Brecht [2], so I learnt a lot from her when I met her again in Munich. She induced me to go for two weeks to Berlin and to look at the rehearsals at the Brecht-Theater in East-Berlin. I was admitted without any problem. Unfortunately Brecht was at the time in Paris but Helene Weigel and Fritz Busch were there. It was just wonderful. In Munich I also went to the carnival. All these beautiful women. Together with another young man of my age I tried to entice one. But we both failed. In the end she disappeared. So we, the competitors, talked together. Later when back in Freiburg, I met him in the student building. I began to talk to him, he had no time, he was going to a meeting of the SDS (German socialist students). Would I like to join him? I did so and for the next years I lived in the atmosphere of the SDS [3]. The young man, Rolf Bo«hme was his name, later became and is still mayor of Freiburg. In the SDS we discussed the bad past of the German universities. Heidegger! We showed an exhibition of trials in Nazi Germany. We presented the film by Resnais ‘‘Nuit et brouillard,’’ which was forbidden in Germany. We presented East German films. We discussed the Algerian war. We were just a dozen, two of us were Jewish, the parents of many of us were social democrats or communists. Some had seen the concentration camps. There I learned that the German universities had a bad past in Nazi Germany.


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But it was all rather abstract. What could I do when Dr Otto Ambros from the former IG Farben was speaking on CN-chemistry in the chemistry institute, and when he was introduced by our director: ‘‘Oh, how happy we are, Dr Ambros, that you are again among us:’’ Ambros had been convicted in the IG-Farben trial for his involvement in the construction of the IG-Farben Werk in Auschwitz with the help of slave workers from the concentration camp. Now he was back in science. I went to hear Robert Kempner, a German Jewish emigrant and prosecutor of the Nuremberg trials. He spoke in the second largest lecture hall of Freiburg University. All seats were taken, some people stood. Kempner began to speak. Daniel, in the lion’s den. Two or three professors of law stood up, left the lecture hall banging the doors. The students were more or less quiet until the end. Then all hell broke loose. Almost all students showed their indignation by making noise with their shoes (‘‘scharren’’). There was no discussion. My friends often came to visit me in my tiny room. My father had given me free access to our wine cellar, so we were sitting there drinking wine and listening to records like ‘‘Brigitte Bardot, Bardot, c a fait chaud, chaud, chaud’’ (Brigitte Bardot, this is hot, hot, hot) and songs of the Spanish civil war. We heard Helene Weigel saying ‘‘Was spricht gegen den Kommunismus, er ist vernu«nftig. . .’’(What speaks against communism, it is reasonable. . .). Or some unknown singer from the DDR singing ‘‘Nur noch ein wenig fegen, dann ist alles rein.’’(just a little more sweeping, then everything is clean). I knew so little at the time. I was interested in biochemistry. When the moment came where I had to look for an advisor of my diploma-thesis, I followed the general opinion that Otto Westphal was the best possible biochemist in Freiburg. I did not know that as a student he had been in the SS, and I certainly did not know that the immunology he was doing was not the immunology of the future. I asked Westphal for a job, fortunately he declined. So I went to the only other biochemist, Kurt Wallenfels.

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Kurt Wallenfels had worked with Nobel Prize laureate Richard Kuhn. Like Kuhn he was an organic chemist with interest in biochemistry. Aside from quinones, he worked on glycosides, in particular on galactosides and was therefore interested in -galactosidase produced by Escherichia coli. At the time when I was in his lab, alcohol dehydrogenase of yeast and -galactosidase of E. coli were the main objects of his interest. An Indian postdoc of his, Prakash Malhotra, reported to have shown that the number of active sites of -galactosidase of E. coli doubled from four to eight when -galactosidase was heated from 20 to 37 C. This seemed most exciting. When I repeated his experiment, I noticed that the color of o-nitro-1-thio--D-galactoside, the inhibitor he had used, slowly increased, when incubated at 37 C with -galactosidase. I demonstrated that, in contrast to all expectations, it was hydrolyzed. Wallenfells and Malhotra retracted the manuscript where they had claimed the doubling of binding sites. My sobering result was just mentioned in footnote 78 of an article by Wallenfels [4]. I did not get publicity but, still today, Iam proud of the experiment. In science it is possible to demonstrate convincingly that a particular interpretation is wrong. On the positive side, I demonstrated that acetaldehyde in its nonhydrated form is attacked by NADPH when bound to yeast alcohol dehydrogenase [5]. This can be easily shown, since hydration of acetaldehyde is a rather slow process. Again, nobody cared, but I began to love science. Here simple experiments could lead to novel, unambiguous interpretations. When in 1960, Jacob and Monod published their first paper on the control of the lac operon [6], I was in the midst of my thesis work on the SH-groups of -galactosidase. The Jacob^Monod paper [6] was discussed in the literature seminar of the Wallenfels group. At the time most students in Freiburg could read French, so we all tried to read it. To me it seemed completely convincing. There was however a postdoc in the lab, a bacteriologist from Kiel University, I have forgotten his name, who ridiculed the paper. ‘‘This is all wrong, typical


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French Spitzfindigkeit (subtleties), playing with words. We all know that bacteria have no nuclei, so they have no genes.’’ Wallenfels did not decide whether his postdoc was right or wrong. One may think that this was unique. Many years later, in the mid-1970s, I was part of an expert group judging DFG-grant proposals. There was one proposal dealing with the DNA replication of phage Lambda. Peter Karlson, who was one of the experts, said about this project ‘‘A German biochemist cannot understand phage Lambda.’’ It was impossible for me to react politely to such nonsense, so I said: ‘‘You may not understand Lambda, but some Germans do.’’ Karlson was not amused. When I was working in the Wallenfels lab, I went to two conferences: to the International Biochemistry Congress in Moscow in 1961, and to the FEBS Meeting in 1962 in Vienna. In Moscow I heard Marshall Nirenberg’s say, that his collaborator Mattheai had shown that poly U codes for poly-phenylalanine. I also heard Alfred Gierer present his way to the genetic code, via a systematic analysis of nitrite mutants of the tobacco mosaic virus. Nirenberg’s analysis was as elegant as Gierer’s was hopeless. In Vienna I heard the introductory talk of Erwin Chargaff. He spoke about the past and the emigration of the Jewish scientists. It so happened that I met Chargaff in the tram leaving the conference. I said to him: ‘‘You ought to write your autobiography.’’ ‘‘Never!,’’ he said. Sixteen years later, there it was [7]. And then he gave me the title of the best book on Jewish emigration, which would appear soon. It took years [8]. I bought it and learned a lot. My time in the Wallenfels lab was coming to an end. I still had a paid position after I received my PhD. Then Wallenfels offered me a postdoctoral position in the Rickenberg lab in Bloomington, Indiana. Rickenberg and Wallenfels had a joint NATO grant. They wanted to show that Lac permease is part of -galactosidase. Wallenfels had already offered the job to another collaborator who had declined. Shortly thereafter

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Wallenfels had fired him. Knowing this, I immediately accepted his offer. I knew that the identity of Lac permease and -galactosidase was all nonsense. I would never spend a second on this project. What would I do? Study the specificity of the lac induction system with all the glycosides of the Wallenfels lab! So I took with me samples of all glycosides synthesized in the Wallenfels lab. In Freiburg I knew I was not old enough to marry and have a wife and children. The Munich experience however encouraged me to meet several beautiful women. I mention here Nanna Michael who went to Paris to become a model, whose photographs, in the 1960s, were everywhere in Paris; Angelina Heidrich, the charming soprano of the Freiburg opera who introduced me to Mozart; and Barbara Schneider, the sister of the writer Peter, who went to New York with the aim to start there the cultural revolution and who disappeared there.

With Howard Rickenberg in Bloomington, Indiana, 1963^1964 In order to reach Bloomington, Indiana, I took a ship that went from Holland to New York. From New York I took the night train to Chicago and from there a train to Bloomington. It was snowing when I arrived. There was no train station. Howard Rickenberg met me at the train. I thought I had ended somewhere in the desert. Howard drove me to the guest house of the university. Visits in the rooms were strictly forbidden. My neighbor was a lady who warned me about the red network, which was active everywhere. I feared that Howard would insist on the identity of -galactosidase and lac permease. He did not. He accepted my proposition to test the specificity of induction. So I did this [9]. At the time I was there, there were two restaurants in Bloomington. There was one bookshop and one bar. But there was also H.J. Muller, the great Drosophila geneticist.


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A year after I arrived in Bloomington, I took ten days off. I drove with my car, alone, to New Orleans. Somewhere in Mississippi a private car suddenly stopped in front of me. We almost collided. It then speeded up and disappeared. Some minutes later I saw a car standing and the driver apparently asking for help. I stopped to help him. It was a policeman. He had his hand on his gun. My papers? ‘‘May I put my right hand into my pocket?’’ I showed my green card and driver’s licence. What was I doing here? ‘‘Pleasure.’’ My luggage? I had almost nothing. The policeman told me ‘‘Get the hell out of here!’’ I did. The next day I bought the New York Times. There I read that three students from New York who had come to Mississippi to demonstrate for equal rights for Blacks had just been shot by the police. I had been lucky. I was rather lonely in Bloomington. There was one German student with whom I had some contact. The American students lived in a different world. For example, when I once said ‘‘in the local supermarket one can only buy two types of cheese,’’ I was countered: ‘‘There is only one type of cheese.’’ The female students never looked you into the eyes when I looked at them on the campus or on the street. This happened only once and ended right in bed. I met a Chinese student Chiu Hsia who introduced me into Chinese novels and poems. So I read the King Ping Me and the Dream of the Red Chamber. What kind of worlds! Years later I was prepared to buy and read the bilingual Moundarren collection of Chinese poets like Tu Fu, Su Tung po, Wang Wei, and Han Shan. Chiu hsia and I always kept some distance, but we never went apart. We are still friends. In Bloomington I learned that many of the Professors of molecular biology were Jewish, and many of them emigrants, who had been driven from Europe by Hitler. There was Howard Rickenberg himself. He came from Nuremberg. His original name was Hans Reichenberger. In Biochemistry was Felix Haurowitz. Close by in Urbana was Sol Spiegelman, who was born in the US. It was not easy to enter this world as a German. One wrong sentence and one was excluded. It was

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there when I first thought that it would be most worthwhile to research and write a history of biology and of chemistry under Hitler. What had happened? How did it happen? Such research would be a major enterprise. But now I had no time. The spectre of history had to disappear. Now I wanted to get Lac repressor and nothing else. My time was running out. To go back to Freiburg or to Germany? Never! In 1964 I went to the International Congress of Biochemistry in New York to present my results and to look for a new position. During my visit in New York, I had an experience which changed some aspect of my life. During an intermission of the Congress I left the hotel and went out on Fifth Avenue. There I saw people marching down the street. They had signs indicating that they were demonstrating against the Vietnam War. It was all quiet and serious. Blacks in the front row, and many women with small children. It was impressive. The next day I read a report on the demonstration in the New York Times. It was matter-of-fact, named the names of those in the front row, and gave some numbers. A week later I bought as usual ‘‘Der Spiegel,’’ the German weekly, read by all German intellectuals. It had an article on the demonstration. Its headline was ‘‘Wascht euch mal!’’ (‘‘Go wash yourself!’’), and described the demonstrators as a crowd of criminals, drug dealers, and whores. The general outcry of the New York public was ‘‘Wascht euch mal.’’ I was so astonished that I called the correspondent of Der Spiegel in New York. He informed me that his phone conversations were tapped. I asked him how he could have written this text: ‘‘I did not write it, my articles are, like all articles, rewritten in Hamburg.’’ Since then I stopped reading Der Spiegel, which was the source of information for all intellectual young Germans. The other magazines and news papers did not seem to be much better. Years later I bought an original copy of Die Fackel of Karl Kraus, and read night after night the 150 copies in their original red envelopes from 1910 to the bitter end in 1936. So my view of the German Press became and still is abysmal.


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Back to the Congress. I thought that Jim Watson’s lab in Harvard would be the best for me. So I tried to speak to him in New York before he gave his talk. When I came to the hall where he was scheduled to speak I saw that it was impossible to enter. A huge crowd waited outside, unable to enter, the room was already overcrowded. So I gave up and drifted through the halls of the hotel where the meeting was held. Suddenly, I saw Watson. There he stood all alone. I went immediately to him, told him I was interested in Lac repressor and that I was looking for a position. He told me that he had none, but that he had a collaborator, Walter Gilbert, who had such a position. Gilbert was in England but in two months he would return. Gilbert and I met then in the Biolabs. I told him about my experiments and failure to isolate Lac repressor. (I had tried to bind Lac repressor covalently to 2-diazo-phenyl--D-fucoside, and thus make it incapable of being induced by IPTG). He told me about his failure. We had the same interest. He hired me. He was just one year older than me.

In the Watson^Gilbert Group 1965^1968 The three years I spent in the Watson^Gilbert group were the most productive in my life. I had just one goal: to isolate Lac repressor. This was dangerous. If I failed, that was it. If somebody else succeeded, and many tried, that was it, too. It was an all or none gamble. The same was true for Wally. He was a physicist. At the time I joined the group he was still teaching theoretical physics at Harvard. In the group were about six to ten graduate students and two postdocs. The principle was that they would publish their results without the coauthorship of Jim or Wally, with the one exception when one of their advisors had participated actively in doing the relevant experiments. Jim never did any experiments and Wally concentrated his effort on the Lac repressor. So he did not become a coauthor of the papers his

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students produced. And they produced excellent results. Here are the names of some of the graduate students who were in the lab while I was there: Jerry Adams, Joan ArgetsingerSteitz, Mario Capecchi, Bill Haseltine, Mary Osborn, and Jeffrey Roberts. While in Bloomington I had learned and developed some tricks on how to select lac constitutive and lac negative mutants in E. coli. So Wally suggested that I should isolate a nonsense mutation in the lacI (repressor) gene. Its existence would prove that Lac repressor is active as a protein, and not as RNA as Jacob & Monod had suggested [10]. I isolated 187 lac constitutive mutants and found among them two, which could be suppressed by an amber nonsense suppressor. Wally was not a coauthor of the paper [11]. When I showed my manuscript to Jim he said ‘‘heavy and Teutonic,’’ and rewrote it. Yet I was the sole author. The Watson^Gilbert group was a most amazing unit. Jim was absent most of the time. He was writing his autobiography. When he was present, he seemed so far away that I did not dare to speak with him about experimental problems. If I had a result I would tell it in one or very few sentences. Then he would smile and say ‘‘good,’’ and that was it. So Klaus Weber, a German postdoc I knew from Freiburg, and I had a competition who had more conversations with Jim? After two years Klaus had a total of about 21 minutes where I had only about 15 minutes. This was Jim. Wally was different. He arrived every day around eleven o’ clock and left every night around midnight. He always had time to discuss an experiment or aspects of a theory. It was most pleasant to talk with him about experiments. He loved to try new techniques and new machines. He had no fear of the unknown. Like Wally I worked twelve hours in the lab. But unlike him I stayed at home on Sundays. I needed a day to relax and to read books. At the time, there were still many bookshops around Harvard Square in Cambridge. They have now almost all disappeared. In particular I liked ‘‘Schoenhof’’ who sold German


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books. There I discovered the books of the German emigrants and Chekhov, Turgeniev, and Montaigne. During the years in Cambridge, I discovered another type of movies I loved: ‘‘Key Largo,’’ ‘‘Casablanca,’’ and ‘‘To be and not to be.’’ And then I discovered a rockband, which called itself the Fuggs. They sang a song which hit me: It began ‘‘Sunday nothing, monday nothing,’’ praised everything as nothing and ended with ‘‘Stalin less than nothing.’’ Wally was married. His wife Celia was a painter and poet. They had three children. One was dying of leukemia. Wally had to teach. How did he do all this? It was admirable. And then I met Celia’s father, I. F. Stone who wrote and published ‘‘I. F. Stone’s Weekly.’’ There he published, like Karl Kraus, his articles against the Vietnam war. He discovered that the attack of a Vietnamese boat on the US American fleet had never happened. It was a lie. Yet it allowed the Americans to bomb North Vietnam. He also had written several interesting books. His last was on the trial of Socrates [12]. This was journalism at its best. Izzy, as he was called by the family, was a convinced defender of American democracy. What a man! There was nobody else at Harvard who worked on the Lac repressor. But on the fourth floor of the Biolabs, in the lab of Mathew Meselson, worked Mark Ptashne who tried to isolate the Lambda repressor. He planned to use specific operator binding as a test. This rationale we could not use since, at the time, no phage carrying lac operator had been isolated. Thus, we concentrated on inducer binding. The best candidate was isopropyl--D-1-thio-galactoside, which in 1966 was sold by a French company with an S35 label. There was good reason to assume that there were no more than ten molecules of Lac repressor in one E. coli cell. From the in vivo data one could calculate that the binding constant was about 6 106 M. In a crude extract one would never be able to demonstrate this binding. Thus, I looked for mutants binding tighter IPTG. I isolated one such mutant, which bound IPTG 2^3 times tighter than wild-type. I constructed the diploid. But even this seemed

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hopeless. We had calculated that we needed at least an increase in binding by a factor of ten. Yet Wally tried and he found specific binding, which was absent in I or I s mutants. How come? We had assumed that Lac repressor is a monomer with one inducer and operator binding site, yet it is a tetramer with four binding sites. It was this unexpected factor of four which saved us [13]. Almost at the same time, just some weeks later, Mark Ptashne isolated the Lambda repressor [14]. We had obtained our results just before the Cold Spring Harbor Symposium. Wally and I went there, and told our story during the intermissions to everyone who wanted to hear it. We were present when Francis Crick received for his fiftieth birthday a huge box as present from Jim. Francis opened it and out came an almost naked lady.‘‘Francis likes beautiful models,’’ was Jim’s comment. At this time Mark Ptashne, who was a Harvard Junior Fellow, invited me to come as a guest to a dinner of the Junior Fellows. About twenty young men of all fields (at this time women were not yet allowed to participate) met every Tuesday evening for dinner and talked with each other about their work. What lively discussions! I was deeply impressed. Such mutual interest and discussion were lacking in German universities, and still are lacking. I have later been a guest again. Every time I have been there my admiration deepened. There was nothing like this in Germany. Some years ago, in Cologne the rector of the university opened a cafe¤, a small dining room, for the faculty in the main building. A year later it had to be closed. Nobody ever went there. It was always empty. I had two most pleasant experiences with Mark. One was at Harvard. I was in the lab, outside it was dark, about eight o’ clock. Suddenly the lights went out. The same happened in the other rooms on our floor. Also across the street, everywhere in Boston, everywhere in New York. Was this the end of the world? Had the first atomic bombs been thrown? No, it was just a gigantic power failure, which turned off the lights along the entire East Coast. I met Mark on the dark floor.


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We went home to his apartment. There we drank some wine. We listened to some beautiful music from a battery radio in the light of two candles. When the lights came back I went back to work. The other experience was in Tiblisi, in Georgia. We met there by chance. Mark had a guide and knew where there was a natural bath. We went there, it was Friday afternoon. It was like a small swimming pool filled with warm brown water. There we sat naked and talked about experiments and private life in the most friendly and intimate manner. What a pleasure. We went to a cafe¤ closeby and were invited by other guests. We considered going to the synagogue, it was paradise. Coexisting with Mark was not always easy. That I was German did not make things easier. Once, in the Biolabs, Mark told me: ‘‘Lambda repressor is my repressor, Lac repressor is Wally’s repressor.’’ After he had isolated Lambda repressor he gave a seminar for the Biolabs. In the discussion a very old professor(I did not know his name) asked him:‘‘Who did these experiments?’’ Mark answered ‘‘I did it all alone.’’ We all knew that he had an undergraduate technician, Nancy Hopkins. So after the discussion we asked him to apologize and to give her some flowers, which he did. At a party of Mark’s I met Barbara, an American citizen, my future first wife. She was most elegant and she could speak for half an hour, or even longer, with Jim! They knew each other. She did not talk science with him. So I learned, if I wanted to talk with Jim I had to talk to him not only about science but also about other things, for example, about paintings. And I began to do so. After Wally and I isolated Lac repressor I felt safer. So I allowed myself the luxury to go from time to time with Barbara to New York for a weekend. There we went to the Guggenheim, the Museum of Modern Art and to Jazz clubs on the lower East Side, to hear Thelonius Monk and others. There, somewhere on Avenue I, close to the 11th Street, I saw in the window of a frame shop a beautiful painting. I entered. There

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were more beautiful paintings, portraits, scenes from the lower East Side. The painter was running the shop. His name was Ira Kaufman. I bought a painting. Over the years I bought again and again. Ira became religious and moved to Israel. Whenever I went there I visited him and I bought some paintings. He was completely unknown and remained unknown. Then in the late 1980s, an attempt was made by a Jerusalem gallery to make him known. Recently, he returned to New York to repair cars. For me he is a Jewish van Gogh. In Boston most people I met were Jewish, but almost none was religious. Once I visited the Boston Genetics Club, or whatever it was called. They met once every two weeks in a restaurant, and one of the thirty or so members gave a talk on his work. Salvador Luria was the president. When I was there he suddenly looked around and said: ‘‘Is it not wonderful, we all are Jews!’’ I was too shy to correct him immediately, but then it was too late. The Vietnam War became worse, and the opposition grew stronger. Jim had the habit of reading the New York Times during seminars which were not interesting. On the first day of the Tet Offensive, Ethan Signer gave a talk on his work on variants of the ph80 phage. Jim sat in the first row and read the New York Times during the entire talk. No trick of the speaker helped. We learned, the war was more interesting than this seminar. In April 1967 I went to New York to the big demonstration in Central Park. Once I participated in a demonstration which went from Mass. Avenue Cambridge all the way down to Boston. I marched next to Jon Beckwith who told me about his Lambda h80 dlac construct. We demonstrated against the war and talked all the time about experiments! In fact this was the DNA construct, which Wally Gilbert used in 1967 [15] to show that Lac repressor bound specifically to lac operator. At this time I was trying to repress in vitro the transcription of lac promoter with purified Lac repressor. This experiment did not work. In retrospect it is clear why: CAP/CRP was missing


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and without it transcription from the lac promoter was just much too weak. So Wally had a positive result and I had nothing. Wally was magnanimous to make me coauthor, I who had done nothing with this experiment [15]. What was lacking was an overproducer of Lac repressor. Here I had the idea to isolate it as a revertant of a I TS temperature sensitive lac constitutive mutant. This could be a true revertant, but also an overproducer! And indeed, I isolated a 10-fold overproducer. This coupled with the Lambda h80 dlac genome, which increased the number of copies effectively 10- to 20-fold, increased the amount of Lac repressor about one to two hundred-fold [16]! This opened the door to the biochemical analysis of Lac repressor. So Lac repressor was for several years the first and only transcription factor available in large amounts for biochemical analysis. In 1968, just before I left Harvard, Jacques Monod visited the Biolabs. I knew that he would give a talk at the medical school, but I had no time to go there. Suddenly he stood in my lab. He said Hello. Then he said ‘‘After all, Benno, it was pedestrian,’’ turned around and left. I was so astonished that I could not answer. Now, after more than thirty years, having no witness, I ask myself again, what did he mean? Today I think he wanted to say ‘‘it was ordinary science,’’ and he was right. There he had failed and we had won. But perhaps I misunderstood him. Winnie Sippel who read this manuscript suggested that he may have said ‘‘predestined.’’ I will never know. In November 1967 I got a letter from the Kultusministerium in Du«sseldorf offering me a professorship at the Genetics Institute of the University of Cologne. This came as a real surprise. I had not applied for the job. At the time professorships were not announced and one did not apply. I had never studied genetics, I had never heard a lecture series on genetics. I understood something about bacterial genetics, but I had never given lectures or taught genetics. I had no experience in organizing a group to do research. I would just have to do all this without any experience. I showed Jim the letter. He said,‘‘You have to accept.’’

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So I sat down and wrote that I accepted the offer. This showed that I was incapable of dealing with the Ministerium, the institute and the university for the best possible offer.Yet this did not matter. There was lots of money for research in the DFG [the German institution corresponding to NSF) and elsewhere. Whenever I needed money for research I got it. I could do all experiments I wanted to do. My thirty years until my retirement were spent in paradise.

The First Ten Years in Cologne 1968^1977 In the morning of April 18, 1968, I arrived in Cologne airport with my wife Barbara, a cat in a box, and two pieces of luggage. In the afternoon, I gave my first lecture on bacterial genetics in front of five students. Barbara, the cat, and I stayed in the guest room on the sixth floor of the institute, until we found a beautiful apartment in an old house in the center of Cologne. I still live in this place. To return to Germany was not easy. This country haunted me. Directly after my return, I bought the drawing ‘‘In Gedanken’’ of George Grosz in the Galerie Nierendorf in Berlin. It shows a man, an officer?, sitting at a coffee table. What is he thinking about? Murder? Somewhere in a distance is a stupid looking man, a soldier, his servant? In Cambridge I had already bought an aquarelle by Grosz showing a nude woman. And then I discovered the paintings of Josef Scharl (born 1896), a painter who had left Munich in 1938 to go to New York [17]. He never came back, he died in 1954 in the States. I bought in the Galerie Nierendorf his portraits of two judges, a student, a business man, a priest, and a blind man. This was Germany, I should never forget that when going to the Institute. On every floor of the Genetics Institute there was a glass door leading to the Zoology Institute. All these doors were locked. The geneticists had no keys. I add here: little has changed in


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the last thirty years! Five of six doors are still locked. Zoologists have little interest in speaking to geneticists. Max Delbru«ck had founded the Genetics Institute in 1961. He had convinced the university and the ministry that there should be several, equal professorships in the institute. This was in contradiction to the typical German situation: one professor and possibly one or two associate professors without rights in each institute. Max Delbru«ck left the institute in 1964. Then all professors, with one exception: Peter Starlinger, left too. Just one group leader, Klaus Rajewsky, stayed in the Institute. The first new professor was Walter Vielmetter, I was the second. We then succeeded to convince the faculty that Klaus Rajewsky should get tenure. Walter Do«rfler was the fifth professor. The institute was unique in Germany that it lacked an omnipotent elderly director. We were all young. The institute worked well, because we all had just one interest: science. One could say the truth. So we criticized our experiments pitilessly, but there was not much cooperation. Klaus Rajewsky and I became friends, we had a mutual interest in literature. Max Delbru«ck had had close relations to some theoretical physicists. We inherited these friendships. So I felt closer to Janos Hajdu and Bernd Mu«hlschlegel, two theoretical physicists, than to my other colleagues in genetics. Peter Starlinger was a genius in organization. He managed that the institute had first one, later two Sonderforschungsbereiche (SFBs, special research units) to which money from the Deutsche Forschungsgemeinschaft was flowing easily. Peter Starlinger was also a democrat. So he arranged that we, the professors, gave our power away to an assembly of N professors þ N postdocs þ N graduate students þ N personnel. Miraculously their decisions were in general for most of us professors acceptable. But after several years, one day, this assembly died. Why? The people were not interested any more to waste their time with boring sessions. So the power came back to the professors.

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I was the successor of Hans Zachau, who had moved to Munich. I inherited all his material. When leaving, his collaborators had left everything in their labs on the third floor of the institute. So I spent the first weeks in Cologne investigating and emptying every drawer on the third floor of the institute. Some things I could use, others I had to throw away. I was completely alone these first weeks and months. No student, no postdoc, no lab technician. At this time I received an invitation to give seminars in East Germany at the Academy of Sciences in Berlin-Buch, at Gatersleben, and at the university of Rostock. I went there with Barbara with the aim of having a closer look: perhaps it would be worthwhile to move there. The Vietnam war and the Western student unrest encouraged me to investigate. Two experiences there completely discouraged me. In the Institute in Buch, I saw a seal on the lock of a door. ‘‘Has one of your colleagues gone bankrupt?’’ I asked jokingly. ‘‘No’’ I was informed. ‘‘In this room we keep in a safe the plans for future research. The safe is guarded by a member of the safety police. If he goes for lunch or to the toilet he seals the room.’’ That was too much for me. They had no interesting results. They had almost nothing. Their plans were nothing. And then they put this nothing in a safe and sealed the room! Never, ever would I work under such conditions. I told them so. My outburst seemed to have a small effect. The guard stopped using the seal. Then in Rostock we were asked to go to the police to get a stamp in our passports to document our presence. We went to the building and asked for the room, where we had to go. The room was locked. We banged at the door. Nothing happened. We asked some official for help. He encouraged us to bang again.We did so and finally the door was opened by a man in military uniform. He asked what we wanted. We explained. He asked us in, studied our papers meticulously. At this moment somebody else banged at the door. The military man did not react. So Barbara went to the door and opened it. There was a very old woman waiting. The police officer screamed ‘‘Close the


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door!.’’ Barbara closed it. Then she burst out ‘‘Scheisskerl’’ (shit guy). The police officer did not react. He treated us politely. This was not the bureaucracy I wanted to deal with. I decided to remain in Cologne. In Cologne, slowly, slowly my lab filled with collaborators. Bruno Gronenborn, Magnus Pfahl, and Dietmar Kamp were the first undergraduate students. It took some time until we could publish the first paper [18]. Bruno Gronenborn and Magnus Pfahl began to analyze the lac I (repressor) gene. They could show that strong negative dominant constitutive mutations (I d-mutations) were only found in the region coding for the extreme N-terminus of lac repressor [19,20]. This implied that only the extreme N-terminus is involved in operator binding. I s mutants in contrast map in the rest of the gene, i.e. the region coding for inducer-binding [19,20]. In 1969 my first postdoc, Konrad Beyreuther arrived. He was an expert in protein sequencing. He wanted to sequence Lac repressor. The Volkswagenstiftung funded the machines needed for sequencing. At the time one needed rather large amounts of protein for such a sequence. Using the mutant I had isolated at Harvard [16], we produced about 11 (eleven!) grams of Lac repressor in Cologne. The sequence was finished in 1973, Konrad Beyreuther published the work without my name as coauthor [21]: I followed the example of Jim Watson and Wally Gilbert. In those years we gave milligram amounts of Lac repressor to various people. In particular, we were interested in the X-ray structure. At that time making crystals was an art, which did not pay the artist. The first crystals of Lac repressor were described in 1990 [22], the first X-ray structure in 1995 [23], 20 or 25 years after we gave away our samples of Lac repressor. The sequence of Lac repressor was the first and only repressor sequence for several years. It did not illuminate its function. For this, a functional genetic analysis of the lac I (repressor) gene had to be done. My students concentrated on the positions of strong negative dominant (I d) mutations [19,20]. Negative dominance

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indicates that the mutant repressor is still able in forming a tetramer, but is unable to bind to operator. All the I d-mutant exchanges occurred in a region, which corresponded to the first 50 N-terminal residues of Lac repressor [24], the head piece as it is called today. I published together with my collaborators a paper on the modular structure of Lac repressor [24]. In this paper I predicted that Lac repressor recognizes the DNA sequence of lac operator by placing an alpha helix in the deep groove of DNA. Furthermore, I predicted that this particular alpha helix would begin with residue 17 of Lac repressor [24]. Later NMR [25] and Xray work [23] supported this prediction. I went even further and tried to predict the DNA sequences recognized by this alpha helix.There the predictions were wrong: an alpha helix has more than one way to fit into a major groove. To me all this seemed clear. But there was Kathleen Matthews. For thirteen years she claimed that Lac repressor uses its core to bind to operator (for a detailed analysis of her claim see Ref. 26, p. 99^101). Kathleen Matthews never retracted her conclusions. This was for years a rather disquieting situation. In 1970 Jeffrey Miller came to work for two years in my lab as a postdoc. Together with a student of mine, Ju«rgen Schrenk, and the help of Geoffrey Zubay they isolated Trp repressor [27], the third repressor to be isolated. Jeffrey’s main interest was the lacI gene and the Lac repressor. From codon 2 to codon 330 he replaced every codon by an amber codon and tested these amber mutants then with all amber suppressors that were available. It took also a long time for Jeffrey to finish his mutant analysis of Lac repressor. The last paper of this series appeared as a joint effort between his and my lab in 1996 [27]. More than four thousand Lac R mutants were analyzed for their function! In the 1970s I became part of the group of German molecular biologists, organized by Hans Zachau, which went every two years to the Soviet Union for a meeting with Soviet colleagues. The other year we met somewhere in Germany. It was interesting to learn how science was organized in the SU.


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A very few directors determined the production of very many scientists. For example Ovchinnikov, who was director of the Institute of Bioorganic Chemistry, had about 600 (six hundred!) collaborators. His biography ought to be written. I was mainly interested in protein DNA recognition. So I was most interested in the work of Georgi Gursky who proposed that Lac repressor recognizes DNA in the major groove as sheet. I tested several of his predictions and they all failed. Yet Gursky stuck to his idea (for a detailed description see Ref 26, p. 102^103). Once, I was invited by Gursky to dinner at his home. There I noticed a beautiful painting high on the wall: a vase with flowers on a table. ‘‘Who painted this beautiful painting?,’’ I asked. He did not know. The painting belonged to his wife. She did not know either. She had inherited it from her mother. I asked whether I could have a closer look. This was allowed, so I took the painting in my hands and read in the right lower corner ‘‘Jawlensky 1908.’’ The Gurskys had never heard of Jawlensky. I told them that the painting was worth at least 100,000 dollars. They did not believe me. The next day I left and went back to Germany. So I told them: ‘‘please, please guard this painting as a treasure.’’ When I returned two years later, it was gone. They had sold it for 5000 roubles. I would have easily bought it for double this price. Sometimes I have been really stupid. Perhaps the prettiest discovery I made with my own hands in those years was the isolation of a fusion between active Lac repressor and active -galactosidase [28]. Protein sequence analysis showed that Lac repressor was missing residues 330^360 and that -galactosidase was missing residues 1^17 [29]. Thus, -galactosidase could be used as a reporter and dimeric Lac repressor was the unit, which recognizes lac operator [28]. These were the years of the student revolt in theWestern world. I had one graduate student, Alex Klemm, who worked in the group of Konrad Beyreuther who went to the extreme. First he told me that he would leave my lab and accept an offer elsewhere, unless I would pay him the salary of a postdoc (BAT II).

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He had the collection of tryptic peptides of Lac repressor. Should he leave and destroy the collection, we would lose two years. It was a mistake, but I gave in. Then he called me several times in public an idiot who did not understand science. Finally, he sent a manuscript to Nature, which contained data of the entire group. Then I told him in the presence of another professor that he would get his doctorate degree, but that I would not like to see him ever again in the lab. His time in my lab had come to an end. So he went on vacations with a couple working with Klaus Rajewsky. They went skiing in the Swiss alps. There they had been warned not to use a particular route: it was dangerous because of avalanches. Alex Klemm said he knew how to deal with avalanches. They followed the dangerous route, the avalanche came and killed all three of them. If it would have been only Alex Klemm who died, I could have quoted Jesus Sirach XXV, 7 with full support. Life these years was not easy. I was working all day at the bench in the lab. I had little time for my wife Barbara. So she discovered during her first Cologne carnival in 1969 a guitarist who played modern music and who had ample time for her and drugs. Here I could not compete. So one year later she left with our car and what I thought was our child, but who was his child. So I went to the carnival. On the third evening I met Rita, the daughter of a poor farmer and a student of French literature, with whom it was most pleasant to talk. She stayed, we got married. In the first years of our marriage my private life was chaotic. To paraphrase Brecht, It was a good but not the best of all times. Whatever happened, I always thought Rita to be the center of my private life. She was the sign of order. So we are still happily married to the present day. During those years I taught the Cold Spring Harbor course on bacterial genetics together with Jeffrey Miller and Julian Davies. I was in Cold Spring Harbor in 1972 when my father phoned me. My mother was dying. I felt unable to leave the course and to come immediately. So I did not see her alive


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again. I arrived just for the funeral. Science seemed more important than my mother. After my mother’s death, my father lived in their old apartment until his own death. He fell in love with a young woman, then twenty-three years old. Miraculously it became mutual love. It lasted until my father died in 1977 when he was 92 years old. There I was present. The older I get, the more I appreciate his character: charming and peaceful. A friend of Michel de Montaigne and Laurence Sterne. One has to age to appreciate some central aspects of human life. During those years I began a friendship with two persons whose books interested me. One had written a book about his time in Vietnam [30]. There he had worked as a psychiatrist under the guidance of Horst-Gu«nther Krainick who had been a co-author of my first publications. I had heard Krainick say how much he looked forward to working in this peaceful Vietnam. Now the Tet offensive had ended his life. He and the other German doctors were executed by the North Vietnamese during the fight over Hue. The author, who called himself Alsheimer, had been on vacations in Germany. The book seemed both honest and courageous. It painted a rather dark picture of the American army. The Suhrkamp Verlag forwarded my letter to the author who turned out to be a psychiatrist, Erich Wulff, working in Giessen. The other person was a painter, Werner Hilsing, just a few years younger than me. I bought one of his pictures at the Cologne Art Market in 1969. And I have bought many of his paintings thereafter. He changed his style several times. He is still not accepted as the great painter he truly is. His life and paintings merit description in a separate book. At that time I also left the protestant church. What their preachers said seemed just empty talk. At the same time I began to read the Old Testament and Talmudic texts. I knew I would never convert. Most of the people I felt close to were not German. They were French, British, Russian, Swedish, and or Jewish. I was turning into a cosmopolite.

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In 1972 I got the strong feeling that bacterial genetics had come technically to its end. So I went for half a year to the lab of David Suzuki in Vancouver, Canada, to learn Drosophila genetics. Suzuki was the most interesting person. As a child he had been interned with his parents in a Canadian concentration camp. It did not help that his parents had Canadian citizenship and passports: they were of Japanese origin. Only this counted. So they lost their house and earnings and had to stay for years somewhere in a camp. Suzuki was losing interest in research. He became more interested in explaining science to the public. And I realized that Drosophila would not become the object of my research. In 1974 I took a course in tissue culture in Cold Spring Harbor. There the rumor spread that Paul Berg had cloned a piece of SV40 DNA on a plasmid in E. coli. Was this not terribly dangerous? From the beginning I did not believe so. The technique revolutionized the analysis of all genes, also of bacterial genes. It also changed my scientific life: I understood the techniques, so I introduced them in my lab immediately, but I never used them with my own hands. Ultimately I stopped working in the lab. In the 1970s one was not allowed to teach at a German university or at a German high school if one was a member of the communist party or an active fellow traveller. Moreover, the Verfassungsschutz (FBI) had to comment on any person hired by a university. I thought this went too far and asked the faculty formally to protest against this act, which I found incompatible with a university. When the issue came up, the faculty voted not to discuss the matter. This move certainly did not qualify me for administrative jobs. I have had virtually no such positions in the last 30 years. I have never been asked to give my opinion on a molecular biologist being hired by a German university. I have received no honors from a German institution. I do not mind and sometimes I am even proud of it. So what is the bottom line? It may astonish the reader, but I think that I had entered a paradise, where I could do in science


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and in my private life what I wanted to do. I or my students could test any idea. The paintings I loved were cheap enough so that I could afford them. I had access to any book. Truly I was living in paradise.

The Next Ten Years in Cologne 1978^1987 In 1977 my policy of never being coauthor on the papers of my students or postdocs, broke down. My postdoc Bruno Gronenborn constructed together with Joachim Messing, a postdoc of Peter-Hans Hofschneider, a variant of phage M13 that carried a piece of E. coli DNA coding for the alpha peptide of -galactosidase. Hofschneider insisted on being coauthor of the PNAS paper. What should I do? If I would not be coauthor, this would indicate that I was uninterested, which I was certainly not. I had discussed the experiment with Bruno from beginning to end. So I gave in and became coauthor[31]. Having fallen once, I changed my policy. Had it been a good or a bad policy? I have been told often that it was bad and stupid. I still do not think so. But certainly it was hopeless. It was impossible to maintain it. In the summer of 1978 something terrible happened. The strong negative dominance of I d mutants suggested that all four subunits of Lac repressor are needed for full repression [32]. But two subunits were sufficient to recognize one operator [33]. Thus, I thought that tetrameric Lac repressor would recognize O1 with two subunits and O2 with its two other subunits (I forgot to consider O3!). Thus, there would be a loop between O1 and O2. So I predicted that a deletion of O2 would lead to semiconstitutive production of transacetylase. We had a series of LacZ deletions, which ended beyond or not beyond O2. So in 1978 I asked a student to test these mutant extracts for transacetylase activity. She found what I suggested. With some deletions the transacetylase activity went up, with others it remained low. I wrote a paper for PNAS and had it accepted [34].

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Just then I got a call fromWally Gilbert. He had sequenced one of the deletions. The transacetylase activity was high, but O2 was still present according to the DNA-sequence analysis (or was it the other way around: I do not remember any more). I went to the lab to inspect the notebooks of the student. She confessed that she had made up the values. I had been too demanding. I called PNAS and retracted the manuscript. I was completely confused. Had I understood that only a double mutant O2^, O3^ is constitutive, it would have made life easier for me. So the problem was solved twelve years later [35]. We sequenced DNA the moment the knowledge about the Sanger and the Maxam^Gilbert techniques spread. Our first two sequences were interesting: the lacY (permease) gene [36] indicated that Lac permease consisted of several lipophilic regions. Today we know twelve lipophilic alpha helices [37]. Then we sequenced the gene coding for Gal repressor. What a surprise to see that Gal repressor and Lac repressor have similar protein sequences [38]. And then we bought the first DNA synthesizer in Germany. This allowed us to analyze how Lac repressor uses its recognition helix to bind specifically to lac operator. We showed conclusively which changes in the recognition helix were recognized by which changes in lac operator [39^42]. We showed all possible interactions. We showed which of them were additive and which were not. The Lac repressor lac operator interactions were elucidated before they were analyzed by others with NMR or X-ray techniques [23,25]. During my travels in 1978 I had two interesting experiences. First I visited some institutes in China together with a group of German molecular biologists. The cultural revolution had come to its end. In one institute the Lysenkoists were still in power. In other institutes we met victims of the cultural revolution. They told their stories. Reality of the cultural revolution truly differed from the beautiful fiction so many of us had admired. One of our group members came crying to my room: he had praised the Chinese cultural revolution as a great step forward.


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Directly after the visit in China I had been invited to a conference in Ku«hlungsborn in East Germany on philosophy of science. I had been asked to speak about the risks and morals of DNA-cloning. I had answered that I would like to come but that I could not speak on this topic, since I saw no risks and no moral problem. I would be delighted to listen and participate in the discussion. When I arrived in Ku«hlungsborn in the evening before the first day of the conference, I was given the program. I was the first speaker on the first day on the topic ‘‘risks and morals of DNA-cloning.’’ So I gave a spontaneous talk. I began with what I thought was a quote of Diderot (it is not) ‘‘philosophy is the opium of scientists’’ and continued that it was ridiculous to worry in East Germany about the possible dangers of DNA-cloning as long as two problems were not solved: 1. No such experiment had been done so far. The young East German scientists should be allowed to spend some time in laboratories of the West to learn the techniques. 2. The custom that a list of all material to be bought in the West should be made and forwarded once a year on December 31, with the expectation that some of the material may be delivered at best by September the following year should be abolished. The institutes should receive instead 50 percent of the foreign money they got now for free use. My talk created a scandal. The chair person tried to rip the microphone out of my hands. One of the professors yelled at me ‘‘provocateur’’ and almost beat me up. From then on to the end of the conference I was almost alone. Only in the toilet some whispered to me that what I had said was fine. My talk had an effect. For the next ten years I was not anymore invited to the Academy Institutes in East Germany, that is, until the autumn of 1989. Yet my talk was published [43]. The paragraphs dealing with travel of the young scientist to the West and payment of material bought in the West were deleted. They were replaced by a line of points and a footnote saying that the talk had been shortened with the consent of the author. This was not true. I had not been asked. Yet I have to say here, that I prefer this type of direct

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censorship to the extensive copy editing, which is common today in most journals and which is most difficult to fight. I should add here, that in those years I published three articles in the East German literary journal ‘‘Sinn und Form.’’ One about my dreams [44], the other on a book of an anonymous German emigrant I had discovered, which I thought to have possibly been written by Ernst Weiss [45], and the last one on Mengele [46]. All these manuscripts were published as I sent them to the editor. No changes, no cuttings. In the winter of 1977^1978, when the cloning and sequencing of DNA was in full swing, I went for a half year sabbatical to a lab where this was not done. I went to Gif sur Yvette (a suburb of Paris) to the CNRS lab of Kissel Shulmeister. I did not work in his lab, but spent my time reading Aristotle and other classical authors on the phenomenon of life. Originally my interest centered around Friedrich Engels and his Anti-Du«hring. These texts and their early interpretations seemed interesting to me. Then came of course the fraud Lysenko and Stalinism. The question was, could one save the essential ideas of Engels? In order to do so I had to read Engels in the context of all other interpretations. So I read with great pleasure the books on embryology and zoology by Aristotle in their bilingual French^Greek editions. I read Hegel and realized that he did not understand science. I read Diderot. And then I read the authors of the twentieth century. There seemed to be a line leading from Mendel and Darwin into the center of the ideology^philosophy^religion of the Nazis. Not much had been written about this development. So I read as much of the originals as I could get hold of. Then I realized that I had to read all German scientific journals and books written in the twentieth century and dealing with genetics. A world unknown to me opened itself before me. When the time came in 1981 to publish my manuscript under the title of ‘‘Die Philosophen und das Lebendige’’ (the philosophers and the living matter), genetics in Nazi Germany had become the most interesting last chapter [47]. I realized that much was lacking. So in the following years I spent a substantial


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amount of time visiting archives and reading relevant documents. I discovered that a large part of the records of the Deutsche Forschungsgemeinschaft was kept in the Bundesarchiv Koblenz. The people of the DFG I had asked did not know. The archivist at Koblenz told me that I was the first person ever to look at these records. There I discovered the reports of von Verschuer on the project spezifische Eiweissko«rper [48] where he called Mengele ‘‘mein Assistent’’ (my assistant) and ‘‘mein Mitarbeiter im Konzentrationslager Auschwitz’’ (my collaborator in the concentration camp of Auschwitz). There Hillmann, a student of Adolf Butenandt, is mentioned as being involved in the project [48]. And then Robert Ritter’s attempt to organize genocide of the Gypsies can be found there [49]. I also interviewed all the German human geneticists I could find. About half of them agreed that our conversation could be published. All this material was included in my book ‘To«dliche Wissenschaft’ (Murderous Science), which appeared in the summer of 1984 [1]. Its appearance was greeted with silence in Germany. In particular, the historians of the Max-Planck-Gesellschaft were silent. They did not review the book. No review appeared in any daily or weekly like Der Spiegel or Die Zeit. Yet the book sold well. Until it went out of print in 1989 it sold altogether 15,000 copies in Germany. In February 1985, on my birthday, a favorable two page review appeared in Nature [50]. What more could I want? I sent copies of the Nature review and the book to the German newspapers. There was just one response. The Frankfurter Allgemeine published a review. All the other journals remained silent. Before the book appeared, I was invited to give a public lecture in Mu«nster at the annual meeting of the German Society for Anthropology and Human Genetics on October 5, 1983. I did so under the title ‘‘Dream and Horror of Genetics.’’ Widukind Lenz, the son of the eugenicist Fritz Lenz had invited me. He was just about to retire and step down from his position as president of the society. It was his last action.

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I do not have a manuscript of my talk.The beginning was probably similar to the introduction of ‘‘To«dliche Wissenschaft’’ [1]. I quote it here: ‘‘How has genetics affected man? There is a special branch of genetics, which concerns itself exclusively with human beings: human genetics. However, genetics also plays a fundamental role in many other branches of human biology: in anthropology, psychiatry, and psychology for example. In these sciences it is easy to think that only what is new is true. But when I think today of the story of how genetics was once put to use in anthropology and psychiatry, I see a wasteland of desolation and destruction. The blood of human beings, spilt million times over, is completely and resolutely forgotten. The recent history of these genetically oriented sciences in action is as full of chaos and crime as a nightmare. Yet many geneticists, anthropologists, and psychiatrists have slipped from this dream into the deep sleep of forgetfulness. I, myself, am trying to wake from this sleep. . ..’’ My talk was not well received: ‘‘here you defend the Jews.Will you now defend the Turks?.’’ ‘‘This is all well-known stuff, but very badly presented.’’ ‘‘Your motives are as dirty as your clothes.’’ The discussion lasted long.Widukind Lenz and Helmut Baitsch came to my aid but the general impression was not favorable.When the discussion ended and the lights went out, several people rushed to me to say that, finally, I had said what should have been said years ago. One person told me that years ago he had to leave the genetics institute, where he was working as postdoc, because he had showed some interest in the past. He was now working in a ministry. The book ‘‘To«dlicheWissenschaft’’ was a success. It was translated into English, Dutch, French, Italian, Spanish, Portugese, Hebrew, and Japanese. Jim Watson wrote an afterword to the English Edition published by the Cold Spring Harbor Laboratory Press. I was invited by geneticists and historians of science to speak on the topic in the US, in many European countries, in Israel, and in Japan. In Germany I was invited too, but mainly by students and not by professors. The honors I received (honorary fellow of the Hebrew University and an honorary doctor degree of the Technion in Haifa) had something to do with this book.


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Through ‘‘Murderous Science’’ I met many people, I would otherwise have never met. I name here Eva and George Klein, whose Swedish books and manuscripts I could read, Dan Bar On whose attempts to understand the children of the perpetrators I found most interesting, Henry Friedlander, who like me saw a continuous process from the euthanasia murders to the death camps of the final solution, Rudi Vrba and Jacob Steiner whom I met through George Klein and so on, and on. There is one person from a totally different field, Josef Weizenbaum, I should mention here. After I read his book ‘‘Computer power and human reason’’ [51], I thought I had to meet this man. We met in Cambridge and then he came to Germany. It is always good to know people who look with critical eyes at their so different field. He is now a friend of the family.

The Last Ten Years Before my Retirement in Cologne 1988^1997 During the last ten years before my retirement, I concentrated my research on the lac system of E. coli. We showed that operator^Lac repressorôperator looping can be quantitized with gel shift experiments [52]. My student Stefan Oehler demonstrated that two such loops can be formed with 01: one with 02 and the other with 03. Thus, only when 02 and 03 are destroyed, repression goes down 70-fold. When 02 or 03 are destroyed, it goes down twofold [35]. Stefan Oehler by chance also isolated a mutant Lac repressor, which was an active dimer. It carries a frameshift mutation in codon 330 [53]. An inspection of the C-terminus of Lac repressor indicates two heptad repeats [54]. So we showed that the heptad repeats of Lac repressor are involved in tetramer formation [54]. We predicted that the tetramer is formed by a four helical bundle [55]. Later X-ray analysis indicated we were right [23]. We then showed indirectly that loop formation depends on the exact position of the two operators.

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To explain the function of the auxiliary operators, we proposed an increase in local repressor concentration [56,57]. There are about 10 molecules of tetrameric Lac repressor in one E. coli cell. When Lac repressor is bound to 01 it competes with RNA polymerase [58]. Either Lac repressor is bound to 01 or RNA polymerase is bound to the promotor. The stronger Lac repressor binds to 01 the stronger the repression. The local concentration of Lac repressor dimer is increased about fifty fold for 01 when tetrameric Lac repressor is bound to 02 or 03. This increases repression on 01 about fiftyfold. So dimeric Lac repressor represses much less well than tetrameric Lac repressor. In vitro, tetrameric Lac repressors binds faster to lac operator buried in Lambda DNA than when it is placed in a short piece of DNA. This effect is not seen with dimeric Lac repressor [59]. Moreover, Lac repressor that has the rather unspecific recognition helix of Gal repressor binds slowly to operator buried in Lambda DNA but normally to operator placed in a piece of short DNA [60]. Thus, highly specific tetrameric Lac repressor is speeded up when recognizing the operator where less specific tetrameric Lac gets lost in the jungle of DNA. When I retired in February 1998, I had worked almost forty years on the lac system of E. coli. Almost all my colleagues who had worked on systems of E. coli had abandoned their objects, under the assumption that these systems have been solved. I think this is far from true. The closer one looks at an object of the living matter the more wonderful it becomes. Now, when industrialized proteomics will be the main type of research for the next twenty years, many will leave the eucaryotic systems and with similar arguments move to the last bastion of the living matter: the human brain. Again I think this is a mistake. The lac system of E. coli is an excellent example of such an abandoned object. I am glad that I wrote a book about it [26]. Sydney Brenner made an interesting comment in his review: ‘‘When molecular biology finally comes to an end, and a massive crash of the computer system in the library of


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Alexandria corrupts most knowledge, I hope that historians of science will unearth a copy of this book. They will surely debate whether all of the stories are true and whether they could have been written by the same person, and might even suggest that Mu«ller-Hill was at least two people. What will certainly puzzle them is that it is full of characters they have never encountered before, and that even the famous ones are hard to recognize. The only thing they will learn about Watson and Crick is that the former produced a naked lady for the latter’s fiftieth birthday. Was this perhaps part of some obscure symbolic ritual enacted each year to celebrate the birth of another model?’’ [61]. In the eighties Konrad Beyreuther received an independent C3 (associate) professor position at the Genetics Institute. He worked in a laboratory next to mine on the third floor of the Institute. He was trying to isolate the DNA coding for the precursor of amyloid of Alzheimer’s disease. His collaborators were protein experts but they had no particular expertise in DNA cloning. So they tried to isolate the chromosomal DNA coding for the amyloid. I saw this and thought it would never work. I had students who were experts in preparing cDNA. And indeed my student Jie Kang isolated the cDNA and another student, Hans-Georg Lemaire, sequenced it in six weeks. Then we told Konrad about our success. His student Michael Salbaum then determined on which chromosome the gene was located. We published some joint papers [62,63]. Konrad then went to Heidelberg and continued with amyloid. We soon gave up and returned to E. coli. During those years I had not lost my interest for the history, philosophy, and sociology of science. Ute Deichmann began to work as a graduate student with me. She wrote a history of Biology in Germany 1933^1945 [64]. To do so she followed my advice: first list all people involved, then look at their personal data in the Berlin Document Centre (now part of the Bundesarchiv). Then read all their papers and books. Finally read their reports and grant applications to the Deutsche Forschungsgemeinschaft (DFG).

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Now, that the history of Biology in Nazi-Germany was written, chemistry was still missing. So I was happy when Ute told me after she received her PhD in science on her dissertation on the history of Biology that she wanted to do the same type of analysis with chemistry. She did so ^ and got her Habilitation in genetics. Her book just appeared [65]. Again I am more than happy that this excellent book has been written. Ute Deichmann drew my attention to Emil Abderhalden who discovered in 1909 the defence enzymes (Abwehrfermente), specific proteases that supposedly are formed in mammals after injection of foreign proteins. The defence enzymes did not exist. They were at the beginning a misinterpretation but in the end they became a fraud. Ute and I published a paper on the subject, which seems to me a disquieting case of science as a social construct [66]. The Abwehrfermente only disappeared in 1950 with the death of their inventor. Abderhalden, President of the Leopoldina, the oldest Academy in Germany, and professor at the University of Halle had an excellent reputation during his lifetime. Something really went wrong. During those years I organized in 1987, with DFG support, an international conference ‘‘Medical Science without Compassion’’ [67]. Occasionally, I invited speakers to give seminars on this topic in our Genetics institute. One of these speakers was Reimar Gilsenbach who spoke in 1989 about the eugenic ideas of the plant geneticist Erwin Baur. This was the time when the enemies of genetics put bombs in front of our institute and the Max Planck Institute fu«r Zu«chtungsforschung nearby Cologne. Erwin Baur had been the founder of the precursor of the Max-Planck-Institute. He died in 1933. Since 1938 he was commemorated in the full name of the institute, ‘‘Erwin-BaurInstitut fu«r Zu«chtungsforschung.’’ To associate the institute’s founder with German eugenics did not put the institute in a good light. Furthermore, I mentioned in my introduction of Gilsenbach that the institute had asked the city of Cologne to rename the street in which the institute was situated ‘‘Erwin-Baur-Strasse.’’ The city declined following the advice of


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the Verein fu«r Christlich Ju«dische Zusammenarbeit who had read ‘‘Murderous Science.’’ One of the directors of the institute Heinz Saedler was present during Gilsenbach’s talk. Some days later the Graduiertenseminar of the Genetics Institute was meeting. There three directors of the MPI participated. At this particular meeting I was by chance absent, but Jeff Schell was present. He gave a fifteen minute speech with the bottom line that I had encouraged the enemies of genetics to murder the Max-Planck-People, that their blood should come over me and that I should disappear from science. All professors present, with exception of Klaus Rajewsky, were either silent or took the position of Jeff Schell. Klaus arranged that Jeff and I should meet. I told Jeff that I defended the right to have lectures at the university on the bad past of human genetics in Nazi Germany and that I asked him to apologize. If not I would fight to the bitter end. I would make it known internationally, that he wanted to end the discussion about the crimes of the German geneticists in Nazi Germany, and thereby their antisemitism, with the argument that this would endanger true science. And that I was against this. Jeff left open what he wanted to do. Shortly thereafter he went to Israel to receive a major prize. I sent him a fax, he must decide right now. He answered with a fax, which could be understood to mean that he apologized. I read the letter to the students of the Graduiertenseminar, and that was it. Years later, in 2000, I met Jeff. He had just experienced the disaster, the fraud of several papers on auxin [68] and he was sick. Then he came to me and apologized profusely for what he had said in 1989. Had I been a student or a postdoc at that time my carrier would have been ruined. I would have been lost. So I just made it. It was truly on the edge. The Max-Planck-Gesellschaft was not only irritated by the fact that I reminded them of their bad past (the Verschuer-MengeleHillmann-Butenandt connection). Additionally, I reminded them that the present organization also had some problems. At the moment in Germany, at least half of the research was

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done in pure research institutes. It was just the right moment for me to interest Heinrich Herberts, a student of sociology, to make a detailed study of the costs of literature citations in various institutes. It was generally accepted that the citation impact factor or the number of citations is an indication of the quality of research. But, of course, more money means more citations. So Heinrich Herberts made a detailed study comparing the cost of citations of university and Max-PlanckInstitutes. He conducted the study manually since at the time no computer program was available, which would allow the subtraction of self citations. The citations produced by the Max-Planck-Institut fu«r Biochemie in Munich were twice as expensive as those produced by the Cologne University Institute of Genetics. The bottom line was: the Cologne Institute of Genetics was more productive than the Max-Planck-Institutes in Munich or Berlin [69^71]. In retrospect, it seems deplorable to me that these days candidates for professorships are only screened for their number of publications in Cell, PNAS, Nature, and Science, and for the amount of grant money they get, but that nobody knows anymore what the candidate discovered. This was certainly not what I had in mind when I was doing the study. Then I collided with Hilger Ropers, Director of the MaxPlanck-Institute for Molecular Genetics in Berlin, an institute that can be traced back to the Kaiser Wilhelm-Institute of Anthropology where von Verschuer and Mengele had been active. Ropers had discovered that a nonsense mutation in the MAO A gene, which is located on the human X chromosome, turns its male carriers into violent criminals [72]. It was and is my conviction that genes should not be connected with crime and therefore with moral values [73,74]. One may say the actions of the carriers are spontaneous, that they do not think the way they act, but one should not claim a direct connection between the mutant gene and crime. Precisely this, I pointed out, had been done by the members of the predecessor institute for example with Gypsies, to legitimize and organize


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their genocide. Ropers interpreted this as an attack on his person [75]. Years later I gave an interview to an Israeli journalist. Unfortunately, I did not insist on reading what I was quoted to have said. I went on vacation and could not be reached. The journalist mixed up several things, he did not understand the difference between Max-Planck-Gesellschaft and the MaxPlanck-Institutes. So I was quoted as having said that brains Hallervorden had collected were in the Ropers institute [76]. When Ropers was shown the text his comment was ‘‘the author is incurably insane’’ [76]. I wrote to Ropers and never got an answer: Was he misquoted as I had been misquoted? Then I called him. He seemed to understand that there were misunderstandings. Considering all these activities, it is clear that I failed when I had anything to do with administration. For example I was once asked to join a large ethics committee of Nordrhein Westfalen, which should think about genetics and gene technology. When the committee met for the first time, the minister was present. A position paper was given to us, which should be the basis of our activities. I found the paper extraordinarily stupid and thus useless. I said so ^ and learned later that the minister had written it herself during the Christmas vacations. Of course I was never invited again. When I say that I had nothing to do with administration, this is not absolutely true. From time to time I had to be acting director of the Genetics Institute. I recall one decision, which determined the course of the institute where my guidance was decisive. In December 1983, when a new building was planned for about half of the people of the institute, two possibilities were discussed: Peter Starlinger, the man who thus far had guided the institute, proposed that the new building should be built close to the Max-Planck-Institut fu«r Zu«chtungsforschung (plant genetics) just outside Cologne. I was for the other option to build it in the center of the campus close to the institutes of physics and chemistry. I feared that the institute located outside

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Cologne would lose contact with students and the university and would eventually became part of the Max-PlanckGesellschaft. This could, of course, also be seen as a positive development. I tried to convince the other directors Klaus Rajewksy and Walter Doerfler of the other option. I was succesful. Was I right? Was I wrong? I still do not know. Then one of our professors, Walter Vielmetter, had offered one of his positions to an independent group leader. The applications had come in, the applicants had given seminars. Just then Vielmetter had a nervous breakdown. What should we do? Should we postpone the decision? I decided that we should choose the group leader from the applicants. We did so. I signed then the contract with Albrecht Sippel. Finally, I tried to convince our faculty to give a doctor honoris causa to the biochemist Rudi Vrba for his Auschwitz report. Rudi Vrba, then a young man twenty years old, had escaped Auschwitz in May 1944 together with another prisoner. They had written a report, which listed the numbers of the people murdered there. I found this list extraordinary. The report ^ finally ^ led in July 1944 to stopping the transport of the Hungarian Jews to Auschwitz. I proposed that Vrba had used a scientific approach, calculating all transports and extrapolation from them. Todays calculations indeed indicate that the numbers of the report are somewhat too high. My proposal was controversial. A mathematician said to me ‘‘just adding up numbers, this is not mathematics’’ and others feared that the faculty may lose its good name if it accepted the proposal. In the voting Vrba lost, he did not get the 75 percent of the votes of the entire faculty he would have needed.

The First Four Years after my Retirement 1998^2001 When I reached retirement age in February 1998, my group was still productive and I did not want to stop my research. For the first three years I still had a large SFB grant, for


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the next two years I got a small DFG grant. I concentrated on Lac repressor. A student, Steffi Spott, tried to change the specificity of dimer formation. She ^ accidentally ^ succeeded with D278L. D278L forms dimers with itself but no heterodimers with wildtype 278D [77]. A postdoc, Lily Gerk, then looked for a mutant Lac repressor that is more heat stable than the wild type. She found one, K84L, which increases the heat stability by 40 C (!) from 52 to 92 C [78]. The existence of this mutant and the mutant mentioned above indicate how little one understands of protein structure and that real discoveries still can be made there. Then we tried to design an operator^Lac repressorôperator loop, which is larger than 1000 bp or for that matter even larger than 3000 bp. The idea was that the protein bound to one operator should be unique in its structure and not occur in solution. We failed with Lac repressor but we succeeded with Lambda repressor. Lambda repressor is a dimer at physiological concentrations [79]. Only at much higher concentrations it forms tetramers and octamers [80]. So I argued that Lambda repressor bound to the two operators ORI and ORII may form there the same tetramer that is formed at high concentrations in solution. Two such tetramers, unique on the two operators and not occurring in solution would then form an octamer. This would increase repression. The loops should be seen with EM. And indeed Bernard Re¤vet showed that they occur in large numbers. In addition, we showed that the loop increases repression fivefold [81]. This has been recently confirmed in vitro by X-ray analysis [82] and in vivo by mutant analysis [83]. Many years ago, when I met Mark Ptashne in Tiblisi I had suggested such a loop between the left and the right operators of phage Lambda DNA. At that time we had just done the in vitro experiments showing lac0^LacR^lac0 loops. Mark laughed and thought the whole idea was nonsense. But now here it was. I am immensely happy that these experiments could be done after my retirement. It is a good experience to end

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experimentation with a beautiful experiment that works and which can be generalized. I have decided to close my lab at the end of 2002. Thus, no new students will come to work with me. They know that my time has run out. Some of the problems I would have liked to solve will not be solved. That is another way of looking at the situation. Both views are valid.

Outlook To sum up: if I look back on my life in science and history of science, I see spirals of chance and luck and chance and luck. I was fortunate to be born in a bourgeois family where science, industry, and the arts, mixed with some anarchy, prospered and where I was put on the path of science. I was lucky with my choice of teachers: Wallenfels, Rickenberg, Gilbert, and Watson. The object I studied for forty years, Lac repressor, remained interesting for decades. The book I wrote about human genetics in Germany 1933^1945 paved the way for others. And where I gave up I found a student who did the work: Ute Deichmann, who wrote books about biologists [64] and chemists [65] under Hitler. I did not mention that I had for a long time one or two graduate students working on Plasmodium falciparum. We were trying to clone a piece of chromosomal DNA, which would encode a protein one may use for vaccination against malaria. I collaborated with Luis Pereira from the Institut Pasteur [84]. We failed in our goal, last not least because our group was much too small. Yet an enlargement would have been against my policy not to have more than twelve collaborators. So in hindsight, I would not have changed my effort. I did what I wanted to do. I was not always succesful, but who is? My story has come to an end. Soon my lab will be closed. I will retire to my library. So a question that may be asked is ‘‘Were there real mistakes? Would you do things differently?’’


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This is an embarassing question for a scientist. Scientists are used only to report their successes. Failures are never mentioned. Scientist sing in a chorus ‘‘Nous ne regrettons rien.’’ While I am writing this, a paper just appeared documenting a working Lac repressorôperator system in mouse cells [85]. The trick was to replace all replaceable CG sequences in the lacI gene and to avoid splicing. The absent CG methylation and splicing allowed then excellent expression of Lac repressor. Some years ago I had a graduate student who was to work on the LacR/0 system in mouse or human cells. In the midst of his thesis he left: He had received an offer from industry he could not reject. Projects that are abandoned often remain abandoned. I have no idea whether we would have solved this problem. We just knew that the exact positions of the lac operators are essential. Whatever, I am most pleased that the problem has been solved. Then there are minor and major mistakes. It was a minor mistake not to isolate the I q mutant instead of the I T mutant. It was a major mistake to assume that 03 has no function and thus to predict that the destruction of 02 should have a major effect on the expression of transacetylase. This mistake in thinking misdirected our experiments for years. Moreover, the Lambda repressor experiment with long loops could have been done much earlier. We misinterpreted our gel shift experiments with Trp repressor [86]. Paul Sigler corrected our mistake [87]. I do not know of other similar mistakes. So the science record could be somewhat better, but not much better. I can clearly say ‘‘Je ne regrette rien.’’ But what about my other activities? I abandoned administration and power for writing books and articles outside my field. I would do it again. All this happened during a time when grant money for research was easy to get. It was a paradise I was living in. I could do what I wanted, I always got the necessary money for research. What more can a scientist want? My book ‘‘Murderous Science’’ finally made an impact on the Max-Planck-Society. On June 7^8 2001 they invited some of the Mengele twins in order to apologize to

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them. Finally they had stopped denying that Mengele and Verschuer were part of their past [88]. I am still happily married to Rita. Our two children Sarah and Jakob study medicine and physics, respectively. So with Jesus Sirach I can say: I am a happy man having pleasure with my children and not only that [89]. There is just one aspect I find disquieting. During these years I read so many books. But I have not transmitted to the students or a larger audience what I read. All the five thousand books of my own library are somehow lost in my own brain. I cannot imagine spending the years, which are left travelling from one place to another, or sitting quietly in the garden viewing the sun rising and setting. What I can imagine is reading again and again in those books and writing about what I read.

REFERENCES [1] Mu«ller-Hill, B. To«dliche Wissenschaft. Die Aussonderung von Juden, Zigeunern und Geisteskranken 1933^1945, Reinbek, Rowohlt (1984) English translation: Murderous Science, Elimination of Jews, Gypsies and Others, Germany 1933^1945, Oxford, Oxford University Press (1988) Cold Spring Harbor, Cold Spring Harbor Laboratory Press (1998). [2] Kesting, M. (1959) Bertolt Brecht in Selbstzeugnissen und Bilddokumenten. Rowohlt, Reinbek. [3] Mu«ller-Hill, B. (1998) Warum wurden wir Mitglieder des SDS, 1946^1960? Zeitschrift fu«r Sozialgeschichte des 20. und 21. Jahrhunderts 13(2), 172^189. [4] Wallenfels, K. and Malhotra, O.P. (1961) Galactosidases. Advances Carbohydr. Chem. 16, 239^298, Footnote 78. [5] Mu«ller-Hill, B. and Wallenfels, K. (1964) Ist Acetyldehyd oder sein Hydrat Substrat der Alkoholdehydrogenase? Biochem. Z. 339, 349^351. [6] Jacob, F., Perrin, D., Sanchez, C. and Monod, J. (1960) L’ope¤ron: groupe de ge'nes a' expression coordine¤e par un ope¤rateur. Comptes Rendus Acad. Sci. 250, 1727^1729. [7] Chargaff, E. (1978) Heraclitean fire. Sketches from Life Before Nature. New York, The Rockefeller University Press. [8] Fleming, D. and Bailyn, B. (eds.) (1969) The Intellectual Migration. Europe and America, pp. 1930^1960. Cambridge Mass, Harvard University Press.


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[9] Mu«ller-Hill, B., Rickenberg, H.V. and Wallenfels, K. (1964) Specificity of induction of the enzymes of the lac operon in Escherichia coli. J. Mol. Biol. 10, 303^318. [10] Jacob, F. and Monod, J. (1961) On the regulation of gene activity. In: Cold Spring Harbor Symposia of Quant. Biol. 26, 193^211. [11] Mu«ller-Hill, B. (1966) Suppressible regulator constitutive mutants of the lactose system in Escherichia coli. J. Mol. Biol. 15, 374^376. [12] Stone, I.F. (1989) The Trial of Socrates. New York, Doubleday. [13] Gilbert,W. and Mu«ller-Hill, B. (1966) Isolation of the Lac Repressor. Proc. Natl. Acad. Sci. USA 56, 1891^1898. [14] Ptashne, M. (1967) Isolation of the phage repressor. Proc. Natl. Acad. Sci. USA 57, 306^313. [15] Gilbert,W. and Mu«ller-Hill, B. (1967) The Lac operator is DNA. Proc. Natl. Acad. Sci. USA 58, 2415^2421. [16] Mu«ller-Hill, B., Crapo, L.H. and Gilbert, W. (1968) Mutants that make more Lac Repressor. Proc. Natl. Acad. Sci. USA 59, 1259^1264. [17] Scharl, J. (1999) Monographie und Werkverzeichnis. (Firmenich, A. ed.) Ko«ln,Wienand-Verlag. [18] Pfahl, M. (1969) Escape synthesis of -galactosidase in strains lysogenic for lambda C1857h8ot68dlac. Mol. Gen. Genet. 105, 122^124. [19] Pfahl, M. (1972) Genetic map of the lactose repressor gene (I) of Escherichia coli. Genetics 72, 393^410. [20] Pfahl, M., Stockter, C. and Gronenborn, B. (1974) Genetic analysis of the active sites of Lac repressor. Genetics 76, 669^679. [21] Beyreuther, K., Adler, K., Geisler, N. and Klemm, A. (1973) The aminoacid sequence of Lac repressor. Proc. Natl. Acad. Sci. USA 70, 3576^3580. [22] Pace, H.C., Lu, P. and Lewis, M. (1990) lac repressor: crystallization of the intact tetramer and its complexes with inducer and operator DNA. Proc. Natl. Acad. Sci. USA 87, 1870^1873. [23] Lewis, M., Chang, G., Horton, N.C., Kercher, M.A., Pace, H.C., Schumacher, M.A., Brennan, R.G. and Lu, P. (1996) Crystal structure of the Escherichia coli lactose operon repressor and its complexes with DNA and inducer. Science 271, 1247^1254. [24] Adler, K., Beyreuther, K., Fanning, E., Geisler, N., Gronenborn, B., Klemm, A., Mu«ller-Hill, B., Pfahl, M. and Schmitz, A. (1972) How lac repressor binds to DNA. Nature 237, 322^327. [25] Boelens, R., Scheek, R.M., van Boom, J.H. and Kaptein, R. (1987) Complex of Lac repressor headpiece with a 14 base pair lac operator fragment studied by two-dimensional nuclear magnetic resonance. J. Mol. Biol. 240, 421^433. [26] Mu«ller-Hill, B. (1996) The lac operon. A Short History of a Genetic Paradigm. Berlin, de Gruyter.

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[27] Zubay, G., Morse, D.E., Schrenk, W.J. and Miller, J.H.M. (1972) Detection and isolation of the repressor protein for the tryptophan operon of Escherichia coli. Proc. Natl. Acad. Sci. USA 69, 1100^1106. [28] Mu«ller-Hill, B. and Kania, J. (1974) Lac repressor can be fused to -galactosidase. Nature 249, 561^563. [29] Brake, A.J., Fowler, A.V., Zabin, I., Kania, J. and Mu«ller-Hill, B. (1978) -galactosidase chimeras: primary structure of lac repressor--galactosidase protein. Proc. Natl. Acad. Sci. USA 75, 4824^4827. [30] Alsheimer, G.W. (i.e. Wulff, E.) (1968) Vietnamesische Lehrjahre. Sechs Jahre als deutscher Arzt in Vietnam, Suhrkamp, Frankfurt a. M. [31] Messing, J., Gronenborn, B., Mu«ller-Hill, B. and Hofschneider, P.H. (1977) Filamentous coliphage M13 as a cloning vehicle. Proc. Natl. Acad. Sci. USA 74, 3642^3646. [32] Kania J. and Mu«ller-Hill, B. (1977) Construction, isolation and implications of repressor--galactosidase hybrid molecules. Eur. J. Biochem. 79, 381^386. [33] Kania J. and Brown, D.T. (1976) The functional repressor parts of a tetrameric lac repressor--galactosidase chimera are organized as dimers. Proc. Natl. Acad. Sci. USA 73, 3529^3533. [34] Mu«ller-Hill, B., Hobson, A., Mieschendahl, M. and Triesch, I. (April 1977) Two operators control the lac operon: lac repressor binds to both. Proc. Natl. Acad. Sci. USA Accepted but retracted. [35] Oehler, S., Eismann, E.R., Kra«mer, H. and Mu«ller-Hill, B. (1990) The three operators of the lac operon cooperate in repression. EMBO J. 9, 973^979. [36] Bu«chel, D.E., Gronenborn, B. and Mu«ller-Hill, B. (1980) Sequence of the lactose permease gene. Nature 283, 541^545. [37] Kaback, H.R., Sabin-Toth, N. and Weinglass, A.B. (2001) The kamikaze approach to membrane transport. Nature Reviews Mol. Cell. Biol. 2, 610^621. [38] Wilcken-Bergmann, B.v. and Mu«ller-Hill, B. (1982) Sequence of galR gene indicates a common evolutionary origin of lac and gal repressor in Escherichia coli. Proc. Natl. Acad. Sci. USA 79, 2427^2431. [39] Lehming, N., Sartorius, J., Niemo«ller, M., Genenger, G., WilckenBergmann, B.v. and Mu«ller-Hill, B. (1987) The interaction of the recognition helix of lac repressor with lac operator. EMBO J. 6, 3145^3153. [40] Sartorius, J., Lehming, N., Kisters, B., Wilcken-Bergmann, B.v. and Mu«ller-Hill, B. (1989) lac repressor mutants with double or triple exchanges bind specifically to lac operator variants with multiple exchanges. EMBO J. 8, 1265^1270. [41] Lehming, N., Sartorius, J., Kisters-Woike, B.,Wilcken-Bergmann, B.v. and Mu«ller-Hill, B. (1990) Mutant lac repressors with new specificities hint at rules for protein-DNA recognition. EMBO J. 9, 615^621.


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[42] Sartorius, J., Lehming, N., Kisters-Woike, B.,Wilcken-Bergmann, B.v. and Mu«ller-Hill, B. (1991) The roles of residues 5 and 9 of the recognition helix of Lac repressor in lac operator binding. J. Mol. Biol. 218, 313^321. [43] Mu«ller-Hill, B. (1981) Bemerkungen zur Ethik der Genmanipulierer. VII Ku«hlungsborner Kolloquium. pp. 101^106. Genetic Engineering und der Mensch, Berlin-Ost. [44] Mu«ller-Hill, B. (1989) Nachts. Sinn und Form 41, 637^650. [45] Mu«ller-Hill, B. (1986) Autor unbekannt. Oder: Warum ich Deutschland verliess. Sinn und Form 37, 1317^1320. [46] Mu«ller-Hill, B. (1985) Kollege Mengele - Nicht Bruder Eichmann. Sinn und Form 37, 671^676. [47] Mu«ller-Hill, B. (1981) Die Philosophen und das Lebendige. Campus, Frankfurt a.M. Italian translation Garzanti (1984). [48] Bundesarchiv Koblenz R73 -15342 Akte von Verschuer. [49] Bundesarchiv Koblenz R73 -14005 Akte Robert Ritter. [50] Trevor-Roper, H. (1985) Seas of unreason. Nature 313, 407^408. [51] Weizenbaum, J. (1976) Computer power and human reason. In From Judgement to Calculation. (Freeman,W.H. ed.), San Francisco. [52] Kra«mer, H., Niemo«ller, M., Amouyal, M., Re¤vet, B.,Wilcken-Bergmann, B. and Mu«ller-Hill, B. (1987) Lac repressor forms loops with linear DNA carrying two suitably spaced lac operators. EMBO J. 6, 1481^1491. [53] Lehming, N., Sartorius, J., Oehler, S., Wilcken-Bergmann, B. and Mu«llerHill, B. (1988) Recognition helices of lac and lambda repressor are oriented in opposite directions and recognize similar DNA sequences. Proc. Natl. Acad. Sci. USA 85, 7947^7957. [54] Alberti, S., Oehler, S., Wilcken-Bergmann, B.v., Kra«mer, H. and Mu«llerHill, B. (1991) Dimer-to-tetramer assembly of Lac repressor involves a leucine heptad repeat. The New Biologist 3, 57^62. [55] Alberti, S., Oehler, S., Wilcken-Bergmann, B.v., Kra«mer, H. and Mu«llerHill, B. (1993) Genetic analysis of the leucine heptad repeats of Lac repressor: evidence for a 4 -helical bundle, EMBO J. 12, 3227^3236. [56] Mu«ller-Hill, B. (1998) The function of auxiliary operators. Mol. Microbiol. 29, 13^18. [57] Dro«ge, P. and Mu«ller-Hill, B. (2001) High local concentrations at promoters: strategies in prokaryotic and eukaryotic cells. Bioessays 23, 1^6. [58] Schlax, P.J., Capp, M.W. and Record, T.M. (1995) Inhibition of transcription initiation by lac repressor. J. Mol. Biol. 245, 331^350. [59] Fickert, R. and Mu«ller-Hill, B. (1992) How lac repressor finds lac operator in vitro. J. Mol. Biol. 226, 59^68. [60] Barker, A., Fickert, R., Oehler, S. and Mu«ller-Hill, B. (1998) Operator search by mutant Lac repressors. J. Mol. Biol. 278, 549^558. [61] Brenner, S. (1997) A night at the operon. Nature 386, 235.

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[62] Kang, J., Lemaire, H.-G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschick, K.H., Multhaupt, G., Beyreuther, K. and Mu«ller-Hill, B. (1987) The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell surface receptor. Nature 325, 733^736. [63] Lemaire, H.G., Salbaum, J.M., Multhaupt, G., Kang, J., Bayney, R.M., Unterbeck, A., Beyreuther, K. and Mu«ller-Hill, B. (1989) The PreA4695 precursor protein of Alzheimer’s disease A4 amyloid is encoded by 16 exons. Nucl. Acid Res. 17, 517^522. [64] Deichmann, U. (1996) Biologen unter Hitler. Vertreibung, Karrieren, Forschung. Campus, Frankfurt. a. M. (1992) Biologen unter HitlerPortra«t einer Wissenschaft in der NS-Zeit. Fischer, Frankfurt a.M. (1995) Englische u«bersetzung, Cambridge, Mass, Harvard University Press. [65] Deichmann, U. (2001) Flu«chten, mitmachen, vergessen. Chemiker und Biochemiker in der NS-Zeit.Wiley-VCH.Weinheim. [66] Deichmann,U. and Mu«ller-Hill, B. (1998) The fraud of Abderhalden’s enzymes. Nature 393, 109^111. [67] Roland, C., Friedlander, H. and Mu«ller-Hill, B. (eds.) (1992) Medical Science without compassion past and present. Stiftung fu«r Sozialgeschichte des 20. Hamburg, Jahrhunderts. [68] Schell, J. et al. (1999) Reevaluation of phytohormone-independent division of tobacco protoplast derived cells. Plant J. 17, 461^466. Abbott, A. (1998) German technician’s confession spurs check on suspected data. Nature 393(293). [69] Mu«ller-Hill, B. (1991) Funding of molecular biology. Nature 351, 11^12. [70] Herbertz, H. and Mu«ller-Hill, B. (1993) Qualita«t und Effiziens. Molekularbiologische Institute im internationalen Vergleich. Futura 8(3), 8^21. [71] Herbertz, H. and Mu«ller-Hill, B. (1995) Quality and efficiency of thirteen research institutes in molecular biology: an international comparison. Research Policy 24, 959^979. [72] Brunner, H.G., Nelen, M., Breakefield, O., Ropers, H.H. and Oost, B.A. van (1993) Abnormal behavior associated with a point mutation in the structural gene for monoamino oxydase. Science 262, 578^580. [73] Mu«ller-Hill, B. (30.3.1994) Humangenetik der Gewaltta«tigkeit, Frankfurter Allgemeine. [74] Mu«ller-Hill, B. (7.6.1994) Die Macht der Wo«rter. Zum Bo«sen veranlagt? Genetik und die deutsche Vergangenheit. Frankfurter Allgemeine. [75] Ropers, H.H. (14.4.1994) Mit genetischen Unterschieden verantwortungsvoll umgehen. Frankfurter Allgemeine. [76] Levy, D. (23.6.00) In the name of science. Haaretz. [77] Spott, S., Dong, F., Kisters-Woike and Mu«ller-Hill, B. (2000) Dimerisation mutants of Lac repressor. II: a single substitution, D278L, changes the specificity of dimerisation. J. Mol. Biol. 299, 805^812.


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[78] Pereg-Gerk, L., Leven, O., Mu«ller-Hill, B. (2000) Increasing the thermostability of Lac repressor by 40 C. J. Mol. Biol. 299, 805^812. [79] Senear, D.F., Laue,T.M., Ross, J.B.,Waxman, E., Eaton, S. and Rusinova, E. (1993) The primary self-assembly reaction of bacteriophage lambda CI repressor dimer is to octamer. Biochemistry 32, 6179^6198. [80] Rusinova, E., Ross, J.B.A., Laue, T.M., Sowers, L.C., Senear, D.F. (1997) Linkage between operator binding and dimer to octamer self-assembly of Bacteriophage CI repressor. Biochemistry 36, 12994^13003. [81] Re¤vet, B.,Wilcken-Bergmann,B.v., Bessert, H. Barker, A. and Mu«ller-Hill, B. (1999) Four dimers of repressor bound to two suitably spaced pairs of operators form octamers and DNA loops over large distances. Current Biol. 9, 151^154. [82] Bell, C.E. and Lewis, M. (2001) Crystal structure of the repressor C-terminal domain octamer. J. Mol. Biol. 314, 1127^1136. [83] Dodd, I.B., Perkins, A.J., Tsemitsidis, D. and Egan, J.B. (2001) Octamerisation of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Genes and Dev. 15, 3013^3022. [84] Koenen, M., Scherf, A., Mercereau,O., Langsley, G., Sibilli, L., Dubois, P., Pereira da Silva, L. and Mu«ller-Hill, B. (1984) Human antisera detect a Plasmodium falciparum genomic clone encoding a nonapeptide repeat. Nature 311, 382^384. [85] Cronin, C.A., Gluba, W. and Scrable, H. (2001) The lac operator-repressor system is functional in the mouse. Genes and Development 15, 1506^1517. [86] Staake, D., Walter, B., Wilcken-Bergmann, B.v. and Mu«ller-Hill, B. (1990) How Trp repressor binds to its operator. EMBO J. 9, 1963^1967. Corrigendum EMBO (1990) J. 9,3023. [87] Haran,T.T., Joachimiac, A. and Sigler, P. (1992) The DNA target of the Trp repressor. EMBO J. 11, 3021^3030. [88] Symposium in Berlin. Biomedical Sciences and Human Experimentation at the Kaiser Wilhelm Institutes - The Auschwitz Connection. Speeches given on the Occasion of the Opening. Max Planck Research Supplement Issue 3/2002. Press and Public Relation Department of The Max-PlanckSociety (2001). [89] Sirach, J. 25,7^11.



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Chapter 10

A Dark Side of Science in Difficult Times BENNO MU«LLER-HILL Institut fu«r Genetik der Universita«t zu Ko«1nWeyertal 121, D-50931 Ko«ln, Germany

Twenty five years ago I began to investigate the past of Human Genetics in Nazi Germany. When in 1984 I published my book ‘‘To«dliche Wissenschaft’’; [1] it was greeted with silence in Germany. The Max-Planck-Gesellschaft (MPG) and the Deutsche Forschungsgemeinschaft (DFG) pretended that this was not part of their past. This is changing slowly. The President of the MPG, Hubert Markl, founded a committee which is to investigate the past of the MPG. Furthermore in June 2001 he invited some of the twins who had been the objects of Mengele’s research in Auschwitz, to apologize for the half of a century of silence of the MPG. The two articles that follow have been written within the last three years. They are about the center of evil in Genetics. I wished I could say an end is in sight. [1] The book has been translated into English: Murderous Science. Elimination by Scientific Selection of Jews, Gypsies and Others in Germany, 1933^1945, Oxford University Press 1988 and Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1998.

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Hist. Phil. Life Sci., 21 (1999), 331^365

The Blood from Auschwitz and the Silence of the Scholars* BENNO MU«LLER-HILL

Institut fu«r Genetik der Universita«t zu Ko«lnWeyertal 121, D-50931 Ko«ln, Germany

Abstract The Kaiser Wilhelm Institute for Anthropology, Human Genetics and Eugenics in Berlin-Dahlem was the centre of scientific racism in Nazi Germany. Its bad history culminated in a research project to analyse the molecular basis of racial differences in the susceptibility to various infectious diseases such as tuberculosis. Josef Mengele, a former postdoc of the director of the institute, Otmar von Verschuer, collected blood samples and other material in Auschwitz from families and twins of Jews and Gypsies. The blood samples were analysed by Gu«nther Hillmann in the Berlin laboratoryof Nobel Prize winnerAdolf Butenandt. Butenandt had just moved to Tu«bingen. The project was paid for by the Deutsche Forschungsgemeinschaft. Butenandt, Hillmann and von Verschuer made scientific careers in the Federal Republic. To the present day this past has not been acknowledged by the Max-Planck-Gesellschaft as part of its history. *A German version of this article has appeared in ‘Geschichte der KWG im Nationalsozialismus’, ed. by D. Kaufmann, Wallstein Verlag, Go«ttingen, Vol. 1, 189^227, 2000. Reprinted with permission from The History and Philosophy of the Life Sciences, Vol. 21. 0308-7298/90 $ 3.00 1999 Taylor and Francis Ltd

A DARK SIDE OF SCIENCE IN DIFFICULT TIMES

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To do science is one of the great pleasures of our time. If you hit upon something truly new and important, its beauty and truth can be overwhelming. If you predict correctly, other scientists may quickly extend your experiments and demonstrate that what you said and wrote was, and is true. In fields like medical genetics a discovery may be applied to help many patients. But science has a dark side too. A dark period was the time of the Nazi government in Germany, 1933 to 1945. A particularly dark field then was human genetics (eugenics, race hygiene, race biology, psychiatric genetics). Genetics was used to define genetically handicapped, different and inferior Germans. Geneticists apparently demonstrated the genetic difference and inferiority of Jews, Gypsies, Blacks and others. They supported the claim of politicians that total or final solutions had to be found and carried out for these different people. The directors, sub-directors and post-docs of the Kaiser Wilhelm Institute of Anthropology, Human Heredity and Eugenics (KWI for Anthropology) were deeply involved in giving scientific advice to the government and thus lent authority to its claims and operations. This merits detailed analysis. I will concentrate on just one enterprise which appeared at the end point of this process: the research project that the second director of the KWI for Anthropology, Professor Dr Otmar Freiherr von Verschuer, pursued in 1943^1944 with his post-doc Dr Dr (MD, PhD) Josef Mengele in the concentration camp Auschwitz (1). All persons and institutions that were actively or passively involved in these crimes have subsequently tried their best to discourage investigation. They are all dead by now. The picture which can be put together from bits and pieces of surviving documents and by witnesses is indeed most frightening. It explains and justifies the profound doubts of segments of the German and international public about the morals of today’s genetic research.

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Von Verschuer, Excellent Scientist and Outspoken Anti-semite In the thirties von Verschuer was highly respected internationally for his scientific work. I mention as evidence that the Rockefeller Foundation funded his research on twins in the KWI for Anthropology in Berlin-Dahlem from 1932 to 1935 (2). In 1939 von Verschuer, a speaker at the International Congress of Genetics in Edinburgh, was invited to give a talk on twin research before the Royal Society. His paper appeared after the war had begun in the Proceedings of the Royal Society (3). Von Verschuer was an excellent scientist. He was an aristocrat and a protestant who went to church every Sunday. He joined the Nazi party only in 1940. But right from the beginning he accepted the value system of the Nazis which was based upon biological, racial, i.e. genetic differences among individuals and races. In particular, he had no problem accepting the violent anti-Semitism of the Nazis. He had been an anti-Semite all his life. Still a student, in 1922, he published an article on ‘Genetics and race science as the basis of vo«lkische politics’: ‘The first and most important task of our internal politics is the population problem, i.e. the care for the future of the GermanVolk, the care of keeping our race. This is a biological problem which can only be solved by biological-political measures’ (4). In 1936, as a newly hired professor of the University of Frankfurt, he gave a talk, at the university, on ‘Race Hygiene as Science and Task of the State’ in which he praised Hitler and his racial politics (5). He said: ‘Men make history. It is a man who has shaped the immense history of our present time, and ^ so we pray ^ will continue to shape it for many years.That he came is grace.The miracle of national socialist renewal which he performed is a masterpiece of education. It was possible due to the richness of the valuable genes (Anlagen) of the German people. This educational work is solidly founded on the youth organisations, on the SA and the SS, on theArbeitsdienst and on the militaryservice.The state of Adolf Hitler has, for the first time, brought into action hereditary


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and racial administrative care. It is a state which like no other state has concentrated on the education of the people’. In May 1937 vonVerschuer participated in the second workshop of the Reichsinstitut fu«r Geschichte des neuen Deutschland. His talk, ‘What can the historian, the genealogist and the statistician contribute to the research of the biological problem of the Jewish question?’ was published in Volume II of the series Forschungen zur Judenfrage. There he wrote (6): ‘The race politics of our state has as its goal the conservation of the gene pool of our people (Volk) as the biological basis of German Volkstum and German culture.This positive attitude with regard to theVolkstum cannot be touched by any considerations of superiority or inferiority of a race which is foreign to ours.We therefore say no to a foreign race mixing with Jews just as we say no to mixing with Negroes and Gypsies, but also Mongolians and people from the South Sea. Our vo«lkisch attitude to the biological problem of the Jewish question (Judenfrage) is therefore completely independent of all knowledge of advantages or disadvantages, positive or negative qualities of the Jews. The results of such research cannot change our fundamental position. Our position in the race question has its foundation in genetics.’ One year later he participated again in a conference organised by the same institute on the same topic. This time he talked about ‘The race biology of Jews’ (7). His list of differences between Ashkenazim Jews and Caucasian non-Jews in the frequency of certain mutant genes leading to genetic diseases (Gaucher, Niemann-Pick) or leading to some forms of cancer is reasonable and has been confirmed by molecular analysis. His claim for differences in mutant genes determining typical rational Jewish behaviour may be seen as reasonable in part. However it is not presented as a hypothesis but as a fact. Moreover it is so amalgamated with the Nazi value system that it can only be accepted fully by anti-Semites.Verschuer notes, in a footnote, that in 1937, at the International Congress of Population Science in Paris, several Jewish scientists claimed, indeed without any evidence, that all human behaviour (intelligence

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etc.) is due exclusively to nurture, i.e. to education, a notion which is alive to the present day as a response to Nazism. It is informative to quote the last sentences of Verschuer’s article (7): ‘The Jew is a specific type of the city man (Stadtmensch), i.e. a man who has no more inner connections to the natural basis of life, who does not live any more out of the instinct of the subconscious, but who believes and takes for his world only what he can understand with his reason...’. This sounds positive to our modern scientific minds but not to von Verschuer. He continues: ‘The danger which Jewry meant for the body of the German people (Volk) was (sic!) twofolds: 1. Through racial foreignness, the maintenance of the essence of our people (Volk) was directly endangered. The complete racial separation between Germans and Jews was therefore an absolute necessity. 2. The spiritual Jewish influence (U«berfremdung) was about to introduce a form of life and selection principles which were positive for- the maintenance of the Jews, but which would have meant ruin for our people (Volk). The racial separation between Germans and Jews had the vo«lkische separation as a necessary starting condition. Von Verschuer does not spell out here in detail the legal consequences of this vo«lkische separation. Step by step the Jews lost all rights. This was indeed an essential part of Nazism: German people with different, ‘inferior’ genetic backgrounds had first fewer and then no rights, irrespective of whether they were full German citizens in 1933. The true Germans had full rights, others, the Jews, very restricted rights or no rights at all. Jews were not allowed to study or teach at German universities. They were not allowed to be active as lawyers or medical doctors. Their synagogues could be burnt. Their property could be taken away. They had no right to live on this planet. Von Verschuer, who accepted all this as a pious protestant Christian and pure scientist, became a grandiose hypocrite.


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In 1941 von Verschuer came back to this theme in his textbook, ‘Primer in race hygiene’ (Leitfaden der Rassenhygiene) (8). There he lists on six pages the racial genetic differences between Jews and Germans and the various forms of separation which were, at the time, imposed on Jews and Gypsies. The Gypsies were indeed in a similar situation to the Jews, but since they were at the bottom of society they were seen, compared to the Jews, to be much less of a problem. Verschuer writes about the Jews: The historical attempts to solve the Jewish question can be grouped chronologically into three periods: 1. Absorption of the Jews, as has been attempted by the western Goths in Spain. 2. Exclusion of the Jews in the Ghetto, the kind of solution attempted in Europe from the fifth to the nineteenth century. 3. Emancipation of Jewry which became widespread in the nineteenth century. All these efforts must be regarded as failures. The political demand of our time is a new total solution (Gesamtlo«sung) of the Jewish problem. This he wrote at the time when the expulsion of all European Jews from Europe was being publicly announced. Half a year later, in January 1942, Verschuer published an editorial in his journal Der Erbarzt. There he wrote: ‘. . . never before in history has the political relevance of the Jewish question become as clear as now. Europe and East Asia, led byJapan, fight against the Jewish dominated English-American-Russian world power.The people who are united with us recognise more and more that the Jewish question is a race question and that it must find a solution as it has been started by us, first in Germany. In the meantime many. other countries like Italy, France, Hungary and Romania have enacted racial laws. This demonstrates that the Jewish question is already a European affair. The final (endgu«ltige) solution is one of the world questions which will be decided in this war (9).

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Von Verschuer published a second, expanded edition of his textbook in 1944 (8). The section on Race Biology shows only minor changes. The sentences I quoted above remain unchanged. In 1944 he published a talk that he gave for the Friends of the German Academy (Freunde der deutschen Akademie) in Berlin (10). There he stated ‘The present war is also called a war of races, when one considers the fight with the Weltjudentum’, then he repeated the key sentence: ‘The political demand of our time is the new total solution of the Jewish problem (Gesamtlo«sung des Judenproblems)’. By then Verschuer must have heard of the actions of the Einsatzgruppen, and his collaborator Mengele may already have told him about his participation in this total solution.

Von Verschuer as a Practising Anti-Semite and Racist One might argue that these anti-Semitic texts were just fashionable at the time, but were without any real importance. This disregards completely the importance of science and the status of institutions like the Kaiser Wilhelm-Gesellschaft and the German universities in the Third Reich. If the best German scientists legitimised the anti-Semitic measures (denial of the rights to study or to teach, to practice as a doctor or a lawyer, to marry a non-Jew) by stating that they were scientific necessities, then they must have been so. Von Verschuer did not only produce propaganda. I present now material about von Verschuer which indicates that his writing had real importance for the lives of particular persons: 1. In 1937 vonVerschuer’s Frankfurt institute was the place where several hundred coloured German children were analysed to determine whether they were indeed the illegitimate children of coloured members of the French occupation force, which occupied parts of Germany after WW I. When this was confirmed by the experts, they were ^ illegally ^ sterilised.


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2. In 1937 von Verschuer was asked, as an expert on paternity, to give his expert opinion on the following case (11). A young man was accused of having offended against the Nuremberg laws by having a love affair with a non-Jewish German woman. He had a Jewish father. He claimed that his Jewish mother had told him already, when he was a child, that during WWI she had had an affair with a non-Jewish German officer, and he was the son of this officer. All the neighbours knew it. So he was only a half-Jew and not to be accused.The officer was dead, and only a very few photographs existed. Von Verschuer was asked to give his expert opinion on the paternity. Von Verschuer asked his Assistent Mengele to do the job. Mengele did so and came to the conclusion that the legal father was the biological father. But the court believed the young man, his mother and his neighbours and set him free. Von Verschuer could have accepted this defeat. But he did not. He wrote an angry letter to the secretary of justice (Justizminister) complaining bitterly that science had not been heard in this case and outlining the disastrous consequences for the German race if such non-scientific arguments were to become general practice (11). The worse the situation became for the Jews in Germany, the more of them claimed that their legal father was not their biological father, and that their biological father was a non-Jew. These cases had to be decided as paternity cases by a human geneticist. Von Verschuer was one of these experts. Did he follow strictly scientific principles? Did he try to save some Jews against scientific evidence? According to his own testimony, the Frankfurt institute produced 448 paternity expert opinions between 1935 and 1941 (12). His former technician, Irmgard Haase, told me that he never cheated to help a person (1). It would be revealing to analyse the data. When I was writing my book I was told by Widukind Lenz, the successor of von Verschuer in Mu«nster, that all the RasseGutachten had disappeared (see below!).

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3. In 1941 Dr Hans Grebe, an Assistent of von Verschuer in his institute at Frankfurt University, was asked to analyse a Gypsy woman (13). A German soldier wanted to marry her, so she needed a document stating that she was fit to marry (Ehetauglichkeitszeugnis). Grebe stated that she was not fit because, as a mixed Gypsy, she was feeble minded (schwachsinnig). So the Genetic Health Court in Frankfurt (Erbgesundheitsgericht) had to decide whether she had to be sterilised. The court decided that she was not mentally retarded. Von Verschuer appealed. The higher court decided again that she was not mentally retarded and thus not to be sterilised. Von Verschuer knew that a law was in preparation which would demand the sterilisation of all mixed Gypsies. This law had been stopped by the Department of Justice. So the Department of the Interior, which pushed the law through, relied in the meantime upon the trick of calling all mixed Gypsies mentally retarded. Von Verschuer alerted the Department of the Interior to the scandalous behaviour of the Frankfurt court. Dr Herbert Linden from the Department of the Interior indeed wrote a letter asking for the sterilisation of all mixed Gypsies on the grounds of a special type of mental retardation, sometimes difficult to diagnose. Von Verschuer sent a copy of this letter to the Frankfurt court and so asked a third time to sterilise the woman. Now the Frankfurt psychiatrist von Kleist was asked to state mental retardation. He could not find mental retardation. So the court came, in its third and final decision, to the conclusion not to sterilise the Gypsy woman (13).

Von Verschuer, Director, and Mengele, Former Post-doc, Now Guest and Collaborator, in the Berlin Kaiser Wilhelm Institute for Anthropology In October 1942 von Verschuer became director of the Kaiser Wilhelm Institute of Anthropology in Berlin-Dahlem, and so


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successor of his teacher Eugen Fischer. Among the scientists who came with him from Frankfurt to Berlin was DrAyres deAzevedo, a Portuguese post-doc. Azevedo had come to vanVerschuer’s lab in Frankfurt in July 1941. He stayed with von Verschuer in Berlin until August 1943. He analysed the sera of twins for differences in their blood groups. The bottom line of his research was that identical twins had identical blood groups (14). In other words, blood group analysis was the best possible technique to determine whether twins were identical or not. Azevedo published his findings in the July/August 1944 issue of Der Erbarzt. He did not indicate where the twins came from. It makes sense to assume that some if not all of them were Gypsies. Gypsies had been analysed before by various members of vonVerschuer’s and Fischer’s institutes. Almost all German Gypsies were transferred to Auschwitz in the spring of 1943. In January 1943 Verschuer’s former post-doc from Frankfurt University, Josef Mengele, came to his Berlin institute as a guest. As an SS-officer, he was still formally Assistent at Frankfurt University where he had worked with von Verschuer. So his salary was paid by Frankfurt University. As von Verschuer wrote to his friend de Rudder, a paediatrician at Frankfurt University, he planned to give Mengele a post-doc position in his new institute later, after the war (15). Mengele had been successful as a researcher. Between 1937 and 1940 he published three research papers, a meeting report and several book reviews (16). He had joined the SS in 1938. He was drafted in 1940 and in 1941 was sent to the Russian front. In June 1942 he was wounded in the region of Stalingrad, but was lucky enough to be flown out. In January 1943 he was sent to Berlin to do office work and to recover. There he joined, as a guest scholar, the KWI for Anthropology, where his teacher, von Verschuer had just become director. So Mengele’s name appears on the birthday list of the Berlin Institute (Fig. 1). During these months in Berlin, Mengele was offered. the position of camp doctor in the concentration camp of Auschwitz, as an alternative to going back to the front. It is reasonable to

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Fig. 1. Birthday list. Professors, post-docs, and guests of the institute and the employees of the entire institute and of the department of human genetics. Nachlass vonVerschuer, Universita«tsarchiv Mu«nster.

assume that he discussed this opportunity with von Verschuer. Both must have realised that Mengele would become involved there, in one way or another, in what was officially termed either the ‘total’ or the ‘final solution of the Jewish. question’ (die Gesamtlo«sung oder Endlo«sung der Judenfrage). Reading the articles and the text book of von Verschuer, Mengele must have come to the conclusion that his teacher was all for it. In fact Dr Hans Mu«nch, the only SS-MD in Auschwitz who had not participated in selections, said, after the war, that Mengele, when they had dinner together in Auschwitz, argued exactly as von Verschuer had in writing, about the scientific necessity


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of the gassings in Auschwitz. They were part of the total solution of the Jewish problem, for which there was no other solution (17). And what opportunities and possibilities there were for anthropological, genetic research in Auschwitz! On May 30, 1943 Mengele started to work as camp doctor in Auschwitz.

The Documents of the Auschwitz Research Project So what was the research project of Mengele and von Verschuer? The research project has to be reconstructed from the bits and pieces of documents which describe it from various angles: 1. The grant proposals which would be most revealing have disappeared. Von Verschuer must have destroyed his copies. The copies which he sent to the Forschungsrat/Deutsche Forschungsgemeinschaft have disappeared along with all other proposals from the period. However, the interim reports von Verschuer sent to the Deutsche Forschungsgemeinschaft in September 1943 and in March and October 1944 have survived the war there (18). 2. There are letters of von Verschuer to his friend Professor Bernhard de Rudder, a paediatrician in Frankfurt, in which he outlines the rationale of the experiments (15). De Rudder was an expert; he had published on race and infectious disease (19). 3. There exists a book and a confession by the HungarianJewish pathologist Dr Miklos Nyiszli on his work as a slave assistant of Mengele in Auschwitz between June 1944 and January 1945 (20). 4. Finally there are other witness reports on the work of Mengele in Auschwitz. These various sources allow a general

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reconstruction of the research project. The goals of the research project of Mengele and von Verschuer were: 1. to decipher genetic differences among Jews, Gypsies and others in resistance to various infectious diseases, in particular to tuberculosis and typhoid/typhus (In non-scientific, colloquial German there is a confusion between Flecktyphus ¼ typhus, which was prevalent in Auschwitz and Typhus ¼ typhoid). Every experiment could be done. Every person could be killed since all Jewish and Gypsy prisoners had already been condemned to death. And 2. to assemble as much material as possible from genetically affected twins or families.

Mengele in Auschwitz I begin by citing how three witnesses saw the activities of Mengele in Auschwitz. Nyiszli, Mengele’s slave assistant, testified before a Jewish committee half a year after his liberation (20). His statement is the most direct description of the horror: I arrived on May 22, 1944 from the ghetto of Aknaszlatina together with 26 colleagues to Auschwitz. We were all medical doctors in public service (Kreisa«rzte). After the selection, I was on the right side. They sent me to Buna, where 14,000 prisoners were assembled. I had completed about 12 days construction work, pouring concrete, when the head physician of Buna summoned all other physicians. All 50 assembled. He told us that all those who knew about pathology could get easier work. Of the 50 doctors, 2 applied. I, because I felt that I would die sooner or later through the construction work. After a detailed oral examination, we were accepted. I had studied medicine in Germany and had worked as a pathologist for some time. I was accepted without difficulty, as was my colleague, who has worked at the University of Strasbourg.... Dr Mengele came after several hours and examined us for about 1 hour.


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He immediately gave us some work. We were to examine some people who were selected because of certain bodily anomalies. We examined them, then they were shot by Oberscharfu«brer Muszfeld (i.e. Erich Muhsfeldt, BMH) with a small bore rifle. We were then asked to dissect there and to prepare a meticulous protocol. We finally treated the corpses with hypochlorite and sent the clean bones to the Berlin Dahlem Anthropological Institute. Such cases happened sporadically, until we were awakened one night by SS personnel. We went into the section room where Dr Mengele waited for us. In the room next to the section room were 14 crying Gypsy twins. Dr Mengele prepared silently one 10 -mL and a few 5 -mL ampoules. He put a box of Evipan ampoules together with 20 -mL chloroform ampoules on the section desk. Then he brought the first twin, a 14 -year-old girl into the section room. Dr Mengele ordered me to undress her and to put her on the section table. Then he injected Evipan into her right arm. After the child had fallen asleep, he felt for her left ventricle and injected 20 mL of chloroform. The child was instantaneously dead and was brought into the mortuary. This way the 14 twins were killed. Mengele asked us how many corpses we could dissect per day; he thought we could do about seven or eight. We said that if he asked for precise work from us, we could dissect four. He agreed.

Nyiszli wrote a book about his time in Auschwitz. It was published in Hungary in 1946. Translated into English it became available to a greater public only in 1960 (20). I quote: I finished dissecting the three pairs of twins and duly recorded the anomalies found. In all three instances the cause of death was the same: an injection of chloroform into the heart. Of the four sets of twins, three had ocular globes of different colors. One eye was brown, the other blue.This is a phenomenon found fairly frequently in non-twins. But in the present case I noticed it has (sic!) occurred

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in six of the eight twins. An extremely interesting collection of anomalies. . .. During the afternoon, Dr Mengele paid me a visit. . . . He gave me instructions to have the organs mailed and told me to include my report in the package. He also instructed me to fill out the ‘Cause of Death’column hitherto left blank.The choice of causes was left to my own judgement: the only stipulation was that each cause should be different. . . . I shuddered to think of all I had learned during my short stay here, and of all I should yet have to witness without protesting, until my own appointed hour arrived. The minute I entered this place I had the feeling I was already one of the living-dead. But now, in possession of all these fantastic secrets, I was certain I would never get out alive. Was it conceivable that Dr Mengele, or the Berlin-Dahlem Institute, would ever allow me to leave this place alive? I had to keep any organs of possible scientific interest, so that Dr Mengele could examine them. Those which might interest the Anthropological Institute in Berlin-Dahlem were preserved in alcohol. These parts were specially packed to be sent through the mail. Stamped ‘War Material ^ Urgent’, they were given top priority in transit. In the course of my work at the crematorium I dispatched an impressive number of such packages. I received, in reply, either precise scientific observations or instructions. In order to classify this correspondence I had to set up special files. The directors of the Berlin-Dahlem Institute always warmly thanked Dr Mengele for this rare and precious material.

When Dr Nyiszli wrote about the ‘Directors of the BerlinDahlem Institute’, he probably meant the Director of the KWI of Anthropology, Professor von Verschuer, and some department or group heads. Nyiszli began his book in 1946 with a declaration which -ended ‘. . .it is most likely that the protocols will be found in the archive of this institute’ (20). We will see however that they were in fact destroyed. The description by Nyiszli may be complemented by the following description by Dr Ella Lingers-Reiner, a non-Jewish


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MD from Vienna who had helped Jews and was therefore incarcerated (21): The most nauseating type of S.S. to me personally were the cynics who no longer genuinely believed in their cause, but went on collecting blood guilt for its own sake.Those cynics were not always brutal to the prisoners, their behaviour changed with their mood. They took nothing seriously ^ neither themselves nor their cause, neither. us nor our situation. One of the worst among them was Dr Mengele, the Camp Doctor I have mentioned before. When a batch of newly arrived Jews was being classified into those fit for work and those fit for death, he would whistle a melody and rhythmically jerk his thumb over his right or his left shoulder ^ which meant ‘gas’ or ‘work’. He thought conditions in the camp were rotten, and even did a few things to improve them, but at the same time he committed murder callously, without any qualms. His interest was centred on his anthropological research on Jewish twins. He would always single out and keep alive such pairs of twins when he was on duty at selections for the gas chamber. He probably also knew that we cheated him and presented brothers or sisters with a strong family likeness as ‘dissimilar twins’. In the summer of 1944 he often looked regretfully at the scientific material he had collected with the help of a large staff of prisoner-collaborators, and said with a laugh: ‘What a pity it will fall into the hands of the Bolsheviks!

Perhaps the earliest description of Mengele as a camp doctor in Auschwitz available to the general public was given in 1946 by Weinreich in his book ‘Hitler’s professors’ (22): Dr Joseph Mengele, assistant at the Institute of Hereditary Biology and Race Research founded in 1934 by Professor Dr Otmar von Verschuer (see p. 27), was known to be particularly severe and sometimes used to send whole transports to the gas chambers immediately on their arrival. But he was a scholar in his field and even while seeing Fu«hrer and Reich he did not forget his studies on

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twins. Prisoners who worked at the reception tracks sometimes managed to whisper to the new arrivals about Dr Mengele’s racialbiological research. In no time children who somewhat resembled each other were brought together and taught new names, and then they introduced themselves to the Germans with the formula: ‘We are twins’. Usually they were put aside for Dr Mengele’s studies and some of them have survived. How many real twins there were among the alleged two hundred children whom Mengele selected nobody knows. ‘At any rate’, Dr Friedman grimly observes, ‘racial science in this case did not build upon very firm foundations.

Finally a book must be noted which contains reminiscences of surviving twins (23). This is Mengele’s side of the story, and now to von Verschuer’s side.

Von Verschuer’s research Activities in the Records of the Deutsche Forschungsgemeinschaft On October 1, 1942, von Verschuer began his post as director of the Kaiser Wilhelm Institute of Anthropology, Human Heredity and Eugenics in Berlin-Dahlem. Under his predecessor, Eugen Fischer, the institute had received 40,000 RM/year (today about 200,000 US dollars/year) from the DFG. On March 23, 1943, von Verschuer sent an application for exactly this sum to the DFG (18). This was supposed to pay for the research of the already existing groups of the institute. Two months later, on May 24, 1943, this was granted. (Please note how fast the DFG operated then!) Every half year a report had to be written. Thus von Verschuer sent reports to the DFG on the progress of the projects on September 27, 1943; on March 20, 1944; and on October 4, 1944. The reports still exist (18). According to the file cards, an additional project was commissioned on August 18, 1943. It was called ‘Specific proteins’ (Spezifische Eiweiko«rper). A further research project was commissioned on September 7, 1943. It was entitled ‘Eye colour’


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(Augenfarbe). The interim reports were sent on March 20 and on October 4, 1944.The applications have disappeared. Extra money (RM 10,000/year) was requested for the research project of highest priority,‘Tuberculosis’ by vonVerschuer on February 25,1944.This was granted on April 6, 1944.Two of the projects were handled by researchers who were already working at the KWI under Fischer: the project ‘Eye colour’ was handled by Dr Karin Magnussen, a biologist, and the project ‘Tuberculosis’ by Dr Karl Diehl. The project ‘Specific proteins’ was run by vonVerschuer. Mengele needed some basic equipment for his anthropological and genetic research. Nyiszli describes having had three microscopes in his laboratory in Auschwitz (20). It is reasonable to assume that they were paid for by the Deutsche Forschungsgemeinschaft. However the relevant documents are missing.

Project I: Genetic Defects in Families and Twins On December 16, 1942, two weeks before Mengele turned up at the Berlin-Dahlem Institute, Himmler had given the general order that all Gypsies, with the exception of a very few racially pure families, ought to be transferred to Auschwitz. A detailed order was given on January 29, 1943. Before, most German Gypsies had been held in various camps. The Gypsies had been an object of study for students and post-docs of vonVerschuer and Fischer. In particular Dr Georg Wagner, a graduate student working in the Dahlern Institute under Fischer, had written his dissertation on them (24). He had published a paper on eye anomalies of some Gypsies in 1944 (25). Dr Karin Magnussen, a biologist and collaborator of Fischer, had specialised in eye defects. She was particularly interested in heterochromatic eyes. Wagner had briefly mentioned the prevalence of this trait among German Gypsies (24), so it made sense to continue the work after all Gypsies had been concentrated in Auschwitz. Magnussen mentions in her paper (26) that she also received eyes from the Berlin anatomist Prof. Dr Hermann Stieve: Stieve had access to the corpses of all

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persons executed in Berlin (27). Nyiszli describes the murder of the twins with eye anomalies (20). In her DFG report of October 1944, Magnussen mentions that she has a paper in print in the Zeitschrift fu«r induktive Abstammungs- undVererbungslehre on heterochromatic eyes. The article never appeared. The publishing house was destroyed in the war. Another post-doc at the institute, Dr Hans Grebe, was interested in inherited malformations. One may assume that the skeletons of the two Jews who, according to Nyiszli, were shot by Muhsfeldt, were sent to him in June 1944. Grebe received a professorship at the University of Rostock in the autumn of 1944. Scientists are known to be curious. Did von Verschuer ever ask Mengele what went on in Auschwitz? Did he ever ask how Mengele came to send the eyes of an entire Gypsy family to his institute? The answer is apparently no. It is the absence of curiosity about the fate of the twins which is so disturbing. The connection between Verschuer’s Dahlem Institute and Auschwitz was tight. Mengele came for several visits to the Dahlem Institute. A post-doc of the institute, Dr Siegfried Liebau, who was also a member of the SS, testified, as a witness at the Auschwitz trial in Frankfurt, that he went at least once to Auschwitz to collect Gypsy material that Mengele had prepared (27). The judge did not ask for any further details.

Project II: Genetic Differences among Jews, Gypsies and Others in Resistance to Infectious Diseases On 16 November 1944 von Verschuer gave an invited talk at the Academy of Sciences in Berlin on ‘The effect of genes and parasites in the human body’. The abstract of his talk, but not the talk itself, was deposited at the Archive of the Academy of Sciences (28). The abstract concludes as follows: ‘The task of future research involves analysis of all those infectious diseases where parts of the defence mechanism are genetically determined. Individuals with a particular defect in this defence


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mechanism could be supplied with a factor identified in such experiments’. Thus he envisaged molecular analysis and therapy based upon the results of this analysis. Truly a high goal. Von Verschuer published in 1948 what he claimed to be his 1944 talk before the Academy. There he lists in his references Abderhalden’s book on defence enzymes (see below) ^ although he never mentions them in the text (28). We know of two infectious diseases which were analysed by Mengele: tuberculosis and typhus. Von Verschuer mentions in his textbook that (Ashkenasy) Jews are less susceptible to tuberculosis than Caucasian non-Jews. This may sound like complete nonsense to the layman, yet it is reasonable. I quote here just the most recent study on extreme susceptibility of .West Africans to tuberculosis resulting from a mutant NRAMP1 gene (29). Von Verschuer knew the recent work of his Berlin KWG colleagues, Nobel prize winner Prof. Dr Adolf Butenandt and Prof. Dr Alfred Ku«hn. They had suggested that genes synthesise enzymes which convert tryptophan to an eye pigment in the fly Ephestia. The addition of an intermediate repairs the defect and thus leads to correctly coloured eyes in mutant flies. Von Verschuer had learned from Prof. Dr Emil Abderhalden, president of the oldest German Academy of Science, the Leopoldina, that defence enzymes, specific proteases, were produced against infectious agents (see below). So he assumed that the type or amount of defence enzymes varied among persons of different races in certain cases. Abderhalden may have informed von Verschuer that his son, Rudolf Abderhalden, who worked in his father’s lab in Halle, had begun to analyse the defence enzymes of human tuberculosis (30). Von Verschuer had already analysed tuberculosis in monozygotic and dizygotic twins together with Karl Diehl in 1933. He had found significant differences in the severity of tuberculosis in dizygotic twins but not in monozygous twins. Diehl had developed an animal model, the rabbit. He had crossed a rabbit strain, which was more susceptible to tuberculosis than another strain. Diehl and von Verschuer had published a book and

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several papers together (31). Now the test for Abderhalden’s defence enzymes apparently offered a molecular analysis and, as von Verschuer hoped, the isolation of a defence enzyme and thus molecular treatment of tuberculosis. In his DFG report of September 27, 1943 he states: ‘Our research on tuberculous twins is being continued. Further material is being collected . . .’. Nyiszli indeed mentions that Mengele worked on tuberculous twins. Weinreich mentions in his book (22) a similar project: ‘Another Oswiecim scholar, unfortunately not mentioned by name, was tempted to investigate the extent of biological difference between Eastern and Western European Jews; ‘racial science’ at times hinted at differences among the Jews themselves (Sephardim and Ashkenazim). Therefore two groups were given typhus injections. The Westerners really turned out to be much less resistant than the control group of Eastern-European Jews and nearly all of them died within ten days’. It is reasonable to assume that this study was performed by Mengele too. There is a witness, Dr Jan Cespiva, who stated, as a witness for the Frankfurter Auschwitz Prozess, that typhus (32) experiments were done by Mengele- with Gypsy twins: ‘During the time I worked in the Gypsy camp, I often encountered Dr Mengele and was able to observe what he was doing. . . . I was able to see with my own eyes how he infected twins with typhus (Typhus) in the sickroom of the Gypsy camp so as to observe whether the twins reacted in the same way or differently. A short time after being infected, they were sent to the gas chambers...’ I will now document the progress of this project by quoting from von Verschuer’s reports to the DFG (18) and letters to his friend de Rudder (15) and from IG Farben director Mann. *August 18, 1943: The DFG informs von Verschuer that his two projects ‘specific proteins’and ‘tuberculosis’ will be funded. If we assume that the DFG worked with its usual efficiency, then von Verschuer must have sent them his proposal in the first days of June 1943, i.e. a few days after Mengele left his institute in Berlin and began his work in Auschwitz.


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*September 27, 1943, first interim report to the DFG on the project ‘specific proteins’. ‘Now after all material necessary for performing this research has been obtained, we have begun with the first trial tests.We tested the method after consultation with Gebeimrat Abderhalden, Halle. This work has been interrupted due to the partial move of our institute to Beetz, but now the laboratories are ready and work can begin’. (On Professor Emil Abderhalden and his method see below). *November 19, 1943. Letter of Wilhelm R. Mann, member of the Direktorium der IG Farbenindustrie, from his Berlin address to von Verschuer: ‘Dear professor, I thank you very much for giving me the opportunity to have met your colleague, Dr Mengele. I was most impressed by his second talk. You can be assured that I, as I told you already, will discuss in the IG Farben the financial support. (I include a first cheque). The experiments of Dr Mengele should be pushed in any case, there we are of the same opinion. Best regards, Heil Hitler, signature’ (33). *March 20, 1944, second interim report to the DFG, on the sub-project ‘specific proteins’: ‘New difficulties arose in establishing the method. They have been solved together with Geheimrat Abderhalden, Halle...’ ‘My post-doc (Assistent) MD, PhD Mengele has joined this part of the research as a collaborator. He is employed as an SS-Captain and camp doctor in the concentration camp of Auschwitz. With the approval of the Reichsfu«hrer SS, anthropological studies have been carried out on the very diverse racial groups in this camp, and blood. samples have been sent to my laboratory for processing’. *October 4, 1944, third interim report to the DFG on the subproject ‘specific proteins’: ‘The research has been intensively pushed forward. Blood samples of more than 200 persons of various races have been worked upon. Substrates of the blood samples have been made. Further research is being carried out with Dr Hillmann, a colleague from the KWI of Biochemistry. Dr Hillmann is a biochemical specialist in protein research. With his help, Abderhalden’s method has been optimised. So now we can begin with the real experiments with the rabbits’.

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Please note that the experiments with sera of rabbits and humans are interchanged here. Gu«nther Hillmann was a biochemist who had begun his career with Professor Karl Hinsberg. Hinsberg had worked on the Abderhalden defence enzymes in the diagnosis and possible therapy of cancer (34). His Berlin institute was destroyed during the war. He had accepted a position in an institute in Posen which was about to be built there and where chemical warfare was to be studied. Hillmann stayed in Berlin. Professor Adolf Butenandt, Nobel Prize winner of 1939, accepted hire as a collaborator. Butenandt had written letters to the DFG asking successfully for a stipend for Hillmann. Hillmann had developed a new test for the products of the Abderhalden reaction, i.e. peptides (35). When Hillmann began to collaborate with von Verschuer, Butenandt had just moved with almost all his staff from Berlin to Tu«bingen, which seemed a place safe from bombing. October 4, 1944, third interim report to the DFG on the subproject ‘tuberculosis’. ‘...We continued the work to find out why some of the animals die from tuberculosis while the others do not get lung tuberculosis. Results have been obtained which open up new possibilities of research. To continue the research with biochemical methods we established contact with Professor Butenandt.’ Butenandt had just moved to Tu«bingen with all his collaborators except Hillmann. So von Verschuer claims to have written to Butenandt about the ongoing experiments. I just note here that this letter exchange, which presumably exists in the Max Planck Archive, will not be made public until the year 2025. On October 4, 1944 von Verschuer also wrote to his friend de Rudder, the paediatrician from Frankfurt University: ‘Precipitates have been prepared from the plasma of more than 200 individuals of various races, some twin pairs, and some families. Abderhalden’s method has been used and supplemented by a method newly discovered by Hillmann (who has joined


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as a collaborator). So we can begin our real research very soon. The aim of our various efforts is now no longer to establish that the influence of heredity is important in various infectious diseases, but rather how hereditary factors act and what kind of events take place in their action’ (15). January 6, 1945 von Verschuer wrote to de Rudder: ‘You will be interested to know that my research on specific proteins has finally reached a decisive stage, now that we have overcome some considerable methodological difficulties with the help of one of Butenandt’s collaborators, Hillmann, who is a protein chemist’ (15). This was the end of it. On January 26, 1945 Auschwitz was liberated. Mengele fled and went into hiding. On February 15, 1945 von Verschuer used the opportunity of the sudden availability of two trucks to move his lab, library and personal belongings west, to the small town of Solz, where his family had a house. Prof. Dr Hans Nachtsheim, a sub-director of the Verschuer institute, who stayed in Berlin, wrote to him on March 12 at his new address in Solz (36): ‘I heard from Miss Jarofski [Nachtsheim’s secretary, BMH] that a mass of documents (Akten) have been left here which should be or have to be destroyed, should the enemy ever come close to here. I have not looked at what and how much it is, but I assume that Miss Jarofski knows exactly.You talked to her. I would have suggested that you take the stuff with you to Solz. In any case, we should not choose a moment when it is too late to destroy them. I feel myself entitled to make the relevant decision.’ So it is reasonable to assume that Nachtsheim destroyed von Verschuer’s correspondence with Mengele and all lab notes from the project. Hillmann must have done the same. And finally Butenandt, later president of the Max-Planck-Gesellschaft, in whose laboratory Hillmann had worked, secreted all his correspondence in the Max-Planck-Archiv, to be held inaccessible for 30 years after his death in 1995, i.e. until 2025. So what were the tests Hillmann did with the sera of the infected twins and families? According to the letters and DFG

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reports of von Verschuer, Hillmann tested the sera for the presence or absence of Abderhalden’s defence enzymes. Abderhalden claimed that specific proteases are formed in the serum of infected humans, or animals, against the infectious agent. Hillmann had developed a more sensitive test for the resulting peptides (35). So he must have tested the proteolytic action of the sera of the infected persons on the infectious agents of tuberculosis or typhus. The expectation was that sera of Ashkenasy Jews reacted stronger than sera of Sephardic Jews or Gypsies in the case of tuberculosis. Abderhalden had published his first paper on defence enzymes in 1909 (for a history of the defence enzymes see 37). They were first used for a pregnancy test. The test seemed to work in many gynaecological clinics. Some years later it was shown by three groups that the test gave the same answer whether a woman was pregnant or not; it was all an illusion. Specific defence enzymes did not exist. Abderhalden then refined the test of the non-existing defence enzymes and found new customers among clinicians. In 1931 he became president of the Leopoldina. Defence enzymes were still thought of as real science in Germany, although a very few scientists regarded them as illusory. The word fraud was not mentioned (for details see 37). Today we have to conclude that the method of molecular analysis used in the research project of Mengele-von Verschuer was fake, i. e. pseudoscience.Von Verschuer and Mengele did not know at the time; they were no insiders. But what about Hillmann and Butenandt? Did Hillmann realise that the defence enzymes did not exist? Had he possibly left Hinsberg for this reason? Had he ever discussed the problem with Butenandt? The Butenandt letters in the Max Planck Archive may give a hint. Mass murder and truth are incompatible, they do not go hand in hand. The excitement of performing mass murder for science led von Verschuer and Mengele to overlook the fact that Abderhalden’s defence enzymes did not exist, i.e. that they were a fraud. The experiments of Mengele and Hillmann in the


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laboratories of von Verschuer and of Butenandt were thus pseudoscience. And what an effort it must have been later for these scientists to stop all investigation into these experiments. Truth had truly disappeared.

1945^1951: Von Verschuer has to Wait for a Professorship When the war ended, von Verschuer was in the house of his family in Solz. His friend de Rudder, now dean of the medical faculty in Frankfurt, wrote him that he had a good chance of returning back as professor to the University of Frankfurt (38). Von Verschuer’s successor, Prof. Dr Heinrich Kranz, a convinced Nazi, had committed suicide, and he,Verschuer, was after all the best German human geneticist. The possibility of such a development was destroyed by a brief note in the Neue Zeitung. On April 15, 1946 there had been a one page article about ‘expelled scientists’. In the first part of the article, some, former Jewish members of the KWG were listed and their fates recorded; in the second part of the article the ‘inner migration’ (Binnenwanderung), i.e. the flight westward, of several German KWG members was noted. Among others, von Verschuer was mentioned as being stranded in a small town near Bebra. Some days later, on May 3, 1946 ‘corrections’ were published. I quote the one about von Verschuer: ‘von Verschuer was a race fanatic. Professor Robert Havemann, the current president (according to the Russians, BMH) of the Kaiser WilhelmGesellschaft, wrote to us the following: Verschuer .... had a distinguished post-doc (Oberassistent) who was active as camp doctor in Auschwitz and who worked only rarely in the KW Institute. This collaborator sent him in regular intervals blood samples and pairs of eyes of whole families, all Gypsies, who supposedly had died natural deaths in Auschwitz. With these 13 to 15 pairs of eyes, von Verschuer analysed the tuberculosis of the kidney (sic). He also had collaborators at the Rassenpolitische Amt who had no medical knowledge and who

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did harm to the institute.’ Please note that the notice does not mention the name of Mengele! Tuberculosis and eyes are interchanged. The writer, Havemann, was a physico-chemist. As a communist he had been incarcerated by the Nazis until the end of the war and afterwards was made president of the KWG by the Russians. This had not been accepted by the rest of the KWG in the western zones. Havemann later became one of the first outspoken intellectual dissidents of the German Democratic Republic. He lost his professorship and died in isolation. A few days later, on May 10, 1946, von Verschuer sent a statement under oath to Otto Hahn, according to the British (39) president of the western Kaiser Wilhelm-Gesellschaft. There he wrote: ‘already before 1933, but also later, I took personal risks and attacked, as a scientist, in speeches and in writing, the race concept of the Nazis. I criticised fundamental points, I argued against attributing values to races, I warned against the high estimation of the nordic race, and I condemned the misuse of the results of anthropology and genetics to support a materialistic and racial point of view of life and history. . .. A post-doc of my former Frankfurt Institute, Dr M., was sent against his will to the hospital of the concentration camp in Auschwitz. All who knew him learned from him how unhappy he was about this, and how he tried over and over again to be sent to the front, unfortunately without success. Of his work we learned that he tried to be a physician and helper of the sick...’ (39). Lies, lies, lies. ‘After I went to Berlin I began research on the individual specificity of the serum proteins and the question of their heredity. To do so I used the reaction of Abderhalden’s defence enzymes. For these experiments I needed blood samples of people of different geographic (sic!) background. . . . at that time my former post-doc Dr M. visited me and offered to obtain such blood samples for me within the context of his medical activity in the camp Auschwitz. In this manner I received ^ during this time, certainly not regularly ^ a few parcels of 20^30


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blood samples of 5^10 ml...’ (39). Please recall that in the letter to de Rudder and in his interim report he mentioned blood samples of more than 200 individuals. Von Verschuer also wrote a letter which he asked Otto Hahn, the president of the KWG, to sign. I quote: ‘... Professor von Verschuer is an internationally known scientist who has kept away from all political activity... With the Nazi offices he collaborated only as far as it was necessary for the protection of his scientific research. Within his institute he protected collaborators who were anti-Nazis. Professor von Verschuer had nothing to do with the errors and misuses of the Nazis, by which his scientific field was particularly hit. He kept his distance from them and, whenever he was confronted by them, he criticised them courageously, several times in public lectures. Von Verschuer’s personality and work ^ also recognised outside of Germany ^ will be capable of reconstituting the honour of his scientific work, which has been touched by the political misuse...’ Hahn did not sign it (39). Havemann went on to organise a committee of KWG scientists in Berlin, who had to judge the von Verschuer case. The committee used the article in the Neue Zeitung and comments by von Verschuer, Nachtsheim and Muckermann (a catholic eugenicist who had been thrown out of the institute by the Nazis). When the committee came to its conclusion, it consisted of Havemann, two sub-directors of the Verschuer-Institute (Nachtsheim and Gottschaldt), and Otto Warburg, the biochemist, Nobel prize winner and first a half, then a quarter Jew, according to the Nazis. The majority of the committee (but not Nachtsheim) agreed essentially with all points of the article (40). If von Verschuer had published what he had published without being a race fanatic, this seemed even worse. He should not have accepted blood or any other material from prisoners. However, the committee did not ask what the experiments were all about. They did not ask for the laboratory records, They did not ask the role of Hillmann and Butenandt. Nachtsheim, who must have known, was silent. He also defended

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the use of the blood samples from ‘the hospital’of Auschwitz and the ideological publications of von Verschuer. Why did Nachtsheim defend the use of the blood samples from Auschwitz? Why did he not ask for details, from whom they were obtained, what the tests were about? I think the reason for this is simple. Nachtsheim knew that von Verschuer knew that he, Nachtsheim, together with a post-doc of Butenandt, Dr Gerhard Ruhenstroth-Bauer, had experimented in 1943 with epileptic children from the lunatic asylum in Go«rden. They had tested whether the children had epileptic fits under low oxygen pressure (i.e. corresponding to an altitude of 6,000 m) and whether this test could be used to differentiate between hereditary and non-hereditary epilepsy (41). They had used a low pressure chamber of the Luftwaffe. Go«rden was known as a place where extensive use was made of the patients as objects of science before they were killed nearby with gas as part of the euthanasia programme (1). Finally the committee decided not to make their report public. So nothing was published for the next fifteen years about von Verschuer and Mengele. But the report made it impossible for von Verschuer to become part of the newly reconstructed Max-Planck-Gesellschaft. It also made it impossible for a local government to agree to the hiring of von Verschuer by a university. So, in September 1949, a new committee became active to help von Verschuer out of this dilemma (42). It consisted of Butenandt, Max Hartmann (a biologist, Berlin), Wolfgang Heubner (a pharmacologist, Berlin), and Boris Rajewsky (a biophysicist, Frankfurt). They came unanimously to the conclusion that von Verschuer was not a Nazi, that he was no race fanatic, that he was tolerant with his collaborators, that he did not know what had gone on in Auschwitz, that in fact it was not even proven that his assistant Mengele knew at the time what went on there, that there was nothing wrong with receiving blood from Auschwitz, but that some of the sentences he published should be heavily criticised. In the end the report concludes, ‘that von Verschuer has all qualities


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which destine him to be a researcher and teacher of the academic youth’. The referees end by stating that it would seem ‘ pharisa«erhaft’ to note single events against a brave and honest man like Verschuer (42). It may be pointed out that, according to Christian tradition, the ‘Pharisa«er’ applauded the crucifixion of Jesus. The report could not have been better. Auschwitz had disappeared from the agenda. Von Verschuer had always performed normal science. About one year later, in the autumn of 1950, the medical faculty of the University of Mu«nster decided to offer von Verschuer a full professorship at the Institute of Human Genetics. Butenandt as Witness at the IG-Farben Trial in Nuremberg Butenandt did not only help von Verschuer. On the morning of February 2, 1948, Butenandt served as a witness in Nuremberg for his friend, the accused Prof. Heinrich Ho«rlein (43). The question was, what was the status of the experiments which had been done with prisoners in various concentration camps, including Auschwitz, using various chemicals or drugs produced by IG-Farben. Butenandt made the point that Menschenexperiment, the term which was generally used in the letters between the camp doctors and Ho«rlein, the head of medical research at IG-Farben, should be translated as ‘clinical trial’. What were called Menschenexperimente in German were in fact medical tests. The judge became sufficiently confused ^ and freed Ho«rlein from the charge. I quote now some questions to Butenandt and his answers under oath: Q: What did you know of the existence of concentration camps and what went on in them? A: I had almost no knowledge of concentration camps. I knew that some existed. Before the end of the war I heard the names Dachau and Oranienburg. The names Auschwitz, Belzec,

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Buchenwald etc. I heard for the first time after the war. What went on in concentration camps, I did not know at all. During the war, while in foreign countries, I heard rumours that crimes were committed there. I did not believe it, because these things were totally outside my intellectual world.This is all I have to say. Q: Did you know that a MD who was active in a concentration camp had to get into conflict with his conscience and his ethics? A: No, I did not know that. Q: Did you know that experiments were performed on prisoners in concentration camps? Perhaps this question is superfluous after what you said, but I want to pose this question anyhow. A: I have never heard anything about that, not even the faintest detail. Von Verschuer et al. Back to Normal Academic Life Von Verschuer was a founding member of the Mainz Academy in 1950. In 1951 he became professor of Human Genetics at the University of Mu«nster. In 1954 he became dean of the medical faculty. In 1956 he gave an invited talk at the international congress of genetics in Tokyo and Kyoto on ‘Tuberculosis and cancer in twins’ (44). In the same year his Italian colleague Luigi Gedda gave a talk where he called von Verschuer a ‘Master and Example’ (45). Von Verschuer received several other honours (see Table 1). In 1961 a German anthropologist, Karl Saller, who had been fired in 1935 because of his left-wing friends and unorthodox view on race and who was reinstated after the war, published a book on the race concept of German anthropologists during the Third Reich (46). The book contained ample citations of the anti-Semitic publications of von Verschuer. The book was reviewed in the German press and von Verschuer was called an anti-Semite. So von Verschuer wrote a letter to the Gypsy expert Dr Hermann Arnold in which he complained about the attack. He wrote that he had


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TABLE 1 Memberships and honours of Professor von Verschuer 1934 1940 1943 1949 1949 1953 1953

Member, Academy Leopoldina, Halle Member, American Eugenics Society, New York Member, Prussian Academy of Sciences, Berlin Member, Academy of Science and Literature, Mainz Corresponding Member, American Society of Human Genetics. Honorary Member, Italian Society for Genetics Member, Scientific Council of the German Society for Population Studies 1955 Honorary Member, Anthropological Society,Vienna 1956 Honorary Member, Japanese Society for Human Genetics 1959 Corresponding Member, Austrian Academy of Sciences,Vienna

intended to sue the author, but after some consideration he had decided not to do so since, he felt after all, there was no justice in present day Germany (47). Von Verschuer retired in 1965. In April 1966 an investigation of von Verschuer was begun by the prosecutor’s office (Staatsanwaltschaft) of Mu«nster. Two years later it was closed since no evidence for any crime could be found. Von Verschuer died in 1969 from a car accident. In 1998, when I asked to see the material of the Staatsanwaltschaft, I was initially informed that it had disappeared (48). Then, by accident, it was found again. I inspected it at the Staatsarchiv Mu«nster (49). Robert Kempner, the prosecutor at the Nuremberg trials, had received an anonymous letter from New York. Its author accused a professor van Verschuur (sic!) of being particularly culpable with respect to Gypsy victims. Nachtsheim was named as the witness ‘who knew everything’. Kempner sent the letter to the prosecutor in Frankfurt, and the prosecutor in Frankfurt sent it to his colleague in Mu«nster. They interrogated Nachtsheim. Nachtsheim mentioned the three or four pairs of Gypsy eyes which had been sent to Magnussen from Auschwitz, but he did not know the cause of the Gypsies’ death. So Magnussen was interrogated. She said they died a normal death, and that

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Verschuer had nothing to do with it. Nachtsheim briefly mentioned Mengele and the blood samples he had sent to von Verschuer from Auschwitz. And he added that none of the donors had presumably died from the blood donation. Nachtsheim did not say a word regarding in which context and for which purpose the blood samples were taken and. the interrogators did not ask. Two other witnesses who. were asked about Verschuer did not know anything. The Frankfurt prosecutor who had investigated Mengele was asked whether there was a connection between Mengele and von Verschuer. The Frankfurt prosecutor stated that he could not see one. So the investigation was closed and von Verschuer was never interrogated. So what happened to the others who were in one way or another involved with the blood from Auschwitz? Hillmann became apl. professor of clinical biochemistry at Tu«bingen University in 1962. He became the first president of the German Society for Clinical Biochemistry. He never said or published a word about the blood from Auschwitz which he had analysed in search of Abderhalden’s defence enzymes. He died in 1976. Butenandt became president of the Max-Planck-Gesellschaft in 1962. When, in 1983, I told him the story of the Auschwitz blood analysed in his laboratory, he said that this was the first time he ever heard about it. He had moved in September 1944 to Tu«bingen, just around the time his collaborator Hillmann started the work with the sera. I think it is rather unlikely that neither von Verschuer nor Hillmann spoke with him or wrote to him then or later about the subject. Butenandt has given his correspondence to the Archive of the MPG under the condition that it may not be touched for 30 years after his death (1995). So perhaps our children will know in 2025. Nachtsheim was chosen in 1949 by the Max-Planck-Gesellschaft to become director of an MPI for Comparative Genetics (vergleichende Erbbiologie), and thus became the successor to von Verschuer in Berlin at the Max-Planck-Gesellschaft. Originally a professor at the Humboldt University in East Berlin, he fled in


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1949 to the Free University (Freie Universita«t) in West Berlin when the Soviets propagated Lysenko’s fraud in East Germany. The building of von Verschuer’s KW Institute was given to the Free University which installed in it the Institute of Political Sciences. Nachtsheim died in 1979. Mengele died peacefully in exile in South America (50). According to Posner and Ware (50) the Mossad lost all interest in catching him by the end of 1962, two years after Butenandt became president of the Max-Planck-Gesellschaft: A public trial of Mengele in the sixties would indeed have ended in disaster for the science of genetics; and biochemistry as practised in Germany.

The Silence of Von Verschuer and his Colleagues, Scientists and Historians After the war von Verschuer and almost all his colleagues in human genetics remained silent about the past until their deaths. None of them ever wrote an article or a book about the past. At best they said or wrote a very few sentences. And the scientific institutions like the Max-Planck-Gesellschaft or the Deutsche Forschungsgemeinschaft were silent too. They were uninterested in the dark sides of their past. The brief remarks on the past by those geneticists active in Nazi Germany are worth quoting in detail. They are written in a language which is difficult to understand. They are difficult to translate. They have so many meanings. I therefore quote the German originals with the references. I begin with vonVerschuer. He mentioned the past just once, in the lecture he gave on the occasion of his retirement, a text which exists only in manuscript (51). He spoke about the possibilities of misuse of modern human genetics. And then he said: ‘Because of the experience of our past ^ misuse of eugenics through political demons (die Da«monie) ^ we ought to be shielded against proposals of any ideological eugenics’. The central phrase is ‘misuse of

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eugenics through political demons.’ So, according to von Verschuer, German scientists and science were‘misused by political demons’. But not a single word about how they were perverted, what went wrong. Not a single word about his own anti-Semitism. ‘Demons’ which cannot be understood or even described serve here as the explanation of a scientist! Von Verschuer wrote a piece in 1955 on Eugen Fischer as a scientist (52). There he does not mention the period between 1933 and 1945 at all. On the occasion of the death of Eugen Fischer in 1967, his former student Prof. Johann Schaeuble wrote an article (53). I quote: ‘... a generation of researchers, who wanted the eugenic best for the future of their people, who in German reality however had to see how their welldesigned and truthful projects ended in crime.’ One has to read this carefully. The sterilisation programmes, the Nuremberg laws, the firing of all Jews from university positions etc., all these were well-designed and truthful projects? Were they not crimes in themselves? How and where did they end in crime? Schaeuble does not give an answer, he continues: ‘The tragedy of this part of anthropology, human genetics and eugenics, has not been written as far as I can tell. It should just be mentioned; it has thrown deep shadows on the life of Eugen Fischer.’ Schaeuble clearly regards Saller’s book (46) as non-existent or wrong. What is missing, according to Schaeuble, is a convincing extensive theory. I suspect that as long as anti-Semitism does not again become part of the thinking of the German university establishment, it cannot be written in the manner Schaeuble et al. would like to see it. It is of interest how the great old man, Eugen Fischer, the teacher of von Verschuer and director of the KWI for Anthropology, Human Heredity and Eugenics from 1927 to 1942, saw the past. In his autobiography (54) he excluded it completely. He touched upon the subject just once (55). I quote: Eugenics was certainly not involved when in National Socialism a wicked (heillos) and criminal misuse disregarding all genetic facts


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and all human dignity was carried out. It is deeply deplorable that eugenic research was hit upon strongly, most heavily of course in Germany. International eugenics is not accused. One does not accuse the Christian Creed, when one believes that the horrors of the inquisition, the witch trials, the bloody religious persecutions and religious wars are human errors and crimes.

These sentences should be compared to sentences Fischer published in March 1943: It is a rare and especially good fortune for a theoretical science to flourish at a time when the prevailing ideology welcomes it, and its findings can immediately serve as the policy of the state. The study of human heredity was already sufficiently mature to provide this, when, years ago, National Socialism was recasting not only the state but also our ways of thinking and feeling (Weltanschauung). Not that National Socialism needed a ‘scientific’ foundation as a proof that it was right (ideologies (Weltanschauungen) are formed through practical experience and struggle rather than created by laborious scientific theorising) but the results of the study of human heredity became absolutely indispensable as a basis for the important laws and regulations created by the new state (56).

And what about Prof. Dr Fritz Lenz, the former sub-director of the Kaiser Wilhelm Institute under Fischer, and professor of Human Genetics in Go«ttingen after the war? In 1953 he wrote for the first and last time about the subject (57). He pointed out correctly that science describes the world as it is and that values do not rise legitimately from science. He touched briefly on the German past: ‘The persecution of the Jews was not caused by genetics; the persecution of the Jews was undertaken by political fanatics who understood little or nothing of genetics’. I may recall the fact that in 1931 Lenz had written a favourable review of Hitler’s Mein Kampf. Its last sentence may be quoted: ‘Hitler was the first politician of significant influence who has understood that race hygiene (eugenics, BMH) plays a

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central role in all politics and who wants to promote it actively’ (58). It was all forgotten; did not Eugen Fischer, the director of the Kaiser Wilhelm-Institute and rector of Berlin University 1933^ 1934, sign the letters to the Jewish professors of Berlin University informing them that they were fired because they were Jews? Did not Fischer ask already in 1933 for the Nuremberg laws? Were the Nuremberg laws not applauded by von Verschuer? What did Lenz write in the Baur-Fischer-Lenz textbook about the Nuremberg laws in 1936? Did he not applaud the fact that Jewish medical doctors and lawyers were not allowed to practice? Did von Verschuer not write positively about the forthcoming ‘total solution of the Jew problem’? Finally one may ask, did these men privately write each other letters about the past in which they honestly confessed? Kro«ner has analysed their letters (38). What he found is a repeat of the same; they did not know anything. They were good Christians. How could something end so badly, that had started so well?

Other Examples of the General Disinterest in the Inglorious Past When I was writing and publishing my book (1) in 1983^1984, I was most interested to read the letters Fischer, and others, had written while at the Dahlem Institute. Clearly von Verschuer had not taken everything with him when he fled Berlin, and Nachtsheim may not have destroyed all the rest. So I talked to various people. Where could I find these letters? The Berlin biophysicist Prof. Dr Ernst-Randolf Lochmann told me that Nachtsheim formerly had worked in the building where he (Lochmann) was now working. When Nachtsheim retired, the bacterial geneticist Prof. Dr Fritz Kaudewitz became his successor as director of the MPI, now called the MPI of Molecular Genetics. A very few years later, when Kaudewitz moved to Munich, his successors Thomas Trautner, Heinz Schuster and


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Heinz-Gu«nther Wittmann moved to a new building of the MPI of Molecular Genetics in the Ihnestrae, not too far from von Verschuer’s old institute. Then the order was given ^ it is unclear by whom ^ that the attic of the Nachtsheim Institute, which was full of letter-boxes, was to be cleaned. All the letterboxes were thrown in the garbage. A student, Bernd Ku«pper, had kept just one box out of curiosity. Lhochmann kindly gave me Ku«pper’s address. So I contacted Ku«pper who had become a biology teacher. Ku«pper sent me the box. It contained the letters which had been exchanged between Fischer and members of the international eugenics community. I made copies and sent the originals back to Ku«pper. I then asked Trautner and Schuster about the KWI for Anthropology in Ihnestrae 24, the predecessor of their own institute. They had never heard of it, and did not know about Fischer’s letters either. When my book appeared in 1984 it was greeted by silence in Germany. It was not reviewed in the Max-Planck-Spiegel, the official journal of the MPG. The historians of the MPG did not comment on it, yet it contained the first quotation of the DFGreport of von Verschuer on his collaboration with Mengele. In 1986 the Freie Universita«t of Berlin planned to put a memorial tablet on the building of the former KWI for Anthropology, which they had bought from the MPG. The secretary general of the Max-Planck-Gesellschaft, Dietrich Ranft, was informed. On July 1, 1986, Ranft wrote a letter to the secretary general of Berlin University, Detleff Borrmann (59). There he stated that there was no evidence that there had been murders for the research of the Kaiser Wilhelm-Institute. ‘The role which Dr Mengele played during the time when Professor v. Verschuer was the head of the institute is not clear at all. The Max-PlanckGesellschaft would appreciate if the history of this rather problematic institute would be worked out fully and free of emotions . . . . until such a history exists, we ask you to understand that the Max-Planck-Gesellschaft is unable to take the responsibility for the proposed or any other text...’ (39). What a statement: the MPG will not care until the history is worked out

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‘fully and free of emotions’. The history will never be worked out fully, too many of the relevant documents have been destroyed. And what a demand: to tell this mass-murder story without emotions, to claim that it is not acceptable when emotions are not completely silenced! In 1990 Helmut Albrecht and Armin Hermann (60) mentioned Mengele and quoted the DFG documents for the first time a voluminous official history of the KWG. They call Mengele a collaborator (Mitarbeiter) of von Verschuer. In contrast, in a short, official article published for the general public in 1993 (61), it is stated that Mengele was not part (Angeho«riger) of the KWI of Anthropology although he sent blood and organs from Auschwitz to the institute. The numerous articles of von Verschuer and others supporting the anti-Semitic measures of the Nazis are not mentioned. The words Jewish or Jew are never used. Anti-Semitism is not used either. All the unmentioned is summarised in the word ‘Verstrickung’ (entanglement, mess). A similar view can be found in a recent article by Dr Doris Kaufmann, now heading up the project ‘History of the Kaiser Wilhelm Society during National Socialism’ of the Max Planck Society. According to her, von Verschuer’s institute produced top science (Spitzenforschung). Anti-Semitism is not mentioned. The Mengele-von Verschuer story is told in one sentence: ‘The last director of the Institute (for Anthropology), Otmar Freiherr von Verschuer, who via his former post-doc Josef Mengele was entangled (verstrickt) in the human experiments in Auschwitz, received in 1951 the first full professorship for Human Genetics in Mu«nster’. (62).Verstrickung is passive, You do not do anything. The others entangle you. I would just like to document this by a paradigmatic quote: ‘He (Martin Heidegger, BMH) has never denied his Verstrickung in the movement (Bewegung) of that time’, so writes. Hermann Heidegger, the son of Martin Heidegger, the master of the German language (63). And now to my last example: in Autumn 1998 I heard from Hans-Peter Kro«ner that the race paternity expert opinions still existed in the Institute of Human Genetics in Mu«nster. I wrote


541

to its present director, Ju«rgen Horst, and asked to see the material. Horst informed me that this was not possible (64). My visit would endanger the rights of the patients of von Verschuer. Only after all patients had been anonymised could the material be shown to me. And at the moment this was technically impossible. It is truly bizarre. The Jewish victims are now called patients. Now they have rights: But their rights prohibit that now, more than half a century after the war, the writings of their henchmen can be analysed.

Is there a Bottom Line? Von Verschuer’s scientific record and his scientific career are deeply disturbing. Equally disturbing is the disinterest of his colleagues in this past. A scientific community which is unable to face its own crimes, and which prefers to look constantly away, works towards its own destruction. It is late but not too late. Von Verschuer was the director of a KW Institute and thus belongs to the history of the MPG. The documents which I present show unambiguously that von Verschuer considered Mengele to be his collaborator (Mitarbeiter) in Auschwitz. It is irrelevant that he later claimed not to be informed about the murders committed by Mengele. The murders happened and they are thus part of the history of the KWG/MPG. The MPG ought to acknowledge this. I suggest that the MPG does so, and invites the last surviving twins to a conference held at the successor institute, the MPI for Molecular Genetics, in Berlin-Dahlem, Ihnestrasse. I suggest that the MPG officially apologises.

REFERENCES [1] The story was first told in Benno Mu«ller-Hill, To«dliche Wissenschaft. Die Aussonderung von Juden, Zigeunern und Geisteskranken, Reinbek: Rowohlt,

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1984. The historians of science and the scientists of the Max Planck Society greeted the book with silence. Translated into English by G.R. Fraser: Murderous Science. Elimination by Scientific Selection of Jews, Gypsies, and Others an Germany 1933^1945, Oxford: Oxford University Press, 1988; Paperback edition with an afterword by J.D. Watson, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1998. [2] Ku«hl, S. The Nazi Connection. Eugenics, American Racism and German National Socialism, NewYork, Oxford: Oxford University Press,1994: 20^21. [3] Von Verschuer, O.‘Twin Research from the Time of Galton to the Present Day’, Proceedings of the Royal Soiety (B), 128 (1940), 62^81. See also: M. Teich,‘The Unmastered Past of Human Genetics’. In: Teich M., Porter, R. (eds). Fin de Sie'cle and its Legacy, Cambridge: Cambridge University Press, 1990: 296^324. [4] Von Verschuer, O. ‘Vererbungs- und Rassenwissenschaft als Grundlage vo«lkischer Politik’, Akademische Blotter (des Vereins deutscher Studenten), 16(1922): 202^205. Reprint in: Heither H., Golltschaldt E., Lemlin M. (eds), Wegbereiter des Faschismus. Aus der Geschichte des Marburger Vereins Deutscher Studenten, Marburg: Marburger Beitrage zur Geschichte studentischer Verbindungen,Vol. 1, 1992. [5] Von Verschuer, O. ‘Rassenhygiene als Wissenschaft und Staatsaufgabe’, Johann Wolfgang Goethe-Universita«t. Frankfurter Akademische Reden Nr. 7, Frankfurt: H. Beckhold Verlagsbuchhandlung, 1936, 3^11. Translation BMH. [6] Von Verschuer, O. ‘Was kann der Historiker, der Genealoge und der Statistiker zur Erforschung des biologischen Problems der Judenfrage beitragen?’. In: Forschungen zur Judenfrage, Bd. 2 (1937): 216^222, Hamburg: Hanseatische Verlags anstalt. Translation BMH. [7] Von Verschuer, O. ‘Rassenbiologie der Juden’. In: Forschungen zur Judenfrage, Bd. 3 (1938): 137^151, Hamburg: Hanseatische Verlagsanstalt. Translation BMH. [8] Von Verschuer, O. Leitfaden der Rassenhygiene, Leipzig: Georg Thieme Verlag, 1941 & 1944. Translation BMH. [9] Von Verschuer, O.‘Der Erbarzt an der Jahreswende’, Der Erbarzt, 10 (1942): 1^3. Translation BMH. [10] VonVerschuer, O. ‘Bevo«lkerungs- und Rassenfrage in Europa’, Europa«ischer Wissenschaftsdienst, 1 (1944): 11^14. Translation BMH. [11] Bundesarchiv, Koblenz R22^486, 112 ff. [12] VonVerschuer, O.‘DieVaterschaftsgutachten des Frankfurter Universita«tsinstituts fu«r Erbbiologie und Rassenhygiene’, Der Erbarzt, 9 (1941): 25^31. [13] Sandner, P. Frankfurt. Auschwitz Die nationalsozialistische Verfolgung der Sinti und Roma in Frankfurt am Main, Frankfurt a.M.: Brandes & Aspel, 1998, 212 ff.


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[14] de Azevedo, A. ‘U«ber die Erblichkeit der Quantita«t der Blutgruppensubstanzen’, Der Erbarzt, 12 (1944): 85^90. [15] Nachla von Verschuer, Universita«tsarchiv Mu«nster. Translation BMH. [16] Mengele, J. ‘Rassenmorphologische Untersuchungen des vorderen Unterkieferabschnittes bei vier rassischen Gruppen’, Morphologisches Jahrbuch, 79 (1937), 60^117; zu ‘Sippenuntersuchungen bei Lippen-KieferGaumenspalte’, Zeitschrift fu«r Menschliche Vererbungs- und Konstitutionslehre, 23 (1939): 17^42; Zur ‘Vererbung der Ohrfistel’, Der Erbarzt, 8 (1940): 59^60; ‘Tagung der Gesellschaft fu«r physische Anthropologie’, Der Erbarzt, 4 (1937): 140^141. Review: L. Stengel von Rutkowski, ‘Grundzu«ge der Erbkunde und Rassenpflege’, Der Erbarzt, 8 (1940): 116. Review: G. Venzmer, ‘Erbmasse und Krankheit’, Der Erbarzt 8 (1940): 214. Review. G. Pressler, ‘Untersuchungen u«ber den Einflu der Grostadt auf die Kopfform sowie. Beitra«ge zur Anthropologie und Stammeskunde Hannovers’, Der Erbarzt, 8 (1940): 47. [17] Lifton, R.J. The Nazi Doctors. Medical Killing and the Psychology of Genocide New York: Basic Books, 1986. Hans Mu«nch is named Ernst B. in Lifton’s book. [18] Bundesarchiv, Koblenz R 73 ^ 15342. Reports to the DFG. File von Verschuer, Document Center, DFG File card. Translation BMH. When I was looking for the DFG-files in 1983, I was told by officials of the DFG that, first of all the DFG was not called DFG in Nazi Germany (wrong), and second that I would not get access to the files because they were eternally confidential. When I discovered their existence in the Bundesarchiv Koblenz I was told that I was the first person ever wishing to look at them and that I was free to see them. So much for the DFG and German historians of science and medicine. [19] de Rudder, B. ‘Rasse and Infektionskrankheiten’. In: Schottky J. (ed.), Rasse and Krankheit, Mu«nchen: Lehmann, 1937, 68^85. [20] Nyiszli, M. Protocol, July 5, 1945, before the State Committee for the deported Hungarian Jews. I thank Dr FrancisWiener (Stockholm) for providing a copy of the Nyiszli document, and Anita Hajdu for its translation. Dr. Mengele Boncolo¤orvosa Voltam AZ Auschwitz I Krematoriumban. A cimlap Ruzicskai Gyo«rgy munko«ja, 1946. English translation: A Doctor’s Eyewitness Account. New York: Frederick Fell Inc., 1960. German translation: Im Jenseits der Menschlichkeit. Ein Gerichtsmediziner in Auschwitz, Berlin: Dietz Verlag, 1992. [21] Lingens-Reiner, E. Prisoner of Fear, London: Victor Gollancz Ltd., 1948. [22] Weinreich, M. Hitler’s Professors. The part of scholarship an Germany’s crimes against the Jewish people, New York, NY: Yiddish Scientific Institute-YVO, 1946.

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[23] Lagnado, L.M. and Cohn Dekel, S. Children of the Flames, New York: William Morrow and. Company, 1991. E. Kor, ‘Nazi experiments as viewed by a survivor of Mengele’s experiments’. In: Caplan A.L. (ed.), When Medicine went Mad, Totowa, NJ: Humana Press, 1992, 3^8. Kor E.M., Wright M., Echoes from Auschwitz. Dr. Mengele’s Twins. The story of Eva and Miriam Mozes, Terre Haute, IN: Candles Inc., 1995. [24] Wagner, G. Rassenbiologische Beobachtungen an Zigeunern und Zigeunerzwillingen, Dissertation, Berlin, 1943. [25] Wagner, G. ‘Partielle Irisfarbung (Hornhautu«berwachsung), ein neues Merkmal’, Der Erbarzt, 12 (1944): 62^64. [26] Magnussen, K. ‘U«ber eine sichelfo«rmige Hornhautu«berwachsung am Kaninchenauge und beim Menschen’, Der Erbarzt, 12 (1944): 60^62. [27] Klee, E. Auschwitz die NS-Medizin und ihre Opfer, Frankfurt: S. Fischer, 1997. [28] Von Verschuer, O. Die Wirkung, von Genen und Parasiten im Ko«rper des Menschen, Abstract seines Vortrages auf der Gesamtsitzung der Akademie der Wissenschafen am 16.11.1944. Archiv der Akademie der Wissenschaften. Translation BMH. Von Verschuer published his speech after the war ^ without mentioning the experiments. he had done with Mengele and Hillmann using Abderhalden’s method. O. von Verschuer, ‘Die Wirkung. von Genen und Parasiten’, Arztliche Forschung, 2 (1948), 378^388. [29] Bellamy, R., Ruwende, C., Corrah, T., McAdam, K.P.W.J., Whittle, H.C. & Hill, A.V.S.‘Variations in the NRAMP1 Gene and Susceptibility to tu«berculosis in West Africans’, The New England Journal of Medicine, 338 (1998): 640^644. [30] Abderhalden, R. Report to the DFG, April/September 1943. Bundesarchiv Koblenz R73^10002. [31] Diehl, K. and Von Verschuer, O. Zwillingstuberkulose, Jena: Fischer, 1933. K. Diehl, Das Erbe als Formgestalter der Tuberkulose, Leipzig: Barth, 1941. [32] Criminal case 4Js444/59, Public Prosecutor’s office, Frankfurt (Auschwitz trial). Translation BMH. The text says ‘Typhus’ which is typhoid, but in German there is confusion between Flecktyphus ¼ typhus, and Typhus ¼ typhoid. Given the fact that typhus epidemics were ravaging Auschwitz it may have very well been typhus. [33] Letter of Wilhelm R. Mann to von Verschuer, November 19, 1943, reproduced in Peter-Ferdinand Koch: Menschen-Versuche. Die to«dlichen Experimente deutscher A«rzte, Mu«nchen, Zu«rich: Piper, 1996: 179. Koch gives as reference: ‘das Mengele Schreiben entstammt einem Moskauer Archiv’. He does not say from which archive. Moreover Koch’s book is confused, full of errors and wrong evaluations. He worked before for the


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magazine Der Spiegel. It is imperative that this document is tested for its authenticity. If authentic, it would indeed be most revealing. [34] Hinsberg, K. and Schleinzer, B. ‘U«ber die Anreicherung und Spaltung derAbderhaldenschen Abwehrfermente bei Carcinomkranken’, Zeitschrift fu«r Krebsforschung, 53 (1943), 34^46. [35] Hillmann, G. ‘U«ber die Fluorescenzreaktion des o-Diacethylbenzol mit Eiwei und Eiweiabbauprodukten und ihre Anwendung auf die Abderhaldensche Abwehrfermentreaktion’, Hoppe-Seyler’s Zeitschrift fu«r physiologische Chemie, 277 (1943), 222^232. [36] Max-Planck-Archiv. Nachla Nachtsheim. Translation BMH. It would have been interesting to interview Ms Jarofski. When writing this, I had a look at the telephone book of Berlin.There was just one entry ‘Jarofski’. I called. Liselotte Jarofski told me that her sister Ruth had indeed been the secretary of Nachtsheim. Ruth died in 1997. I could not ask her anymore. I was too late. She and her sister had often discussed the past. [37] Deichmann, U. and Mu«ller-Hill, B.‘The fraud of Abderhalden’s enzymes’, Nature, 393 (1998):109^111. [38] For a detailed description of the post-war period see: H.-P. Kro«ner,Von der Rassenhygiene zur Humangenetik. Das Kaiser-Wilhelm-Institut fu«r Anthropologie, menschliche Erblehre und Eugenik nach dem Kriege, Stuttgart: Gustav Fischer, 1998. [39] Max-Planck-Archiv. II Abt. Rep. 0001A. Personalien v.Verschuer. [40] Max-Planck-Archiv. von Lewinski an Heubner 23.12.1946. Nachla Heubner III Abt. Rep. 47, Nr. 1505, Bl. 1^2. Translation BMH. [41] Mu«ller-Hill, B.‘Genetics afterAuschwitz’, Holocaust and Genocide Studies, 2 (1987): 3^20. U. Deichmann,‘Hans Nachtsheim, a Human Geneticist under National Socialism and the Question of Freedom of Science’. In: Fortun M., Mendelsohn E. (eds), Yearbook Sociology of Science, Vol. 19, Dordrecht, Holland: Kluwer Academic Publishers, 1999, 143^153. When I wrote the first version of my book, I mentioned there briefly Nachtsheim’s experiments.The lawyers of Nachtsheim’s son and Ruthenstroth-Bauer informed me that they would sue me if Icalled the tests experiments.Theyclaimed the children came from an orphanage and the director of the orphanage had given the consent that the test could be done. Only several years later I discovered the truth: the children came from the lunatic asylum Go«rden. It may be added that Ruthenstroth-Bauer was at that time Director emeritus at the Max Planck Institute for Biochemistry, Munich. [42] Abschrift. Denkschrift betreffend Herrn Prof. Dr med. Otmar Frhr. v. Verschuer. I thank Prof. Butenandt for giving me a copy. Translation BMH. [43] Milita«rgerichtshof NrVI, Nu«rnberg, Deutschland 2. Februar 1948, Sitzung von 9:30 bis 12:30. Institut fu«r Zeitgeschichte, Mu«nchen. REP 501

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Kriegsverbrecherproze VIII Nr. 38 BuerginVI. In 1982 I saw a file card in the archive of the Munich Institut fu«r Zeitgeschichte indicating that Butenandt had served as a witness in the IG-Farben Nuremberg trial. Then I had no time to inspect the document. In the meantime Butenandt had told me that the MPG might sue me when the book To«dlicheWissenschaft appeared. Several years later when I attended a conference at the Munich Institute, I wanted to have a second look at the file card. There was none. I thought that I had hallucinated. Some days later I called a person I knew in the Munich Institute and asked whether such a document existed. It did. Somebody had ripped out the file card. [44] VonVerschuer, O.‘Tuberculosis and cancer in twins’. In: Proceedings of the International Genetics Symposia 1956, Tokio & Kyoto. Suppl. Vol. of Cytologia, 1956, 92^101. [45] Gedda, L. ‘Un Maestro e un Esempio’, Acta Geneticae Medicae et Gemellogiae, Supl. Prim. 5 (1956): 241^248. [46] Saller, K. Die Rassenlehre des Nationalsozialismus in Wissenschaft und Propaganda, Darmstadt: Progress-Verlag, 1961. [47] Von Verschuer, O. letter to H. Arnold, in Nachla Hermann Arnold Bundesarchiv, Koblenz. The Nachlass Arnold does not exist any more in the form I saw it in 1983. It has been separated into the Gypsy papers of the Reichsgesundheitsamt and the Nachlass proper. Both have been transferred to Potsdam and supposedly from there to Lichterfelde. [48] Letter of the leitende Oherstaatsanwalt, July 17, 1998 to BMH.‘Zu meinem Bedauern mu ich Ihnen mitteilen, da die Akten. . .. jetzt nicht mehr auffindbar sind, so da von einem endgu«ltigen Verlust ausgegangen werden mu.’ [49] Staatsarchiv Mu«nster. Verfahrensakte 35 Js 74/68, Bestand Staatsanwaltschaft Mu«nster Nr. 438. [50] Posner, G.L. and Ware, J. Die Jagd nach dem Todesengel, Berlin: Aufbau Taschenbuch Verlag, 1998: 224. Engl. Ed.: Mengele, the complete story, New York, 1986. [51] Von Verschuer, O.‘Lecture on the occasion of his retirement’, February 25, 1965. Manuscript. I thank W. Lenz for a copy.‘Aus der Erfahrung unserer Vergangenheit ^ Mibrauch der Eugenik durch die politische Da«monie ^ sollten wir gefeit sein gegen solche Vorschla«ge jeder ideologischen Eugenik’. Translation BMH. [52] Von Verschuer, O. ‘Eugen Fischer. Der Altmeister der Anthropologie, der Pionier der Humangenetik, der Begru«nder der Anthropologie’. In: Schwerte H., Spengler W. (eds), Forscher und Wissenschaftler im heutigen Europa. Erforscher des Lebens, Oldenburg: Gerhard Stalling Verlag, 1955: 308^316. Translation BMH. Schwerte was an SS officer who changed his


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name after the war and made a career as scholar and president of the Technical University, TH Aachen. [53] Schaeuble, J. ‘Eugen Fischer’, Badische Heimat, 47 (1967): 89^94. Translation BMH. [54] Fischer, E. Begegnungen mit Toten. Aus den Erinnerungen eines Anatomen, Freiburg i.Br.: Hans Ferdinand Schulz Verlag, 1959. [55] Fischer, E.‘Die Wissenschaft des Menschen’. In: Gestalter unserer Zeit,Vol. 4: Erforscher des Lebens, Oldenburg: Gerhard Stalling Verlag, 1955, 272^ 287. Translation BMH. [56] Fischer, E. ‘Erbe als Schicksal’ Deutsche Allgemeine Zeitung, 28.3.1943. Translation BMH. [57] Lenz, F. ‘Diesseits von Gut and Bo«se. Bemerkungen u«ber das Verha«ltnis von Genetik und Glauben’, Deutsche Universita«tszeitung, Heft VIII/23, 1953, 9. Translation BMH. [58] Lenz, F.‘Die Stellung des Nationalsozialismus zur Rassenhygiene’, Archiv fu«r Rass. u. Ges. Biol., 25 (1931): 300^308. [59] Ranft, D. & Borrmann, D. 1.7.1986. MPG Archiv, II. Abt., Rep 0001A, Personalien van Verschuer. [60] Albrecht, H. and Hermann, A. ‘Die Kaiser-Wilhelm-Gesellschaft im Dritten Reich (1933^1945)’. In: Vierhaus R., Brocke B. vom (eds), Forschung im Spannungsfeld von Politik und Gesellschaft. Geschichte und Struktur der Kaiser Wilhelm-/Max-Planck-Geseilschaft, Stuttgart: Deutsche Verlags-Anstalt, 1990: 304^406. [61] Dahlem, Doma«ne der Wissenschaft. 3. Kaiser Wilhelm-Institut fu«r Anthropologie, menschliche Erblehre und Eugenik. Max-PlanckGesellschaft’, Berichte und Mitteilungen, 3 (1993): 36^42. [62] Kaufmann, D.‘Eugenik, Rassenhygiene, Humangentik’. In: Erfindung des Menschen, Du«lmen R.von (ed.),Wien, Ko«ln,Weimar: Bo«hlau, 1998. 347^365. [63] Heidegger, H.Vorwort. In: Die Selbstbehauptung der deutschen Universita«t. Das Rektorat 1933^34, Frankfurt: V. Klostermann, 1998. See also: W.Wette, ‘Was ist Verstrickung’, Die Zeit, Nr. 8, 18. February 1999: 11. [64] Horst, J. Letter to BMH of November 13, 1998.

Selective Perception: The Letters of Adolf Butenandt Nobel PrizeWinner and President of The Max-Planck-Society* BENNO MU«LLER-HILL Institute for Genetics, University of Cologne,Weyertal 121, D-50931 Cologne, Germany E-mail: [email protected]

Abstract Adolf Butenandt was one of the great biochemists of the last century. He was also a successful organizer of science. Which price did he have to pay to be a success in Nazi Germany? He persistently avoided seeing or hearing the blatant injustice, around him. Injustice, he knew nothing about, did not exist. So he became a perfect model for the postwar generation of German scientists. Is he still a model today?

Introduction Adolf Butenandt was one of the great biochemists of the past century. His work about human sexual hormones and the work of his collaborators about the genes of the insect Ephestia and of the tobacco mosaic virus belong to the best of their time. *The German original text is in press in Hist. and Phil. of the Life Sciences. Reprinted with permission.


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Butenandt was also a successful organizer of science, first as director of his institute and then as president of the MaxPlanck-Society during the period 1960^1972.This is all well recorded in a biography written by his student and admirer Peter Karlson [1]. In Karlson’s biography some aspects are missing. They will be discussed here.

Pre-History Twenty years ago, I realized that the history of human genetics in Nazi Germany was not written. Then, like today, I was an active molecular geneticist. Still I could not resist the temptation to investigate that totally abandoned field. So I used a sabbatical (i) to read all relevant journals and books which appeared between 1933 and 1945, (ii) to look for relevant material in all archives I could visit, and (iii) to talk with all German human geneticists (or race hygienicists as they then called themselves) which had been active at this time. Out of this came a book ‘‘To«dlicheWissenschaft, die Aussonderung von Juden, Zigeunern und anderen 1933^1945’’ which appeared in 1984 [2]. Three years later, an English translation appeared at Oxford University Press ‘‘Murderous Science. Elimination by scientific selection of Jews, Gypsies, and others, 1933^1945.’’ The book was translated in seven languages. My, perhaps, most important discovery was, that part of the papers of the Deutsche Forschungsgemeinschaft (DFG) was present in the Bundesarchiv in Koblenz. The reponsible registrar told me that I was the first historian who inspected the papers. Present were the reports on projects financed by the DFG. The grant proposals did not exist. It is unclear whether they were destroyed during the war or whether they were destroyed later or whether they were perhaps hidden in an archive of one of the Allies. Two of the DFG-reports were particularly explosive (i) the reports of Otmar von Verschuer from the Kaiser Wilhelm-Institut (KWI) fu«r Anthropologie, menschliche Erblehre und Eugenik

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(Anthropology, Human Genetics, and Eugenics) out of the years 1943 and 1944 (they will be analyzed here) and (ii) the reports of Robert Ritter from the Reichsgesundheitsamt (National Institute of Health) on his planning of the genocide of the Gypsies (this will not be discussed). The reports of vonVerschuer on the project‘‘specific proteins’’[3] were revealing: ‘‘March 20, 1944.’’ ‘‘New difficulties arose in establishing the method. They have been solved together with Geheimrat Abderhalden, Halle. . . My post-doc (Assistent) MD, PhD Mengele has joined this part of research as a collaborator. He is employed as an SS-Captain and camp doctor in the concentration camp of Auschwitz.With the approval of the Reichsfu«hrer SS (Himmler, BMH), anthropological studies have been carried out on the very diverse racial groups in this camp, and blood samples have been sent to my laboratory for processing.’’ ‘‘October 4, 1944.’’ New interim report to the DFG: ‘‘research has been intensively pushed forward. Blood samples of more than 200 persons of various races have been worked upon. . . . Further research is being carried out with Dr Hillmann, a collaborator of the Kaiser Wilhelm-Institut of Biochemistry. Dr Hillmann is a biochemical specialist in protein research. With his help, Abderhalden’s method has been optimized. So now we can begin with the real experiments with the rabbits.’’ These two reports of von Verschuer show two things [4]: 1. von Verschuer saw Josef Mengele, his former postdoc from Frankfurt University, who was now active in Auschwitz, as collaborator in his DFG-Project ‘‘specific proteins.’’ 2. Gu«nther Hillmann, a collaborator of Butenandt, advised von Verschuer about the test and was collaborating. What was the topic of the project ‘‘specific proteins’’? To make this clear I have to disgress. In the KWI for anthropology a scientist, Karl Diehl, had bred two races of rabbits. One was highly susceptible, the other much less susceptible to tuberculosis infection. A similar phenomenon had been observed with


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humans. Twin studies had shown that susceptibility towards tuberculosis is in part genetic [5]. It was also known that some human ethnic groups, for example Ashkenasi Jews, were generally less susceptible against tuberculosis infection than non-Jews. What was the molecular basis for this observation? von Verschuer assumed that the defense enzymes, discovered by Emil Abderhalden [6] were responsible for this effect. Thus the qualityand quantityof defense enzymes against tuberculous bacteria should be measured in the two rabbit races and in various human races. Mengele thus sent the sera of infected and noninfected twins and families of various racial backgrounds from Auschwitz to Berlin. They should then be analyzed in Berlin. Rudolf Abderhalden, the son of Emil Abderhalden, who worked in the lab of his father, had successfully asked for DFG money to study the defense enzymes against tuberculous bacteria [7]. In his report of the half year April^September 1943, he wrote: ‘‘Experiments to make a substate of tuberculous bacillae were pursued energetically. However, no success has been so far observed.’’ It is not documented but rather likely that von Verschuer was informed about these experiments. The project, however, had a snag, which apparently was not seen by those involved: The defense enzymes did not exist! [8]. They were a phantasy product. Abderhalden had claimed their existence first in 1909. In the years that followed, he remained their faithful defender inspite of the devastating critique of several biochemists who had demonstrated their nonexistence. Abderhalden always found some German medical scientists who claimed to have found defense enzymes, in spite of the fact that they were an illusion. Even Hillmann apparently did not know the truth about the defense enzymes. His former Chief, Karl Hinsberg, believed in them and wanted to use them for cancer diagnosis. Hillmann had optimized the test for peptides which were the supposed products of the reaction with the defense enzymes.When I wrote my book I did not know all this. What did Butenandt know about this? I do not know. Before my book appeared, I had a conversation with him. He said

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that Hillmann was a guest in his institute and not a collaborator. He said that he heard from me for the first time that Hillmann helped in the analysis of blood sera from Auschwitz. Finally, he did not allow that our conversation be printed in the book I planned, whatever his right to make any correction of the text. Among the DFG reports of the KWI for Anthropology, I also found the report of one of the subdirectors, Hans Nachtsheim, about epilepsy in rabbits and man. Nachtsheim had bred two races of rabbits; one was susceptible to epileptic fits, the other was not. At low air pressure, the susceptible rabbits had fits which sometimes ended with their death. The younger the animals, the more susceptible they were. How was this with humans? Were there similar genetic differences? Nachtsheim described experiments which he had done together with a postdoc of Butenandt, Gerhard Ruhenstroth-Bauer. Against all expectations, none of the epileptic children had an epileptic fit at low air pressure, corresponding to an altitude of 6000 m. I had mentioned these tests as experiments with people who had lost their right, in a lecture and in the manuscript of my book. The lawyers of the son of Nachtsheim and of Ruhenstroth-Bauer informed me that the children came from an orphanage and that the director of the orphanage speaks for their parents and that thus the tests had been legally legitimized. Some years later, I found a document proving that the children came from the insane asylum in Goerden, an institution which was well known to have participated actively in the euthanasia murder of its patients. Thus I published, without any legal difficulties, about the experiments [9]. Whatever, Butenandt and the Max-Planck-Society disliked my identification of Mengele as a collaborator of von Verschuer in Auschwitz and my mentioning of Hillmann as a collaborator of Butenandt who participated in the von Verschuer^Mengele project. In the following years no further documents of the Verschuer^Mengele experiments were found.The MPG was silent about the existing material and stated internally that it was


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not sufficient. When I heard that the Butenandt correspondence had been deposited in the Max Planck Archive, I asked for access. This seemed impossible since the Butenandt letters were declared inaccessible for 30 years after his death in 1995. However, Hubert Markl, president of the MPG, found a solution. He created a committee of the president which had access to the letters. The committee coopted first the historians of science, Robert Procter and Paul Weindling, and finally me for limited time. Procter’s excellent report about the letters has appeared [10]. In regard to the questions that interested me, he showed two things: 1. Butenandt exchanged letters with von Verschuer at the relevant time. Butenandt knew in general about the collaboration of Verschuer with Hillmann. 2. Butenandt called Hillmann his replacement in the Berlin Institute, after he himself had moved to Tu«bingen. So he ordered Hillmann to destroy the Geheime Reichsachen (documents Top Secret). He thus trusted Hillmann completely. Beyond that, Procter gave deep insights into the personality of Butenandt. What was left for me? I went once more through the entire scientific correspondence from A to Z, all together 18 meters, the diaries and the scientific reports. The results of my investigation will be reported here. First: Did I deceive myself when I remembered that Butenandt had asked me to delete the name of Hillmann from my manuscript? No, I found a letter of his of April 19, 1984 to the widow of Hillmann, Dr Anneliese Hillmann, in which he wrote ‘‘ Mr Mu«ller-Hill was not willing to follow my wish to delete the mentioning.’’ The same day he wrote to Dr E. Marsch of the Generalverwaltung of the MPG: ‘‘Of course several sentences about the KWG stay in the manuscript which are difficult to bear. But one may expect, that this really bad book, which cannot claim scientific quality, will find only few readers.’’ And now to the letter exchange between Butenandt and von Verschuer.

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Letters Exchanged between Butenandt and Von Verschuer: September 1944^September 1945 How did the collaboration von Verschuer^Butenandt^Hillmann get started? The simplest explanation is that Von Verschuer told his neighbor Butenandt, their villas in Dahlem were next to each other, of his project. Perhaps they met for lunch in the Harnackhouse closeby. Then Butenandt may have told von Verschuer, that he had a collaborator, Hillmann, who was an expert in the test of defense enzymes. In the first letter of Von Verschuer (Berlin) to Butenandt (Tu«bingen), collaboration is mentioned as if it were self-evident. Butenandt had left Berlin for Tu«bingen late in the summer 1944.There a large part of the institute had been moved. During November 1944, he came for some days to Berlin.This was his last visit in Berlin.Then he stayed for good in Tu«bingen. I will now quote in chronological order the letters to give the reader a maximal insight. September 30, 1944: Letter of Von Verschuer (Berlin) to Butenandt (Tu«bingen). ‘‘Many thanks for your lines of the 22 of this month and for your greetings from the lovely Tu«bingen . . . Mr Hillmann has spent a day in Beetz for the first joint work. I am very happy about the success.We follow his advice and will include some methodological improvements. Then it can go on. Diehl waits to meet you at your next stay in Dahlem and to talk with you about his experiments. I am very glad over this scientific connection between you and me. With affectionate greetings, also from my wife to you and your wife, always yourVonVerschuer.’’ Comment: The letter of September 22, 1944 of Butenandt to Von Verschuer is missing in the collection. Part of the laboratory of Von Verschuer had been moved to Beetz. That there was a collaboration between Butenandt and von Verschuer through Hillmann is documented in this letter. October 4, 1944: Von Verschuer reports to the DFG and mentions the collaboration with Hillmann. November 2, 1944: letter of VonVerschuer (Berlin) to Butenandt (Tu«bingen) ‘‘The analysis of kynurenine is a glowing example of


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the successful collaboration of biochemistry with genetics. I hope very much, that the collaboration with you and Diehl in the tuberculosis research will reach the goal we hope.’’ Comment: The animal model has to be solved first. The goal to find a molecular basis of the different susceptibilities is high, indeed. November 16, 1944: von Verschuer gives a lecture on ‘‘the effect of genes and parasites in the human body’’ before the Prussian Academy of Sciences. It is strange that no letter exists with the manuscript or at least the abstract. Did they meet in the Academy? The abstract of the speech, but not the speech itself, exists in the Academy. In the von Verschuer collection in Mu«nster, both the abstract and the manuscript of the talk are missing. In his talk von Verschuer spoke about the project ‘‘specific proteins.’’ I quote from the abstract: ‘‘Thus future research will have to make a pheno-genetic analysis of those infectious diseases from which we know that inheritance is a central issue; the important details of the defense mechanisms will be then analyzed. Individuals with a defect in the defense will then be given artificial defense material as we have shown in principle in the case of lepra and tuberculosis.’’ The next letter of von Verschuer comes from Solz, where he had fled from Berlin. February 19, 1945: letter of von Verschuer (Solz near Bebra) to Butenandt (Tu«bingen): ‘‘. . . At the moment we cannot think to install laboratories with complicated machines. May I turn to you with my Sorgenkind (problem child), protein research. My technical assistant Mrs. Haase has left for the time being, the institute. Mr. Hillmann wanted to come to me for a last discussion, but this did not take place. So I do not know what happened with him. I assume that he may have joined you in Tu«bingen. I am unable to know anything about your position in Tu«bingen. Yet I would be grateful if you could consider the possibility that Mrs. Haase could continue her work together with Mr. Hillmann in Tu«bingen. Then our laboratory, with all which belongs to it, should be joined to a transport which you may

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realize from Berlin to Tu«bingen. Mrs. Haase has left the laboratory with the special rabbit cages to collect the urine in the Dahlem institute. I have just taken with me the particularly valuable and irreplaceable protein substrates. I wait for your comment and answer until I will become active.’’ Comment: Verschuer said goodbye to Mrs. Haase before he left Berlin. Mrs. Haase was the technical assistant who, guided by Hillmann, should analyze the sera and had begun with this analysis. When I wrote my book I found her and talked to her. She confirmed that she had worked in the KWI for biochemistry for Hillmann she could not tell what the experiments were all about. This seems rather normal.‘‘The particularly valuable and irreplaceable protein-substrates’’ were taken by von Verschuer to Solz,‘‘valuable and irreplaceable’’ indeed. January 19, 1945: only very few days were missing before Auschwitz was liberated. Febuary 25, 1945: Letter of Butenandt (Tu«bingen) to von Verschuer (Solz). ‘‘I am so glad that your letter of February 19 reached me . . . Mr. Hillmann is still in Dahlem. There he is my replacement. He has just two young collaborators. He will stay there as long as this is still possible. He just wrote me, that he will stay to the last moment. Only then he will try to reach us. I left it open for him to decide whether he wants to come to Tu«bingen or to join a smaller group in Go«ttingen. You can thus contact him about the protein research in Dahlem. As long as Hillmann is there the experiments on the protein research could continue. What will happen then is difficult to say . . .. I have also consulted with Hillmann independently of you with letters and I will do whatever I can to make it possible that you continue your work.’’ March 21, 1945: letter of von Verschuer (Solz) to Butenandt (Tu«bingen) ‘‘For your letter of February 28 I thank you very much . . .. I wrote to Mr. Hillmann to Dahlem, but so far I received no answer. If it were possible to reconstruct the laboratory in Go«ttingen, I would be very glad. But I do not know whether, at the present state, one may even only think about that.’’


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September 25, 1945: Letter of vonVerschuer (Solz) to Butenandt (Tu«bingen) ‘‘Just a few days ago I received your letter of June 24. Our joint scientific work has to pause unfortunately at the moment. The protein research cannot be continued, Mr. Hillmann is in Dahlem and Diehl in Sommerfeld. But I hope that the dividing border which separates us, will fall and that reunification of the separated parts will be possible and research will start again.’’ Comment: The letter of Butenandt to von Verschuer of June 24 is missing. Thus, at least two letters of Butenandt are missing. The letter exchange Butenandt^von Verschuer is not complete. The letters which do exist demonstrate that Butenandt was informed that his collaborator Hillmann helped von Verschuer how to determine the defense enzymes in cases of tuberculosis. It is evident that rabbit sera were used. In the letters human sera are not mentioned explicitly. Verschuer uses the code word of his DFG grant ‘‘specific proteins.’’ There they are clearly mentioned. Here they are called ‘‘particularly valuable and irreplaceable.’’ It makes sense to assume that von Verschuer and Butenandt talked about the project, when Butenandt was in Berlin. It makes sense to assume that Butenandt wanted to know what kind of sera they were and where they came from. It may have been sufficient for him to hear that they came from a ‘‘Lagerlazarett,’’ a camp hospital. Many people did not want to know specifically what this was all about. I will come back to this. And now to the letter exchange Butenandt^Hillmann.

Letters Exchanged between Butenandt and Hillmann: the Destruction of the Top Secret Papers The letter exchange Butenandt^Hillmann begins, when Butenandt’s second hand in Berlin, Ulrich Westphal, moves with his collaborators from Berlin to Go«ttingen. Butenandt’s group in Tu«bingen consists now of forty collaborators. The group directed by Hillmann is small in comparison. It is just

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a group of a dozen collaborators, only one of them having a PhD. Hillmann wrote on February 3, 1945 about the work done in Berlin by the collaborators of Butenandt. There is no mentioning of the project of Verschuer where Hillmann advised. In another aspect the letter is, however, remarkable. Thus Butenandt writes to Hillmann on February 5, 1945 before he had received Hillmann’s letter: ‘‘My desk in my office can stay as it is. However, we have to think about saving or destroying the ‘geheime Reichsachen’ (top secret material) which are in the safe at the weighing room on the first floor. Moreover, there are, very few publications of the Academy of Aviation which are marked ‘for official use only’ in the book case next to the door of my office. I ask you to save these documents or to destroy them. There are only a very few of the many copies.’’ Hillmann answers on February 15: ‘‘At the moment, I see the situation in Berlin very realisticly, very awake, but also calmly. I thus ask you to give me freedom to decide.You can be sure that I will do your business, save your books or possibly destroy the secret material.’’ On February 27, 1945 Butenandt writes to Hillmann (Berlin): ‘‘I am astonished about Dr Pirquet’s behavior. I have not yet received a report of his work, just a report on Ruhenstroth’s bad health. Pirquet wrote just a few lines about planned experiments, nothing but that. I am not informed about Ruhenstroth’s dealings with the Air Force, about the movements and the plans of the research group of Ruhenstroth I am not informed . . . . I am unable to take the responsibility for the research project of the Air Force, when I am not informed about the details, and when it has been changed without informing me. If possible, speak with Bieneck and Ruhenstroth about this topic.’’ On March 3, 1945 Hillmann writes to Butenandt (Tu«bingen): ‘‘If during the next week a truck could be found, Dr Bieneck will help me with the transport of the relevant material. Otherwise he will go back to Tu«bingen at the end of this week. He will then take with him part of the chemicals and the secret material.’’


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On March 8, 1945 Hillmann (Berlin) writes to Butenandt (Tu«bingen): ‘‘I give this letter to Mr. Bieneck who will bring everything to Tu«bingen. The most important secret material will be brought to Tu«bingen by Bieneck, the rest I would take with me. . ..’’ Thus a large part of the secret material was brought to Butenandt in Tu«bingen. A careful view through the letters of Butenandt shows no trace of this material. Butenandt must have destroyed it entirely. The same is true for the material left in Berlin. Hillmann must have destroyed it.

War Important Projects of Butenandt What was the content of the papers labeled ‘‘Geheime Reichssachen’’? I presume that it were war important research projects, which should not be left to the enemy, because they would help the enemy or compromise the German reseachers. Such projects start small. They have precursors. They grow. In this context the war important projects of Butenandt will be analyzed. Before I begin, I will quote extensively a lecture Butenandt gave on January 23, 1941, at the high point of German military successes, before the Prussian Academy of Sciences [11]. This lecture is not quoted in the publication list of the biography of Karlson [1]. It is also not part of the 906 pages of Butenandt’s speeches and lectures on politics and science [12]. It is worth to be remembered. ‘‘When in times of war the soldier protects the borders of his country or by winning space advances beyond them, then the cultural products of the Nation give the fight a deeper sense, but the products of scientific work determine the quality of the weapons the soldiers use. Our present life is in an equal manner characterized by the use of the soldiers’ lifes, science and technique. Scientist and soldier are close to each other in the first front of our life and war.’’

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‘‘Both fulfill their duty in their self-evident effort. They see the wage for their work in the success of their work and know about the deeper sense of the words of Napoleon: ‘the man of science and good military men are priceless. No state is rich enough to pay or to reward them. All what one can do for them, is to prove, that one estimates them extremely.’’’ ‘‘No time has demanded in such an extensive manner the use of all reserves like the young National Socialist epoque of our people. This is particularly true for the fields of science. Among them chemistry has the largest task. This area of science dominates the picture of the world in growing manner. It is said correctly again and again that we live in the century of chemistry. . .’’ ‘‘We are certain about the continuous progress of science, as long as we try to guard the flame of the pure desire to understand, and as long as we give it intact to the coming generations. To guard this flame and to increase its light this is our vocation! To remember this duty is our vow that we love to renew at the day which serves the memory of one of greatest thinker and politician (Frederic the Great, BMH) and at a day, which situated between the 18th and the 30th January serves to remember the founding of the second, and our long desired, and loved Great German Empire (Reich).’’ The lecture was a success. Butenandt notes in his calendar: January 23, 1941: ‘‘Lecture before the Academy . . . preparation . . . My lecture‘the biological chemistry in the service of the people’s health’ . . . strong echo. In the evening at home we celebrate with wine and music.’’ Butenandt’s collaboration with the Air Force starts a year later, in January 1942, when the German advance in the Soviet Union came to a sudden stop at Leningrad and Moscow. A fast victory was now out of question. Butenandt followed the invitation of Theodor Benziger, the chief of the Experimentation Station of the Air Force in Rechlin. I quote Butenandt’s calendar notices: January 7, 1942: ‘‘Travel to the Experimentation Station Rechlin . . . visit of Rechlin. Scientific experiments until the


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evening. Dinner at Benziger’s with Ro«pke. 9:15 am Start of Junker air plane in fog and snow. 10:10 landing in Berlin’’: January 13, 1942: ‘‘Visit of Benziger. Discussion of work plans’’ Janurary 15, 1942: ‘‘Visit of Heusner. Discussion of work plans.’’ At the same time Butenandt receives an honour from the Air Force. January 22, 1942: the chancellor of the German Academy of Aviation writes to Butenandt: ‘‘I have the honour to inform you, that the president of the German Academy of Aviation, Reichsmarschall Go«ring has promoted you to a corresponding member of the Academy with the beginning of March first.’’ We do not know the research project of Heusner. Another postdoc of Butenandt, Dr Ruhenstroth-Bauer began to work on a problem of aviation research in 1943. He was trying to isolate the hormone hematopoietine, which was thought to increase the number of erythrocytes. The use of this agent, thus was the opinion, should make it easier for pilots to survive in great heights. On March 1, 1944 Butenandt reported on this topic to the Generalverwaltung of the KWG.‘‘In the Kaiser Wilhelm Institute for Biochemistry including the workstation forVirus Research of the Kaiser Wilhelm Institutes of Biology and Biochemistry the following war and state important research problems are worked upon . . . [8] Research on agents of blood forming. Codeword Ha«mopoietines. Work number Az 55Nr 3011/42 (Lin 14,2 IIB) of the Air Force.The experiments which are done together with the Experimentation Station of theAir Force in Rechlin have the goal, to gain knowledge about the chemical compounds which are important for the growth and doubling of the red blood cells. Knowledge of such substances would open up new possibilities under anomalous low air pressure (flight in high altitude, low pressure in submarines).The experiments are in a first stage, the physiological basis is so far not well understood.’’ Four reports of Ruhenstroth-Bauer on this topic are in the Butenandt papers. One is without date, the others have the dates of January 15 to June 30, 1943; January 15 to April 14, 1943. July 1, 1943 to March 31, 1944. From then on there are no more reports.

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There is extensive documentation of animal experiments with rabbits. Part of the experiments deal with antibody formation at low oxygen pressure in the low pressure chamber of the Air Force. Most of the experimental animals died after antigen injection in the low pressure chamber in contrast to the controls. Experiments with humans are mentioned in two reports. One in a report dated and signed by Ruhenstroth-Bauer 1943 ‘‘On the relation between low protein diet and immunisation:’’ ‘‘I therefore began to treat people sick with protein-oedemas with an unspecific push (Stoss) immunisation. I injected them several times highly antigenic proteins every fifth day intravenously. I choose first virus and then bacterial proteins as antigen, in particular after I found out that the virus protein immediately after injection led to strong convulsions of the belly, without further symptoms. As most active protein I choose pneumococcal protein of type I . . . After several preliminary experiments it seemed best to deliver the antigen in the following manner: On the first evening 2 ccm are injected intravenously, the next morning 5 ccm. These injections are repeated after 5 days in a similar manner. If necessary the injections are repeated a third time after another 5 days. Until now 48 oedema sick Russians have been treated. With 8 patients I could follow the state before and after the treatment. In regard to the others I rely on the report of other doctors . . . As a result can be reported that of 48 patients 35 were either fully capable of doing work after 8 to 12 days of treatment, or they were capable to do light work; controls which were untreated, needed weeks to become slowly better. . . . It was not possible, to observe all patients over a longer period after the Stoss-immunisation. Apparently none got again oedemas, even when the food and work conditions did not change.’’ It is not stated where the experiments where done. The ‘‘patients’’ were probably prisoners of war. It may be recalled that more than half of the Russian prisoners of war died from hunger. Another collaborator of Butenandt, Ulrich Westphal, reports in a letter of August 22, 1944 from the Heeresgebirgssanita«tsschule


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(mountaineering hospital school of the Army) St. Johann in Tyrolia, where he spent three weeks: ‘‘We made several experiments with humans on the metabolism at low temperature (in cold water) before and after the injection of cortiron or testoviron. The calculation of the values is not yet ready. In the meantime Miss Bork has become familiar with the determination of cholesterol according to Schmidt-Thome. Everything works fine. Now begins the analysis of the various organs and blood of the experimental animals during adaption to the cold.’’ A report on these experiments does not exist. It is unclear who were the human persons tested. When Butenandt transferred the main part of his laboratory to Tu«bingen,Westphal stayed first in Berlin.Then he was responsible for the destruction of the secret material.Thus exists in the correspondence of Butenandt^Westphal a page: ‘‘Confidential for the chief of the institute: When danger of the enemy coming close, all secret material has to be destroyed according to plan.’’After Westphal left, Hillmann became responsible. It is worth noting that Westphal continued his career after 1945 in a laboratory of the American Army, Fort Knox, Kentucky. Benzinger too went to the US where he joined an Army Institute. Finally, the collaboration with Professor Waldmann, President of the State Research Institute in Riems near Greifswald, should be mentioned. Butenandt and Schramm together visited the institute in Riems. Schramm then worked there several months and introduced a collaborator of Butenandt in the techniques. The goal was to prepare large amounts of some human and animal virus. Mentioned are three animal virus: the foot and mouth disease virus, the fowl plague virus, and the Teschen virus. It is not known, how these viruses were tested or if they ever were used as weapons. I summarize. The main research done in Butenandt’s laboratory was fundamental research. I have not written about that. The applied research directed by Butenandt and done by his collaborators cannot be described clearly, since most reports are missing and this work is thus badly documented.

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The applied work of Butenandt’s laboratory was honored by state agencies. November 26, 1943: Ernst Telchow, the director of the KWG wrote to Butenandt: ‘‘I have been asked by the Reichsminister fu«r Bewaffnung und Munition (secretary of armament) and by our president to inform you that der Fu«hrer (Hitler, BMH), decorates you with the date of October 1943 with the Kriegsverdienstkreuz I Klasse. Herr Reichsminister (the secretary of armament) wants to decorate you personally with this honor.’’

Butenandt and National Socialism Not only Mengele, but also von Verschuer was a product of National Socialism. How did Butenandt see the National Socialists? How did his view change in 1933 when they got power and then in the years until their defeat? How did he see them as president of the Max-Planck Gesellschaft (MPG) in the Federal Republic of Germany? Before I will try to present the view of Butenandt, I will quote from two letters written to him by his teacher, Nobel Prize winner Adolf Windaus. It is revealing that Butenandt’s answers are not kept. Windaus wrote to Butenandt on April 13, 1933 from Freiburg i.Br.: ‘‘Unfortunately I did not have the inner calmness to enjoy the days. To see injustice and being unable to help is difficult to bear. I just came back from the Kaiserstrasse (Freiburgs mainstreet, BMH). A man stood there and screamed without stopping ‘The Jews will be annihilated.’ Even if he did not imply physical annihilation, it moved me to see policemen letting him stir up hatred. It is shattering to see how easily bad instinct may be awakened, brutality, hatred, envy and how fast the feeling for justice and humanity gets lost.’’ ‘‘That Schoenheimer leaves is a big loss. Do these people know that Haber secured nitrogen production and that he organized gas warfare! What do they know, how respected Willsta«tter is abroad and what he has done for German science and economy. At this moment the people carry posters on which the most


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incredible and meanest slander is printed. I am curious, whether the chiefs of industry are right when they say: Hitler is going to act like Mussolini, he will separate himself from the extremist elements which brought him to power.’’ On July 20, 1933, Windaus wrote once more to Butenandt: ‘‘What you write about the professorship of Dr Mentzel is incredible. That a man who lacks entirely scientific interest, like Mentzel, gets a professorship only because he is a party member is the ruin of our science! I never experienced anything similar under earlier governments.’’ ‘‘In Switzerland I read often the Manchester Guardian and a newspaper from Saarbru«cken, now the most read newspaper outside Germany (‘Deutsche Freiheit’). What I read there was shaking, of course I do not know whether is is true’’. ‘‘I received a letter from the Verein Deutscher Chemiker (the association of German Chemists) a letter in which I was asked to inform who of our local group belongs to the NSDAP ( the Nazi party, BMH). I will not write such a letter and I leave the local group. Please give the papers I include to our chief, Professor Jander.’’ How, did Butenandt, the student of Windaus, see the coming National Socialsm? In 1933 Butenandt was asked to send his personal data to the Deutsche Fu«hrerlexikon (the who is who of German Leaders). On November 10, 1933 he wrote about that to his parents: ‘‘Yesterday I was surprised: the government edits in collaboration with the party a book ‘the leaders (?) [Butenandt’s question mark, BMH] of the new Germany.’ This book should contain what is worth knowing of those personalities whose name have importance, so they write. I have been informed that I should be part of this book. I thought about it seriously and agreed. Is this a change, that even people who are not in the party get called? ^ Today Schro«dinger got the Nobel Prize, after he unfortunately yesterday exchanged Germany against England. We have to collaborate.’’ Thus a short biography of Butenandt appeared in the Deutsche Fu«hrerlexikon 1934/1935. He calls himself ‘‘rein arisch’’

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(purely arian). Glancing through the volume, one finds that only very few scientists are included. How did Butenandt get this honor? Nowhere in the letters of Butenandt written between 1933 and 1945 I found a critique of the antisemitic measures of the Nazis. There are also no antisemitic sentences. If one had just the letters of Butenandt one would not know that Jews existed and that they were persecuted. The only critique I found about the Third Reich deals with the dissolutions of the ‘‘studentische Corps,’’ the old student fraternities. On October 4, 1935 Butenandt wrote to his parents: ‘‘Here the new teaching period begins soon, and we still do not know how many students will come. On the other hand the colored student caps will disappear forever. The Corps were dissolved and so they got what 14 years of social democracy was unable to obtain.’’ And how saw Butenandt the end of National Socialism? Manyof his collaborators were drafted during the war. His group was very large. Including the female collaborators, there were about fifty persons. Butenandt fought for every single one. He asked those, who were drafted, to write letters to the Arbeitsgemeinschaft (working group).Thus he wrote on January 10, 1944 to his drafted collaborator Lieutenant Hellmann: ‘‘You write in your letter that you do not understand why we do not all go toTu«bingen. Certainly in many respects we could work there more calmly and leisurely, and it might be even good for the work. But we have a duty to keep the workingplace in Dahlem, under all conditions. It would certainly fall apart if there were not enough people willing to act and to repair. Besides that it is something with a great and important moral side. I am not happyabout the here and there observed flight into personal security which is then presented as doing the duty. Do you not understand, as a soldier, that one does not give up the front, as long as there is reasonable reason, that one may be able to defend it? Why should we all leave, when millions of Volksgenossen (Germans, BMH) have to stay like the soldiers at the front? By the way, the terror-bombings have only bettered our mood.This is very good and it is apparent also in our small work-group. The often


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quoted word ‘soldiers at the front are better people’ is true too where the Heimat (the home, BMH) is entitled to compare itself with the front.’’ Butenandt kept this spirit to the end. So he wrote on December 21, 1944 from Tu«bingen to his collaborator Danneel, then Go«ttingen: ‘‘We are most happy about the offensive in the West, last not least because this led the less attacks by the allied airforce. Hopefully this continues as we wish it.’’ And how saw Butenandt National Socialism after it was defeated? In a letter of January 9, 1947 to the Swiss Emil Abderhalden who had moved from Halle to Zu«rich he wrote: ‘‘The following could be said about my position in National Socialism: Before 1933 I defended publicly the democratic state and I signed a public call against the election of Hitler as President.Thus I had to suffer after the Nazis took power.’’ Procter [10] mentions a call to vote for Hindenburg which Butenandt had signed in the spring of 1932 in a journal of Go«ttingen. Butenandt was a national conservative and thus not the ideal man for the Nazis of Go«ttingen University. This is documented in a report [12]. He was not ideal but he was accepted by the Nazis. October 16, 1936: W. Blume of the NS Dozentenbund (the Nazi organisation of the university professors) wrote to the Kreisleitung der NSDAP (to the organisation of the Nazi Party) in Go«ttingen. ‘‘Report on Professor Dr Phil. Adolf Butenandt.’’ ‘‘Science: Butenandt is in his field one of the greatest.’’ ‘‘Education: I never observed anything dubious or negative. He has an excellent record.’’ ‘‘Character: charming, smooth, a smooth man of the world.’’ ‘‘Politics: Butenandt should be evaluated with great precaution. Before we took power he was a democrat and fits therefore perfectly the general view of the Chemical Institute of Go«ttingen. After we took power he did not make the slightest connections with National Socialism. He is as foreign as before. A Colleague of the Dozentenbund from Danzig told me in Altrehse, that Butenandt refuses in every possible manner National Socialism

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like the SA-Dienst (working as a stormtrooper). Yet he makes all this in such an elegant manner, that it is impossible to catch him.’’ ‘‘In spite of all that we will not be able to do without Butenandt the scientist. Heil Hitler.’’ When the KWG discussed the possible successor of Neuberg, Professor Mentzel of the ministry involved said that Butenandt was unacceptable for political reasons. It is not clear if, when and how Mentzel eventually changed his mind [14]. This is how the Nazi elite saw Butenandt. But how did he see them? Butenandt continues in his letter to Abderhalden: ‘‘In the year of 1935 I and about 250 persons of the public life of Danzig were asked by the Gauleiter (party chief ) of Danzig to join the list of people asking to become members of the Nazi party. Thus we would declare our belief in the idea of Great Germany. After considering this seriously I agreed and thus I was on the waiting list for the Party membership since 1936. Perhaps this step seems inconsequential, but I believe that under similar circumstances I would do it again.’’ ‘‘The ideology of National Socialism was a mosaic of diverse elements: to promote the good and to fight the bad within the boundaries of my work area seemed to me only possible when in touch with the Party. This particularly in Danzig which had then a national socialist government. As I experienced later, my view of the world was not wrong.’’ These are remarkable sentences. What was the ‘‘good in the National Socialism which should be promoted?’’ I assume the good was the fact that in Nazi Germany much more money was spent for fundamental research than in the Weimar Republik. Thus, the Third Reich offered a general chance for scientists. In addition, it offered the chance to young scientists to enter the positions of the Jews which had been fired. Butenandt himself profited from that. When Carl Neuberg had not been fired from his post as the director of KWI, his institute would not have become available for Butenandt. How saw Butenandt the NSDAP? ‘‘After I moved to Dahlem in the autumn of 1936,’’ so he wrote to Abderhalden in the letter


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quoted above, ‘‘I had no close relations to the party. I never became a member of the Party. I did not give the oath. I was thus on the waiting list until 1945. This was my only formal incrimination:’’ The Butenandt papers in the Document Center in contrast, show him as a party member. Butenandt’s diary shows a notice ‘‘February 27, 1940: 20:15 pm general member meeting of the Party group of the NSDAP in Dahlem: Partyman Wa«chter speaks.’’ Finally, there exists a letter of Butenandt from October 17, 1946 to the Dozent (assistant professor) Dr Robert Haul. Haul had asked Butenandt for a letter supporting him. He reminded him that they first met in Danzig, then Berlin. Butenandt answered: ‘‘Unfortunately I cannot help you by writing a letter in support. I would have liked to do so but you know, of course, that I was myself a party member. Therefore I think it is not OK and also not good for you when I write such a letter. With this argument I have denied repeatedly to write letters to other colleagues in a similar situation. I hope that you understand and that you get in every sense again a restart of your scientific career.’’ And Butenandt’s lectures outside Germany during the war? ‘‘I believe,’’ Butenandt wrote to Abderhalden, ‘‘that story is clear. The claim that I have made propaganda outside Germany cannot hit me. I was often outside Germany always trying to build bridges, to forward understanding and to enhance collaboration. I always followed invitations from foreign countries. I was never willing to let me be sent. Therefore I declined four times the wish of German officials that I may give lectures in occupied France.’’ For the true scientist the real political world disappears.What counts and remains is science. Everything else is not perceived and therefore does not exist. The past was scientifically fertile, that is it was good, or it does not exist. I will now quote from several letters of Butenandt’s. On February 13, 1946 Butenandt wrote to Hillmann and gave him advise for the future which is also his own: ‘‘I am certain that today many people think about the past. One should not do it

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too much. Only those will survive the massive changes who are willing and able to work with all their force for something new.’’ On October 3, 1949 he wrote to Telchow, the former Generaldirektor (general director) of the KWG, and now Generaldirektor of the MPG: ‘‘My regard to you, dear Mr. Dr Telchow, has not changed. All what I said before to you and what I wrote to you is still my opinion. I am happy that I could help you in the difficult time after the war, and that I had occasions to defend you when different opinions turned into dangerous points. I have forgotten nothing what you have done in the last 13 years of my membership of the Kaiser WilhelmGesellschaft for my institute and for me. I also regard highly what you did for the existence of the Kaiser Wilhelm-Gesellschaft during and after the war.’’ Georg Graue, the Dozentenfu«hrer der freien Forschungsinstitute (the (Nazi) leader of the professors of the free research institutes, i.e. the Kaiser Wilhelm-Institutes) wrote on November 13, 1948 to Butenandt: ‘‘After a pause of about almost four years comes the first sign of life from me, the man believed to be dead. It is a miracle that I survived the years. There are not so many who come back. Most of them are broken in body and soul. I have been rather stable.’’ Graue asked Butenandt for reprints. Butenandt answered on August 10, 1949: ‘‘. . . it was a real great pleasure for me to hear that you overcame the many difficulties which were in your way and that you are reunited happily with your family . . . Of course I fulfil your wish and send you the relevant reprints . . . with affectionate greetings from house to house . . .’’ The letter exchange continues. On February 19, 1975 Butenandt wrote to Graue: ‘‘. . . we hope very much to see you and your wife again at the next Hauptversammlung (general meeting) of the Max-Planck-Gesellschaft. Then we can talk with each other and discuss what we think necessary and important for the future of Germany. With best wishes from house to house. . .’’ On February 12, 1947 Butenandt wrote to Eugen Fischer, the former director of the Kaiser Wilhelm-Institute for Anthropology,


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Human Genetics, and Eugenics: ‘‘It was a great pleasure for me to hear through your letter of January 18 from you and to find confirmed that you and your wife are well. How beautiful that you have the peace to write a new book . . .’’ Butenandt apparently did not recall that Fischer had been the rector of Berlin University in 1933/1934, that he fired the Jewish professors and assistants, and that he, in the years that followed, again and again supported the racial measures of the Nazi regime. On March 26, 1975 Butenandt wrote to the anthropologist Gieseler: ‘‘. . . It was a really great pleasure when I opened your little package last week, glanced at your book ‘The fossile history of man’ and read your friendly lines . . . What a pleasure to recall the joint happenings on good and bad days which come back to me through your lines and what a pleasure to accept your present and your dear words . . .’’. Finally, I quote from a letter to Karin Magnussen [15]. Magnussen had received her PhD under Ku«hn with work on the eye color of some insect. She then got a stipend of the DFG to work at the Kaiser Wilhelm-Institute of Anthropology first under Eugen Fischer, then under von Verschuer. In 1944 she received several pairs of heterochromatic eyes of a gypsie family which was murdered by Mengele in Auschwitz. Butenandt knew about that. He wrote her on August, 20, 1982: ‘‘I was enchanted to hear that you continue your scientific work. Moreover the work on the formation of eye pigment in rabbits reminded me about the old times spent in Dahlem.’’ Butenandt supported his collaborators. Ruhenstroth-Bauer who worked all his life on erythrocytes and as I understand never published a real breakthrough, became in 1967 MaxPlanck-Director in the Munich Institute for Biochemistry. This had consequences. On November 16, 1961 Ruhenstroth-Bauer answered for Butenandt a letter of Heinrich Matthaei who asked for the position of a postdoc in Butenandt’s laboratory. Ruhenstroth-Bauer wrote: ‘‘As far as I can see the only possibility would be a postdoc position under the direction of Dr Zillig. The problem of a fast Habilitation will meet various difficulties.

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Only after a longer time spent actively at the institute and after relevant productions this could be ripe for decision. . .’’ Ruhenstroth-Bauer wrote this to a man who just had made one of the best experiments of those years. Matthaei had solved and published a fundamental problem of the genetic code: Poly U codes for polyphenylalanine and Poly C codes for polyproline. The letter of Ruhenstroth-Bauer demonstrates that he did not understand what Molecular Biology was all about. I was alerted that Butenandt had perhaps other reasons not to accept Matthaei. Whatever, in any case, it was a bad decission.

Butenandt: Master of Selective Perception We all use the technique of selective perception. It is a technique which enables to survive. Who does not use it is lost. The horrors of the world are so large that they destroy those who do not look away from time to time. No success in life, craziness or psychiatry are the answer for those who consequently refuse to look away from time to time. I do not argue that Butenandt has done something what is completely alien to scientists. Selective perception is used everywhere. But it is something else when the owner of a restaurant, a hair dresser, or a dentist use it than when one of the greatest scientists does it. When it is about science it is destructive. Conversation ends. Butenandt pushed selective perception to far. I will quote now an extreme example. In 1949, Butenandt together with the biologist Max Hartmann, the pharmacologist Wolfgang Heubner, and the Biophysicist Boris Rajewsky composed a paper which was to liberate von Verschuer from all accusations. I quote four central sentences: ‘‘The assumption that he (von Verschuer, BMH) would have been informed about the mass destruction in this camp by Dr Mengele seems to us an over-subtle, unfounded and incredible construction.’’ In a copy this sentence is slightly changed:‘‘The assumption, that he (von Verschuer, BMH) was informed about the mass


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destruction in this camp by Dr Mengele seems to us unfounded.’’ The second sentence says: ‘‘How far Dr Mengele himself knew at the relevant time, ^ i.e. during the time when the blood samples were sent ^ about the atrocities and murders in Auschwitz, is not clear in the documents we inspected.’’This claim together with the disinterest to find out why the blood samples were taken from whom, indicates a deep disinterest in truth and thus science. The authors conclude: ‘‘It would be pharisaean, would we today choose single events as unpardonable moral faults of a man, who was honest and valiant on his difficult path and who has demonstrated often his humanitarian and noble convictions. We signatories believe together that Professor von Verschuer has all the qualities, which predestine him to be a researcher and teacher.’’ This wrote Butenandt and his colleagues in the autumn of 1949. In 1988 Cristian Cobet, a bookdealer and sociologist from Frankfurt, wrote to Butenandt and asked him whether he would still think that the paper about von Verschuer was okay, Butenandt answered Cobet on October 12, 1988: ‘‘After studying again all the documents available to me I declare that I confirm today each word of the document signed by me and that will not have to change anywhere my way of thinking. I am convinced, that the three other signatories (Wolfgang Heubner, Max Hartmann, and Boris Rajewsky) who were trusted and venerated because of their character strength and their general attitudes, would give the same declaration, if they were still alive and if they could have had the chance to evaluate the facts and the knowledge of the years from 1949 to 1988.’’ In the letter exchange Butenandt^von Verschuer, two letters of Butenandt are missing. Is the missing of these letters a random event or is it due to selection and cleaning? In this context it seemed interesting to study another letter exchange. So I inspected the letter exchange Butenandt^Neuberg. The letter exchange begins with a letter from Neuberg to Butenandt from November 8, 1949. Neuberg thanks Butenandt ‘‘The publisher of the Zeitschrift fu«r Naturforschung sent me a copy of an issue in which you together with W.Weidel and H. Schlossberger dedicate

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me your article to my seventieth birthday. I felt surprised and honored by this attention. I thank you deeply.’’ Butenandt answers on December 5, 1949. ‘‘You made me a big pleasure with your letter of November 8. It is a pity that the issue which was dedicated to your 70th birthday came out so late through the difficulties of our time. But it made us all happy that our attentiveness could become effective for you.’’ Glancing through the letters, I noticed a letter of Theodor Bersin to Butenandt of January 7, 1949.: ‘‘How I understand from a correspondence with C. Neuberg, New York University, he feels pain that in Germany, to which he feels attached, no notice is taken of his 70th birthday, not to talk about any Wiedergutmachung (compensation) or invitation. Could you possibly, as his successor at the KWI, find a form to present a little pleasure for the old man?’’ Butenandt found the way. But this is not all. Among the letters of and to Neuberg in the Max Planck Archive the first letters are missing. The true letter exchange begins with a letter from Butenandt to Neuberg on January 29, 1947. The letters of the first years are painful to read. Are they therefore missing in Butenandt’s collection? The interested reader may consult the Neuberg papers in the Archives of the American Philosophical Society in Philadelphia, where the entire letter exchange can be consulted. All the letters cited so far were not written for a broad public. How saw Butenandt as president of the KWG/MPG the past? In his lecture before the Hauptversammlung (general assembly) of the Max-Planck-Gesellschaft of January 8, 1961 he said: ‘‘Today’s meeting as part of the 12. General Assembly of the Max-Planck society is under the sign of the 50th birthday of the Kaiser-Wilhelm-Gesellschaft, which was founded January 11, 1911 here in Berlin.’’ He speaks then about the KWG in the first World War and in the Weimar Republic. Then he comes to the years of the Third Reich: ‘‘Only the time after 1933 brought to the KaiserWilhelm-Gesellschaft irreplaceable personal losses of highly esteemed scholars and their schools and so also of gifted young scientists which are so important for the advance of science.


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Now the principle which was put into action in 1911 to have a broad distance between the state and its reglementions, paid off. Until the war began in 1939, we were able to have growing contacts with foreign countries and to stay with colleagues, who had to leave Germany, in scientific exchange of thought.’’ ‘‘We may book several successes in science until the year of 1945, the year of the entire breakdown, and end which led to a new beginning . . . Wrong thoughts about the work done in institutes, combined with a resentment against the name of the Society made the continuation of the old as difficult as the start of the new. I have to think gratefully about those foreign colleagues which due to deeper knowledge of the essence of the Kaiser-Wilhelm-Gesellschaft helped efficiently, to abolish all resentments which existed against the KWG.’’ Here and in the few speeches where he mentioned the past, the injustice done to the Jewish scientist is summarized as‘‘irreplaceable losses for the MPG.’’ That it was injustice done to Jewish scientists, which was only corrected in part and slowly after 1945, is said nowhere. That some institutes, like the KWI for Anthropology, the KWI for Psychiatry, and the KWI for Brain Research legitimized, propagated, or even commited crimes is never said. It is not mentioned anywhere. The words Jew, Jewess, Jewish, and antisemitism do not exist in the letters or speeches of Butenandt. If only his letters and speeches were left over, one would not know that Jews existed and that they were persecuted in Germany. This nonperception of the injustice committed was general in many parts of the society of the young Federal Republic. Butenandt was here a child of his time. But this attitude made discussion with Jewish scientists difficult. Science lives from discussion.Thus Molecular Biology started late in Germany.

Another View: Opportunisms and Power Hunger I have so far described Butenandt as if his opportunism was the price he had to pay to make scientific research. The ‘‘scientific

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letter exchange’’ which I was allowed to read suggested such a point of view. But is this the entire truth? Only after I had left the Archive I realised that the ‘‘private’’ letter exchange which did not interest me was not the only other form of letter exchange. There must have been a letter exchange with state agencies of all types, with firms, like Schering or IG-Farben. There was the letter exchange with the Nobel committee, with universities about professors to be hired. All these letters I had not seen. Moreover, after I left the archive, I learned that 25 meters of letters had so far not been worked upon [16]. I had not seen these letters. So I asked to be allowed another two days visit to have a look at these 25 meters. I was given access by the Generalverwaltung. So I worked myself through a mass of new letters. There was nothing earth shaking hidden among those letters. They were more of the same, I had seen before. Then and now power-hungry oportunists existed, who were excellent artists or scientists. I name here the actor Gustav Gru«ndgens, who was described perfectly by Klaus Mann in his novel ‘‘Mephisto’’[17], I mention the architect Albert Speer [18], the conductor Wilhelm Furtwa«ngler [19], and the rocket builder Werner von Braun [20]. Is Butenandt one of them? I think yes. Butenandt had power: He was able to animate the IG-Farben that they paid his virus institute. At the end of the war, he had still thirty collaborators in Tu«bingen. Twelve years he was president of the Max-PlanckGesellschaft. He had influence who got which academic job in biochemistry in Germany. So his personality has many sides. He was not only the great scientist but also the power-hungry opportunist who made every necessary compromise with the enemies of truth and justice of Nazi Germany. He had (Nietzsche’s) will to power. I know that Butenandt was the teacher and great model of many of my colleagues. A good teacher becomes a model, which is defended by the student. To make good science and to solve difficult problems is one of the greatest pleasures. The rest of


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the world here disappears. I understand that without difficulties. But for me, the scientist Butenandt has pushed selective perception of the world and opportunism too far. Many German scientist did not think differently then. But few were so extraordinary productive as Butenandt. He was a bad model. It is time that his students of the coming generations see this gulf. A discussion should be enlightening.

ACKNOWLEDGMENTS

I thank the Pra«sidentenkommision of the MPG to give me the possibility to inspect the letter exchange of Butenandt, Professor Eckart Henning, and Dr Ulrike Kohl of the Archiv zur Geschichte der Max-Planck-Gesellschaft for competent help, Dr Carola Sachse of the MPI fu«r Wissenschaftsgeschichte and the Harnack-Haus of the MPG for hospitality. Finally, I thank my friend George Klein, who alerted me on power, an aspect missing in the original text. Sources: Archiv zur Geschichte der Max-Planck-Gesellschaft, Berlin (MPG Archiv) Nachlass Adolf Butenandt II Abteilung, Rep84. When I inspected the letters they were not yet ordered in detail. I thus do not give detailed numbers. Bundesarchiv Berlin, fru«her Koblenz and Berlin Document Center (BDC) Parteiakten Butenandt (formerly BDC). Reichsforschungsrat, Korrespondens, Bundesarchiv Koblenz.

REFERENCES [1] Karlson, Peter (1990) Adolf Butenandt. Biochemiker, Hormonforscher, Wissenschaftspolitiker, Stuttgart. [2] Mu«ller-Hill, Benno (1984) To«dliche Wissenschaft Die Aussonderung von Juden, Zigeunern und Geisteskranken 1933^1945, Reinbek, RowohltVerlag. [3] Bundesarchiv Koblenz R73^15342. [4] Mu«ller-Hill, Benno (1999) Das Blut von Auschwitz und das Schweigen der Gelehrten. In: Geschichte der Kaiser-Wilhelm-Gesellschaft im

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Nationalsozialismus. Herausgegeben von Doris Kaufmann, Wallstein Verlag, Go«ttingen 189^227, (2000). English translation: The blood from Auschwitz and the silence of the scholars. History and Philosophy of Life Sciences 21, 331^365. [5] Verschuer, O.v. (1942) Zwillingsforschung und tuberkulose. Beitra«ge zur Klinik der Tuberkulose 97, 317^331. [6] Frewer, Andreas (2000) Medizin und moral in der weimarer Republik und Nationalsozialismus. Die Zeitschrift ‘Ethik’ unter Emil Abderhalden. Frankfurt a.M., New York, Campus Verlag. [7] Abderhalden, Rudolf: Bericht an die DFG April, September 1943, in: BA Koblenz, R73^10002. [8] Deichmann, Ute and Mu«ller-Hill, Benno (1988) The fraud of Aaderhalden’s enzymes. Nature 393, 1098^1111, Siehe auch: Kaasch, Michael (2000). Sensation, Irrtum, Betrug? Emil Abderhalden (1877^ 1950) und die Geschichte der Abwehrfermente. Acta Historica Leopoldina 36, 145^210. [9] Mu«ller-Hill, Benno, (1990) Genetics after Auschwitz: Holocaust and Genocide Studies 2, 3^20. German translation: Genetik nach Auschwitz. In: Die zweite Scho«pfung (Herbig, J, and Hohlfeld, u.R. eds.), pp. 79^105. Hanser, Mu«nchen. [10] Procter, Robert N. (2000) Adolf Butenandt (1903^1995) Nobelpreistra«ger, Nationalsozialist und MPG-Pra«sident. Ein erster Blick in den Nachlass. Ergebnisse 2.Vorabddruck aus dem Forschungsprogramm Geschichte der Kaiser-Wilhelm-Gesellschaft im Nationalsozialismus. Ed. Carola Sachse, MPI f.Wissenschaftsgeschichte, Berlin. [11] Butenandt, Adolf (1941) Die Biologische Chemie im Dienste der Volksgesundheit. Festrede am Friedrichstag der Preussischen Akademie der Wissenschaften am 23.1.1941 gehalten. In Preussische Akademie der Wissenschaften, Vortra«ge und Schriften, Heft 8, Berlin, De Gruyter & Co. [12] Butenandt, Adolf (1981) Das Werk eines Lebens Bd.I/1 (1043 pages); Bd.I/2 (1000 pages); Bd I/3 (861 pages) Wissenschaftliche Arbeiten. Bd II (906 pages) Wissenschaftspolitische Aufsa«tze, Ansprachen und Reden. Vandenhoek Ruprecht, Go«ttingen. [13] Akte Adolf Butenandt, fru«her Doc Center, Berlin. Jetzt Bundesarchiv Berlin. [14] Sitzung des Verwaltungsausschusses der KWG am 9.4. 1936. Archiv zur Geschichte der MPG Signatur I. Abt., Rep. 1A. Ich danke Prof. Dr Henning fu«r den Hinweis. [15] Hesse, Hans (2001) Augen aus Auschwitz. Ein Lehrstu«ck u«ber nationalsozialistischen Rassenwahn und medizinische Forschungen. Der Fall Dr Karin Magnussen. Klartext-Verlag, Essen.


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[16] Frau Dr Marion Kazemi, Archivarin im Archiv zur Geschichte der MaxPlanck-Gesellschaft, schrieb mir am 31.10.01: ‘‘Wie gross die ungefa«hre Zahl der bisher noch nicht geordneten Briefe (Butenandts, BMH) ist la«sst sich nur schwer beantworten. Es handelt sich um Ordner mit Sachakten und um lose, un Kartons liegende Unterlagen, insgesamt etwa 25 laufende Meter, die nicht nur, aber eben auch Korrespondens erhalten.’’ [17] Mann, Klaus (1936) Mephisto. Querido. Amsterdam. [18] Sereny, Gitta (1997) Albert Speer Sein Ringen mit der Wahrheit. Droemer/ Knaur, Mu«nchen. [19] Kater, Michael (1997) The twisted muse. Musicians and their Music in the Third Reich. Oxford, Oxford University Press. [20] Neufeld, Michael (1996) The Rocket and the Reich. Cambridge Mass, Harvard University Press.

The Fraud of Abderhalden’s Enzymes* BENNO MU«LLER-HILL

Earlier this century, the German biochemist Emil Abderhalden deceived the scientific world with his spurious ‘‘defence enzymes’’. Unless there is a change in clinical thinking, such a fraud could happen again. Ute Deichmann and Benno Mu«ller-Hill Is science a social construct, as some sociologists of science claim [1], or is its structure independent of social conditions? In the forefront of science, where everything seems possible, data may be misinterpreted and errors litter the path of science. In physics and chemistry such mistakes are usually quickly corrected by colleagues or the authors themselves. But what about fraud? Fraud assumes intention, which is difficult to prove. We think the deliberate invention and interpretation *Nature 393, 109^111, 1998. In the two preceding papers Mu«lller-Hill mentioned Abderhalden’s ‘‘Abwehrfermente’’ in connection with the, say, ‘‘dubious’’ research of Verschuer and Mengele. This third paper by Mu«ller-Hill recounts the story of these ‘‘defense enzymes’’ which, in fact, do not exist. The story is remarkable and instructive in more than one respect. It lasted more than half a century; it arose and was kept alive by a blend of self-deception and fraud. An overly authoritarial system (to put it mildly) is particularly prone to sustain this kind of false truths to the point of making them into a nearly untouchable myth. (The mileu in which Abderhalden opearated was perhaps congenial to his personality). But self-deception and fraud lurks in every scientific lab even now. Many of us know of junior collaborators who feared to face the disappointment or the anger of the powerful boss if he were to be told that his working hypothesis was not right, and began to make ‘‘a critical appraisal of the experimental data’’ ^ i.e. to ‘‘select the right experiments’’ ^ the first step towards outright forgery. As the Managing Editor of the FEBS Letters for nearly 14 years I was too often discouraged at seeing how fragile ethical behaviour can be. (Note of the Editor, GS). Reprinted by permission from Nature 393: 109^111. Copyright (1998) Macmillan Publishers Ltd.


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of data in science is a social construct. In physics and chemistry such social constructs are extremely rare; they are quickly detected and they have half-lives of just a few years. But we propose that this is different in medical science, in which science and social constructs may peacefully coexist. We will demonstrate this by presenting the case of the nonexistent Abwehrfermente (defence enzymes) created by Emil Abderhalden (1877^1950), who was professor of physiology and physiological chemistry at Halle University from 1911 to 1950, president of the Leopoldinathe oldest German academy of sciencefrom 1931 to 1946, editor of several journals, and author of several books and more than 1,000 research papers [2].

Abderhalden’s Fraud Abderhalden was born in 1877 in Switzerland. He received a medical education at the University of Basel, and in 1902 he went to Berlin to work with the great organic chemist Emil Fischer on the synthesis of peptides and the action of proteases, which are enzymes that break down proteins. In 1908 he became professor of physiology at the Tiera«rztliche Hochschule in Berlin, and three years later became professor of physiology and physiological chemistry at the Univeristy of Halle. He was due to become director of the Kaiser WilhelmInstitut for physiology in 1914, but the First World War intervened. As a kind of compensation, the Kaiser WilhelmGesellschaft financed his research with substantial grants until 1944. Abderhalden’s fame as a biochemist was achieved through two types of experiments. First, with Emil Fischer he began to synthesize and isolate peptides, and in his career he synthesized and isolated more than anyone else in Germany. Unfortunately, little use was made of them. In 1909 he published his first work on the second topic, the Schutzfermente (protection enzymes) or, as he called them later, the Abwehrfermente. In 1912 he published

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a book about them, and considered them to be his most important discovery [3]; three new editions appeared before the end of 1914. According to Abderhalden, animals and humans produce specific proteases, called Abwehrfermente, when they are challenged with foreign proteins. For example, the serum produced by pregnant women contains proteases specific to proteins of the placenta. The test for this claim is straight forward. Placenta is boiled, and the denatured, insoluble placental proteins are treated with serum from a pregnant woman. Peptides that arise through the action of the defense enzymes in the serum are dialysed and then identified by Biuret or ninhydrin reactions. Sera from non-pregnant women and men supposedly do not show this reaction. This test intrigued gynaecologists and biochemists worldwide. Between 1912 and 1913, more than 25 papers from various gynaecological laboratories appeared that dealt with Abderhalden’s pregnancy test, most of them with positive results [3]. In 1914, the directors of German university women’s hospitals were asked by a medical journal to describe their experience with this test. Of the 15 that replied, all had more or less positive results, and none had negative results [4]. The excitement increased. In the fourth edition of Abwehrfermente (1914), Abderhalden quotes 451 papers, many of them in non-German journals, which describe various uses of his test. As well as in pregnancies it was used successfully in three other contexts: the diagnosis of sarcomas and other carcinomas; the diagnosis of infectious diseases such as syphilis; and the diagnosis of psychiatric diseases such as schizophrenia. Cancer therapy using Abwehrfermente seemed just around the corner.

Why No One Stopped Him We have to keep in mind that all these medical scientists deluded themselves: defence enzymes do not exist! It was a case


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of the emperor’s new clothes: when everybody sees and admires his elegant clothes, just one child can destroy the social construct by pointing out that the emperor is naked. This ‘child’ was the German^Jewish biochemist Leonor Michaelis. In 1913 he had just published with Maud Menten the seminal paper on enzyme kinetics. Working in the biochemical laboratory of a hospital, he was asked by its director to establish the validity of Abderhalden’s pregnancy test. He found that he and his collaborator were unable to repeat Abderhalden’s experiments, despite spending a week in Abderhalden’s laboratory in Halle. There was no difference between the sera of pregnant or nonpregnant women or between women and men: the pregnancy test did not work. In 1914, Michaelis and his collaborator published their negative results [5]; it marked the end of his academic career in Germany. But Michaelis was not the only biochemist who could not repeat the results. Donald van Slyke from the Rockefeller Institute and Florence Hulton from the Univerisity of Pennsylvania failed too [6,7]. In 1920 Jacques Loeb wrote [8] to Michaelis, who was still in Germany. ‘‘Nobody speaks of the Abderhalden reaction any more in the United States and I am very much surprised to see that in his journal Abderhalden still continues that myth’’. The reply from Michaelis [8] seems timeless: ‘‘In Germany one can succeed only when one presents practical, applied science, however bad it may be. Anyone who wants to work on pure science is regarded a crank, and so he finally stops working’’. About Abderhalden he wrote [8]: ‘‘For me his type of work is disgusting. My position in Germany has suffered because of my opinion against his pregnancy test. There may be many who see through him, but nobody dares to say anything against him’’. Michaelis left Germany in 1922 to become visiting professor at a Japanese university, and later became a lecturer at Johns Hopkins University in Baltimore, Maryland, and then a member of the Rockefeller Institute in New York. But how could Abderhalden continue with the Abwehrfermente from 1915 until his death in 1950? His strategy

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was simple and straightforward. He must have had collaborators who found what he wanted them to find (he dedicated the second edition of his book about the Abwehrfermente to his ‘‘faithful collaborators’’). In general, he argued that the pregnancy test and other tests had worked in a large number of laboratories; so many scientists could not have deluded themselves. He conceded that in some laboratories the test did not work properly, and claimed that this demonstrated that the method was difficult, that it was not properly used, and ^ alas ^ let it was not perfect. So Abderhalden and his co-workers continued to streamline his method. The test became technically more and more complicated, and supposedly safer and safer. But the material to be tested became simpler. His research received a fresh impetus when he published a paper saying that the pregnancy test could work with urine, which was much easier to obtain than blood and supposedly contained more specific Abwehrfermente than blood. And so a second wave of users appeared.

Sinister Applications In the 1930s and 1940s, a wide range of topics were analysed in half a dozen German institutes with the help of the Abwehrfermente [9]. There were tests for various forms of cancer; the final objective was cancer therapy. Tests for psychiatric diseases such as schizophrenia were developed; the Abwehrfermente were also used for effective shock treatment of psychotic patients. Tests were worked out to diagnose the various psychological types proposed by the psychiatrist E. Kretschmer [10]. (To test how patients dealt with fear, guns were fired behind their heads and pictures were taken; one schizophrenic patient is quoted as saying over and over again: ‘‘We want poison gas. . . why do they not give us poison gas?’’). The Abwehrfermente were used by Abderhalden’s son Rudolf [9] to diagnose various infectious diseases. They were even used to distinguish races of


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sheep [9]. Remember that none of these diagnostic tests could possibly work because the Abwehrfermente do not exist, so the therapeutic value of the injections was dubious to say the least. Some uses of the Abderhalden reaction were particularly disturbing. In November 1942, the human geneticist Otmar von Verschuer was appointed director of the Kaiser WilhelmInstitute for Anthropology in Berlin. His former postdoc Josef Mengele joined him there in the winter of 1942^43. Mengele had been wounded in the war and spent some months convalescing in Berlin before moving in April 1943 to the Auschwitz concentration camp to become camp doctor. He must have discussed the scientific possibilities of Auschwitz with his teacher, because, when Mengele left, von Verschuer immediately applied for a grant from the Deutsche Forschungsgemeinschaft to finance Mengele’s work in Auschwitz on the Abwehrfermente produced by members of various races deliberately infected with infectious diseases. They planned to use the Abderhalden reaction to demonstrate racial differences, and von Verschuer sent a technician to Abderhalden’s laboratory in Halle to learn the technique [11]. Aware that the technique was not easy, von Verschuer called on Gu«nter Hillmann to supervise the tests. Hillmann was an experienced biochemist who had worked in the laboratory of Karl Hinsberg, who had set out to improve the test of the Abwehrfermente for cancer cells; indeed, Hillmann himself had synthesized and tested a chemical that allowed a better quantification of the peptides released by the Abwehrfermente [12]. But he had difficulties with Hinsberg, and in 1943 he moved to the laboratory of Adolf Butenandt, who had won the Nobel prize for chemistry in 1939. Mengele sent blood samples from infected Jewish and Gypsy twins to Hillmann, who began analysing them. On 4 October 1944, von Verschuer wrote to a friend [11]: ‘‘Precipitates have been prepared from the plasma of more than 200 individuals of various races, some twin pairs, some families. Abderhalden’s method has been used and supplemented by a method newly

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discovered by Hillmann (who has joined us as collaborator). So very soon we can now begin our real research. The aim of our various efforts is now no longer to establish that the influence of heredity is important in various infectious diseases, but rather how hereditary factors act and what kind of events take place in their action’’. A workshop on Abwehrfermente was held in 1947 in Tu«bingen, chaired by Butenandt. In a two-page report on the meeeting [13], Gerhard Mall, a collaborator of Kretschmer, wrote that Mall, Hinsberg, Kretschmer and Bersin had found specific defence enzymes. But Butenandt asked for the use of chemically homogenous proteins to re-test the claims. That was not the last word on the Abwehrfermente, however. After Emil Abderhalden’s death in 1950, his son Rudolf declared that they were the perfect diagnostic tools to determine the optimal cell type for the Frischzellen-Therapie (fresh cell therapy) invented by Paul Niehans [14], and so for a few years the Abderhalden reaction was used again. But two clinicians demonstrated that it made no difference whether the patient was healthy or sick; the sera reacted just the same [15]. We do not know when the Abwehrfermente finally disappeared as a diagnostic tool. Last year the Frischzellen-Therapie was outlawed in Germany by the Department of Health, but this ruling has been contested by doctors who claim it infringes the rights of both doctors and patients. The Supreme Court of Germany is pondering the problem, but this is likely to take a long time.

Behind the Fraud It is worth noting that Abderhalden was a convinced eugenicist. Given that his main scientific activity was based on self-deception and fraud, it is interesting that between 1922 and 1935 he edited a journal about ethics (Ethik). He wrote a textbook of biochemistry that appeared in 28 editions between 1906 and


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1948 and was translated into four languages. He was president of the Leopoldina Academy from 1931 to 1950. After 1933, the notes ‘‘membership ended’’ or ‘‘membership extinguished’’ were secretly added to the file cards of the more than 90 Jewish members [16] of the academy; the members were not informed, but they no longer received the academy’s journal or invitations to events. All candidates for new international membership were discreetly vetted by the German foreign office to see whether or not they were Jewish [16]. After 1945, Abderhalden claimed that no Jewish member was ever expelled from the Leopoldina. Truth was not his business. Historians of science have largely ignored Abderhalden and his Abwehrfermente. The biochemist Peter Karlson wrote [17]: ‘‘Emil Abderhalden certainly did not ‘invent’ the Abwehrfermente, he worked in many fields, he was a distinguished professor. . . and he certainly did not need to increase his fame through dubious publications. . . presumably one will have to classify the literature on Abwehrfermente as ‘unconscious collection of wrong data’, some kind of auto-suggestion’’. Theodor Wieland, a peptide chemist, calls Abderhalden [18] ‘‘the founder of scientific biochemistry’’. About the Abwehrfermente, he wrote [18]: ‘‘Defence enzymes raised great hopes for theoretical biochemistry and also for practical medicine, which, however, in spite of intensive work, mostly with the participation of his son Rudolf, were not fulfilled. Thus they did not succeed in the isolation and characterisation of a defence proteinase’’. The immunologist Otto Westphal told one of us (U.D., private conversation) that his colleague Hans Brockmann wanted to work with Abderhalden of the Abwehrfermente: ‘‘Brockmann tested one or two systems, but was unable to reproduce them. He went to Abderhalden and told him that it worked the first time but not the second time. Abderhalden asked him why he repeated an experiment that worked well once. Brockmann left the institute immediately, considering Abderhalden to be a fraud’’. Westphal adds: ‘‘I had no doubt in the beginning myself, in fact I wrote a review on the Abwehrfermente in 1939. In 1942 or

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1943 I spoke to Brockmann. The whole Abwehrfermente story was a fraud from beginning to end’’. Never repeating an experiment that worked once, or discarding controls that did not work, is not science but pseudoscience or fraud. Abderhalden must have known this. It was his way of trying to seduce a young scienctist to join him in the fake world of science as social construct. But Brockmann was a real scientist and fled. Westphal is listed as a participant at the Tu«bingen workshop on the Abderhalden reaction [13]. Was he critical? Did the participants use a double language that allowed the true scientist to abandon the non-existent defence enzymes and the believers to continue their social construct?

Can it Happen Again? Abderhalden was a pure biochemist, but most of those using his method were medical, clinical biochemists. Such researchers often work during the day with patients in a hospital, and their experiments are confined to the afternoon or more likely the night. The senior author of research papers may be the director of a hospital. Clearly the universe of science is rather different from that of patients, who prefer to hear an optimistic diagnosis rather than the simple truth. It is difficult enough for the director of a large laboratory to validate all the experimental details of his collaborators, but for a physician, the director of a clinic who is clearly not possible. He must trust his collaborators, yet he is an authority. Truth may discreetly disappear. Clinicians were offered the possible advantages of the Abwehrfermente for diagnostic breakthroughs. Most of them failed to admit that their tests did not work. Excellent non-clinical biochemists such as Butenandt and Kuhn kept silent too, at best stating that the specificity of the Abwehrfermente was not rigorously proven. The existence of the Abwehrfermente was seriously questioned only by Michaelis, van Slyke and Hulton,


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and the possibility of fraud was never mentioned in public. In medical biochemistry, ideas or hope may be stronger than experimentally proven reality. It is true that some antibodies have catalytic properties, but the question is whether the existence of specific defence proteases can be measured and proved in repeatable experiments. Here Abderhalden and almost all the people in the field failed. At the time, Germany was regarded by many to be the leading country for medical science. The story is disturbing when we realise that it did not end in 1950 with the death of Abderhalden. The Abwehrfermente disappeared from the literature in the 1960s but nobody wrote a clarifying obituary. The elite of today are loyal students of old elite, and they have learned and internalized the old values. Has medical, clinical science in Germany today really changed that much? We doubt it. The Brach^Herrmann^Mertelsmann affair [19] provides a brief glimpse into the abyss of medical science in Germany. Will it be soon forgotten by the German medical elite, or will there be a real change in the spirit of true science? œ Ute Deichmann and Benno Mu«ller-Hill are at the Institute of Genetics at Cologne University, Weyertal 121, 50931 Ko«ln, Germany.

ACKNOWLEDGEMENTS

This work meinschaft.

was supported by

Deutsche

Forschungsger-

REFERENCES [1] Latour, B. and Woolgar, S. Laboratory Life. The Social Construction of Scientific Facts (Sage, Beverly Hills, 1979). [2] Anonymous Z.Vitamin. Horm. Fermentforsch. 4, 1^18 (1951^52). [3] Abderhalden, E. Die Schuftzfermente (Springer, Berlin, 1912).

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[4] Berlichte u«ber Krankheitsfa«lle und Behandlungsverfahren: Bedeutung der abderhalden’schen Untersuchungsmethode. Med. Klinik. 11, 453^455 (1914). [5] Michaelis, L and Lagermark, L.v. Deutsche Med. Wochenschr. 7, 316^319 (1914). [6] Van Slyke, D.D., Vinograd-Villchur, M. and Losee, J.R. J. Biol. Chem. 23, 377^389 (1915). [7] Hulton, F. s. J. Biol. Chem. 25, 163^171 (1916). [8] Library of Congress (1920, 1921). [9] Harrer, G. Z.Vitamin. Horm. Fermentforsch. 1, 484^503 (1947^48). [10] Kretschmer, E. and Mall, G. Fermentchemische Studien zur klinischen und konstitutionellen Korrelationsforschung speziell zur psychiatrischen Endokrinologie (de Gruyter, Berlin, 1941). [11] Mu«ller-Hill, B. Murderous Science (Oxford University Press, 1988). [12] Hillmann, G. Hoppe-Seyler’s Z. Physiol. Chem. 277, 222^232 (1943). [13] Mall, G. Z.Vitamin, Horm. Fermentforsch. 2, 47^48 (1948^49). [14] Abderhalden, R. in Die Zellulartherapie (ed. Niehans, P.) (Urban & Schwarzenberg, Munich, 1954). [15] Kanzow, U. and Schulten, H. Die Medizinische 13, 447^450 (1957). [16] Gerstengarbe, S. Leopoldina (R3) 39, 363^410 (1994). [17] Karlson, P. Naturwissenschaft Rundsch. 39, 380^389 (1986). [18] Wieland, T. and Bodansky, The world of peptides. A Brief History of Peptide Chemistry (Springer, Berlin, 1991). [19] Schiermeier. Q. Nature 389, 105 (1997).


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Chapter 11

The Sarcoplasmic Reticulum Ca -ATPase and the Processes of Energy Transduction in Biological Systems 2þ

LEOPOLDO DE MEIS Departamento de Bioqu|¤ mica Me¤dica, Instituto de Cie“ncias Biome¤dicas, Centro de Cie“ncias da Sau¤de, Universidade Federal do Rio de Janeiro, Cidade Universita¤ria, RJ, 21941 590, Brasil E-mail: [email protected]

Introduction Science in an institutionalized form began in the nineteenth century in Europe and USA and a few decades later in Japan. Since then science has grown exponentially and the large amount of new knowledge generated during the past 200 years has led to profound changes in everyday life. Most of this knowledge was, and still is, produced in a few countries: the USA, Britain, Germany, Japan, France, Canada, Russia, and Italy. At present, scientists in these countries are responsible for about 75% of the scientific papers published in the most important journals each year [1]. In many developing countries, including Brazil, there has been an increasing awareness of the need to develop science. In these countries, institutionalization of science is recent when compared to Europe and USA. In Brazil, the first federal agency to support science, the National Council for the Development of Science and Technology (CNPq), was created in 1951. When I entered medical school in 1956,

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there were very few active scientists working in Brazil. At the university most teachers had a whimsical view of science and the vast majority of students (including myself ) did not know what science was about. Starting in the late sixties and extending up to the present day, there has been a dramatic change. Now science is an institutionalized activity and in the main Brazilian universities, professors are required to maintain a significant scientific productivity in order to progress in their careers. Up to the late sixties, there were no formal courses leading to a further degree such as an MSc or a PhD. However, despite the absence of formality or a title, the training of scientists occurred, albeit on very small scale. Some scientists were trained in the country while others went to Europe or the USA. The creation of graduate courses in Brazil, served as a catalyst for the formation of new scientists [2] and this in turn led to an exponential growth of Brazilian science (Table 1). My generation has witnessed this transition and in this chapter, I will try to describe the scientific atmosphere that we lived in during that time. I will start by describing general events and personal memories without focusing on the specifics of the research

THE SARCOPLASMIC RETICULUM Ca2þ -ATPASE

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TABLE 1 The growth of science in Brazil Indicator

Year

Brazilian Courses MSc. PhD. Degrees awarded in Brazil M.Sc. PhD. Published articles (ISI*) World Brazil Brazilian contribution

1968

1987

2000

^ ^

861 385

1,537 837

^ ^

3,865 18,374

1,005 5,344

364,723 53 0.01%

907,051 3,648 0.40%

1,164,595 12,667 1.09%

*Institute for Scientific Information. For details see references.

work. In the second part, I will try to summarize how we came upon to the main biochemical findings in my laboratory and how this was influenced by both the local ‘‘scientific atmosphere’’ and by interactions with colleagues from other countries.

Personal Memories The Origins and the Choice of a Career Both my parents were Italian, from Naples. My father was a musician (a cellist) and so were my grandfather and greatgrandfather. When I was born in March, 1938, my parents were in Egypt. The second world war started shortly afterwards and my family moved back to Naples when I was just a few months old. My memories of Italy were nice but not very happy. Although my parents did all that was possible to protect the family, the shadow of war was always present and this included the lack of food, the continual menace of bombs, and fear of the

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fascists. Naples was a strategic port and was severely bombed, first by the Allies and later in the war by the Nazis. Fortunately, my father was not recruited by the Italian army because one of his legs was several centimeters shorter than the other. However, he did not like the fascists and made his political views public. Thus, he had to hide frequently and had difficulty in getting jobs. After the war, it was difficult to earn a living as a musician in Italy and in February, 1947, my family emigrated to Brazil. I was 9 years old when we landed in Rio de Janeiro. My father intended to stay in Brazil for only a couple of years ^ but in fact, we never returned to Italy. When I was 18 years old I had to decide whether I was an Egyptian, an Italian, or a Brazilian. I decided for the Brazilian citizenship and never regretted it, not because of any grudge toward Italy or Egypt, but simply because I grew up in Rio de Janeiro and liked it. At the age of 18, I was admitted to the medical school of Rio de Janeiro’s Federal University. At that time, I was a young fellow, very sure of myself; and during the first two years of medical school, I was absolutely certain that I would never be a biochemist and totally convinced that I was going to be a surgeon. Biochemistry was taught all through the first semester and among the students there was a legend that the professor was a great expert in porphyrins and would soon be awarded the Nobel Prize for Medicine. During his classes he used to draw dozens of formulas elegantly distributed across the blackboard, without ever consulting notes. His major feat was the heme ring and that astonished all of us: he would draw the structure using both hands simultaneously and finish it in perfect synchrony by inscribing Fe2þ in the center of the heme ring ^ Fe with the left hand and 2þ with the right hand. The horrible thing was that in the written examination we were required to reproduce all these formulas by memory and after a few classes I was absolutely sure that I would never be a biochemist. Years later I became the professor of biochemistry at the very same university I studied (so much for my youthful convictions) and found out that my famous predecessor never published a paper in


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a journal with referee, but in spite of this brutal truth, every year, between the months of September and October, he would not allow the staff to use the only telephone available in the department ‘‘because he was expecting an international call from Sweden.’’ He was a very decent person and in his own fashion was dedicated to his classes and students, but at least in relation to biochemistry and science, somehow fantasy prevailed over reality in his mind. Every year, the medical school used to dedicate one week to various cultural activities. During this period, classes were suspended and distinguished personalities were invited to deliver lectures to the students. The final event of the week was a dance. Every year I use to participate enthusiastically in those events (the dancing in particular), and it was during the cultural week organized in my first year at the medical school that I met Dr Walter Oswaldo Cruz, the man who led me into science. At the time of his conference, I did not know that he was one of the rare true scientists working in the country. The title of Dr Walter’s lecture was ‘‘The scientific career.’’ The lecture was quite interesting and at the end he announced that there were three positions available in his laboratory for medical students. The selected students were expected to work 12 hours per week during the academic year and full time during vacations. The part of the announcement that really captured the interest of most of the audience was that each student would receive a stipend equivalent to about 200 American dollars per month. Selection would start at his home, 2 weeks after dance, and I immediately signed up for it, not because the lecture had stirred up in me the desire to be a scientist, but because I desperately needed money. The economic situation of our family was quite precarious ^ my father was a musician and they were badly paid in Brazil. I was dismayed to find out that dozens of students had enlisted for the selection and I feared that I would not stand a chance since most of the candidates were far more advanced than I in the medical course, and some of them were nearly at the end of it. The first test was an interview with Dr Walter at

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his home. When I arrived, there were about a dozen students waiting for their turn in the living room. Eventually, I was called into his office, and after a very brief social conversation he started to show me several cartoons selected from the American magazine ‘‘New Yorker’’and I was supposed to identify the humorous point of the cartoon.When I left Dr Walter’s home I was puzzled. During the interview, he did not ask about my academic performance at the university, nor did he care to know if I had any previous laboratory experience. Only a small fraction of the interviewed students were called for the second test and much to my surprise, I was one of them. The second test was in his laboratory at the Oswaldo Cruz Institute, where he worked (the Institute was founded by Dr Walter’s father). Now he showed us complex pieces of equipment and asked what they were for. When I answered that I had not the foggiest idea he would say ‘‘good, now make a guess’’ and we were supposed to infer what they were for. At the end of the selection, I had the feeling that in Dr Walter’s view, the main ingredients for science were a good sense of humor and a lot of intuition. Well, maybe I had these qualities because I was one of the selected candidates. Thus, I entered a research laboratory at the beginning of my second year at the medical school, when I was 19 years old. In the following years I used to advertise loudly in the laboratory that soon I would leave because I was going to be first a surgeon, and later a clinician. Nobody seemed to care much for my bombastic statements. I got very good scores in anatomy and somehow I was able to attend rounds in an abdominal ward and to sneak into an operating room at the end of my second year, first to watch and then as a scrub nurse, handing the surgeon the tools he needed during surgery. I remember clearly my first entry into the operating room ^ all dressed up and sure that surgery was my destiny. At that time the opening and closing of the abdominal cavity took most of the time allotted for the surgery and these were standard procedures repeated in practically all surgical intervention, without much of a change. Thus, after a few months and attendance at many surgical


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procedures, I was horrified to find that the process had a soporific effect on me and I started to feel sleepy. It did not take much time to realize that a sleepy surgeon could not possibly be a good professional. But then clinical classes fascinated me and right in the beginning of the fourth year, I started to work on a ward and to boast in Dr Walter’s laboratory that now I had found my true vocation. As in the previous years, this did not make much of an impression on the laboratory staff, giving me the feeling that maybe I was not that indispensable for the laboratory. In the ward we had to interview each new patient admitted to the service and write down the patient’s medical history in the most detailed possible manner. This was called anamnesis and during the initial couple of months I found it interesting, but unfortunately, it was repeated over and over again. After so many anamneses I started to feel sleepy again and sadly I had to conclude that I had no talent for medical practice. At the beginning of the fifth year, Dr Walter informed me that it was time to make a decision: either I would be a scientist and stay, or a medical doctor and leave, in order to make room in the laboratory for a new student. I never felt sleepy in the laboratory; so quickly I answered that I would stay and be a scientist. Dr Walter did not seem to be surprised at my decision, as if he already knew it. Years later, I saw the same sequence being repeated in my laboratory several times ^ bright young medical students stating emphatically that they were going to be surgeons or something of the kind, and later becoming addicted to science. Once having decided that I would become a researcher, during the last two years at the university I dedicated most of my time to the laboratory. DrWalter usually arrived at the Institute between 9:30 and 10:00 am carrying his lunch bag. Most of his working time was spent at the bench and we were not allowed to interrupt him during experiments. He never raised his voice with us, but all of us feared and respected him. Each senior student had an assignment such as taking care of the stock of drugs, watching over of the animal house, etc. In addition, we were responsible for the

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maintenance of the few pieces of equipment available in the laboratory. I was in charge of the balances and pH meter. Once a week we had a seminar that took the whole afternoon. Seminars started with a short report on the status of the equipment and other administrative matters and then each student had to present a paper of his choice. Dr Walter fostered competition among his students. Somehow he was able to convince us that the most desirable thing in the world was to inherit his laboratory, and at regular periods of time, he would select one of us to be the future heir. The heir would be treated with deference and publicly praised but after a month or so he would be deposed and a new one appointed. Dr Walter was very democratic about his choices and all of us were eventually appointed future heirs. Every year, fresh new students were admitted to the laboratory. These were assigned to work under the supervision of the older students who had already decided to follow a scientific career. We were fully responsible for them, deciding their working schedule, the experiments that they would perform etc. If something went wrong, such as a paper not well presented at the weekly seminars, then DrWalter would not criticize the younger students but instead would complain to us for not having properly supervised our charges. If the new student remained in the laboratory for more than 2 years, then he would be directly supervised by Dr Walter. This was what we later called ‘‘supervision in cascade’’ and I, Peter von Dietrich, Me¤cia Maria de Oliveira, and Jose¤ Reinaldo Magalha‹es were the first four students selected by Dr Walter to start the process. Dietrich became a well-known biochemist for his excellent publications on sulfated polysaccharides and Me¤cia Maria for her work in lipid metabolism. The senior students were invited to Dr Walter’s house on Fridays for dinner and work. We went at 7 pm directly from the laboratory to his house, where his wife, Dr Silvia, a medical doctor, would receive us warmly and announce that we would have lasagna for dinner. One week we were informed that she had prepared lasagna because she knew it was my preferred


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dish, the second week because it was Peter’s preferred dish, the third week because of Reinaldo, and on the fourth week I was again the one who according to her view, loved lasagna.Thus, she systematically prepared the preferred dish of all of us without never changing her Friday menu. After attending dinner for two years at Dr Walter’s house, somehow lasagna became indeed one of my favorite dishes and up to the present day, whenever I eat a good lasagna I cannot avoid remembering with warmhearted nostalgia Dr Silvia’s dinner. The Friday working schedule was long and Dr Silvia would discreetly clear the table shortly after dinner and leave us for a long session, which would never end before 3 am of the following day. We usually began by presenting to Dr Walter a brief report on the activities of the young students whom we supervised, followed by a report on our own work. To finalize, we had a study section. Dr Walter selected books that he felt we should read. Most of them were on biochemistry.We would divide the chapters of the selected books among us (Dr Walter included), to study during the week and present to the group on following Friday meeting. We went through several excellent books. One that I especially liked was ‘‘Dynamic Aspects of Biochemistry,’’ by Ernest Baldwin.Through this book I discovered a new biochemistry, totally different from the one I was taught at medical school. Baldwin described how new knowledge was discovered, made clear the frontier between what was known and what was not known, and accomplished all of this with a limited number of formulas. Contrasting with this approach, most of the teachers at the university taught as if all that should be known was already available and compacted in their classes, not making it clear that there was still a lot to be discovered. At the end of the Friday session we used to leave Dr Walter’s house exhausted but quite excited and could hardly sleep. Thus, we would go play snooker until 6 am, when the first popular cafe¤s opened, and it was only after a cheap but generous breakfast that we headed home for sleep. Needless to say, the Friday meetings had a crucial role in my training. The Oswaldo Cruz Institute was a large facility of the federal government, dedicated to

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health research and production of vaccines. In DrWalter’s laboratory, we were given the impression that there was no other place in the state of Rio de Janeiro where we could have a proper training in science. Although most of the employees worked on vaccines, we knew that there were other research laboratories in the Institute, but we were not encouraged to visit them, not even to borrow drugs or use their equipment.Years later I found out that there were excellent research laboratories headed by Dr Haity Moussatche¤, Dr Fernando Ubatuba, Dr Herman Lent, and a few others. At the university where I studied, we knew that in the Department of Biophysics (later called the Institute of Biophysics Carlos Chagas Filho) there were people doing research, but on the rare occasions that they were mentioned on our Friday evenings, the notion was passed along that ‘‘they were not as good as they claimed to be.’’ Later I found out that the Biophysics Department was an excellent research center and probably the best in Latin America.This quality could not be perceived during the course of biophysics taught in the first year of medical school because the major effort of the staff was directed toward research, and teaching was not given proper emphasis. The biophysics classes were tedious and were given in a dark classroom right after lunch, making it practically impossible not to doze during the class. The attitude of not encouraging any interaction with the very few active research units nearby was a general feature among research leaders and not an exclusive attitude of DrWalter’s.The few scientists available in the country used to compete quite a lot among themselves and I never understood why. Perhaps it had to do with the competition for the meager funds available for science or with the difficulty in attracting talented students to research laboratories, but whatever the reason, the fact is that competition did not facilitate communication within the small scientific community, and this may have delayed the growth of science in Brazil. In retrospect, I was extremely lucky to have attended Dr Walter’s lecture during my first year of medical school and I am sure that this was the most important event of my professional


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life. The hard fact that even now I find it difficult to admit, is that at the time I was not able to recognize the real meaning of his lecture for what it was worth: it was greed for the stipend and not the awakening of an intellectual facet of my personality that drove me to his laboratory. In spite of all the beauty that I see in the practice of medicine, if I had not attended Dr Walter’s lecture probably I would have been a frustrated and very mediocre medical doctor. From the data of Table 1, it can be inferred that there were very few people in Brazil who were able to publish one or two scientific papers in good journals per year and the statistical chances of a student interacting with one of them were quite small. I do believe that within my generation there are many colleagues who would have been excellent scientists if they had had the opportunity to be exposed to the practice of science as I was. In spite of the fact that the medical school I attended was one of the best in the country, most of my colleagues did not know what science was about. During my last year at the medical school, I made a survey among students from the first to fifth year of the medical course, asking what they knew about research and whether or not they were considering the possibility of pursuing research as a career. None of them contemplated the possibility of being scientists and the majority mistook a research laboratory with a laboratory for clinical analyses.

Tininho Mr Isaltino Rodrigues Soares was a magic personage who was a dear friend for many years and a great partner in the laboratory. He was a small but strong man with a preciously trimmed moustache and when I met him, he was about 45 years old. Mr Isaltino was one of the technicians working in Dr Walter’s laboratory and at that time, he was considered a very lazy employee. He would register his arrival in the morning and then disappear during the rest of the day. After my first year

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in the laboratory, Dr Walter decided that Mr Isaltino would be my technician and from then on I was responsible for his activities. At first I was horrified with the decision ^ how could I possibly be responsible for someone that I could not even find during the whole day? But then Mr Isaltino kindly told me that there was no need to be upset. He would sit quietly near me at the bench for a while, and day by day leave me in peace for progressively longer periods of time, until Dr Walter would forget about his decision and I would leave the laboratory to become a famous surgeon. I got the message and immediately agreed, but it did not work quite that way. Mr Isaltino was a very skilled technician, trained in his youth by a group of epidemiologists sent by Rockefeller University to work and train people at the Oswaldo Cruz Institute. While sitting near me he taught me a lot of small things that were very useful. Then we started to eat lunch together, to discuss the last soccer game and one day, during an informal conversation, I found out that his salary was outrageously low. I was astonished and decided to ask Dr Walter for a raise. I was sure that Dr Walter would ignore me, but I thought that Mr Isaltino would appreciate this gesture. Thus, I went to his office ready to put up an argument, but after I stated my request, Dr Walter simply said ‘‘OK ’’ and Mr Isaltino started to receive an addition to his salary, paid from Dr Walter’s meager grants. From then on I started to call Mr Isaltino by his nickname ‘‘Tininho’’ and we never parted until he was 71 years old and left all of us forever.

Postdoctoral Training in the USA After we completed our medical course, Dr Walter insisted that we should work abroad for a period of one to two years and helped us in all possible ways to get a fellowship. At that time I was coauthor on several of Dr Walter’s papers, published in good journals such as Blood and the American Journal


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of Physiology, and this played a decisive role in obtaining a fellowship from the U.S. National Institutes of Health, one that included a grant of 10,000 dollars to be used when I returned to Brazil. Thus, I had the good fortune to work under the supervision of Herbert and Celia Tabor at the headquarters of NIH in Bethesda for a period of 18 months between 1962 and 1964. One of the many things I found out in the USA was the importance of the environment on the learning process. The first scientific meeting I attended was an unforgettable shock. It was the meeting of the Federation of American Societies for Experimental Biology held in Atlantic City. I knew that there were a lot of scientists working in the EUA, but I did not know there were so many ^ thousands of people presenting their data in a most cordial atmosphere. At NIH, I usually had my meals at the cafeteria in the medical center (building 10), which was always crowded with people discussing science. The tables were large and it was unusual to be seated alone. Thus, I could not avoid to overhear what others at the table were discussing. On one of these occasions, I could not refrain from asking a question of a group of three scientists who were engrossed in conversation about the genetic code, and much to my surprise they kindly interrupted their conversation to enlighten me on the subject. From then on I no longer felt ashamed to ask questions and at the end of my fellowship, I found out that I had learned far more biomedical science at the NIH cafeteria than I did during my whole medical course in Brazil. Herbert and Celia Tabor’s research subject was the metabolism of polyamines. It was a subject that I knew nothing about, quite different from the more physiological work I was trained to do in Brazil, and to make things worse, I did not have Tininho around to help me out of the several experimental difficulties I run into during my work. Celia and Herbert were always very solicitous and ready to discuss any kind of result I managed to get, but I was not able to get much and at the end of my fellowship all that I was able to show back home was a small chapter in ‘‘Methods in Enzymology,’’ written by the Tabors, where I figured as the

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middle author. In spite of this apparently meager result, it was in the Tabors’ laboratory that I really learned what biochemistry was about and it was at the NIH cafeteria that I discovered the vast horizons of the biomedical sciences. But, this was difficult to describe in reports and there was a certain disappointment about my performance at NIH. When I left for the USA I was considered a bright young fellow who had published several papers with Dr Walter, and in the USA, the land of plenty, I was expected to blossom and publish much more. It was difficult to explain how important it was for my scientific formation to be ‘‘graduated’’ by the NIH cafeteria. A final important statement ^ if I had not had the opportunity to work in Dr Walter’s laboratory during my medical course, I would never have been able to take advantage of what was offered at NIH, and I would not have understood the meaning of the professional attitude of the NIH scientists.

The Military Regime and the Hard Years Back Home I returned from the USA in 1964, shortly after the military took over the government. The next two years were the bleakest of my life. The new political regime selected as director of the Oswaldo Cruz Institute a man who claimed to be a vehement anticommunist and very loyal to the new government, i.e., one who would have made a successful career either during the fascist period in Italy or during the McCharthy’s reign in the USA. The new director was a former investigator of the Institute, but in fact he did not know what science was about and worst, deeply disliked the few true scientists of the Institute whom he classified as arrogant people who lived in‘‘ivory towers.’’ A month after my return I was called in by the security branch of the Navy for interrogation. I was received by an officer who in a very educated manner asked me if I knew about the communist meetings held in Dr Walter’s department every Wednesday afternoon. I was shocked and explained that there must be


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a mistake ^ on Wednesdays we had our weekly seminar to discuss scientific papers and as far as I knew, no one in Dr Walter’s laboratory was a communist. The officer seemed not to be surprised and informed me that he was going to have a cup of coffee in the next room and meanwhile, he would not mind if I read the dossier he had on his desk. As he left, I immediately started reading and was horrified at the accusations that the new director had made against Dr Oswaldo Cruz and the few fine scientists working at the Institute. At the end of the dossier, there was a letter from the high command stating that the Navy knew that the new director was a bad scientist and a very mediocre administrator but, because he was a wellknown anticommunist, his charges should be carefully examined. One of the director’s initiatives was courses on chemical and bacteriological warfare to be taught separately to army sergeants and captains. The research staff of the Institute was ordered to give these courses. Dr Walter refused and so did all his collaborators, including myself. The tension between the scientists and the director grew rapidly, and most of the space occupied by Dr Walter’s laboratory was taken away by the director, who, assisted by the Institute police and in the presence of Dr Walter and his students, personally sealed most of the rooms of the laboratory, with all the equipment inside. The atmosphere at the Institute became unbearable and finally I resigned and went to the university to work in the Biophysics Institute directed by Professor Carlos Chagas Filho. One year later Dr Walter had a stroke and died. A short time later, the military regime issued an ‘‘institutional act’’ prohibiting politically undesirable persons from working in public service or any agency having a connection with the government. The director of the Oswaldo Cruz Institute prepared a long list of names and most of the few good scientists working there were fired, as were many good scientists from different research institutes and public universities of Brazil. Most of them found very good positions in American and European research institutions. Had I stayed at the

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Oswaldo Cruz Institute, probably I would have been on the list ^ not because of my political ideas, but because I worked in Dr Walter’s laboratory, which surely would have headed the list prepared by the director. Most of the people who were fired were not communists, and had no interest in politics. As time passed, the communists were no longer the bad guys and the undesirable persons were the ‘‘subversives’’ who become the true enemies of the state. To my mind, one of the major mistakes of the military regime was to appoint mediocre civilians to important administrative positions. As in the case of the director of the Oswaldo Cruz Institute, they were trusted because they claimed to be warriors in the quest against the subversive world, and it did not matter that they had no technical skills needed for the position they occupied. Naturally, many of them defined as subversives the competent people that they could not understand or tolerate. The quality of my life greatly improved after I went to the university. In addition to being a good scientist, Professor Chagas had great diplomatic ability. His family is one of the most traditional and influential in the country and he was able to protect the Biophysics Institute without ever compromising with the military regime. There were several excellent research laboratories at the Biophysics Institute, and I was fortunate to work in Anto“nio Paes de Carvalho’s unit. A well-known electrophysiologist, he not only gave me space in his laboratory, but also shared with me the small grant he had, and revised the manuscripts I wrote, and all of this without ever accepting my offer to include his name as coauthor on the papers I published. This was also the time when I met a beautiful and highly talented geologist. We fell in love, married, and had four children who to this day I proclaim to be my best contribution to biology. It was impossible to ignore the nightmare prevailing during the worst period of the military regime but Regina and the Biophysics Institute were two refuges of happiness that made life bearable. The worst was when the guerrilla started movement to fight back, against the new regime. What follows


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are two short reports that illustrate the fear instilled by the regime and the dignity of the Biophysics staff.

Report 1 The agents of the repression usually came at night, between 11 pm and 1 am, to pick up ‘‘suspects,’’ cover their heads with black cloth and take them for ‘‘interrogation’’ in a unknown place. Most of the ‘‘suspects’’ were brutally tortured in order to confess their subversive activities and to give names of associates. Many innocent and apolitical people were brutalized and at the height of their pain they would admit whatever the torturer wanted and give any name that came to mind. Thus, any citizen could be arrested and so treated. Many students were arrested and frequently, when we arrived in the morning for work at the University, we would find distressed parents asking for our help. The routine was to go to different military installations and ask for the arrested person. In all of them we had to wait a long time before being received. The inquiring person had to show his identity cards, which would be carefully copied, and then he would be informed that the subject we were looking for was not there. They would never admit that they had arrested anyone and we had to look for the students in different places. It was known that if the repression agents were convinced that the arrested had in fact a connection with the guerilla movement, then he would never return, but if after the ‘‘interrogation’’ they concluded that they were not involved, then after torture, the suspect received medical care and was kept prisoner until the wounds healed, only then would he be released. If different people searched for the suspect right after arrest, then it was believed that the torture period would be reduced and the innocent person released sooner. Most of the professors at the Biophysics Institute including myself did go in search of missing students, and we all felt both outraged and terribly scared.

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Report 2 Dr Herta Meyer was one of the senior investigators of the Biophysics Institute. She was a German Jew who with the help of Professor Chagas flew to Brazil on the eve of the second world war. Dr Chagas helped several people from France and Italy to escape the Nazis, and after the war received the ‘‘Legion d’honneur’’ from the French government for his deeds. The only electron microscope available in the city was in Dr Herta’s laboratory. She kindly allowed any one to use it during a part of the day and there was a waiting list for the use of the equipment. One day, an officer of the Navy came to Dr Herta’s laboratory and informed her that he needed to use the microscope. She politely told him that there was a long waiting list. The man loudly announced that he was an officer of the Navy. Dr Meyer was not impressed by the tone of his voice, and looking him up and down coolly answered ‘‘We are all employees of the government,’’ and that was it. The officer did not expect an answer, went away, and never returned. This seems a simple story, but those who lived through that period know that a large measure of bravery was needed for Dr Herta to stand her ground and not let the officer have his way.

Heidelberg Aristides Pacheco Lea‹o was a distinguished neurophysiologist with an international reputation for the discovery of spreading depression. He was both the president of the Brazilian Academy of Sciences and one of the leading scientists of the Biophysics Institute. In August 1969 Aristides quietly suggested that Regina and I should leave the country for one or two years. He had heard unsettling rumors about us and felt we should leave while things cooled down and possible misunderstandings could be cleared up. The very same day we started writing letters and sending our curriculum vitae to the USA and Europe in search


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of positions. We selected cities where good research centers could be found in both my field and Regina’s. We were determined not to let adversity spoil our research careers and the deal was that if one of us got a position, the other would work even without a salary until better times came and we could go back home. One of the cities selected was Heidelberg, in Germany. Regina worked in geomorphology and at the University of Heidelberg there was an excellent group working in her field of interest. At that time I had already several publications on Ca2þ transport by the sarcoplasmic reticulum of skeletal muscle and decided to write a letter to Wilhelm Hasselbach, head of the Max Planck Institute in Heidelberg. Hasselbach had discovered the Ca2þ pump of skeletal muscle in collaboration with Madoka Makinose and I had read with respect and admiration most of their publications. We had positive answers from about 20% of the letters sent and in two of them we were offered positions in the same cities, one in the USA and the other in Heidelberg. The happy memories of my postdoctoral period at NIH and the ghosts of the war that haunted me since my childhood emotionally predisposed me to accept the USA offer, but Regina was adamant on the subject. She simply asked me how much different I thought what we were living in Brazil was now from what my parents had experienced in Italy during fascism and the Germans had suffered under the Nazis. That settled the question, and a few weeks later we went to Heidelberg for a period of one year. It was a wonderful time. For a start, we could sleep peacefully at night without being worried about who might be knocking on our door. Heidelberg is a marvelous city and we used to go picknicking with our children almost every weekend. At the Max Planck I had a bench and a nice desk in Makinose’s laboratory. I discussed my results regularly with Hasselbach, the most brilliant man I ever met. His mind was amazingly fast ^ he would glance at the graphs of my experiments and instantaneously understand what I had done without any explanation. Makinose was a marvelous man, always very helpful in the laboratory; and I could

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discuss science with him any time I wished. We both shared a common interest in music. Makinose played violin and every week he used to play with two of his nieces, a pianist and a cellist. I was admitted to the group to play recorder and spent many delightful evenings at his house. Dr Hasselbach’s wife found out about my ‘‘musical talent’’ and because she also played recorder I was invited to play duos once every two weeks at their house. Hasselbach apparently did not appreciate our performances. He usually disappeared while we played and after a while (never more than an hour) he would come back stating that we had had enough music, and invite me for a glass of wine. During the conversation that naturally accompanied the wine I discovered a whole new dimension of science. I confess that I used to do my best to suit Frau Hasselbach’s musical aspirations so I could be invited for a glass of wine with Hasselbach. After a year and a half we heard from our friends that it was safe to return. Thus, we decided to go back home. It was not an easy decision. Hasselbach had offered me a position to stay at the Max Planck and Regina was very happy at the University of Heidelberg. Our life was very pleasant and productive, but we missed our families and somehow felt that to stay was a sort of treason toward our colleagues at the university who had decided to teach and work in science in the country no matter what adversity had to be faced. Thus, hesitantly, we went back to Rio de Janeiro.

The Bonanza and the Graduate Courses The military regime promoted the development of science in Brazil. The government wanted to enter the nuclear era and decided to finance science on a scale that was unprecedented in Brazilian history. At the beginning, the bonanza was focused on physics, chemistry, and mathematics, but shortly afterwards the support was extended to all branches of science. One of the


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most fruitful initiatives was the creation of graduate courses. MSc and PhD students were granted fellowships that allowed them full-time dedication to their studies. The nuclear project never matured and fortunately we still do not have nuclear weapons, but the surge of science in the country shown in Table 1 is one of the few good results of the military period. When we left for Germany, our salaries were low and the working conditions at the university were very poor. In order to balance our budget at home we used to spend long hours at night translating texts from English to Portuguese. The scenario changed during our sojourn in Heidelberg and after our return we found that the salaries were good and there was money for research, so in a couple of years we were able to equip our laboratories in a manner that we had never before dreamed possible. Up to the sixties, before the institution of the graduate courses, the number of students interested in science was small and the possibilities of getting fellowships for them were poor. Starting in the seventies, the number of MSc and PhD students steadily increased and the working conditions greatly improved. This combination led to a progressive rise in Brazil’s scientific productivity. The Biophysics Institute grew at a very fast pace without losing its positive working atmosphere, a blend of professionalism and camaraderie. The years passed by rapidly as I was deeply involved in my work, until, in 1978 when I was faced by my major professional challenge ^ to replace my old professor of biochemistry, the one who used to draw the heme ring with both hands.

The Department of Medical Biochemistry Until the end of the 1970s the university appointed only one full professor for each department and referred to him as ‘‘catedra¤tico.’’ This was a position that was highly placed in the university hierarchy. The ‘‘catedra¤tico’’ was entitled to be the chairman of the department for life and had full powers

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over the rest of the staff. In 1978 the old catedra¤tico who had waited in vain for his Nobel Prize retired. After a complicated selection process I was appointed to fill his position. When one of these positions became available there were usually several candidates to compete for it and the selection process resembled a medieval contest. The committee in charge of the selection consisted of the retiring ‘‘catedra¤tico’’ and four other ‘‘catedra¤ticos’’ of biochemistry invited from different universities. The selection process included a written examination, a practical examination, a lecture, and the defense of a thesis describing an original piece of research, never before published. The ‘‘curriculum vitae’’ and the list of previous scientific publications of the candidates had practically no importance in the choice of the new professor. The topics for the written and practical examination were selected from a long list of subjects that covered all the known topics of biochemistry and were revealed to the candidates right before the test. There were two candidates for the new position, myself and a colleague several years older than me. After an exhausting week I was appointed the new ‘‘catedra¤tico.’’ I would like to think that my appointment was related to my experience in science, but in fact, it was decided in the practical examination. The topic selected was to demonstrate electrophoresis of plasma proteins. We were supposed to start by collecting the blood from a volunteer. Due to my experience in Dr Walter’s laboratory I knew how to collect blood and I had no difficulty in finding a volunteer among my friends at the Biophysics Institute. The test started in the morning and by lunch time I had my electrophoresis showing all the expected bands. The other candidate, however, did not have much experience in drawing blood and was not able to find a volunteer. In these cases, according to the statute, the department had to provide a ‘‘donor,’’ which in this particular department used to be a tall, fat man who cleaned the classrooms. As soon as he heard of the topic selected, he simply disappeared ^ and it was only in the


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early afternoon that the man was found and a price was agreed upon for his volunteering. The second electrophoresis was completed late at night but with several protein bands missing. At the end of the day the selection committee was exhausted and in a very foul mood, and I was appointed to the position. A week later I was called into the Dean’s office. He was dressed in a sober suit and wearing elegant turtle-rimmed glasses. In a very severe tone he informed me that he was very disappointed with the result of the selection and that he ‘‘would keep an eye on me.’’ During the military regime I had learned not to be easily shaken by threats, so I asked him ‘‘how thick were his glasses and which one of his two eyes he intended to use.’’ This was the beginning of a long struggle to change the mentality of the biochemistry department from its morose conservative ways of thinking to the open mentality created at the Biophysics Institute by Professor Carlos Chagas Filho, the difference being that I never learned the diplomatic skills of Professor Chagas. Years later, the same Dean publicly complimented me several times for the excellent development of what he baptized as the ‘‘new biochemistry department.’’ I went to the new department accompanied by Antonio Luis Vianna, my best friend and former student from the Oswaldo Cruz Institute: a postdoctoral fellow; and several MSc and PhD students. We were received by a group of more than 20 professors ranging from assistant to associate professor, all of them determined to ignore the new arrivals. During the next three years, most of them were transferred to other departments, and only a minority remained until retirement. They were replaced by professors with good research experience selected on the basis of their curriculum vitae. Several of my former PhD and postdoctoral students became professors of the ‘‘new biochemistry department,’’ and they have trained new students who in turn have won positions as professors in the department. At present, the department has nine full professors and 44 associate professors and as a result of the collective effort, it is considered one of the best in the country. Each of the new professors has contributed in his own

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way to shaping the present department, but beginning in the eighties, two of them played a particularly important role: Martha Sorenson, an American colleague who has taught several generations of new biochemists how to write a scientific paper in English; and Jorge A. Guimara‹es, a man with a natural gift for academic politics, who played a key role in the transition from the old to the new scientific mentality. The ‘‘catedra¤tico’’ position has disappeared during the different administrative changes of the Brazilian university and the chairman is now appointed democratically by the staff of the department. I was chairman of our department for no more than 6 years. Thus, except for the administration of my own grants, I have dedicated almost all my time to research and education, two activities that I truly enjoy.

Strategies and Great Friends I like to do my own experiments, and I have always spent most of my time at the bench, alone or with students. In only a small fraction of my publications, I limited my participation to discussing of the experiments and writing the manuscript. Whenever I start a new research line, I like to do the experiments alone, without students. I have always had a large number of students in my laboratory with whom, I am proud to say, I have had a very good relationship, and from some of them I learned a great deal. However, students have quite a limited amount of time to complete their theses and I usually suggest a research project that I already know has a good chance of yielding publications, so necessary for the beginning of one’s career. Another successful strategy has been to bring a visiting professor to work in my laboratory for a period varying from a week up to a month. Up until a few years ago, there was only a small number of scientists in Brazil and it was very rare to have two of them working on related subjects. Thus, it was difficult to have a productive discussion with a colleague


615

unless one went to international meetings or visited colleagues abroad, but even then the amount of time for serious talk is limited because there is always much to do and little time to spare. Thus, I usually invite people to come to my laboratory where I can talk with them as much as I wish. Most of the time, I learned ways of thinking and technical details that would have taken months or years to discover by myself. I had several illustrious visitors, but with four of them I maintained a long collaboration. We started working together at the bench and from then on, in addition to our common scientific interests we became very good friends. These were Giuseppe Inesi from the University of Maryland, Armando and Marieta Gomez-Puyou from the Autonomous University of Mexico (UNAM), and Andre¤ Goffeau from the University of Louvain la Neuve in Belgium. I met all of them at international meetings. The longest collaboration I ever had was with Giuseppe Inesi, who became my dearest friend. We met in 1978 and since then have collaborated on several projects, resulting in various publications. Whenever I have an experimental doubt or I need to discuss a result that I do not understand, I usually call Giuseppe by phone, a practice that has proved to be very productive. When I met Armando and Marieta GomezPuyou, they worked with both the mitochondrial F1 ATPase and the membrane bound pyrophosphatase of Rhodospirillum rubrum. I went to Mexico several times and they came to Rio, although not so many times as I wished. The three of us liked to work at the bench, and our experiments were made with six hands. At the end of the day we used to go for a beer and frequently embarked on long conversations during which we navigated through wildly improbable dreams, but Marieta always brought us down to earth when we had to decide the specifics of the experiments. Armando is the only Mexican I know who prefers French fries to tacos. He usually dresses informally, but when we planned an experiment that we thought would definitively prove our premises, Armando would show up in the laboratory wearing an elegant tie

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and proclaiming that his elegance would undoubtedly contribute to the success of the experiment. I think he had a collection of ties just for these specific events. I never told him, but with time I started to fear Armando’s ties. I am not superstitious, but somehow I started to believe that the nicer the tie, the bigger the chances of the experiment going sour. Finally I met Andre¤ Goffeau, an expert on yeast HþATPase and well known for coordinating the resolution of the yeast genome. Andre¤ interacted with several laboratories in Rio de Janeiro and helped to organize the genome project of Xylella fastidiosa, the first genome ever sequenced in Brazil.

The Research Work The First Steps As a medical student I performed the experiments designed by Dr Walter O. Cruz and was a coauthor for several of his publications dealing with the hemostasis of small blood vessels. During this period I did publish a couple of papers of my own but these were minor points. During my postdoctoral training at NIH, I had learned a lot but accomplished very little in terms of research. It was only when I moved from the Oswaldo Cruz Institute to the Biophysics Institute that I became productive. When I went to work in Paes de Carvalho’s laboratory I was interested in muscle contraction. With the Tabors I did learn that muscle contains significant amounts of spermine and spermidine, but at that time their physiological role was unknown. Thus, I tested the effects of these two polyamines in muscle and it turned out that both promoted muscle relaxation [3^5]. My ‘‘debut’’ in the field of Ca2þ transport was testing the effects of polyamines on the rate of Ca2þ uptake by vesicles derived from skeletal muscle sarcoplasmic reticulum [5]. The work with polyamines resulted in three publications that got


617

lost in the literature, but they were extremely important for my self-esteem. After a long depressing period with no publications I had begun to doubt my talent, and with these three papers ^ ‘‘I did prove to myself that I could do it.’’ From then on I published regularly and focused my attention on Ca2þ transport. At the beginning there was not much correlation among my publications except for the fact that they dealt with Ca2þ transport. For instance, in one of my early papers I described a Ca2þ pump in brain microsomes that had kinetic properties similar to the muscle Ca2þ -ATPase [6]. Then I went back to skeletal muscle and found that the Ca2þ -ATPase could use acetyl phosphate as a source of energy to pump Ca2þ, and the kinetics of transport varied depending on whether ATP or acetyl phosphate was used as a substrate [7^9]. The work with acetyl phosphate played an important role for my sojourn in Heidelberg during the military regime in Brazil. In 1970 Hasselbach and Makinose were in the process of discovering that the Ca2þ -ATPase could use the energy derived from a Ca2þ gradient to synthesize ATP from ADP and Pi. In the course of their experiments they needed vesicles loaded with Ca2þ in a suspension free of ATP and ADP. This was not easy to attain using ATP to load the vesicles, but with acetyl phosphate it was possible to fill the vesicles with Ca2þ in the total absence of nucleotides.Years later I learned from Makinose that my letter asking for a position reached Hasselbach’s office shortly after they read my work on acetyl phosphate, and this apparently contributed to the generous offer I received from Hasselbach. When I returned from Heidelberg, I focused my work on the mechanism of energy transduction in biological systems and the Ca2þ -ATPase was my main experimental model. Unlike from my earlier publications, now each work was related to the other and the sequence slowly formed a coherent history that grew with the years. What follows is a brief description of the main topics we studied and Refs. [10^17] are selected reviews where these topics are described in more detail.

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Phosphoenzyme of High and Low Energy In 1966 it was known that the catalytic cycle of the sarcoplasmic reticulum Ca2þ -ATPase is initiated by phosphorylation of the enzyme by ATP [10]. In this reaction an aspartyl residue located in the catalytic site of the enzyme reacts with ATP (1) and the acylphosphate residue thus formed is hydrolyzed in a subsequent step (2). During these two intermediate reactions, two Ca2þ ions are translocated across the membrane.

E þ ATP

ADP

HOH

ð1Þ

ð2Þ

!EP

! E þ P1

Reaction 1 was known to be fully reversible [10,11] and in water an acylphosphate residue similar to that formed in the catalytic site of the Ca2þ -ATPase has the same energy of hydrolysis as ATP. Thus, up to 1973 it was thought that the energy needed to pump Ca2þ through the membrane was made available to the enzyme during the hydrolysis of the aspartyl phosphate residue, i.e., during reaction 2 shown above. In 1961 Peter Mitchell [18,19] proposed the chemiosmotic theory to explain the mechanism of ATP synthesis by mitochondria. According to Mitchell’s hypothesis, the energy needed for the synthesis of ATP was derived from the Hþ gradient formed across the inner membrane of the mitochondrion. In 1967 Garrahan and Glynn [20] showed that in accordance with the chemiosmotic theory the (NaþþKþ) ATPase could use the energy derived from the gradients of Naþ and Kþ to synthesize ATP, and in 1971 Hasselbach and Makinose published three short reports in FEBS Letters [21^23] demonstrating that the Ca2þ -ATPase could also use the energy derived from a Ca2þ gradient to synthesize ATP from ADP and Pi. This was demonstrated by incubating vesicles from muscle sarcoplasmic reticulum previously loaded with Ca2þ in a medium containing excess EGTA, a Ca2þ chelating drug. The energy derived from the


619

Ca2þ gradient thus formed across the vesicle membranes was used to reverse the catalytic cycle of the Ca2þ -ATPase and to synthesize ATP from ADP and Pi. In 1972 Makinose [24] demonstrated that the synthesis of ATP was initiated by phosphorylation of the enzyme by Pi, forming an acylphosphate residue at the catalytic site (reaction 2 in reverse). In accordance with what was described for the Ca2þ uptake, it was concluded that during reversal of the pump, the energy derived from the gradient was captured by the enzyme to form the acylphosphate residue from Pi. These observations had an important impact on the bioenergetic field because they were a clear demonstration of the chemiosmotic theory previously proposed by Mitchell. In fact, it was not possible to obtain with mitochondria such a clear experimental demonstration that a membrane-bound enzyme could convert osmotic energy into chemical energy. After my return to Rio de Janeiro I was fortunate to supervise the theses of a group of very bright young students and one of them was Hatisaburo Masuda, who had studied biology and went for his MSc at the Biophysics Institute. I was fascinated by the reversal of the pump, and together Masuda and I decided to reproduce the phosphorylation of the enzyme by Pi described by Makinose [24]. This was easily done with Ca2þ loaded vesicles. Then, as a control we used leaky vesicles, i.e., vesicles permeabilized with a small amount of ethyl ether. Because there was no gradient, i.e., no source of energy available, these leaky vesicles should not have been phosphorylated by Pi. Surprisingly, we found a small but significant level of phosphorylation, about 1/10 of what could be measured with Ca2þ -loaded vesicles. We then decided to vary the experimental conditions in an attempt to increase the level of the phosphoenzyme measured in the absence of a gradient. The best results were attained when the pH of the medium was adjusted to 6.0 and the Pi concentration in the medium was raised from 1 to 2 mM to the range of 5^8 mM. In these conditions we could obtain the same level of phosphoenzyme as that measured with

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the gradient by Makinose. Different tests confirmed that the acylphosphate formed in the absence of the gradient was the very same acylphosphate residue as that formed with the intact vesicles and gradient [25,26]. Masuda’s MSc thesis dealt with the phosphorylation by Pi in the absence of a gradient. Masuda’s heart was really in biology and after his PhD he slowly drifted toward the biochemistry of insects. After his postdoc in the USA he joined forces with us at the new biochemistry department, where he became a full professor and organized an excellent group working in Insect Biochemistry. In the meantime, I found out that the apparent Km for Pi increases several fold when a Ca2þ gradient is formed across the membrane or when the pH is decreased from 7.0 to 6.0 [10,11,27]. Thus, the gradient simply increased the enzyme affinity for Pi. The phosphorylation by Pi measured both in the presence and absence of a gradient were inhibited by the addition of Ca2þ to the medium in the same concentration range as that needed for the activation of ATP hydrolysis. There was, however, a major difference between the phosphoenzyme formed in the presence and absence of a gradient and this was the sensitivity to ADP ^ only the phosphoenzyme formed in the presence of the gradient was able to transfer its phosphate to ADP, leading to the synthesis of ATP [10,11,28]. Thus, we had the same acylphosphate residue with two different energetic levels, one of low energy that could be formed spontaneously in the absence of a gradient but could not transfer its phosphate to ADP and a second of high energy that was formed in the presence of the gradient and could transfer its phosphate to ADP, forming ATP. This led to the conclusion that the energy derived from the gradient was not needed for the phosphorylation of the enzyme by Pi but it was required to convert the phosphoenzyme from ‘‘low energy’’ into ‘‘high energy.’’ In 1973, Kanazawa and Boyer [29] found that a small but significant fraction of the enzyme was phosphorylated by Pi when intact vesicles not loaded with Ca2þ were incubated in a medium containing EGTA and Mg2þ. The amount of phosphoenzyme


621

measured was 20 times lower than that measured by Makinose with Ca2þ -loaded vesicles. The low phosphorylation level measured with Pi was abolished when the membranes of the empty vesicles were disrupted with the detergent Triton X-100. It was already known that sarcoplasmic reticulum vesicles contain a small amount of endogenous Ca2þ [29^31]. On the basis of the low amount of phosphoenzyme obtained and its inhibition by Triton X-100, Kanazawa and Boyer [29] concluded that phosphorylation by Pi was promoted by the small transmembrane Ca2þ gradient formed between the contaminant Ca2þ inside the vesicles and EGTA in the medium. Later, Kanazawa [32] showed that the Ca2þ -ATPase was unstable in the presence of EGTA and Triton X-100, thus explaining the earlier findings with Boyer. After the addition of Triton X-100, a significant phosphorylation by Pi could only be observed during the first seconds after the addition of the detergent, after which the enzyme was denatured and was no longer phosphorylated by Pi, giving the wrong impression that there was no phosphorylation in the absence of a gradient. We were lucky to have chosen ethyl ether to permeabilize the vesicles, a treatment that does not damage the Ca2þ -ATPase: otherwise we also would have missed the formation of the ‘‘low-energy’’ phosphoenzyme. Our paper showing phosphorylation without a gradient and that of Kanazawa with Boyer were both published in 1973. After reading their paper I wrote to Paul Boyer explaining that I had not quoted his work because mine was already past the galley proofs and I could no longer add references. Paul kindly answered and finally I invited him to come to Rio. Fortunately he accepted and we repeated in Rio de Janeiro the experiment with leaky vesicles. Paul was convinced that in fact there could be phosphorylation without a gradient and we started to collaborate at a distance, measuring Pi $ H18 OH exchange catalyzed by the Ca2þ -ATPase under different conditions [33,34] ^ the experiments were performed in Rio and the analysis of O18 in Los Angeles. I learned a lot from Paul Boyer and started to admire him

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not only because of his excellence in science but also for his kind and unassuming personality. During his visit Paul explained to us his Pi $ H18 OH exchange experiments with mitochondria indicating that ATP could be spontaneously synthesized at the catalytic site of the mitochondrial F1ATPase without the need for energy. The ATP thus synthesized was ‘‘tightly bound’’ and could not be dissociated from the enzyme. According to his view, first published in 1973 [35], energy was needed not for the synthesis of ATP but for the dissociation of the ‘‘tightly bound’’ ATP from the enzyme. Recently Paul Boyer was awarded the Nobel prize for the discovery of the mechanism through which the mitochondrial ATP-synthase works.

Binding Energy and Synthesis of ATP in the Absence of a Ca2þ Gradient In 1971 Makinose [22] observed that the Ca2þ -ATPase catalyzes simultaneously the hydrolysis and synthesis of ATP when the vesicles are filled with Ca2þ and a steady-state between Ca2þ influx and Ca2þefflux is reached. This was referred to as the ATP $ Pi exchange reaction and was one of the parameters used to characterize the interconversion between osmotic and chemical energy. The disruption of the vesicles integrity with either phospholipase A or diethyl ether was found to abolish the synthesis of ATP. This led to the conclusion that at steadystate the energy derived from the hydrolysis of ATP was used to maintain the Ca2þ gradient and at the same time, the energy derived from the gradient was used to synthesize ATP from ADP and Pi. At that time it was a general belief that the presence of a gradient was an absolute requirement for the ATP $ Pi exchange reaction because the osmotic energy was needed to drive the synthesis of ATP measured during the exchange reaction. This assumption was derived from studies in mitochondria where it was not possible to measure


623

ATP $ Pi exchange in the absence of an Hþ gradient. For the Ca2þ -ATPase the synthesis of ATP is initiated by phosphorylation of the ATPase by Pi and we had already shown that this reaction is observed both in the presence and absence of a Ca2þ gradient [25, 26]. The possibility was then raised that in absence of a gradient there was no ATP synthesis because the phosphoenzyme formed from Pi could not be converted from ‘‘low’’ into ‘‘high’’ energy. When a gradient is formed, the Ca2þ concentration inside the vesicles reaches the range of 2^10 mM. We then theorized that the conversion of the phosphoenzyme from low into high energy could be related to the binding of Ca2þ to a site of the ATPase located in a part of the protein facing the vesicles’ lumen. To test this hypothesis, we prepared leaky vesicles and incubated them in media containing various Ca2þ concentrations, from a range similar to that found on the outer surface of the vesicles during transport (micromolar) up to the range found in the vesicles’ lumen during Ca2þ accumulation (millimolar). The result of this experiment was very rewarding. In the presence of low Ca2þ concentrations (micromolar), the ATPase was able to catalyze only the hydrolysis of ATP, but as the Ca2þ concentration was raised to the millimolar range there was a simultaneous inhibition of ATP hydrolysis and activation of ATP synthesis. The Ca2þ concentration needed for both halfmaximal inhibition of ATP cleavage and half-maximal activation of ATP synthesis was in the range of 1^2 mM [36,37]. From these experiments we concluded that (i) during catalysis the enzyme was able to conserve some of the energy derived from ATP cleavage in such a manner as to utilize it to synthesize a new ATP molecule from ADP and Pi; (ii) the mechanism of the energy conservation involved the binding of Ca2þ to a low-affinity binding site of enzyme facing the vesicles’ lumen (Ks 1^2 mM); (iii) the standard free energy derived from the binding of two Ca2þ ions to the low-affinity site is 8.2 kcal/mol. This is sufficient for the synthesis of 1 mol of ATP for each mol of enzyme. According to these findings, the energy needed for the conversion of the phosphoenzyme from low into high energy and the

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subsequent synthesis of ATP was derived from the binding of Ca2þ to the low-affinity site of the enzyme. Thus, the mechanism by which the enzyme recognizes the gradient for the reversal of the catalytic cycle is related to the asymmetrical binding of Ca2þ on the two sides of the membrane. In order to phosphorylate the enzyme by Pi and form the low-energy phosphoenzyme, it is necessary that the Ca2þ concentration at the outer surface of the membrane be low enough so that binding to a high-affinity site of the protein facing the outer surface of the vesicle cannot occur, and in a second stage, to convert the phosphoenzyme from low into high energy, Ca2þ must bind to a low-affinity site of the enzyme located on the inner surface of the membrane. In the conditions used to measure ATP $ Pi exchange there is no net synthesis of ATP; the rate of ATP hydrolysis is always faster than the rate of ATP synthesis. A year after our reports on the exchange in the absence of a gradient, Knowles and Racker [38] reported that the Ca2þ -ATPase could catalyze the net synthesis of ATP in the absence of a Ca2þ gradient after a single catalytic cycle. This was achieved using leaky vesicles and a two-step procedure where initially the enzyme was phosphorylated by Pi in the absence of Ca2þ and then ADP and 3^4 mM CaCl2 were added to the medium. After the Ca2þ jump, half of the phosphoenzyme phosphate was transferred to ADP, forming ATP. Shortly after that we confirmed this experiment and in addition showed that different perturbations could drive the synthesis of ATP. These included a sudden change (jump) of pH, temperature, or water activity of the medium [39^41]. There are several similarities between the (NaþþKþ)ATPase and the Ca2þ -ATPase and discoveries with one enzyme frequently could be extended to the other. In 1975, Taniguchi and Post [42] reported that similar to the Ca2þ -ATPase, the (NaþþKþ) ATPase could be phosphorylated by Pi and could catalyze an ATP $ Pi exchange reaction in the absence of a transmembrane gradient. At this time, Robert Post visited our laboratory for a 3-week period. He is a very kind and gentle


625

man and we learned a lot from him. Robert is an excellent athlete and one of the things I took up after his visit was jogging.

The Reaction Sequence The study of the reversal of the Ca2þ pump led to the proposal of a basic reaction sequence [10,11,43^45] shown in Figure 1. During catalysis the enzyme cycles through two distinct conformations, E1 and E2. The enzyme form E1 binds calcium with high affinity on the outer surface of the vesicles and can be phosphorylated by ATP but not by Pi. The enzyme form E2 binds calcium with low affinity at the inner surface of the vesicles and can be phosphorylated by Pi but not by ATP. The key feature of this cycle is the mechanism by which energy is transduced. In earlier models it was thought that the energy of hydrolysis of the different phosphate compounds would be the same regardless of whether they were in solution in the cytosol or bound to the enzyme surface, and that energy would be released and become available to the enzyme when the phosphate compound is hydrolyzed. The finding that the energy of hydrolysis of the phosphoenzyme formed from Pi varies during the reversal of the

Fig. 1.

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Ca2þ -ATPase indicates that the energy becomes available for the translocation of calcium through the membrane before the cleavage of the phosphoenzyme. Thus, during the catalytic cycle there is a large change in the equilibrium constant for the hydrolysis (Keq) of the acylphosphate residue of the phosphoenzyme and the work is linked with this transition of Keq and not with the hydrolysis of the phosphate compound (Table 2). After the description of the phosphoenzyme of ‘‘high’’and ‘‘low’’energy in our laboratory and the ‘‘tightly bound ATP’’ of the mitochondrial ATPase described in Paul Boyer’s laboratory, in several laboratories it was discovered that the energy of hydrolysis of various phosphate compounds varies greatly depending on whether they are in solution or on the enzyme surface (Table 2).

Solvation Energy and Phosphate Compounds of ‘‘High’’ and ‘‘Low’’ Energy The concept of ‘‘energy-rich’’ and ‘‘energy-poor’’ phosphate compounds was formalized by Lipmann in 1941 [46]. On the basis of the knowledge available at that time, Lipmann proposed that the energy that could be derived from the hydrolysis of a phosphate compound would be determined solely by the chemical nature of the bond that links the phosphate residue to the rest of the molecule. In this early work, the possibility that some energy might be derived from the interaction of reactant and product with the environment (solvent, cations etc.) was not taken into account. It was only in 1970 that George and coworkers [47] proposed that interaction with the solvent might play an important role in determining the Keq of a reaction. According to their view the energy of hydrolysis of a phosphate compound could be determined by the difference in solvation energies of reactant and products. Solvation energy is the amount of energy needed to remove the solvent molecules that organize around a molecule in solution. Later, Hayes et al. [48]

Enzyme

Ca2þ -ATPase, (NaþþKþ) ATPase F1-ATPase, Myosin Inorganic Pyrophosphatase Hexokinase

Reaction

Solution (Before Work)

Enzyme-Bound (After Work)

Keq (M)

G (kcal/mol)

Keq (M)

G0 (kcal/mol)

Aspartyl phosphate hydrolysis

106

8.4

1.0

0

ATP hydrolysis PPi hydrolysis

106 104

8.4 5.6

1.0 0.5

0 0.9

2 103

4.6

1.0

0

ATP þ glucose ! Gluc-P þADP

0


TABLE 2 Variability of the energy of hydrolysis of phosphate compounds during the catalytic cycle of energy transducing enzymes*

*For details, (see Refs. [14,15]). The relationship between the standard free energy of hydrolysis (G0) and the equilibrium constant of the reaction (Keq) is: G0 ¼ RT In Keq, where R is the gas constant (1.981) and T the absolute temperature.

627

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calculated the energy of hydrolysis of several phosphate compounds in gas phase and the values found were quite different from those measured in water, thus supporting the proposal of George et al. [47]. According to these calculations the energy of hydrolysis of an acylphosphate residue, which in water is in the range of 8 to 10 kcal/mol, decreases to þ 5 to þ 32.5 kcal/mol in gas phase, i.e., it is a ‘‘high-energy’’ phosphate compound in water but it has a very ‘‘low-energy’’ in gas phase. Based on these theoretical estimates, we raised the possibility that in the E2 conformation the catalytic site of the enzyme is hydrophobic so that Pi and the aspartic acid would interact as if in a gas phase, i.e, the energy for the hydrolysis of an acylphosphate residue would have a positive value and the reaction would occur spontaneously. In this case, the major thermodynamic barrier for the formation of the acylphosphate residue would be not the formation of the covalent bond (k2/k2) but the binding of Pi to the enzyme (k1/k1), i.e., the partitioning of a hydrophilic ion (Pi) from the assay medium into the hydrophobic environment of the catalytic site. E2 þ Pi

k1

k2

k1

k2

! E2 P1

! E2 P

Factors facilitating this partition (k1/k1) should also facilitate the phosphorylation of the enzyme by Pi. In Figure 1 these two steps are simplified as reaction 5. The phosphoenzyme E2 -P formed from Pi would not be able to transfer its phosphate to ADP because of the large difference in the energies of hydrolysis of the acylphosphate and the ATP in a hydrophobic environment. The binding of Ca2þ to the low-affinity site of the enzyme (reaction 4 backwards in Figure 1) would then promote a conformational change in the protein that would allow the entry of water into the catalytic site, with subsequent solvation of both the acylphosphate residue and ADP (reaction 3 backwards). As a result, the energy values for the hydrolysis of the acylphosphate and ATP would become equal and the synthesis of ATP would


629

proceed spontaneously (reaction 2 backwards). Experimental conditions, which reduce the entry of water into the catalytic site, should also impede the synthesis of ATP. According to this hypothesis the existence of ‘‘high-energy’’ and ‘‘low-energy’’ forms of the phosphoenzyme would be related solely to the activity of water in the catalytic site. We tested this hypothesis by measuring the phosphorylation of the enzyme by Pi and the synthesis of ATP in the presence of various organic solvents (dimethyl sulfoxide, glycerol, and N,N-dimethylformamide) [40]. In aqueous mixtures, these solvents markedly increase the partition coefficient of Pi from the aqueous medium into an organic phase containing isobutanol and benzene. The phosphorylation of the enzyme by Pi demonstrates saturation behavior, indicating the occurrence of a phosphateênzyme complex prior to the phosphorylation reaction (k1/k1). If the catalytic site of the enzyme is hydrophobic, then the partitioning of Pi from the assay medium into the catalytic site should be facilitated when the difference in hydrophobicity of these two compartments is decreased by the addition of organic solvent to the medium and this should promote a decrease in the apparent Km for Pi. Accordingly, replacing 40% of the water in the assay medium by dimethyl sulfoxide promoted a l000-fold decrease in the apparent Km for Pi. The phosphoenzyme formed in the presence of 40% dimethyl sulfoxide was not converted to a high-energy form after the binding of Ca2þ to the low affinity site of enzyme, i.e., there was no synthesis of ATP after the addition of Ca2þ and ADP to the medium. The inhibition of ATP synthesis was related to the decrease in water activity caused by the organic solvent because the synthesis of ATP was restored if, after the addition of ADP and Ca2þ, the dimethyl sulfoxide concentration was suddenly decreased from 40 to 2%. The hypothesis proposed states that ‘‘high’’ and ‘‘low’’ energy forms of the phosphoenzyme are correlated with the availability of water at the catalytic site of the enzyme. The experimental results were consistent with this hypothesis [40]. As predicted, phosphorylation of the enzyme by Pi was facilitated when the hydrophobicity of

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the medium was increased by the organic solvents and, after the addition of Ca2þ, the phosphoenzyme was only able to transfer its phosphate to ADP if the water activity of the medium was increased by dilution of the organic solvent with water. These data were reproduced in different laboratories and extended to other enzymes such as the mitochondrial F1-ATPase and the (Naþ þ Kþ) ATPase. In these two systems, the addition of 40% dimethyl sulfoxide promoted both a large decrease of the apparent Km for Pi and an increase of the equilibrium level of either the tightly bound ATP (soluble mitochondrial F1-ATPase) or the level of the phosphoenzyme formed from Pi (Naþ/Kþ ATPase) [14,15,41]. Finally, the recently reported crystal structure of the Ca2þ -ATPase indicates that the conversion of the E2 into E1 forms involves structural changes in the region of the catalytic site that are compatible with the hydrophobic/hydrophilic transition proposed for the conversion of the phosphoenzyme from ‘‘low’’ into ‘‘high’’ energy [49^51].

Pyrophosphate of High and Low Energy A direct manner to study the role of the solvent in the energy of hydrolysis of a phosphate compound was to measure the Keq and the G0 in media with different water activities. The water molecules that organize around a protein in solution have properties that are different from those of the medium ‘‘bulk water’’ ^ for example, a lower vapor pressure, a lower mobility, and a greatly reduced freezing point. Similar changes in the properties of water are observed in mixtures of solvents and water. The simplest known ‘‘high-energy’’ phosphate compound is PPi. In totally aqueous medium, the G0 of PPi hydrolysis varies between 3.5 and 4.0 kcal/mol. We found [52^54] that the energy of hydrolysis of PPi decreases when different organic solvents are included in the medium, reaching values of G0 þ 2.0 kcal/mol, i.e., it is possible to convert PPi from ‘‘high energy’’ (G0 4.0) into ‘‘low energy’’ (G0 þ 2.0) simply by modifying


631

the water activity of the medium. In Boyer’s laboratory it was shown that similar to ATP and the mitochondrial F1-ATPase, the energy of hydrolysis of PPi varies greatly depending on whether it is in solution or bound to the yeast inorganic pyrophosphatase. In the presence of organic solvent, it is possible to decrease the energy of hydrolysis of PPi to values even lower than those measured on the surface of inorganic pyrophosphatase (Table 2). Shortly after that, Wolfenden and Williams [55] found that the energies of hydrolysis of PPi and ATP in wet chloroform are significantly smaller than those measured in totally aqueous medium. After these findings, I felt much more confident about our proposal that the change in the energy of hydrolysis that occurs during the process of energy transduction was indeed related to a change in the solvent structure around reactants and products in the catalytic site of enzymes (Table 2). Thus, I decided to venture into studying other enzymes besides the Ca2þ -ATPase. The aim was to see if the correlation between water activity and changing energies of hydrolysis at the catalytic site could be extended to different enzymes involved in energy transduction.

Role of Water Activity in the Process of Energy Transduction by Different Enzymes The chromatophores of the photosynthetic bacteria R. rubrum retain a membrane-bound inorganic pyrophosphatase, which is able to catalyze both the synthesis and the hydrolysis of PPi. When illuminated, the chromatophores use the energy derived from light to form a proton gradient across the membrane and the membrane-bound pyrophosphatase uses the energy derived from the gradient to catalyze the synthesis of pyrophosphate from inorganic phosphate. With Armando and Marieta GomezPuyou [54,56] we were able to show that: (i) similar to the Ca2þ ATPase, inorganic pyrophosphatase is able to catalyze simultaneously the synthesis and the hydrolysis of PPi in the

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absence of a proton gradient; (ii) organic solvents promote a decrease in the apparent Km of the pyrophosphates for Pi; and (iii) with soluble pyrophosphatase it is possible to synthesize the same amount of PPi as that synthesized by the chromatophores under light solely by changing the water activity of the medium. After our studies on the effect of organic solvents on the Ca2þ ATPase, Sakamoto [57,58] reported that the affinity of the soluble mitochondrial F1-ATPase for Pi increases by severalorders of magnitude when part of the water of the medium is replaced by an organic solvent. This finding was reproduced in different laboratories [14,15]; and in collaboration with the Gomes-Puyou’s [59,60], we were able to reproduce this finding and further showed that when the water activity of the medium was decreased there is a slow exchange between the ATP tightly bound to the F1-ATPase and Pi in the medium. It was only possible to measure this exchange when the apparent affinity of the ATPase for Pi was enhanced by the addition of organic solvents.These findings indicate that, as proposed for the Ca2þ -ATPase, during oxidative phosphorylation the catalytic site of the F1-ATPase undergoes a hydrophobic^hydrophilic transition. In his Nobel Prize lecture, Boyer quoted our proposal as the probable mechanism that promotes the spontaneous synthesis of the tightly bound ATP [61]. Embedded in the plasma membrane of yeast there is an Hþ-ATPase that uses the energy derived from ATP hydrolysis to pump Hþ across the membrane. The catalytic cycle of this enzyme is similar to that of the Ca2þ -ATPase and (NaþþKþ)ATPase (E1 E2 ATPase). In collaboration with Andre¤ Goffeau [62, 63] we found that similar to the Ca2þ ATPase, the yeast Hþ-ATPase is also able to catalyze the synthesis of ATP (ATP $ Pi exchange) in the absence of an Hþ gradient and that a decrease in the water activity of the medium leads to both a decrease in the apparent Km for Pi and a severalfold increase in the rate of ATP synthesis. Post’s laboratory [42,64] reported that similar to the Ca2þ ATPase, the (NaþþKþ) ATPase could be phosphorylated by Pi and could catalyze the synthesis of ATP in the absence of


633

a transmembrane gradient. A few years later [65^67], it was found that in the absence of a gradient, a 200-fold decrease in the Pi concentration needed for half-maximal phosphorylation is observed when 40% dimethyl sulfoxide is added to the assay medium. The phosphoenzyme formed in the presence of a low concentration of organic solvent can transfer its phosphate to ADP to form ATP when 400 mM NaCl is added to the medium. The synthesis of ATP is inhibited when the concentration of dimethyl sulfoxide is raised to 60%. These findings indicate that similar to the Ca2þ -ATPase, the conversion of the phosphoenzyme from a ‘‘low-energy’’ into a ‘‘high-energy’’ form of the (NaþþKþ) ATPase is also related to a hydrophobic^hydrophilic transition at the catalytic site The Thermogenic Function of the Ca2þ -ATPase: Uncoupled Ca2þ Efflux and Uncoupled ATP Hydrolysis The catalytic cycle of the ATPase varies depending on the Ca2þ concentration in the vesicles’ lumen. When the free Ca2þ concentration inside the vesicles is kept in the micromolar range, the reaction cycle flows as shown in Figure 1. The main feature of this cycle is that the hydrolysis of each ATP molecule is coupled with the translocation of two Ca2þ ions across the membrane (4^7). This stoichiometry is only measured when the Ca2þ concentration in the vesicles’ lumen is kept below 0.2 mM [68^71]. Under physiological conditions, however, the Ca2þ concentration inside the reticulum rises to the millimolar range and in this condition the stoichiometry falls to a value varying between 0.3 and 0.6. Evidence obtained in different laboratories indicates that the enzyme cycles through two more sets of intermediary reactions when the Ca2þ concentration inside the vesicles rises to the millimolar range. These are ramifications of the catalytic cycle and are denoted as dashed lines in Figure 2. In one of them, a part of the Ca2þ accumulated by the vesicles leaks through the enzyme without catalyzing the synthesis of ATP.

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This is referred to as uncoupled Ca2þ efflux and is represented by reactions 7, 8, and 9 in Figure 2 [72^76]. In another ramification of the catalytic cycle, the progressive rise in the lumenal Ca2þ concentrations promotes ATP hydrolysis without Ca2þ translocation [71,77]. The uncoupled ATP hydrolysis is derived from the cleavage of the phosphoenzyme form 2Ca: E1 P (reaction 10 in Figure 2). Until recently, it was assumed that the amount of heat produced during the hydrolysis of an ATP molecule is always the same, as if the energy released during ATP cleavage were divided into two non-interchangeable parts, one for Ca2þ transport and the other converted into heat. In my laboratory [14^17, 78^85] we found that during Ca2þ transport, a fraction of both chemical energy derived from ATP cleavage and osmotic energy derived from the Ca2þ gradient is converted by the ATPase into heat. Thus, depending on the conditions used, the amount of heat released during the hydrolysis of ATP may vary between 7 and 32 kcal/mol. We found that this variability is derived from the uncoupled Ca2þ efflux (reactions 7, 8, and 9 in Figure 2) and from the uncoupled ATPase (reaction 10). When

Fig. 2.


635

the Ca2þ concentrations on the two sides of the membrane are kept below 0.1 mM, the amount of heat released during the hydrolysis of each mol of ATP varies between 9 and 12 kcal/mol. This was measured using leaky vesicles that are not able to accumulate Ca2þ. In this condition, there is no gradient, and no ramification of the catalytic cycle, and the cleavage of ATP is completed after the hydrolysis of the ‘‘low-energy’’ phosphoenzyme E2 -P. The yield of heat increases to the range of 20^ 32 kcal/mol when intact vesicles are used and the Ca2þ concentration in the vesicles’ lumen rises to the millimolar range; a part of the Ca2þ accumulated leaks through the ATPase leading to the conversion of the osmotic energy into heat [77,82]. The major source of heat, however, is derived from the uncoupled ATPase (reaction 10). The high intravesicular Ca2þ concentration forces the reversal of reactions 5 and 4 leading to accumulation of the ‘‘high-energy’’ phosphoenzyme 2Ca:E1 P, which is then hydrolyzed. During reactions 4 and 5 in Figure 2, a part of the chemical energy derived from ATP cleavage is converted into osmotic energy. If the hydrolysis of ATP is completed before Ca2þ translocation through the membrane (reaction 10 in Figure 2), then there is no conversion of chemical into osmotic energy, and during catalysis more chemical energy is left available to be converted into heat [77,84,85]. The rates of uncoupled Ca2þ efflux and uncoupled ATPase activity are modulated by different drugs, temperature and water activity of the assay medium [78,80,81,83^85]. When taken together, these findings indicate that the Ca2þ -ATPase is able to handle the energy derived from ATP hydrolysis in such a way as to determine how much is used for Ca2þ transport and how much is dissipated as heat. In this view, the total amount of energy released during ATP hydrolysis is always the same, but the fraction of the total energy that is converted into work or heat seems to be modulated by the Ca2þ ATPase. At present my work is concentrated on the possible thermogenic function of the Ca2þ -ATPase. The aim is to see if there is a correlation between heat production by the Ca2þ -ATPase and control of heat production and thermogenesis

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in animals. The general interest in this subject has increased during the past decade due to its implications in health and disease. Heat generation plays a key role in the regulation of the energy balance of the cell, and alterations of thermogenesis are noted in several diseases, such as adiposity and thyroidhormone alterations.

Science and Education I have always enjoyed teaching. Since the beginning of my carrier at the university, I have taught medical students ^ first biophysics, and then biochemistry. The students are always 18^20 years old and thus, in spite of the fact that every year I get older, I am usually familiar with the way of thinking of this younger generation, and this has been a great help in raising my children. In 1987 I decided to get involved with science education at schools. Since then, I have progressively left all administrative activities in order to have more free time for education and at present, in addition to my teaching medical students, I dedicate about 20% of my time to science education in schools. It all started with an experimental course for young school students that I organized with my students during the summer vacations. A broad theme was selected, for instance photosynthesis or muscle contraction, and starting from the first day, we stimulate the students to design an experiment to elucidate an aspect of the theme that they would like to know about, and then we would assist them in the execution of the experiment. Usually, a group of 5^6 students select the same sort of experiments and work together in carrying them out. During the courses there are no theoretical classes and we avoided answering questions raised by the students that are not related to the experiments being performed. The course lasts for about 40 hours and took a whole week. On the last day we organize a mini-congress, where each group of students presents the results of their experiment for general discussion. After that


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there is a party with cake and soft drinks. From the very beginning the success exceeded our best expectations and since then we receive two to three groups of students during summer and winter vacations. Now, the courses are given by our MSc and PhD students under the supervision of one of the professors of the department and my participation is limited to hunting for money to cover the expenses. After a few rounds of these courses, we decided to expand our activities and started the same sort of course for school teachers, with two weeks’ duration. During the first week they perform experiments and in the second week they assist our graduate students in the course given to school students. After the first year, the graduate students started to pressure me to expand the program further. In each course there was always a group of school teachers and students whom they felt were particularly bright, and it was a pity to just drop them after a few days’ work. Thus, I had to find more money to expand our activities and now, after each course we select one or two school teachers to work in the laboratory for a period of a year, receiving a small stipend. From the school students, we also select two or three that have done well during our course and invite them to work in the laboratory. Since the beginning we directed our activities preferentially to public schools that are attended by children of families with low income, and in the selection of the students, preference is given to students of low-income families. These students work under the supervision of a PhD student, and receive enough money for transportation and food, plus any expenditure related to their education. This include books, and parallel courses in English, computers, or whatever the PhD supervisor deems necessary. A high school student and a PhD student work in a sort of a symbiosis. The high school students helps the PhD student in his experiments and in return, the PhD student supervises his studies at school. The aim is to prepare the high school students to enter a public university. These are the best universities in the country and are free. The problem is that there is a stiff selection to enter and only a small fraction of the students who

638

L. DE MEIS

apply are selected. The school teachers selected to work in the laboratory are engaged in research project dealing with either education or science sociology. Some of them select a biochemistry research project and work in the bench. Since the beginning of the project, more than 2500 school students and 800 school teachers have participated in the courses organized in our department. From the students selected to work in the laboratory, about 20 have made it to the university, and my best PhD students at present did start in one of these courses. Among the school teachers, three of them went all the way up to a PhD degree and recently were admitted as associate professors in our department. One of them decided for biochemistry and is now in postdoctoral training in California (USA), two of them got their PhD’s in science education and are now in charge of this part of the program inventing new ways to increase the interaction between the school and the university.

Science and Art Illustrated Books Since the beginning of my activities with schools, I have been astonished at the unattractive aspect and low-quality of the books given to school students, mainly to those of low income families attending public schools. Thus, I started to look for young artists to prepare educational material that would seduce the students by its beauty. I was fortunate to find a very talented cartoonist, Diuce“nio Rangel, a graduate from the art school in our university. With Diuce“nio, we prepared two illustrated books that have been very well received not only by schools, but also by our scientific community. The title of the first book produced is ‘‘The Scientific Method’’ and the second, more recent, is ‘‘Respiration and the First Law of Thermodynamics.’’ In these two books we recount the story as it was discovered, but using cartoons; 90% of the pages are


639

colorful drawings with short sentences. We are now in the third edition of ‘‘The Scientific Method’’ (each edition comprises 4,000 books) and preparing the second edition of the ‘‘Respiration and the First Law of Thermodynamics.’’ About 70% of these books were distributed (free) in reading rooms of public schools and the rest were sold at a cost price.

Theater Based on the two illustrated books, in collaboration with Diuce“nio we mounted a theater play on the ‘‘scientific method.’’ The play is actually a condensed story of science. I, Diuce“nio, and between 10^12 graduate students performed on the stage for about 90 minutes. The play was intended as the closing event of our vacation courses, but we have been invited to several schools, universities, and even scientific congresses (annual meetings of the Brazilian Societies of Biochemistry and Chemistry, and the Brazilian Society for the advancement of Science). Different universities in different states of Brazil (Sa‹o Paulo, Rio Grande do Sul, and Espirito Santo) have organized excursions in different cities to perform both for university students and school students.

CD and DVD This is our most recent activity. Working with Diuce“nio and two students of the school of art who are experts in graphic computation, we have just finished our first animation, 23 minutes long. It is about the mitochondrion and is presented in three formats. In the first we use a language similar to that used in movies. In the second, didactic languages is used, as in a classroom; and finally, the third version is an artistic trip through the mitochondrion.

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Final Statement To end this story, it is necessary that I publicly confess ^ thanks God for Walter O. Cruz’ lecture (as I mentioned before, I would have been a very mediocre surgeon). And honestly. . . I have had, and I am having, a lot of fun working in science and education. REFERENCES [1] [2] [3] [4] [5] [6]

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[26] de Meis, L. and Masuda, H. (1974) Biochemistry 13, 2057^2062. [27] de Meis, L. (1976) J. Biol. Chem. 251, 2055^2062. [28] de Meis, L. and Carvalho, M.G.C. (1976) J. Biol. Chem. 251, 1413^1417. [29] Kanazawa, T. and Boyer, P.D. (1973) J. Biol. Chem. 248, 3163^3172. [30] Duggan, P.F. and Martonosi, A (1970) J. Gen. Physiol. 56, 147^176. [31] Chevalier, J. and Butow, R.A. (1971) Biochemistry 10, 2733^2737. [32] Kanazawa, T. (1975) J. Biol. Chem. 250, 113^119. [33] Boyer, P.D., de Meis, L., Carvalho, M.G.C. and Hackney, D. (1977) Biochemistry 16, 136^140. [34] de Meis, L. and Boyer P.D. (1978) J. Biol. Chem. 253, 1556^1559. [35] Boyer, P.D., Cross, R.L. and Momsen, W. (1973) Proc. Natl. Acad. Sci. USA 70, 2837^2839. [36] de Meis, L. and Carvalho, M.G.C. (1974) Biochemistry 13, 5032^5038. [37] de Meis, L. and Sorenson, M.M. (1975) Biochemistry 14, 2739^2744. [38] Knowles, A.F. and Racker, E. (1975) J. Biol. Chem. 250, 113^119. [39] de Meis, L. and Tume, R.K. (1977) Biochemistry 16, 4455^4463. [40] de Meis, L., Martins, O.B. and Alves, E.W. (1980) Biochemistry 19, 4252^4261. [41] de Meis, L. and Inesi, G. (1982) J. Biol. Chem. 257, 1289^1294. [42] Taniguchi, K and Post, R.L. (1975) J. Biol. Chem. 250, 3010^3018. [43] Tanford, C. (1984) Crit. Rev. Biochem. 17, 123^151. [44] Carvalho, M.G.C., Souza, D.O.G. and de Meis, L. (1976) J. Biol. Chem. 251, 3629^3636. [45] de Souza, D.O.G and de Meis, L. (1976) J. Biol. Chem. 251, 6355^6359. [46] Lipmann, F. (1941) Adv. Enzymol. 1, 99^162. [47] George, P.,Witonsky, R.J.,Trachtman, M.,Wu, C., Dorwat,W., Richman, L., Richman,W., Shurayh, F. and Lentz, B. (1970) Biochim. Biophys. Acta 223, 1^15. [48] Hayes, D.M., Kenyon, G.L. and Kollman, P.A. (1978) J. Am. Chem. Soc. 100, 4331^4340. [49] Toyoshima, C., Makasako, M., Nomura, H. and Ogawa, H. (2000) Nature 405, 647^655. [50] Danko, S., Yamasaki, K., Daiho, T., Suzuki, H. and Toyoshima, C. (2001) FEBS Lett. 505, 129^135. [51] Hua, S., Ma, H., Lewis, D. and Inesi, G. (2002) Biochemistry 41, 2264^2272. [52] de Meis, L. (1984) J. Biol. Chem. 259, 6090^6097. [53] de Meis, L., Behrens, M.I., Petretski, J.H. and Politi, M.J. (1985) Biochemistry 24, 7783^7789. [54] Behrens M.I. and de Meis, L. (1985) Eur. J. Biochem. 152, 221^227. [55] Wolfenden, R. and Williams, R. (1985) J. Am. Chem. Soc. 107, 4345^4346. [56] de Meis, L., Behrens, M.I., Celis, H., Romero, I., Gomez-Puyou, M.T. and Gomez-Puyou, A. (1986) Eur. J. Biochem. 158, 149^157.

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[57] Sakamoto, J. and Tonomura,Y. (1983) J. Biochem. (Tokyo) 93, 1601^1614. [58] Sakamoto, J. (1984) J. Biochem. (Tokyo) 96, 475^481. [59] Gomez-Puyou, A., Gomez-Puyou, M.T. and de Meis, L. (1986) Eur. J. Biochem. 159, 133^140. [60] de Meis, L., Go¤mez Puyou, M.T. and Go¤mez-Puyou, A. (1988) Eur. J. Biochem. 171, 343^349. [61] Boyer, P.D. (1998) Biosc. Rep. 18, 97^117. [62] de Meis, L., Blanpain, J.P. and Goffeau, A. (1987) FEBS Lett. 212, 323^327. [63] Goffeau, A. and de Meis, L. (1990) J. Biol. Chem. 26, 15503^15505. [64] Post, R.L., Toda, G. and Rogers, F.N. (1975) J. Biol. Chem. 250, 691^701. [65] Moraes,V.L. and de Meis, L. (1982) Biochim. Biophys. Acta 688, 131^137. [66] Jorgensen, P.L. and Skriver, E. (1982) Ann. N.Y. Acad. Sci. 402, 207^225. [67] Moraes,V.L.G. and de Meis, L. (1987) FEBS Lett. 222, 163^166. [68] Inesi, G., Kurzmack, M. and Verjovski-Almeida, S. (1978) Ann. N.Y. Acad. Sci. 307, 224^227. [69] Inesi, G., Kurzmack, M., Coan, C. and Lewis, D. (1980) J. Biol. Chem. 255, 3025^3031. [70] Inesi, G. (1985) Annu. Rev. Physiol. 47, 573^601. [71] Yu, X. and Inesi, G. (1995) J. Biol. Chem. 270, 4361^4367. [72] McWhirter, J.M., Gould, G.W., East, J.M. and Lee, A.G. (1987) Biochem. J. 245, 731^738. [73] Inesi, G. and de Meis, L.J. (1989) Biol. Chem. 264, 5929^5936. [74] de Meis, L. (1991) J. Biol. Chem. 266, 5736^5742. [75] de Meis, L. and Inesi, G. (1992) FEBS Lett. 299, 33^35. [76] de Meis, L.,Wolosker, H. and Engelender, S. (1996) Biochim. Biophys. Acta 1275, 105^110. [77] de Meis, L., (2001) J. Biol. Chem. 276, 25078^25087. [78] de Meis, L. Bianconi, M.L. and Suzano, V.A. (1997) FEBS Lett. 406, 201^204. [79] de Meis, L. (1998) Biochem. Biophys. Res. Comm. 243, 598^600. [80] de Meis, L. (1998) Am. J. Physiol. 274 (Cell Physiol. 43), C1738^C1744. [81] Mitidieri, F. and de Meis, L. (1999) J. Biol. Chem. 274, 28344^28350. [82] de Meis, L. (2000) Biochem. Biophys. Res. Comm. 276, 35^39. [83] de Meis, L. (2001) Biosc. Rep. 21, 113^137. [84] Reis, M., Farange, M., Souza, A.C.L. and de Meis, L. (2001) J. Biol. Chem. 276, 42793^42800. [85] Barata, H. and de Meis, L. (2002) J. Biol. Chem. 277, 16868^16872.

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Name Index Abderhalden, E., 112^114, 486, 521^524, 526, 569^580, 582^589 Abderhalden, R., 521, 553, 584, 586, 587 Abdullaev, N.G., 348 Abir-Am, P.G., 48, Adams, J., 462 Adler, J., 350, 353 Afanasiev, D., 326 Agalarova, M.B., 397 —gerup, B., 192 —gren, G., 157 Aharonov, A., 250 —kerstro«m, G., 177 —keson, —., 59 Akusja«rvi, G., 78 Alberts, B., 243 Albrecht, H., 540 Allge¤n, L.-G., 150 Ambler, R., 65 Ambros, O., 455 Ameisen, J.C., 377 Andersen, B., 298 Andersson, B., 204, 205 Andreyev, A.Yu., 330 Anfinsen, C.B., 111 Anseth, A., 152, 153, 160 Appella, E., 65, 67 Argetsinger-Steitz, J., 462 Arison, T., 226 Aristotle, 480 Arnold, H., 531 Arnstein, H.R.V., 275 Arruda, P., 332

Arseniev, P., 394 Ashcroft, F.M., 426, 430 Ashcroft, S.J.H., 426, 429, 430, 434, 436 Ashour, S.J., 438 Astbury, W.T., 109 Auhagen, E., 274 Avron, M., 240, 243, 256

Bagger-Srensen, M., 301 Bahr-Lindstro«m, H., 78 Bailey, K., 109, 415 Baitsch, H., 482 Bakalkin, G., 375 Bakeeva, L.E., 344, 354, 356, 375 Balazs, E.A., 141, 142, 145, 147, 148, 152, 156, 178, 187, 211 Baldwin, E., 415, 599 Baltimore, D., 244 Ba¤ra¤ny, E., 157 Barker, H.A., 295 Barnard, E.A., 279 Barre, H., 328 Baryshev, V.A., 350 Bates, H., 246 Bauer, W., 155 Baur, E., 486, 538 Beadle, B., 39 Beadle, M., 40 Beck, J., 423 Beckmann, J.S., 252, 253 Beckwith, J., 466 Begg, G., 63, 121^123, 125

644 Beggs, M., 440 Bell, D., 416 Belozersky, A.N., 340, 341, 346, 386, 387 Belozersky, A.S., 341 Ben-Gurion, D., 221, 232, 234, 259 Benzer, S., 279 Benziger, T., 560, 561 Benzinger, T., 565 Berg, P., 236, 248, 278, 299, 476 Berger, P.J., 437 Bergman, M., 112 Bergman, T., 83 Bergman, M., 112 Bergmann, E.D., 232^235 Bergstro«m, B., 107 Bergstro«m, S., 85, 92, 151 Bergstrand, C-E., 137, 139, 196 Bernadotte, L., 207, 208 Bernadotte, S., 209 Bernhard, C.-G., 194 Bernstein, C.S., 223 Bersin, T., 574, 586 Bert, B., 71 Bertsova, Y.V., 367 Berzelius, J.J., 92, 129, 140, 194 Bessman, M., 236 Betz, H., 271 Beyer, R.E., 325, 326 Beyreuther, K., 471, 473, 485 Bianco, A.C., 332 Bibikov, S.I., 350 Bieneck, 557 Bjo«rk, I., 151, 152, 160 Blix, A.S., 328 Blix, G., 156, 157 Bloch, K., 235 Bloemendal, H., 245, 246 Blomba«ck, B., 103, 115, 146, 151 Blomba«ck, M., 57, 121, 129, 146 Blume, W., 567 Blumenfeld, L.A., 337, 338

NAME INDEX Blundell, T., 278 Boedtker, H., 243 Bogachev, A.V., 361, 363, 367 Bogdanov, A.A., 388 Bo«hme, R., 454 Boltzmann, L., 44 Bonhoeffer, F., 454 Borek, E., 249 Borg, H.C., 353 Borisov, A.Yu., 399 Bork, 563 Bornstein, J., 420 Borrmann, D., 538 Borsodi, A., 279 Bosch, L., 246 Boss, O., 335 Bostro«m, H., 146 Boveris, A., 372, 376 Bowden, J.A., 438 Bowker-Kinley, B.J., 438 Bowles, J.T., 370, 378, 380 Boyd, V., 69 Boyer, P., 206 Boyer, P.D., 620 Bra«nden, C.-I., 74, 80 Brach, F., 589 Bradeser, D.E., 379 Brand, M., 334, 335 Brecht, B., 452, 454 Brecht, B., 474 Brenner, S., 58, 252, 484 Bresson, R.A., 377 Brettenbach, M., 378 Brockmann, H., 112^114, 587^589 Brodersen, R., 289 Brown, I.I., 359 Brustovetsky, N.N., 330 Buchanan, J., 297 Bu«cher, T., 274 Buck, I., 229, 232 Buck, N., 229, 232 Buckley, C.E., 20

645

NAME INDEX Burk, D., 293 Busch, F., 454 Bushkin-Harav, I., 272 Butenandt, A., 481, 487, 501, 502, 520, 523^527, 530, 534, 535, 548^577, 585, 586, 588 Butenandt, G., 556 Butler, J., 58 Buzzell, J., 12

Campbell, P., 275 Capecchi, M., 462 Carvalho, A.P., 606 Caspersson, T., 144 Catterall, W.A., 279 Cech, T., 288 Cederlund, E., 69, 97 Celsius, A., 193 Cespiva, J., 521 Chagas, F.C., 605 Chain, E., 416, 417 Chance, B., 342 Changeux., J.-P., 15 Chappell, J.B., 342, 369 Chargaff, E., 240, 457 Chase, J.F., 429 Checkley, G., 176 Chekhov, A., 463 Chen, Z.-W., 57 Chen, L.B., 356 Chentsov, Yu., 354 Chesnecopherus, J., 165 Chibnall, A.C., 415, 417, 419 Chiu Hsia, C., 459 Clarke, A.E., 278 Cleland, R.L., 172 Cobet, C., 573 Coddington, A., 301, 302 Cohen-Tannoudji, C., 213 Cohn, E.J., 6^19, 13, 14, 29 Cohn, J.T., 9

Cohn, M., 236 Comper, W., 175^177 Conant, J.B., 13 Consden, R., 110 Cooper, R.H., 431, 436 Coore, H.G., 423, 426, 428, 430 Cori, C.F., 237, 416, 422 Cori, G., 236, 237, 416 Cornelis, P., 256 Corte-Real, M., 377 Courtois, J., 274 Cox, R.A., 242 Craword, I., 236 Crick, F.H., 46, 58, 241, 242, 247, 279, 298, 464 Cruz, S., 595 Cruz, W.O., 595 Curstedt, T., 75

Do«rfler, W., 469 Dahl, L., 185 Dalton, H., 367 Dalziel, K., 65 Daniel, V., 245, 251, 252 Danielsson, H., 170 Danielsson, O., 81 Danneel, H., 567 Danon, D., 243 Davidson, B., 58, 60 Davies, J., 474 Dayhoff, M., 83 de Azevedo, A., 511 de Baetselier, A., 266 Debye, P., 16, 17 Dedukhova, V.I., 396 de Gennes, P.-G., 213 Deichmann, U., 485, 486, 492 Delbru«ck, M., 469 De Mars, R., 236 de Meis R.M., 606 de Montaigne, M., 475

646 Dent, C.E., 418, 419 Denton, R.M., 428 Denyer, H.R., 438 de Rudder, B., 511, 513, 522, 524, 525, 527, 529 Deutscher, M.P., 245 Dibrov, P.A., 361, 363 Dick, A.P., 420 Diderot, D., 479, 480 Diehl, K., 518, 520, 549, 553, 556, 557, 559 Dimroth, P., 360, 363 Dixon, M., 416 Dizengoff, M., 223 Djerassi, C., 196 Dodemont, H.J., 266 Doerfler, W., 490 Dohlman, C.-H., 152 Donehower, L.A., 382, 385 Dorfman, A., 156 Dorothy, N., 292 Doty, P., 241, 243 Drachev, L.A., 348, 349, 356 Drenth, P., 198 Drury, D.R., 425 Dudai, Y., 279 Duester, G., 78

Eastman, G., 39, 40 Eberson, L., 204 Edblad, S., 198 Edelman, G.M., 22 Edelman, M., 243 Edman, A., 105 Edman, G., 63 Edman, P.V., 61^64, 83, 103^109, 111^116, 118^122, 124^131 Edman, V., 105 Edsall, J.T., 6^19, 14, 21, 29 Effron, M.B., 322 Ehrenberg, A., 59

NAME INDEX Einstein, A., 16, 38, 233 Eisenberg, A., 229 Eisenberg, E., 240 Eisenberg, H., 229 Eklund, H., 74, 80 El-sayed, R., 191 Elson, D., 240 Engberg, E., 288 Engelberg-Kulka, H., 377 Engelhardt, V.A., 321^325, 352 Engels, F., 111, 480 England, P.J., 425, 429 Engstro«m, L., 157, 186 Engstro«m-Laurent, A., 184 Epstein, W., 359 Erlander, T., 107 Ernster, L., 275 Espinal, J., 439, 440 Eva, E., 483 Evans, F.A., 112

Fadock, V., 371 Fatania, H.R., 438, 439 Faulkner, W., 452 Fedoroff, N., 391 Fellous, A., 266, 267 Fenn, J., 86 Ferlin, N., 107 Fernandez, J., 236 Fischer, E., 48, 511, 517^519, 536^539, 570, 571, 581 Fischer, E.H., 273 Fish, W., 31 Fishbach, G., 279 Fisher, S.K., 279 Fleissner, E., 249 Fleming, A., 416 Fleury, C., 332 Flodgaard, H., 312 Flodin, P., 159 Florey, H., 416

NAME INDEX Forte, M., 377 Foster, J.E., 21 Fowler, A., 78 Fro«hlich, K.-U., 377, 378 Francescatti, Z., 234 Frankfurter, F., 40, 41 Franklin, R., 241 Fraser, J.R.E., 181, 182, 184^187 Frederic the Great, 562 Frederiksen, S., 302, 303, 305, 307 Fresco, J., 243 Fridkin, M., 254 Friedkin, M., 298 Friedlander, H., 483 Friedman, 517 Friend, C., 260 Fruton, J.S., 113, 128 Fuchs, S., 250 Fuller, N.J., 438 Furtwa«ngler, W., 576

Galen, 376 Gardell, S., 146 Garland, P.B., 425, 428 Garlid, K.D., 330, 331 Garrahan, P.J., 618 Garty, N., 272 Gedda, L., 532 Gelfand, I.M., 342 Gennis, R., 362, 363 Gentner, W., 235 George, P., 626 Gergely, J., 148 Gerk, L., 491 Germaine, P., 198 Ghosh, D., 75, 80 Giacobino, J.-P., 332^334, 336 Gibbs, J.W., 11 Gibbs, M., 296 Gierer, A., 243, 457 Gieseler, W., 571

647 Gilbert,W., 461, 462, 464^466, 467, 471, 478, 492 Gillam, I., 248 Gilsenbach, G., 487 Gilsenbach, R., 486 Ginzburg, I., 256, 266^269 Giovanni, M., 261 Giveon, D., 268 Glagolev, A.N., 350, 353, 354, 360 Glick, M.C., 261 Glynn, I.M., 618 Goelet, P., 279 Goethe, J.W., 449 Goffeau, A., 615 Goldfarb, A., 390, 391, 392, 394 Gomez-Puyou, A., 615 Gomez-Puyou, M.T., 615 Gonano, F., 249 Gonza'lez Duarte, R., 80 Gorbachev, M.S., 388, 395 Gordsky, G.M., 426 Goridis, C., 279 Go«ring, H., 561 Gottschaldt, K., 529 Gozes, I., 264^266 Gra«slund, A., 205 Gro«nvall, A., 158 Gru«ndgens, G., 578 Gru«ndgens, G., 574 Granath, K., 178 Granit, G., 91 Granot, D., 377 Gratzer, W., 27, 46 Graue, G., 570 Grav, H.J., 328 Gray, W.R., 119 Gray, W., 61 Grebe, H., 510, 520 Green, D., 23, 345 Green, N.M., 22, 23 Greene, H., 35 Grefrath, S., 35

648 Greville, G., 416 Griffin, B., 250 Griffin, S., 269 Grimberg, J.I., 252 Grinius, L.L., 344 Grodsky, G.M., 423 Gronenborn, B., 471, 477 Gronenborn, B., 477 Gronowitz, S., 204 Gros, F., 279 Grosfeld, H., 258 Gross, J., 155 Grosz, G., 468 Grunberg-Manago, M., 237, 238 Guarino, A.J., 303 Guimara‹es, J.A., 614 Gulevich, V.S., 321 Gursky, G., 473 Gustafsson, B., 170 Gustavsson, A.-M., 70 Gwynne, J., 29^31 Gyllenhammar, P., 191

Haase, i., 508, 509, 554^556 Haber, E., 24 Haberland, M., 78 Hade, E.P.K., 26 Hagel, P., 112 Hagg, M., 437 Hahn, O., 235, 528, 529 Haines, C., 292 Hajdu, J., 469 Hales, C.N., 424, 426, 428 Halle¤n, A., 174 Hallervorden, J., 489 Hammarsten, E., 140, 143, 144, 149, 151, 165 Hammarsten, O., 109, 144, 165 Han Shan, 459 Handler, P., 29, 36^38 Hanley, M., 279

NAME INDEX Hannon, J.P., 325, 326 Hansma, P., 94, 95 Hargittai, I., 196 Harington, C., 418 Harman, J.B., 419 Harper, A.E., 438 Harris, I., 56, 58^60, 62, 63, 68, 79 Harris, R.A., 438 Harter, B., 451 Hartley, B., 58, 61, 62 Hartley, B.S., 119 Hartmann, M., 529, 572, 573 Hartree, E.F., 415 Haseltine, B., 462 Hasselbach, W., 610 Haul, R., 569 Haurowitz, F., 459 Havemann, R., 527^529 Hayaishi, O., 295 Hayes, D.M., 626 Hedbys, B., 152 Hegel, G.W.F., 480 Heidegger, H., 539 Heidegger, M., 454 Heidrich, A., 458 Heinrich, H., 488 Heldin, C.-H., 190 Heldin, P., 186 Helenius, A., 34 Hellmann, H., 566 Hellsing, K., 162 Hellsing, U., 147 Helting, T., 174, 175 Henning, E., 577 Henningsen, J., 308 Henschen, A., 64, 118, 123, 126, 127, 132 Heppel, L., 239, 295 Herberts, H., 488 Hermann, A., 540 Hermann, J.M., 458 Herrmann, F., 589

649

NAME INDEX Herzberg, M., 252 Heubner, W., 529, 572 Heusner, 561 Heyden, V., 307 Hill, C., 448, 453 Hill, R., 416 Hillman, O., 556 Hillmann, A., 552 Hillmann, G., 481, 487, 501, 502, 522, 523, 526, 528, 534, 550^554, 557^561, 563, 565, 569, 583, 584 Hillmann, H., 525 Hilsing, W., 475 Himmler, H., 519, 549 Himsworth, H., 418 Hindenburg, 566 Hinkle, P.C., 345 Hinsberg, K., 524, 526, 550, 583 Hiragashi, K., 363 Hirs, C.H.W., 119 Hitler, A., 503, 536, 565 Hoff-Jrgensen, E., 297 Hoffmann-Ostenhof, O., 273, 274 Hofmeister, F., 48 Hofschneider, P.H., 477 Ho«glund, N.-J., 169 Hogness, D., 236 Holley, R., 244 Holme, T., 151 Holmgren, H., 141, 143 Hood, L., 62, 63, 83 Ho«o«g, J.-O., 70, 78^80 Ho«o«k, M., 188 Hopkins, F.G., 417 Horecker, B.L., 295 Ho«rlein, H., 531 Horst, J., 540 Houcker, B.L., 296 Houssay, B., 421, 422 Howard, R.D., 370, 384 Huang, B., 438 Huez, G., 255

Hughes, W.A., 439 Hulton, F., 585, 590 Hummeler, K., 262 Hunkapiller, M., 62, 63 Hunt, D., 87 Hurwitz, J., 236 Hutson, N.J., 437, 438 Hutton, J.J., 314 Hyman, A., 377, 378

Imae, Y., 353, 361 Inesi, G., 615 Ingelman, B., 158, 178 Ingram, V., 118 Inouye, H., 250 Isaacs, A., 242 Isherwood, A., 292 Israel, M., 279 Iverius, P.-H., 174

Jacob, F., 456, 462 Jacobsen, 311 Jacobsen, J.C., 48 Jacobson, B., 143, 145, 146, 149 Jacobson, C.-O., 195 Jander, G., 565 Jarofski, R., 525 Jasaitis, A.A., 344, 345, 361 Jawlensky, A., 473 Jeffery, J., 74 Jensen, H., 112 Jensen, K.A., 286, 287, 289 Jezek, P., 331 Jim Watson, J., 461, 462, 464, 466, 467, 471 Johannes, H., 355 Johansson, J., 63, 75, 83 Johansson, S., 184, 188 Johnson, E.A., 36 Johnson, G., 390

650 Jolle's, P., 63 Jones, G., 58 Jonsson, A., 88 Jorpes, E., 56, 107, 115, 128, 140, 143, 144, 146, 153, 164 Jo«rnvall, H., 60, 70, 74, 77^82, 84, 89 Jovin, T., 309^311 Julian, M.M., 48 Kader, J.-C., 329 Kagawa, Y., 345 Kakinuma, Y., 363 Kalckar, H., 237, 289, 290, 292, 293, 297^299 Kaminska, I., 251 Kamp, D., 471 Kanazawa, T., 620 Kandrach, A., 346 Kang, J., 485 Karas, M., 86 Karlin, A., 28, 35, 279 Karlson, P., 457, 548, 558, 587 Kashiwagi, A., 376 Katchalski (Katzir), E., 231, 247, 278, 432 Katchalsky, A., 231^233, 278, 279 Kaudewitz, F., 537 Kaufman, I., 466 Kaufmann, D., 539 Kaufmann, G., 253, 254 Kaulen, A.D., 348, 349 Kauzmann, W., 7, 29 Kawahara, K., 26 Kay, C., 244 Kaye, A., 265 Keilin, D., 415 Kelmers, A.D., 248 Kempner, R., 455, 533 Kendrew, 60 Kerbey, A.L., 431, 436, 437 Kesting, M., 454 Ku«hn, A., 521, 571

NAME INDEX Khokhlov, R.V., 387 Khorana, H.G., 294, 348 Kierkegaard, P., 205 Killander, J., 159 Kimhi, Y., 240, 250, 253, 256, 260, 261, 267 Kirkwood, J.G., 14, 16, 17 Kirpichnikov, M.P., 394 Kirsch, J., 270, 271 Kisselev, L., 325 Kjeldgaard, N.O., 290 Kjelle¤n, L., 188 Klein, E., 483 Klein, G., 483, 577 Kleinschmidt, A.K., 257 Klemm, A., 473, 474 Klenow, H., 285^317 Klingenberg, M., 329^331 Klingenstierna, S., 193 Knowles, A.F., 624 Kodama, T., 361 Kohl, U., 576 Kolarov, J., 330 Kondrashova, M., 399 Konstantinov, A.A., 349 Konstantinova, T., 396 Kornberg, A., 236, 237, 239, 248, 295, 298, 307^310, 312 Kornberg, R., 255 Kornberg, S., 236, 239 Korshunov, S.S., 365 Kozak, L.P., 333 Ku«pper, B., 540 Krainick, H.G., 475 Kramer, M., 242 Kranz, H., 527 Kraus, K., 460 Kraus, K., 460, 463 Krebs, H., 416 Kretschmer, E., 584, 586 Kroemer, G., 371 Kro«ner, H.P., 538, 539

651

NAME INDEX Ko¤sa¤ry, D., 198, 199 Kuggie, N., 71 Kuhn, R., 456, 588 Kunitz, M., 109

Ladenstein, R., 75, 80 Laemmli, U., 65 Lagerkvist, U., 144 Lancet, D., 279 Langmuir, I., 33 Lapanje, L., 26 Lardy, H.A., 323, 328 Larsen, P., 286 Lassen, U.V., 305 Lau, K.S., 439 Laurent, B.E., 137 Laurent, A., 152, 172, 179 Laurent, B.M., 151 Laurent, C., 151, 214 Laurent, E., 160 Laurent, T., 138, 151, 160 Laurent, U., 147, 148, 152, 162, 172, 179^ 183, 186, 196, 198, 207, 214, 216 Laursen, R., 64, 70 Lazarides, E., 279 Lazdunski, M., 279 Lea‹o, A.P., 608 Lechevalier, H.A., 306 Leder, P., 248, 251 Lehman, R., 236 Lehninger, A.L., 323, 324, 342 Lehrfreund, B.W., 224 Lehrfreund, R., 225 Leick, V., 288 Leloir, L., 294 Lemaire, H.-G., 28, 485 Lenin, V.I., 389 Lennerstrand, G., 210 Lent, H., 600 Lenz, F., 536^539 Lenz, W., 481, 482, 508

Levi-Montalcini, R., 237 Lewis, K., 350, 377, 384 Lewis, M., 471 Lie¤becq, C., 274 Lie¤becq, S., 274 Liberman, E.A., 341, 342, 344 Lichtler, E., 298, 300 Liebau, S., 520 Lieberman, I., 236 Lindahl, U., 160, 188 Lindberg, B., 307 Linden, H., 510 Linderstrm-Lang, K., 12, 16, 310 Lindqvist, I., 193 Lingers-Reiner, E., 516 Linn, T.C., 430 Linnaeus, C., 166, 192, 193 Lipmann, F., 234, 246, 362, 416, 626 Littauer, D., 230 Littauer, F.S., 224, 226 Littauer, M., 230 Littauer, S., 223 Littauer, T., 230, 239 Lochmann, E.R., 537 Loeb, J., 583 Logunov, A.A., 88 Lohse, B., 465, 468, 470, 474 Lowell, B.B., 333 Lowry, O.H., 424 Lozier, R., 397, 398 Lukretius, 452 Luria, S., 466 Luzzati, V., 241 Lynen, F., 235 Lysenko, T.D., 227, 388, 480, 534

Maale, O., 298 MacNutt, W., 298 Madeo, F., 377 Magalha‹es, J.R., 598 Magnussen, K., 519, 520, 532, 571

652 Magnusson, S., 58, 59, 70 Makinose, M., 609 Malakhovskaya, O.O., 396 Malatesta, F., 369 Malhotra, P., 456 Mall, G., 586 Malling, H., 304 Malmstro«m, B., 169 Mann, K., 31, 576 Mann, T.R., 416 Mann, W.R., 522, 523 Manon, S., 377 Marbaix, G., 255 Marchington, D.A., 438 Marchington, D.R., 438 Marchington, P.B., 438 Marcussen, M., 315 Markl, H., 552 Markovic›, O., 74 Marks, P., 261 Marsch, E., 552 Martin Heidegger, M., 454 Martin, A.J.P., 109, 114 Martinose, M., 609 Marton-Lef e'vre, J., 208 Marx, K., 111 Maslov, S.P., 325^327 Masuda, H., 619 Matschinsky, F.M., 430 Matthaei, H., 457, 571, 572 Matthaei, J., 246 Matthews, K., 472 Maurice, D., 152 Maxam, A., 76, 478 McCaslin, D., 28 McCourt, P., 184 Medawar, P.B., 370 Mehler, A., 295 Meister, L., 270 Mendel, G., 480 Mengele, J., 480, 481, 487, 488, 493, 494, 502, 503, 509, 511^523, 525, 526,

NAME INDEX 528, 530, 534, 535, 539^541, 550^552, 564, 571^573, 585 Mengele, M., 520 Menten, M., 583 Mentzel, R., 565, 568 Merrington, T., 419 Mertelsmann, R., 589 Meselson, M., 463 Meshkova, N.P., 323 Messing, J., 477 Metchnikoff, I.I., 385 Meyer, H., 608 Meyer, K., 156, 169 Meyerhof, O., 223 Mu«hlschlegel, B., 469 Michael, N., 458 Michaelis, L., 583, 588 Michel, H., 349, 390 Milbauer, R., 249 Mildner, P., 274 Miller, J., 472, 474 Miller, W., 449, 450, 453 Milstein, C., 58, 62 Mirsky, A.E., 17 Mishima, S., 152 Miskin, R., 263 Mitchell, P., 339, 342, 343, 357, 359, 618 Mu«ller-Hill, D., 449 Mu«ller-Hill, J., 494 Mu«ller-Hill, R., 494 Mu«ller-Hill, S., 494 Mu«ller-Hill, W., 449, 450, 453 Mu«nch, H., 512 Mokhova, E.N., 330, 399 Moliere, J.B., 384 Monard, D., 279 Monk, T., 465 Monod, J., 25, 27, 456, 462, 467 Montaigne, M., 452, 463 Moore, S., 66, 67, 110 Moreno-Sanchez, R., 336

653

NAME INDEX Morgan, H.E., 424 Morris, H., 211 Moskalewski, S., 429 Moussatche¤, H., 600 Muckermann, N., 529 Muench, K., 248 Muhsfeldt, E., 515, 520 Muiz, W.M., 370, 384 Mulder, G., 48 Muller, H.J., 458 Mullis, K., 206 Munch-Petersen, A., 299, 301 Muntyan, M.A., 362 Mussolini, B., 565 Mutt, V., 56, 57, 68, 70, 146 Myasoedova, K.N., 340

Nachmansohn, D., 223 Nachtsheim Ins, H., 540 Nachtsheim, H., 524, 529, 532, 533, 535, 539, 551 Najjar, V.A., 294 Napoleon, 559 Nedergaard, J., 333 Needham, D., 292 Needham, J., 292 Nelson, C., 20 Nernst, W., 44 Neuberg, C., 233, 568, 573, 574 Neuman, D., 268 Neurath, H., 37, 68, 69 Newmeyer, D.D., 371, 374 Newsholme, E.A., 424, 425, 428 Newsholme, P.J. 426 Nicolson, G., 34 Niehans, P., 586 Nietzsche, F., 576 Nirenberg, M.W., 246, 247, 259, 260, 279, 457 Nishizuka, Y., 278 Nobel, A., 92

Noelken, M., 22 Norde¤n, B., 204 Northrop, J.H., 109 Novogrodsky, A., 247 Nudel, U., 255 Nunez, J., 279 Nyiszli, M., 513^516, 519, 520, 522

O«brink, B., 173^175, 190, 191 O«brink, K.-J., 166 O’Brien, P.J., 433 Ochoa, S., 237, 239, 250, 257, 273 Odyssey, R., 439 Oehler, S., 483 Oesterhelt, D., 347, 350 Ofengand, J., 236 Ogston, A.G., 154, 158, 159, 170, 172, 176 Okun, L.B., 391, 392 Olhagen, B., 171 Oliveira, M.M., 598 Olovkinov, A.M., 380, 387, 398 Onsager, L., 18 Oppermann, U., 78, 80, 81 Osborn, M., 462 stergaard, E., 315 Otmar, 503 Ottesen, M., 310 Otting, G., 75 Ottoson, D., 209, 210 Ovchinnikov, J., 348, 473 Overgaard-Hansen, K., 301, 304, 305, 315 Ozrina, R.D., 397, 398

Pale¤us, S., 65 Palfrey, C., 260 Palmberg, C., 97 Palme, O., 194 Palmstierna, H., 144, 151

654 Pan, G., 332 Papa, S., 365 Paray, P., 234 Pare¤s, X., 80 Parker, P.J., 439 Parks, C.R., 423 Passonneau, J.V., 424 Patston, P.A., 439 Pauling, L., 17, 20, 23, 39, 69 Paxton, R., 439 Peacocke, A., 242 Pease, R.N., 6 Pedersen, K., 173 Pellicci, P.G., 382 Pereira, L., 492 Perham, R., 60, 63 Persson, B., 80, 81, 83 Persson, M., 83 Pertoft, H., 162, 176, 177, 182 Perutz, M.F., 58, 275, 415, 416 Peters, R.A., 416 Petrovsky, I.G., 340, 346, 386 Pettersson, A., 169 Pettersson, U., 78 Pettit, F.H., 431 Pfahl, M., 471 Phelps, C., 158, 159 Philipson, L., 77, 176 Phillips, D., 74 Picasso, P., 452 Pietruszkiewicz, A., 152 Pilch, P.F., 332, 334 Pirquet, 557 Pleasure, D., 270 Pleij, C.W., 257 Plesner, P., 299 Pletyushkina, O.Yu., 374, 375 Polhem, C., 193 Pollock, M.R., 242 Poltorak, D.M., 397 Ponstingl, H., 269 Popov, K.M., 438

NAME INDEX Porath, J., 66, 159 Portenhauser, R., 431, 436 Post, R.L., 624 Postgate, J.P., 367 Pressman, B.C., 328, 342 Preston, B.N., 172, 175, 176, 179 Priestman, G.S., 438 Procter, R., 555, 567 Ptashne, M., 463, 464^491

Rabin, Y., 228 Racker, E., 328, 345^347, 624 Radcliffe, P.M., 437 Raff, M.C., 279, 377 Ragnar, G., 92 Rajewksy, K., 469, 474, 487, 490 Rajewsky, B., 529, 572, 573 Ramel, S., 207 Randall, W.H., 279 Randle, E.A., 426 Randle, P.J., 421, 424, 426, 429, 431, 432, 437 Ranft, D., 538 Rangel, D., 638 Rasmussen, A.H., 305 Reed, C.H., 423 Reed, L., 429 Reed, L.J., 436 Reed, R., 186, 189, 190 Rehberg, P.B., 287 Reich, E., 312 Reichard, P., 140, 144, 156, 157, 164, 169, 274, 301 Revel, M., 249, 255 Re¤vet, B., 491 Reynolds, J., 9, 28, 35, 42, 43, 45 Rich, A., 239, 241 Rickenberg, H., 457, 458, 492 Ricquier, D., 329, 330, 332, 336 Rigler, R., 87, 94 Riltman, M.L., 333

NAME INDEX Rimington, C., 418 Ringselle, 105, 106 Ritter, R., 481, 549 Roberts, J., 462 Robertson, D., 33 Robinson, N., 35 Rode¤n, L., 146, 156, 160, 173 Rommel, E., 228 Roosevelt F.D., 18 Ropers, H., 488, 489 Ro«pke, 561 Rose¤n, N., 193 Rosenheim, M., 419 Rubin, K., 189, 190 Rudall, K.M., 109 Rudbeckius, O., 165 Rudolf, R., 586, 416 Ruhenstroth, G., 557 Ruhenstroth-Bauer, G., 530, 551, 561, 562, 571, 572 Rybak, S., 266, 267 Ryde, U., 74 Ryhage, R., 144 Ryle, A.P., 65

Sachse, C., 576 Sadovnichii, V.A., 387, 388, 389 Saedler, H. 487 Saffran, M., 294 Sakamoto, J., 632 Sakmann, B., 278, 279 Salam, A., 199 Salbaum, M., 485 Saller, K., 531, 536 Salomon, R., 250, 255, 257 Saltykov, B.G., 390, 391 Samartsev, V.N., 330 Samuelsson, B., 85, 91, 207 Samuilov, V.D., 344 Sanger, F., 56, 60, 62, 63, 76, 110^112, 114, 276, 311, 415, 423, 478

655 Sappho, 452 Sarid, S., 251, 252 Sartre, J.P., 452 Sattath, D., 250 Savill, J., 371 Scarano, E., 294 Scatchard, G., 10, 11, 17, 26 Schaeuble, J., 535 Scharl, J., 468 Scheele, C.W., 193 Schell, J., 487 Schellman, J.a.Ch., 48 Schepens, C., 143, 147 Scheraga, H., 19, 20 Scherson, T., 266, 268 Schlenk, W., 234 Schlossberger, H., 573 Schmitt, H., 258, 264, 266 Schneider, B., 458 Schneider, P., 458 Schoenheimer, R., 564 Scholefield, P.G., 328 Schramm, G., 565 Schro«dinger, E., 565 Schrenk, J., 472 Schuster, H., 537^540 Schwartz, M., 80, 268 Scott, J., 149, 150, 152^154 Seaborg, G., 170 Segerstedt, T., 165, 168 Sela, M., 250 Semenov, A.Yu., 349 Semenza, G., 319, 396 Severin, S.E., 321^323, 325, 327, 328, 341, 377, 378, 397, 398 Severina, I.I., 344, 338, 347, 354, 355, 386, 399 Severson, D.L., 431 Sharon, N., 253 Shipp, J.C., 425 Shnol, S.E., 338 Shooter, E., 279

656 Shugar, D., 250 Shulmeister, K., 480 Sigler, P., 493 Signer, E., 466 Silman, I., 279 Simmonds, H., 291 Simms, E., 236 Simon, E., 426 Simon, S., 247 Simons, K., 34 Simonyan, R.A., 333, 335 Singer, S.J., 34 Sippel, A., 490 Sippel, W., 467 Sirach, J., 474 Sjo«quist, J., 115, 118, 120 Sjo«vall, J., 85 Skou, J., 206 Skulachev, D.P., 393 Skulachev, I.V., 347 Skulachev, K.V., 347 Skulachev, M.V., 347 Skulachev, P.S., 319 Skulacheva, N.A., 319 Skulacheva, T.V., 340 Slater, E.C., 277, 278 Smedsrd, B., 174, 182, 184 Smith, E., 68^69, 78 Smith, G.H., 424 Smith, J., 252 Smith, M., 78, 206 Smith, R.E., 325, 326, 328 Smyrniotis, P.Z., 296 Soares, I.R., 601 Socrates, 463 Sohlman, M., 207 Sorenson, M., 614 Soreq, H., 254, 255, 257, 263 Soros, G., 390^392 Spackman, D., 66 Spatz, D., 454 Spector, I., 260

NAME INDEX Speer, A., 576 Spiegelman, S., 235, 236, 251, 459 Spiegelman, S., 459 Spirin, A.S., 243 Spott, S., 491 Stace, P.B., 438 Stanley, W.M., 109 Starkov, A.A., 365 Starlinger, P., 469, 489 Staudinger, H., 13, 47 Stein, W.H., 66, 110 Steinberg, I., 279 Steiner, J., 483 Stenhagen, E., 144 Stern, R.R., 224, 248, 249 Sterne, L., 475 Stieve, H., 519 Stockmayer, W., 11 Stoeckenius, W., 35, 347, 350 Stone, F.I., 463 Strindberg, A., 452 Strong, M., 199 Su Tung po, 459 Sudgen, P.H., 437 Sugden, M.C., 438 Sumner, J.B., 109 Sundelo«f, L.-O., 168, 172^175, 181 Suzuki, D., 476 Svedberg, T., 9, 48, 109, 173 Swedenborg, E., 193 Synge, R.L.M., 109, 114 Szent-Gyorgi, A., 341, 416 Szer, E., 251

Tabor, C., 603 Tabor, H., 295, 603 Ta«ckholm, V., 139, 200 Tal, J., 245 Tandberg, O.G., 198 Tanford, C., 1, 160 Taniguchi, K., 624

NAME INDEX Taylor, J., 419 Telchow, E., 564, 570 Temkin, M.I., 337, 338 Tengblad, A., 180, 181, 185 Teorell, T., 144 Tham, C., 203 Theo, H., 91 Theorell, H., 56, 107, 144, 165 Thoenen, H., 279 Timasheff, S.N., 26 Tiselius, A., 48, 109, 157^159, 166 Titov, G., 246 Tjian, R., 278 Tolkovsky, A.M., 374 Torbern, T., 138 Totter, J.R., 298 Trautner, T., 537^540 Truman, J.T., 306, 307 Tryggvason, K., 88 Tsujimoto, Y., 377 Tu Fu, 459 Tubbs, P.K., 429 Turgeniev, J., 463

Ubatuba, F., 600 Ulvsson, J., 168 Unckel, P., 201, 202 Unemoto, T., 360, 361, 363 Ungerstedt, U., 96 Ussing, H.H., 287

Valentine, R.C., 22, 23 Vallee, B., 58, 70, 71, 78, 81 Vanderkooi, G., 33 van Gogh, 466 van Slyke, D., 583, 588 Varney, P., 236 Vercesi, A., 332 Verkhovskaya, M.L., 361 Verma, I.M., 243

657 Verschuer, A., 556 Vianna, A.L., 613 Vielmetter, W., 469, 490 Villon, F., 452 Visser, L., 35 Vogel, Z., 279 Volcani, B., 235 von Bahr-Lindstro«m, H., 78 von Braun, W., 576 von Dietrich, C.P., 598 von Euler, H., 141 von Euler, J., 141 von Euler, U., 141 von Heijne, G., 205 von Kleist, K., 510 von Verschuer, O., 481, 487, 488, 494, 502^514, 516^518, 520^526, 538^541, 549, 558, 564, 571^573, 585 Vrba, R., 483, 490

Wa«chter, 569 Wagner, G., 519 Wahren, J., 89 Waldmann, 565 Walker, J., 206 Walker, M., 265 Wallander, J., 209, 210 Wallenfels, K., 455^457 Wallenfels, W., 492 Wang, X., 371 Warburg, O.H., 292, 293, 296, 528 Wargentin, P.W., 193 Wasteson, A., 173, 175, 189, 190 Watson, J., 46, 199, 249, 298, 299, 394, 461, 466, 471, 482, 492 Webb, E.C., 416 Weber, K., 279, 462 Wei, J.Y., 382 Wei, W., 459 Weidel, W., 573 Weigel, H., 454, 455

658 Weindling, P., 555 Weinreich, M., 516, 521 Weisgal, M., 234 Weismann, A., 369, 370, 376, 378 Weiss, E., 480 Weizmann, C., 233^235 Wells, J., 177 Wenner-Gren, A., 210 West, I.C., 359 Westermark, B., 189, 190 Westphal, O., 455, 587^589 Westphal, U., 556, 562, 563 Wewdling, R., 474 Whelan, W.J., 274 Whitehouse, S., 431 Whitney, P., 24 Wieland, O.H., 430, 436 Wieland, T., 587 Wiesmeyer, H., 236 Wik, O., 179, 185 Wikstro«m, M., 400 Williams, R., 631 Williamson, D.H., 434 Williamson, J.R., 425 Willmer, N., 415 Willsta«tter, R., 233, 564 Wilm, M., 87 Wilsta«tter, R., 233 Windaus, A., 564, 565 Winkler, E., 331 Winnick, T., 240 Witney, P., 20 Wittenberg, J., 297 Wittmann, H.G., 223, 539 Wittmann-Liebold, B., 63, 64, 69, 96

NAME INDEX Wojtczak, L., 331, 342 Wolfenden, R., 631 Wolff, H., 419 Wols, 452 Woods, K., 61 Wrinch, D., 48 Wu, P., 438 Wulff, E., 475 Yablokov, A.V., 390 Yaguzhinsky, L.S., 399 Yalow, R., 423 Yamashina, I., 115 Yankofsky, S., 247 Yeltsin, B.N., 387, 390, 391, 394, 396, 397 Young, F.G., 419, 421, 422 Yu-Cang, Du, 111 Yueh-Ting, Kung, 111 Yun, D.-J., 377 Zabin, I., 68, 77 Zachau, H., 244, 470, 472 Zakharov, V.E., 394 Zamecnik, P., 313 Zare, R., 211 Zetterqvist, O«., 157 Zillig, W., 571 Zisapel, N., 261 Zoratti, M., 373 Zorov, D.B., 355, 356, 373 Zotterman, Y., 210 Zubay, G., 472 Zutra, A., 263, 270

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