Magnesium - A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References

MAGNESIUM A M EDICAL D ICTIONARY , B IBLIOGRAPHY , AND A NNOTATED R ESEARCH G UIDE TO I NTERNET R E FERENCES

J AMES N. P ARKER , M.D. AND P HILIP M. P ARKER , P H .D., E DITORS

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ICON Health Publications ICON Group International, Inc. 4370 La Jolla Village Drive, 4th Floor San Diego, CA 92122 USA Copyright 2004 by ICON Group International, Inc. Copyright 2004 by ICON Group International, Inc. All rights reserved. This book is protected by copyright. No part of it may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without written permission from the publisher. Printed in the United States of America. Last digit indicates print number: 10 9 8 7 6 4 5 3 2 1

Publisher, Health Care: Philip Parker, Ph.D. Editor(s): James Parker, M.D., Philip Parker, Ph.D. Publisher's note: The ideas, procedures, and suggestions contained in this book are not intended for the diagnosis or treatment of a health problem. As new medical or scientific information becomes available from academic and clinical research, recommended treatments and drug therapies may undergo changes. The authors, editors, and publisher have attempted to make the information in this book up to date and accurate in accord with accepted standards at the time of publication. The authors, editors, and publisher are not responsible for errors or omissions or for consequences from application of the book, and make no warranty, expressed or implied, in regard to the contents of this book. Any practice described in this book should be applied by the reader in accordance with professional standards of care used in regard to the unique circumstances that may apply in each situation. The reader is advised to always check product information (package inserts) for changes and new information regarding dosage and contraindications before prescribing any drug or pharmacological product. Caution is especially urged when using new or infrequently ordered drugs, herbal remedies, vitamins and supplements, alternative therapies, complementary therapies and medicines, and integrative medical treatments. Cataloging-in-Publication Data Parker, James N., 1961Parker, Philip M., 1960Magnesium: A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References / James N. Parker and Philip M. Parker, editors p. cm. Includes bibliographical references, glossary, and index. ISBN: 0-597-84026-1 1. Magnesium-Popular works. I. Title.

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Disclaimer This publication is not intended to be used for the diagnosis or treatment of a health problem. It is sold with the understanding that the publisher, editors, and authors are not engaging in the rendering of medical, psychological, financial, legal, or other professional services. References to any entity, product, service, or source of information that may be contained in this publication should not be considered an endorsement, either direct or implied, by the publisher, editors, or authors. ICON Group International, Inc., the editors, and the authors are not responsible for the content of any Web pages or publications referenced in this publication.

Copyright Notice If a physician wishes to copy limited passages from this book for patient use, this right is automatically granted without written permission from ICON Group International, Inc. (ICON Group). However, all of ICON Group publications have copyrights. With exception to the above, copying our publications in whole or in part, for whatever reason, is a violation of copyright laws and can lead to penalties and fines. Should you want to copy tables, graphs, or other materials, please contact us to request permission (E-mail: [email protected]). ICON Group often grants permission for very limited reproduction of our publications for internal use, press releases, and academic research. Such reproduction requires confirmed permission from ICON Group International Inc. The disclaimer above must accompany all reproductions, in whole or in part, of this book.

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Acknowledgements The collective knowledge generated from academic and applied research summarized in various references has been critical in the creation of this book which is best viewed as a comprehensive compilation and collection of information prepared by various official agencies which produce publications on magnesium. Books in this series draw from various agencies and institutions associated with the United States Department of Health and Human Services, and in particular, the Office of the Secretary of Health and Human Services (OS), the Administration for Children and Families (ACF), the Administration on Aging (AOA), the Agency for Healthcare Research and Quality (AHRQ), the Agency for Toxic Substances and Disease Registry (ATSDR), the Centers for Disease Control and Prevention (CDC), the Food and Drug Administration (FDA), the Healthcare Financing Administration (HCFA), the Health Resources and Services Administration (HRSA), the Indian Health Service (IHS), the institutions of the National Institutes of Health (NIH), the Program Support Center (PSC), and the Substance Abuse and Mental Health Services Administration (SAMHSA). In addition to these sources, information gathered from the National Library of Medicine, the United States Patent Office, the European Union, and their related organizations has been invaluable in the creation of this book. Some of the work represented was financially supported by the Research and Development Committee at INSEAD. This support is gratefully acknowledged. Finally, special thanks are owed to Tiffany Freeman for her excellent editorial support.

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About the Editors James N. Parker, M.D. Dr. James N. Parker received his Bachelor of Science degree in Psychobiology from the University of California, Riverside and his M.D. from the University of California, San Diego. In addition to authoring numerous research publications, he has lectured at various academic institutions. Dr. Parker is the medical editor for health books by ICON Health Publications. Philip M. Parker, Ph.D. Philip M. Parker is the Eli Lilly Chair Professor of Innovation, Business and Society at INSEAD (Fontainebleau, France and Singapore). Dr. Parker has also been Professor at the University of California, San Diego and has taught courses at Harvard University, the Hong Kong University of Science and Technology, the Massachusetts Institute of Technology, Stanford University, and UCLA. Dr. Parker is the associate editor for ICON Health Publications.

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About ICON Health Publications To discover more about ICON Health Publications, simply check with your preferred online booksellers, including Barnes&Noble.com and Amazon.com which currently carry all of our titles. Or, feel free to contact us directly for bulk purchases or institutional discounts: ICON Group International, Inc. 4370 La Jolla Village Drive, Fourth Floor San Diego, CA 92122 USA Fax: 858-546-4341 Web site: www.icongrouponline.com/health

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Table of Contents FORWARD .......................................................................................................................................... 1 CHAPTER 1. STUDIES ON MAGNESIUM ............................................................................................. 3 Overview........................................................................................................................................ 3 The Combined Health Information Database................................................................................. 3 Federally Funded Research on Magnesium ................................................................................... 6 E-Journals: PubMed Central ....................................................................................................... 60 The National Library of Medicine: PubMed ................................................................................ 66 CHAPTER 2. NUTRITION AND MAGNESIUM ................................................................................. 111 Overview.................................................................................................................................... 111 Finding Nutrition Studies on Magnesium ................................................................................ 111 Federal Resources on Nutrition ................................................................................................. 117 Additional Web Resources ......................................................................................................... 117 CHAPTER 3. ALTERNATIVE MEDICINE AND MAGNESIUM ........................................................... 127 Overview.................................................................................................................................... 127 National Center for Complementary and Alternative Medicine................................................ 127 Additional Web Resources ......................................................................................................... 142 General References ..................................................................................................................... 159 CHAPTER 4. DISSERTATIONS ON MAGNESIUM ............................................................................. 161 Overview.................................................................................................................................... 161 Dissertations on Magnesium ..................................................................................................... 161 Keeping Current ........................................................................................................................ 168 CHAPTER 5. CLINICAL TRIALS AND MAGNESIUM........................................................................ 169 Overview.................................................................................................................................... 169 Recent Trials on Magnesium ..................................................................................................... 169 Keeping Current on Clinical Trials ........................................................................................... 173 CHAPTER 6. PATENTS ON MAGNESIUM ........................................................................................ 175 Overview.................................................................................................................................... 175 Patents on Magnesium .............................................................................................................. 175 Patent Applications on Magnesium .......................................................................................... 214 Keeping Current ........................................................................................................................ 254 CHAPTER 7. BOOKS ON MAGNESIUM ........................................................................................... 255 Overview.................................................................................................................................... 255 Book Summaries: Federal Agencies............................................................................................ 255 Book Summaries: Online Booksellers......................................................................................... 256 The National Library of Medicine Book Index ........................................................................... 264 Chapters on Magnesium ............................................................................................................ 265 CHAPTER 8. MULTIMEDIA ON MAGNESIUM ................................................................................. 267 Overview.................................................................................................................................... 267 Video Recordings ....................................................................................................................... 267 Bibliography: Multimedia on Magnesium................................................................................. 268 CHAPTER 9. PERIODICALS AND NEWS ON MAGNESIUM .............................................................. 269 Overview.................................................................................................................................... 269 News Services and Press Releases.............................................................................................. 269 Newsletter Articles .................................................................................................................... 274 Academic Periodicals covering Magnesium............................................................................... 277 CHAPTER 10. RESEARCHING MEDICATIONS................................................................................. 279 Overview.................................................................................................................................... 279 U.S. Pharmacopeia..................................................................................................................... 279 Commercial Databases ............................................................................................................... 280 APPENDIX A. PHYSICIAN RESOURCES .......................................................................................... 285 Overview.................................................................................................................................... 285

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NIH Guidelines.......................................................................................................................... 285 NIH Databases........................................................................................................................... 287 Other Commercial Databases..................................................................................................... 290 The Genome Project and Magnesium ........................................................................................ 290 APPENDIX B. PATIENT RESOURCES ............................................................................................... 295 Overview.................................................................................................................................... 295 Patient Guideline Sources.......................................................................................................... 295 Finding Associations.................................................................................................................. 297 APPENDIX C. FINDING MEDICAL LIBRARIES ................................................................................ 301 Overview.................................................................................................................................... 301 Preparation................................................................................................................................. 301 Finding a Local Medical Library................................................................................................ 301 Medical Libraries in the U.S. and Canada ................................................................................. 301 ONLINE GLOSSARIES................................................................................................................ 307 Online Dictionary Directories ................................................................................................... 308 MAGNESIUM DICTIONARY .................................................................................................... 311 INDEX .............................................................................................................................................. 401

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FORWARD In March 2001, the National Institutes of Health issued the following warning: "The number of Web sites offering health-related resources grows every day. Many sites provide valuable information, while others may have information that is unreliable or misleading."1 Furthermore, because of the rapid increase in Internet-based information, many hours can be wasted searching, selecting, and printing. Since only the smallest fraction of information dealing with magnesium is indexed in search engines, such as www.google.com or others, a non-systematic approach to Internet research can be not only time consuming, but also incomplete. This book was created for medical professionals, students, and members of the general public who want to know as much as possible about magnesium, using the most advanced research tools available and spending the least amount of time doing so. In addition to offering a structured and comprehensive bibliography, the pages that follow will tell you where and how to find reliable information covering virtually all topics related to magnesium, from the essentials to the most advanced areas of research. Public, academic, government, and peer-reviewed research studies are emphasized. Various abstracts are reproduced to give you some of the latest official information available to date on magnesium. Abundant guidance is given on how to obtain free-of-charge primary research results via the Internet. While this book focuses on the field of medicine, when some sources provide access to non-medical information relating to magnesium, these are noted in the text. E-book and electronic versions of this book are fully interactive with each of the Internet sites mentioned (clicking on a hyperlink automatically opens your browser to the site indicated). If you are using the hard copy version of this book, you can access a cited Web site by typing the provided Web address directly into your Internet browser. You may find it useful to refer to synonyms or related terms when accessing these Internet databases. NOTE: At the time of publication, the Web addresses were functional. However, some links may fail due to URL address changes, which is a common occurrence on the Internet. For readers unfamiliar with the Internet, detailed instructions are offered on how to access electronic resources. For readers unfamiliar with medical terminology, a comprehensive glossary is provided. For readers without access to Internet resources, a directory of medical libraries, that have or can locate references cited here, is given. We hope these resources will prove useful to the widest possible audience seeking information on magnesium. The Editors

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From the NIH, National Cancer Institute (NCI): http://www.cancer.gov/cancerinfo/ten-things-to-know.

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CHAPTER 1. STUDIES ON MAGNESIUM Overview In this chapter, we will show you how to locate peer-reviewed references and studies on magnesium.

The Combined Health Information Database The Combined Health Information Database summarizes studies across numerous federal agencies. To limit your investigation to research studies and magnesium, you will need to use the advanced search options. First, go to http://chid.nih.gov/index.html. From there, select the “Detailed Search” option (or go directly to that page with the following hyperlink: http://chid.nih.gov/detail/detail.html). The trick in extracting studies is found in the drop boxes at the bottom of the search page where “You may refine your search by.” Select the dates and language you prefer, and the format option “Journal Article.” At the top of the search form, select the number of records you would like to see (we recommend 100) and check the box to display “whole records.” We recommend that you type “magnesium” (or synonyms) into the “For these words:” box. Consider using the option “anywhere in record” to make your search as broad as possible. If you want to limit the search to only a particular field, such as the title of the journal, then select this option in the “Search in these fields” drop box. The following is what you can expect from this type of search: •

Magnesium Chloride Slows Gastric Emptying, But Does Not Affect Digestive Functions Source: Alimentary Pharmacology and Therapeutics. 16(8): 1571-1577. August 2002. Contact: Available from Alimentary Pharmacology and Therapeutics. Blackwell Science Ltd., Osney Mead, Oxford OX2 OEL, UK. +44(0)1865 206206. Fax +44(0)1865 721205. Email: [email protected]. Website: www.blackwell-science.com. Summary: An inverse relationship has been established between serum magnesium and serum lipid levels. This article reports on a study in which, by means of breath tests, the authors tested the hypothesis that magnesium inhibits intraluminal lipid digestion and subsequently causes changes in lipid metabolism. The authors also investigated the influence of the administration of magnesium chloride on protein digestion and gastric

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emptying. Five healthy volunteers performed simultaneous breath tests for gastric emptying and intraluminal lipid digestion, and six others for gastric emptying and protein digestion. Each test was performed in basal conditions and after the intake of 800 milligrams of magnesium chloride dissolved in water. The oral administration of a single dose of magnesium chloride resulted in a diminished rate of intraluminal lipid and protein digestion. The most pronounced effect of magnesium chloride, however, was a decreased gastric emptying rate of both test meals. After correction for gastric emptying, no differences were noted in intraluminal lipid or protein digestion. Therefore, the lower lipid levels noted after magnesium supplementation are unlikely to be the result of altered lipid assimilation. The authors conclude that magnesium chloride slows gastric emptying but does not influence lipid digestion. 8 figures. 2 tables. 20 references. •

Magnesium and Diabetes Source: Practical Diabetology. 10(2): 1-5. March-April 1991. Summary: Despite the frequency with which magnesium deficiency occurs in diabetes mellitus, symptoms are rarely present. This article describes the case example of a patient with uncontrolled diabetes mellitus and magnesium deficiency. Focal neuromuscular irritability, characterized by involuntary rhythmic myoclonic contractions, was caused by magnesium deficiency. The problem was resolved after magnesium repletion. Magnesium deficiency and its relationship to diabetes is reviewed within the context of this case. The author also discusses the clinical usefulness of a magnesium-loading test and gives a therapeutic outline. 2 tables.

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Magnesium and Diabetes: A Call for Concern Source: Practical Diabetology. 14(1): 7-11. March 1995. Contact: Available from R.A. Rapaport Publishing, Inc. 150 West 22nd Street, New York, NY 10011. (800) 234-0923. Summary: In this article, the authors call attention to the problem of inadequate magnesium levels in people with diabetes mellitus. Topics covered include a description of magnesium and its role in the body's metabolism; magnesium deficiency; magnesium and glycemia; macrovascular disease, diabetes and magnesium; microvascular disease and magnesium; and magnesium intake. The author concludes that subtle magnesium deficiency may play a significant role in diabetes and its complications. The article concludes with an editorial comment by Dr. Joel Zonszein, who focuses on determining which patients with diabetes should receive magnesium replacement or supplementation. 2 tables. 16 references.

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Magnesium Deficiency and Diabetes Source: Diabetes Educator. 18(1): 17-19. January-February 1992. Summary: Magnesium plays a key role in many enzyme systems in the body and is required for all enzymatic reactions involving the nucleotide adenosine triphosphate. This article discusses magnesium deficiency and diabetes. The authors report that magnesium deficiency is seen in as much as 25 percent of the diabetes population. The authors discuss magnesium loss and diabetes-associated complications, including hypertension, retinopathy, dyslipidemia, focal seizures, and reduced release of insulin. In addition, they note that magnesium deficiency may influence the binding of insulin to peripheral tissue and has been associated with ventricular arrhythmias and sudden

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cardiac death. Two final sections cover hypomagnesemia with hypokalemia and magnesium repletion. 1 figure. 25 references. •

Serum and Dietary Magnesium and the Risk for Type 2 Diabetes Mellitus: The Atherosclerosis Risk in Communities Study Source: Archives of Internal Medicine. 159(18): 2151-2159. October 11, 1999. Contact: Available from American Medical Association. Subscriber Services Center, P.O. Box 10946, Chicago, IL 60610-0946. (800) 262-2350. Fax (312) 464-5831. E-mail: [email protected]. Summary: This article describes a prospective cohort study that examined the association between serum magnesium level and dietary magnesium intake and the subsequent risk for incident type 2 diabetes in a cohort of 12,128 middle aged adults without diabetes from the Atherosclerosis Risk in Communities Study during 6 years of followup. Fasting serum magnesium level, categorized into six levels, and dietary magnesium intake, categorized into quartiles, were measured at the baseline examination. Incident type 2 diabetes was defined by self report of physician diagnosis, use of diabetic medication, fasting glucose level of at least 7.0 mmol/liter, or nonfasting glucose level of at least 11.1 mmol/liter. The study found that, among white participants, a graded inverse relationship between serum magnesium levels and incident type 2 diabetes was observed. From the highest to the lowest serum magnesium levels, there was an approximately two fold increase in incidence rate. This graded association remained significant after simultaneous adjustment for potential confounders, including diuretic use. Compared with individuals with serum magnesium levels of 0.95 mmol/liter or greater, the adjusted relative odds of incident type 2 diabetes rose progressively across the following lower magnesium categories: 1.13, 1.20, 1.11, 1.24, and 1.76. In contrast, little or no association was observed in African American participants. No association was detected between dietary magnesium intake and the risk for incident type 2 diabetes in African American or white participants. The article concludes that, among white participants, low serum magnesium was a strong, independent predictor of incident type 2 diabetes. That low dietary magnesium intake did not confer risk for type 2 diabetes implies that compartmentalization and renal binding of magnesium may be important in the relationship between low serum magnesium levels and the risk for type 2 diabetes. 1 figure. 5 tables. 52 references. (AAM).

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In Review: Magnesium Deficiency and Celiacs Source: Celiac Disease Foundation Newsletter. 3(4): 1. Winter 1993. Contact: Celiac Disease Foundation. 13251 Ventura Boulevard, Number 3, Studio City, CA 91604. (818) 990-2354. Summary: This brief article reviews the role of magnesium deficiency in people with celiac disease. Topics addressed in the article include the metabolism of magnesium and how it is used by the body; current research; diagnostic tests used to determine magnesium absorption; and the role of magnesium in the prevention of osteoporosis, particularly in people with celiac disease. The article concludes with a description of a research study to establish baseline data on people with celiac disease, confirming that there is an increased incidence of osteoporosis in celiacs. The article includes the telephone number of the Celiac Disease Foundation for readers who are interested in obtaining more information about this study.

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Role of Magnesium in Diabetes Mellitus: A Possible Mechanism for the Development of Diabetic Complications Source: Journal of Diabetes and Its Complications. 6(2): 143-149. April-June 1992. Summary: This review article investigates the role of magnesium as a possible mechanism for the development of complications in diabetes mellitus. Topics include magnesium homeostasis; hypomagnesium, insulin resistance, hypertension, and ischemic heart disease; hypomagnesemia and diabetes complications; possible mechanisms for the action of magnesium; mechanisms linking hypomagnesemia and diabetes complications; and magnesium and inositol transport. The authors conclude that the recognition that hypomagnesemia is associated with essential hypertension, insulin resistance, hyperinsulinemia, hyperlipidemia, and ischemic heart disease may provide mechanistic support for the currently proposed theory that the association of these conditions constitutes a distinct clinical syndrome. 72 references.

Federally Funded Research on Magnesium The U.S. Government supports a variety of research studies relating to magnesium. These studies are tracked by the Office of Extramural Research at the National Institutes of Health.2 CRISP (Computerized Retrieval of Information on Scientific Projects) is a searchable database of federally funded biomedical research projects conducted at universities, hospitals, and other institutions. Search the CRISP Web site at http://crisp.cit.nih.gov/crisp/crisp_query.generate_screen. You will have the option to perform targeted searches by various criteria, including geography, date, and topics related to magnesium. For most of the studies, the agencies reporting into CRISP provide summaries or abstracts. As opposed to clinical trial research using patients, many federally funded studies use animals or simulated models to explore magnesium. The following is typical of the type of information found when searching the CRISP database for magnesium: •

Project Title: ANALYSIS & CONTROL OF NONSYNAPTIC EPILEPTIFORM ACTIVITY Principal Investigator & Institution: Durand, Dominique M.; Professor; Biomedical Engineering; Case Western Reserve University 10900 Euclid Ave Cleveland, Oh 44106 Timing: Fiscal Year 2001; Project Start 04-SEP-2001; Project End 31-AUG-2005 Summary: (provided by applicant): Epilepsy is characterized by the abnormal synchronization of large numbers of neurons. The synchronization and propagation of epileptic seizures are thought to rely on synaptic transmission. However, non-synaptic mechanisms such as neuronal swelling, electric field effects, potassium diffusion, gap junctions and glial cell function also contribute to the generation and spread of epileptiform activity. Non-synaptic epilepsy is generated by lowering calcium in the extracellular space thereby eliminating synaptic transmission. As a result, the clinical relevance of non-synaptic mechanisms has been questioned. We have recently generated

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Healthcare projects are funded by the National Institutes of Health (NIH), Substance Abuse and Mental Health Services (SAMHSA), Health Resources and Services Administration (HRSA), Food and Drug Administration (FDA), Centers for Disease Control and Prevention (CDCP), Agency for Healthcare Research and Quality (AHRQ), and Office of Assistant Secretary of Health (OASH).

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novel models of non-synaptic activity in the presence of normal calcium and normal synaptic transmission. We propose to analyze the role of non-synaptic mechanisms in neuronal synchronization in order to understand and potentially develop novel therapies to prevent abnormal neural activity. We have recently shown that the frequency, amplitude and duration of non-synaptic epileptiform events can be controlled independently suggesting that different mechanisms are responsible. In particular, preliminary experiments show that gap junctions are not responsible for the propagation of non-synaptic events generated in zero-calcium medium, but that potassium diffusion (potentially mediated by the activity of glial cells) plays a crucial role. The goal of this proposal is to analyze and control non-synaptic epileptiform activity. Specifically, we propose to 1) determine the common mechanisms underlying three models of non-synaptic epilepsy, 2) establish the conditions sufficient for the generation of non-synaptic epileptogenesis, 3) analyze the mechanisms underlying the propagation of non-synaptic epileptiform activity, 4) develop a computer model of nonsynaptic propagation to test hypotheses not directly testable by experimentation, and 5) develop methods for controlling epileptiform activity. Multi-disciplinary experimental approaches such as computer simulation and fluorescence imaging will be combined with pharmacology and in-vitro slice electrophysiology to achieve these goals. Current therapeutic agents are not capable of controlling seizure activity in 25 percent of all epileptic patients. The results of our studies should provide valuable insight into mechanisms underlying epileptogenesis as well as new tools for the control and suppression of epileptic seizures. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ANALYSIS OF A KINASE DOMAIN WITHIN A NOVEL ION CHANNEL Principal Investigator & Institution: Nadler, Monica J.; Beth Israel Deaconess Medical Center St 1005 Boston, Ma 02215 Timing: Fiscal Year 2002; Project Start 15-APR-2002; Project End 31-MAR-2007 Summary: Little is known of calcium channels underlying flux in lymphocytes and other non-excitable cells. I have recently cloned a novel ion channel, designated LTRPC7, that is expressed in many types of non-excitable cells including all immune system tissues. This channel is a member of the LTRPC TRP sub-family and is unique in that it contains a catalytic kinase domain homology to eEF-2K at its C-terminus. Preliminary data indicate that this protein is bi-functional: it is a cation channel that is permeable to calcium and magnesium, and it has kinase activity. We have observed that the ion permeability of LTRPC7 is sensitive to and suppressed by Mg-ATP. These data suggest that the kinase activity and/or changes in conformation of the kinase domain upon phosphorylation could be critical to the gating and/or modulation of this channel. Experiments are proposed to fully characterize the enzymatic potential of this domain (Aim 1). In addition, experiments are proposed to explore the regions of LTRPC7 that mediate and influence the Mg- ATP suppression effect (Aim 2). Finally, preliminary functional characterization of LTRPC7 demonstrates that over-expression of it in HEK293 cells is toxic and/or induces growth arrest. In addition, production of a double allele LTRPC7 knockout cell line in the DT-40 B-cell system results in lethality. These results suggest that LTRPC7 function is linked to basic cellular processes required for cell survival. Experiments to address the unique structural features of LTRPC7 that could account for these functional effects and assessment of its ability to affect intracellular calcium are also proposed (Aim 3). Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: RELEVANCE

AROMATIC

AMINE

DNA

STRUCTURES--MUTAGENIC

Principal Investigator & Institution: Broyde, Suse B.; Professor; Biology; New York University 15 Washington Place New York, Ny 10003 Timing: Fiscal Year 2001; Project Start 01-SEP-1997; Project End 31-AUG-2006 Summary: (provided by applicant) The human population is routinely exposed to a large number of environmental chemicals: some of them may initiate cancer while others, only slightly different in structure, are harmless. One prominent route by which carcinogens exert their effects is to react with DNA in a way that leads to a mutation in a vital cellular target. Insight into the mechanism by which a carcinogen-damaged DNA produces mutations is needed in order to identify potentially hazardous substances. In this project, intensive computer modeling is used to explore this process. Our efforts here are targeted particularly to frameshift mutations, whose contribution to carcinogenesis has perhaps been underemphasized. In particular, we will attempt to relate chemical structure to mutagenic effectiveness within the framework of the slippage/misalignment theory. This theory has successfully explained the sequence dependence of many frameshift mutations. We will work with four aromatic amines, members of a chemical class that has demonstrated an exceptional ability to induce frameshifts. Our selection includes acetylaminofluorene (AAF), chosen because of the extensive data based concerning its mutagenicity, 2-amino-1-methyl-6phenylimidazo(4,5-b)pyridine (PhIP) and 2-amino-3-methyl-imidazo(4,5-f)quinoline (IQ), carcinogens that are formed during the cooking of protein-rich foods, and 1aminopyrene (AP), the transformation product of a common pollutant present in diesel engine exhaust, urban air particulates, and a number of other sources. We will follow the behavior of modified DNA primer-template complexes as they proceed through the steps of extension, blockage, and/or misalignment within the active sites of selected polymerases for which suitable crystal structures are available. Our methods include the use of the programs DUPLEX (for molecular mechanics with modified DNA) and AMBER for molecular dynamics simulations with DNA in solution or in a polymerase. DUPLEX permits an extensive search of conformation space without the use of assumptions concerning the final structure. The molecular dynamics studies include explicit solvent and salt, and provide animation, but are more restricted in their search. Molecular dynamics trajectories yield ensembles of structures that will be used to compute free energy differences between conformers in solution, and binding free energies of polymerase-primer-template complexes. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ASSEMBLY OF HIGHER ORDER STRUCTURE IN GROUP I RIBOZYMES Principal Investigator & Institution: Doudna, Jennifer A.; Professor; Molecular Biophysics & Biochem; Yale University 47 College Street, Suite 203 New Haven, Ct 065208047 Timing: Fiscal Year 2001; Project Start 01-AUG-1997; Project End 31-JUL-2002 Summary: The three-dimensional structure of RNA is critical to its cellular function. Catalytic RNA molecules, called ribozymes, fold into complex globular structures to produce active sites that promote chemical reactions independent of protein facilitation. Although the molecular basis for such structures is currently unknown, their formation involves the assembly of short helical elements via direct RNA-RNA and metalmediated contacts. An understanding of the molecular interactions that give to correctly

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folded RNAs is fundamental to understanding the structural and mechanistic principles of RNA catalysis. A detailed knowledge of the principles of higher order folding in RNA will enable rational design of efficient ribozymes for therapeutic purposes and will illuminate the role of RNA in such fundamental biological processes as protein biosynthesis and messenger RNA splicing. Like proteins, large catalytically active RNA molecules are often composed of independently folding structural domains. In the wellstudied group I self-splicing introns, the catalytic core is comprised of helical components residing in two separate domains. The crystal structure of one of these, the independently folding P4-P6 domain, revealed that conserved helices of the core lie parallel to helices in an extended subdomain. In addition to two specific sets of tertiary contacts, divalent metal ions and 2' hydroxyl groups of riboses in the RNA backbone stabilize this remarkably close helical packing. This exciting structure also provides tantalizing clues to the organization of the complete intron catalytic core, and is thus the basis for probing structural interactions both within the P4-P6 domain and between P4P6 and the rest of the intron. The specific aims of this proposal are threefold: 1. Determine the affinity and specificity of magnesium ion binding sites clustered in an adenosine-rich corkscrew motif, the A-rich bulge, that is central to P4-P6 domain folding. 2. Define the classes of divalent metal ion binding sites in the P4-P6 crystal structure, and correlate them with functional data for P4-P6 folding and intron catalysis. 3. Measure the energetic contributions of individual tertiary contacts within the P4- P6 domain, involving magnesium ions, ribose 2' hydroxyl groups and nucleotide bases, to domain structure and global intron folding and catalysis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: BICARBONATE REGULATED ADENYLYL CYCLASE Principal Investigator & Institution: Buck, Jochen; Pharmacology; Weill Medical College of Cornell Univ New York, Ny 10021 Timing: Fiscal Year 2001; Project Start 01-FEB-2001; Project End 31-JAN-2005 Summary: Mammalian soluble adenylyl cyclase (sAC) is structurally, biochemically, and physiologically distinct from G protein coupled transmembrane adenylyl cyclases. sAC activity is not regulated by the known modulators of transmembrane adenylyl cyclases activity, such as G proteins and forskolin, but it is directly stimulated by the bicarbonate- ion. Multiple physiological processes (i.e., breathing, blood flow, cerebrospinal fluid and aqueous humor formation, spermatocyte development) are modulated by carbon dioxide and/or bicarbonate. With this grant application we plan to test the hypothesis that sAC is the physiological bicarbonate/carbon dioxide sensor in biological systems. Aim 1 will study the direct interactions of bicarbonate with purified recombinant sAC protein using enzyme kinetics, binding assays, and limited proteolysis studies. Aim 2 will study whether bicarbonate activated sAC activity is present in bicarbonate/carbon dioxide regulated physiological systems. Aim 3 will determine with the help of sAC knockout studies in mice whether SAC activity is essential for bicarbonate/carbon dioxide regulated physiological systems in vivo. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: BIOCHEMICAL INVESTIGATION OF P-GLYCOPROTEIN Principal Investigator & Institution: Senior, Alan E.; Professor; Biochemistry and Biophysics; University of Rochester Orpa - Rc Box 270140 Rochester, Ny 14627 Timing: Fiscal Year 2001; Project Start 01-JAN-1994; Project End 31-DEC-2004

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Summary: Multidrug-resistance is a situation encountered in cancer patients in which the tumor becomes resistant to a variety of cytotoxic anti-cancer chemotherapeutic agents. It often involves enhanced expression of P- glycoprotein (Pgp), a plasma membrane protein. Involvement of Pgp in resistance to anti-AIDS drugs is also stronglyindicated. Pgp consists of 1280 amino acids, arranged in two repeated halves, each of which contains six predicted transmembrane helices and one ATP-binding site. It acts in an ATP-dependent manner to exclude drugs and a wide range of other hydrophobic compounds from cells, displays substantial drug- stimulated ATPase activity, and is now widely-believed to act as an ATP- driven drug-efflux pump. A catalytic cycle involving alternating catalytic sites and a mechanism for coupling of ATP-hydrolysis to drug-transport, presented by our laboratory, has become widely-adopted as a working model. We recently made a breakthrough, namely the development of a large- scale method for preparation of pure, detergent-soluble, mouse and human Pgp, using Pichia. Not only wild-type but also mutant Pgp may now be obtained in quantity, facilitating a broader range of structural, biophysical and biochemical approaches. The aim of this proposal is to characterize structure and function of Pgp. Structure will be determined by electron-microscopy and X-ray crystallography. Catalytic mechanism will be studied by specific insertion of fluorescent probes to monitor nucleotide binding parameters and occupancy of catalytic sites, and by mutagenesis of critical catalytic site residues. Coupling of ATP hydrolysis to drug transport will be investigated. The two halves of Pgp will be purified separately and reconstituted, to facilitate understanding of interactions between catalytic sites and membrane domains. Basic knowledge of this kind will be invaluable in devising ways to disable P-glycoprotein and overcome drugresistance in patients. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: BIOCHEMICAL METABOLISM

MECHANISMS

OF

POLYPHOSPHATE

IN

Principal Investigator & Institution: Kornberg, Arthur; Emeritus Professor of Biochemistry; Biochemistry; Stanford University Stanford, Ca 94305 Timing: Fiscal Year 2002; Project Start 01-FEB-2002; Project End 31-JAN-2006 Summary: (provided by applicant): Inorganic polyphosphate (poly P), a polymer of hundreds of phosphate residues linked by high-energy bonds, is found in every cell in Nature-bacteria, fungi, plants and animals. Among poiy P functions are: kinase donor to glucose and nucleoside diphosphates, phosphate (P1) reservoir, divalent metal (Ca2+, Mg2+, Mn2+) chelator and component of a membrane complex in bacterial transformation. Our recent studies have disclosed three major roles: 1) "alarmone" in response to stresses and deficiencies, 2) adaptations for survival in the stationary phase and 3) virulence in some pathogens. Thus, poly P has a variety of functions depending on the cell and circumstances. The multiplicity of functions and the plasticity of microbial organisms has complicated the search for the biochemical mechanisms responsible for each action. The proposed research will focus on mechanisms of metabolic regulation. Based on promising preliminary studies, we intend to pursue two major lines: 1) Regulation of protein synthesis and turnover by the supply of amino acids by biosynthesis and protein turnover, and 2) Operations of phosphate uptake and efflux and concomitant divalent metal ion transport. In addition, we will be mindful of control of in vitro transcription of genes known to be activated by poly P in vivo and a possible role for poly P as a donor for protein kinases. The widespread conservation of the enzyme, poly P kinase (PPK), responsible for poiy P synthesis in bacteria, including many of the major pathogens, has led us to knockout ppk in several of these pathogens.

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In Pseudomonas aeruginosa poly P is essential for motility, quorum sensing, bioflim formation and virulence in mice. We expect that the proposed biochemical studies will reveal mechanisms that will assist in the current search for antimicrobial drugs aimed at PPK as a target. The research may also provide clues to the biosynthesis and metabolism of poly P in eukaryotes about which little is known. In view of the ubiquity of poly P in animal cells and subcellular organelles (particularly nuclei), it seems likely that poly P serves multiple functions in human metabolism that affect growth, differentiation and disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: BIOMATERIALS (MG/ZN/F-BCPS)FOR OSTEOPOROSIS THERAPY Principal Investigator & Institution: Legeros, Racquel Z.; Biomaterials and Biomimetics; New York University 15 Washington Place New York, Ny 10003 Timing: Fiscal Year 2003; Project Start 30-SEP-2003; Project End 31-JUL-2007 Summary: (provided by applicant): Osteoporosis is a 'silent' progressive and debilitating disease characterized by bone loss, thinning cortical bone and disorganized trabecular bone leading to bone fragility and fracture. Osteoporosis results when the processes of bone formation and bone resorption become uncoupled and the rate of bone resorption becomes much greater than that of bone formation. FDA-approved pharmaceutical interventions have antiresorptiive properties. Some of these drugs have serious side effects. Fluoride (F) therapy as sodium fluoride is the only one shown to consistently increase bone mass, but was also reported to increase fracture risk. The goal of the proposed research is to develop novel materials incorporating magnesium (Mg), zinc (Zn), F ions in a calcium (Ca) phosphate system (Mg/Zn/F-BCP). Separately, these ions have been associated with bone formation, biomineralization and osteoporosis therapy. Specific aims are to: (1) prepare and characterize the crystallographic, morphologic, and chemical properties of a series of Mg/Zn/F-BCP materials; (2) determine the short and long term initial dissolution rates and release of Ca, Mg, Zn, P and F ions of the materials prepared in Aim 1; (3) determine in vitro response of bone forming (osteoblasts) and bone resorbing (osteoclasts) cells to Mg/Zn/F-BCPs of various compositions using human osteoblast-like cells and rat osteoclast-like cells; (4) determine the effect of orally administered various Mg/Zn/F-BCPs on (a) bone properties (mechanical strength, density, quality, composition, and histomorphometric parameters and bone mineral (crystallinity, composition and dissolution) of adult and aged female and male rats; and (b) on the development of osteoporosis (deficient-diet induced) in adult rats; and (5) determine therapeutic effect of various injected Mg/Zn/F-BCPs on ovariectomized rats using biomechanical, histomorphometric measurements and chemical analyses on bone and plasma analyses. Hypothesis: Biomaterials with Zn, Mg and F ions in a calcium phosphate matrix (used as dietary supplement and ion releasing injectible 'implants') will improve bone health. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: CA2+DEPENDENT K+CHANNELS: ALLOSTERIC GATING Principal Investigator & Institution: Cui, Jianmin; Biomedical Engineering; Case Western Reserve University 10900 Euclid Ave Cleveland, Oh 44106 Timing: Fiscal Year 2002; Project Start 01-APR-2002; Project End 31-MAR-2006 Summary: (provided by applicant): The long-term objective of this research is to understand the molecular mechanism of the voltage, Ca2+, and Mg2+ dependent activation of large-conductance K+ channels (BK channels). BK channels have road

12

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physiological functions, including the modulation of neurotransmitter release and the control of blood vessel diameters. As a consequence of these physiological functions BK channels are of significant clinical importance. For example, abnormal activity of BK channels has been associated with hypertension in animal models; their increased activity may reduce the incidence of ischemia- reperfusion-induced cardiac arrhythmia. In the activation of BK channels voltage induces movements of the voltage sensor in the channel, Ca2+ or Mg2+ binds to the channel to cause conformational changes in the channel protein to open he activation gate. Now the structure of the K+ channel pore has been solved; protein sequences underlying he activation gate, the voltage sensor, and the Ca2+ binding site have been identified. However, the manner n which voltage sensor movements, Ca2+ or Mg2+ binding are coupled to the opening of the activation gate remains unknown. Until the structural and energetic basis of these couplings is elucidated, how voltage, Ca2+ and Mg2+ sensitivities are modulated in various BK channels to subserve their physiological functions cannot be understood. Based on previous studies, we hypothesize that a structural domain of the channel protein that is physically close to the activation gate (the RCK domain for Regulating the conductance of K+ channels) is central in these couplings. Recently, the X-ray crystal structure of the RCK domain has been solved. Guided by the structural data we will perturb the channel structure using molecular biology and determine its impact on the energetic contribution of Ca2+, Mg2+, or voltage to channel opening using our recently developed electrophysiological approaches. We will also use approaches of protein biochemistry and nuclear magnetic resonance spectroscopy (NMR) to map specific intramolecular protein interactions that nay be altered during channel activation and hence control channel function. These experiments will provide a foundation for understanding how various BK channels play their role in physiological processes and define targets on BK channels for therapeutic purposes. They will also contribute to our understanding of ion channel gating in general. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CALCIUM ACTIVATED MEMBRANE TARGETING BY THE C2 DOMAIN Principal Investigator & Institution: Falke, Joseph J.; Professor; Chemistry and Biochemistry; University of Colorado at Boulder Boulder, Co 80309 Timing: Fiscal Year 2001; Project Start 01-APR-2001; Project End 31-MAR-2005 Summary: Description (applicant's description): The conserved C2 domain has been recognized in over 500 proteins, where it plays a central role in targetting proteins to new cellular locations during Ca2+ signals. The most common type of targetting driven by this ubiquitous motif is Ca2+-triggered membrane docking, which initiates critical signaling processes including neurotransmitter and hormone release, activation or inactivation of phosphorylation and G protein signaling cascades, inflammation, and cell cycle control. The present new research proposal aims to develop a molecular picture of C2 domain function, mechanism and structure. The five broad goals of the research are as follows. (i) Distinct classes of C2 domains, differing in their Ca2+ activation parameters and even their mechanisms, will be resolved by comparative equilibrium and kinetic studies of isolated C2 domains from functionally diverse proteins. (ii) The mechanisms by which these different C2 domain classes dock to membranes will be elucidated, and residues essential for membrane docking will be identified. (iii) A medium resolution structure of the protein-membrane interface will be determined via a novel strategy. (iv) The mechanism by which Ca2+ triggers membrane docking will be investigated. Finally, (v) activation parameters and mechanistic models

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developed by studies of isolated C2 domains will be tested in multi-domain proteins and in living cells. To achieve these goals, a range of methods will be employed. Equilibrium dialysis, fluorescence titrations and stopped flow kinetics will be used to quantitate the equilibrium and kinetic features of selected C2 domains. Scanning cysteine mutagenesis and careful solution measurements will identify critical residues and forces that drive membrane docking. A novel combination of EPR distance measurements and constraint-based modelling will reveal the structure of the proteinmembrane interface, and will probe the Ca2+ triggering mechanism. Finally, hypotheses arising from in vitro studies of isolated C2 domains will be tested in multi-domain proteins and in living cells. Overall, this research will provide the first detailed molecular portrait of one of the most prevalent signaling motifs in nature, and will develop new methods to probe the challenging protein-membrane interface. Furthermore, comparative studies of C2 domains will provide information crucial to genomic analyses of many signaling pathways. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CELLULAR MAGNESIUM AND CALCIUM ION HOMEOSTASIS IN HEART Principal Investigator & Institution: Scarpa, Antonio; Professor and Chairman; Case Western Reserve University 10900 Euclid Ave Cleveland, Oh 44106 Timing: Fiscal Year 2002; Project Start 15-FEB-2002; Project End 31-DEC-2006 Summary: Despite the abundance of Mg2+ in the body and within tissues in the body and within tissues, little is known about the regulation of cellular and plasma Mg2+ homeostasis. Studies conducted during the last nine years of finding to this Project, as well as those many other laboratories, have shown that cellular Mg2+ homeostasis is very active, has sophisticated and multiple forms of regulation and may provide, directly or indirectly, a novel role in regulating cell function and metabolism. Specific hormonal stimulation and changes in intracellular second messenger level induce the transport of large and rapid amounts of Mg2+ from heart into the extracellular milieu and ultimately into the bloodstream, or vice versa. This proposal continues to use a large variety of models (perfused hearts, myocytes, other isolated cells, permeabilized cells, isolated organelles, purified proteins) and experimental approaches (31P NMR, Electron Probe, Microanalysis, cell imaging, isotopic potentiometric techniques to acquire or integrate the knowledge on MG1+ homeostasis in heart. The objective of this application is o test several major hypothesis: That in myocytes there is a multiplicity of Mg2+ transporters mediating Mg2+ uptake and release, as well as a redundancy of signaling pathways activating and inhibiting Mg2+ transport. That cellular Mg2+ release may be a major part of the alpha1 or beta adrenergic response. That Mg2+ efflux from myocytes increases interstitial Mg2+ concentration in the myocardium and stimulates adenosine production. That in the heart a fraction of the large efflux of Mg2+ is coupled to Ca2+ uptake through a novel Mg2+-Ca2+ anti-porter Hence, Mg2+ efflux stimulated by catecholamines may be an additional pathway of Ca2+ entry. That the accumulation of Mg2+ in heart and other tissues is unaffected by extracellular Mg2+ but is independently regulated by specific signaling pathways through protein kinase C. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: CELLULAR MAGNESIUM DYSFUNCTION IN DIABETES Principal Investigator & Institution: Romero, Jose R.; Brigham and Women's Hospital 75 Francis Street Boston, Ma 02115

14

Magnesium

Timing: Fiscal Year 2003; Project Start 20-SEP-2003; Project End 31-AUG-2006 Summary: (provided by applicant): This small grant application has been prepared by a new investigator in order to obtain the preliminary data necessary to prepare a competitive RO1 grant application on endothelial cell dysfunction in insulin resistantstates. The objective of this application is to identify the regulatory mechanisms involved in insulin-stimulated Na+/Mg2+ exchange activity in cells from normal and diabetic patients. Our long-term goal is to define the cellular mechanisms that lead to insulin resistance and diabetes. The central hypothesis for the proposed research is that low levels of Mg2+ attenuate the intracellular signal generated following insulin binding to its receptor or antagonize binding of insulin to the receptor. This hypothesis is based on preliminary findings obtained in human microvascular endothelial cells and red blood cells. We propose to test our central hypothesis and accomplish the overall objectives of this proposal through the following specific aims: Aim 1: Identify the role of insulin in cellular Mg2+ regulation in human microvascular endothelial cells. On the basis of our preliminary data, we hypothesize that insulin regulates cellular Mg2+ levels via activation of the exchanger, which in turn regulates nitric oxide production in human endothelial cells. We will characterize the intracellular signaling pathways that are downstream of PI3-kinase activation and modulate the activity of the exchanger as well as nitric oxide production in these cells Aim 2: Identify the mechanisms for insulinregulated Na+/Mg2+ exchange activity in ex vivo human red cells from normal subjects. Erythrocytes have been used as ex vivo models of what may occur in target tissue of insulin resistance's pathophysiology. We hypothesize that the insulin receptor is functionally coupled to the exchanger in human red cells via PI3-kinase activation. Therefore, our studies are designed to characterize PI3-kinase activity as well as phosphorylated Akt levels in erythrocytes upon activation with insulin. Aim 3: Identify the mechanisms for regulation of Na+/Mg2+ exchange activity in ex vivo human red cells from Type 2 diabetes mellitus patients. We hypothesize that elevated Na+/Mg2+ exchange activity in the red cells of diabetic patients in comparison to normal subjects explains the low cellular Mg2+ levels observed in patients with diabetes. This suggests an uncoupling between the insulin receptor and the exchanger in these patients. Therefore, we hypothesize that PI3-kinase activity is altered in diabetic red cells when compared to normal. We will compare PI3-kinase activity and phosphorylated Akt levels in erythrocytes from normal and diabetic subjects. We expect that these studies will identify novel cellular mechanisms underlying insulin resistance as well as characterize cellular Mg2+ regulatory mechanisms. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CHARACTERIZATION OF THE STRUCTURE AND FUNCTION OF DMP2 Principal Investigator & Institution: George, Anne; Associate Professor; Oral Biology; University of Illinois at Chicago 1737 West Polk Street Chicago, Il 60612 Timing: Fiscal Year 2002; Project Start 01-JAN-2002; Project End 31-DEC-2006 Summary: The proper mineralization of bones and teeth has great importance in normal human growth and development including musculo-skeletal functions. The mineral phase in these tissues contributes to the hardness and comprehensive strength of the structure and also has a major physiological role as the metabolic reservoir of calcium and magnesium. Problems in the mineralization process are evident in a number of skeletal pathologies. One of the most interesting questions in mineralized tissue research is how, within the physiological environments, circulating calcium and phosphate and other mineral phase ions can be concentrated in specific, localized organs or tissues. Up

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until this point we have defined little about the process by which this cascade is initiated and regulated. However, the organic matrix has been implicated to have a major role in regulating the mineralization process. We have focussed our efforts primarily on dentin mineralization because it is a simpler system than bone, but the two mechanisms are probably closely related. Our basic hypothesis for matrix-mediated mineralization, is that acidic macromolecules first bind within the collagen matrix and these are responsible for nucleating and starting the mineralization cascade. Probably these acidic macromolecules also regulate the size of carbonated hydroxyapatite crystals. In the search for the gene encoding for the principle acidic noncollagenous protein (NCP) of dentin, namely phosphophoryn (PP) we identified 2 clones one representing phosphophoryn now named DMP2 (dentin matrix protein 2) and the other DMP3 (dentin matrix protein 3) which is a compound of dentin sialoprotein and a mini "phosphophoryn"like domain. The amino acid sequence deduced from the partial DMP2 cDNA is of special interest because it clearly represents an aspartic acid and serine rich acidic protein of the type to be expected of a dentin matrix component. This gene has been tightly localized to mouse chromosome 5q21, equivalent to human chromosome 4Q21. This chromosome location is especially interesting because of the linkage of human chromosome 4q13-21 with the dentin mineralization disorder dentinogenesis imperfecta type II. In order to expand our understanding of the DMP2 gene structure and its function we propose the following specific aims: (1) To determine the complete primary structure of rat DMP2 (2) To delineate DMP2 promoter sequences and identify elements involved in tissue -specific regulation (3) To clone the human DMP2 gene in order to ultimately identify gene alterations in patients with Dentinogenesis Imperfecta Type II (4) To examine the temporal and spatial patterns of DMP2 expression during tooth development (5) To determine the calcium binding property of DMP2. The longterm goal is to understand the regulatory mechanism of DMP2 in dentin mineralization. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CINCINNATI COMPREHENSIVE SICKLE CELL CENTER Principal Investigator & Institution: Joiner, Clinton H.; Professor; Children's Hospital Med Ctr (Cincinnati) 3333 Burnet Ave Cincinnati, Oh 45229 Timing: Fiscal Year 2003; Project Start 11-JUL-2003; Project End 31-MAR-2008 Summary: The Cincinnati Comprehensive Sickle Cell Center, based in the Division of Hematology/Oncology. at Children's Hospital Medical Center and affiliated with the University of Cincinnati College of Medicine, provides a full range of clinical services to people affected by sickle cell disease. Building on the finding that elevated homocysteine (Hcy) in sickle cell patients is associated with pyridoxine deficiency, an inter-Center collaborative research proposal examines the correlation of elevated Hey with activation of coagulation and endothelial cells in patients, and tests whether pyridoxine supplementation corrects these abnormalities. An interactive research team with expertise in molecular biology, membrane transport, in vivo cell kinetics, and clinical research will pursue projects focused on SS RBC dehydration, a key factor in sickle cell pathology, which results from two abnormal transport pathways, KCl cotransport (KCC) and sickling-induced cation leaks. The clinical study of Project 2 tests the therapeutic potential of dipyridamole, which inhibits sickling-induced cation leaks, and magnesium, which inhibits KCC, to improve SS RBC hydration in vivo. Project 3 examines the rate and mechanisms of hydration changes of SS RBC in vivo, using biotin label techniques unique to this Center. Project 4 explores the molecular basis of posttranslational regulation of KCC by cell volume in normal and SS RBC. Project 5 focuses on the erythroid isoforms of KCC and their transcriptional regulation. A Clinical Core

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Magnesium

includes Pediatric and Adult Progra ms to provide comprehensive services to over 375 patients, to support basic and clinical research efforts, and to participate in inter-Center collaborative trials. An Education and Patient Services Core provides educational and psychosocial support for patients and families, and includes a Transition Program bridging the Pediatric and Adult Clinical Programs, in collaboration with the Sickle Cell Awareness Group and the Urban League of Cincinnati. An Administrative Core provides overall fiscal and programmatic management and serves as the focal point for Center activities, programs, and communication. The projects and cores in this proposal will be integrated with independently funded programs of newborn hemoglobinopathy screening follow-up, education and counseling, a Hemoglobin Diagnostic Laboratory, and other basic science and clinical research projects. The Cincinnati comprehensive Sickle Cell Center is poised to lead in the national effort to develop and test new therapies for sickle cell disease to improve the lives of affected individuals. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CLINICAL RESEARCH IN PEDIATRIC SICKLE CELL DISEASE Principal Investigator & Institution: Mueller, Brigitta U.; Pediatrics; Baylor College of Medicine 1 Baylor Plaza Houston, Tx 77030 Timing: Fiscal Year 2001; Project Start 01-SEP-2000; Project End 31-AUG-2005 Summary: Clinical research in cell disease (SCD) has reached a new developmental milestone. Ironically, the recent success of hydroxyurea and other new treatments makes the work of clinical investigation harder. In order to move the field forward, we now need to develop clinical investigators who excel in the arenas of clinical investigation and sickle cell disease. Dr. Brigitta U. Mueller is an ideal person to grow into this role. She is a pediatric hematologist/oncologist who early on focused on clinical trials related to pediatric HIV disease at the National Cancer Institute's (NCI) intramural program. We have recruited her to Children's Hospital so that she can shift her attention to sickle cell disease while expanding her training and experience in clinical investigation. The goal of our five-year development plan is for her to become an independent investigator - lead sickle cell clinical research at our institution, and direct multi-institutional studies. Dr. Mueller will pursue her career development under the mentorship of Dr. Orah S. Platt, an experienced investigator in sickle cell disease, and Dr. Carlo Brugnara, an expert in design of innovative therapies in sickle cell disease. She will focus her efforts in four major areas: 1) Throughout the 5 years she will see and discuss patients with Dr. Platt, and meet with Dr. Brugnara to discuss research ideas and review data. 2) She will conduct and analyze the proposed clinical trial - evaluating the effect of Mg, a known inhibitor of K-Cl cotransport, in patients with Hb SC disease. Our hypothesis is that oral Mg will block K- Cl cotransport, prevent cell dehydration, and reduce polymerization-induced vasoocclusive complications. 3) She will design and implement a research infrastructure that will be used as a resource for future proposals. 4) She will do course work at the Harvard School of Public Health and obtain the MPH degree, concentrating on research design, implementation; and analysis. The trial of Mg for the prevention of pain crises in patients with Hb SC disease that we propose, will serve as a template for bringing a variety of treatments designed in basic laboratories to clinical trial. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: CONTROL OF CALCIUM RELEASE IN SKELETAL MUSCLE FIBERS Principal Investigator & Institution: Schneider, Martin F.; Professor; Biochem and Molecular Biology; University of Maryland Balt Prof School Baltimore, Md 21201

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Timing: Fiscal Year 2001; Project Start 01-JUL-1985; Project End 31-DEC-2002 Summary: The overall objective of this project is to gain deeper understanding of the mechanism controlling Ca/2+ release from the sarcoplasmic reticulum (SR) in skeletal muscle. Depolarization of the transverse tubules (TT) in a muscle fiber activates SR Ca/2+ release channels at triad junctions. We have recently used the Ca/2+ indicator dye fluo-3 with laser scanning confocal microscopy to detect discrete SR Ca/2+ release events ("Ca/2+ sparks"), which may arise from the opening of a few SR Ca/2+ channels or perhaps even from a single channel in functioning muscle fibers. These events, which originate at triad junctions and can be discerned at low levels of SR release activation, can be gated either by fiber depolarization or by physiological cytosolic ligands. They thus offer a unique window into the operation of SR Ca/2+ release channels within the normal structural and molecular environment in the muscle. We will characterize the frequency and pattern of occurrence of Ca/2+ sparks under either ligand- or voltageactivation in order to determine the effects of cytosolic [Mg/2+] and [Ca/2+], which are major physiological modulators of both voltage- and ligand-gated events. By determining the concentration dependence and interaction of these two divalent cations on event frequencies and patterns we will develop and test gating schemes for both types of activation and determine whether the same mechanistic steps underlie both ligand- and voltage-gated events. Determining the frequency of sparks under various conditions in these studies will provide the first characterization of the opening rates of SR channels in functioning muscle fibers. We will also use a diffusion and binding model, including diffusion of Ca/2+, CaDye and the sarcomeric distribution of Ca/2+ binding sites, to stimulate sparks and to estimate the amount of Ca/2+ related in a spark. Finally, we will determine whether a spark is generated by the opening of more than one SR Ca/2+ channel. These studies will provide deeper insight into the cellular and molecular mechanisms underlying regulation of Ca/2+ release in skeletal muscle in various physiological states as well as under possible pathological conditions. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CONTROL OF NEMATODE DEVELOPMENT Principal Investigator & Institution: Lambie, Eric J.; Biological Sciences; Dartmouth College 11 Rope Ferry Rd. #6210 Hanover, Nh 03755 Timing: Fiscal Year 2002; Project Start 01-AUG-1993; Project End 31-AUG-2006 Summary: (provided by applicant): The general goal of this work is to understand how cell division, differentiation and survival are regulated in response to extracellular and intracellular signals. The specific focus of this work is to determine how these phenomena are regulated within the gonad of the nematode, Caenorhabditis elegans. The work described in this proposal will focus on a set of genes that mediate the response of the somatic gonad progenitor cells to growth regulatory signals. Two genes, gon-2 and gon-11, have been identified that are required for gonadal cell divisions, beginning in the first larval stage. gon-2 encodes a predicted protein of the TRP cation channel family. The molecular identity of gon-11 is not yet known. In addition, four different gem (gon-2 extragenic modifier) loci have been identified by screening for revertants of the temperature-sensitive allele, gon-2(q388). A convergent line of work has resulted in the discovery of a new gene, neg-1, mutation of which leads to pleiotropic defects that include necrotic death of the gonadal precursor cells. neg-1 and weak alleles of gon-2 can be suppressed by increasing the level of magnesium in the medium. However, gon-11 and strong alleles of gon-2 show little or no suppression by increased magnesium levels. gon-2 is proposed to be expressed within the somatic gonadal cells, where it is activated in response to developmental signals and mediates

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the influx of Mg2+ and Ca2+ These cations would then act in combination to promote cell division and maintain cell viability. The activity and downstream effects of gon-2 are regulated by the gem loci, gon-11 and neg-1. There has been no genetic analysis of how LTRP channels are regulated or how they control cell proliferation/viability. Similarly, there has been no genetic analysis of the regulation of metazoan development in response to the increases in intracellular Mg2+ and Ca2t Therefore, this work will provide a framework for understanding which cellular components are involved in the regulation of LTRP activity and how cells respond to increases in intracellular Mg2+ and Ca2+. The molecules that regulate cell division in response to extracellular signals have been well conserved evolutionarily and aberrancies in their regulation contribute to the onset of cancer in humans. Therefore, the results of this work are likely to be highly significant with regard to the etiology of human disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CORE--INTER CENTER COLLABORATIVE CLINICAL RESEARCH Principal Investigator & Institution: Steinberg, Martin H.; Proofessor of Medicine and Pediatrics; Boston Medical Center Gambro Bldg, 2Nd Fl, 660 Harrison Ave, Ste a Boston, Ma 02118 Timing: Fiscal Year 2003; Project Start 01-JUL-2003; Project End 31-MAR-2008 Summary: At birth, about 1 in 830 African Americans has HbSC disease. These patients have the same vasoocclusive complications as sickle cell anemia---only less often. Clinical descriptions of HbSC disease abound and substantial insights into its distinct pathophysiology have been gained. Nevertheless, few published trials of its treatment exist. Perhaps this is clue to the mistaken perception that HbSC disease is benign or that its rarity precludes conclusive therapeutic trials. We submit that: HbSC disease complications often merit acute treatment; a safe preventive treatment might forestall the disease complications that develop with age; novel means of reversing the pathophysiology of this disorder are available; sufficient patients exist to carry out a successful therapeutic trial. HbSC disease exhibits a characteristic erythrocyte dehydration compared with sickle cell trait or HbC trait erythrocytes. Cell dehydration plays a crucial role in the pathophysiology of HbSC disease because it allows for the intracellular HbS to reach concentrations that induce clinically significant HbS polymerization and cell sickling. K-CI cotransport is highly expressed in HbSC erythrocytes and determines their characteristic microcytosis and dehydration. Thus, HbSC disease represents the ideal target for therapies aimed at preventing K-CI cotransport mediated cell dehydration. Pilot studies showed that hydroxyurea and Mg affect erythrocyte hydration in HbSC disease. Our hypothesis is that oral Mg and hydroxyurea will increase intracellular Mg, block K-CI cotransport, prevent cell dehydration, and reduce polymerization-induced vasoocclusive complications. Accordingly, we propose a double-blinded, placebo-controlled trial to examine the effectiveness of hydroxyurea, magnesium pidolate and hydroxyurea + magnesium pidolate in reducing cell density in HbSC disease. As a secondary endpoint, because of the required brevity of this trial, we will examine the effectiveness of this treatment in preventing sickle cell disease-related vasoocclusive episodes. Other secondary endpoints include HbF level, F-cell numbers and hematologic measurements. The cellular effects of hydroxyurea and magnesium should modulate favorably the course of this disorder and the results of this study will provide the framework for a definitive efficacy trial. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: PROTEINS

CRYSTALLOGRAPHIC

STUDIES

OF

ELECTRON

19

TRANFER

Principal Investigator & Institution: Rees, Douglas C.; Professor; None; California Institute of Technology Mail Code 201-15 Pasadena, Ca 91125 Timing: Fiscal Year 2003; Project Start 01-JAN-1991; Project End 31-MAR-2008 Summary: (provided by applicant): Nitrogen fixation, the reduction of atmospheric dinitrogen to ammonia catalyzed by the enzyme nitrogenase, is the sole biological process for replenishing the nitrogen that is used in the biosynthesis of cellular materials. This enzyme system consists of two metalloproteins, the Fe-protein and MoFe-protein, that mediate the coupling of ATP hydrolysis to substrate reduction. Nitrogenase is a prototypic example of an enzyme with multiple and varied iron-sulfur clusters that participate in electron transfer and substrate reduction, as well as providing an excellent model for energy transduction of ATP hydrolysis. We will utilize crystallographic, biochemical and spectroscopic approaches to investigate the enzymatic and metallocluster assembly mechanisms of nitrogenase. Special emphasis will be placed on establishing the atomic identity and mechanistic significance of the light-atom ligand we recently observed in the center of the FeMo-cofactor, and in assessing the structural framework for the nucleotide-mediated gating of electron transfer processes in nitrogenase. Towards these objectives, we will address: 1. Very high resolution (