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

ACIDOSIS 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., 1960Acidosis: 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: 0-497-00013-X 1. Acidosis-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 acidosis. 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 ACIDOSIS ................................................................................................... 3 Overview........................................................................................................................................ 3 The Combined Health Information Database................................................................................. 3 Federally Funded Research on Acidosis ......................................................................................... 7 E-Journals: PubMed Central ....................................................................................................... 62 The National Library of Medicine: PubMed ................................................................................ 69 CHAPTER 2. NUTRITION AND ACIDOSIS ....................................................................................... 115 Overview.................................................................................................................................... 115 Finding Nutrition Studies on Acidosis...................................................................................... 115 Federal Resources on Nutrition ................................................................................................. 119 Additional Web Resources ......................................................................................................... 119 CHAPTER 3. ALTERNATIVE MEDICINE AND ACIDOSIS ................................................................. 121 Overview.................................................................................................................................... 121 National Center for Complementary and Alternative Medicine................................................ 121 Additional Web Resources ......................................................................................................... 131 General References ..................................................................................................................... 132 CHAPTER 4. DISSERTATIONS ON ACIDOSIS................................................................................... 133 Overview.................................................................................................................................... 133 Dissertations on Acidosis........................................................................................................... 133 Keeping Current ........................................................................................................................ 133 CHAPTER 5. PATENTS ON ACIDOSIS ............................................................................................. 135 Overview.................................................................................................................................... 135 Patents on Acidosis.................................................................................................................... 135 Patent Applications on Acidosis ................................................................................................ 161 Keeping Current ........................................................................................................................ 172 CHAPTER 6. BOOKS ON ACIDOSIS ................................................................................................. 173 Overview.................................................................................................................................... 173 Book Summaries: Federal Agencies............................................................................................ 173 Book Summaries: Online Booksellers......................................................................................... 181 Chapters on Acidosis.................................................................................................................. 181 CHAPTER 7. MULTIMEDIA ON ACIDOSIS ...................................................................................... 183 Overview.................................................................................................................................... 183 Video Recordings ....................................................................................................................... 183 CHAPTER 8. PERIODICALS AND NEWS ON ACIDOSIS ................................................................... 185 Overview.................................................................................................................................... 185 News Services and Press Releases.............................................................................................. 185 Newsletter Articles .................................................................................................................... 187 Academic Periodicals covering Acidosis .................................................................................... 189 CHAPTER 9. RESEARCHING MEDICATIONS .................................................................................. 191 Overview.................................................................................................................................... 191 U.S. Pharmacopeia..................................................................................................................... 191 Commercial Databases ............................................................................................................... 192 Researching Orphan Drugs ....................................................................................................... 192 APPENDIX A. PHYSICIAN RESOURCES .......................................................................................... 197 Overview.................................................................................................................................... 197 NIH Guidelines.......................................................................................................................... 197 NIH Databases........................................................................................................................... 199 Other Commercial Databases..................................................................................................... 201 APPENDIX B. PATIENT RESOURCES ............................................................................................... 203 Overview.................................................................................................................................... 203

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Patient Guideline Sources.......................................................................................................... 203 Associations and Acidosis .......................................................................................................... 206 Finding Associations.................................................................................................................. 206 APPENDIX C. FINDING MEDICAL LIBRARIES ................................................................................ 209 Overview.................................................................................................................................... 209 Preparation................................................................................................................................. 209 Finding a Local Medical Library................................................................................................ 209 Medical Libraries in the U.S. and Canada ................................................................................. 209 ONLINE GLOSSARIES................................................................................................................ 215 Online Dictionary Directories ................................................................................................... 216 ACIDOSIS DICTIONARY........................................................................................................... 217 INDEX .............................................................................................................................................. 309

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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 acidosis 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 acidosis, 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 acidosis, 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 acidosis. 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 acidosis, 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 acidosis. 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 ACIDOSIS Overview In this chapter, we will show you how to locate peer-reviewed references and studies on acidosis.

The Combined Health Information Database The Combined Health Information Database summarizes studies across numerous federal agencies. To limit your investigation to research studies and acidosis, 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 “acidosis” (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: •

Hepatic Failure and Lactic Acidosis Due to Fialuridine (FIAU), an Investigational Nucleoside Analogue for Chronic Hepatitis B Source: New England Journal of Medicine. 333(17): 1099-1105. October 26, 1995. Summary: In this article, the authors describe severe and unexpected multisystem toxicity that occurred during a study of the antiviral nucleoside analogue fialuridine (FIAU) as therapy for chronic hepatitis B virus infection. Fifteen patients with chronic hepatitis B were randomly assigned to receive fialuridine at a dose of either 0.10 or 0.25 mg per kilogram of body weight per day for 24 weeks. They were monitored every 1 to 2 weeks by means of a physical examination, blood tests, and testing for hepatitis B virus markers. During the 13th week, lactic acidosis and liver failure suddenly developed in 1 patient. The study was terminated on an emergency basis, and all treatment with fialuridine was discontinued. Seven patients were found to have severe

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hepatotoxicity, with progressive lactic acidosis, worsening jaundice, and deteriorating hepatic synthetic function, despite the discontinuation of fialuridine. Of these seven patients, five died and two survived after liver transplantation. The authors conclude that the toxic reaction was probably caused by widespread mitochondrial damage and may occur infrequently with other nucleoside analogues. 5 figures. 1 table. 43 references. (AA-M). •

Renal Tubular Acidosis: Pathophysiology and Diagnosis Source: Archives of Internal Medicine. 156(15): 1629-1636. August 12-26, 1996. Summary: In this article, the authors review the pathophysiology and diagnosis of renal tubular acidosis (RTA). They review the literature on this topic and then present a case that illustrates the diagnostic difficulties that may be encountered in a patient with suspected RTA. The principles of renal acid excretion are then discussed. The diagnostic tests available are covered in regard to both their general principles and to interpretation of the results in the context of the various RTA subtypes. Finally, a schematic approach to the diagnosis of RTA is presented to illustrate that only a few relatively simple tests are required to diagnose RTA and to differentiate the pathophysiological subtypes.

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Alkali Therapy in Renal Tubular Acidosis: Who Needs it? Source: JASN. Journal of the American Society of Nephrology. 13 (8): 2186-2188. August 2002. Contact: Available from Lippincott Williams and Wilkins. 12107 Insurance Way, Hagerstown, MD 21740. (800) 638-6423. Summary: Renal tubular acidosis (RTA) is a renal (kidney) tubule disorder that causes acidosis by restricting the reduction of urinary pH and thereby the titration of urinary buffers and the excretion of acid. The acidosis of type 1 RTA (classic) results in osteoporosis and other disorders of bone demineralization. Alkali (base pH) therapy has been given to children with RTA to heal osteopenia and to encourage normal growth. This article considers the use and indications of alkali therapy in adults with RTA. The authors review recent studies in this area, including studies investigating the impact of high net acid dietary load. The authors also consider the therapeutic potential of alkali in the increasingly large number of elderly people who are likely to be impaired by RTA. The authors conclude that with the scope of its recognized expression so expanded, the frequency of its consequent occurrence so increasing, and the potential societal benefits of its treatment so enormous (and so inexpensively achieved), RTA may be coming of age as a disorder of public health importance. 41 references.

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Inherited Renal Tubular Acidosis Source: Current Opinion in Nephrology and Hypertension. 9(5): 541-546. September 2000. Contact: Available from Lippincott Williams and Wilkins. P.O. Box 1600, Hagerstown, MD 21741. (800) 638-3030 or (301) 223-2300. Fax (301) 223-2400. Website: www.currentopinion.com. Summary: The past few years have witnessed great progress in determining the molecular basis of inherited renal tubular acidosis (RTA). This article summarizes the current understanding of the genetic causes of primary inherited RTA. The authors also evaluated the ability of known functional and biochemical properties of the mutant

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proteins to explain the pathophysiology of associated renal acidification defects. Molecular genetic study has demonstrated that inherited RTA is associated with mutations in genes, with expression restricted almost uniquely to the kidney. Consistent with the physiologically defined importance of multiple gene products in urinary acidification, heritable renal tubular acidosis is genetically heterogeneous. Autosomal dominant distal renal tubular acidosis has been association with a small number of mutations, although the pathophysiologic mechanisms behind these mutations remains unclear. Rarely, autosomal recessive distal RTA is caused by homozygosity or compound heterozygosity for the loss of function mutation. A larger proportion, often accompanied by hearing loss, is associated with a different gene mutation. Another gene mutation has been recently identified in proximal renal tubular acidosis with corneal calcification. The authors conclude that although these mutations have been discovered during investigation of the genetic causes of inherited renal acidification defects, studies of these naturally occurring mutations will advance the understanding of the normal urinary acidification process, as well as of acid base transport processes in other organ systems. 2 figures. 1 table. 33 references. •

Hereditary Distal Renal Tubular Acidosis: New Understandings Source: in Coggins, C.H.; Hancock, E.W., Eds. Annual Review of Medicine: Selected Topics in the Clinical Sciences, Volume 52. Palo Alto, CA: Annual Reviews Inc. 2001. p. 471-484. Contact: Available from Annual Reviews Inc. 4139 El Camino Way, P.O. Box 10139, Palo Alto, CA 94303-0139. (800) 523-8635. Fax: (415) 855-9815. PRICE: $47. ISBN: 0824305450. Summary: The primary or hereditary form of distal renal tubular acidosis (dRTA), although rare, has received increased attention recently because of dramatic advances in the understanding of its genetic basis. This review article discusses several recent important studies that have begun to unravel the genetic defects causing different types of primary or hereditary dRTA (the authors do not discuss acquired dRTA). The final regulation of renal acid excretion is effected by various acid and base transporters localized in specialized cells in the cortical collecting and outer medullary collecting tubules. Inherited defects in two of the key acid or base transporters involved in distal acidification, as well as mutation in the cytosolic carbonic anhydrase gene, can cause dRTA. The syndrome is inherited in both autosomal dominant and recessive patterns; patients with recessive dRTA present with either acute illness or growth failure at a young age, sometimes accompanied by deafness, whereas dominant dRTA is usually a milder disease and involves no hearing loss. Hypokalemia (low levels of potassium in the blood), metabolic acidosis, nephrocalcinosis (calcium phosphate in the tubules of the kidney, resulting in kidney insufficiency), and renal calculi (kidney stones) are seen in both autosomal recessiave and dominant dRTA but tend to be more severe and more common in autosomal recessive dRTA. Growth retardation is also much more common and severe in recessive dRTA, probably because the recessive form occurs earlier in life and causes severe metabolic acidosis. The severity of the acidosis manifesting at an early age is the key determinant of delayed growth. 2 figures. 1 table. 64 references.

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Diabetic Ketoacidosis Source: Practical Diabetology. 10(3): 1-5, 8. May-June 1991. Summary: This article describes the pathophysiology and therapy of diabetic ketoacidosis (DKA), from initial presentation to discharge. Topics covered include the role of insulin, increased production of free fatty acids, increased hepatic production of

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glucose and ketoacids, dehydration, the diagnosis of DKA, interpretation of the diagnostic tests used to confirm DKA, treating DKA (replacing fluids, insulin, and potassium), monitoring the patient during therapy, and other clinical and laboratory considerations. The author also mentions potential complications of DKA, including hypoglycemia, hypokalemia, insulin resistance and prolonged DKA, hypoxemia, obtundation and brain edema, lactic acidosis, and hypercholermic acidosis. 1 figure. 3 tables. •

Value of Calcium Carbonate in Treating Acidosis, Phosphate Retention, and Hypocalcemia Source: Nephrology News and Issues. 5(7): 16, 18-19, 32. July 1991. Summary: This article discusses the value of calcium carbonate in treating acidosis, phosphate retention, and hypocalcemia. The author notes that calcium carbonate is easily available in high and varied dosage forms and is inexpensive. Topics include indications for calcium carbonate in the renal patient, guidelines for the use of calcium carbonate, and complications arising from the use of calcium carbonate, including hypercalcemia and constipation. The author suggests that continued problems with high serum calcium and phosphorus in some patients using calcium salts may necessitate the intermittent use of aluminum binders. 43 references.

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Treatment Strategies for Diabetic Ketoacidosis in Children and Adolescents With Insulin-Dependent Diabetes Mellitus Source: Clinical Diabetes. 11(5): 102-106. September-October 1993. Summary: This article reviews the current thinking on treatment strategies for diabetic ketoacidosis (DKA) in children and adolescents with insulin-dependent diabetes mellitus (IDDM). Topics include a brief review of DKA and how it happens; rehydrating patients; replenishing potassium; lowering the glucose level; correcting acidosis; treating other DKA symptoms; avoiding cerebral edema; continuing insulin therapy; and monitoring patients. 2 tables. 11 references.

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Diabetic Ketoacidosis and Hyperosmolar Nonketotic State: Gaining Control Over Extreme Hyperglycemic Complications Source: Postgraduate Medicine. 99(6): 143-148, 150, 152. June 1996. Summary: This continuing education article focuses on the evaluation and management of diabetic ketoacidosis and hyperosmolar nonketotic state in adults. The distinctions between these conditions are clearly defined, and the authors stress that accurate recognition is important for proper treatment. Topics include the differences between insulin-dependent and noninsulin-dependent diabetes (IDDM and NIDDM, respectively); assessment considerations; factors related to decompensated hyperglycemic states; characteristic findings in diabetic ketoacidosis and in hyperosmolar nonketotic state; treatment modalities; and recommendations for followup of treatment. The authors contend that the two conditions may represent variants of decompensated hyperglycemia that differ only by the magnitude of dehydration and the severity of acidosis. 4 tables. 21 references. (AA-M).

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Federally Funded Research on Acidosis The U.S. Government supports a variety of research studies relating to acidosis. 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 acidosis. 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 acidosis. The following is typical of the type of information found when searching the CRISP database for acidosis: •

Project Title: A CHEMICALLY SYNTHETIC ANTI-TOXIC VACCINE AGAINST MALARI Principal Investigator & Institution: Schofield, Louis; Walter and Eliza Hall Inst Medical Res of Medical Research Victoria, Timing: Fiscal Year 2002; Project Start 15-SEP-2000; Project End 31-AUG-2003 Summary: (Adapted from the Applicant's Abstract): Plasmodium falciparum is a global health problem because of the morbidity and mortality associated with infection. Much of this morbidity and mortality is believed to arise from the actions of a malaria toxin. The toxin initiates a systemic inflammatory cascade involving cytokine excess, which may result in disseminated intravascular coagulation, hepatic dysfunction, acute renal failure, multi-organ inflammation, hypoglycemia, lactic acidosis and death. The toxin may further contribute to organ-specific and cerebral disease syndromes by hyperactivation of the vascular endothelium. Clinical immunity to malaria is acquired considerably earlier than anti-parasite immunity, and it is possible that this is mediated by anti-toxin mechanisms. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: A ROLE OF ACID-SENSING ION CHANNELS IN ISCHEMIA Principal Investigator & Institution: Simon, Roger P.; Director, Neurobiology Research; Emanuel Hospital and Health Center Portland, or 972083950 Timing: Fiscal Year 2002; Project Start 01-JAN-2002; Project End 31-DEC-2003 Summary: (provided by applicant): Ischemic injury causes an immediate and marked fall in brain pH, the consequences of which are multiple and complex and incompletely understood. The recent discovery of proton-gated cation channels (acid-sensing ion channels, ASIC) in the brain offers a novel opportunity to explore hitherto unknown aspects of biology relevant to understanding the response of the brain to ischemia. The ASIC system does respond during ischemia. Our preliminary data show up-regulation of the brain-specific ASIC2a subunit following ischemia and co-localization of another

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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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ASIC subunit to brain mitochondria, a major site of perturbation during ischemic necrosis and apoptosis. In patch-clamping experiments, cultured neocortical cells showed an acid-evoked current matching that of ASIC1a, whose properties changed after in vitro ischemia, and neuronal (PC12) cells displayed an acid-evoked current that was blocked with an ASIC subunit-specific antagonist. We therefore propose to 1) characterize the expression of all ASIC subunits after focal and global ischemia; 2) identify and compare ASIC subunit-protein interactions in normal and ischemic brain; 3) describe the sub-cellular location of the ASIC subunits; and 4) assess electrophysiologically the effect of ischemia on ASIC function and the effect of ASIC activity on the outcome of ischemic injury. These novel studies are likely to offer new understanding of the biology of brain ischemia and offer new targets for therapeutic intervention. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ALDOLASE-MEDIATED REGULATION OF V-ATPASE Principal Investigator & Institution: Lu, Ming; Medicine; University of California San Francisco 500 Parnassus Ave San Francisco, Ca 941222747 Timing: Fiscal Year 2003; Project Start 30-SEP-2003; Project End 30-JUN-2005 Summary: (provided by applicant): Vacuolar H+ATPases (V-ATPases or H+ATPases) are highly conserved proton pumps that couple hydrolysis of ATP to proton transport out of the cytosol. They are essential for renal acid-base homeostasis, for sorting of newly synthesized proteins in the Golgi, and for acidification and normal function of the yeast vacuole. Although a central question in the field is how V-ATPase is regulated under physiological conditions, until recently little was known about the underlying mechanisms. The glycolytic enzyme aldolase has been identified to interact with three subunits of V-ATPase by our lab. This represents the first example of physical association between the ATP-generating glycolytic pathway and an ATP-hydrolyzing ion pump. Deletion of the aldolase gene in yeast cells results in complete disassembly of and a dramatic reduction in V-ATPase. These abnormalities can be fully restored by aldolase complementation. Our data suggest that disruption of the interaction between aldolase and V-ATPase results in malfunction of V-ATPase, which leads to renal tubular acidosis found in patients with hereditary fructose intolerance, an autosomal recessive disorder caused by mutations in an isoform of aldolase. In this proposal, we will carry out molecular genetic analysis in yeast cells to examine the structural basis and regulation of the interaction between aldolase and V-ATPase, and test the hypothesis that aldolase mediates V-ATPase assembly, function and stability. The specific aims of this proposal are: 1) to generate aldolase and V-ATPase subunit mutants that lack binding for a specific interaction but retain aldolase enzymatic activity and/or binding to other V-ATPase subunits; 2) to express the aldolase and V-ATPase subunit mutants in yeast deletion mutant strains lacking either aldolase or a single subunit of V-ATPase, and examine the effects on V-ATPase assembly, function and stability; 3) to examine the parameters required for aldolase to bind intact V-ATPase and disassembled V-ATPase sectors. These studies will provide important insight into the molecular basis for metabolic control of proton transport. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: ATP SYNTHESIS IN MITOCHONDRIAL DISORDERS Principal Investigator & Institution: Manfredi, Giovanni; Assistant Professor of Neuroscience; Neurology and Neuroscience; Weill Medical College of Cornell Univ New York, Ny 10021

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Timing: Fiscal Year 2002; Project Start 01-SEP-1999; Project End 31-AUG-2004 Summary: Mitochondria are the main sources of energy in the cell. They contain their own DNA (mtDNA), whose genes encode components of the respiratory chain/oxidative phosphorylation system. They are essential for the normal functioning of all cells in the body, and are absolutely critical for the function of those tissues that are highly dependent on aerobic metabolism, especially muscle and brain. Since 1988, both mtDNA point mutations and mtDNA rearrangements (i.e. large-scale deletions and duplications) have been associated with a heterogeneous group of sporadic, mendelian, and maternally-inherited mitochondrial encephalomyopathies. These mutations generally cause an impairment of the respiratory chain, with a reduction in ATP synthesis. However, very little is known about how affected cells cope with the reduced ATP production: for example, which ATP-dependent cellular functions are preserved and which are down-regulated or abolished. Understanding ATP distribution inside mutant, as well as normal, cells would be extremely important for the interpretation of the biochemical and clinical phenotype of mitochondrial disorders. This Career Development Award Application proposes to investigate the effect of mtDNA abnormalities, on the intracellular ATP pool in different cell compartments, with particular emphasis on the mitochondria, the cytoplasmic membrane, and the nucleus. We plan to study the ATP content in cytoplasmic hybrids of human mtDNAless cyss ( rho o cells ) repopulated with mitochondria derived from patients tissues, by targeting a recombinant firefly luciferase to different cell compartments. Utilizing a similar experimental approach, we will also attempt a novel genetic strategy for treatment of point mutations in the mtDNA ATPase6 gene, that are responsible for a maternally- inherited form of Leigh syndrome (MILS): to recode the ATPase 6 gene to contain the universal genetic code by in vitro mutagenesis, to fuse a mitochondrial targeting sequence to the recoded sequence, and then to transfer this construct into the nucleus, in order to express the gene from nuclear DNA and target it back to mitochondrial ( allotopic expression). Allotopic expression of the recoded wild-type genes should partially restore the APT synthetic function in mutant cells. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ATPASES IN MICROVASCULAR ENDOTHELIAL CELLS AND DIABETES Principal Investigator & Institution: Martinez-Zaguilan, Raul; Physiology; Texas Tech University Health Scis Center Health Sciences Center Lubbock, Tx 79430 Timing: Fiscal Year 2002; Project Start 01-SEP-2000; Project End 31-JUL-2004 Summary: Angiogenesis is important for vascular remodeling and is strictly controlled under normal conditions. In pathological states such as cancer and diabetes, angiogenesis is altered. The process of angiogenesis requires growth and invasion/migration of endothelial cells through extracellular matrix proteins. Microvascular endothelial cells are exposed to an acid and anoxic environment, conditions that are unfavorable for growth and survival. Thus it is paradoxical that microvascular endothelial cells grow under these conditions. It is known that the extracellular environment of tumors is acidic and anoxic, yet these cells grow and survive in this hostile environment. A vacuolar type proton ATPase (pmV-ATPase) has been identified in highly invasive and metastatic tumor cells. The cells utilize pmVATPase to maintain an aalkaline cytosolic pH permissive for growth. Because microvascular endothelial cells also invade and migrate through extracellular matrix proteins, it is hypothesized that microvascular endothelial cells exhibit pmV-ATPase that is greater at the invading/leading edge of the cells, and that pmV-ATPase activity is

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exacerbated by chronic acidosis. It is also hypothesized that pmV-ATPase is decreased in microvascular endothelial cells from diabetic models where angiogenesis is impaired. To test these hypothesis, pmV-ATPase will first be analyzed in microvascular endothelial cells by evaluating the role of pmV-ATPase for pHcyt regulation using timeresolved fluorescence spectroscopy and ion substituion/pharmacological approaches. The presence of this pump's activity at the plams membrane will be analyzed by immunocytochemistry, immunogold electron microscopy and Western blot analyses of subcellular fractions. The relevance of pmV-ATPase at the leading edge of the cell will be understood by studying pHcyt regulation in single cells from leading to lagging edge using line scanning laser confocal resolution respectively. The physiological relevance of pmV-ATPase will be evaluated using in vitro invations mibration assays, in vitro angiogenesis models and the wounded monolayer model. This will be done in the presence and absence of V-ATPse inhibitors. The relevance of pmV-ATPase in angiogenesis and diabetes will be understood by using microvascular endothelial cells from a spontaneous model of diabetes with inborn defects. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: BICARBONATE TRANSPORT BY THE MATURING RENAL TUBULE Principal Investigator & Institution: Schwartz, George J.; Professor; Pediatrics; University of Rochester Orpa - Rc Box 270140 Rochester, Ny 14627 Timing: Fiscal Year 2002; Project Start 01-JUL-1990; Project End 30-JUN-2004 Summary: (Adapted from the Applicant's Abstract): Membrane-bound carbonic anhydrase (CA) is essential for HCO transport in the proximal tubule and collecting duct. Clinically, problems in neonatal renal acidification may be due to delayed membrane CA expression. Membrane CA includes CA isoform IV, which is anchored to the luminal membrane via a glycosylphosphatidylinositol (GPI) linkage. However, two anti-CA IV peptide antibodies show that CA IV is expressed on both apical and basolateral membranes of the proximal tubule, the latter not expected for a GPIanchored protein. Other membrane CA isoforms (CA XII, and XIV), could reside on the basolateral membrane and cross-react with these antibodies. The identity of the basolateral CA, the mechanisms for its targeting, and its regulation during acidosis and development will be examined in rabbit kidney. Aim 1 is to determine which CA isoform resides in the basolateral membrane of the proximal tubule. A PCR-based approach will be used to obtain cDNA probes for CAs XII, and XIV in rabbit. Peptides will be prepared from these sequences to test specificity of the CA IV antibodies and to generate CA XII- and XIV-specific antibodies. Another anti-CA IV peptide antibody (with no homology to CAs XII or XIV) will be made. The apical basolateral CAs will be compared and characterized. Aim 2 examines the targeting of membrane CAs. Membrane CA cDNAs will be FLAG-tagged and transiently transfected into immortalized mouse proximal tubule cells. The polarity of expressed membrane CAs will be examined and compared with the targeting of the C-terminus. This will establish whether GPI-linkage confers apical polarity and the C-terminal hydrophilic segment confers basolateral polarity to the membrane CA. The regulation of these CAs in response to metabolic acidosis and maturation will also be examined. Aim 3 investigates physiologic correlations. The presence of basolateral CA activity on HCO transport and cell pH will be examined in perfused proximal tubules. The regulation of basolateral CAs in response to acidosis and development will be studied. Finally, the mechanisms by which acidosis causes resistance to CA inhibition will be studied in outer medullary collecting ducts, which absorb HCO despite a luminal disequilibrium

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pH. The objective is to better understand the role of membrane carbonic anhydrases (CAs) in mediating renal acid-base transport. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: BIOCHEMICAL DEFICIENCIES

GENETICS

OF

CARBONIC

ANHYDRASE

Principal Investigator & Institution: Sly, William S.; Chairman and Professor; Biochem and Molecular Biology; St. Louis University St. Louis, Mo 63110 Timing: Fiscal Year 2002; Project Start 01-APR-1988; Project End 30-NOV-2003 Summary: The twelve known carbonic anhydrases (CAs) and CA-related proteins (CARPs) play important roles in diverse physiological processes including respiration, bone resorption, renal acidification, gluconeogenesis, signal transduction, and formation of cerebrospinal fluid and gastric acid. The recently discovered CA IX and CA XII are related to oncogenesis and are over-expressed in certain cancers. The broad goal of this research is to study the functional genomics of this gene family to determine the importance of individual members to health and disease. We have five specific aims: 1. Complete studies characterizing the biochemical and molecular genetics of CA II deficiency. CA II deficiency is the basis for the human inborn error of metabolism producing osteopetrosis, renal tubular acidosis, and brain calcification, novel studies are also proposed on the CA II-deficient mouse. 2. Characterize the mouse doubly deficient for CA II and CA IV and determine what other CA in kidney compensates for CA IV deficiency. CA IV is the GPI-anchored membrane CA on surfaces of epithelial cells in kidney and gut and of capillary endothelial cells. The CA IV null mouse lacks the expected renal defect. 3. Characterize the CA VA gene knockout mouse, the newly discovered CA Vb, and candidates for CA V deficiency. CA VA is the mitochondrial CA thought to be involved in gluconeogenesis and ureagenesis. 4. Characterize CA IX and define its role in the regulation of cell proliferation and in oncogenesis. CA IX is a tumor-associated CA that is over-expressed in several cancers and expressed in normal stomach. 5. Characterize the properties and the functional genomics of CA XII. CA XII is a newly discovered, transmembrane CA that is over-expressed in several cancers and expressed in normal kidney and intestine. These studies should enhance our understanding of how thee individual carbonic anyhydrases contribute to normal physiology, how single CA deficiencies produce disease, and why the newly discovered CAs IX and XII are over-expressed in certain cancers, and should also suggest new targets for isozyme-specific CA inhibitors. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CELL ADHESION MOLECULES IN CEREBRAL MALARIA. Principal Investigator & Institution: Van Der Heyde, Henri C.; Microbiology and Immunology; Louisiana State Univ Hsc Shreveport P. O. Box 33932 Shreveport, La 71103 Timing: Fiscal Year 2002; Project Start 01-JUL-1997; Project End 31-AUG-2002 Summary: (provided by the applicant): Despite decades of effort to conquer malaria, it remains a leading cause of death due to a single infectious agent. The advent of drugresistance by Plasmodium falciparum and insecticide resistance by the mosquito vector jointly makes development of therapy against malaria highly desirable. While a vaccine to prevent infection is a laudable goal, the development of therapy to control the pathological consequences of malaria may represent a more achievable solution. To develop such a therapy, we need a detailed understanding of the pathophysiology of malaria. Results from clinical studies of P. falciparum patients indicate that these

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individuals develop impaired consciousness with posturing (cerebral malaria), respiratory distress with lactic acidosis, anemia and (rarely) acute nephritis. Development of shock is, according to the WHO, a prognostic indicator of poor outcome. In addition, patients with P. falciparum have activated endothelium with increased expression on endothelium of a number of cell adhesion molecules (CAMs). Collectively these findings indicate that the processes of circulatory shock are occurring in patients with P. falciparum malaria. Recent work from our laboratory indicates that P. berghei-infected mice, a well-recognized model of cerebral malaria, also develop circulatory shock and respiratory distress with lactic acidosis. This model is therefore useful to mechanistically dissect the role of CAMs in the development of cerebral malaria and respiratory distress. We will use the recently developed dual radiolabel technique to assess CAM expression on endothelium and flow cytometry to assess CAM expression on T cells during P. berghei malaria. CAMs with increased expression during P. berghei malaria will be tested for their role in cerebral malaria and respiratory distress by using CAM0/0 and anti-CAM mAb-treated mice. Pro-inflammatory cytokines are often needed to increase CAM expression, so we will determine whether selected pro-inflammatory cytokines function in pathogenesis of malaria by regulating CAM expression. Our preliminary data indicate that both CAMs and pro-inflammatory cytokines are indeed required for pathogenesis of P. berghei malaria. We will test whether inhibition of an intracellular signaling pathway (specifically NF-?B) abrogates cerebral malaria and respiratory distress by preventing increased CAM expression and T cell adherence to endothelium. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CELL CYCLE PROTEINS IN RENAL GROWTH, INJURY, AND REPAIR Principal Investigator & Institution: Preisig, Patricia A.; Professor; Internal Medicine; University of Texas Sw Med Ctr/Dallas Dallas, Tx 753909105 Timing: Fiscal Year 2002; Project Start 15-AUG-2000; Project End 31-JUL-2005 Summary: Renal epithelial cells are normally quiescent, but can increase their growth rate. In some situations, such as following an acute renal injury, the cell growth is essential to repairing the damaged epithelium, and appears tightly regulated. In contrast, unregulated, and ultimately destructive, growth occurs in renal cancer, polycystic kidney disease, and the progressive loss of renal function associated with loss of renal mass, diabetes mellitus, and most forms of glomerular injury. The growth changes that occur involve both hyperplasia (resulting in an increase in cell number) and hypertrophy (resulting in an increase in cell size). Using in vitro systems, two mechanisms of renal epithelial cell hypertrophy have been characterized; One involves regulation of the cell cycle process (referred to as being cell cycle-dependent) and is mediated by growth factors and cytokines, and the other is independent of cell cycle processes and mediated by agents that alkalinize intravesicular compartments, such as NH4C1. Using in vivo renal growth models, we have shown that: 1) Compensatory renal growth following uninephrectomy is a hypertrophic growth process that involves primarily activation of cyclin D kinase, without an increase in BrdU incorporation, and is not affected by inhibiting ammoniagenesis, suggesting that a cell cycle-dependent mechanism is involved; 2) Diabetes-induced renal growth involves an initial hyperplastic growth phase associated with activation of both G, kinase and an increase in BrdU incorporation, followed by a hypertrophic growth phase associated with continued activation of cyclin D kinase, inhibition of cyclin E kinase, and inhibition of BrdU incorporation; 3) The renal growth associated with chronic hypokalemia can be

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reversed when ammoniagenesis is inhibition by an alkaline diet, suggesting a cell cycleindependent mechanism of growth; and 4) In transgenic mice in which the endothelin B receptor has been knocked out, uninephrectomy does not lead to hypertrophy. Aim 1 will continue to characterize the role of cell cycle proteins in compensatory renal growth. Aim 2 will focus of diabetes-induced hypertrophy. Studies will examine the regulation of the cell cycle proteins in the switch between a hyperplastic and hypertrophic growth pattern. Studies will be done in 3 models of diabetes mellitus: streptozotocin-induced type I, in Nod mice (type I), and in db/db mice (type II). Studies will also be done to determine the role of the endothelin B receptor in diabetes-induced renal growth. Aim 3 will determine the mechanisms involved in the activation of cyclin D kinase and inhibition of cyclin E kinase in cell cycle-dependent hypertrophy. Aim 4 will determine if the endothelin B receptor plays a role in chronic metabolic acidosis and chronic potassium deficiency, models of renal hypertrophy thought to be mediated by the cell cycle-independent mechanism. Together these studies will afford us the opportunity to determine if direct regulation of cell cycle processes provides an avenue for therapeutic advances that will either improve upon the beneficial effects or reduce the detrimental sequelae of renal injury. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CELLULAR MECHANISMS OF MEDULLARY SEROTONERGIC NEURONS DURING DEVELOPMENT Principal Investigator & Institution: Richerson, George B.; Associate Professor; Children's Hospital (Boston) Boston, Ma 021155737 Timing: Fiscal Year 2003; Project Start 07-JUL-2003; Project End 31-MAR-2008 Summary: The overall goal of this program is to define the cause of SIDS. With the recent discovery of abnormal LSD binding in the medulla of SIDS infants, we have focused our efforts on the medullary serotonergic system, to define what specifically happens to serotonergic neurons to cause them to malfunction. The goal of Project 5 is to define the cellular properties of serotonergic neurons, as a means of providing a neurobiological explanation for the link between serotonin and SIDS. We have previously demonstrated that serotenergic neurons closely apposed to large arteries in the rat ventral medulla increase their firing rate in response to acidosis, and we have proposed that they are chemoreceptors that stimulete breathing, arousal and other CNS changes to restore pH homeostasis: We now plan to use a rat model to determine whether the cellular properties of serotonergic neurons can explain the three risk factors in the Triple Risk Model for SIDS. We wiil use a combination of multielectrode arrays and patch clamp recordings from serotonergic neurons in culture and in brain slices. We will: 1) Study how different subsets of serotonergic neuron respond to acidosis, hypoxia, temperature and glucose. 2) Examine how muscarinic receptor activation leads to enhancement of chemoreception. 3) Define the changes in chemosensitity of serotonergic neurons as they undergo development. 4) Determine the effects of acute and chronic (during pregnancy) nicotine exposure on the function of serotonergic neurons. 5) Compare the cellular properties of serotonergic neurons from rats with those of piglets and mice, and determine whether serotonergic neurons from these species, as well as human infants, are closely associated with large arteries of the ventral medulla. These experiments will provide information critical to a full understanding of the role of serotonergic neurons in brain function, and will lead to specific testable hypotheses about how their malfunction or maldevelopment could lead to death during sleep. URimately, the results of these experiments may provide important insights that could lead to diagnostic and therapeutic tests for those infants at highest risk of SIDS.

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Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CEREBRAL ISCHEMIA IN THE FEMALE Principal Investigator & Institution: Hurn, Patricia D.; Professor and Vice Chairman for Research; Anesthesiology/Crit Care Med; Johns Hopkins University 3400 N Charles St Baltimore, Md 21218 Timing: Fiscal Year 2002; Project Start 01-FEB-1995; Project End 31-JAN-2004 Summary: In this animal research protocol, the investigators propose to ascertain the cerebral blood flow and metabolic effects of the presumed neuroprotective female hormones during and following global cerebral ischemia. Specifically, the proposed study will determine the effect of global cerebral ischemia on cerebral blood flow (CBF), energy metabolism, and pial vessel reactivity in female animals compared to their male counterparts. The investigators also will evaluate whether or not the female hormone, beta- estradiol, plays an important role in recovery mechanisms from ischemia. Unneutered males also will be studied to determine if there are important gender-specific recovery responses during ischemia/reperfusion and if any therapeutic benefit from reproductive steroid administration is limited to females. The proposed experiments will explore two specific mechanisms of ischemic injury in vivo; specifically, 1) acidosis leading to depressed recovery of energy metabolism, loss of pH regulation, and related iron- catalyzed oxidant injury; and 2) microvascular endothelial dysfunction. To study these variables, the investigators will use magnetic resonance (MR) spectroscopy and intravital microscopy to determine if estradiol acts via specific cellular mechanisms. Four Specific Aims are presented. In Specific Aim #1, the investigators will test the hypothesis that ischemic acidosis is less in females, with consequently more complete recovery of brain energy phosphates compared to males; and that chronic estrogen therapy further improves post-ischemic recovery of energy metabolism and intracellular pH. Specific Aim #2 will examine the effect of pre-ischemic hyperglycemia and its consequent exaggerated tissue acidosis on metabolic recovery, testing the hypothesis that the anti- oxidant activity of estradiol decreases vulnerability to hyperglycemiamediated reperfusion injury. Intravital microscopy will be employed in Specific Aim #3 to determine if post-ischemic pial vessel reactivity to endothelium-dependent pharmacologic agents is impaired in females and estradiol-treated animals. Finally, Specific Aim #4 will test the hypothesis that chronic estradiol therapy increases brain cGMP, nitric oxide synthase activity and pial vessel responsivity to NO-mediated agents in a dose-dependent manner. The information derived from these experiments should contribute to our understanding of vascular function in females at decreased risk for cerebrovascular disease relative to males and of the role of estrogen as potential neuroprotective therapy for patients of either sex. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: CHARACTERIZATION OF A NEW FAMILY OF PROTEIN KINASES Principal Investigator & Institution: Popov, Kirill M.; Assistant Professor; Molecular Biology and Biochem; University of Missouri Kansas City Kansas City, Mo 64110 Timing: Fiscal Year 2002; Project Start 01-JUL-1995; Project End 30-JUN-2004 Summary: (From the application abstract:) The mitochondrial pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate, the reaction that determines the metabolic fate of carbohydrates. The enzymatic activity of the mammalian PDC is regulated by reversible phosphorylation. The specific kinase (pyruvate dehydrogenase kinase or PDK) converts it to an inactive form that can be

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reactivated only by a specific phosphatase. The hyperphosphorylation of PDC observed in diabetes, ischemia, and metabolic acidosis directly contributes to the morbidity and mortality associated with these conditions. It is generally believed that the hyperphosphorylation is due, in part, to enhanced kinase activity. Recently this laboratory provided the first data indicating that, in humans and other mammals there are multiple isoenzymes of PDK. The physiological significance of multiple isoenzymes is currently unknown. The results available thus far strongly suggest that the isoenzymes are functionally different. The isoenzyme PDK2 is likely to be responsible for the * short-term regulation of PDC activity. The inducible isoenzyme PDK4, in contrast, may be mainly responsible for long-term control. Its over-expression in diabetes is likely a leading cause of the hyperphosphorylation of PDC that, in turn, contributes to hyperglycemia. This proposal is aimed to further elucidate the structure, function, regulation and physiological significance of the multiple isoenzymes of PDK. Its major goals are: 1) to determine the three dimensional structure of pyruvate dehydrogenase kinase; 2) to elucidate the molecular basis for catalysis and substrate recognition by pyruvate dehydrogenase kinase; 3) to further define the molecular mechanisms responsible for regulation of pyruvate dehydrogenase kinase activity; 4) to characterize the molecular interactions between isozymes, as well as between isozymes and pyruvate dehydrogenase complex under normal conditions, as well as under starvation and diabetes. These goals will be achieved though a combination of structure/functional analysis, biochemical characterization, as well as more physiologically oriented studies of isozymes under conditions such as starvation and diabetes. This will allow us to understand how this structurally unique protein kinase functions. It will also allow us to take the first step towards the design of isoenzymespecific drugs that may alleviate some of the symptoms and prevent complications associated with diabetes, ischemia and acidosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: SYNDROME

CLONIDINE

TREATMENT

FOR

NEONATAL

ABSTINENCE

Principal Investigator & Institution: Gauda, Estelle B.; Associate Professor; Pediatrics; Johns Hopkins University 3400 N Charles St Baltimore, Md 21218 Timing: Fiscal Year 2003; Project Start 30-SEP-2003; Project End 31-JUL-2006 Summary: (provided by applicant): In the United States, as many as 20,000 babies a year are born to opioid ("narcotic") addicted mothers. Like their mothers, these infants are opioid dependent. Following birth, the infant is removed from its opioid source, inducing a withdrawal syndrome in these infants. Withdrawal symptoms in newborns include vomiting, diarrhea, poor feeding, tachycardia, hypertension, diaphoresis, restlessness, insomnia, irritability, tremors, clonus, hyperphagia with poor growth and acidosis, reversible neurologic abnormalities, and even seizures. This complex of signs and symptoms is referred to as neonatal abstinence syndrome (NAS). Reinstitution of opioids followed by a slow tapering protocol is currently the standard of care, necessitating prolonged hospitalization from weeks to months. Clonidine is a nonnarcotic central alpha2-adrenergic receptor agonist that blocks the effects of overexcitation of the sympathetic nervous system and is an approved treatment for opioid withdrawal in adults. We currently have a physician sponsored IND (#63,781) to study the effect of clonidine as adjunct therapy to opioids for the treatment of NAS. This proposal will test the hypothesis that combination therapy of clonidine and opioids is 1) safe and efficacious, 2) allows reduced amount of opioid drug use, and 3) results in shorter time of treatment and hospitalization. This will be accomplished in a

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randomized, placebo controlled double blind clinical trial comparing diluted tincture of opium (DTO) combined with a placebo (control) vs. DTO combined with clonidine. Additional sub-studies include determination of 1) pharmacokinetics and pharmacodynamics of DTO and clonidine in the enrolled cohort and 2) further safety evaluation by evaluating developmental outcome on the Bayley Scale of Infant Development (BSID) at 6 and 12 months of age. Pharmacokinetics will be determined by measuring serum concentrations of clonidine and morphine and analyzing volume of distribution, elimination half-life and clearance. These results will have important clinical implications and may change the standards of care not only for management of infants with severe NAS, but also for the management of infants and children, after long-term iatrogenic opioid exposure for instance following prolonged analgesia for mechanical ventilation or multiple operations. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CONTROL OF BREATHING DURING PHYSIOLOGIC CONDITIONS Principal Investigator & Institution: Forster, Hubert V.; Professor; Physiology; Medical College of Wisconsin Po Box26509 Milwaukee, Wi 532260509 Timing: Fiscal Year 2002; Project Start 01-JUN-1986; Project End 31-MAY-2005 Summary: Several theories on the neural control of breathing that were based on data from reduced preparations were not supported by our recent findings in awake and asleep goats on the effects of rostral medullary neuronal dysfunction and/or carotid body denervation (CBD). Some findings mimicked the altered breathing found in obstructive sleep apnea (OSA) and congenital central hypoventilation syndrome (CCHS). The mechanisms that mediated these effects are not established, but one likely mechanism is through intracranial chemoreceptors for years thought to exist only near the ventral medullary surface (including the retrotrapezoid nucleus RTN)). However, findings in reduced preparations of chemoreceptors at widespread brain sites have raised questions related to the location and role of chemoreceptors that affect breathing in awake and asleep states and whether brain chemoreceptor sensitivity is altered by CBD. One recently identified site of chemoreception is the medullary raphe nuclei (MRN) whose role in the control of breathing during awake and asleep states remains speculative. Accordingly, to study chemosensitivity and the role of the RTN and MRN in the control of breathing, we will implant microtubules into these nuclei of goats to: a) create a focal acidosis by dialysis of mock cerebrospinal fluid with different PCO2's, or b) induce neuronal dysfunction through injection of glutamate or serotonin receptor antagonists or agonists, or a neurotoxin. Major hypotheses are: 1) focal acidosis (equivalent to that breathing 7 percent inspired CO2, delta brain pH approximately -.05) in the RTN will increase breathing in awake, but not asleep states, while acidosis in the MRN will increase breathing in asleep, but not awake states, 2) at RTN sites where focal acidosis increases breathing, neuronal dysfunction will attenuate whole body CO2 sensitivity, but not alter rest and exercise breathing, 3) neuronal dysfunction in the MRN will attenuate CO2 sensitivity and rest and exercise breathing, 4) during the first 10 days after CBD, the effect of RTN and MRN focal acidosis will be attenuated but 15 plus days after CBD, the effect of focal acidosis will be accentuated. and 5) at most RTN and MRN sites, the acute effects of neurotoxic lesions will be hypoventilation (rest and exercise) and attenuated CO2 sensitivity; the acute effects of these lesions will be greater in CBD than in intact goats, but recovery after lesioning will be greater in intact than in CBD goats. Our unique studies are important because hypotheses generated largely from reduced preparations will be tested in awake and asleep states to enhance the understanding of medullary chemoreceptor contribution to the control of breathing and

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how abnormalities in this contribution may underlie diseases such as OSA, CCHS, and the Sudden Infant Death Syndrome. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CONTROL OF RENAL GLUTAMINASE INDUCTION DURING ACIDOSIS Principal Investigator & Institution: Curthoys, Norman P.; Professor and Chairman; Biochem and Molecular Biology; Colorado State University-Fort Collins Fort Collins, Co 80523 Timing: Fiscal Year 2002; Project Start 01-OCT-1989; Project End 31-MAR-2006 Summary: During chronic metabolic acidosis, increased renal ammoniagenesis and gluconeogenesis from glutamine are sustained, in part, by a cell specific increase in expression of the mitochondrial glutaminase (GA) that results from the selective stabilization of the GA mRNA. Previous experiments identified an 8-base AU- sequence within the 3'- non-translated region of the GA mRNA that functions as a pH-response element (pHRE) when introduced into a chimeric reporter mRNA. This sequence was used as an affinity ligand to purify and identify zeta-crystallin/NADPH; quinone reductase as the pHRE-binding protein. The functional characterization of the pHRE was carried out in LLC-PK1-FBPase+ cells, a porcine proximal tubule-like cell line that expresses multiple forms of GA mRNAs. The corresponding porcine GA cDNAs were cloned and sequenced. Only the 4.5-kb GA mRNA contains pHREs that are identical to the sequence identified in the rat GA mRNA and only this form of GA mRNA is stabilized and increased by incubating the cells in acidic medium (Ph=6.9, 10 mM HCO3). Thus this cell lines provides a system to further characterized the molecular mechanism of GA mRNA stabilization and the associated signal transduction pathway. Finally, the 4.50kb GA mRNA is the ortholog of a newly identified isoform of the human kidney-type GA that is generated by alternative splicing of exons within the GA gene. This isoform contains a unique C-terminal domain of unknown function. The specific aims of the proposed research are: to express and characterize the isoforms of the kidney-type GA; to characterize the mechanism of GA mRNA turnover; to characterize the role of zeta-crystallin/NADPH; quinone reductase in the stabilization of the GA mRNA; and to identify the signal transduction pathway that leads to enhanced binding of zeta-crystallin/NADPH; quinone reductase to the pHRE. The results of the proposed experiments should significantly increase the understanding of the molecular mechanism that regulates this essential adaptive response and may provide insight to improve the clinical treatment of chronic acidosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: CORE--LABORATORY OF INTEGRATED KIDNEY FUNCTION Principal Investigator & Institution: Wang, Tong; Research Scientist; Yale University 47 College Street, Suite 203 New Haven, Ct 065208047 Timing: Fiscal Year 2003; Project Start 01-DEC-2003; Project End 30-NOV-2008 Summary: Recently, the use of genetically altered animal models became an important tool to examine the contributions of individual channels, transporters and proteins to the maintenance of physiological function and pathophysiology of disease. We are now able to perform renal clearances and in vivo and in vitro tubular microperfusion studies in mice. These techniques will be used to gain insight into the physiological role of specific transporter proteins to tubule and overall renal function. To facilitate the goal of the Program Project to extend our fundamental knowledge about cellular and molecular

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mechanisms regulating renal tubular function in normal and diseased states, we propose to continue the Small Animal Physiology Core, with the following specific aims: (1) Provide a variety of rat and mouse animal models, such as adrenalectomy and hormone replacement, metabolic or respiratory acidosis and chronic diuretic treatment (furosemide, thiazide). (2) Provide a metabolic measurement of plasma and urine electrolytes and examine the dietary modulations in different animal models, including knockout and mutant mice provided by each PI. (3) Perform renal clearance experiments in rats and mice to examine renal phenotypes in mutant animals and to assess the physiological roles of ion channels, proteins and transporters, such as ENaC, ROMK, CFTR and NKCC2. (4) Measure arterial blood pH, pCO2 and HCO3- and urine pH and HCO3- to evaluate the acid-base status in these animals. (5) Perform in situ microperfusion of the proximal tubule and loop of Henle of rat and mouse to assess segmental tubular function and measure electrolyte content of nanoliter sized samples (Na+, K+, Cl-, total CO2, Ca2+ and Mg2+) of tubular fluid collected from these different sites. (6) Perform in vitro microperfusion of isolated proximal tubule and collecting tubule to examine the electrolyte transport in these segments. (7) Measure tubular absorption of proteins such as albumin to investigate endocytosis-related tubular functions. This core will be utilized by most of the PPG projects. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CSF AND THE CENTRAL CHEMICAL CONTROL OF BREATHING Principal Investigator & Institution: Nattie, Eugene E.; Professor of Physiology; Physiology; Dartmouth College 11 Rope Ferry Rd. #6210 Hanover, Nh 03755 Timing: Fiscal Year 2002; Project Start 01-DEC-1981; Project End 31-MAY-2004 Summary: Appropriate breathing requires 1) feedback concerning the level of CO2 from central chemoreceptors, and 2) a tonic 'drive', partly from CO2, and partly from other sources including the rostral ventrolateral medulla (RVLM). Recent work established that 1) central chemoreception is present at many brainstem locations, and 2) the retrotrapezoid nucleus (RTN) is a key RVLM site that provides both chemoreception and a tonic drive to breathe. We ask: Why are there so many central chemoreceptor sites? How do they work? What is the physiological role of the RTN in the control of breathing? We will evaluate chemoreceptor and RTN function during sleep and wakefulness in a chronic unanesthetized rat model using a microdialysis probe to deliver substances to the RTN (or other site). The probe tip is 1 mm in length and 240 mum in diameter, a volume of 45 nl. It allows repeated application of neuroactive substances at the same site in the same animal with continuous measurement of ventilation and oxygen consumption (whole body plethysmograph), arousal state (EEG, nuchal EMG), body temperature and blood pressure (telemetry), and, in some cases, blood gases and pH. Approximately 2/3 of the experiments use this model; 1/3 an anesthetized, ventilated rat with phrenic activity as the measure of respiratory output. For studies of chemoreception physiology, we produce focal tissue acidosis by CO2 microdialysis in both models. For studies of mechanism, we alter neural function by injection/dialysis within the focal region of acidosis in the anesthetized rat. For studies of the RTN, we inhibit neurons reversibly by dialysis with muscimol, a GABA-A receptor agonist, in the chronic model. Central chemoreceptor physiology is significant; CO2 is a key component of the respiratory control system and CO2 retention in disease causes morbidity. Chemoreception and RTN function vary with arousal state, and thus are likely to be important in sleep disordered breathing, and the RTN is hypothesized to be an animal homologue for the arcuate nucleus, described as abnormal in SIDS victims. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: EFFECTS OF ACID ON BONE Principal Investigator & Institution: Bushinsky, David A.; Professor of Medicine and Physiology; Medicine; University of Rochester Orpa - Rc Box 270140 Rochester, Ny 14627 Timing: Fiscal Year 2002; Project Start 01-APR-2000; Project End 31-MAR-2005 Summary: Metabolic acidosis, present during many clinical disorders, has significant effects on bone. The proposed studies are designed to test hypotheses relating to the effects of increases in proton concentration ([H+]), acidosis, on the ionic composition of bone mineral and on bone cell function. We propose to study how acidosis affects mass spectroscopy and to study how acidosis alters signal transduction pathways and bone cell expression. With the microprobe we obtain sensitive, mass resolved images and spectra of the ionic composition of bone mineral. We propose three Specific Aims: 1) To test the hypothesis that there are specific changes in bone ion composition with respect to time and location after exposure to acidosis. To pursue this aim we will test the hypothesis that in vivo acidosis initially causes a preferential loss of mid-cortical bicarbonate, Na and K due to physicochemical dissolution, with subsequent additional loss of phosphate due to cell-mediated resorption. We will test the hypothesis that the magnitude of H+ buffering by bone increases with age as the carbonate content increase and the phosphate content decreases and that there are labile pools of Ca and K preferentially released during in vivo acidosis. We will test the hypothesis that mice deficient in osteopontin (OP), matrix gla protein (MGP) or c-src have altered basal bone ion composition and an altered osseous response (Jca and JH) to acidosis. 2) To test the hypothesis that the mechanism of acid-induced cell-mediated bone resorption involves activation of signal transduction pathways such as protein kinase A (PKA), protein kinase C (PKC) and/or mitogen-activated protein kinase (MAPK) by using specific inhibitors of PKA and PKC and measurements of PKA, PKC and MAPK during acidosis. 3) To test the hypothesis that metabolic acidosis selectively inhibits osteoblastic extracellular matrix gene expression via distinct transcriptional mechanisms, by testing acidosis effects on OP and MGP RNA transcription initiation. We will identify sequences in the mouse OP and MGP genes which are capable of conferring pH dependence to a reporter gene. These Specific Aims are interrelated as the effects of acidosis at the level of the gene lead to alterations in bone cell function which subsequently alter bone mineral. Our long term goal is to develop a model to describe how acidosis affects bone in order to devise therapy to preserve mineral while maintaining the H+ buffering properties of bone. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: EFFECTS OF ACIDOSIS ON EXERCISE INDUCED MUSCLE PROTEIN SYNTHESIS Principal Investigator & Institution: Johansen, Kirsten L.; Assistant Professor; University of California San Francisco 500 Parnassus Ave San Francisco, Ca 941222747 Timing: Fiscal Year 2002; Project Start 01-DEC-2001; Project End 30-NOV-2002 Summary: This abstract is not available. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: EFFECTS OF BIER BLOCK W/ BRETYLIUM ON RHYTHMIC FOREARM EXERCISE Principal Investigator & Institution: Sinoway, Lawrence I.; Professor of Medicine; Pennsylvania State Univ Hershey Med Ctr 500 University Drive Hershey, Pa 170332390

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Acidosis

Timing: Fiscal Year 2002; Project Start 01-DEC-2001; Project End 30-NOV-2002 Summary: The goals of this study are to determine if the withdrawal of sympathetic tone leads to greater blood flow to exercising muscle. We will also determine if this greater flow leads to less muscle acidosis and less sympathetic discharge. We will also determine if in subjects with heart failure, the effects of sympathetic withdrawal are greater than they are in age matched controls. We hypothesize that in heart failure, sympathetic blockade will increase limb flow, reduce muscle acidosis, and attenuate the muscle reflex response to exercise. During the last GCRC period, we have perfected the bretylium bier block procedure. We have studied the effect of this block on limb blood flow during forearm exercise sympathetic discharge, and muscle metabolism. Sympathetic blockade produced a two-fold increase in forearm blood flow and vascular conductance. (P