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

CHLORINE 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 2003 by ICON Group International, Inc. Copyright 2003 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., 1960Chlorine: 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: 0-597-83865-8 1. Chlorine-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 chlorine. 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 CHLORINE ................................................................................................. 3 Overview........................................................................................................................................ 3 The Combined Health Information Database................................................................................. 3 Federally Funded Research on Chlorine......................................................................................... 4 E-Journals: PubMed Central ....................................................................................................... 37 The National Library of Medicine: PubMed ................................................................................ 39 CHAPTER 2. NUTRITION AND CHLORINE ....................................................................................... 75 Overview...................................................................................................................................... 75 Finding Nutrition Studies on Chlorine ....................................................................................... 75 Federal Resources on Nutrition ................................................................................................... 77 Additional Web Resources ........................................................................................................... 78 CHAPTER 3. ALTERNATIVE MEDICINE AND CHLORINE ................................................................. 79 Overview...................................................................................................................................... 79 National Center for Complementary and Alternative Medicine.................................................. 79 Additional Web Resources ........................................................................................................... 82 General References ....................................................................................................................... 83 CHAPTER 4. DISSERTATIONS ON CHLORINE ................................................................................... 85 Overview...................................................................................................................................... 85 Dissertations on Chlorine ............................................................................................................ 85 Keeping Current .......................................................................................................................... 88 CHAPTER 5. CLINICAL TRIALS AND CHLORINE ............................................................................. 89 Overview...................................................................................................................................... 89 Recent Trials on Chlorine ............................................................................................................ 89 Keeping Current on Clinical Trials ............................................................................................. 89 CHAPTER 6. PATENTS ON CHLORINE ............................................................................................. 91 Overview...................................................................................................................................... 91 Patents on Chlorine...................................................................................................................... 91 Patent Applications on Chlorine................................................................................................ 116 Keeping Current ........................................................................................................................ 152 CHAPTER 7. BOOKS ON CHLORINE ............................................................................................... 155 Overview.................................................................................................................................... 155 Book Summaries: Online Booksellers......................................................................................... 155 The National Library of Medicine Book Index ........................................................................... 160 Chapters on Chlorine ................................................................................................................. 161 CHAPTER 8. MULTIMEDIA ON CHLORINE .................................................................................... 163 Overview.................................................................................................................................... 163 Bibliography: Multimedia on Chlorine ...................................................................................... 163 CHAPTER 9. PERIODICALS AND NEWS ON CHLORINE ................................................................. 165 Overview.................................................................................................................................... 165 News Services and Press Releases.............................................................................................. 165 Newsletter Articles .................................................................................................................... 167 Academic Periodicals covering Chlorine.................................................................................... 167 APPENDIX A. PHYSICIAN RESOURCES .......................................................................................... 171 Overview.................................................................................................................................... 171 NIH Guidelines.......................................................................................................................... 171 NIH Databases........................................................................................................................... 173 Other Commercial Databases..................................................................................................... 175 APPENDIX B. PATIENT RESOURCES ............................................................................................... 177 Overview.................................................................................................................................... 177 Patient Guideline Sources.......................................................................................................... 177

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Finding Associations.................................................................................................................. 180 APPENDIX C. FINDING MEDICAL LIBRARIES ................................................................................ 183 Overview.................................................................................................................................... 183 Preparation................................................................................................................................. 183 Finding a Local Medical Library................................................................................................ 183 Medical Libraries in the U.S. and Canada ................................................................................. 183 ONLINE GLOSSARIES................................................................................................................ 189 Online Dictionary Directories ................................................................................................... 191 CHLORINE DICTIONARY ......................................................................................................... 193 INDEX .............................................................................................................................................. 263

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

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

Water and Electrolyte Absorption and Secretion in the Small Intestine Source: Current Opinion in Gastroenterology. 7(2): 215-219. April 1991. Summary: This article provides a brief overview of recent studies dealing with small intestinal electrolyte and water transport. Topics covered include sodium, chlorine, and bicarbonate transport; the role of inflammatory mediators; the Nippostrongylosis model of malabsorption; calcium, magnesium, and phosphate transport; iron transport; the mechanisms of mineral transport and the influence of diet and membrane composition on mineral transport; and the role of intestinal transferrin receptors in iron absorption. 16 annotated references. (AA-M).

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Identifying and Managing Common Nail Problems: Longitudinal Ridging, Onychoschizia, Onycholysis, Green Nail Syndrome Source: Women's Health in Primary Care. 1(1):34-36. February 1998. Summary: This journal article for health professionals discusses the identification and management of common nail problems, including longitudinal ridging, onychoschizia, onycholysis, and green nail syndrome. Longitudinal ridging is very common in the elderly. Onychoschizia, which is split and brittle nails, is common in adults and may be treated with methods similar to those for dry skin. Onycholysis may have both external and internal causes. External causes include irritants, trauma, fungal infections, and drugs that act as phototoxic agents, and internal causes include psoriasis, inflammatory skin diseases, neoplasms, and subungual warts. It may be prevented and treated by keeping nails dry and short, using nail polish sparingly, and avoiding unnecessary manipulation of nails. Green nail syndrome is a consequence of onycholysis and involves a painless discoloration under the nail. It may be treated by soaking the affected nail twice daily in a mixture of either one part chlorine bleach and four parts water or of equal parts acetic acid and water. 4 photographs.

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

Project Title: A CCD CRYSTALLOGRAPHIC X-RAY DETECTOR WITH LENS OPTICS Principal Investigator & Institution: Westbrook, Edwin M.; Director; Molecular Biology Consortium 835 S Wolcott (M/C 790) Chicago, Il 60612 Timing: Fiscal Year 2002; Project Start 15-MAR-2002; Project End 28-FEB-2005 Summary: We propose to study lens coupling between the phosphor and the CCD sensor in X-ray detectors for macromolecular crystallography. In this project we will develop three technologies: large aperture lenses; high gain phosphors; and backilluminated, large-format CCDs. We have developed a prototype big lens, and we are now characterizing it. We have developed a new phosphor (ZnSe:Cu,Ce,CI) with

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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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significantly higher gain and less afterglow than conventional Gd2O2S:Tb. We propose to study and improve on this technology, specifically to find a phosphor that is even faster and brighter, and does not contain selenium. We have begun a program to thin a 61mm CCD for back illumination, thus increasing light conversion efficiency in these large CCDs. We propose to develop ever larger CCDs with faster readout and lower noise, that are back-illuminated. Lens-coupling optics eliminates zingers, dead spaces, and the inherent defects of fiberoptic coupling optics (chickenwire, shear distortion, nonuniform optical transmission). Lens coupling reduces spatial distortions and positional nonuniformities relative to fiberoptic coupling. The point response should be better. With improvements in CCD efficiency and phosphor gain that we expect to realize, and the optical transfer efficiency that these very large lens systems can achieve, the overall system gain (electrons stored in the CCD/incident X ray) should be comparable to or superior to fiberoptic coupling. The detective quantum efficiency (DQE) and dynamic range should also be equivalent or superior to modular mosaic CCD detectors. In production, this technology should be substantially cheaper than mosaic CCDs. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: AIRWAY RESPONSES FOLLOWING CHLORINE GAS EXPOSURE Principal Investigator & Institution: Solomon, Colin; Medicine; University of California San Francisco 500 Parnassus Ave San Francisco, Ca 94122 Timing: Fiscal Year 2001; Project Start 04-SEP-2001; Project End 31-JUL-2004 Summary: Chlorine gas exposure occurs in occupational environments. Chlorine gas inhalation results in dose-dependent for acute and chronic pulmonary responses to chlorine exposure are unknown decrements in pulmonary function and respiratory tract irritation. The risk factors and controlling mechanisms. The specific aims of this study are to determine: (1) the effects of asthma with pre-existing airway hyper reactivity on the pulmonary function and airway reactivity and inflammation responses to chlorine; (2) the effects of chlorine concentration on the pulmonary function and airway reactivity and inflammation responses to chlorine concentration on the pulmonary function and airway reactivity and inflammation responses to chlorine; and (3) to assess the time dynamics of the pulmonary function and airway reactivity and inflammation responses to chlorine. It is hypothesized that both airway hyperactivity and higher chlorine concentration will result in larger changes in pulmonary function and airway reactivity and inflammation responses, and that these changes will have differential time courses following chlorine exposure. This study will use two controlled human exposure human exposure experiments, utilizing a single-blind, repeatedmeasures, and counter- balanced design. The two subject groups for both experiments will consist of 21 individuals with no airway hyperactivity, and 21 individuals with both asthma and airway hyperactivity. In Experiment One, subjects will be exposed for 15 min separately to each of: (1) chlorine at 0.4 ppm; (2) chlorine at 1.0 ppm; and (3) filtered air (Control). Pulmonary function and airway reactivity will be measured immediately pre-exposure and 1 and 20 h post-exposure. Airway inflammation, as determined by cellular and biochemical components from sputum-induction, will be measured 65h pre-exposure and 20 h post-exposure. In Experiments Two, subjects will be exposed for 15 min separately to each of: (1) chlorine (concentration determined from Experiment One); (2) filtered air (Control). Pulmonary function and airway reactivity will be measured immediately pre-exposure and at 3 and 72 h post-exposure. Sputuminduction will be performed 65h pre-exposure at 3 and 72 h post-exposure. The results of this study will provide information on a major irritant chemical relevant to

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occupational environments. Specially, the susceptibility of a large sub-population at increased risk, the dose-response effects, and the post-exposure time dynamics of the effects of chlorine gas will be determined. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ALTERED GENE EXPRESSION BY MGCL2 IN TARGET ORGANS Principal Investigator & Institution: Mansour, Mahmoud M.; Tuskegee University Tuskegee Institute, Al 36088 Timing: Fiscal Year 2002; Project Start 01-JUN-1978; Project End 30-JUN-2006 Summary: (provided by applicant): Mercury is a significant contaminant of the environment and poses a serious systemic toxicity risk to humans, animals, and the aquatic food web. Published data indicate that inorganic mercury accumulates and induces strong renal toxicity and adversely affects spermatogenesis and fertility in male rats. Such effects underscore the threat to human health because mercury is ubiquitously present in the environment and is widely used in the industry. Hence, the long-term goal of this project is to understand how environmental exposure to mercury induces toxicity. The specific aim of this application is to determine the effects of mercuric chloride on differential gene expression in the kidney and two male reproductive organs. We hypothesize that the toxicity of mercuric chloride largely results from the loss of control of differential gene expression. This hypothesis is formulated based on published reports which indicated that mercuric chloride induced apoptosis, and transcriptional activation and/or suppression of several genes in the kidney, and based on our initial studies, that mercuric chloride down-regulated estrogen receptor alpha mRNA expression in the efferent ductules, and induced apoptosis in the testis of adult male rats. To test this hypothesis, we will test the effects of oral administration of mercuric chloride on differential expression of genes (repressed versus induced) using differential display PCR and relevant toxicity endpoints. Using micro-array, we will determine the effects of mercuric chloride on unique genes related to phase I and II metabolism, apoptosis, inflammation, DNA damage, cell transport, and oxidative stress in the kidney, efferent ductules, and the testis. Semi-quantitative PCR will be used to determine the effect on steroid hormone receptors (estrogen and androgen) in the efferent ductules and testis. The proposed experiments will identify pathways by which mercuric chloride causes toxicity, and identify molecular biomarkers for exposure to inorganic mercury. Such approach will be valuable in the identification of new drug targets for treatment of mercuric chloride. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: ANALYSTS OF DBP DNA ADDUCTS Principal Investigator & Institution: Giese, Roger W.; Professor; Pharmaceutics; Northeastern University 360 Huntington Ave Boston, Ma 02115 Timing: Fiscal Year 2001; Project Start 01-MAR-2000; Project End 28-FEB-2002 Summary: Comprehensive detection and some structural elucidation of disinfectant byproduct (DBP) DNA adducts formed in vivo will be undertaken in this project. The analysis will rely on a combination of fluorescence post- labeling, capillary electrophoresis-laser induced fluorescence detection (CE-LIF), HPLC and mas spectroscopy (MS) techniques. CE-LIP detects nucleotide standards at the low amol level (same ultra-sensitivity as 32P- post-labeling). Tissue samples (furnished by NTP) from animals exposed to four DBP chemical (bromodichloromethane, sodium chlorate, 3- chloro-4-(dichloromethyl)-5-hydroxyl-2 (5H)-furanone and dibromoacetic acid) will

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be tested. Because the dye for adduct labeling has significant mass, the resulting dyeDNA adduct conjugates can be detected with high sensitivity (low fmol level) by matrixassisted laser desorption ionization (MALDI)-MS. Both time-of-flight and Fourier transform MS instruments will be used, with the latter providing accurate mass and (MS)n measurements to enhance the partial structural elucidation of unknown DNA adducts to be discovered in this project. Comprehensive, definitive detection of DNA adducts in these exposed animals as proposed, with simultaneous monitoring of carcinogenicity, is a good, first step towards understanding the corresponding risk to humans from exposure to these chemicals. The ultra-sensitivity of CE-LIF makes it likely that the methodology will be useful, as needed, for the analysis of human samples in the future. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ANESTHETICS, GABA AND THE INJURED BRAIN Principal Investigator & Institution: Warner, David S.; Professor; Anesthesiology; Duke University Durham, Nc 27706 Timing: Fiscal Year 2003; Project Start 01-FEB-2003; Project End 31-JAN-2007 Summary: (provided by applicant): Perioperative stroke remains a major risk during surgery. Volatile anesthetics protect against experimental brain ischemia but the mechanism is not defined. We hypothesize that volatile anesthetics potentiate GABAergic neurotransmission and enhance CI- influx. This hyperpolarizes neurons delaying time to ischemic depolarization and Ca2++ influx. This hypothesis is derived from observations that volatile anesthetics potentiate GABAA receptors and bicuculline, a GABAA antagonist, reverses isoflurane protection in vitro. We also hypothesize that volatile anesthetic GABAergic properties are more important to protection than glutamate receptor antagonistic properties. We propose these Specific Aims: 1) Define a dose-response for isoflurane protection during rat forebrain ischemia/Compare with efficacy of muscimol, a GABAA receptor agonist/Compare with time to onset of ischemic depolarization and pre-ischemic cerebral metabolic rate; 2) Determine if isoflurane neuroprotection against severe forebrain ischemia is permanent; 3) Compare the relative neuroprotective effect of selective NMDA/AMPA receptor antagonism to isoflurane, which also possesses GABAergic potentiation; 4) Determine the role of GABAA potentiation in isoflurane protection against severe forebrain ischemia. For Specific Aim #4, we will examine: a) if isoflurane protection against forebrain ischemia or striatal NMDA microinjections is reversed by GABAA antagonists (flurothyl, bicuculline, flumazenil), b) if isoflurane delays time to ischemia induced Ca2++ influx and if this is reversed by GABAA antagonists, c) if correction for this delay, by extending ischemia duration, equivalently reverses neuroprotection, d) effects of GABAA beta subunit-targeted deletion (knockout) or striatal antisense oligonucleotide microinjection on in vivo isoflurane protection, e) the extent to which isoflurane provides protection in organotypic hippocampal slices against NMDA excitotoxicity or oxygen/glucose deprivation and respective effects on CI- uptake, f) the extent to which this is reversed by antagonists of the GABAA, GABAB, and strychnine sensitive glycine receptors, and g) relationships between isoflurane and GABAA antagonists on CI- and Ca ++uptake in NMDA stimulated synaptoneurosomes. We believe that GABAAergic pharmacologic properties of volatile anesthetics known to be critical for anesthesia are the same properties that confer cerebral protection. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: BIOLOGICAL BACTERIA/CATALYST/ENVIRONMENT

DECHLORINATION--

Principal Investigator & Institution: Smith, Geoffrey B.; New Mexico State University Las Cruces Las Cruces, Nm 880038001 Timing: Fiscal Year 2001 Summary: The subject of this proposal is the biological removal of chlorine groups from halogenated compounds that are of public health concern. Through complete dechlorination of chlorinated aliphatic compounds, the toxicity of these compounds is eliminated. The work proposed here involves investigation of a dechlorination reaction identified in anaerobic bacteria from an aquifer contaminated with halogenated compounds. The isolation and characterization of the apparently novel dechlorinating bacterium is proposed, as well as the purification of the catalyst that is responsible for chlorine removal from dechlorinating bacterium is proposed for chlorine removal from trichlorofluoromethane (CFC-11). The hypothesized dechlorinating bacterium is present in a co-culture of two other aquifer bacteria and has resisted traditional isolation attempts because of its inability to grow on agar. We propose to isolate this bacterium by using a robotics microscope in which bacterial cells can be physically isolated through the use of laser tweezers. With the bacterium in pure culture, we propose to purify the dechlorination catalyst using medium pressure liquid chromatography. The hypothesis that the dechlorination catalyst is one of the enzymes of a novel sulfate reduction pathway will guide the purification steps. The goal of this part of the work is to identify and characterize a novel bacterium with a unique ability to simultaneously use two electron acceptors, one physiological, the other a synthetic contaminant (CFC11). It is also proposed to do dechlorination work at the microbial community level using samples from an aquifer and a wastewater digester. We have documented dechlorination activity in both these samples to propose to investigate the role which the dechlorination reaction plays within the microbial community present in these anaerobic samples. Short-term batch incubations and long-term column flow-through systems will be used to test the effect of pollutant exposure. Metabolic activities will be assayed by liquid and gas analyses for pollutant and metabolites, and pollutant effects on specific populations will be assayed using a combination of Most-Probable-Number techniques with molecular analyses. One molecular analysis will use 16S rRNA probes specific for methanogenic and sulfate-reducer populations. Another method will target these groups as well as other, less defined populations through the use of PCRamplification of bacterial and archaeal conserved 16S targets, followed by denaturing gradient gel electrophoresis (DDGE) analysis of amplified populations. To resolve some of the microbial diversity present in these types of types of samples, extracted DNA will be fractionated using bis-benzamide gradients before PCR amplification. The goal of this environmental level work is to understand the role which dechlorination plays within the environment that is exposed to halogenated pollutants. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: BIOLOGICAL VANADIUM--MODELS OF STRUCTURE/FUNCTION Principal Investigator & Institution: Pecoraro, Vincent L.; Professor; Chemistry; University of Michigan at Ann Arbor 3003 South State, Room 1040 Ann Arbor, Mi 481091274 Timing: Fiscal Year 2001; Project Start 01-JUN-1990; Project End 30-JUN-2003 Summary: (adapted from applicant's abstract) Vanadium was once considered an oddity in biology with ill defined structures and even more obscure functions. In the past two

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decades, scientists have realized that this element is found in marine enzymes that may be responsible for many useful natural products and ultimately may serve as a new therapeutic regiment for oral treatment of diabetes. During our previous three years of funding, we have focused on preparing functional models for the vanadium haloperoxidases. Early last year, we presented the most efficient haloperoxidase reactivity model compounds yet reported. Unlike previous models, our compounds are functional as mononuclear, monoperoxo species, exactly as proposed for VHPOs. About six months ago, we made a major breakthrough in that we can oxidize chloride at a measurable rate. We estimate that our reaction is at least 10 (and more likely 100 fold) faster than any previous vanadium based chloride oxidation catalyst. We also now have data that may address the functional role of amavadin which we have suggested may be a rudimentary bromo- or iodoperoxidase. The studies proposed for the next funding period build on these major advances. 1) Probe the mechanism of vanadium haloperoxidases by establishing the factors that lead to activation of our bromide and chloride oxidation catalysts. As necessary, we will develop and characterize new ligand sets that serve as more efficient agents in these reactions. 2) Expand our studies of the reaction of V(IV) species with hydrogen peroxide since these may be important interaction that define the reactivity of amavadin and the cellular metabolism of vanadium insulin mimics. 3) Through collaboration with several spectroscopists, develop new methodologies that will allow scientists to define to higher chemical resolution V=O binding sites in proteins. Since V=O substitutes for many divalent ions, this approach can be broadly applied to a variety of metalloproteins. We propose that high molecular weight acid phosphatases can convert to VHPOs in the presence of vanadate. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CADMIUM, COBALT, NICKEL TRANSDUCTION IN SHARK RECTAL GLAND

EFFECT

ON

SIGNAL

Principal Investigator & Institution: Forrest, John N.; Mount Desert Island Biological Lab Old Bar Harbor Road Salisbury Cove, Me 04672 Timing: Fiscal Year 2001 Summary: This application seeks support through the Center for Membrane Toxicology Studies (CMTS) at the Mount Desert Island Biological Laboratory for studies on the effects of three heavy metals (cadmium, cobalt and nickel) on signal transduction pathways of hormones regulating chloride secretion in the shark rectal gland. The shark rectal gland is a homogenous, single cell type, highly specialized epithelium that is a model system for hormone regulated chloride secretion. Our hypothesis is that the toxic effects of heavy metals results from interactions at different specific sites of stimulatory as opposed to inhibitory hormonal signal transduction pathways. We have determined that cadmium reversibly blocks receptor-mediated inhibition of chloride secretion and that a major component of this effect occurs by a novel and unexpected mechanism- and augmentation of the response to stimulatory hormones. We will determine the specific site(s) of this metal-protein interaction by distinguishing between effects of cadmium on (a) an extracellular receptor for Cd mediating activation of inositol triphosphates and release of intracellular calcium; (b) direct effects on specific isozyme of cyclic nucleotide phosphodiesterases; and (c) a post-receptor/kinase mechanism- translocation of DFTRchloride channels from an intracellular site to the apical plasma membrane. In contrast to Cd, cobalt and nickel inhibit VIP and forskolin stimulated chloride secretion in the perfused rectal gland. In collaboration with others in the center we will determine the protein interactive site(s) of these metals and distinguish between toxic effects on the

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calcium messenger system and direct actions on apical DFTR channels. Studies will be carried out in the in vitro perfused rectal gland, in primary culture monolayers of rectal gland cells, and in Xenopus oocytes expressing the DFTR chloride channel. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CHLORIDE TRANSPORT IN HUMAN CILIARY EPITHELIUM Principal Investigator & Institution: Crook, Richard B.; Ophthalmology; University of California San Francisco 500 Parnassus Ave San Francisco, Ca 94122 Timing: Fiscal Year 2001; Project Start 01-DEC-1993; Project End 30-NOV-2002 Summary: Glaucoma is a blinding eye disease. A major treatment strategy for this disease is the reduction of aqueous humor secretion with B-adrenergic antagonists such as timolol and other drugs. Although such treatments are effective, their molecular mechanisms are largely unknown. An understanding of ion transport mechanisms which underlie aqueous humor secretion and its regulation might lead to improved glaucoma treatment protocols. Considerable evidence suggests that timolol may function as a hypotensive agent by inhibiting diurnal adrenergic elevation of aqueous humor inflow. The PI has reported that Na+, K+, Cl cotransport in ciliary epithelium is stimulated by B adrenergic agents and inhibited by timolol. This is a promising lead in understanding diurnal adrenergic regulation of aqueous humor inflow in humans. The PI proposes to investigate the molecular mechanisms underlying stimulation of Na+, K+, Cl cotransport by adrenergic agonists, including the roles of second messenger pathways and phosphorylation dephosphorylation pathways. He also proposes to test the hypothesis that other hormones known to either increase or decrease aqueous inflow might exert their effects through modulation of Na+, K+, Cl cotransport activity. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: CLC-2 IN MEDIATING EPITHELIAL CHLORIDE SECRETION Principal Investigator & Institution: Bear, Christine E.; Assistant Professor; Hospital for Sick Chldrn (Toronto) 555 University Ave Toronto, Timing: Fiscal Year 2001 Summary: There is compelling evidence for variability in the severity of Cystic Fibrosis (CCF) disease amongst CF patients and differences in the nature of the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene mutation cannot account for all of this variation. Using a murine model of CF, our group has shown that secondary genes can ameliorate the severe intestinal phenotype, the major cause of mortality in this species. These modifier genes have not yet been identified. However, as our comparative studies of the intestinal mucosa of CF mice with mild disease and CF mice with severe disease show a correlation between disease amelioration and chloride ion secretion, we suggest that disease modification may occur through modulation of the chloride channel(s) on the apical membrane of intestinal epithelial cells is a fundamental to developing our understanding of how disease severity can be ameliorated in intestinal epithelial cells. So far, our findings suggest a correlation between ClC-2 expression and intestinal chloride secretion. Specifically, we found that chloride (Cl) secretion by intestinal mucosa obtained from CF mice with mild disease can be blocked by inhibitors of ClC-2 function and the ClC-2 protein is localized toward the apical pole of these epithelial cells. Hence, ClC-2 protein is appropriately localized to mediate chloride secretion. However, ClC-2 can only mediate secretion if it is activated in epithelial cells and so far, we have only a poor understanding of how ClC-2 channel activity is regulated. Our overall goals of this study are to understand how ClC-2 is

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regulated and to determine if this channel protein can mediate secretion in intestinal epithelial tissue. Specific Goals: 1) to study the regulation of ClC-2 channel function in vitro and in vivo. We have determined that purified, reconstituted ClC-2 protein can mediate Cl ion electrodiffusion. Our reconstitution system will permit detailed analysis of its permeation and gating properties. We also found that Clc-2 is endogenously localized at the apical surface of the Caco-2 cell line ane have developed tools to study its regulation in the model epithelial cell line. 2) to determine whether ClC-2 in Cl secretion in Caco-2 cells by modifying its expression through transfection. Furthermore, the role of ClC- 2 in secretion by native tissues will be investigated by generating transgenic mice over-expressing ClC-2. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: COMBUSTION PROCESSES--EMISSIONS, MONITORING, AND INTERVENTION Principal Investigator & Institution: Koshland, Catherine P.; University of California Berkeley Berkeley, Ca 94720 Timing: Fiscal Year 2001 Summary: Superfund sites have arisen in part because systems were developed for materials processing, energy production, manufacturing, and waste management without sufficient consideration of the health and environmental impacts of byproducts or waste from processes or product use and disposal. Combustion systems are used for the treatment and destruction of wastes, and dominate the production of energy and materials. In all these applications, they have the potential to produce air pollution and hazardous byproducts. Combustion processes are also the major source of ultrafine particles, especially those smaller than 1 micron. The long-range goals of this project are to improve high-temperature processes, particularly combustion processes, and to provide a means of on-line real time monitoring of air emissions. These efforts can contribute to the reduction of direct and indirect exposures to toxic combustion byproducts, especially to metals, chlorinated hydrocarbons (CHCs), and small particles. In the proposed research, we seek understanding of the processes and mechanisms that result in the evolution of particles and gas-phase seek understanding of the processes and mechanisms that result in the evolution of particles and gas-phase byproducts in high temperature environments. In addition, combustion may be regarded as a model system for applying environmental metrics during the design phase to improve performance and reduce environmental health impacts. One additional dimension of the project will be to extend the application of excimer laser fragmentation-fluorescence spectroscopy (ELFFS) to the measurement of toxic species in soils or solids; this work may provide additional resources to other program projects, which involve the fate and transport of toxic metals and CHCs in the ground. The specific aims for this project are: 1) Continue development of non- intrusive monitoring techniques (such as ELFFS and in situ Fourier transform infrared (FTIR) spectroscopy) for application to metals and other hazardous species produced in flames or in the post-flame environment. 2) Study the behavior of metals and metal species in flames and in the post-flame environment with the goal of identifying conditions that produce specific metal compounds and particles. 3) Expand our studies of the chemistry and interactions of chlorinate hydrocarbons to mixtures where chlorine, metals, and oxygenates are present. 4) Use the information generated in aims #2 and #3 combined with toxicity metrics to explore possible design and control strategies to reduce the amount and toxicity of species emitted to the environment when high temperature systems are utilized. 5) Extend our laser diagnostic methods to the detection of metals and metal compounds in solids and soils.

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

Project Title: CORE--MASS SPECTROMETRY Principal Investigator & Institution: Deinzer, Max L.; Oregon Health & Science University Portland, or 972393098 Timing: Fiscal Year 2001 Summary: The broad objective of this Mass Spectrometry Core is to provide high- end mass spectroscopy techniques to support the biomedical and non- biomedical projects in this Superfund Basic Research Program and to develop and apply a new, highly specific and sensitive mass spectroscopy technique to detect and quantify environmental compounds and their metabolites. These compounds will include tetrachloroethene, 1,1,2- trichloroethene, cis-1,2-dichloroethene, vinyl chloride, chloroacetaldehyde and chloroform. The development of this new instrument will require the construction of a gas chromatograph electron monochromater-time-of flight-mass spectrometer (GC-EMTOF-MS) to analyze negative ions using the EM for the production of monoenergetic beams of ionizing electrons in combination with pulsed extraction. To support the other biomedical and non-biomedical projects in this application, we will develop and apply mass spectrometric methods to analyze adducts formed between proteins and hydrocarbon solvent metabolites. We will develop mass spectrometric methods for analyzing protein and peptides resulting from reaction with 2,5-hexanedione, and analogs, 3-methyl-2,5-hexanedione, 3,4-dimethyl-2,5-hexanedione, 3-4- diethyl-2,5hexanedione, 2,9-dimethyl-4,7, decanedione and 1,2- diacetylbenzene. The effects of gamma-diketone hydrocarbon adducts on the thermal stability of neurofilament protein structures will be a assessed by hydrogen-deuterium exchange (H/D) exchange and equilibrium thermal denaturation studies in conjunction with electrospray mass spectroscopy. The effects of hydrocarbon adducts on the conformation of neurofilament protein subunits will be determined by kinetic H/D exchange experiments, together with electrospray mas spectrometry. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: DEVELOPMENT OF TOOLS FOR MONITORING IN SITU BIOREMEDIATION Principal Investigator & Institution: Alverez-Cohen, Lisa; University of California Berkeley Berkeley, Ca 94720 Timing: Fiscal Year 2001 Summary: The overall objective of this study is to develop non-culture based tools for evaluating the progress of in-situ bioremediation of chlorinated solvents in the field. Two specific approaches will be used to achieve these objective, the first based upon stable isotopic analyses and the second based upon application of molecular biological tools. The hypothesis that we propose to test in the first approach is as follows: stable isotope ratios of chlorine and carbon components of reactants, products and microbial cells involved in the biological degradation of chlorinated solvents can be analyzed to determine the dominant metabolic pathways and redox conditions of the biodegradation reaction. The specific goal is to broaden our understanding of the fractioning of chlorine and carbon stable isotopes during the metabolic, co-metabolic, and abiotic degradation of chlorinated solvents such as perchloroethylene (PCE), trichloroethylene (TCE), and carbon tetrachloride (CT) under aerobic and anaerobic conditions. Although a number of studies have been performed to measure the fractionation of stable carbon isotopes during the aerobic and methanogenic

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degradation of organics, only a few studies have been performed to evaluate carbon fractioning during the degradation of chlorinated solvents under anaerobic conditions only, and chlorine isotope fractionation has not been evaluated under any biodegradation scenario. Also, there is no published information on either carbon or chlorine isotope fractionation during abiotic degradation of chlorinated solvents. The hypothesis that we propose to test in the second approach is as follows: tools based upon molecular biology and direct microscopy and direct microscopy can be developed for application to subsurface environments in order to quantify microbial community activity and structure during in situ bioremediation. The specific goal is to adapt tools such as fluorescence in-situ hybridization (FISH) techniques for use in evaluating the relative contribution of different physiological groups of bacteria to the degradation of chlorinated solvents inc contaminated subsurface soils. FISH techniques using a series of rRNA- targeted oligonucleotides probes specific for chlorinated solvent- degrading microorganisms will be developed and applied. While several previous studies have used FISH techniques for characterizing subsurface soil community structure, they have not been used to evaluate microbial populations associated with degradations of chlorinated solvents in contaminated soils. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: DIELS ALDER REACTIONS IN ROOM TEMPERATURE MOLTEN SALTS Principal Investigator & Institution: Lee, Carlos W.; Tennessee State University 3500 Centennial Blvd Nashville, Tn 37203 Timing: Fiscal Year 2001 Summary: This abstract is not available. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: DISINFECTING DRINKING WATER VIA HYBRID OZONE SYSTEMS Principal Investigator & Institution: Bowser, John J.; Compact Membrane Systems, Inc. Wilmington, De 18904 Timing: Fiscal Year 2001; Project Start 30-SEP-2001; Project End 28-FEB-2003 Summary: The ozonation of drinking water is attractive for small communities that employ local wells or surface water sources provided it can be done at a reasonable cost. Although chlorine is widely used and is cheaper to use that ozone, its use results in the formation of carcinogenic trihalomethanes. Also chlorine has been found to be ineffective in the treatment of Cryptosporidium and viruses. Under an SBIR Phase I grant from the NIH, Compact Membrane Systems (CMS) demonstrated at lab-scale that CMS' membrane technology could be used to effectively ozonate water contaminated with organics, Cryptosporidium, humic acidd, bacteria, and viruses. Recently, CMS identified an ozone hybrid process that is 50-400 times more effective than ozone alone in destroying organic contaminants (no data yet on biologicals). In Phase I, CMS will build and evaluate this hybrid system for removal of Cryptosporidium, nitrobenzene, and humic acid. Long term (7+ days) testing and economic evaluations combined with hybrid system performance will provide understanding of system potential for enhancing cost effective production of quality non-carcinogenic (trihalomethane) drinking water. Focus will be initially on smaller facilities (serving 50, 000 people or less). PROPOSED COMMERCIAL APPLICATION: The proposed ozone hybrid system

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will provide clean drinking water free of organics, Cryptosporidium, humic acids, bacteria, and viruses in a cost effective way for small communities. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: EFFECT OF LOW DOSE PERCHLORATE ON THYROID FUNCTION Principal Investigator & Institution: Braverman, Lewis E.; Chief, Section of Endocrinology,; Brigham and Women's Hospital 75 Francis Street Boston, Ma 02115 Timing: Fiscal Year 2001 Summary: The purpose of this study is to determine the effect of low doses of oral potassium perchlorate on thyroid function in euthyroid male volunteers. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: EFFECTS OF CADMIUM AND COTRANSPORTER IN SHARK RECTAL GLAND

MERCURY

ON

NA-K-CL

Principal Investigator & Institution: Kinne, Rolf; Mount Desert Island Biological Lab Old Bar Harbor Road Salisbury Cove, Me 04672 Timing: Fiscal Year 2001 Summary: Cadmium and mercury are extremely nephrotoxic and the understanding of their mechanism of action is one essential step in preventing or reversing their detrimental effect on the kidney. This study is designed to provide insights into the action of heavy metals on critically involved in the urinary concentrating process. Using as a model isolated plasma membranes of the rectal gland of the shark, which are an abundant source for this cotransporter, their mechanism of action will be first characterized kinetically with regard tot he ion transport and bumetanide binding sites. Then the amino acid side chains involved in this interaction between heavy metals and the specific reagents with that of the toxins. Finally, the amino acids carrying these heavy metal- sensitive side chains will be localized at the molecular level. These studies will contribute substantially to the understanding of the action of heavy metals at the transporter protein level and thereby provide important information on potential target sites, putative receptors and possible binding motifs of heavy metals and their chemical nature. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ENTERIC PATHOGENS Principal Investigator & Institution: Guerrant, Richard L.; Professor; University of Maryland Balt Prof School Baltimore, Md 21201 Timing: Fiscal Year 2003; Project Start 01-AUG-2003; Project End 31-JUL-2008 Summary: Research Project V includes two Sub-Projects focused on key new aspects of the genomics, pathogenesis and vaccinology of the two leading Category B low-dose enteropathogens, and on their detection in clinical and environmental samples. These include the highly chlorine-resistant Cryptosporidium parvum, the most fearsome Shigella threat (S. dysenteriae 1) and the largely untreatable, Shiga toxin-producing enterohemorrhagic E. coli. These three pathogens all pose serious risks as low infectious dose agents of bioterrorism as well as major national and global health endemic and epidemic challenges. The team of investigators for this project has a very strong track record of working with these organisms. Led by experienced investigators with international reputations in enteric diseases, Richard L. Guerrant and James B. Kaper, this project builds upon highly productive expertise and upon longstanding and new

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cross-institutional synergies at UVa, UMd, VCU, VT, USUHS, UVt, and JHU. Our first Sub-Project V.1, on "Cryptosporidium genomics, pathogenesis and vaccinology" builds upon the near complete sequencing of the human (type 1) C. parvum genome by the VCU group, the published tissue culture, animal and field experience with Cryptosporidium by the UVa group, the plant-based production of mucosal vaccines at VT and on studies of the genetics of susceptibility at UVa, UVt, and JHU to identify and express type 1 (human) C. parvum candidate genes, define their roles in pathogenesis and immunity, express promising candidates and define genetic determinants of human susceptibility and thus optimal approaches to vaccine development. Sub-Project V.2 will engage UMd, USUHS and UVa colleagues to construct novel Shigella dysenteriae and enterohemorrhagic E. coli (EHEC) vaccines and develop novel therapeutics for EHEC disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: GABAERGIC INSECTICIDE TOXICOLOGY Principal Investigator & Institution: Casida, John E.; Professor of Entomology; Environmntl Sci Policy & Mgmt; University of California Berkeley Berkeley, Ca 94720 Timing: Fiscal Year 2001; Project Start 01-DEC-1998; Project End 30-NOV-2002 Summary: The long-term objective is to define the fundamental basis for the selective toxicity of insecticides acting at the gamma-aminobutyric acid (GABA) receptor of mammals and insects. This is the target of major neurotoxic insecticides acting as both blockers and activators of the GABA-gated chloride channel. More than 5,000,000,000 pounds of these channel blockers have been used for pest control in the past 50 years and they range in chlorine content from 52-73%. The major channel blockers used at present, representing 6% of the insecticide market, are endosulfan and lindane and this market share will increase with expanded use of the newly-commercialized polyhalogenated fipronil. The activators such as avermectin and moxidectin are also used in ever increasing quantities as insecticides and anthelmintics. More specifically, the goal is to provide toxicological profiles and maps for the insecticide blocker site and the insecticide activator site by designing and using high-affinity radioligands (the insecticide itself or a closely-related model compound) and photoaffinity probes to study binding site interactions and localization in the brain and chloride channel. Emphasis will be placed on the discovery of differences between the GABA receptors of mammals and insects that confer preferential sensitivity to insecticides and safety to mammals. The proposal is to prepare suitable radioligands and photoaffinity probes and use them to localize the binding sites as to brain region, receptor subunit and specific derivatized amino acid(s) in the chloride channel of mammals (bovine) and insects (Drosophila); the chemistry to achieve this end comes largely from discoveries in this laboratory. The research also involves rat cerebellar granule cells in primary culture to study radioligand binding and chloride flux in intact cells, localization of radioligand binding in mouse brain slices following in vitro and in vivo exposure to unlabeled toxicants, and receptors derived from Sf9 cells transfected with cDNs of human GABAa receptor subunits. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: INDIVIDUAL FACTORS IN NASAL IRRITANT SENSITIVITY Principal Investigator & Institution: Shusterman, Dennis J.; Associate Clinical Professor; Medicine; University of California San Francisco 500 Parnassus Ave San Francisco, Ca 94122

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Timing: Fiscal Year 2001; Project Start 01-SEP-2000; Project End 31-AUG-2003 Summary: (Adapted from the Investigator's Abstract): A variety of symptoms linked to indoor air pollution, including eye, nose, and throat irritation (as well as reflex nasal congestion, rhinorrhea, and sinus headache) are either mediated (or triggered) by trigeminal chemoreception. The premise that humans exhibit significant inter-individual variation in nasal trigeminal irritant sensitivity is one that has been suggested on both clinical and epidemiologic grounds, but experimentally has been incompletely investigated. The purpose of this series of experiments is to systematically explore the influence of personal factors -including age, gender, and allergic rhinitis status- on nasal irritant sensitivity, using stratified samples of non-asthmatic subjects aged 18-69 years. Operationally, "nasal irritant sensitivity" will include both perceptual acuity (the ability of an individual to detect an irritant gas or vapor) and physiologic reactivity (the tendency of individuals to experience reflex-mediated physiologic changes when exposed to irritants). For perceptual acuity, two distinct experimental systems will be employed: detection thresholds using odorless irritant (CO2), and localization thresholds for an odorous volatile organic compound (VOC). Nasal physiologic reactivity will be studies by examining changes in nasal airway resistance (NAR) after both chemical irritant (low-level chlorine) and pharmacologic (aerosolized histamine) provocation. Finally, biochemical markers of mast cell degranulation (tryphase) and neuro-immune modulation (nerve growth factor) will be assayed in nasal lavage fluid pre- and post chemical provocation in a subset of subjects. Issues of test-retest stability and cross agent generalizability of sensory tests will be examined, as will the degree of correlation between individual perceptual acuity and physiologic reactivity. The overall goals include: 1) to better understand heterogeneity of upper airway symptom reporting in polluted environments; 2) to evaluate the relationship between functional subcomponents of "nasal irritant sensitivity"; 3) to further standardize psychophysical and provocation testing protocols for possible use in clinical and/or epidemiologic settings; and 4) to explore the pathophysiology of the nasal response to irritants, including selected interactions between the sensory and immune systems. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: INTESTINAL ADENOSINE A2B RECEPTOR Principal Investigator & Institution: Sitaraman, Shanthi V.; Pathology and Lab Medicine; Emory University 1784 North Decatur Road Atlanta, Ga 30322 Timing: Fiscal Year 2001; Project Start 01-SEP-2000; Project End 31-AUG-2005 Summary: (adapted from the application) The overall aim of this project is to better define the characteristics of intestinal epithelial cell-neutrophil interaction as it relates to fluid and electrolyte secretion. Many intestinal disorders, particularly the acute flare of inflammatory bowel disease (IBD), are characterized by migration of neutrophils across the intestinal epithelium into the lumen to form 'crypt abscess'. Crypt abscesses are pathognomic of active IBD and infectious colitis and correlate with severity of disease as well as clinical symptoms. We have previously shown that neutrophil migration into the intestinal lumen elicits electrogenic chloride secretion (secretory diarrhea) and the major effector of this chloride secretory response is the neutrophil-derived secretagogue, 5'AMP. Intestinal epithelia express an ectonucleotidase, which converts 5' AMP to adenosine which then interacts with intestinal epithelial adenosine 2b (A2b) receptor to elicit chloride secretion. Thus the A2b receptor plays a central role in orchestrating chloride secretion induced by neutrophils. An understanding of the regulation and signaling mechanism of A2b receptor may therefore lead to designing of novel treatments for this component of IBD. In this proposal, I intend to characterize the

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biology of intestinal A2b receptor using two intestinal epithelial cell models: T84 cells and Caco-2 bbe cells transfected with the A2b receptor. A2b receptor is the only adenosine receptor in T84 cells while Caco-2 bbe cells lack A2b receptor. First, I will study the polarity of surface expression, distribution, kinetics of turnover, structural requirements for ligand binding, desensitization, and G-protein recognition. Second, I will analyze the existence of ectonucleotidase and A2b receptor in signaling membrane microdomain such as caveolae. These microdomains represent invagination of plasma membrane enriched in glycosphingolipid, which have been shown to contain signaling proteins. Third, I will study the role of adenosine receptor in the modulation and feedback regulation of neutrophil-epithelial interaction including transmigration and chemokine secretion. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: INTESTINAL SECRETION & INFLAMMATION - IMPACT OF AMMONIA Principal Investigator & Institution: Matthews, Jeffrey B.; Christian R. Holmes Professor and Chairm; Surgery; University of Cincinnati 2624 Clifton Ave Cincinnati, Oh 45221 Timing: Fiscal Year 2001; Project Start 01-SEP-1996; Project End 31-AUG-2005 Summary: Numerous diseases affecting the GI tract, ranging from secretory diarrhea to cystic fibrosis, are characterized by dysregulation of epithelial C1- secretion. This project originally identified that ammonium ion (NH4+, normally present at high concentrations in the colonic lumen) may be a novel endogenous regulator of C1secretion via effects via effects on K+ channels and begins to define the interaction of NH4+ with the basolateral membrane K+ transporters also required for Cl-secretion. Based on work already accomplished, the current application considers how altered K+ channel regulation may influence various intestinal disease states. Preliminary data indicate that the ammonia-derived oxidant monochloramine (NH2Cl) may contribute to the diarrhea of colitis by potentiating Ca2+- dependent K+ channels. Experiments also suggest that docosahexaenoic acid (DHA, a component of fish oil) can augment Ca+2dependent K+ channels, finding of particular interest as DHA begins clinical evaluation as therapy in CF. Preliminary findings suggest that the actin cytoskeleton an functionally alter Ca2+-dependent K+ channels, and conversely, that these K+ channels can modulate cell functions such as epithelial restitution that involve actin remodeling. Three sets of studies are proposed. First, the impact of ammonia on colonic epithelial transport will be further characterized in cultured epithelial ells and in human colonic mucosal preparations, with attention to the interaction of NH4+ with the basolateral Na+-k+-2Cl- co- transporter, Na+_K+ ATPase, and K+ channels. Second, potentiation of basolateral Ca+2-dependent K+ channels by cAMP and NH2Cl will be explored using cultured epithelial cells as model systems with the goal of defining a common mechanism for K+ channel potentiation by these seemingly diverse stimuli. The potential for therapeutic modulation of basolateral K + channels will be explored, specifically examining wheth4r docosahexaenoic acid (DHA) can augment Ca2+dependent Cl- secretion in T84 cells and human colon, and, if so, to determine its mechanism of action. Finally, the studies will define the effect of chemical manipulation of F-actin on Ca2+-dependent K+ channel regulation and extend preliminary findings suggesting that K+ channel regulation affects the actin-regulated process of epithelial restitution. These studies highlight the importance of basolateral K+ channels in the regulation of secretion and other epithelial functions and reinforce their potential as targets for new drug design. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: INVESTIGATING THE BASIS FOR COLLAGEN STABILITY Principal Investigator & Institution: Danielson, Mark A.; Biochemistry; University of Wisconsin Madison 750 University Ave Madison, Wi 53706 Timing: Fiscal Year 2001; Project Start 01-SEP-2001 Summary: Collagen is the principle structural protein in vertebrates. Defects in collagen have been associated with a wide variety of diseases, and knowledge of the atomic basis for the structure and stability of collagen is an important step in developing treatments for these diseases. The structure of collagen is stabilized by hydroxylation of certain proline residues. The means by which the hydroxyl stabilizes the structure is not clearly understood. Preliminary evidence supports a hypothesis in which the inductive effect of the hydroxyl favors the trans conformation of the proline, which is necessary for proper folding of the collagen triple helix. The proposed research will test this hypothesis as follows. 1) Several halogenated collagen mimics will be synthesized containing fluorine, chlorine, bromine, or iodine in place of the hydroxyl on the modified proline. 2) The 4(R)-hydroxyls of a collagen-like peptide will be modified with the electronwithdrawing acetyl, mesyl, or trifluoromethyl groups. 3) The thermodynamics and energetics of all of the synthesized or modified peptides will be measured and correlated to the thermodynamics and kinetics of cis-trans isomerization of the peptide bonds of related small molecules. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: IRRITANT-INDUCED PATHOGENESIS

ASTHMA:

EPIDEMIOLOGY

AND

Principal Investigator & Institution: Malo, Jean-Luc; Hopital Du Sacre'-Coeur De Montreal De Montreal Montreal, Timing: Fiscal Year 2001; Project Start 30-SEP-1999; Project End 29-DEC-2002 Summary: The general aim of this proposal is to explore the following questions related to irritant-induced asthma from both epidemiological and physiopathological approaches: 1) Do single irritant exposures (Reactive Airways Dysfunction Syndrome-RADS- and multiple irritant exposure (Irritant-Induced-Asthma--IrIA--) exposures result in equivalent consequences for airway structure and function? 2) Are baseline characteristics (atopy, airway caliber and responsiveness) relevant to susceptibility of developing IrIA and RADS? RADS and IrIA represent up to 15 percent of all cases of occupational asthma. It is also a condition of general interest for asthma per se. It has been estimated that 60 000 inhalational accidents occurring at home and identified as RADS are reported in U.S. emergency rooms on a yearly basis. RADS was originally defined as the development of respiratory symptoms in the minutes or hours after a single accidental inhalation of high concentrations of gas, aerosol, or particles, after which subjects are left with asthma like symptoms and airway hyperresponsiveness. Multiple exposures to high concentrations of products such as chlorine in pulp and paper mills can also cause IrIA, a more general term to describe an asthmatic syndrome that results from single or multiple exposures to irritant products. We will examine and follow new employees at risk of acute exposure to chlorine and serially assess their characteristics (atopy, airway caliber and responsiveness, smoking, nasal symptoms) and exposure events. In a subsample, we will also examine induced sputum. In a rat model, we will 1) examine the response to single and multiple exposures to chlorine vapor; 2) test the importance of background characteristics of the rat strain (atopy, airway muscle) to the response; 3) quantify the degree of airway remodelling in a susceptible and resistant rat strain; 4) assess the effects of neurokinin antagonists on the

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airway damage; 5) evaluate the role of airway epithelial chemokines such as RANTES and eotaxin in the inflammatory response. This work will identify, both in an epidemiological survey and in an animal model, the effect of single and multiple exposures to a respiratory irritant and the host predisposing factors. It will also further our understanding of the physiopathology of RADS and IrIA as well on the natural history and physiopathology of occupational asthma and of asthma induced by irritants. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ISOTOPIC PROBES OF ENZYME REACTION MECHANISMS Principal Investigator & Institution: Raushel, Frank M.; Professor; Chemistry; Texas A&M University System College Station, Tx 778433578 Timing: Fiscal Year 2002; Project Start 01-DEC-1984; Project End 31-DEC-2005 Summary: (provided by applicant): The broad long-term objectives for the research described in this proposal are aimed at a determination of structure-function relationships in catalytically active proteins and enzymes. The primary focus of this proposal will be directed towards the enzyme phosphotriesterase and related members of the amidohydrolase superfamily. This group of proteins catalyzes the nucleophilic attack of an activated water molecule on a diverse set of substrates. Phosphotriesterase catalyzes the detoxification of organophosphorus nerve agents while other members of this superfamily catalyze the hydrolytic cleavage of a carbonyl group attached to oxygen, nitrogen or chlorine. Since highly toxic organophosphate molecules are of rather recent environmental origin, the elucidation of the catalytic functions within phosphotriesterase, and the related proteins of the amidohydrolase superfamily, will provide unique insights into the mechanisms for the evolution and development of novel enzymatic activities. To define and articulate the factors that contribute to the determination of substrate specificity, random and site-directed variants of phosphotriesterase, and selected members of the amidohydrolase superfamily, will be constructed. The elucidation of the chemical reaction mechanism will be addressed by the construction of metal-substituted variants, site-directed mutants, and substrate analogs. The kinetic properties of the mechanistic probes will be utilized in the formulation of a self-consistent description of the catalytic events within the active sites. To more fully understand the factors that direct the evolution of new catalytic activities, the mechanisms of action and the three-dimensional structures of prominent members of the amidohydrolase superfamily will be determined. These studies will provide a historical window for how new catalytic activities can evolve from pre-existing enzymes. These investigations will complement concurrent efforts to direct the evolution of new and enhanced activities from present-day proteins. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: MECHANISM OF VISUAL EXCITATION Principal Investigator & Institution: Ebrey, Thomas G.; Professor; Botany; University of Washington Seattle, Wa 98195 Timing: Fiscal Year 2001; Project Start 30-SEP-1985; Project End 29-SEP-2003 Summary: (Adapted from the applicant's abstract): The long-term goal of this work is to elucidate the biophysical and biochemical mechanisms underlying visual excitation and adaptation. A deep understanding of the visual system itself is, of course, highly desirable in order to understand how it might be affected in abnormal states brought on by disease and genetic defects. In addition, the visual system is probably the most accessible of a very large family of signal transduction systems that are found in all

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vertebrates and invertebrates; these signal transduction systems are all based on cell surface receptors that are coupled to GTP binding proteins (G proteins). Visual pigments are examples of such G protein-coupled cell surface receptors. The findings will, therefore, have important ramifications to the understanding of odorant, hormone and neurotransmitter signaling. The approach is to use both the broad range of naturally-occurring visual systems, not only the rhodopsin/rod system, but long wavelength cone systems, mixed rod/cone systems like gecko, very short wavelength cone systems like human blue cone, and invertebrate systems such as octopus. The investigators seek to understand the similarities and differences between vision based on these distinct categories of pigments. They will focus on the events leading up to the formation of the active state of a visual pigment. They will utilize low temperature, flash-induced kinetics, FTIR (Fourier transform infrared) spectroscopies as well as lightinduced photocurrents to study the transformations that a visual pigment undergoes leading up to its active state. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: MECHANISMS OF ANION TRANSPORT IN SALIVARY GLANDS Principal Investigator & Institution: Melvin, James E.; Interim Director; Oral Biology; University of Rochester Orpa - Rc Box 270140 Rochester, Ny 14627 Timing: Fiscal Year 2001; Project Start 05-DEC-1990; Project End 30-JUN-2005 Summary: (Adapted from the Investigator's Abstract): The quality of life for millions of Americans is adversely affected by salivary gland dysfunction. A variety of etiologies contribute to the existence of dry mouth, including the use of xenogenic medications, Sjogren's syndrome, radiation therapy, or systemic diseases such as diabetes mellitus. The development of interventions to improve function for these individuals requires a thorough understanding of the molecular physiology of salivary glands. The current fluid secretion model predicts that saliva is produced as a two step process whereby acinar cells initially secrete an isotonic plasma-like fluid, which is subsequently modified by ductal cells to conserve NaCl. Chloride (Cl) channels and chloride/bicarbonate (Cl/HCO3) exchangers are key transport mechanisms involved in transepithelial Cl movement, the driving force for both the secretion of fluid and the resorption of NaCl. At least four distinct types of Cl channels and three different anion exchangers have been detected in salivary gland cells. Although the general biophysical properties of these different classes of proteins are known, major gaps exist in our knowledge of their regulation and the contribution each makes to the overall secretion process. Thus, to verify and refine our understanding of the function of these proteins in salivary gland cells, Aim 1 will examine the regulation of these proteins by secretagogues. It is hypothesized that those Cl channels and Cl/HCO3 exchangers necessary for secretion will be activated during stimulation. Aim 2 will test the hypothesis that the localization and expression of each protein is consistent with its predicted function. The cell type in which each transcript is expressed and the distribution of each protein in the plasma membrane should reflect the role that the transporter plays in fluid secretion. Aim 3 will directly test hypotheses as to which Cl transport proteins are essential to the fluid secretion process through the study of animals lacking functional expression of a specific gene. This multidisciplinary approach will define the essential Cl transport mechanisms involved in the production of saliva, critical information in the development of treatments for salivary gland dysfunction. Furthermore, the results of these studies will provide a foundation for future studies to analyze the in vivo structure/function relationship of a given Cl transporter.

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

Project Title: MECHANISMS OF COOPERATIVE PROTEIN/DNA INTERACTIONS Principal Investigator & Institution: Brenowitz, Michael D.; Professor; Biochemistry; Yeshiva University 500 W 185Th St New York, Ny 10033 Timing: Fiscal Year 2001; Project Start 01-APR-1988; Project End 05-DEC-2002 Summary: (Adapted from abstract): The formation of unique ensembles of protein-DNA and protein-protein interactions determine which of three RNA polymerases transcribe a gene in eukaryotic organisms. Quantitative studies ar proposed to describe the thermodynamic driving forces and kinetic pathways of these interactions. A focus is the central role of the TATA binding protein (TBP). Aim I seeks to correlate the energetics of DNA sequence-specific bindin by TBP with the atomic resolution 3D structures of TBPDNA complexes that differ in their affinity for TBP. An analysis of TBP binding to DNA molecules of altered conformation will assess the contribution of DNA conformation to binding. Aim II seeks to determine the kinetic mechanism describing formation of the TBP-DNA complexes using complementary stopped-flow fluorescence resonance energy transfer (FRET) and synchrotron x-ray hydroxyl radical footprinting techniques that monitor global and local changes in DNA conformation. The generality of the kinetic mechanism determined will be assessed by conducting selected kinetic studies using TBP from Homo sapiens an Arabidopsis thaliana. Aim III proposes to determine the contributions of protein-protein and protein-DNA interactions and DNA conformation to the assembly of the minimal pre-initiation complexes specific for RNA polymerase I and III. Protein mutants that specifically affect the intrinsic and cooperativ interactions in the TBP-TFIIB-DNA ternary complex will be analyzed. The proteinprotein association reactions linked to the cooperative formation of ternary complexes will be quantitatively analyzed by a new method of protein footprinting. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: MERCURY IN CHLORIDE TRANSPORT IN SHARK RECTAL GLAND & RABBIT THICK ASCENDING LIMB Principal Investigator & Institution: Silva, Patricio; Mount Desert Island Biological Lab Old Bar Harbor Road Salisbury Cove, Me 04672 Timing: Fiscal Year 2001 Summary: The intent of this proposal is to study the mechanism of action of the toxic effect of mercury on transport by chloride transporting epithelia. Mercury inhibits chloride transport in both the shark rectal gland and the mammalian thick ascending limb but its mechanism of action mechanism of action is unknown. In the proposed set of experiments we will study the effect of organic and inorganic mercury on the transport of chloride by the shark rectal gland and compare it with that in the thick ascending limb of the loop of Henle. The mechanism of the inhibition of mercury, mercurial (both inorganic and organic) or of any compound on chloride transport by the rectal gland or the thick ascending limb can be evaluated with reference to our present understanding of transport by these cells. Inhibitory effects are possible at five different sites: 1) The site of entry of chloride into the cell, the Na-K-2CI carrier; 2) Inhibition of Na-K-ATPase with the resulting reduction in the driving force for entry of sodium, chloride and potassium via the cotransporter; 3) Inhibition of cellular metabolism resulting in insufficient ATP to power the pump; 4) Inhibition of the exit of chloride at the apical membrane by a reduction in the apical membrane conductance for chloride; 5) inhibition of potassium recycling through the potassium conductive pathway. These

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possibilities can be distinguished using specific transport inhibitors in combination with mercury. An additive effect of mercurial to inhibit chloride transport in the presence of specific inhibitors of these transport steps will suggest a site of action of distinct from that of the specific inhibitor. Because we have found unexpected and striking differences in the response of these two epithelia to inorganic and organic mercurial, we believe that the comparative approach will be particularly fruitful. We will examine the mechanism and the cellular site of action of mercury by determining its effect in the isolated perfused rectal gland, isolated rectal gland tubules, rectal gland membrane vesicles, rectal gland plasma membranes, thick ascending limb cells, thick ascending limb vesicles, thick ascending limb plasma membranes. Specifically we will examine the binding of mercury by plasma membranes and cytosolic proteins; the effect of mercury on the chloride cotransporter, the efflux of potassium, the chloride conductance, and its effect on transport related enzymes. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: MERCURY INTERACTION WITH THE TAURINE TRANSPORT SYSTEM OF RED BLOOD CELLS Principal Investigator & Institution: Preston, Robert L.; Mount Desert Island Biological Lab Old Bar Harbor Road Salisbury Cove, Me 04672 Timing: Fiscal Year 2001 Summary: The overall objective of the proposed research is to identify and characterize the molecular targets and mechanisms of interaction of mercury with membrane transport proteins. We will focus specifically on the interactions of mercury with the Nadependent taurine transport system in the hemoglobin containing coelomocytes (red cells, RBCs) of the marine polychaete, Glycera dibranchiata. This transport system is similar to taurine transport systems in mammalian tissues such as heart, kidney and brain. In Glycera RBCs, taurine is maintained at exceptional gradients (950:01; 190 mM intracellular taurine: 0.2 mM extracellular taurine). We have shown that this transport protein is very sensitive to inhibition by mercurial. A one minute exposure to 20 muM HgC12 inhibits taurine influx 50%. Studies using reduced sulfhydryl reagents of different molecular size indicated that the reactive sites on the transport protein appear to be partially occluded by the membrane since complete reversal is obtained only with small molecules. Since HgC12 in high chloride media is present predominantly as anionic complexes that are impermeant to membranes, we propose the operational hypothesis that the nonionic HgC12 complex is the form that reacts with taurine sulfhydryl groups lying in membrane spanning regions of the protein. We will test this hypothesis by addressing the following specific aims: (1) Physiologically characterize the form of HgC12 that interacts with the taurine transport system and identify the transport proteins involved in cell volume regulation that are sensitive to interaction with mercury. (2) Clone the Na-dependent taurine transporter from Glycera RBCs. (3) Characterize the interaction of mercurial with the taurine transporter expressed in Xenopus oocytes. Flux measurements on intact RBCs and on oocytes expressing the taurine transport protein will be done using radioisotope methods. The molecular procedures will depend on reverse transcription and PCR of poly(A)+RNA with taurine transport activity identified by expression of taurine transport activity of the mRNA fractions microinjected into Xenopus oocytes. Physiological studies will measure solute contents and net fluxes with a variety of techniques including ion selective electrodes, atomic absorption spectroscopy, HPLC and radioisotopic methods. The results of this study will provide important basic information on the molecular mechanism of

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interaction of mercury with the taurine transporter that has a hole in many animals tissues including human cardiac, nerve and kidney tissue. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: MIBG ANALOGUE RADIOPHARMACEUTICALS Principal Investigator & Institution: Vaidyanathan, Ganesan; Associate Research Professor of Radiolog; Radiology; Duke University Durham, Nc 27706 Timing: Fiscal Year 2001; Project Start 08-SEP-1997; Project End 30-JUN-2003 Summary: (Adapted from Applicant's Abstract): The radiopharmaceutical metaiodobenzylguanidine (MIBG) has been used in the detection and therapy of neuroendocrine tumors, especially neuroblastoma and pheochromocytoma. Although it is a satisfactory agent for diagnostic applications, the outcome of MIBG therapy is inadequate. The goal of this proposal is to develop an agent which is metabolically more stable, clears faster from normal tissues, and yet is sequestered and retained in tumor. The initial objectives of this proposal are to develop synthetic methods for the preparation of various radioiodinated and [At-211]-labeled MIBG derivatives and to systematically evaluate them in vitro and in vivo. The structural alterations planned include introduction of chlorine, bromine, iodine, nitro and sulfonic acid moieties at the 4-position in the benzene ring of MIBG and replacement of the benzene ring itself with a pyridine ring. Solid-phase synthetic methods will be developed for promising agents. The uptake and retention kinetics in vitro and the effect of several pharmacological agents will be studied using a panel of neuroblastoma, pheochromocytoma and medulloblastoma cell lines. Although not a neuroendocrine tumor, specific uptake of MIBG has been demonstrated in several human medulloblastoma cell lines, and this neoplasm is well suited for [At-211]-therapy. The therapeutic potential of these new [I131]- and [At-211]-labeled MIBG analogues will be evaluated using thymidine uptake and clonogenic assays in monolayer and spheroid models. Tissue distribution of these agents will be determined in normal mice and athymic mice hosting neuroblastoma, pheochromocytoma, and medulloblastoma xenografts. Strategies to augment tumor-tonormal tissue ratios will be investigated. The outcome of this study should help improve the endoradiotherapy of neuroendocrine tumors and possibly of medulloblastoma. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: MINIATURE SYSTEM FOR REMOVAL OF DISINFECTION BYPRODUCTS Principal Investigator & Institution: Kim, Jinseong; Lynntech, Inc. College Station, Tx 77840 Timing: Fiscal Year 2002; Project Start 01-JUN-2000; Project End 31-MAY-2004 Summary: (provided by applicant): The availability of safe drinking water is a significant health concern. The introduction of chlorination as a disinfection method caused a large drop in mortality from water borne diseases and was considered a major public health advance. Unfortunately, disinfection byproducts (DPBs) are formed when chlorine and other disinfectants are used in water treatment plants. Many disinfection byproducts can have serious health effects and are potential carcinogens. Current treatment options to minimize DPBs in drinking water are either inadequate or too costly to implement. A potential means to combat exposure to DPBs is through the widespread implementation of treatment methods near to the place where water is consumed (i.e., point-of-use treatment). In Phase I, we demonstrated a point-of-use water treatment device based on photocatalytic oxidation. The method has a high

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potential to degrade DPBs to benign end products and provide safe drinking water at a cost lower than current treatment options. Significant accomplishments of the Phase I study included demonstration of the device's potential for mass production as well as its multiple applications, including control of microbial contaminants. The Phase II will focus on optimization of the device's design and capabilities. PROPOSED COMMERCIAL APPLICATION: There are many economic advantages to the point-ofuse water purification method described in this proposal. The proposed device can be used as a stand alone treatment method, or its commercial potential may be enhanced by integrating it with siilar water purification devices that are used by millions of consumers in the U.S. The market opportunity is substantial, given the large number of point-of-use devices that are used in health care facilities, homes, restaurants, food service establishments and hotels. The market is estimated to be worth over $1.5 billion annually in the U.S. Lynntech is well positioned to commercialize this technology, having secured intellectual property covering many aspects of the invention. Additionally, Lynntech's management team is experienced in commercializing new technology related to water treatment, healthcare and consumer products. PROPOSED COMMERCIAL APPLICATION: There are many economic advantages to the point-ofuse water purification method described in this proposal Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: NA CARDIOPLEGIA

TRANSPORT

INHIBITION

IN

NEWBORN

HEART

Principal Investigator & Institution: Anderson, Steven E.; Human Physiology; University of California Davis Sponsored Programs, 118 Everson Hall Davis, Ca 95616 Timing: Fiscal Year 2001; Project Start 01-APR-1997; Project End 31-MAR-2003 Summary: This abstract is not available. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: NEUROTOXIC AND MUTAGENIC ACTIONS OF SUPERFUND CHEMICALS Principal Investigator & Institution: Kisby, Glen E.; Oregon Health & Science University Portland, or 972393098 Timing: Fiscal Year 2001 Summary: Organochlorine solvents are a common contaminant of groundwater and drinking water and drinking water supplies and, therefore, pose a particularly important long-term health hazard to humans. Of the many organochlorine solvents, vinyl chloride poses the greatest threat to humans because it is highly prevalent, a common metabolite of many organochlorine solvents (e.g., TCE, DCE), and tentatively linked with long-term neurological dysfunction and brain cancer. The identification of biomarkers to determine the relative health risk associated with human exposure to vinyl chloride is a high priority of the EPA and a long-term objective of this proposal. The focus of the present study is to identify the neurotoxic and neuro-oncogenic mechanisms of vinyl chloride and to use these as tools or biomarkers for risk assessment. The major metabolite of vinyl chloride monomer (a Superfund chemical on the ATSDR priority list) is chloroacetaldehyde (CAA), a known human and rodent genotoxin with neurotoxic, mutagenic, and oncogenic properties. Because our previous work indicates that alkylating agents like CAA (e.g., methylazoxymethanol, MAM) are neurotoxic, damage DNA, perturb DNA repair, and are mutagenic, we propose that CAA induces its neurotoxic and mutagenic effects by a similar mechanism. Experiments

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are proposed to examine the relationship between the formation of etheno base DNA adducts and neurotoxicity or mutations. Neuronal and astrocyte cell cultures will be developed from different brain regions (e.g., cortex, hippocampus, midbrain, cerebellum) of DNA repair proficient and deficient mice (i.e.,k N-methylpurine DNA glycosylase Aag) and examined for acute and delayed CAA-induced neurotoxicity. Separate sets of astrocyte cell cultures will be developed from Aprt heterozygousdeficient mice and examined to determine the spectrum of CAA-induced mutations. Findings from these studies are expected to provide important information about the neurotoxic and mutagenic mechanisms of vinyl chloride. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: NEUROTOXIC PRODUCTS OF CHLOROALKENE AEORBIC COMETABOLISM Principal Investigator & Institution: Semprini, Lewis; Oregon Health & Science University Portland, or 972393098 Timing: Fiscal Year 2001 Summary: Accidental spills, leaking underground storage tanks, improper disposal, and landfill leachates are often the sources of chlorinated aliphatic hydrocarbons (CAHs) in groundwater. Trichloroethylene (TCE) is the most frequently found contaminant in sites of the National Priority List, and cis-1, 2-dichloroethylene (c-DCE) and vinyl chloride (VC) are commonly present due to the transformation of higher chlorinated parent compounds. Aerobic co-metabolism is a potential biological process for treatment of CAHs both in situ and ex-situ. Here microorganisms stimulated on a primary electron donor fortuitously degrade the contaminant. The process involves relatively nonspecific oxygenase enzymes that subsequently oxygenase enzymes that subsequently result in a one-step transformation of the CAHs. These oxygenase enzymes typically have low half-velocity co-efficients, and, as a result, achieve in low contaminant concentrations upon remediation. Co-metabolic degradation of CAHs, however, is known to produce toxic intermediate compounds. This resulting toxicity is an important process in the selection of the co-metabolite process because of its effect on the degradation kinetics. In addition, these toxic intermediate compounds are of concern because of their potential health of ecological impacts if they remain in the treated aquifer. In the proposed research we will expand the understanding of these toxic metabolic intermediates in relation to their identify, their impact upon CAH degradation. Studies and the mechanisms by which toxicity is exhibited. We will focus on the co- metabolism of VC, 1,1-DE, and c-DCE since less we know less about transformation toxicity for these compounds, compared to TCE, will be performed with pure bacterial cultures grown on ammonia, toluene, and butane, to access how cometabolic transformation of the selected CAHs will be determined. The resulting toxicity will be assessed based on loss in growth substrate utilization activity. Also oxidized products, such as alcohols and organic acids, and oxygen consumption. The mechanism(s) of co-metabolic toxicity will be evaluated in Task 2 using 14C radiolabeled CAHs coupled with protein assay methods. Products of the co-metabolism will be tracked using 14C measurement methods. The distribution of the CAH intermediates will be determined by protein binding assays using the 14C-labeled CAHs. The effective of active substrate metabolism upon the degree of co-metabolic activity and the ability of cell to recovery from toxic effects will be evaluated in Task 3. In Task 4 the impact of o-metabolic toxicity on the composition of consortium in a mixed culture constructed of three different pure cultures, growing on a single substrate

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(touene and butane) will be evaluated. The changes in the community will be tracked using molecular methods and protein assay methods. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: NEW MEMBRANES FOR CARCINOGEN REMOVAL IN WATER TREATMENT Principal Investigator & Institution: Nemser, Stuart; President; Compact Membrane Systems, Inc. Wilmington, De 18904 Timing: Fiscal Year 2001; Project Start 17-JAN-1997; Project End 31-MAR-2004 Summary: (Adapted from Applicant's Abstract): The ozonation of drinking water is attractive for small communities that employ local wells or surface water sources, provided it can be done at a reasonable cost. Although chlorine is widely used and is cheaper to use than ozone, it's use results in the formation of carcinogenic trihalomethanes. Also, chlorine has been found to be ineffective in the treatment of cryptosporidium. Under a SBIR Phase I grant from the NIH, Compact Membrane Systems (CMS) demonstrated at lab-scale that CMS' membrane technology could be successfully used to achieve efficient ozone levels that would be commercially practical, while membrane lifetimes in excess of 1300 hours when exposed to ozone and water were also observed. In Phase II the investigator will a) scale-up device size for pilot-scale water treatment operations, b) employ CMS' membrane technology to enhance the device performance and longevity, c) demonstrate efficient ozonation of water streams spiked with Cryptosporidium with the aid of animal studies, d) undertake pilot-scale experiments at the local water treatment plant, and e) develop detailed economics with respect to system size and calculate cost trade-off for system size versus costs per 1000 gallons of water treated. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: NOD-H2H4 MICE AS A SENTINEL MODEL FOR AUTOIMMUNE THYROID Principal Investigator & Institution: Burek, C Lynne.; Associate Professor; Pathology; Johns Hopkins University 3400 N Charles St Baltimore, Md 21218 Timing: Fiscal Year 2001; Project Start 30-SEP-1999; Project End 29-SEP-2003 Summary: (Taken from the Investigator's Abstract) Autoimmune thyroiditis is a multifactorial disease in which genetic predisposition combines with environmental factors to induce disease. In humans, the thyroid can be compromised by diet, drugs, and other synthetic chemicals. Excess iodine may be partially responsible for the increasing prevalence of autoimmune thyroiditis. Other environmental chemicals may also be implicated, but there is little documented evidence as to which chemicals may be involved. Candidate pollutants include polyaromatic hydrocarbons, polybrominated biphenyls, and polychlorinated biphenyls. Infectious agents are also known to trigger autoimmunity. In this proposal the investigators will 1) develop standard reproducible conditions for obtaining a 50% incidence of iodine-exacerbated autoimmune thyroiditis in NOD.H2h4 mice and 2) use this mouse model to study the effects of environmental chemicals in food, industrial products, or infection on development of autoimmune thyroiditis. Methylcholanthrene (MCA) will be used as an example of a polyaromatic hydrocarbon, KBr will be used as an example of a polybrominated biphenyl, and theophylline will be used as an example of a drug which can increase iodine uptake. In Aim 3, lipopolysaccharide (LPS) will be used as a surrogate for infection to determine

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the role of infectious agents in autoimmune thyroiditis. The genetic predisposition of the NOD.H2h4 mouse to autoimmune thyroiditis will be used to study the potential additive effects of the above compounds on autoimmune disease. The NOD.H2h4 animals are an ideal sentinel model to examine the potential interactions of genetics and environmental agents on autoimmune disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: NUCLEOSIDE TRANSPORT AND INTESTINAL CHLORIDE SECRETION Principal Investigator & Institution: Ward, Jeffrey L.; Medicine; Johns Hopkins University 3400 N Charles St Baltimore, Md 21218 Timing: Fiscal Year 2001; Project Start 01-SEP-1999; Project End 14-JUN-2002 Summary: Na+-independent nucleoside transport systems are facilitated-diffusion systems, and two distinct classes have been identified according to their sensitivity to the inhibitor, nitrovenzylthioinosine (NBMPR). Both NBMPR- sensitive [ENT1 designated the cloned transporter; ES designation refers to physiological studies (Equilibrative NBMPR-Insensitive) systems are of broad selectivity, transporting both ubiquitously expressed, whereas the EI system has been described only in intestine, leukemia cells and cardiovascular tissues/cells. Both isoforms appear to be involved in scavenging nucleosides, which is especially important in cells that are unable to synthesize nucleosides de novo, such as those of the intestinal epithelium. Adenosine, a substrate of both the ES and EI nucleoside transport systems, is released during ischemic episodes and stimulates chloride secretion. Activation of pro-secretory adenosine A2b receptors may occur when normal basolateral nucleoside uptake mechanisms (scavenging mechanisms) are overwhelmed and local concentrations of adenosine increase, a response that may contribute to the diarrheal response associated with mesenteric ischemia. In T84 cells, a model for intestinal chloride- secreting crypt cells, Na-independent nucleoside transporters on the basolateral membranes have been synthesized to help in maintaining extracellular concentrations of adenosine below the threshold of activation of the adenosine A2b with respect to nucleoside transport and protein kinase regulation in a transporter deficient cell line, PK-15NTD. In addition, structure/function studies re proposed to understand how the ENT1 transport functions at the molecular level, including the role of glycosylation, the role of cystein residues in regulating function, and whether the transporter functions as a monomer or an oligomer. To address the physiological roles of ENT1 and ENT2 in regulating intestinal adenosine-mediated chloride secretion, T84 cells will be mutated to obtain cells lines that express only ENT1, or cell lines deficient in both ENT1 and ENT2. In short-circuit current experiments, wild-type and mutant cell lines will then be studied to elucidate the contribution of both transporters to the regulation of chloride secretion. Our proposed studies should provide insights into the molecular mechanisms and regulation of Na+-independent nucleoside transporters, as well as their role in scavenging adenosine in inflammatory bowel disease and certain infectious causes of diarrhea. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: ORTHO SUBSTITUTED PCB ON CALCIUM HOMEOSTASIS IN APLYSIA BAG CELL NEURONS Principal Investigator & Institution: Laessig, Susan A.; Marine Biological Laboratory 7 Mbl St Woods Hole, Ma 02543 Timing: Fiscal Year 2001

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Summary: Polychlorinated biphenyls (PCB's) are a family of persistent and ubiquitous environmental contaminants which originated from heavy industrial use. There are 209 separate compounds containing variable numbers of substituted chlorine molecules. Congeners without ortho-chlorine substitutions can assume a coplanar conformation and elicit toxicity similar to dioxin. Ortho-chlorine substituted PCB's are believed to be responsible for the neurotoxicity elicited by PCB mixtures. Neurotoxic effects of PCB's include developmental delays, cognitive deficits, altered motor function, and decreased brain neuro-transmitter levels, dopamine in particular. Recently, a potential mechanism by which ortho-substituted PCB's alter dopamine content in brain and pheochromocytoma (PC12) cells has been proposed. They have been shown to elicit toxicity through calcium-selective channels, ryanodine receptors, which regulate the release of calcium from the endoplasmic reticulum (ER). Changes in intracellular calcium provide important signalling information to cells and calcium gradients across cell membranes play a key role in regulating cell-signalling in the nervous system. PCBinduced increases in calcium transport have been demonstrated to occur through direct interaction with ryanodine receptors in microsomes from mammalian brain, skeletal, and cardiac muscle, and cause increased intracellular calcium in cerebellar granule cells. The opistobranch mollusc, Aplysia, is a well-studied model of simple learning and has well-characterized neural circuits. The aim of this study was to determine if orthosubstituted PCB's can alter intracellular calcium levels in a manner similar to that observed in mammalian cells. Aplysia bag cell neurons used were in culture for 2 days and had adhered to the surface of poly-lysine coated coverslips and formed neural processes. The cells were filled with the calcium indicator dye Fura-2/AM by bath application for 1 hour followed by three 5 minute washes with artificial sea water (ASW). Cells were imaged on the Attofluor imaging system after calibration of the system with calcium standards. 2,2',4,4'-tetrachlorobiphenyl (TCB) (100mM) was applied to the dish during imaging and images were recorded for