IOTIN A M EDICAL D ICTIONARY , B IBLIOGRAPHY , AND A NNOTATED R ESEARCH G UIDE TO I NTERNET R EFERENCES
J AMES N. P ARKER , M.D. AND P HILIP M. P ARKER , P H .D., E DITORS
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ICON Health Publications ICON Group International, Inc. 4370 La Jolla Village Drive, 4th Floor San Diego, CA 92122 USA Copyright ©2004 by ICON Group International, Inc. Copyright ©2004 by ICON Group International, Inc. All rights reserved. This book is protected by copyright. No part of it may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without written permission from the publisher. Printed in the United States of America. Last digit indicates print number: 10 9 8 7 6 4 5 3 2 1 Publisher, Health Care: Philip Parker, Ph.D. Editor(s): James Parker, M.D., Philip Parker, Ph.D. Publisher's note: The ideas, procedures, and suggestions contained in this book are not intended for the diagnosis or treatment of a health problem. As new medical or scientific information becomes available from academic and clinical research, recommended treatments and drug therapies may undergo changes. The authors, editors, and publisher have attempted to make the information in this book up to date and accurate in accord with accepted standards at the time of publication. The authors, editors, and publisher are not responsible for errors or omissions or for consequences from application of the book, and make no warranty, expressed or implied, in regard to the contents of this book. Any practice described in this book should be applied by the reader in accordance with professional standards of care used in regard to the unique circumstances that may apply in each situation. The reader is advised to always check product information (package inserts) for changes and new information regarding dosage and contraindications before prescribing any drug or pharmacological product. Caution is especially urged when using new or infrequently ordered drugs, herbal remedies, vitamins and supplements, alternative therapies, complementary therapies and medicines, and integrative medical treatments. Cataloging-in-Publication Data Parker, James N., 1961Parker, Philip M., 1960Biotin: 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: 0-597-84355-4 1. Biotin-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:
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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 biotin. 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 BIOTIN ....................................................................................................... 3 Overview........................................................................................................................................ 3 The Combined Health Information Database................................................................................. 3 Federally Funded Research on Biotin............................................................................................. 5 E-Journals: PubMed Central ....................................................................................................... 59 The National Library of Medicine: PubMed ................................................................................ 75 CHAPTER 2. NUTRITION AND BIOTIN ............................................................................................. 99 Overview...................................................................................................................................... 99 Finding Nutrition Studies on Biotin ........................................................................................... 99 Federal Resources on Nutrition ................................................................................................. 104 Additional Web Resources ......................................................................................................... 104 CHAPTER 3. ALTERNATIVE MEDICINE AND BIOTIN ..................................................................... 107 Overview.................................................................................................................................... 107 National Center for Complementary and Alternative Medicine................................................ 107 Additional Web Resources ......................................................................................................... 122 General References ..................................................................................................................... 125 CHAPTER 4. DISSERTATIONS ON BIOTIN ....................................................................................... 127 Overview.................................................................................................................................... 127 Dissertations on Biotin .............................................................................................................. 127 Keeping Current ........................................................................................................................ 128 CHAPTER 5. PATENTS ON BIOTIN.................................................................................................. 129 Overview.................................................................................................................................... 129 Patents on Biotin........................................................................................................................ 129 Patent Applications on Biotin.................................................................................................... 160 Keeping Current ........................................................................................................................ 195 CHAPTER 6. BOOKS ON BIOTIN ..................................................................................................... 197 Overview.................................................................................................................................... 197 Book Summaries: Federal Agencies............................................................................................ 197 Book Summaries: Online Booksellers......................................................................................... 198 Chapters on Biotin ..................................................................................................................... 198 CHAPTER 7. PERIODICALS AND NEWS ON BIOTIN ....................................................................... 201 Overview.................................................................................................................................... 201 News Services and Press Releases.............................................................................................. 201 Academic Periodicals covering Biotin........................................................................................ 202 APPENDIX A. PHYSICIAN RESOURCES .......................................................................................... 207 Overview.................................................................................................................................... 207 NIH Guidelines.......................................................................................................................... 207 NIH Databases........................................................................................................................... 209 Other Commercial Databases..................................................................................................... 211 The Genome Project and Biotin ................................................................................................. 211 APPENDIX B. PATIENT RESOURCES ............................................................................................... 215 Overview.................................................................................................................................... 215 Patient Guideline Sources.......................................................................................................... 215 Finding Associations.................................................................................................................. 217 APPENDIX C. FINDING MEDICAL LIBRARIES ................................................................................ 219 Overview.................................................................................................................................... 219 Preparation................................................................................................................................. 219 Finding a Local Medical Library................................................................................................ 219 Medical Libraries in the U.S. and Canada ................................................................................. 219
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ONLINE GLOSSARIES................................................................................................................ 225 Online Dictionary Directories ................................................................................................... 225 BIOTIN DICTIONARY ................................................................................................................ 227 INDEX .............................................................................................................................................. 319
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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 biotin is indexed in search engines, such as www.google.com or others, a nonsystematic 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 biotin, 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 biotin, 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 biotin. 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 biotin, 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 biotin. 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 BIOTIN Overview In this chapter, we will show you how to locate peer-reviewed references and studies on biotin.
The Combined Health Information Database The Combined Health Information Database summarizes studies across numerous federal agencies. To limit your investigation to research studies and biotin, 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 “biotin” (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: •
Comparison of the Nutritional Composition of Diets of Persons with Fecal Incontinence and That of Age-and Gender-Matched Controls Source: Journal of WOCN. Journal of Wound, Ostomy and Continence Nurses. 27(2): 9097. March 2000. Contact: Available from Journal of WOCN, Mosby-Year Book, Inc. 11830 Westline Industrial Drive, St. Louis, MO 63146. (800) 453-4351. Summary: In clinical and research experience, persons with fecal incontinence (involuntary loss of stool) anecdotally report altered diet intake to avoid incontinence. This article reports on a study undertaken to compare the dietary intake of 39 persons with fecal incontinence living in the community with that of age and gender matched control subjects who had normal bowel function. The diets of both groups were
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compared with recommended dietary allowances (RDAs) for their constituent nutrients. Subjects prospectively recorded the type, amount, and method of preparing all foods and beverages ingested for 8 consecutive days; diet records were analyzed using a nutrition software program. There were no significant differences in the intake of total kilocalories, protein, fat, dietary fiber, caffeine, or lactose by the fecal incontinence and control groups. The fecal incontinence group had a greater intake of carbohydrates, manganese, and vitamin B1 compared with the control group. Diets of both groups exceeded 100 percent of the RDA for protein, phosphorus, iron, sodium, potassium, Vitamins B1, B2, B3, B12 and C, and folate. Diets of both groups had less than 50 percent of the RDA for biotin, chromium, copper, and manganese but did not differ significantly. The authors conclude that the diets of persons with fecal incontinence were similar to those of control subjects with normal bowel function. Both the fecal incontinence and control groups may improve their nutritional patterns by lowering sodium and protein intake and increasing dietary fiber and monounsaturated fat intake. Calcium and vitamin D supplementation may improve dietary deficiencies and lower disease risks. Including a nutritional assessment and consultation in the care of persons with fecal incontinence to improve their general health and prevent disease is recommended, but consideration must be given to altered diet patterns perceived by the patient to prevent fecal incontinence. 4 tables. 36 references. •
Water-Soluble Vitamin Status in Patients With Renal Disease Treated With Hemodialysis or Peritoneal Dialysis Source: Journal of Renal Nutrition. 1(2): 56-73. April 1991. Summary: Patients with end-stage renal disease (ESRD) who are receiving some mode of dialysis treatment comprise a subset of the population for whom water-soluble vitamin therapy may be indicated. This article examines this population and the uses of vitamin therapy. Factors that contribute to a need for vitamin supplementation include limitations to the intake of a well-balanced diet, metabolic derangements due to uremia and associated syndromes, drug interference with absorption and/or action of specific vitamins, changed requirements in the aged population, and the process of dialysis itself. The author reviews the literature in this area and suggests that daily replacement with the US Recommended Daily Allowance is indicated for B1, B2, B12, niacin or niacinamide, pantothenic acid, and biotin. 3 figures. 4 tables. 63 references. (AA-M).
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Treat Your Nails With Kid Gloves Source: Healthy Skin and Hair. p. 6-7. Winter 2001. Contact: Available from Quadrant HealthCom, Inc. 26 Main Street, Chatham, NJ 079282402. (973) 701-8900. Fax: (973) 701-8892. Summary: This journal article discusses how the health of the nails reflects the overall health of the body and provides tips for keeping nails healthy. Fingernails and toenails are made up of protein layers called keratin. Nails grow quickly in children. Aging and menopause slow nail growth and tend to make nails thicker and brittle. Splitting, peeling, or chipping of nails may indicate a vitamin or mineral deficiency. Ridges, white spots, and spooning can indicate severe illness, thyroid disorder or fungus, and iron deficiency, respectively. Dry and peeling nails may be due to nutritional deficiencies brought on by starvation and yo-yo diets. To keep the nails healthy, they should be kept clean and in dry weather soaked in warm water and then rubbed in a cream or emollient. Gloves should be worn when using detergents or chemical solvents. Onychomycosis, a fungal infection occurring more frequently in older people and
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patients with diabetes, should be treated with topical and oral medications. Older patients should take a multivitamin plus biotin and vitamin A. •
Synovial Membrane Inflammation and Cytokine Production in Patients With Early Osteoarthritis Source: Journal of Rheumatology. 24(2):365-371; 1997. Summary: This journal article for health professionals describes a study that examined synovial membrane histopathology and cytokine content at various stages in the development of osteoarthritis (OA). Synovial membrane samples were obtained from the knees of 63 patients at the time of arthroscopy for unexplained knee pain or at the time of joint replacement surgery. Evaluations of synovial membrane variables, including thickness of lining layer, vascularity, and inflammatory cell infiltrate, were by a blinded observer. In a subset of 20 patients, production of interleukin 1alpha (IL1alpha), interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and IL-1 receptor antagonist (IL-1ra) at the mRNA and protein levels was determined using in situ hybridization with biotin labeled riboprobes and immunohistochemistry. Results show that there was evidence of thickening of the lining layer, increased vascularity, and inflammatory cell infiltration in synovial membranes from patients with all grades of OA. The most marked changes were seen in synovial tissue from patients with advanced grades of OA. Similarly, production of IL-1alpha, IL-1beta, and TNF-alpha was present in synovial membranes from all patients with OA, regardless of the degree of articular cartilage damage. There was a trend to decreased levels of IL-1ra in synovial membranes from patients with OA that did not attain statistical significance. Similarly, there was a decrease in the ratio of Il-1ra to IL-1alpha and beta with increasing grades of OA. Results suggest that significant inflammation occurs in the synovial membrane that may contribute to the continued damage of articular cartilage as a result of cytokine production. 24 references, 4 figures, and 2 tables. (AA-M).
Federally Funded Research on Biotin The U.S. Government supports a variety of research studies relating to biotin. 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 biotin. 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 biotin. The following is typical of the type of information found when searching the CRISP database for biotin:
2 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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Project Title: A NEW HEPATOCYTE-SPECIFIC LIGAND FOR GENE DELIVERY Principal Investigator & Institution: Wong, So M.; Mirus Corporation 505 S Rosa Rd, #104 Madison, Wi 53711 Timing: Fiscal Year 2003; Project Start 15-JUN-2003; Project End 31-MAY-2004 Summary: (provided by applicant): We have recently identified a protein, the T7 phage tail fiber protein (p17), which initiated the rapid and selective uptake of the intact T7 phage head, a 60 nm DNA containing icosahedral protein shell, by mouse hepatocytes in vivo. We mapped the hepatocyte-targeting function to the central part of the protein a triple helix forming rod domain. Both the full-length p17 and rod domain alone were able to initiate the accumulation of various protein fusion partners into hepatocytes in vivo. We postulate that this protein will be a powerful new targeting signal for liver gene delivery. The overall goal for this phase I proposal is to determine if the highly efficient p17 mediated hepatocyte targeting of the relatively large T7 phage can be capitalized for hepatocyte specific delivery of heterologous DNA particles. In order to evaluate the potential of pl 7 as a targeting ligand, the first technical hurdle is to develop linkage technology for attaching p17 to DNA particles so that the function of the protein is also preserved. Once a functional attachment method is developed the utility to deliver Virus' proprietary DNA particles systems will be assessed. Successful outcome of this proposal will form basis for further development. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: A NEW PEPTIDE LIGAND FOR TARGETING HEPATOCYTES IN VIVO Principal Investigator & Institution: Wolff, Jon A.; Professor; Pediatrics; University of Wisconsin Madison 750 University Ave Madison, Wi 53706 Timing: Fiscal Year 2003; Project Start 01-JUL-2003; Project End 31-MAY-2006 Summary: (provided by applicant): Hepatocytes are an important target for the delivery of pharmacological agents. Greater efficacy and decreased toxicity would be gained by delivering these therapeutic agents, small and large, more selectively to hepatocytes. Our preliminary results describe the discovery of a new protein determinant, within the p17 protein of T7 phage that enables the highly efficient delivery of fusion proteins and phage particles to hepatocytes in mice in vivo. Our hypothesis is that this ligand is a promising ligand for delivery of both particulate and soluble agents into hepatocytes in vivo because it interacts with a hepatocyte-specific receptor. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: ABC-TRANSPORTER BINDING PROTEINS AND TRAFFICKING Principal Investigator & Institution: Ortiz, Daniel F.; Physiology; Tufts University Boston Boston, Ma 02111 Timing: Fiscal Year 2003; Project Start 01-APR-2003; Project End 31-MAR-2006 Summary: (provided by applicant): The ABC (ATP-Binding-Cassette)-type proteins SPGP and MDR3 are essential for bile formation. SPGP mediates ATP-dependent transport of conjugated bile acids across the canalicular membrane and MDR3 is a phospholipid flippase that mediates transfer of phosphatidylcholine to bile. The transporters, which reside primarily in the canalicular membrane, can be recruited or removed from the apical domain in response to signals such as bile acids, cAMP or changes in osmolarity. Mobilization and targeting of the transporters to and from the canalicular membrane is probably mediated by association with proteins that link SPGP
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and MDR3 to sorting and trafficking networks. However, outside of the interaction between CFTR and NHERF, which is essential for polarized sorting of the chloride channel, little is known about proteins that bind and regulate trafficking of ABCtransporters. We have identified two proteins that specifically bind MDR3 and SPGP. GST-pulldowns from liver homogenates, FRET analyses and co-immunoprecipitation of transporters with associated proteins, confirmed the validity of these interactions. The goal of the proposed research is to study the role of these binding proteins in regulation of SPGP trafficking in polarized cells. Experiments in Aim 1 will use immunofluorescence microscopy and FRET to establish the sites in hepatocytes where SPGP interacts with binding partners, and the effect of stimuli which induce SPGP recruitment to, or retrieval from, the canalicular membrane. Aim 2 focuses on determining the function of the interacting proteins vis-a-vis SPGP trafficking. The specific amino acid motifs in SPGP which mediate its association with interacting proteins will be identified using yeast two hybrid assays. These moieties will be mutated with the objective of generating mutant transporters that do not bind interacting proteins. Trafficking of the SPGP mutants will be studied in polarized cell model systems to determine the function of the association with the interacting proteins. Mutations which cause abnormal trafficking of canalicular ABC-transporters have been associated with cholestasis of pregnancy and Dubin-Johnson syndrome. Therefore, elucidating the pathways that govern transfer and recruitment of ABC-transporters to the apical membrane, and identifying proteins which control these processes, will provide critical insight into mechanisms underlying cholestasis and suggest targets for therapeutic drug design. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: AMINOGLYCOSIDE ANTIVIRALS TO COMBAT ARENAVIRUSES Principal Investigator & Institution: De La Torre, Juan C.; Associate Professor; Scripps Research Institute Tpc7 La Jolla, Ca 92037 Timing: Fiscal Year 2003; Project Start 01-AUG-2003; Project End 31-JUL-2005 Summary: (provided by applicant): The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an excellent model to study the molecular and cellular biology of arenaviruses that cause important and severe human diseases like Lassa fever virus (LFV) and the South American viral hemorrhagic fevers (SAHF). In addition, weaponized forms of these viruses pose a real threat as agents of bioterrorism. No licensed vaccine is available in the US, and currently there is not efficacious therapy to treat these viral infections. Therefore the importance of developing novel effective antiviral drugs to combat pathogenic arenaviruses. We have developed a reverse genetic system for LCMV. This system provides us with a robust platform for the development of novel strategies to target specific steps of the Arenavirus life cycle. We have molecularly characterized the Arenavirus genome promoter. Disruption of the interaction between the viral polymerase and promoter is predicted to abrogate virus multiplication. RNA molecules can form intricate structures that can be targeted by selected specific high-affinity antagonists. This proposal will explore the use of aminoglycoside-based small molecules to target and functionally disrupt the Arenavirus genome promoter, thus inhibiting virus infection. Aim 1 is the screening of combinatorial aminoglycoside libraries to identify small molecules that bind to the LCMV genome promoter. For this, biotin-RNA conjugates of the LCMV promoter will be attached to streptavidin-coated sensor chips and used as a ligand to screen the libraries using surface plasmon resonance. Aim 2 will assess the antiviral activity of binder molecules (Aim 1). Selected aminoglycosides will be tested for: (i) their effects on
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RNA synthesis mediated by the LCMV polymerase using our LCMV minigenome system; (ii) their ability to inhibit LCMV multiplication in cultured cells. Finlly, Aim 3 will assess the generation of viral variants resistant to antiviral aminoglycosides. Emergence of resistant variants will be assessed based on production of infectious virus and intracellular levels of virus RNA synthesis during serial passages in the presence of aminoglycosides with anti-LCMV activity. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ANALYSIS TO ENHANCE SENSITIVITY FOR MEMBRANE PROTEINS Principal Investigator & Institution: Hanash, Samir M.; Professor and Director; Pediatrics & Communicable Dis; University of Michigan at Ann Arbor 3003 South State, Room 1040 Ann Arbor, Mi 481091274 Timing: Fiscal Year 2002; Project Start 28-SEP-2001; Project End 31-AUG-2003 Summary: (provided by applicant) For quite some time, the standard approach for comprehensive profiling of protein expression in tissues an cell populations has been two-dimensional (2-D) gel electrophoresis and silver staining, coupled more recently with mass spectrometry for protein identification. This approach is currently in use in the PI's laboratory, fo NCI funded projects whose objectives are the molecular analysis of tumors to devise novel classification schemes of cancer, and the identification of novel protein markers for early cancer diagnosis. The standard 2- approach generally allows detection of 1000-2000 protein isoforms in a tissue sample, which is far short of the number of protein isoforms contained in a cell or tissue type. In particular, the 2-D analysis of whole cell lysates yields few proteins from cellular compartments such as membrane proteins. The tagging of proteins b, biotinylation has been extensively utilized for the selective capture and analysis of individual proteins an. antigens and has the potential for providing a substantial increment in sensitivity for the analysis of whole subsets of proteins. Preliminary data obtained by the applicant group demonstrate a remarkable increase in sensitivity and in the yield of low abundance membrane proteins using this approach. The objectives of this application are to develop procedures for the systematic separation, quantitative analysis, identification anc databasing of biotinylated membrane proteins from cells and tissues, that take advantage of the increased sensitivity of this approach. The goals of the R21 phase are to evaluate the utility of the membrane. biotinylation procedure by demonstrating that biotinylated proteins indeed represent membrane proteins; anc that their 2-D patterns are informative with respect to cellular lineage and differentiation state; and that it if feasible to apply this procedure for the selective biotinylation of membranes in intact tissue. Achievement o these milestones will lead in the R33 phase to further optimization of biotinylation procedures; to the development of a database of identified membrane proteins and of their expression patterns, to serve as; resource for other investigators; and to the development of liquid chromatography based procedures for the separation of biotinylated proteins coupled with their mass spectrometry based identification. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: ANTIBODY RESPONSE TO BINDING REGION OF HIV-1 P17 TO REC Principal Investigator & Institution: Popovic, Mikulas; None; University of Md Biotechnology Institute Baltimore, Md 212023101
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Timing: Fiscal Year 2003; Project Start 01-APR-2003; Project End 31-MAR-2005 Summary: (provided by applicant): The HIV-1 matrix protein (MAp17) plays an essential role in the viral life cycle. In addition, this structural protein also has extracellular functions manifested in vitro mainly by enhanced T-cell proliferation, HIV1 replication and proinflammatory cytokine production. We have found that the MAp 17 exerts its biological activities via binding as yet to an unidentified cell surface receptor expressed on peripheral blood-derived mononuclear cells. Mouse antibodies generated to the receptor-binding region (RBR) of the MAp 17 blocked binding to the receptor and abrogated the extracellular effects. Moreover, asymptomatic HIV-1 infected individuals, in contrast to AIDS patients, frequently recognized a potent B cell epitope located at the N-terminus of MAp17, which overlaps with the RBR. Based on these observations, we hypothesize that HIV-1 positive asymptomatic individuals with slower progression towards AIDS generate antibody responses to the RBR of the MAp 17, and these antibodies block MAp 17 binding to its receptor. To address the role of these blocking antibodies in the pathogenesis of AIDS a sensitive serological assay will be developed. The assay will be based on measurements of biotin-conjugated MAp 17 binding to cells using flow cytometry. The establishment of the methodology will proceed as follows: (1) development of a binding assay suitable for assessment of sera from immunized animals to block the specific binding of the MAp 17 to its receptor. Important steps in this stage of the binding assay development are: (a) identification of a permanent cell line suitable for MAp17binding; (b) optimization of blocking the MAp 17 interaction with its receptor using antibodies to the MAp 17; (c) assessment of antibodies that recognize the functional/B-cell epitope region, termed also functional RBR, in MAp17pep scan analyses; and (d) testing of these antibodies to neutralize MAp 17 capacity to induce proinflammatory cytokine production; (2) evaluation of applicability of the binding assay for detecting antibodies with capacity to block MAp 17 binding to its receptor in sera from individuals positive for HIV-1p 17/p24. The binding assay for assessment of human sera will include the following important steps: (a) using human sera, we will reassess the optimal conditions for blocking of MAp 17/receptor interactions that were determined in the testing of sera from immunized animals; (b) testing the capacity of sera to recognize the functional RBR in the MAp 17 pep scan analyses; and (c) evaluate RBR positive human sera to neutralize capacity of MAp 17 to induce the proinflammatory cytokine production. The binding assay development using sera from MAp 17 immunized animals will be performed in the first year and its applicability for testing HIV-1 (MAp17) positive human sera will be performed in the second year of the project. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ASPECTS OF BIOTIN NUTRITION Principal Investigator & Institution: Mock, Donald M.; Professor; Biochem and Molecular Biology; University of Arkansas Med Scis Ltl Rock Little Rock, Ar 72205 Timing: Fiscal Year 2002; Project Start 01-JUL-1985; Project End 31-MAR-2007 Summary: Long term goals of this project are to determine the biotin requires for normal individuals in circumstances in which biotin status may be impaired and to investigate the consequences and pathogenic mechanisms for marginal biotin deficiency. We recently demonstrated that marginal biotin deficiency is common during normal human gestation and have demonstrated that marginal deficiency is quite teratogenic in mice. Thus, the following five specific aims are relevant and timely. In Specific Aim #1, we will test the hypothesis that maternal biotin deficiency causes abnormal development of fetal skeletons and palate by causing deficient fetal activity of
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the biotin-dependent enzyme acetyl- CoA carboxylase which leads in turn to deficiency of arachidonic acid and prostaglandin. In fetal palate and limb bud explants from biotin deficient and sufficient CD-1 mice, we will quantitative fetal arachidonic acid component and synthesis rates and will examine the malformation ameliorating effects of supplementation of arachidonic acid and prostaglandin and the amelioration blocking effects of cyclooxygenase inhibitors. Analogous studies will also be conducted in vivo. In Specific Aim #2, we will test the hypothesis that infants with cleft plate or limb shortening have significantly reduced biotin status compared to normal infants. In a case-controlled study, biotin status will be assessed in cord blood using odd-chain fatty acid composition in red blood cell membranes and plasma and lymphocyte activity of the biotin-dependent enzyme propionyl-CoA carboxylase. In Specific Aim #3, we will clone and sequence a biotin transporter recently discovered in our laboratory. In studies of cells from the first individual with biotin transporter deficiency, we will investigate the molecular nature of the genetic defect. In Specific Aim #4, we will confirm promising new indicators of biotin status and investigate the validity of the expression of particular biotin- related genes (e.g., carboxylases) as indicators of marginal biotin deficiency in healthy adults rendered marginally biotin deficiency by egg-white feeding. In Specific Aim #5, we will determine the subcellular localization of the enzyme(s) responsible for catalyzing the beta- oxidation of biotin to the inactive metabolite bisnorbiotin and characterize this pathway. Understanding of this pathway is important because accelerated biotin catabolism may be the major cause of biotin deficiency in pregnancy and anticonvulsants. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ASSEMBLY AND AXONAL TRANSPORT OF NEUROFILAMENT PROTEINS Principal Investigator & Institution: Brown, Anthony; Associate Professor; Neurobiotechnology Center; Ohio State University 1960 Kenny Road Columbus, Oh 43210 Timing: Fiscal Year 2002; Project Start 01-APR-1999; Project End 31-MAR-2003 Summary: Neurofilaments are thought to play a central role in the etiology of a number of human neurodegenerative diseases, most notably amyotrophic lateral sclerosis. These disorders are characterized by massive accumulations of neurofilaments in the axons of affected neurons, leading to axonal degeneration. The accumulation of neurofilaments in these diseases is thought to be caused by changes in the mechanisms of slow axonal transport which move cytoskeletal and cytosolic proteins along axons from their site of synthesis in the cell body. However, these mechanisms are poorly understood and controversial. The principal issue concerns the site of assembly of cytoskeletal proteins and the form in which they move. The polymer transport hypothesis proposes that the cell body and proximal axons are principal sites of assembly of cytoskeletal proteins and the form in which they move. The polymer transport hypothesis proposes that the cell body and proximal axon are principal sites of assembly and that cytoskeletal proteins are transported in the form of moving polymers. In contrast, the cytoskeletal proteins are transported in the form of subunits of oligomers that assemble locally along the axon and the axon tip. To test these hypotheses, the assembly and axonal transport of neurofilament proteins will be investigated in cultured neurons, which are advantageous because of their accessibility to direct observation and experimentation. The proposed experiments will address two specific aims. For Specific Aim 1, immunofluorescence and immunoelectron microscopy will be combined with quantitative digital image analysis to determine the sites of assembly of biotinylated and
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endogenous neurofilament proteins in neurons. For Specific Aim 2, novel strategies that include constriction that includes constriction of axons will be combined with direct observation of fluorescent neurofilament proteins in living cells to determine the form in which neurofilament proteins are transported. The long-term goal of this research is to determine the mechanism by which neurofilament proteins move in axons and the mechanisms that lead to the accumulation of neurofilaments in certain neurodegenerative diseases. By testing specific hypotheses on the assembly and axonal transport of neurofilaments, the studies proposed here represent an important step toward this goal. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ASSEMBLY AND FUNCTION OF BIOLOGICAL IRON-SULFUR CLUSTERS Principal Investigator & Institution: Johnson, Michael K.; Distinguished Research Professor of Chem; Chemistry; University of Georgia 617 Boyd, Gsrc Athens, Ga 306027411 Timing: Fiscal Year 2002; Project Start 01-JAN-2001; Project End 31-DEC-2004 Summary: (Adapted from applicant's abstract) Iron-sulfur clusters are present in more than 120 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. Although their primary role lies in mediating biological electron transport, iron-sulfur centers are known to constitute the active sites of numerous enzymes and to have important structural and regulatory roles. However, the functional diversity of biological ironsulfur clusters has yet to be fully defined, and the mechanism of cluster biosynthesis, which is central to cellular iron homeostasis and the regulatory roles of iron-sulfur clusters, is still poorly understood. The long-term goal of this project is a molecular-level understanding of cluster biosynthesis and of the newly emerging roles of biological iron-sulfur clusters in disulfide reduction, initiating radical reactions in Sadenosylmethionine-dependent enzymes, and providing the sulfur for biosynthesis of biotin and lipoic acid. Ultimately this will lead to enhanced understanding of iron homeostasis and human diseases related to iron overload and defects or inhibition of respiratory chain enzymes. The approach involves using molecular biology techniques to effect large scale expression and/or site-specific changes in the target enzymes and proteins, biochemical and enzymatic assays, and the application of biophysical spectroscopic techniques (electron paramagnetic resonance, absorption, magnetic circular dichroism, resonance, absorption, magnetic circular dichroism, resonance Raman, Mossbauer and mass spectrometry) that can probe the nature and detailed properties of iron or iron-sulfur centers during catalytic cycling or cluster biosynthesis. The specific systems to be investigated include the proteins involved with nitrogenfixation-specific and general iron-sulfur cluster biosynthesis in Azotobacter vinelandii, biotin synthase from Escherichia coli and ferredoxin:thioredoxin reductase from chloroplasts. The objectives are to establish the mechanism of NifU/NifS- and IscU/IscS-mediated iron-sulfur cluster biosynthesis, determine the role of the ironsulfur cluster in ferredoxin:thioredoxin reductase in mediating reductive cleavage of the active-site disulfide, characterize the cluster transformation that is responsible for providing the sulfur for biotin biosynthesis, determine the mechanism of iron-sulfur cluster-mediated reductive cleavage of S-adenosylmethionine in biotin synthase, and develop an in vitro catalytic system for biotin biosynthesis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: BINDING PROTEINS FOR COMPOUNDS WITH VITAMIN A ACTIVITY Principal Investigator & Institution: Ong, David E.; Professor; Biochemistry; Vanderbilt University 3319 West End Ave. Nashville, Tn 372036917 Timing: Fiscal Year 2002; Project Start 01-AUG-1983; Project End 31-AUG-2006 Summary: (provided by applicant): The long-term goals are to understand the roles of specific binding proteins in vitamin A absorption, transport, and metabolism. A novel retinoic acid (RA) synthesizing system, co-expressed with cellular retinoic acid binding protein (II) (CRABP(II), has been identified in the female reproductive system and in human mammary epithelium. CRABP (II) and cellular retinoic acid binding protein (CRABP) have been demonstrated to be associated with the mitochondria. Specific studies to build on these observations are: (1) to determine the distribution/regulation of this and other RA synthesizing systems in rat tissues, and to characterize the RA synthesizing enzymes in human mammary epithelium to examine defects in tumororigenic cell lines; (2) to delineate the novel mechanisms of mitochondria association of the two retinoic acid binding proteins; (3)to access the role of mitochondria in retinoid metabolism; and (4) to reveal the participation of CRABP (II) in these processes. Experimental designs and methods are: (1) sites of expression of CRABP (II) and RA-synthesizing enzymes I epithelial linings of rat tissues will be revealed by immunohistochemistry and in situ hybridization. The effect of vitamin A deficiency on expression will be examined. (2) Sequences involved in mitochondrial association will be identified for CRABP and CRABP(II). Alanine-scanning mutagenesis will define the residues essential for association. Mitochondrial association will be followed by confocal microscopy of the pattern of immunofluorescence of proteins or of peptide fusions with green fluorescent protein for FLAG tag. Sequences established as important will be chemically synthesized with a photoactivatable group and attached biotin, incubated with mitroochondria and cell extracts and cross-linked to allow identification of proteins involved in recognition. (3) Appropriate mitochondria will be tested for ability to metabolize retinoids. Metabolities will be identified, including possible novel metabolities that might served as ligands for the retinoic acid nuclear receptors. (4) CRABP(II) will be mutated to ablate targeting to the mitrochondria to see if this "loss of function" affects retinoid metabolism and movement in cells normally expressing this protein. These studies will increase our understanding of the essential role of retinoids in maintaining many epithelia, particularly epithelial prone to pathological conditions such as cancer. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: TRANSISTORS
BIOCHEMICAL
DETECTION
WITH
SINGLE-ELECTRON
Principal Investigator & Institution: Brousseau, Louis C.; Quantum Logic Devices 7801 N Lamar, Ste D-98 Austin, Tx 78752 Timing: Fiscal Year 2002; Project Start 17-JUL-2001; Project End 30-SEP-2003 Summary: (provided by applicant) This application describes the extension of singleelectron transistors (SET's) to biochemical detection. Theses devices are sensitive to even single-molecular binding events and use very little power. Fabricated with standard silicon micromachining techniques, they are rapidly and inexpensively produced, and easily adaptable to specific application requirements. This project will demonstrate their use in monitoring the coupling of biotin/steptavidin, as well as DNA oligomer hybridization in order to demonstrate the effectiveness of SET's for biological research
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(microarrays), medical diagnostics, and biochemical warfare defense. PROPOSED COMMERCIAL APPLICATION: This device will have commercial potential in the areas of point-of-care medical diagnostics, biological research, genomics, and biochemical warfare defense. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: BIOSPECIFIC POLYMER ENZYME CONJUGATES FOR DRUG DELIVERY Principal Investigator & Institution: Kwon, Glen S.; Associate Professor; None; University of Wisconsin Madison 750 University Ave Madison, Wi 53706 Timing: Fiscal Year 2002; Project Start 08-JAN-2001; Project End 31-DEC-2004 Summary: (Applicant's abstract) Once a target location has been identified for a drug, protein or gene, proper spatial and temporal control in the delivery of these molecules is a fundamental problem in biomedical engineering. In one promising approach for anticancer drugs called antibody-directed enzyme prodrug therapy (ADEPT), a monoclonal antibody (MAb)-enzyme conjugate selectively binds an antigen expressed on tumor cells, and the enzyme moiety releases drug from the subsequently injected prodrug at the target site. ADEPT is in clinical trials. However, drawbacks of MAbenzyme conjugates limit ADEPT. They express low chemical and physical stability, short blood-half life, immunogenicity and low tumor to blood ratio. Attaching a common water-soluble polymer, methoxy-terminated poly(ethylene glycol) (PEG), onto MAb-enzyme conjugates enhances stability, prolongs blood circulation and reduces immunogenicity, but with no marked increase in tumor to blood ratio. Our research will focus on biospecific polymer-enzyme conjugates and their role in ADEPT. We attached a biotinylated PEG on a model enzyme, carboxypeptidase A (CPA). A biotin moiety at a chain end of PEG may mediate several useful functions for the first time for a PEGenzyme conjugate. A biotin moiety may mediate the separation of PEG-CPA conjugate by affinity chromatography, fractionating in terms of number of attached biotinylated PEG on CPA using an immobilized monomeric avidin. A biotin moiety may tether an antibody in conjunction with biotin and streptavidin. Lastly, a biotin moiety may bind a clearing agent (e.g., streptavidin) in blood, an interaction that may mediate the clearance of biotinylated PEG-CPA conjugate by the liver and an increase in its tumor to blood ratio. The specific aims of the proposal: (1) To prepare a biotinylated PEG-CPA conjugate with controlled levels of biotinylated PEG at varied molecular weight by reductive amination and by affinity chromatography with immobilized monomeric avidin. (2) To study the catalytic activity and the stability of fractionated biotinylated PEG-CPA conjugates. (3) To study the immunogenicity and the plasma profile of fractionated biotinylated PEG-CPA conjugates in mice, focusing on their clearance by streptavidin. (4) To tether an IgG1 (174H.64) together with biotin and streptavidin on biotinylated PEG-CPA conjugate with optimized properties, purify the conjugate to obtain a 1:1 complex, study its stability, and assess target cell binding in vitro. (5) To study the plasma profile and the biodistribution of antibody-biotinylated PEG-CPA conjugates ("active" targeting) and biotinylated PEG-CPA conjugates ("passive" targeting) in tumor-bearing mice (KLN-205), assessing the effect of injected streptavidin. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: BIOSYNTHESIS AND NOVEL FUNCTIONS OF FE-S CLUSTERS Principal Investigator & Institution: Huynh, Boi-Hanh V.; Professor of Physics; Physics; Emory University 1784 North Decatur Road Atlanta, Ga 30322
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Timing: Fiscal Year 2002; Project Start 30-SEP-1992; Project End 31-MAR-2005 Summary: (provided by applicant) Fe-S proteins are a group of functionally diverse proteins that contain prosthetic groups composed of Fe and inorganic sulfur of various structures, termed Fe-S clusters. In addition to the well-established role of electron transport, Fe-S proteins are involved in a diverse range of non-redox processes including sensing and regulatory functions. In this application, we propose to employ a combined spectroscopic/rapid-kinetic approach to investigate the biosynthesis of Fe-S clusters and to study the newly discovered functional role of Fe-S cluster in stabilizing radical intermediates. It has been established that a pair of the nitrogen fixation gene products, NifU and NifS, are essential for the assembly of the Fe-S clusters for the nitrogenase enzyme system. Homologs of NifS and NifU, termed IscS and IscU, respectively, are found in a wide spectrum of living organisms ranging from bacteria to human, and thus, have been proposed to be involved in the general assembly/repair of Fe-S clusters in biology. Here, experiments are proposed to investigate the mechanism of Fe-S biosynthesis and to establish the roles play by NifU/NifS and IscU/IscS in this important biological process. For the purpose of enhancing our understanding of Fe-S cluster functions, three functionally diverse proteins were chosen for the proposed studies: pyruvate formate-lyase activating enzyme (PFL-AE), ferredoxin: thioredoxin reductase (FTR) and biotin synthase. PFL-AE activates pyruvate formate lyase (PFL) by catalyzing the generation of a glycyl radical in PFL. FTR catalyzes the reductive cleavage of disulfide groups in thioredoxins for enzyme activations, and biotin synthase converts dethiobiotin to biotin. Evidence accumulated so far suggests that all three enzymes employ a 4Fe-4S cluster-mediated site-specific u(3)-S(2-) based chemistry for their respective functions. The proposed study is designed to evaluate the validity of this suggestion and to determine the detailed mechanistic steps involved in the catalytic cycles. The methods of choice for the proposed studies are Mossbauer and EPR spectroscopies, which are particularly suited for the study of Fe-containing proteins. Rapid freeze-quench kinetic techniques will be used to trap reaction intermediates for spectroscopic characterization and for kinetic investigations. Whenever possible, other complementary techniques, such as resonance Raman, ENDOR, and EXAFS will be used to obtain further structural information on the reaction intermediates. Site-specific variants will be engineered, produced and subjected to similar kinetic/spectroscopic investigations for the purpose of defining the functional roles of specific residues. Detailed mechanistic insights at a molecular level are expected to emerge from the proposed investigations. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CANCER THERAPEUTICS THAT ANCHOR PROTEINS TO MEMBRANES Principal Investigator & Institution: Peterson, Blake R.; Chemistry; Pennsylvania State University-Univ Park 201 Old Main University Park, Pa 16802 Timing: Fiscal Year 2003; Project Start 01-JAN-2000; Project End 31-DEC-2007 Summary: (provided by applicant): Multidrug resistant (MDR) cancer remains the primary impediment to curative cancer chemotherapy. MDR cancer cells differ from typical tumor cells by dramatically upregulating production of several factors including the drug transporter P-glycoprotein, the cholesterol binding protein caveolin, and components of lipid raft microdomains of cellular plasma membranes. These raft domains are enriched in cholesterol and sphingolipids and play key roles in signal transduction processes. The distinct composition of plasma membranes of MDR cancers may enable selective chemotherapy targeting these cancers. During the last grant cycle,
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novel small molecules were synthesized that comprise cholesterylamine covalently linked to protein ligands such as biotin. These compounds bind lipid rafts in plasma membranes of cancer cells.Treatment of cancer cell lines with a synthetic biotincholesterylamine ligand (ligand #1) and the protein Streptavidin (SA) efficiently targets SA to lipid rafts, resulting in rapid clathrin-mediated endocytosis of this protein-ligand complex. This novel system mimics penetration of cells by Cholera toxin, which binds ganglioside GM1 in lipid rafts. This project is based on the hypothesis that ligand #1 will regulate endocytosis of SA linked to endosome-activated toxins daunorubicin and exotoxin in cancer cell lines. Since ligand #1 binds lipid rafts, selective delivery of SAlinked toxins to lipid raft-rich MDR cancer cells will be investigated in vitro and in vivo in murine cancer models. The effectiveness of ligand #1 at enhancing endocytosis of Satoxins fused to neuropeptide Y, which targets specific receptors on neuroblastoma cells, will also be evaluated. This novel approach directed at enhancing endocytosis of surface receptors by targeting to lipid rafts with small molecules could address the major problem in immunotherapy of non-internalized tumor antigens. The hypothesis that ligand-regulated delivery of SA to antigen presenting cells (APCs) will stimulate immune responses will also be tested. Novel immunostimulants will be investigated by fusing SA to the ovalbumin antigen, regulating endocytosis in APCs with ligand #1, and analyzing T-cell activation. This approach could control immunostimulation at the molecular level and yield novel tools for vaccine development. Recruitment of intracellular avidin fusion proteins to plasma membranes by ligand #1 will also be studied in an effort to conditionally regulate cellular growth and death Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CANNABINOID RECEPTORS ON IMMUNE CELLS Principal Investigator & Institution: Klein, Thomas W.; Professor and Vice Chair; Medical Microbiol & Immunology; University of South Florida 4202 E Fowler Ave Tampa, Fl 33620 Timing: Fiscal Year 2002; Project Start 15-FEB-1998; Project End 31-JAN-2004 Summary: It is now clear that the brain and immune tissues have a cannabinoid system of receptors and ligands. However, it is not clear how this system is structured and how it affects human physiology and health. This is now a critical issue because the medicinal use of marijuana is gaining political and public support and is now recommended for use in AIDS wasting disease. The overall goal of our research is to understand the immunological effects of cannabimimetics and the natural function of the "immunocannabinoid" system. This proposal is a continuation of a project started in 1998 on defining the cannabinoid receptor (CBR) mRNA expression in murine immune cells. We have been able to show that immune subsets from mouse and human differ in their expression profile ranging from no expression to high expression. Furthermore, activated immune subsets modulate CBR mRNA expression. The present application is an extension of these findings. In Aim 1, we will define the distribution profile of CB1 and CB2, both mRNA and protein, in T cells, B cells, NK cells, and macrophages from mouse tissues and human PBMCs. It is our hypothesis that immune subsets have different basal levels of CBR expression commensurate with the putative role of these receptors in the immunobiology of the cell. Purified subsets will be analyzed by RTPCR, flow cytometry, Western blotting, immunoprecipitation, and biotin labeling of surface proteins. We expect to document the CBR phenotype of key immune tissues. In Aim 2, we will define the changes in the CBR profile following cell activation and possible mechanisms of these changes. It is our hypothesis that CBR gene activity and receptor expression are regulated by the gene activation sequence in immune cells.
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Subsets will be mitogen and cytokine activated and analyzed for receptor expression by protein analysis and real-time quantitative PCR. Because there are at least 5 different mRNAs associated with CB1, variations in exon usage in different immune subsets will be analyzed by transcription analysis, mRNA usage by RT-PCR and RNase protection, and 5' end mapping with 5' RACE. We expect to document changes in the CBR expression profile in activated subsets and to discover the CB1 exon usage pattern. In Aim3, we will define the putative role of the cannabinoid system in cell proliferation, TNF production, and IL-12-induced Th1 activity. It is our hypothesis that these functions are regulated by the "immunocannabinoid" sytem. The experiments proposed will increase our understanding of the imnunobiology of the cannabinoid system and shed light on the health consequences of medicinal marijuana use in HIV disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CHEMOSENSORY TRANSDUCTION Principal Investigator & Institution: Van Houten, Judith L.; Professor and Chair; Biology; University of Vermont & St Agric College 340 Waterman Building Burlington, Vt 05405 Timing: Fiscal Year 2002; Project Start 01-SEP-1990; Project End 31-MAR-2004 Summary: This is an application for renewal of funding study chemical sensing in Paramecium. This organism is amenable to multiple techniques for dissecting chemosensory transduction mechanisms in chemoreceptor cells: biochemistry, molecular biology, transmission genetics, electrophysiology, behavioral analysis of populations and individual cells. After we disrupt the cell and study its transduction components in vitro, we can often return our study to the level of the intact cell and ask whether our findings have relevance for the physiology of the cell. Also, we know the identity of stimuli, which bind to distinct receptors. In the past funding period, we have accomplished most and gone beyond some of our original specific aims which were to apply molecular, immunological, biochemical and fluorescent dye techniques to the study of the cAMP receptor and the plasma membrane calcium pump, which appears to participate in transduction pathways. We have partial clones of the cAMP receptor gene, and the full length gene for the plasma membrane calcium pump. We have subcloned and studied the pump's calmodulin binding domain. Highlights include There are not 1 but at least 3 transduction pathways, 2 of which involve the calcium pump; attractant stimulus NH4CI has no receptor and rapidly alkalinizes the cell; while all attractant stimuli hyperpolarize the cell, only glutamate increases cAMPI and very rapidly; some chemoreceptors probably are GPI anchored and not integral membrane proteins; biotin is an attractant and its receptor likely is GPI anchored cloned; attractants hyperpolarize cells and the conductances initiated by biotin and acetate have been partially characterized. There is a sustained outward conductance (possibly the pump current) and a large inward conductance for the off-response in biotin; there is an initial inward calcium and outward K conductance in acetate with a small inward conductance for the acetate off-response. The calcium pump appears to sustain the hyperpolarization in both biotin and acetate. We used mutants with conductance defects to test these ideas. We propose to test three hypotheses that arise from our studies. 1) Chemoreceptors for biotin and glutamate are GPI anchored proteins. 2) Chemosensory transduction in 2 pathways in modulates a calcium pump to generate a hyperpolarizing conductance. Gene structure suggests that these modulations involve PKA, PKC or calmodulin. 3) Biotin and acetate induce distinct sets of Conductances, which we will test using voltage clamp analysis of normal, mutant and transformed cells. We will use electrophysiological, biochemical, molecular biology, and immuno-techniques in a
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collaborative effort between two laboratories. These studies should inform the research of others who study sensory transduction in olfaction, taste, chemotaxis and other signaling stems. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CHIRAL CATALYSTS DESIGNED TO CATALYZE ORGANIC REACTIONS Principal Investigator & Institution: Jacobsen, Eric N.; Professor; Chemistry and Chemical Biology; Harvard University Holyoke Center 727 Cambridge, Ma 02138 Timing: Fiscal Year 2002; Project Start 01-JAN-1991; Project End 31-DEC-2005 Summary: (provided by applicant) This program has as its objectives the discovery, development, and application of selective catalytic reactions of use in organic synthesis. In particular, we seek to identify catalysts for asymmetric reactions of broad synthetic utility, to elucidate the reaction mechanisms of these reactions, and to illustrate their utility through their application in the efficient synthesis of useful building blocks and complex targets. The ultimate goal of this effort is to advance the field of organic synthesis through the development of truly practical catalysts that will find application in both industry and academia for the enantioselective synthesis of biologically important compounds. Four major projects are outlined in this application. We propose to develop novel multimeric metal catalysts that operate on the basis of cooperative reactivity. This will be accomplished through the preparation of conformationally constrained oligomeric complexes wherein the relative orientation of catalyst units can be tuned through conformational control of the linker units. We will also explore the possibility of generating synthetically accessible self-assembling multimeric catalysts that display cooperative reactivity. In a second project, we plan to test a new hypothesis for asymmetric catalysis wherein two different chiral catalyst complexes function cooperatively to catalyze nucleophile-electrophile addition reactions. The goal of this effort is to establish whether reinforced stereoinduction and reactivity effects may open the door to valuable new reactions. A third project is focused on the investigation of the mechanism and scope of a novel class of non-metal catalysts. We recently applied a parallel library approach to the discovery of extraordinarily general non-metalcontaining catalysts for imine hydrocyanation. Preliminary studies indicate both a fascinating enzyme-like mechanism of action of these novel catalysts, and a highly promising level of generality in other synthetically useful reactions. In a fourth area of effort, we plan to investigate new classes of chiral catalyst systems, and two new research directions are described. One involves the design of effective chiral imidazolium carbene ligand systems for late-transition metal catalyzed reactions. A second is focused on the discovery of self-assembling biomimetic oxidation catalysts, and their development into synthetically useful systems for enantioselective epoxidation of alkenes with hydrogen peroxide. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CORE--ELECTROPHYSIOLOGY AND HISTOLOGY Principal Investigator & Institution: Yoshikami, Doju; University of Utah Salt Lake City, Ut 84102 Timing: Fiscal Year 2003; Project Start 01-JAN-2003; Project End 31-DEC-2007 Summary: The electrophysiology component of this Core will screen conotoxins for their functional activities against known ion channel targets as well as identify possible new targets for novel conotoxins. This will be pursued at three levels, with each
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succesive level having decreased scope but increased specificity. A) Extracellular recording from skeletal muscle and motor, sensory, and sympathetic nerves in isolated tissue preparations will be used to obtain a global assessment of a toxin's activity. B) Whole-cell voltage clamping of dissociated neurons will be used to focus in on the general nature of the channel affected by the toxin (e.g., Navs K; fast- vs slowinactivating, etc.). C) The specific channel isotype targeted by the toxin will be pinpointed by examining the toxin's effect on cloned channels expressed in Xenopus oocytes. Levels B and C will also address the mechanism of toxin-action. The histology component of the Core will identify the sites of conotoxin binding in excitable tissues by light microscopy. Toward this end, conopeptides will be labeled with the following reporter groups: (a) fluorophores, for direct viewing by fluorescence microscopy; (b) 125I, for use with autoradiography and viewing by dark field microscopy; and (c) biotin, for use with avidin conjugated to HRP for histochemical staining and avidin conjugated with fluorophores for fluorescence microsocopy. These experiments assessing the locations of the cellular and subcelluar binding sites of toxins will complement the physiological experiments assessing the toxins' effects on target function. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CORE--IMAGING AND MICROSCOPY Principal Investigator & Institution: Kim, Ji-Yong Julie.; University of Illinois at Chicago 1737 West Polk Street Chicago, Il 60612 Timing: Fiscal Year 2002; Project Start 02-MAY-2002; Project End 31-MAR-2007 Summary: The goal of the Imaging and Microscopy Core is to provide assistance with histological tissue processing, in situ hybridization assays, immunofluorescent staining, and image analysis. Training opportunities in modern histochemical methodology will also be available. By providing these services as well as reagents, the core will facilitate progress toward each project proposed by the PI's/Co-PI's of this Center grant. A centralized core will provide standardized results and savings in time, costs and invaluable team resources. The Imaging and Microscopy Core will: 1. Provide equipment and technical services for the acquisition of frozen and fixed tissue sections. The investigators provides the tissue and the Core than has the microtomes, cryostat, dry ice, and other supplies needed to cut the tissue. 2. Provide a "Tissue-In-Slide-Out" service for frozen or fixed tissues. For immunocytochemistry (ICC), the investigator would provide the primary antibody and the experimental tissue. Detailed protocols for immunohistochemical (both fluorescence and avidin-biotin complex (ABC) staining) and in situ hybridization histochemical techniques will be available for those who wish to do the processing themselves. A fully automated tissue processor and computerized staining system will be purchased. 3. Provide a bank of baboon and human tissues for histochemical processing. Frozen sections and plastic or paraffin embedded tissues will be available. 4. In liaison with the Tissue Procurement/Cell Culture Core, the Imaging and Microscopy Core will fix human endometriosis tissues as needed and provide paraffin embedded sections. 5. Provide facilities for image processing and data analysis through state of the art image analysis equipment, including a state-of-the-art SPOT digital camera that is linked to a PC based imaging analysis system (Image Pro Plus 4.o). The microscope is equipped for bright field, dark field, and immunofluorescent analysis. 6. Provide training in immunohistochemistry, and digital image capture, processing and analysis. Dr. Harold Verhage will also provide consultation in immunohistochemistry, morphological analyses, and tissue processing. 7. Prepare tissue samples for investigators who wish to use confocal microscopy and/or electron microscopy at the
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Research Resources Center (RRC) of the University of Illinois, which is fully equipped with state-of-the-art equipment. 8. Future development in conjunction with the Laser Capture Mcrodissection (LCM) facility. As a future development, we will become conversant in the use of this machine and ultimately provide services for the appropriate preparation of tissue samples to used with the LCM. This technique requires special preparation and fixation of tissues to optimize single cell analysis at the molecular level. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CORE--IMMUNOLOGY Principal Investigator & Institution: Witztum, Joseph L.; Professor; University of California San Diego 9500 Gilman Dr, Dept. 0934 La Jolla, Ca 920930934 Timing: Fiscal Year 2002; Project Start 01-APR-2002; Project End 31-MAR-2007 Summary: (provided by the applicant): Hybridoma Core (Scripps): The responsibilities of the Hybridoma Core are to generate, characterize and maintain monoclonal antibodies for use by all investigators. Specific services include: 1) immunization and bleeding of mice; 2) cell fusion; 3) primary and secondary screening assays (in collaboration with UCSD as needed); 4) cell cloning; 5)ascities fluid production; 6) cell maintenance, freezing and storage; 7) cataloging antibody storage; 8) antibody purification (in collaboration with the UCSD facility); and 9) Immunoglobulin heavy chain identification. Detailed methodologies for each of these techniques have been published previously. More than 250 hybridomas have been generated, cloned, characterized and maintained over the lifetime of this SCOR collaboration, and more than 24 in the last 4 years. Immunology Core (UCSD): The Immunology component at UCSD is responsible for preparation of immunogens used to generate antisera, as well as those used to immunize mice at the Hybridoma Core at Scripps. We use guinea pigs almost exclusively for antisera generation as we have found that they reliably yield high-titered antisera. In fact, we routinely use only 2 guinea pigs per immunization. The Core lab immunizes guinea pigs and determines titers, characterizes the pre- and postimmune antisera, purifies specific immunoglobulins when necessary and stores and catalogues them. Antigens range from isolated apoproteins to fusion proteins to putative receptors, etc. Often such highly purified antisera prove superior even to monoclonal antibodies, for example for cell-injection studies conducted by Drs. Glass, Rosenfeld and colleagues. The UCSD component of the Core also prepares a wide variety of immunological reagents to be used by the other Units, such as primary or, secondary antibodies to be used in immunoassays or Western blots or FACS analysis, and also participates in the purification of antibodies as needed. In addition, the Core will facilitate the selection of polypeptides to be synthesized to make reagent-specific antibodies, which are generated by conjugation to appropriate carriers, such as KLH. For example, we generated peptide-specific antibodies to macrosialin as noted above. During the past four and a half years, we have used 226 guinea pigs to generate antisera to 113 antigens for use by SCOR investigators. We also label antibodies with enzymes, such as alkaline phosphatase or with biotin which can then be used by the different Units for various chemiluminescent assays, Western blots, or for FACS analysis. Finally, an important component of the Immunology Core is to assist investigators and to train new postdoctoral fellows in immunological techniques, both by providing reagents as well as assistance in setting up assays and instructions in use of the chemiluminescent methodologies that we have adapted for all assays. For example, we have utilized commercial reagents for analysis of cytokines, and by use of chemiluminescent techniques greatly increased the sensitivity and linearity of the assays.
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Biotin
Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: COVALENTLY REACTIVE CD4 MIMETICS FOR INHIBITION OF HIV Principal Investigator & Institution: Hanson, Carl V.; Research Scientist; Public Health Foundation Enterprises City of Industry, Ca 90638 Timing: Fiscal Year 2004; Project Start 01-FEB-2004; Project End 31-JAN-2006 Summary: (provided by applicant): Therapeutic administration of soluble CD4 is theoretically an attractive means of competitively inhibiting the binding of HIV to its primary cellular receptor, but was a failure in clinical trials more than a decade ago. We propose the creation of novel CD4 analogs to overcome the stoichiometric and affinity limitations of that initial approach. Using electrophilic phosphonate ester probes, we recently discovered that HIV gp120 contains activated nucleophilic amino acids. In the proposed studies, CD4 (and CD4 peptide mimetic, s) will be modified by the addition of activated electrophilic phosphonates, which will result in specific covalent bonding of these constructs to nucleophilic sites on gp120 on the surface of HIV virions. Such an "infinite" affinity interaction will result in irreversible blocking of HIV infectivity. The concept is based on our successful analogous work on catalytic antibodies, in which nucleophilic immunoglobulin sites bind to electrophiles in the substrate ligand. Thus the Specific Aims of this proposed Innovation Grant are (1) to create covalently reactive CD4 and CD4 peptide mimetics; (2) to determine the potency, intra- and intersubtype breadth and irreversibility of HIV neutralization and inhibition of cell-to-cell spread of HIV infection via cell fusion by these covalently reactive CD4 analogues; (3) to demonstrate the irreversibility of binding of the constructs to gp120 and to intact virions; and (4) to determine the role in the virus neutralization that isplayed by the covalency (vs. conventional noncovalent antibody binding). Successful results should lead tofollow-up testing in primate models and human clinical trials. The proposed studies may also help to definecovalent and pseudocovalent forces as a novel mechanism of protein-ligand interactions. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: DEFERIPRONE THERAPY FOR SICKLE CELL DISEASE Principal Investigator & Institution: Olivieri, Nancy F.; Director, Hemoglobinopathy Program; Uhn Toronto General Hospital 200 Elizabeth St, Ccrw1-800 Toronto, Timing: Fiscal Year 2001; Project Start 30-SEP-1996; Project End 31-AUG-2004 Summary: (Adapted from applicant's abstract) This research study is designed to determine if administration of the orally active iron chelator deferiprone can ameliorate the chronic hemolytic anemia of sickle cell disease by inhibiting iron-induced oxidative damage to the sickle erythrocyte membrane and decreasing red cell destruction. The cytoplasmic surface of sickled cells has been shown to carry abnormal deposits of free iron, capable of generating free hydroxyl radicals that induce protein thiol oxidation and lipid peroxidation leading to cation leak, cell dehydration, reduced erythrocyte deformability, and premature red cell destruction. Removal of iron from the red cell membrane would be expected to reduce the generation of hydroxyl radical, and represents a novel approach to the therapy of sickle cell disease. Preliminary studies with the orally active iron chelating agent deferiprone (L1) have demonstrated the utility of this agent in the removal of free iron deposits from membranes of red blood cells in vitro and in vivo. In the proposed studies the dose, schedule of administration, and pharmacokinetic profile of deferiprone that will be most effective in the removal of
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erythrocyte membrane free iron and that which achieves maximal sustained plasma drug concentrations of deferiprone will be established, and improvement in biotin red cell survival, ferrokinetic measurements of erythron transferrin uptake, and abnormalities associated with oxidative denaturation of hemoglobin and lipid peroxidation within red cells in patients with sickle cell disease treated with an extended period of deferiprone under the optimal dosing regimen will then be examined. The combination of determinations of red cell survival using biotinylated erythrocytes and of ferrokinetic measurements of erythron transferring uptake will provide a comprehensive assessment of red cell production and destruction in patients with sickle cell disease, before and after extended therapy with deferiprone. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: DEVELOPMENT OF NOVEL GOLD BINDING FUSION PROTEINS Principal Investigator & Institution: Irani, Meher; Biohesion, Inc. 1208 Ne 100Th St Seattle, Wa 98125 Timing: Fiscal Year 2003; Project Start 01-APR-2003; Project End 31-MAR-2004 Summary: (provided by applicant): The ability to directly and uniformly immobilize proteins onto detection surfaces in a robust and simple way, with full retention of biological activity is a highly coveted goal in the field of bioengineering. It leads to the development of high value devices and applications which will have a tremendous impact on basic research, drug discovery, diagnostics and biodetection. An excellent way to achieve this is to use the property of known gold binding peptides to strongly adhere to gold surfaces under very mild binding conditions and to covalently attach any desired target protein to such peptides by recombinant means. The specific aims of this proposal are: a) to test the feasibility of producing gold binding peptides in Escherichia coli as fusion proteins with molecules of biological and commercial relevance such as protein A and streptavidin; b) to optimize the expression and purification conditions; c) to characterize and optimize gold binding of the fusion proteins; d) to construct and characterize biosensors with these recombinant fusion proteins. The long-term aim of this project is to expand the expression capabilities to a host of other molecules of biological and commercial significance and develop biosensing devices and assays for a wide variety of applications of considerable commercial potential. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: DIPYRIDAMOLE/MAGNESIUM TO IMPROVE SICKLE CELL HYDRATION Principal Investigator & Institution: Kalinyak, Karen A.; Medical Director; Children's Hospital Med Ctr (Cincinnati) 3333 Burnet Ave Cincinnati, Oh 45229 Timing: Fiscal Year 2003; Project Start 11-JUL-2003; Project End 31-MAR-2008 Summary: Vaso-occlusivc episodesare common among patients with sickle cell anemia (SCA), causing pain and chronic organ damage. SCA is characterized by the presence of dense, dehydrated sickle red blood cells (SS RBC), which are rheologically abnormal and are selectively trapped during vaso-occlusion. Strategies to prevent cellular dehydration would offer important therapeutic options that might decrease vaso-occlusive episodes. SS RBC dehydration results from cation depletion mediated by two cation transport systems, a sickling-induced (SI) leak pathway and the KCI cotransporter (KCC). Previous work at this Center has shown that di-pyridamole inhibits the SI fluxes of Na, K and Ca in vitro. Increasing cellular magnesium inhibits KCC activity and increases cellular hydration in animal models of SCA. A small clinical study in SCA patients
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Biotin
demonstrated that Mg supplementation increased cellular Mg, reduced KCC activity and improved red cell hydration. This study will test the hypothesis that significant reduction in SS RBC dehydration will be seen in patients with SCA treated with either dipyridamole or magnesium. An additive, and possibly synergistic, effect on dense cell formation is hypothesized in patients treated simultaneously with both agents. A prospective, randomized, crossover, repeated measures design will be conducted among 48 patients with SCA, ages 12 years and older. Patients will be recruited from the Cincinnati Comprehensive Sickle Cell Center and the Sickle Cell Program at Wayne State University in Detroit. This design will allow for efficient comparison of the three treatment options; dipyridamole alone, magnesium alone or a combination of both. We anticipate that these therapies will be well tolerated by the patients. Primary outcome measures include the number of dense cells, assessed by automated cell counting and phthalate density gradients, cellular cation content, cell volume and hemoglobin concentrations. Using the biotin label technique pioneered in Cincinnati, measurements of red cell survival and rate of dense cell formation will be made in six patients in each treatment group, and will shed light on the mechanisms underlying SS RBC dehydration and its postulated inhibition by dipyridamole and Mg. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ELECTRON PARAMAGNETIC RESONANCE SPECTROMETER Principal Investigator & Institution: Warncke, Kurt; Associate Professor; Physics; Emory University 1784 North Decatur Road Atlanta, Ga 30322 Timing: Fiscal Year 2003; Project Start 01-APR-2003; Project End 31-MAR-2004 Summary: (provided by applicant): The objective of this application is to request a Bruker BioSpin Corporation Elexsys E560 continuous-wave electron paramagnetic resonance (EPR) spectrometer with 10" electromagnet/12 kW power supply that will operate at X-band (9-10 GHz) and Q-band (34 GHz) microwave frequencies, and include the electron-nuclear double resonance (ENDOR, TRIPLE) accessories for X- and Q-band. Also requested are cryostats for operation at liquid helium and nitrogen temperature, to adjust to the different relaxation properties of the paramagnets, and a goniometer for single crystal studies. The new instrument would replace an aging EPR spectrometer system. The user group includes the PI, an experienced EPR spectroscopist, and three other Major Users and two Minor Users. The instrument will support the NIH-funded research of four users (5 R01 grants), the pending NIH-funded research of one user, and the preliminary studies of another. The proposed EPR/ENDOR projects include the following: (a) determination of the molecular mechanism of electrondeficient radicalmediated catalysis in coenzyme B12-dependent enzymes, (b) investigation of the emergent novel biological functions of iron-sulfur clusters in biotin synthase, pyruvateformate lyase activating enzyme, and ferredoxin:thioredoxin reductase, in combination with Mossbauer spectroscopy, (c) the mechanism of biotransformation of aromatic hydrocarbons by toluene monooxygenases, by examining metal site structure in single crystals in combination with protein X-ray crystallography, (d) the structure and function of flavoenzymes, (e) determination of the role of divalent metals in the mechanism of fibrillogenesis in amyloid formation, and (f) characterization of the mechanism of radical-mediated DNA strand scission and histone modification by novel organometallic complexes. Operation at Q-band is justified by the need for enhanced resolution of both EPR and ENDOR spectra, and the dual X- and Q-band operation is essential for success of multi-frequency approaches to constrain spectral simulation analyses. The projects supported by the requested EPR spectrometer cover investigations of the wide range of recognized biological radical reactivity, from
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"programmed" essential radicals in enzymes, to deleterious effects of free radicals and metals, to the use of radical reactivity as a therapeutic tool. A sustained high impact of the EPR spectrometer on biomedicine is therefore anticipated. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ENHANCED T AND B CELL RESPONSES VIA RECOMBINANT PROTEINS Principal Investigator & Institution: Gosselin, Edmund; Assistant Member; Alld Health Education Programs; Albany Medical College of Union Univ Albany, Ny 12208 Timing: Fiscal Year 2001; Project Start 01-AUG-2000; Project End 31-JUL-2004 Summary: (Adapted from Applicant's Abstract) A safe and successful vaccine against HIV will likely require the simultaneous priming of both cellular and humoral immune responses, and will preferentially involve the use of recombinant proteins. Targeting immunogens to Fc gamma receptor type I (FcgRI) on antigen presenting cells (APC) significantly enhances T cell activation in vitro, and antibody production in vivo. In addition, it can also lead to simultaneous priming of both cytotoxic and helper T cell responses. Furthermore, by combining the administration of antigen with cytokines, T cell activation can be further enhanced, and T cell subset development modulated. It has also been demonstrated that targeting antigen (Ag) to FcgRI on APC can eliminate the need for traditional adjuvant, easing difficulties associated with vaccine preparation and distribution. Therefore, developing a strategy which facilitates antigen targeting to APC, and the use of cytokines in vaccines, is likely to have a significant impact on current vaccine technology, in particular as it applies to HIV. We propose to utilize molecular techniques, and FcgRI-specific constructs, to create and test the ability of a prototype two component (modular) immune targeting system to stimulate enhanced humoral, CD4 helper T cell, and CD8 cytotoxic T cell responses in vitro and in vivo. Components will consist of a humanized divalent FcgRI-specific biotin-binding targeting element, and biotinylated functional elements including Hepatitis B Ag, gp120 Ag, and IL-2. The ability of the two component immunogens to modulate human CD4 and CD8 T cell responses in vitro, and murine B cell, CD4 T cell, and CD8 T cell responses in vivo, will be examined. In the latter instance, transgenic mice that express human FcgRI will be immunized with two component immunogens. Following immunization, CD4 and CD8 T cell responses, as well as the generation of Ag-specific antibody will be measured. These studies will provide a novel and safe approach for simultaneously priming humoral and cellular responses in vivo using recombinant proteins. This approach will not only provide an effective means for controlling the spread of HIV, but many other infectious organisms as well. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: ERYTHROPOEITIN PHYSIOLOGY AND PHARMACOLOGY IN INFANTS Principal Investigator & Institution: Widness, John A.; Professor; University of Iowa Iowa City, Ia 52242 Timing: Fiscal Year 2002; Project Start 01-APR-2002; Project End 31-MAR-2003 Summary: The many red blood cells (RBC) transfusions administered to preterm infants as treatment for anemia are expensive and pace them at risk for blood-borne infections and other transfusion related complications. The long-term objectives of Project #1 is to develop effective strategies for optimally administering recombinant human erythropoietin (r-HuEPO) in the treatment of anemia of prematurity. Achieving this
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Biotin
goal is important and timely because r-HuEPO remains an evolving clinical therapy without a clear consensus of how, when and for whom it should be used in these patients. Our objective will be accomplished by performed mechanistically based EPO pharmacokinetic (PK) and pharmacodynamic (PD) studies in sheep and human subjects using sensitive and accurate tracer methodologies. Achieving maximal stimulation for erythropoiesis relies in part upon developing a comprehensive understanding of EPO's thus far elusive in vivo metabolism and relies in part upon developing a comprehensive understanding of EPO's thus far elusive in vivo metabolism and its complex PK and PD behaviors. Because plasma EPO concentrations in anemic preterm infants are reduced relative to those in anemic adults, inadequate EPO production has been suggested as a primary underlying mechanism for the anemia these neonates inevitably develop and as justification for r-HuEPO treatment. Based on direct animal and indirect human data, we suggest that inadequate EPO production is not the primary on direct animal and indirect human data, we suggest that inadequate EPO production is not the primary mechanism, but that other mechanisms may be more important. The basis for this speculation is an expanding body of data indicating that EPO's in vivo disposition occurs primarily via a saturable, receptor-mediated process. As an overall hypothesis we speculate that EPO's in vivo PK and PD are determined by the number and affinity of EPO receptors (EPO-R's) located predominantly on the body's expandable pool of erythroid progenitor cells. This hypothesis is consistent with the observation that EPO's PK behavior is non-linear, that EPO elimination is 3 to 4 times greater in premature infants than adults, and that the red marrow occupies a 3- to 4-fold greater volume per kg body weight in young children than adults. Because of their sensitivity and accuracy, PK methodologies utilizing labeled EPO tracers are ideally suited for investigating EPO's non-linear behavior and for directly measuring its endogenous production rate. In addressing our proposal's three aims, 125I-r-HuEPO and biotinylated r- HuEPO and biotinylated r-HuEPO (Bio-EPO) will be administered as tracers in advanced system analysis-based PK and PD studies performed in sheep and humans, respectively. In Aim #1, methods for analysis-based PK and PD studies performed in sheep and humans, respectively. In Aim #1, methods for biotinylating r-HuEPO retaining its PK behavior and for measuring Bio-EPo in tracer amounts will be developed and validated. In Aims #2 and #3, 125I-r-HuEPO tracer methodology will first be applied in vivo experiments in sheep to determine the effect of perturbations in erythropoiesis (i.e., by bone marrow ablation and by phlebotomy-induced anemia and r-HuEPO treatment) on EPO's PK, PD, and production rate. When Bio-EP becomes available as a tracer, similar studies will be conducted in human infant and adult study groups. Knowledge gained about EPO's PK and PD will permit more optimal use of r-HuEPO thus leading to a reduction in the multiple RBC transfusions typically recovered by preterm infants. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ESSENTIAL ROLE OF BIOTIN IN CELL PROLIFERATION Principal Investigator & Institution: Zempleni, Janos; Nutritional Sci & Dietetics; University of Nebraska Lincoln Lincoln, Ne 685880430 Timing: Fiscal Year 2002; Project Start 01-MAY-2001; Project End 31-DEC-2004 Summary: Background: For many human cell types including those of the intestinal epithelium and the immune system, rapid proliferation is essential for normal function. Proliferating cells exhibit increased growth and DNA synthesis which generate nutrient demands. In particular, we have shown that rapidly proliferating lymphocytes increase the uptake of the vitamin biotin five fold, suggesting that cell proliferation generates a substantial increase in biotin demand. This demand might arise from increased
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synthesis of biotin-dependent carboxylases or from biotinylation of histones (or both). Consistent with this hypothesis are our preliminary studies indicating that biotin deficiency causes reduced proliferation and cell cycle arrest. We hypothesize that biotin has an essential role in cell proliferation in some ways analogous to the role of folate in methylation of DNA regulating the cell cycle. Long-term objectives: To elucidate the role(s) of biotin in the regulation of cell cycle progression and to characterize effects of the cell cycle on cellular biotin homeostasis. Specific hypotheses: (1) At certain phases of the cell cycle, lymphocytes increase expression of the biotin transporter gene leading to increased synthesis of transporter and increase of biotin uptake. (2) In addition to holocarboxylase synthesis, biotin is required in a biotinidase-catalyzed, cell-cycle dependent biotinylation of histones. Specific aims: (1) To quantitate biotin transporter mRNA and transport activity in lymphocytes at various phases of the cell cycle. (2) To quantitate biotinidase mRNA, cellular biotinidase activity, and biotinylation of histones at various phases of the cell cycle in lymphocytes from healthy adults and, for purposes of comparison, from biotinidase-deficient patients. (3) To investigate the intracellular trafficking of biotin and carboxylase activities. (4) To characterize the effect of extracellular biotin concentration in the culture medium on the proliferation rate of lymphocytes. Benefits: The proposed research will likely lead to novel insights into the mechanisms that generate the increased biotin requirement of proliferating cells. These studies are the initial step to determine whether biotin plays an important role in histone modification and cell cycle regulation. The basic knowledge generated may well be relevant to teratogenesis and immune dysfunction caused by biotin deficiency. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: FOURIER OLIGOSACCHARIDES
TRANSFORM
MASS
SPECTROMETRY
OF
Principal Investigator & Institution: Lebrilla, Carlito B.; Professor; Chemistry; University of California Davis Sponsored Programs, 118 Everson Hall Davis, Ca 95616 Timing: Fiscal Year 2002; Project Start 01-MAY-1993; Project End 31-DEC-2002 Summary: The analysis of oligosaccharide heterogeneity will be addressed by the development of several complementary analytical methods based on Fourier transform mass spectrometry. Oligosaccharides are involved in a host of biological functions including cell-cell and cell-matrix recognition, hormonal actions, inter- and intracellular trafficking, and protection. However, unlike DNA and proteins where sequence provides nearly all the primary structure, oligosaccharides are characterized by their sequence, linkage, and stereochemistry. Additionally, the large diversity in the monosaccharides due to chemical modification and isomerism, the labile nature of the glycosidic bonds, and the poor intrinsic basicity all combine to make the structural elucidation of oligosaccharides significantly more difficult than other biopolymers. There is currently no analogous method for oligosaccharides with the sensitivity, reliability and accuracy of the Edman degradation for proteins. We propose to develop analytical methods to rapidly elucidate structures in oligosaccharide libraries. The methods will be developed in the study of the jelly coat of the South African toad, Xenopus laevis. X. laevis is an important model for the study of the early stages of fertilization. The jelly coat plays a role in several important processes including the block to polyspermy, prevention of cross fertilization and protection. Collision induced dissociation will be employed to determine structures of the released oligosaccharide alditols. A catalog of structural motifs with their corresponding CID fragmentation pattern (an oligosaccharide fingerprint) will be produced using the structures of known oligosaccharides. From this catalog, the structure of the minor components will be
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determined. Alkaline degradation (AD) will be used to obtain linkage and sequence information of unknown oligosaccharides. AD coupled with matrix-assisted laser desorption/ionization and Fourier transform mass spectrometry has recently been demonstrated in this laboratory to provide sequence and linkage information on a host of model compounds. The method will be further refined for greater sensitivity and shorter analysis time. It will also be coupled with electrospray ionization. This method will be tested with the oligosaccharides of X. laevis. However, its application is aimed towards libraries where little structural information exists. Finally, a method will be developed to eliminate the tedious process of separation prior to analysis. Strong biotinavidin interaction will be the basis for the development of an analyte specific MALDI probe. A probe with immobilized avidin will be used to extract biotinylated oligosaccharides directly from solution for immediate MALDI-FTMS analysis. This method bypasses the long separation process necessary for the isolation of released oligosaccharides. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GATING MEMBRANES
OF
PROTEIN
CHANNELS
IN
LIPID
BILAYER
Principal Investigator & Institution: Finkelstein, Alan; Physiology and Biophysics; Yeshiva University 500 W 185Th St New York, Ny 10033 Timing: Fiscal Year 2002; Project Start 01-JAN-1981; Project End 31-DEC-2004 Summary: This abstract is not available. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GFI-PROTEINS IN HEMATOPOIETIC DEVELOPMENT Principal Investigator & Institution: Orkin, Stuart H.; Professor; Dana-Farber Cancer Institute 44 Binney St Boston, Ma 02115 Timing: Fiscal Year 2004; Project Start 01-DEC-2003; Project End 30-NOV-2008 Summary: (provided by applicant): Growth Factor Independent (Gfi)-genes, Gfi-1 and Gfi-lb, are sites of retroviral insertions in murine lymphomagenesis and encode zincfinger transcription factors. Homologs of Gfi-proteins in C. elegans and Drosophila regulate cell fate decisions in the nervous system. Within the mammalian hematopoietic system Gfi-1 and Gfi-1 b are expressed in a complementary pattern with Gfi-1 highly expressed in granulocytic and lymphoid cells, and Gfi-lb within erythroid and megakaryocytic lineages. Gene targeting experiments from this laboratory demonstrated that Gfi-1 is essential for granulocyte maturation and for down-regulation of macrophage properties. Gfi-lb is required for both erythroid and megakaryocytic differentiation, resembling in some aspects of GATA-1 deficiency. The overall goal of this proposal is to use a combined genetic and biochemical approach to link the Gfifactors to specific regulatory networks and pathways within hematopoietic stem cells (HSCs) and their progeny. Aim 1 focuses on genetic dissection of functions of the Gfiproteins in hematopoiesis. This will involved a detailed structure-function analysis of Gfi-1 and Gf-lb using rescue of gene targeted cells or mice, assessment of interchangeability of these factors through targeted gene knock-ins in mice, evaluation of the role of a novel isoform of Gfi-1 b (designated Gfi-lbL), and determination of the effects of Gfi-1, Gfi-lb, or combined deficiency on HSC competitive repopulation. Aim 2 focuses on biochemical approaches to the role of Gfi-proteins in hematopoietic cells. A method for in vivo biotinylation of expressed proteins will be used to facilitate efficient purification of proteins associating with Gfi-1 in myeloid cells and with Gfi-lb in
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erythroid and megakaryocytic cells. Gfi-associated proteins will be identified by massspectrometry microsequencing. Candidate interacting proteins will be further validated by coimmunoprecipitation assays and permit the Gfi-proteins to be placed within known regulatory pathways. In a second approach direct target genes of the Gfiproteins will be sought by chromatin-purification from cells expressing biotin-tagged Gfi-proteins. Candidate target DNA regions will be identified by sequencing and will be validated by conventional chromatin-immunoprecipitation assays. The significance of target genes will be further assessed using gene targeted cells and mice. This research program should elucidate how Gfi-proteins control hematopoietic differentiation and how their activation contributes to oncogenesis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GLYCOLIPID-SPECIFIC T LYMPHOCYTES Principal Investigator & Institution: Bendelac, Albert S.; Professor; Princeton University 4 New South Building Princeton, Nj 085440036 Timing: Fiscal Year 2003; Project Start 01-DEC-2002; Project End 30-NOV-2007 Summary: Glycosylceramides perform important immunological functions as T cell antigens presented by CD1, a family of lipid-binding MHC-like molecules. For example, alphaGalactosylCeramides (alphaGC) specifically activate regulatory NKT cells which modulate, through the release of either Th1 or Th2 cytokines, various disease conditions, and other glycosylceramides may elicit adaptive T cell responses involved in Multiple Sclerosis. Thus, the program project uses glycosylceramides as a prime model antigen family to explore fundamental issues of glycolipid antigen presentation by CD1 molecules with potential clinical applications. The project brings together several new tools and methodologies developed in a multidisciplinary collaboration. Dr. Savage (Project #1) is generating an extended set of synthetic glycolipids modeled around alphaGC whose physical and structural interactions with CD1d and TCR are studied by Dr. Teyton (Project #2), and which we will use in this proposal to systematically explore, in the mouse system, antigen presentation by CD1d and the corresponding TCR repertoire and T cell functions. The specific aims are 1) To study T cell recognition of glycosylceramides in vivo using CD1d tetramer technology, and compare their adjuvant effects; 2) To study the Th1 vs Th2 modulatory properties of selected alphaGC variants in IDDM, EAE and rejection of liver metastasis; 3) To study how glycosylceramide structure dictates intracellular trafficking and access to endosomal compartments, using fluorochrome and radio-labeled glycolipids and developing new methodologies for the detection and imaging of CD1d-alphaGC complexes. Because glycolipids do not mutate and CD1 molecules are highly conserved, these basic studies in mice lay the foundations for the development of universal lipid-based vaccines as well as the rational design of glycosylceramide adjuvants which can modulate autoimmunity, cancer and infections. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: GONADOTROPIN ACTION Principal Investigator & Institution: Moyle, William R.; Associate Professor; Ob, Gyn and Reproductive Scis; Univ of Med/Dent Nj-R W Johnson Med Sch Robert Wood Johnson Medical Sch Piscataway, Nj 08854 Timing: Fiscal Year 2002; Project Start 01-AUG-1983; Project End 31-MAR-2006 Summary: This abstract is not available. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Biotin
Project Title: HIGH THROUGHPUT FTICR FOR MOLECULAR ANALYSIS OF CANCER Principal Investigator & Institution: Smith, Richard D.; Battelle Fellow, Chief Scientist; Battelle Pacific Northwest Laboratories Box 999, 902 Battelle Blvd Richland, Wa 99352 Timing: Fiscal Year 2002; Project Start 16-JUN-2000; Project End 31-MAY-2004 Summary: In this four year two phase (R21/R33) project we will integrate and apply new approaches for obtaining broad systems level views of protein expression in cancer research. The overall approach will advance the study of proteomes by more rapidly identifying proteins, precisely measuring the relative abundances for all detected proteins, and providing much greater sensitivity than existing methodologies. Our approach will utilize proteome-wide stable isotope and biotin labeling of cysteinecontaining polypeptides combined with new approaches that use ultra-high sensitivity Fourier transform ion cyclotron resonance mass spectrometry. The approach will be at least 2 to 3 orders of magnitude more sensitive than existing 2-D PAGE methodologies and able to rapidly identify and measure relative expression levels for thousands of proteins in a single analysis. Phase 1 of this project will integrate and provide an initial demonstration of methods that include the sample processing for mouse B16 melanoma cells from culture, validate the use of new accurate mass tag and multiplexed-MS/MS methods for protein identification, and demonstrate the precise determination of relative protein abundances for all detected proteins from B16 and B16BL6 cell populations. Phase 2 will involve the pilot application of the technology to the study of proteome changes that occur as cells progress from low or nonmetastatic states, to a highly invasive and metastatic phenotype, using the B16 melanoma system as a model. The technology to be applied will enable ultra-sensitive (attomole level, and anticipated to be better) proteome-wide precise profiling of proteins from cells maintained in culture and from tissues (obtained by micro-dissection). The results will provide an abundance of new information on protein expression, and enable precise measurements of differences in relative protein expression levels as a function of cell type, developmental stage, metastasis, etc. A product of this research will be the first application of a high throughput technology for obtaining precise proteome displays that may be expected to illuminate the complex mechanisms and pathways relevant to cancer. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: HIV ASSOCIATED NEPHROPATHY IN DRUG ADDICTS Principal Investigator & Institution: Singhal, Pravin C.; Professor; Long Island Jewish Medical Center 270-05 76Th Ave New Hyde Park, Ny 11040 Timing: Fiscal Year 2002; Project Start 01-AUG-1998; Project End 31-JUL-2003 Summary: (Applicant's Abstract) The goal of the present proposal is to determine the role of HIV and illicit drugs such as opiates and cocaine in the development of renal injury which manifests itself as focal glomerulosclerosis (FGS) and tubulointerstitial lesions (TIL). We hypothesize that HIV-I in combination with the drugs interacts with monocytes and tubular cells (TC) and mesangial cells (MC) to induce TIL and expansion of the mesangium (precursor of FGS). Tubulo-interstitial lesions, a unique feature of HIV-associated renal injury, set the pace of the progression of renal failure. To examine the role of HIV-I and drugs in both glomerular and interstitial lesions we plan to 1) study the effect of HIV and drugs on the migration of macrophages (Mphi) into the interstitium and mesangium 2) determine the effect of HIV-l/drugs and Mphi interaction products on MC proliferation and matrix accumulation, 3) examine the effect
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of HIV-1 and drugs on the migrated Mphi and proliferated MC, and 4) study the effect of HIV-l/drugs and tubular cell interaction products on kidney fibroblast (KF) proliferation/apoptosis and matrix accumulation. Transmigration of Mphi across the endothelial cell layer will be determined as well as across a gelatin coated filter. Thymidine incorporation studies will be used to evaluate MC/KF proliferation. Western blots will be used to measure the laminin, fibronectin, proteoglycan and collagen components of matrix and Northern blots to measure mRNA expression for growth/cell death related genes and matrix components. A biotin-avidin assay and zymography will be used in the measurement of gelatinolytic and stromelysin activity. To examine the effect of HIV-1 gene expression, studies for cell proliferation and matrix synthesis will be carried out on MC derived from mice transgenic for HIV-I genes. In addition, these mice will be treated with drugs and acceleration of renal cortical and medullary mRNA expression of matrix components and cytokines will be evaluated. The hypothesis for the role of HIV and drugs in the development of renal injury will be tested based on our preliminary findings of increased MC/KF proliferation, and matrix accumulation when HIV-I proteins, HIV-1 protein-Mphi/TC interaction products are added to MC/KF in culture. Moreover, our results show that HIV and MC secretory/gpl20 and TC products enhance the migration of Mphi into the interstitium and mesangium. We believe that tubulointerstitial lesions determine the accelerated course of renal failure in drug addicts with HIV infection. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: IDENTIFICATION OF CASPASE SUBSTRATES FROM HUMAN PROTEOME Principal Investigator & Institution: Liu, Rihe; Medicinal Chemistry and Natural Products; University of North Carolina Chapel Hill Office of Sponsored Research Chapel Hill, Nc 27599 Timing: Fiscal Year 2004; Project Start 01-JAN-2004; Project End 31-DEC-2008 Summary: (provided by applicant): This proposal is directed at scanning the human proteome to identify the downstream targets of caspase-2, -3 and -8 using a novel technique called mRNA display. Specifically, human proteome domain libraries displayed on their own mRNAs are generated and immobilized on the solid surface via the biotin residue specifically introduced near the N-terminus of each protein. Upon incubation with a purified caspase of interest, protein sequences that are specifically cleaved by the caspase are released and enriched, with the intact mRNA still covalently attached to the C terminus of each cleaved protein fragment. The selected protein sequences are then regenerated for iterative round of selection, by PCR amplification followed by in vitro transcription/translation, until the pool is dominated by sequences whose protein portions can be cleaved by the caspase. The identity of each protein is readily determined from its mRNA, by sequencing or cDNA microarray. To analyze the proteolysis of selected proteins by the caspase, free protein fragments and full-length proteins are transcribed/translated in vitro and incubated with the purified caspase of interest in the presence or absence of a specific inhibitor; and/or with cell-free extracts prepared from nonapoptotic and apoptotic cells. Such confirmed potential caspase downstream targets will be characterized to determine their cleavage sites using different approaches, including a focused mRNA displayed protein domain library generated by randomly priming the selected cDNA. Potential novel caspase substrates that have been demonstrated in vitro will be further analyzed in vivo, by western analysis of the potential caspase substrates in lysates from apoptotic cells. The specificity of their proteolysis will be determined by in vivo inhibitor studies. The biological
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significance of each confirmed novel caspase substrate will be studied by correlating the extent of its specific proteolysis with the degree of cell death and with the proteolysis of other important caspase substrates. The effect of over expression of the proteins on apoptosis will also be addressed. The proposed research expected to allow a systematic examination and identification of the downstream targets of caspase-2, -3 and -8 on a proteome-wide scale. It will have significant implications in understanding the molecular mechanisms that govern the caspase-induced cell death, whose malfunction or dysregulation may result in cancer, neurodegenerative diseases, or other pathological conditions. The simplicity and high throughput of the methodology involved will make the approach broadly applicable to rapid scan the human proteome for downstream targets of any other caspases. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: IDENTIFICATION OF IMMUNOPHILLINS, & CLIENT PROTEINS
HSP90
COCHAPERONES,
Principal Investigator & Institution: Blagg, Brian Sj.; University of Kansas Lawrence Youngberg Hall Lawrence, Ks 660457563 Timing: Fiscal Year 2002; Project Start 30-SEP-2002; Project End 31-AUG-2007 Summary: The 90 kDa heat shock proteins (Hsp90) belong to a family of chaperones that regulate intracellular functions and are required for the refolding of denatured proteins following heat shock as well as the conformational maturation of a large number of key proteins involved in cellular processes. This proposal outlines a novel proteomics approach toward the identification of Hsp90 client proteins, cochaperones, immunophilins, and multiprotein complexes that fold nascent polypeptides. Geldanamycin derivatives will be synthesized to contain both photolabile and nonphotolabile biotin linkers, which are expected to bind to the N-terminal ATP binding region of Hsp90 and stabilize the complex association of Hsp90, client proteins, cochaperones, and immunophilins. In the presence of ubiquitination and protease inhibitors, these Hsp90/biotinylated GDA complexes will be removed from crude cellular lysate and the multiprotein components analyzed. The identification of proteins and multiprotein complexes that are bound to Hspg0 will provide insight into the role Hsp90 plays in the maturation of regulatory pathways by the identification of Hsp90 associated proteins and could provide additional targets for cancer chemotherapy. The identification of Hsp90 client proteins will provide evidence for the rational design of future inhibitors that are selective against individual client proteins and help determine the ramifications of inhibition of the entire Hsp90 process. A derivative of geldanamycin (17-AAG) has entered clinical trials, however, it is unlikely to proceed to phase II studies due to cytotoxicity unrelated to the inhibition of Hsp90. Using the multicomponent assay described in this proposal, it is possible to identify proteins other than Hsp90 to which geldanamycin binds, providing additional controls for the development of future Hsp90 inhibitors. Using similar techniques, Hsp90 dependent proteins from various cancer cell lines will be profiled. Protein profiling of Hsp90 dependent proteins will provide additional insight into the identification of proteins that are under or overexpressed in different cancer cell lines for future applications of Hsp90 inhibition. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: IDENTIFICATION OF SUBUNIT INTERFACES IN PROTEIN COMPLEX Principal Investigator & Institution: Prevelige, Peter E.; Professor; Microbiology; University of Alabama at Birmingham Uab Station Birmingham, Al 35294
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Timing: Fiscal Year 2002; Project Start 30-SEP-2002; Project End 31-AUG-2004 Summary: (provided by applicant): The objective of this proposal is to develop a general methodology for the rapid identification of intersubunit interfaces in protein complexes, and utilize it to identify the subunit/subunit interfaces in immature and mature HIV-1 capsids, The strategy will be to synthesize a family of biotin-tagged chemical crosslinkers with both specific and non-specific cross-linking activity and use them as distance constraints to position subunits of known atomic structure (in this case the structural proteins of HIV) relative to one another in space, To achieve the necessary structural resolution the cross-linked amino acid residue pairs will be identified by mass spectrometry. This project extends the parent grant (AI44626) whose mission is to utilize hydrogen/deuterium exchange studies to identify intersubunit interfaces and dynamic motions in HIV capsids. The novel component of this proposal is the synthesis of biotin tagged cross-linkers, which can be rapidly purified using strepavidin affinity techniques coupled with mass spectrometry for identification of the cross-linked peptides. Such cross-linkers are not commercially available. Our preliminary data indicates that while the cross-linking data can be used to pack subunits, its widespread applicability is limited by the inherent difficulty of detecting a small number of cross-linked peptides against a background of a large number of uncross-linked peptides. Biotin tagged crosslinkers can be rapidly purified from the complex digests and analyzed by mass spectrometry resulting in increases in sensitivity and throughput. As a result the technique will become a generally applicable tool for merging data from the structural genomics and proteomics initiatives. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: IMAGING MALIGNANT MELANOMA WITH RADIOLABELED ALPHA-MSH PEPTIDE ANALOGS Principal Investigator & Institution: Quinn, Thomas P.; Assistant Professor; University of Missouri Columbia 310 Jesse Hall Columbia, Mo 65211 Timing: Fiscal Year 2003; Project Start 01-AUG-2003; Project End 31-JUL-2008 Summary: (Revised Abstract) (provided by applicant): This application outlines a research effort to develop and evaluate radiolabeled peptides for malignant melanoma imaging. Malignant melanoma is a serious public health problem due to an increase in its incidence and resistance to conventional chemotherapeutics and external beam radiation therapy. Early detection is critical for proper therapeutic management. There is a clear need to develop new and efficacious imaging and therapeutic agents. Our laboratory has developed a new class of metal-cyclized peptide (CCMSH) that target the a-MSH receptor present on melanoma cells. The CCMSH peptides will be radiolabeled with 99mTc, 111In for SPECT and 64Cu for PET imaging of melanoma in murine and human melanoma mouse models. Mice bearing solid tumors and metastatic melanoma in the lungs will be imaged at various times during the course of their disease to determine the detection sensitivity and specificity of the radiolabeled peptides. Micro SPECT and micro PET imaging studies will also be compared to determine the optimal combination of peptide, radionuclide and detection modality for melanoma imaging. The CCMSH imaging agents are envisioned to be part of a matched pair strategy for melanoma imaging and therapy in which the same melanoma targeting vector (CCMSH) can be radiolabeled with radioisotopes that possess imaging or therapeutic properties. We also plan to employ bacteriophage (phage) display technology to discover new melanoma targeting vectors. The random peptide libraries, displaying 5150 peptides per particle, will be selected in human melanoma bearing mice for tumor targeting peptides. In vivo selection strategy should closely simulate the complex
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targeting environment an imaging agent encounters thus allowing us to select superior melanoma avid phage. We propose to use the tumor avid phage particles themselves as targeting agents in a pretarget approach. The tumor avid phage will be conjugated with 500 kilobase pairs). Excerpt(s): This invention relates to a method for attaching nucleic acids, such as DNA, to microscopic beads or other support structures using a terminal transferase. Recent technical advancements in nanomanipulation have allowed the mechanical behavior of single DNA molecules to be studied. These techniques include the use of microspheres, magnetic beads, microfibers, microneedles, optical traps, and hydrodynamic flow. The attachment of microspheres to DNA has proven useful to manipulate DNA for placement or immobilization on a selected substrate or mechanical support, where the DNA strand can be confined in an extended conformation. Once the DNA is affixed to a substrate, a variety of processes (e.g., laser tweezers, scanning probe microscopy) can be used to sequence or map gene locations of the DNA. In addition, tethering microspheres to DNA may be useful in purification or separation methods that selectively isolate labeled or tagged DNA fragments. Conventional techniques of tethering or attaching DNA to microspheres rely on hybridization and ligation of manufactured, labeled single-stranded DNA probes to known DNA sequences. U.S. Pat. No. 5,674,743 to Ulmer discusses methods in the art for attaching the DNA to a microscopic bead and is incorporated herein by reference. One method is to first couple specific oligonucleotide linkers to the bead using known techniques, and then to use DNA ligase to link the DNA to the linker on the bead. Oligonucleotide linkers can be employed which specifically hybridize to unique sequences at the end of the DNA fragment, such as the overlapping end from a restriction enzyme site or the "sticky ends" of bacteriophage lambda based cloning vectors. Web site: http://www.delphion.com/details?pn=US06420112__
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DNA fragments containing biotin biosynthetase gene and use of the same Inventor(s): Akimaru; Jiro (Nishinomiya, JP), Mitsuda; Satoshi (Takarazuka, JP), Mukumoto; Fujio (Toyonaka, JP), Nishio; Shoichi (Toyonaka, JP) Assignee(s): Sumitomo Chemical Company, Limited (osaka, Jp) Patent Number: 6,410,293 Date filed: December 3, 1998 Abstract: A DNA fragment containing a gene concerned in biotin biosynthesis and derived from a microorganism belonging to the genus Sphingomonas, a plasmid containing said DNA fragment, and a biotin-producing transformant containing said plasmid. There is provided a technique for utilizing a gene concerned in biotin biosynthesis and derived from a microorganism belonging to the genus Sphingomonas, for breeding of a biotin-producing micro-organism by genetic engineering. Excerpt(s): This application is the national phase under 35 U.S.C.sctn.371 of prior PCT International Application No. PCT/JP98/00858 which has an International filing date of Mar. 2, 1998 which designated the United States of America, the entire contents of which are hereby incorporated by reference. The present invention relates to a DNA fragment containing at least one gene concerned in biotin biosynthesis and utilization thereof. Biotin is an essential vitamin for human beings, animals, plants and some microorganisms and is useful as a food additive for human beings or animals. As a process for producing biotin by using a microorganism, there have been known a process using a streptomyces or a micro-monospore (JP-B-41-21756), a process using a sporobolomyces (JP-B-42-3074), a process using a bacillus, a chromo-bacterium or a pseudomonas (JP-A-56-160998), a process using a sphingomonas (JP-A-6-133790), etc. There have been also proposed processes for breeding a microorganism in which a gene concerned in biotin biosynthesis and isolated from a microorganism capable of producing biotin is introduced into another microorganism by a genetic engineering technique to promote the expression of the gene concerned in biotin biosynthesis, whereby the activity of an enzyme capable of catalyzing biotin biosynthesis reaction is increased to improve the biotin productivity (JP-A-61-202686, JP-A-2-27980, JP-A-7231789, etc.). Web site: http://www.delphion.com/details?pn=US06410293__
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Fermentation process Inventor(s): Anderson; Kevin W. (Indian Springs, OH), Wenzel; J. Douglas (Cincinnati, OH) Assignee(s): Cognis Corporation (gulph Mills, Pa) Patent Number: 6,569,670 Date filed: January 4, 2001 Abstract: A fermentation medium containing: (a) a source of metabolizable carbon and energy; (b) a source of inorganic nitrogen; (c) a source of phosphate; (d) at least one metal selected from the group consisting of an alkali metal, an alkaline earth metal, a transition metal, and mixtures thereof; and (e) biotin, substantially free of particulate matter and bacteria. A fatty material may be added to an aqueous suspension containing at least one dicarboxylic acid to modify the rheological characteristics of the suspension.
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Excerpt(s): This invention relates to an improved fermentation medium and process for making an aliphatic polycarboxylic acid using said medium. Long-chain alpha, omegadicarboxylic acids, i.e., those having a carbon number of 9 or higher, are used as raw materials in the synthesis of a variety of chemical products and polymers. Diacids with carbon numbers greater than four are currently produced almost exclusively by nonbiological conversion processes. These types of chemical processes for the production of diacids have a number of limitations and disadvantages. Each process is restricted to the production of diacids of specific carbon chain lengths, based on the starting material used. For example, the dodecandioic acid process begins with butadiene, therefore the products of this reaction process are limited to acids with chain lengths in multiples of four. In addition, the processes are based on nonrenewable petrochemical feedstocks, and the multireaction conversion process produces unwanted byproducts which result in yield losses, heavy metal wastes, and nitrogen oxides which must be destroyed in a reduction furnace. Web site: http://www.delphion.com/details?pn=US06569670__ •
Galenic formulation containing biotin Inventor(s): Bunger; Joachim (Gross-Umstadt-Heubach, DE), Driller; Hansjurgen (GrossUmstadt, DE), Huschka; Christoph (Halle, DE), Motitschke; Lothar (Hilden, DE), Neubert; Reinhard (Halle, DE), Wohlrab; Wolfgang (Halle, DE) Assignee(s): Merck Patent Gmbh (darmstadt, De) Patent Number: 6,660,251 Date filed: July 27, 2001 Abstract: The invention relates to galenic formulations which contain a) one or more compounds selected from biotin, its physiologically compatible salts, biotin esters and the steroisomeric forms of these compounds; and b) one or more compounds selected from urea and urea derivatives. The above formulations are suitable for use as topically administered formulations for the cosmetic and/or medical treatment of the skin, hair and/or nails. Excerpt(s): The invention relates to topically applicable galenic formulations which contain one or more compounds selected from biotin, the physiologically tolerable salts of biotin, biotin esters and the stereoisomeric forms of these compounds and one or more compounds selected from urea and urea derivatives and are suitable for the cosmetic or medicinal treatment of skin, hair and/or nails, for example for the treatment of diseases or functional disorders of human or animal skin, hair and/or nails. The efficacy of biotin in skin, hair and nail diseases has been assessed in different ways in the past. Although, by means of continuous biotin therapy, it was possible to show the efficacy in the case of soft, brittle and splitting nails, different forms of alopecia and atopic and seborrheic dermatitis in a detectable manner, use remained sparing, as this is associated, despite no side effects at all, with the necessity of an extended treatment period of several months (R. Bitsch et al. (1994) Biotin, Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart; V. E. Colombo et al. (1990) Treatment of brittle fingernails and onychoschizia with biotin: scanning electron microscopy. J. Am. Acad. Dermatol. 23: 1127-1132; G. L. Floersheim (1989) Behandlung bruchiger Fingernagel mit Biotin [Treatment of brittle fingernails with biotin]. Z. Hautkr. 64: 41-48; G. L. Floersheim (1992) Prufung der Wirkung von Biotin auf Haarausfall und Haarqualitat [Testing the action of biotin on hair loss and hair quality]. Z. Hautkr. 67: 246-255; W. Gehring (1996) Der Einfluss von Biotin bei reduzierter Nagelqualitat. Eine
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plazebokontrollierte doppelblinde klinische Studie [The influence of biotin on reduced nail quality. A placebo-controlled double-blind clinical study]. Akt. Dermatol. 22: 20-25; U. Siebert et al. (1996) Zur Dosierung und Wirkung von Biotin bei Nagel- und Haarwachstumsstorungen [The dosage and action of biotin in nail and hair growth disorders]. Zeitschrift Hautnah 6: 438-443). It is known that topically applied biotincontaining formulations having a biotin content of 0.25% and 0.50% lead to a visible reduction in the development of wrinkles on the skin of old people (L. Gilli et al. (1995) Beeinflussung der Faltchenauspragung bei Altershaut durch topisch appliziertes Biotin [Influencing the development of wrinkles on the skin of old people by means of topically applied biotin]. Z. Hautkr. 70 (6): 419-425). In these investigations, it was not possible to show any epithelial and histopathological changes at all in the areas treated. Web site: http://www.delphion.com/details?pn=US06660251__ •
Genes encoding SCIP-1 orthologs and methods of use Inventor(s): Hendrick; Carol A. (Des Moines, IA), Hu; Xu (Urbandale, IA), Lu; Guihua (Urbandale, IA) Assignee(s): Pioneer Hi-bred International, Inc. (des Moines, Ia) Patent Number: 6,660,907 Date filed: July 10, 2001 Abstract: Compositions and methods for enhancing the resistance of plants to pests and for altering the level of biotin in plants are provided. Nucleotide sequences isolated from soybean, rice, maize, and wheat are provided. The nucleotide sequences encode orthologs of sunflower SCIP-1. Also provided are the SCIP-1 proteins encoded by such nucleotide sequences. The methods involve transforming plants with the SCIP-1 nucleotide sequences to enhance the plant's resistance to plant pests or to alter the level of biotin in the plant. Transformed plants, plant cells, tissues, and seeds are also provided. Excerpt(s): The invention relates to the genetic manipulation of plants, particularly to transforming plants with genes that enhance disease resistance. Throughout their lives, plants are routinely subjected to a variety of stresses, which act to impede or alter growth and development processes. Stresses to plants may be caused by both biotic and abiotic agents. For example, biotic causes of stress include infection with a pathogen, insect feeding, and parasitism by another plant such as mistletoe, and even grazing by ruminant animals. Abiotic stresses include osmotic stress, excessive light intensity or insufficient light intensity, cold temperatures, warm temperatures, synthetic chemicals such as those used in agriculture, and excessive wind. Because a stress negatively impacts plant growth and development processes, stress to agricultural plants has a negative economic impact expressed in the form of reduced yields, increased expenditures for pesticides or both. Developing crop plants that are better able to tolerate or even avoid stresses is desirable and will most certainly improve agricultural productivity. Given the world's both increasing human population and diminishing land area available for agriculture, improving agricultural productivity is a paramount challenge. A thorough understanding of the mechanisms used by plants to avoid or tolerate stresses may help in the development of new strategies for improving the stress tolerance of agricultural plants. Web site: http://www.delphion.com/details?pn=US06660907__
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High density array fabrication and readout method for a fiber optic biosensor Inventor(s): Albertson; Donna G. (Lafayette, CA), Gray; Joe (San Francisco, CA), Pinkel; Daniel (Walnut Creek, CA) Assignee(s): Medical Research Council (london, Gb), The Regents of the University of California (oakland, Ca) Patent Number: 6,417,506 Date filed: August 17, 2000 Abstract: The invention relates to the fabrication and use of biosensors comprising a plurality of optical fibers each fiber having attached to its "sensor end" biological "binding partners" (molecules that specifically bind other molecules to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotinavidin, etc.). The biosensor preferably bears two or more different species of biological binding partner. The sensor is fabricated by providing a plurality of groups of optical fibers. Each group is treated as a batch to attach a different species of biological binding partner to the sensor ends of the fibers comprising that bundle. Each fiber, or group of fibers within a bundle, may be uniquely identified so that the fibers, or group of fibers, when later combined in an array of different fibers, can be discretely addressed. Fibers or groups of fibers are then selected and discretely separated from different bundles. The discretely separated fibers are then combined at their sensor ends to produce a high density sensor array of fibers capable of assaying simultaneously the binding of components of a test sample to the various binding partners on the different fibers of the sensor array. The transmission ends of the optical fibers are then discretely addressed to detectors--such as a multiplicity of optical sensors. An optical signal, produced by binding of the binding partner to its substrate to form a binding complex, is conducted through the optical fiber or group of fibers to a detector for each discrete test. By examining the addressed transmission ends of fibers, or groups of fibers, the addressed transmission ends can transmit unique patterns assisting in rapid sample identification by the sensor. Excerpt(s): This invention relates to the fabrication and use of biosensors comprising biological "binding partners" (molecules that specifically bind other molecules to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc.) linked to optical fibers. Specifically, batches of optical fibers are mass processed with the same species of binding partner, singulated from their particular batch, regrouped with like optical fibers from other batches having other species of binding partners. Upon regrouping of the optical fibers, high density arrays are formed which can simultaneously interrogate samples for a multiplicity of analytes for sample identification and processing. Biosensors are sensors that detect chemical species with high selectivity on the basis of molecular recognition rather than the physical properties of analytes. See, e.g., Advances in Biosensors, A. P. F. Turner, Ed. JAI Press, London, (1991). Many types of biosensing devices have been developed in recent years, including enzyme electrodes, optical immunosensors, ligand-receptor amperometers, and evanescent-wave probes. Updike and Hicks, Nature, 214: 986 (1967), Abdel-Latif et al., Anal. Lett., 21: 943 (111988); Giaever, J. Immunol., 110: 1424 (1973); Sugao et al. Anal. Chem., 65: 363 (1993), Rogers et al. Anal. Biochem., 182: 353 (1989). Biosensors comprising a biological "binding molecule" attached to an optical fiber are well known in the prior art, most typically as evanescent wave detectors (see, for example, U.S. Pat. No. 4,447,546 to Hirschfeld and U.S. Pat. Nos. 4,582,809 and 4,909,990 to Block et al.). In order to maximize sensitivity and selectivity such biosensors typically utilize a single species of biological binding molecule affixed to the face of the sensor.
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Web site: http://www.delphion.com/details?pn=US06417506__ •
Labeled vitamin D compounds and the use thereof Inventor(s): Holick; Michael F. (Sudbury, MA), Ray; Rahul (Wayland, MA) Assignee(s): A & D Bioscience, Inc. (sudbury, Ma) Patent Number: 6,455,714 Date filed: March 19, 2001 Abstract: Biotin, fluorescent and chemiluminescent labeled vitamin D compounds are disclosed as well as their use in assays for the presence of vitamin D, its metabolites and vitamin D analogs in biological fluids. Excerpt(s): The present invention relates to non-radioactive vitamin D compounds and methods to assay for the presence of vitamin D, vitamin D analogs and their metabolites which may be present in milk, blood or other biological fluids. The assay methods employed in this invention may be enzyme linked immunoassays (ELISAs) (with biotin containing compounds) and fluorimetric and chemiluminometric assays (with fluorescein or chemiluminiscence containing compounds). It is well-established that cutaneously synthesized vitamin D.sub.3, a seco-steroid, undergoes sequential metabolic conversions to 25-hydroxyvitamin D.sub.3 (25-OH-D.sub.3) in the liver and to 1,25(OH).sub.2 D.sub.3 in the kidney. 1,25(OH).sub.2 D.sub.3, the dihydroxylated metabolite of vitamin D.sub.3, is the most active form of vitamin D hormone which is intimately involved in calcium and phosphorous homeostasis (Holick, M. F. (1989), "Vitamin D: biosynthesis, metabolism and mode of action." In Endocrinology, vol. 2, Degroot et al. (eds.), Saunders, W. B., Philadelphia, pp. 902-926). In addition to vitamin D.sub.3 (synthesized in the skin), another chemical form of vitamin D.sub.3, called vitamin D.sub.2, exists in nature. Vitamin D.sub.2 is metabolized to 25-hydroxyvitamin D.sub.2 (25-(OH).sub.2 -D) and 1,25(OH).sub.2 D.sub.2 in a manner similar to vitamin D.sub.3. Vitamin D.sub.2 is obtained primarily from diet and vitamin D supplementation, and can be as little as 5-10%, or as high as 100% of the circulating concentration of 25-OH-D depending on the relative amounts of vitamin D.sub.2 present in the diet and cutaneously-produced vitamin D.sub.3 by exposure to sunlight (Holick, M. F. et al. (1986) "Calcium, phosphorus and bone metabolism: calcium regulating hormones," in Harrison's Principles of Internal Medicine, 13th Ed., Braunwald el aL (eds.), McGraw-Hill, New York, pp. 2137-2151). In the following discussion, it may be assumed that vitamin D, 25-OH-D and 125(OH).sub.2 D will represent the total pool of vitamin D and its metabolites, unless otherwise mentioned. Biosynthesis of 25-OH-D and 1,25(OH).sub.2 D and their metabolism are regulated by the factors that control mineral and skeletal metabolism (Holick, M. F. (1989)). As a result, the serum 1,25(OH).sub.2 D level is an important pathophysiological indicator in several diseases. For example, production of 1,25(OH).sub.2 D is strongly influenced by a number of diseases such as acquired or inherited disorders of vitamin D-metabolism, including renal osteodystrophy, certain metabolic bone diseases, sarcoidosis, hypercalcemia associated with chronic granulotomous disorders, and vitamin D-dependent rickets types I and II (Holick, M. F. et al. (1986)). Web site: http://www.delphion.com/details?pn=US06455714__
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Labelling and selection of molecules Inventor(s): Vaughan; Tristan John (Cambridge, GB), Derbyshire; Elaine Joy (Royston, GB), Johnson; Kevin Stuart (Caldecote Highfields, GB), McCafferty; John Gerald (Babraham, GB), Osbourn; Jane Katharine (Cambridge, GB) Assignee(s): Cambridge Antibody Technology Limited (cambridgeshire, Gb) Patent Number: 6,489,123 Date filed: January 23, 2001 Abstract: A method of labelling molecules which includes providing in a common medium a label molecule, a marker ligand able to bind a member of a specific binding pair, such as an antigen, a sbp member, an enzyme able to catalyze binding of the label molecule to other molecules, the enzyme being associated with the marker ligand; causing or allowing binding of the marker ligand to the sbp member; and causing or allowing binding of the label molecule to other molecules in the vicinity of the marker ligand bound to the sbp member. The marker ligand may be an antibody or any specific binding molecule, such as a chemokine or cytokine. A complementary member of the specific binding pair may be included, e.g. an antibody, or a diverse population of such sbp members, e.g. antibodies, may be included within which those which bind the counterpart sbp member, e.g. antigen, may be labelled and subsequently isolated for manipulation and/or use. Suitable labels include biotin-tyramine with signal transfer being catalysed by hydrogen peroxidase. Cells, virus particles and other moieties may be labelled, for identification or obtention of proteins which interact or are in close proximity with a particular sbp member, or of cells of interest, or for enhancement of labelling, e.g. for cell sorting. Excerpt(s): The present invention relates to labelling and selection of molecules, such as members of a specific binding pair (sbp) able to bind a complementary sbp member of interest, especially though not exclusively a complementary sbp member for which an existing ligand is available. In exemplary embodiments, the present invention relates to selection of antibodies, or polypeptides comprising an antibody antigen binding domain, specific for an antigen of interest for which an existing binding molecule, which may be an antibody, such as a monoclonal antibody, is already available. It involves deposition of a label or reporter molecule, such as biotin-tyramine, on molecules in the vicinity of a "marker ligand" which comprises for example a monoclonal antibody (specific for an antigen of interest) in association with an enzyme which catalyzes such deposition. Molecules labelled in accordance with the present invention may include binding members such as antibodies which bind the same binding target (e.g. antigen) as the marker ligand if such binding members are included in the reaction medium, the target molecule to which the marker ligand binds, which allows for identification and/or purification of unknown antigen targets, and/or other molecules in the vicinity of the binding target and/or the marker ligand when bound to its binding target, e.g. on a cell surface on which the binding target is found, including molecules complexed with the binding target, allowing for identification of novel protein-protein interactions. There are also various advantages in labelling cells or other particles using the present invention, especially when the process is reiterated to augment the extent of labelling. Further aspects and embodiments of the invention are disclosed herein. Numerous kinds of specific binding pairs are known, as epitomised by the pair consisting of antibody and antigen. Other specific binding pairs are discussed briefly infra and may equally be employed in the various aspects of the present invention disclosed herein. For convenience, however, most of the discussion herein refers to "antibody " as the type of (first) specific binding pair (sbp) member whose selection is sought in performance of
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methods of various embodiments of the invention, "antigen" as the complementary (second) sbp member of interest for which specific binding molecules may be sought to be selected and marker ligand as the pre-existing binding molecule known to be able to bind the complementary sbp member of interest. Generally, the marker ligand comprises an antibody antigen binding domain specific for the complementary sbp member of interest (e.g. antigen). Other suitable marker ligands include hormones, cytokines, growth factors, neuropeptides chemokines, enzyme substrates and any other specific binding molecule. Also present is a label or reporter molecule and an enzyme that catalyses binding of the label to other molecules in the vicinity. Bearing this in mind, the present invention (in some embodiments) can be said to have resulted from the inventors having identified a means to select for antibodies binding to an antigen, e.g. on cell surfaces, other solid supports, or in solution, using a marker ligand for the antigen to guide the recovery of antibodies binding in proximity to the marker ligand. This provides means to label molecules which bind in close proximity to a given defined ligand by transfer of a reporter molecule or label to the binding molecules. The defined ligand occupies a specific epitope on the antigen and generally blocks that particular epitope, and epitopes overlapping it, from binding other antibodies. Thus, antibodies which are selected for are usually those which do not bind to the marker ligand epitope, but are those which bind neighbouring epitopes. Antibodies which bind the same epitope as the original marker ligand may be obtained by an iterative process--using an antibody obtained in one round of the process as a second marker ligand in a further round--or by using appropriate conditions, as discussed further below. Web site: http://www.delphion.com/details?pn=US06489123__ •
Ligands added to adenovirus fiber Inventor(s): Curiel; David T. (Birmingham, AL), Engler; Jeffrey A. (Birmingham, AL) Assignee(s): Uab Research Foundation (birmingham, Al) Patent Number: 6,683,170 Date filed: July 13, 2001 Abstract: The fiber protein of adenovirus has been genetically altered via attachment at the carboxyl end of a peptide linker, preferably up to 26 amino acids in length which forms a random coil, which can be used to attach a non-adenovirus ligand altering the binding specificity of the fiber protein. Examples of ligands include peptides which are selectively bound by a targeted cell so that the modified fiber protein is internalized by receptor-mediated endocytosis, and peptides which can act as an universal coupling agent, for example, biotin or strepavidin. The linker is designed to not interfere with normal trimerization of fiber protein, to avoid steric hindrance of binding of the fiber protein to a targeted cell, and to serve as a site to introduce new peptide sequence. Excerpt(s): The present invention generally relates to modification of the adenovirus fiber protein and methods for use thereof to modify cellular attachment by the fiber protein. In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one
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intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis is reviewed by Brown and Greene, DNA and Cell Biology 1991 10:6, 399-409. A number of viruses infect cells via a receptor-ligand interaction. Adenovirus is an example of a virus that utilizes receptor-mediated endocytosis to internalize infectious virus. Gene therapy requires transfer of recombinant nucleic acid constructs into cells. Although a number of different methods for gene transfer have been proposed, one of the most promising remains by utilizing recombinant viruses. The development of recombinant adenoviruses for this purpose has had a number of applications, based upon the unique advantages of this system. Web site: http://www.delphion.com/details?pn=US06683170__ •
Method for introduction of reporter groups into bacterial lipopolysaccharide-derived carbohydrates and the subsequent coupling of such derivatives onto solid surfaces Inventor(s): Boas; Ulrik (Copenhagen, DK), Heegaard; Peter M. H. (Copenhagen, DK), Jakobsen; Mogens Havsteen (Vanlose, DK), Stenbaek Jauho; Eva Irene (Copenhagen, DK) Assignee(s): Exiqon A/s (vedbaek, Dk) Patent Number: 6,436,653 Date filed: December 14, 1999 Abstract: The present invention provides a method for immobilizing a polysaccharide (PS) to a solid surface, said polysaccharide having a keto-carboxy group (--C(.dbd.O)-COOH) or a ketal or hemiketal group corresponding thereto, e.g. derived from KDO (2keto-3-deoxy-D-manno-octonic acid)), the method comprising the steps of: (a) forming a covalent bond between the carboxy group of the polysaccharide and a reporter molecule (RM), thereby forming a polysaccharide-reporter molecule conjugate (PS-RM), said reporter molecule comprising a recognition/substrate site (e.g. biotin or an anthraquinone); and (b) immobilizing the polysaccharide-report molecule conjugate by forming a specific bond (e.g. by photocoupling or formation of an affinity pair) between the recognition/substrate site of said reporter molecule and a reception/reagent site of the solid surface. The present invention also provides a solid surface thus obtainable and the use of such solid surfaces for diagnostic purposes, e.g. for the detection of bacterial infections from Gram-negative bacteria that are human or veterinary pathogens, e.g. enterobacteria, respiratory bacteria, urogenitial bacteria, and neuropathogenic bacteria such as Salmonella sp., Actinobacillus sp. Excerpt(s): The present invention relates to a method for immobilising special classes of polysaccharides to solid surfaces. Such a method is highly valuable in the construction of reliable assays for the detection of an antibody corresponding to the polysaccharide antigen. The present invention also relates to modified solid surfaces and to the use of such surfaces in various diagnostic assays. Furthermore, the present invention relates to novel KDO derivatives which are valuable intermediates in the construction of such modified solid surfaces. Bacterial lipopolysaccharides (LPSs) are characteristic outer membrane constituents of Gram-negative bacteria. LPSs are widely used as antigens in diagnostic assays specially designed for the specific detection of antibodies in serum, plasma, meat juice, saliva or other body fluids, originating from bacterial infections in humans and animals. LPSs are highly immunogenic and comprise one of the epitope characteristics for a given bacterial strain. In fact, the definition of a serotype is often based on the LPS and/or capsular polysaccharide (CPS) antigenicity. The antigenic
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specificity of the LPS molecule resides in the polysaccharide part of the LPS, the Oantigen, whereas the toxicity of the LPS is caused by residues contained in the lipid part of the LPS, called the lipid A. LPSs are highly amphiphilic compounds because of the joint presence of a hydrophilic O-polysaccharide group and a hydrophobic lipid group in the LPS molecule. Most of the characterised LPSs have the same principal structure which is especially conserved in the lipid A and in the inner core parts of the LPSs. The core is the part of the polysaccharide that comprises the bond between the O-antigen and the lipid A. This bond is invariably comprised of a ketosidic bond between the hemiketal function of the innermost KDO-residue and a hydroxyl-group of a GlcNAcresidue of the lipid A. The O-antigen of a specific bacterial serotype varies with respect to numbers of repeating units and may contain non-stoicheometrical substitutions with acetyl, phosphate, glycosyl or other groups. Generally, LPS-molecules without Oantigens, that is carrying only (parts of) the core saccharides in addition to the lipid A are called "rough" LPS, while LPS-molecules carrying O-antigens are called "smooth" (Raetz, C. R. H. in Escherichia coli and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C. E. A., ed.) Vol. 1, 2nd Ed., pp. 1035-1063, American Society for Microbiology, Washington D.C., 1996; Hitchcock et al, 1986, J. Bacteriol. 166, 699-705). Previously, LPSs have generally been immobilised onto a solid surface without any modification of the molecules since the hydrophobic lipid A part of the molecules functions as a fairly efficient "anchor" binding the LPSs to the surface via non-covalent hydrophobic interactions leaving the hydrophilic O-polysaccharides pointing outward accessible for interactions with binding components, e.g. antibodies. However, it has been shown that the efficiency by which the LPSs are immobilised onto hydrophobic surfaces depends on both the nature of the surface and the equilibrium between free LPSs and formed LPS micelles. The equilibrium between free LPSs and formed LPS micelles depends on the amphiphilic nature of the LPSs and varies between LPSs from different bacteria strains as well as between different LPS serotypes. Certain types of LPSs have shown to be very difficult to immobilise onto solid surfaces by non-covalent bonds without addition of various micelle-dispersing agents (detergents) to the coating solution. Web site: http://www.delphion.com/details?pn=US06436653__ •
Method for producing biotin Inventor(s): Hauer; Bernhard (Fussgonheim, DE), Schroder; Hartwig (Nussloch, DE) Assignee(s): Basf Aktiengesellschaft (ludwigshafen, De) Patent Number: 6,436,681 Date filed: January 11, 2000 Abstract: A gene construct comprising a biotin gene having the sequence SEQ ID No. 1 or SEQ ID No. 3, organisms which comprise this gene construct, the use of these sequences or of the gene construct for preparing biotin, and a process for preparing biotin are described. Excerpt(s): This application is a 371 of International Application No. PCT/EP98/04097 published as WO 99/05285 which has an international filing date of Jul. 2, 1998. The invention relates to a gene construct comprising a biotin gene having the sequence SEQ ID No. 1 or SEQ ID No. 3, to organisms which comprise this gene construct, to the use of these sequences or of the gene construct for preparing biotin, and to a process for preparing biotin. Biotin (vitamin H) plays an essential role as coenzyme in enzymecatalyzed carboxylation and decarboxylation reactions. Biotin is thus an essential factor
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in living cells. Almost all animals and some microorganisms have to take biotin in from the outside because they are unable to synthesize biotin themselves. It is thus an essential vitamin for these organisms. Bacteria, yeasts and plants by contrast are themselves able to synthesize biotin from precursors (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295-326, DeMoll, E., Escherichia coli and Salmonella, eds. Neidhardt, F. C. et al. ASM Press, Washington D.C., USA, 1996: 704-708, ISBN 1-55581-084-5). Web site: http://www.delphion.com/details?pn=US06436681__ •
Multiflavor streptavidin Inventor(s): Cantor; Charles (Del Mar, CA), Reznik; Gabriel O. (Boston, MA), Sano; Takeshi (Needham, MA), Smith; Cassandra (Boston, MA), Vajda; Sandor (Medfield, MA) Assignee(s): The Trustees of Boston University (boston, Ma) Patent Number: 6,368,813 Date filed: March 23, 2000 Abstract: Compounds and methods are described for producing streptavidin mutants with changed affinities. In particular, modifications to the sequence of the natural streptavidin gene is described to create amino acid substitutions resulting in greater affinity for biotin substitutes than for biotin. Excerpt(s): The present invention relates to compounds and methods, and in particular, modified streptavidin having affinity for biotin substitutes. The biological sciences have been employed since early times by mankind to modify living organisms or their constitutive elements for a variety of purposes, such as the production of foods and therapeutic agents. However, only during the last fifty years there has been progress at the genetic level to gain a much better understanding of the essential component of living systems. This has led to the understanding that nucleic acids, in the form of DNA and RNA, store and distribute genetic information that determines the sequences of amino acids that characterize proteins; proteins contribute to the structure of an organism and execute most of the tasks required for its function and that even proteins form part of the mechanism by which they are synthesized (e.g. chaperones); polysaccharides, linear and branched polymer of sugars, provide structural elements, store energy, and when combined with peptides or proteins play an important role in cellular recognition; lipids, which include molecules such as fatty acids, phospholipids, and cholesterol, serve as energy sources and are the most important components of the membrane structures that organize and compartmentalize cellular function. However, proteins are the biological macromolecules with the greatest functional diversity. Proteins catalyze most reactions that occur in living cells, or serve as inhibitors of enzymatic reactions. They transport oxygen, electrons and energy to specific regions in the cell. Other proteins protect living organisms by recognizing and binding to foreign substances. There are also proteins that have a structural function such as collagen, the main constituent of connective tissue fibrils and bones, or have functional roles such as actin or myosin, which are involved in muscle dynamics. Web site: http://www.delphion.com/details?pn=US06368813__
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Multi-vitamin and mineral supplement Inventor(s): Cooper; Kenneth H. (Dallas, TX), Grundy; Scott Montgomery (Dallas, TX), Jialal; Ishwarlal (Dallas, TX), Selhub; Jacob (Brookline, MA), Willett; Walter Churchill (Cambridge, MA) Assignee(s): Cooper Concepts, Inc. (dallas, Tx) Patent Number: 6,361,800 Date filed: April 13, 2000 Abstract: This invention is directed to a multi-vitamin and mineral supplement which supplies the right amount of the right micronutrients at the right time to assure adequate intake of micronutrients needed for disease prevention and protection against nutritional losses and deficiencies due to lifestyle factors and common inadequate dietary patterns. The multi-vitamin and mineral supplement is comprised of vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, niacinamide, vitamin B6, vitamin B12, biotin, pantothenic acid, iron, phosphorus, iodine, magnesium, zinc, selenium, copper, chromium, potassium, choline, lycopene, and co-enzyme Q-10. Excerpt(s): This invention relates to multi-vitamin and mineral supplements. In particular, this invention relates to multi-vitamin and mineral supplements for improving health by insuring adequate intake of micronutrients needed for disease prevention and protection against nutritional losses and deficiencies due to such factors as lifestyle patterns and common inadequate dietary patterns. Vitamin and mineral preparations are commonly administered to treat specific medical conditions or as general nutritional supplements. Micronutrients are elements or compounds which are present in foods in small or trace amounts and includes vitamins, minerals, or other elements, and compounds found in foods for which a Recommended Daily Allowance (RDA) has not yet been determined. The macronutrients consist of carbohydrates, fats, and proteins which supply nutrients and calories. Some elements such as calcium, sodium, potassium, chloride, and phosphorus are consumed in relatively large amounts, while many such as iron, iodine, and zinc are consumed in small amounts. vitamins such as B12 and folic acid and the minerals cooper, selenium, and chromium are consumed in very small or trace amounts. In as much as the human body does not synthesize many compounds which are essential to the human body, these specific vitamins and minerals can be obtained from only two sources: food and supplements. The primary source of all nutrients is food. However, the majority of people do not meet the RDA of the foods containing these essential compounds and elements. Thus vitamin and mineral supplementation has become a recognized method of meeting accepted medical and health standards. An international panel of diet and cancer experts announced in London on Sep. 30, 1997, that as many as 30 to 40 percent of all cancer cases worldwide--3 to 4 million a year--could be avoided if people ate a healthy diet and got enough exercise. USA Today, Oct. 1, 1997. However, for some nutrients, the amounts proposed as being healthy apparently cannot be provided by a reasonable quantity and variety of natural foods. Thus nutrient supplements may be important for health promotion and prevention of chronic diseases. Journal of the American Medical Association, May 7, 1997. Web site: http://www.delphion.com/details?pn=US06361800__
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Nutritional composition made from conventional foods for mixing onsite in a blender and treating patients with hepatic disorders Inventor(s): Muszynska; Julia (108 Center St., Staten Island, NY 10306) Assignee(s): None Reported Patent Number: 6,524,610 Date filed: February 26, 2001 Abstract: A nutritional composition made from conventional food mixed on-site in a blender and treating patients with hepatic disorders. The composition includes a vitamin A enriched conventional food, a vitamin D enriched conventional food, a vitamin E enriched conventional food, a vitamin K enriched conventional food, a vitamin C enriched conventional food, a thiamine enriched conventional food, a riboflavin enriched conventional food, a niacin enriched conventional food; a pyridoxine enriched conventional food, a folic acid enriched conventional food, a pantothenic acid enriched conventional food, a vitamin B12 enriched conventional food, a biotin enriched conventional food, a chloine enriched conventional food, a sodium enriched conventional food, a potassium enriched conventional food, a chlorine enriched conventional food, a calcium enriched conventional food, a phosphorus enriched conventional food, a magnesium enriched conventional food, a copper enriched conventional food, an Iodine enriched conventional food, a manganese enriched conventional food, and a zinc enriched conventional food. Excerpt(s): The present invention relates to a nutritional composition. More particularly, the present invention relates to a nutritional composition made from conventional foods for mixing on site in a blender and treating patients with hepatic disorders. The liver, and its proper functioning, is of utmost importance to the survival of a patient. Because it is responsible for the metabolism of nearly all nutrients, and is the primary site for the inactivation of numerous toxins, the liver is one of the most important organs of the body. For example, the liver accounts for approximately 20% of the body's basal metabolism. The liver extracts a majority of the amino acids, carbohydrates, lipids, vitamins, and minerals from portal circulation. These nutrients, extracted by the liver, are used as substrates or cofactors in all metabolic processes carried out in the liver. Synthesis of plasma proteins and bile secretion are additionally important processes carried out by the liver. Web site: http://www.delphion.com/details?pn=US06524610__
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Nutritional system for nervous system disorders Inventor(s): Foreman; David J. (Chesterfield, VA) Assignee(s): C & D Foreman, Inc. (chesterfield, Va) Patent Number: 6,399,114 Date filed: May 24, 2001 Abstract: A novel composition for treating nervous system disorders. The composition is formed by preparing a mixture comprising an effective amount of vitamin B-6, folic acid, vitamin C, magnesium, vitamin B-3, copper, probiotics, fructo-oligosaccharide (FOS), betaine, pancreatin, papain, pepsin, vitamin B-1, vitamin B-2, vitamin B-12, biotin, pantothenic acid, chromium polynicotinate and a digestive support ingredient selected from the group consisting of dandelion root, juniper, aloe vera, burdock, ginger
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root, artichoke, and kelp. Other ingredients may include: beta carotene, vitamin E, selenium, zinc, sea vegetation, alfalfa, trace minerals and molybdenum. Excerpt(s): The present invention pertains to the field of nutritional formulas. Specifically, the present invention pertains to an improved formula for nervous system disorders. There are many disorders that affect the proper functioning of the nervous system. Examples of these disorders include autism, ADD, ADHD, hyperactivity disorder, and depression. People who suffer from these disorders often have common secondary symptoms including allergies, sluggish digestion, weak immune function and poor diet. Treatment for these various nervous system disorders include the use of synthetic drugs. Specifically, for ADD and ADHD mild central nervous system stimulant drugs such as Ritalin.RTM., Cylert.RTM. and Dexedrine.RTM. have been used. These drugs are not always successful. Moreover, such drugs may lead to undesirable side effects such as loss of appetite, insomnia, headaches, stomachaches, drowsiness and cardiac arrhythmia. Web site: http://www.delphion.com/details?pn=US06399114__ •
Polynucleotide portions of the biotin operon from B. subtilis for use in enhanced fermentation Inventor(s): Hohmann; Hans-Peter (Upper Montclair, NJ), Mouncey; Nigel John (Verona, NJ), Schlieker; Heinrich Winfred (Bloomfield, NJ), Stebbins; Jeffrey W. (Nutley, NJ) Assignee(s): Roche Vitamins, Inc. (parsippany, Nj) Patent Number: 6,656,721 Date filed: August 8, 2000 Abstract: The present invention provides a process for producing a target fermentation product. This process includes providing a fermentation medium containing a recombinantly-produced microorganism that over-produces a fermentation product and contains a mutation which causes auxotrophic growth of the microorganism wherein the auxotrophy within the microorganism does not compromise the ability of the microorganism to produce the fermentation product. The medium is then supplied in excess with all substrates required for production of the fermentation product and in growth limiting amounts with a substrate complementing the auxotrophy. Host cells, vectors, and polynucleotide sequences used in the process are also provided. The polynucleotide sequences of the present invention include sequences derived from the biotin operon of B. subtilis, and in particular the bioFDB gene cassette. Excerpt(s): The present invention relates to a process for producing a target fermentation product. More particularly, the present invention relates to a process for over-producing a target fermentation product in a microorganism having a mutation which causes auxotrophic growth of the microorganism, but that does not compromise its ability to over-produce the target fermentation product. Host cells and polynucleotide sequences used in the process are also provided. Many commercially valuable products are produced by fermentation reactions. For example, riboflavin, which is an essential vitamin that is required by all bacteria, animals, and plants, is synthesized by plants and bacteria, however, it cannot be produced by higher animals, which must acquire it from their diet. Riboflavin is produced commercially for use as a food and feed additive by, for example, fermentation reactions using Ashbya govsypii, Eremothecium ashbyii, or Candida flareri cells. (See e.g., Ainsworth, G. C. and Sussman,
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A. S., The Fungi, Academic Press, New York (1965); Heefner, D. L., et al., WO 88/09822; Hickey, R. J., Production of Riboflavin by Fermentation, in Industrial Fermentation (Underkofler, L. A. and Hickey, R. J., eds.) pp. 157-190, Chemical Publishing Co., New York (1954); and Perlman, D., et al., Fermentation Ind. Eng. Chem. 44:1996-2001 (1952). Web site: http://www.delphion.com/details?pn=US06656721__ •
Process for the fermentative preparation of L-amino acids using coryneform bacteria Inventor(s): Eggeling; Lothar (Julich, DE), Eikmanns; Bernd (Ulm, DE), Mockel; Bettina (Bielefeld, DE), Pfefferle; Walter (Halle, DE), Sahm; Hermann (Julich, DE), Tilg; Yvonne (Mettmann, DE) Assignee(s): Degussa AG (dusseldorf, De) Patent Number: 6,379,934 Date filed: July 29, 1999 Abstract: The invention provides a process for the fermentative preparation of L-amino acids using coryneform bacteria, in which the subunit carrying the biotin-carboxyl carrier protein domain and the biotin-carboxylase domain of the nucleotide sequence encoding the enzyme acetyl-CoA carboxylase (accBC gene) is amplified, in particular is overexpressed. Excerpt(s): The invention provides a process for the fermentative preparation of Lamino acids, in particular lysine, using coryneform bacteria in which the accBC gene is amplified. L-amino acids, in particular L-lysine, are used in animal nutrition, in human medicine and in the pharmaceutical industry. It is known that these amino acids are prepared by fermentation using strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to the high degree of importance of these products, a constant effort is made to improve the method of preparation. Process improvements may be based on fermentation engineering steps such as, for example, stirring and supplying with oxygen, or the composition of the nutrient medium such as, for example, the concentration of sugar during fermentation, or the working up process aimed at obtaining the product itself by, for example, ion-exchange chromatography or the intrinsic power of the microorganism itself. Web site: http://www.delphion.com/details?pn=US06379934__
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Process to control the molecular weight and polydispersity of substituted polyphenols and polyaromatic amines by enzymatic synthesis in organic solvents, microemulsions, and biphasic systems Inventor(s): Akkara; Joseph A. (Holliston, MA), Ayyagari; Madhu (Brighton, MA), Kaplan; David L. (Stow, MA) Assignee(s): The United States of America AS Represented by the Secretary of the Army (washington, Dc) Patent Number: 6,362,314 Date filed: February 4, 1999 Abstract: A process of controlling the molecular weight and dispersity of poly(pethylphenol) and poly(m-cresol) synthesized enzymatically by varying the composition of the reaction medium. Polymers with low dispersities and molecular weights from
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1000 to 3000 are synthesized in reversed micelles and biphasic systems. In comparison, reactions in bulk solvents resulted in a narrow range of molecular weights (281 to 675 with poly(p-ethylphenol) in a DMF/water system and 1,400 to 25,000 with poly(mcresol) in an ethanol/water system). Poly(p-ethylphenol) was functionalized at hydroxyl positions with palmitoyl, cinnamoyl, and biotin groups. Excerpt(s): The present invention relates generally to the preparation of phenolic and aromatic amine polymers, wherein the reaction conditions are controlled such that high product yields, molecular weight, and a uniform molecular weight distribution are obtained. Phenolic and aromatic amine polymer resins constitute a very important and useful class of chemical compounds. They have a number of uses, e.g., as coatings and laminates that provide a number of functional advantages. Besides possessing good thermal properties, these polymers can be doped to make them electrically conductive, making them a key component of integrated circuit (IC) chips. At present, these polymers are prepared by chemical synthesis, e.g., as from phenol and formaldehyde. The polymers's linearity/network structure (and, by extension, their functional properties) varies depending on the monomer and type of reaction conditions used. However, the use of certain constituent chemicals, such as formaldehyde, is being restricted in the chemical industry because of their toxicity. Accordingly, the enzymemediated synthesis of polyphenols and polyaromatic amines offers a viable alternative to the currently used chemical synthesis of such commercial phenolic resins. Web site: http://www.delphion.com/details?pn=US06362314__ •
Purification of oligomers Inventor(s): Sproat; Brian S. (Adelebsen, DE) Assignee(s): Ribozyme Pharmaceuticals, Inc. (boulder, Co), Yale University (new Haven, Ct) Patent Number: 6,410,225 Date filed: June 27, 1997 Abstract: Compositions and methods are disclosed which facilitate purification of oligomers and other compounds. The disclosed compositions are silyl compositions that can be directly coupled, or coupled through a linking group, to a compound of interest, preferably to an oligomer at the end of oligomer synthesis. The silicon atom includes between one and three sidechains that function as capture tags. In one embodiment, the capture tags are lipophilic, which allows a derivatized oligomer to be separated from failure sequences by reverse phase chromatography. In another embodiment, the capture tags are compounds with a known affinity for other compounds, which other compounds are preferably associated with a solid support to allow chromatographic separation. Examples include haptens, antibodies, and ligands. Biotin, which can bind to or interact with a streptavidin-bound solid support, is a preferred capture tag of this type. Excerpt(s): The disclosed invention is in the general field of oligonucleotide synthesis and purification. Oligonucleotides are being pursued as therapeutic and diagnostic agents. Examples of such oligonucleotides include antisense oligonucleotides, aptamers, triplex forming agents, external guide sequences, catalytic oligomers, and ribozymes. Synthetic oligonucleotides based on oligo(2'-O-allylribonucleotide)s which can cleave, or induce cleavage of, specific RNA molecules are showing great promise as a new class of rationally designed therapeutics. Such chemically modified oligonucleotides are useful
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for cleaving an unwanted or overexpressed RNA in a highly specific fashion, thus preventing the synthesis of the corresponding protein which is causative for a particular disease state. Such an oligonucleotide having RNA cleaving activity has been successfully used in mice (Lyngstadaas et al., EMBO Journal 14:5224-5229 (1995)). Several other examples of such compounds have been shown to possess reasonable pharmacokinetics (Desjardins et al., J. Pharmacol. Exp. Ther. 278:1419-1427 (1966)). Versions of these compounds that are stable enough for in vivo applications, yet have reasonable catalytic activity, contain five residual purine ribonucleotides at critical positions in the hammerhead catalytic core and are generally in the range of 34 to 38 residues in length (Lyngstadaas et al., EMBO Journal 14:5224-5229 (1995)). Synthetic methods for preparing oligonucleotides generally include solid phase synthesis using phosphoramidite coupling chemistry. This chemistry generally involves coupling the 3'hydroxy group of a first nucleotide to a solid support, and reacting the 5'-hydroxy group with subsequent monomers containing protected 5'-hydroxy groups. After each coupling step, the 5'-protecting group is removed, freeing up a subsequent 5'-hydroxy group for subsequent coupling with an additional monomer. The coupling chemistry has a relatively high yield for each step, but about one to two percent of the hydroxy groups are not successfully coupled at each step in the synthesis. These un-reacted hydroxy groups must be prevented from further reactions, to avoid the preparation of unwanted sequences. To accomplish this, the hydroxy groups are capped, for example, using acetic anhydride. The capped sequences are typically referred to as failure sequences. Web site: http://www.delphion.com/details?pn=US06410225__ •
Recombinant inactive avidin mutants Inventor(s): Brandstetter; Hans (Chelsea, DE), Deger; Arno (Pernzberg, DE), Engh; Richard (Munchen, DE), Kopetzki; Erhard (Penzberg, DE), Muller; Rainer (Penzberg, DE), Schmitt; Urban (Oberhausen, DE) Assignee(s): Roche Diagnostics Gmbh (mannheim, De) Patent Number: 6,391,571 Date filed: August 4, 1999 Abstract: The present invention concerns muteins of avidin and streptavidin with a reduced binding affinity for biotin as well as their use as interference elimination reagents in methods for the determination of an analyte e.g. in diagnostic tests such as for example immunoassays and nucleic acid hybridization assays. In addition the invention concerns the use of muteins of avidin and streptavidin as systems capable of regeneration for binding biotin e.g. for the analysis of biotinylated molecules, for examining receptor ligand interactions as well as for the affinity purification of biotinylated molecules. Excerpt(s): The present invention concerns muteins of avidin and streptavidin with a reduced binding affinity for biotin as well as their use as interference elimination reagents in methods for the determination of an analyte e.g. in diagnostic tests such as immunoassays and nucleic acid hybridization assays. In addition the invention concerns the use of muteins of avidin and streptavidin as systems that can be regenerated for binding biotin for example for the analysis of biotinylated molecules, for investigating receptor-ligand interactions as well as for the affinity purification of biotinylated molecules. In detection methods for the determination of analytes such as immunoassays and nucleic acid hybridization assays the analytes are often determined
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by means of high affinity interaction between the partners of a specific binding pair. A typical example for a specific binding pair is the avidin/streptavidin-biotin complex. When using the avidin/streptavidin-biotin binding pair its high binding affinity is used. In this process a solid phase coated with avidin/streptavidin is for example used to which a biotinylated complex of analyte and specific receptor can bind. In other test formats avidin/streptavidin can also be used in a soluble form. However, apart from specific interactions. side reactions often also occur such as for example undesired interactions and unspecific binding reactions between the test components and additional components present in the sample or on the solid phase. In particular other substances present in the sample often bind to immobilized or soluble avidin and streptavidin and thereby cause false positive or false negative test results. Furthermore these interactions can also cause an increase in the background signal and an increased scattering of the signals which decreases the sensitivity and specificity of the respective test. Web site: http://www.delphion.com/details?pn=US06391571__ •
Recombinant streptavidin-metallothionein chimeric protein having biological recognition specificity Inventor(s): Cantor; Charles R. (Berkeley, CA), Glazer; Alexander N. (Orinda, CA), Sano; Takeshi (Albany, CA) Assignee(s): The Regents of the University of California (oakland, Ca) Patent Number: 6,391,590 Date filed: October 21, 1991 Abstract: Streptavidin-metallothionein chimeric proteins with biological recognition specificity in which the streptavidin moiety provides high affinity biotin binding and the metallothionein moiety provides a high affinity metal binding. The binding affinity of the streptavidin-metallothionein chimeric protein both for biotin and heavy metal ions allows specific incorporation into, conjugation with, or labelling of any biological material containing biotin with various heavy metal ions. Excerpt(s): This invention concerns streptavidin-metallothionein chimeric proteins which possess biological recognition specificity. Recombinant streptavidinmetallothionein chimeric proteins containing various metal ions are molecules which have a great potential in preventive and therapeutic medicine in both humans and animals as well as for diagnostic use. While each individual molecule, i.e., streptavidin and metallothionein have been known and described previously, their composite molecule has never before been constructed. Biochemistry of metallothionein, particularly its amino acid sequence in various species, its metal binding sites, metal thiolate clusters and spatial structures are described in Biochemistry, 27:509 (1988). Ann. Rev. Biochem., 55:913 (1986) is directed to the gene structure, organization, amplification and transcriptional regulations, and describes also some genetic engineering applications, such as for example, metallothionein-rat growth hormone and metallothionein-human growth hormone genes expressed in transgenic mice, or conferring resistance to copper toxicity via the CUP 1 copper-metallothionein gene. Web site: http://www.delphion.com/details?pn=US06391590__
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Inventor(s): Asanov; Alexander N. (Starkville, MI), Oldham; Philip B. (Starkville, MI), Wilson; W. William (Starkville, MI) Assignee(s): The Uab Research Foundation (birmingham, Al) Patent Number: 6,511,854 Date filed: June 12, 2000 Abstract: An improved electrochemical method for disassociating a biological binding partner from a corresponding second biological binding partner associated with a waveguide surface, the electrochemical method involving the application of an electrical potential to said waveguide surface (118), the improvement comprising: applying the electrical potential to the waveguide surface (118) as a square wave polarization function. Preferably, the waveguide surface is comprised of indium tin oxide. The biological binding partners are selected from the group consisting of antigen-antibody, avidin-biotin, enzyme-substrate, cell receptor-substrate/analog, antibody/antiantibody, DNA, RNA, and fragments thereof. The antigen may be comprised of an epitope. The epitope is produced by a solid phase peptide synthesis performed on said waveguide surface (118). Excerpt(s): This invention relates generally to a diagnostic apparatus and to related methods for using that apparatus in rapidly analyzing samples for analytes of interest, and more particularly to an apparatus and associated methods which provide simultaneous fluorescence detection and electrochemical control of biospecific binding. Investigation of the interactions between biomolecules has attracted increasing attention in recent years. An understanding of these interactions and the ability to control them are critical for a variety of objectives, such as the determination of structure-function relationships and protein crystallogenesis, drug design and development of targeted drug delivery systems, and biomolecular engineering and design of biosensors. Total internal reflection fluorescence (hereinafter "TIRF") techniques have proven to be wellsuited for investigating biomolecular interactions. Such techniques generally utilize optical waveguides, either planar or cylindrical, having a portion of one surface of the waveguide carrying an immobilized binding agent, such as a specific binding partner (e.g., an antibody or antibody fragment). A light beam is introduced into the waveguide wherein the light beam travels in the waveguide. The light beam is totally internally reflected at the interface between the waveguide and a surrounding medium having a lower refractive index than the waveguide. A portion of the electromagnetic field of the internally reflected light beam penetrates into the surrounding medium and forms an evanescent light field. The intensity of evanescent light drops off substantially exponentially with increasing distance from the waveguide surface. In a fluoroimmunoassay, evanescent light can be used to selectively excite tracer molecules directly or indirectly bound to the immobilized binding agent, while tracer molecules free in solution beyond the evanescent light penetration distance are not excited and thus do not contribute "background" fluorescence. The use of evanescent field properties for fluorescence measurements is sometimes referred to as evanescent sensing. For a glass or a similar silica-based material, or an optical plastic such as polystyrene, with the surrounding medium being an aqueous solution, the region of effective excitation by evanescent light generally extends about 1000 to 2000.ANG. (angstroms) from the waveguide surface. This depth is sufficient to excite most of the tracer molecules bound to the capture molecules (antibodies, receptor molecules, and the like, or fragments thereof) on the waveguide surface, without exciting the bulk of the tracer molecules that
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remain free in solution. The resulting fluorescence reflects the amount of tracer bound to the immobilized capture molecules, and in turn the amount of analyte present in the aqueous solution. Web site: http://www.delphion.com/details?pn=US06511854__ •
Serum free medium for chondrocyte cells Inventor(s): Cancedda; Ranieri (Genoa, IT), Dozin; Beatrice (Rapallo, IT) Assignee(s): Consorzio Per LA Gestione Del Centro DI Biotechnologie Avanzate (genoa, It), Istituto Nazionale Per LA Ricerca Sul Cancro (genoa, It) Patent Number: 6,617,159 Date filed: June 11, 2001 Abstract: Serum free media for growth and proliferation of chondrocytes and mesenchymal stem cells in culture are provided. A serum free medium for growth of chondrocytes includes a serum free composition comprising FGF-2, linoleic acid, ascorbic acid, B-mercaptoethanol, transferrin and dexamethasone. Further the composition comprises EGF, PDGFbb, insulin and albumin. A method for growing chondrocytes in a serum free medium comprising the composition is also provided. Also provided for mesenchymal stem cell growth, is a serum free medium which includes a composition comprising FGF-2, LIF, SCF, pantotenate, biotin and selenium and method, therefore. Excerpt(s): Bone and cartilage transplantation is an absolute need in reconstruction of bone and cartilage segments in plastic surgery, traumatic surgery or after the removal of neoplastic lesions, etc. Typically, material of human (autologous, from donors or from cadavers) or animal origin has been used for this purpose. Given the increased demand from clinicians for transplant tissues, the increased need for microbial safety in tissue transplantation, the advances in cell biology, cell differentiation and tissue engineering, the concept of rebuilding tissues from autologous or allogeneic cells expanded in vitro has become a growing field in the world of biomedical sciences. Cellular sources for skeletal repair include chondrocytes and cells committed to the chondrocyte lineage, and mesenchymal stem cells, the former specific for cartilage, the latter multipotential and therefore having the potential to be used to replace bone, cartilage and other tissues. Mesenchymal stem cells (MSCs) are found in bone marrow as well as in blood, dermis and periosteum. Although these cells are normally present at very low frequencies in bone marrow, these cells can be isolated purified and culturally expanded, for example, as described in U.S. Pat. No. 5,486,359. Typically, the ill vitro expansion of chondrocytes and MSCs takes place in culture medium supplemented with bovine serum or optimally with autologous serum from the patient. However, the presence of animal or autologous serum in chondrocyte and MSC cultures has certain disadvantages and limitations in view of the potential therapeutical applications of these cultures. Web site: http://www.delphion.com/details?pn=US06617159__
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Streptavidin mutants having secondary functional domains Inventor(s): McDevitt; Todd C. (Seattle, WA), Nelson; Kjell E. (Seattle, WA), Stayton; Patrick S. (Seattle, WA) Assignee(s): University of Washington (seattle, Wa) Patent Number: 6,413,934 Date filed: August 25, 1999 Abstract: Streptavidin molecules are disclosed that contain a secondary functional domain. In preferred embodiments, the secondary domain is a cell adhesion peptide incorporated in the streptavidin amino acid sequence at a site not interfering with biotin binding. In a preferred embodiment, the cell adhesion peptide is arginine-glycineaspartate (Arg-Gly-Asp) (RGD). The peptide is preferably placed on an exposed loop of the streptavidin molecule, such as within the loop defined by residues 63 to 69. The mutant streptavidin molecule can have other characteristics such as reduced biotin binding due to a modification of an amino acid at the biotin binding site. Preferred uses for the disclosed streptavidin molecules are as adaptors to bring, via a streptavidin/biotin interaction, the secondary functional domain into proximity with a cell or molecule to be affected and as a coating for substrates such as vascular devices or prostheses. Excerpt(s): The present invention is directed to streptavidin molecules having secondary functional domains such as a cell adhesion domain. Streptavidin, a protein produced by Streptomyces avidinii, forms a very strong and specific non-covalent complex with the water soluble vitamin biotin. Streptavidin is a tetrameric protein that binds biotin with an affinity that is among the highest displayed for non-covalent interactions between a ligand and protein, with an association constant (Ka) estimated to be in the range of 10.sup.13 M.sup.-1 to 10.sup.15 M.sup.-1. This binding affinity is strong enough to be essentially irreversible under normal physiological solution conditions, and provides the basis for streptavidin and biotin's usefulness in a wide variety of clinical and industrial applications. See, Green, Adv. Prot. Chem. 29:85-143 (1975). Both streptavidin and the homologous protein avidin, which shares its high affinity for biotin, have been studied as paradigms of strong ligand-protein interactions. The X-ray crystal structures of streptavidin and avidin, both in their apo and holo forms, have been described. The sequences of both have also been reported, as have the construction of several streptavidin fusion proteins (Sano and Cantor, Biochem. Biophys. Res. Commun. 176:571-577 (1991); U.S. Pat. No. 4,839,293). Web site: http://www.delphion.com/details?pn=US06413934__
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Substituted 2-[-6-benzyl-5-oxo-3-phenyl-(3s,7s, c][1,3]thiazol-7yl] compounds
7aR)-perhydroimidazol[1,5-
Inventor(s): Chavan; Subhash Prataprao (Maharashtra, IN), Chittiboyina; Amar Gopal (Maharashtra, IN), Kalkote; Uttam Ramrao (Maharashtra, IN), Kamat; Subhash Krishnaji (Maharashtra, IN), Ravindranathan; Thotapallil (Maharashtra, IN) Assignee(s): Council of Scientific and Industrial Research (new Delhi, In) Patent Number: 6,486,328 Date filed: October 12, 2000
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Abstract: A novel route has been developed for substituted 2-[-6-benzyl-5-oxo-3-phenyl(3s,7s,7aR)-perhydroimidazol[1,5-c][1,3]thiazo l] compounds; crucial intermediates for D(+)-biotin of formula (7) which involves simple, efficient, practical and cost effective protocol. These are crucial intermediates for commercially important D(+)-biotin preparation. These compounds are more stable and are produced by non-hazardous methods. Excerpt(s): D(+) Biotin is prepared in the prior art from amino acids viz., cysteine, cyctine and serine. These processes involving L-cystine as the precursor, incorporate intramolecular radical cyclization (E. J. Corey, M. M. Mehrotra, Tet.Lett., 29, 57 1988) as the key step is construct the tetrahydrothiophene moiety of biotin. Another prior art process involves intramolecular cycloaddition (3+2) of derivatives of L-cystine (E. G. Baggiolini, H. L. Lee, G. Pizzolato and M. R. Uskokovic, J.Am.Chem.Soc., 104, 6460, 1982), and L-cysteine (, H. L. Lee, E. G. Baggiolini and M. R. Uskokovic, Tetrahedron 43, 4887, 1987). In another process starting from L-cysteine, a bicyclic imidazolidine is the key intermediate leading to D(+)-biotin (E. Poetsch and M. Casutt, EP 242,686 1986 CA:108:1612077k 1988; Chimia 41, 148 1987). In an totally different and novel approach. L-cysteine was converted to its thiazolidine derivative which on treatment with bromine is converted stetreospecifically to a bicyclic intermediate as a single stereoisomer and eventually transformed to D(+)-biotin (P. N. Confalone, E. G. Baggiolini, D. Lollar, and M. R. Uskokovic, J. Am. Chem. Soc., 99, 7020 1977). Process for stereospecific synthesis of D(+)-Biotin from sugars of suitable configuration are known. [From Mannose Tet. Lett., 32, 2765 1975]. Web site: http://www.delphion.com/details?pn=US06486328__ •
Wildlife nutritional supplement Inventor(s): Fuhr; David R. (Winigan, MO), Hauser; David (Winigan, MO) Assignee(s): 4 Seasons Wildlife Nutrition, Llc (winigan, Mo) Patent Number: 6,572,903 Date filed: May 8, 2002 Abstract: The present invention is a wildlife nutritional supplement for free ranging ruminants including about 7.5-8.5% calcium, about 3.5% phosphorus, about 32-37% salt, at least one "B" series vitamin is selected from a group consisting of pantothenic acid, folic acid, riboflavin, niacin, thiamine, cobalamin, and pyridoxine hydrocholoride, about 16-19% sodium, about 0.15% magnesium, about 0.15% potassium, about 2.5% sulfur, about 1,200 PPM iron, about 20 PPM copper, about 105 PPM manganese, about 45 PPM zinc, about 5 PPM cobalt, about 1 PPM selenium, about 1 PPM iodine, about 50,000 IU/LB Vitamin A, about 20,000 IU/LB Vitamin D, about 50 IU/LB Vitamin E, about 134 MG/LB biotin, about 60 MG/LB ascorbic acid, oxytetracycline and fenbendazole. Excerpt(s): This invention relates to wildlife nutritional supplements, and more particularly to a ruminant feed supplement having enhanced palatability and immunesystem bolstering effects. The reduction of habitat due to human development has left a noticeable impact on the health and vitality of wild ruminant animals. Ruminant animals such as deer, elk and the like, suffer diminished reproduction, decreased weight, smaller antlers, and susceptibility to disease and parasites. There are seven major minerals that have an important effect on wildlife: (1) calcium aids in the growth of bones, teeth and antlers and is important in the function of muscles and nerves; (2) phosphorus aids in the growth of bones, teeth and antlers, enhances energy metabolism
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and enzymation as well as proper protein utilization; (3) potassium is integral in the function of nerves, enzyme processes, as well as mineral and water balance; (4) sulfur is an essential component of some proteins; (5) sodium is vital to the function of muscles and nerves and also maintains water balance; (6) chloride of sodium forms hydrochloric acid in the abomasums which aids in protein breakdown; and (7) magnesium is an important component is almost all body processes. Web site: http://www.delphion.com/details?pn=US06572903__
Patent Applications on Biotin As of December 2000, U.S. patent applications are open to public viewing.9 Applications are patent requests which have yet to be granted. (The process to achieve a patent can take several years.) The following patent applications have been filed since December 2000 relating to biotin: •
22325, a human biotin-requiring enzyme family member and uses therefor Inventor(s): Tsai, Fong-Ying; (Newton, MA) Correspondence: Steven A. Bossone; Millennium Pharmaceuticals, INC.; 75 Sidney Street; Cambridge; MA; 02139; US Patent Application Number: 20030096276 Date filed: August 20, 2002 Abstract: The invention provides isolated nucleic acids molecules, designated 22325 nucleic acid molecules, which encode novel biotin-requiring enzyme family members. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing 22325 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a 22325 gene has been introduced or disrupted. The invention still further provides isolated 22325 proteins, fusion proteins, antigenic peptides and anti-22325 antibodies. Diagnostic and therapeutic methods utilizing compositions of the invention are also provided. Excerpt(s): The present invention relates to an enzyme belonging to the superfamily of biotin-requiring enzymes. The invention also relates to polynucleotides encoding the enzyme. The invention further relates to methods using the enzyme polypeptides and polynucleotides as a target for diagnosis and treatment in enzyme-mediated disorders. The invention further relates to drug-screening methods using the enzyme polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and treatment. The invention further encompasses agonists and antagonists based on the enzyme polypeptides and polynucleotides. The invention further relates to procedures for producing the enzyme polypeptides and polynucleotides. Biotin is an essential co-factor for a major class of enzymes that are involved in lipid, amino acid, and carbohydrate metabolism (Moss et al. (1971) Adv. Enzymol. 35:321-422; Wood et al. (1977) Annu. Rev. Biochem. 46:385-413; Wood et al. (1985) Ann. N Y Acad. Sci. 447:1-22; Knowles (1989) Annu. Rev. Biochem. 58:195-221). The biotin-requiring enzymes (BREs) of aerobic organisms are either carboxylases or transcarboxylases. The carboxylases catalyze the transfer of a carboxyl group from bicarbonate to a metabolite such as pyruvate, propionyl-CoA, acetyl-CoA, or 3-methylcrotonyl-CoA. In a first step the biotin co-factor
9
This has been a common practice outside the United States prior to December 2000.
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is carboxylated in a reaction that requires ATP, Mg.sup.2+, and bicarbonate. The carboxyl group is then subsequently transferred from the carboxybiotin intermediate to the metabolite that is specific for each BRE. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Arrays using polymerized monomolecular films and methods for using and manufacturing the same Inventor(s): Bednarski, Mark D.; (Los Altos, CA), Guccione, Samira; (Hillsborough, CA), Hobbs, Susan K.; (Fremont, CA), Shi, Gongyi; (Dublin, CA), Yang, Yi-Shan; (Saratoga, CA) Correspondence: Bozicevic, Field & Francis Llp; 200 Middlefield RD; Suite 200; Menlo Park; CA; 94025; US Patent Application Number: 20030104451 Date filed: October 31, 2002 Abstract: Devices and methods of use and manufacture for the identification and characterization of analytes, e.g. proteins, are provided. The subject devices are characterized by having a substrate with a polymerized monomolecular film over at least a portion of the substrate, the monomolecular film having at least one ligand or specific binding pair member associated therewith. Preferably the monomolecular film is stable to the laser intensities employed in MALDI-MS. In certain embodiments, the ligands are biotin, integrin antagonists, antibodies and antigens. In using the subject devices, a subject device is contacted with a sample. If present in the sample, a member of the binding pair of interest binds to its complementary ligand and, once bound, can be analyze by mass spectroscopy techniques. Also provided are kits, which include the subject devices. Excerpt(s): A current emphasis in proteomics is the development of materials for use in immobilized microarrays for protein detection similar to that of gene expression analysis. Currently, materials for microarray development have focused on arraying biomolecules in a manner that retains their biological activity. Antibodies, small molecules, peptide ligands, purified recombinant proteins, and whole tissue preparations have been non-specifically arrayed on different materials. A more difficult problem is to design materials that can capture specific proteins and limit nonspecific binding to the surface. So far, the investigation of small molecule or peptide arrays have been limited to model systems that contain only purified proteins binding to their respective ligands displayed on the material surface. In addition, the captured biomolecule is often present at low concentrations and must be accurately detected and quantified. No materials have demonstrated the capability to both reproducibly capture specific proteins from complex protein mixtures and be amenable for rapid mass spectral analysis. For example, one current approach to protein identification involves isolating protein using two dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by enzyme degradation of the isolated protein spots on the gel and the preparation of peptide maps and bioinformatic searches. To perform 2D-PAGE, at least hundreds of femtomoles to low picomoles of isolated protein must be present on a single gel spot in order to identify the protein and/or characterize it. Thus, significant drawbacks to 2D-PAGE include, but are not limited to, time consuming processes, large sample requirements, time consuming sample preparation, low protein solubilization, separation of low abundance proteins from complex mixtures difficulties, high sample loads needed to evaluate co-translational and post-translational modifications and
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difficulties when used with highly basic proteins and very large (greater than 150 kDa) or very small (less than 10 kDa) proteins. In addition to 2D-PAGE, methods such as 2Dliquid chromatography and capillary zone electrophoresis have been employed. However, these also suffer from similar problems, i.e., lengthy processes, etc. Recently, chemical modification of surfaces by organic molecules or films has been successfully used to identify and/or characterize analytes such a proteins in a sample. Such chemically modified surfaces are commonly known as arrays (also known as microarrays). These arrays, in which a plurality of ligands or members of a specific binding pair are deposited onto a solid support surface in the form of an "array" or pattern, find use in a variety of applications, including, but not limited to, proteomic identification and analysis, gene expression analysis, drug screening, nucleic acid sequencing, mutation analysis, and the like. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Biotin derivatives Inventor(s): Nilsson, Rune; (Lund, SE), Sandberg, Bengt E.B.; (Hjarup, SE), Wilbur, D. Scott; (Edmonds, WA) Correspondence: Smith, Gambrell & Russell, Llp; Attorneys AT Law; Suite 800; 1850 M Street, N.W.; Washington; DC; 20036; US Patent Application Number: 20020159994 Date filed: June 15, 2001 Abstract: A method for the conditioning of an extracorporeal device is described, as well as a method for extracorporeal extraction of toxic material from mammalian body fluids in connection with diagnosis or treatment of a mammalian condition or disease, in which methods reagents having the ability to extract toxic material from mammalian body fluids are involved, and an extracorporeal device comprising said reagent. Excerpt(s): The present invention refers to a method for the conditioning of an extracorporeal device and to a method for extracorporeal extraction of toxic material from mammalian body fluids in connection with diagnosis or treatment of a mammalian condition or disease, in which methods reagents having the ability to extract toxic material from mammalian body fluids are involved, and to an extracorporeal device comprising said reagent. Toxic materials may be introduced into the blood of humans by accidents, from disease states, from bacterial or viral infections, or from administration of substances for treatment of certain diseases (e.g. cancer therapy). Many of these toxic materials may do considerable damage to body tissues such as kidney, liver, lung and bone marrow, and may even be fatal. It is desirable to remove such materials from the blood as quickly as possible. Although the body has natural defense mechanisms to remove unwanted toxic materials, those methods can be ineffective in many examples. Thus, certain toxic materials are best removed from the blood in an extracorporeal device. An example of such a device is the kidney dialysis machine, where toxic materials build up in the blood due to a lack of kidney function. Other medical applications where an extracorporeal device can be used include: [1] removal of radioactive materials, [2] removal of toxic levels of metals, [3] removal of toxins produced from bacteria or viruses, [4] removal of toxic levels of drugs, and [5] removal of whole cells (e.g. cancerous cells, specific hematopoietic cells--e.g. B, T, or NK cells) or removal of bacteria and viruses. In order for the extracorporeal device to function in toxin removal, it must have a chemical entity bound on it that has a high binding affinity with the toxic material that is to be removed from blood. Rather than
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binding that chemical entity directly to the column matrix in the extracorporeal device, it is preferentially bound through another binding pair of molecules. This arrangement of binding is used to make the toxin binding moiety more available in the blood and to make the device more generally applicable to a variety of toxic materials. A column matrix material is used that provides a high surface area while not restricting the flow of blood through it (Nilson, R. et. al. EPC 567 514). The column matrix has a protein (avidin or streptavidin) bound to it that has a high affinity for another molecule (e.g. biotin). That column is conditioned for use in a particular medical application by conjugation of a moiety that has a high affinity for the toxic material with two molecules of biotin such that attachment to the column matrix can be readily achieved. This conditioning reagent contains two biotin moieties rather than one as this configuration provides a higher degree of stability to the column matrix. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Biotin-peg-substrate for a lipase assay Inventor(s): Fowler, Anne; (Forest Farm Estate, GB), Hawes, Calvin Richard; (Forest Farm Estate, GB), James, David Martin; (Forest Farm Estate, GB), Poulsen, Fritz; (Bagsvaerd, DK), Price-Jones, Molly Jean; (Forest Farm Estate, GB), Tornqvist, Hans; (Bagsvaerd, DK) Correspondence: Amersham Biosciences; Patent Department; 800 Centennial Avenue; Piscataway; NJ; 08855; US Patent Application Number: 20040014133 Date filed: December 9, 2002 Abstract: Disclosed is a compound of Formula (I), wherein: L is a linking agent; B is a binding agent; X is an atom or group suitable for attaching L to the glycerol chain; and R is a straight chain saturated or unsaturated alkyl group having from 8 to 30 carbon atoms, substituted with M' or M" wherein at least one of M' and/or M" is a detectable label. The compound can be used as a lipase substrate in a solid phase-based assay system, such as a scintillation proximity assay, to detect lipase enzyme activity. Excerpt(s): The present invention describes a novel substrate for use in an assay for lipase enzyme activity. In particular, this novel substrate can be labelled and used in a homogeneous assay. Lipases are enzymes that catalyse the hydrolysis of triacylglycerols in the first step in recovering stored fatty acids for energy production. The sequence of hydrolysis from the three positions on glycerol depends on the specificity of the particular lipase involved. Lipase enzyme activity is an important function and its strict regulation is necessary to ensure healthy metabolism. For example, lipases in adipose tissue are key enzymes for the release of major energy stores. Their activity is under hormonal control to ensure that triacylglycerol hydrolysis is balanced with the process of triacyglycerol synthesis to assure adequate energy stores and yet avoid levels of fatty acids becoming so high as to cause adverse effects. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html
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Biotinylation of proteins Inventor(s): Zhang, Lin; (O'Fallon, MO) Correspondence: Donald R. Holland; Harness, Dickey & Pierce, P.L.C.; 7700 Bonhomme, Suite 400; Clayton; MO; 63105; US Patent Application Number: 20040033603 Date filed: August 19, 2002 Abstract: A eukaryotic bicistronic expression system for in vivo biotinylation of a protein is disclosed. The bicistronic expression system is based upon a polynucleotide which comprises a nucleic acid encoding a fusion protein made up of a selected protein and a biotinylation peptide, a nucleic acid sequence coding for an internal ribosome entry site and a nucleic acid sequence encoding a biotin ligase. Also disclosed are vectors and host cells containing the nucleic acid as well as methods for preparing a biotinylation protein and kits comprising the nucleic acid. Excerpt(s): This invention relates generally to the biotinylation of proteins, and, more particularly, to nucleic acid molecules, vectors and cells which can be used to produce biotinylated fusion proteins. Modem biochemistry and cell biology depend heavily on the ability to covalently attach affinity tags to biomolecules such as proteins. An affinity tag, when used with a binding partner for the affinity tag, provides an aid to detection or isolation of a biomolecule. One of the most commonly used affinity tags is the biotin moiety. Biotin, a naturally occurring vitamin, has several high-affinity binding partners, such as avidin, streptavidin, monomeric avidin, and anti-biotin antibodies (both polyclonal and monoclonal). The binding between biotin and avidin is among the strongest non-covalently attachments between molecules known, having a K.sub.d of approximately 10.sup.-15 (Wilchek, M., and Bayer, E. A. (1988). The avidin-biotin complex in bioanalytical applications. Anal. Biochem. 171: 1-32). Because of this unusually high binding affinity, and because biotinylation of a biomolecule can often be accomplished without altering the protein's activity, biotinylation is one of the most important tools available for tagging proteins. For example, streptavidin immobilized on a solid support can be used to separate a biotin-tagged protein from a mixture: the biotinylated protein attaches to the immobilized streptavidin, and the mixture can be washed away, leaving the biotinylated protein attached to the solid support. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html
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Cell-based assay for detecting activation of poly (ADP-ribose) polymerase Inventor(s): Szabo, Csaba; (Gloucester, MA), Virag, Laszlo; (Lugossy, HU) Correspondence: Pennie And Edmonds; 1155 Avenue OF The Americas; New York; NY; 100362711 Patent Application Number: 20020164633 Date filed: April 18, 2002 Abstract: The present invention relates to methods of detecting poly(ADP-ribose) polymerase ("PARP") activity in cells and tissue by contacting a cell with an effective amount of biotinylated NAD.sup.+ and detecting the presence of incorporated biotin within the cell. The invention also relates to the identification oxidatively stressed tissues and cells by detecting PARP activity. The invention also relates to identifying a patient suffering from a disorder that causes PARP overexpression.
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Excerpt(s): This application claims the benefit of U.S. Provisional Application No. 60/285,175 filed Apr. 20, 2001, which is incorporated herein by reference in its entirety. Poly(ADP-ribose) polymerase ("PARP") is a nuclear enzyme that becomes activated in response to DNA damage (de Murcia & Menissier de Murcia, 1994, Trends Biochem Sci 19:172-176). Activated PARP cleaves NAD to nicotinamide and ADP-ribose, and polymerizes the latter on to nuclear acceptor proteins such as histones, transcription factors and PARP itself. Poly-ADP ribosylation contributes to DNA repair and to the maintenance of genomic stability (Wang et al., 1997, Genes Dev 11:2347-58; SimbulanRosenthal et al., 1999, Simbulan-Rosenthal et al 1999, Proc. Natl. Acad. Sci. U.S.A. 96:13191-13196; and Muiras & Burkle, 2000, Exp. Gerontol. 35:703-709). During inflammation, ischemia-reperfusion or shock, free radical/oxidant-induced DNA single strand breakage triggers the over-activation of PARP, which leads to depletion of NAD (Szabo & Dawson 1998, Trends Pharmacol. Sci. 19:287-298 and Szab, 2000, Cell Death: the Role of PARP. Boca Raton, Fla., CRC Press). In an effort to re-synthesize NAD+, ATP will also be consumed, resulting in necrotic type cell death (Schraufstatter et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:4908-4912; Virag et al., 1998, Immunology 94:345-355 and Virag et al., 1998, J. Immunol. 161:3753-3759). This PARP mediated suicidal pathway has been implicated in the cell death of immune stimulated macrophages, as well as in peroxynitrite- or hydrogen peroxide-induced dysfunction or cell death of thymocytes, macrophages, endothelial cells, neuronal cells and fibroblasts (Soriano et al., 2001, Nat Med 7:108-13; Virag et al., 1998, Immunology 94:345-355; Szabet al., 1998, Proc Natl Acad Sci U.S.A. 95:3867-3872; and Zingarelli et al., 1996, J. Immunol. 156:350-358). Inhibition of PARP activity by pharmacological inhibitors or the absence of functional PARP enzyme in PARP knock out animals provides significant protection in animal models of a wide variety of diseases including various forms of inflammation, shock, stroke, myocardial ischemia, diabetes and diabetic endothelial dysfunction (Szabo & Dawson 1998, Trends Pharmacol. Sci. 19:287-298 and Szab, 2000, Cell Death: the Role of PARP. Boca Raton, Fla., CRC Press). Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Chromium/biotin treatment of dyslipidemia and diet-induced post prandial hyperglycemia Inventor(s): Greenberg, Danielle; (Waccabuc, NY), Harpe, Jon De La; (New York, NY), Juturu, Vijaya; (Dobbs Ferry, NY), Komorowski, James R.; (Trumbull, CT) Correspondence: Knobbe Martens Olson & Bear Llp; 620 Newport Center Drive; Sixteenth Floor; Newport Beach; CA; 92660; US Patent Application Number: 20020197331 Date filed: February 27, 2002 Abstract: A method for treating dyslipidemia and/or post prandial hyperglycemia by administering a combination of a chromium complex and biotin to an individual in need thereof is disclosed. The two compounds are administered orally or parenterally in daily dosages which provide between 25.mu.g and 1,000.mu.g of chromium and between 25.mu.g and 20 mg biotin. A method for reducing the glycemic index of food is similarly provided. Excerpt(s): The present application claims priority to Provisional Application 60/271,881 entitled CHROMIUM/BIOTIN TREATMENT HYPERCHOLESTEROLEMIA, filed on Feb. 27, 2001. The subject matter of aforementioned application is hereby incorporated by reference in its entirety.
No. OF the The
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present invention relates to the improvement of blood cholesterol and triglyceride levels. More specifically, the invention relates to methods of lowering LDL cholesterol, increasing HDL cholesterol, and decreasing triglyceride levels in the blood by administering doses of chromic picolinate and biotin. Additionally, the present invention relates to methods and compositions for reducing post prandial hyperglycemia and for lowering the glycemic index of foods. Dyslipidemias are disorders of lipoprotein metabolism, including lipoprotein overproduction or deficiency. These disorders may be manifested by elevation of the serum total cholesterol, low-density lipoprotein (LDL) cholesterol and triglyceride concentrations, and a decrease in the high-density lipoprotein (HDL) cholesterol concentration. Each year, millions of human beings suffer from the sequelae of hypercholesterolemia. Examples of the afflictions include hypertension, coronary artery disease, congestive heart failure, peripheral vascular disease, aneurysms, and death due at least in part to these conditions. Elevated blood cholesterol is one of the major modifiable risk factors for coronary heart disease (CHD), the leading cause of death in the United States. Kannel, W. B. et al. Declining Cardiovascular Mortality. Circulation 70:331-336 (1984). CHD is responsible for roughly 490,000 deaths each year. National Center for Health Statistics. Annual summary of births, marriages, divorces, and deaths: United States, 1993. Monthly vital statistics report; vol 42 no 13. Public Health Service, 1994. Nonfatal myocardial infarction (MI) and angina are similarly a source of substantial morbidity. Secondary physiological effects of hypercholesterolemia include cerebral strokes, compromised liver function, renal artery blockage, senility, male impotence, and arteriosclerotic aneurysms. The risk of such diseases can be reduced by increasing the level of HDL cholesterol in the blood and/or decreasing the level of LDL cholesterol in the blood. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Colorimetric in situ hybridization detection methods Inventor(s): Fletcher, Jonathan; (Brookline, MA), Xiao, Sheng; (Boston, MA) Correspondence: Helen C. Lockhart; Wolf, Greenfield & Sacks, P.C.; Federal Reserve Plaza; 600 Atlantic Avenue; Boston; MA; 02210; US Patent Application Number: 20030044822 Date filed: April 26, 2002 Abstract: The invention is a method for genomic subtractive hybridization. Specific nucleic acid sequences are removed from a sample of nucleic acid sequences by specifically hybridizing the sequences to a complementary nucleic acid sequence bound to a target molecule such as biotin. The target molecule is then contacted with a binding partner such as avidin and separated from the sample of nucleic acid sequences. As the target is separated from the sample the hybridized nucleic acid sequences are also removed from the sample. The method preferably involves the removal of repetitive nucleic acid sequences from a nucleic acid sample to generate a library of probes that are substantially free of repetitive nucleic acid sequences. Excerpt(s): The present invention relates to a method for genomic subtractive hybridization. The invention also relates to methods for generating probes for in situ hybridization techniques. In particular, the methods for genomic hybridization are useful for generating probes for techniques such as in situ hybridization. Chromosomes within a living cell encompass all of the DNA of a particular organism. The number and structure of chromosomes within a cell are indicative of various normal and abnormal
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developmental traits. For instance, the presence of a particular chromosome type or number of a particular chromosome may be indicative of an abnormal condition. Humans having three copies of chromosome 18 (trisomy) develop a fatal disorder causing death within one year of birth and subjects having three copies of chromosome 21 (trisomy 21) develop Downs syndrome. In general chromosome abnormalities can result from extra or missing individual chromosomes, extra or missing portions of a chromosome, or chromosomal rearrangements, including translocations, deletions, and inversions. The trisomies discussed above involve addition of chromosomal material. A translocation involves the transfer of a piece of a chromosome to another chromosome. An example of a disorder involving an exchange of chromosomal material is chronic myelogenous leukemia which involves a translocation of chromosomal material from chromosome 9 to chromosome 22. An inversion involves a reversal in polarity of a chromosomal segment. Dicentrics produce a chromosome with two centromeres. Characteristic chromosome abberations have been described in a wide range of tumors. Specific oncogene and tumor suppressor gene targets affected by these chromosomal abnormalties have been characterized in some tumors but most of them remain to be studied. A major goal in studying human cancer chromosome and gene aberrations is to elucidate biological pathways responsible for neoplastic transformation. Several such pathways have already been identified through characterization of particular cancer chromosome aberrations (1, 3). Another goal in cancer chromosome evaluation is the identification and validation of novel diagnostic and prognostic markers (4, 5). Cytogenetic markers have utility as histological adjuncts, and methods that delineate diagnostic chromosomal aberrations or associated molecular changes are becoming increasingly important in experimental pathology (6-8). Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Compositions and methods for prophylactic and therapeutic supplementation of nutrition in subjects Inventor(s): Balzer, Charles; (Lavalette, NJ), Giordano, John A.; (West Orange, NJ) Correspondence: Mckenna & Cuneo, Llp; 1900 K Street, NW; Washington; DC; 20006; US Patent Application Number: 20030012826 Date filed: October 19, 2001 Abstract: The present invention relates to compositions without added iron and methods for prophylactic nutritional supplementation and therapeutic nutritional supplementation. Specifically, the method involves administering to an individual a composition comprising carotenoids, vitamin E, vitamin D, vitamin C, thiamine, riboflavin, niacin, folic acid, pyridoxine, biotin, pantothenic acid, cobalamin, magnesium, manganese, zinc, selenium, chromium, copper, alpha lipoic acid, and lutein, wherein the composition is free of added iron. Excerpt(s): The present invention is related to and, in accordance with the provisions of 35 U.S.C.sctn.119(e), claims the benefit of provisional patent application Serial No. 60/301,443 filed Jun. 29, 2001, which is expressly incorporated fully herein by reference. The present invention relates to compositions comprising various vitamins and minerals, and without added iron, and methods for using these compositions for prophylactic nutritional supplementation and therapeutic nutritional supplementation in, for example, physiologically stressful conditions and to minimize the effect of exogenous iron supplementation. Nutrition plays a critical role in maintaining good
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health. Proper nutrition prevents dietary deficiencies, and also protects against the development of disease. Proper nutrition plays an increasingly important role as the body faces physiological stress. For example, as the body ages it suffers significant physiological stresses. Specifically, as the body metabolism shifts to accumulating larger fat stores and decreasing lean body mass, this increase in body weight may lead to obesity and associated conditions such as diabetes, cardiovascular disease, hypertension, osteoarthritis, and cancer. Other conditions, such as anorexia, malnutrition, gastrointestinal disorders, chronic alcoholism, chronic infections, acute infections, congestive heart failure, hyperthyroidism, poorly controlled diabetes, cheilosis, gingivitis, stomatitis and dietary restrictions, often result in physiological stresses that may be exacerbated by poor nutrition. In particular, these disease states may result in increased oxidative stress or elevated homocysteine levels that further compromise health. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Device and method for integrated diagnostics with multiple independent flow paths Inventor(s): Barski, Stanislaw JR.; (Limerick, ME), Clark, Scott M.; (Cape Elizabeth, ME), Kepron, Michael R.; (Standish, ME), Suva, Robert H.; (Windham, ME), Workman, Erwin F. JR.; (Cape Elizabeth, ME) Correspondence: Mcdonnell Boehnen Hulbert & Berghoff; 32nd Floor; 300 S. Wacker Drive; Chicago; IL; 60606; US Patent Application Number: 20030040021 Date filed: April 3, 2002 Abstract: Devices and methods for performing assays to determine the presence or quantity of a specific analyte of interest in a fluid sample. In devices according to this invention two separate flow paths are established sequentially in the device with a single user activation step. The first flow path delivers the analyte of interest (if present in the sample) and conjugate soluble binding reagents to the solid phase. If analyte is present, an analyte:conjugate complex is formed and immobilized. The volume of sample delivered by this first path is determined by the absorbent capacity of the solid phase, and not by the amount of sample added to the device, relieving the user from the necessity of measuring the sample. The sample/conjugate mixture is prevented from entering the second flow path because the capillarity and the surface energy of the second flow path prevent it from being wetted by this mixture. The second flow path allows a wash reagent to remove unbound conjugate and sample from the solid phase to the absorbant, and optionally to deliver detection reagents.The invention may be adapted to many assay formats including, sandwich immunoassays, colloidal gold, or sol particle assays, heterogeneous generic capture assays and competitive assays.In one embodiment, sandwich assays can be performed by immobilizing an analyte binding reagent on the solid phase, and drying a labeled analyte binding reagent in the first flow path. In a competitive assay embodiment, the first flow path would contain labeled analyte that is dissolved by the sample, and the analyte binding reagent is immobilized on the solid phase. In each of these embodiments, the assay can be further modified to run in a "generic capture" format, where the solid phase binding reagent is instead conjugated to a generic ligand such as biotin, and dried in the first flow path (either together or separately from the other assay reagents), and a generic ligand binding reagent (such as avidin) is immobilized on the solid phase.Another aspect of the present invention includes a subassembly for the immunoassay device that is comprised of a
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plastic housing and a means for delivering fluid and/or wash solution. This subassembly comprises a structure formed from a hydrophobic polymer selectively treated with a water insoluble surface active agent that has been applied as a solution in an organic solvent rendering portions of the surface hydrophilic. When the surface is contacted with an aqueous liquid, it flows only along the treated areas, creating a defined fluid flow path, thereby delivering sample/conjugate solutions to said solid phase. Excerpt(s): This invention relates to devices and methods for performing assays to determine the presence or quantity of a specific analyte of interest in a fluid sample. Devices of this invention assay a measured amount of sample employing at least two separate and distinct flow paths which are initiated simultaneously with a single user activation step. These paths are timed for the sequential delivery of assay reagents to the reaction zone, followed by wash or substrate and wash reagents to that zone. These inventive devices and methods may be used for qualitative, semi-quantitative and quantitative determinations of one or multiple analytes in a single test format. They may be practiced with ELISA, sol particle and other assay formats, and are particularly suitable for simultaneous multiple analyte assays. These inventive devices and methods provide for the controlled, self delivery of reagents with no timed steps, and minimal user intervention, in most instances a single activation step. Many prior art assay devices and systems require the user to measure or control the amount of sample added to the device, for example, by dilution. Many prior art assays and systems also require the user to perform a timed sequence of steps and/or to make multiple physical interventions to the device in order to perform the assay. Buechler et al., U.S. Pat. Nos. 5,458,852 and 5,885,527 (1995 and 1999, respectively), disclose diagnostic devices which do not use porous membranes. In assay methods using such devices, fluid flow is unidirectional and reaction and detection occur in distinct zones. Excess sample/conjugate mixture is used to wash the detection zone. The use of a separate, non-sample wash is not taught or suggested, limiting the versatility of assays of this invention. Likewise, Buechler does not teach or suggest the creation of a second flow path. The time gate in Buechler functions as a delay mechanism in a fluid path, not to redirect fluid flow or to permit different fluids to flow sequentially through a reaction zone. The methods and devices of Buechler et al. do not allow one to use multiple reagents, different wash and substrate reagents and cannot be used in an enzyme amplified assay. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Devices and methods for isolating and recovering target cells Inventor(s): Bernstein, Steve; (Los Olivos, CA), Ross, Amelia A.; (Laguna Niguel, CA) Correspondence: Oppenheimer Wolff & Donnelly Llp; 840 Newport Center Drive; Suite 700; Newport Beach; CA; 92660; US Patent Application Number: 20030175818 Date filed: September 18, 2002 Abstract: A cell isolating device and method is provided to concentrate or isolate cells with specific characteristics from a mixture of different cell types. One embodiment may comprise two subtypes of antibodies that are directly conjugated to biotin (Ab.sub.b) and conjugated to a fluorescent molecule (Ab.sub.f). The conjugated antibodies (Ab.sub.b+Ab.sub.f) bind to the target cells in a mixed cell suspension. The cell suspension is then passed over an immobilized avidin or streptavidin substrate on a
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glass microscope slide. The biotinylated target cells adhere to the avidin/streptavidin substrate, while the unbound cells are washed off and collected in a wicking member. Captured cells on the avidin/streptavidin substrate may then be visualized directly using a fluorescent microscope or detected and enumerated via an on-board fluorescent detection device. Additional chemicals and/or physical manipulation may then be applied to the device to release viable target cells for subsequent analysis. Excerpt(s): This application claims the benefit of U.S. Provisional Application No. 60/364,679, filed Mar. 15, 2002, and whose entire contents are hereby incorporated by reference. All living bodies are comprised of individual cells, each cell defining an environment where various biological and chemical reactions take place. In particular, each cell contains a cell membrane that separates the internal environment of the cell from the external environment and thereby controls the entry and exit of various nutrients and waste. Additionally, the cell membrane includes various proteins, sugars, and other molecules that "identify" a particular cell type, these identifying molecules commonly being referred to as antigens. In order to better understand the function and pathologies of cells, numerous methods have been developed to isolate and concentrate a desired target cell population from a mixed cell population so that the target cell population can be further analyzed. One such method is based upon cell density wherein a mixture of cells is spun at high speeds in a centrifuge so that the higher density cells become separated for the lesser density cells. Although this method is effective at separating different cells, centrifugation does not have good cell-separation specificity as different types of cells may have the same or similar cell density. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Diagnosis kit for mycobacterium species indentification and drug-resistance detection and manufacturing method thereof Inventor(s): Kim, Hyung-Jung; (Gyonggi-do, KR), Kim, Jeong Mi; (Seoul, KR), Kim, Na Young; (Seoul, KR), Park, Mi Sun; (Busan, KR), Yoon, Sung Wook; (Seoul, KR) Correspondence: Frank Chau; F Chau & Associates; Suite 501; 1900 Hempstead Turnpike; East Meadow; NY; 11554; US Patent Application Number: 20040038233 Date filed: July 7, 2003 Abstract: The present invention relates to diagnosis kit for Mycobacterium species identification and drug-resistance detection and manufacturing method thereof, which can discriminate a Mycobacterium Tuberculosis rpoB gene point mutation relating to the Mycobacterium species identification and drug-resistance swiftly, exactly and in large quantities using an oligonucleotide chip. The diagnosis kit for Mycobacterium species identification and drug-resistance detection in accordance with the present invention consists of an oligonucleotide chip including a Mycobacterium tuberculosis complex probe, a Mycobacterium species identification probe and a drug-resistance detection probe of a Mycobacterium tuberculosis rpoB gene, and a fluorescent material containing a biotin-binding protein so as to detect hybridization of amplified products of a specimen marked as biotine and the corresponding probe. Excerpt(s): The present invention relates to a diagnosis kit for Mycobacterium species identification and drug-resistance detection and a manufacturing method thereof, and more specifically, to a diagnosis kit for Mycobacterium species identification and drugresistance detection in which point mutations of Mycobacterial rpoB gene related to the
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drug-resistance can be discriminated speedily and accurately in large quantity by using an oligonucleotide chip, and a manufacturing method thereof. About two million people die from tuberculosis worldwide each year. The increase in immigration, the spread of HIV/AIDS, and the emergence of drug-resistance strains are enhancing the mortality of tuberculosis. AIDS patients and newborns with weak immune system can develop tuberculosis from not only Mycobacterium tuberculosis infection but also MOTT (Mycobacterium other than tuberculosis) infection, particularly, Mycobacterium aviumintracellulare (MAI), Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium kansasaii, Mycobacterium xenopi, Mycobacterium marinum, Mycobacterium scrofulaceum, and Mycobacterium szulgai. Tuberculosis is normally treated by chemotherapy with various anti-tuberculosis drugs. Since there are numerous different strains of Mycobacteria with diverse drug-susceptibility, detection and identification of the causative bacterium is important for the effective treatment. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Dioxin derivatives and method of measurement therewith Inventor(s): Kodaira, Tsukasa; (Tokushima, JP), Yanaihara, Noboru; (Toyonaka-shi, JP) Correspondence: Oblon, Spivak, Mcclelland, Maier & Neustadt, P.C.; 1940 Duke Street; Alexandria; VA; 22314; US Patent Application Number: 20030108955 Date filed: November 1, 2002 Abstract: The invention provides a highly sensitive measurement and detection technique for dioxins, which has advantages inherent to immunoassay; and markers to be used in the technique.Disclosed are a biotinylated dioxin derivative of formula (1): 1(wherein X represents a hydrogen atom or a chlorine atom; R.sup.1 represents a biotin residue; R.sup.2's, which may be identical to or different from one another, independently and individually represent an arginine residue or a lysine residue; n is an integer from 1 to 5 inclusive; and m is an integer from 1 to 3 inclusive), and an immunoassay method for dioxins characterized by using the derivatives as a marker. Excerpt(s): The present invention relates to a novel biotinylated dioxin derivative, and to an improved immunoassay method for dioxins, inter alia, 2,3,7,8-tetrachlorodibenzo-pdioxin (hereinafter referred to as "2,3,7,8-TCDD"), making use of the derivative. Dioxins collectively refer to polychlorinated dibenzo-p-dioxins (PCDDs) and their analogues, polychlorinated dibenzofurans (PCDFs). Dioxins include numerous isomers which differ from one another in terms of the positions and numbers of chlorine atoms. Dioxins are discharged from factories and incinerators when, for example, chlorinecontaining organic compounds are incinerated, and spread into the air, rivers, and soil. Dioxins have attracted attention because of their toxicity; in particular, 2,3,7,8-TCDD is highly toxic, adversely affecting organs and physiological functions of humans, livestock, and poultry, and raising serious social problems in both Japan and overseas countries. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html
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Directed enzymatic modification of analytes for affinity capture and analysis Inventor(s): Schulman, Howard; (Palo Alto, CA) Correspondence: Swanson & Bratschun L.L.C.; 1745 Shea Center Drive; Suite 330; Highlands Ranch; CO; 80129; US Patent Application Number: 20030003512 Date filed: June 27, 2002 Abstract: Proteins are selectively extracted from complex biological mixtures for subsequent analysis by methods such as mass spectrometry. Protein-binding (e.g., PDZ or SH2) or cellular compartmentalizing (e.g., membrane-targeting) domains are provided to target specific proteins, and targeted proteins are enzymatically modified by attachment of modification molecules. For example, targeted proteins can be biotinylated by the enzyme biotin protein ligase. The protein-targeting domain and protein-modifying enzyme can be immobilized on a solid surface (macroscopic planar surface or particle surface), provided as a fusion protein, or chemically coupled. The modified proteins are then exposed to complementary molecules (e.g., avidin or streptavidin) that bind strongly to the modification molecule, resulting in affinity capture of the selected proteins. Because the protein-targeting domain brings the protein in proximity to the active site of the enzyme, the method effectively broadens the substrate specificity of the enzyme, allowing affinity capture of any desired protein subset. Excerpt(s): This application claims the benefit of U.S. Provisional Application No. 60/301,683, "Targeted Biotinylation for Affinity Capture in Multiplexed and Quantitative Mass Spectrometry of Analytes," filed Jun. 27, 2001, incorporated herein by reference. The present invention relates generally to qualitative and quantitative analysis of components of biological mixtures. More particularly, it relates to a method for selectively enzymatically modifying subsets of proteins in a mixture to enable their subsequent affinity capture and analysis by methods such as mass spectrometry. Recent developments in electrospray ionization (ESI) mass spectrometry (MS) and matrixassisted laser desorption ionization (MALDI) MS enable the quantification and structural elucidation of novel proteins in complex biological samples. Although MS can be used for multiplexed quantitative assays over a broad range in molecular weight, interpreting mass spectra becomes difficult when the samples contain a large number of components of similar mass. Furthermore, the wide concentration range of proteins in biological samples makes it difficult to detect low-abundance proteins, because highabundance proteins overwhelm or saturate separation systems. These problems can be addressed by separating the sample in a manner that reduces the complexity of the proteome and simplifies the acquired mass spectra. One useful sample separation method is affinity capture, in which proteins are selectively extracted from solution onto a surface containing molecules with which the proteins interact. The surface can be a single surface (e.g., planar) to which the sample is applied or the surfaces of nano- or microparticles dispersed in the sample and recovered after capture. The captured proteins subsequently can be eluted from the surface (if necessary) for analysis by MS. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html
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Enhancement of intracellular delivery and tissue targeting of drugs and genes Inventor(s): Albelda, Steven M.; (Bala Cynwyd, PA), Muzykantov, Vladimir R.; (Warwick, PA) Correspondence: Licata & Tyrrell P.C.; 66 East Main Street; Marlton; NJ; 08053; US Patent Application Number: 20030206911 Date filed: May 28, 2003 Abstract: A method for enhancing intracellular delivery of effector molecules is provided. The method involves modifying selected antibodies with biotin and streptavidin, conjugating these antibodies with an effector molecule, and delivering the conjugated effector to an intracellular target specifically recognized by the antibody. Excerpt(s): This application is a continuation of U.S. application Ser. No. 09/623,822 filed Oct. 25, 2000, which claims the benefit of priority from PCT Application Serial No. PCT/US99/05279 filed Mar. 10, 1999 and U.S. Provisional Application Serial No. 60/077,375 filed Mar. 10, 1998 each of which are herein incorporated by reference in their entirety. Targeting of drugs or genetic material to defined cells, tissues or organs increases the specificity and effectiveness of drug therapy and reduces the incidence of potentially harmful side effects. Intracellular delivery and proper intracellular processing are required for specific and effective therapeutic applications of certain classes of drugs including, but not limited to, immunotoxins, antioxidants, NO-donors, antibiotics, antisense oligonucleotides, nucleic acids and intracellular hormones. Further, intracellular delivery of gene therapy products is crucial to successful treatment. In the case of antioxidants, immunotoxins, antisense agents, hormones, gene therapy agents and other therapeutic compounds, referred to herein as "effectors", only limited spontaneous cellular internalization typically occurs. Accordingly, strategies to facilitate or enhance internalization have been developed and include chemical modification with polyethylene glycol (Abuchowski et al. J. Biol. Chem. 1977 252(11):3852-3586; Abuchowski et al. J. Biol. Chem. 1992 252(11):3578-3581; Beckman et al. J. Biol. Chem. 1988 263:6884-6892), encapsulation in liposomes (Freeman et al. J. Biol. Chem. 1983 258:12534-12542; Briscoe et al. Am. J. Physiol. 1995 12(3):L374-L380), and conjugation with ligands of internalizable receptors (Wagner et al. Adv. Drug. Del. Rev. 1994 14:113-135; Chen et al. FEBS Lett. 1994 338:167-169). Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html
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Finely particulate functional metal and finely particulate functional semiconductor each with dispersion stability and process for producing the same Inventor(s): Ishii, Takehiko; (Washinomiya-Mati, JP), Kataoka, Kazunori; (Nakano-ku, JP), Nagasaki, Yukio; (Moriya-mati, JP), Otsuka, Hidenori; (Kawasaki-shi, JP) Correspondence: Sherman & Shalloway; 413 N Washington Street; Alexandria; VA; 22314; US Patent Application Number: 20040038506 Date filed: February 28, 2003 Abstract: A stabilized dispersion of metal fine particles comprising, fine particles of metal which is obtained by reducing at least one metallic acid or salt thereof selected from the group consisting of haloauric acid, haloplatinic acid, silver nitrate and halorhodic acid by a reducing agent in the aqueous solution of (1) R-PEG-SX [R is a
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functional group selected from the group consisting of acetal, aldehyde, hydroxyl group, amino group, carboxyl group, active ester group, azide group, biotin group, monosaccharide, oligosaccharide, amino acid, nucleic acid, allyl group, vinyl benzyl group, methacryloyl group and acryloyl group, PEG is --(CH.sub.2CH.sub.2O).sub.n--, X is H or pyridylthio group] or (2) R-PEG/PAMA (given structural formula A), and said fine particles load a polymer having PEG unit possessing above mentioned functional group on the surface. Excerpt(s): The present invention relates to a stabilized dispersion of metal fine particles having high functional group at the voluntarily end of a compound having a PEG unit mentioned below, block copolymer having said PEG unit and unit of structural formula A or graft polymer of structural formula B, obtained by existing a compound having a PEG unit which possesses a functional group disclosed in claim 1, a compound having a PEG unit which possesses a functional group disclosed in claim 3 and block polymer of structural formula A or graft polymer of structural formula B having a PEG unit which possesses a functional group disclosed in claim 5 during the producing process of monodispersed metal or semiconductor fine particles (colloidal particles), further relates to the method for production of said stabilized dispersion of metal fine particles. In general, metal or semiconductor colloid (ultra fine particle) is an important industrial material which is broadly used in various fields, for example, medical treatment [as a medicine (medicine of ultra fine particles having penetrating ability to membrane internus), various inspection agents or DDS], dye (coating), foods or catalyst. Especially, metal particles of several tens to several hundreds nano meter size (called as submicron: meso size) and having narrow distribution is expected as a high functional materials such as diagnostic which uses Raman spectrum or microwave or a labeling agent for electron microscope. For example, Assay method, which uses colloidal metal particles has been developed. Said method is characterized as follows. That is, since colloidal metal is used as a label, the specific bonding site to be labeled is bonded on colloidal metal by absorption and said labeling material is detected and measured (JP Laid Open Publication 6-116602, hereinafter shortened to Document A). Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Forming and modifying dielectrically-engineered microparticles Inventor(s): Becker, Frederick F.; (Houston, TX), Gascoyne, Peter R.C.; (Bellaire, TX), Sharma, Susan; (Sugarland, TX), Vykoukal, Daynene; (Houston, TX), Vykoukal, Jody; (Houston, TX) Correspondence: Michael C. Barrett; Fulbright & Jaworski L.L.P.; 600 Congress Avenue, Suite 2400; Austin; TX; 78701; US Patent Application Number: 20030119057 Date filed: December 20, 2001 Abstract: Engineered microparticles, libraries of microparticles, and methods relating thereto. The microparticles are distinguishable based on differences in dielectric response to an applied electric field. In different embodiments, the dielectric differences may be engineered through, but not limited to, dielectrically dispersive materials, surface charge, and/or fluorescence. Gangliosides may be incorporated with the microparticles to control aggregation. Vesicles including erythrocyte ghosts may be used as a basis for microparticles. The microparticles may utilize a biotin streptavidin system for surface functionalization.
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Excerpt(s): Methodology of the current disclosure may be used with the apparatuses and methods described in U.S. Pat. No. 6,294,063, which is expressly incorporated herein by reference. Other patents and applications that may be used in conjunction with the current disclosure include U.S. Pat. No. 5,858,192, entitled "Method and apparatus for manipulation using spiral electrodes," filed Oct. 18, 1996 and issued Jan. 12, 1999; U.S. Pat. No. 5,888,370 entitled "Method and apparatus for fractionation using generalized dielectrophoresis and field flow fractionation," filed Feb. 23, 1996 and issued Mar. 30, 1999; U.S. Pat. No. 5,993,630 entitled "Method and apparatus for fractionation using conventional dielectrophoresis and field flow fractionation," filed Jan. 31, 1996 and issued Nov. 30, 1999; U.S. Pat. No. 5,993,632 entitled "Method and apparatus for fractionation using generalized dielectrophoresis and field flow fractionation," filed Feb. 1, 1999 and issued Nov. 30, 1999; U.S. patent application Ser. No. 09/395,890 entitled "Method and apparatus for fractionation using generalized dielectrophoresis and field flow fractionation," filed Sep. 14, 1999; U.S. patent application Ser. No. 09/883,109 entitled "Apparatus and method for fluid injection," filed Jun. 14, 2001; U.S. patent application Ser. No. 09/882,805 entitled "Method and apparatus for combined magnetophoretic and dielectrophoretic manipulation of analyte mixtures," filed Jun. 14, 2001; U.S. patent application Ser. No. 09/883,112 entitled "Dielectrically-engineered microparticles," filed Jun. 14, 2001; and U.S. patent application Ser. No. 09/883,110 entitled "Systems and methods for cell subpopulation analysis," filed Jun. 14, 2001, each of which are herein expressly incorporated by reference. Yet another application that may be used in conjunction with the teachings of the current invention include those described in "Micromachined impedance spectroscopy flow cytometer of cell analysis and particle sizing," Lab on a Chip, vol. 1, pp. 76-82 (2001), which is incorporated by reference. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Homogeneous immunoassays for multiple allergens Inventor(s): Brown, Christopher R.; (San Mateo, CA), Murai, James T.; (San Bruno, CA) Correspondence: Townsend And Townsend And Crew, Llp; Two Embarcadero Center; Eighth Floor; San Francisco; CA; 94111-3834; US Patent Application Number: 20030109067 Date filed: December 6, 2001 Abstract: A homogeneous immunoassay method and system for quantitative determination of total immunoglobulin E and specific antibody levels to a plurality of allergens, in which a relatively small sampling of blood is required. The method utilizes relatively small microparticles in suspension with 1-5.mu.L of undiluted sample. The immunoassay procedure is an immunometric sandwich procedure preferably utilizing biotin-streptavidin signal amplification techniques and R-phycoerytherin fluorescent labels. Excerpt(s): This invention relates to a homogenous immunoassay method for determining specific antibody levels to a multiplicity of allergens from a blood sample, or for determining total immunoglobulins E levels in such a sample, for the purpose of diagnosing allergy. Confirmatory diagnostic testing may be conducted by in-vivo skin testing, in-vivo provocation testing, or in-vitro testing for the presence of circulating allergen-specific antibodies from blood samples. Direct provocation, by direct inhalation or ingestion of possible offending allergens, while relevant, is unpleasant, possibly dangerous and cannot be performed for multiple allergens at one sitting. Skin testing
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(also referred to as skin prick testing or scratch testing) is an in vivo procedure that involves applying an allergen sample, or more generally a multiplicity of allergens, directly to a patient's forearm or back via a small needle scratch and measuring the size of the inflammatory reaction (wheal) at the applied site on the skin. Skin prick testing is widely used, is reliable under optimal testing conditions, can be painful, is subject to large differences in technique and interpretations, and cannot be used on patients taking certain drugs or patients with skin problems. Furthermore, both provocation and skin prick in-vivo diagnostic methods have the potential for sensitizing patients to new allergens and, in extreme cases, eliciting a life-threatening anaphylactic reaction upon direct exposure to the offending allergen(s). Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Insect resistant transgenic plants expressing biontin-binding protein gene Inventor(s): Bailey, Michele; (College Station, TX), Howard, John; (College Station, TX) Correspondence: Arent Fox Kintner Plotkin & Kahn; 1050 Connecticut Avenue, N.W.; Suite 400; Washington; DC; 20036; US Patent Application Number: 20030213011 Date filed: April 11, 2003 Abstract: Methods of controlling insect infestation of plants using a nucleotide sequence encoding biotin-binding protein to produce high levels of biotin-binding protein in the plant, i.e., a mean concentration of.gtoreq.100 ppm. Excerpt(s): This application claims priority under 35 U.S.C.sctn.119(e) to U.S. provisional application Serial No. 60/204,639, filed May 17, 2000. This application is directed to methods of controlling insect infestation of plants using a nucleotide sequence encoding a biotin-binding protein to produce high levels of the protein in the plant. The output of world agriculture must increase if the demands of a rising population are to be met. To preserve more of what is grown, genetic engineering has been utilized to produce transgenic crops that contain proteins detrimental to critical life functions of pests and pathogens. See, e.g., Hilder and Boulter, Crop Protec. 18, 177-191 (1999). One of the first commercially successful transgenic food crops is maize, which is resistant in the field to corn borers because it contains toxins produced by the soil bacterium Bacillus thuringiensis (Bt). See, e.g., U.S. Pat. No. 5,322,687. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html
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Magnetic fine particles having lower critical solution temperature Inventor(s): Furukawa, Hirotaka; (Yokohama-shi, JP), Kataoka, Kazunori; (Tokyo, JP), Ohnishi, Noriyuki; (Yokohama-shi, JP), Ueno, Katsuhiko; (Tsukuba-shi, JP) Correspondence: Wenderoth, Lind & Ponack, L.L.P.; 2033 K Street N. W.; Suite 800; Washington; DC; 20006-1021; US Patent Application Number: 20030165962 Date filed: February 21, 2003 Abstract: The present invention relates to magnetic fine particles having a lower critical solution temperature to which at least one substance selected from biotin and avidin is immobilized, and a method of converting a substance, a method of separating or
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concentrating a microorganism, a method of modifying a denatured protein, a method of detecting a nucleic acid, a separating agent, and a method of separating a biological substance using the same. Excerpt(s): The present invention relates to magnetic fine particles having a lower critical solution temperature, and a method of converting a substance, a method of separating or concentrating a microorganism, a method of modifying a denatured protein, a method of detecting a nucleic acid, a separating agent, and a method of separating a biological substance, each using the magnetic fine particles. As described in Clin. Microbiol. Rev., 1994, pp. 43-54, there are many attempts to separate a variety of biological molecules and microorganisms by immobilizing antibodies or pairing bases to magnetic fine particles. As the magnetic fine particles for use in these methods, those having a particle size of 1.mu.m or more are usually used in consideration of the time for recovering them with a magnet. However, since the surface area decreases as the particle size increases, the efficiency is a big problem in the case that a small molecule such as a protein or nucleic acid is tried to separate. As a method for solving the problem of particle size, Applied. Microbiol. Biotechnol., 1994, Vol. 41, pp. 99-105 and Journal of fermentation and Bioengineering, 1997, Vol. 84, pp. 337-341 have reported that magnetic fine particles having a lower critical solution temperature (hereinafter referred to as "LCST") and a particle size of about 100 to 200 nm are obtained by polymerizing polyisopropylacrylamide having an LCST in the presence of magnetic fine particles and that the magnetic fine particles is possible to recover easily through their aggregation caused by raising the solution temperature to the LCST or higher even though the particle size of the magnetic fine particles is smaller than that of conventional particles. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Method and apparatus for recognizing molecular compounds Inventor(s): Han, Shubo; (Alhambra, CA), Ralin, David; (San Gabriel, CA), Rassman, William; (Los Angeles, CA), Zhou, Feiming; (Temple City, CA) Correspondence: Marvin H. Kleinberg; Kleinberg & Lerner, Llp; Suite 1080; 2049 Century Park East; Los Angeles; CA; 90067; US Patent Application Number: 20030224370 Date filed: May 31, 2002 Abstract: A probe-target reaction is made more recognizable by the provision of a massenhancing and/or evanescent-field-perturbing amplifier element which reacts uniquely with and binds to the probe-target pair to provide increased mass. Where the probetarget pair is hybridized dsDNA, a suitable mass-enhancing amplifier is anti-double stranded DNA mouse IgM. In examples with sufficient sequence pairs in the probetarget combination, a sequence-specific minor-groove-binding polyamide can be used that carries biotin which can be amplified by streptavidin in a suitable carrier. In a preferred embodiment, a plurality of probes are immobilized at the sites of a microarray, each probe being specific to a different target. Optics utilizing total internal reflection are described for observing perturbation of the evanescent field. Excerpt(s): The present invention relates to methods and apparatus for detecting, qualitatively and quantitatively, molecular interaction. To help describe the prior art and the present invention, it is deemed useful to provide a glossary of terms that will be used herein. A "probe" is intended to mean a known entity which may either be
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immobilized or free floating. A "target" or "ligand" is an "unknown" entity which has a known, specific complementary or reactive relationship with a specific probe. Probetarget combinations may be referred to as "complexes" or "pairs". A "primary amplifier" or "mass enhancing unit" is a "bulky" entity which has an interaction or bonding with a pair, or with a linking element that bonds to the pair. A "secondary amplifier" interacts with the primary amplifier or with the combination of primary amplifier-probe-target pair complex to form a new combination. A "tertiary amplifier" is any successive mass enhancer which can interact with a primary amplifier-probe-target pair complex or amplifier. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Method for detecting biopolymers Inventor(s): Iwao, Kanako; (Tokyo, JP), Kuwabata, Susumu; (Osaka, JP), Morita, Toshiki; (Tokyo, JP), Nakao, Motonao; (Tokyo, JP), Sato, Keiichi; (Tokyo, JP) Correspondence: Reed Smith Hazel & Thomas Llp; Suite 1400; 3110 Fairview Park Drive; Falls Church; VA; 22042-4503; US Patent Application Number: 20030162215 Date filed: February 26, 2003 Abstract: A technique is provided that easily detects biopolymers such as a DNA or a protein by utilizing semiconductor nanoparticles having different excitation wavelengths and fluorescence due to differences in particle size. By binding the semiconductor nanoparticles with avidin (or biotin), detection of biopolymers labeled with biotin (or avidin) is enabled. Excerpt(s): The present invention relates to a technique in which a semiconductor nanoparticle is bound to a molecule for detection, such as avidin, streptavidin or biotin, for detecting, as a fluorescent substance, a biopolymer such as a polynucleotide or a protein or the like. First, RT-PCR of mRNA is performed using reverse transcriptase. At this time, Cy3-dUTP or Cy5-dUTP is incorporated and unreacted dUTP is removed to prepare the target cDNA. Next, hybridization of the target cDNA with cDNA on a DNA chip is conducted. Finally, a laser beam is irradiated onto the DNA chip to detect fluorescence with wavelength. A laser beam having an excitation wavelength of 552 nm is irradiated for Cy3, and a laser beam having an excitation wavelength of 650 nm is irradiated for Cy5. However, in the above-described method, Cy3 and Cy5 are individually excited by their respective lasers, and it is only possible to detect one fluorescence with wavelength at a time. In other words, it is only possible to detect the fluorescence with wavelength corresponding to one excitation wavelength. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html
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Method for producing micro-carrier and test method by using said micro-carrier Inventor(s): Chang, Rong-Seng; (Chungho City, TW), Chao, Yu-Chan; (Chungho City, TW) Correspondence: Richard E. Fichter; Bacon & Thomas, Pllc; Fourth Floor; 625 Slaters Lane; Alexandria; VA; 22314-1176; US Patent Application Number: 20030059804 Date filed: May 29, 2002 Abstract: The invention provides a method for producing a microcarrier, which includes patterning pluralities of bar code on a mask; exposing the bar code to a substrate coated with photoresist; etching and removing residual photoresist and electroforming to a nickel plate; placing a bead coated with biotin or poly-L-lysine between two-nickel plates, and compressing the bar code on the surface of the bead to form a microcake-like particle with bar code; and combining the is particle with the corresponding biomolecule thereof to produce a micro-carrier with a label. The invention also provides a test method for identifying a bio-molecule, which includes mixing several micro-carriers with the labeled unknown bio-molecules; and identifying the bar code on the microcarrier via image recognition system, wherein the numbers and types of the known micro-carrier can be flexibly adjusted. Excerpt(s): The present invention relates to a method for preserving and testing biologically genetic information, and particularly to a micro-carrier and a test method for identifying unknown bio-molecules such as DNA or proteins by using a code labeled micro-carrier. Biotechnology has been developing quickly in recent years. Various products can be produced using molecular biology, biological cells, or other metabolites thereof by this technique, which can be extensively applied in the fields of pharmaceutical, pesticide, environmental protection, process development, and aquaculture. The combination of biotechnology with electric technology is a trend; wherein the most attractive is the biochip and DNA chip (i.e. gene chip). In addition to silicon, the material of those chips can include absorbent materials such as glass, plant cellulose, gel, and organic polymers. The gene chip has various gene fragments neatly aligned and adhered onto a nail-sized chip, in which thousands upon thousands of gene fragments are accommodated. Users can select different kinds of gene chips based on their purposes. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html
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Method for sequence specific biotinylation Inventor(s): Ambrosius, Dorothee; (Muenchen, DE), Lanzendoerfer, Martin; (Muenchen, DE), Schraeml, Michael; (Muenchen, DE), Watzele, Manfred; (Weilheim, DE) Correspondence: Hoffmann-la Roche INC.; Patent Law Department; 340 Kingsland Street; Nutley; NJ; 07110 Patent Application Number: 20030092073 Date filed: September 20, 2002 Abstract: A method of preparing a biotinylated polypeptide in a cell-free peptide synthesis reaction mixture by contacting, under suitable conditions, a polypeptide to be biotinylated, with a reaction mixture that includes ribosomes, tRNA, ATP, GTP, nucleotides, biotin and amino acids, and a polypeptide that includes an enzymatically
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active domain of a BirA enzyme. The polypeptide to be biotinylated includes a BirA substrate sequence tag, and the polypeptide to be biotinylated and the polypeptide comprising an enzymatically active domain of a BirA enzyme, are expressed in situ in the reaction mixture, by at least one nucleic acid molecule encoding the polypeptide to be biotinylated, and the enzymatically active domain of a BirA enzyme, respectively. Excerpt(s): The invention relates to an improved method for sequence specific biotinylation of polypeptides. The enzyme biotin haloenzyme synthetase of Eschericia coli ("E. coli"), is a biotin ligase (hereinafter also referred to as "BirA") that is the product of the qrA gene (Cronan, J. E., Jr., Cell 58 (1989) 427-429). BirA catalyzes the covalent addition of biotin, in vivo, to the.epsilon.-amino group of a lysine side chain in its natural substrate, biotin carboxyl carrier protein ("BCCP") (Cronan, J. E., Jr., et al., J. Biol. Chem. 265 (1990) 10327-10333). BCCP is a subunit of acetyl-CoA carboxylase and, in E. coli, BCCP is biotinylated. Biotinylation of proteins using a biotinylation enzyme by recombinant means is described, e.g., in WO 95/04069, incorporated by reference herein. Sequence specific enzymatic biotinylation, (also referred to herein as "specific biotinylation" or preparing, "specifically biotinylated" polypeptides) using BirA is also described for recombinant polypeptides during expression in E. coli (Tsao, K.-L., et al., Gene 169 (1996) 59-64), incorporated by reference herein. Altman, J. D., et al., Science 274 (1996) 94-96, incorporated by reference herein, describe the enzymatic biotinylation of isolated polypeptides in vitro, using also BirA. However, such a method is very laborious, requiring considerably more purification steps compared to conducting the biotinylation in vivo. First, the protein must be prepared, isolated and purified. Subsequently, biotinylation is performed, and thereafter, another purification is carried out. Parrott, M. B., and Barry, M. A., in Biochem. Biophys. Res. Communications 281 (2001) 993-1000, incorporated by reference herein, describe the metabolic biotinylation of secreted and cell-surface proteins from mammalian cells using the endogenous biotin ligase enzymes of the mammalian cell. Saviranta, P., et al., in Bioconjug. Chem. 9 (1998) 725-735, incorporated by reference herein, describe the in vitro enzymatic biotinylation of recombinant Fab fragments through a peptide acceptor tail. The proteins were recombinantly produced in E. coli, purified and subsequently biotinylated in vitro with BirA. After the removal of non-biotinylated Fab fragments, the overall yield of biotinylated Fab was 40%. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Multiple antioxidant micronutrients Inventor(s): Haase, Gerald M.; (Greenwood Village, CO), Prasad, Kedar N.; (Denver, CO) Correspondence: Ostrolenk, Faber, Gerb & Soffen, Llp; 1180 Avenue OF The Americas; New York; NY; 10036-8403; US Patent Application Number: 20030147996 Date filed: August 28, 2002 Abstract: A method for optimizing the health of humans according to their age and sex is disclosed wherein the method comprises administering to said humans a daily dose of a multiple antioxidant micronutrient composition comprising vitamin A (palmitate), beta-carotene (from natural d. salina), vitamin C (calcium ascorbate), vitamin D-3 (cholecalciferol), natural source vitamin E including both d-alpha tocopherol and dalpha tocopheryl acid succinate, thiamine mononitrate, riboflavin, niacinamide ascorbate, d-calcium pantothenate, pyridoxine hydrochloride, cyanocobalamin, folic
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acid (folacin), d-biotin, selenium (l-seleno methionine), chromium picolinate, zinc glycinate, calcium citrate, and magnesium citrate.For persons over the age of about 51, the composition preferably further comprises one or more of co-enzyme Q.sub.10, Nacetyl cysteine, and alpha lipoic acid. Preferably, also, vitamin D is added for women over the age of about 36. Excerpt(s): We claim the benefit under Title 35, United States Code,.sctn.120 of U.S. Provisional Application No. 60/315,523, filed Aug. 29, 2001, entitled MULTIPLE ANTIOXIDANT MICRONUTRIENTS FOR OPTIMAL HEALTH. In the beginning, the earth's atmosphere had no oxygen. Anaerobic organisms, which can live without oxygen, were thriving. About 2.5 billion years ago, blue-green algae in the ocean acquired the ability to split water into hydrogen and oxygen and this chemical reaction initiated the release of oxygen into the atmosphere. The increased levels of atmospheric oxygen caused extinction of many anaerobic organisms owing to oxygen's toxicity. This important biological event also led to the evolution of multicellular organisms, including humans, who utilize oxygen for survival. The content of oxygen in the air gradually increased to the current amounts of about 21 percent in dry air and about 34 percent in water. The use of oxygen by any organism generates free radicals that are toxic. Therefore, during this period of atmospheric oxygenation, organisms must have struggled to survive the sudden exposure to oxygen toxicity. There must have been enormous rearranging of nucleotides in genes to produce specific proteins that could protect organisms against the damage produced by free radicals. This process eventually led to the production of three antioxidant enzymes. Superoxide dismutase (SOD) requires manganese, copper, or zinc for its biological activity. Mn-SOD is present in mitochondria, whereas Cu-SOD and Zn-SOD are present in the cytoplasm and nucleus of the cell. All three can destroy free radicals and hydrogen peroxide. Another enzyme, catalase, requires iron for its biological activity and it destroys H.sub.2O.sub.2 in cells. Human tissue also contains glutathione peroxidase which requires selenium for its biological activity. It is also responsible for removing hydrogen peroxide. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Multi-vitamin and hormone replacement supplement Inventor(s): Fox, Dorothy Jean; (Chesapeake, VA), Schloss, Caroline Maxine; (Knotts Island, NC) Correspondence: Kimberly A Chasteen; Williams Mullen Clark & Dobbins; One Iod Oyster Point Road; Suite 210; Newport News; VA; 23602 Patent Application Number: 20030096018 Date filed: September 23, 2002 Abstract: A supplement is disclosed for use by naturally or surgically menopausal women. The supplement includes: Estrogen, Selenium, Zinc, Chromium, Calcium, Copper, Phosphorus, Magnesium, Molybdenum, Iodine, Beta Carotene, Ascorbic Acid, Vitamin D, Vitamin E, Vitamin K, Thiamin, Riboflavin, Vitamin B6, Vitamin B12, Folic Acid, Iron, Pantothenic Acid, and Biotin. The supplement provides hormone replacement therapy along with nutritional supplements. Excerpt(s): The present invention relates generally to a pharmaceutical supplement for menopausal women and more specifically to a pharmaceutical supplement which combines the hormone estrogen with daily supplemental vitamins to treat menopausal women and women who have undergone complete hysterectomies as more fully set
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forth in the below specifications, drawings and claims. It is well known that estrogen is critical to a woman's health in that it helps to protect the cardiovascular system, helps protect against bone loss and aids mental sharpness. At menopause or subsequent to a complete hysterectomy, the estrogen levels decline significantly thus, the protective aspects of estrogen are significantly reduced for these women. Because heart disease is a major cause of death in women, this creates an increased risk for menopausal and posthysterectomy women. Further, loss of the protection against bone loss can lead to osteoporosis, another major problem for these women. The impairment of cognitive abilities can be another side effect of the significant estrogen loss suffered in menopause or post-hysterectomy. Additional side effects have been linked to reduced estrogen levels such as urinary incontinence and weight gain. Many women are treated with hormone replacement therapy to help reduce these symptoms. The treatment generally consists of supplemental estrogen. This reduces the problems noted above, heart disease, bone loss, loss of cognitive ability, urinary incontinence, weight gain, as well as other well-known symptoms such as hot flashes. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Nutritive media and manufactured seeds comprising same Inventor(s): Carlson, William C.; (Olympia, WA), Grob, James A.; (Auburn, WA), Hartle, Jeffrey E.; (Milton, WA), Heilesen, Mollie K.; (Tacoma, WA), Salatas, Katherine M.; (Tacoma, WA) Correspondence: Christensen, O'connor, Johnson, Kindness, Pllc; 1420 Fifth Avenue; Suite 2800; Seattle; WA; 98101-2347; US Patent Application Number: 20030167684 Date filed: February 20, 2003 Abstract: Manufactured seeds are disclosed that comprise a unit of totipotent plant tissue and a nutritive medium. The nutritive medium can contain a number of different components selected from the following: a gel solute, charcoal, a carbon source, urea, KNO.sub.3, NH.sub.4NO.sub.3, CuCl.sub.2, CuSO.sub.4, KI, KH.sub.2PO.sub.4, CaCl.sub.2, MgSO.sub.4, Na.sub.2EDTA, FeSO.sub.4, ferric citrate, MnSO.sub.4, MnCl.sub.2, H.sub.3BO.sub.3, ZnSO.sub.4, CoCl.sub.2, Na.sub.2MoO.sub.4, (NH.sub.4).sub.2MoO.sub.4, thiamine, riboflavin, pyridoxine, HCl, Ca-pantothenate, nicotinic acid, biotin, folic acid, and myo-inositol. The nutritive medium can also include any of various protein amino acids, any of various polyamines, any of various oxygen-absorbing compounds, any of various non-protein amino acids, and/or a smoke suspension. Excerpt(s): This application is a divisional application of U.S. patent application Ser. No. 09/529,933, filed Apr. 21, 2000, now abandoned, which claims the benefit under 35 U.S.C.sctn.365(c) of PCT/US98/24820, filed Nov. 20, 1998, which claims the benefit under U.S.C.sctn.119(e) of U.S. Provisional Patent Application Serial No. 60/066,232, filed Nov. 20, 1997, the disclosures of all are hereby expressly incorporated by reference. The invention pertains to nutritive media, methods for using such media in the production of manufactured seeds, and manufactured seeds comprising same. In many instances it is desirable to grow large numbers of genetically identical plants. These plants can be selected and grown based on their particular qualities, such as their ability to grow in a particular climate, or their ability to produce a particular type or quality of fiber. Unfortunately, in many cases the production of such plants through standard breeding is not feasible.
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Patterned biological molecules on inner surface of enclosed tubes Inventor(s): Craighead, Harold G.; (Ithaca, NY), Orth, Reid N.; (Ithaca, NY), Turner, Stephen W.; (Ithaca, NY) Correspondence: Schwegman, Lundberg, Woessner & Kluth, P.A.; P.O. Box 2938; Minneapolis; MN; 55402; US Patent Application Number: 20030153010 Date filed: January 9, 2003 Excerpt(s): This application claims the benefit of priority under 35 U.S.C. 119(e) to U.S. Provisional Patent Application Serial No. 60/347,622, filed Jan. 10, 2002, which is incorporated herein by reference in its entirety. Many micro and nanotechnology bioassay applications such as biosensor/chromatography systems require protein patterning to operate effectively. Biological samples must be fixed in place on a desired surface. Several methods have been developed to fix such samples on glass surfaces. However, some such techniques require large quantities of the biosample. Attempts have been made to apply the samples, and then enclose them with a glass plate. Unfortunately, the adhering process used to achieve adequate sealing also produced high heat, that adversely affected such samples. Biomolecular photo-based patterning methods utilize avidin-biotin technology to immobilize functional proteins on the inner surface of silica glass tubes in desired patterns. The methods are useful for nanofluidic affinity biosensor/chromatography systems and on silicon dioxide substrates for biosensor applications. The resulting patterns are optimized based on the application. In one embodiment, a zebra shaped pattern is utilized for an affinity chromatography system. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html
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Plant biotin synthase Inventor(s): Allen, Stephen M.; (Wilmington, DE), Kinney, Anthony J.; (Wilmington, DE), Miao, Guo-Hua; (Johnston, IA), Orozco, Emil M. JR.; (West Grove, PA) Correspondence: Connolly Bove Lodge & Hutz, Llp; 1220 N Market Street; P O Box 2207; Wilmington; DE; 19899 Patent Application Number: 20030192073 Date filed: May 7, 2003 Abstract: This invention relates to an isolated nucleic acid fragment encoding a biotin synthases. The invention also relates to the construction of a chimeric gene encoding all or a portion of the biotin synthases, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the biotin synthases in a transformed host cell. Excerpt(s): This application claims the benefit of U.S. Provisional Application No. 60/172,929, filed Dec. 21, 1999. This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding biotin synthase in plants and seeds. Biotin is an essential component for all living organisms even though many, including humans, cannot synthesize biotin and are dependent
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upon its uptake from their environment or diet (Eisenberg (1973) Adv Enzymol 38:317372, Pai (1975) J Bacteriol 121:1-8). Biotin serves as a cofactor that covalently binds to carboxylases and facilitates the transfer of carboxyl groups during enzymatic reactions involving carboxylation, decarboxylation and transcarboxylation (Dakshinamurti and Bhagavan, eds., (1985) "Biotin", Ann NY Acad Sci 447:1-441; Knowles (1989) Ann Rev Biochem 58:195-221). Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Polynucleotide motor, a motor system, their preparation and uses Inventor(s): Firman, Keith; (Portsmouth, GB) Correspondence: Nixon & Vanderhye P.C.; 8th Floor; 1100 North Glebe Road; Arlington; VA; 22201-4714; US Patent Application Number: 20020160485 Date filed: November 26, 2001 Abstract: A polynucleotide motor is disclosed, comprising an enzyme capable of binding to a nucleic acid sequence, which enzyme is also capable of translocating the nucleic acid sequence without causing cleavage thereof. The motor may be associated with a substance bound to the nucleic acid sequence so that the bound substance, such as a magnetic bead, biotin, streptavidin, a scintillant or the like, can itself be translocated, relative to the region of binding of the enzyme, during translocation. Such a system has applications in the screening or testing for a pre-determined biological, chemical or physical activity; for example, in screening for new pharmacologicallyeffective ligands. Excerpt(s): The present invention relates to a nucleic acid sequence having bound thereto a particular complex involving a subunit of a restriction endonuclease, which complex is capable of translocating the polynucleotide without causing cleavage thereof; and its use, inter alia, in a molecular machine system. Molecular machines have been described as molecules--on a nanometric scale--that have moving parts and do useful work. A molecular machine system may therefore be a multi-component molecular machine. For such a machine or machine system to operate successfully, it must be based on a compact, stable molecular structure. Accordingly, theoretical studies of molecular machine systems have focused on inflexible, covalent structures, such as graphite- and diamond-like materials, working in a vacuum. However, it is unlikely that such theoretical systems can be built, in practice, in the near future. On the other hand, the art of preparing polymeric structures is comparatively well-advanced. The drawback of these, however, is that they must fold appropriately in order to provide a usable structure. Protein folds, for example, are difficult to design in view of the lack of strong, natural complementarity of individual amino acids. Contrastingly, work has been carried out which shows that it is possible to design DNA-based structures, so that nucleic acids could be engineered to serve as scaffolds for complex molecular motor-and other--systems. The problem then is to provide a suitable motor or machine system that can appropriately interact with a DNA-based structure. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html
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Polysaccharides with antithrombotic activity comprising at least a covalent bond with biotin or a biotin derivative Inventor(s): Duchaussoy, Phillppe; (Toulouse, FR), Herbert, Jean-Marc; (Tournefeuille, FR), Petitou, Maurice; (Paris, FR), Savi, Pierre; (Seysses, FR) Correspondence: Sanofi-synthelabo INC.; 9 Great Valley Parkway; P.O. Box 3026; Malvern; PA; 19355; US Patent Application Number: 20040024197 Date filed: March 21, 2003 Abstract: The present invention relates to novel synthetic polysaccharides with antithrombotic activity exhibiting at least one covalent bond with biotin or a biotin derivative and to a process employing avidin or streptavidin which makes it possible to neutralize these polysaccharides. Excerpt(s): The present invention relates to novel synthetic oligo- and polysaccharides exhibiting at least one covalent bond with biotin or a biotin derivative and having the anticoagulant and antithrombotic pharmacological activities of heparin. Heparin catalyses, in particular via antithrombin III (AT III), the inhibition of two enzymes which are involved in the blood coagulation cascade, namely factor Xa and factor IIa (or thrombin). Preparations comprising low molecular weight heparins (LMWHs) comprise chains formed of 4 to 30 monosaccharides and have the property of acting more selectively with respect to factor Xa than with respect to thrombin. It is known that the inhibition of factor Xa requires attachment of heparin to AT III via the antithrombinbinding domain (Domain-A) and that inhibition of factor IIa (thrombin) requires attachment to AT III, via the Domain-A, and to thrombin via a less well defined binding domain (Domain-T). Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html
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Pretargeting methods and compounds Inventor(s): Axworthy, Donald B.; (Brier, WA), Reno, John M.; (Brier, WA), Theodore, Louis J.; (Lynnwood, WA) Correspondence: Seed Intellectual Property Law Group Pllc; 701 Fifth Ave; Suite 6300; Seattle; WA; 98104-7092; US Patent Application Number: 20030129191 Date filed: April 17, 2002 Abstract: Methods, compounds, compositions and kits that relate to pretargeted delivery of diagnostic and therapeutic agents are disclosed. In particular, methods for radiometal labeling of biotin, as well as related compounds, are described. Clearing agents and clearance mechanisms are also discussed. Excerpt(s): This application is a continuation-in-part of U.S. application Ser. No. 163,184, filed on Dec. 7, 1993, which is a continuation-in-part of pending PCT Patent Application No. PCT/US93/05406, filed Jun. 7, 1993 and designating the United States, which, in turn, is a continuation-in-part of pending U.S. patent application Ser. No. 07/995,381, filed Dec. 23, 1992, which is, in turn, a continuation-in-part of pending U.S. patent application Ser. No. 07/895,588, filed Jun. 9, 1992. All of these applications are incorporated by reference in their entirety herein. The present invention relates to methods, compounds, compositions and kits useful for delivering to a target site a
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targeting moiety that is conjugated to one member of a ligand/anti-ligand pair. After localization and clearance of the targeting moiety conjugate, direct or indirect binding of a diagnostic or therapeutic agent conjugate at the target site occurs. Methods for radiometal labeling of biotin or other small molecules, as well as the related compounds, are also disclosed. Clearing agents and clearance mechanisms are discussed, which agents or mechanisms facilitate a decrease in the serum half-life of targeting moiety-ligand or targeting moiety-anti-ligand conjugates. Conventional cancer therapy is plagued by two problems. The generally attainable targeting ratio (ratio of administered dose localizing to tumor versus administered dose circulating in blood or ratio of administered dose localizing to tumor versus administered dose migrating to bone marrow) is low. Also, the absolute dose of radiation or therapeutic agent delivered to the tumor is insufficient in many cases to elicit a significant tumor response. Improvement in targeting ratio or absolute dose to tumor is sought. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html •
Process for preparation full-length cDNA and anchor used for the same Inventor(s): Jang, Mi-Sook; (Chungbuk, KR), Jeon, Jin-Tae; (Chungbuk, KR), Park, HanOh; (Chungbuk, KR) Correspondence: Foley And Lardner; Suite 500; 3000 K Street NW; Washington; DC; 20007; US Patent Application Number: 20030049637 Date filed: November 21, 2001 Excerpt(s): The present invention relates to a process for the preparation of full-length complementary DNA (cDNA). More particularly, the present invention is directed to a process for selective amplification of full-length cDNA, which comprises: i) a step for preparing a hybrid composed of a messenger RNA (mRNA) strand and a cDNA strand of which three (3) or four (4) deoxycitidinemono phosphate (dCMP) are combined at 3' end, by treating mRNA with reverse transcriptase; separately from the above step, ii) a step for adenylating single strand anchor of which biotin or phosphate group is combined at 3' end, and phosphate group is combined at 5' end; iii) a step for ligating said adenylated single strand anchor to 3' end of full-length cDNA strand of said cDNA/mRNA hybrid to select full-length cDNA/mRNA hybrid; and iv) a step for amplifying only the full-length cDNA/mRNA hybrid through polymerase chain reaction (PCR) which employs a primer of which base sequence is complementary to that of said anchor. Recently, new techniques for mass production of various genetic engineering products such as proteins, have been developed through identification of novel genes and determination of base sequences thereof, and then characterization of their biological properties. In addition, some methods for the treatment of various diseases caused by inappropriate expression and/or suppression of a specific gene or by the influence of foreign substance such as carcinogen or teratogen, can be developed through the analysis of the base sequences of the gene. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html
Patents 187
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Production of biotin Inventor(s): Asakura, Akira; (Fujisawa-shi, JP), Hoshino, Tatsuo; (Kamakura-shi, JP), Kiyasu, Tatsuya; (Fujisawa-shi, JP), Nagahashi, Yoshie; (Fujisawa-shi, JP) Correspondence: Mark E. Waddell, ESQ.; Bryan Cave Llp; 245 Park Avenue; New York; NY; 10167-0034; US Patent Application Number: 20030096382 Date filed: March 22, 2001 Abstract: The present invention provides process for making biotin from desthiobiotin either contacting desthiobiotin with an enzyme reaction mixture containing bioB gene product and nifU gene product and/or nifS gene product and isolating the biotin or cultivating a microorganism transformed with DNA encoding the bioB gene product, nifU gene product and nifS gene product and isolating the biotin. Excerpt(s): Biotin is one of the essential vitamins for nutrition of animals, both human and non-human, plants, and microorganisms, and very important as a medicine or food additive. There are many studies on fermentative production of biotin. Escherichia strains are known as microorganisms which can be used for the above process [see Japanese Patent Publication (Kokai) No. 149091/1986, WO 87/01391 and Japanese Patent Publication (Kokai) No. 155081/1987 ]. In addition to the above-mentioned strains, Bacillus strains [Japanese Patent Publication (Kokai) No. 180174/1991), Serratia strains [Japanese Patent Publication (Kokai) No. 27980/1990] and Brevibacterium strains [Japanese Patent Publication (Kokai) No. 240489/1991] are also known. But these processes have not yet been suitable for industrial use because of the low efficiency of carbon recovery from the nutrients into biotin and, in some cases, the accumulation of the direct intermediate, desthiobiotin. It is therefore desirable to improve the efficiency of the conversion of desthiobiotin to biotin. A conversion reaction of desthiobiotin to biotin using the resting cell system of Escherichia coli (Antimicrob. Agents Chemother. 21, 5, 1982) and one using cell-free extract of Escherichia coli [J. Biol. Chem., 270, 19158 (1995); Biosci. Biotechnol. Biochem., 56, 1780 (1992); Eur. J. Biochem., 224, 173 (1994); Arch. Biochem. Biophys., 326, 48 (1996)] are known. According to these publications, it has been clarified that protein factors such as ferredoxin-NADP reductase and flavodoxin together with biotin synthase are involved in the biotin formation from desthiobiotin. Nevertheless, only limited effect has been observed for biotin production from desthiobiotin under these conditions. It was simply speculated that another unknown protein should be involved in this reaction to more efficiently convert desthiobiotin to biotin. Furthermore, a conversion reaction by using the purified biotin synthase of Bacillus sphaericus with photoreduced deazaflavin as an artificial electron donor instead of using physiological electron transfer system of ferredoxin-NADP reductase and flavodoxin has recently been reported [Biochem. Biophys. Res. Commun., 217, 1231 (1995)]. But the reported reaction efficiency is not high enough for the reaction to be usable in the industrial production of biotin. Web site: http://appft1.uspto.gov/netahtml/PTO/search-bool.html
188
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Biotin
Secretions of proteins by encapsulated cells Inventor(s): Akselband, Yevgenya; (Brighton, MA), Jesperson, Diana; (Malvern, NJ), McGrath, Patricia; (Cambridge, MA), Moen, Phillip T.; (Foxborough, MA), Trnovsky, Jan; (Saugus, MA) Correspondence: Townsend And Townsend And Crew, Llp; Two Embarcadero Center; Eighth Floor; San Francisco; CA; 94111-3834; US Patent Application Number: 20030143642 Date filed: May 28, 2002 Abstract: The invention provides methods of analzying a secreted protein from a cell encapsulated in a microdrop. The microdrop is formulated with biotinylated matrix molecules at a reduced ratio of biotin to matrix molecules compared with previous formulations. The reduced ratio is advantageous for improving the resolution of detection and allows simultaneous detection of multiple secreted proteins and/or multiple cell surface markers. The invention further provides inter alia methods of isolating IgG isotype antibodies that have switched from IgM isotype. Excerpt(s): The present application derives priority from U.S. S No. 60/293,822 filed May 26, 2001, which is incorporated by reference in its entirety for all purposes. The gel microdrop (GMD) secretion assay involves encapsulating cells in a biotinylated matrix, followed by capture and detection of cell-secreted molecules with fluorescent markers (17-34). This technology differs from other encapsulation methods in that the small size of the microdrop (e.g.,
