Report of the Council for Tobacco Research

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Report of the Council for Tobacco Research - Usa Inc. 850000

Date: 19850000/P
Length: 220 pages
85702113-85702332
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Author: Hobbs, W.D.
Alias: 85702113/85702332
Type: PSCI, SCIENTIFIC PUBLICATION; ABST, ABSTRACT; BIBL, BIBLIOGRAPHY
Area: LEGAL DEPT FILE ROOM
Site: N14
Named Organization: American Cancer Society; Anticancer Research; Archives of Pathology + Laboratory Med; Beth Israel Hospital; Biochemical Pharmacology; Biochemical + Biophysical Research Com; Blood; Brigham + Womens Hospital; Cancer Cells 3 Growth Factors + Transf; Cancer Research; Center for Blood Research; Charles A Dana Research Inst; Cold Spring Harbor Laboratory; Columbia Univ; Creighton Univ; Ctr, Council for Tobacco Research; Dana Farber Cancer Inst; Duke Univ; Elsevier Science Publishers Bv; Eppley Inst for Research in Cancer + A; European Journal of Biochemistry; Fonds De La Recherche Scientifique Med; Frederick Cancer Research Center; Genentech; Gladstone Foundation; Haemotology + Blood Transfusion; Hahnemann Univ; Harvard Medical School; Heredity Cancer Inst; Huntington Medical Research Inst; Irvington House Institute; Johns Hopkins Oncology Center; Johns Hopkins Univ; Journal of Biological Chemistry; Journal of Immunology; Journal of Interferon Research; Karolinska Hospital Uppsala; Karolinska Inst; Keio Univ; Laboratory of Developmental Pahrmacolo; Lab of Molecular Oncology; Lajolla Cancer Research Foundation; Leukemia Society of America; Ministere De La Politique Scientifique; Mi Cancer Foundation; Modern Trends in Human Leukemia; Molecular + Cellular Biology; National Inst of Arthritis Diabetes Di; Natl Heart Lung + Blood Institute; Natl Institute of Child Health + Human; NCI, Natl Cancer Inst; NC United Way; NIH, Natl Inst of Health; Nordisk Insulinfond; Nucleic Acids Research; Ny Univ; Proceedings of the Natl Academy of Sci; Research Inst of Scripps Clinic; Salk Inst; San Francisco General Hospital; Science; Scripps Clinic + Research Foundation; Somatic Cell and Molecular Genetics; Springer Verlag; Stanford Univ; Swedish Medical Research Council; Swedish Society of Medicine; Tissue + Cell; Transfer + Expression of Eukaryotic Ge; Tx Agricultural Experiement Station; Tx Agricultural Experimental Station; Tx A + M Univ; Unite De Biochimie; Univ Hospital Uppsala; Univ of Az; Univ of Ca Berkeley; Univ of Ca Davis; Univ of Ca San Diego; Univ of Ca San Francisco; Univ of Co; Univ of Helsinki; Univ of Liege; Univ of Ne; Univ of Ne Medical Center; Univ of SC; Univ of Southern Ca; Univ of Wa Seattle; Uppsala Univ; Usda, U.S. Dept of Agriculture; US Public Health Service; Veterans Administration; Veterinary Toxicology + Entomology Res; Veterinary Toxicology Research Laborat; Virchows Archiv B; Virology; Walton Jones Cell Science Center; Wayne State Univ; Wistar Inst; Alan R Liss; Albert Einstein College of Medicine
Named Person: Ambrogio, C.; Anagnou, N.P.; Antoniades, H.N.; Babiss, L.E.; Barbis, D.P.; Beaune, P.; Bengtsson, M.; Bertics, P.J.; Bing, R.J.; Bowden, D.H.; Brennan, M.J.; Bresnick, E.; Brodin, E.; Bullock, P.; Busbee, D.L.; Chen, L.B.; Chen, Y.T.; Civin, C.I.; Clark, R.; Clawson, G.A.; Cochet, C.; Colbert, D.; Coltoffschiller, B.; Damjanov, I.; Davis, S.; Degraeve, J.; Deisseroth, A.; Ding, J.H.; Duesberg, P.H.; Duvivier, J.; Dvorak, H.F.; Feldman, J.D.; Fisher, P.B.; Ford, D.H.; Frankel, J.W.; Friedberg, E.C.; Gallo; Gardner, W.U.; Gielen, J.E.; Gill, G.N.; Ginsberg, H.S.; Glaister, D.; Goldfischer, M.; Goldfischer, S.; Greaves; Helder, J.; Hermo, H., J.R.; Hess, J.; Hines, R.N.; Hoffman, R.M.; Honn; Horton, E.J.; Howley, P.M.; Irvin, T.R.; Jacobson, L.O.; Jacobs, G.A.; Janka; Joe, C.O.; Kan, N.; Karnovsky, M.L.; Karpatkin, S.; Kato, T.; Kim, H.L.; Kremers, P.; Leach, M.F.; Leary, A.G.; Lehto, V.P.; Letawegoujon, F.; Levin, J.; Lewis; Liao, Ycj; Liaw, W.S.; Lim, E.; Lindgren, P.G.; Lisanti, V.F.; Lundberg, J.M.; Lynch, H.T.; Marks, B.; Martial, J.A.; Mcallister, H.C.; Mcdonald, J.R.; Miettinen, M.; Montelius, J.; Nadakavukaren, K.K.; Nadakavukaren, J.J.; Nebert, D.W.; Neth; Norheim, I.; Norman, J.O.; Nunn, M.; Oberg, K.; Ogawa, M.; Omiecinski, C.; Papas, T.; Paye, M.; Pearlstein, E.; Perezstable, C.; Pierce, G.B.; Pinto, I.M.; Pollack, R.; Poncin, J.E.; Powers, S.; Ragsdale, S.; Regenstein, J.; Rydstrom, I.; Sato, G.H.; Schultz, R.A.; Shen, Ckj; Smuckler, E.A.; Sommers, S.C.; Song, J.; Stipanovic, R.D.; Stone, D.; Strauss, L.C.; Sylvia, V.L.; Tatemoto, K.; Taylor, J.M.; Terasaki, M.; Theodorssonnorhaim, E.; Thompson, D.M.; Vancantfort, J.; Vannice, J.L.; Virtanen, I.; Wang, A.; Warburg; Weber, W.; Weiher, H.; Weiss, M.J.; White, H.T.; Wieder, S.; Wilson, R.; Winters, A.L.; Wong, J.R.; Yuan, T.Y.; Zhou, R.P.; Ziprin, R.
Date Loaded: 12 Feb 1999
Master ID: 85702113/2332
Author (Organization): Ctr, Council for Tobacco Research; Scientific Advisory Board
Litigation: Stmn/Produced
Characteristic: OVER, OVER SIZE DOCUMENT; PARE, PARENT
UCSF Legacy ID: jag40e00

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eonard PU V alan a ssne andAssr,c iategIna 13-LINCOLN ROAD -P,-0 , BOX 223_GREATNECK,NY: 11022 (518)4833715- FOR:- THE COUNCIL FOR -~'OBACCG RESEARCH-iJ.S:A.-, Inc. FOR RELEASE FRIDAY, SEPT._ 12, -19$6 CC?UNCIL'S 1985- REPORT HAS-- RECORD NUMBER_OF ABSTRACTS - New York -- A record number of 327- abs trac ts appears -in the 1985 annual report of -the-Council for Tobacco Research, issued today. These abstracts, with those-in previous ar,nual rep-orts; -bring to at -least 2,852 t-he number of -reports-acknowledging- Council support that have appeared in the medical and scientific litera- ture since 1954, when-the Council-began funding research by inde- pendent scientists into_ smoking and-h_ealth. The Council's ongoing research program is one of- the most extensive- of its lcind~-any- Harold T. 1985 from joined the Scientific Joining where. The report also _ noted that Dr. i~iilliam- U. Gardner retired in the Scientific Advisory Board-- to t-he Council. He- had Board in-1972 and also had served- as-the Council's Director. the Board- in 1985 was-D-r-.-Manfred L. Karnovsky, who is White Professor of Biological Chemistry at Harvard Medical School in Boston. By the end of 1985, the Council had provided -more than $100-million in research support to fund 551 scientists conduct- ing--924 original studies in 290 medical- schools, hospitals and research institutions. - The Council does not operate any research facility. The Scien- tific Advisory Board, a group of distinguished investigators, meets regularly to evaluate research applications,-,judging them solely on the basis of scientific merit and relevance. - BLIC RELATIONS COUNSEL -

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1985 REPORT of THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., INC. THE COUNCIL FOR TOBACCO RESEARCH-U.S.A.; INC. 900 Third Avenue, NEw-York, N.Y. 10022 -

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SCIE NTIFIC ADVISORY BOARD to The Council for Tobacco Research-U.S.A., Inc. - as of December 31, 1985 LEON O. JACOBSON, M.D., Chairman Joseph Regenszein Professor of Medical and Biological Sciences (emeritus) Professor of the Department of Medicine (emeritus) University of Chicago Chicago,-Illinois RICHARD J. BING, M.D. Director of Experimental Cardiology and Scientific Development Huntington Medical Research Institutes, Pasadena, California Visiting Associate California Institute of Technology, Pasadena - Professor of Medicine (emeritus) University of Southern California School of Medicine- Los Angeles, California - DRUMMOND H. BOWDEN, M.D. Professor and Head -- - Department of Pathology University of Manitoba Health Sciences Center Winnipeg, Canada MICHAEL J. BRENNAN, MD. - President and Medical Director Michigan Cancer Foundation Professor of Medicine Wayne State University School of Medicine Detroit, Michigan JOSEPH D. FELDMAN, M.D. - -Editor-in-Chie1, Journal of Immunology San Diego, California - - Member (emeritus), Research Institute of Scripps Clinic Scripps Clinic and Research Foundation La Jolla, California PETER M. HOWLEY, M.D. Chief, Laboratory of Tumor Virus Biology National Cancer Institute - Bethesda, Maryland 4 k

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MANFRED L. KARNOVSKY, Ph.D_ - - Harold T. White Professor of-Eiological Chemistry Harvard Medical School Boston, Massachusetts - - HENRY T. LYNCH, M.D. PrAofessor and Chairman - -- Department of Preventive Medicine and Public Health Professor_of Medicine President.-Heredity Cancer Institute Creighton University-School of Medicine Omaha, Nebraska -- G. BARRY PIERCE, M:D: - American Cancer Society Centennial Research Professor University of Coior-ado Health Sciences-Center - Denver, Colorado - - GORDON H: SATO. Ph.D. - Director W.-Aiton Jones Cell Science Center, Inc. - Lake Placid, New_ York SHELDON C. SOMkSERS,_?v1.D. - Scientific Director - The Council for-:Tobacco Research-U.S.A., Inc. Clinical-Professor of Pathology College of Physicians & Surgeons of Columbia University New York, New York. - Scientific Staff of The Council SHELDON C. SOMMERS, M.D. Scientific Director DONALD H. FORD;-Ph.D: HARMON C. McALLISTER, Jr., Ph.D. Associate Resear~:es Director - Associate Research Director VINCENT F. LISANTI, D.M.D. - DAVID STONE, Ph.D. -Associate Research Director - Associate Research Director tz I

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- CONTENTS Introduction . . . : . . . . . . . . . . . . . . . . . . . . . . . . 5 Abstracts of Reports . . . . . . . . . . . . . . . . . . . . . . 7 I. Cancer-Related Studies . . . . . . . . . . . . . . . . . 7 II. The Respiratory System . . . . . . . . . . . . . . , . , 32 III. Heart and Circulation . . . . . . . . . . . . . . . . . 52 IV. Neuropharmacology and Physiology . . . . . . . . . . . . . 89 V. Pharmacology and Biochemistry . . . . . . . . . , ... . 117 VI. Immunology and Adaptive Mechanisms . . . . . . . . . . . 146 VII.- Metabolic Studies . . . . .. . . . . . . . .. . . . 172 VIII. Epidemiology . . . . . . . . . . . . . . . . . . . . . . 180 Active Projects . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Completed Projects : . . . . . . . . . . . . . . . . . . . . . . . . 198 Index of Principal Investigators . . . . . . . . . . . . . . . . . . 216 ~

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INTRODUCTION - This report records several noteworthy events in regard to The Council. First, it-sets a Council record for a single_year because it contains abstracts of 327 published scientific documents acknowledging Council support. Most of these reports were published in 1985 while others were published less recently. At least 2,852 reports acknowledging Council support have appeared in the - literature since The Council began providing funds to independent investigators doing research in smoking and health. - This indicates that The Council's ongoing research program is one of the most extensive of its-kind anywhere. One of the abstracts represents what may be another first publication of a Council-supported study in a scientific journal in the People's Republic of China. During 1985, William U. Gardner, Ph.D., retired from the Scientific Advisory Board which he joined in 1972; he also served subsequently as The Council's Scientific Director. - Joining the-Board in 1985 was another distinguished scientist, Manfred L. Karnovsky, Ph.D., who is Harold T. White Professor of Biological Chemistry at Harvard Medical School, Boston. - - Since its establishment in 1954, The Council has made available more than $100,000,000 for research by 551 scientists for 924 original projects in 290 medical schools, hospitals and research institutions. - - .

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ABSTRACTS OF REPORTS I. Cancer-Related Studies PLATELET-DERIVED GROW i H FACTOR: A LINK TO MALIGNANT of the biologically active molecule of the dimeric PDGF. osteosarcoma cells is shown to code for the carboxyterminal region of the PDGF-2 chain specific PDGF atntiserum. The partial sequence of c-sis cDNA from human gliblastoma, fibrosarcoma, and- osteosarcoma cells, which express the proto-oncogene (c-sis) homolog of v-sis, produce and secrete mitegenic-molecules that are recognized by shares st-uctural, functional and immunological properties with the predicted protein product of the viral oncogene (v-sis) of the simian sarcoma=virus (SSV). Human Platelet-derived- growth factor (PDGF) is the -major mitogen of human serum. It TRANSFORMATION Pasadena. Antoniades, H. N. et al. - Cancer Cells !/Growth Factors and TransJorrnation. Cold Spring Harbor Laboratory, 145-151, I985. _ Other support: National Institutes of Health, the American Cancer Society and the Leukemia Society of America, Inc. From the Center for Blood Research, Boston; Department of Nutrition, Harvard School of Public Health, Boston; and the Division of Biology, California Institute of- T echnology, MOLECULAR BIOLOGY OF CYTOCHROMc P-450 The cytochrome P-450-dependent monoxygenases are widely recognized as playing a primary role in the detoxification and/or- activation of xenobiotics in hepatic- and extrahepatic tissues. In the experiments reported here, it is shown that pretreatment of animals with various inducing agents results in- increases in_ the activity and specific content of particular cytochrome P-450 -isozymes and may result in decreases in- the specific content and activity of others. Preliminary investigations in several laboratories have shown that the increase in cytochrome P-450c, the major 3-methylcholanthrene inducible isozyme, and cytochrome P-450b, the major phenobarbital inducible isozyme, is preceeded by an increase in specific mRNA levels, suggesting a role for transcriptional regulation. Investigations are continuing into the mechanism of regulation for the induction of these two proteins. Hines, R. N., Bresnick. E.. Omiecinski, C., and Levin, J. In: Rydstrom, I., Montelius, J. and Bengtsson, M. (eds.): Extrahepatic Drug Metabolism and Chemical Carcinogenesis. New York: Elsevier Science Publishers B. V., 1983, pp. 419=422. Other support: National Institutes of Health and the National Cancer Institute. From the Eppley Institute for Research in Cancer and -Allied Disease, - University -of Nebraska Medical Center, Omaha, and the Department of Environmental Health Sciences, University of Washington, Seattle. 7 t

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DNA POLYMERASE ACTIVITY IN A-REPAIR=DEICIENT-HUMAN CELL LINE A human low-density-lipoprotein {LDL) receptor-deficiant diploid fibrobtast cell line (GM1915) was determined to be short patch competent (DNA polymerase-P) and long patch deficient (DNA polymerase-a) for DNA excision repair. Analysis of DNA from GM1915 cells or froni WI38 control cells, following trestment-w.ith a mutagen - known to initiate- long patch excision repair, showed that GM1915 -cells exhibited decreased resynthesis of oligonucleotide segments excised during repair. When cells deficient in DNA polymerase-a: activity were permeabilized to-permit LDL entry, repair - synthesis immediately increased. These data suggest that DNA polymerase-o is not - activated by mutagen- :reattnent in GM1915-cells and that introduction of -LDL into the= cells results in activation of-the enzyme. - -- -- - Joe, C. O., Norman, d. O., Irvin, T. R., and Busbee. D. L. Biochemical and BiopSysical-Researcn Communications 128(2):754-759, 1985. Other support: National Institutes of Health, U. S. Department of Agriculture, and the Texas Agricultural Experiment Station. - - From the Department of Anatomy, and Department of Physiology and Pharmacology. College of Veterinary Medicine, Texas A & M University, College Station, and the Veterinary Toxicology and Entomology Research Laboratory,_ -U.S. Department of - Agriculture, College Station, TX. - POLYCHLORINATED BIPHENYL' UPTAKE AND TRANS_PORT BY LYMPH AND PLASMA COMPONENTS The uptake and vascular transport of ingested Aroclor 7242, an isomeric mixture of polychlorinated biphenyls (PCB), was investigated in experimental animals. High concentrations of ingested PCB were found in the chylomicron fraction of thoracic duct lymph. When the lymph flo-w was exteriorized. PCB were not subsequently found in the -- vascular circulation. When lymph was not exteriorized plasma PCB concentrations - reached maximal levels 6 hr after ingestion. -Less than 1% of total plasma Pi.B was detected in--cellular -fractions -of blood over a 10-hr period- following ingestion. Chylomicrons contained 31% of total plasma PCB 30 min after ingestion, decreasing to -less than 6% at 4 hr. A maximum of 10% of plasma PCB at l-hr, and <5% at 6-hr, after - ingestion was associated with very low densi.y lipoproteins (VLDL) or low density lipoproteins (LDL). Although PCB enter the vascular circuiation -with -thchylomicron fractions of lymph,-delipopioteinated plasma contained 52% of the total PCB in blood collected 30 min after ingestion. This level increased to 78% after 2 hr and remained constant at about 80% for an additional 8-hr period. High performance liquid chromatographic exam7natiosss of delipoproteinated plasna from- blood taken 6 hr after PCB ingestion showed elution of > 95% of plasma PCB to coincide with the albumin peak. Etectrophoretic examinations of delipoproteinated plasma showed the association of PCB with albumin to be noncovalent. The results suggest that apolar - PCB are absorbed into intestinal epitnelial cells from which they are secreted into the lymphatic drainage sequestered within the apolar core of chylomicrons, that these PCB transit the thoracic duct and enter the vascular circulation within-chylomicrons and are metabolized or otherwise released from chylomicrons during hepatic chylomicron clearance, and that resulting PCB or PCB derivatives circulate in association with plasma albumins. --- Busbee. D.L. et al. ~ 8 ~ ~ ~ {s,

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Proceedings of the Society for Experimental Biology and Medicine 179:116-122, 1985. Other support: National- Institutes of- Health, Texas Agricultural -Experimental Station, and the U. S. Department of Agriculture, College Station, TX. From the Department of Anatomy and Departments of Physiology and Pharmacology, College of Veterinary Medicine, Texas A & M University, College-Station. ALKYLATION OF_DEOXYG_UANOSaI+fE BY THE SESQUITERPENE LACTONE HYMENOXON Hymenoxon, _ a toxic sesquiterpene lactone found in the ruminant forage plant Hymenoxys odorata, _ binds deoxyguanosine yguanosine in a cell-free system and forms adducted guanine residues in sheep lymphocyte DNA. Mitogen-stimulated DNA synthesis in lymphocytes was inhibited by hymenoxon at concentrations greater than 100 µM. Unscheduled DNA synthesis in lymphocytes - was initiated by hymenoxan concentrations exceeding 50 pM, and inhibited by concentrations above 100 µM. We describe an HPLC method which separates unmodified hymenoxon and deoxyguanosine from the hynienoxon-deoxyguat.osine adduct and allows the quantitation of adducts in hymenoxon-treated cells. Sylvia, V. L., Joe, Ci O., Stipanovic, R. D., Kim, H. L., and Busbee: D: L. -- - Toxicology Letters 29:69-76, 1985.- - Other support: National Institutes of Health, U. S. Department of Agriculture and the Texas Agriculture Ezperitnent Station. - From the Department of Anatomy and Department of Physiology and Pharmacology. College of Veterinary Medicine, Texas A 6c M University, College Station, and the Veterinary Toxicology Research Laboratory, U. S. Department of Agriculture, College Station, TX. UPTAKE AND VASCULAR TRANSPORT-OF INGESTED AFLATOXIN - The uptake and vascular transport of gastrically instilled aflatoxin Bi (AFBI) was investigated in sheep. Aflatoxin uptake was compared with that of _dalmitate, a water- insoluble -oil known to be-absorbed_ into the_ intestinal lymphatic drainage which bypasses the liver to enter the peripheral vascular circulation via the thoracic duct.- After_ instillation in animals-with exteriorized thoracic duct 47ow, AFBI was detected in inferior vena cava blood within 30 min, while palmitate was not detected in vena cava blood at- any time. Pslmitate-was detected in-thoracic duct lymph after approximately 2 hr. _ Greater than 95% nf palmitate in lymph was associated with the chylomicron fraction, while aflatoxin in either plasma or lymph was not detectably associated with any of the circulat'sng lipoproteins. In addition, AF-B1 did not partition into plasma or lymph lipoproteins in vitro. Toxic lipophilic xenobio.ics, such - as benzo(o)pyrene and polychlorinated biphen_ yls, do partition into lipoproteins, are absorbed into the intestinal drainage, bypass- the liver to directly enter the peripheral vascular circulation, and are not specifically hepatptoxic. These data indicate that the mode of AFBz absorption from the gastrointestinal system results in its immediate transport to the -iiver, which may - contribute to AfB1 hepatotoxicity. - - - - - Wilson, R., Ziprin, R., Ragsdale, S., and Busbee. D. 9 t 5

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Toxicology Letters 159(1-2):69-76, 1985. Other support: National institutes of Heaith,-U.S. Department of Agriculture, and the Texas Agriculture Experiment Station. From the Veterinary Toxicology Research Laboratory, U.S. Department of Agriculture, College Station, TX, and-the Department of Anatomy, -and Department of Physiology and Pharmacology, College of Veterinary Medicine, Texas A & M University, College Station. MITOCHONDRIA IN TUMOR CELLS: E-FFECTS OF CYTOSKELETON ON - DISTRIBUTION-AND AS TARGETS FOR SELECTIVE KILLING The basis for the difference in-mitochondrial rhodamine -123 retention between carcinoma cells and norm_al_epitheliai celL is still under investigation. However,- it is likely one of the factors involved is the higher mitochondrial- membrane potential in- carcinoma cells. It is of interest to_ note that this is the reverse of what has -been observed in normal and transformed mink fibroblasts. -Despite the lack of understanding _ of the basic mechanism, this=work confirms, in a-partial and-prelimi nary way, Warburg's intuition that paying attention to mitochondria in tumor cells may ultimately help the treatment of cancer. Chen, L. B. et al. ln: Levin, A. J. et aE (eds.): The Transformed Phenotype: Cold Spring Harbor Laboratory, 1984, pp. 75-86. Other supportr National Cancer Institute and the American Cancer Society. From the Dana-Farber Cancer Institute and the Department of Pathology, Harvard Medical School, Boston. LOCALIZATION OF ENDOP-LASMIC RETICULUM IN-LIVING -- - AND GLUTARALDEHYDE FIXED CELLS WITH FLUORESCENT DYES Certain fluorescent dyes-, previously reported to localize mitochondria, when used at higher concentrations al.c-7-localize-a confinuous cet-like-structure -in both living and - glutaraldehyde-fixed cells. -- A similar reticular structure can be detected by phase-contrast microscopy -and- -.vhole-mount electron microscopy in -potassium permanganate-fixed cells as well. This structure is mostly tubular, with some patchlike areas, and-is dikely to-be the endoplasmic reticulum. The organization of the- reticular structure is sensitive to colchicine and rotenone but not to cytochalasin B, taxol, monesis, the calcium ionophore A23187, 12-O-tetradecanoylphorbol 13-acetate, or hydrocortisone. Terasaki, M., Song, J., Wong,-J. R., Weiss, M. J., and Chen.-i:. B. - Cell 38:101-108, 1984.__ Other support: National Cancer-]nstitute. From -the Dana-Farber Cancer Institute and the Department of Pathology, Harvard ~ Medical School, Boston. _ ~ ~ 10

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MITOCHONDRIAL AND PLASMA MEMBRANE POTENTIALS CAUSE - UNUSUA-L-ACCL:MULATION AND RETENTION OF RHODAMINE-123 BY HUMAN BREAST ADENOCARCINOMA-DERI1 ED tiiICF-7 CELLS Quantitative studies of MCF=T cells (derived from human breast adenocarcinoma) and CV-1 cells-(from normal African green monkey kidney epithelium),°- using the permeant cationic compound- tetraphenylphosphonium (TPP), in conjunction with fluorescence microscopy using rhodamine 123 (Rh123), indicate that the mitochondrial -and plasma membrane potentials af fect both uptake and retention of= these compounds. Under conditions that depolarize the plasma membrane, uptake and retention of TPP and Rh123, driven onlyby-tt,e mitochondrial membrane potential, are greater in- MCF-7 than in CV-I. An ionophore that dissipates the mitochondrial membrane potential -of MCF-7 cells causes them to _resemble CV-i- cells by decreasing uptake and retention. Hyperpolar izing the ` mitochondriat- membrane- of- CV-I increases accumulation and prolongs retention; hypergolarization of the= plasma membrane further heightens this affect, causing the uptake of CV-1 cells to resemble that of MCF-7 cells even more closely. The greater uptake and retention by -MCF-7 appear to be a consequence of elevated mitochondrial and plasma-n:embrane potentials.- The plasma membrane potential affects mitochondrial retention of TPP and Rh 123, and its role in enhancing the effect of a-difference in mitochondrial membrane potential is explained. Davis, S., et al. (Chen. L. B.) The Journal of Biosogical-Cherrtistry 260:13844-13850, 1985. From the Dana-Farber Cancer Institute and the Department of Path_ology, Harvard Medical School, Boston. MITOCHONDRIA.IN LIVING CELLS: EFFECTS OF GROWTH FACTORS AND TUMOR PROMOTERS. ALTERATIONS IN CARCINOMA CELLS, -AND- TARGETS FOR THERAPY Rhodamine 123 is useful for localizing mitochondria as well as for reflecting electFochemical gradients across mitochondria in living cells. The latter is substantiated by tetraphenylphosphorium (TPP) a widely --usecf- membrane potential probe. 12-0-tetradecancylphorbol- 13-acetate (TPA) converts filamentous mitochondria into -a -granular shape but-does not-affect-the electrochemical gradient. Platelet-derived growth factor_leads to an- increase in mitochondrial membrane potential as well as to-a clustering of mitochondria at the perinuclear region 3 hr after treatment of resting -BALB/c-3T3 cells. Whereas the majority of tumor cell lines do not have altered electrochemical gradients across mitochondria, the forms of expression -between membrane potential and pH gradient can be varied. For numerous adenocarcinoma-derived cells; ~this gradient appears to be preferentially expressed as a membrane potential. Thus, the mitochondrial membrane potential is higher-in=adenocarcirroma -cells than in normal epithelial cells. These carcinoma cells take up 5-fold to 20-fold more rhodamine 123, TPP or other lipophilic, cationic compounds that can reach mitochondria than do normal epithelial cells. Tnis phenotype may be-exploited for chemotherapy of adenocarcinoma cells. A lipophilic, cationic drug, dequalinium chloride; widely used for sore throat irn humans, is highly toxic to carcinoma cells and exhibits a significant anticarcinoma activity in animals. Chere. L. B. st a1. CANCER CELLS 3/ GrowtA Factors and TransJormution. Cold -Spring Harbor Laboratory, 1985. f il-

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Other support: National Cancer Institute. From the Dana-Farber Cari=er Institute, New England DeaconesrHospitat Brigham and Women's Hospital, and Harvard Medical School, Boston. INCREASED RHODAMINE 123 UPTAKE BY CARCINOMA CELLS The total cellular content of the fluorescent rtttitochondrial-specific dye, rhodamine 123 (Rh-123), was quantified by butanol extraction as a function-of-time of exposure- and dose for a variety of cell lines. These results were compared with observations made by fluorescence-microscopy on dye localization and mitochondriak morphology. There appeared to-be two categories of cell types based on Rh-123 uptake: those which progressively accumulate the dye, such as Ehrlich ascites tumor -cells, carcinoma-derived lines MCF-7, PaCa-2; i,-HeLa, and normal fibroblast line CCL 64; and those which appear to equil'abrate- with-the-extracellular dye within 1 h of incubation in Rh-123 (1 µg/ml)-wiih a minimal level of uptake, such -as the normal epitheliai=derived lines CV-1 and MDCK and the transformed fibroblast line 64F3.= Within the first category, the absolute value of uptake per cell correlated with the concentration of Rh-123 in the- medium and with the period of exposure to the dye up to a point of apparent cellular saturation. The length of time required for apparent saturation depended on the cell type. In the second category, equilibration-was very early, and the total uptake was a function of the extracellula:"=concentration of Rh-123. This probably does not represent a saturation level of dye content in the non-accumulating, low uptake cell lines. Fluorescence - microscopy -revealed that ' Rh-123 localization was- initially mitochondrial-specific for --al1 the cell lines examined. Over time, alterations in mitochondrial morphology and_ cytoplasmic fluorescence was observed in the high uptake cell lines but not in the mir:imal uptake cell lines: Incubation of the high -uptake HeLa cell line with- ihe-= mitochondrial membrane' - potential inhibitor, p-trifluoromethoxyphenylhydrazone, substantially -decreased Rh=123 uptake. These observations may indicate a transformation-related characteristic of carcinoma cell mitochondria. It may be possibie -to exploit the mechanism -respbnsible for the progressive accumulation -=-of Rh-123 by carcinoma-derived celi- types for chemotherapeutic approaches to ce.-tain types of carcinomas. - From the Dana Farber Cancer Institute, Boston, and the Department- of Pathology, Harvard Medical School, Boston. Nadakavukaren, K. K., Nadakavukaren, 3. J., and Chen. L. B. Cancer Research 45:6093-6099; 1985 Other support: National Cancer Institute. IDEN i IFICA T ION-AND QUANTIFICATION OF A MESSENGER-RIBONUCLEIC ACID INDUCE-D BY-POLYNUCLEAR AROMATIC HYDROCARBONS - USING A CLONED HUMAN CYTOCHROME P-450 GENE The researchers have isolated four overlapping human genomic clones associated with the polynuclear aromatic hydrocarbon (PAH)-induced form of- cytochrome P-450. The form of P-450 most closely associated with PAH induction has been defined as P1-450. These four overlapping genomic clones span a total of 31.0 x 103 base pairs in 12

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length with the coding sequence lying in the center of these clones. Translation in vitro -of 3-methylcholant)trene-induced mRNA, selected with- the human P,-450 genomic clone, detects a protein with Mr-52000, which is immunoprecipitabie by- the anti-(mouse P,-450) antibody. - The - isolated human P1-450_ - gettomic clone - hybridizes to 3-methylcholanthrene-induced mRNA from monkey tiver, benzanthracene and 3-methyicholanthrene-treated human mammary tumor cells (MCF-7), but not to isosafrole-treated human cells. - Upon treatment with PAH there is a positive correlation- between induced arylhydrocarbon hydroxylase (flavoprotein-linked monoxygenase) activity and the amount of mRNA that hybridizes to the isolated human genomic clone - for P1-450. The size of mRNA, induced from human cells and monkey liver by .PA-H, is around 3.3 x_10' -base pairs, which is the same as the larger of two mRNA_ induced by PAH in the inbred strain of mouse (C57BL/6N). Their data also showed that the isolated DNA clone - can -, detect a- mRNA -size of 3.3 x 103 = base- pairs from phytohemagglutinin-activated benzanthracene-treated human lymphocytes. Densitometer scanning indicated the presence of a 3.6-fold variation (highes-lowest) in the levels of lymphocyte Pl-450 mRNA contents among six individuals studied. Kato, T., Ding, J-H._ and_Chen, Y-T European Journal of Biochemistry 151:489-495, 1985.- Other support:-North-Carolina United Way. - From the-Department of Pediatrics, Duke University Medical Center,-.^.urham, NC. DISPARATE DIFFERENTIATION IN HEMOPOIETIC COLONIES DERIVED FROM HUMAN PAIRED PROGENITORS The researchers analyzed_the differentiation of hemopoietic-colonies derived from human paired daughter cells. Candidate progenitor cells were-isolated by use of a micromanipulation technique from cultures of- My-10 antigen-positive cord- blood cells. Nine to 36 hours later the paired daughter cells were separated with a micromanipulator and allowed to form colonies in-methylcellulose medium containing erythropoietin, phytohemagglutinin !eukocyte-conditioned medium, and platelet-poor plasma. The cellular composition of the colonies was determined by differentiating all the cells of the May-Grunwald-Giemsa-stained preparation. Of a total of 75 evaluabie pairs of colonies, 35 consisted of 28- types of disparate pairs revealing nonhomologous lineage combinations. Forty pairs were homologous in lineage expression. However, the proportions of the individual cell lineages were significantly different in th-e members of some of the homologous pairs. Some pairs revealed significant differences in- colony size. These observations are similar to those reported for murine paired-progenitors and are consistent with the stochastic model of huma stem cell differentiation. Leary, A. G., Strauss, L. C., Civin, C. l., and Ogawa, M. Blood 66(2):327-331,1985. Other support: Veterans Administration and National Institutes of Health From the Veterans Administration Medical Center and the Department -of -Medicine, Medical University of South Carolina, Charleston, and The Johns- Hopkins Oncology Center, The Johns HoD9cins- University School of Medicine, Baltimore, MD. 13

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STRUCTURE OF THE RAT a,-ACID GLYCOPROTEIN GENE The complete nucleotide_sequence of the rat a3-acid glycoprotein gene has -been determined from an isolated lambda recombinant bacteriophage. Southern blot-analysis- and DNA sequencing indicate that there is only one gene per genome; it contains six ezons_ and is located within- a 3,200-base-pair fragment starting- from a-TATA box and extending to the polyadenylation signal AATAAA. Transcription starts 37 base- pairs upstream from the beginning of the translation-codoa ATG. The TATA box (TATAAA) lies 26 base- pairs upstream from this site. The gene contains several potential- glucocorticoid receptor-binding sites,-both inside and outside the structural gene. - Molecular Biology, Genentech, Inc., South San Francisco. Francisco; Gladstone Foundation; San Francisco General Hospital; and Department of From the Department of Pathology, School of Medicine, University of California, San Liao, Y.-C.J., Taylor, J. M. !/annice,- J. L., Clawson, G. A., and Smuckler, E. A. Molecular und Cellular Biology S(12):3634-3639, 1985. -- INTERMEDIATE-FILAMENT PROTEINS IN PARATHYROID GLANDS AND PARATHYROID ADENOMAS The intermediate-filament proteins of normal,- hyperplastic, and adenomatous -parathyroid glands were analyzed immunohistochemically and by immu_nobloiting with monospecific antibodies. In both normal-and adenomatous parathyroid glands, we found keratins with molecular weights of 52, 45, and 40 kilodaltons (Nos. 8. 18, and 19, respectively. Vimentin-proteins could be identified only in stromal cells,-while glial fibrillary- acidic proteirn was not found. in normal parathyroid glands, neurofilament positivity was seen only in nerve axons. In five of 15 parathyroid gland adenomas some keratin-positive cells expressed neurofilament-like immunoreactivity also. In cytoskeleta_1 extracts of one adenoma, the -200-kiloda;ton neurofilament protein was identified by immunoblotting. Thus, it appears that some parathyroid gland adenoma cells may acquire neurofilament proleir.s and coexpress cytokeratin and neurofilament polypeptide in a way comparable with that reported in certain neuroendocrine tumors. Miettinenti M., Clark, R., Lehto, V p.f Virtanen, I., and Damjanov,-i. - Archives of Pathology and Laboratory Medicine_109:986-989, 1985. From the Department of Pathology and Laboratory Medicine, Hahnen:ann University School-of Medicine. Philadelphia, and the Department of Pathology, University of Heisinki, Helsinki, Finland. ORIGIN OF LAMININ IN THE EXTRACELLULAR MATRIX OF HUMAN TUMOR XENOGRAFTS IN-NUDE MICE - Monoclonal antibodies reacting exclusively with laminin of human origin and a polyclonal antibody reacting with both murine and human laminin were used to immunohistochemically study the extracellular matrix of four human tumors - grown a; xenografta in nude mice: a lung carcinoma and a yolk sac-carcinoma because they produced cell associated larrrinin in vitro, and two hepatocellular carcinomas which did not produce cell associated laminin in vitro. The extracellular matrix of the xenografts of 14 CL

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the lung carcinoma and the yolk sac carcinoma contained la;nir.in of both human and murine origin. Xenografts of liver carcinoma contair,e3 cniy laminin of= mouse origin. glycoprotein to -the extracellular matrix of the solid tumor formed-by thesn iri vivo. -- Thisshows that the malignant cells capable of laminia-production irt vitro contribute this Phiiadelphia,-and La Jolla Cancer Research Foundation, 4.a Jolla, CA. From the Hahnemann University School of Medicine -an, the Wistar Institute, Darajanov. /.-et aF,--=- Virchows Archiv B-[Cell Pathology]49:45-52; 1985. Other support: National Institutes of Health. REGULATORY FACTORS SPECIFIC FOR ADULT AND EMBRYON:C GLOBIN GENES MAY GOVERN THEIR EXPRESSION IN ERYTHROLEiIK£IvIIA CELLS - In order to test if trans-acting regulatory factors specific for globin genes of the adult and embryonic stages of development exist in erythroid cells, transcriptionally active embryonic and adult globin genes on the same_ chromosome were transferred by cell fusion from the human leukemia cell K562 into phenotypically adult mouse erythroleukemia cells. Restriction-fragment-length polymorphisms of the K562 ; embryonic giobin- genes were- used to establish that all -three--sopies- -of human chromosome 16 present io the-K5h2 cell showed the-sa:ne pattern of human gfobin gene expression after transfer to the mouse erythroleukemia cell. Adult (a) but not embryonic (f) human globin mRNA was detected in all nine of the independently derived mouse erythroleukemia hybrid cells, each=--of which contained human chromosome 16. Restriction endonuclease studies of the K562 a and S-globin genes after transfer into the mouse erythrolemia cell showed no evidence of rearrangements or deletions that- could explain this loss of, ~-globin gene expression. -These data suggest that regulation of globin gene expression in these erythro:eukemia cells involves trans-acting -regulatory factors specific for the adult-and embryonic stages of development. Anagnou, _N. P., Yuan,- T.- Y., Lim, E., Helder, J.; 1EJieder, S., -cilaister, D.,- Marks, B., Wang, A., Colbert, D., and Deisseroth, A. Blood 65(3):7C5-7i2, 1985. - Other support: University of California, San Francisco, the-Veterans Administration, and the National Heart, Lung and Blood Institute. From the University of California, San Francisco Hematology/Oncology Unit at the San Francisco Veterans Administration Medical Center. CHARACTERIZATION OF A UNIQUE RNA 7NITIA T ED IMMEDIATELY UPS-'1REAM FROM-HUMAN-al GLOBIN GENE IN VIVO AND IN VITRO: POLYMERASE-II-DdPENDENCE, TISSUE SPECIFICITY, AND SUBCELLULAR LOCATION - canonical -cap- site of human a1- globin gene in bone tr.arrow-ceils and in COS-7 cells t_ansfected with an-¢1 globin gene-containing plasmid. Similar to the major al globir, transcript, this upstream RNA is preser.t almost exclus-ively-ire the cytoplasm of the trans- The authors have identified an- abundant -transcript initiated upstream from the 15 ~

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fected COS-7-cells. It is ab.. synthesized efficiently in xitro by-RNA poiynterass- il in_ _ the nuclear extracts -prepared from a Heia cell line and aff -crythroieulcemia cell line, K562. RNAs isolated froru-these cell liues; however do not contain this upstream - transcript. The putative 5' end of the al globin upstream-RNA is mapped by primer ._ extension to base -45, which is located in between the CCAAT and TATA boxes. The synthesis of_ this RNA in -r::ro and in vivo, and the close proximity of its 5' end to the _gene suggest a common mechanism regulating the _ promoter of the al globin transcriptional initiation of both the upstream and the major a1 globin RNAs. Hess, J., Perez-Stable, C., DeissProt;h, A.; and Shen, C-K. J. Nucleic Acids lQesearc.513(;7;:6059-6075, 1985. Other support: - National 7nstitutes of Health, American Cancer Society and V-eterans Administration Merit Review. From the Department of Genetics, University of California, Davis,-aid the Department- of Medicine, Division of Hematology and Oncology, University- of California; -San Francisco. AVIAN £RYT-[iROBLAST--OryfS VIRUS T26: NUCLI;OTIDE_-SEQITENCE OF-THE - TRIPARTITE on2 GENE AND-OF THE LTR,_ AND ANAL_ :'SIS OF Ti-[E- CELLULAR PROTOTYPE OF THE VIRAL ets SEQUENCE - An intact 5.7-kb prcvirus of the avian erythrob;astosis virus E26- has been molecularly cloned for comparisons with avian myeloblastosis -v-irus (AMV) and other _ avian tumor viruses. E26 and AMV transforr.. hemopoietic ceft sxclusively; Both cause _ myeloblastosis, but E26 also causes erythroblastosis. Sequence analysis of the proviral DNA-showed that (i) The tripartite transfornnrir.g gene-of E-26-forms a contiguous_ reading frame of 1046_codoas, including 272 gag, 283 mybE. and 491 ets codons. No- subgenomic ets-specific mRNA was detected in E26-infected cells. By c ontrast, the onc gene of -AMY consists almost entirely of a asyb' - sequence expressed via subgenr:nic mRNA that extends over the 5' and-3' ends of rnybE.= (ii) mybE is only slightly - diverged from the myb'A homolog of AMV and even less from the- cellular prote-rnyb sequence withpo-characteristic_ mutation that sets apart the twa viruses from proto-5nyb: (iii) The U5-segion of the long terminal repeat (LTR) of E26 and AMV are colinear and differ only in scattered point mutations. The U3 region of the E26 LTR is different - from that of AMV but-is colinear and-closely related with that of avian carcinoma virus MH2 and-also with that of Prague Rous sarcoma virus (RSV), except for an unexpected 15-nucleotide substitution of 22 RSV nucleotides. Upstream of the 3' LTR, the -c region of E26 appears to be the-same as that of RSV for 70-t.ucleotides and very similar to those of AMV and MH2-for about 20 to 30 nucleotides. Since the U3s of E26, MH2 and RSV are very closely related and neither MH2 nor RSV shows a particular erythroblast tropism, it is possible- that the U3 does -not play a critical role in the erythroblast tropism of E26. _Eiectrophoretic size-analyses of chicken DNA digested with restriction enzymes indicate that DNA fragments totaling over 50 kb hybridize with- - viral ets DNA. Nunn, M., Weiher, H., Bullock, P., and Duesberg, P. Virology 139:330-339, 1984: Orher support: National Institutes of Health. 16 !

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Molecular Biology and Virus Laboratory,--University- o ACTIVATED PROTO-ONC CrE NF.S: SUFFICIENT OR NECESSARY FOR-CANCERI Proto-onc genes are normaLs.elluiar genes that are related to the transforming (onc)- genes of retroviruses. Because of this relationship, these genes are now widely believed to be potential cancer genes. In some-tumors,-proto-onc genes are mutated or expressed more than in normal cells. -Under these conditions, proto-onc genes are hypothesized to be active cancer-genes in one of two possible ways: The one gene-onc cancer hypothesis suggestF that one activated proto-onc gene is sufficient to cause cancer the muitigene-onc cancer hypothesis suggests that an activated proto-onc gene is a necessary, - but not a sufficient, cause of cancer. However, mutated or *.rrnscriptionaliy activated- proto-onc genes--are not consistently associated- with the tumors in which they are occacionally- found and do not transfor-nt primary ceils.- Further, no- set of an activated proto-onc gene a-ad a complementary cancer gene with transforming function has yet been isolated from a tumor.- Thus, there is still no proof that activated proto-onc genes are sufficient or even necessary to-cause cancer. Guesibrg. P.H. Science 223(d70t2):~ti9-677, 19803. _ Other support: National Institutes of Health. _ From the Department of Molecular Biology, University of California, Berkeley. MU'Y'ACENESIS OF AVIAN CARCINOMA VIRUS MH2: ONLY-ONE OF TWO - POTENTIAL TRANSFORMING GENES (6gag-myc) TRANSFORMS FIBROBLASTS Avian carcinoma virus MH2 contains= two potential -transforming genes, Qgag-mfi: and 6gag-myc: Thus, MH2 may be a model for two-gene carcino- genesis in which transformation depends on two synergistic genes._ Most other directly oncogenic virus_gs contair. single, autonomous transforming (ahc) genes and are models for single-gene carcinogenesis. To determine which role le each potential on.c gene of MH2 plays in oncogenP_ sis,-we have prepared deletion and frameshift mutants of each of- the two MH2 genes by in vitro mutagenesis of cloned proviral DNA_ and have tested transforming function and virus production in cultured-primary quail cells. We have- found that mkt z:eletion mutants and wild-type virus transform- primary cells and that myc deletion and frameshift mutants do not. The morphologies of cells transformed by the mhf deleticn _mutants and by wild-type MH2 are similar yet vary considerably. -Nevertheiess, typical mutant transformed cells ean often be distinguished from cells transformed by wild-:ype- MH2. We conclude that the Sgag-myc gene transforms_ primary cells by itself, without-the second potential onc gene. This myc-related gene is the smallest that has- direct transforming function. Agag-mht is without detectable transforming function-but may affect transformation by dgag--myc. Thus, MH2 behaves _ like a virus with a single onc gene, although it expresses two potential onc genes, and it appears not to be- a model for two-gene carcinogenesis. - Further work is necessary in _ order- to determine whether the Ggag-mht gene possibly enhances oncogenic function of 6gag-m)c or has independent oncogenic function in animals. 17

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Zhou, R. P., Kan, N., -Papas, T.; and Dues6erg, P. Proceedings of the National Academy of Sciences of the United States of America 82:6389-6393, 1985: Other support: National Cancer Institute. - From the Department of Molecular Biology and the Virus Laboratory, dJniversity of California, Berkeley; -and the Laboratory of - Ivlolecular Oncology, National Cancer Institute, Frederick, MD. ARE ACTIVATED PROTO-oncC~iENES CANCERGE-NESP - animals. The efficiency of- the assay to identify cancer genes unrelated to that the test is insufficient to determine whether proto-onc genes cause tumors in and because in some cases they precede tumor formation and in other they evolve during tumor progression. Despite its effectiveness to transform 3T3 cells, it would foliow that mutated proto-ras is not a dominant singular cancer gene, similar to a viral onc gene, and coincidental or consequential mutations rather than cancer causative mutations occurring in tumor cells, because the mutations are not consistently correlated with -specific tumors that they are not relevant for tumor formation. - Available data suggest that these are proto-ras mutations found by the 3T3 assay do-not transforra-primary cells, it is possible detected in human tumors -signals -another limitation of the 3T3 assay. Since the that the assay failed to detect them. That only ras _related proto-onc genes have been described in this paper suggests that either no genes have caused-thenegative tumors or The preponderance_ of 3T3 ceil-t.ra_nsf ormation negatives among _ the tumor proto-onc- genes remains te-bee determined. - Duesberg. P. N. et al. In: Neth, Gallo, Greaves,=Janka (eds.~lfaemotology and Blood Transfusion Vol, 29, Modern Trrnds in Kuman Le-ukemia Vl,Springei-Verlag Berlin-Heidelberg, 1985, pp. A27. Other support: National Institutes of Health. From the Department of Molecular Biology, University of California, Berkeiey; The Salk Institute, San Diego, CA; Laboratory of Molecular Oncology, National Cancer Institute, Frederick Cancer Research Facility; Frederick, MD; and Genentech, Inc., South San Francisco. REGULATION OF EXTRAVASCULAR COAGULATION BY MICROVASCULAR PERMEABILITY - Extravascular coagulation is a prominent- feature- of such important pathological processes as cellular immunity and neoplasia and has been thought- to result from procoagulants associated with the inflammatory or-tumor cells peculiar to these entities. It was found that increased microvascular permeability alone is sufficient to induce equivalent extravascular coagulation in several normal tissues. The results indicate that saturating levels of procoagulant are present even in normal tissues and that microvascular permeability is a rate-limiting step in extravascular coagulation. - Science 227:1059-1061,1985. -

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Other support: U.S. Public Health Service and Monsanto Corporation. From the Department of -Pathology, Beth Israel Hospital, Har vard Medica_l_ School and Charles A. Dana Research Institute, Beth Israel Hospital, Boston. - MUTATIONAL ANA LYSIS OF THE FUNCTIONAL DOMAINS OF THE Ad5-Eia GENE PRODUCTS EFFECTING CELL TRANSFORMATION I-n this paper, the data described using viruses wittr specific mutations in the portion of the genome enc-oding the Ela 51 kd protein imply it is the viral gene product that - is necessary to maintain- the recognized - properties -of transformed cells. - The -conditionally lethal, cold-sensitive phenotype expressed -by the mutated genomes, whether introduced into CREF cells by infection with virions or by transfection with intact -genomes or plasmids, implies that the Ela 51 kd protein interacts with a host macromolecule- ar the Ela 48 kd polypeptide. The he cold-sen_sitive phenotype may reflect a decreased binding constant at 32' C between the mutants' truncated proteins and a still unidentified host and/or viral macromolecules. Iden-tification of the putative host component involved- and the characteristics of the viral protein=-host macromolecule interaction should :eveai critical clues to understandir.g the mechanisms - of - viral transformation. Babiss, L. E., Fisher, P. B. and Ginsberg, H. S. In: Transfer e-nd Expr ession of Eukury oti crenes; 247-262, 1984. Other-support: U. S. Public Health Service. _ From the Department of Microbiology and Comprehensive Cancer Center, College of Physicians & Surgeons of Columbia University, New York. CHEMICAL-VIRAL INTERACTIONS IN CELL TRANSFORMATION: - - ENHANCEMENT OF HUMAN ADENOVIRUS TRANSFORMATION OF CLONED RAT EMBRYO FIBROBLAST (CREF) CELLS BY ALKYLATING CARCINOGENS A major advance in studying carcinogenesis has been the development of well defined cell culture systems which mimic in vitro carcinogenesis as it occurs in vivo. Utilizing these systems, stages analogous to initiation, promotion and progression have been identified and important insights into the molecular events involved in these processes should be forthcoming. In this review we describe some of our recent studies using the highly transformable CREF cell line to investigate the-interactions between MMS and Ad5 in regulating the -frequency of viral transformation and expression- of the transformed phenotype. Our findings - indicate - that carcinogens enhance Ad5 transformation, but do not alter the pattern of viral DNA integration into cellular DNA or the qualitative expression of the transformed state. A likely hypothesis is that certain carcinogens exert their enhancing effect on DNA virus transformation by increasing the- proportion -of cells ;n an infected culture which can stably integrate viral DNA=and thereby become transformed. In other cases, carcinogens may enhance DNA virus transformation by altering the expression of viral and/or cellular genes required for establishment of the transformed state_ Human T-cell leukemia virus, herpes viruses and Hepatitis- B virus have been implicated in the development of specific human malignancies, but a direct link between viruses and the majority of human neoplasms has not been demonstrated. A-possible reason for this apparent negative correlation may be that in most human malignancies, putative viruses cause cancer via synergistic interactions with initiating chemical 19

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I E carcinogens, tumor - promoters, hormones or other yet to -be_-_-defined cofactors. Well-characterized- cell culture systems should-prove instrumental in elucidating the role of multiple-factor interactions in the etiologyy of human- cancer, as well as lead to-_ aa1 _ understanding of the-basic,principies underlying the processes of tumor promotion and progression. - Hermo,-H., Jr., Babiss, L. E., Liaw, W, S., Pinto, I. M., McDonald, R. J., and Fisher, P. B: In: Molecular Basis of Cancer. Part A: MaeromoJecular Structure, -Carcinagens, and Ortcogenes. New York: Alan R. Liss,-Inc.; 1985,_pp. 489-512. From the Department of Microbiology, Cancer Center/! nstitute of Cancer Research, College of Physicians & Surgeons of Columbia University, New York. ANALYSIS OF THE REDUCED GROWTH FACTOR DEPENDENCY OF SIMIAN - VIRUS 40-TRANSFORMED 3T3 CELLS The authors have measured in a defined seruin-free medium the platelet-derived- growth factor (PDGF) and insulin requirements of normal Swiss 3T3 cells, simian virus _ 40-transformed 3T3 cells, and partial revertants of simian virus 40-transformed 3T3 cells. Swiss 3T3 cells displayed strong requirements for both PDGF and-insuiin. Both of these requirements were significantly diminished in simian virus 40-transformed 3T3-- cells. Analysis of the PDGF and insulin requirements of the revertants indicated that the loss of either of these two gTowth factor requirements was not necessarily linked to the other; rather, the growth factor requirements were specifically associated with other parameters of transformation. The reacquisition of a PDGF requirement cosegregated with reversion to density-dependent growth inhibition, whereas reacquisition of a normal insulin requirement cosegregated with reversion to a normal growth dependence on calf serum. Anchorage dependence was dissociable from both growth factor requirements. The relationship between the -PDt;F requirement and density-dependent growth inhibition was further analyzed in normal 3T3 cells by ineasuring the PDGF requirement at different cell densities. At high cell densities, the- requirement f or PDGF - became significantly greater. We suggest that at least in part the ability of transformed cells to grow to high saturation densities results from their loss of a requirement for PDGF. Powers, S., Fisher, P. B., and Pollack; R. - Molecular and Cellular Biology 4: 1572-1576, 1984. Other support: U. S. Public Health Service and National-Institutes of-Health.- - From the Department of Microbiology, Cancer Center/Institute of Cancer Research, Columbia University College of_Physicians & Surgeons, New York. DEPRESSED INTERFERON SYNTHESIS IN SKIN FIBROBLASTS FROM LUNG CANCER PATIEN T S - Skin fibroblast cell cultures derived from male adult lung cancer patients, an adult control population and a newborn population, were examined for their susceptibility to transformation with Kirsten murine sarcoma virus and their ability to respond to an interferon inducer (poly I-poly C). An association between sensitivity to viral 20 -4

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transformation and_-induction -of interferon was observed. = Cultures derived from lung cancer patients demonstrated an -increased sensitivity to virus -transformation and a -decreased ability td- .-esportd to interferon induction as -compared- with=- age-matched controls and newborns. - Winters, A. L., Leach, M.-F.; Horton, E. J., and Frankel.-J. W. Journal of Interferon Research 5:465-470,1985: From the Department of Health and Rehabilitative Services, Veterans Administration Medical Center, Tampa; FL. STUDIES ON GENE TRANSFER AND REVERSION TO UV RESISTANCE IN XERODERMA PIGMENTOSUM CELLS The authors have examined several parameters which address the feasibility of complementing the UV-sensitive phenotype of xeroderma pigmentosum (XP) fibroblasts by gene transfer. We present a con-rparative study which demonstrates that relative to immortalized cells, - human diploid cells are poor recipients for gene transfer. As measured= by both transient and stable expression -_assays, diploid fibroblasts- were completely refractory to DNA transfer by calcium phosphate coprecipitation -and- exhibited substantially reduced levels of expression following gene transfer by fusion- with E. coli protoplasts. We also examined the significance of reversion of the phenotype- of UV sensitivity in SV40-immortalized XP-A cell lines. In- addition -to confirming a previous report of reversion to wild-type levels of UV resistance at a frequency of L- ]0-T, we have attempted to facilitate the identification of XP-A cells complemented with genomic DNA by employing less- stringent selection schemes and cotransfection of a selectable marker. Under these conditions, we observed an increased freque ncy of = reversion and were unable to identify true transfectants. Schultz; R. A., Barbis,-D. P_ and Friedberg, E. C.- Somatic Cell and Molecular Genetics Il(6):617-624, 1985. Other support: U. S: Department-of Energy. From the Laboratory of Experimental Oncology, Department of- Pathology, Stanford -University, Stanford-, CA._ PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST PHENOBARBITAL-INDUCIBLE C-TOCHROME P-450 Monoclonal antibodies against cytochrome -P-450 -were prepared from phenobarbital-induced rat liver microsomes. The immunoglobulin classes and subclasses, as well as the binding capacity to cytochrome P-450, of the different antibodies were characterized. -- Their specificity- was verified by various techniques and seemed to correspond to a- single form of cytochrome P-450, the ma-jar Dher.obarbitaf-indncible form._ However, the= antibodies were unable to inhibit completely the monooxygenase activities investigated.- These antibodies may constitute very specific and powerful analytical tools for characterizing and quantifying cytochrome P-450 isoenzymes. Letawe-Goujon, F., Kremers, P., Beaune, P., Paye, M, and GieFen. J. E. Biochemical and Biophysical Research Communications Ii9(2):744-750, 1984. ~ 21 ~ ~ N G= .;_ ...

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Other support: - Fonds de la Recherche Scientifique- Medicale. From the Laboratory of-Medical-Chemistry, Institute of Pathology; University of Liege, Liege, Belgiuet BEN2A(ajPYRENE METABOLISM IN MOUSE LIVER: ASSOCIATION OF BUTH 7,8-EPOXIDATIOiv AND COVALENT BINDING OF A_METABOLITE OF THE - 7,8 DIOL WITH THE A LOCUS The 7,8-epoxidation_-- of- benzo[a]pyrene, and- the- 9,IQ-epo;.idation of benzo(njtrons7,8-dihydrodiol coupled with covalent binding of the highly reacYive diol-epoxide,-are two key P-45a=mediated reactions believed to be important in cancer initiation, mutagenesis aad teratogenesis. New assays for -these two reactions were developed with mouse Iirer-microsontes: These=two-activities have apparent K. values- (approximately 6 pM) similar to that of aryl hidrocarbon hydroxylase activity. Twenty six individual3-methyicholanthrene-treated Ah /Aha and Ah a/A_.hd -- progeny of the (C57BL/6N) (DBA/2N) F, x JBA/2N backcross were studied. Both of the- newly described activities appear- to represent P-450 protein(s) that are responsible for -aryl hydrocarbon hydroxylas_e activity and that are coordinately controlled by the Ahb allele. - - Van Cantfort, ;., Ceelen: ~i.=E. and Nebert, D. W. -- - Biochemical Pharmscology 34(I4j:182i-i826, 19E5. Other support: Fonds de la Recherche Scientifique Medicale.- - From the Laboratoire de Chimie Medical, Institut de Pathologiea Unite -de Biochimie,- Liege, Belgium; and Laboratory of Developmental Pharmacology, National Institute of - Child Health and Human Development, National Institutes of Health, Bethesda, MD. THE EXPRESSION i7F DIFFERE CYTOCHROME P-450 IN NEON A HEPATOCYi'ES IN CULTURE- NT MONO.~2. X1'CrENASES SUPPORTED BY TAL-RATS AND IN TRI?+IARY FETAL In rat liver, the perirtatal_ development ent of various monooxygenase activities follows different patterns, depending upon the reaction studied. The ontogeny of tlte-6,8-, 7a- and 16a-testosterone hydroxylase activities differs very significantly. - -Aldrin epoxidase and steroid-metabolizing monooxygenases are expressed in primary fetal rat liver cells in culture after- treatment in vitro - with dexamethasone. Testosterone is not metabolized by the control cells and is hydroxylated on the 6,8 and 16a positions following the addition of corticoids to the-culture medium. The dose and-time -curves vary accordiflg-to Ahe hydroxylated position-of tlre-steroid. Aldrin epoxidase activity is nearly undetectable in-the control cells but is-present and is inducible by - phenobarbital following treatment with the corticoid. -Phenobarbital--induces= aldrin epoxidase in the absence cf-dexamethasone in the culture medium, providing that- the cells are pretreated with the corticoid for 48 h: --The use nf antibodies against the main cytochrome P-454-species.purified-from adult and phenobarbital-treated rats confirms- that a similar cytochrome P-450 can be induced in fetal cells in culture. - The perinatal regulation of bioiogical- events,- such as the expression of the monooxygenases, can be reproduced in fetal rat liver cells in culture; such a model constitutes a unique tool for studying the biochemical mechanisms which control these phenomena. 22 ,

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ii I - Kremers,-P., Letawe-Goujon, F., DeGraeve, J., Duvivier, J., and-Gielen, J. E. Europ?an Jour,al of t?ioche_mistry-13`:603-60g, 1983. Other supporr: - Fonds de la Recherche Scientifique Medicale. - From the Laboratory of Medical Chemistry, Institute of Pathology, University of i.ibge, L-itge-, Belgium: CLONING AND STRUCTURE ANALYSIS OF 'I'IIE RAT APOLIPOPROTEIN A-I cDNA Apo!ipoprotein A-/, the major protein in mammalian high -density- lipoprotein, acts as a cofaetor for lecithin-cholesterol acyltransferase during the formation of cholesterol ester and as such, is -thought to promote cholesteroi efflux from per ipheral cells- to the liver. In this paper, the authors report the partial purification-0f rat- liver apolipoprotein A-I mRNA by a polysome immunoadsorption=iechnislue and its cDNA cloning. Isolation of two overlapping -cDNA clones enabled the researchers to derive the whoie- rat apolipoprotein A-I eDNA coding sequence. Comparison of the-deduced protein sequence with its human counterpar t reveals a striking homology between the prepropeptide -precursors. Both mature protein amino-terminal regions are very homologous, suggesting that this particular domain could be involved in lipid-protein binding or lecithin-cholesterol acyltransferase activation. Poncin, J. E., Martial, J: A.-and Gielen, -J. E. European Journal-9f Biochemistry 140:493-498, 1484: - Other support: MinistZ7e-de !a Polit-ique Scientifique~ Belgium_ From the Laboratoire de Chimie Mbdicale, Inst;tut de Pathologie, Universite de Li6ge. Belgium, and Laboratoire de Genie Ger.etique, Institut de Chimie, Belgium. STRUCTURE AND REGULATION - OF= THE HE EPIDERMAL vROINTI'1 FACTOR RECEPTOR The epidermal -growth- factor (EGF)- receptor is -a transmembrane,= glycosylated phosphoprotein with'--intrinsic protein-tyrosine phosphotransferase activity.. _ The arninoterminal cell-surface part -of the EGF receptor, which contains amino-linked carbohydrate, has a sing!e EGF-binding site_ The carboxyterminal cytop!asmic domain corresponds to erb-E, a known- oncogene structurally homologous - to other protein-tyrosine-kinases.-_Self-phosphorylation-of the EGF-rec-eptor, which occurs on its carboxytermin.al_ domain via an intramolecular mechanism, enhances catalytic activity. Phosphorylation of the-EGF receptor by protein kinase C=suppresses-its protein-tyrosine kinas activity. --- Becadse phosphatidylinositot kinase activity can _be sepa-rated, protein-tyrosine kinase seems to be the only catalytic activity intrinsic to purified EGF receptors. In-human epidermoid caecinoma-A431 cells, the-extent of amplification of the EGF-receptor gene tietermines=_ the cellular c,..tcant- of EGF-receptor protein. - The amount of EGF recelstQrs in c!onal varian: A43!-cells dictates their-growth responses to EGF. Gill. G. N., Bertics, P. .h, Thompson, D. M. Weber, 1Y.; and Cochet, C. Cancer Cells 3JC°rowth Factors and Transformation, 11-18, 198 5. 23 ~.:

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Other support: National Instit°ute of Arthritis, Diabetes,-Digestive and Kidney Diseases, and American Cancer Society. p_3 gy, ni s ersi y o rizor.a, ucsen, and Laboratory of Molecular Biology and Virology, The Salk Institute, La Jolla. From the_Department of Medicine,-Division of Endocrinology and Metabolism; School of di i iv M c U i f C li" i : n Di S L J lla i r, e c n ers ty e a o n a , at,,. a ego a o , A RELATIONSHIP BETiVEEN PRODUCTION OF EPIDERMAL GROWTH FACTOR RECEPTORS, GENE AMPLIFICATION, AND CHROMOSOME ?- TRANSLOCATION IN VARIANT A431-CEI LS Synthesis of the epidermal growth factor (EGF) receptor has been analyzed -in a ;_ series of variant A431 human -epidern-ioid carcinoma cell clones reported to -eontain different amounts of EGF binding sites.- The amount of-_ EGF- receptor- ~rotei~, quantitated by immunaaffini~y chromatography, and EGF recep:or mRNA, quantitated by cDNA hybridization, were closely correlated to the extent of EGF receptor gene - amplification. This correlation existed in variants selected for reduced EGF receptors and in revertants from those varianu with increased EGF receptors. There was also a ~ correlation between the frequency of translocation of chromosome 7, containing the EG -receptor gene, and EGF receptor protein. These results support gene amplification as the mechanism enhancing A431 c_el': EGF receptor protein and determining growth responses. Gill, G.Ivl et a1. ~ 1 Somatic Cell and Moletulari:enetics il(4):309-3i,1985. ~ ~ Other support: ~Iational Institutes of Health and the-American Cancer Society. `- From the Department of Medicine, Division- of Endocrinology and Metabolism, I University of-California at San Diego, School_of Medicine, La Jolla, CA; Department of Molecular Biology, Keio_University School of Medicine, Tokyo, Japan; Department of - ~= Cellular and Develo r'enta' Biolo -U '~ 't f A' T SELF-PHOSPHORYLATIOI`' ENHANCES THE PROTEIN-TYROSINE KINASE- ACTIVITi! OF THE EPIDERfi4:.L GROWTH FACTOR RECEPTOR - The effect of self-phcaphory:ation on the protein-tyrosine kinase activity of the = epidermal growth factor receptor has been investigated using-immunoaffinity-purified protein. Enzyme was first incubated for various_tizaes with excess ATP to phosphorylate _ it to differing extents; -the ability of the enzyme to phosphorylate exogenous pepn de substrates was then-measured as a function of its-self--phosphorylation state. Increasing - self-phosphoryla*.ion to= 1.3=__8 - mcl of phosphate- nol'1 of epidermal growth factor receptor enhanced protein=tyrosine kinase aetivity_ 2-3-fotd:--Coaparison of- the kinetics of protein-tyrosine -kinase activity at different ATP concentrations revealed' significant differences between unphosphorylated and phosphoryiated enzyme. At low levels -of ATP, a double reciprocal plot of the -protein-tyrosine kinase -activity- of the unphosphorylated enzyme was hyperbolic, suggesting-that ATP may act as an activator of I the enzyme. At higher ATP :oncentrations; where greater levels of self -phosphorylation ( occurred during the reactior; the kinetics appeared linear and similar to those of the r phosphorylated enzyme. Dose-respotue- studies using three different peptide substrates I (angiotensin II, gastrin, and a synthetie peptide corresponding to the self-phosphorylation- i

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site in p60`1~) showed that-exogenous-substrates inhibit receptor self-phosptiorylation- in each case_-~ haI_f-maximal inhibition was observed at a pertide concentration approximately equal- to the substrate's Am A kinetic analysis comparing peptide- phosphory-lation using unphosphorylated and prephosphyrylated enzyme indicated that the self ~hosphorytatior; site can act as a c?~mpetitive inhibitor (alternate substrate) versus peptide substrates. These results -suggest that silf-plrosphorylation of the epidermal growth factor receptor removes a competitive constraint so that exogenous substrates can be more readily phosphoryiated.- Bertics, P=.-J. and Gr13,. G. N. The_Jourenl of Biological Chemistry 250(27):1464?-14h47, 1985. From _ the - Department of Medicine, _ Division of -Endocr'enology and Metabolism, University of California, San Diego, School of Medicine, La Jolla,-CA.,- MICROFIBRILS, ELASTIC ANCHORING COMPONENTS OFTIfE - -= -- EXTP.ACELLULAR MATRIX, ARE ASSOCIf.T-ED WITH FIBRONECTIN-IN THE ZONULE OF ZINN .; ,ND-AORTA Microfibrils are striated tubules that play a role in the formation of elastin fibers by providing - a scaffold upon which newly -synthesized elastin is deposited. Ultrastructural-a-nd staini ng studies also demonstrate microfibrils that terminate where elastin is sparse or absent in basal laminae, plasma membranes, and the collage-nous matrix. The most striking accumulation -of- microfibrils is ~ound in the zonule of Zinn, .he transparent and elastic suspensory ligament of the -lens; which contains- ho- etastin. Application of immunocytochemical=staining with a peroxidase-antiperoxidase (I'AP) procedure demonstrates that fibronectin is associated with the microfibrils of the zunule and aorta. Agg_regates-of microfibrils are identical to-_oxytalan-("acid enduring') fibers that have been-describedt in peridontal membranes and other sites subject to mechanical stress and they can be found in sites as disparate as the rabbit zonule, rat= hepatic stroma and human cardiac papillary muscle, indicating that microfibrils-are a-widely distributed_ connective tissue element, witlr a function that extends beyond elastogenesis; their association with f ibronectin and localization suggests that they serve- as an elastic anchoring component of the e-traceilular-matrix- - GoldJucher-. S., Coltoff_-Schiller, B., and Goldfischer, M. - Tissue ~ Lell I7(4):441-450, 1985. Other support: Nationai Institutes of Health. - From the Depart-ment=of Pathology, Albert Einstein College of Medicine, T'he_ Bronx, NY. ALTERED METHiOrdINE METABOLISM AND T_RANSM_ E T HYLATION - IN CANCER Methio nine mQtabolis_2 and transmethyqation are f requently altered-in cancer cells. The alteration is often expressed as an inability of the- cancer -cells to grow when methionine is replaced by hon:ocysteine in the culture medium, a condition that allows the growth of normal cells. - This metabolic defect is -termed methionine dependence. Methionine dependense- may reflect an overall imbalance in -transmethylatron which results_in the overmethylation of some substances and undermethylation of others within 25

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cancer cells. Many carcinogens affect_ various -stages of ruethionine,/transmetiytation altered metabolism. prevention of altered meth-icnine/transmethylatioA metabolism -or compensation of the process. The known protective effect of inethionir,e against cancer may be- due to metabolism may be the disruption of the regulation-of genes involved in the oncogenic metabolism The ultimate effect of the -altera:ion of - methionine/transmethylation -` for Cancer Research. From the Department-of PMiatrics, University of California, San DiFgo, La Jolla. Other support: The National Cancer Institute and the -George A. Jacobs Memorial Fund #!o/fman. R. M. Anticancer Research 5:1-30,_1985. irrelevant antiimmunoglobulin ant_ibody- in 136 control animals. With Lews- lung 71%) (l24 experimental-animals), when compared to the effec,- of noaimmune serum or ° 47 to 6596) and decreased the mean rtodule volume of tumor per _ung by 37% (range, 0 to tumor cells decreased the mean number of CT 26 tumor nodules per lung by 57% (range, - level for 24=hr. Antiplatelet antibody given 6 hr pre- and 18 hr post-i.v. injectips of decreased the platelet count from 1.5 to 0.12 x 10°/pl at 6 hr, which remained at this - development - Qf= pulmonary metastases. - Antiplatelet antibody, when in jectxd = i.p., - and C3TBL/6J) to test-the effect of=rabbit- anti-mouse platelet antibody-on the - carcinoma, and B16 amelanotic melanoma, were-injected into syngeneic- mice- (BALB/c Three different nturine tumors, CT26 colon- adenocarcinoma-, Lew:s- lung EFFECT OF ANTIPLATELET ANTIEODY ON THE DEVELOPMENT OF- PULMONARY METASTASES FOLLOWING INJECTION OF CT26 COLON ADENOCARCiNOMA, LEWIS LUNG CARCINOMA, AND E16 AMELANOTIC MELANOMA-TUMOR CELLS INTO MICE carcinoma, antiplatelet antibody decreasPd- the mean number of tu:ao: nodules bs 6=2% (range; 57 to 78%) and decreased the mean nodule volume oi` tumors by 64% (range, 60- given s.c., antiplatelet antibody given 6 hr pre-injection, IS_hr post-injection and every 48 hr thereafter also -decreased the mean number of metastases by 42% in 14 experimental and 15 control animals. - With EL6 amelanotic melanoma, antiplatelet antibody given-6 hr pre- and 18 hr post=imjection decreased the mean number of tumor- nodules by 85% and decreased the mean nodu':e volume of tumors by 66% using 9 experimental-and 9 control animals. Similar results were obtained when all three- tumors were injected 6 hr after the=injection of ant_ip?atelet antibody. However, negative results were obtained if-antiplatelet antibody was-injected 6 hr after the injection of tumor cells. Since antiplatelet antibody has its maximum effect at 6 hr, it is likely that platelets play their role in the development of pulmonary metastases during -the first 12 hc of tumor inoculation.- to 77%) using 48 experimet tal animals and 65 controi animals. Whern tumor cells were Pearlstein, E., Ambrogio, C., and itarpatkin, S. Cancer Research 44:3884-3887, 1984. From the Departments of Medicine and Pathology and Irvington House institute, New- York -University Medical School, New York. 26 A k

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LACK OF EFFECT OF !N-VIVO PROSTACYCLIN ON THE DEVELOPMENT OF PULMONARI'=M£TASTASE.S IN MICE F-0LLOWING-INTRAVENOUS- INJECTION OF C~3'26 COLON CARCINL)MA; LEWIS LUNL'i CARCINO- MA, - {3R-Bt6 AMELANOTIC MELANOMA CELLS Honn et al have recently reported a 93% reduction in the development of metastases of B16 amelanotic tumor cells given-i.v. following a single dose of prostacyclin- (PGI=) and theophy.line (100 pg each) 30 min prior to the injection of tumor cells. The present authors have been unable to reduce pulmonary metastases induced by the i.v. injection of CT26- colon adenocarcinoma, Lewis lung carcinoma, or B16 amelanotic melanoma cells with a similar regimen. Thus, PGI= and theophylline given prior to injection of tumor cells and 2 hr post-injection had- no effect on the number or volume of pulmonary- tumor nodules for CT26 cells, using 15 experimental and 14 control animals; L ewss lung cells, using -14 experimental and- 13 control- animals; or Blfi amelanotic cells, using-26 experimental and 12 control animals. The -PGI= used was shown- to be active-in vitro, inhibiting tumor-induced plate!et aggregation by all three tumors at 10'9 M and in vivoby inhibition of Lewis lung-induced thrombocytepenia at 1 hr, using 100 pg PGh priorto the injection of tumor cells. Karpatkin. S., Ambrogio, C., and Pearlstein, E. Cancer Research 44:3880-388"s, 1984. Other support: National Institutes-of Health:_ From the Departments of Medicine and Pathology and Irvington House Institute, New York University Medical Schnol_; New York. NEUROPEPTIDE K: A MAJOR TACHYKININ_ IN PLASMA AND-Ti7MOR TISSUES FROM CARCINOID PATIENTS Evidence- is presented for - the presence of an entire - family - of tachykinin-immunoreactive peptides-in plasma and tumor tissues from patients with carcinoid tumors.-- The peptides include, in addition to substance P and neurokinin A, neurokinin B; an eledoisin-like peptide, and neuropeptide K. a 36 amino acid long tachykinin which contains neurokinin A at its C-terminus. Neuropeptide K seems to be the tachykinin which is present in highest concentrations in plasma as well as in acetic acid eatraLts-of tumor tissues. It is highly biologically active and may therefore contribute to the clinical symptoms of carcir+.oid tumors. Theodorsson-Norhaim, I., Norheim, I., Oberg, K., Brodin, E., Lundberg, - nl., Tatemoto, K., and Lindgren, P.G. - Brochemical and Biophysical Research Communications !31(I):77-8"s, 1985. Other support: Nordisk Insulinfond, Swedish Society of Medicine, Karolinska Institute. Uppsala University, and Swedish Medical Research Council. F-rom the Department of Clinical Chemistry, Karolinska Hospital; the Department -of Pharmacology and Biochemistry Ii, Karolinska Institute, Stockho!m; and the Departments of Internal Medicine and Roentgenology, University Hospital, Uppsala, Sweden. -

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HEREDITARY OVARIAN CARCINOMA:-EIOMARKER-S'hiJDIES ' - - - Three ovarian-cancer-prone kindreds were= studied, two- of which contained identical twin sisters concordant for ovarian carcinoma. In one kindred, both identical 28 t candidates for intensive surveillance/management programs.- - -- and specificity, would ena6le_ identification of individuals who would be prime with the eventual development of a risk factor profile which, given acceptable sensitivity controls (P-0.04 and-°-0~00-2, respectively). These findings are discussed in context significant lower levels in= 50% risk individuals when compared to spouse and published (i) in- rrtro hyperdiploidy in dermal monolayer cultures, and (2) lower serum- levels of alpha-L-fucosidase ( 5 275 lU/me)`=in al_ cancer-affected patients and statistically documents on cancer-of-ail anatomic sites. Putative biomarker determinations inciuded: included detailed questionnaires -with° retrieval of primary medical and pathology autosomal dominant mode of inheritasce.- -Medical=genetic= survey of- each family showed vertical -transmission irt all three families in a pattern- consonant with an identical-twin sisters had an- ovarian-canzer-affected daughter: Ovarian carcinoma twin sister had daughters with ovarian carcinoma. In another kindred, one of the Lynch. H. T. et a!. Cancer 55(2):410-415, 1985. Omaha; and Cornell University Medical College, Ithaca, NY. - Omaha, NE; Boystown National In stitute for Hearing and Speech Disorders -in Children,=- - From the Creighton University School of Medicine and the Hereditary Cancer Institute, -Other suppost: Nebraska Division, Fr-aternal Order of Eagles. FAMILIAL EMBRYONAL CARCINOMA IN A CANCER-PRONE KINDRE.D- elucidation of cause and control is-discusseds remains elusive. Priority -attention to biomarker research in families of this type for malignant- melanoma- and -urinary- bladder carcinoma). Hereditary -syndrome designation-- embryonal carcinomaa in this woman's maternal half-brother (their mutual mother had the son of a cancer-free but putative obligate gene carrier mother, and-the adult form- of -F'amilial testicular cancer is rare. -- This report describes a family with an- unusual cancer spectrum xhat included the infantile form of embryonal carcinoma of the testis in - Lynch.ll. T. tt a1. - -Tl+e American Journal eJ Medicine 7g:891-89e, 1985. - From the Department of -Preventive- Medicine/Public Health and the Department of Pathology, Creighton University School of Medicine, and the -Hereditary Cancer Institute; Omaha, NE. COLORECTAL CANCER IN A-NUCL-EAR FAMILY: FAMILIAL OR - - HEREDITARY? Because of the high incidence of colorectal cancer, familial aggregations of- this disease are common: -Differentiat-ion - between etiologies contributing - to familial - clustering (which may have resulted either from common environmental exposure or from mere chance) and primary genetic factors may prove vexing to the physician. This _ report deals with the myriad problems encountered when attempting_ to make such

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etiologic distinctions in order to provide appropriate surveillance and management, based upon tumor spectrum and natural history, for patients at increased cancer risk. ® I Lynch. H. T. et al. -- Diseases oJ-the Colon_& Rectum 2g(5):31Q-316, 19g5. From the Departments of Preventive Medicine/Public Health, Surgery. Pathology, and Radiology, Creighton University School of Medicine and the Hereditary Cancer Institute, Omaha, NE. THE SARCOMA, BREAST CANCER, LUNG CANCER, AND ADRENOCORTICAL CARCINOMA SYNDROME REVISITE-D: CHILDHOOD CANCER We studied-twa children-who had rhabdomyosarcoma and glioblastotna and who were from a family_ with a hereditary cancer syndrome that was characterized by sarcoma, breast cancer, brain tumors, lung cancer, laryngeal carcinoma,-leukemia, and adrenocortical carcinoma. The deleterious genotype has now been expressed through the fourth generation of this large kindred. The pedigree -emphasizes the need for- an extended history of several generations to arrive at a hereaitary-syndrome diagnosis. A limited pedigree may result in nonappreciation of the genetic component. The pedigree illustrates that, in certain circumstances, the highly specific varieties of cancer may occur in children before -it is expressed in the parent who carries the putative gene. Pediatricians, in evaluating the causes-of childhood cancer; must be cognizant of cancer among adult relatives;,-since this recognition may aid in the-diagnosis of those hereditary cancer Yyndromes -that are characterized by cancer occurrence in children as well as adults. Lynch; H. T., Katz, D. A., Bogard, P. J., and Lynch, J. F.- Ame6icart Journal of Diseases of Children 139:134-136, 1985. Other support: Nebraska Division, Fraternal Order-of Eagles. From _the Departments of Preventive Medicine,/Public -Health; and of Pathology,- CreigMon University School of Medicine, and Hereditary Cancer Institute, Omaha, NE. THE CUTANEOUS EVOLUTION OF-NEVI IN A PATIENT WITH FAMILIAL,ATYPICAL, MULTIPLE-MOLE MELANOMA SYNDROME- For almost two decades we have followed a kindred with the familial, atypical, multiple-mole --r.telanoma (FAMMM) syndrome. We first evaluated the proband's 14-year-old daughter when she was age 5 years. We documented the evolution, - both clinically and histologically, of the FAMMM phenotype in this girl for eight years. - Lynch. H. T. et al. _ Pediatric Dermatology 2(4):2-g9-293, 1985. From the Departments of _ Preventive Medicine/Public Health- and Dermatology, Creighton University_ School of-, Medicine, Omaha, NE; the - Department- of Internal Medicine, University_ of- Nebraska Medical Center; Omaha; the- Hereditary Cancer - Institute, Omaha; -the- Dermatology Department,_ The Polyclinic, Seattle; and the Department of Patholpgy, Permian G-eneral-Hospitai,-Andrews, TX. 29 :,

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BIOMARKER STUDIES-lcjl HEREDITARY OVARIAN CARCINOMA Ovarian cancer has been increasing in-frequency during the past several decades, particularly in Western industrialized nations. It has the ignominious distinction of being the major cause of death from genitourinary cancer in women in the United States. A smalt but significant fraction of patients- with ovarian cancer=have family histories that are compatible with a primary genetic factor. The hereditary variant is heterogeneous but these pedigrees reveat-a high predictability of cancer. Therefore, families that are prone to ovarian cancer merit the highest priority for biomar k¢r investigations. When considering the generally poor surveillance measures available to us for detecting ovarian cancer sufficiently early to improve prognosis, the search for biomarkers of high sensitivity and specificity assumes major significance. In turn, such biomarkers may shed light -0n the etiology and pathogenesis- of ovarian cancer in the =general population and more importantly provide mechanisms echanisms for early detection and prevention of death from this tumor. Lynch. H. T.. Schuelke, G. S., and Lynch, !. F. Cancer Detection and Prevention 8:129-134, 19$4. Other suppo:t: Nebraska3?ivision, Fraternal-Order of Eagles. From the Department of Preventive Medicine/Public Health, Creighton University School of Medicine, and the Hereditary Cancer Institute, Omaha. CANCER GENES, MULTIPLE PRIMARY CANCER, AND VON HIPPEL-LINDAU DISEASE Von Hippel-Lindau disease is inherited by an autosomal dominant gene that may show marked expressive variability of cancer phenotype in certain patient/families. We describe a patient with a strongly positive family history of this disease who at age 28 - underwent craniotomy with removal of a cystic cerebellar hemangioblastoma; at age 48, he- developed syringomyelia of the- spinal cord, became quadriplegic, and had -a progressive downhill course. At autopsy, hemangioblastomas of the cerebellum and spinal cord were found, as well as aieft renal cell carcinoma, an-oat cell carcinoma of the lung, a-hepatocellular- carcinoma, and an atypical thyroid adenoma. This tumor spectrum appears to be unique, although chance-canno2 be excluded. It is possible, however, that these findings- might represent an expression of the deleterious genotype- that became evident becau-se- of this patient's prolonged survival from his initial cerebellar hemangioblastoma. - Lynck. H. T.. Katz, D. A., Bogard, P., and Lynch J. F. Cartcer Oenetics and Cytoger.etscs 16:13-19, 1985. Other support: Nebraska Division, Fraternal Order of Eagles. From the Departments of Preventive Medicine/Public Health and - of Pathology, Creighton University School of Medicine, and Hereditary Cancer 3nstitute, Omaha, NE. 30 Cli

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PANCREATIC CARCINOMA-AND HE-REDITARY NONPOLYPOSIS COLORECTAL CANCER. A FAMILY STUDY - This paper describes a kindred with vertical transmission of cancer through 5 generations which showed features-of hereditary nonpolyposis colorectal cancer (HNPCC) in concert with pancreatic cancer. -Tlte proband_ is a 55-year old white -dale with verified pancreatic-carcinoma. This patient, and subsequently his= available -relatives, fiiled out detailed - medical-genetic questionnaires. Their signed - per mission forms enabled the authors tti-corrosorate family, medical, and cancer (all anatomic sites) history through secured primary med: al and pathology documerus._ HNPCC is beconing more frequently recognized ° than its dominantly inherited counterpart, familial multiple polyposis coli (FPC),-a fac-t which has contributed to the recent increased interest in this disease (Lynch et al, -1481 p. There are at least two forms of HNPCC: (9) hereditary site-specific colonic-cancer, referred to as Lynch_syndotne 1; and (2) the Cancer Family Syndrome, referred to as Lynch syndrome II (Boland and Troncale, 1984). In both disorders, one finds multiple primary cancers of the colon with an excess of involvement of the proximal colon. Lynch syndrome II includes cancer of other anatomic sites, particularly the - endometrium and ovary and- possibly = the pancreas. Genetic heterogeneity with respect to variation in tumor spectrum has become increasingly more evident in HNPCC (Lynch et aI., 1982). -Neuroblastoma, a lesion more- characteristic of _ childhood, in the pseband's son at age 22 is puzzling. There is a need for more biomarker and pedigeee studies with documentation of cancer of all anatomic sites in HNPCC kindreds (Danes and Lynch, 1482 j. i,yncF.. H. T. et al. British Journal of Cancer 52:271-273, 1985. From the Department of - Preventive Medicine/Public Health, Department of Internal - Medicine, Division of Gastroenterology, Creighton University School of Medicine and the Hereditary Cancee-dnstitute, Omaha; and The McGreevy Clinic, Sioux Fa1ls, SD. FAMILIAL _MESOTI-EFLI3MA-: RbVIEW AND FAMILY STUDY To- date, with few exceptions, primary attention to the etiology,- of- malignant mesothelioma has been focused on environmental factors. = However, several repor ts of familial aggregations of- mesothelioma strongly support the supoosition that host f actors, in concert with -environmental- exposure, particularly asbestos,- may contribute etiologically to an as yet unknown fraction of occurrences of this disease. However, in evaluation of familial mesothelioma, -it is important to consider the possibility of household exposure to asbestos. We report a family in which two brotherJ with prolonged occupational asbestos exposure manifested malignant pleural rnesot.heliomas- of similar histology:_ _ Lynch, H. T.. Katz, D., and Markvicka, S. E. - Cancer Genetics and Cytogenetirs SS:25-35, 1985. From the Departments of Preventive Medicine/Public- Health and Pathology, -Creighton University School of-Medicine, and the Institute for Famiiial-Cancer Management-and Control,-Omaha, NE. 31_

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ALVEOLAR MACROPHAGE FUNCTION AND INFLAMMATORY STIMULI IN SMOKERS WITH AND-WITHOU"I` OESTRUC-E IVE LUNG DISEASE - To explore-possible cofactors in,the=de-velopment of chro-r_ic obstructive pulmonary-- _release_ by macrophages froni_smokers with COPD was significantly higher (p :_- 0.016) than - was eiastase release by macrophages from -normal - smokers. There were -sd differences betweert cl;emoattractiveness nf alveolar macrophage supernatants for one- _ person's poiymorphonuclear leukocytes among the groups of smokers and there -was no detectable C5/C5a in these supernatar,ts (limit of detection of-C64 greater than 1 ng/ml). There were _no significant differences in numbers-or species of bacteria in aerobically and anaerobically cultur_ed bronchial -brushings:- There was-nv difference in alveolar __ macrophage superoxide anion-releasr with particulate or membrane-perturbing stimuli for_the smokers. Alveolar macrophages from the 3 groups of subjects had- similar limited - microb_icidal-ability foa=the .obligate intracellular protozoan, Toxoplasma gandii- and similar numbers of elastase receptors-and-affinity for eiastase. disease (COPD) in smokers, the authors performed bronchoalveQlar lavage in 6-smokers - with normal pulmonary function, 6 smokers with_COPD (FEV,jFi%C :5 65%) matched for smoking history and age;=and_ 9 age-matched-norsmoking control subjects. Elastase McLeod, R., Mack,-D. G., McLeod, E. G., Campbell. E. J., and Estes, R.G. American r?eviees of Resp:rato_ry Disease t31:377=3g4, 19g5. National Heart, Lung an- d - Blood Institute, and the Research, Education and Teaching in Pulmonary Medicine Fund of--Michael Reese Medical Center, Chicago. -- - Research Institute-Council,-_the National :nstitute-of Ailergyand Infectious Diseases, the - Other .tupport: ?he Te.sner and- Kenzer Foundations and the Michael Reese Medical -From the Department of Medicine, -Mic' hael Reese Hospital, Chicago; the University of Chicago Pritzker Schooi: of_ Medicine; The Jewish Hospital at Washington tiniversity-- Medical Center, St. Louis. _ _ Concomitant with the appearance of pulmonary injury was the generation of and histologic evidence of a predominantly neutrophilic leukocytic iniltration: untreated animals to 3-.74 mg/ml and 6.64 mg/ml in FNLP- and Pit4A-treated animals, respectively, the appearance of a diffuse pulmonary infiltrate in chest roentgenograms, -mean protein content of -bionchoalveolar- lavage (BAL) fluid from 0.51 mg/ml -ir- phorbol myristate avetate-(PDiA). Indicators of pulmonary injury=incl_uded an increase in by the intrabronchial instillation of the-fnrmyiated peptide norleu-leu-phe (FNLP) or = EXPERIMENTAL PULMONARY INFLAMMATORY INJURY IN THE 11riONKEY- Inflammatory pulmonary injury was induced in-Macacca rrtulatta ihesus monkeys _- proteases-and oxidants in the BAL fluids. Neutrophil elastase, bound to al-protease acrease from 0.47-µg/ml in untreated monkeys to 0:9y pg/rnl in inhibitor- was found-to i human prekallikrein, instilled-for 2 min into the inflammatory site and retrieved by lavaging, was found to have undergone proteolytic cleavage; this cleavage was not cor,sis- _ FNLP-treated animals and= 1.23 - µg/znl in monkeys- receiving PMA. Radioiodinated 32 M ® i

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® ® I tentiy inhibitable with the inclusion oT antibody to elastase. BAL fluids were- shown t_o- contair: an - amidelytic activity when tested on the synthetic - substrate Ei-1?=pro-phe-arg-pFIA. This activity was partially iahibitable with-known inhibitors of active Hageman - factor and kallikrein. fl-Glucuronidase levels- in the BAL fluids increased fror 0.85 iJ/mi to 4.36 U/cii-and 8.25 U/mi in FNLP- and PMA-treated animals, respective':ya-- Myeioperoxidase (MPO) levels also i ncreased from 1.37 OD L U/ml min ro=15.5ci and 30:45_Ov^ U/ml min in the same groups of animals. Oxidant generation was also assessed in several different ways. The specific activity of the oxidant-sensitive inhibitor_a,-PI recovered in the BAL fluid_ decreased from 0.80 in control samples to 0.57 and 0.65 in FIf,P- and PMA-traated-animals. That this inactivation-wa3-due to oxidant injury of the molecule was confirmed by the return to full-activity_of four of five BAL samples after their incubation with -the reducing agent dithiothreitol- in the presence of methionine suIfoxide_ peptide reductase. ___ i iee specific activity of_.catalase- in the BAL fluids of animals-given 3-amino, 1,2,4 _triazole (-AT) I h before Iavaging showed drops from 0.97 in_ unereated monkeys to 0.04 in FNLP=t-reated-and 0.49 in P-MA=rreated monkeys._ MPO levels alw feli_in t;5e AT-treate;'r- izjured animals from-16.5-i to 0.85- A QB/min ml in 1=_lVi,P animals in the absence and presence of AT, and-30.4-t_to 0.60 a OD%min ml in PMA-treated animals. Inhibition of MPO by AT was shown in -_-vitro to be hydrogen peroxide dependent._ Total giueathione - levels in the BAL fiuid=.did not change appreciably a.`ter-F?tLP or PMA treatment. These studies_-pr,esent-substantial evidence of the generation of-both proteases and oxidants during the establishment of_ acute pulmonary inflammatory injury - it+n an experimental primate model. Revak; S. D., -Cochran<, C. G. et ai. Journal of C7ir1c?I _Inrest*gation 76:1182-1192, 1985.= Other support: V. S: P;;biic Health Service and Office of Naval Research. From the Departmenuof Immunology, Scripps Clinic and Research- Foundation, La Jolla, CA and the Departrr.entof Surgery,-Harporv-iew Medical Center, Seattle. , -ISOLATION OF TROPOELASTIN a FROM LATHvRITIC-C[-tIC.lC AORTAE - Tropoelastin a- was isolated from lathyritic chick aortae by using severe denaturing conditions for the initial_ extraction. The amino acid composition of this new species of tropoelastin is elastin-like in its high proportion of proline, giycine, alanine and valine.- However, it differs_ from authentic tropoelastin b in containing a higher percentage of polar amino acids and cysteine residues. -In addition, Yhe= am_ount o proline -pfelastln b. hydroxylation i-s-3 times ):igher than-that found -in_chick_tro Rieh, C. B.- and Foster1. A. r7iochemrstry Journal 217:581-584, 1984. Other support: National Institutes of Health. From the Department-of Biology, Syracuse L'niversity, Syracuse, Nf. 02 i 33 0

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EFFECT OF HYDROXYL RADICAL SCAVENGING ON ENDO T OXIN-L*1DUCEIi-LIJNG INJURY - The release of oxygen radicals, in particular the hydroxyl radical, from sequestered neutrophils produces acute lung injary_after a aumber of insults. Our purpose was to determine whether hydr_axyl , radical OH is respo-nsible for the lung injury- from endotoxin characterized by (i), pulmonary leukostasis. (2) increased thromboxane production leading to pulmonary hypertension and hypoxia, and (3) increased protein permeability. This ihypo;:resis was tested by infusion -of a selective Ohi sravenger, dimethyl thiourea-(G.75 gm _kg), into unanesthetized sheep- before- end-otoxin"and comparing the response to .nat-seen with endotoxin alone. Pulmonary vascular- integrity was measured by the use of'hsng lymph floiv, Q~, lymph protein transport, Thromboxane - A= was measured as TxB4, acd prostacyclin as 6-keto-PGF;1L. We found ng difference in the degree of leukopehis_ and hypoxia after endotoxin or the levels of TxB 6-keto-PGFt~ and pul~pary lypertension with dimethyl- thiourea, compared with endotoxin alone. The permeability injury was also iden,ical, with a two-fold to three-fold increase in protein-rich lymph seen in both groups. It appears that OH- does not play a major causative role in either phase of endotozin_lung injury. Wong, C., Fox, R., and 13em:ing, R. H. Surgery 97(3):300-3D6, 1985. _ Other support: National Institutes of Health. From the Longwood Area Trauma Center, Beth Israel Hospital, Harvard-MEdical School, and Brighain and ii%cmen's Hospitais: and the Children's Hospital, Boston: PROSTAGLANDINS AND INTRACELL_U_LAR CYCLIC AMIrIN RESPIRATORY SECRETORY CELLS Prostsglandins- are known to affect ion trans_port and mucus secretion in the trachea, and at least part of their effect is thought to be- mediated by cyclic AMP. Because no prostagtandin ,receptor-assay is currently available, we have used an immunocytochemical probe for t he intracellular ;ocalization of eyclic-AMP to identify those specific cell types in dog and cat trachea_ that respond to prostagiandins. Using tracheal explanrs- similar io those used by other investigators for in -vitro studies of ion transport and glycoprotein secretion, we examined the effect of endogenous and exogenous prostagianc3ins on immun3react-ive cyclic A?yiP. Endogenous prostaglandins,- which may be secreted in response to minor distortion of tissue membranes, stimulated immunoreactive cyclic AMP in ciliated epithelial cells and in both serous and mucous submucosal gland cells. This was-not observed in tissues assayed 60 min after dissection or dissected in the presence of indomethacin or of BW755C. Exogenous prostaglandin T increased cyclic AMP in these same cell types. We conclude that prostaglandins stimulate cyclic AMP in specific cell types in the trachea, and that endogenous prostaglandins play _ a major role in many in vitro preparations. Lazarus, S. C. Basbaum, C. B. and Gold, W. M. American Review ol Respiratory Disease 130:262-266, 19L4. Other support: National Heart, Lung and Blood Institute. 34 r

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From the Cardiovascular Research Institute -and the -Departments oT Medicine and Anatomy, University of Californ'ia,- San Francisco. - AIRWAY RESPONSIVENESS TO IN's-hlLEU-ANTIG£N, HISTAMi_N, AND METHACHOLINE IN INBRED. RAi.rWEED-SENSITI?ED DOGS - The authors studied-the responses to antigen in animals selected iront a colony of -inbred dogs sensitized to specific aligrgen; to-determine if theyhad characteristics similar to those of human asthmatics.- They were im_r.iunized- with- ragweed and grass - pollen extracts {10 pg in alum) i:dmediatelv-after routine -vacciFatipn with attenuated live virus {disteinper and hepatitis) and killed -bacteria (Leptospira) at-4, 8, and 12- wk of age. - Subsequently,-ragwerdand grass injections were repeated every-2 months. Immunized dogs made specific -IgE -antibodies in serum averaging-3 to- 4 times-that of control animals (no immunizhtion with-pollen or-vaccine). 'I`hey-shoxed positive-skia responses to the injection of ragweed pollen extract, whereas control dogs did not- respond to ragweed- po!len by quantitative skin test or inhalation challenge. In immunized- dogs under barbiturate anesthesia, air-flow_ resistance of the total respiratory system increased from 0.60 ± 0.07 (mean ± SEM) before to 12.6 ± 3.4- cm I?EO(ips 5 mir_ after the start of antigen_aerosol; respiratory resistance remained increased for 20 min and was associated with hypoxemia -and increased arterial plasma histamine. _ In- addition, airway responsiveness to both inhaled histamine and methacholine was greater in immunized dogs than in ronima:unized- dogs of comparable age. Airway responses to each agonist were- highly reproducible on repeated testing. T hese results- indicate that physiologic responses to antigen-by inbred, ragseeed-sensitizQd dogs resemble human asthma closely and that thes-e-dogs appear suitable for a variety of experimental studies of asthma with respect to pathogenesis, diagnosis, prevention, and treatment. Mapp, C., Hartiala, L, Fricic, O. L. Shields, R. L., and Cst_d, W. H. Americar: Review oJ iespiratory Disease 132:292-298, 19&5. Other support: - National Lung and Blood Institute; National Institute of Allergy and Infectious Diseases;-and the-Nortliern California Allergy Society. From the Cardiovascular Research Institute and Departments of_ Medicine and Fediatrics,- University of Califcrnia; San=Fraocisco, and the Animal Resources Services- Facility of the School of Veterinary Medicine, University of California, Davis.- -_ CI-tARACTERIZATI-ON OF PURIFIED DOG MASTOCYTOMA CEL-[:S: AUTONOMIC MEMRRANE= R ECEPTORS AND PHARMACOLOGIC - MODULATION OF HISTAMINE RELEASE - - There is conflicting evidence as to which° autono._,ic receptors mast cells possess - and whether the receptors are capable of modulating mediator release. -The authors have studied dog mastocytoma cells because they are available in large-numbers in a relatively pure form, unlike as:,nal-dog mast cells. Mastocytoma nodules from a dog were excised and disaggregated-witA collagenaseYo provide a cell suspension of mastocytoma cells of > 92% purity. The pr$sence of autonomic receptors wax assessed by both radioligand binding assays and by evaluating pharmacologic modulation of mediator release. In the radioligand binding assays; beta-adrenergic receptors were estimated by fsl:)prazosirn binding and cholinergic receptors by f,3Hjquinuclidinyl benzilate binding_ Nonspecif'_c-_ binding was determined in each case by incubation in the presence of the specific 35 - ~ ~ ~- 1~- ~.~ 0 I

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antagonists propranolol, - phentolamine, and atropine, respectively. The effect of autonomic agonists on immunologic and nonimmunologic histamine release was examined using the beta-adrenergic agonists isoproterenol and terbutaline, the alpha-adrenergic -agonist phestylephrine with a-nd without propranolol, and the- cholinergic agonist acetylcholine. Dose-response curves were constr~;cted both for the autonomic agonists and the histamine-releasing agen*_s. Results from the radioligand binding and the pharmacologic studies were-concordant. These dog mastocytoma cells had a high density of beta-receptors (21,500 ± 3,300; mean ± SE beta=receptors/cell, n=5) of which- the predominant subtype appeared io-be beta:. No evidence was found for the presence of alpha-adrenergic or cholinergic receptors either by direct receptor binding or by their actions on histamine release_ Phillips, M. J., Barnes, P. J., and Gold, W: M. -- Amerisan Review of Respiratory Disease 132:1019-1026, 1985. OOther support: National fleart,-Lutig and Blood= Institute,-the Strobel Medical- Research- Fund of the American Lung Association of Sa.rF"rancisco; the University of California Research Evaluation and Allocation Committee, and the California Research and Medical- Education Fund of-the American Lung Association of California. From the Cardiovascular Research Institute and Department of Medicine. University of -California,-San Francisco. - - - NICOTINE-INrUCED RESPIRATORY EFFEi I S OF CIGARE T TE SMOKE IN E4CKGS The authors report that nicotine is responsible for both a blood-borne stimulation of the respiratory center and a direct effect on intrathoracic airway tone ia dogs. They introduced cigarette smoke inio - the lungs of donor dogs and in jected arterial blood obtained from them into :he circulation of recipient dogs to show that a blood-borne material increased breathing and airway smooth muscle tone. Smoke 4'rom cigarettes containing 2.64 mg of-nicotine was effective; that from-cigarettes containing 0.42 mg of nicotine was not- Nicotine,- in doses comparable to the amounts absorbed from smoke, also increased breathing and tracheal smooth muscle tension when in jected into the vertebral circulatio n of recipient dogs. -Finally, blockade of nicotine receptors in the central nervous system and in the airway parasympathetic ganglia inhibited the effects of inhaled cigarette smoke and intravenous nicotine-on the respiratory center and- on bronchomotor tones. The authors concluded that nicotine absorbed from cigarette smoke is the main cause of cigarette smoke-induced bronchoconstricion. It caused central sespiratory stimulation, resulting in increased breathing and airway smooth muscle tension, and had a direct effect on airway parasympathetic ganglia as well. Hartiala, J. J., Mapp, C., Mitcheli, R. A.. and Gold. W. M. Journal of Applied Physiology 59(1):64-'. i; 1985. Other support: National Heart, Lung, and Blood Institute. From the Cardiovascular- Research Institute and- Departments of Physiology, University of California, San Francisco. edicine and ~ (+" ~ 36

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AN ULTRASTRUC'EIJRAL -Ale'AL-YSIS OF DOG MASTC~: Y°fOMA CELLS-AND- _ - NORMAL MAST CELLS - - blue, and weresimilar in ultrastructure to normal dog mast cells. The proportion of mast cells -in- this tumor averaged 67%; eosinophiis; fibryblasts, plasma cells, and macrophages also were present. Tr-2 mean diameter of mast cells (12.79 pm) and the mean diameter of their cytcplas^tac granules ,f47j' - nm} were similar to those reported for mast cells and ma.stocytoma- celis from various species.- -The heterogeneity in appearance of the mastocytoma-granules-wa-s consistent with a variable degree-of granule maturation. After disaggregation ar pe_icds of culture ranging from-2-days to 4 week-it, the mean granule -diameters were b59r :arger than those measured _in the intact mastocytoma cells, though other morphologic _featurEs remained unchanged. Although the cells retained their distinct morphologicai featu-res for at t least 4 weeks, some of their physiological responses were lost,after I week in culture. This study showed that dog mastocytomas can be a source of a large, -relatively homogeneous population of cells that are useful for -elucidating some of the structurai and f unctional properties,of mast cells. _ morphometric, histochemical, and biochemical methods. The ultrastructure of cells irn the intact tumor waz compared to the mo; pho':ogy of cells disaggregated f rom the tumor and cultured for periods_as-long as 4 weeks and to normal dog connective tissue mast cells. Most of the tumor cells contained -histami ne (mean = 5.$1 pgJeell), demorstrated chloroacetate esterasa_activity histochemically, stained metachromatically_ with toluidine - A-well--differentiated dog_mastocytorna was characterized uitrastructurally-using Calonico, L. D.. Phillips, M. J.,-MeDonald, D. M., andGo:d,-W. ltfi. The Anatomic Record 212:399-407, 1985. Other support: National- Heart; Lung and Blood Institute, the Strobel Medical Research - Fund of the American Lung Association of San Francisco, and the University of California Research and Medical Education Fund of the American Lung -Association of California. From the Cardiovascular Institute and Departments of Anatomy -and Medicine, University of California, San Francisco. IM~.I~lUNOLc`',GI_ C`',GI_C CHALLENGE AND EPITHELIAL ION TRANSPORT IN CANINE TRACHEA In this study the authors used measured changes in short-circuit current to reflect ion transport across the canine tracheal epithelium and have shown that mediators released from lung fragments by immunologic challenge stimulate ion transport in the trachea. The presence of histamine irn the supernatants, although the concentration is too low to account for the short-circuit current effect, indicates that mast cell degranulation has occurred_ Particularly, the observation that the increase in short-circuit current can be blocked by pretreatment of the epithelial tissue with the cyclooxygenase inhibitor indomethacin suggests that the mediator effect on short-circuit current is indirect and= is produced by activating the cyclooxygena-se pathway in the epithelium. - -- Lazarus, S. C., Leikauf, G. D., McCabe, L._ J., Paige, K., Chung, K. F., Nadel, J. A., and v^o/d. W. M. Chest 287S:188S-189S, 1985. 37 4

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- - - Other suppors: National }ieart; Lung and Blood _Institote;- the California Research =and - Medical Education Fund of- :he American Lung Association of California, and the- Research Evaluation and AllocaFion Committee of the University of California SchooE of Medicine, San Francisco. From the Cardiovascular-_Researc_h_Institute and Department of Medicine, University of- California, San Francisco.-=-= ~ [~ _ PROTECTIVE REFLEXES-AND VASCULAR EFFECiS IP'FHE NASAL MUCOS ELICITED BY ACTIVATION OF_CAPSAICIN-SENSITIVE - SUBSTANCE P-IMMUNOREACTIVE TRIGEMINAL NEURONS The aim of the-_preie-r,t investigation was to study the structpre= and functional characteristics _of- the sensory pathways wtiich= provide protective reflexes in the nasaL - animals. Tne use- of capsaicirf de3ensitiz.ation as a= mucosa in various experimental - possible treatment against-nasal irritation has also been evaluated. The following specific aspects were stz;_died. - (I) The ongin- and distribution _of SR-I=R- nerve fibres in the nasal mvcosa- were- investigated in several-species; including man. Also the_eCfect of- systemic capsaicin _ pretreatment on SP-containing neurons was studied. (2) The effect of antidromic- triyeminaa and parasympathetic nerve stimulation on cat nasal blood -fl:rw was compared with-the response seen-follo;ving-local infusions of SP,- VIP and capsaicin. (3)- The effects of antidromic trigeminai- nerve stimulation, local-application_ of SP or - capsaicin on plasma extravasation ire the nasal mucosa were studied_ in_ control _ and capsaicin-pretreated rats. (4) -- The-effect- of cigareae smoke and its-various components on nasal mucosai-_ permeability was studied in=the ra*- - Additionally, the sensory mechanisms underlying the avoidance- behavior (nose-wipings) induced by cigarette smoke was investigated in the - guinea-pig. (5) The mechanisms underlying sneezing responses to toth-chemical irritants and tactile stimulation were studied followir_-g capsaicin pretreatment. (6) The acute and long--tcrm_ effects of local systemic capsaicin pretreaiment- and cryosurgery on the nasal mucosa were studied. -Ihe-experimenG-examined the inhibition of nasal irritation, loss of SP-IR neurons, neurogenic plasma extravasation, local tissue damage=and systemic-effacts following these treatments. (7) i he importance- of _ capsaicin-sensitive sensory nerves- for = the_ cardiovascular _ responses induced by local application of nic®tine-and capsaicin to the nasal -mycosa was -,- ala investigated. - ~ Lundblad, L.-(Lundberg, J. M.) I Acta Physiolvgica-Scandinaric>s Supplement 529:1-42, 1984. Other suPport: The Swedish- Medical Research Council, the Swedish '7°obacco Company, - the Astra Foundation and the-Karoiinska institute. 38 t

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From the Department of Pharmacology, Karolinska Insr_itute,- and the Department of -_ 39 . c 7::~ .. . r .. Oto-rhino-laryngplcigy, Karoliitska H-ospital, Stockholm, Sweden. "_EVIDENCE FOR SUBSTANCE P-IMMLTNOREACTIVE SPINAL AFa-ERs.NTS_ THAT MEDIATE BRONCHOCONSTRICTION- - disappeared in the Iower airways. The -bronchoconstriction_!nciuced by capsaicin was Immunohistochemical analysis, howevei,-did not-reveal any-clear=cut change -in the number and distribution of SP-IR fibres in the lung of these animals. After combined stellatectomy plus -local capsaicin treatrftent of the _ vagal nerves, mos? SP-IR nerves Stellatectomy caused a significant reduction of SP-IR in the lung and pulmonary artery. immunoreactive (IR ) nerve f ibres in the lower airways we-re studied in - the guinea-pig. The origin and functional role of capsaicin-sensitive substance P-(SP-) stimulation of afferent fibres traversing_the stellate gang!ion. In conclusion, the present absent after systemic capsaici:± pretreatment, suggesting that it was due to aniidromic which was resistant to cholinergic or adrensrgic receptor blockade. This response- was had been chemically_sympathetctomi7ed by 6-OH-dopamine caused bronchoconstriction, - treatment of-vagal nerves and stellatectomy. Stel!ate ganglionstimulation_in animals that was not influenced by stellatectomy but markedly reduced by combined -sapsaicin significantly reduced after stellatecte`n.; and abolished after stellatectomy plus capsaicin pretreatment of the vagal ne=ves. Ether- inhalation caused- bronchocbnstrict_ior, which bronchial smooth muscle and around blood vessels. both vagal ane-spinal origin. These sensory fibres seen: to have branches both within the data suggest that tfie=!ower airways receive SP-IR capsaicin-sensitive C-fibre afferents of Saria,-A.; Martling, C.-R.; Dalsgaard, C:-7., and Lundberg, ,l. M. .tcta Phy-siologica Scandinavica 123:407-414,-19g5. Other support: The Swedish Tobacco Company, the-Gustaf V. Foundation, the -Astra Foundation, the Petrus and Augusta Hed!unds Fonds, the Karolinska Institute, Austrian Scientific Research Funds, and the Swedish Medical_Research Council. -_ From the Department of Experimental and Clinical Pharmacology, University of Graz, Graz, P.ustria; the DFpartments of Pharmacology and Anatomy, Karolinska=Institute; and- - the Department of Anesthesiology, Karolinska Hospital, Stockholm, Sweden. SEQUENCE OF PATHOLOGIC CHANGES IN THE AIRWAY MUCOSA OF GUINEA PIGS DURING OZONE-INDUCED BRONCHIAL HYPERREACTIVIT-Y- The authors assessed- the nature and progression of airway mucosal disease and muscarinic bronchial reactivity in guinea pigs studied in groups of 4-_at 2h, 6h, 14h,- I day, 2 days,- or 4 days after ozone exposure (3.0 ppm for 2h), and in I control group. Muscarinic reactivity was determined by measuring specific airway resistance as a function of- increasing doses of intravenous acety!choline in 31 intact,- unanesthetized; spontaneously breathing animals. After-testing, each group was killed to obtain tracheal tissue for light microscopic examination. We found that airway hyperreactivity to acetylcholine occurred in- 96% of the animals exposed to ozone. Its degree at 2h was substantial. Complete remission- was not observed until the fourth day. In association with the acute bronchial hyperreactivity found at 2h, a marked decrease in airway- mucosal goblat cells and-an increase in mucosal mast cells occurred. Neutrophilic infiltration occurred -later and lasted longer, despite- remission of the hyperreactivity.

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Our results indicate that acute, ozone-induced bronchial hg~perreactivity is' related to _ signs-of airway mucosal injury and mast cell in.'iltration. After this early- phase of consequence of the damage ratyer than a cause of the increased-airway reactivity after ozone exposure. Murlas. C. and Roum, d: H. '- - American Review of Respiratory Disease 131:314-320, 1985: Other support. National Heatt, Lung-and Blood Institute. From the Department of Medicine, University of Cincinnati, Cincinnati, OH.- OZONE=IN1.'UCED CHANGES IN MUSCARIIVIC-B-FiONCHIAL REACTIVITY BY-DIFFtRE-N`I' TESTINa- METHODS suggests mechanisms other-t_hsn airway mucosal-hyperpermeability are responsible for employed. The consistent occurrence of hyperreactivity after 03 to- parenteral agonist 0s-induced hyperreactivity to in-haled-agonist may be a consequence of the technique persisted. Our results indicate that variability in the -occurrence and time course of - day~ later, hyperreactivity to inhaled agonist had remitted; that to intravenous drug agonist occurred consistently, but not to inhaled agonist. Hyperreactivity demonstrable by either route was similar in magnftude and time course within 14h of exposure. Two reproducible results to intravpnous agonist:-Aftee exposure, hyperreactvity to parenteral - spontaneously breathing ani=ttals.- -Bef'ore exposure, we observed trtore' gradual and =- - acetylcholine-- -and/-0r-- aercsolized methacholine challenge _in - 34 unanesthetized, _ determined by measuring spec7ic=air.vay resistance upon administration of intravenous - delivery. Reactivity before and froin-4h to 2 days after tf. exposure (3.0 ppm, 2h) was- - We examined the effect of ozone (03) on-nuscarinic bronchial reactivity in -the - guinea pig and compared reactivity determined -by two different routes -of agonist.- this hyperreactivity. -lournal of Applied Physioloes 57(6):1783-1789, 1984. Other support: National Heart, L ung and Blood Institute, National Institutes of Fiealth and American Lung Association. - - From the Departments of Medicine, Physiology and Biophysics, University of California, Irvine, and Department of Medicine, University vf Cincinnati, Cincinnati, OIt -- We-i3vegtigated the-ef:ects of BW 755C, an inhibitor of both the cyclcoygenase and- liPOxygenase pathways-ef arachidonic acid-meta'oolism; FPL 55712, a selective antagonist of slow-reacting substance of anaphylaxis; and indomethacin, a cyclooxygenase inhibitor, on bronchial reactivity-after ,zone exposure. Guinea pigs in groups of 5 were -treated - with BW 755C (t0- mg/kg -given- intrave.n.ously),- FPL 55712 - (5 = mg/kg given intravenously), or lndomethacin (30 mg/kg-given intraperitoneally) and studied before and 30 min after a 15-min exposure to 3.0 ppm ozonee- These animals were compared -- OZONE-INDUCED BRONCHIAL Y.YPERREACTIVITY--IhE GUINEA PIGS IS ABOLISHED BY BW 755C OR =FPI_= 55712 BUT NOT BY INDOMETHACIN 40 ,= airway damage, neutrophilc infiltration -occurs and -persists, suggesting that it - is- a-

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with a similarly exfsesPd group-that_was untreated (n=10):_ Reactivity was determined -by measuring specifis-aerway resistance (SRaw) upon intravenous acetylcheline infusion in unanesthetized, sp?rtaneously-breathing animals. Prior to ozone exposure, we -found that at drug treatment did not affect either SRaw or ctusearir_ic reaetivity- After exposare to arachidonic acid metabolism post-ozone. This effect may b mediated in parr by lipoxygenase products derived from lung treated with eitherBW 755C or FPL 55712. -We conclude that ozone-induced bronchial hyper-reactivity in the guinea pig rapidiy develops after a brief, high-level exposure. indomethacin-treated animals, marked elevations-occurred in SRaw after-ozone. In contrast, no change in SRaw or muscarinic reactivity occurred after ozone in any animal Indomethacin, trea_-me.n.t did not inhibit this e-ffect. Furthermore, ir_ the 3.0 ppm, all untreated guinea pigs showed substantial muscarinic hyperreactivity. From the Departments of Medicine and-Environmental Health, University of Cincinnati School of Medicine, Cincinnati; OH. - -- Lee, H. K, and Mzr.a.s, C. - American Review o; ;Resprratory Drse4se°132:1005=1009, 1985. Other support: National Ileart; Lun_g and Blood Institute. U-60,257 INHIBITS 03-INDUCED BRONCHIAL HYt ERREA_CTIVITY IN THE GUINEA FiG We studied the efle_ct-on ozone-induced airway hyperreactivity of U-60,257, a pyrroloprostacyclin shown to inhibit leukotriene C/D biosynthesis in vitro._ A group of 5 guinea pigs-was pretreated with U-60,257 (5 mg/kg IV) and studied before and 30 min after a 15 min exposure to 3-0 ppm ozone. These animals were compared to a similarly sxposed_ group that was untreated (ng10). _ Reactivity was determined by measuring specif ic airway resistance (SRaw) upon intravenous acetylcholine infusion in ozone exposure: high level exposure. -This effect may be mediated in part by leukotrienes generated upon occurred after ozone in any animal pretreated with U-60,257. - We conclude that the ozone-induced bronchial hyperreactivity in the guinea pig rapidly develops after a brief, unanesthetized, spontaneously breathing animals. We found that U-b0,257 treatment prior to ozone exposure did r,ot affect either SRaw or muscarinic reactiviFy. After exposure to 3.0 ppm, all untreated guinea pigs showed substantial muscarinic hyperreactivitye ln co_t.trast,-no significant_change in SRaw or muscarinic reactivity Murlas. C. and Lee, H.K. Prostag-tandins 30(4):563-568, 1985. Other support: National Heart, Lung and Blood Institute. From the Departments of Medicine and Environmental Health; University of Cinc-innati _ School of Medicine, Cincinnati, OH. -IND.7b`.ETHACIN INHIBITS THE AIRWAY HYPERRESPONSIVENESS BUT NOT THE NEUTROPHIL INFLUX INDUCED BY OZONE IN DOGS To determine whether oxygenation products of arachidonic acid may be involved in the airway-hyperresponsiveness induced by ozone exposure, we studied-whether 6

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ozone-induced hypanesponsiveness in dogs could be inhibited by the prostagiandin synthetase inhibitor, ihdomethacin. Airway responsiveness was -assessed with dose- response- curves of acetylcholine aerosol versus pulmonary resistance-in 2 sets of experiments: in one set,-5 dogs-were given no indonFethacin treatment and were studied both before at3d after ozone exposure (3.0 ppm, 2 h); in another set, the same dogs were studied before indomethacin treatment or ozone exposure and then during treatment (: mg/kg every 12 h for 4 days) both before and-ater ozone exposure. On each occasion, we also determined the number of neutrophils in biopsies of the airway epithelium. When the dogs were not treated with indonsethacin, _ozone caused a marked increase in responsiveness to acetylcholine and a marked increase in the number of neutrophils in the airway epithelium. When the dogs were given indomethacin, responsiveness was no different during treatment than before treatment, but more importantly, responsiveness did not increase significantly- after they_ were exposed to ozone. Interestingly, indomethacin treatment did not affect either- the baseline- number of epithelial neutrophils before ozone exposure or the increase in the number of neutrophils after exposure. Th e results suggest that oxygenation products of`arachidonic acid that are sensitive to inhibition by indomethacin play a role in ozone-induced hyperresponsiveness without affecting the influx of neutrophils. O'Byrne, P. M., Waiters, E. H., Aizawa, H., Fabbri, L. M., Holtzman. M. )., and Nadel. J. American Review of R4spiratory Disease 130:220-224, 1984. Other support: National Heart, Lung and Blood Institute, Fis=, ns Corporation and California Air Resources Board: From the Cardiovascua: Research Institute and Departments of Medicine and Physiology, University of C`~ifort;ia, San Francisco. - CONTROL OF NEUROTRANSMISSION BY PROSTAGLANDINS IN CANINE TRACHEAL.IS SMOOTH MUSCLE ° Contractile responseg-"of canine _tracheal -smooth- muscYe to electrical fieid - stimulation diminished -over a 2-h- period of incubation. However; addition of indomethacin (It?sM) for asimiiar time not only paevented-tRis inhibitiorn of" contrac?ile response, but actually increased markedly -the reiponse to electrical field -stimulation. _ suggesting that prostaglandina were responsible for the time-dependent inhibition. -- Measured prostagiandin Es- increased in the-tissue bath over 2-h in- control_ tissues. Addition of prostaglandin- £; to the -tissue produced similar inhibition - of - contractile responses to electrical field stim>>lation in a conten'aIation-deyendeni manner. In contrast, incubation alone, treatment with indomethacin or addition of prostaglandin E2 had little,_if an-y, effect on =ontractions induced by acetylcholine. We conclude tha*t the release of prostaglandins from car,i ns tracheal smooth-muscle that occurs with time has a predominantly :nhibitory effect on cholinergic neurotransmission at a preJunctional site. WaltersFE. H., O'Byrne,-P;`M., Fabbri, L. M., Graf, P. D., Holtzman, K 3. and Nadel, J. A: Journal of Applied Physiology S?(():129-134, 1984. Other support: Nat-ional_ Heart, Lung and Blood Institute, Fisons Corporation and California Air Resources Board. From the Cardiovascular Research Institute and Departments of - Medicine and Physiology, University ofCalifornia, San Francisco. 42 0 a ill ® go ~!! ~ 8 ~ Z ~ 1

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ANANTI-INFLAM1vtATORY DRUG (BaN755C)-INHIBITS AIRWAY HYPERRESPONSIVENESS INDUCED BY OZONE IN DO:rS-_ - - To follow up our previous observation that airway hyperresponsiveness induced by ozone is linked to airway inflammation, we investigated the effect of =BW75SC, an anti-inflammatory i]rug, on ozone-induced -hyperresponsiveness in dogs. Airway responsiveness was assessed with dose-response curves of acetyichot'ane aerosol versus pulmonary resistance- in two sets -of -experiments. In one set (placebo treatment), five dogs were given only saline solution treatment and_ were studied before treatment- or ozone exposure and then after treatment both before and after ozone (3.0 ppm, 2 hours); in another set (BW753C treatment), the same dogs were studied before BW755C treatment or ozone and then after-treatment (]0 mg/kg intravenously) both before and after ozone. When the dogs were not given BW755C, ozone induced a marked increase in airway responsiveness to- acetylcholine. When the dogs were given BW755C, responsiveness was no different during treatmen: -than before- treatment but, -more importantly, responsiveness did not increase significantly after ozone. It is concluded that DW755C markedly inhibits ozone-induced airway hyperrespor.siveness in dogs, probably by inhibiting the formation of oxygenation products of arachidonic acid. Fabbri. L. M., Aizawa, H., O'Byrne, P. M., Bethel, R. A., Walters, E. H., Holtzman, M. J., and Nadel, J. A. - Journal of .tllergy and Clinical lrnmurtaingy 76:1t;-2-166, 1985. Other support:- Nationar Institutes of Health, Fisons Corporation, California Air - Resources Board, and tkP Italian National Research Council. -- From_ the Cardiovascular Research Institute and Departments of Medicine and Physiology, University of California, San Francisco. PREDOMINANT GENERATION OF 15-LIPOXYGENASE Iv1E T ABOLITES OF- ARACHIIK3NIC ACID BY EPITHELIAL CELLS FROM HUMAN TRACHEA Epithelial cells of 99% purity and 9296 -yiability-weTe isolated from human tracheas obtained post mortem, and the cellular pathways for lipoxygenation of arachidonic acid were examined- in ri!rv. The lipoxygenase metabolites were identified by comparison - with synthetic- standards during reversed-phase -and -straight-phase high pressure liquid chromatography, t tY spectroscopy and -gas -chromatography/mass spectrometr y: Epithelial cells--incubated without arachidonic acid failed to generate detectable quantities of metabolites, while cells incubated with arachidonic acid at 1-50 µg/mf for -i-30 min invariably generated - predominantly 15-lipoxygenase products-, - including 15-hydroxyicosatetraenoic acid (15-HETE), four isomers of 8,15-dihydr-0xyicosatetraenoic acid- (two -8, 15 diHETES and two 8,15-leukotrienes), at least one isomer of 14,15-dihydroxyicosatetraetroic acid, and smaller amounts of 12-HETE and 8-HETE,-but little or no detectable 5-HETE or 5,i2-diHETEs: The capacity of epithelial cells from human pulmonary airway to selectively generate l5-lipoxygenase metabolites of arachidonic acid suggests a potential role for the products as mediators of airway epitheiial function. Hunter, J. A., Finkbeiner, W. E., Nadel, J. A.. Goetzl, E. J., and Holtzman, M. J. -- ~ C1~! ~ 43 Q IPA 1 U. 7^ ®

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Proceedings of the __National Academy-_oJ Sciences of the--Cfnited States of America 82:46-33-4637,-1485.-_ _- From the Cardiovascular I?esearch_ Institute; Howard Iiughas_ Medical Institute;--and Departments of Medicine,_ Pathology- and Ph;;iology_, University iversity of_ - Califor_r.ia, San Francisco. Other support: National institutes of Health and Cystic-Fibrosis Researclr Foundation. responsiveness and irn thromboxane Bs but did not alter neutrophil chemotaxis. - Thus we speculate ehat leukotriene B4 causes_ Reutrophil chemotaxis and _release of thornboxane Bs. which increases airway responsiveness. O'Byrne,-P_. M., Leikauf, G.-O., Aizawa, H.,_Bethel, R. A.,-Ueki, I. F., Holtzman, M. 7., metabolites in brortchoalveo!a;-lavage-fluide in dogs. -Inhalation of leukotriene BS aerosols had no effect on resting tota!-puimonary resistance but increased- airway_ responsiveness, _ and effect that was maximum in -3- h and- that returned_to °control levels witnin- l- wk. Three hours aftee-?eukotrieoe B*, the number of neutrophils and the concentration of thromboxane B= recovered _ in_ lavage fluid increased markedly:- Pretreatment with the thromboxane synthase inhibitor OKY-046 prevented the increases -in airway to inhaled acetylcholine aerosols and- on- the- cellular components and- cyclooxyngenase _ LEUKOTRIENE B4 INDUCES AIRWAY HYPERRESPONSIVEI`dESS IN DOGS _ Tb:e authors-studied thueffect_of leukotriene B4 aerosols oa airway responsiveness Journal of Applied Physiology- -596(G):1941-1940; 19g5. and Nadel. J. A. Other support: National Heart, Lung and Blood Institute, Fisoas Corporation, and the California Air Resources Board. From the- Cardiovasc_ular_ Research institute and- -I)epar-.ment_s of_- Medicine _ and Physiology, UniversiTy of California, San Francisco. PROSTAGLANDIN Fsa-INCREASES-RESPONS'._=iElt`ESS OF-PULMOI`1ARY - AIRWAYS IN_ DOGS. - The authors studied the -effect of prostaglandin F3a (PGF2Q) on the responsiveness of pulmonary_airways in dogs. Airway respo nsivehess was assessed-by determining the, bronchocanstrictor response to increasing = concentrations - of- acetylcholine aerosol delivered to the airways. In -each of five dogs, we determined - responsiveness during - treatment with physiologic satine,-hi3tamir.e, or PGF=Q aerosols. The doses -of -histamine - and PGF=. were determinedd by establishing- the_ largest dose of each which could be given to the-dog without carsing broachoconstriction (subthreshol; doses). We found= - that airway responsiveness-_was not significantly different-during histamine treatment _ than after saline; however, FesponsivenesE increased during treatment with PGF2Q. In addition, the hyperresponsiveness induced by PGF. Q was prevented by pretreatment with the ganglion blocking-drug_hexamethonium (5 mg/kg given intravenously). The results _ show that PGF=a specifically increases the responsiveness of puimonary-airways in doses that do not cause bronchoconstriction, and-suggest that the hyperresponsiveness involves a neural mechanism such as increased responsweness of airway sensory nerves. _ 44

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I O'Byrne, P. M., Aiza%va, H., Bethel, R.-A., Chung; K. F:; Nadel. J: A-., and Holtzman, Frnstagtandirts 28(4):537-5431984.- Gther support: IEatibnal Heart, Lung_aad B3ood Iiistitute,-the-Caufornia Air Resources Board and Fisons Corporation. - From the Cardiovascular Research Institute and Depar.ments of Medicine and _ Physio!ogy, University of California, San Francisco.-- NEi1TI~LOPHIL DEPLEiTON INHIBIiS AIR1_!%AY H?'PERRESPOIv`SRr'E=`4ESS INDUCED BY OZONE EXPOSURE The authors studied whether ozon?=induced-hyperresponsiveness could be-inhibited by neutrophil depletiob in dogs. Recpo_r.siveness was-ass2ssed with dose-response curves of acetylcholine aerosol versus pulmonary resistance; depletion was assessed by counting ®eutrophils ir,_ vesous bload and in biopsies_ of the airway epithelium. Responsiveness and neutroghil-nur,bers were determined 5 days-and I day before ozone and i k after- ozone (3.0 ppm, 2 h' in Cs-untreated dogs and in 6 _dog'; treated wwith_ hydroxy!irea (200 atg/kg-ciaity for 5 days starting 5 days before ozone =:n uiptreateri- dogs, responsiveness - _ and neutrophilnumbers-5 days and I day-beI`ore ozone did r.oa change, but re?ponsiveness= and epithelial neutrophii: increased markedly after- ozone. In treated dogs, circulating neutrophils decreased from 88.9 +_ 2:2 to 0.6 ;^v_.01 X 10' per mm3 (mean ± SEM),- and responsiveness before ozone did not -:hange -Furthe.rmore; increases in responsiveness and epithelia'_ neittropl:ils did--not occur after -ozone.- Six wk after-stopping hydrozyurea, responsiveness and epitheiial_ neutrophils increased markedly after ozone. The results- _ suggest_ that- ozone-induced hyperrespor.siv¢ness -may depend on the mobilization of neutrophils into tne airsvays. O'Byrne, P. M., Waiters, E. If., Gold, B. D., Aizawa, A:, Fabri, L. M., Alpert, S. E., Nadei. J. .+.. and I-Ioltzman,_M. d. Annerrcan Review of Resprratory Disease-Y30:214-219, 1384. Other support: National Heart, Lung and Blood Institute and-Fiscns Corporation. From the Cardiovascillar Research Institute, and Debart-tnents of- Medicine and Physiology. University of California, San Francisco. -- AIRWAY HYPERRESPONSIVENESS AND CI-iANGE.S-IN CELL COUNTS IN BRONCHOALVEOLAR LA:'AGE AS`fER -OZONE EXPOSURE IN DOGS The authors stddied _ whether - airway hyperres;_ onsi-v_eness induced by ozone exposure is associated with _ changes in the nua5bers of different types of cells in- bronchoalveolar lavage In dogs. Airway responsiveness to acetylcholine_ and the numbers of cells_ in lavagefluid -wrere determined i wk before and-then I h and I wk - after 2-h -exposures _to filtered `air and to ozone (3.0- ppm) in each of 5 dogs- Airway responsiveness and the ~umbers of cells irr Eavage fluid did not change after exposure to- -- filtered air. By contrast, airway responsiveness increased markedly I h after exposure to ozone and returned to control IeveIs I wk later: In addition, the_ numbers -of neutrophils -- and of ciliated epithetiai cells in lavage increased markedly 1 h after ozone -and returned to control levels I wk later. Our previous study showed that airway hyperresponsiveness I '.! 45 ~ i - ~ ,.~ " 0

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induced by orone is associated w,ti-, an- infiua- of netttroghits into the most- central -_ airways; the present results suggest that the hypcrrespo:uiveness is also accornpanied-by an inft-u-a of neutrophils into more distal airways -and by desquamation _ of airway_ epitheiial celL. Fabri, L. M., Nadel. J. A. et ~t. Aneerican_Rereew of Resyrratory Disea. :e 129:2g8-291, i984. Other ss;,port: National Hesrt; Lung and Blood Institute, Fisons Corporation' and _the California Air Resources Board. - From the Cartliovascutar Research ° Institute, Departments of Medicine and Physiology, University of California, Saii_=Francisco. OCCURRENCE AND DIS-i RiBUT7ON OF-Rt.GU_ LA3'ORY PPT. iDES_ IN THE _ RESPIRATOR_Y TRACT- A multimembered system_ of active° regulatory -peptides has been fsur.d--iri the respiratory tract. Srsdies ori the neuroendocrine marker;- neuron-specific enolase, indicate that there are still further types of trtucosal endocrine-cells and nerves whose products remain to be elucidi;te_d. This is not surprising in view of the rate of discovery -_ of novel regulatory peptldi- by -new~ hiochemic-al methods and recombinant DNA- ° technology. It iwetl known that the lung possesses f unctiors in addition to mere respiration and these functions-may well be regulated by the various regulatory peptides- _ present-in the respiratory tract. Many diseases of the lung are still joor?y- understood. The discovery of rep_u:atory peptides involved in revulating n;ucosecretion,-blood-ffow, and muscle tone will-unuonbtedly contiibute to-the further understanding -0f t+hese - diseases. - The basic mechanism of tumor growth and the biology of tumor cells will also be better understood after further study fl: the active regulatory -,eptides,-htany -of which play an important role as autocoids in the maintenance of tumor growth. t'ol4k. !. Af. and Bloom, S_ R. -_ Cancer Research 99:1-i5,-1985. From the Departments of Histochemistry and Medicine, Royal Posrgraduate Medical School,-Hammersmith l-Zospir_+i, London,=E_r,gland. - BRONCI-1ODILATOR-RESPONSE DURING .3CU-TE VIRAL BRONCI-IIc7LIT-I-S iN - INFANCY _ Bronchodilator responsiveness wwas assessed-- bY- measuring- specific- respiratory _ conductance before and after inhaiation of aerosoiized bronchodilator in 50 infants -whc - had acute bronchiolitis due tc- respiratorv syncy*.iai virus infect-ion. 'I'fiirtypercent of the--- infants s howed an improvement in specif ic conductance. Responders could - nz;t be differentiated from nonresponders by family -hiszories- of atopy, eosinep}hil counts or immunoglobulin levels_ in blood and nasal secretions. Eighty-th-ree percent of the families and 54% of the mothcrs_of the infants were smokers. Babies-of smoking snothers had lower specific conductances than did those-of nonsmoking mothers but -showed no differences in bronchod:ls,tor _response: - The clinicat significance of-' this bronchodilator-responsive subgroup has yet to be defined. -Soto, M. E., Sly, P., Uren, E., Taussig, L. M., and Landau, L. 46 1

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Pediatric Pulmonology i(-2;:o3-g61985. 0:5er support: -Natiena] Inscitutes of Health, =rom the -Professional -Depar tment of Thoracic Medicine, Royal ChildrPn's Hospital, - Melbourne, Austraii -UTILIZATION OF A PEROXIDASE ANTIPEROXIDASE COMPLEX IN-AN ENZYME-LINKED IMMtJNOSOR°ENT ASSAY OF ELASTIN-DERIVED PEP'1'II3ES IN HUMAN PLASMA Chronic obstr-uctave pulmonary disease (COPD), a= m3jor-cause of morbidity and death in the smoking pejpulatioz:, develops insidiously over many years, and significant impairment joi lung f unction usually occurs before the disease is diagnosed.- Because lung = elastin degradation sppears-to-be- a prerequisite for the- development of -the -disease, immunol.;gic detection of elastin-derived peptides in the blood might -be an effective approach to the early detection -and monitoring of the disease. We here report an -improv-ed enzyme-linked- immunosorbent assay for - elastin peptides using a- perozida=-e-antipefoxidase complex as the reporSer " group. = The assay is -sensitive to 2ng/ml elastin peptides. ` We show that for optimal, reproducible results the assay should be carried out at l°6c C rather than at room tempErature-a9d t:`_a*t deier:iinstions -should be made on plasma oontaining protease inhibitors rather than on -se=um._ The=-le-vels of elastin-derived pel`t.ides appeared to-reinain relatively constant when multiple samples _- were taken during a;- to 10=wk period from individual subjects. In addition, patients with tiOPD had e!dyated etastin pepi:de ievels (127±47ngjml), whereas_ normal smokers_ -N.ad values intermediate between the 2 groups= (mean peptide levels of 76±42ng/m1) _ -compared with levels -in normal nonsmokers (38±17ng/mtJ. _ A small group _ of normal - smokers ;20'18) had elevated pept':de levels similar to those it: the emphysema group and -_ may represent that groups of smokers at risk of developi ng obstructive lung disease. - = Kucich, U., fi'einbaunt, v.: et ol. - American Review oj-lZespirctory Disease 131;700-713, 1985. Other sup port National Institutes of Health. _ - From the Department_ of Medicine, Graduate Iespital; Al'aert-t=instein-Medical Center- and Center fi?r- Oral Iiealth` Research, Schqol- of Dental Medicine. University of - -_ Pennsyivania, Fhiladeiphia. -AiitINO ACID DERIi'EI)-LATENT ISOCYANATES: IRREVERSIBLE INACTIVATION OF PORCINE PANCREATIC ELASTASE AND HUMAN LEUKOCYTE-£LASTASE - -Several amino acid-derived azolides have- been-syr.thesized and investigated for their inhibitory actiy:Fy toward human leukocyte elastase and porcine-pancreatic elastase. The inhibitory activity_was _found to be dependent on the nature of the precursot-amino- acid ester. Thus, compounds derived from L-valine methyl ester 3, L-norvaline methyl ester 5. DL-norieucin- methyl ester 9, and L-methionine methyl ester 10, were found to inhibit irreversibly both enzymes. Compound-14-vva& found to be a specific and selective inhibitor of human leukocyte elastase. In contrast to these, inhibitors derived from _ glycine methyl ester 1,-D=val,ne methyl ester 4, and D siorvaline methyl ester 6 were 47 r E

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found to be -ioactive: - The results _ of the - present study show that latent isocyanateF _ -_derived frone Qppropriate amiao acids can serve as selective inhibitors uf serine-proteases- - and are of potential-pha_rmac o';ogical value. Groutas; W. C., Idein!+a;rrrt. G.. et at.- jmurna! of Medicinal Chemistry 28(2).204-209, 1985. Other support: American Chemical Society, American Lung Association and - Nationa': University of Wisconsin, Eau=Claire. Veterinary Biosciences; Univprsity of Illinois, Urbana; andthe Department of Chemistry, Hospital. Department of Me:icine,-Research Division, Philadelphia; the-Departmer.t-of- -_ From the Department of Cheznzstry, Wichita State Upiversity;-Wichit?, KS; the Graduate Heart, Lung and Blrsod-Institute: CGLLAGENASE IN THE LOWER RESPIRATORY TRACT OF PATIENTS WITH ADULT RESPIRATORY DIS=TRESS SY1vDROME- - 17 and Type HI collagenase =was-detectable in 1II_o,r _13 patients with ArDS. The 10 control subjects had no detectable- ?'ype- I or III _sollage?ase activity. Total and di[ferential white ~ell counts we ~-ar,a.iyzed in the_ lavage fluid. Although _ the total counts did not differ befween patients with A:2D3=aed control subjects, the percentage - of 'neutrophils=was increased=-niore than 25-fold and the percentage of tr:a5ruphages was _ reduced almost 10-fold inthe -ARDS patients.- Serial-collageru`e aEtivity-was-followed in i ARDS survivor. In-this patient Type III collagenase_ac_tivity peaked before the Type I collagenase activity or serine pretease activity reached their maximums. Both the last two enzyms-activities paralleled *.he-tvtae recoverable cells in the BAL. and-against Type--I-iI collagen (13_ patients). Svrine_DroteasL activity was also measured against Type III oollagen (i3°patients).__Type I collager.ase activity was detectable in 12 of __ Collagenase activity ih the bronchoaiveolar lavage _(EAL) . of- patients with adult - respiratory distress syndrome- (ARDS) was-mea,cured against Type-I_-collagen (1'-patients) Christner, P., !1'eihbavm, i.;; el -a! -- wmerican. Review of r2es¢iruwry Disease-13f:F90-o46, 1985: Medicine, Graduate Hcspital, Philadelphia. Histology, University of Pe nnsylvania; and the Division of Research, Department of Center, New York; Temple University Health Science Center; the i:epartme*;t- of Dental From -the Division of I'ulnionary and Critical Care Medicine, Albert Einstein Medical Other sunport: - U. S: Public Health Service. SECRETION OF ELASTIN IN_THir EMBRYONIC CHICK AORTA - AS VISUALIZED BY IMM`?NuELECT-r2ON MICROSCOPY Recently, significant advances have been made in- characterizing _the pathway of elastin biosynthesis from the biochemical point of view and- a 70,000 dalton protein, designated tropoelastin, appears to be-the primary translation product and soluble intermediate of the insoluble elastin. However, relatively little is known concerning the intracellular-secretory pathway of tropoelastir.. We-previously-developed- an electron microscopic technique usin-g-elastin-specific ar4tibody-and ferritin-conjugated secondary I

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antibody- to identify, -intracellu!ar elastin- acd- to- identify, provisionally, -intrace!lular +esic!es containinQ, ei_astin. However,- the method did not permit loca}ization of elastin in other intracellular organeiles. We-now- describe an improved post-e: jbedding-lechnique using the pero>;idase-antiDeroxida<Fe method 3o detect the p:imary elascin anti"oody; we have iocalized- elastin in_ both the endotheeial and medial cells of the embryonic- chick aorta. Specific staining was visualized 4-the cisternae o: the endop!astric reticul_um.- in -the Golgi apparatus,- and in vesicles forming on the trans sidf o the Go!gi. Some of these smaller vesicles appeared to fuse, forming larger vesicles which may have a storage function: Both types of vesicles were see : fusing _with the ce!I plasma- membrane, suggesting that elastin is secreted by an exocytotic process. These results-suggest that tropoelastin follows the c'_assical-pathway for -protein secretion. Damiano, V., Tsang, A. L., l5'edr6oum.=G., Chrstfer, F.; and fZosenbloorts, 7.= Coitagen Related Researck 4:153-164, 1984. Other support: National Institutes of Health. From the Center for=_=Ora1-Health ltesearcir,School of Dental kfedicir+.e; of Medicine, Graduate Hospital; and-'_'niversity-of Pennsylvania, Philadelphia. PROTEIi`ASE IN)=IIlsITOR`¢ FUATC T1ON IN INF'LAMMA 3OR1 LUNG DISEASE _ 1. ACUTE SAC'tt!'cIAL PNEUt~fONIA patients with $cutg bacterial pneumonia (n=13) and makes a comparison with a controi group (?1-5). _.-Thz proteinase inhibitory capacity was examined and f aund - to be composed pritrar ily. of aipl-ia,-proteinase inhibitor (Pl,_ a!pha,-antitrypsin) -a.^.d, to a lesser extent, bronchial mu.cosal inhibitor_ - Although- the average-PI concentration svas- elevated- approximately 3-fole in the-pneumor.ia group,_its inhibitory functioA-agairst e!astase- was decreased 15-fold when compared with that in the control group. T ke pneumonia group - showed an increased concentration of immunologically identified elastir.-derived peptides. - Some of-=the BAL fluid from patients with pneumonia showed elastolytic activity against amorphous insoluble lung elastin. The majority of the e-!astase appears to be of neutrophil origin. Bronchial mucosal inhibitor is shown to be a component-of both normal-and pneunro:,ia -iyAL fluids by both immunologic quantitation and by its resistance to pe_rch3atic acid inactivation. Compared with those from control subjects, BAL samples from patients with acute bacterial pneumonia 3howed a deE::rsased proteinase inhibitor _unctiYn and both increased elastolyr+c activity-and e!astirt-derived peptide concentration, This study examincs= bronc5ia! ,lveolar lavage (BAL)-saanples- f rnm a group of Abrams,-lY. R., Weinheum, G: et-al American Review-oJ Respiratory Disease 129:735-%41, 1984. - Other support: National Heart, Lung and Bloolnstitlte. From the Graduate Hospital, Department of Medicine, Research Division; Albert Einstein Medical Center, Department of Medicine, Pulmonary and Critical Care Divisions; Temple University Health- Science Center, Pulmonary Disease Section, Philadelphia, and-the Pulmonary Disease Department, Saga Medical School, Japan. -49 - the Department

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NEVTRL)PHIL ELASTASE-RELEASING FACTORS IN BR()NCHvALVEQLA_R _ I AVAGE FROM PATtENTS1J-T.TH-ADC?LT RFSPIRAMORi DisTRESS-S-`_'t:DW3IvIE Bronchoalveolar lavagA fi.tid (BA L) :1as _ obtained fratF patients with adutt - _ respiratory distress sy-ndronE tARDS;. Con.ro+s included BAL troaa no-mai subjects and ~ f rom patients with sarcoidcsisso.- ptlmora, y fibro3es. 2r`eutropt+il elastase n:easured -all BAL samples, but it was strikingly greater in _ EAL immunologically -was foi;n~ I from rom patients- with ARDS = than in - the BAL fiom normal sui ~eru or pa*.ients with ~ sarcoidosis. There v~~ Te significant difference in the neutrophil elastase nnngn concentrations BAL samples from pat ents with ARDS and those wi h_ _p ulmt,nary ~ fibrosis. No el :n astolyuc_ activity was found in either group. -The alphs-l ' a titrypsin and -_ ~- _ Ltie bronchiaFmucus inhibitor were greater in f3AL from patients with ARDS, There w;s a highly significant correlation between the alveolar-arterial oxygen tension -differencF ~ _ - paE;eIIS -with ARDe. ~ and the neutrophil etastase=concentratidn in -BAL from the f_ , I_ Kallikre_in, prerekallikrein, factor XIa-like activity, and high molecular weight kininogen ~ antigen were found in BAL =;f-patierts with-ARI-'.-S, suggesting-that the kaltikrein-kinin cascade may be activated in the lungs_of patients with r.It.DS. ;allikr_in-like- activity in ~ the BAL=from the patients with ARDS was significantly correlated with the number of utrs last e co. -ntration hil the BAL h -n hii c and the bi!it th RAL t i f * -p _ e e _ , -a y o ~ neu rop s n _ e - _ e , to release elastase ir0m cytoct[alasin_-if-treated neutropntis-. tlrere was no correlation _ between - these variables and C5a concentration. -- These 3tudiea demonstrated an associat;on-between BAL neutrophil elastase and-the clinical state of patients with ARDS. Idell, S.; Weintau,r.. O. et al. American Review of Respir.tory Disease 132-:1098-11C5, 1985. Other support: National Heart, Lung and Bit ic±d Institute, Natio ,31 Institutes of }:ealth- and American Heart Association, Pennsylvania Affiliate. F~rom- the - Pulmonary Disease aca` Hematology-Oncoiogy Sections, Department - of Ivledicine,- and Thrombosis Research Centar, Temple University Medical- School; Pulmonary- Disease Section; Albert Eirstein Medical -Center, :Vorthern- Division, Philadelphia. JXI~3ANT$ INCREASE FAI~'ACyLLITLAR PERMEAItIi.-ITY IN A= CULTURED EPITHEI.IAL` 'c.LC -i.INE. Inflammation of epithelia is an important -stEp in the pathophysiology of a wide variety of diseases. Because-r?active oxygen-metabolites are important effector molecules of acute inflammation, we examined the effect of oxidants on the barrier function of a cultured epithelium. Madin Darby Canine Kidney cells, by measuring the transepithelial electrical conductance, G, qf monolayers grown on permeable supports. We found that HsDs, added directly ot-gYneraied with glucose okidase, increased r~. Similar effects ~.ere observed with addition of xanthine -and xanthine oxidase, a system which enzymatically generates auperoxide _radical Oa-: The oxidant-induced increase in conductance was- reversible if-the exposure to oxidants was not proionged t s 20 min), $nd if the concentration of H=Or was < 5:. lo -s M. - The etecrease_ in Cz --uggested that oxidants increase the permeability of the paracellular pathway, aiuggestion supported by an oxidant-induced increase-ib the permeability to 14C-mannitol; whic:i primarily crosses epithelia via the extracellular route. 5C--

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In addition to- : ur,ctioteai changes io=the cpitheli3l_ monolayer, oxidants changed the cell morphology; after Ha(%a es'._bos>;re, the cells tended to-pull apart, most prontinently at =--_ _` their basolateral surfaces. _ These changes were heterogeneous, with most areas showing no changes.- Some of the morphoiogic c_hangcs could be reversed if_the exposure to Ii30, was limited. - We a:so, observed _a disruption - of the - r~oraral pattern of- -the actia-cyto<keletonz yarticularly in the area of cell to- ce9i junctioos, as-demonstrated by ---_- -fluo_resceht sta_ining of f-actin with raodamine phai:icidia. These functional and 3tP3cttlra_l- findings indicate fhat -ozidants increase the permeability iif thg_ parac-eiliflar pathway in-a cultured epitheliu?i.- The- char.ges can be reversible and_ are _accdmpanied by alterations in organization of the cell- Cytoskeleton._ These-studies demer:=trate the dynamic nature of the interaction betwee n epithelial cells - and oxygen mQtabo7ites. -.Iourr:al of Cdst: ;:. : rtres,igateor '13:113144b$,-19g5. [9ther support: Natipral Institutes of Health, American Heart Association and Veterans Administration: l:rpm the Laboratory oi_ Epitheiial Transport aod-Pul:nonary-Bivision, Department of Internal Medicine, University of Iotia College of ?1Cedicine, and Veterans rkdrsinistration - Hospital, Iowa City. -

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CHARACTERIZATION C-F LIPOPROTEIN PARTICLES ISOLATED ATED BY IMMUNOAFFINITY CHROMATOGRAPHY Two populations -of A-I-containir.g - lipoprotein particles: A-I-containing lipoprotein with A-II (Lp (A-I with A-Ii)), ano A-I containing -lipoprotein without A-II _ (Lp (A-I w.ithout A-II))_ have been isolated from plasma of 10 r.ormolipidemic subjects byF immunoaffinity chromatography and -- characterized. Both types of particles possess- a-electrophoretic mobility and - hydrated -density in -the range of plastna high-density-lipoproteins (HDL). Lp (A-I without A-II) and Lp (A-I with A-H) are heterogeneous in size. Lp (A-I without A-II) c9mprised two distinct -particle sizes with mean apparent molecular weight and Stokes diameter of 3.01 x 106, and 10.8 nm for Lp (A-I without-A-II)l, and-f.64_x 105, and 8.5 nm for Lp (A-I without A-II);. Lp_ (A-I with A-Ii) usually contained particles at least three distinct- molecular sizes with mean apparent molecular weight and Stokes diameter of 2.28 x 105 and 9.6 nm for Lp (A-I with A-II)1, 1.80 x 10s and-8.9 nrtt for Lp (A-I with A-iI)2, and 1.25 x- 105 and 8.0 nm for Lp (A-i with II)3. Apoproteins C, D and E, and lecithin_-cholesterol acyltransferase (LCAT) were detected in both -Lp (A-I without A-II) and Lp (A-I with A-II) with most of the apoprotein D, and E, and LCAT (EC 2.3.1.43) in Lp (A-I with A-ir) particles. Lp (A-i without A-II) had slightiy higher lipid/protein ratio than Lp (A-I with A-II). L p (A-i with A-Ii) had an A-I/A-II molar ratio of approximately 2:1. The percentage of- plasma A-1 associated with Lp (A-I ~.vi3hout- A-L) was highly correlated with the A-I/A-II ratio of-plasma (r = 0.96, n- 10). The variation in A-I/ A-II ratio of HDL density subfractions therefore reflects different proportions of two discrete types of particles: particlei containing A-I and A-Il in- a nearly constant ratio and particles containing A-I but no A-IL Each type of particle is heterogeneous in size and in apoprotein composition. Cheung, M. C_ and Albers. J. J. The Journal of Biological Chemistry 259(19):12201-1220a, 1984. Other support: National Inst :utes of Health and the American Cancer Society. From the Department of Medicine, University of Washington, Seattle. - PLATELET-DERIVED GROWTH FACTOR ANITMAL7t'iNANT TRANSFORMATION Studies on human platelet-derived growth factor (P-D-GF) described in this--paper represent the collective effort of several teams of investigators over the past-10 years. These studies produced an abundance of information concerning the nature and structure- of PDGF, its role in cell growth _and its diverse functions affecting cell- migration, metabolic processes and receptor modulation. This work also led to an important portan*. discovery linking this potent mitogen to the transforming protein of the simian sarcoma virus, providing a basis for the understanding of the processes involved in transformation induced by the SSV onc gene. Specificially, the-findings described here provided the missing link for the understanding of the=mechanism of cell transformation -induced-by the-onc gene of the simian sarcoma virus. This onc gene,-v-sis, was shown to encode a PDGF-like polypeptide which is a potent mitogen for fibroblasts, arterial-smooth muscle 52

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® i 1 cells and glial cells. Activation of- sis transcription may cause the sustained abnormal _ proliferation of cells responsive to the -mitogenib effects of the PDGG like_ molecule. Overall, -these findings are consistent- with the suggestion that sis activation rnigkrt -be involved-in the-process leading normal cells of certain types towards malignancy. Antoniades. H_N: _ Biochemica! Pharmacoiogy 3(18):2823-2828, 1984. Other support: U.S. Public Health Service and the Auierican- Cancer Society. -From the Center for-Blo_od-Researcn and the Department of Nutrition, Harvard School of Public Health, Boston. _ SYNTHESIS AND SECRETION OF PROTEINS RESEMBLING -_ STO:".3A_ PLATELET-DERIVED GROWTH-FAC`fOR BY HLT-NiAN GLIOBLASTOMA AND FIBROSARCOMA CELLS IN CULTURE Immunoprecipitation of proteins extracted frdm_ metabolically :abeled- human gliobl_astosna and fibrosarcoma cells-.v-ith -antiserum to platelet-derived growtit_ factor (PDGF) showed that these -cells express and secreze_ proteins that- are recognized specifically by-the antiserum. The r;;olecular-n:asses of immur9precipitated-proteins in the lysates-of the malignant cells ranged from 16 k?>a_to 140 kDa. Both-cell lines secreted -a 3l-kDa_polypeptide=with-struciural, immunological and biological properties simiiar to those of human-PDGF-. These cell lines were shown to synthesize a 4.4-kb mRNA that contained sequences from all the six currently identified exons of the-human c-sis gene. These _ data suggest -that the PDGF-like - proteins in the -two mesench-yTie-derived transformed cells are encoded at least in part by the c-sis locus. In this report we describe the intraceHular _synthesis -of PDGF-like proteins in cultures of human glioblastoma and fibrosarcoma cells and correlate the presence of these proteins with a c-sis mRNA species transcribed by the human c-sis- locus. The intracellular PD ;F-like proteins appear to be precursor and processed molecules of a major secreted protein of 31 kDa that i3 structurally, immunologically _and functionally similar ;o human-PDGF. Pantazis, P., Pelicci P.G., Dalla-Favera, R;, and Antoniades, H.N. Proceedings of the National Academy of Sciences of t?:e _ United States of America g2:2404-2408, 1985. - Other support: National Institutes of=Health and the American Cancer Society. From the Center for Blood Research_ and Department of Nutrition; Harvard School of Public He-alth, Boston, and- the Department of Pathology, Kaplan Cancer Center, r,- New York University-School of Medicine, New York. PLATELET-DER1_VED GROWTH FACTOR POLYPEPTIDES IN HUMAN MEGAKARYOBLASTIC-LIKE CELL LINES Novel cell lir.es-derived from human peripfieral blood were shown to synthesize polypeptides recognized by specific antisera to human plateiet-derived g_rowth factor (PDGF). Metabolically labeled intracellular polypeptides were immunoprecipitated by ® PDGF antiserum and were analyzed by SDS-polyacrylamide gel electrophoresis. The ® 53 Q ,- ~ ~ ® ~ ,

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molecular masses of the_ intraceilular-forms nf PrGF-:ike polypeptides in these cell ]iaes ranged from- 12,000 to 48,000 daltons (12-48 =kD), suggesting the presence of -PDGF - precursor and processed molecules. - i.rietabolically=-labeled polypeptidPs _ immuno- precipitated by PDGF -antise_a- were also recognized in the_-conditioned media of one of the celi lines obtained from-a leukecic patient. The molecular masses of the secreted PDGF-like polypeptides were 12 kD, 3.1 kD, and A6 kD. Upon reduction, the 31-kD polypeptide was reduced to 16 kD, suggesting t hat it represents a disulfide-linked dimer similar to that described for FDGF.- Pantazis, P., Morgan, D.A., -Brodsky,_I., and .intor:iodes, H.N. In: CANCER CELLS 3/Crowth _ Factors and T'ramsJormatron: Cold gpring- Harbor Laboratory, 1985, pp. I53-157. Other support: National Institutes of Health and, American Cancer Society. From the Center for Blood- Research and Department of Nutrition, Harvard School of Public Health, Boston, and the Department of HematologyJc:ncology,- Hahnemann University, Philadelphia_ _-=_ EFFECT OF CALCICIM OK : fiE sl'ABILITY-OF THE PLATELET IvIEMBi;ANE- GLYCOPIIOTEII!I IIb-IIIa Lc7MPL J.` Platelet membrane giyr.oproteirs Iib and_ IIIa form- a Ca2'-dependent heterodLmer complex that contains binding sites for fibrinoge-n, von_ Willehrand- factor, and fibronectin following platelet stimulation. The authors have studied the effect_of Ca2+ on the stability of the IIb-Ilta. complex using a-IIb-Illa cotriplex _ specific monoclonal antibody ArAa to detect the-presence of the complexes. Soluble IIb and [Ila_inieracted with A=A,-Sepharose only,in-the presence of Ca2+ _with 5096 Ilb-IIla--binding requiring 0.4 µM C-ai'. In contrast,-at.25'C the binding of radioiodine labelled A:A9 -_ to - intact unstimulated _platelets- suspe_r.ded- in - buffers containing -EDTA or---ethyle nr glycol bisCB-aminoethyl ether)_- N NXX,-tetracetic acid was independent , of the presence of-- Ca='. I-Iowever, the effect of Ca=4 chelators on 1351-AEAo binding varied with temperature. At 3TC,-'=a1-A:P.9_ binding to intact _platelet_ became Ca2'-dependent with 50% binding requiring -4.4 pM -Ca`''_. This effect of temperature was not due to a change in platelet membrane fluidity because enrichment or depletion of _fllatelet membrane cholesterol did not influence antibody binding. Also, 1l6I-A:Ao binding to- intact platelets at 25' C beca me Ga3`-dependent when the pH was :ncreased above_ 7_4. At I nM Ca2+ and 25'C, 50% antibody binding occurred at pH 9.0, These studies demonstrate that Caz'- dependent l;b-I',Ia -complezes--are° present on -unsti.~iulated platelets and that the Ca2+ binding sites- responsible for the stability of these complexes are located onthe external platelet surface.- These e-zperimEnts also suggest that chynges in platelet cytosolic Ca2+ do not regulate the formation of IIb-llia-comp?e--es. Brass, L. F., Sh attil, S. J., Kunick, T. J., and Bennefl. A.-S.- .Tke Journal of Biological Chemistry 26t3(13):787S=7g81,-19R5. Other support: National Institutes of Health and the American fieartAssociation. 54 4

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From--the Hematolor?y-Oncology Section and the - Department-.of Pathology and _ Laboratory Medicine-;-- University--of Pennsylvar.ia-School of ;`",edicine. Philadelphia; and the Blood Center of Southeastern Wisconsin. Milwaukee. ° THE TETRAPEPTIuE. ANALOGUE OF T H£ CELL ATTAC:NMENT SITE OF FIBRONECTIN INHi3ITS_PLATELET AGGREGATION AND FIBRINOGEN BINDING TO ACTIVATED PLATELETS- Fibrinogen binding to receptors oa activated platelets is a prerequisite for platelet- aggregation. Howeve.r-, the -regions of fibrinogen interacting with these receptors-have not been comp!etely- characterized. Fibronectin also binds to platelet fibrinogen receptors. Moreover,-*.he_amino acid sequence Arg-Gly-Asp-Ser, corresponding to the cell- attachment site _-of fibronectin, is- located near=ihe carboxyl-terminal region of the a-chain of fibrinogen. The researchers have-examined the ability of-this tetrapeptide to inhibit platelet aggregation and fibrinoge n- binding to- activated platelets. Arg-Gly-Asp-Ser, but not the peptide Aig-Gly-Tyr-Ser=Leu-Gly, - inhibited - platelet aggregation stimulated by ADP, collagen, and 7-thrombi-is without inhibiting platelet shape change or secretior,.- At a concentration of 60-80 µM, Arg-Gly-Asp=Ser inhibited- the aggregation of ADP-stimulated gel-filtered platelets ft 50%. Arg- Gly-Asp-Ser, but not Arg-Gly-Tyr-Ser-Leu-Gly, also inhibited fibrinogen binding- to -ADP-stimul_ated platelets. This inhibition was competitive with a Ki of about 25 pM but was incomplete even at higher tetrapeptide concentrations, indicating that Arg-Gty-Asp=Ser is a partiai competitive inhibitor of fibrinogen binding. These data suggest that a region near the _ carboxyl-terminus of the a-chain of fibrinogen interacts with the fibrinogen receptor on- activated - platelets. -_ The data also support - the concept that- the sequence Arg-Gly-Asp-Ser has-been conserved for-use in a variety of cellula: adhesive processes. Gartt.er, T. K. and Bennett, A. S. The journa: oJ=Biologieal Ckemistry 2(0(22):I1g91-I1g44, 1985. Other support: National Institutes of Health. - From the De-partment- of- Biology, Memphis State- University, Memphis, and the Hematology-Oncology Section and Cancer Center, University of Pennsylvania School of Medicine, Philadelphia._ CARDIAC3NOTROPIC RESPONSE OF A NEW #-I-AGONIST (TA-064) WITH LOW SARC'OLEMMAI -ADENYLAT£ CYCLASE ACTIVATION These experiments were concerned with an investigatior of the effect of a- new selective P- I -adrenergic compound, TA-064, on the function of- purified sarcolem7nal- vesicles prepared from perfused canine heart preparations. Special emphasis was placed on the- effect of TA-064 on adenylate cy clase activity, on ATP-dependent Ca_'° uptake and on phosphorylation of purified sarcolemma (SL). Per fusion was carried out in a supported canine heart- preparation with a-perfluoiochemical (FC-43) as per-Cusate. TA-064 (317 µg) was injected into the left atrium. As previously reported, this res-ulted in - a marked positive inotropic and chronotropic effect which- was abolished by propranolol. Purity of the SL -preparation was confirmed with marker er.zymes-for mitochondria, sarcopfasmic- reticulum and -SL. In addition-, Na`Ca"-exchange characteristic for SL was determined. Sidedness of the vesicles was ascertained by means 55 -11 l

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of adenylate cyclase act°vity using SL _preparations with _ and svithour ala:nethicin. TA-064s as compared -io 1=isoprctereno_l,- ezerted little effect on adenylate cyclase activit~: -The addition of-TA-064 either to the perfused whole heart preparation or to prepared SIL vesicles resulted in small eievations-of adenyiate cyclase activity -relative -to 1-isoproterenol. TA-064 increased ATP-dependent Ca' uptake and phosphor_ylation. - The relative minor response of adenylate cyclase to TA-064 was apparently sufficient to elevate both_ myocardial i~cntractility and phosphorylation and Ca'* dependent ATPase activity. Therefore, TA-f3d4 belongs together with-prenaltero'-in a group of ,6-1=agonists with full inotropic activity but relatively diminished capacity to activate adenyfate cyclase. The pharmacologic and clinical implications of these findings were discussed. Bing. R: J. et al. CLrrent Therapeutic Researck 36(6)'1127-1144, 19a4. Other suppprt: Hoover Foundation and the Charles A. Lindbergh Fund: From the Huntington_ Medical Research Institutes, Huntington Meznorial H-o-spital. metabolism has been influenceir by biochemistry and biophysics by separation of subcellular particles-and oijaneltes: -Renewed stimulation for the study of the isolated mammalian heart has come from the laboratory of Neely ot a!, whose perfusion technique has been particularly useful in the undertanding yf-the-reguiatiorn of glycolysis in the ischemic and anoxic myoc=rdium. Neely's perfusion studies were primarily carried out on the rat heart. This has limited the option s for the study of glycolytic interanediates and glycolytic flur, excluding - determinations `of- high energy -4hosphates, which necessitatespeciniens of greater weight. The use of perfluorocheinicais has now made it possible to perfuse largFr mammalian hearts using Neely's working heart preparation. The remainder of t his discussion is concerned with hemodynamic and metabolic data on -t.he different types of the_ fai;ing rabbit heart perfused in vitro with Fluosol-43. The_ effect of TA-054, a new;f&si;ive inotropic agent, i3_ briefly_ mentioned. In addition, this paper deals with a new s3proach-to the problem of coronary spasm, using the isolated heart t'erfused with Fluoso?-43 oxygenated with a bubble oxygenator. Metabolism. Alew Vork: F;enum Publa`shingCo:poration, 1984, pp. 23.'-2g1:- Bing. R. J. In- Ferrari, R., Kata, A., Stiug, P..,-ar,d Visioli, 0. (eds.): Ffyocaraial fschernia and Lipid Other support: Hoover-Foun datior-i. From the Huntington Medical Research Institutes-- and Huntington Memorial Hospital, Pasadena. CA. 56 t Pasadena. CA. - CARDIAC PERFUSION, PAST AND PRESENT course of cardiac metaboliz.-n. Determination of the _nutrition of -the human heart ig si results were dwelt uport; according to the author, because they determined the -futvre lipids :n relation to cardiac metabolism and function. The work of pioneers ia this area, including Tigerstedt; Ludwig, .amgt-^dorf, Wild, and Ringer, is noted: These early This paper was presented at a sympos:ur which .*+as-devoted in part to the role of- - r was a direct and logical e~;ension of their work. More recently the course of cardiac

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EFFECT OF INTACT ENi^ATHELIUM AG%AiNST PLATELET-INDU_CEi) CORONARY ARTERY SPASM IN ISGi,isTED 1°iABBIT-i?;I;ARTS m: with about 500,000 plateles/pI) nottrested with collagen failed to_constrict coronary-- arteries either with intact or deriuded endothelium. In contrast, injection of platelet =-- suspension immediately after activation with collagen caused vasocoustriction -of'-- -- denuded obtuse marginal coronar-y arteries in 10 of ; cases. In 6 preparations, occlusion - was complete, lasting up to 16 minutes.- In arteries with intact endcthelium, no coronary - - - - vasoeonstrictio3 ccyurred. In hearts with coronary artery spasm, total coronary vascular - - resistance increased sig nificantiy. _ This study f urrishes additional- -evidence_ that=_ ___ andothelial iesiot?-i-are a contributory factor for large coronary artery-spasm and that endothelial cells possess a protective function against vasoconstrietor- substances released from aggrega°.ing j)Fatelets. patent blue a]ve). E pdotbelial-denudatior of the obtuse r:arginal artery was accomplished - by scraping the lumen -with a roughened plastic_tubing. Injection of washed platelets (10, - coronary arteries was determined by means of gated=cofor-arteriography (injection-of -- Effecs of Eollager-ac~ivated_waslsed rab3it p[_aceiets on coron-ary arteries with abd_=- withoeit intact enalothelium -Were- stud;ed it- a supported rabbs*_ heart _prepar ation _ using -a perfluorocarbon (FC=43) as- perfusate. The vascular- diameter of obtuse marginal _ American Journal of Cardiolo,gy-55.1596-1600, 1985 From the- ilsntington Medical Research Irstitptgs, _ Huntington Vlemorial Hospital, - Other slpport. Hoover i="oundation and the Charles A. Lindbergh Fu:id. -Pasadena; CA. - THE EFFECT OF NIMODIPINE,_A CALCIUM ANTAi,ONI__ST, ON INTI;t:iSCClRTICAL AItTERIOLES IN THE CAT BRAIN The purpose of this study was to test fne -effect of the calcium antagonist aimodipinine_ oz the ;Atracartical arteriYles =and- on _ cerebral biood= floev - in_ the anesthetized cat. A.teriolar, diameter, red cell velocity an-d_regionai cerebral blood flows were determined- by a technique employing transilfirm ination and high speed cinematography. °6lobal-cerebtai--blood flow was n:easured using-tlte_ 5ticrosphere- tecKnique.-- After a five-minute infusioa= of nimodipine (0~25 µg/kg), there was a significant increase in cortical arteriolar diameter (p < 0.001) and in regional blood flow (p < 0.01). Red ce[F velocity increased- (p-< 0.01). GCobal cerebral blood- flow increased (p < 0.02) while mea. arterial blood pressurz declined (p < 0.05).- Therefore_, direc*t observations confirm=that aimodipine dilates intracortical a_rierioles and increases red cell -velocity in these ves_-els: Schmidii, !., Santillap, G. G=, Saeed, M., Palmieri, D., and Bing, R. !. (;rvrem Tiserape,ctic kesearch 38(1)-94 .03. 1985. Other sscpYOrr: hioover Foundation and-the Charles A. Lindbergh Fund: From the Huntington _Medical _Research institutes,_ -Hu ntington_ Memorial Hospital, - - - _ Pasadena, CA. 57 ~

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BETA-I-ADREhIOCEP`fOR---AOO?dIS`t'S WITH LOW AJ£NYL.4TE_CYCLASE= - ACTIVATIC~?~ -- ~°HEOR£T!CAL=AAid? CLINICAL-It-__-PLiCAT1La ~ -_ Several partial beta=agonists such as TA-064, - prenalterol, IC?89,9G3, and ICII ;9,033 possess prenoe:n; d positive inotropic effects; but induce - only - limited - activation of myocardial adeaylate _cyclase; this resuiis in low cyclic AMP IFvels. -Partial agonist+, as compared to isaproterer:ol, need }iigh receptor occupancy for 3riaxitnal- physiologic2l response _ ano demonstrate 'snadeqLate coupling _ betweere recptor and adenylate cyc!ase: _ Ho.vsrer,_ the biochemical mechanisms which generate the -final biological effects originatirrg from cyclic AMP are tissue-specific, and for-that_=-reason; can maintain a pronounc ec4 positive inotropic -action. ' Clinical data with _ TA-064 -=at_d- j prenalterdl have-estab4shed pve inytropic effects without signeficant ch3rge3 in heart rate. Partial beta-agonists w h diminished cyclic AMP may be less arrhythmogepic than - full beta-l-agonists. 'I'he_ clinical importance of partial beta=l=agonist3 remains to be established, partic~larly with relev3nce to long-term exposure and inherent beta blockade. Chemnitius, i.Ir:. and Bing, P. j_ Ce.nsdiast Journal of Cardiology ](3):180-190, 1985. Otner supflort:= Hoover- Foundation and the Charles A. I,indbergh_ Fund. From - the Huntington Medical Research Institutes, Huntington Memorial Hospital, -Pasadena, CA.. CI?YS"I'AL-LOID ANB PERF:,JORflCI-IE1vIiCAL PERFtJSATES IN ," M ISOLATEi; WORKI?dC'i RABBIT HEART PREPAR.A i ION Krebs-Henseleit buffer_ (KH)-and -a perfluorochemical (FC-43) were _ compared as perfusates ir!- an - isolated= *orking rabbit heart -preparation _= Both -pergusates were oxygenated in-an identical manner-u-sing an infant bubble oxygenator. After 60 min -of -perfusion, no difference could _be-detecaed-in the ratio o:` veet to-dry- heart :veighs between KH- and_FC=-43-perfused hear*v_(I~H, o?Sa_ U.`s; F'C-43, 5.99 +_0.20).- Left ventricular-systolic pressure,_maximaI rate of left-ventriculyr pressure rise, mean aorti_c systolic pressure, cardiac output; aortic floiv, left ventricular power-=and myocardial Oi consumption (MVos) were sig nificantly higher in FC-43-perfused hearts -throughout the time of perfusio_n. [-lr,wever, there were no differences in resistance to cardiac output and heart_ rate. In KH and FC=4's=periPused heaits,-YsSVo_ i-and left °ventricular potver were closely correlated (KI-I,_ 0.'.93; FC-43, r -0.831). Significantly higher coronary - flow of KH-perf used .hearts could be attributed to the lower viscosity of K_ H-(i.05 Pa-s) compared with FC-43 (1?>til_ Pz-s) compare3 with FC-43 (1.91 Pa-s). Increased O3 extraction during KH perfusion could not compensate for low O:-carryiog -capacity - of KH buffer (345 compared lvith-705 nmol 0= mj-1 in FC-43_ em~!_sir.n). --A postis_~iemic increase of corcnary f7ovv,was_observed only in FC-43-perfused heart-s (28%). These results demonstrate a different 3esponse of perfused-heart preparations to FC=43 and KH buffer. - L'hemnitius,_d. M., Burger, W., ar.d-Ri-ng, R. .'. American Iournal o,t-Fhysiojody 249(Hrart Circ. Physio!.):H285-H292, 1985. Other tuyport: Hoover Foundation and the Charles A. Lindbergh Fund, 58 - I

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From the Huntington X;edicai Research tnstitutes,_ Huntington Memorial Hospital, Pasadena, CA. , _ A NEW METHOD F.^_---R `1`Hr''. VISUALIZATION OF Si1BEF;CAFE.DIAE, CORONARY - AR'i:I't1ES IN SMA-L L iSOLAT-ED-IviAMIv1ALIARI HEARTS - A-model is desc.-ibed which permits direct-visualiration df-Iarge_coronary arteries in a supported ra!odifirj perf used heart preparation, -using a-,perfluoroehFmical-- (FC-,3) as_ perfusate. Fillir.g-of -a large coronary artery with Patent Blue Dye is recorded by gated photography (color azteriography). The technique is applicable to -ahe study of reactivity (spasma of-cdronazy -arter_ies in _hearts of_ small-and large animals (rals, rabbits, dogs). The techniquR has the following advantages: preservation of vascular endothelium, adequate ozygenation, avoidsnce of major surgical intervention to implant sensors ior the detyctiorr of changes in coronary diameter, eter, quantitative evalyation- -of time-dependent - coronary vessel and tota;-coronary vascular resistance. _ changes in geometry of large coronary arteries, and simultaneous :neasursments of -large Pasadena, CA. Frem- the Huntington Medicai- Research Institutes, Il'untington Memorial Hospital, Burger, W., Chgmnitius, J. M., and-Bing,_ It. I. - Proceedings of tiu Society for Experimental Biology ardMedir-ine 1'8:309-312, 1985 Other support: Hoover Fo-undaiion and-the Charles A. f,indbergh Fund._ --'I'HE EFFEC'I' OF CARDIOPULMONARY B`tPAsS ON CEREBRAL BLOOD FLOW _- - diameter showed aa tendency of ar-terioiar flow to increase. 7t,g condition is-remini3cent cortical arterioles increased. Volume_ flow ~caleulated from red cell velocity and arteriolar sortical- arterioles. -Ev-en- though blood pressure remained unchanged,- the diameter of microvasculature by means of high-spe-ed-cinematpg,raphy-revealed dilatation of cerebr-al -- interdependent and showed direct proportionalitv. Direct observations of the= cortical cerebral blood Ilow and blood pressure -in--animas treated -as_ a- group became resistance declined wiiite_ blood pressure_ did not change significantly. Durirg bypass, that during cardiopulronary_-bypass; serebral-bload flow- rose and cerebral vascular immediately following cardiopulmonary bypass._ Studies on global cerebral flows showed tension and pH were :.cntinously monitored. Measurements were carried out pr'_or -to and changes in caliber of cerebral arterioles -and of red cell ve:ocityr -Arterial blood gas radioactive ntic_rnspbizfes, regional cerebral_ flow - by- direct- observation through - traflsiliutnination-and high-speed cinematography.- The iatter permitted observations of - These experiments deal. with the effect of total cardiopulmonary bypass on global and regional cerebral 6!oc,d -flow. -_ vloba;-cerebral blood fiow was deter :uaed with of the 'luxury perfusio5 symdrome' (overabundant cerebral flow relative to -metabolic - needs of cerebralftissuei_ Santilian, r. E., Chemnitit!s, :_ M.,_and Bing. R. J. Brain Research 345:1-9. 1985.- Other support: Hoover Foundation and the Charles A. Lindbergh Fund. From the Huntington Medical Research Institutes, Huntington Memorial Hospital, Pasadena, CA. 59 No& S

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A MODEL FOR THE-S3'UD Y OF L.ORONAR'd-SP%sS14t-IdVDUCE:? CHANGES - - IN CARDIAC 1`fETABOLIShz - A model is described which permits the rtu0iE of localized and -ger.eral'azed arterial _ spasm in the intact working:pcrfused _ rabbit-heart_ with a_ pert Funrochemical (FC-43) as perfusate. Coronary ar3eries ~vere visualized ~;+-in*.raatriai_injection of Patent Blue Dye with gated photography. Localized spasm resulted- from= topical- spray- of histaarine (40 pmols) nn the epicardial =surface overlying an obt.use marginal artery. Before and- following topical administration ~f ~ist_amine,- regional coronary flow was -detPrmined _ using radioisotope-labeie;i =anicrospheres. Ge .zralized arterial spasm was initiated by-_ intraatrial injection of- histamine (10 4irr.ols'-. - After topicnt-_admiK"rEtsation. -obtuse marginal artery diameter decreased by 5'?9i<; largg vessel resistance rose 32-fo1d;-2045 rise ` of tor?I coronary resistance resulted i, a-s_;gh reduction of total coronary flow (164s?): Heart rate, cardiac output. -dP/ dtM" ae.d myocardial orygen_ sonsum; tio, did not chang_e_-However, regional coronary flow.in the rieyocardism-supplied-by the affected -_arterv diminished 21%. resulting in-isrhem_ ;c_eha..ges in redcu-t+airs. Aftet iztt?aoirial injecrioes of histamine, changes v.ere-more pronounced--Obtuse n±arginai arter~ -diameter declined by 88%, resuiting-ii: 3300-fold rise_rf large_ vessel :esistance. Total- coronary resistance= increased 130%, coronary `low and cardiac output diminished (16% and 24%). Both heart rate and dP/dt., increased-(1696 and 779b).- Generalized _coronary spasm- __ after intraatr.al histan:ine- injection resulted_in several metabolic effects: Myocardial oxy-gen consumption -(-48%); ATP (-29%); creatine phosphate (-34%); redox -ratios, a-glycerophosphate/dihy_droxyacetone phosphate and -lactate;pyrtivate, increased _ by 449% and 11404, respectivelsK. The Tindings -illustrate- that lo:alized- sr.d general - coronary spasms can be- produced and quantitated ;n-a working heart :nodel: ___ Burger, Rl.,Cheanr,itius; J. M., I~etz, M. 2., and Bing. R. !- --- From- the- Huntington- MeJ:cai Research Institutes, Huntington Memorial Pasadena, CA. i THE EFFECT OF ISCHEMIA AND PEf'ERF-USION ON SARCOLEMMAL FUNC T ION IN PERFUSE3-CANINE HEA-RTS- Hospital _ This report deaL- with the effect _ of ischemia and reperfusion- on purified sarcolemma- obtained from-canitr&-atyocardium_of p_erfused heart~reparation:= PerTusion ` was carried out--with a perfluorochemical (F0-$3).- , Ischemia was produces: _ by intermittent total clamping-of inflow and outflow-fo'los:e-~ by relaa~e until the decrease in dp/~~,., had become stabile--Rurity of_ sarcolemmal vesicres, was_asceziained with --_ markr.r enzymes: succinate cytochrome- c reductase (for mitochondria}, 3C°=sti:nulated - p-nitrophenylphosphate (K'-pNPPase), (Na+/K j A;Fase and 2denylat- cyciase (for SL).- in addit7on Na¢/Ca't=exchange c-haracterisr.cs for SL weTe determined: Sidedness -of vesicles was ascertained by means of adenylate cyclase activ'sty using sarcolemmal = -preparations treated _ and - Gntreated -.vith aiamath'scir:: Emph3sis was placed on ATP-dependent Ca+} uptake, phosphorylation of sarcoler_s?Yal vesisles-and-yield of SL proteins. Ischemia and reperfusion_resulteE in a-sign:f:cantreduction in adenylate 60 V I 1

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cyclase activity: Th-iE-o'ecline was significant follo;ving iEchernia and reperfusicr_. The -_ yield of pr;;tein recc;vered-from SL vesicles from ischerr.ic-reperfused heart-preparations was also signifitatttly- decreased. Both initial Eate_ of ATP-dependent Cat` uptake and maximal Cai+ uptake fP'sign?ficsntly ollewing ischerhia and rperfusion._ The initial re-af phosFhoylation a]se dropped significanay.- TSese-siisturbarces in SI Ca;+ transport following iachemia and reperfusion are probably_a-party of the general deficit- in Ca;+ translocation. Chemnitius; J. Art. S4saki, Y., T3i;rge_,-1V., and Bing R. J. Journal of Molecular and Cellular Cardiology 17(12).!-139-1150, 1985. Other szpport: Hoover Foundation and the Lindbergh Fund. From the I-Iuntingtor Ie!edicel---Reseacca-- Institutes, Hur:tin gton _vlemorial -_:ospital; - - ° -- Y--sadena, CA. CEEAYAf'sE OF F:UMAN-H[GH-MCs`L.ECLJ;.,AR-tV_EiGH_T KINPvOC_EN BY FACTOR Xta !N Y1TRc'J -1Ve have_ secrnt?y( demon~trated that human high -molecu!ar weight kininogen (HMWK) is a prss-co-_'actor that is-cleaved_by kai?ik-rein to- yield a two-chain cofactor (fIh:WKa} and -the ras:apeptide braC;ki:.ir'.- This proteo:ysis eniances- its associatio:r with an activating surl'ace, ati_event-necessary for-expression of its cofactor activity. We now report that factor XIa_i-s capable of -!-..ydro,'y-Ling HMWK and releasing mradykinitr in apu:ified systein as well as_ cleaving and inactivating H^4`:dK in -a plasrna environment during the contact-attivation process-. T he profile of proteolysis differs from- that produced by-kallikreif and-by factor Xlla i.nn that the first cleavage by factor XIa yield- 75--and-45-kGa po}ypeptides, whe:ea: both factor Xlla-and kallikreirr initially-produce 65-~-and 56-kI}a species. Further proteolysin- by all three enzymes eventually pyoduces_ similar heavy chains-(Mr - 65,000) and-light chains (45,000). Howevers the amount -of factor XIa generated in plasma during contact-activation furt'r.er degrades the light caain of HIvISYX, eve_r,tua!ty destrovyi_ttg its- ;oagulan; activity. Furthermore, in- a purified - system, enhancement of the degradation of HMWK coagulant-activity by !'acto7 XIa_ was achieved when kaliikie=n was included in the-incubaticn mixture, suggesting- that the preferred-substrate f2-factor XIa_is the_active form of HIvIWK--(I-1-MW_Ka) and not the pro-cofactor. These data suggest that factor-XIa has the poten*.ial to act as a regulator of eontact-activated coagulation by virtue of-its avility to destroy the cofactor function of HMWK after its generation by either kaliikrein, factor Xlia, or to a iesser,extent, factor Xla itself. ° Scott;-C, F., Silver, I.,_D., Pu7don-,-A. D., and L'olman R.=1-.''. The JouFnql of EiolagirACl'h.emistry 26fijl§):10856-10E63,-19g5.- Ocher support: -National Institutes of Health. - From the Thrombosis Research Center and the Hematology/Oncology Section of the Department of N:ediciue, Temple University Health Sciences Center,-Ph(ladelphia. 61 t

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HUMAN PLASMA I6ALLIKRIN AND C! INHIBITOR FORM ACOMPLEX -POSSESSING AAl EPt;''J'rE-THAT IS Nd'3'I`DE-TEGTAELE ON THE PARENT , MOLECULES: DEIv13INST¢ATION USING_-A k2Ot.i9C-LONAL-ANTIBODY -_ The inactivation hf-hvman plasma kaIlikrein (-PC 3,4,21;$)_ by the inhibitor of -activated complement-- component 1(C!_ inhibitor)_ induces the formation = off a 1:1 stoichiometric kallikrein-C1 inhibitor complex and a proteolytica!!y modified form of Cl inhibitor. We have produced a monoclonal antibody that recognizes the kallikrein-Cl - inhibitor complex as -3vell~as modif;ed C] inhibitor, but failsto react with- virgin -C! inhibitor or native plasma- kallikrein. _ This observation constitutes ar unequivocal demonstration that the reactioti-betweer plasma kallikrein and Cl inhibitor leads to the emergence of an epitope that is undetectable on the parent enzyme and inhibitor molecules. Agostini. A. de,-Schapiro, M:, Wachtfogel, Y. T., Col=nsm-R:!}'., and-Carre!, S Proceedings of the National -£cadem.y of Sciences of -the United States of - America 92:5194-5193, 1985. ~Other support: Swiss Nationzl Ssience Foundation and L'.S. National Institutes of Health. From the Division de` Rhumatologie, Hdpital Cantonal Univer,itaire; Geneva, Switzerland; Thrombosis -R esearch Center and_ Hematology/Oncology Sec-tie:n, the Departrnent of kiedicinP; ?emp!P University Schoo'-af Medicine, Philadelphia; and the - Ludwig Institute-for Cancer-Research_ Lausanne Branch, Epalinges, Switzerland. - PLASMA KALLIKREIN AND DRORENIN1N-PA-TIENTS WITH HEREDITARY ANGIOE-DEMA- -- Recent evidehce indicates that plasma ka1_likrEic is activated during acute attacks of ° hereditary -an$ioedema. Plasma -lcallikrein is known to -convert inactive-_renin,- or_ prorenin, into an acuve--Droteol_ytic enzyme -in plasma ekposed to ac-id or Ioww temperatures as well as in pzrified systems. To establish whether plasma kallikrein could activate prorenin -_under physiologic or p3thologic- conditioe,s, prorenin---to- - rFnitt_ conversion was assessed at neutral pH in _p!a:ma deficient in _C1- inhibitor -(he-editary angioedemap. - In these pl3srha sampl?s lacking the s_wo major inhibitors of ka"llik{ein and possessing <_ 10% of the inhibitory -activity of_normal plasma, prorenin `+as"not;converted to an active enzyme- despite conditions under which pr$kailikrein- was completely = activated to- plasma kal:i-krein, and - despite -normal prorenin cancent_ratiens_ and-_ activability. - Purdon, A. D., Schapira, M., Agostini, =A. de and Co1mdn. R. 1P_ - -T'he-lournzl of Lahnratory=a3rd [:.tinica!-k1o_dicine ?0S(6):69e-699, 1985. Other suypwrt--. National Institutes of--liealth and the Swiss National Science Foundation. From the Thrombosis Research- Center and -Hemato!ogy/Oncology- Section of the - Department-of Medicine, Temple University School of Medicine, Philadelpfaia, and the- Divisiofl of Rhumatol3gie,-I3>;pita: Ca-atonal Universitaire, Geneva, Switzerland. 62 r.'

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PLATELET Cl INHII3I T OR: A SECRETED ALPHA-GRANULE PROTEIN In order tox Iearacterize-which protyins ef the contact phase of coagulatiotr interact -with- platelets;- humaA piatelets- were studied i:-,attursr,ci:emically and functionally to determine if they_rontain C-1 -irihibitar. -P; means of -nnonospecific_ antibody *_o-CI- inhibitor, a competitive enzyme-linked tmmunosorbent assay (CELISA) was developed to measure directly-platelet- Ci inliibitor_ With the CELISA; £fom=33 to 115 ng of Cl inhibitor antigen per '1pt platelets from 15- ns?rmal-donors -was qoantified in lysates of washed human platePets solubilized in nonionic detergent. The mean concentration in 10" -platelets was 52±33--ng (SD).=Plasma CI inhibitor-either in the 9latelzt -susgension -medium or on the surface-of the-platelets could account foi only f_ror" 6.5 to 1b4fr of the total antigen measured in - the -solubilized - plate-lets: - Upon functional studies, platelets contained 8d#38 ng (SIi) of Cl inhibitor activity in 10a platelets. As assessed by the C1;LISA, platelet CI inkibitor- antigen was imrnunochemi=ally identical- to plasma and purified Cl- inhibitor. in contrast, the mean concentration of platelet Cl inhibitor antigen in -platelets-fron? four aatients-evith classical hereditary angioedema was $.3_ og/10° platelets (range 5.3 to 11.3 ng/108 platelets). 25 % and 31%-of the total platelet Ci inhibitor was secrete-d-withovt cell lysis from normal platelets after exposure to collagen (20 µg/ml) and thror:Ibin (I"tJ/mlj respectively, and -this secretion was blocked by metabolic inhibitors. Platelet subcellular fractionation s3owed that plateler C- inhibitor resided mostly in alpi:r,-granules, -si-miiar to the location A-f platelet fibrinogen. Thus; human platelets contained C1 inhibito. that became available by platelet secretion. The identification of platelet CI inhibitor suggests that platelets may-modulate the activation of tha pFotein-s of early blood coagulation and the classical complement Rat-hwaysf -- Schmaier, A. H., Smith, P. M. and -Coi_man; :2 -W-- .lourna: oJ C:in:_al inresfigat;on 75;24Z-250, llfc5. Other support: National Heart, Lung and Blood In_r;itute; a-Temple- University Biomedical Research Support--Grant;= iRme_ricari Heart- Associa2ion;- and a Specialized- -Centers of Research grar.t. From the Hematolog-;,/ Oncology Section. Department of Medicine, Thsori:bosis Resea_rch Center, Temple-L?niver-sity-Senoot of Ivledicine,-Philadelvlr=a: EFFECT OF HEPARI?V ON THE INACTIVATION RATE OF HUMAIt ACTIVATED FACTOR XII-BY ANTIT-HROi4,BiN III Human antith-rombin III (ATIII) is a plas ;ia=inhibitor of several serine proteases o the blood coagulation -Aystem. -Previous investigations have *eported-that the presence of heparin has-a multif-otd accelerating effect on the inhibition of factor Xila and XIIf, the active species derived from f ac_ ior -XII. -Recent studies from our laboratories have -confirmed-that ATIII -inacteva,e3 factor X11a atrd-factor X;If, but- only contributes 2%-to 3% to the inhibition of activated factor XII species in plasma.- The ma;er inhibito is-C-I inhibitor. _ThereforP, we have reeaamined_the heparin effect on the rate of- inhibition of factor X11a and-factor XIIf irn purified-systems. We also- have studied the- effect cf heparin on the itiactivation of both factor XII-deri;red active species by_ various plasmas. Using purified factor X11a and kTIII, we found that heparin (0.7 to 34.0 LI-/mi.) increased the rate of inhibition of factor XIIa. 63 Tl -. =z-k ~.., r. ,

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However, at heparin 3oncentrations-usualiy achieved during= anticoagulant therapy (0.7 CI/ mL), the inhibition was accelerated only four-fold. This implies onty a 6% contribution to the-inhibit-wy tffect-of p!a`rna: TFeis-suggesticn was confirmed by the observation tha* h~p.a=iii (#:5 if,/n:L) added to facior X;Fa did not producz- a detectable - enhanceinent vf the-rate of inhibition of factor XIIa. Furthermore, using- purified factor XIIf and antithrombin HI. 'tiepatin {3,6 -to=57.2 U/mL) increasvd the inactivztion rate constant of factor Xilf by-1.6_ to 14.O tirres- This small-effect was confirmed by the - observation that heparin at a_ concentration -greater thad that sufficient for° anticoagulation (1.4 -U/mL) =did not modify the- inactivation rate of factor Xllf by prekallikrein-deficient plascba, and thus Ci inhibitor remains the major inhibitor even in the presence of-Leparin: From this study ardour-previous ir:vestiga_ tions on the-effect of t,eparia an the inhibition of kallikreis and factor Xla, we conclude that-heparin -does not significantly affect the protease <ctivity o.-purified contact activation factors o= the- activities expressed-by these proteases in plasma. Pizley,-R._ A., Schapira, M., and Cofr.mn, R. W. Other support: American Hezrt Association, Souiheastern Pennayiv-ania- C_hapter; National Institutes of Health Individual National Research Service Award; and Swiss National Science Foundation. From t} eThrombogis Research Center, Temple = LI_r.iv?rsity School - oU Medicin_. Philadeiphia, and Division de Rhumatologie, H6pit4l Cantonal i.ir.iverc_itaire,_ Geneva, Switzerland. - - -_ --- - ` ' -- - - - THE REGULATION OF H-UMAN-FAL'iOR Xl?a BY PLASMA PROTEINAf£ INI{IB;TORS Studies of the inactivation of factor Xll-a by plasma protease inhibitors in purified systems and in -plasri3-wgre initiated to detein:ine the rp!at;ve importance of these inhibitors to the neutralization of factor XIIa. Factor Xlta was measured by the grnidolysis of H-D-prolyl-L-ph enyl.alanyl-L-arginine-p-nitroa.iiide dihydroch7oride or by coagulant activity. Cl inhibitor (C!lNH), o,-an*.ip!a_s-r.in (Lr,AP),-n,-macroglobuiin - (a,!vi), and antithrombin ai (ATIIiI inhibityd factor -XIIa with -seyond-or{er rate- cpnstants of 2.2 X IOb, 1.1-X=104, 5.0 %-!0s,-anr 1:3 X 10s IvFlmin-t. Factor XIIa activity was not affected by dt-proteinase inhibitor: Incubation of ts¢I-radiolabeled factor XIIa _ resulted in LI stoichionietric complexes with CIINH (Mr-190.000), ATIII (Mr i25,000), and rr=AP (~fr 150;t~~ and_ i25;000) _ using sodiurr dodecyl aulfate-poiyacrylamide- gel electrophoresis.- Incubation of 12sI-Factor XHa with Qa!vt resulted in a component oE` IH; 85,000 on a reduced sodium'dodecym sul*'a*.e-pclyacrylamide gel, indicatir_g that a subunit of factor XII` was cevalen*iy tiound=to a_proteolyzed portion of o2IvI. - The reiativ¢ efectiveness of each inhibitor at plasma concentrations waz 61:2:?:I for C!INI-I, aaAP, ~Z1VI, and XTIiI, respectively. 'i.inetic-studies of the inactivation of -purified iactor--XIia incubated with plasma confirmed that_t`actor-Xlla-CIIIv`H was the_ major comple.. Analysis by sodium dodecyl-ylilfate-polyacry!amide gel e.ectrophoresis indicated that the complexes in plasma- had the same molecular size as those wish -purified inhibitors. CIINH functions as the predominant inhibitor of actor XIIa in plasma. added-to various plasmas containi: g different concentratio ns of CIIN.Li- yerified the predictions from the purified systems. - Gel filtration of -radiolabeled- factor XII.a

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Pixley, R. A., Schapira; M., and Caiman. 1@. W. ° The JourrF~l oJBrolagrcal Chemistry 260(3):1723-1729, 19>?5: OtAer suvQor7.= Natioisal institutes of He~t#t; Ame_ricair Heart aE.ociationF SrutFieasierr. Pennsylvania Chapter; and Swiss-Na3iotral Science Fourdation-- From the Thrcmbosi; Research Center -and the- Department of Medicine, Temple L3niversity School of =-ledicine; ?hil_delphia, and the Division de_ FCt;untaz6iogie, Hopjtal Cantonal- Universitaire: Geneva, Switzeriand. EFFECT OF CLEAVAGE OF T14E H-F-AVY CLIAINOF HUMAN PLASMA KA1 LIKIIEIN-OP` ITS FUNCTIONAL °ROt ErcTI-ES Human plasma kallikrein consists of- an N-terminal heavy chain of molecular weight (moi wt) 52,000, lilked-by disulfide-bonds to two light ehain variants (mol wt 36,00G or 33,000). Althopgh the activa catalytic site- of kallikrein resides - on - the C-terminal Iightchaiai the role of the hi-ter:_^.ir:al heavy chain-iY less clear. We -therefore studied an-enzyme designated-t3=kaLikrein, cauntaining a-single -cleavage in _tfie_ heavy chain (mo-1 wt 28,0,00 + 1S,0L~_)) ar.d-cornpared it to the enzyrner 0-kai;ikrein. -with -an intact -heavy chain, ` The rates of inactivation by CI-- inhibitor- of _-p__asnta- a= and fl-kallikreins were =kinetica7ly identical, as nieasured-§y :esidual arnidolytic activity,- after various times of incubation with the inhibitor.= Both enzymes reacted completely _ with C-1 inhibitor after 18 hours an1 formed identical C1 inhibitor-kallikrein complexes of moi wt 1950t)). The rate nf activation of factor-XII by a-ka:iikrein and 6-ka:likrein was similar. I., cor_ras3, the rate _pf :ieavage of high molecular weight kininogen (I-It:3`aK) by a-kallikrein was at least fivefoid-4aster aad-the-ratio of coagulant activity to amidolytic activity was fourfold greater than for y-kallikreii. Plasma a-kallikrein, at concentrations poten*.iziiy achievable in pias,,,a>-induced aggrega:iort of neutrp;,hils, but -,6-kallikrein failed to elicit this response.- In additior_, human neutrophils__tF.at had been pretreated with cytochalasin B released-2.25 ± 0.i0 µg/lQ7 cells of elastase antigen, but 0 -kallikrein -released only 0.25 ± 0.10 µg/10' celis.- The_se observations suggest that _ cleavage-of the heavy chaininfluenees the rate of cleavage _o:`-HMti'K and -decreases- : s eoaoilart activity. Mureover;_ su intact -heavy chain appears to be requisite to suppor t- the ability of kallikrein. to aggregate neutrophilsand release elastase. Colman, R. W. et ,l. - - $lood65(2):311-318, 19g5. Ot-her support,`IVational Institutes of I-leaith and the Swiss NnticnalScience Foundation. From - the -`d'hrombosis_ Research Center and the-- HematoioggY-Oncolog}~ Sectio_n, Department of !u=ediciti=, T'eTpde University School of Medicine, Philadelphia, -and the _ -- Graduate Hospital -:~esearch Center, - Division de R humatologie, Hc~ita=_ Cantonal -Universitaire, Geneva, Su%itzerland. I I ~ ~ 65 ~ ~ ~ ~ ® a

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REGULATION OF T°eIE_CO_Au`.JLr1N T A-'~'-TaVI i i-AND SURFACE BINDING OF HIt`iI: MOLECULAR W:-:!:a!-iT KININOGEN Events in the formation of the enzymes in the intriusic coagulatiort pathways can- be conceptualized as three reactinn clusters- = =T-b.e procofactors of the two later _ 2teps; __ factors VHI and V-, afe each tzroteo3yzgc# by thrombin to active cofactors, factors- VIIIa _ and Va, which are then bound to phospholipid-mice!les or platelet membranes. Each which the bindingef XII and HMWK on the su_r_"ace is,coordinated. XIIa_first cleaves _ PK, which -in turn converts .the -procofactor,_ HMWK, to an-active- cofactor, I-IMWKa,- with the= ability to bind to-an activating surface a nu express coagu!a*tt activity by bringing PK and X3-intv proper appositioo-for cleavage by X/ia. XII_a then cleaves XI to factor--XIa, by activated factor XfI. We -have recently delineated the _ me:.hanismi by- factor-XII to a negatively charged surface where autoactivation occurs for form factor- XIIa. The substrates -of factor XIIa, prekall;krein (PK) and factor XI. , exist in bimolecular complexes with the contact -system procofactor, high - molecuiar weigFt kininogen (HMWK), which LF required to tra nsport them to an activating eurfaca. Here, on the surface, these zyniogens_are-converted to the active enzyr;3es, xal!ixrein_lK) aad_ studies indicate that the -pr=.cofactor of -the contact- system is similarly subject to activation by kallikrein to an active cofa_ctor prior to binding to an activating surf ace. The activation of the contact system is probably initiated by the binding of plasma cofactor then serves as a`receptor'_ to which the_enzymes, factors IXa andXa, bind prior - to activating the substrates, factor X and prothpomt3in.- The cofactors, factors_ _3!illa and - -Va, are then-irnactivated by another-enzyme_ protein, activated protein C. Our- recent XIa. - -C`oEman R. W --Silver L I7 ,_ Purdon, ,^. -D., -C}-.ang-i. J., _and Scott, C.F. - - Transactions of -!:e Assoezatioa of Am_ericar Phycirla:~s XCVII:113-123,-1484= Utker supnors: National-_Instatutes-of HeaCth and-SyEcialized_ Center_of- Research on Thrombosis. From the Tfirombosis ResEarch_- Cer-.ter_ and-_ the Hematology-Oncology - Section, Department of Medicine, Te;^p.e University ScF,co! of Medicine,=rhiladeiphia. -__ _ PLASMA PREKALLIKREIN ASSAY: REVERSIBLE INHIBITION OF CI INHIBITOR BY CHLOROFORM AND ITS USE I-N MEFESURINC', PREKALLIKREIN IN DIFFERENT MAMMALIAN SPECIES examines -t-he effect of -seauential_-preioeubation_of plasma_ with- chloroform and-__ met:iylamine on the plasma preka!Iikiein assay._ -Ch!oroform_was-demonst_rated to- be a chemical inhibitor of ourifi_ed -Cl-_ inhibitor, but ds-macroglobutin was not. Chloroform inhibition of Cl inhibitor -was not caused by precipitation of the protein into the- interface between the water and organic solvertt-phase. Greater than 955% of Cl inhibitor antigen was recovered in the supernatant of chloroform-treated pur ified C: -inhibitor, previoiesly been employed _zo -selectively- inactivate c=-macrog3obulin., Our ytudy -- The assay of plasma prekal!ik.*ein requires activation of prekallikrein to ka!likre3i; and sufficient inactivation- oP the plasma protease inhibitors of kallikrein to accurately measure -the generated- irallikrein activiav. _ One method of elimination of - the plasr:3a protease-iahibitors to kall kre-in :s to chemically pretreat the piasma. Methylamine has- 4ll ~ ~ ~ _

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- and chloroform-saturated tuffe_r- inhibited purified C! inhibitor. Ckloroform did riot - -di-mciate apreformed comp7ex of kaafkreirt and Ci inhib3tor, but its irshibition of C+ inhibitor was- revers.ible_ The addition of inethylami.,e to plasma pretreated-mith chloroform in- the _p!a<sma- prekallikrein assay ailowe. for cnly--a-siight increase in the - -- ari!9unt of kaliikeein= °measyred a--1 =minute' kaolin activation tirna, -but provided for - sustained measurement of-activated-piekaFlik-rein when kaolin activ-ation times were 5 to - 7-minutzs. _. Without- chemical pretreatment, prekallikrein was not measurable in rabbit pla;ma.--Both=rabbir and-pig plasma prekallikrein were-aieasurable after exposure of-the plasma- to ch7o-rof3rnr and methylansine; although the peak-acttvation tiE:?es and the contribution of each aniraal's protease-=inhibiiors- varied with- the spe,ies. -(:rr results show that chloroform is a-reversible inhibitor of Cl inhibitor, and- that the plasma prekallikreia assay in which it is used is useful for the ireasurament of prekalli: rein in nonhuman mammalian plasana-samples.=- Sihmaier. A. I-I.; Oustatson,-E., Idea, S: a nd CoPnraan. R. iV - ; he Journal of Laboratory and Crtnica; Medicine 104(6):88i-8I2, 1984, Other support: hiational-Heart, Lung and Blood Institute, American Heart Association. and National Institutes of Health. - From the Thrombosis Research C'enter, Hematology/Oncology -and Pulmonary Sections, Department of-Medicine, Temple University School of Medicing,-Philadelphia. - - THE EFFECT OF HIGH MOLECULF.['c WEIGHT KININOCaEN ON - SUItF'1aCE-Af3SOR$ED EIERINO: EA1 High m-oleculai 'weight ka:.inoger (HMWK) ?lays an important role in altering the -:ssociation of plasma=iGrinoger, with surfaces. Plasma initially dmposits -fi6rinogen onto most materials, but on hydrophilfic surfaces within !0 min adsorbed plasma fibrinogen cannot be -detected= cB the surface by anti=;brinogen antisera. - However, using HMWK-deficient plasma, fibrinogen remains immunologically identifiable. T:e- inter-relationship of adsorbed plasma fibrinogen with kin.inogen, on-_hy3rophilic sLrf aces- is studied further using glass slides stained for protein wit;~ Coomassie Blue, and cxidiz',d silicon crystal slices in an automated ellipsometer. On glass slides when plasma - that is deficient in both low molecular-weignt=»ininoge® (LMWK) and HMWK is reconstituted - with HMWK (0.40 Units/ niI); fibrinogen is no ?onger- detected on-the surface. This - f inding is specific for IiMWX;-sinee, when the same plasma is reconstituted with LMWK (220 ug/m!), the amount of fibrinogen detected on the- surface is unchanged. Thee alteration of_ surfac&adsorbed fibrinogen by-HMW;'K is_ not due to plasmin=induced fibrinolysis, since it _cccurs-in plasminogen-free=plasma. In the eli'spso:r.eter, ,.eter, surface adsorption of normal plasma is associated with- a significantly less (p <-0.C005) thick - protein_ layer (LeiS _± t.0S _degr eE change in azimuth) than Wiasmas def icient in - HMWK =_ (2.32 ±-0.11). Using-ellipson:etry, HMWK ;n plasma is-strown to shorten the -ti::e in which irrimunologicaily detectable surf ace-adscrbed fibrinogen was remov¢r or altere?. These s:udies in a whole plasma system present further evidence thae HMWK specifically = -modifies thea.ssu;.iation of plasma fibrinogen with hydrophilic- surfaces. Schmaier, A. H., Silver, L., Adams~ A. L., Fischer, G. C.. Munoz, P. C. -Vroman, L., and Cblman, R:W. =- - Thrombosis Research 33:51-67, 1984. - 67

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Other support: National Heart, Lung and Blood Institute, American-Heart AssoYiation, a Tample UniversitySroredi~t-ltesevrch SuppG~rt_..`irantxs:id Nat;:.r~al In_tihutesjf Iaaltls.- Fiom the He_rnatology/Oncology_ Ses.Eion_ and_ Thr-0:nbcxis Research -Center, Temple - -University School of-=Metficine, Phr!adelphia: and the__ Interface Laborato.ry,= Yeterans_ ~ Administration Medical Center, BrooRi-y _New York. CHROMOSOMAL TRANSFER IN MUUSE-EItYTHROLELs`-KE_hi_iA CE_L.LS_SH(3:v"S THAT TRANS REGULATION N'EGIAT-ES TISSUE.SPECIFI... P"_NDDEVELOPMc.NTALLY SP:- IFIC-PATTERNS rJF_CI,t;BIN-GENE EXPRESSIDN The data presented in this paper have suggested that the activation of embryonic globin genes in the_ K562-cell line arose throqgh ;.-singie mutation wlsich led *_o altered expression of a d'affusibi regulatory factor capable of activating embrycnic g!obi* genes. _To test the hyp.;thesis that embrvonic giobin ge_rre-activati_on had occurred in the K562 celi through altered eAwression of a trans regulato,y factor, rather than a mutation in-cis to the gene,_it was-decide4 to intro_du_e_'the cranscription_.lly active_ zeta gloGio---= genes of -the X~.b2 cell-_into-_ the tetraploid Fr iend mouse erythrole::kemia-cell irn which - adult-but not embryonic gioyir, gene expression occurs. In this study the authors also used restriction- fragments 'sength polymorphisms _to show that hoth-?iomo!ogces - of - chr3mosome-16 which are_:present in=the K362_cells conta_in= arpKa:ar~d_zeta--glob:n- genes -- which behave in an ident-ical manner when transferred to the mouse erythrolexkemia- cells. T he results show that the mutation -which led to the activation of- ht:ma n embryonic globin=genes in-:fie K 562 cells is asyntenic to the zeta-globin -genes. These ~ 5 results-alsc; suggest that -.he sC562 -cell_ e:rnr.ains a- trans--regu!awary._ factor which is t - competent to support the exrrressiori=of the human vmbryonic gio bin genes. ~ - Ds3sserotfi, A. B. It? Staattatoyannopoulof, £a7 and N}enhuis, A._ (eds.): £xperirr,ent3l ~4pproae}es_ for !h~ - Study aj Hemoglobin Sxi~itthsng, New York-: 4lan-I?. i,iss, _i-9i;2, 293-353. - Other sapportc- Veterans Ad_r_:in:stratsen an<iN4tional Heart, Lung and BlUod-Institute. 1-=- From the Department of. Medicine, University -of California =at San F ncsc.-o,__ Hematology/ Onc.ology _ dJnit and _ t-he-- Veterans - Acministration Medical -Center _ San _- ~ Francisetz PLASMA PROTEIN AND LIVER-mItNA LEVELS OF TWO JJ CLOSELY RLATED_ MURINE al=2RJTEAS °iNI-iiITURS iiUl?IlY~'r THE ACUT~~'::ASE RET~: i IUN_ 1 1-D" Plasma- leves o_t ~-.-.-I(T; _ at.d ~1-PI(E),- two cosely _ related ntursne -aipha 1-protease inhibitors, having affini,ies fcr trypsin = and, elastase,_ respective!;, -were compared to ~~:anges in specific liver mRN-A levels after induction of the ac,~a-phase - reactior_-by subcutaneous injection of turpentine. In_earlier,-qualiEa:ive experimeats,- an inc_rease_in plasma levels-of ol-Pd(E) but not ol-PI("I') disring the acute-phase Yeaction had been shown. It is ,noiv=-shown that sti_mulation of plasma al-;'1(E) levels reaches a__ - maximum of 35-50% above baseline_ 12h after induction of the acute-phase response using either a- functional or immunological assay to measure protease inhibitor -activity- Consistent with earlier ebservations;=!ittle or no change in plasma levels of chl-PI(T) is 6a rx V ~:- r , ~ae i

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® seen. Determinatiuf of mRNA_ levels in -t,e- mouse liver specific- for QIFI,'E;_ and -e1-FhT) was _a;con,plishgd using 3= cell free=_-trazslatioq system followed b-y- - immtrfio precioitation __,t' the I-S-Iabeied= prot:ase-in,hib;;ors. The apparent molecular weights of-oI-,PI;E,l=aa8.ai=Pl(T).synthesized_rn vitro are 4ZiG-and_ 46K, respectively. Qppare=t_~olecular_ av- igJ:ts of _thu native prote]ns_-ia_~l^sma =are_ _{5K = an~i 65K. bTnex*~ecredl-y, mRNA,3e-ve:s for both ni--I'ItE; and cl-l'I(Tj were_found to increase after induction _ef the atwe-phas; reacti". Ivteximal stimulation for both mRNAs was approximately 300% and occurred -9h after tuapentine administratio n. Under these = conditions, levels of translatable albumin mRNA in the_mouse liver decreased to 40% of baseline in 6-9h.= F razer,_J. M., Nathoo{;;. A., Katr, J., Genetta, T._L-and finlay; :'.-H. Archives a; Jiocl:ewist;yami__BrL3physics 39(:):Ii2-119, l9$5. Otn<r support: blatitina: S.:ience FouadatFcdm the Departmen_ of.-Jbstetrics and-Gynecology; New York University Medical Center, New York. _ _ 1REvULATION QFTL4tii`-`tA F r+iCTG? :{III BI'S.r.;INCr-:)V _VIi`Rc`3 - The binding of p:asma fa, tor?LIIi to fibrinogen o `ibritr that has bPen-chemically= - or enzymatically induced-to--polymerize- w:s-;tudied._-- Fac*dr-XIII bin3i n3 was assayed- using_a 3Ii-pvtrescina -_ncorporation-assay and an-t3Sl-plasma-factcr XIIi binding ass,_y. More_than .0Cr+b-of the nat:ve and radio[aaeled plasma a ctor X ilLwas bound to fibrin I formed by reptuase in E;7TA, citrate, or heparin anticoaguiate(F plasma. Sodium dodecyl- su;fate-~lyacryiamide gel electrophoresis (SJS-PAr~F) of r'-I-plasma factor Xiil bound to fibrirt 1 ar- fibrin II fornied by reptilase or thrombin ia the presence of EDTA dertaonstra*.ed the ba=Ettbunit -remained bound to -the a c;tains -or thrombin-cleaved a-zhains. In the presence fi:_ calcium chloride and throrn;zin, t,he_ bi-subulit aissociated-- and factor XIiIa was bound. -Protamine sulfate taused fibrinogen polymerization -in the absence of divalent-.tatiors and red~ced-_both plas:~a-_factor XIII and immur~oiogic- fibrinoger,-IevRls. fibrinogen polymerized by protamine sulfate ixound_ p:asma- facior _ XiI and the a:-suburat of 327:-piatvlet_factor XIII. Feasma-.actor-XIIi _cvas-also bound to _ sonic-ated non-cei3ss-liriY d_ ibrin II in tither -normal_ plastria ur afibrinogenemic plasma. Plasma _Ievels -of several coagulation -prnteins were unchanged after the addition of reptilase, protamine sulfate or sonicated fibriu to plasma. These results dmonstr,te that a specific binding site fo= the a:-se;bur,it of plasma actar XIII is present on polymerized fibrinogen, fibrin I and fibrin lI. Furthern:ore,-the presence of divalent cations, thrombin-cleavage of plasma factor XIII_ and release_ of fibrinopeptides A vr-_B are not required for plas;r:a factor XIII binding=to-pclymerized=ibrinogenanu-fibrin. -- .^,reerfkerg;_C._S'..,Dobsom, J. V., and Miraglia, C.fi,, Blood'_36(SY102-3034,19a5. C`ither support: American Heart Ass2ciation. From the- Departmen-ts of -Medicine and °athology,-Duke University- Medical Center, -Durham, NC. _ 69 ® ® I ® M I

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MEASiJ'Rz'Mr:I+IT OF _ Bi.OOI: C.r.AG i1LATION =FA%TOIi - XIIIa FORMATION IN PLASMA CONTAINING t::LY(;"I.-~ PRULaIL-:.-ARGIt~]YL-L-RROLINE A method is described- t_o-directiy measure the formation of- blood coagulation Factor 3EIiIa in - platelet-poor _plas.ma un,::cdifi?d _by==heat "1'lre - syathetic peptide glycyl-L-p_rolyi-L-arg;,:yl-L-proline, a f:brir poly,:_erizatiorf inhibitor, was- _used- :o _ preveni clotting of Qlateiet-pxr pla.s,rta. _Qlasma was diEuted to a_final conce:nratinn of 2.5% (v/v) in- 0.1 M'I:'ris-:ikl, pfi-$.5,- buffer crntainin-g 25% gl;cerol, 5- mM calcium 'B 1 chioride,_ and 0.25 mM g!ycl-:.-prolyi-L-arginyl-L-proline and -then activated by - thrombin (20 lI/ml) for 15 n,in. The Factor XIIIa-catalyzed incorporation hHlDutrescine into Hammersten casein wasused to measure Factor XIIla formation. The assay detected Factor XIlla in 2.5 to SG µi of tlirombin-treated plasma. When purified Factor Y[lI was added to Factor XIII-def icie_nt plasma, there was complete recovery of the -Factor XIII added. Glycl-L-prolyl-L-areinyl-L=proline did not inhibit Factor XIIIa activity -in - thro:nbin-treatEd plasma or, purified platelet Factor XIIIa: G lyce-rol stabilized Factor - XIIia activity--in thrombin-treated plasma and- buffer for 60 min. The presence of fibrinogen in plasma did -not modify the - assay results. The time course of thrombin-catalyzed Factor XllIa- formation in -platel-et-poor plasma _ containing glycyl-L-proiyl-L-arginyl-L-proline-was directly- measured using the assay. Miraglia, C. C. and Greer.ber; ,-C- S. Analytical Biochemistry 144:165-1?1,-19g5. Other sLppsrt: National Institutes of Health. From the Department of Medicine, Duke rtJ*iiversity Medical Center, Durham, NC. THE EFFECT OF FIBRIN POi.Y_r1ERS- ON THROMBIN-CATALYZiD PLASMA FACTOR XIIIa FORMATION_ The effect of fibrin polymers on thrombin-catalyzed factor X1IIa-formatic?n was- studied iff afibrinogenemic plasma. Fibrin polymers derivec# from des A- fibr;_r-ogeit and des A,B fibrinogen increased six-fold the rate of throm5in-catalyzed factor Xlila formation in_the presence of-EDTA: Calciam chloride accelerated iactor XIIIa formation i4-fold ,a the presence of des A_,i;-fibririogen without increasing the rate of ihrombin formation. Fibrinopeptides ; A and -B had no effect on f actor -XIIIa -formation in afibrinogenemic plasma. Des A,B fibrinogen reduced by 20- to 40-fold the thrombin concentration required to activate factor XIII. Glycyl-L-prolyl-L-arginyl-L-proiine - (gl_y-pro-arg-pro), a fibrin polymerization=inhibitor, inhibited des A and -des A,B _ fibrinogen-from enhancing -thrombin-catalyzed factor Xliia formation. - Gly-pro-arg-pro did not-modify factor XIIIa formation in afibrinogenemic plasma and did- not inhibit thrombin cleavage of the chromogenic substra-e 5-223g. These results demonstrate that fibrin-polfmers accelerate thtombin-cata!y-zPd plasria factor XIIIa formation. Greenberg. C. S. and Miraglia,-C. C. Blood 66(2):466-469,14g5. - Other support: IQational-Institutes of Health and American Heart Association. From the Departments of Medicine aed Pathology, Duke University, Durham,-ItiC.

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RADIOIMiVIUNOAAY FOR NEURUPEPTIL?F. Y(Nl'_Y-Y ChIROIuIATU<`~I?API:If' -trIIARA~-I'£RiZA.TION--GFIM7rI~TOREA£TIi';TY IN_-PLASNIAA~NDT.SSUE ' - £n T RAC'IS Q sensitiv __n!!d specifi; - razioimrounoassa;_- wa; deveioped te _ deiersrsine the occurrence and concen_trati4n of -nturopeYt:de- y- (N; Y] in -Ffasma a ntr tissue esEtracts. --_- Furthermore,_NPy sikg immunoreactivity (I*3PY-I.i) was characterized by means of three different chromatographic syftems. - The NFY--ar.tiserum used (NIYdid_not cross-react with- related pepiides- -of the- pancreatic polypeptide family ezcept avian - pancreatic polypeptide (.% cross-reactivity;. Unextracted plasma contained high molecular weight proteins wrhich interfered in the-assay. -Acid-zth-arroi extraction removed this protein interferenccaz;owing a909fo recovery of NPY-Li.- The cqn*.ent-of NRY=Ia-_in -hunsan plasma f rorn :ealtn, shbjects was close -to or below the detection limit (~22 pmol/11, Cympathetic_ herve_ -stiti,ulation _ in _ the- -cat-- increased the° output of NPY-r i_ fro:a the sp!enic vein saggesting- tYe re[ease of this peptsde upon sympathetic activation. -The major paa;: of NF:-Li in spleen_ extracts= a-~d _ spienic_-vein plasma co-e?pted -wi=h synthetic porcine-)`I'Y and a miFor peak with iargsr-3tokes radius Kas-slso-present. the present- radioimmuncassay enables furt-her_ studies_= on the_ physiological and _ pathophysiological ri;le_pf NPY. Theodorsson-Norheim, £., l=Iemsen, A. and Lunvi>Qrg. J. ~S`a. - Scandinavian Journd.t af Clinical & Laboratory Investigatiox 43¢35-5-365, I°8r. OtAer-support: The Swedish _33edical Resyarch-Council,- the Karolinska Institute and tFe- Astra Foundation. From the i3epartm-ent of Clinical Chemist_ry, Karolinska IIospital; and the DerartmeUt of Pharmacology, Karolinska Institute, Stockholm, Sweden. iBENTIFIEA TYON OF HUMAN PLATELET MEMBRANE FIBRINOtiEI3 _ RcCi PT0°,S BY Ii)F;iiL7NOCHEMICA.. Tt.(:.I3NIQ£JES-- The filbrinoge,e_ rscej;tors of piate7ets -:vere inye_stigated with the use of three types of anti-platelet nternArane=antiaoliPsan-d three types of platelets. We found that antisera ra raised_ in rabbits $gai nst merttbrar.es prepared from human intacc, chy;nor.rypsin-_ or pronase-treated ,.iatelets_ inhibited the - fibrinogen-inzfruced,- -aggregations of ADP=si7ottilaied ista_t- p-Iatele:s, chymotryps3n-ireated platelets and pronase-treated piatelets. These _ar:iisera also blocked tlie-binding df_liad-fibrinogert to -ADP-stimulated intact,_ chymotrypsin-treated. and pronase-treated pia.tele_ts._ -T hese results suggest that all - three antisera blocked the interaction of fibrinogen with its receptor on the- surface of the th_ree-types. of platelets studied. Fibrin clot ret_raction .bx intact platelets-wa+ also inhibitAd by thgse-_3h_ee-aneib_ odies indicating an impordani role of platelet -mF:nbrane proteins in c!ot - retraction. As demonstrated by techniques using :3:1-syrface labeling, Staphylococcus - aureus im rnunoprFeipitation, SOS-pnlya_cr yiamide gel electrophoresis and autoradiography, anti-intact platelet inembrane anrcibody immurtoprecipitateu he- membrane glycoproteins= GPIlb, GPIII -9nd_a protein w'h h an apparent molecular weigAt_ of 66,000 trom detergent solubilized surface 1S`l-ic-x_iinat2d chytnotrypsin-.reated= _Dlatelets.- Anti-chymotrypsin and anti-pronase=treated platelet membrane antisera ii_n=rnunoprecipitatee mostly GPIri-and the 66,000 molecular weight protein from detergen-i-soluE:lized, surface 2 ESI-iodinated chymotrypsin-treated platelets.- 71 --

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I- The 66,000 Mr protein was--;ot found on the surface of intast (urastiift4ulated) =pl3teleta - which do not -bind ;=1I-fib=anogen and are not aggregated by fibrinogen svithout= tht- prior addition of F,DP. The ability of anti-;;lateiet memabrane antibodies to b3ock- - fibrin5ten-induced p!atele3 aggregatiop a nd fibrinogen biad:ng to pEa.e;ets ;,orrelated with their ability to it^anu:rs~recipitate ~ 66,6Li6 >~r prot-ia=fraar ths-p:a;elet surface. It is proposed thal the 66,:)Ct0 1;1r protein may be-the_ fibrinogen b;nding domain of GPIiI which becomes permanently expused on the surface of chyntotrypsin and ~roFase-ireated -_ platelets foliowing profeolyris and which becomes exposed upott ;timutarirn of intact --- platelets by-agents such as ADP. - Kornecki, E., :'uszynski, G: P., and Niewiarcwski,-S.- Other sapport: N3tionaI-It-,fautes of Health and -Nationai-Resea- Research 4-.erv:ce: -- Fror:. the "I'hrombosisIF sea ,h _ Cen -r, Tenple CTniversity FfeaStt Sci_`nc_s Center, Philadelphia. MEMBRANE FLiJIDITY ANL`i PI,ATELET-FIBRINOGEw RECEP'I'GR- E-XPc`jS{IP,F- BY PP.QTEGErTIC ENZYMES Incui?a iot.- of olatelew with pr3nase _er c-hy-rrorrypiin- sults_ in the exposure _vf =- - fibrinogen receptors. - We dztermir.ed-that these enzymes did not affect the membrane - fluidity as evaluated _bK _`s4e_ d;.polarizatiotr o!'- the fTuoresce nce--of_ I,6-diphenyl-S,»5 hexatriene (DPIt).- There ka-ne-{ig_r,;icant d:fferenceir. et her the dep:,lariWion- or in -- iu temperature dependence t&r=contrul, pronase or chymotryps:n-treated platelets. - Thus, it can be concluded that the exposure of_fibiinogen receptors on the olatei3t-surface by proteolytic enzymes does not depend on the changes of rnembrar:e=f luidi*_y. We ano propose that-the proteoly,-iF anzyrzes do no: caase a major aiteTatiorr in the extear-of _ protein chains-embedded in the lipid layers ot-the platelet membranes. Simons, E., k'hitin_J. C., MociAelfi,-T. A., Stewart,{'r. J:, and Niewiaroruski, S_ E hrom6osis- Research 39 91-36; 1485: Ot%:er support _- National Insi=iates-of I-Ieai-th. -From theThro?rtbosis Resear>hCenter and Department of Physiplogy, Te!riple UnivPrsity- Health Sciencas Center, Pkiladelphia, and the- Department of B:ochemistrp - Eostcn_ University School of Medicins, Boston. AGGI'ceGA T IOPI OF CHYM(")TRYPSIN- TREATEEt THROMBASTHENIC - Pi,ATtLE-TS-'.S hiEDIATEP--`3Y-F-I_BRINCGE?i BINDING .^.-ING TO G-L-Y£a'JPRGTEINS Iar AhiDIII2- Previous experiments - demonstrated = that chymotrspsin, hut- not adenosine diphosphate (ADP;, e,~po;ec~ f ibrinogen binding sites- on platelets from patients with - Glanzma.nr.'s thrombastheniT. Three of- these patients have been reexamined, and =_ previous-observations were confirmed. - The quantity of iodine -I25--la$eleif glycoprotein IIb (GPIIb) -and -glycoproteia llla (fPllla)- on the platelets of these patients was- _ 72 t

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considerably less th au normal but- was detectable by -inemunoprecipitation, sodium aggTegat-iorn of enzy?r3e-tr_eated plateiets.-'I'z:e treatment of washed platelets of a fourth specific for the GPIIb-GPilla complex blocked both the fibrinogFn- binding and the pronase resulted in fibriPogen biriding and platetet agg-regation:° -Ivtoac-colonal antibodies - stimulation with- AI;R However, ir;cubatien-nf'-ti;ese` platlets-with chymptrypsin- or patients did not aggregate with fibrinogenn and did t:o=t- bind - 136I=fibrioogen on = antFb^.,dles was between 3% and IL4'rs of the r:orcnal-vaiue.- Platelet suspenslons frosr.-these ~ - residkal~:Pllb and G11ltIa as measured by e;iniing studies with radiolabeled-monozlonal d!sdecTl sulfate=pcyz=yryla~eint gele'e:r~rr,phnresis, ara=aotbrt.d=agrajhi= The amount of the platelet surface.- - hliewlarowski,-S. et al E+ggregation of_chyrno,rypsiri-treated throribasthenic platelets by fibrinogen appears to represent a sencitive'-test for detection of functionally act`rfe GPIIb-1PIIIa complex. on thrombasthenic` plate's by chymotrypsin are derived from- GPIIb-GPIIIa _ moleculzs: to < 0.35% to 03%of normal valres.- In conclusion, fibrinogen binding sites efposed on binding and aggregatjon. -Kowever, t#:-e-ievel_of GPllb-and `PI:Ia-on these platelets as measured by a Western blot techniq,e and by monoclonal a.-.tibody- binding amounted - thrombasthee.ic-patie*tt with ;;: I' or wiih-chymotrypsin failed to result ir: fibrinogen -- .lmonal of Laboratory-ard Clinicai Medicine 1C6(6):651-660, 1985. OOther support: National Institutes of Health and tbe-hrngricaa Heart Association. From the T hrombosis Re,earc'n_ Center and I3epar.messtif Physiology, Temple university-- I:ealth-3ciences-Ceafyr, Philadelphia Yne 4;emstologyrCOcotogy _Sect ioss= Departtnent- uf :3edicirie, L=-niversity=VT_Pe~.r,syisari;: Sckool-of-friedicne, Philaijelphia;a d-ths ;i3pitai -- Laribn_isiere, Paris, Yr: nce. ` IrJENTiEICATIrN Ci= PtAI ALLOANTIGEN DOMAI;;7 ON P. 66 kDa PROTEIN CT£I2] DERIVED r ROM GLYCOPROTEIN IIIa OF-I-IIlh-ral`7 PLA'I': .gTS - - - Identified on GP IIIa (but- not on vP IIb,;- and on a 66 kDa protein- by means of immunoblot procedures using platelet-T-riton _3i-114 extracts arid these purified proteins. Anti-P.Alaatiserutn di± u,t recognize CP IIIa or he slrface df intact (untrealcd; rbiIatelets nor the 6fF icDti protein-on the surface-of chytnoir;+psin -treated- piatelets PiA-negative ind-ividuals- The present data-demonstrate directly that the 66 kDa protein -ks derived from GP III, and contains the-PI-Al ail-oantigen. - - plateie}.s by human -a^ti-PlAI antisera. iiioreover, the presence of the P,Aj antigen -was - from detergent extracts of soiubilized,- sur-face radielabeied chymet, ypsir.-treated Is^.cl.batiofi of platelets- with -chsnpotrypsin leads te the- exposure of -fibeinogen receptors and to the appearance of a- ~ kI)a n;ambrane ~ornponent on-the_ surface of platelets: Both glyc~-promir. Ila (vP IIIa;r and-a 66 kra component were precipit_a;ed- Itornecki, E., Chung, S.=Y,- I-Iolf 3 C., Cierniewski, C. S:. T uszynski, t;. -and Niewiarowsici, S: Bioc4emfca e: 3iopi!y*iea Acta gig?$3-?9Or:yo5. - I r . - - -< Other sbpport: = National Institutes- of Health and :-;:e National Neart, -i.ung and Elood - nsti_u_e- 73 l

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grorn-:he Thrombosis Research Cente:-, Temple- Ln.versi:y--I'iea:th _Scier_e -Cen:er, Philadelp_hia, and the = Iiey.ar trt~ent= of `Psyc4i3t:; ; I<eurr~sc6ence IlesEarch Unit, University o Vermyrtt, ALrrltngtGn. -_ THE INi E RACTION O. `dU:'.'IAN PLATELET ; HRt3I:93OSPONDIN WI T H FIBRINOGEN: `I ~.#RO.MEOSPONDIN_- PtJFtIEICAT.O?J AND - SP E CIFICITY O1= II+IT;=.RAC i ION these interac:ions do_ not require cat: :um or ntagnesiuna ions. --- --- fibrinogett-Sepharpse. T-SP preferentially bound to uncross-linked fibri :, suggesting that- the TSP-fibrinogen i3in ding site is unavailable in cross-iinked fibrin. These results indicate that TSP_binds spe4if:catly ;o irrmmo?~ilize dilized fibrinogelr ~r uncros_s-linked fibrin = through determinants present in the carbozyl-tescinal- portion of the a chai-- and that yield of TSP was I,13 *_ng Q-r ap9*3zimately_ 22% of that present in platelet-rich plasena as - detertniFied by radl'JI%L1tt`.u?;oassay. It an al-zed on discontinuous sodium didP yI sulphate - gels a a single rand having apperent rnolecula= a+eights oi 180,000 and -& 4040,000 under -reducing and nonreducing eonditions; rgspecc:rely. Amino acid- sna' s:s-ga?+e- results similar to previot:sly published values. Antibodies ?aiaed'in r-bbitJ were - monospecifis; as eva:uated I y radioimr.;tnoassay. In double irnr-r:u..odiffusio1 test~,- these a.:.ibe~ies gave one line-of id2_atity against TSP purified by this procedure and TSP purified by- - publyh-ed Vrocedures, confirming the identity of the_ material -isolated. The--protein possesses no lectin-L'.ke activity. The specificitq of t}c TSP-fibr.noge- interaction was investigated. TSP binding io -fibrinogen-Sepharose occurred in the presence of-EDTAS indicating that cal_ciur-. and magnesium ions are not required foF interaction of-TSP wlti: fihrinoge5. Thn biooing of TSP io :;br=nogen-Sepha4oae wa3 -quYntitatively _ttoclied by pretreatmL:,t- with an - -anti~y to the cyanogen bromidP '-eavage ragcs-:~r~--pcsed-ol.'-residues 2-ti--4716 of the carbozyt-ter~tina: erid o#` _lia aclsain of iibrinogen:_ ?it3tiboeies aga nst :he I} and F-= dou,aitss rf fibriLaett bs,r-n-)- e__'fect on he binding. Excea;-fioinogeti (30 m-g;`ms added to -plateret extract =quantitatively inhibited binding oE TSP - to =- chrotnatography on fibrinogen eounied to c 3anogen bromide=2ctifated SeDharose. The - I3uman - platelet- t,=rombospondi:, (TSP) was purified to ->rio mcgeneity i I - -Tuszynski, O- P. Srivas: t S., Sw;taisks, H. :olt, J- Cie niiewsls:; r l ~ and R rewiarowska. S. - I - -- _ The joF:-rrra~' o! 8:olog~enr ~~err~=,tr; 360(sl t'<40 :?~45, IasS Other _aupport: U SDepart_ment of 7-Ieal*h and Human Services _and the American_ ~ Heart Asso: iation, Pennsylvania Keystone Affilrate-- ~ i - ~ ~- From the Thromtosis-R2s?arch Center and tie-Department--of Physiotogy; Temple University Schoel of viedisine, Phiiadelphi~ LOSS OF FIBRINOGEN RECEPTORS FROPrt TTzE PLATELET SURFACE ( _ DURING S3vI-UL=RTED EXTRA[ORPC ?EAL CIRr ILi?ION F= - In :rtro recirculatian of fresh human heparinized blood in a*r eztracorporeal circuit- with a membrane oxygenator decieased - f"sbrir.ogen-ind,iced platelet aggregation and- - diminished the number of fibrinogen rece; tors and giycoprciein IIh/IIIa (OPiib{'rPIIIa) antigenic sites on the platrlet ~urface. ~n seven ezperiaients, the mean 3 SD- I':, value for fibrinogen (i.e., molar concentration of fib;-inoge_rn required to cause 50% of- the maaiTat rate- of aggregation) was 1.58 x!0'' mol/L ± 0.65- u_ i"u-z mol/L. After recirculationt this value increased to 3.g x-10'' mol/L ± 1.94 x 10-' mol/L-(P = 0.035). I 74

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I ® The maximal aggregation-rate of chymotrypsin-treated pIatelerFS airrcreaaed by 40% afte: 2 howrs-c= rezculat=on (P t_ p.0'S). The nu,0ber o= -fiorinogen -:eceptors on- DlateleEs, which _wKrz rreaied_ with cltyanE?yT+sin after recircu,at+vn, decreased frcn: 41,370 ± 24,000 to 13,230-± 10:230/platelet under the same conditions (P < 0.075)._ The number of antigenic_rites for etscnoclonaE antibody reacting weth vPIIb/GPIIia com-Plex of adenosin,~-diphoskiwte-stimNiatet platelets dereased from 84,200 s 5,440 to 19,5u0 ±- 9,iE0-/plat:,let after rec,.culatie (P 3 0025,).: Prostagtandin El (0.3 µmol/F,1- in the perfosem circuit preserved_the=ab-ihty of platelets to react with fibrinogen. in conclusion, re lcss of fibrinogen _ecea:ors fron -the surface of platelet membranes results r:am the interaction of plat-aets with thy surfaces of per.`_nsion-circuits V-= Adusiar1, J., 1:'iewiaroxskt, _S., Htrshock,- D., Morinelli, T. A.; Coln:an,-_R. _ W., =and = - Edmu. ~,-L. H ] : . _ - Joi:rna_1 eJ La_boralAr~_,nd Clinical Medicine 105:514-522, 1985, Other support: Na ional .nstitute - of 3-Eeaitn.- From :he Departzent= of Cardiov;scular- Surgmy, E3nivWity. of Pennsylvania Hospital and the Throanbosiw- Research esearc.h Center, Temple University Health Sciettces -i.enter, Pl:iiadelphia _ RAD/flIMMfJNDF.SSA S' OF I;"llN!-AN PLATELET T;idi`JNtBOSF~:NDIN:= DIFFEItENT PAT''E3??3~S -OE-Tl-ikOMBvSPON:iIIv' AYN):i-A=1:23i3A:@f?- .~'a- - LG`Bif'_=_I"; PlKTIvE;i-SEGRE.?'I7N AND Ci.Ets.RRlVC1TFE4'-,IL'- T;iE CIP.C'~--'.i ATIGi, A method for raitioimniunoassav-_of human thrombosponrin _ was developed- Iy4onospecifit p ec piiating anti-human ihrombusp;,ndin antibody was raised in_ rabbits after iniecti_on 6f thFonibosponQin purii ied by fibrinogen-agarose chromatography and preparative sod',um-d-x'ecyl- sulfate-polyacrylnn3ide ge1 electrophoresis T'r.e linear portion of the thro:ali :,spondin radioitr:°ninoassay staLdard curve was 0.5- to 20 r.g/ml. Normal rlatelet~ a:_d-lat7et-Root plasma contained 26,900 ±14,500 -ng thrombospondin per 10° -platelets _and 60.6 ± 10.7 ng/ntI (besa r SD), respectively. Using radioimmunoassays for ,q thirmboglobulin and thsombospo ndirs antigen=, we compared platelet location and secretion- of these pro=eins. B'sth antigens shared _ similar distributions in platelet subcellular fr3ctio ns- with the -largest -amount localized to platelet o-gcanu:es. With thiombin (0.25 U/ ml`! as a platelet agonist, b2.4% and -19.5% of total fl-thromboglobul:n and thrombospondin, respectively,, were secreted_ from suspensions of washed hun-an platelets. Because oniy 20% of the total platelet thrombospondin was secreted, further studies were initiated to determine whether-the remaining thrombospondin became- - Iocalized ot_+= the - activated platelet mPmbrane. r=sE-Fab antithrAmbospondin specifically bound to activated platelets- but not to unstimulated platelets. In contrast, _t=rl-Fab ar.ti-B-thromboglobuiin-did not_ bind to activated platelets. Plasma clearance of human fl-thrombogl-obulin (half-life- fast 7.6 minutes, slow 56.6 mihutss)_and of-hum9n thrombospcndin (hal:-iifefast 29.9 minutes, slow 190 minutes) foiiowed a Esiphasic exponential curve. In conclusion, both fl-*_hromboglobul:n and thrombospondin are located in platelet a-granules, but they show a-difl`erent pattern of secretion and -e-xpression on tne- platelet membrane_ and plasma clearance. Switalska, H. I., NiewiarDwski. S. et at. Jmrnal-oj Laboratory and Ctir.ical Medicine 106:690-700, 1985. 75 ® 1+. 1 -

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Other supPcrt, National Irts-Litutes oi' Heat-h and the- jacmerican Heart-Association_ _ From the "I':romhosis= Research= Center, Departnjents of-=Fhysiology an;r-_Medicine, Ti enepfe Universit y HealthScienees=Center, Philadeiphia. _ r= agents that have been causally associated with- heart disease. Thi3_ study was undertaken to determine if cigarette use intensifies the abnormalities of myocardial funztion_ and coniposinon-ozserved in ezp_tiriment-a3 alc6holism-over att-lg-h-,dnth period. Young adult 1=__ male beaglRs :vith-tracheostonty were d_ ivii<ed_inio_ four groups. There were 10 controls t- -- (group 1); 9 smoked oked seven tigarettcs. per day (group 2); 7 --vere fed ethanol as 20% of - thanol and cigarettea ;group ti1.= After a period ~ calories ?group 3), and 6 received both e Chronic cigarette use -is common in-persons_who habitually= use other cardioactive -- _ , of Ig months, left yentricu,'ai'- function was assessed under anesthesia. Heart rate. -leFt __ r- ~= ventricular end-diastolic pressuresand -vrlumes (indicator-diiution)did-not differ in- the-- four group&, An indez_of contractility derived by-nor:.,alizing peak dP/dt for pre-_ and = ~ after-load was reduced significantly below the level of 2.a ± 0.7 cm/s in control -dogs- to 1.41 ± 0.35 in group 2, 1.= + 0.3g= in group 3, and.2g :_ 0.1, in the ethanol- cigarette -~- ~LOStp (each p< n.~,7~). _-~~-.rie[ial p`essldres Werei..[iderately eie`.~alc; above-group 1 in all- - ~ three ezperimentaL gro- p< .vlthr ut e+iderc,e of left vent lar hype troph} In, contrast - to smoxsng,- whrc--r elr r d- =o abnormalities of +mvoca dial ea! cionAoSit on, e:hanoI reduced myocardial rx~t ss;u~ and sodrtm in- g,oup 3w:CFioest a 5l.. of '.ter =:rnten*. " ~-_- In group-d, no further decline of tissue c,t!ont was observed. Thus, cigarette use when V _ combined with ethanol over a-=rFlatively- long period, produced no- greater myocardia# t_- abnc-rnalities- thare athanel-__a;one and -ma}; ;-not=-_oe essential to the-- genesis of~ ~- cardiomyopathy in alcoiwliss.=_- F I _ - _ Ahmed, :: S., Torres, R.; and-nega.. ?.-J_.-_- _ INTERACTION-Ds CHRONIC CIGARETTE A?ND-EPHANOL-L?SE ON MYOCARDIUM F rar-a the Division -of Cardiovascular Diseases, Departrnent of Medicine and Deniewy of New Jer-sey-tviw Jersey Medical School, N.-vark. Methods for isolation,-:dentifi.ation and cuiture=of pe:Jmonary endothelial~cells_are = now routine. In the past, methods of isolation -have-used proteolytic enzymes- to- detach = cells; thereafter, traditional__ methods--- for_ eell' passaging -have_ used , trypsin;-EDITA __ mixtures. Cells isolated and passaged ~sing_-proteelytic_ enzymes==hase-=been useful in - establishing the-fie-ld and in-verifyi:,g cer-tain endotheliai properties. -However, there is- a- growing-awareness_of the role of-e.ndethelia=: cells a.n.-process:ng -vasoactive substances,-in- _ -responding to_hormones and other agonists, and-.in cell_-cell-inter-actior.s ivith- oiher- cell types of the va;cular-wall, with blood cells and with cellular' products. Consequentl J,-a _ new requirement has arisen for cells in vitrr that maintain thee differentiated properties of their counterparts irr vivo. =._,"he deleterious effects of trypsin and other proteolytic enzymes commonly used in-cell culture on surface structures of endothelial-cells such as enzymes, receptors andd junctional=proteins, as well as on-eztraceiiu!ar layers such _ as the - glycocalyz-or 'endotheEial fuzz," have 1Fd to the development of ntethods-that avoid use of proteolytic enzymes 3t bot: the isolation step and daring-sufiseque nt subcs:ture.- This ISOLATION AND_CUJT:Jr2-E OF PULMt--~-NAItY-TNI)OTiiEL1AL CELLS 76

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-chapter describes -v=anit_ionai--methoaf for lisolati ng -pulmonary -endothelial cells but emphasizes -newer ar:)roacnes usir.g-zrtechaaical har vest-and sca<e-up=using ¢n:crr,car-riers_ The nem~ metfiods elow maisenaw.c:, of Ic~rg-ternz,=.arge-sc3~_:altu cs -of ce::s that retain tns fut( comivie.~:.er,t=uf-sur~sc e.er,t=uf-sur~sce properties and that ;n airtain 'he- cobblestone identificatiot of isclated cells are thereFcre also considered as methods for_vaii-4ation of _ monoEayer- morphology and diCferentiated functional properties. Methods _ for - cultures 3uring their irt vitro 1 sesprn. Ryan, J. S. - E'nyeroKrnemtal Fleotch rersrec:ives 5b:1C3A14,-i9gk. Other support: -*Ia*.io::ai_ in+ticutes of_ Eiealth- and the John A. Hartford F"oundatiot-.-- From the ]r3epartmeri- of-Meyicine, LTnivera'its of-Miari:i Sshool_of Mrdieir.e, Miami, FL. - Endohelial_ can be-havested rronisegnrvr._ts of adult human saphenous vein in =v=5RICOSE V3 N§.AS-A _S-OURCE OF ADULT [iUMAlv -E ND01TDELIAL CELLS a varicose crtsc:itipn-Fernoved fro:n-paTiefl-ts -having sir.gie-oi bilate.al-veiri--lig-ation and- - or Primaria iasks-and--are passag^: --b_v_"removal with- a-ruFilber -palicetnan- - Tl:e_ca;ls- 3teipping. The-celis are_harvested by scraping-Aith a-scalpel, seeded on, to gelatin coated w-ith growth _fartors _ The cells grow with a tLbbiestoner monolayer morphology, possess = sultured ir thi; ~aaaer are maintained- in a gr.?wth_ -medii::a~-that is not s:p-plententaAl a5giotens-i3 co,.verting-enzymP aetivity-a-nd-react with aytitodiesTO Fpctoe 1-'lI: antigen. The cells E3uc resee zr:ghH; after rea ti_ow w~th rcio oeIo:.ai antihpdies-spceii iw for human endothelial-=cells_ "~'?~.us, siripped~ar:rv~ vea: 3egrnx.Y~ provide a re-adily -availa5;e_ t i c ceiis. -s urce o endotte -F;yarr. U. S. and White, 1.. A<- Tissur & Cell 17{l):Ii1-1'6, i985. i-ro:n the_Depa..tTient_of-Madicme, 'Urtiversityo uf kfiarr.i Shrol-of MEdic-z , Miami,_,=)e. - CJ-Ii»i-ESE HAMS-TEit UVARY i-EL-LS -A-PC7SSiBI=E REGULATORY EOLE OF SQUALENE EIsGXI-DASE IN Growth o. Cb=r~ese harnster ovaryr=Fells-I,CHtt) in _the- presence- of- 20% Iipij depleted serum (LDS} f cr_ prily -Znr -resu:ts in an :ncrease in the_syn=htsi3 b: ~j -sterols from [;4C].mevzkottate=and f:ern ,I*Qsqua7ene compared with cel's-growrL uncler normal ~ result in enhanced il`Cl sterol-cynthesis from -["-C] 2,j,-ozidosqualene. - Incul,3tion of these cells with [']rievalcnatc 1'esuited-in the accur*.tulation Q#C] squalene regardless -cells -frrr time neriod up to 42.52hr te a growth -medium _containing 20% LDS-did _ not syn thesis increases witR_tirne ot-ezposure-oP the -celis to LDS. Kowevec, exposing _these growth conditions =int the presence of-1G'.£ fetal c?..lf -seru-nn (g-C~). _ This---enhanceo' s-terol -_ - of the presence-pf ei=1=: r_LD& or FCS: = These rvsuJts suggest that=sq_ ua:ene_ epoxidase is a- regulatory enzyme ih-tne choiestercl biosynthetic pathway=in CH4. -- - Eilenberg, H. and Shechte! f. - _

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Lip=ds-19(7):539-543, 1984.- F'om the Depariment of B,ocnemsstry; the George S. Wise Facuity of Life Sciences, Tel Aviv University: Israe!_ _ UPTAICE OF HICrfi-DE;ISITY LIFOPROTEIh1-ASSOCIATED APOPROTEIN A-1 _ AND CHOLESTEROL ESTERS BY 16 TISSUES OF THE RAT_ :M VIVO AND BY ADRENAL CELLS AND IY:EFATOCY'I'ES IN YITRO- Vliichotestero- ether and -he-ratio of ether-upta-ke :o-apo-A=I uptake were greater in - moieties in both cell tyl>ea showed sa,urability.- Both=the absolu;e rate of aptakN of -The disp:oportionate uptake of HDL--cholesterol ether relative-to HDL apo A-i was also observed in primary cultures _i`if rat-adrenal ceHs and hepatocytes. - Uptake -of both cholesterol ether and-apo=A=l were about equal in-the- other tissues (except kidney). ovary and- liver:-7-fold, 4-fold, and 2-fold greater, respectively. The rates of uptake of -- Cholesternl ether wastaken-;:p at a greater-fsactionai rate than apo-A-1 by adrenal, - counterparts,-and wem well trapped at theie_ sites-of uptake: associated -with the - HDL fraction in the p!asa.a; adequately traced their unlabeled -- apo A-I, and f5H]cholesteTyl linoleyl ether traced cholesteryl es-ters. Both labels remaiaed labeled with nondeeradab!e tracefs; covalently attached- 13sl-tyramine-cellobiose traced - The uptake of high-density lipoprotein (HDL)-associated tpolipopretein A-I (apo A-I)-and cholesterol esters was estimated in 16 tissues of the rat using rat i-IDL double its recognition by receptors. However, -apo-A-I ;:ptake was decreased, suggesting that apo-E mediates the uptake of particles-containing apo-A-I but dces-aot contribute to the disproportionate uptaks of- ['H]cholestery! ether - adrena!-cel!s than i:rhepat.ocytes, consonant with the ir-vivo observations- Very zimilar--_ - results were- obteined using HDL biologically labeled with ;s jclio!esterol-esters.- _The- - disproportionate uptake of ;sH]cho!estergl ether was not significantl-y- decreased by depletion- of apo-E fror.r the HDL, nor fiy rEd,tctive *.aethylatioa of the apo-E to block The Journal o! Zio:ogica! Chernistry 260(2):744-750, I9S5.-- _ Other sup,oort: - A!ational-_syear3 Lung and Blood In3titute and Nation-al- InstitL4e -of - Geneca! MedicaL Sciences. - From the Departnient of Medicine, Division_ of Endocrinology and -Metabolism,- 'Jniversity of Califor nia at San Diego, La Folla-. ROLE OF THE L-OlrlDEINSITYLIPJI'R'JTEIti RECEPTOR-Ihl PENETR-ATION - OF LOW DENSITY -LIf't`?EROTEIN-INTO R ABBIT AO R TIC WALL _ --The_present study was designeti to'dete*snine _.vlrether binding cf :ow density lipoprotein= (LDL) to -endotheliat LDL reee-pcor;, contributes significantly -ic-the r- -penetraiion of LDL.into-normal-rabbit aorta. initial flux rate was used as a measure -of -- uptake of LDL. - Reduct;vk=me3ltylatiotj -of LDL is -known rto block= its recognition by the {- - LDL receptor. T.p.erefore -ihe difference- ih-x`iux-rates of native LDL and-reductivelji L - methylated f.I3L (*iet,iyl-LD ) was assumzd to represent - the receptor-dependent _ uptake. Native LDL and methyl-LDL were labeled with diff reM isotopes-(-=6I or 13ii) A b th- w th e ini ct t ltar e~ sl i ~- d ir t i bhi i 3~ 60 ~ Af _ er _ _ ar o e e s i u u y n o sa ne r a t. ter _ to m nutes, trichlr,rncetie acid-precip;t6ble counts were determined in-aortic specimens. The initial_ W flux- rates, expressed as plassma clearance (nl; g/hr ), were 17&2-for native LDL and 1924 -_ ~ 78 ~ ~ ...- ... - f ~ S1/

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for methyl-LDL. The difference was-not significant, which suggests that the flux of LDL into the aorta-is not significantly dependent upon, or re$ulated-by, endotheli3l LDL receptors, but i.t mediated by ather mechanisms. Wiklund; 0., Carew, T, E. and Steinberg, D. Arteriosclerosis 5:135-1iI, 1985. Other support: National Heart Lung and Blood Institute_ and the -National Institutes of General MedicatSciens;es. From the Department of Medicine, University of-Califcrnia at San Diego, La Jolla. CONTRIBUTION OF ACTIN TO THE STRUCTURE OF THE CYTOPLASMIC _ MATRIX The realization that actin is a constituent of naamuscle cells -(I) and the- identification of actin filaments in the periphery of such cells (2) opened up a new world of molecular biology. For the-first time, it became possible to explain in detail a body of - phenomena described over two centuries-conceining important aspects of cell shape, movement; and consistency. Although actin comprises only one of several intracellular _ -fiber systems of-cells,-lt is an extremely important one. This essay briefly reviews_some present concepts of thesontribution of actin to the 'cytoplasmic enatriz." - - Stossel, T. P. The Journal of Cell Biology 99(1):155-215, Ft.2, July, 1984. Other support: U. S. Public Health Service, the Muscular Dystrophy Association, and the Edwin S. Webster Foundation. From the Hematology/Oncology Unit, Massachusetts General Hospital, Department of Medicine, Harvard Medical School, Boston. NONMUSCLE ACTIN-BINDING PROTEINS _ Because the workings of actin-binding proteins depend upon the characteristics of actin itself, the first section of this article summarizes current knowledge of actin assembly. The ensuing -review of actin-binding proteins- classifies these proteins according-to their functions, as has been done previously (Schliwa 19g1; Weeds _1982; Craig & Pollard 1983; Stossel 1984). There are actin-binding proteins that tind predominantly to actin-monomers ar,d-inhibit-their self-assembly into-filaments. Other i,roteins bind to one or more actin monomers_ when they are in the form of filaments, either to the ends of the- filaments or in some instances to monomers within the filament, causing the filament to break. They then remain bound-to one end of the fractured filament. Still other proteins bind simultaneously to actin monomers in different filaments, which causeS the actin filaments to be cross-linked into_ particular conformations relative -to one another. The word 'predominant' is very important here because as work progresses on particular actin-binding proteins, their functions tend_ to become less 'pure' and more- complex. This review emphasizes biochemical data concerning the structural and functional properties of actin-binding proteins and their interactions with-actin in vitro. Some effort is made, however, to speculate on how these interactions might operate in vivo. 3iossel. T. P. et al. 79 8 . :=;i~

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Annual Review of Ce;!-Bioli,gy 1:353-402, 1985.- Other support: U. S. Public HzalthServ:ce and the Edwin S.-Webster Foundation. _ From the Hematology-Oncology Unit, Massachusetts General Hospital, Department of Medicine, Harvard Medical S<hool, Ltostcn. COAGULANT A'TIVITIES_OF PLATELETS IN_CORONARY - - ARTERY DISEASE Platelets have been implicated in the pathogenesis of coronary artery disease, and a number of studies have examined platelet function and coagulation parameters in patients with such disease. We have exantined platelet coagulant activities, volumes, and aggregate ratios in 23_ patients with chest pain, seven of whom had normal coronary angiograms (group 1) and 16 of whom had angiographically proven coronary artery disease (Group II). There were no significant-differences in the mean values for platelet volume or platelet aggregate ratios between een the two groups. The platelet roagulant= activities concerned with initiation and the early stages- of intrinsic coagulation were- significantly increased in patients in group 11 as compared with those in group 1.- No I significant differences were -r.oted between the two groups with respect to prothrombin i time, partial thrombopiastir time, and plasma levels' of fibrinogen and coagulation factors V and VIII. However, the mean activity in plasma of antithrombin III (but not the level of antithrombin III antigenj was significantly lower in patients of group II compared with group 1. Overall, our observations provide evidence for an enhanced contribution to the intrinsic coagulation system In patients with coronary artery disease. The platelet coagulant hyperactivity not4d in these patients may reflect a role of platelets in the pathogenesis of coronary artery disease or may be secondary to the underlying arterial disease. Rao, A. K., Wn?sh, P. N. et-al. CirculQtion 69(1):15-21, 1984. Other support: Ciba-Geigy Corporation and U. S. Department of Health and Human Services. Medicine, Temple University Health Sciences Center, Philadelphia. - From the Thrombosis Research Center and the Cardiology Section, Department of _ ACTIVATION OF COAGULATIDN_ FACTORS ON THE SURFACE OF PLATELETS Platelet physiology and coagulation biochemistry have traditionally been studied and conceptualized as separate and distinct entities.- This -briet - summary of platelet-coagulant protein - interactions presents an alternative viewpoint which emphasizes the effects of platelets in coagulation and the effects of coagulation proteins on platelet physiology. These interrelationships are so intimate and important that any consideration of the hemostasis mechanism that fails to take them into account is meaningless. Walsh. P. N. Haematologia 17(l):55-66, 1984. 80 C.^

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Cther support: -Put,lic Health-SErvice. - From the Thrombosis _Researoh Center, Department of Medicine, Temple University School of Medicir.e,-Philadelphia. - PLATELET COAGULANT ACTIVITIES IN DIABETES MELLITUS: EVIDENCE FOR RELA'I7ONSHI_P-BETWEEN PLATELET COAGULANT HYPERACTIVITY - AND PLATELET VOLUME - Platelets have been shown to be-capable of initiatin g and promoting-some of the reactions in intrinsic coagulation and have-been implicated ir:- the pathogenesis- of vascular-lesions in diabetes mellitu-s. We have eza_mined-the contribution of olatelets-to the coagulation reactions in 39- patients- having diabe?es mellitus with- and without retinopathy [background retinopathy (BR) of proliferative retinopathy (PR)1: 18 patients with nonvascular eye diseases (patient controls),-and 38-normal subjects, In comparison with-control yatients;_significant elevations were noted in four-of the-five assays for platelet coagulant ac.ia=ities in diabetic patients with- PR and in three of five assays in patients with BR. Thus, platelet coagulant- hyperactivity is present in- diabetics with retinopathy_ Significant elevations weie noted in two--of the assays in -patients without retinopathy (NR), suggesting that plateltt-coagulant hyperactivity may not necessarily be secondary to the vasculopathy. The predominant abnormalities noted were in- assays reflecting_ the contribution of platelets to the early stages of intrinsic coagulation rather than to later stages of prothrombin activation. in addition, the mean platelet volume was found_to be significantly greater in patients with PR and=Bl'c but not NR than in normal controls.- A positive_linear correlation (r = 0.81)-was-ncted between the ?nean of the coagulant acti-vities-_and platelet volume - in- diabetic subjects, _ suggesting a hitherto undescribed relations-hip between platelet coagulant activities and platelet volume. Rao, A. K., Goldberg, R. E. and Walsh. P. N. The Journal of Laboratory and Clinical.Medicine 103(1):82=92,1984. Other support: National Institutes of Health. From the Thrombosis Research Center and Department of Medic-ine, Temple University School of Medicine, and Retinal Vascular Unit, Wille Eye Hospital, Philadelphia. THE POSSIBLE ROLE OF PI,ATELETS IN BYPASSING THE CON T-ACT PHASE OF BLOOD COAGU°LATION- - Data presented -herein and previously -support an active role for p-latelets in promoting the interaction and activation of the coagulation proteins of the contact phase of intrinsic coagulation. T-he-platelet membrane, activated by ADP collagen or thrombin, can promote the proteolytic activation of factor XII to factor XIIa in the presence of kallikrein and high molecular weight kininogen. The zy_m__ogern factor XI associates with high molecular weigh kininogen in plasma and becomes bound to a site-on the membrane of thrombin or collagen act;vated platelets. Thereafter, platelet bound factor XI can be proteolytically activated to factor XIa either_in the presence of factor-XIIa or in the presence of kallikrein. These observations could ezplain the absence of bleeding complications in -patients with -factor XII deficiency. - In addition, platelets- contain a molecule which has a higher molecular weight_than plasma factor XI and possibly consists - ~0 Gt

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of a tetramer of four idantical subur:its of 52,000 daltons each of which is functionally and immunologically similar to plasma factor XL Since this molecule is present in the platelets of patients with-severe plasata-factoi XI deficiency and no evidence -0_f bleeding; we postulate that platelet facto: Xl can substitute_ for plasma factor XI in hemostjsis and -possibl,v_ account for the considerable variability in clinical severity observed in -patients with factor XI deficiency. Walsk, P. K., Tuszynski, G. P.,°Greengard, 1. S., and Griffin, J. H.- ffaenratologia l7(2):169-178, 1954. -Other support: U. S. Department of Health, Education and Welfare. From - the- Thrombosis Research Center, Temple University School of MediciAe,- Philadelphia, and Scripps Research Institute, La Jolla, CA. ASSOCIATION OF FIBRIN W%TH-THE PLATELET CYTOSKELETON The authors have -previous!y postulated that surf ace membrane proteins =become specifically associated with-_the internal platelet cytoskeleton upon platelet activation.- - Four lines of evidence support this general hypothesis since we now show that elateiet surface receptors for _ fibria = become specifically- associated with the platelet-= _ Triton-insoluble cytoskeletons:. 1) -Fibrin was detected immunologically in the washed ' Triton-insoluble cytoskeietons`af= thrombin-activated platelets under conditions where fibrin polymerization and resultant precipitation_were blocked with Gly--Pro-Arg-Pro,- a synthetic peptide that_ inhibits pol-ymerization of fibr in monomer. 2) Radiolabeled-fibrin bound to throia bin-activited= platelets and became associates3 with the cytoskeleton. 3) The amount of radiolabeled fibrin bound to thrombin-activated- thrombasthenic plateiets- and their cytoskeletons- amounted to about- 20% of the fibrin bound to thrombin-activated control platelets and their cytoskeletons. 4) The association of fibrin with cytoskeletons and with the platelet surface was nearly_quantitatively blocked by- an I antibody prepared against cytoskeietons (anti-C), an antibody against isolated mentbranes ~-- of Pronase-treated platelets (anti-MI), and a monoclonal antibody to the platelet surface giycoprotein complex, GPII4-GPIII-(anti-GPIII)- These antibodies blocked ADP and thrombin-induced platelet aggregation as well as thrombin-in.iuced clot retraction:- -Analysis of-the immunopreciRitatesobtained with-anti=C; Anti-M1; and a5ti v^-IJI from detergent extract of ri9I-surface -labeled--piatelets- revealed that these antibodies recognized GPIh-GPIII. These data suggest that thrombin activation of platelets res;:lts in the specific association of fibrin- with the platelet cytoskeleton, that this association may be mediated by the GPIib-GPIII complex, and that these mechanisms may play an important role in platelet aggregation and clot reiraction-induced by thrombin. Tuszynski. G. P., Kornecki, E., Cierniewski, C., Knight, L. C., Koshy, A., Srivastava, S., Niewiarowski, _S., and bValsh,-P: N:_ _ The Journal oJ Biological Chemistry 259(8):5247=32t4,-1984. - Other support: U. S. Department_of_ Health and Human Services. From the Thrornbosis Research Center, -Temple University School of Medicine, Philadelphia. 82 Q

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AMIDOLYTIC ASSAY_ OF_ HUMAN FACTOR XI7N PL_ASMA: - CO1vfPARIS Oh.' WI_TH A-COAGULANT ASSAY AND A NEW RAPID RADIOIMMUNOASSAY The traditional coagulant assay for plasma factor XI suffers from a relatively high coefficient of variation, the need for rare congenitally deficient plasma, and a poor- correlation between precision and se nsitivity. We have developed a simple functional amidolytic assay = for factor XI - in plasma using the chromogenic -substrate PyrGlu-Pro-Arg-p-nitroanalide -(S-2366). After inactivation of 01-antitrypsin, Cl inhibitor, and other plasma protease inhibitors with CHC!s, plasma-was incubated with kaolin, in the absence of added calcium, which iimited the enzymes formed to those dependent on contact activation.. Soybean trypsin inhibitor was used to mimize the action of kallikrein on- the substrate. Once the reaction was complete, co=n- trypsin inhibitor was lssed-to -inactive factor Xlla, the enzyme generated by exposure of plasma to negatively charged_surfa_ses which had activated the factor XI. The assay is highly specific for factor Xl, since plasma total deficient in that zymogen-yie!ded only i%-3% of the enzymatic activity in normal plasma under identical conditions. The requirements for complete conversion of factor_XI to XIa in plasma within Fi0 min were, respectively, factor XII, 0.6 U; ml. Preka!likrein was not an absolute requirement for complete activation but diai~ccelerate-the reaction. The intraassay coefficient of variation was 3.4%, and the mean_ of 35 normal plasmas was 1.00 U ± 0.24 SD. _ In addition, a new rapid radioimmunoassay was devised using staphylococcal protein P as the precipitating agent for a complex of.factor XI-antigen with monospecific rabbit antibody. The mean was 1.01 U± 0.30 SD. The correlation c-0efficienu for- amidQlytic versus coagulant and amidolytic versus,redioimmunoassay were _ =_ 0 Q5 for the former and 0.96 for the latter. -Thus, a simple, accu_ ate- amidolytic assa-y_ ar,d a-radiounrsunoassay have been devised -for measuring factor XI in plasma that correlate, well with the coagulant activity of factor XI, as determined in our laboratory. Scott. C. F., Sinha,-D., Seaman, F. S., lYYelsh,--P. N., and Colman, R. W. Blood 63(!_):42-5'0,1984. - -- - - Other support, National _insti*.utes-0f Health. - From the Thrombosis Research Center and Hematology-Oncology Section, Department of Medicine, Terhple- University School of Wted-icinE, Philadelphia. INTERACTION OF COAGULATION PROTEINS WITH PLATELETS: - POSSIBLE SICiNIF'.CANCE FOR IvfAIhITENANCE_OF HEMOSTASIS AND PATHOGENESIS OF THROMBOEMBOLISM - - The purpose-of this chapter is to present some of the evidence that platelets possess binding sites for a variety of coagulation proteins and that these specific- platelet coagulant protein interactions can promote zymogen activations at various stages of intrinsic coagulations including the initiation of intrinsic-coagulation: In additior: studies are presented that are consistent with-the_ hypothesis that these interactions of coagulation prote-irs with platelets are important physiologically in promoting normal hemostasis-and pathologically in predisposing to certain venous and arteiia4 thrombotic diseases. Walsh. P. N. - ln: Atherosclerosis Reviews 12, Raven Press, NY, 199-4, pp 129-143. 8.i - 1- l'

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Other support' U. S. Department of Health; Education 3,.d Welfare. _-1 From the Throinbosis-Re';earck, Institute, Department or_Medicine, Temple University _ School of Medicine, Philadelphia. ACTIVATED HUMAN PLATEi-ETS DISTINCT FROM THAT FOR FACTOR Xi - BLOOD COA-OULySTI-OIe~-FACTOR Xla BIND3' SPECIFICALLY TO ASIT-E ON binding was detected. -Factar- XIa-binding to-p atelets was partially reversible and was - saturable at=concentrations of added Factor-Xla of 0.2-0.4 ugjml- (1.25-2.5 yM).ThF number of Factor XIa- binding sites-on activated- platelets was=estimated to- be- 225 per platelet (range 110-450). --W-e conclude that specif ic, high affinity, saturat;ie binding sites - for Factor -XIa are present on activated platele-s, are distinct from-those previously demonstrated for Factor XI,=attd require the presence of higk molecular weight kiniaogen.= platelets, were-incubated with Factor XIa, ortly a low level of nonspecific, nonsatuvable- -- 50- to 100-fold- molar excesses of=-unlabe3ed Factor -Xla prevented- binding, avF.eteas Factor XI, prekallikrein,_-=_actor. XIL, and prothrombin -did not:- When washed - erythrocytes, added at-concentrations calculated_to provide=an equivalent surface-area to - Binding of- 12617-Factor XIa to platelets required-the presence of high molecular weight kini_r.oger:, was enhanced avhern platelets- were-stimulated with thrombin, and reached a plateau after 4-f_+ rttin of incubation=at 37C.- Factor XIa binding was specific: Ot!,er- support: !'dational- Institutes of Health and American Heart - Associaiion, Pennsylvania Affiliate. Sinha-, D., Seaman, F. ;, Koshy, A:; -K-night, L. C., and- L='alsh: P. N. - Journal of Clinical Investigatdon 73:1550=1556, 1984. From the Thrombosis -Resear-ch Center, Section of Hematology/Oncology, Department of Medicine, Temple University School of Medicine, Philadelphia. FACTOR V: A PLATELET CYTOSKELE T AL-ASSOiriA-TED PROTEIN cytoskeleton through receptors present on the platelet ;et.°face. The following evidence- supporu this hypothesis: (a) Prio: secretion of Factor Va is necessary for the association of Factor Va-on the cytoskeletorr -(b)-Fteagents that-inactivate Factor Va on the surface of the platelet, such as-EDTR and proteolytic enzymes, also inactivate the Factor Va on -the cytoskeleton. (c)--T-h:e piate-tet Factor Xa binding- sites= (i.e:,- Factor - Va) are quantitatively retained- on the platelet cytoskeleton. (cbj Platelet cytoskeletons prepared from platelets that are thought to be deficient in_Factor Va binding sites contain 2391, of Factor `i a- activity of dtorm.a-l -platelet-cytoskeletons-and platelet cytoskeletons prepared from platelets-deficier.t-in-Factor Va contain no Factor Va activity but-regain control levels of Factor Va only whetr Factor Va is added to the platelets prior to the preparation Platelet cytqskeletona - prepared - from thrombin activated plateiets contain specifically assoctated Factor Va. We postulate that Factor Va associates with the of cytoskeletons. Tuszynski, G. P. and Wolsh, F. N. Haeraatologia 17(1):67-76, 1984. 84 f., ..;,. a

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Other support: - U. S=Department of Hea-lth-and Human Services. From the_ Thrombess ' Rese_arch Cetster,--T_e_mple University - School - of Medicine, Philadelphia. THE PLATELET CYTOSKELETON CONTAINS ELE_ MENTS_!f`iF-_= THE-PROTHROMEINASE COMPLEX Triton-insolubl_e cytoskeletons prepared from thrombin-activated platelets were - found to potentiate --the -activation-- of_ prothrcrnbin-- (prothromb'snase -_ activity). Cytoskeletons prepared from red ceils-or lymphobl_asts contained fio- ~prot-lesombinase activity. -The platelet prothrombinase activity w~ss dependent-on -cytoskeletal-associated Factor Va, and ezogenously added Factor Xa and prothromisin. Cytoskeletons contained _ 38% of the total platelet prothrombinase activity: Both platelets and sytoskeletons displayed I+alf-mariral -a ctivtities a`.- similar prothrombin concentrations: - The role of lipids in the cytoskeieta]- proYhrombinase activity was _investig2ted. Cytoskeletons were iound to contain 1.8% of _the total-plafelet-phospholipids, consistin; _5f the following lipids expressed as-percentage_of_total presE-Zt-in platelets: 6.0% sphingomyelin, 3.g°.~, phosphatidylcfioiine, 2.9% nhosphatidya--ethanolamine, 4.4% phosphatidylinositol, and -2.2% -phosphatidylserine. The cytoskeletal- prothrombinase activity- and the lipid ith phospfiorus- content of cytoskeletons- derreased_ -after_ treatment of cytoskeleto_ns wh various doses of phospholipase C. Incubation of -c;toskeletor:s with the highest _ concentrations tested:.(10 pg/ml) resulted irt a 72% loss of phosphatidylserine and R4% loss of cytoskeietal prothrombinase activity. [ ytpskeletal prothrombinase activity destroyed- by phospholipase C-_treatment could be restored to control levels by tre3tment- of hydrolyzed cytoskelet-ons with total cytoskeletal lipid or mixtures of phosphatidylserine/phosphatidylchoIine (25:75ti, by-weightj. 'These-results suggest-that the cytoskeletal prothrombinase complex in addition to containing Factor Va, as has been - previously shown, contains - a lipid cofactor activity consisting in part of phosphatidylserine. _ _ Tuszynski, v. P., Maucos, O. P., Koshy A., Schick. P: K., and 4Volsh.-P. N. The lournsl of Biological Chemistry 254(1 ! ):6947=69Sl, :984. Other support: U: S. Department of Health and Human Services. Frota-the Thromtrosis- Research_ Center, :emple=-University School _of Medicine, Philadelphia. COMPARISON OF=FLEEDING-TENDENCY, FACT OFL XI COAGULANT ACTIVITY,_AND FACTOR XLANTIGEN IN 25 FACTOR Xl-DEFIi_IENT_-- _ KINDREDS - The relaiionshin of c4irical-bleeding tendency and-fastor XI antigen (XI:Ag) in factor XL def iciency was studied in 78 -members of 25 factor X-[-deficient kindreds. Factor- XI:Ag was measured in a competitive radioimmunoassay using monospecific, heterologous anti-factor XI antibody, 1S6I-labeled factor XI, and staphylococcal protein A as the precipitating agent. Deficiency of factor XI clotting activity (XI:C), < 0.62 :J/ml occurred in 48 individuals, 22 of whom experienced postopera*_ive or posttraunnatic bleeding. Their mean factor XI:C was 0.21 ± 0.04 tJ/ml (SEM) and factor XI:Ag was -85 a

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0.23 ± 0.04 U/ml. The ren?aining- 26 had no clinical bleeding, many despite surgical _ challenge. Their mean -factor XI:C -was 0.30 ± 0.04-3J/ ml and factor JCI:Ag-was 0.34 ±__ -control. In conclusion,-bleeding tendency_ap_nears to_be consistent within a given kir,dred and-is not determined eacltisiyely-by factor XI:C or factor XI.Ag levels r differ from control a5d all points feit within 95% confidence limits .~.enved from line for factor XI:C and factor XI:Ag data-poi*:ts in t_he 7g individuais tested did not - appeared to be positive for- _r_e-ss-reacting material (CRM). The slope of the regression had none with deficient hemostasis. Two heterozygous factor XI-deficisnt individuals - 0.05 U/m1. In all, i3 kindreds had_ between 1 and_ 11 members with bleeding; the other 12= -- Ragni, M. V., Sinha,-D., Seaman,-F., Lewis, J. I?., Spero, 3. A. and, Walsh, P. N. _ Blood 65(3):719-734, 19E5. From _the Department of _Medicine, University of ?ittsburgh; Central- Biood_ Bank, Pittsburgh; Thrombosis Res=arch _-Center, Temple University _Schooi -_of -lvledicin_e, - Philadelphia. Other support: National Instit:rtes_of Iyealth: FUNCTIONAL C'1-IATtAC'I'EFcIZzTION OF HUtdIAN-BLOOD-CQAGULATION FACTOR XIa USING I:YBRIDOMA ANTIBODIES. During the initiation_ of intrinsic coagulation, factors - XI-, and- XIa _interact intimately with several other -coag:iiation proteins (factor Xlls, high M; _ kininogert,' and- - factor IX) as -well as_ with_ ~the-platetet snrface. To, help__ elucidate , these complex - intramolecular _interactions, _~e have prepared a eoilecticn_-of, mcnoclo: al -antibodies directed againsc various epitopes in Factor XI. We have utilized these reagents to--isolate -- ?'actor X: and_ the--light chain of factor XIa ort affin'rty- columns and to probe - stracture-function relationshipa-involved in the_ interactions-of factor-XIa< with-fac*.or IX. The isolated light chair._ of factor Xia retained > 90% of its amidolytic activity against the oligopeptide substrate pyro-Glu-Pro=Arg-pNA (5-2366), but only 3.08% of -its clotting activity in a factor XIa assay and 1%-of its factor IX activating activity- in an activation peptide release assay. -TF.is suggests that regions o:' the heavy chain are required for development of _coagulant activity and specifically- for the interaction of factor XIa with factor IX. To test this tiypothesis, the effects of three of the=monuclonal antibodies (5F4, IFl_, and-3C/) on-the function of-I'actor XIa were etami ned. The results show that in a clotting assay the light chain-specific antibody (51=4 j inhibits-I0i1% of thc factor XIa activity, whereas_of the heavy, chain-speeific_-antibodies, one_ (3CI) inhibits 75% and another (IFI) only i7'16. Similariy,-in the factor IX activation peptide release assay antibody 5F4 inhibits 100% of-the factor XIa activity, whereas 3C1 _ inhibits 75% and i F1- inhibiu 33%. We conclude that regions located-in the heavy chain, ir-s addition to those in the light chain, are involved in the in*,eraction of factor XIa with factor IX and in the expression of the coagulant activity of factor XI. Sinha, D..-ICoshy, A.; Seaman -.£., and Wulsh. P. N. The Journal of Biological Chemistry 26DC19):107 1 4=197 19, 1985. - OOther support: -U. S. Departinent of Health and Human Services, W.W. Smith Charitable Trust, American Heart Associatiob_ Biomedical Research, and Division of- --Research- Itesources, National Institutes of Health. From the Thrombos3s Research Center, Department of Medicine, Temple- University School of Medicine, Philadelphia, PA: 86 ~ ~ ~ ~ ~

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PLATELET-MEDIATED COAGULANT PROTEIN INTERACTIONS IN HEMOSTASIS From the evidence reviewed here, it-_is-p3ssible_to censtfuct an overview of the- I.emostatic mechan>sm-e.Lphasioing_ -tI:e -essential fiontributions _cf platelets in zymogen activations at_ taelt sta$e of intrinsic coagulation. -Specific high=affirity recr_ptors for various coagulatior, proteins are- exposed- cn _ the platelet surface - membrane. The coagulation proteins- so far shown to bind the platelet membrane ineEude the factor XI-higft-mol wt kininogen_ compiex, - factor XIa, the factor= Xa--tactor --Va complex, thrombin, and= fibrinogen. -As a c£nsequence- of these interactions, platelets promote the activation of prekaliikrein, factor Xii, factor XI, factor IX, factor X. and prothrombin. :'iatelets also°appear-to part:cipate-in a variety-of ai3ernative reaction mechanisms that may minimize or obviate the requirement for certain coagulation protei ns, including factor XII and factor X'. This function of piatelets may -account for the absence of hemostatic defects 3n patients with deficiencies of some of the proteins involved 'an- the contact phase -of= iiltr=:nsic= coagulation. Finally, platetets appear to protect certain coagulation proteins, such as factor XIa and factor Xa, from inactivation by plasma prote5nase inhibitnii. -T'nese various functions of- platelets are seet;- as localizing coagulation reactions-to the kemostatic plug 3hat occurs at loci of blxd-vessel:injury. Walsh, P. N. Seminars in i#erats+logy, 22(3):-176-196-13g5. __ Other support. National I-nstitutrs of Health and American Hear t Association. From the Thrombosis_ Research Center, Temple Urzi`ersity_ School = of---Medicine, Philadelphia. POTEN MATION OF THE ACTIVITY OF COAGULATION .JLATION FACTOR XI BY HUMAN = PLATELETS The -presebt studies-demonstrate that when- washed-platelets or platelet membranes are incubated with ;mrified_factor XI and the mixturesare assayed for factor XI activity, a 20-fold potentiation of-coagular;t activity is observed. This synergism is dependent on the presence of active -factor XI and intact platelet membranes since it is- abolished either by- preincubation of -factor - XI with monospecific anti-factor XI antibody, or-- by preincubation of platelet membranes-with phosphotipase C. - The saturable nature of the potentiation of purified factor XI by platelet membranes suggests the possibility that it represents a functional correlate of the saturable, specific binding of factor XI to platelets previously demonstrated_ This suggestion is supported by the demonstration that platelets enhance the rate of proteoiytic activation of factor XI in the presence of either factor XIIa or kallikrein. Walsh; F.-N., and Tuszynski, G. P. Thrombosis Research 40:257-266, 1985. Other support: National Institutes of Health, American Heart Association and W.W. Smith Charitable-Trust. From the Thrombosis Research-Center, Department-of Medicine, Temple University School of Medicine, Philadelphia. g7- , I

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EFFECT_OF PROfTANQIDS ON THE :.=ORONARY=CIRCULATION-= An accurate evahla_ ot. of the physiological and _ pathophy3ioiogicaL role of = endogenous prostanoids iwthe human cor,r__ary_ sirculation has to- rely upon the join*. results fromstudiPs usingdifferen, techniques._ In-the present paper some data-obtained in this laboratory are presented, which are-aimed to elucidate-the physiological_ and patnophysiological :nvolvement=of-endogenousprostanoicis -in the reguiation of_ coronary blood-flow. -Futhermore, some data on the pharmacology of PGI2 in-patients--wish acute myocardial infarction-(ALciI)_ are included. -Base_ d on the data-presented- here and on -the -_ considerations discussed it seems obvious that coronary prostanoid formation is -limited both in hez-khy sub jecra ard in patients with IHD. This :s evidenced- both by the lack of -- detectable release of PGHz, the main coronary prostanoid formed, under -normaL - conditions and under-physiologioal stimulation -o.`- coronary tlood-'low, as-.vell as under-- conditions- of=mat.ifest caidiac=ischaemia. On=the other hand, the lack of significant release of PGL= is not due to a limited capacity to such- formation; as evident from_ the - marked release-of 6-ke*.o-P_Gc,Q in patients subjected to- cardioplegia. _ Although the eata -_ presented here do nat support the concept of a significani role for co^ronary prostanoids _- in- health and- disease the_:.-resuLw =presettted -on interaction -between adenosine : and prostanoids in the regulation of forearm blood flow may be of significance also in the coronary circulation, implying that prostanoids may play the role of -a- vasodilator messenger which transfers the-signal to vascular _relazation-from the endothelium to t?:e - vascular smooth muscxe: LVennrnalm_. A. et 41- I i In: :UPHa,R 9tr5 int. Cong.-oJ Pharrnacel.-London. 1984. = Proceedings, voL. 3, pp. 13-19, _ ~ - Macmillaq Press, London, 1984.- _ Other support: The Swedish Medical Research CounciL-and the National Research Council of Italy. From the Department of Clinical Physiology, Karolinska institute, Huddinge University Hospital, -and Karoli-riska Hospital, Stockholm, -Sweden; and Department - of Pharmacology, Catholic University. Rome, Italy. 8$ -

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IV. Neuropharmaco39gy°aQd Physiology - LEVELS AND TURNOVER IN VARIOUS IiVPOTHAL!UvIIC CATECIfOZA1vYl'NE_ NERVE TERN4INAL SYSTEMS A*?GON-T`HE SECRETION OF PITUITAItY = HORMONES IN THE MALE RAT - ° EFFECTS OF~CIIROIt3:C E-XPOSURE TO CIGARETTE SMOKE Ol4 AMINE Male rats w_ereeaposed to the smoke fron: 2 cigarettes every morning for a total period of 9 days. 'I'h~ next day they were decapitated immediately af ter the exposure- to the smoke from 4 cigarettes (Kentucky referertce' 71't-1 type) burned- at 30-min intervals. Control an'smals were=exposed to air alone or to -nicotine -free cigarette smoke (Cambridge -glass fiber fiiceri?' `:n co irrast to chronic expc suie to f;;teeec~ smoke, efposure_ to unfiltered smoke resulted in- a 10% increase in catecholamine (CA} levels (4uahtitative histofluorimetry) within ihe 7ateraF palicade zone, -the posterior periveittricular aypothalamic nucleus and within- the dorsomedial hypothalamic nucleus. There was also an increase in -amiere turndver (tyrosine = bydroxylase inhibition by a-methyl-dl-p-t~osine methy:ester (aMT) in the cloparriine (DA) systems of the medial- and lateral palisade zones and in -the periventricular noradrenaline -(NA) hypothalamic systems. - Chronic exposure to nnfilterefi cigarette smoke resulted in - reductions of prolactin, LIH and FSH levels (radioimr:unoa3say). -Eollowing nlviT treatment, chronic e_xpasure to unfiltered cigarette-srr3oke stil! led to reduced prolactin serum leveis: In addition, an increased vasopressin seru~n concentration wa s found. T ne effects of chronic exposure to cigarette smoke_ _on- neuroendocrinr function- and on hypothalamic CA systems are -s::agested to be mediated via- i:icotir__e. Combined with the results from a previous study, the present results indicate that toler ance does not -deveop __ with regard to t_he inhibitory effects of exposure to cigarette smoke on-prolactin, LIi and FSH secretions. The same is true for the`stimulatory-effects on the tubero-i - n=fundibular DA neurons and the periventricular NA systems. - But chroi-ic exposure to cigarette smoke seemed tc indece tolerance with-regard to its stin:u!atory effects cr- subependymal dorsome- a! and paraventricular hypothalamic NA systems' a_nd- on corticosterostesone release. Andersson, K_, Enero+h,=P:, Frxe. IC.,-Mascagni,-f., and Agr,ati, L. F. 14'Frsroandck:ri .;;,ogy 4-i(5):4b2-466, 19855: From the Department of ]-Iistology, Karolir.ska izystitute, and Research and -Deve!opment Laboratory, Department of Obstetrics and Gynecology, Karolinska L'_ospital; Stockholr_r_; Departrnentf of Human Physiology and Endocrinolr,gy, University of h4odena. Modena, - Italy. EFFECTS OF ACUTE CENTRAL AND PERIPHERAL ADMINISTRATION OF NICOTiNE-ON AS-C-ENDING DOPAMINE PA°I'HWAYS IN THE MALE RAT - BRAIN.-EVIDENC£-r°"OI-2 NICOTINE INDUCED INCREASES OF DOPAMINE TURNOVER IN- VARIOUS TELENCEPHALIC DOPAMINE NERVE TERMINAL - SYS T EitiiS. The actions of irttraventricular injections and intravenous infus':ons of nicotine- were studied orn dopamine stores -and turnover in discrete areas of the forebrain of normal male rats. This was donetty measuring the decline of the dopamine stores after tyrosine hydroxylase inhibition using c-methyl-tyrosine methyl ester (H 44/t~8}. The dopamine concentrations in the various-telencephalic dopamine nerve_ terminal systems were measured using the Falck-I=illarp methodology- - involving --c:iantitative 89,

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1 ffiicroflyorin:etry. The catecholattFine-concentrati6ns in the anteromedial f-rortai corte== were measured bic.=hemicaliy, using high pressure liquid chromatography sombined with electrocl±e>itical detectio::: - Intraventricular ex?erirnents. - T he dopamine levels in disc7 _ete -areaF of -nuc: caudatus and nuc.' accucitben.s were significatt:Ty -reduced- evPr w:th the lowest dose of nicotine (I pg,/rat,. Intraventricular injections of=nicotine- in a dose of 100 yg/rat produced significant increases of doramine turnover in various types of dopamine nerve terminal systems in the nuc. caudatus, nuc, accumbens, and tuberculum olfactorium, and _ following a dose of IO pg/rat, increases of dopamine turnover _wera observed in the- >redial part of the nu_. cauefiatus. Furthermore, nicotine 1It'A pg/rat) sigr.if;cantly increased noradrenaline bat not dopaminP turnover with the anterofrontal cortex.- Intravenous experime dts. The doparrtine levels were selectively= _redueed by - nicotine (1000 -µg/kg) in the cholecystokinin positive and-negittive_ oopam,_r.e nerve- term=ina7 systems of -the nuc. accutrtber_s.- 9n-the other hand, -dopamir.a levels itt the anterotaedial frontal cortex were increased after this dose of nicotine_ Intravenous infusions of nicotine f10-1Cw0 kg%kg; produced dose-related lated iricreases of dopamine - turnover in the various dopamir-e nerve terminal systems a nal; zed in the -teier.cepha!on. These effects became significant with a dose of 1000 ug/-k-g/h. The dopaznine terminals - in the nuc. caudatus showed a higher sensitivity to int-ravena4s= infusions of - nicotine, - being affected by 10-100 Isg/kg of nicotine. These- findings suggest that relatively low doses oses of jjecotine via an activation= of- central nicotir.e-like cholinergic receptors can reduce dopamine- concentration =and --- -increase dopamine -turnover in discrete lin.bi_c anj strietai areas. These aFtions may _iL part represent-the neuroehemical basis for the rewarding actions_of nicotine and- for ricotine dependence in man: Anderssop, K., Fuxe, K.: Agnati, L. F., and Eneroth, P. -Medi-car Biology 54:170-173, 14$1: Other supporl. Swedish Tobacco Company. -From-the Department of Histology, Karolinska7nstitute, Stockfiolm; Sweden, and the University of Modena, Mode-na; Italy. EFFECT OF MATEP..NAL NICOTINE ON THE DEVELOPMENT OF SITES FOR [3H]NICOTINE BINDINC'r IN THE FETAL BKAIN The sites -for ['H]nicotine binding in fetal brains-- were examined after administration of nicotine into pregnant rats. Administration of unl_abeled nicotine into the pregnant- rats increased B.. values for the sites for tririared nicotine binding without affecting Kd valui3-in the fetal brains. Treatment with this regimen, however, did not show any significant change-in_ the sites for ['H]quinuclidinyl benzylate binding. In addition, treatment with this regi:nen_ increased Bn~ values _ of the sites for ['H]nicotine binding-in the brains of pregnant rats. :r-Bungaro_toxin had no effect on the sites for j?H]nicotine bind'ang.- It is inferred, therefore, that a similar response is elicited by nicotine binding sites to administered nicotine in both the fetal and maternal brains. Furthermore, a possible effect of nicotine in pregnant rats may be the facilitation of the development of nicotine- acetylcholine receptors in the fetal brain. Hagino, N, and Lee, y. W. International jow.rai of Developmental Neuroscience 3(5):567-57:: 1985. From the Department of Cellularand Structural Biology, The University of Texas Health Science Center at San .4ntonio. 90 - i ~ -~ ~ ~ 0 ALIT

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THE ROLE OF MA'I'ERNAL- NICf3'TINE-IN TFIE_DEVEL OP7.4ENT OF NICOTINIC AL`ETYLCHOLiNE RECEPTORS-AND HYPOTHALAMIC 1?OP_AMIlyE4GIC NEf iRONS IN THE`.FFTAL-B;iAIN It has- already - been reported that_ delayed development of hypothalamic dopamineagic-( T ID-A) r._eurons in the fetas brain caused a delay of _ differentiation of prolactin cells and pu'erty orset i:~ offspring uf rats. From observztions mentioned, it seem_s likely- that maternal neurotrat.s!anters -and/or hormotres tnuid- influence the development of_ TIDA tteurons- and-_differentiation of 3rolactic cells in offspring. Preliminary results ind icaL _that-mate:nal and placental acetylchoiit.e ;Ach1 seem to act on nicotinic sites to_activate the nicotinic acetyicholine- receptnrs (nAchR1 :~nd inflsence - the development-of_TIDA _neurotts in the fetal brain, This study tends to lend support to_ a vvorking--ltypothesis thar.nti_hlss appear ie the fetal biain di,r Lg nn earlier stage of gestation. During the tast week of gestatifla, maternal and-placenta7 Ach act on nicotinic sites to activate Ca="=fsua .~hich-stimulates TIDA neurons throughh activation o('- protein kinase and phosphor:,latio:£. _7 hue; t;aters.al nicotine could irri7uenye the_ growth and development of -'i IDA -r-euroas in tha fetal brain. _ /isrg?Ro: N. In: Caciagii, F.,_ G-iacnbini, E.,- and Paoletti,_ R. ;eds.Z Developmental Ne4rosrience. Physrological, Fnarrrtacotogica!- and Clinical .fspeets, hlew- Yorlr. Elsevier Science Publishers _B. V., 19$2.-pp- i 2'1=1 3i;. From the I2eparknipnt of Cellular and S.rnciural Biology, The-Uaiverity of-Tezas Health Science Center, San Antonio. [3H}NICOTINE BINLSINv SITES IN DEVE:..OPING FE T AL CR.'~I?:S IN t`tic iS [sH]leticotine binding sites were examined in developing fetal brains in rats. The fetal brain- membranes bo'?nd_(?I-i}nicotine w ith a~n affinit, si_pila to that of adult brain membranes.- This binding_ was displaced by unlabeled nico: ne or earkaenylcholine,- the inhibition concentrations being approximately the same for fetal and adult brain preparations. a-Bungarotoain had no effect in [=H}nicotine_ binding to fetal brains membranes as- as eo adult brain rrsparatio ,s: _The specific (sH}n_icotine binding _ v as first detectable on day io of gestation and increased several fold until birth. _- Sttgiyama, H., lragi-to. "1. MoorP G.s and Lee, :. W. ' Neuroscrence Resea? c_ti-2.387-392, 14is5. I - _ i From the Department-of Cellular and Structural Biology, The University of= Texas Health -Science Center at San Antonio, and the Department of Cellular Physiology: National Institute of Physiological Sciences, Okazaki, Japan. GUANINE NUCLEGTILSES REGULATE [?~I1,S:JIISTA_NCc: P BINDING IN RAT _ SMALL INTESi%NE - The binding of ('H}substance P(SP)_to membranes of the rat small intestine demonstrates specific binding-to-recep_tors_having more than=one affinity for SP.- The values of the binding parameters for the high-affinity site obtained from a non-linear - 91

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regression analysis are as follows: K5 = G,2<S nhl, I;, ~ - 149:5 =frnolJmg pretein. Inhibition c:irves of 3H-sP-binGir_g using various unlabeled t'°^hykir.ins show frat the high-affinity receptof is o the I?-subtype -ha..ng the I<ighe<_t affinity for SF and 'cwer affinities for eledoisin and kassinir, -C=uanine nucleotides and sodium independently recep.or u li:.lCed to an ef .ector- by a GTP-bin3!r;g regulatory protein. reduce thee bi nding ef'sH-SP to the liigh-rffi .;y-:eceptor in a dos--relatvd n:atner GTP and GDP are more potent thag ON1P. -The reduct;oi of specific SP binding by GTP can be ascribed primariiy to an=i^crease :n the of f-rzte. The effects of guanine-_ nuc :eotides on 3H-SP binding to=mami?raoes of rat smail_intest3ne suggest thai the high=affinity r -- Smith Kv a d`tl s. iY F. - _ n . os ,. Regu{ator-y Peptides-i1:2?S-2g5, 1985): Other support: National Institutes of Health. From the Center for Erair_-=Research, University of Rochester, Schcol of Medicine and Dentistry, Rochester, NY. CHARACI`ERiZA~FIr~N OF MUSCARINIC CHOLINERGIC_ RECEF'FO.S IN THE 13RAINS OF t-OPPER=DEFICIENT RATS muscarinic receptors in-the central nervous system, it is suggested-thai copper, because of its ability-to form complexes with=srrm_ e proteir:s; may-hav-e an endoger.ous_role in the with honiogenates from nur:nal= animals in the presence of co-,~er. Since copper deficie.,c,- has dramatic effzcts on both receptor-number-ar_d the binding o3 agonisis to increasing IIJao and_derived-dissociatior_ constants. The addition ef copper to the- assay _ medium caused an appaienYreverraf of the in vivo effect of copper deficiency cn agonist binding,-decreasing II)so and derived dissociatio: constants to t°al~:es near those observed= to copper tr eatment of con trol homogenates. Minimally ele_f ic'ser.t rats displayed verv __ similar changes in- recepter' properties compared with -the -inoie severely de:'icie?:t_ animaRs. Minimal copper deficiency produced robust-effects on !he-birding of-agonists, In order !o assess a pqssible_ ro`e for copper as- a regulator -of- muscariric=_ receptors in vitro, the eee~Pter was character ..ec-+-_ in rats made copp deficient by- a dietary- iegin:en: in=foreh;3in, regions 3iiere-was a_ decrease in both the affinity of the receptors for VHj-l-quinuciidiny, benzilate ° and the density of receptors in the copper-deficient animals compared with control anirnals.- Copper treatment in vitro of homoge_nates_ from deficieW an3ma's did riot reversee th¢ -in -vivo, ef fects on antagonist binding but, rather, decreased receptor occupaacy_and ligand affinity ir a manner similar - regulation of ;he receptor. Farrar, J-R., Hoss, lf`.: Herndon_ R.-M., and I'=uzmiak, M. The lournal vf Jlteuioscie:rc-a=S(s):11_!g3-I0c°9, :985.- e7ther support: Nationsl- Institutes of Health. From the Center- for Brain_Research, University of Rochester School of Medicine and Dentistry, Rochester, NY.= PROT-;:IN PHOSPHORYLATION AN-'J;.IEIl-RONAL FUNCTION - Studies -in the past=;everal years have provided direct evidence that protein phosphorylation is involved in the regulation of neuronal function. -Elect:-o- 92 N

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physiological experinents have demonstrated tha*t three distinct classes =o:= protein kinases, i.e., cycl',c-Atvir-depentfRnt protein kinase,'pr_ntein k;nase_-C.,un1-CaM kinase Ii, - -modulate physiological processes in neurons-. Cyclic A.MP-depen!ter;t-pras_ei,^: kinase--and kinase C have been 3howo to modify potassiuin and calcium channeis, and CaM_ kinase 11 has-b2en 3hown-to-enlriance neurotransmiite_r_ reiea_se_- A large-number of substrates for these -prbtein _kinase& have_ been found in - neurons. .n- some cases (e.g., tyrosir hydroxylase, acetylcholine -receptor; sodium c-hannel-) _ these proteins have_ a- known -function,-waereas most of-iherri (e.g.; synapsin-l) had_:eo known function when they were first identified as=phosphoproteins: ln- the case-of_ synapsin- ;, evidence now suggests that-it reg;:Ntes-naurotr_nsmitter release. These studies of synapsin I suggest that :he characterization of ;,reviously unknown neuronal pfiospioproteins will lead to the elucidation_of-previously unknown regulatory pr[?-c?sse5 ?n nelFr4ns. - Browning, M. D., Huganir R., and Greengard, P jounal of NFeurochenrtstry i3(1):11-23, i9g5. Other suppor:: National Science Foundation, U. S. Public Health Service, and a contract from the U. S. Air Force School of Aerospace Medicine. From- the Labc_ratory of -Molecular and Ceisular---Neuroscience; The Rockefeller University, New York. EFF_ EC"i OF_NIvOTtNE ON CERE_ BRAL PF2CDTE[N METABOLISM Di_>R1Nts DEVELOPMENT -=- -__ _ Several studiesan the effects of nicotine_ on fet-al growth have suggested that altered protein syntiis_s is the underlying meshanisr_ of changes in ~etation21_ length, brain weight, or behavior. =Piscrepa ,cies were found in gross structural changes; in some instances fetal size a¢s9 brain weights were not found _io be different after exposure to nicotine. However, -most r_esults irdicate behav7oraLchangPs in the_offspr ing. _ T-he results presented here suggest= that nicotine does affect cerebral protein n-,etabolisrK irn both Geveloping-and-adult-brain.__-These researchers @zan'ened the rates of protein-metabolism -in the offspr_ing~of-rats-ad::?incstere_d-ntcotine during ges at?ofi to assess-whPthPr- changes- in weight-or protein content occur and whether they are related to slterati5ns- of protein trietabolum.- Results showed that there were.-no gross_ m-orphologicaL changes-in the offspring of rats administered nicotine during geftation. _Cortical_and-_cerebeliar brain weights were similar-to sontrol_values at 2 and 4 days after_birth. Total protein-content was also similar. However, measured synthesis rates were 10% and 5% lower in cortex of nicotine-treated- animals at 2 and 4 days after birthr, respectively. Calculated degradation rates (peYcent synthesis per h minus percent protein increase per-h; are therefore-lower ir, the nicotine-animals -to account for the lack of change in protein increase in -these attimals. Therefore,_ci the basis of-this study, it appears that nicot;le influences brain function, and that protein degradation and s_ynthesis processes in fetal-newbc>rn rat brain are affected hy--nicQtine. _ ° dn: Caciagli, F., Giacobini, -E., and Paoletti, R. (eds.) fle>elopmen.tal -?:-euroscience: Physiological. Pharmacological arid- Clinical Aspects, Nesv_-York: Elsevier Science Publishers B. V., 1984 pp. 119-122. From the Center for Neurochemistry, Nathan S. Kline Institute for Psychiatric Research, iJard`s Island, New York.

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NICO3'INR BINDING SITES IN BRAIN - In the past-lu yea-rs, many-atteTpts have been made-go identify sites in brain`that bind labeled nicotine. -Howewe_r, in -these studies rto- estimat-ion wac :rrade of saturable - - versua non-saturabte binding. More -recenx studies show curvilinear -S:a .hifr d_b1o-ts: suggesting the presence of multiple-sit2s: Nicotinic or muscaririic antagonista have little- _ or no affinity to the binding site, which has-been-suggested bvRomar,o and Goldsteit+n to be dug to an agonist-induced shift= 5f the receptor to a-hi,h=aff'snity_ agonist selective - state.- The kinetics of-binding indicate a high-affinity component with a K. in the range of 0.1-60 nM,-and -a low-affinity component wiSh af-fir_iiies thaf vary widely froni orte preparation to another.- The present investigators have contpa3ed-the binding of (±1-{sH] nicot-ine and [-H] azetylclt-ol-ir:e-in various brain regions Although ::-idbrai:,-showed the highest and cerebellum the low4st binding for both nicotine and acetylcholine; the ratio of nicotinefacztylcholine I)inding showed-a three-fold regional variation. Although - there is still much varialbi:ity in the resuits of binding studies from various laboratories, nicotine binding`in -brain is, p obably not solely related to the classical nicotine receptors. Multiple binding sites have been reported, and may be related to the multiple effects of - nicotine on the cen-tral nervous system. - Sershen, H., Reith, M. E. A., ar,-d Lajiha, A= .n: Caciaglia F:.-Cracobini, L'.,- and Paoletti. R. (eds.) Developrtental- Neurossience. Pbysiogical, Pharrracological -and Clinical Aspects, _New 1'ork: lsevier- Science _ Publishersa.Y., 1984, pp. _47--13U. From-:he Center for-,Neurachemisfv~-Nathan S.-Kline Institute for Psychiatric- Research,- Ward's=IslarC; New York. FRBi-RAi'iIC.AI MEDIATED k.L'I'ERAi7ONS OF THE SYNAPSE- cortexj were prepared and exposed to-HRP. In the absence of K+-depolarization; synaptosomes exposed to°HRP for 45 minutes take up 70% less [f'C]OA-EA. Observation of this HRP-induced reduction of syt.aptosomal neurotransmitter uptake in relation to the hlgh-affinity net:rotransmiiter uptake and synaptic vesicle tnembraIte. T h'c fact that non-K'-stimulated cerebra, cortex slices (no synaptic vesicle-plasma membrane fusion) _ which are-exposed to HRP (in HEPES) for 60 minutes_ exhibit no -reduction of - [i*C]^ABA uptake indicates that fused s~esiFte membrane i.; succeptible to HRP damage _ and that damage-tu these membranes-alte,s the neuroeransmittr transport system. In order to isolate and better study the functional synaptic unit, synaptosomes (cerebral - - morphological and biochemical data _points--to the-exis!encg of a relationship betweerr contaip.ing HRP, followed by--- 60-minute- incubation in HEPES (synaptic= vesicle recovery), exhibit- a 30% °-reduction of ["C]GA.BA uptakl and the- appearance of nonsynaptic vesicle = membrane in the nerve terminal. Ttte - combination of these acid (C,ABA) -uptake and in easily distinguishable-vesicular rnetrbrane alterat=ions=aftei recycling. Brain slices initially- incubated foc-60 minutes in depolarizing (K+) - buffer cortex- slices- -esults in both the reduction of high-affinity ['4C]-gatttma-arninobutyric recycling. Similar HR-P ertpost!rY p= the synaptic=vesicle membranT=of ():1 mm 'yerebra! - membrane to horseradish-pe.-ov:ide-(HRP` leads to=vesicular-men:brane a1-terations during containing b15 mhf -K+-propionate- and=that the exposure of tkis- exte~nylized vFsitular -_- _ Previous work from- -th-is laboratory-dentanstrated that in the frog cutaneous pectoras-neuromuscular junction,-synaptic ves'scles can-be induced to reNersibly fuse with the plasma membrang-crf-Zh_ nerve terminal by=the application of a Ringer's soiution 94 ~ ~

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IIRP -induced -syeaptic- =vesicle membrane alterations observed in mouse- cerebral cortex slices indicates that the plasma membranes of' these isclated synaptosorna units- are therqselves_ susce&tiblc i3RP-iaduced_ damage a?td_?hat_ t! r_damag nl th s ensmbrane_ also alters ueueotransr_,~-=tter uptake. Debler, E:-A., Sershen,=H., Lajtha. A-, and=Gennaro, J. F., Ir: Annals o-j1ke_New York Academy of Scieacfs_435:140-144, 1984. FFrom the iTepart:aen;-_ of Bioiogyr New York -1<fnirersity,_ and the C°}nter- for ' ' ees± York. Neurochemistry, iVard s-fsland, Iy EiLDSX`Eh1OUS,Mr1TEICIslLIN BRAIN IN?`_I_BIT.N(_s 1-?:]NICC2TIN1= AND - _[3HIAC£TYLCHDI.1_IY:_I3I-NTJINCr - The supe,rnataQt _-0btained from mouse brain honnoger.-a+es contains material _that iuaibiu the saturable binding of sh]nicotine in mouse cerebral corte--. This inhibi.ory material_was-fuether purified by heat denaturation, ulirafiltration-through an Amicon- PIvI-10 membrane filter, and_ ge1 chromatography on Sephadex G--;8. Tne_ mater~'. inhibited the binding-of ]'HSace*_ylcho:ine with the same r+otency as :t did that of ;sI}]nicotine._- It also ,~ad-so:ne- affinitY for the sites that seecif ical-_y bind_- rsH]D-A.a, D-Leu -enkephalin, -bit had_ nsuch _ lower affinity for the binding sites for tritiated quinuclidinyi benailaa'e-i;?NBr, zpiropgrid-31,-nalozone,-or _aaiP-r?*fi.ir_e: Accid ::ydro!ysiF _ destroyed the activity. -Yhese preliminary- results suggest the presence in brain of `nicotiae-like' substances, one of w_nich_may be the endogencus Iigar_d for =the zites that speclflcally bind k]I?]3icotlne, Sers1hea,-H., Reith, M. E. A., iashi m, A.,-and-Loji.4a. A. iorsrnal of Neuroscience APsearcis >; 2(4):563-569,_ 1984. _ -- - Orthee support: New York State Health_Research Council. - From the C'enter=for Neurnchen:istry, Nathan S. Kline Iustitute for Psychiatric Research;_ YK3rd's I3land, NL_'-w iFork. CDMPAFtISl1N OF f,=Kd14TIC:;~"II`~F AND '3Ii]fiC_I~t~CLCI-In1-.INF E?NUING ITv'- MDUSE BI'cAIN: REGIONAL DIS; RIBUT%-`JN In a continuina_siudy_of 6icotine-binding_s'stes, the authors delermined tlie reiative amount o!' nicotine bindingaT.d acetylcholir.e binding in. variou.s brain resipns of_ C5?/B::, and of DB-A mice. Fcthor+gh midbrain showed the highest ar:& cerebellum the lowest binding for both 13H]nicotice-and_[3 I]acetylcholine, the ratio_;,f_n:cyti_ree to acetv:c6ciine bir~ding--showed a thre~-fo!d r4giv;tai ;~ariation. Acety:c?ie:ind ishi;it rn_:f [=t'r]n:coti;te binding indicated-t:a;-8 portion of nicotine_ bisding was roi_i_r,h:bited-15y 2cetylEh.3aie. 'I'hese results indicat6-Isr.port+yt d+_fferenes between the bindir;g_of _(i)-(_3E?, .icotine and- that of [3H]acetyEchdlirse. -_ Sershen, H., Reith, M. E. A., Hashirn, A., and Lajtlsa. A. - Research Cornr.;unrcatiorrs in_ Chemical_Pathology and Qharmacol+_zgy 4g(3):145-s_5>, lyn. From the Center for Nsurochemistry, N2thzn S. Kline Institute for Psychiatric EzPsearch, Ward's Island, New York, 95 -

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LOCAL AND SYSTEMIC £`Al vAICIN PRETREATMENT INHIR'7`S SNEEtiING- ANI? THE I1sCREASF-.N NASfi:,_VA _ SC-GLAR PE :-MEAL$LITY~ INDUCLL BY rERTAIN-CRIEMIC:4; IRRITAPfT'S = l. _ I he- ettects ot local exposure to cretnicat irritants and mechanical st,mulation ~ on sneezing reflexes have t~enssudied it- normal and caGsa_cin-preirvated, conscinus guinea-pigs. The influence- oltocal and systemic capsaicin pretreatment -on vascular ~ permeability to plasma-protems- and the cardiovascular tffects of-local application of I capsaicin to the nasal mucosa have also been-studied itr ar,aesthetize8 a_r:inials. 2. Local applicat,o o capsa3cit± jt; reshotd dose 3 µM) ni otine -tthreshoid dose - 300 pM) or fcrmalin to the i:9saz mucosa induced reflex sneezing discharges. Systemic or local cacsaicin pretreatment-atolished or reduced the sneezing r2sp,nses_t_s capfaicin and formalin. The resDoese to nicotine was also reduced following local pr-6treatment with capsaicirr; whiie the response tG systemic pretreatment with capsaicin was- only slightly affected. The sneezing response to _rnechanical stimu:ation was not affected by capsaicir. - pretreatnsent. 3. Pretreatment with- °- locaF anaes*_laetiL enduced a- similar dose-dependent inhibition-of .he-sneezing tesponses to_both-cagiaiciT a nd nicotine. _- - 4. Local applicatiott of dis-~dium erur~oglyEate- to the nasal ni>zcosa ~ed_ ueed tkf sneezing'respo*rse. to `capsaic.n, but not that to nicotire: ` _ 5; Local pre*reatriart with ttie 3 rn1vF atr_d 30 mM capsaicen solution =irihibited the increase in vascular per-meaniTity to plasma-protec ns in the nasal macosa induced _ by -i.v. _ - induce sneezing. Furthermcre, thereseems to be at least two types of af_ferent nerves in the nasa - n:ucosa= which -respond--to specific chemical irritation. One type, capsaicin. Local prexreatn-iettt with capsaicin did= not rsuli in= any reductiar: ir_ tht= capsaicin-indticed permeabii-ity in the ureter, suggesting that such treatmeat did not have any majt3r systemic toxic effecs However, a smal#, acute .rcrease in re3piratory- in_=ufflatr.,:r press-u-re, -:ndicating = bronE4Lyonstriction,-e,a; seen when t:ie 30mM capsaicin-solution :va3 applied to the -riasal- _- mucosa. The application of capsaicin (3 mM arrd 30 mM) to the-nasal mu_osa -resulted i n an increase in arter:al- blood lprvawre - and tachycardia due to reflex syr?tpathetic activation. 6.-=Exposu_re oLnoratal guinea=pigs to an atmos9here saturated with ether caused excited avoidance behaviour and intense nose wipings with the fore paws. T his response _ -was abolished by- systemic yretreatrnent -with capsaicir, and reduced by local capsaicin pretreatment_ - 7. -- Local application of= 3erotanin,- #rista:nine,=° leukotr;ene -CS bradykiniit, phenyleiguan-;de, substance E_ (SP) or ;d_-Arg, d-^rrr- d-Trp, Leu]SPs and SP-antaoonist, did not induce any sneezing. High -conc=..,trations-of compounl 4£{g-~ caused_ a-smal't sneezing response. Local- pretreatment with the Ss-2ntagenist_ (7 x 7Q IR1) did not influence the sneezing res~on.ses te nicotine o~_c~~saicin. g.= it is concluded that onlv_substances that are knowrs to act'svate sensory nerves capsaicin-sen=.itive nerves:- which resper,d- to zapsai:'n, io:,,.alin, stlrer and nicotine, while another type of afferert-nerves :n:olved in snneezi:-8 ref!axes is=largely resistant to capsaicin pretreatment_ar,d_=is activated by sticotine: l ocal applicatian of capsaicin to_ the- IIasal mucosa may -thus_=ae a selective wa-y-of rediicirtg nasal- reacsiiy to -ce-rtatn- chemical irritants without causing s;stemic degeneratiot.-of capsaicin-sensitive C-fiber afferents. -' Lundblad, L., Lundbzrg; 1. A9., and Anggard, A. Archives of P#arsnaf;ol6Ey's`76:2;4-241; 1q34. Qtl¢e: -su9?ort: Swedish B+Iedical ?tesearcL Council; the Swedish Tobacco-C?mpa-ny; the- Astra -Foundatiar:; thL- Swedish Society of _ Medical Szience; and t-he_ Funds of the Karolinslca-Institute. 96

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From -the Depaftnient -of Oto-Rhino-haryngology, Karolinska --Hosprtal, and- the DeDartment of Pt~aiolog*® IC=ar3Finka :rrsti nte ;S[oeklso:?~, Swederz - EFFECTS Ci= NEUROPEPTIDE YYbI!?`-')_GN I~E('HAi3iCA1__ P,.t. ~:'`I~'-Y._AItiF3 =N£~iR6TRRNSAfaSSSION IN THE HEART, VAS DEFERENS AND URINARY BLADDER OF T1-IE GC7INEA xI-G' '_ The effects bf-yreincubation-:'c-r 10 min ivith synthetic porcine neuropeptide Y - (NPY) ost muscle t,ne- 2nd eutonc;mic transmission in the guiriea-pig right atrium; _vas deferens, urinary=bladde ; portal-veiA and-trachea w~ere"analysed in vitro, _ NP1' indused - a metoprolo?=resistant, long=la3ting; positive : otropic and cronotropic ef fect--_2er- se in the spor,taneobslyb,,-at:ng rsght atriom F;rthErn=iore, NPY casced a reversible inhibition _ of both the metopr6Iol and arropiRe-se-.aitive auricle resp~nse~ to field °tiinusl,tion (2 I-Iz- or 4 Hz for '_; v<i:hout affecting the re?rorse_te exogenous noradrenaline (NA) or -- a_cetylzholine (ACii)- -_ -= NPY did n-ot= induce any contraction of- the -vas defe_renss but inhibited both the rapid witch response and the- sustained tdni cortsa=eicn_induced by field stimulation. The NP f-induced irshibition of the tonic coestr3c:ion was`more iong-la.:ting than than of the twitch re ponsa=- The tonic contraction wa` blocked_b3 phentclamine_ard_ the twitch response by a-, P-methylene ATP -tachyphyla--is. tiPt d'ed r:c,i inhibit the_ contractile ont:actile effects of NA, ATP or a=, a-methylene ATP. NPY afso`i;,duce.d a reyersible= reduction - of thL non-cholinergic, rlot-adre.^.ergic- Co:;tra::tile response to field stimulation of the urinary bladrler. Ia ihe-poftal Yein,_NPY (up to 3rlO-7.') did no .nhi5i the spontaneous_ _ motility or the pheLtelamine-se:~sitive contractile respon_ses-to field-s,ir:iulation-and NA. ilt. atropi,.e=sensit:ve cdntracf_en o£ the' trachea or the nor.-adrzr grgic, _ non-c; oiirergic =relazL-#id5 induced by fiQ-ld stimelation vvas ne+_signifisently_znf;ueticed by NPY in doses up to S~eiO-r'A.-- I:i condiusic:i, the present data show that in the guinea-pig. NPY -ezcrts positive- chronotrop:c- and- inotroaic effects or the right -atrium -of the heart. Furthermore, NPY may rtave presynaptic effects on a_drenergic, __ cholirzrgic and non-adreneigic-n~ _holirergi¢ neurittansftsission. l.irrdberg J. Af., Hyti, X.-Y_, and r'_rancd-Cere:.eda..A 4cto Physioloxica Scn.ndinar%ca ; 21a25=332, i9E4-, tJrSer= support; Sw¢dish Medical Research Cm:rail, the S::edis't Tobacco Company, Astra -Foundation, and t:±e'Rarolinsk-a Institute. - Frotri-the Departnien't of PharrLacblogy, 1Carolinska ifistttutf, Stockholm, Sweden, CAPSAICIN-IND'aTCED STIMULATION OF THE GUINEA-PIG ATRIUM: INVOLVEMENT OF A NO_s E L- SENSORY T2ANSMITTcR OP A i)i12ECT- AC'CION ON M; OCYTE;" - l. The u:echanism underlying the positive inotropic ar!d chro:~otropic effects of- -capsaicin were investigated-rsing the spontaneously beating guir:ea-pig_atrium in vitru. 2. Capsaic-in induced a long-tasting stimulatory effect (threshold dose- a0-4~-M). -_Tetrodotoxin, phentolaming, 6-OHDA, mepyramine plus cimetidine, methysergide-, indomethacia-, somatostatin- or morphine pretreatment and= local --treatment -Kith capsaicin on the vagal nerves did not reduce the capsaicin response while it was abolished up to-i month after systemic capsaicin pretreatment. -- 97 -

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3. The capsaici~ response was subjec, to a-rapid-tachyphylaxis: During capsaicin= tachyr,hylaxis-, the positive inotropic ard clir5hotropic_effects_vf°noradrenal_ine, serotonin and histamine were uncha::aed. 4. Varioua neuro'-ptides were i.vestigated---wi.h regard -ta cardiac activity. Physalaemin, eledoisin and somatostatin had negative ittotropic and chronotropic effects. - SubstanceP, bombesin, ha.sinin, C{'{:=8 or PI-II-(up to i~-s M=o f each) did not :,=ause -ny detectable response on-.~te'guinea-pig aaricle, while :I:e- substance P antagor:-ist [D-Aig, D-Pro, IT-Trp, #.eu]SP and nced, a long-lasting stimulation of heart activity. VIP also stimulated the heart. = 5. _Yarious -adenyl compounds `were- also tested.- Adenosine;' A-taP; ADF,= ATP and ,4-, ry=rinethy-lene ATP pad`negative chronnotopic and inotropic efects; while a-, 0-trtet- -- --, - - hylene ATP- taduced -a_stitnulatory response. - During a=, t?=methyleoe_ ATP -- - cannot be excluded. AdoaeusitiYation pheaomenon-.vould-then-also ec-ci_r on possible receptive-s'_tes for capsa;c=n on the myccytes. iapsaicitt Fretreatmmn:=rrtay tisss i?duce - very long-lasting, sfiecific; f uncticnal changes in hea ; furction: . transmitter, including substar.ce P and ATP. The=c-apsaic'sn-response was- abolished by --capsaic_i_n, pretreatment,_whi..i is ktt;wrn to cause_degeneratlon .T chemosensifive nerves ° in the heart. This_sugge3ts Ahat tapsaicin may re7emeo*her bioactive subs*.aTces- thatr substances P from -sefisort nerves. AdiTect action of- capEatcin-'o: cardiac nlyocytes -- tachyphylaxis; tha' auricles s_aI- respondad to' _capsai<.in. The inhsbitor3' efftects of adenosine and ATP xnaingaes were antagoniced by theophylline -and g-p-suiropi:e~syl theophylline. Capsaicir induced a- sttjal! - release of' - Iabeled -=--nucleotides from 3(H)-adenitle=prela_5_ e:Pd atria -from- corltrot,=-but not - f r_om capsaici.T-rr et-r eaEed =.-_- - animals. 6. GTP, aspartate and kainic acid fup-co It3~4M; had no effect or the guinea-piB a_trium, while $lutamate haa a Qegative inotropic'-action. ' In eonclusion, the 6resent fi_r:dings show a~ecifiu stimulatory ac;ion of capsaicin on heart function.- This effect-does not seer to be mediated via any - classical A.rchsvesoJ Pharmacology _12-5:I76-182,i984_ Lw:dbera. J W Hua, Y., _ar, Fredhntrm-, B. B. :.ther-suyQort: Swedish l6d;cal Research Couneil,=the Swedish Tobacco Compatsy,-t?±e i-ians oc.h Loo rJstermans Foe=ndation, the Astra Foundatio n, a nd the R.arolirski-InstiTute. From the Beparement of Pl:ar-n:acolbgy, Karoli.ska institute,_Sto,Yholat, Sweden. - COMPARATIVE i--pi:!"L]NONISTOCI-IEIviICAL AN-~-s BtOs^.HE?:;ICA:.. ANALYSIS- - OF PANCREATIC POLYPEPTIDE-LiICE PEPTIDES WITH SPECIAL REFERENCE TO PRESENCE OF NEUROPEPTIDE Y IN CENTRAL AND PERIPHERAL NEUTRONS Antisera raised a-gaifiit= porcine -rFeuropeptide Y(?IPY)-and peptid=e- YY ir'YY; were - characterized with regard tc itninuno~istoctiomisal- staining, cross-react_ivity -to several pancreatic polypeptide (PPi-related peptides, and radioimnuu nz=assayab=.e tissue levels in the rat and pig. The- N-PY 3ntiserum (102R) reacted Yvith nerves in many areas of both-- the-cent_ral and peripheral oervo-us systems, but it did not ;tai,- endocrine--cells- of the`= pancreas or intestine. i+Io--e4idence for any sross-reactivity of the-NPY aatiserurrt avith- related peptides -of the PP f-amily, such as avian PP, bovine PP, =PYY: 7-hQSH, _ FMRF amide, or avian PP (1't=s6), waF-obtained. The Tv'-PY arrt7serum was N ierrttinal!y -_ directed, and regional level3_of NPY as seen by=radioimmuttoassay paralleled welf ihz - wcurrence of NPY=-immuobrreactive-structures- seen in the dmm-unohistoche~mical study. _= High pressure liquid chroT?.ography analysis revealed that the NrPY-itnmunoreactivc 98

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ntaterial= from cerebral cortex and vas_deferens had elution_profile- sim-ilar- tc-- those _ of standard ~or_ire R`P3':' t~.-:E PV;' antisetusri -rr:ain!y-staiaed tndotrine--_cells- in the pancreas and intestine as v:ell as a -small neuron system in- the braistem of the -rat. Although this a*itiseium -fiad_a slight crosa-rm-a; tiv-i;y to :dPY in radioigtn:unoassay- the neurenal PYY stsinia&_wa.s-sep,r4te fiomthat of NPY. High levvls of PYY were foufid in the_intestine, and-ievgls aboveYhe thresholdweee also seen in the dorsal vagal complex of the-eat. Thevther antisera invest_igated-(raised against avian PP, bovine ,P1; ry-1vISH, and ; h4;:F-aei,iie) caused neurona! staining ths-t- was-abolished -bu= preat;sorpttoe- with NPY. This was also s_en even if no detectable-cross-r;=activity rvith N^Y-wa` found in radioimmunoassoy. These latter antisera also stained endocrine cells in the pancreas and intesti ne with con:plep-eross-reactivi'y.eelationships; suggesting ihe presence of intestinal PP-like peptides in addition to PYY and P:PY, Lundberg. J. M., Terenitrs, L., Hokfe!t,-T. and Tatemoto, K-.- _ The-lourna! of Neuroscieosce 4/?:2376-235, 194, Other support: Karolinska Institute, Astra Foundation, Nationb!-Institute of -Neurological and Comcnunicate Disurders and-Stroke,_and the U. S. National Institute of _Menial Health. Frorrm the Departments of Pharmacology, Histology -and Biochemistryr_ Karo!inska Institute, Stockho!m, - Sweden; and- the Department of_ Pharmacology,= University of Llppsala, Sweden. CORELEASE OF VASOACTIVE INTESTWAL POLYPEPTIDE AND PEPTIDE HISTIDiNE-ISOLELTC!NE IN-RELAT-IQN TO ATRCPINE-RF.SISTANT VASvDILATItlN-IN CAT SUBN!ANr-~IBL7LAR SALIVARY GLAND Parasympathetic nerve- stimulation of the submandibular salivary gland in the cat caused salivary secretion, vasodilation and a corelease of vasoactive intestinal polypeptide (VIP;-and pep:ide histidine isoleucine (PHI) immunoreactivities (IR) into t~e vtnous effluent, as indicated by an increase-in output. The ratio between the released_ VIP-IR and PHl-IR was close to 1:1. .r',~e!-perr.~xaticn-chromatograp hy of-plasma -from the submandibular venou- eff!uent-indicated that the released VIP-1R acd PHI-IR were -very-- similar to- porcine VIP a?±d-PHI, respec:iveiy.- Atropine pretreatment enhanced_ output of both VIP-IR-and Pl:l-?R during the parasysnpathetir-h-erve-stimulation to- a similar extent f,about-5-fold, compared -tcr control stimulationE. T his increase could be due to an _ inhibitory presynaptic muscarinic receptor regulation of VIP and P:{l release. Since VIP and PHI are present_ir the same postganglionic parasympathetic ner3es in the g'.and- and both peptides have va;odilator activity, the present-data suggest that both 1~1P-and P'_i, may- contribute to the atropine-resistant vasodilation seen- upon stirf?ulation of -the _ chorda-lingua nerve. T he parasympathetic control of salivary gland function may thus involve- a- multimrssenger- system with the classical- transmitter acetylcholine and the peptides VIP and PHI-;_ LundOerg, 1. M., Fa?rrenkrug, J., Larsson, 0., and Anggard, A. Neuroscience LeBters 52:37-42, i9g4. Other support: P:-CarL Petersen's Fund, N4:'i.) Foundation, Swedish Medical Research Council, Swedish Tobacco Company, Astra Foundation, Swedish Society-for kledical- Sciences, Karolir±ska institute, and Swedish Patent Revenue Research Fund. - 99 ~

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From - _the _= I?epartr!tent_ _ 2o:'. -Pharmacology; - - Karoiinska° Institute, Department of tlinical Chemistry,-Bispebje_g Hospital. Copenhagen, Denmark: - Oto-iti.ino-Laryngol_ogy,= Karo fi2si;a IiosDital,- -Stockfiolrn, Sweden; L3epartme~t ~f COMPARISON OF CARDIvirA-SCLiLAR AND BRCrNCI:C7CONS'I"R1C`1t;R EFFECTS OF_SJ$STANCE P,-StJ-BST,4ENCE K AN'S UTI-lyR TACI-IY/CINtrIS Hua, X.=-Y., : srnaitij~g. J. M., i_r.eoslorsscn Norheidr; ~.; and Brod',n. E: have rather similar-hypcten_sive action, while the vascular permeability increase _tp-plasma -_ proteins _is especially pronounced -after physal?eenin-and -eledc~isor: _SK, kassi:iin and eledoisin have prominent qronchoconstr.ctdr effects- Neuromesfin K, _howeysr, dispiay- -_ poo; activity in the present mode#s.°_ T he -existence of novel tachykinins suclr as SK in _ addition_ to SP in- arammaiiarr tissues- suggests- that -effects _ seen- "upor -antidromic- stimulation_of 3ensory iierves.rttay be'caused by-seve:al structurally related peetides: __ a.- Bombesin increas-ed- insu-f ;ation vressure_wit-h-no clearcut effect on vascular_ permeability. ?. It is concluded that in the same -species; r d, guinea-pig, several- t6chykin-irs permeability in various organs-than=`5;' and S1i. The maxiral pcrrr~eabifity-in-~ra_asing - effect c(_Sk was-stnaller t~:an =that of SP, altho~3gh the potFncy was similar. - e-ff6ct.- -Physaiaemin and eledoisin were- generally more potent in_ increasina- vascular increasing respiratory- irsuf iation pressuse.- The effect of SK had a particularly ior.r, duration. Neuromedin-K o_rAy induced a a.eak-incrRase-in ir[suffiation_pressure at a very _ . - _ _ - - -` - -- high dose. - 5. Ah tachykinirs except neuromedin IE induced an increase in vascula-r permeab'llity to plassra pro_eeins in many visczral_ organs,-as indicated by-E.-`ns -blus eztravasation. The trachea and ureter were the most 3ensitive organs with regard to this - 4. SK_, kassinin and_--gledoisin were more-potent-thai SP-and physaiaemia in tachycardia. Neuromedin K.(up to 40-nmol x kg-1) did not=influence-`heart rate. -_ 1. The effects- of substance F-(SP} substance K(;K); physalaemir_., eledoisin, kassinin, neuromedin K- and bombesin --Jn -blood press:~re; F.eart rate, respiratory - insuff!ation pressure and plase!a _extraEasa-tion were stt:died in the goinea-pig. 2. AIl-tacfiykinins except neuromedin K caused a fall in b?ood pressure w:th rather - similar potency. The hypotensive respo nae- after p!:ysaiae:ren wes comparatively more long--lasting. 3.-_S1; and eledoisin (2,3 nmol -x kg`' i.v_) caused an initial br3dycardia which- then changed into -tachycardia.- The other tachykinirs induced a slowly de}eloping Other sa:pport: Swedish Medical -Research Council, -the Swedish Tobacec" Company, the Astra Foundation; and the-Ka_olinska Institute: Archives cj Pharmacdlogv 32gJ9b-201,_1y84.- From the Department of P~rarmacol,;gy, Karo:iuska institute, and the Department iof Clinical f hemistry, Karofinskylqospi-tal, Stocklolm, Sweden. ° (IR) occurred in the same autonomic neurons in the upper respiratory tract, -tongue and C9 EXISTENCE OF PEQI1DRIiI (PFIi) AND VIP IN NERVES REGULATiNG BLOOD FLOW AND BRflNCHiA-L_SMCOTIi-MUSC LE TttN-IN-VARf(SLTS MAMMALS INCLUDING MAN By immunohistochemistry it was found that_ PII-I- and `JIi like- in;mur.oreactivity 100 0=

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- sa-l:vary_ glands= i,ritk-ass,Gciated- ganglia in rat, guinea pig_ cat, pig and man: VIP-- and FHi~ilcs in~rraunbrea:~,ivit3 were aise found ir sirt:il_ar locaiions in=the human heart. "he lV-tez r inally_directert, but not the C`-trr:ninallV-~irected. Prli antiserum or ehe- VIP antiserum stained endocrine cells in the pig duodenum. m. This suggests the existencE- of- an additional PFII-iike peptide. Ligation of nerves -acstely caused marked averlarping asona: accum.ulatiors of PHI- and VIP-IR _ centrat to the tesion. Two weeks after transection of the= t;erves; both types -of _imr_r,tinoreactivities_ were still ebserved in _ accumulatiotts both in the azons as well as in the corresponding cell bodies. The levels of PI-I-I- and VIP-IR-ice_-nocral tisspes frc:rn the cat were around 10-50 pnscllg with a molar raseo of about I_ to 2. Systen:ic administration of PIi1 and VIP- irduced- hypotension, probably due_to perijheral vasodilatation in both guinea pig and cat. Further?rz-ore, both 3HI and VIP caud?j an inhibition of the vagally induced increase in respi_-atory - insuffiation-pfessure_in guinea pig. Flil and Vie relaxed the guinea pig trachea i r rrtrU, - suggesting a direcfaction on tracheobronchial snioothrrsuscTe. VIP-was abost- 3-I0 times more potent than_:'IiI with regard to hypotensive effecu and 2-3 fold, considering respiratory s,uooth°rnuscle-rela--ana effects in the guinea pig. °HI was aborr. -50-fold less p:,tent in i_nducing_ hypot_e_r,sion in the-cat than in the guinea pig. Ali hougk species _ differences seem to-exist as regards bioiogic=l potzncy; PHI should also be considered when examining thg=-role of VIP as a n auto-r_omic ne-,:rot_ransmit,e.. Pekttd?s3:3p3°5D6, liS4-, Otker_suypert: =Swedish=lk.edieal Pesearcn Cosincsi, ~svei±ish 'i`o#iaccc Cotnp2ny; NOVO - FoundatioQ A rs FuundaticAn, a:rct-er .:arulinsliQ Instituze. --RELEASE OF SUBSTANCF: P- AND SUPSTANCE-K-LII~E IMiv.~GTi~~TIES FZOIl~1 TI-iE ISOLATE-i~ PEpFt?SEL~ - GUINEA-FIG LUNG In this study; the authors offer_direct e-vidence for a-release-of=SP- -and- Si4-like- -- ime unoreactiyities (I l; -bv -cFesniea'- irritation ieYn the =isolated perf sed guinea-pig lung_ Chemical irr ;a,ion of tissu?s was achieved by perfusion v3-ith- b::`rfer-co-tainieg- _ _ _ , 1 µtil capsa icin. SP-LI-waslneasured=with th; _rrtibodyK'1vrhi:h-does-r;ot cross-react to SK. SK-i Iwa; _rpeasured with tha_ antibody K12-g307 which does _no;- show eress reactivity to S, . Infusion of capsaicin indufen'a several-fold -increase irr the outflow of SP-L I and SK-LI. Capsaicin, a strong irritant of the_respiratory tra.=t, excites and, irn high doses~ selecuvely destroys -sensory C-fibers. Furthermore, a calcium reo,uirrng_ release oFSP from ser._so, y neurons of the -guin_ea-pag ureter by capsaicin has been _previously ~e.^ons_trated. Tlius, the pYESent-rESu'_tS indicBtE tha$ 5K-LI in -the respiratory t_ract is, like SF= c9^taiFed-in primary afferent C-fibers. Capsaicin-sensi:ive-_ C-t"ibers-sre respo nsible for both bronchoconstricti2n and tracheobronchiai ed_ma after -che:nical irra* t on.= SP has be°F postulated as urie med'iator=of these responses rnainfy because of_ the inhibitory effects=of. SP-antaganis.s wv hich, however, inhibi t also actions of ethertachykinins. ' _Sa?ia, A., Theodorsson-Norneira, E.,-C;arse, R., and Lurrur7erg.7_ n uropeart J-nurnal of Pharmlico'pgy t-G6:Z0?=2Jg_ 1935. Othgr sud,oort: _ A-;strian Sciertific Research Fund,-Swedi_h Tobacco Con:pany, Pe3rus and Augusta Hedlt:-r_irts- Foundation and the Sweiish Medical Pesearch CounciL - -101

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From-the Detrartr:_et3t-of .'_Exderi.aerztal--anQ_Clinical $hsr_rtEaeclogy, Jnivcrsira_ of_ Graz, Graz, Austria; Departmert- of~Clirrkat 1hemristry; Karolnslca :Iospitgl ano_ Department of Pharmacology, Karofiins3tt Instii,cte, Stockholm, Sweden. DIFFERENTIAL EF~"FEM GF RESRPINE AND 6-HYDRtJXYD(;PAMINE GP+- ItTiJP.GP-EP_'TIi'-E Y(A:PYj AND NC3ItAI)RENALINE IN PERIPHERAL NEURONS !. The-effects of ti-hydroxydopam3nP (6-OHDA) and reserpioe-pretreatment__on _ peripheral neurooeptide° Y (NPY)- and h_ cradreriai:n@- (NA)-containing neur oe±s wEre studied in guir;ea-B gs- 2. Ten days -after 6-GHDA pretreatment a 6i-a046 red.:otion_ of thR NA content was observed in the_°right atrium of the heart; stellate ganglioh and spleen. The content _ of NPY=iike -immunoreactivity (LI)-vvas reduced by about S0~ in_ fFi_e h~art, .arsd. was unchanged in the sfslse.:-wlzile it i*:csased to 200% of co ntrol-_inthc sietlate ganglior.. Immunohistechemis*_ry showed a= pronounced ` loss of Ni' Y-_ arid tYrosineF.ydroxylase- (TH)-immunoreactive (IR) nerves i:r-the heart but not in the spleen. Increased NPY-IR was seen in axons and cell bodies of-the stellate ganglio n. -_ 3. Reserpine pretreatnent (thres hold dose 0.5 mg X kg-') caused a dose- and time-dependent reduction of the content of NPY-LI in the heart. A- maximal tiepietion - of NPY-LI {abdut 80%) --vas obser ved 5 days -after reserpine. R eser pine- pretreatment als~ reduced the co nter:t o.`- NFY LI in t:e spleen, wi:ile -,~o sign~--idant--changA was observed in the ad-enai gl-asd or vas defrrns=- The levels o? NPY-L.i increased -ia the stellate ganglion to about t-00% of oontrol-5 days after reserpine. Itnmunohistochemicai analysis teveaied an altnov total loss -of NPY-1R nerve fibres- in the -hear t as well as around blood vessels in the lung and skeletal -muscle. No detectable -changes yeere observed in perivascular .NPY-IR nerves in the spleen, vas deferens or kidney. - TH-IR nerves remained unchanged after reserpine. indicating that the observed loss of NPY-IR nerves was due- to a depletion of NF Y and not a degeneration. _ 4. No change in the-levels of substance P-Li was observed in the right atrium 5 days after reserpine. - -- 5- NA was, ia-contr2st to NPY, markedly depleted_i-r._ali tissues investigated -afte-r reserpine treatment. The'.deple;icn-of -NA was more extensive, and occi:_rred more rapidly anct at-much lower doses as cornpared to the effects on NPY-LI. - -- - 6: Ligations of the sciatic nerve revealed that NPY-LI was transpor-tel axonally with a rapid rate (3-r-im/h;r. Reserpine pretreatment significantly inereased the-amcur,t of accumulated NPY-iR above the ligation, suggesting-an increase in axonal transport. -Z. High performance liquid chromatography revealed that the NPY-LI consis*.ed_o` _ two major peaks in the ste,late Ranglia, while only one peak closely corresponding to- porcine NPY was seen in the right~triurrj: g. In-conclusion, 6-OHDA pretreatment depletes NPY-LI in certain terminal regions and incra..ases NPY-Ll -in ganglia. _-Reserpine =induces a tissue- and- -dose-dependent depletion of NPY-LI in-certa-ir: terminal-areas, while corresponding cell body content and axo.ml transport of the peptide seem to increase. - Lundberg. i. M. et al: - Archives of Pharmacology 339:331-340, 1S&5: - Other support: Swedish Medical Research Council, Swadish Tobacco Company,- Astra Foundation, Swedish Society for Medical Sciences, the K-arolinska Institute, and Austrian -- Scientific Research Funds 102 -

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From the Departments of Pharmacology-and Histology, Karolinska Institute, Stockhoim,= Other- .rr.ppors: Swedish Medicai- Ftesearch Gouncil; hviedish--ToYEcFe Company, and P esrus and August-a=Hedlunds Foundation. Lundblad, L., L_undb4rg,J. Af.,Anggard, A., and-i-et'eistran,;l- 01. European Journal o_1-Phar,nQco[`ogy 11t3?E,1-3's-2ri985.- -- -, From the I3epartryents of Oto-Ithino-Laryngoiooy and Thoracic Medi_'sne, Karo!inska_ Hos);ital,-a^d :xtartn:eat-of Fharmacoiogst lt:aroiinska?nititute, Stoc3):olrn,Sxeden. - i $weden; Departtnrat= of £z~rimental=aed iainicai Pharnacoiogy. U~ivtr ity of Graz, -Gr.aa, Atsstr-ia; Department of Pharmaccibgy-, 'F3*siversity of CJpvsala, -gwefen;- and the °- = I3epa_rtment ef-PsychiaTry, New Y ork University Medical Cente-r,- New- York. CUTANEOUS ALLERGIC REAC!-:Gt7 IN MAN CAFSAICIIv= PRETIREAT1vIE1Ti' INHIBITS TI:E-FLAI?-E Ct?Iv?PO?dENT-.~`-,r THE pretreated 3ocalis -with capsaicin. :.apsaicin-pretreatrnent produced a- burning sensatioo and47are reaction-but no wi-,e^sI,==lnizction of rat"allar,en into cotftrotsxin-area-e3ualted- - - In this study the authors have investigated whether capsaicin pretreatment coulr impairthe cutaneous triple response reacti::,n to tintiget: challenge_i:::n=an.- The-skia of 6 volunteers ,3 males,- 3 females age- 29-40)- with an established aller;y° to -rats was in instant ilchiflg-follo:ved by a=progressiveiy dsveloping-flare and- wheal react:oa. -Capsaicin _aretreatntent of t?:e- skin thus almost totaliy abolished the acute fsare comvonent and reduced the-itching sensation of-the cutaneous alle.rgy-reactiijn in rran. This suggests-that-capsaicir-serisitive°sensor3 nerve endings are-activated -by mediators released upon ma3t- cell degranul,t=2n_r by allergen exposure. - Nerve activation then probably results-in Yhe release of vasoactive peptides such as sursta nce P- which mediate the flare reaction:: The wheal==responses however, was unchanged after -capsaici n pret;.eatmen-t: This suggests that activation=cf _sensory nerves- plays-a major role in the- fiare=and- itching reaction__ whi:e the wheal response is apparently to a=~tor extent :9depende-nt=of me-diato: release fro8r c.apsaiei.i=sensitive nerves, -- SU3C£-LL1tLA)?.STL?RACE AND A aTJNAL- T;cANSPC7ItT CiFIS'EL%RAD~r-EPTI^ Y (NPY)-IN RELATION TQ-CATECISGLAIviINF:S Ilt.? THE CA T smaller extent in-lig:Rter-fractio=ns.=-Llsing-reversed-phase IiPLC, one molecular fros:i of NPY-LI corresponding to°porcine NPY was found-in the-coeliac ganglion, while the - -adienal medulla also contained minor peaks with NPY-LI in addition to the main iorrr, _ The suhcel'-ular storage of -neuropeptide Y-':ike imn:unoreactixity (NPY-LI) in peripheral aympathetic-neurons and adrenal gland as well as its axonal transport in the - sciatic-nerve-was st-ydied 'in relatior_-to-catechoiamines, in-the cat. I`i the suticeilular fractions from different parts-of sympathetic neurons, f.e cell fiodies-(4oeliac gang!iz), =azons (aciatic nerve) and `terminals-(sblzea), the= NPY=Li -was fou,nd - ioget :er with - noradreoaline=INA)=irt heavy fractions -assumtd-to-contain large dense-cored-Yesicies. In addition, minor lighter fractions in the voeliac ganglion_ contained NPY-LI. The mola? - -ratia between vesicular `dn and NPY was high =ri tie terrninal regions (! 30 to- 1) and much lower in-aaons and cell bodies (10 to I); thus reflecting the different n:echanisms- o resupply for classicai=transmitte.- and peptiae. In the adrenal gland the N: Y-LI - was mainly-locatey in the catecholamine-storing chrornaffin--gra.n.uie-fraetion and also to-a 103 t ~ .~ Y

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whicb co-eiuted iivith $orciae NP-Y-.-- NA-was stored `+otb in light and heavy fractions -in the-spleen;-while 4_t-w%aa ~ai-nly fbund-ir: t.eavie: fractions in the-sciat:c nerve-. In the coeliac ganglion, most ot-the-norad.-enaiine was prese:rr in-a -non-partisuiate-f3rns - The- anterograde-trans.art-rate-for NPV-iI inlhe sciatic serve-=was estimeied *.o-be about 9 whicir implicates differences in the storage, turnover and -retease of these co-eaisting resupply of NPY to teiminais_is; in contrast to NA, most likeiy by ar.ona transport. with small -dense-cored vesicles probably- contain NA tut not NPY-I<I.- ` The ma-in - NA in heavy fractions corresponding *.;; large dense-cored vesicles, while- light fractions mm-h-t: A minca retrograde transport of NP`--'-i.I was also detecied . In cona,:sion; the present data suggest that-NPY, a peptide with sympathoactive actions, is co-stored with substances in the ayrttpathoadrenal s.+stern=. Fried, G.; -L>o.°rdbe.'g: _r: AL,and Theodorsson-?.°r-orheiraj E. - nlcta P3tyszologrca Scandiaravica 125:145-154,1985 C)tke- support: 3we':slr-a3edical Research Cnuncil; Swedish Society f<i: Medical Sc:entes, Swedish Tobacco-Contpa_ay, I-iedlund`s-FoundatioF, and the Karoiinska Itest :ute -- From he- Fyepa=rtmet:t--of Physa`dloay--ard -I=harmatology,- KaroIinska Institute and Department of-Clinicai Chemistry. Karoanska 4iospital~ ~toekhglr.:, Sweuen. _ VIP AND PHI IN CAT-3?-EL`Rt-`SNS:- CO-LFJCAEIZATIO'b] BUT VARIABLE TISSUE C(5NT-RNT POSSiWL -E vUETO DIFFERENTIAL PR7'~CESSI?'G - - low VIP/PHI ratio. In csnclus-ion:- VIP and PHI seem -to co-exist in most -neuronal systems_ Altfiough_ the ratio oi--VIP-ar:d-Pf':# on the precursor gene- is a:l, = dif ferences in- posttranslational processir,g=may create a considerably highercontent of VIP than P'-3I- in considerably more nzfves were PHI- than VIP-IR.- This observation was in parallel to a immtinohistozr`emica? analysis revealed that `~Ir - and PHI-I R were ~re,eni irr the same ganglion cells io- the intssine, pancreas; uterus and sympathetic gar:glia. Furthrmore, the terminal networks for these two geptides were very sirnr~sr in the perip'r=-ery.- In the median eminence of tho==hypotiialam3s= and in the posterior -lobe -cf the piruitary. The concentrations o1 vaso:a -tive i,=ize<Ci;alpolyDept;ife (VIP) and the peptide with NH==terminal histidine and COOI-i-ter?nibai isoleucine (PHI) in various peripheral tissues and some--areas in the CNSof the cat :were compared with tlteir i. trriunohistqchetrrical localiza*,ion. The VIP Ie:eis in the gastro'sntestinra tract were 3 to 6 times higher than PHI levels. Much (up to ?ff-fold) higher VIP than PHI ievels were-also observed- in- the ` gettitourirary tractas wT11as in the'ung°and heart= In the neurohypaphysis,-howexer, the VIP/PH: ratio-was-c:0se to-'--. [3el-per;neatiozt chromatogral;hy revealed tFat--VIP- and-PHI=immunnreactivity(IRyin the intestine, pancreas and brain consisted of three-= larger-rnolecular -fora:s in idd"¢tion to -tfie -standazd" pep.ides These larger ° _forttis -w#iic.l` -had- overlapping Elu.ion positiorts- ntay- represent--prepro=VIP/PHI forrins: The- most terminal areas. °- _ - _- - _ - -_ Yahrenkrug,-:, Bek, T., Lundber_g:7. M:, and Hokfe_t, T: - Regulatory-PeLtidFs 12:21-34, 1'iY5: tlae Swedish Tobacco Coanparry, Petrus=-oci- Augttsta= -I-ledlunds `Stif:else, -and the ~ _ Hospital Foundation for Medicai Research, ttieSwedish Council for Medical Research; - Karolinska Institute. - c. ~

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-Erom the Department=-of -CEiriicai C4errtisFry, $ispebjerg-Ffospital, Copenhvgen, I?enma,=k, a=nd--the Departm-etits= of Pharmacology --attd- Hi.=stolog}!,- theiCarolit=ska- lnst_tute,- Stockholm, Swede6,=- , _ _ -- -- - E41iJEI~iCE.:_Fii~_= SI }CIc1C- NEi3is.OPEPI'1~Y-_n1NDtN.r, =iN ` RAT _BRrS1N SYNAPTOSOMES is an important=facto: :t>-n_ettrotransraissior'. 9ne cf- the major pe-Wides -in the brain- and per ip,heral a~ervoi:s systera: -!JP:'- -a -exists wit-h s:oradrenaline;NA) in neurons in both the CNS and periphery but is also present in non-catecholaminergic systems: Sympathetic activation induces co=release of NPY and NA, -subseaueettly . these _ t-wo _ agents_ cooperatF in -funtti,nal _:esponses such as vasoconstr:ction. NPY has therefore bee nproposed to be a co-transmitter together with NA. In th6== pesent= study, = fUrther evidence is provided for NF Y being a tt2lif=_tfalc°-.tnitt?: S.HCe-yp2i.ffiC binding Sit2s for ?251-N~'Y txist in brain :'tenlbFnes of the rat: =As detronstratedir< tass-studyz the al'3ine.;-of'1-9-NPY_to rat_- brain syttptoso^tes and t}:e_3iumber of=binc3ing sites- afe of -aa magnitude _simil:r so that fbr_ receptors described for other -putative pe,:tidergic trarism_ itier=. 1251_rp~ -bit~di;tg could be c~ispla°e~+-b;v-unfiabelet# NPY and PYY, b_utltct by other sFructurally_related prptiriec from the pancreatic polypeptide family or-neur_otensin. Ther f^r:, the :nvestigated binding sites show a-hig_h ,pekifici4y for NPY a.^a PY-Y. Poweves=, NPY is- probably the endogenous ligand -f or these binding s:=tes, since the_ K ~ value -f or-NPY _was_ h~ighz: than that for PY Y a-nd,=r:tareoYe: PYY -doe's-Lot-seem tote,T:resent_i_It.msss apa:of the- rat *:ain. =°1he Getrot-stafiort af.soecific, higG-afinii_y birdin_, z fv= 'r51-NPY, whir-h have c;.aracte: istics of pharaeacological rceptors: f urther supports ti eproposal t'r.at= NPY Neurypeptide=Y__(NYPY,;=a neuropeptide with N-,and-C-terminal tYro,ine=t:Y;, is - - Saria, A.,-Theodorssoti-Nar#seim~, E.,=and Lurdberr-. J. 1.:.- Ettropean Journal of Pkarmacof8gy,if17:105-1Qi, 19E58 of Pharmacology, Karolir.ska-;rtstitute, Stockhoi?n. Swed-en. btker support: =Ewzd 3h hied~cal°Resear_ci Council, Swedish Tobacc~= Comparfy and Petrss and Augpsta-l4edlu-ds Eounjatiott..-=- E`rotn t}e- Department of Experintentai and Clin,c,al Pha ra asology, University of Gra-_, Graz; Au;tria; Department-of Clinic2l-Cbei:::c_cy,_l~,rolinska Mospital; and Department - -YIvC-REASE UPON _ct=A _DRENtKEP-TC12 A_CTl4_'A_TI_C:N _AND-DIRECT -PRESSOR EFFECTY-3N-PIT-HED RPTS- hlEUROPEPTi7E Y -ENIi-ANCEtvf;EN'T-OE BLOOD P1z ESSU2 E activitv in viro. The effects of neuropeptide Y (NPY) on blood pressure and heart rate were studied in pithed rots.- System infusion of NPY in a dose (230 pmcl X kg'1 it) .vhich-rer -se 3id not affect b_o^d pre_ssur; en%a_nced'.he prLssar-re=ponse -ta phenyiepnrir_e (50 rmal X - kg`S i.v.) and that to electrical stimulation of Vie sympathetic r,utflow= in highYr- doses,'NPY -caused a:presscr-effect-per sen, which was Aose-dependent:y attiLeonifed by nifedipine but tot by adrenocaptor antagonists- In - conciusiort; -NPY enhanced the -~-adrer.~eptor_!nediate~_ response and 6ad-- Ca~°-Cepend_ent= -vasoconstrictor Pafiiof; C., Bah14f, P., and Lu tdberg, J. M. European Journal of Pirarr,tacology 1d9:2$9-292, 1985. _

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Other suPport.° Swedish Medical Research Council, the Swedish Tobacco Company, the ICaroli nska-Inst=tLte, the Swed=izh Society for Medical Sciences, and the Astra Foundation. From the Department of Clinical Pharmacology, Sahlgrer'sHospita;, -University of Goteborg, Goteborg; a_nd - the- Departmen t of- Pharfl:acoiogy, Kar3linska Institute, Stockholm, Sweden. NEUTROPEP'I:DE Y AND-SYMPATHETIC CONTROL OF HEART CONTRACTILITY AND CORUNARY-VASCULAR TONE - - The effects of neuropeptide Y (NPY) on contractiity of the spontaneously beating guinea-pig atrium - and- -transmural nerve stimulation (TNS)-induced -effiuz af tritium-no_radrenaline (s:3-N-Aj were studied in vitro: -NPY induced a mlderate poa:tive chronotropic and inotropic atrial response which was resistant to metroproiol. - TNA at 2 Hz for 2 s caused an increase in rate and contractile force. These -ef_fects were _- sigr:eficantly-reduced by NPY. NYP aiso-reduced the TNS-induced (2 H-z for -20 r),_ fractional [3I-I]*; Arelease by 40% without affecting the contractile response_ The contractile effects of exogenous NA ott the guinea-pig atrium were_ not affected -by NFY. _ NPY- caused a tong,lastir.g--increase in coronary perfusion pressure and= also, in high doses, an inhibition-of-ventri=culaT contractility in the isolated, perfused -guinea-pig heart. The perfusior, pressure increase- to NPY, which most likely reflects coronary vasoconstriction, was resissant-to a- anti-,6-adrenoceptor blockade but se nsitive to- the calciurn antagonist nifedipine. A 50% reduction cf the vascular hIPY response occurred at 1Ce'° M--difedipir:e,-which did not influence-cardiac contractility per se- or the _ contr3ctile effects of _NA.-- NPY did not modify the increase-in ventricular contractility- -induced by NA.- Noradrenaline did not influence- coronary -perfusion =pressure - after fl-biockade_ Since-NPY is present together with NA in cardiac nerves,-it may -be- suggested that NPY is in+olved in the regulation of NA release as well as the sympathetic control of atrial contractility and coronary blood flow. -_ From the Department of Pharmacology, Raroli:.ska Institute, Stockholm, -and t_trt Astra Foundation; tI!e_ Swed-s : Society-for- Medical-Sciences, alld :he K arolins'k.-. Instituiz. Other.i<pport: -Swedish-Medical Research_Council,-the Swedish Tcibaccd Com6any; the - Franco-L-`erece3a, A., Lund;,2rg.-L. M.. and Da3rlof, C-. -- ,leta P_nysroiogica Seandir_in:aca I2s:3Ei-369, 1985. Department of Ciinicai-Pharm.acology, Sahlgren's Hospit-al,- Gothenburg, Sweden. -- MECHANISMS UA_ 1DEP3 YINC CIiANC'sES IN THE= CQNTENTSOF ° NEUROPEP''I^.E Y IN CARDIOVASCULAR-N£RVES AND ADRENAL CrLAND-INDUCED BY SYM3'ATHOE-tTIC DF1;TCiS-` -present study, the aut_hors,report that pretreatment with sytnpi'_tholytic agents influences the tissue levels aFNPY-like immunoreactivity-(NPY-LI) in-the guinea pig. Thus, 2d h_ after reserpine, noradrenaline (NA) and aiso NPY=_ LI were depleted in the -heart, saleen and the adrenal gland., The= levels of NPY-Li-in the vas deferens and _stellate ganglia, however, were unaffected by reserpine in spite-of marked depletions of NA. The reser= Ne;:rgpeptide Y fNP.'1is erscentiy isolated vasoactive peptide:wl-iich is presen.;- together_with-catecholamiae3,-ir: sympathetic nerves-and in the adrenal meduila. An the- - 0

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pine-induced depletion of NPY=1-I--evas prnbab:y cau3et# by _ nhancey-necve-im^ti flov~ ''``_ and subsequent release from cardiovascular nerves in excess of resupply, since it could be prevensed by =the gangliccic-blockin& agent chEorisonda,.,anee-_ Long-term _ (6 _-days) treatment with chlorisonda~ir.e reduced ~e'eve-ls of FdP'f-Lt .--,he_ _ tellate -gang?ica. Short-term treatmert (48 h) with guanethidine partially prevented the reserpine-induced depletion of- NPY-LI, probably due tc inhibition of NPY release. Long-term guanethidirte treatment depleted--not only NA, but also NPY-LI from the spleen. Pretreatment with the alpha-recepor-antagonist -phenoxybenxam-ine did not influence the NA levels but seduced the c,dntyn; of NPY-Li in the spleen _via a mechanism that was deDendeni on- intact=-ga-nyliorric- tiensrnissaop-.- Since NPY has several cardiovascular actions, changes in NPY- mechanisms may contribute to the pharmacological - and therapeutical ef fects of=symp3thufiy3ic `agents: Lundberg, 3. M. et aF. Ac!a P-~ysiologira Scandiqavica'.14:503- ol I, 7985. vt4.er suppor : -Swedish- Medical - Research-.-Councal Swer±_sh Tobacco -ompanv, '1Carolirrska Institute, Astra Foundation, and the Austrian Scientific -Research Funds. From the Departme ,i of Pharmacology, Karolrnska -Ins.tst -te,= StocAholrn-, Sweden; the Department of ExperimentaY and Clinical Pharmacology, Garversity, of Graz, Graz, Austria; and the Department of Clinical=Chemistr-yf-Karolinska Hospitai,.5teck.i:olrn, NEJKOPr-PTIDE Y-(NPY) Yi=EC)tJCES FIELD -S"I'I-MULATICN-E`yf..3KD RELEASE OF NOR;4EXEIRENAI,INE AND ENHANCES FOkCE=CF CO,N T? A:'I'IflN _ _- IN THIc RAT PUR-i-ALVEIN l. The E£:'ect of neurop eptide Y{NPY) -on fractional t_;itium-norad3en6Yine (3H-NA) release and-contrac?ile aetivity-was sttidied-i, the isolated porta'- vein of SHR and WKY rats. 2. -NPY (5 X 10-3M) enhanced the force of the spontaneous contractile - =octlvity_ by about =4P%. =-3. The fr<<'~1]onal 3I_-re_;ease el5cFte:;-ay - transrnuFaf rve - stimulation (;'i;lS), which -mainly reflect4-'H-~:A, was -reduced by ai~ur 40% ~after preincubation w+it4 { K 10-sM NPY-in_portal veires-frum_botlt _SItR=and i?,CY rats. The inhibitory effect of_=`NPY= on TNS-evoked sH-relPase ~ was _aeore sl_owly , reversed by v+ashout than the_facil:catory-action on spontan eous contractile force. 4. The contractile= response to- field stimuls-tion °was--not rduced- by-NPY; but rather tended to _ be increased. 5. It is concluded that NPY -exerts a dual action=in-the YH& and WKY portal vein, ths:s enhancing the smooth muscle contractions and inhibiting sympathetic neyrotrarsmss3on. The inhibitory effect ot NPY on TNS-evoked-NA e`f1ux, which is present i=, both SHR and WKY rats, i; most likely due to a presynaptic site-of action. 1}ehlof,,C.; ilahiof, -P=; 'I'atemotc, K.,-and_Lundberg; ,-.-Y: __ - ArcEh:ef of Prarmacoeogr328329-330, 19E5. aeher supgorC Swedish Medical Research Council, thr=Swedis5- Tobacco company.- Karotinska Institute,_and the Astra-E'oundaiion= From the Departmeqt of Clinical -Pharmaeology, Sahlgrer-'s Hospital;-Deparcment of Pharmacology, University of-Gotefiorg,-6otelzorg,-_Sweden;- and '._he-Departm_ents of - _Biochemistry and Pharmacology, Karolinska Institute, Stockholm, Sweden. - - 107

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CALCITONIN GENE-R`E~JATED PEPTIDE AND ITS E9NDINE,_-SITES_IN'I`YE HLFIvfAP-' CENTRAL NEKVOUS_ Sl'STE3, AI-'D=PI"FUITRR E-- _ Binding sites for syrith tie= human= ±a:-:abelea calcitot.._ _ger.a-releted peptide - (1=^I-C.c~iR=F) have been -demonstrated in membranes of the _hu?zan nervous ,- system. _--_- Binding was_high in the-cp:ebellar.cortes_(1.35 + S?.2i fmo!/~ng of tissue; mean b SEiz3), spinai cord-(1.05 f 0.2Z_ ty-1.27 T- 02s-frno1/mg)9 and--nucleus_ dentatus_ ;l.0z ._-_9.l5 - f mol/mg), =nte:med ate in_the inferior colliculus (O.8t1 ± 0.14 -fmol;`mg) and_ substaraia_- rzigra (0.75 i 0.14 fdtol/etig), ;ow -in- the neocortex, globus us pallid:is, nucleus caudatus, hippocampus, a$tygda,a.- tuperios collicu!_s, -::ia:arnus.- ano- hypothalarrius-= ,O.+_5-Q.32_ fmol,'sng), _and negligible in--spi nZl ard--sytnpat'r.etii_ ganglia and _pituitaiy--(- c-,C.I34 fmol/mg). _ fiuioradiography showed distinct--'S6I- GRP binding over the -molecula and Purkinje cell layers of the ceret`e'ler cortex and over the sybsea.,tia galatinosa posterior of the spinaic_ord: Theltighest ievels_nf CGRf'-Iike components were recognized_ite the dor=al part of the spinal cord and the pituitary gland. In the ventral part of the spinal cor3 as well as in the piwitary and= thvroid_ glands, CGRP values were higher when- measured by radiorecepto> assay as compared to RIA, indicating that at least two _ C;RP-like components ar . present:- -'I-he predontinant-jCGR: -Iike= peak on HPL°C had the retention -time of syt<thetic-human CG1;P. - Imtnunahistoche:rustry_ revealed the presence of a dense plexus of C.GRP smrlunorecct:ve ner ve fibers in the _dorsai horn of -- the spinal cord-. - Lundberg, J_Y_ et ttl. Proceedings -o> the tiatmona! Academy of Scien:es of the United States America 82;248-252, 1985. _ - llth_or support: Swiss Nat.onal Science Foundation; t'ree- CQnton of Zurich, Switzerland; Ciba-Geigy Company,; Swedish Medical Research Council. From the Research Laboratory for Calcium Metabolism, Departmenfs of- Orthopedic Surgery and Medicine, the- Institute of Pathology, University_ of _ Zurich, Switzerlanda -- Depattments-of Histology and rharmacology, Karolinska Institute, -Stockholm, Sweden, _ anct the Department of Anatomy,-Universiiy, oof Oxford, Oxford, England. . LEUK-OTRIEl`7-ES, C,, D,t ,4ND E, {'AUSE WIDESPREAD AND E XTENSIVE- PLASMA -EXTRAVASAT-ION IN THE GUINEA PIG _ -1. Intravenous injection of ieukotriene C4 (1 nmol/kg) caused substar.tial- plasma exudation in anesthetized ga:inea-pigs, as evidenced by -marked hemoconcentration (!5% in 5 min) and significant extravasationaf Evans blue. -_ - 2. Fluorometric quantit_ation of Evans blue content in 3S selected- tissues documented that leukotriene C4 caused signif icant plasma exiravasation throughout the body, except_ for-the brain, stomach, duodenum, colot:, and-gonads.- -- 3.- In particular, the respiratory and the -uro-genital tracts, but--also the conjunctiva, the esophagus, thV bile ducts and the umbilica! _ligaments, -were very- sersitive to the edema-prortirting effect of leukotriene C4. - -4. Intravenous injection of leukotriene D4 (I nmol/kg) or E4 (5 nmol/kg) evoked plasma extravasation with a_distribution-and magnitude_that-was similar to -t_hat _induced by leukotriene C~: _ 5. Ie is concluded that-the three major constituents of slow-reacting substance of - anaphylaxis (SRS-A), leukot-ienes C4, D~ a,.c E~ cause- a generalized andextensive-plasma exudation_ that is consistent__ with-- the proposal that these leukotr ienes are - important mediators of inflammation. ~:

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Hua, X.-Y., Dah!en,-S. E., Lundberg, ;. M.; Hammarstrom, S.; and Hedqvist, P. Archivec of Pharmaca-log; 335.1,6-141, 1985. Other support: Swedish-Medical Research ^ouncil; Swedish Society Against Heart and- Chest Biseases; Astra- Foundntion; Harald and Greta Jeanssoe.s FoL-ndatior; -Swedish -- - Tobacco Company, Petrus and Agusta Hedlunds F_oundaSion; Karolinska Institute; and National Institute of Environmental Medicine. From the Departments oT Pharmaco!ogy; ' Physiology -and Physiological Chemistry, - Karolinska Institute9 and the National Institute of Environmental Medicine. Stockholm, Sweden. CAPSAICIN TREATMENT DECREASES TISSCIE-I.EVE;.S_ O_F-NE-`EIRC'K_INiN Ft-_ LIKE IM-M'ilNOREACTIVIT"Y /N T-tIE GUINEA TIf'i The tachy'xinins are a-family of naturally occurrir.g bioactive peptides which share a similar C-te_rminal sequence of amino acid residues and have similar biological activities. Until recently, substance- P (SP) was-thz only tachykinin known to occur in mammalian species: = Substance P-like i.-nmunoreactiv-ity (SPLI) has been found in the central nervous system-and- in sensory- neurons innervating many peripheral organs. Now; in additiom to SP,- mammalian tissues_havx -been found-to contain- tac!:ykinins-_- relaced to kassinirrand SP; neurokinin A(NK_ (NKA) and neu-rokinin-B WK_B). The-purpas of the present study was to investigate whether pretreatment with capsaicin also _- decreases the-concernra*ion of mamma!ia.n tachykinins other than SP in-some peripheral - organs. The results prese nted here ir.dicate-that capsaie:in pretreatment depletes certain tissues of tachykinins other than SP. As has previously been shown for SP, the largest relative d-,r!etions_wer=e found-ir the tissues c4ntainir,g:a high density of primary sensory - afferents (ureters; urinao-y bladder. These results indicate a close relationship between SP- - and other mammalian tachykinins such as NKA and NKB, not only-in -terms of similar tissue distribution, but also=in terms of similar reactions to -c3psaicin,-suggesting that NKA and NKB are additionai peptides of sensory origin. It is-therefore interesting that NKA, but not NKB; has a very potent broncho-constrictor_ action and causes protein extravasation in the tissues, where acute ad:ninistratioa of capsaicin reduced the nettrokinin concentrations. Neuro!cinin A may therefore contribute, together with SP, to - the neurogenic inflammatory-response. - - -Theodorsson-NorhPim. E., Hua; X., Brodin, E., and Lundh2rg. J. A!. Acla Physaalogrca Scandinavica 124 139-131, 1g8S:- Other sspporr: -Swedish Medical Research Council, Swedish Tobacco Company, Petrus and Margareta Y,"ed!unds Foundation, Ivlagnus Bergvalls Stifte!se, -thg Astra Foundation, the Nordisk Insulinfond, and the Karo!inska Institute. From the Department of_Clinica; Chemistr}!,-Karolir.ska Huspita!, -and Department Yf- Pharmacology, Karolinska Institute, Stockholm, Sweden. _ NON-CHOL{NERGIC VASODILATION IN T Hz. TRA CHEOBRONCHIAL TREE OF THE CAT iNDUb:ED BY VAGAL NERVE S5'IMtILATION The tracheobronchial m-ucos-a-ts eatensively-vascularized and it-a!so receives a dense parasympathetic and-sensory innervation. Chemical or mechanical irritation of the N T1.~ «.~ e

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c:ucosa- induces p;rotective reflexes, sLC;_ as mugh and 'oronclespas:?. - ln addition, the- irritation leads to a clinically-and experi^3ecttally-wel1-kr:c~:n hype=err_ia and rnlcosal swelli_n.F,, _ T he_-increased vasc-ular pertneability to-macronolee,ules seerttc to- be dependent on a..tivation of syrsor-_, n ve which release mediator: s-each -as substance P. ~:, the presentstudy, the authors have tried tr establish the existence of a_vagal r.on-cho?inergic vasodilation in the lower 2i~~ays_using the-r-adioactive ri:crespTere_tzchniyue to,nieasure° blood flow in-atropiniaed cats: The experiments were _per?o rn_ed in se;en aaalc cats. The data presented trm thiw-pzper esta5iish the Px-sten ;.e of a non-cholinergic vasodilation upon vagal nerve stimulae:or, in the tracheobronchial tree using the microsphere technique. In the nz-!sal mucosa it has,bee., Fussib,e to elicit non-cht'_ine:gic v;rsodilation in two-- principally °diffre t ways: (D by parasympathetic pregati-giioric ::erve stimulation, where the i:asodilatior is abolished by h9xaiuethonium; and (2) by vasodilation; which iritex:methoniura resistant and-is elicited by antidromic stimitlation - of sensory nerves using high threshoid stimulation parameters (Lundberg 1984). The_ parasympathetic non-cholinergie vaso.aitatior. =i3_ probably- due to the release- off peptides such as VIP and_PNI=From postganghotric neurons (LFndkerg e: al 19EGa), while the second type is due to antidrGmic impulse propa-0ation and the release of tachykin:ns such as substance P (Lundberg 19i:4),- _ -- Martling,-O-R., Anggarr,-a:, and t'.,,rdberg. i. M.- _ Acta ?hysr;,logrca ScanrirsiavEca 125:343-346, i985. Othev-tt;pp< ° Swed!s: Medieal_Kese , t=Councilr5we+isn Ts;bacco -Coatpagy; Fe:rus -ani Augusta=Hedlur.d; Foa::-J-at=n-n. and?;:e R=;3liaska I?istit-i~te. _1?Iospitai, and Department c!`-pharrracolbg°i, Karolittska Institi;e, St,~--khn'm,-Sweden: From t`r_e -De~;artmen'~ of AyStFesokgy_-a. JrA R h:ao-:..aFyngoaogy,_ Ka-n]intka (23-54%) and the response to NPY was_ almost unaffected 5y the a-receptor blockade. The remaining effect of sympathetic stimulation after phsntotantine_ -was abo<iahe_d -by gua nethidine.- However, t-he retpor,se to -NFY was not changed by the latter d-ruS. -_ dn conclusion, the -yasocohst: ictor response-in the dental pulp and oral mucosa -causei by activation of-sympathetic nerves is more resistant to phentolamine than the response - blood flow of_ a similar maj;titude in- both tissses _ F.Cte: _a-adrenocept5r blockade with phentr,la^.ine, the vasocords'r.ctcr effect -of n3iadrerialine was abolishe+: - Fiuwever, the tffe=s - of syiapa-thet.ic stimulation af ter phesi:olaeninc-- was -only partically _ reduced- electrical sti mulatiot,_ of _ tlie'a.ympathetic-nerve_ or during oFose intra-arttriai inEUsion of nofadre_^.aline or-Nl'Y_ Ail three"procedures-sesultect in a prr<r:uuced decrease i* local the -main peak of NPY-:'_ke imm:rnoreactivity ;-sund-'in the superior tervicat ganglion -co-chrontatographed with syr,thetic-por,cit.e_NPY. Changes in blood-f'_ow in-dent_ai pulp or_oral->itucosa were trem'-d-red indi:etlv-b,v recording local clearanee of --;=1 during Neuropepside Y (NPY) iminunoreact:v;ty (-IR) -as f.~:rt?d to- be presert_ iR periva<culaz nerves in ihe cat nenta] -pulp and oral raucosa= Ma :y _gang,ivn cells ist the _ sur2erior cervical gangli nalsu_t<or~iai n?d NPY-IR._ Ligation of-thE inferior -al-veolyr or lingual nerves produced an -ac;-unsuia:iei:-oi' N.PY-fR in azons proximal to= the site of - ligation'-_suggestirig-ar: anterograde axoaal transb^rt of the peptide. After unilatera`r aympatyrectomy the NPY-l;i- disappeared in the dental pulp and-n:al rnucosa on the ipsilateral-side. Reversed phase high_per_for,naFce liquid _hrornar;,graphy- -s,Sowedthat IN ORAL itrrJCOS-A AND DENTAL PiJL1' iN THE CA f Nf L'ROPEFTID£ Y-{NPY) _AW i S a Iv1PATiiL Tii, COti T RDi - OF BLOOD FLOW 110-

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induced by infusion of exogenous- neradrerneline Since NPY_ is- -prdhably-- co-localized with ndradfen line ia the sympathetic periyzscu'ar nerves and ivPY red::ces iocaz-_blood flow..?t is pror>os°.3 3h 3,is-,-..-pc:de is involved ;n -syrr.,athetic v-ascula-r control in aFri-..l tissues Edwall,-B., Gaze'_i-us B., Fazekas, A.,-Thodorsson-Norheim, E., and Lunlberg, r. M. .lcta Physiologica Hungarica i25:253-26Q, 19$5. Other supPort.~ Swedish Medical Research Council; School- of Dentistry, iCartilinska Ins*.itute; Swedish Tobacco Company; Hedlunds Foundation. From the-d?epartment of Pharmacology, Knroiinska-in,titute; Stockholm, Sweden. PRE- AND PDSTItFNCTIONA"L- EFFECTS OF NPY-ON SYMPATHETIC CONT?ROL-OF RAT FEMORAL ARTERY Netiropeptide-Y (NPY,I-like immunoreactivity-(LI) has recently been demonstra?ed in -periva-scu:ar,= noradrenergic (NA), sympathetic neurons. - NPY is a potent vasoconstrictor agent in vivo while the contractile effects of NPY on isolated blood vessels are variable. For this=study; therefore, the authors have ar.alyz9d possible -pre- and-postjustctiot+.ai effects of NPY in relation to noradrenergic mechanisms in the rat femoral artery. 'he data presented in this paper -show that, except for the vasoconstrictor activity per se, N=FY-enhar.ces the contractile effects of NA on arterial vascuiar-sraooth musvle. _`I'hz-NPY effect may be related ta changes ir:--the influx of extracel;uiar-calcium, -since it was absent after -nifedipine: The contractile response to transmurai nerve stimuiation iTNS; was also increased somewhat by_I`IP3. However, in --- the present study, NPY in-the same co:cc-ntration caused a ma: ked-(abou! 50%) reduction of TNS-evoked ':T=INA efflux. Thus, it is striking that a marked _depress;on-_of stimulus-evoked 3H-NA secretion can occur simuitatteously- with an enhanced -vascular smooth muscle-contraction.- This indicates that postjunctionalcontracti!e-and facilitatory mechanisms of NPY have a more profound effect on sympathetic neeer-otrar.smission than a SO'96 reduction of NA release: T hus, NPY may have an important role in -thE regulatinn of sympathetic vascular cor,trol.- Z. und berg, J. M. pt -ar'. Acta Pkysiologica Scandinavica 123:511-513-,-1985. - Other suppori: Swedish Medical Rese2rch Council, the Swedish Tobacco Company, Petrus and Augusta iiediunds Foundation, the-Astra Foundation and the-Karoiinska Institute. From the Departmens-of -Pharmacology and Biochemistry, Karolinska Institute, Stockholm, and Department of Clinical Pharmacology, Sahlgrens 1-Iospital,_Gothe nburg, Sweden. AMINO ACIDiJPTAKE- BY HUMAN b1.ACENTA:-Ai,TERA'1'IONS BY- - Nt-COTiNE-ANO_-T,3EACCCJ 11v1CKE-COiv1F(iNEN'i'S-9NI) THEIR rIvtPLICATIONS ON FETAL GROW. Thi The tetus of_the smoking mother may partially be subjected-to amino acid- defici+.s due to the decreased uptake by trophoblast and the - transfr: of_ arrriro aeids_ from trophoblast toletal circulation. l, i

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Sastry. B. I<, Rama. In Caciagli F., .;riacobir;i, E. _ and Panletti _ i;= -(eds.): 0etialopme:r as 1'Iei.roscience: Pnysias-ogicaf, Rnarma_4;ag'caL -and Clinical Aspects. Elsevie: = Science Publishers, Amsie*.dam, pp. 137-140. 1984. Other support: _ U. =S. Lnaed- States -I'ubiic Health Service and National Institutes of - Heaith. From the Department of ~harmaceecl-y; Vanderbilt U+niversity, _NAar,vitle, Efr"FECTSGF CALCILM CHANNEL BLOCICING A~idNTS ON r'v:Eiv(BRANE P.?ICROVI,.~OSI TV AND CALCIUM IN THE LIVER OF THE CARBON TETRAC I-IL^RIJE TREA I'ED itA I_ Membrane microvisc,sir;i wa= determined fro±n- ti,e-polarized fluorescence of - diphenylhexatriene in plasma membranes an d micro-sarc,es-prepased from the liver-,of administration of the earban tetrachloridg. Depression of microviscosity was also seen in carbon tetrachloride treated rats. It was greatl; depressed btweert 12 and 2a hr -after ihe- the iiposomes which were' prepared from these re:nbrar.es, There were decreases iri phosphoiipid content and phcspholipid itiethy-itransferas2 acti^ y, but these changes did nct appear to explain- the_ decreased microviceosity.= A large accumulation of zalcium- - occurred in ths li+et c-el:x`betweeri 1Z-and 24 hr aater the administration of carbon __ ~ tetrach+or-ide Chlorpromaz:=Ye- verapam l a7d nafadi3's e when adm-ni tered pi.o -*o the _ carbon tetrarhhoiide paT-t atir- r,.duced'ct -later a°umulat on of calcium and reatd ed the degree of hEstological damag- ohE _rved -Vw'hen these agents ~sr admiai ,erei# 12 hr aft.er ~ the admittisrration of aroon tetrac.kloride, they did -not reduce the - subsequent- accumulation accumulztion of calcium. When- administered prior to- and 7- hr after carbon- --- ' tetrachlo_ride; they had a small but potentially significant effect on the microviscosity_- l change. It is-suggested that ae low levels of microviscosity,-a critical threshold - may -exist below which entry ot caacium into the cell is poorly controlled and that calciur,r channel ~_ - blocking agents may- be ineffective if administered at- a time when- membrane microvisc;sity is very losv-. -'I'issue cal-cium sccumulatioa was associated wit're visib'e cell damage. Landon, £. J., Jaiswal, R. K., Kaukam, R. J. and Sastry, B. V. Rania - BiocHemicai Pharmacology 33;22):3553=3540,1ci84:- Other suFport: -U-. S. Public Health Service. From the Department of Pharmacology, Vanderbilt Unive:sity, School of- Medicine, Nashville, TN._ C:-1O- LINERGIC PROPERTIES {5F CFi J'LiNE ETHERS - Effects of nine cholineethers, (CHS)~<1`CI13CIi(R)=0-R', on the muscarinic and nicotinic receptors of longitudinal smooth muscle of guinea pig- ilyum were studied to understand the role of electronic; steric factors- at the ether-oxygen in -stimulating the cholinergic receptors. -Their EDsos to cause-contraction of the ileum in presence of hexamethonium (37 x 10-$ivl) were 2 to 307 times higher than that of-acetylcholine (ACh;-__ 2.9 x i0'f.d). The relative maximal effects of 5 ethers (1.20 to 1.34)-were higher than that - of ACh (F.0), while 4 exhibited lower maximai-effects (< 0.71) These ethers exhibited ~ 112

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- no significant inhibition of cholineacetvl-iransferase-a.nd eholinesterase activities from the longittedinal-nuscle at Eheir £D;0s. Hezamehoniunz-signifiyantly i:creased the -£D,as -o f ~tholir.e ethers. -Tl~te_EDEGs af some of-~hee±hers also were significantly in creased by treating the _ atutsc?e with phy;ostig:rtine _(38.5 x_ MY-aki) g? p`rysr tigidige and hezarffethaniu:n.- 4s'zo;ine (> 1 a I~-etw) bl~rked th= r:o5tr~etions-indbeed by these ethers. The steric :.inderance caused- by the f-methyl and,/or O-alkyl groups- and the electron density around the -e-*_her-oxygen-_ are --iiritia gitiag the- mts-carinic. as well as 3icoticic; pr_:tencies of these-cho1_ine ethers. -i.holi,e ethers possessing- the beta methyl and O-n-propyi, :sc-propyl and ter-butyl groups presumably release ACh at a site- - eausing inhibitory potential through a secondary pathway. Chaturved3, A=.-IE,-and Scstry. B. V. s'rIImw. Archimes :ntgrnateon4l!es de=Bhormacody"zatnie et We Turapiz 2-57(1):15-27, 1985. OtJrer support: - U.S. Pablic Healtf: Service, National Institutes of 7-le_alth and North - - - Dakota Divisio n of the American Cancer Society. _ From the D2partmen i of _Pl:arryaceatical ScienLes%TO,oldg_ y; -C?llega_of Pharmacy, North Dakota -State= University Fargo, a ad the Deparment -of- Pharmaco!Qgy, School- of kledicine; i~anderbilt University, Nashville, TTd = AUTOFcE zU=LATION OF ACE TVI.C:`svi,iN°_l:: RELEASE: ROLE LE -: F SUBSTANCE yTHST_APtC.E P- A-N`D A1E=t F!:0 F.`'tNE ENiLEPr--'ALttti_ A-n_- ace yichgl*ne__;AC1r,-4aaplification o-posi e a feedbaek _h:eti;attism apd a negative fegdback_--n. echanistn- wetg_ proposed for--rclP.ase- of _A_Ch from oholi nergic - um ions and two= endo$eaous--peptides, - Iter JEs. -~it rate - of inf lu~ os extracellular calc- ' sne-enkephalin -(MEK) were-_ also_ known to- regulate ACh substance P(aP) and methion 5 release= Therefore, these-studies we7e_ unde~ken ta delireate the various components of the above feettoack-systenis. Mouse -cerebral_ slicess were inct:bated in- a Krebs King-ei bicarbonate buffer containing imethY:-y;:) e}o1isF (fl.l atRl 0-223 Cifm"t: for Fe ani3. Thty= were filterFd;- washed, and-tranFfezsed to-a m=crobat:~ set up for ssF.erfosioz--witYr 'he"_r.bove buffer. The release of 3H-ACh, SP and IyfE;; into the superfusate was measured a.; describsi: elsewher$_ -_'d'he follE;wing results were observed: (a) The rate of ACl> release increased initially for the firsf 5 min and then declined exponentially. The highest rate-of release for SP was obs_erEed-wiih 2 min preceding the peak release of AC"h~ A broad peak for the release of MEK followed that of ACh. (b;SP (v.6-p'a) increased-ACh release (35%) and Ca{t uptake 1i3-4%). _.-he_ long acting enkephalin (D-a+a-enkepi-.alinamide, 34 nvl)_ decreased the re:e ase- of ACh (6:%) and svell as Ca++ uptake (48%). (c) S-Hydrozyme*.aylf urfurylt=rimethylamr,ioniusn (L9 n.*vii decreased Ai h-release by 50% in a -mediumcontainin-g Ce+-f2.6 m?h). :t_ blocked ACh= release induyed by K' (20 mM) I and disteroylphos_vhaiidic acid-(43 p!v). - It-increased both spontaneous (14 tianes) and =i evoked (2-1 times) release of SP. it--decreased spontaneous (16 times) and evoked (20 -' I times) -release_ of -IaIE?c. --i nese results suggest that the positive f eed'aack for ACh release may operate tfiroag h a muscarinic receptor, SP and activation of- Ca'{ influx, and =the- negative4eedback through a_nt>jscarinic receptor, b,EK and inhibition of ^aa'+ ihfius_ In: Su~stJrce_=~-lt+etoco!isr_rs_ and Bio.ogico7_ Aetiars. Jo dan, C.C. -and £3eh i.e; P._ (tds.: Taylor and Francia,_ London, 1985, p. 243. i

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Ct1:e, suPrort: i?. S: Public Health Ser:ice: : rom the Department of Ph..rrsacology, `r`an3e, bilt- i3n,versscy~ School tsf _ Med cinz,= Nashville, "I'N: NIC;3TMR = -The mediation of the-befiavioral=eft'ecs =associateu witr aicotitve and s-moking has been examined in humans and in several aninEa' species. In general. -an improvement in behavior or a reduction in-stress has been reported. - Since-rrietabolism and dosages have been shown to depend on the species exaniined anri on the -r3t=of administration,- it i= difficuit to speculate on the orocess-s in hbFians that are influenced by nicotine. Aside - from the possible addictive__ properties of-nicotine._ its behavioral eff`cts-_su6gest that nicotine may function as-a =n-ea:r_otransmitter. The action of nicotine on t~ nervous- systen: nsay---be mediated t'r.r=ough a specific hinding-site as yet poorly identified. 'Ihe- nicotine bindittg siie in brairr probably differs -from the -classical- nico;irtic_ choliriergic receptor site as descrih<d =a.'- the periphrral--aervous systern, sincekinding_ studies with-_- labeled acetyc_holinc,-o ~!igarotoaia, or-nic-ot<ne suggest that these ligsnds bind to different sites in=brain. Nieotizc is of part-icuiar interest because of-its w1duse-_ by maa, because it is among the compounds that penetrate the brain very rapidLy, and because- it affects behavior. In: Lajtha. A. ;ed.): ifar:ahuv_1c-oz`1JeuRoefrrrs_ist:~.youl. y,-New Y_vrk:_- P;_c_t utn Publishing c;orporation,1933,-pp _263=273. rrom the Center for Neurochemi=_try; Nathasi_S. Kline Research : Institute, Warc t Island,= - _ Al ~( l flw or C. PROTECTION .4GAINS`('-?-M£TI-FYL-4=P!-IE_NYL-},2,3,6- T ET3A__?IYI3R_D_ PYR-IDIN _ NEL1R^ TOXICIT=f $Y Q H)r- ANTI<JXIDANT ASC()RB-IC ACID Administration of I-irebvl-4=pr.eny:-1 236-Ye.trnhyisroa;riiine- (MPTP; 2-x -g- _ mg/kg tetio-orbi?al) to Ba LE/ cBy mice reduced- ;iIflmazind<= binding t-o_ 3triataL. _ membranes by 50%. Reactive o:.ygen.deriaatives have been suggested to be involved_in_- 14iPTP neurotoxic_ity;=t,erefo_e;_these researchers exatai:ied the efi'ects of ascorbic acid-- (,ast antioaidant)_ Ascorbic acid , lu0 iag/kgi given 20 m;n pricr io Mi TP administratioet apnreciably preve?tted- the rtd_ct;on of [?K]maz:ndc: binding._ _ The involveme :t_ of _ oxidative processes in the mechanism of .KiP`I-P neurotoxicity Tiay-suggest=a relationship - to the-etiology=af Parkinson 's disease and the possible benefit of treatment-with ascorbis_ ec',d. Se; slran, H: el a1. Neu;-o5harrnr.calogy-24(12):125;=-255, 1985. From tae_Center fcr-Neuroch=mistrys Nathan S. 1G1ine Research_Institute, Ward's Isand, New York. 14 f t

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~COMPARiSON OF PI-I]NICOTINE AND ['H]ACETi'LCHOLINL BINDING IN- MOUSE BRAINR1e;,IONALDISTI?IBUTION _ showed a three-fold regional variation._ Acetylcholine inhibition of ['H]nicotine binding indicated that a portion of nicofine binding was nor inhibited by acetylcholine. These results indicate important diff_eren tes-betweerr the birding=of-(±)-pH]nicatine ard that of fsH]ac,ety1chol irie: In a continuing stEldy_oT nicotine bi:~di.:g sizes, we deter~_ ned the relati~-amcunf o:` nicotine binding a nd acetylch oli-r_e- oin ding in various brain_regions of C$ 7 fBL and of DBA mice_ F+Ithcugh ^idbrain showed the highest and cere4ellurn- the lowest binding --- _ for both ,sH]nqcotins` and ]sH_iacetylcholine, the ratio_of-nicotine-to acety_lcholine binding Sers h2r,. H:, Reith, -K.-E. A:;=}Iashim, A.,-and L.ajtFa~-A. Research C':smrrrunFcalionsin Chemical Pathology r,rd Pharmacology 4S(3) 45-3j2, 1985. -Froei the Center-fof Nesrot:hemistry; Nathan S.. Kline institute !'o: Psychiatric ;vesearch, Ward's lslartd,' Ne .v '= or=k. - = EFFE,~_".rOF ,,VMEl'I3YE=i-PiiENYL-I,g,,-,h-TETkA"t-iYDROP Y RIDINE (M-?TP) ON AGE-RE' ATED CHAiVGES IN DDPAMiNE TURN©°-'ER AND TRANSPORTER fUNCTIO?E LN THE MOUSE STRiATUM- decreased dopamine-level (26% iowerln agzd_MIPTP-trea-£:.3 anir*.sals- versus young MPTP treated animals). -Altogether thi; data indicate ?v[PTP aa'ministration to mice results i n a critical losa of dopamanergic cells (40-64%), necessitating-both an increase -in synthesis - and-ird release (turP-orer) of dopamirte-fro.r~-the _sur.iving.neurnns. - MPTP ne_rotoxicity =- is greater irn the older mouse passibty-azsesiated-=with an age==reiated increase in the susceptibilit_y-of drnsarn:nyrliccells to oaidative damage;=_inGicatinfc that aged mice can be -useful in studying rr~echar.isms- o-MPTi~neurotoxicity_ to examine MPTP eur9toxicity_ in the aged mouse aL wh,ch--_antioxidant -mechan+.sms- may be comprised. The present study shows that in MPTP treated animals, ['H]mazindol binding was-decreased by 24q6 more in aged mice as compared to young mice, and a similar increased sensitivity to the ,.eurotoxic effects_ of MPTP was observed in a Dama-ge to neuronal cells, either during the normal aging process or related to IvIP'I'? 5eurotoxics`t,-'.iave implicated oxidativa free-rad-icat rr:pc_ranisms. =-.^tu*.athione (c natural reducing agerit) is s enlflcantly-lower=in the substantio nigra compared to other brain-regions and virtually absent in the nigra of Parkinsonia -p,t.;ents (Perry e, ri., -19g2). If MP"I°P neurotox;cify invu:ves free-Tac#ica! sediated-ksrocessey; it was ef interest Serafien, f/.. Mason, M. F., -I-lashim,- A:, and-Lajt;na, A. `Lruropea-..io,:rnnl of Pharmacology 113-135-13b, 5093.= - Fro:r t_k,e Center for ",eurochemistry, Nathan S. Kline Institute,Al'ard's Island, New York. ACTH PEPTUDES AS ORGANIZERS OF_NEURONP.L-PA7°TERNS (Iti pg/kg/day IP) or ACTH/MSH 4-9 (Org 2756) (0.01 pg/kg/day IP) were compared to neuromuscular junction (ianj). Maturatio.n.al-changes evoked by ACTH;%MSH-4-10 - SEM was used to visualize the rormal-postr.atal develotment- of the neonatal rat -IN DEVELOPMENT; MATURATION OF TI-SERA'I' NEUROMUSCULAR JUNCT23N ASSEN BY SCANNING ELECTRON-MfC scOSCOPY- 115 I ..

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controis-and to pups trezted -wi-th-nicotine during p:enata; and_ postnatil life, -or snly during the gastationr-period- Pregnant fe;nales received-i.23 mg/kg 2x daily_E?; neonates- - 0.05 mg/kg/day SC. Th_ Desaki and Uehara and Fahim et a!. methods reyeale, the n,z:j - o:, the extensor digi2orntr-_muscle,c-be cpvered-by,a delicate draperg of psstj:rnctinnal' folds that surround the i::N-na*tr.e endFj+ate-region. - By the=second week of fiostnatal life; these-£olds-become ~nore-tomple; and cove: a-iarg-er area. Upon ihaturatisn the fblds ° descend and invaginate int~ the muscle f;-ber: Peptide treatment with -either AC:H/NISH- 4=1u or ACTH/MSH p-9 acselerates !naturation of the- endptaie as demonstrated -by-tlie increased convolutions of the folQs, Similar effects follow nicotine administratio n. The observed chat:ges in_ morphology-of the developing nmj subjected to nicotina- may be mediated through nicotine-evoked ACTH release. - Frischer, R. E., E1-Kawa, Sl. M. and Strand, F. L. Peptides 6(2),13-i9, 1985. Other support: Biomedical Research Support, Grant Program Divisio=n of Research Resources,a_nd National institwes of Health. From the Biology Department, New YbrkUniversity, New York.

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SPECIFIC BINDING AND MET-ABOLISJvS vF (-)- AND (+)- [3HjJ-NiCV^-TI_NE-Ifd ISOLATED RAT=H.EPAT'JCY-TES AND HEPATOCYTE MEMBRANES -'I=he specif-iv-binding of (-)- and (o)--sH -nicotine,-as well as-tlteir relative r_ ates of metabolismT yre_.-e_-_lavtst_ioated in isolated rat hepaiocytes and-hzpatocyte membranes. A Scatchard plot of both tritiated e,-,ahtiomers revealed two campcrien*.s; with the higher affinity si.te havint_ Kd va'urs of = s W'c and ? z 10-e M and Bm,~ values of 5 z 10-tr' (+;-enantio~ Ers, respectively. The and 4 x- 10. is moie3,mg protein for the ! 3- and ;ovrer affirtity site for both en3ntiomers had a K_a o= 4 x 10'g M and B~~ of S x 1 C'~ moles/mg protein. -;'he pH opfi-Anum-of bi nding was in ilie higher pl-f- rar:ge,- i- contrast to -brain-membranes where_-tne optimum was 8.5 involving- the protoneated form of- _gccrd°-correlation _was observed between tte pharmacoicgir petency= of a -Licotine.- A -group of nicotine analosues and their ability- to bind--to intact and hepatocyte membranes. The shte of co:,version of nicciifie_ to cotir.ine in heGatocytes is -related to -the degree of binuing- and accumulation. The results are discussed in- terms of _ the possible relationship of the nic-otir.e binding and translocation to its-me,aboiism ADood. L.G. et at. arckiyes interrationa'es de Ftiarrmaroayaamr_- et de Taeraple 2i3(1-):bi-'3, 1985. - Other supDort: DA0014S4. From the Center ior Brai- Research at:~-Fefartment of _i3;~scheristry, University Rochester Medica! Center, Rochester, NY. EFFECTS OF CHRCNI;;AI<LY ADMi°riiSTEREGS NICOTINE AND SAi.INE ON MOTOR a C TIYITY IN RATS This study ins~;stigated the d-iffere:,ti=i effccis-of chr;;nically admi;.istered nicotine and sa=ine_on motor activity on the-rat. Nicotine was administered via a subcutaneously implanted osmotic mit±ipu :np to effect an-8 hour off, 16 hour on flow. Subjects were 48 male and 48 female albino rats, each about 165 days old. Activity was monitored every ~our fo?-192 ~ ns~cutive 4ours. Results indicated that the female snimzis ~were more active than the ma!1s and that-anitnals receiving nicotine were significantly more active on the first two day7p drug administration than control animals; hbwever, by the fourth day there were no significant differences b-etween the-act_:vity levels of animals -that receivednicotine and those of conirol anir=ia-:s. Cronan, T., Conrad, 3., and BrysoR.-:.. Pharmacology Bioc;temistry & Behayror 22(5j 897-899, 1985. From the Department of Psychology, San Diego State University, San-IYsego, CA. i i i

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CFLARACT'£I'cIZE! T Ii'iN OF ANTI£ODIES Tt?-:NiC-'-3TWE--- - The re-Aarchers studied -the specificity of- antibodies _prcduced against four different hapten-carrier conjugates: (D,L)-6-(p-aminobenzamido) nisotir.e-bovine serunro albumin (ESA;, (t.,L)-6-(r-aminxaparamido,t nicotine-$SA. -(D,i.)=b- (p-aminoben=mid) nicot'sne-ESA and (D,L)-b=aminvnicotine-E3A. All of the- produced- antibodie3-shoeved about equal specificity for both-D- and=L-isomers o3-nii.otine. The carbon length of the spaciag - mp!ecufe between the hapten and-carrier molec:aie did _*:ot given substantial_ influence-tta the specificity for ihe-=pyratidine= portion of= the vicotine molecule. _The specificity 3n-pyridine prtiosr-wa.: i-ricreasg,i-as the_length -of- the spacing- molecule increased_.- All of the produced antibo_dies dic1 not z:9ss=react significantly (less that 0.=.%) with cotinine, N-me.},y.-=pyridiniutrr compounds of = (..)-N'ritrosonorn cotit.e. _ Studies on nicotine levels ic.~the- biological f-tu:ds= of rnatr and- rabbita indicated -that the radinimrrtunoassay tec};t}iax,A deveioped-in t;ie-p-esRit studies could dete_izs low -as 2-3 ng/mi. ' _ Castro. A.. Munji, N,:-Aii,-;I>, Bowman, F. R., and Mc._en^is, i BrochernicL.t-A-rcl:ives 1(4)173-i33, 1985, _ From the Department of Pathology, Hormone Researcir Laboratory, Jttiversitx of Miami _ School of Medicine, Miar.ii, FL and the Division of Biochemical Pharmacology, Medica-t College-of Yirginia; Ric4mo= nd. 'SEMi-RICyIr` AND "FLfSZ=$LE' LINKAGES IN f4hi-is'ODY-PRODUCTIvN FOR D:TEI!LMINATIO74' OF .N!: -.`.TINE - - to couple racemic-6-amincn:cotine t3=bovire serum albiirir. -_- --- functionatized-hh?ten,-nico3ive antibodies suitable tor use in nicotine de:erminatiozs have been produced from_anti,gens in which both 't ezible' and 'semi-rigid" chains serve for vari4us reaosons.- In the=-:pmsest s'udy= of -the use=of racemic 4rninLnicctine -as a conc!uded that 2- and-6-aminonicot:ne were-not suitable functionalized nicetine-haptens certain unique advantages with srnal: samples ans tow-nicoti ne-concentratir_-ns. In the first published_study of a rldioimmunyasgay for d=ter:tina;ior_ -o nicotk.e.-it= was disad-vantagec Hcwever; radioimc:unoassays and related p.ocedures have=proven to have Asi- increasing interesd, ir:- precise methods for deter^-ina_tion pf- n-ico-ti:,x 7evels- ir body fl;:ids-has led Ha deve:opment -w:_-numerous procedures, a s of which have some Cas_tro, A., Pv:cKennisx #=.;lk_;dvlonja, N., and FBawa:atit E. R. BfocfleTHcQ:-ArcFrives](4):205-r1A, 1953. Other support: The Americar, Tobac-eo Company; . - College of 3 irginia; Richmor.d. School of Medicine, Miami,-FL, and the Division oi-i3iochemical Pharmacolsgy, Aiedic?l- Frocri the-_Dgpartment of Pathology, Hor:fione Research Laa-aratory, -University of Miami P7-IOSPP;OR3CLATION OF A CHROMAFFIN GRAN'JLE-$INDIN_ v PRC3TEIN BY PROTEIN KINASE C -_ Protein kinase C:vas dAtected in a group of CaZ`-depend-ent ;.hiomaffin granule atembrane-bir.ding proteins (chromcbindira) on the basis of CaZ`-, ph-osphatidylserine -1

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l,2-dio!ein-, and-phornol-myristate_ acetate stimulated histone kinare activity. When the -- chrots!obindi5s were incubated-witK j~;-s4Pj.~'I'P, calciut~ and ShosphaiiCy!seri:~e s=P was incorporated predorninantly-into-a protein of mass 3?-4_ 1-kilp<ialtons-fcasornohindin- S, or CB9y. -Phofphcry!atias,-of this` protein -.vas-also-sti'tnulated by dieleitr arrd phort+ol myristate acetate, indicating that it=is a-suEstrate-for the -protein-kin?se C a; tivity- pesent it: the chrorri9bi^dins: Mazirnuat phnsp!:-stg incorporation into CB97 iP -the Presence- of -- r 1mh4 calciuat; '3- µg,/-atI of -ohbsphatidylserifv--~~2.3 yg;'m' o~ dio:ein, an-1;2.i µg(ml of t 3°P-la_boled phosfhop'ptides dithiothreitol= was 0;53 mat/;ol of CB9 ia 5 min: F`igh were resolved in-twn=-di=mens":oval e!ecirophr,-retic ttta;s of trypsin digests of CB7. - Phosphoamino= acid aralysis revealed that phospho, y!atiob was exclusi3ely oE~ ser ine E94%: and eh-reonine (6%)- residues: I>?cu4at_io n cf dhy chrotnobiiRins with chromaffin granule meu,l;canFs-Yn the presence of [ry-3Pj,w,'fP resulted in_ the irccsp4raticn of '-x° into eight atiditional_~szc~teins 5e_ides CB~ that cccld-ie separated from the rnemlzrart=.s by -centai.rugytien ih- the prese--~e ` ef eti:yleaP glycot = bis;hela-aminveth=y!: ether)- = Iv,N.N',N'-3etr-aace2 acid. We suggest that phosphofyla.ien - of _ CB9 or these additional eight pro=eins m=ay regu,zte eveats undvrlyin4 exocy*.osis in ,1;e c:rom-afiiil c_!l. _ Summers, T. A: arrd=Cieutz. C. E; __ Tirrlourn41 oj°Biodogi-cat i.her;7stry_260(4):Z-437-2A@3, 143. Other suDPort.°.Natienal Inst;tufes 6- = Hea-lth; National Science Foundation and University of Virginia Diabetss-,Research and-Training Cenig.. From the Department of Pharmacology, L'niversity of V:rginia,-C harlottesvilie. P'fIOSPHATIDYLINOSITOL-S'ECIFIC- PHOSPht,'LIPP S;: C_ ACI°IYI'f'Y - OF CHROMAFFIN aaRi3N:JLE-BINDIAi:,r PROT EINS: -Lising (FJ='4C]=I`,--osptsatidy!inositol-as =substrate: Ca;+-depender:t p?hspliCrlipase- C activity was detected in a group o,f- 't>ovine adrertal -pned,ilary-prot-ei>=w that bind to - chromaffin grar,ule=ntembranes in ghe presence of Cas* Echromohind`_ns;' CrFutz; C.E., Dowling, Lfi.; Sando, I.~.;-'::Ilat-!kalasi,_C., Whippie, 3`rl.; and Zaks. `,esr.=~(~g3) J. Biet. tsvity was- t~aasmal at nettiral yII and _ rep:eserfted an Cherrt. g58,-i46o!! .i4~. 7he a _ 80- to 240-fold eif3ichmeni of adrenal eteduilary -cytosol phespholipase= C- activity- ~ measura1 at pIi 7.3: T1t ~ti*rtulaYir~ rif aetivi;y ~y Cas* was cornplex no activity was ~_ present- in ihe absence of calcittm, 3Ss"s activation occurred_at i'M -Ca`t, and-full ~ activation at 5 mM C.a=*: Tbe--enzyme bound to c!:rotaaffin granule membr;:nes in the presence of 2 rrrM Ca3+ but was-reiease i at 40 µM, suggesting that intrinsic enzyme I activity -may be reguladed-°by r_a'*; _ at 1 pM but additional- activation at higher concentrations of calcium is seen in vitro as a result of CaZ*-::Fpettdeni oiuving o, the _ active enzyine--to :al;strate-containing membranes. Tltis_ enzyn:e- may- generate __ diaeylgiyterol aad_ p!tosphorylated inosiio: to act a; iatracellular messengers irl the vieinf*.yof thez3t;~affin°gra==-~le ~eehisrane during ;^e-process of eicocytosis. Creutz. C.. i2owlii L, Kyger, E., and Franson,_R. C_ The lownat of-Biofogica! Ch9rnisiry 25t?;12 j:'1'.7l-7173, 1933. ~ Other suyport:-Nationa!-Iostitutes of Health and National Science Foundation. - 119

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From the Departrneat of Paarrstacofogy; Uari3ersity o Virginiat-Charlc*.tesvilie, and the Departrrt~ra of ~iechemi~t;5, ~Medicad c:ol:ege-o` _~'irgin:a, ~sc~ona. _- and forskolin.- The major soldble phosphoprotein to be affected by these-agents had a relative mo1 wt -of- 55-57 kQa':tois o:- sodiusn-dodecyl sulfate-gels a?d- corresyonded io tyrosine hydroEylase-, which is-a specifftc n]ar-k°r- enzyme in _ the nTrerial f" ch,roii3attin - - cells. We propose that-bov+: e adrPnia_1 ch:Cmaff ii± ce11s-eErl'ss- A' T~r?--a receptors iOtich_ _ are ;.oupled- to aderrylatg cycl??e. _ While na acLCe effect of ACT-H s was_ f:,u~_! o-ri- catecholaenin.e secretion, AC-1-:I may play a direct r_olei_e the reguiatio.^n of _catecho}aming_ synlhesis b; - stimuiating- ~ the-_ phosphorylation = of = tyrosine- _ hydroxylase by cAMP-depenrebt protein k-inase. "a rnanner indistinguishabl~--iro?t i} at--irduc~~. by carbachol _ io 32P-labeled- BAM cells in ir.crease in cellstIaf- cAMP without -an effect: on `cateehofar::ir.z secr;.tion_, sug5sting -cortica°- cell-ct+atarr,ination: Percoll density -separatioo of ~boi, mAM cells arid-adreaal cortical cells revealed thar th, greatest tAMP responses fo AK:Ti:,-corresFcnded td_ the catecholarRine-con-taininK cet.- fractions and no_t- to those density layers where= co-r _ioaf tetts sedimentd. _ BAM=ee!!s isolated on Percoll did not metabolize [;'rC}ci olestero_- to stero:d.c as would be-espected were the=ACT-H-stimu;rted-^,AMP accumulations 1ue to cortical cell contamination of- the c rl,cres- - Ar ft! stimufated _ protei:=, phosphphor}iation sricnal s=secre!ion coupl.ng~- The-additio.^. of .?x"'1'f:-to these cella c-aused a?0- -to 50-!'o1d __ i r- prepared and r:ai: tarn d- iu culture - and used -to examine the role of cA*4P increase in'-ihtracelfular- cAMIP fe'el;f- Bovine adrenal, mez:u_Iiary_ (°AM;- _ca!!s were Reh-easeof adrena:s;ateeho:afn,"n~by -ca;-bacho:-has;oe1t_shown_to-Cxrincide with ay ADrZ-ENx2beE,: s`JLLARX CEf LS:= RDEI+fi?S11-4E 3.5='-Mr)NOPYOSI3HATE- _ DEPEND E1`IT-E'r'tdSPHGR~i=ATIi:N-OF TYRGSiNE_ H-YDR-C '.Xy`:.-A :,r =- D!? Ec!' EFFE^TS-5 rJF -ADE'cE-!-rOCCRTlCOT#tOF1C HG13MC>NE ON B-CtV l--irlE Michener, M. L.; Peack, M. j,;-and Creuiz.--t E Ehd ocraslology 117(2).730=737-' . i,-~s's-5. _ rj- ttar .ti:pyord: Na*.ional irt3tatt`s of Health, a*.t the-Nationa1 -Siy°iY;-0ce--Four±datio.=.- w,~h aam- tln;.,P-;ry nf Researc` and Traioi: e Center and y ascular Smooth Muscle Prograrts_- ¢ro iect.- __ - From the i~patrnent of P:ait:~acofogy, 1,Inivzrsity of 1 ira:n-iaChar attesv i1:e. 4 LrTlrJNIS AND_ilsl MANNITOL AS AN C!" S<"AVENGE''t R=-AQUEGL'S SGL _ BIOLOGICAL SYSTEMS By uing the techniaLO-of -pulse-radi~r'_ysis !o generate J!-l-- radicals, t?:e-_ ~?utnors- have determined through e<<npeticior( with SC :`-,-i` and FWCtti~g4- the reaction ra?e _con-- atanf of manr.itol-with Ji! ryd:cal at pi-f ; 7 to be ~1. t 0.4? R t0~ s.-t s-t: This value - justifies the use of-mabnitoC as an- 01- sc-averiger: Howe~v-ez, in Eiological systents there is some uncertainty concerning the relevant =c^ncentration= of- this-sca-ve=ger. -."' ~,._e - -concen.rtion inside th-ecelfa:ayhe-very different from that in the solution -because of active transport on the one hand or through-metabalisT _or= the other. Even if -the_ authors caa determine the ednceatratii,n-of mannito! ir.side the cell, they are not able to determine the conce ntratiorr of the targets where the dantage occurs. For afl these- 120 -

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reasons, they cgnnot txpect that the effect of-0?- scavengers in biological sys_tems w+ill= be'?ropor .oral t- lf~e=i= - e _onsca ts w<<b ~i:; Croidstein, i:-and Czorrk:. v, ln?eln9tionol Journnt-of Bnslaat% n Brc:qg; 4E(6):'?25:29, q8E.- Other- srpport,- U.S. -L)ep partment of inergy, and Cesel!sc%aft fur Strahlen Forschung Neuhe-rberg,-zVest Gern ia ny. From the -Dep=rtmer:t -of_ Physical Cheinistry, _}iebrew_ Un_iveF=ity of- .~erusai::n, Jerusal_m,l sraet.. KINRTICv=OF C7dEIDA=sIDi:' OF CUPROU&-CJt1!_Pz.:AEs C7f3UB5_';IT-iJiEi3 PfiENANTI-iROLiNI AND °2,2'-9IP;'RIDYi BY MOLECULAR OXYGEN AND -BY HYDROGEN P-ERGXIi E IN AQUEOUS SOLUTISiYv _ The= kinetics arid- the -reactic,n - nsechanistar of -Capper(I) ' Co:npleies - of - 5-ynethyl-l,i0-rshenzt:tniciine, 5achioro-l,I0-p ser.antnroiine, -5-niir I,f~.'.-phenatr- throline, 2°=ditnethvl=,10-phenar.throl ine,- and 2,2"_-ricyridyt w5th-_ oaygen -a nd- - hydrager pert+x=de-?ia~,E been investigated in a_quecus scw?ions witF _use_ of thz- pulse- radiofiy3is technioue. 'i'he axidatio^ b}_ 02 is- _ second =o' dcr_ in th-_c_opper"I) complex, - +i::ile the Oiidation byr I-i;O1 is t i3t Craer-ln the C,-p[ser(i} ColFiplex.-- Both re2: tianS are rst order ihrx ic~a^-ts. 'Fhe icinetic resUlt~ of t=~= o- idation -;f copoer(e) cosiplezes by-= oxygen ~re nierfiretew ~y a mechanism that-qtvceeds v;3 a s oer:,xtde sr.te :ned.ate. Goldstein, Sc. and C'zap;ki; G. lnorg--n.ic -Cherr.istry 24(7)--10R7-iQ92, 19i?5. 0her sup~.e}t: Gesel:3cha:t =~r Strahler. Forschung ?~'e~;.hertie:g -a:~d the 1J.3. L~e; artrect_ of Energy. Fro_m- the i3epartanen, of fhysieal- Chernistuy; Hebrevl--Uniizrsity of krusa:em, Jer~sale~ ,. srael - -THE CC*N'I`RIBUTIDN (3FT?3DC3CiENOiJ$ AND EXOGENOUS EF_FEC'r~ TO RADIATION-INDUCED DAMAGE IN THE BA: TERIAL SPORE Radical scavengers such as polyethylene_Zsycol 400 and 4000 and=- bovine a?bumin- - have been uszd_to define the contr iizutio: of exogenous and endogenons effects to _the gamma-r-adiaion-induced~.'-arnage in alqueous buffered suspe?:sion3 of Bacillus purnitus = spores. -The- reEults in dicate that -this -damabF- in the - faoterial- srors - is- pre- dominantly endogenou': both in the pFetence-of I atmosphere of oxygen and i n anoxia: __ Jacobs, G, P., -Sa:nuni, A., and ezapskr; Z. International ,iournal oE Radiatron Biology 47(5):E21-5Z7, 19&i.- Other_support:-U.-t. Department of Energy. - From -the De;.artment - of -Phar;nacy; School of ftarma:,y,--Department of Molecular Biology; School of_ Medicine, and Department of-Physical Chemistry, H-e-brew Ur.iversity -of JenLisalem, Jerusalen:, israel. 121 - cn

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EFFESTS OF E i HANDLAND_TEMPEItAT'IIR=E JN iLD'COS-ELrT.EIZATiON - IN-iHE IN-V-11t: AND ISG1,A5E--~J-1=Ei4FU3ED_W-DL3SE BRAIN temperature-de3endrnt-m-en;brane phase transitinns;_ brain perf:sions were performed' with a medium cehtaini-g ethanol, vvhich s-known -to increase membrane f'vidity- - i'erfusioy of the mouse braina with a fluia containing gi ntM Pthanol (A001 mgjdl; resulted in decreases in the glue~s ui!i~tion rate_ a: 3 but i:,t -at 2g'C. At 28'C, ethanol- produced-an increase in glucose utilization -ki the teed_ulla-;ons and in the cerebellum of perfused brains. - Fn riro__-ate-s-f of cerebral qE'.:c,se utilization it two 1'sn=s of mice selectively bred -for -diffgreeces- in ethanol=cnd-uced 'slei'p timt- ind'nc.ei that n-o-` differeQces in basal metabolism or following a4-gjt<g-dose of ethanol eaisted between the two iines. Interp:e_vtions of ett.ano't°s differential effects on cerebral glucose- utiliza_ion rates -in the isclased pezfu3ed -mouse-brair, at-2g'C-and 37'C--are prrsated - along with a discussion of the apparent discrepancies observed when in rivo and pesf tised - suggests that a change_in,:he_rate-limiting step of cerebral g:ucose metaf+olis t occurred - between _ 28'C and 32'C. To investigate the possible involve~ert__ of :--A [3A"P-det35Sy-t3-Eiucose tEt:hn3'3iie was ;:sed to investigate the effects of ethanol and temperature-var-iatioe on=the rate oC-giucos:-u:i[ization in the i;olated- perfuged _- mouse brain. Glucose ntili3siion r3CCa in $ci.id=r2gio o( the_itoiet2d perfused mouse brain riro ,;,ouse brain. The at-37'; were similv ~ to the ates in compar~b!z Teg'sons of~he in temperature dependeoc== of the_-gl;:cose utilizatior-rate-in the-pcrfused mouse' -tirain brains are treated with equivalent concentrations 3f ethanol. = Other siJpport:-U. S. Public h_alth SerYice. Atcoholism: Clinical and-LErperimental Research d(-1):11G-i16, 1992e- 2; To,velS, :.-F:; 1;I1; ani! Erwin,-V. O. From the AlcoholReseari,lr yenter, School of .haranacy, University s:f iolorada, goulder- -FFECTS OF NIC_'QT1NIz..0N-fl-ENDOP.1'IIIN, a_IvIS', AND AC?'k4I -_SEe:.RETIOtd_BY--= I.SDhA'fED PERFUSED KO;eSE BRAINS AND PITUITARY GLANDS, IN VI _Tfa --- 'i'he effects of aico.i;~e_on secretion of_the-pituitery peptides-fi-endorphin, a 1dSH-- and AC"j-:I were studied using the is©ia=ed perfused-mouse brain_(11PA+IE) ane isolatEy superfu~edpitul'tirles of C.33': i"iali.E. Nicotine (6:1 ji.'~1" stimulated secretion _ of B-endorphin imm3noreactiviq from C3H IPMB approzimateEy -t-wofoij. Secretion of a'k5.s-I-d_ intmunortactisity-was s3;o3~1v;~_~pgresi~ra;ety tvoa and sixfold -b, 6.1 juM - and 12.Z- tilAl nicotine, tiney res~*i.tival '. --I-Iowevyr , _nicfltinF .(?f1 -pf1:i)= had - no direct effect qL`~ - tfie secretion of B-endorph=n, a MSH, or AC CH :mn;unoreact:vities from the isolated superfused pituitaries.- The-data-suggest aicotine acts in the-brain to stimi'late°pitnitary __ __ sscretiEn_ of a t.S13- aa_el-fl=eRdotpnin. Electrc--coiticograp;ic- (EC`.oG) activity of the _LBrvi13 was monitored. Nicotine- induced bharacteristic-EisoU changes inclueiing -$ reduction of input tr:tege,_ abipbasic response of rapid ticsy_^.chronizarti~.5 foilowd-_ by prolonged synchro,rzation, na seizure ai-i3ig.a de-s.cs (12).2-IaM )_ _ Marty, kt. _Q., £_r_wir. V. G.. Corriell. K., _andZgombitk, ;. Nl. PF.er:!ta3.cofogr. 6iocPrenrrstryA Behavior 42:31'1-311% 1985. Otber sappori U. S. Public-:flea!*.h Service.-= Fromm the Center _fo;-Alcohol Research, Scros: a: Pharmacy, -ilniversitY of Colorado, 13oulder. _ 122

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f -V.A1-dADi:TIE.Et4HAD~L£l+aft-4~T;C- _-e-H-vXkF3AT-O_NtrF?RDH-Ir G2`: £FFF';'S =F PHDSt H ~'E-A Nrz C1==r~ iTi-N ;: b.G dN TS. t -- Th i~ - - rt~ ~^d - _= Xanadate_,;<ta_r~c<dl=y s._sr:ulat?s shti c;idat ntt,.c=` *_TR~LI°by '°, >3oth-phosqf=te -and- Tris ~-:e nhibr.nty ,; but phosphate ai~stsi3nes,tr:e K.~ter !!hi'bito3y effec: ~: T and _tltus -gires the=,~pearance of- stimulating vvnE~-_,dd to T`ris-bu:fered reactioa: mixtures. ::he:agir+.g aoer:ts-moderately_increased-ihe ox:dation__of?~ADH but el;rzinated the much greater catalytic effect _ C'f _ vanadate, -_ Dest'ee'a l'al h%ai the rTisl ef {fb;iv e of the -chelating agents, and Yould be used to-t:trat vanAda,y sf,ectro4hoic,-e_.risally~or in_ terrKs of-the=-dimii.irtion--~sf-it~_catalytic a~vird._-_ This_,ermitied the demon3trat;on that :netavana~ate or _K?hc- at`.ada.c co;ad- forrt- l.-l complexes with _ desferal -and - that-- o a e xyt,c o-, ..a.~ ,.2-,_a.~ tv ~..t+?r.e s~! !z? - Darr, D. and_ Fridovif~. f.-- -irchlves _oJ Bro,. :emistry-nndB+opiysics 343(1):.L2C1-22';,_ i485. _ _ - Faundation, at.d- rlational-:nstitutes vf H4alth. _ r3m the Depart:aerat oC Biochern!s ry; -D! .e UnavetsityIvi_dical Center, Durham, NC_ --c<ther support:_iJnitsl $taea_Army Resesrcr tif-f;ce, Naiidal Science Foundation, _+lP:?t+ -=i.SSEl1 T IAL[: Y-CF -a 3i>=: .-.C -11: F-S1 TE ARGININE RF,',l-DUE FOR THE CHTA£,Y'3'1C-ACTtVITY f3F=Cu,Zn SL7PFROXIDF DI31wTASa., a!;com~anied by the-_c7;_odificatior-of less ihatn two arginine residues ner subun t with no concomita$t locs o:=!s o= Zfl: `I' lie _ehenylglyG!sal-mdd .ied_enzyes require a: least a ~0 -f vid greater conce~=tration-o= cyanide for 50% inniGit:on than dG the c~rrespondF. g , native -etrcymez. -- Fiiyaf,ry-lani,~-~e_-gel elec.rGg_oresis and -actiVity_ staining= of -tne p~enylgiyoxal-i~tacti;at£_d-_en4yfi~.=s demonstrate that the residual a~tiv!!y,i; =la~gely associated with _modi fied-for,tts that bear lower-net positive charge t`a;i the aative superoxide disni2tases_~ - Borders, C.-L-,,r., Saunders, : E., Elec'i D. M.,and-Fritiovich.I = _- £iochemistry >marr.als3Q? 711-7'0; 1995, Otker support __-Retre.eum- Research Fund, -U S. Army Research Office, the_ National = Ssjence _T3ur_diGrr,-P.nd1?e -G-nh_-FCss sdaEiG ..- From the Department of Chemistry, College of Wooste ; Wcoster, -DFi, and the Department of Biochemistry,,Duke University Medical Center. Durham. NC. - ° - - C~!t'IIYicaF n od_iiicat;i~n_ of ` N-m-itne_rred-ye3s: 'C1:,Z_naupiero?.lde_-diE.;%niz a-_ss- ii'+r h - ehenylgiyozaf die :ir,ishes the catalytic activities by _ yg% and treatment of these er3zymes with - butanedione plus borate leads to -> 96% inactivation. The activitv- loss is F

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PSEUDOCATALASE FRt-`j_-~r# LrlCTO~PACIf.LUS!)=,ANT,4P~!J:.': Fz!37"Ni=c -FOR a iiOS-OYF:M1'T-AMErtIC STRUCTURE C31v?Air.it=.C TWO -°.zavlS OF MANGA1SFS':-=~_ - PER SUBUNIT ^n improved procedu for the aolat:oa of the gser?docatalase--of_=#4.,.oac,s'ur ¢lantarum has been devised ard.-the ~uaterqary-structure-and-manga~ese-cotEbtEitt-o-th`ss__--- -_ enzyme nave bgen reezamrrsed. --Sedtmentat:on gqui,rhraum of _the-native- enzv_me- at= everal, salt cotrcentrattons ~ave a molecttlar- weagh: e-_ t72,Q00-. - i ng subun;t, we=_GUt, obtained by yedimentation _eouiliGriuin in SAM guanidiniutti chloride, with or without - prior redu_ctiot, and carbca; met _ylation, was 34 kilodaltons_. The amino -acid cornpos:tion - indicated 150-Arg + Lys,- and -afte-r- exhaustive trvpv.c digestion, 32 peptides were resolved. -.1^e_st' data su.*,gf'st that-the_ f m~dTcual2.4c-is a hom°.)Elereta?:cr: Cross-l'- nl:irag_-- with dieT_ethvl suberimida'&, followed by polyacryl-a:'!1ide R:l eie_ctro;^,horeSis fn. the Pre<-ence-_ of sodium -dodecy su'sfate,- yielded five major bands, another indication of - ©entameric structure. 1 he man garese ,r'.o;)t_eiat Was roun4i to be 1.8-4.4 per- sub':dni`. 3-s. - w-as found to be_9.6S and j; Iu - 7.2, _<uggesting a giobular st_ ucture of Stokes radius 44a: Beyer, W. T.,_Jr. and J'ridar;h:-L. Fiociterrrtistry 2a(23):5460-64G:, 19g5. _ Other 3lpporr National ITstitutes of Health. t1_ S- Arsty ;<esearsh' Of!';cer and_ the ?Iational Science-Foundatio :. From the 1)epar trnent s:f Bic:c;ternistiy, .~r,-uke University MLd:ca'_ Center; Durham. NC. THE SALMCtN=LLA(MAMM AL.iAN MICRC'SOMF AaJ?'r0NICJTY TEST-- COMPARISGINuF_I-iiNv(Atv Al`lER_AT' {.IE'R S aS ALr'T15 ATINCi-SYyT4MS fne --a:utagcnicity .o several test- _ compounds- - was --verified =- by the ._ Salrnonelia%rnicfasori?e{nut3geniclty te-st (Arne_s test). uc'rng=both h;tmat: iivef and rat live_ :- (J3ltre3ted-ar pretr2ate4 iYithA-raicia'3r :254 ) S9- tt;°ider-Identica`r- experimental -clndrt.rF.s -_ A£lat>xin -B1; y-met4; lckolantirreae, ans cig4,e ,:e-_smoke conu'z-rsate- wr_ les3-trrutage,r in the-orese_r.ce of -human=t's-vAr Syt`rao in the=preserace-df rat-!iver S9 (particularly aGter treattnen_t wittz-Aroylqr 12541. -71e oppo,ite was a}3erved w.'t*.h 2-a?Ei."antl-Eracef e and -to a lessee' degree witi. 2=a'n1nofI1jo:a:le, correlation studies indicate --21-at the iwo - compounds were activated i:y the same or by very-similar enzymes, probably cytochrome - P-45Gs. These results clearly indicate that human-liver S9, as an acivating system, - behaves differently than rat-liver S9; therefore, it may constitute a useful, addition al-tool ortheftudy of mutagenicity and, probably, carcinogenicity in man. Beasue, P., L tr,es.tre lornet, R.,1Cren?ers, P., Alaest, A.; and ~Iele~, ~: !lJutati-n_n Research 1S6:L34-146,-1985. _ Other support: INSERM (France's Natio :al Institute of Health and Medi.al Researc}s)- -_ From the LaboratoirE de Chimie Medicale, Institut de Pathologie; Universite de Liege, Liege, Belgium.

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MONOCLONAL AM'FIOnDIES flGAfNIST-Fft-;MAg3 LIVER CYiiCi?RCIVIE P-450 hihnoc!onal hyhridomas which produce antibodies a_gait:st 1hu:ran liver tnicroso :ra3 cytochrome P-450 were developed. Three similar hybridomas_produced antibodies which- recognized an epitope specific to a family of human P-450 isozymes (P-450e). This epitope was also present on cy tochrome P-450- PCN-E (; regneno!or.e-16.--car'rxinitrile induced) from rat liver microsomes,_but=this isozyme diff ered from the human P=450e by its molecular weight.- - These antibodies enaSEed_ th e- authors to quantify cytochrome P-450s - human liyer microsonres and :o demonstrate an -irnport-ant quantitative polymorphism in the human-liver monooxygenase sys%e3r,. Beaune, P., Kremers, P.,-Letawe-Goujon, R., and Gr'elen, J. E.- Biocfiernical Ptiarrnaco!_ngy l4~!4):35d7-3552,i9$5. J:?ser sugy-or.: INSLK~%r (France-s-National Institute of Health and Medical `cesearck). From the INSERM, Faculte-de !vledicine-Ne,_eer, Paris, France. -EVIDENCE FOR I;F.G:ILa TIVN OF A_:"T:N Sj'NTIiESIS_ iN CYTY3CfiALAS!N D- - TKEA T Es7 :{Fp-2 CELLS In -HFp-2 cells treated ~r -_th 0,2-or-2 Q pM cy*.^c:.alasi: = (Ci ),the _r: aiive rate of _ actin synthesis increaspd for $bCn: 12h and then reached a plateau; this increase was suppressed by actir,orr.ycin D (AD). When CD was washed f rorn ce!ls -which had_ been treated Ior 20 h, the elevated rate of-actin synthesis declined_to the contro! value within ca 4 h, as rheactin-co^tai::ing c3toskeleeal components rea=ranged-Ey CD recovered their normal morphology. Subsequently, actin synttiesis was depressed below control valuzs -for a prolonged perioy; during recovery from 2 h treatment with CD, this depression_ was of much- shorter dura.ion. Ke-addition of CD to cells after a-3h recovery pe: iod= again induced the cytoskeleta?-aiteations-cha acterist+_c- of-C-r'i :reatrrie~a but did n ot -reverse tie- -prioc decline in the rate of actin synthesis: t-1 HEp-2 rel!s -treated with ;.yclohexirnide during exposure to- C-D for 20h, the relative rate of= acti.n. _ sy nthesis measured after retnoval of tycloheximide was twofold higher thaT= with CD alone- ar.d -such- cells- exhibiteda twofold slow¢r decline-in the rate of actin synthesis during recovery=from CD in the -continued presence of cycloheximide. These effects of cyc:oheximide; which resemble observatlons on 'sun9r-induction,'-suggest -that actin synthesis in- CD-treated -and reoveri$g !iEp-2 cells may be regulated by a repressor protein. -The possibiiify that the proposed repressor protein is actin and that actin may thus be-a feedback inhibitor -of- its own synthesis is discussed. 7annenbaum, 3.s Brett; J. G. and Grdraan:-G. r. - E-xceri,nenEa? Cell i;rsr-arch !f0:435-44$, 1985_ Other au_cPr%t: biuscu 4r-DysE;ophy'Association-and NaYional tnsiitutes of E-!ealth - From the National Science Fo undatio;- and the Department of -Patholcgy, College of Physicians & c Surgeons of Columhia-CJnive mity, New York. 125

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- - MACROVACUOLATION INDUCED BY Cl'TOCI-IALASIN: ITS-RELATION T'D- _- E ~%'TC:C 1-IICELETON ~ +-_ _1 ~+~~ L _ VwE-R-VATIC).`. ~ = A: higher doses- of_ cg:ccha_as,n (e.g s rg,i "o 3-2~= hr), cell; of iha- rat - F fibrotzlastoid --line,--14rF-Fi- unde go- extreme retraction, arborizatiun, and--subse;ueni-- roundrng, anu deveioo big=cystic vacuM es:-fih se vacuo;e3 are always- c osely- inve -ted by microfllarnen4ous-',~T,asses,_'the CD-indueed- der vatLv-es of th +,n-bas°S cytw3keletun; which aggregate in the endoulasm. Vacuolati(n is progressive ;e.g. z9e ceffs at 6'hr; > ~ - t .- ~_- , 4 B ~ . ° enveloping. . - -- .._. .. - __aciia-filiametiu - 3ggregates--are snrrc,unued - by arrays of vimentin-based interm=_diate filaments. A new + mc._,branous compartment with characteristics of=plastna membrane is thus formed :.~iti?in the ceii .;nder the influence of CD. Rounding Grought- aLo}3: by other means caasc's a"Io var'6rolizatlon.-''~---_scroYac-ilnlat+Un, lke-tl_re other :hanees - caused 3y_CD, :s coy rpletely revi°sib.e-o, restora°.ior: of ce?is to normal-nedivtrF. - - ''is::ug & Cel: 160j:311=32a,;484. - Other support: 't+lationa!-Ins.itutes-oI' I:tialt =. ___ _ University, New York. _From the- Department of =Pathologyr College of Physicians & Surgeotts° F: --Colambia' -- Muscarinic cholinergic -receptors_ isolated=frcnt Ll-rosoyni!F head-s: rat and human brain,-- doB heart, and=--ntonkey ~iliary- muscle - were- e-zarnined- for_ _structural similaritiesjdif-ferer-c; s - - by utilizing -isoelectric = #'ocusing~ =s3diu.ndodecyl - - suEfate; polyacrylaniide-- gel,eiectrophoresis,- and Y,onoctonal antibody crossreactivity. Muscarinic receptors were a_ffir:ity labeled with [s;=}propylbenzilylcholir±e- mustard and subjected-'so=isoe?ectric_ f~;cps:ng. Muscarinic secep.or fr:;.% each species- focused witlt an -isoelee*.ric poir.Y of 5.4.- The satre proteiw- all migr--2ed- with ar a5paren; molecular mass of 80,400= daitarss =or=-_ sodiurrm dodEcyt- sulfate gels. siz I+_ybridort;as_ secreting - a'no]oclon_ai antibodies spe-ciiicc for m:rsCari:`ic- receptors ?'y.e-rr devRl-'v~pt-a by 4I-si:g j:urif ipd rat brain irtuscari_r,ic recept-o;rs as_the antigen. -Th4-six - different monaclon3l aneibodiEs _ immunoprecipitated muscarinic receptors from all tissues and species tested, including human and 6rosopnifn-brains,-with equal=efficacy.-These data indicate that muscari-nic -recegtors-are _highly conserved over a- conside7auT evolutienary period. One of the six muscerinis -seceptar -monccional antibodies also immunoprecipitated rat liver a~adrenergic receptors. Furthermore, two out of five monoclonal antibodies raised - 1tiONOCLONAL-r.N-1:E Ol_3I£S nETEC.1T-T-IzE CS,NSEr2VATjt_vi= tvP.JF--CAn-i^iIC !r'1IOL.PfEERy1C RECEPTOR STP.UCTJRI FROM DRf3SnPHiLA -T-Ci f=1i;1NiAht BRAIN AND DETECT fOSSiRLE STftUCT URAL-HCiviOi.OOV =- i:i9'I+ ar-ADRN_rRCslC REi;EfrTOr'tS = - 126 = junctiona; specializations as those found at the tell surface; they lack the rn eni:rane- - V Vacuole membranes __ have tLLe same dimension (85 A), surface marker 5'nuc:eotidase, and ). re?ated 6 at_18 hr),- .o, total dose {concentrat~on x time, anc fo congencr 1c.JJ s%_ 8G m-arkers_assoc:ated with =endo;#sesnbrane= systenes (e.g. AcPase; T-PPase, lDPasei -aad are not lyso{or,.al= VatuoEes reyresent ireterr_a'izcd plysrsa membra e; they apparently result from- retention iz the -en&plasm: and .usion,=-o' vritccytotic vesicles originating ai ti e cell surface. Vacuole membrane is always in intirnate- rela.ion tco the - actii-based micrnfi:arnent aggregates-tltat'surround -the Yac=uoaes;-anP -actin-men:brarie linker-Froteins fodrin- and v;ncutin a =~ ioc ~lized- af thP v^cuoie botihdariAs Va^uoits ard thei ~

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d mu;Far151iC feC?3?~Jr,. _TFieit-Clata 331ggr't that against those 'CCepeo_es iffalt'~_ IloyreCiCita V sottre degren o; structarat ;3oenology_ exists _=betwee:= rnascar:nic_ -c}oline~gic recertor -- and c3-adrenergie receptors. ~'edier,7.F:, Erd;,B; L~t~ L.4f:: a~i#-Fr~er, C.M. _ Proceedings oT tFa=-f+?att'opal Ataderny o~ -S-cizhces of tto lJnfte._~._- _S:ates of 4rtert .~ ~1:172-2'r6, 1~4. - -_ . G:her cuppo; t: Ameriran i-Iea*t Assoc-iatiorF- - Frcnr the Deputinertof=Ksr-,lecular :ritt:unoscgY, 1o=wei1 Park M4roria: Institute, i~e:~ . 1'or_X Ctate_ Department rf Health, Bu~'aIC:, ar~d=the= Department ~f-i3enstis, hloer: Ei ~t~ir: College of hiYd=sioe, ?! e Bro^n °Nv , Q-EtrTNG,z`.IiGTCXC`J_bL!CI;S EXCITATORY SYNAPTIC TRANSMISSIGN, F3ET_CVEa'.F7 CE!iC AL SENSORY NEL'EC21VES-Lti:ND-GiANT INTE.°..NEi1-Ys-ONE" 2__ OF THE CJCIEFIOA~':IrPi<!IlPL.'sf.Ef /. AMi<-RlCA:'dA -'i.- Alitoradiogr;h1?--!rC'-a!IZatirn,..Of .Art-=126i-o,-bSlno3:oTij,in-i7inE' aing-=-CQS:pcne'=2i _ revea!ed=thQt speciffy_oinding was distributed main!y_in the neuropi;v and=to-s_ome -extent _ in- the perig=:ery of the 1er.ninai=a_bdominaE ganglion of_the coc!<m_aCh, Peripianet: americana. 2. -Action -potentiais _ recerded from the axon-o: Gl I under _ curren;-clarnp - t_nditians were not arfected by exposure tc!.G X iG-5M o=hanrarotoz'n.- -- - ~. ExCitatorv poStsyn aDti,WteFtials ?ektrdt°d -fro!*:-Gl =, e-zio!=ed 4y-st:IFeEiiati4n-of - _ers813enzoly oPUrene.3; wefe sezzsFi3ve_tl btbck by_ !e!at-vely !cw_;from I~-~-_cm concentrations of a-.bungar3toxin. =The time-eourse for blockade was found to den-end CG tUxin coilcetttratio:=3f.d fre8lency-of affere?itstiii:eilatlfrR.=-°__ 4-. Excitatory posmy naptic r_+ot?r.tials recorded from GI -1 were not affected - by exposure to 1.9 X 1(Feisd c,uinuclidiny!-henzi!ate. - S. !§'e concludg that at :eas- ? f±ort'sod af -he r"I-a-sung-arotoxin hinding _-pcst;yna, +c - fun: cn at component -rzpresents_ choliner-g ;- -receptoAns which have a s j napse. between 2r al -Eenscry r?eoioness and ;,r 2= : 's~teile, ~. E.~ rla,rour,=l. D., ?-Iue;~8:, -Pelhate, m:;=C--~apnes, a. I, an;1 Ha:,!,_L. A-?.-- Journai of Expersmcn:af Biology 107:47"s-4fi9, 1983. Other support: CibaFeu-ndation Anglo-F.°nck -Exchange Bursary and the l.t. S. National Science Foundaiion: Frcm the A.R.~ Usfit°of -Insect ideuroph}siof6gy an FI:armas-.ology;-_Deyartnse?~f 1f spologyCamhridge t, -iivers;t,; Camhringe, U dt ;--La+o-atoigo_de I'h;Eiciogie. Fa. s!:_<_ -de lv~edecine, U.ii~ersit, a`_Angers; France; and the-Departmeni of Genetics, Aluert Einstein College of ryiedi;,ifle~; The-3ronx,-NY.- CHR6NIC tNI-IALi4TIvN '-;:-:iDiE-',IN-MICE L'r`ACILiTIES-AND- -- - i- ^sAREa -T Y'-E? i; 'E Sf'-AOKE _ ~a r1?IL 'F'C'c?JRE TO E~tJI~'~EEI~'T F`JIc'1?f0$ . . _ . 5 ~ - - , . - Faeiiities a~_~quipr~n: ara described for large_=scale, long-term -'r.ose-on!;~' inha7ation_exeosure of mice to -whole cigarette-smokt.- Exeer:zrienta: =procedurF= and 127

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equiFnner.t were. des_gaed=to 'provide the micew_th Zs_posure condit6ons vrhere(!) the :ung= --- v:as-the ma;or, target org¢a~ for the saroke~ (2) large auantities -oi` f reshi wholy cigarette smoke couid-be geaerated; (3; 1ar$f-r.am:;$rs of snimals z3ui, be exp5=_ed at one time, ;4; rou*_inx, daily xxposures could be-gisen ^wer a^_jo_ portion of -he i:etime of the anirIIa :-(4)-'!14'-nBtCriiig 4-ia:j--aocIlRSerdtatiCln-ws--the q:la5tity LFE smoke pres°nte/} 'v t`--_?- _ -animals was provided during each exposure session, ib) safety sysEzn,s were provided s_ka-t a_sured-eay~sure of the at;imais='a smoke ~nly=urder p:e-5et-~zposure-coaditionz, and (?) cigarette SI50ke was generated under conditions where iaki6rS, such as cigarette tVSe, _ smoke aerosol concentration and smoke pajticie size, were controlled. Henr,-, C. jft a!. f_E;rsrobiologica! a`ss-ociatesj From :he Analytical Chemistry Divisiyn, Oak Ridge National Lab`-ratory, Oak Ridge. TN; Division of Tosiccio¢y and ia`ec!ecutar Eir,-lo$y, Microbiological -Associates, Bethesda, - MD; and Process and Instruments Corporation, Brooklyn. NY. Heitr3ge_aur-i c.;iclcforssh::i~rg !«trrafiona! f3(','; 37-33. s8-5. CALMC.'D1JLlN-DEPETe'DEI4''; NAD KI-i+I4SF_ OF HUMAN NEUTROPHILS ---- totai c-ytlcee?tration of NADP +-NnDPH i^.-the-hLiriaa mgtiirG-',~;i. used 1Ficreas-_d 2..~.-f o.i. _- 'sn response to activation =$j fhorbat myris :: acetate. Fi$aliy3 net#rop`i! NAD xinasz- Fias a M, based upon gel filtration, of l;-',9,800. application of--Red -,A$arosg, ion-zach=anae; and gel-f i:t=ation chromatography. The - - enzvrae has ri broad pH ^ptirnutn, 7,0--9.3.-is strictly depFndenr=vpoe t`e Preixnce of Ir!g{% and iu the_abs"^, o--- ca:` utrr exh:bits-K- ia:ue., o7 0:6 and 0.9 mFf ior IN;-v' and AI'P, _respeCtively.=- _'!A.DI ._ kinsse= astiviiy- ii- extremely sL--tteitlve - tr -free CS-.,Su:P--= concentration, with _i~4-rta~~a1 a:a~it~ df~rv~3 af free sa=ciurn yonc_ent.ations o= approximately 0.4-iM= 4n ceilliar e tt~ac s; cai i~m=d ~ndent` a tisati~-~ ot NAD i_rIase =- increases the maxiinuarve:ocity of-the reaciioti from i`to 5-fe,rdwhile not` affectin$ K..... values for NADD and A:!', The activity of the part .iall~ pur~f ied NAD -kie:ase es stimulated 3.5-foicu by the addit-idn o#' caimodu;=n in the preseyce-of c3.ciu,_r. Th is _ - stimulation is inhibited t1,E-- the addit"sorr of 20 -f-FN _trifiuoperai;ne to the incubatiott. These data are interpreted-as-ir.-ofr,'cating-c_tinndulii3 in ivAD kiaase regulation. - The- NAD--k-inase- from human- neutrophils has beeh partially =pu_rifisd-bv sequeatial- _- Wi:liar,.s, M. B. and Inne.s: H. P. Bra:kemistry ared Biophysiss 23'Si i ):SC;-g7, i93y. 0thfr arpport_ Kroc Fou ndata`oee: From -the-Det-rart-meqnr~ of--3iocFesnisti, " C*s'.2ge= s_.` Medicine, t3dive;sity= o:'. Souti__ Aia-baala. MotiilE. A CvMPAi2A T dVE-ST'JD:' OF NLi1TRCPHIL EuF:FI4AT7(°iN AND FiNCTIO3v Several d ffx:ent methods are cerrxniiy_used by numerous _taboratories for the - isviation=tf human-xus_rcpla;lz: These methods ir±clu_de partial purif:cation by dextran sedimentation- followed by- water-=.ysis. and more roraptete- purificatiat! procecures utilizin$-discontinuous density gradients coupled with dextran sedimentation and in some cases hypototsic lYsis.=So~me investigaiars r?fr=ain_fro-m using certain purificat%o3-schemes __ beEau° certain steps or-reaents tlse-,ad in-a-particuiar metTiolf migtt-adverseiy affect the 129

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- functional parametsrof the nPutrop`:i_tf?y wish to measure. in spite of th?se_ concerns iherF-ha3 been no systen+otie ornir6ir~.,n of the Iunc.ioral-statui of'neutrop.i;s preparrw iiy- the-varsou.met:.~'r,iogies Ir" 2h.E stu' w_'have ccent:u-ed=4 ccmiroalyaszd methods-of iso:at;- wit': -neutroYSii unctioI:. The *esults of -th;s study i:,dicate that while nea:rophil yis:d and vuay were aiete-rnined by isol3tion yroceaure, ote cells were _-ec;lliVaient WItil fega;d :3 _clieaiota~,t!C .".ai[Crn:an^?,- 2bSi ty't~ degrar'lle~:e, and aiil?litY_ to p.~r - di1Ce superoxide ~d _ - ~' - T -- ~'rpOC~='s7r04S aC~._ .firisFa[n, ?+t, W., Eag7erson, i. D., (vlcCOrd,' J. ?i., and fores. h. P.- Jvurrr:f r.I_/rnmurnofoai,af Methads 92;3ij=3120, S98y= _ --_- 0lher_fKgport: The k~?r:caA Heart-~~:so~-iation. From the Deeartrtettt 3f Ii'soch2mist_ry iol;ege of _I~edicine, Univrsity _ of South Alabama, Ivohile.--- . Ac; I7VATION=ASSC'Ci A s ED =ALTEa?. i It`iNS I:Y -NEtJ°: ROPiiiE P:%RIDI.`-: E ANCLE<D'FID-E LE VELS A P1'd'EN'fIA-LItEGULAT=3R Y ROLE FOR CALCIUM = AND CAL'rtf%DULI31 _ The concentratio, of - NADP- +_ NA_LjPH_ in resting_ humaa _reutroghils has been -M$'d`Ffre dd`Ffred -to be -24.L i f ?.iY c°ii- i3a i ac5/Vat C a W1i~ a. -=o31Sen IS':?3o-332 23322 A ,-_ _- _ phorb-ol myristic esetiite fi_ N-a`ormyl:reiliiarl`y:ieucv;php_ vlalae~:e, _Feut.Jrhi: NADP ;P -- iv~F~i~~~-y~!~=ia-crse_ to= 8~3 ~4.0 and _;d0_X ;ala[FoiJ_cIE,_ ~es~c3:Yeiy. iheie ir;c_re¢sgs iA y r:din~ n:1c' -^<-t de =09 oent~3ot: are y'xk~ ° t4e 6d£it__n ;f tfie : a!ciu ~: antagonisi >i f c~-tsmRt}ly3min o;-ocZy:-',~,5 rlreti~~3beazoa? sydocEr =i~~ w3ile calcium ionoQnvre,:23:a'., i~ tl~e_eres~l_cs of ca!Eium w_ iir c: 3ger the ;ncr?ase in tlp- - aBsen_ce_ --ot - other _ _ st,tnu:~__ - Catmodnlirf a:fta$onists== tri]uvptrazinr_ and- ,Y-l,6-amiao`.*~1z-5-chaoro=l-o phtha:efe suYanarnide also- inhibit- s':irnulus- induced- ir,cre;isss-iQ tt:o-_hADP-± i1_AD-PI4 pool. These studies_ are i!:tercrssed xs ,suggestirig a role fcr calcium and calm:odLlin : and _possihfy -pruteilt kirasF C in the -- regulatson ot r-yr:IImR zuctzotrde corei;rai,cn-in tne-activated rseutr_or, - ;- - -S¢arkmaa, TB_ 7ohs T.,-Rrgersor. Ta.nQ .:c'P_ s: H-, P Bioc-hi7nica Ft Biorh;ssc3 Act3 gs6:$--,a, 1935, Other su;.port:-Natio-nal Instituts3 of Heaith. - _ F-rom the--Deyartment= of' --Biochemistry, Collet3e oT=Med.icine, University of South - -Alabama; Mobile. ~_ i=.FFEC I OF Ai.Li1rL.'R' 'flL ON tiEJifiiGP?-I_L SITi=E-RjDXIDF. -mz ~OD17CTIOl'°T, CIt:Eh;il'CAXIS, DR-DE:;I;A`d>;LAI'Dhi = Recent st.ldje: examirhn& -.ne cr:ecV -of tlopuriUt,ol _ on hacte :a1 ki?iit:g by :eiIIEUCytes, I1a:y.. been interpreted by those authors as prctif that xaRthil'e oxidase is the I - _ maj9r sle¢`,rCxide prod1iC5rf,g MZyrne in acsr-a-ted leukocytes. To :BSt the as5e[tiGl t-I:ae - _ i - xanthine oxidAse ts_ .nvoived it:= the produc'irn: o:` suyeoni,.e b---_actiYateci hrmar_ ..edtroghils, the xanthine oxidase s2tse 3 of n°utroAhils was measu~d and the effect of -_~ -llopurln~l= on, ne~:to~hil functions, including Superox ce p oduc:io l><as studied. _ ~ _- NtUtrCiphilS were fOllnd_t(; CoF:t3eIn a leVc, o_-~a e,~i.Ce o>E dase= iC uf fecieJ. to accollPt for ~- _- the flux of superoxid¢-associated with reutrcFh:I activation. Rfiopu[iaoLiid not infiiYit j A r _Z

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ssperoxidg proaluctioFr induced; by opsonized=-°zymosar_s p?rorhboi n:yr_siic a=et-ate- or- _ srperoz;de-meaiacl _sarrageena n-induced foot edema ia the- rat. These --stu2ies ase- an:Pr-prete;i_ m erid;.rcr_ 3I[~: zanthine- oxi~e -:s aoS n= ~iajot supe~siae-gen-e*atirg --` syst m in ac.iva?ed neutrvnttil_=-a< -has-been suggestedby -c-t!rers. _ --lati3n=was affected by allvpurina=: -Al!csnrinhl vas=aiso found ineffective ir blocking - furmyi-methi~»yJleucylpheatyialanine; =-FurtherrnorP, n?ither ehemota-xis ;,_or degranu- lones, ?!- P. et ai. Biochemical 'harsracology-a4(20):36i3-3375,i9a5. at_h2r-si:pport: National Institute of Arthritis, Metabolism, OiRestivf-and ;:idr,ey Disease and the=Fimerican~Heart_As_x1:.iatiora, From tho--Depa-tmenl of _=Itioche_:~i..~ty, _s..i,lege of iviecicire, _-u~versi.*y of- Sou.fi Alabama, Mobile.- _ =INCTtEASED BE'l'A-THIir--'1&.4BOc,"LOELzLIN IN THE SERUM OF SMOKERS fou3d: in_ihe t'i.'6gfio&Ips.- A.1-in`Yei'~-rel=atiorisl:ilj between release and 7-latea@1 Ilun3-ber-was_-$!so_--= - - -platelercontecl of fi-;fa,-ajm-y the cnhcaturatton of-p-TUVer pLite:et *d neerly- =fdentical-= -- _ fG_-~.'nd tU beiigrzifi aritly !<,$her in S6ttoker°s-fih?n -in_3-'J-+tstnOaCeT5_-Thiiinc7eas°-lSz -P:oSu--_-- li!:ey aue_ to nbserved increased -p!atelet-aumhers ia smok$rs raiher--i!'iam ftr i, creased OCC't7RS SE~r-.AUSr-saF INCREASED PLATELET Ct31;hiT3. The amount -of j8-t_b.-arnhoglobulie (P_=3G), -a platelet aipha _ granule pzot released ia the serum f sm.okers hy tts-o m5in ih ;ei duFi..6 whole hiobd=-c!ottiny _-w3s = i,ortgz netLer. Cr.L_ e3 al. = -Frcm the _I3epartrrtnt -oI'- Pharm3.ology; Cdllege o_S- Medicine, University of=nuuth -Alaharaa,_ Iv:ohilz. - ResearEh-Coerrrmnuar:ons in-Substances ot-Atrr.te~5(2);1d7 7-I52, 1984. ----- -- response is dependent upon capsaicen-sensitive affrrer:ts.- Tiie nicotine response-iniolve, (0=3-30 p&t) or nicotine (0.3-30 mR^") induced dase-depertdenr- incrEa es in arterial- blood pressure, mainly dtie-to-an iherease in per'sp:,eral-vasetYar rrsistance:='Ihe-capsaicih and n:rrtine respcnwes _Mere abf :ished_atter=Ictiai Anaest hesaa and markedly reduced (-ty_ about -2(% of control) by -co:a±ined- pretreatment .vith pherusla:~.ine and k:~-~re-eoloi, _ suggesting ref cxogeric_ syn=pathz!ic activaticn_ System ca~s~ci!~ _prstreatTie~~it a-~.,,c,!3ne d,!3ned -.:- - the hypertens%ve_effect-of--eapsai:=iiii-(30 -AiLt) and- reduced the_response -to nicotir.e- applicatioa to about 25% cf _contrcl (p < 0.001):__-Locad= capsaicin- pietieatta?ent- of--the nasal m::eosa- one week -ear!ier al.so sigeifica_nt]y reduced "she-capsaicin_ response (p -< 0.05), while- the-_aico~ine-i~ziuced -)s~~rease in blood pressure was -hot= significantly = changed._ _The present findings a suggest the-presei:te=of -tsvo= affgrent .:iechanisrns in the nasa! _sucesa wh3ch-induce hyper*ension--upo: chemical irritation. - ~'-ti- _capsaicin=--- MECI-IA:?JISMS FOR-R£r -E'~4.°ii/E-HYF'EI'cTEN-S3F>N INDUCED B Y ictX.AL A°EL-ICATION OF CAPSAICIN A-NB NICOTINE TOTI:E NASAL hii.TC0j4 Tht-cardiovascii!ar effects of-locally applied nicot;ne and capsaicin to _the nasal mucosa -were-stsdied- in ac~aesthetized goiFea=pigs. Local -app!ica.ioF of capsaici,3 130

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risainly- capsaicn-sna~itive_ nebronE=a_Tid, in°=add'stiari,--"a- minor -co:-Wnetst Wh-ich- is -resrs,at2t _tq caosaicin~;rPtrea-mes?.- Thbs- °th~_hypertensive effect-df r<i~otitr~-- applie~- :~all} ta ;he nasg' nau: _a setins_ 2io be riai:i:y ined:ated via sensory -._nechar*isr_~ vthe. than t:;e-yr,eeziFg :espo . r which is not 3etieadet::-cn cap.aici: sezcsitive ;teFves.=-= L_itndblad, L.s Hcia; and Lurdbera: J. $3. " Acta Pf:yslologfra-sswna-inavica 12;:2'7-252, 19184. Gt.her=.rupport: -Swedish Medical R-6seafch-Couf:cil.-SsveAish e'obacco t ompany, Astra Foundation, a;d the Karoliris-ftA ;nstitute, : rc ~ the ~pa_ri nen; c~f=Pha.~nEcolog:, Rarofitas=a Insnt te, and the 13eoarrr:;ent o= GGt.o-Fhin_--La.;nge;iogy, IC,re:inska I-:cspisai, Stockholm, Svz°°den - - EFFRCTS OF N:CO'flPvR..~`~--I BLAS STOCYST DEVELOPMENT PRIOR TO :I1,7~LfiN 3-riTFO`?--i~T 1-IE RAT= determined-i.:l'vs ?`rat_ -snd body growth subsequently pleted.-The onset of ,^,ube:ty tsnsior were i?trasu:ed in oseudh,~eattent ra-== Lit e~ s ze`~{ birth ~ig~st were levels were deern:ir~d in treated tml `vs contro: S`1 rats during Ihe -in,.ial 5= days sf >re$Fancy_ aP?_ the :c°Ef ~-=~3 the aikafc.~ on utal~!_e--i`~l.rg- flow ~nT~-f51.rc,>£e'ine ~=~3Cagi-n_ - werP T ^nitarcS _fowJbn_ g-Iertn ~1'.,s!=pi°M?a eff_ n-p_` -n.C ti :~ -°yyTssire G-_= _t6w$',_ aRi -- - 3exilal iZlaturatii3±t. _-ro ln.vesiigate p't3sp5s1F m°dof '"~3cotanE a*`i1C' -n=~ plasma pqogciierrine -- The following stzcdy was under:ake-_to detcrr:ne the effects of ricatine tre_tmera rrr conceptus de+elo{merzduri,n; the initial ; d6E; of p.egt?a,_;: °n additan Eittc;s --_ ~ in females ~wa37 de:eFrn:zedby nc ti-e the day of v3ginai oper_ing. _Re=-ul g show l ai - - tidil iitsiii fr weeay-s.-neeon of n-coine-du?n8 tbrirsi -5 days -of-pegnarcy =Vetards coraeeptus growth as-'_ndicated by the reduced=nwn5er=of cells per b#astocyst. The= same t_eatn:ent had nc~=effee _a litter -sizei-bir,h weight or body grayvter. However, the da; of tagin?E- openir,g was delay-ed.__ Nicotine treatfn_cnt~_gen eraRey ruppressed plasma - pro~ gesterons levels __ during the iai:al 5 days of pregnanc. Fu_rtTaermdre, a sir.~fe injecio~ o: the al3a:3id - :rddced a rakid and sustainei reauctia~ i: lcerine b?ooci -flow _ and a concomitant decrease -in int-ral5ee=ine oSya.en. i;F indTM'~ated by this: -worx;- ni;7tinF -anay- molil y=-- - ~ - - _- _ czin.eptli:f- deVelopm~nt- by =altei5ng - reproductive traG't fi:nEt"sC3P-.-- - NIc-oF,': e--,nduce=a -changes in serum grrogesterone l-evg(v and uterine blaodflow pFobasLy-:esu=.;-pfi3narily = -frotst the-a[-kaoid's _dtverse aed cnrspleg- actions o: the nervous ste?r:: The prese-at- resalts confirm and eztend previou3 znvestr~atirrs 4eport;ng rffect_:-af pre-ir.~~+;anta;ion nicotine exposure oo_-conceFtusAeveloymentand progeste.ure secret'_on. ?n-addition,-the present study demonstrates that -evan-g,-elatively brief 'e{posure=to r.icetine,- f.e., during the ir.itiai_day ofp~~isancy-~s sut;icient=to n:3dify-tne=timing of pnbe-t~sn the rat- t ___ Mitchell f A amf-T-lEr:izner~ R.=--. .. Veuruscfence.-Physealpgte.al.-Ph rmacatagdzral=-ar,d=Clani_ral-_A-saects, New 1'ork: Rlse=ier - Ycience Pubiishers R.`r'.; i9Sv, FP. 33l-1S`S. Rrrom-the Departmee:W =~nato~sy, 1Vaytie State U n:ver3i~y School of Medicine; Detroit. __ lm Caciagri, F., Giacdbiai~ E:; and PaoletdL R. (ed3.):-DevelopmPntai _-

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- EFFECTS OF Iv1CO-TSWP-f:N fl;ttD-yICAI, BLOOD FLt:?V AND E,1 y:tYO =_ DE4'ELJPit1ENIT Fia~ THERA7 zdicotipe_(3.0 mgjkg~-was inmected{sx:-:) twice daily or_ Clar= I dr Da.s 1-4 or s-5 o' _ P<Gt.QI-)_ N tent+_ne enaecta0n also_resu!tcd in a:*tarksd andorc!oneed reduction n oviduct reduced cell number-(sai: ae ys.=nicctirie-42 5 g-L=7 _rs; 30.5 ;D.e, at 12:0C h day on E-ar 5; __ I2:UQ h on Day 3; P<-QtJ3.- However, treatment for even t day (Day " significantli - reduced embryo cell°nui,b,~r (rsaiine rs_nicotine: 43=.3 _ 1.7 vs 23.'- t 11.9 rtuc?ei,/embryo, at _'oregna:cy. Catnt:!at!ve +'a-?es or na'.4tte re'=xt:ed' -eti3b:tG cell cleavage and suS_-tant:a:ly - 31ood:;ew (kretreatmen, v~9J~;in_after nicotirte: t?_ck+- 0~ -~~s iJ37 ± Ci14 rl;tr u.g"'; - ~_ r' < 0.,G-05. The reslES ir'id_ca*e ihat in the :at, even a- brief ewp alire tort_ico e, th;, chief a~kalo d of tobac o, reduces oviducal blood flow and h rate of emb yo cglt p:oltferation The embryo is theref~are strseptible to tne= ftects -of nicotirt >~fdri tmpastatiop. ~-= hlitc.k,ell, J. A. and Fiarit,--ner, R. E. I ~' Jourstal o! Reprodeci on ~ re t 1 Ey 74 7i 7b,-19. Other support: The Reseaic;_=Soriet;F of Sign.a Xi. ~ grottt-the Deparitnenx_o` Ar?atofry, Waytse State-University Schoc-To;-Mesicirte. Dctrett. aetiaity refle_sted selective -acz:vas_ion sf=zhe-pa:tisulate rorm of gttat!yiaie cyclase; hemin inhibite~=t?e soluble forrsa_of gyar}aie c;¢iase =7~i to 9d'~ over a-wice range- of =-- - sonce_r,tratiotis. Activation-was not-secondary ta protPolysis- sinee a-variety of protease inhibitors failed_to alter stimulation by hem:n. Protophorphyr-ir_ iX-h;d little -effect a~: _ particulate -g;:anylrte- ,.°yciase- activity a-nd- sodium bmphyd-ride almost =cotnpletely abolished hemin-slependenrt activaiion.- These data suggest a requirement=for the ferric form of the porphyrin-metal chelate foraciivation. However, agents yvKich interact with_- -_ the irots nucleys of porphyrins, such as cyanidg,__had little effect ota the ability of hernii=. _ to=activate guanylate cyc'ase.= The sti:aulatory effects of hemin were observed-ir~ the presence of deteegents -such as Lubrol-PX, a.nd-highly purified particulate enzyme could be activated to the satn<s extea.t=as ec:zy;are in native_racmlSraoes. _T9ese data ;ugg:.st-that = ttte interaction of porphyrins with particuiate gua:±ytatecvcease-icsor?ptr.z in nature ar.d- Aifferen! from that wit't the soiubie enzyme. hemin with highe_£ coneen~atio_*~s ~~eini~ inhibitory. _~rtivatnin was obsers-s~ when _ Mgl'-t;'f'P but not jvhe:r ~itii2*~%T-P-was used-a3 the-stbstrate. = Increa;2d-enzvmc-_ B103 rat- neuroblastotrra -ceits.---Mazimustt activatied was observed with -50 to it;9- ,a.4 - Cuany_late=_cyclase was=activated.:~ -to-ltJ-fo/ai by hemin ite a= dbse=depepdeni ma;nes-in _tnerribrat+.es ~repared from honsage-nazes pfra_ Iting; Cg_iat 3uc?za -ceils, o SE'sECTi-.'E= ~s:°TIi.~T:i~'. 1~T:i~'.1 13F -~`-'AIt<~"tCvLATE GiJA1~-~'L.:4e~= C 1`~'~_4:S_t BY ; SPEC, tFiC- Ci,t+.«S mJsORM- RJ!Z?S Waldrnan, S.-R., Sinacore, M. S., ;rewic;_i,-J. A., Chang, L. Y. and Murad, F. - The Journal of _Bcolagica1 Cltamistry 339(7):4E33$-4J42, 1984. - l~z

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I I- I 33tt!ef au; pc t ;o.:oAha Institutes of }'e~lEk ant From the jDeaartr m- aff -Medic'_?:e--and P:rarn=eaco!ogy,-3ta_for,_UniyersitY, Stanford, '-_ - - CA and .etyrais ~G_ rosir~tir Nlea~ aieni~_, P::o A'tu. CA -HIOI:LY PURIFIED PARTICULATE GU ANY:.A:E CYCLASE SE FROM IftAT= LUhIG, OHAI'ir1'..'".'PP.IZ=ATIOiJ AND COMPARISON WITH °sOLL7Bi.-: -G;:An.'LA:c OY-_-LASF Guan_ylats cyFla-se was purified 10-00-foli from was ;ed- rat tu=n.?- t;a_rr__,cu!ats frac 3ots toa final sgc fic activity of ~?n nino e= cyclic OMP rradn=s±fm n/mg 5r,tein by a conhinatio- i of detergent e;tractiEn anc -c zrotn-atogravhy- on yo*icariavaiin A-Sevhatnse, _ O'CI_'=agarose, nrha- blue agarose. - Patticulate gu:rny!ate Lyc!a3e has a molecular weigjitt i1f 300,000 diFeti'ini, a- iokes radius of 43A and a--3fali?leni3tiOn cr;efficie ,t of 9.4. ;v~h%!e the soluble fdr L has a ho!zcu!yr weight of !50,0J0~ da!tons, -a -- - Stokes radilFs Qe' 44 ~, a.l:C; a 3ediment2tiCn c3ef lcie:t -7f 7.G, - `'heneas the p-articiit;tE-- - eti:yzle-ts a g!ycatSr;:teit-1 with a specific affini.y for fi~_ncanava!ia A and =~'heat germ aggletinitl, the solu}'v form of gi:anylate cyc_ase did not -bind -to these=lect:ns. Purif i :- - particsilaie g~!artyi~tg-eycla=e_did not-cross-react with a m_mtes of -monoclvaal ant!t;odies generated to the so!Lo!e enzymz._ Witiie both forrn-$ of the enzy:necdu:d be regulai=d by- -- the formation of r?ixzd disu!f ideE, the varticulaie_ enzyme -was _re:ative?y _insensi_ive to inhibition by cyzti_ty: With- i,T_ a& substrate, &,th forms _of the en_zy:F:e-demo*-strated tyGicai kinetics; andevith O i P ana:oEuFE, r.egatiie cooperstivit; was anserved with b;_k enzyme fo:ms. _ PhESr data su-ppor; the ruggestiv., r_h2t-the two forms of gyanyiate cyctase _-:po`.,fes: Siu_.-!ar ataIy_6c te. 1fthough tx-,.li-r~a~ia ru':ure t.- erder~ei.iTresulting ifl- _ differeijces~.^: s~~b-c-e1:31ar distti! _tii,n, phy,ical w Eracte,isic{ ans ar.tigen-iEa;. W:Iddan. S. e4._ Lewicri. !. A:, Chang, L; Y: aai Murerf: F. _ Bfo:ecufar andCelf `i~,. Bar<t?;Firist.vS''.155-I6E; 1483: Other vuFFa-ri~- ?Iaiia~a!anstitutes -if I-;eal*I -znd Veterans Adm"sryiztratiaa: From i3e Depar*rm2ntsoi -.kYedac ne-e.r:cI PtiQrmacol'ag;r,= Stanford ilnivmity; __- ~;anfc~d, ~~ and :'?te :s Administ:a o:3'~iedicatzente:, Pa!o A:to, CA. -- _icNBC?3FILL[C3M-?)EF£!VI'_%ETv'i' AND t=I?Rf3irw3%'-)DIL=wTC%It- IN1:3(3CE=s,- -- -R-EI,AXATION OF VASCULAR JMZnYT}?MU$!'L£: ROI :-rr CYCLIC GMP The n:e~hanism s_by which endc,thetiitrr;-devend=r: re-I,FantE ar d nsirova~dilaters - cause res-at,on of :as uta -s ~ o~ih rnu...,!e has teerf reviewed. -,A-mode! " xelan_ o fhese- = obset-.ztivns_es sam :?r.zed ih F'i_k. 1. 'I'4.` endothe!iusri-depenheat _vnod°laters, -throu6h interaction with their appropriate receptors, arv thought to activate vhcsnholiFSse A3 and ° ---callse !-lie-re!eaie ot= an unsaturated fatty-acid. The r°!eased unsaturated Eattr aCid. or a= _-- = taetabo!ite is t;:ough.*to be the "endothe!ial relaxant factor' that interacts with the smooth _ muscle coraDorseW lE cause-i'e!azati=n. sa`hi_i= the unsaturated fatty acid may be oxidized in either the erdJthe;ia! ce,! or s~oeta musc1A F?*:, fhe 3abi!i:y_ of the endothe!isl re=azant- t`acior sugaests that at aasi some of this processing ovcufs h2fere itr release from the endoihe!irsm~ i~:e nu~el-in Figure : suggests that an ,='sd'_zed f_try acid or a derived `rer -- - radical is-responsiyie fcr activation of sr~f tl: musc!e guanyiate cyclase and increases in cyclic GMP levels. As vaiited ~st above, the use of varlous inhihitor-r of fatty acid 1_3 .~ ~ ~ i~

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-release ac.d me:ahglisu:-::-m- -nor a'!owPd us or othazs to_pFedicstqe structure a?_thp- a_tive -,'12terial:_ To date the oe3t evldFFrce v.lggests that the ualsaturartd fatty acid is a jlre3duc.t_ -of either ths lipexygenas-t or ?-a~~ pathwa,s ?'capoporT: i3 PA. -idd Mw~.~, _F:otuna! of Cyc'ic .3rrleot.°de a nd i6rotein PhospitoryEatior Research 4td-3;:~°1-s95; I9S3. Other suppoi~s. ha*.ioaai ~:st,tutbs'of -Peatth and V-eterans A4tr,n siration. _ From t'he Dspartrneets--nf=Med:cice snd--Pharrr_acoPogy, Stanford University Sch.;f of- _ Medicine, S?attiord. CA,-ard Veteraos Ad:uieistr3.ien R+fedica.! Center, P21_c_ Alte3;,CA._.- __ ATRIAL NATRiLJRE i`IC F=i`'OR SiLEC°:_ ]`fEL:' AC'T3VhT_ES-FF-.R iCi_Ji.ATE_ _- G;JAN:'LATE C`r'CLAS: AND ELEVATES CYCLIC G'+4? 1L RAT TISSUES _ Astcty= was dor; -ea the ffiects en_- guarylaFe cycla;e arc3 _cycl c_ Glk44 a: curn3la:iot! of a synthevis peotide cc ,taining the a-nit,o ac:r _sequence__and-hioiogical - acttvity of atrial aatrure3,' factor fANF ~. A~iF a<-tivatedpartLCu;at.e guanylate cyciasr - in-a conCQtftration= and=t'ict3e°duPettd@at fashion i'-~ crude_ memGran?,3 -obtained _dFo7t[ homogenates o£ --at ksd e-y. _-Activatiom ef-part _ulate guanylate cyclasE. by Al<i' was slsc observed in-partic;,latr fractions from homogatates _of ra? _aorta -teat it:tes*.itfe, ':_urg,_. and lVer; bE" n4t from #,¢a*5 or bra,tr So:vbi e griinylats cy iase_o~~ i. ef f,omm tl,e e--_ -- -tissues-; nc;t activaie,i by Ai~: Tvy p,+cirea meit of ANIF Pr&vt?ted-0 w- acSi~vaiiow .- guaoy'3te cyclase, while lieat treatri -nt h2~T no effes1. Accnm,,~atior: af Y}ci c,MF, , kidney minces and -a ol° ~ras-st~Eu!ytc~ _y-iA~.':~ ~a- as,ac of tuai:yla te c-ycia.,,-- These _ data =#'Jg$est a-*Oe =fo. --0-d_L;,te gllanvla[e-M cycias - iR_ _'re=-.moiecu'Ur= me..harrsr.33 ~ _ -underiying the_6hys,olo~i..a' ~ reets of ANF such a=-va,cu:ar e.axaiion,_natriu-resis a:,cd di;tresis. The lmurna! ~~ J~iofvgrcc~ C:e3r: i'st-y a3f;::3'i:! 433?-.4334, 1984. _ dther .uppari: National I::stitutes-of Hbth a-nd r ete_raPs Adminismatia s= - Fror ;-the _Dep2rtnttnts=of :','edic:ne and-??tariri4m:ogy f-SignforcT 'U::ivetsity-Sc':ooi ot- Medicirne. Stanford, CA, a-dVeterans A3rnir,istratisn-Medicai i.etrter,_Paio-Alte, 'A. -- ATRI_4L 7Y-Arft1? ~R~ET7C FA:,it7R-ELi_:.iTSAN-Itii.JTHEL-[UNI--= - INt'3EgEt3DEi;"I' RELA?-A-T IISN AND ACTIVATES FAR`'ICliLA T E:,GANY_LAT _ CYCLASE IM V _A3CCILAR-eM.00TI-i MLtSCLE-_ !R 13 atr,irsc a sd sy :~}e*ic PePt ~e fragr.n - of a iia: srarra~retic- facter=aiA_IFs -_ relaxed 1-so!ated -rabbit aort e~re gii?emts itt_ `zvnec:; the e^~c hel. m xas _eit?,e~ inract_ or -funct',;,nally dcs~nyee. ='~ha :el~eat,o~s=~~aetemp^rai!v~ss c-atei wi_h- increases t-=- - tevets of cGAt-P -with noc:.an~gc in the leve:s o'' °AM?. - i e AN_--indsced increases in _ cGMP were iso observed ir aortis segments pretreated with caicie::r,-free_ buffer ce the-- __ cG~SP -pho3phuEiestera?e i: h bicoi i~t&$ `2,S4S. _Quaii~ativeLy simi:2r results were-, oTotairted for -sod±w-*_. nitr~russid~. A`a' ~ 1e,,t;vety _,2cfiv2t ~_ t artic~late= ga sy vte cyclase, having no effect nt _thc s:.ub`e form of the- enryme. Ti us :he _ d_irecc _. -(endothe!iutn-iPdepende :t;= yasodi!ato= effect of= ANF may be n:ediated= via increased- tissue _ tissue levels_d% cGMP. ANF appears to increase s±ascillar z°;;M?- lea-s-hy activation et`-_ particulate guanylate cyclase. 134 -

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~-=-_= --~~--~-- - --- -- -. -___ Winquist, R. t--. Faison,_ E, P., Waldmanz S. A., Schwartz, u}_h_iwad F. a?td_ Fapcport_ R. kl. __ Prt'.tet11_T.gs F!; dj:£_';:aSEei3ai -zc,3t:Y-rSi __:; Sciences of sj7e :7ni4d~$.'fil_°s irf Ai'_srf,^.a - 8': O0er scrsprss?.^=Naasna. ;na-t.pt~;~ of Healt-h-a nd _:~ etern~ Admir istratinn. - =From- the -pep3ftfiietit of_ >r~ard',ovascli:a-r shar3tt_1sology, iVi_E.'cli iAstituEe for ; herape-itifi Research, West roii:t; PA; -Departmenr: of rvteeicine- aa;d Pharmaco?ogy, Stanford University, Stanf2r»,=trA~ -yr,d VeteansAdsninistraion-;\iedi_a] Ceaer, Pa:G Alte, CA. 1tilECI-lA NiSMS-i:F .-'\L=Ei?USI?:E TKIP_`r~°'_OS;`:3ATE-, TI-IP«ahr2BxN-a AND TRYP"N-INDSzCEDRLAXis-_`I'It~' OF RaT TI-t_U` ACI[_'_P{JRTA= The nsechayisrns _by which adenosine triphosphate, t;.roinb:F, and t=yvs_in cause_ relaxatiorr ou va_tcu:3s_s.~nAoth tlasc:e wPre~nv~tigat=el. _I~ = elaxat:~ of ti:e rat :horabic aorta- with adetronsioe, triphospr_aie, tirombin,__ and/or tryp.rs was associated with -- increased 1eveis_ -=of _ cyclic _ guanosir.e - rnenephospl:ate in bot-h time- an d conci:ntration-depetdent :n3nn l'rti: ?}3omk?n and tvp3ir did :ot all r cy:Crc ade.iosir.e monophospieate_ IeveE -whereas--aderJslr,e Sr;paosp}iarA :rc.,reased- -C~Vc ic adenossine - --morloghu5phate_ ieve-s-a"r s gf't ic.3ly MasatioTt -occtFrr _-.as. ReRSC-vaI _:i:- the -endotheliEln'E abolished adenosi.e' triphesf+- ste--,= thrFyntrifi=: -an t ir:pci: -i duced zelataticn `an,f the ants£ociatedF`.]'-'c{eas~` -S levels of cy.,:ii:-r.lc'_-0tii'es_ -P-e-l3°a ,ts:_ d.ae t2 1he3e ge'Su was -31sCx 'snhibited-, b-y-sx"liFe- to _ t;ord k)#~gya?zretmc..cid, aL -lipoxygenase iciFiihl;or, -rld- - ticosate+raynoic acid, c=3ipoxygenase- an~-tyc3o=~xygena L- in~, h_,.uT._ ;Fai~f; ett?acin, a= cyc:ooxygenase-inh,bitar,-pi.ientiateri.r_glawat.on to these agent3 ki,erca- the ,r_creased -leveis=- of- cyciir - nuco-eot.- ec.- -dueL -to -a-nesink iripha~F~ate-- wue ° u_naltered= Istornoyhenacyl brm:i;°, a prospYs.ipase A, in:F:bito*; cUecteased- reaxatio:- due to - -adenosin-e triphosphate, thrombi n, and trypsin and the associated `r-cre-ased -ieveis-of =- _ -.".ysli~ nucleU-_;d°S- =- RemM3v31 -of- P v:?relli:la: icc:ciun_, vh ch-also preFur*=b;'~ in iibi3 --- phofphdlipase A_ , prevented the elevated levslls of c!-`Cl-' nucleQiideS ai5c, tlnP i:131ibiiory -- effects of--ader.o_siae _triphosphate- and r,d trypsia_ on- contraction. In vonrast_ sodiumi - r~ troprt:sside induc~:.d relax-tion and%or inereased_ fevels of cyclic g=;anosine -_ aonophosphate ave_e- unaltered by nordi.~;drogasiaretic_ a_id, eicosatetraynoit acid. I bromop,enacyi-bro&aiee, aast removal -of = extraceltular rafci_m. -After °ancuba-tion- of - =:1 intact tAsue with y?P-ozt:_:,ph?spisate, the patternf of :;_rote;; phosph-ary~dti~n yusPd by ~ t~L~pho3i:a?'.CS,.s thrQm_bir?;==~n~_~1ryy-airi wu-,ra ti ose= of =-I -acetyioholine zodiu +n rx uide a,d 3-bro-.qo cy=c gsanoLe c-onUFho=-p` te- AV- I t;ese agents deuhosphoryla.ed -rayosir light fhain hus the p esent study supports the - hyporhesis that relaxa on insuced_ by _ adenosine triphos,,na:e th-romyin and rr- psi- is I mediated th o& the fc rjauo5 ni an ena4tht::a1 factce hiE:,h elevates oyc:ic g;rancsire m-onophospha,e lev~eisand=-cay=-.._ cyzfic guanosir:z in3ro5hc, pha3e-dFpendent protein I pho=_phoryls.ion a.-d depr.,sr-horyiatio.n of .,.yosier ligh-t .ria...: = I Rapop;_Y, PMU rz ;-it- Fi., and ,It.urad, f._,=. C;r;ultzanM ke5--ere_k 25514:ahg--79, k9°4. Otl-tr svp-porr: Na:-iot'.al=-snstituSes of=l-i_ealih, Veterans-fEdmisistratio: , idationaF Research Service Award. ErSra the IIepartrn~ris:of *4edicine and ?:~arrnocolsgy, Stanford Univers;;: Sk:ool of vtediE-ine,-Staafurd, CA, and Vet_ran s Adrinistratior. Medica? Center, Palo Alto, CA. _ 135 ~ _~J

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AT!21OLPEF TiNI EL E v_r~_T ES CY"'<`::.,'.C G119P, ACTI=VATES CYCLIC - s;MP-DEPEIYi,:.PI; PR3 T ESi+1 KINASE-ANI)-CAUSyS_ FtE_LAX ATICi'` IN RAT THORACIC AGRTA- -Synthertic-_atrievep:i=-11; an-atrial : a,riuretsc-factc± with-potent- yascsdfilatory ea`fecf;, was studied in isclated strips of rat thoracic aort3 to deternein-e ics_ actions on-- suggest that cyclic GsT-P_ and. syc!ic-GMP-kinase may °inediate- v-ssti.u:sr aelaz.-tion to a - A?: P,-onc_entrations or cycli_ A?.lP-depen2ent pre:ei^- Kinase sctivity.- --;-hese data _'n- c.clic C-IvfP-kinase_aca:vit;- fatlos: A-trio-p¢5tiry it did nc* Yib,if:ciptlr altz; cyc.lic - an-aC% relazation; an 8-fosd increase in-cycli> GMP coneenarations and a 2-fold increase - concen*.ratio n for all three effects was I nM. Atriopeptin 1!-(IQ r°?st fpr i0--mio)_ produced precontracted with _4.3 - tslvt noreQinophrine- whether or not the - e_n_3oeh_lial- layer was present. - Fie!axation= to-° striopeptirE iI vv?s closely cor relatzd==,n- g' tim --- -and_ cor,ce:.traiion-de;~ndent_ n;ann :yr with- iocrases- i: c-yclic -s~P-c=oncentr3tions az d activatioY of cyclic G-Mi=-tependent protein kir,asse t_yclic iaMP-k:: asej. The threshold nucleotide=depe.drtt pro-leir -kinases. iatriopapti?t il was found-to_ret_r aortic strips - contractility, =cyclic-_ nuc=eot!de -concentrzt:on= and_ en3ogendus ayt:k~ciy of --_yclic -- - natriuretic factors-- irn va;cular relaxation to-r=i-irovascoiiy-tors, e::dotheiism-dependeni vasod',lators and atria! includs-. cyclic GMIP acc=.:n[ulation and activation-oI'-cycli_ ;Iv4f-kinase may°be_involved: vasodilator,_acetylchcline-_ TherPfore,-a common biochemical -mech>nisri' of ifction., tliat_ -- observed Risi, the nitrvvasodilatar,-fcdiur.t nit-.r~,s~sside, and tfse enilot}±e!ium <iependert - riew claas-Of-v~sdzcaive-ageats,-tfie=-atriat rat.rii3i@'~ic factor,. -~Sil^aii-~_r effeCts--Irevt been-- c?thdr s:rpp-rt: -National Irs:itute"f iteaith and Veterans Administration. Fiscus, I'c.- It.., 3apoycrr, R._,%i., eVaidnan, S. A.r a nd °: urad, T_ B,ochimica ei Biophysica Acta 846a79--184, i983. -Ffo*.tr the_ fiapar'anents of RSedicine- and Pharmacology, SianfQrd-LTniuersity, Stanford; CA; and Veterans Ad<-ainistrat-iarMedical ienter,=Pa!o i. 1to;_CA.- A TVG-ST-I:.F PROCEDURE FURLBTAINiNrG' HIGHLY PUi2I-Fi':_D PARTICULATE GUANYLATE CYCLASE FFQIvf RAT LUNC'i - particulate gaiany!ate cyclase about sznG- to S'_QX)d-fe+d_from hc^rcgtinatzs of zat_ f:eng. - Portiors of this work have appeared in abstraz form.- 2aea urr.hi-vsper;n but,-to dat-e; homogeneous preparations of particulate guan;late- cyclase _ from mammalian tissues have notz }ee, obtained. I-IerP we-report our-efforts_an purifying- - - Guanylate cyclase ;G-T P-pyrophosphate lyase (eycrizing); EC 4.6.1.r~ , the enzyme cata!yzing--the-forrnation af cyclic- f'rhiP fiom GTP, exists in=botts soluble--and -membrane-bcund -forms. - These -enzymes-hav$- been ir_7plicated -as- key regulatory cUfipor.ents- in a-variety -of bioloQica# events such as secretion and smooth -r3uscte-- cefaaatien. Understanding the role these enzymes_play in cellular re-gulatien is predicated upon obtaining purified preparations of guanylate cyclase. =-Several- procedures for pu :fyir,g the soiub!e enaynle to apparent flumogeneity from avariety of Cissues 'ave been reported. i'he-particufate en2~yme has been_p:rrifie-33e apparent homogeneity from Wald_rnan, S. A., Chang, i.-Y., and Murad, F. frepara:ivP_Bio0rem:stry!3(-3rj:103=119, 1985.- _ - - - 136 - ~ ~

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Other Wpor1:-=Natid5al dnstitute_ of I-tea:th -aad-Vetcrar.s-Adrr,inistratioc: --From-tl'ie Deya=ttiier:ts of c4edicinv anc: Phu:macalogy: St3nford-Jhives3ity, Stanfo:,:.- - CA, and Veterans AAn:ittistra.ion Medical `:enter, raio A'sto, CA. -=--_ -t)lat- free-= thiol- g,r3;',pS_ or-_= ftEembr3_rir'- _t.'roteins .-__r'_`ayit:portan in - atriope'~.'tIP - thiei-reA=tive _age ji<=b7ocke'-the=_a,_*=iyation-of partic;tla; aua.ryiate yclase, st:ggesting - 7+triopep.in 13 activated pa rzs-ulate gu:tty a~e cycl -e-3-14 t`old in; a-cpncentraion- and tit5e-deptnde*t--fashior in trude_ rc,embranes_of;tained from hoa-.ogenates of rat adrPnal cor;ex or medutla.-_ ;imil_r effects were observed with otncr atriopeptin analogs. cvcase itr=these prepara ns .ve e no.- &c°ivated. Soiuble guanylate cyclase 5-und adenylate Accurnulation= of=_cyclt ' hiP :r ani: s cYf acrenal- o.tex o---medalia was increased 6=g-fold= due -to at*iopept:n Iii: activation of part iculate gu-anylate-- cyc'asa. Several _ EFTE`,.TS OF ATRIOPEPTIN O:'±_ PAR_:i!~.'i:LATE GUANYLATE iYi.LASE FROM RAT ADRENAL _-_- teceptor-guanyla_te cr elase -coupE n. -.- Wald_ntatt; S. k., Rapoport, R_=nrt.,-r Esr_us, f`c.=R_, and Murad, F, Haoc!rirn-i.a et Siop?y3eca-.4.,aWZ9g=343, 19d3_ -- Other suppo:t: Veterans A-dmini=-tration. Vi edicine__Sta:^.ford, CA~ an d V?;:m-_r,_s Administr=.tior-I4ledicaF_Ce nte , Palo A_.. CA. - From the Departmsnt-of Medicine and Pharmacology, Stan;'rA._LJnivervity `:chool -of endothelium presenr.=_fn contrast, the ¢ffetts of the endcatkeii;i ni-depe-nden ag;.nts: on all- -_ a+storadiograpny.- -The- cffects of citrourusside -:ve.e observzd with or wi:heu: the - concegtration- and-time-dependert 3'ashion. With rat aorta seg-r-_aent3: ;hese -agnts also - increase cyclic G-MP-=dependen. protein-tcinas= activity and aEier the a..coTporaticn-oi_ -f? - --:Atc-n'~n?ierous sNl~t`__l-fmtscl2-_proteaS--,_i3e"tica} ,~i'~;telns of= Prot_eii: phosphTrylavon w:.re-observed with both classes 6 telaxanis orn two-dirner_-sionaF gel electrophore-.is e-id RC3:.F OF i:Yi,i.iC-GfViP IN RhL kXAT:JNt {`iF S;-SCLJ`LAR SP~i00TUM`i.`'St:LE Relaxation ~Ef - -rat- ao:t3- .Segm_eww_ - with sodium 3itroprslS3:de and ea;dothe_t:un-depee,de:.:- vasodiiator:,- Sach- as acetylc.tn°.iine; _ hista--ine, - Az31$%-, Ai P, +,ro,nbin, and- trypsin, is associated wih cyciis-GMP (~rvi"F accrmui~{iotn i n a level of guany-late-cyclare- activation _ ancd cGMh accumu!atir,: ._whith - explains the _ common bioche.-.iica_;_and-p?,yanipgical effect_ on s^±o`=t-h muscle Of thesE-iwo -clas-se3 of vasodilators. _ - identified as m yosin_#ig:,tcinain. - A~mo-de-!- is pivser.ted suggesting _+.hat the effects of _ endothrlium-d?pendec:+ va.s-odi:V;crS a nd direc:,y acting ~,t,e~~~,iatcTS corlNesge- at the -decreased- ='P -inccrporation _a±=ter=-3reaasner.t wita _either class--of -relazants-?,as been phosphorylation) _ reqr,_ed- the ts:tegr,tp of--the, er,dothelru_:n.,_ rar:ous ts}ib!tors of phospholipase-.and _lipOxygenase= -preventPtx _-the c-ff'ects-- of the er,c:ot~ I,u:r-depen-dea- - agEnts, suggesting that a-metabolity ~f, arachido~ic- acid_is-- _the- endutrelium-relaz=n3 factor-and respor.sibie- for guarylate=cyclase-acuvation , A 3txrooth-rnusc[e_ protein- with of - these parametFrs- _(crb_-'P,- = cCaMP-defiendent proteii kinase_ and_ prE tein Mu.ad. F.. Rapoport, R. i`I: and FisEus,R. - 3oure,al of Cardiova-wuia? Piran-nuce-logy 7(SuppE. 3F,:Sl l:-Si-13, 105. 137 -

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Other -s::pfoit-'ATatioeal Ins;ie-1tes of 1-1ea1th and Vetera-7sAdministr2 tii:. -= trora the= Departanents=o -114edscirtz and -Ptirmacology, Stanl'ord University School of Ivtedicine; Stanford, sr-A, andi Vetesan= Ad**:izn__r,t:o5 Medicat Cer,=ter;-Palo Alto, CA._ EFFECT OF SD13IPUhd-PO" ASSI?TM PUMP INHIRITORS AND MEMRkANE -D EPvi:,kI1;IZI!fG A -.^3icAITS ON SD:gILiNi - -_ ° NITROPR[JSSIDE-N`~JGTCD REI AXA T IC?FN AND CYCLIC - rUAlE)SI1tIE '_ IviOAlrPKuSP1-IA'I'EA:rC_=iML1LA'i`It'?hl'll:-ILAT jiaFTA - 1 `e pur,5t5Se- vl'- [hs -s21.'dy was to investigate -the-relatiGn3liip betw'se7t sosiiilt.? - - nier9pru sside `:ad s:A.d relaxiarron--; ir_hio tiort o: ~he ,cw.~ni - ;3:utw_- siz.-n p_n3P, nr! tvclic- ~3no53f`.e-m4rtvpllj yhase. sR'~s3rE-C3~i rat a~rra~:c~rrta -m uaYin, _t-y~.taaaa:~= o3___ IDagnesiu~,-fTe` Rrebs-R~ngPr oica:b:~aafe `-sot'*tion: pro~durt_ s~h_:.fi1:°esun'.abl:_ - . - _ . - inhikit the sodi;tsn-p3?assi~:n PurnF,_'or to pnta<>i'3m chlor:de or- tetraethyi_ ,rr.o: ;. urrr, - men;brane_ de,3oiarizirrg agentsr inhi~ited relasation ,o-r-itroprussid-e_- These cond :tionx had-little or -ao_ effect on the etevated cyclic- g_uanosiRe Ynonopho3phate levels at -a _ concentration of nit:opruyside (0.1 Pie;; that r_-la>:ed norep;ifiephring cputracte:i-tissue=_ hy--_ - --- 80%.--Ht?K'2Ner_-at a=lrl`sxi':S[era rer3R-an;_~t3ncefa-~atial-i of nltr~riFSSIde=~7.0 --~: v~t~- tht,'St- - - condit:ons decreased the ele atiotr o` cyclic _guanos~ne_rn,or-hosphate. The innib;tic"-oi r,levated cysiic_gtraricsi>xe `mose,.hosplea*.e-leveis aas-indepenwent o#'-tle endvtheiiurrr, eLiracellitl.9ir- calimiuci!, -a4>< -t:'=e-- c)(cli='~- e'anG..-me -T>-SoP;ipt:o3v-^.?If'-plir`iSpScxhleStereSv'` - inhil:it3r; -M&.7 _63948."'F}+Q tn'll`het3r)y_ _ ffft_i= of onrbai'+ -m n~ of -Pot--.',imm° and mag~e~_ :~-ree solution -~ ihc increased *4~ed levels o c;elis= guan~in- -.-tsun0i~~late-=- - caidsed by 11.0 pM niiroprus<i{jr were aFJCS1:Sh2lwi-en_ tsssLea were itiCt~'Jate4r- -wia'hout _ norfpinephr-ne, or with 5o*p=n e>hrine i n the-presence- of the u-adrenergic biocker; -phentolrsmine. I; ccr:trast,- a$=ad-rene_rg;c tioclctr, prop_ aaolol; -had- no eff ect o~ the oua-bain-induced i.hihitio ~' ~f e'evat?c =yclic -geano:ine rnonop`nosphat eosphate levels, with norepine3hrii±.i-ryresi:at. These resa;ts are consistent w+ith-th? :ypot-h`sis that Fufrrorane events ° r-egulate - cycli^ =guan:,sine monot+hosp?swe - synthzsis: - - ~ t nitro;Fs uss-ide- = concenttatio?s greater ttan-0-, ;cM.=the foi=rcata`on-ot cyc:i<gsanosinc mo=:olkosphate appears to ne-=oupled to the'statusAf-tha smouth tnuscle cel_=-rna-,brane and 31a integr-:ty`-_ ° of the_ 3odadm°-po:as,ii:rti b-.rm-p. -- Fuitliernyore~ --thz--elevatRd°= zydi~ guarcosine monopreosphate _leveis inii<ced= vv- nitropra3s:d¢ may _be-~coinpartms;;ratized = and depending upon -the inlr-acellular -concei:;ratsn; may - ble associated - with -= -several -_ innctions: ih-e,v unctions--m:y _i~cle!~e aetivi<tii;fi of thesEinn-;;o ',ium obmr andjor mzn7brane-~.~lypcrpoiariZatiiSn .aex,fiweV$Y; an g ffect of -cyclis gtlanoSine ]'it3noph-_ 3phatr ofr _ - SoO'i3t11=pCaaSSJiln3 pL'Fxip activity andJOr= ini:C1=-!~ran£ pGtentifil remains to bC-uefr4ori5IFSLed ..-=~ - - - ® - --s:ircuid?-ivn ResenrcF. 33'f(i):E614-190, :9$6--°_=- _ O:her aupport: National Institutes of-l-iealti: anri Ve:erans_ Admiiist atioc. _ Ercm the Departments of--M=ficine'-aaa Phar:r_azoloyy3 St~~?ord- Universir,-Scliaoi c_ f' - Medicine and°Veterane-=Ad,r:-!Ttasc=atien Mcdical Ceas-er Pa15_Rh_, OA_ - i

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CAI..PAO^UhIlti-LAN;'-_-IArv:DE ION Eh:;111AF.i.t'.-iE KINETICS = k~;ow ~ais' apr. _~t~ts s u:?aole t~°_-tt~ study-oE :rig, 4 ~~,rt:ity ~tal 3itt~':-,*~g- _ proteins _'h~ ~en ',..'.l~Q tcl s'~,~y-C2j-u'~u ;i?: , -ettt 2JCCh2~~., k1:1-.tECS. CalF.io~l:::fl labeled with u-l5s and ~~-i,3 wss- dialyzed- aoair~st -~sffsr s:~n*.ai7isg yar ~~as dam~t_ng rneta-ic~iv. ';:.ecrdte of rnetat ..,ciaasge was _nno_,i,ored by a gamma-ray - s{en!lltaiion detectar. The kinetics of exchange are first order-a;td thv rates fall :r:tr, two- categeries: Ca(ll) and _: d(4I)_ in one, and :he-lantLanides -Eu(lllj; Gd(-IIl),-attd l.A(t_ir_)- in -- di!e ather. _ _ Buccig,4ss, ,1. O'Jonne + e., a d,Versor f2, _ ~crerce E sesa. Bei_i<ng, Chena. vp. 4064409, 1981- - 3f Cperac rs+; 6F'~ T?niyrIty. .~-.'1.rCesie=, ?r?FE. - 'Tfo[P O! Cemrirrt--:f mm~aad imersr air -°Y_fter new<=e =sig^ifisant -2 ir.rge_ra±n- g aF?d r#iffe~_,ce ie I3c- The disgesrt:on bl :,irzotine satiruine anA 1..,ot,?te-N-oxide ..a, tnve,tigatey in- rr,als_ r57BL-. D", and _,MAri r4iC4- -it~ q.ca.e (l--4jn:~ia'L~.:},e_-_ - half-hves (t~; -f nicalt._ r ir:, El:,-0- dd 'w-.re ~.~ .a ...9 e ;}n. =-T$e raoid e',imir,rtiYn -;,f _ ~~E-TA$G_:TES Iti' i}I°> EE INBRED `~ PAI~lS r `~ Ihf,Ct - - ~ A CtVMEARAT IVE ST ztDY-OF THE DISPOSITION OF- NIWTINE AND IiS- u1G -ti3r--cCitFaine af - 18S ni.3 ru nicotine I!-Q3 .a'c. -rne 3T-values f'r 11Fat 'le ia 4rai_^.- .4cOilr:e 9'a-ss acc;i .:Pa:fe-a"5 Uly- acCeer::u.a:3o_ -- -iFz _ w;etaa<-:z_LC__-_ .u3Rircia: cQncxrit-a_ fiors 3f :.o-nine in=-blood(234 to 364 at"wsrs achieved in .?= n:in o: d_- 3cot1^ e ,-'c..3. _`+ttE '.~'s- 3S n',nr- TI2~ ti ._:n li:~`~ood ~ -M ~S-a 3.~.>' --WM SinYieai to ihase :df b#Cs--a,_ but the values for the iive( wE-re si.Rhiiy Jar-g2r. C- .3 _ to 9.2 --- $tice-MTigared sa a eQatr_> gr"-p: _ _ -- t~ f~r coiBinie was fo;.1-G- t:e,_r" YabGi`_ety waseli"1=I'iated_f onl ,e-filooa of DBA _ PiliCe at- _ e sia aboui- ore ha !'-__he Mte deterr,~i^ d for .t^.e o.hi=r trair7;__- s ne ,,''o _ riicGti-ne -IV-ozic3e in liver raog..s frem_ 12.7 to 27.3_ min; :he v:l-sseE_ were s:gn;f altly.-fiffes§nt w. ith COSI_- >'D;3A- a:l, ~ C3H mice. St-ast:-rc'at6d d fi-etences were a.so 't±served in = rrvonse to c5 onic ex tsos-ure-to czga:ette sm xe. T;le ?)=o.` snsjecte nicotine to fc-e-s-iightl3r decreased -t57n and hBIA, -,s;c-e but was 3nc-x _ ed ny-6G°% :n livers oF Q:I _ Pesersm D _ P. Z+Ior,at ~ =>r_ and Thou;3 on, !.-A. lp:ug Mewbolrsr-t and D: s1Ts;t:crr_ :3its):i;~Gi-CQ~',_ From t3e_Sch3ol-of Pharmacy, Fi3Yiversity of i:olora ;o; Bouiaer. To investigate_'he ior_~at~oL ahG e~ ~ii7~tu+l _~f *+~..~~:r~ !'de ('yEi(~; - I~-s3i ATI.r.3N ,'-_?dP A;`4=&I i`SIi- :E N-Ci)U:'E=ME-TABC-A-Ii ES;1r''_TE=LT;A.PY A?rU:`dE$; QVAIaT`E'IT.ATIONOF-I+L/CO: iNE- i'-:-CXj~Dt -F mice treated vuith-e :ingfe_ inJectioa of -ci_otir.e,_se+_rsitive- and selective rne.hods were -_- cieveloped to yuanGtzte-this ~lar and heaa-l-ahiie rfie+a~li-i:.. __ hc eornqcurd was isolated redioI2heiing - was _ necessary -to achieve the -se7sitixit}_ r±q;:ired =-for-= 64-76% -from -biological media. Several methods ds of --deteetict', _ v+ere evaluat:,d; - wi_h a -m3hile= phase _coas st:ng of i<ourepa^.oi-w__er.- Ove;ait-rertnve;is: of NNO anslyz`d o: -an a:ri :o or,ded-phase high -perfor_:janci: liquid chrvrnatographrc colurn - from tissue horao$e.nates-as a doue'ry3-sulfate ion pair with Cl extraction cartridges and . W M ~ W 0 1W M W- ---=- s-

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phar3nacokinetic studies in mice. The cis and tsans isomers of NNO .ccFe sersrated o:n a -Partisil PAC cQEur!a _-n_d en-z°vTatic selectiv:ty wa^-eva.uatl~d for the formati-an o°.'- :hase isoiners-ia T7toaips~or!,-I. A., Norris, K . JI aru; Fai-<rser. 0:=k. - :aurrc! oj t &umwtogfaph= 341:34 r3-»~; i98=,° ° - From the t..hooio-_` Phzr~:eacy, U::iversity of C-olorAdc=; S3aider: INlsO'I'IVATICK OF a_ _UIE:-AN 4-1=-PRO"hl_lvXSE _ 1NHIBi TI Ohc BY GA3=PI-IAS_= CIi3AriEI'I`;.: SMOK;` As rrpGTted in A`Yii3 g`aody; direct ex;iiizur, L? }Srm at: al-prJtP'r?:c iE_~iFiiiG: (~3![`:i to the- ga=-pi ase- smoke fnus:r os- cigs=~tte- reEi:P-s -in ~ bi f hasic isactiva=iun o' s i~__ rhe a,~!iors ~bse: ve a rap,~ !r :ial loss ~: ciasta~~ ~: ?+ibit;: f~pacity, the xMourtt of which i.: dapencle;n uoo., th:,-age =of;he smn??ke,-T}ri-"rav+d_, sko-r: t<rm. i^cctsvatr'_cp is not seen when the-erotein is-e=;~,-sed t-a aqueous txtract> sf cigarette sroke (as h: d _ been- done done iE the past)~ - ;3j -Hovfever,-- hoth e;;p~osure=--egimen~ p:oduce a -=second , .s,ow iFactivr~imer 1::ac occ;t=rs ~var -seve=al ~lays=- T-he a!~_hors =suggest_ _t: at tf~e- short-tern: sra tio!rmiay'he auz tc a-penzxyr.it~a--_ ( na si,,.as reec:t:vt sps!ks; i!:aa e~^_rmed_ ~cM= _ ize3..a1_ -~.-. the y--t e b:'st is cl;Ista 7l2 in ?4'ue ott.,° N- ~I;i:i;»a- The a uinQSr3 ^ave -=n E.~'r- ye isenFifi~d a-a-+e=Aes re~os:~i= ~ot- tke"long=tern: inae:ivat*o~:° ahsesved following baih--- _ diref* e-was!'Te-t3_c_Tg,?-T2ita 3nlv.ict- 2tt'~ g~cn'.a;LLfz 'Eitr:'F5. --0.!t;=-~-irs.''.)Y.:cg ~_ . ` " that thy°ie act eYatior, coenes ato~_t as-a tesoa of ihP lcooaidatiur of ' 4_ -IP3 w-;thastea;,dizaa'e `- - smoke compornd.-& thaP dissolve En aq;!eous sjtlit!a:.R. - ° -°,:or. ii'. .4:. Doc:eyt M. ivi., and Cnurstn, =D r.- - = - u~#cth2rnical ~nd ~is p~!}secdf ~.=esrc~ ~o:» mrnrtatisr <= : ~?i2;:fi,fr-o34, i9a~ _ - ot1!~ suogscerf': National =nye.tute_E-rf Iftaloh~ an_" the Nlatiohvl Found. afog= C,:nce_=- Research. _ _ F7or'3 t<-'-e-DeFari~'t-eIt$ of Che-t5i3StFS $ndrair:therr~_s;T Louisw~-- 5tate-EJ9!Lv_er;,3t,v, Eaton - ~t-ouge. NI : R('Sfi'I :ON -Of-Okt:i.4N14" ' HYDROPEROXIDES BY N-1ITROG r-N Dz`JXIL'-EjDINITROGE":-I'ETRAOXI^EE- Cun?y1 and terr outyl h}drcperox!des react rapidly a_th i!'t;,; NE0F, in .rga5fc - solver.tS-ift - tEee -preseCrce o. -am e=to--t~?crf- th..~f,-gariac nitrate I~Di4L2j-1s-=the major -- prodl+ct,`toge_itce w1t1F tSl;i'ieT--a7T!]Fu£ts- of the AcorrejJt°--3n(SirQ_nftrJte- ~I?O*1OY, rI-vOha^,:, - and czrb3ayi compound -;acetopl!et:o-.,e or- acetone frr,s: curayl and trrt=hu:yi hydroperoxide, rPspe- !.+e ,The products azohr -teR,-huty3 h}-droper»xide -are sirnnitgr: whether a-hase i: -pFe3rt _. nfit b~:E E.`=,ose from .umal-hyiroperoxide aTe m?r¢-c,omplex. -The researc,tiers have furrctulated_ne :n itiai-reaction as-a-niticsatioF d? -the -hydror!F_ro_zke:=- _ by -NA to give the peroiiynitrite- ester. --°s i!is latter species -is unstah}e -an~ ei(her- rearr_a ~Fs_ t~ give the n:tratP oi'=dissociates-t~°Iortn alko~cyl radicals and= nitrogen diorxide= - that -ultimately g::es the 'r_-her o+tserved prod!_cts: -'i=he l;iagti,s-of the -reac°ioh were -- studied by stopped flow anj ard complex, but the ir_vesGgatc~s co:!clu:;e they are - -

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s'omisterl: with the ti<t!fisaiio:-r-ri°tia?tEis:t. The Ia?'c Ci:t;St:TIiS at M°C:. a:-e--s.4 x -10r and - - -~ - - --,_ - S ~ -~~ , r _ . - - - x I-Qti fi.r . i-be:'rvl T~a autL s ~`~ - suggeEt that th'sfacsle rPactio.-: of `IO2- -,/N2D4 with hydroper=y;..zes..ay~ate`ir?.~~ ~an# cottseqSieItces with Ii£p°c'Ct lQ the p>>1f33o:'=ry toxicity of !!O6 iC7 smi_1ggy air. From the vE9artftlefts of L.hemistry and Pi4Ci amistry, Louisiana at3te_--`I:versity, I?aton - jotirh4l if the American Chemical _eocrc ,r 10-41:2:'-211'* 19$5.- - - _ `-- _ Otker ssspporF.° jlationa1_Idstituteso: Health. -smokz and our moz:ei systern.- Our data suggest that one pr,ssiFil2 class of species is= 1.leroxyIIttrates: - - - t.getastai5ie Ca7^,poi7i1-m- foixTaed by ralic'.?i sr.7c-P_."sses itl the S2s pSaSe-of both -S.igSreits t:o nor Lppear ttLinaCti:'ate 3iPi.- {'i_e jLc':SSilYiiity i5i~l3t the t:$CtFva?ino s'p?cies` sre- speci&s remE3:z5 to a.^$_deter5tittei_ hizWWe`lerf si'~'1=~li s1ko_vi 2_' ~~^,?~-_ r,?~vl S--d~ral~~g- Lc~ 35-a.~ _ _ ___.~ 3 _ F:__ spi;t-trappable from gas--phase s;nocY as we!E as - sro:rs the NOjair%i3prxne syste; - in 31r Is-fJ~Scd i rl the E~ ~';Qi -tF~ ;t!_~-i:~~ting-pacies`. -'-T}.~.ed`.'-is of_t--_}-tesz- - o-xygen in the gas-stI§anx. sugaestiog that the oxidation of oittiti=cxice ta aiirogAe::-3i!sxidl-- - A mixture a-.3:: ic oxideYi-3O; snd iscFrene ir air ~sas been studied as a tiodPV for gas-phase cig-reue sig-mv _Y We have sltown-th3? thi_< -lodei ;ysfeatr dz<pIicates many of he p-opeiiie_s of the inactivaEiou f~~r: ibhbitrur (ol?a.~. 1-0 this S.uoy. ~i:ffe ~d ~I~ of al, . wc e _pos2d to ; ur o" c~t~rflo~ 300 ~ p~ NO rd G t3 pp~+ t~-pr~ ze ~,h- .ag of ~~:e ~~-~t /is~p'e1?e g~ 'atreaRl IjrEcilyt!:raUg 's7FF:i`e325~ ~I~ts~~ so:~' uPa ..au e- 4'P: tU l7IIdE ~o afn,,: 1ae of iehib.E ,y '-av&-t,L _ °F3l=S ra,. nac: a ot: -is t°c*, ois-ved when ai'r is ezp~.sed - to aqyeous fxtlatts cle c~e-~+~;%a;Ii3s,prehe ~:*ture. Scfh d:_ect expi>s:ile-ar,d exposure *o SQI+°oF2.4 exilaG'e.3, iJwL`-'e', cause aIYI2o undergo a sl@w loss of activ'ty- thal_contit4Lei for several da='s-astl;e proteir_ :s itievboted in the h_I'ferYolutions: Gas-phase ci~are:tr sttiokeiru already t.ee:t show to cause this mme two-phase ittactivatioa-of a1P1. - he inactivation of e1P: by the o~e1 sys?hm is dependent co the presence of HUMAti c=1 OR BY ti Y'kI=y z'bF !ISD3~EiT~ MtXT+J;t1r$ IN A;R - ~4 E-C }jANLS?vCi i F `-IG_ A R `I`,''E-S iv?O'.~-_-I;- TbX tLITY: T:I I?~A CT IV AUn' CF - °;uor. W.A.:Dc~'ii _e; _~K3nd-r~ i.?" ('hemlcy!-Bioloz%c-'fnteroc:iors 34:173-183,1955. t2f:Fc'r silppOri Ra,Ei~pai iDstit[3teS 'or teal'3l aftd- M1Ta iQI15i Foundation foi Cai`ce: Research From the DorparttteE_ts ;,f -Chasist;y and Bio-cher=istry,-1=.ousian3 -S:ate Ut ive. s:Ty, ~aioy Rouge. A

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[1'?AE.":'[E'AI'fO}. OF HUMAN A.LPia._ I=PRC~- EFNA:E i:IiilBI:OR EY -CIGA RE i I'E SMOKE: EFF-FCi'OE I-~NtOKEP!3A,EANB T-ZJf'FER- ?n order to resolve a` discrepancy ir the literat::TF, we ::avf e_ ~T~ined the _ in vitro inactivation of human a1-proteina.;e inhi_ki!or by direct exposures either to whole cigarette smoke or to filtered (i.e., gas-phase) smoke. Wyss-and-coworkers reported that whole smoke dGes not inaCtirate the protein, vAer?as we rep"`tEd t; at gas-~'iha3e s.T.oke does. We now 1`i n: that di-act exposure to gas-phase cigarette smoke causes a- slightly greater ii=activation of zhe.protein than d:4>_d-,yct extrsure-to wa3.?_ ci,areite- smoke, - F: yorr 1+'.-.f. _and risoley, ':-. 1;_ of iris quenches the activa!iQn process almost cor:ip:ete'y. Our expe:itneat3 used - - ifhasp~~a'[e bl3f,er.-'~kE-su~PSt tha: ''':~-is an Ua_titahie b»fer fjr-!we in experaL7rtkii that probe the effects of cigarette smoke. confirming our earlier -s;:ggestivSi t3:at-Ao* st:oke i. less ^'idi=irsg ti3a n is, gas-p}==E smoke. This differe.*ce, however_ does_ not eFS::ain the drarriitic d-i?'fererv_--e het-we=n ou_r prevlou- _-firtClr.Es 2nd r;_sc c~l '~ ss aPd coworktrs. The exp.anasion fei ch d csrepan__cj- lies itt°thr : ature of the bi&?'rers used. Wyss and ;oworkers L_ed iri3 1<lffer-bnd the use ~ tner_ su~;pr~rt. Nationsa_ -Ifstltutes-_o. -flgalth and ~atlonal Foyni:ati.~._ -fo: Cance_r .Snserico.~t R3vtew of R~3r~irc?ary iyl:Q41-4a's, IyE~ - _- _~ -- - _- - _ __- FrC=_a the =sa.tmi33 of ChS'1_ - E: s-`7ai<a aO_a-.'e - a~-iil°'. t-36t`I: -RotJl<Ce. FREE- nADIOAI. ~HE'~.3Iv=._RZ OF CIC,4R c.~#`E SMOKE AN0 !: ~ ~ .~-.~T'l~3~i../A~~~~ . --T'~.~~lllV.L~fi. ~!~'.E .~'~i.l'==AT El0'i:3-_ a ~ tt k r1 ° ' di l ' - re !'s e sC e Gt3 s s tW f vel; fferefl_ p.Dpu aWtes_ OC ' e radicali.o.^C in-fhE _We-alaSS Aiscids`~ t:7e- ti?%la.Sl4itia` R_tr;p16cA:E0n5 IE`ir_ -the rad3ca:&/M s:Ti`3_kP in terttiF_ of a nutnoFr Wr ;;d:ca=-ttfed.ated de~sY p=oces&ps, .ncltd - :n g e. Iph;s Ir,= at.dearce:: Cigarette smoke cW'yixs throls to disulfides; wy'saggest the aetlve ozldantz are KO and:~;0,=-'Fhe eftect3-P'-?,ke ct~ li~:~i~eroxi~aeion a=e-cwrr~pie~, and th i_=is ~'isc_~sed. -fresh stnoXe, . - . - - - - - ~ - _ - - - ra~: Q._ 3-actions sla. -_ _respeseo,e_ror the 1z 3ftlva;l~~ of c a.=10roteinase az._h!h=tor_ by radicals °hat are aiuch more rezctive thaii are_the t2r=r;hase-radican,- These -gas-prase rodicals do not a=ise-in-thz-#':a~,e,=b~t rat:e;-are pro<fucei.-i : i-i: a stea-Cy scate by ;he oxidstio- of f10 10 A-~3, whieh #neh reac-m with reactiv: _pecies-i.: canokee such a; isoprene.-_tie suggest that thPse radical, an:d the reetastahle pro<:ucts derived fEvm these - - _-e- gas phase of garette s-nokL contains ~Ir~a oxygen- and carbsn-centered - the prinsi©2l -ad,cal Iti tar ...rac s with ON'A in vltro, _posslbiyby covate . biFd-ing.- _i _ i. _-.- rea g_to hydrogen_p-roxuig and hydro>tqi 1au:ka:%r addite~ ~ws havy snown tfi3t_ ° system unat rs capapE.e_ ef - rechtiCing moctElar_oxygen to-pQducE tso°r xlje-,_-event-.~l a_- co!>tplex held in th? Ea rv Fa-_t=ix. we-s= egsst-tra trl r~r :_ad ac.~ $ re<<a - - -_ - _ -~-= = _ -- - - ---~ _ _ _ -= - - - -- _ --- -radiCa's: we ~`r._asE -idP 1-rl»fy: prin . yfial-4ad C.al a_: tar- nd one ii3 the=~i3as- _'i'h-*ur =-~h~ ra =rc;az;~etl =*3et: fre~ Church, O. F. and Pryor, W. -Environntental ilealtlz Perspectives 63::11-126, 19g3-

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43rh.el sI[pp?r!. maT3Ltta1 :iUteS t:i- healt.a aRd.'tie Rr?toRai 3'trwIQa;:;i^- iCr S;anCe: = --- _ - iEseaCG . =F:O:.^n ti3e-De[laa't:- ~.115__i!F Chemistry and-Bi.fichnl:stry, I..ai'sdsia_n_a S iate Li[.='cr8=;4. B ato=1 UTERINE 13d0:>; FLOW P.?JI:-C%sTACIIOLAivIIASs. R_y_W~: $E Ty_RE-PETI_T_IVE NICOTINE E7:?OJR) iN THE_PRgLiNANT t\vE- _ In order to sirnulate_kuttlam sri:alking, -eRperianents were designed to--determin= -what dose- of L-iw ire _ it- Me - p---gnan sbeep- xoai±:.- produce those -pfasma- r:icv,ir,e roncen-rzticnv oyservd ,-r: qiman srr.ok-ers anr.a measure we,iRe__b:?od fiow an3_ r,lasma ratet;iela_ ::,es Mr_e3~ n?-to repetitive exposure ;eve:y' -yJ ?aleutes) ) to t#cat tiiCotstae dasse se%ren C, nical_ Ct::terized- pregn3r,, =sheet:_ equipped with - ?iecti3ma~netit ~; wp;obei': aru;ud bot: uter;: a ar,eries, twe observed that a- nicotine ----dose of 0.z It13/_imnta for 5mbfiilEe3resLlts=in aTaea'-1 piasL-3 n:Cwi.r:+e i=oi3C@fit73:5c3:~3 of i.):1- s f. ng/sl SEM (n-~ l7; in n?-dintel, f raowing ixl'isior--. Th~ siose of nicot ,e -wa+ t1re~~n :,;faseci_t:very 3Utes for 4 hosr~; =and ali0;rota=oF blood were draws immediately before and after= ricotin? infusion for_ determination of catee, olarnines- No-- _-=ii$'-ilif=CaR%- adetat'.s tis 2n--pias^3a EGi?IejLhriR£-- atfit-?SorepSnephfire were ~obse}ved-- t:1rou $,hoe3_t cE.`--e7[pi¢rimef[s~ jr ~-$), a: c._ Ro-Sig?S-lI.C2i'.t Gtanges --IO--t3terLle- 3}aod fltJw -oCiurredatan-tir5 dLing the ezper3r. eai We cory;,W-ethat !here 's a species =dif-fefestCe._ betweeL: - sheep --ae3d = - .wIt:_ resp ecE= -to -t: e - L,IC?ti:aiC- ETres-Wd --ir -CateCieola-r7tnC rglease. ~- - - -- ° - _ - _ __ ---- _-_ -- °-- _ _ __ _ P.:3vek. R:; Conover. `,1°, B., Key; T_ , aRd- `Ja:, :?`-3aaltis.4f _- - Amerdce-r--.or<<F;aLof Ob.rretcicc :;i(7),-&85-8"'J, i985-- Ot`.=; _surS>RS7.= U. ._krblic---HealchServeC Fram the Department c` Reproductive-Meci-'scine,-Universixy af-California at-San-Difgo,=-- ~hool o:" Medicin~.-arr,l rh~-~par t:nenY ~f E h, r: is.ry -Pra^deis- rn_~ers%~~aidham- IN. _ - ? 33y4TME F'T:;1F-L7Ai rS;BFA:,IESS FO1<SIMU:.TAN WS i'-I.Fii YsIS_- - - - -Alv"D tO1vCENTRa'110;`:- - Y)la.yses rnersEOranes_--itsed for_ simultsneous_=-d,aty;.;-soneenratzo-r_ _ ieqnireti pFetreatrl<ent to re,.nicve uV -a-asor6in g cosn.pab, d; ,eazhe-d=l':oif-_ the.-rtrenq braRes-and to_ reduc¢ the abso ptO c; pr33e iz to th, -mern5rar.es_ T-F4is wai a,;cs,_rsapiishrd '--wit5 sodinm - carboirae abd pthahui o_r with °sulf:~r-sen:ovai 3clutions,." -- Proteitr- raem:lra?ions were =de :vitira mm=Br~~i- r' pcoteia- r tei-an and with ,v a;:sor a =-__at 2S0 rr ;: SaIuh,e - !hktnb<a5e- =ort5pone - as -coniiibnteg to aberrant itv-spectra= attd altered the ratio ot 1$0i2trlD-nnF- alisorb~ce -Sinnultar.$ousdialysis-a.,d can.ce3tratie>i in tt:e -tr5icio- nrotein dia1~~ coacer~*2-toF ~;,. ara~s,= ce bar._ing, aspect3 -__af_--thin-layee -Aiatysi,= and 911trafilts,'i:5n -ia=rapri rtr.`oya, ot- _alts_rom _ the ,r,rv[@ir;_ so-lit'sor,s. Pri^r - treatment of =neriucanes_ reduced -r ,c;ertaihti;.s_-in retentztz recwvaries, eiir.t'snatd -u:-abscrb-ing cohipof-°rtts of meriEranes, and i~sproved ~ecoveriet oi Qrof:IL= Richr:otd. V. L, St. Denis; R., ani C9hen, E. _ dnciytirai daochemisrry%4S:?43-35u, 140.5. From the F'acific Northwest_I`Cesearch Foundation, Seatt!e, WA. - 143 - - _ ~: - ,~^

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S~':RLJCI-1$ZA_:REGl:1iYki-rE-N:?fOR X1LOST EIRIC 'f4C,°Tl'.~t~TOkS ;°a-RA;= __ LIVER MICROSOMAL 3=L _Y-OItOX3'-i-CFE7HY7-G;-EITARYi-CE:eN'-: h{E A REDUCiASE _ -_ ~.~'verE;ct3vmounus-cJtsi3iniilg ilari-°'us structural iilCietrE$ of- azAt?EPXHj,- were exacninsri aposzible effectors c:f rat liver 3-h;s~ozy-~-r~thyld:utae:i coe:zymE f; - redJCtase ]CzEv_rty, =Mzicr3somall red-zifta_'--e~ :vo = tiv2!¢d ..a+ity'.r :.5 rtiM-Z__==H, 3s8yed ~it-h aubsa.urat~ng NEiDrF- sor=e_tr~t;: n atd rar as's_.g amz;~nt3 ot-:}~` testec co!:pour_fid Under ihese conditions, ths_essentiyl and suffic:en-s:=u:,m*e required to elloszericall-y enhance the activity of ths_ veductase is that of 5'-AMP. When the 2` ;,osition of the nucleotidE u yhcsphorylated,ini3 a'losteric acti;,>tior i;-diirihished: - _-_ - Roitetmar,_3. and B-c-cf mea: iar:d -&rp%~'rsfcu' Re=--.?'7 Cvm.yi:arrcWfO~i; s3(1):Sys=9y-7, t9st. --- Frorn t5s__Depart.iens.of 1:_f,c:ieenis_t~~~ ihe Ce _rge S. Wise Eacrilr; of-Life S`ier ees, TeI Aviv rJniversij*y, Lime%. _ _ = ALLOSTERIC ACT:VATKWWZ'iF RAT L,i"E-R M=,CROS~--~MA_I=- ;~I`a1T;N aWDE .'-~DENI%-~ t.-15Lat_,7f L E-OG `3DES --- ~ _- _- _ _--- -_ ____ 31-HYOROX4'=3-ME-::'iY<.-!y=Lt:~tARr`1, rCE?:Z:°ME A-RED`CTIASE BY = i- -NADH and NAD+- _ art- -_ r:eith.r yubstrates- nor inn:bi'^-r3 - of- _3=-vd-oxy- t = - -3-methvlgltita=yl coen-'yrrie ed~etasF -=a° tottcelf-ra:ions u7=_ to I rr ~t - j_ addition _Qf eithcr- i:aDI?= or NAt.#en_harwed tha activity of rat- liv;r m:croEorn-f reciu ta fye r+r:uH #'aflzd _ o affect the a civr-y -+f t;e t,--eeze-thawsolubil:zec:-er.zyn:e. The degree en5arrcemenr of enzyme acv vty b} NADH decreasew as GSH conctr;tratien =r~ the a sa-y ir:crzaiAd.-nd~it:A~ n'~ ~^rr ~?l~i *: A011 {o_ the as3ay ~orverted t he sigrnQid - ; :-H _ i=f-cce.fi e_er- __2.01, -N A ?I H -de.-vnaent kine ; eu=y= of ;he ?r: rosomE: reductase- into Mishael-s-ntea kinetics-(I`'a! caeff zien*. ~ -,.fr1 F ;rrtber3 nvre, -the - - ~ - _- ~ : ~ kinetic Stir~+es-~'ere ~fte-~= E ._The 5~~-Ft, 7u3in of 500 uM. I,4aDH. Again, this effect-bf NADH was diminished as irSH cot:ee*!?ratiyas increased:~- 3'hesc-- res'_!ti- derror:st,a,e that _ NaD;Hi- ~s -an allos,eric- activator bf ~uc-Lase 'F ~~su~ts _a ro ifiaicat; tna. ~:?~ri~-«t~ re3ue~se has ?EA~H) conceat_aatiQn o!-N?.F;*-1 reqtzired to c,bb+taiF haif-ma--i:.aa velosa=y (SL s) iz_-tTe n_es_ ,ce fC t~c ..M -~ -bi~ d7F F it i n 3'~i IEIA -_ _ ___ i~i-I ~ i i . , . tl d3 ~ ~- ~,.:_s c Fti i a S C 51tc ;'-_ __ (-- - -- - - - - - ' - - - - - Roitelrr_n n,-J:=snd Shec hteF. _ ' _ I~ _ _- _--- _ - - _-' - _ -- -e - -_--`. The .,oueoo~ f~ ~i-¢r3rst ~-2S4t2z~aa(3p9 I4Q}21~~: c From the Departte=nr of E herit G crgt ~. W_, Fxc i:y r;_. - ita-'ei~.:ces = Te= _ ~vl tJnivers.ty-ISraR1- -_-__ _ ---_F -- J- RRDUc EL3 vrXTAt~i-t OI4~. `i~;i:7NE5E HAr.fi?ER, v0. _Aj;tm CE1 ? z I PI`c_.fi'TI'EL"Ir AGAINST r~I 3CTIVA.TIOrv OF3.`!YOR(`XY-3-hfETIIY' CrLJi-A-'.T ~ COENZYvt-E_A DLDOC`T]A_Sr_BY s=N!EI2L.'A°TOET?-IANOL-DL~."I,FIDE - ~- ~- 1- _ i-= When the di3ulfide br r-, er~aptoe:hara: (ESSE; is added to AF medium of _ufltured Ch trese hamraer oviry-(CHO) -ceiIs, LLt me and torrcentratian dei:+?::ay-aa relesse of _s-ffierc3f3toeti 3rlQ: to the medium - is CbservLd. - The _ red;.'ct;t1i. of ESSE to _ - 2=-nrerca~ytoetlar,o# by ce=3s=_ a saturabItrproc=-s~ t3erate be:_;g abprc-ximat-l~-5~ _~...oles _ ~~ - ~- 1~7 !~

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ot' s=sercaptcsethars9l per= . ag cell prntein -fcr an hour up:,ry ex-posure-_to=250 -p;Lt and -thus prevent tt==rrom iarhibiting tfi=' redye~a e- Cultured ~ e;ts respond to E£SF admit-6Y c:evati~_the:r total and=acid--sdlu5leg!utachione levels. The-use of ESSE as a partuban- of the GSH Sta s in cells-is discusse{. --_- when -'isolate.d- frorn-i- Cklf`3--_°/ls- but total - '~!(s --synt1.eSlt fF©ni -{2-34C]-acetate-in intact cells is not affected by ESSi.==at-conce:trations up to 501: pM-- Cvtosolic reduced =g!ttathinone can _s fomtwteously e=,::hattge -Iisudfide bVn&-°wit:, EsSE Rednctic,ts`-rate-of ESSE bycell.ss a:tac-l:ed-tc a-substratrr: -A independent of gi~.-cc-- a_n& - insulir.=f'or pesiehs up-to ~ hcurs.= -:fawever, in-deta_hed cells, swiri_d 'e-- suspen-sion_ addition of glucose and znnulisr is necessar; iL-onde; to obtai :a ii~ava= -;eouct-ion rate of ESSE. The= - iate --- litnitin.-.. ee:ayme. - s =the- -_Ste:o! -=biosynthetic_ - pa-: way, _ ~=h_ydrozt=3-me~?±~!sl~tafy! _Ccerizyn;e A-redueta;e ~~~.=1.1.1-..~4), is inhibited by ESSE DCta-^,, l. and Shec--:er, .. -From theDepartme.t of 3iochym1st=y, the Gecrge S. ?" se Faculty :or Life Sciences, Tel Journal of Ceau-laF _Plsy.ir;tagv I}?:'_a--2C- z-$` Aviv Liniversi+_v, Israel AFID LOW pH 11I_ISCI.A'7=FD-`YLART O: RAMIF - - ADENOSI--iVE-INDCJCED CORONARY REI EASE-GF PROSTACXCLtN AT-Nf3hMAi.- - - - - - - _ 6 - i u + . - was not affected by`perf;~sio IC .of the-heart v.it`t a'enos~n~-containing-ruedium- in hearts -pre-,abeled with T~="-C]-arachidoni_ acid,-aderosi:~e (10 p:;) induced a-specific liberation of labeled lipid-extrartable substances, including 6-k~o-PGF ti. to. From these data we conclude tha[~ adenosine sti_~clates the - liberation of 6-keto-PGF `from ihe-rabbit heart by=i-ncreasing precursor availability and-seibseq~~ent -- formation ot pro;ta'=ycliYt i~ the coronary ceSs.°s. Filr2herr~iE~-t!:e increase in coronary flow in~±uced-b} tis-e_a edosis is not related to an augmented for:natien ef prastacyctin. adenosine-induced in crease in corona~ry fiow was-not facilitated b;~ lovv pli: -- - 4. The base-cororrarv efflux oT-6-keto-PGF.- ~'io;n_thF hearts--was -2.5-=:6 ng/-enin: Ade;tos:ne-[1~-e-1L~`:~:)-significaotfy faci!itat a this efflux, up t~_~~- ng-/rnin= The-e:`flux o:-6=keid-PGF~ was not chsnge~ir by perfusion with acidic medium, eTther i?t the basal state ~r dursng perl'usio.nn with-adenofine, _ - 5~`he basat aersti~ia~ eff~s:- of 6-'~eto-PT=- w~~ 4 F- ~ r 3/Mi~ T~ " ~rft flow, amounting to about 75Sir=at-nornsal pH and -a drtig- c1~cenrratio: of 1'"Sk:. - The -- 1: = Rabbif hearts werp -perfused -by the I..a-ngendorff -rnethod _With- crug-free- ` ~~erfusiot+_=mediunw or- w-ith a me~*ium containing adenosine 00-,=h'° - JO-arl)_ a^d- the - - coronary and=Irans_~yosardia!=yfflux-rates ~--6=xeto-pr:,staY3arrdin -F~ !6=ketn-PGF were measured. -F erFUsion was performed both at pd-: 7.4 anA 6.9. 2=_ In other=experienents the fiearts-were pre-lab;led with- [;fC]-aTachidonic acid _ =_ artd-the -coronary -efflun dF-radioactiv-ity and of labeled lipid"nd 6-keto-PCrFI~ was determined. - 3. The- aasal coronary f!ow was elevated by almost ?0% durin,3 -&ssue acidos-is, in comparison So cont;F:- Adenosine=irrdnced a=dose-dependent iscrea~; -zn--tfie coronary Ciabattori, G. and Wennrnizfm, ~. BritisF. lournaf_o,f tharmacol_r.p--g5;357-5AV3, -1485. _ Other supporl: Swedish Medical Research Council. Department of=Clincal-Phys',-olegy-at Karc:inska Institute, I-I-ucidinge-:.iniv-ersity From the Departrsert _of- Pharmacology, -Catholic Lneyerslty, Rome, Ita1y, Stork4ol.m, Sweden.

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;;aARAC;'EIiIZr'_TIEjN 6s' PIliOTEFtJ K1NAS"F C-A Ci?`lITY IN FNTERF'.`~.F:O1` - _ - GAMMA TREATED ML'R-Ib1E-r -EiifTClNEAf. MACROrHAGyS _- iV3G.rop,'-..3gp--`aCtiYl!!t1ocRf-'Jr [u3o_rlcl°aj- SnIcrisb5c!dai fu`:ctlEn± Caa be ac111eve(? it- .-- __part by trEOtme ;t w`t:~ rea>ora4inar interfererr Samrr.a !IFry~r sn varo: ~4e ~a+~ - previously$e~37b'wrateis tfiat=l:N'y Uzatnleiltof muPii7eper'it6nea;riacr0-phag-e=aresl'.tai-M. _ versus IT?.` t-~reate~_rnae:ont~ages= ~a~ the imagnitude- of the resE o3sA_ ta-DC _^r pt_3A; - difierence which iould=bc-;Iiscertse=ber+vx-.n pr«tein -k:naa:e C de-iv=d frora cor.r.-~'_-_'-- increased both by IE'N; treatment and also by stimulation with I3E3 ti. I?A.A. The _rrtRjoF (elt~leF ~i.`sa- or stin-iLLia'9<I' haYl conFparat`';e Km '-aae~, h?axii t~~*, vc:!-ncit~- ~ F'man ) was concentrytion `cependerrce=iJO-ea.ed _*.hat-bo'si co_*,tro'_-aac! treated- enz-°mz preparatib:rs re5;i1lree_~or~timulatt5u -~,f- actiY.'ty.=- uinc ic~ifai;~~1s =~ the .-ATf (aS--st:b3t-rate )- _ disr-inguished-in ternic:f t_}e-diacyglyczre. (DC'Fer piu;rba9 diester {PiL:A)-cor.cen;ration a two- tc, five-:`vld iricrea_P =ir the_ aCtivi.y Of Ca`* _^v4o3nhoi pid dependent pro.ee :_ KinaSe C. We nowrepo?rr that thlS MfectwaiPCt depen`-e:'-t zipiai So':Sln u:iag .?telrn synthesis, since treatment With-cyceflhtxE-![_ide ih.eW conyitfioLs-wh9re nO,`i211 protein synthesis was inhibited by &eatar than 959i: -had no effect -upvte the developntert of increased enZyr3e acti'vity. FJCar-_inatioa of_ va't an~c _'k`.h:-sphG:il,3i=1 rsyTuireTie!itt re`:ea`eece no d'sfff;relta5 betwee-.vZnZY-.r.?-i3otSiEa'I fr0:?I cC7titrCl-or 's!=P1^3-trRated cet'S -^-ould not be - b.v 3 faE-tCir of tvia= to``fouq~ffl:d. T-hE<_C .'6sL':ts sug.ge3t tha=-I:Nvy -:ie3tm2:°-=Ie y5 iv' bcPMry tre :men= increased .he-stir: =latibr, index ;i.e,ratin ol`basai to stimulated activity) - --- -.__-_ -- -` when exposed to an spprc+ptia.6 _-~verS7~== mQi - 9~Eyn Gi ?s_75i-; g;~J2F 5 x.r;ase-C.e_ehauoit: laseiticu- iWytic activity D.L. Adom=.T-'r.D.; arid-Hamikon - -_ - Becton- T A .--- , _ , - '- - fourrv1l r-JCeUslorPfr;si~togy E25:1485-a9-1, 19985. Other aupo:;t t~nitd STate Y bnc~e itr oe*vece ~ _ From tlse Diepartmn m r,f Ped.atric;, Pat:-..1ogy a..==I 1__robiolog, ri ..u-~ ~'eay: Duke ' t_ LJnlyesStY.-Jurham, N . _ - ~ duLh;TIF,CA T IL~~ u'F T_-',E:UYMPtirJ-K_flhtE SG-LUBLE -°-__M-"iti.Uslii RtSFoNsE SUPPRESSOR iN Lr-RIN£= 0- fiiEPlIRIOTIC _t=1f;1! DREZT -_ _ - - ~ wi ~ ~ i. ~~ -- ^ ' . Patse>3ts th ciange ro I^ ) jiecuerta have --anamumnyh.Uu kS;. e ic =n:or y ~ -suppressed :n-3ivo and rnv:_ro immune responsiveness of uncertain etrolax,=- Secau«- -iQc-reased- suppressor cell activ-t4- 'haE eser `as iate-:! with tt~ - vi_ sase, :?.lnes ' ~ 1v1CN5 gatisnm we e-sE e ried foact +iF c? the lmhkse s:lt:.f °n+m ne_ e-sxn e ~ supprzssor (SIRS), a prnda,.t i-4 Centa-ravali^ A-or ifite.Lerori-ec-ti%,;a;f-d,rppressor_ :' i= cells. Urines from u>s reated MCNS p3:ier,s suppressed polyctcria pisque-forc_r,ir_, cPli responses of cultured spiRracytes. This su-ppres ive acsi.vty was ider.ti=ied as human-= ~° SIRS bythe following fdnctiona: ar.e -+asica3 eri,eria; =a) molecuiarw4igr_ esti:_nated by gel filtration; (3) krae sOr suppres r~n-_(c) i~ sibit-t*n -of suppresslorr by catalaSe, -- - leva?n4SoleR an4! 2-mer^apt- e'1'andol; ~"1 arri;gat:?i oat.iv.ty a,;- asl(5 'vr proieaSe - ~_- ---treatment,T (e) -elution pattern or_ -Irigr_= prrforenance liquid chromatography; and -;#} - i cross-rEactivity with mo_n.o,aoRa_; antimurine-31-RF ,._antibodie3. $upprESS:vF activity t - . . i f oh af symptoms. disappeareu rom uritie=_aftei :,i lat:on of treatmem iaut_ before raanisf

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birinea were tes_ed frorn e~ p3tieMs w.fi_MC4IS, ali-of whom excreted SII?S. -In additiyn, two r.ekhrotic patients w:t h a=ute glor_^.erulonephr:'.s_ano three: -pE re ;c pat!euts w th - sJppressed insMSine 7eSpU:a~-es ofte-U3osF!d ln j epFtr.^,t!C Y'ndrCrtle-. - patients did not-. -i ise results indicate m`st excretion af SIRS .,c12r3 in ce:tsin-case. 'vS Iet~hrotic sy,ndriD:rie an-jthat the pr£_s ^¢ ot SIRS in the ;ne -it not ?ccCUr-ted far solely - t; the presencs<--of- p-rcte ^uri--er nephresEsy= Serum _rr .-: fau rtephrot=c -i;atier,ts -a!so coT!tail7ed S7RS, xhei-eas neither _srein: nor urs frsm -=six- --nermsl subjects had SIRS activity. The systemic presence of SIRS in these four patients and the iventificatiozr of SIRS t:_ urines- froiz_a arger g,eup e patienF .--u ~~est a s:~ss,b?P rale tGr`ziCtS in ta- mert br4*toproiifsra ise cisease xezied S'_RS, o=E other nep; rotics-and all nEOnephro.is - -. Sc1!P=npe=, F.e~__4R'.'. A-yn.eU i~.t.'1..=-- . . Journo of ~frie~:,.Ir~+esa~dfir,r_73,341-349,-!9£3. ` Other suppar Monsanto Coatpany. LabcraYu: y -.~,;eine~, °i`he= 3~is~ :-lospital of rt. From the-Depaitmertt o'.` Pa_tho:ugy and Louis and the Uepartrce nts of Psdiat:il-_ and Pa hology, Wa;hir,gior %niversity._S,x'.t:oL!_ of ° - °_ - - -medic!ne,-St f,o,r s I_`IFfIFi:TI0-N OF 5?iLtPLt fiviMiJI`-t-£ kiSPni`ISE-_StSFPRE` SOR _ isC1'IVITV Sf I'sR ;w.TH FA^.CiR., _ not iGr-or .L!_-- re rsed' -ir.hibit;o.n.- of aatibody sec eion by SI?Sox in a ti:ne- and- added late in-the culture period.- -f.-1-_ I:,=2 and -~f also blocked:-s:=?t<res3ien by jIKSQ~ tio5vei~er,-;_ rsr and 3-.=I b'oc''..ed st3Vrr s34on by SI-~-vOX oFe-° when added 3°s hr _ 3efore addition -. whereas-_EL-2 blockgd suppression by SIP a.w7len added before or up to 3 hr after andition of S-hsO.. I--urthes eva!uation r;iowPd that IL -=, but SIRS . Intrleuki?-,.- (~,-: j, inta-leu~_in s(IL-2) a;td epidernjai growt h_fa.-'_or (EGF) ~ each ;nhibited SIt=S-mediated_ st~~=rezsion _of antibory_-secretion by c,ltured mouse _op+ee7t=cell3. -_ni]ibii3on of-Sifb1 actJvity was rtti~st efieC`._IVe_~'hen groV~'t h fa:ior_- sooe- cell lines. Effects of purified growth fdctors on suppression of antib o3v secretion were - G7Calnit:ed to CeterlTine whether any lVouid oppose ti e i_.lEibitor} effects of SIMS- or _ ' to its activated for.;:_,--S`,_R5,- s, by_ i-iydro ge^-perozid°-produced by marrophag°s. SIRSa. nhioi= antibody sete tio; by °.yEr,p~tocytey and ceil division ?yy r:r,rmal or trassfo:r: ed Soluble itnminE-tespanse syppresso:_ lSIRSt_ a bratei:._af M £ 14,3:e, is "a iXnphol:ir:e produc,ed bv inter`e:on--ar-aon-car,aval=n A-;etivivteai £vp?ressvr T cel._s_and -is oxidizer properties of Si-RS. ,n both he eregenebus an3 horr:ogenecus cel' pe.psTationi-. ~ (9.3-: agjcra)wher-adt d to h?MI-'kg cultures tFr'p fore {tSa~, ;,^.te:fered with I ~_ the ability of S,3E~~ to Irn;yit . a=1 dvis.-. :. Ta en Yg t-`-,z t':Fs- u indicate that _gr'iowtsi f=z'''to,3 .Pa_.E,.s.. ',v` tY tJth the _ T=lTi,.n(3mypre.,siv? and _g.(;wth _ inhibi!ory ~- eoncentrat:on-depende_nt nsanner_: rV;th ~J dt,its 9. aL-2 per v -5-na_ culture, reve:sal was L complete within I t?. The ahilit;+ of growti factors to interfere with inhibition of cell =-_ t division_ by $IRSQ.=°v:e.+_exarnined with tt!e htt?:ian B-ce:S leukerFia ~.cF:I:-17go= This cell I =f -or IL-2. -E;E i line uinds Eur- but s not ~.n,,.vn to have ~el9 surface rYEe2 ors for t! - Aune; T.M.- , Proceedings of the Mationsi .4raderry of Seiencesr of tiie -`lr:eted- _5tates o; America g2-i2bE-b3.,64: 19='. Other support: Science Foundation and the Monsanto Compat.y. 147 ~ '+~`e - I `

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F=ott£ tht Department of 11athology a-~ Lsboraty_y :viediyiae; The =eYis,h 4qt?srital- of-Sw. Louis; and the Depar nse__t of Fatlvolo=y, W ash.. gton: t:,Itravgrsitv Sritoo_ ef "3JIesic v, .9 = Louts, - SCI{!S"I'OSO, ~IA~1S MAN",~`~N I `~NI - SUPPRESSOR (S!RJ)_!i[t~L1~11~=~as101'~i0_E>;FE12i!aEt~TAL t`'TcOb'-.'C.`-t"IZ'iN OF THE '~.Yr1PI3t.Ii-+IE.SOi.°J'-EL-.`.. IA4k!Z; ~!E RESPONSE - Chronic schstosomiasi, mansoni ...a hel_ninrhic ir,fection c?rarasterized- by cel;-n+ed;atect anti-egg oMnulo^:ato3c reactions and a-- variet- sf associared ~ i itnmunoregltatory phenomena. Soluble immune_ response suppressor ~(S!RS; is a lymphokine produce' by activated-suppressor- T lymphocytes in various experir_tental ~= setiiags, ihis report dernonstrates the presence of SII~S-in the sera of-mtce with chrr nic 4chistosti:fiiaSis msnS;tn=i <<E.:e3's: 20 wk of infection), but not lin thes.°,Ia of nllce--wiih earlier_ infections. A]so; c4lturzs of isolated, =intact, hepatic, egg-focus_d -grahulomas from -chronical'y infecee~_,mice--relea<e detetabie_- leveis of S!IraS_ --These ar- the ia:nrunortodu!ated lesions ch_arac:erisr_;c of-ihiF infections. Large, intense, +~ntr:od~~ated -_ granulomas obtained from acutely infected mice -did not release SIRS. There is, therefore, a strong association between the presence of SIRS in the serum, -the production I- of_SI17S by lnta=t lesions;_ and tFe-chronic, immunor_iodulated stage of- schistoso?iias:s mansoni. r ~ - i Au.rTY, .,-Freed~r_:_a§,G.i._: ;r.rand Crliz)r. I5. s. i ~ : he Journui of [rnrnur.otrrgy,i35(t):27Eo-277I , 19g~ = ~_- - C7ther st~ppcrf;-Nazionai Science Fcund-at-ion, Nationat-InstituTes of-i-zealtY and- Vvtrans From theDepartmeat of-Pathology and Laboratory !vledicine,=the iPwish=H-ospiti-_ of=-Sc:- - Iouis; the Department of -?athoiogy-= Wash:ngton-4Jr.iversity Schoc,: of 3vYedicin®; S_.- - Lauis _ Veterans Administration Me3iYa Ce: and- Depar+saent of lvlicrobiology, ~ Vanderbilt l?nivers_fty=Scl?nol.of ikiedicino,_Nl3shv-ilhr TN. i -- - i F - _ TWO DIf'FERENT_ PATHWAYS OF INTERFERON Ml`01ATED SUPPRESSION OF ~ AN T iBOBY SECRETION = - responses by B cells than-by-spieen cells=to the-antigen i'iuoresceiriated-Bruce.'iauhe.-rius (FITC-BA). :fowevr,-suap-essi^n of sp!een_ ce:i responses-coutd be- blocked by aduition of ei:hes -2-mz r-captoet::anol. _le:amis3le - or - rm.,neclonal antibo4ies agains the -l£mphokine.-soluble immune sespcnse- suppre-s`cor (SIRS)-, whereas st:ppression of -B-ce-li- responses by IFNa -was--unaffrted Ey- these agents. Each of _these-agents interferes- with SIRS mediated supprezsion of immune Yesponszs. Addition -of T cells t B cell cuiturea stitnulared with FITC-BA did not affect the tcta!-plaaus-_forming cell -response nor the -- extent of suppression by- iFNa, -but it did restore 2-mercaptoethaner sensit=vity -to-- I !FNa-mFdiated suppression. - As few as,-1 X 106 T--cellE were- :ffective-and it was- 1-- necessary to add T_ells within-3-h of addaion-of IFNa to-confe: 2-mercaptr,ethanol = I - 148 ~ Admmsstratson, Interferon st:ppresres_a variety-of in vi.ro immune responses by2 -=^techanism w?:ich- has-notbeen-well defined. 3oth direct supGaession andac;i-var_ion of-suppressor --. cells have-been st;gge;ted-ss possible mec_hsnisrtts os-ihterferon-actior, =In an atten=.,~r tc,- -' exam~te this questiixts=interferon-Q (IFNe)-med:ated suppress;or: of a plaaue forming _ cell response to a T cell independzn-aatigen_by-spleen-cells or by-L}=cPlls-was: examined: Somewhat _greater o,uantities_ of IFNa were reauirezd to sup~Ier plaque Porruir~g cza _

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ser[sitivity=lo IFNL-mediated siaplSressipo.'?'=csc datl suggest that If'i=7a -ca!: zpppress =mr+u.::e -reypoTs-em by= fwc=tiiff-.er.t-pat..wars and _ that- in :c pre3eiica - of celts, _ activation e; aupp-m-sJr T cells is the dominant pat!ewaJ= The pr;.sence of-T cells- m_ u_t- - 3lx°EPrev!;m d:rict _suGDressitjn 6!'-$L'S`t!F hy i2'1V4._ ,F,mne. -T.Y: - is,-aund_the -Depar:m-5E3I,-Patlto:ogy;-Was+!inoto^ Universiis-Schaoict_` iyieditine, St. =_ Fxo.n the DeDartm-etrt of Partbclo;y-and Laborafory Nia ic-ine; The Jewish Hospital of S:.- _ IntrnatEona! louiie3: of f-mmimcp:rarn:arloiogi '<(i ):55-71,19g5. - Other zuDpos: Mlonsanto Company. - -IN:_IB=.rT:Oiv-OF _N:lT Csl'tA4ls.iiUNG &?` ADIENOSIR'f Fc-I?tCiN[}i.f.LC'i;'luES Tti=- effect= of_--nucieotid:s -on riat_=ai k=3ler"-activity i=n mouse spleer-: w y- that ATP acts lWe-tn-ih@ i~itsc~:vcle: possibly IffirFie'!laieip preccding=thE_progra:r!!t!ing fnr ly~sis. _ - - _ _ - _ t4exirreEics of th. ~TV effe;,t wi;h t!~e eifect=_-_of trifiunge~ ~-in- and quinacrine showed - ffect-is tran=n!itt?E_ by purinrgic receotor.',sj iocat}don the-cell sur-faYV. ^bosya: isen of inhiGitson-_ was high 1; ~~c;fiz __-fo: adef!o=ine r-iboFuc;eet:de~; adenosine= deCXyribotti!0zotid¢;,-g~:onos%!1? and "a°_:JSine F3b-on ttcie-Itides [!ad- =Yery =low,=if ar,y= 3;?!iGitory=ef#'ect: ==Ex-geriments-with s3ecifi~, inhibitors si!owea that the activit75 wlth -a 4alf mazi!iial -inni-bitio ; aa ar+_ ATP-cencentra:ion of 2.0 yiW The investigRted. _ Ad":si,= -fiGonuclel-tii:cs were found 10 -strtingly`inhibit natsrrai -killer - -- hnnirtioiogy Let:grs 7:171-17fr; 19g"s. - _ -i,abtFator-y, tf_i=tiersit~of Cali1'ornia, Eterk?:>ry. Oshe.- 3uppor: _ U.-s.-t'uG:icHaaltn Sersice a5d-die National Cancer_Inst?tute. From -th? Depa rtrnen: of-Mi:_crr_.;,ioluty=and- It;munolog"r, _nd-tse _ Cancer Research _ ANT_I-IvlY-2ti: A MONOCLONAL ANT-:BdDY IN1-iWTiNG li';XtA.-33 ffir.E',OCYTlc'c-CHI:MlLiJM?NE$E:)vIYC-' concentrat-ior} range,-faitd ta geneeate=any deeieate in"granulocyte autoi~e:ozescenc. ~ at!tofW-orescence wa,3= affected hy-anti-my-2$. _h addition, ioncmycin, ove- -a wide 3ecretagogaes- e:?3i°:7- or ionop-iy~cin-;cvici!tm iorores.=an+d pi:ari3ol ntyristat_e aEetatc-- =_AQti=My-t(r. in,`-}i-bitior- z{ -Ci..= .ras rese;s.G;e ar:a was dependent on both secretatogue and r=roPoclunal antibody ccncentration. - This intiibitia-nappeared to -ie- siirected at - the----cGmponeat -of- _ gratiul:,iy te -CL --*.hat irde[;e-ndeht -o - i+lAD(P)ii=ozidase.-:.atalyzed -for:natson of- supQroxid,.° -gniom, _Gee3use= ieither =opsen-ized ;y;r:osan=s*.imuiate: -CL aor the --PiLiA-induced decrease f!i' NAI)- (PiH-associated ° - - -_ - - A+~ti-1+~i~y-2E, a mou-sa r?onoce<~:al lgvl ani~d;~, s,as ~ise~ ~A~ifs3_ fiuman grsncrio~;tes- and has -e`xcn 3how~ t'= inhi3ii l~i!~tia~o:-~nhaacedt -glu-iavevendens -_ chemi{urninesc.ehce '#rL}.,f=h»naii-grenu[ocy= ~v==-=,ofGiyt;a) 3es[or!ding to tho-solubie ozygen=coosu!hpt.ion-:=-Furthsrmorg, arti-iVfy-2h was not rytotc;zi-c : and did not -itself-- ~ - The-!l321IS7-::.ducec-CL At:hiGite2 by anti-lyiy-2b was-sofrelat_6 with its_ cpressi-in of _ -- - _ _ _ -_ _ - - -_ I - I 14 9 i I ~

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induce oxidative- met~lis~ when -Azsed -~_=a _-timu:nnt~_ Bindsg_of anti-[:~1~-25 to pnaltocy:Fc-ells =w= not decreased by pzg-e~-we_t~t eE-s cV eltax A23 ~$7 o Evidence -is -Drexnied to st;ggest -L4 ti:_ b,nding of a.._'r=wty _z~ to r~e=_~ afi liv~-yte- _ surf>ce ,n}=Fats=t~,e or~da°.-,.a res~;sY to ~I~u-r ,c pi<,,re t..,~ P'~A by b,~_ commo~patl!waysi sti,~uiatA--.,-b; these dii'~reRisecrPt=gogues. -_ - ~Var=_en,_en, L. T. and C'i~ :. Ca: - - - - 7nelournaio; I~ma~Iog,-t33(3).ig27-lg3S, 1~&5 Other ,~;~port. National ~ti:ytes of i?._alt~ .alt~. From tr? I~~r_mel es ~, and tnr- I.?=F~~iL:B Of =P~!atilc ~.!7CS) =_-sf, a!t= Johns '7J__G'4_~. _s'4~._s Of15T![3 v Ten T' a f~., T- g- ~~r, n-~v~n! l~-.7`_1-CFn s tJniversity School of l+4edirine,2-aii,tsor¢- - A.'i7-tv,.-=2g,^AI e~F~ a_aG~^T~ va.t~sI~-, ~{~- ~~ ;L4( sl m,tLt:~FsL fa,! ?1.00Y, - _ r-- = BINDS TO A-StJG.4R SEQUENCE ;I, E :CTO-N-NEOTE'I~RAGSE - _ t3-nti-My-2s_-i4 an Ig1VFi,.-It 1tonLti"larYi-ap_t-,vou`.' pkvdtced=buy-a _-):yiuiidam3»-=`7re73iefaa from sFlee,a cells of- aa inouse- ismur:-Ledwith _nor-mal- fiumstt graculocy:es. -- By - _ Emn;unolluor9sEerce Et..b3nds- W -_ Ilil'_nai,==.g3'i&lo-_yC s b-3L- ot -_~C - .a!^Iafi~:gs ani:- I'3'inpl;'.Y'yt=s. yfie treatirI& -?.Is_ tie-3ih _me,ia 'Y"tP._dE_s°. O:gi:.ti-,-_c*y-Y_l=o Inired- _ to- lymphocytes and ma.nocy:cs 9 nd to many eukemic telt a nes and F+-atserr Ixa:kcr!,c- y!ast cells. Qnti-MS=2d-binGs ~-se~Arai neut:a,l glycaiigirs_=Pnci_-vessi;i3 daar;gEios des ot leukocyi°s srr± e throcres ~ i!,o--F~c b;~ ~adi;~in.r-.,eooassay and i_rna:=~rncv~Qi>:in~-oi t:ia tayi--c/`~-roi;aSfgFs:s~'`-It Te.:f}gn3_ s=a-siigffr scqL--^ce-in___lac tdT-i-neo_tttr-aosG~ GIc!VAcfi I-3C',a;p-4G!c. 'Fi:i_ tetrasacrt,ai-i+?e occurs ir the giy~l,p~ds pa.aglo~~,de and _--sialosvivarsrioboside, ;a:ii its d_is`tali-t:isaccksridc_ sgquer:ct.in 5ibghei gIylce:iyiRS - and gl;toprot}ins. _ -- Wgcd66i^.a:319-326; i9315. Oi.~-_'?r 3ap,fiier!~- Nati`Jilal=9nstit;:tes oi ifealCh.==-- Frc?n? the National-Institute of Arthritis, Diabetes, arvd!:igs:ive and Kidney Diseases, Bethesda, MD, and the Divisinn-oi--Pediatric Gncology; the Oncology -Center, The Iol:ns Iiopkins;.7niversity Sclsool tii tytedic±pp, _Ba!t'ensore ~~.. - MIXED GLYCOSIDE Pti;~`I"REiyTy;l:N:-REDL?CES M-UNSPtCIT'lC3[t.Te71_fvG OF 1 ' ` A^ TI~~~iE~?*?FI c(:Zri?-=i~T' E SE sf~Ivs .Ifn+d_rect lRtmtEr'ohl~-.u^herl:cal, stF3i17es.Tii__f+o=.e.l_ mdJ-etlss{. - - S+i;r E+5!=1s`sF - - monoclcma# antibodies yi;i".. in- -ge.,°ra!, auboptirnal results ~rirr,ar, y -Fec us~ =o: - indiscriminate_ 3in:iirg--of sgcrsn,isry=-ar.ti3ody- to all mouse- inimunoglcbul;ris, i.e., -tS th- - monoclonat reagent and to Lndogen_ou&_ imtt:unugioi;uiin ocrso-vcifically-`tta5pe3 i:i-rF;e ` - tissue. To rte;,ce_ tl,is-_Eorsp"ific yt;ai;ing,-Prozen_;eccians-ci_ mouse kidney ~ae @--Ereaed- saz,n,atica'lY. Optimal rtt:;lts srereotitained following-a 2 nr t:eaim;erit with 20 mg>anl - of mixesl=-glycosidasen - :-k1G , _ Thi.V_ tTeatmert'._ reduce3- ih°-_eow - pc!,fi:: 3ackground_ - Etalnln°EY-:f tllia Literst5tial sp3cES'3nd SJlood_-E'easels, vil'r_Jio~ not affr:ct the_ reac'd`_vit} of - structnriily-bouat- immunog!-o ol~in-G {igG} ia the g!u,reru!,-os aitEr a:e fe ^tiritv cf` - 150 N- b,

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metise renal tissu?= to t,e ruo :oclonat antiboiy_ ,h~t - reso~n:zeo_ ~.:_ ehQos9urtaridt antize3C det°Z7tinai3T "ElLEaFsflol?t ,...ZV;r°.c tre?ted fe y---.r< _iSEuE_3e,.2?Fis _cCntai?#.d .. 3SCr_3n2!a irrllntieCr~~^~3~%g 1~~~ i9 tS ef CGTI` _f '; -,vn .j. tl;ese E-s.a- inela.st_- that cIG -- treaiment-;jf frCzen S£cZirs coCld-be'afelyuse.~ t6 i<ed u t;rthe c-^te-4 of z.Cristru4.u ra'hP bCl:n(f i:nrnt.'-nfJF,iobu`71s 1 f[oze n-tlSSue4 and -dh1_ :?prCye the-v3..aI23-t.on '-?~ 3p2,".ifC monoclon aI antabody bi ndng. -_ The Journal rf f'iswSzTistry a._d CEtocTie-w_stry x1(`7):b93-~s'g, 1^g5. Ofher suprort: tdat:oral Irstit=ultes of Health. From the Departme-K, of Pathology ad Laro_ii.or; Med_.ns Is_ah~e nnrn iTt::vers~t; SS.hool-of Medi_ rne~ Philadelpfia: L-Cr'ALIZA'i'ION OF: cA.',r' Itk=~~'iC-- ANI3'CA': T RAi.IIE?r.--EFFL'C'?S OF -_- I+-ADRcNER~'iIC AGONISTS Adenosine nosinz 9;5'-cycl"sc ~r2s op~csp~iate jcsLmPj `is belicved :a :riediate the - transport of- pns, witAr; ,n? mu-cous glycopruteirrs in--the= respiratory tract. $cca>ise- _ - I Femiral me 3ssrri' tip- ' g C. tCz.. iS-ue a_ eh, Cr -A M F-ma~-_.:Ct a.ar_y- .- l2='i ch''a?geS .-".~- S;ecil'l; cCII- t;pes; - we- -ada'Sted -sta!kzrd~ imt?uc,Crra uchesr°r~. a `-- 33;et h---~,~~i= ~ exanit:_: -the- - - - - - ~- cellila: :C4a .?~?:07- of tAMP -~;ff' dog 3 nd ca - trflC.~'eae The fl=-2~~-en~rg:c- -'sg0i.~'sS - -_ t!rbLta:i:o an'.:-sop:`=''eronLCI _napas-ed- i'f_ CMYte-ei--epit^eeia3*-ceaskz= - nf dog and cat _tracheae -an3 in -both serous ~ad ,eiu cu~ Dia .d cells et_ :ai tr~.beae. - Epithelial goblet cill-s did not resprnd_ to-P-adrenergic _agonists in either species.= - f'his study provides infvr:natior about the location-of 6-receptors on individ-al cells in the traci±ea that-es-not sNsilable from chemical assays of eit; er cAMP or #=receptors in these _ tissues==Our results support the hypWihcsis thst secretory functionr, in both serous and s mucous submucosal gland cells and ciliatey epithelial cells, but not goblet =Cells, may ___ -- in-valve cyclic AiviP=dependenr_-mec"f.anisms Lazarus, S. C., 1?asba;rn,, C.-B. and £-:3ld, W. M. -_ Am.erisanlour r>% --f ?hy iff[ogy 247 (Cel' Physioiogy IE1:C32',_-C334s-l4g-4.- Other supPort: - National Heart, Lung and E1ood 3n,titute; and the S_trobel- Iv!ed_ical- research F und of-the- A:nerican Lut?g A3sociation of-Sa-n l='ra:cisc!r. - From the Cardiovascular-Researc!:-Insiitule a nd >;epartrnen,s of F.?¢Aicine and Anatomy, .Jniversitv of Califor e_ia,San Frra,:cisco. IMMUNOLOGIC RELEASE OF :IiSI'itnie#dd F I'cAMM_ CC111fPAI~:~~kiT.~F_ - IN VIVO AND I1-tViTi'tdti-FF_°+PONSES dN THE SAMi ANIi.4Ai ;'Ie purpose of tt is study -was tr compare, for-the-first ti_.c, antiggn -induced histanine-release=fror?: the lun& in the same na;ive-y allergic_doxs both ipu-viero and in. _ sivv. In six dogs, maximal antigen-induced histamine-rel-ea.se frosn the lung correlatei- closely in vitro and in vivo (r=@.44), althoug!r it_varied widely between dogs (0V to 75.5% - of totaf tissue:istamine ccn.ent ;=similarly, the at:tigen ;oncentr-atio : to produce _5C°.~-o~ _ maximal_ histarr~inz- release varied sixfo/4-betweep dogs (40 ag7:nl to 1150 Ngi mi): -it.- each - 151-- lie- - v -- 4- - V .

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~ of- five other dogs, -terbutaline ,sulfat- administered intravenously - caused a dose-dePende?it_ inhibitiOrt-Csf aritigCn-ind1eced histamine release rr+;m lung fiagme= ;- ir'~ ~ v1t-a the maximal ir,hibit on_prtiduced by 1 ong/ag was 60 ± 4.5% (*rsean SEM). tn_ ~- these same dogs, I?-3av1 terbutaline _irrubated with turg fragments in vitro caused inhibitioo- of antigen-induce_d_ histamine releazP _compa,ao,e to _ I mg ; kg -te_ sutaline vivo. lncreasing the dose of terbutaline- in vitro produced maximal inhibition at ItJ-`TA ( with no greater effect of the drug at !0-"M-(71.5±3.8% inhibition): IF both experimental r situations propranolol Faused=a dose-dependent inhibitio n of fi-adrenergic modulation of .i3CariS-ind4;ced release of histamine.. This res7Li su_i3ports the conclusion that terbutaline produced its effects by-acti,rrs mediated by P-adren?rgic 3ecept:;rs on pulmonary mast cells. This experimental approach provides a sEitable preparation in t which to estimate the effective dose of agnnists~ that modulate antigen-induced mast celi j function in vivo. Frey, M. 1., Chesrown, S: F-, Re?d, B. R_, Greenspon, L. W., Shields, R. L-_, Bourfe; R.,-Brorvn, J. i;., and s;ofd. W. M. ~ _ . " ~.^ Clinical ar?3;~rso,ogy-i4(3j.:-S-.c~, 1984 = Other support: U. S. Public Health Service Pulmonary Specialized Cerrter of Research E`rom_the Cardiovascular Research Institute and the Division of Clinicai Fharmxcolegy. Department of Medicine, University of Cslifornia, -S3n Francisco. -_ i iJ{TRACELi.,;31 AR- MATItM b1IP'RGF1ERftS ARE -C?u?nS£T?fi,F CORE ~ -F'FOT_EIh-St,.'CFATED W1Tl:FIBii`{fll_VECT-IN I ExtraceHuiar proteins -af cultured calf aortic srirooth muscle cells - consist 4 -predominantly of -rnicrofibril3 1ii-23 nm in diam.eter-_ typisal= of "elastin -associated'- __- ~ microfibrils described in -many- tissues. C.hers]icai_ -and imrr.unoc :emicai evidence is i -presented that rn'scrof-ibrils consist of at teas_ two_proteias: core protein and fibrOneCtln Insoluble-proteins ol`t:`:e mic-rofibrils were obtained in t'ir fbrr: a~ellgt-and-zrriibodies _ ~ raised in rabbits againsE_3hzze- ccmponen*_s. The antiseFa-reoi'acted witi±-_tfie=inso'--uble __ i L*_icrc.fibr'il!ar proteins- and -w th soluble fib?on ecti^ in enayr-efnked ir--nu5osorbent assay, ana -imsnunostained-. ne c_xtaacell,iiar .-microfibrits iF - c i'tured _ cellg._ An__ I imtttanoglpbulir: (ig), i'raction was prepar-ed and a#a ur_bed with fibn?nec:in. T heff-absorbcd =IgG retained its reactivity wit~i-the micrcfibrialar protein; butyvas no-i<_nger reacti,e= ~ with- soluble fibronectin. _,:nrmusoflucrescence studies -.vere carried out--usi5g the absorbed IgG and_IgG to soluble fibronectin. Gyth-ar.iibodies s-fsO-wed immunoreacnve - -niicrofibrils in the extr~cellular- matr,w of-Cells it Log phage.-:9oweve , with 3ncrea lnp ~ time in cul:ure, as the cells reached confluence, the immrnofiuoresrence of -rr;icrof ibrils - reacting with the_absorbed `gG became lesc intense, whereas that of r::icrofibr:!s reacting- ~_ _ with !gG to fibrcr.ectin is cre sed; in czii luent cells, esseirtially r_3 stairsing-was detected witF_e the absorbed :gG, and a dense network of intensety stained micrtzfibrils- was -seetr- i with FgG to fibronectin: Trzatsnent of these cultirr=s-with urea led to f:artial dissociation of the fib3onectirancf increased visualiza;io m. ot the m-'ir-rofibr ii3 w;h the absorbey -'.gir - doubis-iabei im,..uFofluorescence showed that both p:oteirs -occur_ed ;,n~- the- same- microfiHrils, i he lo:aliratiorr of i:lmunoreactive sites to the -extracellular microfibrils was confir-rned by imrnunoelectror: micros_cony. *:ea1; ye:a-ytrtati°:e cleavage with CN;?r failed-to- dissociate the antigenieally active_f_ragments oE' fibronec~in from = the Cb1Rr- fragments of the core proteins of the :nicrofibrils.= It was concluded that microfibrils _ I contain core aroteins-and fibropectin t't.ai-are codistributed i.n insoiub-le, possibly cov_ a- f I -15< = _j r~. ~

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iently ct`oss-linked; agaPegates.-'I`he core proteins are first deposited sly the-cell and, as w £iticfion of- time in eii*ufe, fihronectin gradmIly cc,am -t,tieir surr`ace. ~,.=~iv~aru, E ; ~ :~#_fisctter. S.. Coltolf ~~~i!le:: B:j a~d ~?t.~cY~:~ C- 0 - `=-. -.T-he .f'olern.G'j-of aitd=Cswhen:ii'rY s3(Q):3tf° ?747. =1995. -&.e Elumenfeld =Ea:niiy MeniorYal Eur:d.s: _ Other st<pyvslNatiataal=Irstitutesof Health ans3 t-he I3Evid -Orxhinsky-i:enry Segal and Medicine, The Brortv..'Y. _ From -the DeFartbieot3- o£==Rixi-tert:istry -3n3 Patholosy~ -A!hert_ rinstein -College of - IN4,%(3JIvE1.!'w;ICINYiBI-i.t<)?? °2F1_1LT-cZS? I(JLET F:ADIra:IOI'?-IN-D;,T-.ED TUMOR SLiPPRESfi:?k CELL A'y : IVI= :. (E-JK;_blis not Att? .132 splenocytes. 'rhvse £ilidir,gss provide=evidence tha° eienseFts . _ E--eli5. T;1sP3-actlv,ty of thi ^.onCri.lt3t1al arn5bwas ei~`^lnatgd by-aC:3CorptF-yL o-'_l. Bht<BR -= or-lo~+ doss oi "Cyclop-ht'~sfl:.af:-:~e-i13F-liYp ;n: ib.ed- t.-~--e=d°c:e.lopTiei2t or activity Cf -t-hes-: - antibodiea to the produia othe I~: sA-regio : of the Ms,or ltistocorapatasrlity- comp!yz a: a-c-lass before ;l<e appearaace-o£ overt tumars. Admarlistratiou of monoclonal _ - f9i"`suppre3sivr=l`-c`.ell3 -lt'is:3=_mcodhi`se ultiaviolet--radiatio:_e-i-.larcer--regreb5or-;kin- a.ancers -- Long-term exposure of C-31i -mice to olraviolet radiation resulted irn the formation their pathogenesis. - ;laerapeUtic akproaches-to tnaligriarGS'es-or-sther disease3iyv~king=stappressor-7- cells iy overt dt;ri~ovrae-nt ai ilitraviole: .3d3atlC'-enducd- skin and 3uggest -`.1o's=e! expressing the I-J tetzrmirtanrare i;3aportant in regulating ?he host response prior fo zhe _ _ _ - - - - - - ~ - - Fouadat~ or.- ~or. - - - - Oher sypp6rr;=-Ri_aisomat Resea=ch Service =Aaard and Arthur G: anI-Guliaa is:: N-llraan Science 214-615-<17,19-84, _ C3ran3?~ir.-Ii':~L~=> Pa=ri~F, i A,?>:cAuEi££e, I3~-1.; `~'altf~bau~h, E'., ttrd= :re?re~=+3.-I. Imrinutioiogv_ NdtFhwestera-Jrtiv rssity !vte#icn3 &_ :oo1,=C-hisago.`= Massa, huseis Genetai Hosvit-at, Ilsmar, and =- Deparanent =- oi = Micrnriolcgy a:,d --_ E`=Lan=-the-=L-epartirents; of Dermatology and =Pati:clcgy Ha-rvard-Meaacalz ~,.~hool and -Thtis, the-skir:'can bconsidered-a corrs;4ea b_gan-in whichcelbs reteYaTt tc-the :nanune iunctionallYvery similar:or identicai tG inter:eukin I iIL !-3,_is prEduced--hy keratinocytes. _ dAndiitic cells- called= epide-mai Langerhans ce9!s- (i~C). AdditionalLy, epiderrrial ceFl-derived, =hycnacyte-aetivat-ir;s -factor (i:TAP:- aa- entity = beochemi^aliy- and lymp:~oid - tissues- (SA-L'I`)= - Immunolo`ir -=E tFCctions of titese elements -=have - been demoi<stratPd te° itrcliade processing- and- presentation of -antifterr to lymphocytes bY actlYe eie:nents °resiri4nt .n -the skFn, whac!a has led to t,~e concept of_ sxzn-assoc:ated In recent-years- Rvidebce=Ras--ascumulatwd=fo- the-presenc1t=- itnnrjr:o!Ra°sa-;y - EPIDERMA-L jk-N7IiFEN-PZESEIs+-i=Ni CELLS IN °k` CTI?`A3`ION OF SUPPCZES5it7?`. iDEtv?`IFICP TIC:V OF A!tiEw- FJ-:VC7`ICMAL TYPE OF __ ULTTcAr;Gi.ET ;e-ADI.Ac;i01-'-RE3Ih.A1ti'i EPiL'ER?~f:AL= CELL system are _representev. l53

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Fxpos_ure-of mice to,-!ow doses of ultrayiu:et radiation_`I<TVRj in i<ito leads -to- a;~ -- inabiiity to sensitize them to contact sensitizing reagcqts ai the siie_;rf irradia,iofi, gn d- administered subcutaneously, and such i^tntunization results in the appearance of . _ suppressor cells. -We report that-the mt!ri-ae epider-:niacontains a previtusly unrecognized antigen=Presenting cel7 (f.i~C) that is requi-ed for :he_aci:v,t-ion of suppressio:e;- and that this APC is resistant to IJVR. The dose of UVR employed is, however, sufficicnt to prevent substantial positive i:+t*_rur.ization of - mice with svngenwic- UV=irradiated hapten-coupled EC. -'T'i hcse _ data- ffplaitt-in -part-_the-changes induced in - Fp idermal aniigert-presenting-functio:+ by UVR, and ha..s co.n.sequPnces- for the understanding- of UVR-induced cutaneous carcinogenesis. (EC) from i3VR-trc~ted mice--are .^.able immunize -mice -eff:cientiy when _ ths depressed sensitization is accompanicd by _ the-formation -of ar_tigeII-speci::e _ T_ suppressor { T s) eelis. - It==,`:_as_ also_ been demor.strated that haYtcn-co,p!ed _epidrmal cells =_ Grar.steir., R. D.,-Lawy, A:, and Oree:te. M.-f. The Journal of frn:u.-alogy a33(s):553-50`, 1925. Other suppor_: National Eye Instituto,-National-Institutes of Health and Arthir-O. a-ni` Gullan M. Wel3ma-n Founda:-ion. From the Departments cf _ ivermato:ogy and Pathoicgy, Harvar-d Medical School, and Massachusetts General Hospital, Boston. - SP"LEI-1IC i-i_REARING aNTIGN-PRF'SENS'INd'6F.`L-._--_~ IN A'I_VA_TIU?Y= imrnunize for a DTH resporse.-It was also fou .d that the previously desc:_ebed sp:enic-_ I-J=bearing AriC needed-_for third-order s_uppressor -cell ('I's3, effector=suppressor cell) activation is adherent and_i3-VR resistant. The sets of I-JI-beari.ng APC appear to be _ crucial lements in the activation-of- srppression ahd- thus in- deter Funi.,g the b3lance between immbnologic_reactivity and urresk=nsiver.ess.ess. FurthermVre, the--UVR resistance of this set of novel APC may be relevant to the in vira effects of UVR exposure ac mice. A set of I-r-bear'rng-murin3 spienic antigen-presenting cells (APC) has been found to be-res.ynslbie for :irst order suppressor cell (TsF, afferent suppressor-ce1l) activarion in .he_ azobenzenearsoaate- (ABA) hapten system after -intraven©us administration. - Suppressor _ceHs induced by this set -of hapten=coup:ed cells do- -not function in the- cff_ereni phase of?_te-de!sysd hypersgns-tivit; (DTH) ::=_t}_vr4-e_ The fur,ctional ae:ivizy- o_f this novel APC to activate afferent suppressor cells was- ri:sistant to a dose of ultraviolet radiation %UVR) su.`ficien to largely abrogat< the ability_of-s,ienic APC ?o- OF SUPPRESSION: -1:=URTHFR CHA,.RACTirUIZr.TIGP: Granstein, R. I?-and Greerre, M. i, C9-fl1fe:-lr,irsrunology-91:i?-i0,-1995. Other srEpport: Arthur (9: aad G;a:an lvi. Wellman-Foundation. trom the Departments of 1--w._,atalogy_ and--Patholegy,_ I-Iar v-ard-_ Medical r;.hool ar;d_- Massachusetts General Hosr:tal, Bosto. . - 154- -

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MJNOC{.GNRir IClOi'OPE\%Aifil?,'r_A=aIfi`-YT-S-?ER?'?COCCEJS _ -- - - P1k!EUisfOiYlAt -INFt=.f'I'iOta=°= A monoclvnzVani-idiotope- ar.abody-_ecuQie-d- to -a arFi -t'i.otem wac-ised -to ir3i31:3n1ie BAi.Bjc ffix-_againit a.-ietl?al-.,}frepioC,.^Ce`7[s- y5. £tlrflotilae inief&iofl - Vaccinated mice-deselopeG, a hish fi er of antibody-to=phosphory-i_hs ;_r,e,-whach is : now. to pro%ec agaitrt-infeeti~?a with_ ~t:-epl~n -rr~s-p~ceu~ro:r:~,e. fv1_asE er°e :t of-the madia .- ies~a- dose of the- bacteria-indicated that anti-idictope immuqization signifis,ayti;-= i73creased -the resistance af- B.llB; c_mi^e_ t© hg-ba_ctFr al challenge. f~rti~dy-to a~ id_it~tc-~=_kafi thus be used as an antigen sub,titute-for the induction of prztPc.i~e imn:=un-;ty. - McNamara, -M K., Ward--R. F -ae.E Kohier. #r = _ S-cIeRCc2-26:1324-e$l16; M .4. --= ---'- ?;t.3.-s4ppaFt: _-Ls.S._Ftib!iA_ d-tFa;rr,-SerE'i~ and tee'rdev: York .,,ate De;:arttner- o= Health AIDS Institute.- ® a Froin_ the 3Y..par tmen- of Mo_ lecular Immunology, Roswel. _ Park Memor ial In=_titute, _ Eefalo, NY. ANALYSIS OF A 'f'$t ° T~? Id_I P; ;t ! E`s'~ f!RCi;T ia thr _gresen-_.st-iid;,=-tl:e =_'_15 idiotype-rfco='1" _fzlpez cell circuit - sas dissected with respest -tc its_ iiomeostasi inter' ti4e s,e£ificlt; Staaslity over time at d effects o:. B cell expressiou. Analysis of *he :y_ yelis by adoptive transfer ezperiEn.nts indicates their sisr IK ed state-$t activrty 4ur~rg ~fi i~2 ~Is are--s;inulaaed. ;.i call- a4-tiV:iy w.,, _a1sc, d1reWy mo;.it.,red, -Gy the use of '?'-1<1?-anti-i-r5 hyFrida.:a- antigetis: was, iq"d that_ TH~° ceaG-are_ deie:ted 'wk a*te' prir..=ng- :+itl=-_P%--ry, wj:erea;-Ti:; cel:5-~ieCGIFe activated after_~=wk G' pr6rriiiSg_C_ofilp2,aiisie analysiS.--~f iHi cells tiy ysino-tw d:ft=erent T'Ny-anti=.:"1y hybr-idarna arti¢en irdn.ates> ''F'~, 3pecif ici*y =- _ _ for-= a -~Elare(~ id!otops - _- T~i$ ital~iilit~f -oYer._?iE. e-_ of_ trie`,-T,,- _ 2T-- . - -£irsu°- was demonstrated by_ com-.a_ ing I'u2 _frec7uencies Iuyouung ar_d olamic: F-sz.ally. we addressed iht- -question of the function of =the tdiotyy-e-r_er.-o~:3Y-in~- T helper cels a,.~--=shovsed --that stirnuiat; o., of i~ii-dot,pe=specfit--~'k2 -ce~ls-_c~.}-ir correlated w'sih -a signifi,.art_ increase- in the pa=ce_r_tage af _:'13 idiotype rn=-art atiti-PC response. ~ollect:vety=,_thse data 3escri3e arr idiatY;,e- peific :_h_eiper cia_=uit a_s Fart of the nt;twor.'`._-b=si3 o.=.1e-IrninCS'_sP SySIe°*`.-.= MeNamara, M., Iz;ang, C.-Y., e nd Kohlcr E: _ The iourna[ oj_irrtmi-molosy 133(3):1603-loCy, !v>:3. _ O:ker_s wor tiatic -fial iastit te orrksMr: From ihe_ Department of Molecular Immunology. Roswell Park-_Me_morial Institute, RuffaloNY.- 155-

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IIN~~'a~lU?YOFrAr'rrV=i`--CA.LC!-TOlslgl=.-GEItiE=it-I:LAT'EC PEPTIDE AND SUBSTANCE P COEXIST -Fai Sr'yScaS. ;S.; !~vi~KQ~1' ~" z [h i ~ ~ 3_;~ THE ~~_ ~1. C~, i; AND (IdTYRAri`T. iN cSPiNAL :EHAYI{%Y.AL. F.eSVi;J3ES-OF THE PAT comt:nation. Thus: S?=(Lp=to 20`Ag) a_or,e-cajsed ' Ba only for a few ninutes after injectior,,-w!rereas SOR-.'I0 pg" v.us CGKr (20 {sgi-caused-a reepo nse witl-~ a=Aturation up to 40 min. CGRF _(20 rg) alone had no effects in this model. These findings protiide sup; ort for aposs=ble _nferF~;,tior of- the- two pe;,lide._ a`r s_ynypses __. :hs dursal hc: n of the spinal cord. behavior was -carried- out- aftes- in-trathecal- administration ef-C`rF ? and SP a!cr_e° or in spinal :sensory neurons. =Ar__akvsis of caudall=y-_diPected-biting--and scratching caicitoniL gene-related pFp,ide (i,G_-=F)= and substance P -(Sr )-lihe iri:mu-oreaaivitf in Jsing i:arnurlhisto=rerrdst=y, = e~~nee - was- oht?rired= -f:yr -:he _oex istenc.e of= WiesenCeld-Hallin, Z.. Ho{fe!t, T., Lundberg, ;. M., For=smann, W. G, Beinecke, M., Tshoirp,: F. A, and- zixher~ j_ A. _ - - - - . - -_ - -- _ - 1Veurssc:cnce Le-lers 5Z:199-zo4,i4&4: - _ f3wher sxppcr:: The-Swedish'~:,,dicai Re,sars.h Council, the Fol-ksam Insurance Gompany, the ICarelinska Institute, anc thr Swiss_National Sciercr Foundation. -From the Department of tr"ical z,e4rophg'siolnoy,-I-lu:?d_nge :?ospital;-L=epar=t;ier!ts of = Histol-ogy and Pba=mlacoiog.,-Karolinslr.a Institu°e, gtoc!clr:fm Oe9artment af An=tor,a III, ilniYefsir_y -of Heidei*±rg,= -;-=ederal I`epubtic of _ i=:erniany, ard- the Research - L Swrato'y ifr Calcium ~4t35oEis.-=; ars__ g-v" isrt - f~ _:rcr~i_ .- hoped;c Surgery and Medicine. IJniversity-e:-=[°:4rich, Switzrriand.- BIOSYNTIIESIS Ol`''iiLYGt'a'I-ii-NiGOi.-Il'-II34-;~=i3Y-IiLJP.3AN lvfirELO!I; _ -LEiJKLMfA,C£E,.Lg synthes~ed hy t§ree-hvman-tn;vioi; 9eukerai~cell-Iine=, K562-, KG 1, and IiL=~, whicb were -rnei,hOli~~lly _ I-ahe:Py---witn= i'~~;gai,ctose t,- ~~aluat~ sh~~ges in neutral glycospringolrp: -syntnes-is <~+rt:, myelosd celi- differe,~tiatiorr. ,=IndFvldua! - neutrai-, glycosphingolipids containing ene= tR four suga=rs -ware-pu. ffied. hy=3-i<om-tsination olf the- - foaowing _m--thods. ° dieth_;~)ar:lt-L:echy!=Seph~dex- crtl_n3r.- ch:oma:ography: aceiyia?ion=_' - 1 tor~=il=coiumn chromatooiaphy= and =Figh-perfo=marcee fiquid ciiro_r.3atebraphy Acsin& ai Iatrobeact ccylumn. -_!'ompounds ~!~+ith -or_e° -sug2r were analyzed i,y_ thir,-layer chromatography on horate 3lates- This analysis showed *.hat_ HL-40 cells synthesize only glucosylceramide, -- wl3ereas ° K-S5: and ` K-G I cells synthAsi?e predominantly gluccsy!cera.ide,-buc also a smaiI amount of ga!actosyiceramide, Compounds with two to four sugars were- thara=rcrized by--treatm-tw with exo- and endoglycosi=la.es. The- _re3alt3 showed ii3'-c K5C2aSlt. Kvi ellr are .._niilar to cells -from patients 4ith acute leuke mia in ezpressing two-3e=-ies (globo a-d`neolacro) of - natural-glycus,~)hirigoiipids,- - whereas the-1=IL-SG cells are similar to Ma-ture humarn myelcid ce!ls-iF expressing only _ one series (neolacto). Therefore, huinart myel,id leukemia cells blocked at different _ stages of diffPrentiation vary in their ability to synthesize neutral g!ycosph; nbaiipi-i'-s: Buehler, J., Qwan, E., ;3eGregor ia, M. W., and M_ acher, B. A. - - Bioc-ke .r-:asiry 22-F9)g=i59g4, 1983. We have performed -_ccimbar>ti-ve studies= of _tke- neutral= glycosphingolioids= 156

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Z'ther sxpport: -T he :.ouis R. L.:rie Epundation-and-tlee National i;ancer_lnstitu-te. 1Fror__ the-Cancer Rese -bh _Institl<*e 3d_ F'paatr:w.,tef -I'ha ink --e-,tica. ihemi~;tr_x.-_ - U ~lYercity of ~allf,r?S3, -t'7a_'-1c3sco; Ci:;hdret'is CanceS-ge5earcy-,*nsatu$e, I'aclfls'. Frtsbyterlan 1`-&Adacal Center, Sar Francasco, REC 0G/'ATiO~_O-r---;-? -. JI°Ltl`(KLh CsALAC"t>SE-at,S1DLIyJ - HUMAN N~TU~AL .v,i`TI-a=i,ALACT'JS~'- Ig(r-II._ THE Sl';<-CIFlC = Galili, IJ.; rhacher. B. 4., Buehler, J.,-aui Shohetf S. B. Journaldf Experir.tente! Y-euiciae 1'3:57"s-5-92, 1985. OOther Suppoit: National Institutes of Health:- iurra-n B-actEve glycolipid, -indicating-that TuLose-linked al - 2-to the penultir:;ate __ golactose prevents_an_ti-Ga: binding. "fhe-anti-Gal=specifici-ty__fc~ RRBC glycolipids also paralleled that of th, a-galactosyl-s:ecific Eandeiroea si.rp7iciJolid lectin. The p_ossible- reaSons-for :1l@ oc&ur_rence_r'_t thi5-unlque astiisody-it's-Ia3ma :3eru.7i a.e disc=a3se~ - -_ with terrainal-a-galactcsyl resi-d+iee-were tested !'o_r- antibody binding. -The antibody - specificsHybound-to glycc~'-ipids svith Galai =~-? iertninal res:jues, and trenmerlt of thestr = glycolipicds with a-gaiastosidase-abc1_isht:d bindi ng.- Tlemagg(u=i na:i .-i inhibition ctudies with ohigr,sacchar ides of-kr_..wn-stsucture also showed that.`F;e antibody binds specificaity to glycocon jugates with atr crl-- 3 ierminal galactose rgsidue. - A-t.ti Gal did not bind _to-a = residues. Two types (glycosidic linxages of i-. 3 vs. 1-f 4) of rabbit red- cells giycolipids - i*lteracts with rabrbit red blood cell (1t-"Cj glycolipids with a-ga;actosyl terminal -- its affinity for normal- and pathological senescent human red cells, the_ antibody readily purified by affinity chromatography o n a column of melibiose-Sepharose. In addition to found in: large amounts {0.3-LC% o<f serum IgG) in all iridfividuals_tested. ]t has been -- - A natidral- lgG.w 3?it,boCy raittl-cialj with 3-g3lactOsyl bi(fding specificity has been From -the MacP+f-i11an-Ca-rgila Hematology- Research Laboratory, Cancer -Research ]nstitute, Departments - of _Pharnlaceutical -Cheirlis:;y-_anu Laboratory- Medicieee, University of-Catifornia, San Francisco. - Fuc lzl - 2GaI,61 - 4(Fuccl _-a +)G1cNAc,YeipectivelY.-In eddition, three nPw types of _ that carry the X aatigei cad others the ~arltigen Galrl 3(Fucc~l - 31G1cNAc an~ -- - S 3GalP@ - 4G:cNAcfl1 -->3Gal ,4! - 4GIc1N1:Rcfl -- 3Ga'-#11 - 4-G1c=Cer.-Scveral species of aeutral glycosphingdlipids witrz six to more tltat; ten monosaccharides were detected antibody Den and chrpmatographed as-the cerair-ide cwtasacchar;det (Ga1ftl-~ 4Glct.lAcfil deffenn; rea_ctior__ patterns with humar_leucocy3es_were tested 's_n_chrort?atogram binding assays -for- reactions with _rrlyeloid- - cell -4lycolipiss der ived- froT- nor;nal- huniaa granulocytes a,d_ chroaic =rlyalogenous feu-ke^nia cells. = Antigenici-ties- were found= exclusively on minor glycolipids which were ba-re-iy-y or not at all detectable with orcinol-sulphu ac acid stain. Among these,-y neutral glycos;,hingoiipid bound thg anti=i - X-or Y, and- seven anti-;nyeioid -artibodiy; (deterrr:inan?; °unknown) selected--for their- GL'fC.'1.SFHINGGL1PlI5 CAIti:IEI'til OF CARBOHYDRATE A NTIGi;NS OF HUMAN 119YELOID CgLLj 1t-EC:OGNd-E,ED B-Y MONLiCLONrEL Ai-4T'iSOpIi=.S Sar.-monoclo_-^.al ar,tibodies with irnc~wn sae-;ficities-for thi_carbonydrate antigens i, ~._ ~ ~

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1 _ - carbohydrate specificities rrer°_dptrsted among the mye:oid cell_glycoiipids.-_'fwo wtre- asSoCiated with Celltral= $lsC1oiipid,E.-. the -firvs_ reca')g ni_ed 23y- anti--~_.'-}'eltid aittil!~!,Cs_ _-- zIf.~f-1 and YIPvi-1C. was e.lres-sed c. - a~stinct set- of =giybalazis with six 4wr - -more monosaccharides, and the sRcand, raco gn-ized hy-VIM=9. =was- ?xpresseA-On gf.colip:dE ---- veith mdre= than ten monosaccharides. = The ihirQ =_pectfic;ty, recognized - by -t;-w- -- apti-nzyeoid antibody V1M-2, was exprev?d on sEow- ri:igrati-g s;alygly<nE:ri s with back-bone structures of the p<;ly-: -acetyilactcsarnine type that are sur eptibie to degradation with endo-f'-gal$ce;sidase: 'I`hus. we con-clude th,t- the i and Y - antigens - occur a~r.g tire gl;colip -ds- of-'cnrma3=:£:ysloidand chroaic mvFaog°r-ous eeijkemia crlls_ end tilit= a high proportion of .hibraa`?-tlIlods'_¢=rassed ==3g'irl;2 - dif:erentia2ion- antigens ea myeloid cells are dibPcted at carbohydrate st:ucrurea. Uemura, 1C.; :tilaches B. it.-,'Def,re;goreo, M., Scudder, -1',-Eiehler__ J-.,-kfiapY; Waad _- - Feizi, T. BtccF:z:c3 ef ifOphysr?a .4 cPL 946:2~`-36, 8983..- O?hersuppvcrt:=-National Caater_I-nstitn_te,l;7orth-At_?antic Treaty 1G==gari3aticn- and Loui4 - R, LLrie FoUnzatinn. From the Applied Imnmunoc,3e?riistry- 12esearch t=sroup;= Division -ocr`- Communicable Diseases, Mec;icatResearch [_'iou :cii's Clirticai Research Center, Middlesex (:jr.K.=1;-Cancer Research lnstitute~---De-Par=rseht of-_ I-'harrnaycu :cal Lherr~i_s=sy, U?iiversity e1= Cali?or ;ia,- .ciaw - Francisco; -Children=s=--Ca= cer-= Res-earch_ ?s§:it'ety;-~ haCif #c- '1liedicai -=Ces.ter; and ° _ Department nf- Internal N4e1ici-rss;- -Institute of Imr:ru"l_ogv;° U-1nrve_rs;ty os= "ienna.= A-ztstria. .: _ fN _ 1'r`J'RO TCELL-~MECIA T ED KILLING OF FSEUUO7SlON-4S AERUO:jVOE,4 1. EVIDENCE 'f3-I4T-A L Y-APiiOK1NE-A;iDIATt;S t=ILL:A?G- - Irrevious-studies -#rave -deroonstra-red-in rlvo that T ceI°s can provide -peotective immunity, in 31he absence of ar.tibo¢y, against = ihfection_-with the= extraceliu!ar Gram-negativi-bact£rium -Im aureot3pF i-(/T-1? fse's~tr~r.~~~.evuor:rso<- :Ye_ estaylicl±ed -_- gn in ritrfi system io-wlri;h, irr:rsune TT cells, afw _ree xt;osrire _ to bacteial--an:igens- and 3o- ~sacropfi3gcs; secrete a product that -iciils the tacteria.- Although macrophages are_ ie4uired for in riiro -kili:ng, they funcfi~n- nsither as -antigen=presenting- nor phagocytic cells in this syste: e. T cells frorn animals ic3muniEed aga-i.st a different_ P.= oerugir:osa im:.runotypr wil` not -kill IT_-I vrgxnisms, but the supernat3ns produced by -i'f-1 irn-h?une T cells aftei exposure to macrophages arid IT-1- P. aerugino-sa organiams are nor,ssecifi:ally effective-7riil;g g un:eiated-bz--ceria=.--Eecauselhe-;operna:artsAf r:im immune T cells lose their bac.e_ricidai-p;roperties-upcn-rninimaJdilution, we conclude that if this- mchanism i=_ active in vrro; it w-,st-plag a:o:R ir-local immuzmty. _- -_-- -- 4;.:rkhars, R. B., Goellner. J>, -and Pier, r. B. -_ - The Journal of imrrtu.nclogu-133i?j:952-96g,=19g4. Other suppori: -&uxiliary of *he Je-Aish=1:ospita!-af St.-Lc;uis; U.S. Pibl _ I:ealtF- Seevice; Natron al inst:tttes of Hea'tl:L a-d U . S. Army Research and Deveopr°tnc Co-a m=.nd: From the=-Departanents of =Medicine-, and Microbiology ~4mmunology,- Washington Universitv Schpol of :aiedicine,-_arEd The Jewis?r--1-ICs;ital of St, Louis; t'hartning-- Laboratory; Harvard Medical School; and- Rrigham-Qasl W5=.3en's Hospital, Boston. - ._ -

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ME_ziBRAN-E ANLr-Q-yTOSK tf.Et ;L CFIANGES=ASStX'f.4TED;VITE1 fgt_-ME t:A?'Ei3 ER.7 G:+:#i<; REiEAe .`'pOM RAF BASOP3#t If ' E4tK;rNHA CLL4 ° B'nrt:~g_ of antiger; to_ I3E rece>?u crmp;Fes o?r hs surEacL -0f :a+ hasophilic `,eukemaa i;et15 rs the firstevient leading.tn,the release _of_=ce_l!ular serotofiin, -_ hrstarr,ene and s he* mediators o'_` al_lerg c,= asthmatic and :nflamrnatorv respca_ses. We have used d:n cr~phenu~-c+5jagared bovine serum albumi----fI3NP-BSP; as- Wes as -the fiuorescent ant $en: I333i -B-pflycoe-yt`irin, and the -- elec,ron-densc - ar.tiue !, __ DIVI'-;3$a-gb!d =to inves .gas dyriarr::._ rcmbrane and cyte3keletal- everatl3:_associated with tis relea - oC [3rlaerdtorln frc:;° an .:-_15 NP-;gE-prirr:ed RBir-2!-Is cells. - These ° - multivalent arlt!gerls -bind raSSgl- to- cell surface igF--ie_e_nti=e- complexes. T heir -_ di3tribytion is initial!y- uniforra, but within 2 tnih 1Lle_P-:3S_A-_ golc_ is found in cs±ated- pit+ and is sursequentay en_,.rnali?ed. Ant e_.-intPrna-.izat.op occurs in the presence and --=absence of ebtrarellUlar i.a_3" The F-actin cy: te?St Gf--t: eActergent-`Atra.26d ce[ matrice= analyzed by SDS ;'A_GE decreases-durine the first !t5-30 s of antigen binding and then incea5eg by :.. Islin to almost 7.oub!e the control !e=els. . A rata:d -an d sustained increase is aisc- observed when total -_ _ae:;n is-_ nuantifeeE - by fisw cyip-lrcetiy af ter bindiag of fF-od~ ~~ Ei ~ha#!oi^.in. The a£ 1igen-u5muiat_d-iecrease in F acti:: coincides with (and rr£ay-cause; the traesformation tf-the ce!1 surface from a finely rnicrovilious to a high:y folded -or plieat§d- topography. Other ear!r niefibrane__ resporsss--_ incfude inccrase3 cell spreading and- a_ 2?--fold increase zi the urta_ke- of fb oresce;: -d~ttrv :-5y f!uid pirpdytosis. The -surface and _ F act;ti -changes shn -_-e saane=-deper+_dence sn M -bIP-prote:n eorcensa* - d as stimulated 13H] -serotoa's_ t re Ya3e; and botn t1=-tree~rane - respoes=es , and tire . release of -rtedia ors -a_re terrnina.ed , by the adcition_.. -of the ° a CS-cr~~=linklng ru;;.'mva!e31t ligar_d- DN.r' iysfne_. ~hese data in4'ic.`:tet e- the saine- _ antiger-stimula!ed transcucti%~g pat~_way- i.cYntrols -boti tree- nembran~'c_;toske!etal-_ ar;d - - e;.retory --- eve?Ity. - ---However, - t?3'e -Sle:n2rane and - -actin = respotESe; fo IgE-racgytor-cros;-!ar.ki-ng _are :ndeQe-ndent -of eztacel!u!ar -Ca-' and xry=--in:icked by = - pkorbor- iiyristzte acetate,_ whereas !ieand-depend-ent rSediEtn, °e!_3se depends or ext:ace!lular -Ca'` ar'.d-s rniinesked by t! aCa'* io:.oph3re A231 g' i':'eifl'er, ;=tilSe graivet_ CY,_ Davis, BH., Deant°+. G_-:r., nnd C11i er 3: Sf - Ts~ of', Other sup part. Am fioan CaF:.r gocrty and N-t-tional Irwtitutes-o_ =#AaWrl. Fi0111--the f3epa°tment Cf Pathoiogy, i.tn.v-siy of-=New :'fex;coSs.l:oo1 _ of -MedicinF, - !tibucue qye, ard tne ~parsnent_of _Patr~ o:cgy, State-~'nivers:tyof New -~'ork; = EJp.tate #~edical f'ente S;racuse: _ riSTRI-rsli"Ifl!s.r Gt= ~!~~.!}3~TIN ?F.%rMUNt;R1A:. IVIT~' _I~=~ Tfi£ L lrS!~I!?A°ORY_ -TRAC"f` OF-P#G, GUINEA PIG, I~cA Tj AND iu' Galanin, a-new!y di3covered-peptide isoiated _rom porcine intes:ine, i~-i;nowrr tri cause- ccntract;er, in rai smooth Tuscle preparati6ns and to induce -hyperglycemia in =_ dsgs: By -the use- of _rad-ioimtnunoassay and- imrnunsSisicchemica!- techniques -the concentration and distriburion r-,f-gala..`?in ;mnwr.o_reavtivit_y---were determined in-severa- areas of the respiratory .r3ct s,f ~ive doos, five guiRea-pigs, five rats; and=two pigsz. Antibodies were raised in rabbits .o whole unconfu,,ated :latural-pvrcine ga!anin. -'I'he- hi$hest gatanin concentrations were found in the bronchus and the trachca of the dog, -- 159 -- - - ~ ~ - ~ _ -!

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guipea_pig; rat j2=-pao1%g in=zQch=caseZ: and Isig {-:=yr~rilgj. -The^:owest ga-lanin Eonce-atratioes-were fouiid=in-the iucig=paretrctiyraa." Oe; c.heo...atog-r3phit-an?lysis ir ihe- pig showed oae molecu!ar iorm of galanin coeiuting with *. ;e porcine galanin ~tandard. By means of the indirect immunofi-ore3cen ce technique on see-ion3 of tissues f ised ~._ ben~uinese-_~iution, ~ian"sr= ~vas fostd to bg -confir.eu to nervr f=it?res -: different _ regions of the respirator_y-tract.'- itt 3he-nasal=muccss-of_the-pig, nrrve fibe.rx~-zo?eta~ssing °_- galanil! -were-distributeb =arou.n-d sem-ttuc-us=glands and hlood_ vessels and beneath= t::~ epit!e{iuns. In tre=traclaea;=brorsebus, and-rajor iatra~ lrnon~r;-airw;=y-of tiie pig, dug; _- and zuinea pig, galaain =im-nsuniireacti-e_iiueis were detected pre;os;~inant=y-=3n-=sm ooth = muscle, as weli-as= arouM serotrtucos -gla.nd3-and in -;?se=__dve:tigia -cf blood _vessels. __ Rarely, gaianin-i!n_munoreactive nerve fibers wera fo°.:z:d in the lung parenchyma.-? few== gaPa atn IffimllnOreacii`JP _g7n~lion -cells also c.,talnir,g ras~~~tlvei7P.lestinal pot}°-paptide `_ were°found-in=t'r-e azivertitii;a of the tracheo4ronchiat ~a_Pt.of the=pig-and dog. =_The - v ascularand secretc:y-f~nytions the ::.r~r,::a :az,=respi:aYi ry tiac-t.= = idis.ribtition o-galaniri suggests that : is pepti& mayr-hav¢ some -influence on airway, - and-ft=oom; 3. _R, _ OheLng A--', :mcsak, j. tia..-8auea, T: E:; Cawiux, A, CatriytofidNs, N. i . Springaii, -0-- i<_, i?ivrnx 4Qi:$89-896; 1985. -- --Oticer-sitp.pori:-Mle3ical-Research Counci (United Icin~gdom): From th-ivp artments-~~=~i~ac~tee.=,is'ry=a=d-~~~zic*ne,'R,a aE=-resgraduate=Med cal = 8choc:,-London, £-nglaad = - I= AivAi<YSIS -OFAa`Ar'ROPesArjL-O!FFEFL-NTI-AT7ON ANB FLTNCT-IOiN =.'r`I3'h- ` &fONOOLONA> ANTIt3OI :)lE& ! _ A large number of a,c rnouse and ant, h=rreyn marrophage/:nonocyte monoclonal ~ antibodies-J?~Ab) _ have recently beea obta:ned that are proving ,nvaluayle reagents of - I extraordinary specificity for the-study a.+f macopnaoe differ:,i?tiation, function, and- - I surface antigen structurz:_ _"F`his chapter summarizes information on st:cra MAb up -tr - February -1983. 3n the w:,ouse, =a; least five antigens _ that -can- be distinguished by- ' molecuiar weight :h ave been found on macrophages but-Fot on lyri:pry?cs. One of the mouse Fc receprors,-present=3n-inacropaages as well as on sorr_e lyr::phocytes, has also been-defined with MAb. Further antigens-have not been defined biochemicallybut = appear to- have distinct di3eributions on functional subp:~puiations. irtac-7 was-*.he first antigen to-be defined by_ MAb that -is present -o+. Tacropi:ages and not on lymphocytes. A second antigen-has been discovered that is distinct fioin M4ac-I in-celi distribgtioti, function, and a-subunit structure, but appears to use the same #-subunit. in the course of studies-on the molecuia=-basis of T-cell fur.ction, ktAb were selected for their ability to inhibit antigen-speci:`ic 'F=iyniphocyte-mediaeed killing. It appearc thai LFA-I is _- d-istinct fro:rr the a_digen-eeceptor,-0ut works together with it in contiibuting=2o the-_ _ avidity_of the CTL for *.hE-targetcel:. LFA-i_is present on Ft lymphocytes and myeloid cells ~ well ~~ T - lvm3hoc vter su~tzes,inR-that it viavs a more general role fh =dhesion j _ than_do-antigen recgptj:s; A si:.3ilar fa.TiiL}`-of related morectilea has been fotina o-n human cells. Other _3ections-of- this study deai with MAC-2 ar.tigezi,=-Mac-3 antigen, Langerhans celis, deedrite:, cells=,_ and macrop=ages,- defi5ing=- r5acrophages by ~neir surface markers and immunolog-ical_evolution- -Springer. T. A_ and Unkeless, !. D.

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In: Adams, D. 0, and Hanna, W. ..''i., lr: (eds.): i.onte~:~rar;~ Topics in _ lrnr,7unobiofogy-Vot 13, New York:~lenum, Publ:shing L.orporation,-19a4, pp. 1-=31.= - Other suptort; t1 S._Public Health Service and t`ne American Cancer Society. - From-the Laborztory of Membrane Immunochemistry, Harvard Medical School, Boston, and the Laboratory of Cellular Physiology and-IC*.:r;:uno'.cgy, The Rockefel3er Llni vrrsity, New York. ~IIIvIACRrFIIAt;E PREPARATION AND USE t)c MONOCLONAL AN ANTIEDDIES; - za rophages were chosen ar1 tlte:r--charae+¢ristit~_ were su nsFarized. ; os~io!f applications of tl=rese_a'=ticudies-are-also described. this section, a-nu.r;ber-of monos.lonal antibodi,°s with ceiatively restricted specifi;.ities Tor from other cells whe;eas-others are associated with distinct subsets of macrophages. In over 40 macrophage antige~ls, some of which are useful for distinguishing macroph9g~s oFdifferentiation. The-advent of hybridoma technology has allowed the identification d - ib anatomical-locaiizoti;,ns, macrophajes can exhibit heterogeneity in functioirand state --cells, and-in almost every_organ, as=Yised-tissue macrophages.' In addition to- variation -moaocytes, iCupffar cells, alveolar macrophages, peritoneal raacrophaNrs, L_tgerhans Macrophages are a diverse famil;+ of ceils n:iginating-fro.,3 pluri;, tent stem celis in the bone mar_ow. Outside the marrow, n:acroEhages- can take the form of blood Methodsin Enzyrnofogy 108:313=32-5,i9d4. Other support: U. S. )<ublic-Health Service. From Harvard Medicaf 5c:hool. Boston. TY.E-LFA-1,-Maca:_Gi.YCOPROiEIN FAhfili f AND I-`I'S ~~EFIC_IENCS' IN AN 3Nk:ERI : EL7 LZiSEASE antibodies, on-inherited disease, LFA=1-, Mac-1 deficiency, was discovered in humans. :rltis deficiency_ has_ confirmed that this-glycoprotein family is of central importance >> ;Eukocyce cell surface adhesion reactions. - identical P subunits-has recently been defined on :eukoc;te cell=surfaces. -Soonafter these rnolecui.es=and ar leastso:ne of their functions had-been defined with -n:o~clor:ai- A family, of-furctionally- important, high=nclecular-weigtl: giycoproteins-_ with FederatiorrProceedi .gs 44:E'iW2663t IS83.- - - Ot!ier- support: National -Institutes nf-Hea9th. - ---= From the Dana-Farbef Cancer Institute, Harvard Pvledical School, Boston.--

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Tf:E SEYERE AISD-MJDER;:E ._PHENc`1i'YPE3_CF :=iERITA$LE :.ac-t, I.t=A-i ~2FFIC_Ef~!C-f: TI~EI~ Q~? +l':~'.T-'ATI6E DEF_'iN`~~NT AND REi:-s5'1F9N- TO - iELfKOC'.-'R-.'E 1`:YfFUh! :IOI: r'nN1'lCI,ltjll.^P<.L FE.`.TTUR S: 1- LFA-l, nr - p1-3Q;95 _(or a co=s binatiQ n.) -= three st: s=ctur ally refateil_ 'adhesive"' surf aF-°_ - andlor delayed urqbilical cord s_e aeratt4e--w_ was _recog .izR4 in four tna.e -anpit four ;easi;.le- patients.- As shown with syblrit-specific mQ oclonai ant:bQCis ~r immybofluor_srence flow cytometry and '-'sI iummustoprecipitzt :n techniQues, _i^n addition to a tritiated -granuloSytes, -minoc-ytes or sodium 69rOhydsld@-gaIa_fiQEe -?Aica_:? -labz!ing asssa;, lymphocytes front these in;:ividu5is had a`ir,oderate" or 'sefere` deficiency- of -Fvfac-l, soft-tifsue-infections,.dirnir=khesipus !'nrAmat:on; isbpaired wound h°a+ing, granulo-cytosis, ~~ An inherited syndro E characterized by- recurrent or progrsssi-ve necrotic glyfQproteins.=--I--.Lo di tint fhenottypys s.'re deair.ed Q.'n t::e s wi_ c h trc Q~i = _ ,~tity -of a:ztigen e=eressed. T hree ?3at nts w h se ae yefuc enc- and 1'.,ur pat's@ ty w th moderate d@fec ency--exprersed c0-3% a ti 2.? S!% of ncrmal 3mQ nts of he-e_.e ol2c -.e's for endothelial n?argina*-ietf& and tihsu? =exiudatem -_- The ri=s,osn__i=;n= p[-phenotvpic= varlatlon--aQlC`Rg patie.tg ,yitf=Mac-E, iFt\_ 1-_zi_"sflf.iei.?'y r?2y.13e-@ .~?Q:dant F_4kth- F~sp-=; -tST_ -~- - thera-pe~2.ic st:ateg?es - - :. - - -- ~~ - - ~ _ - _ --_- -- ._ ~ ---- profvund_-abnermali5es oi-tissue leukocyte rerobili~.atioh, aranulocyte-kirzc';ed ftii&r;tiydrl ---- hyperadherence,- phagocy* se;- of -i(:3b-apsonized part :.ees. and corn pler-sent-- or _- _ antitody-dependrnt c-ytotoac_ic-il*y were -f"ound ia individuals w hsevere, tes-~.uYgared ivith -moderate, ~eficin_y_ _ a is- proppsed_ that in aso _abnor~a!it r& _Qf-- lPuaocyte mobi!iiation refl_Ect_thLL-crltifae rols of Mac-1 glyfoprQt iiis in a33Egsiv@ evenis r¢Guired On grantrlocyie su-tac@s, 3°=rec ttvely. TI?e se_e,ay of c¢inlcal infectious ccM.pl-c?t.onS- among these patients was d1Fc aly F latec~ tp, the degree of_glycop ote:r ;:efhea :_y. Mor~ A ndersdr-, iD. _r. ,~ipr;ag-er. 7`.- , et- :a_ Tlic Iouinal oj' ~rrfectias-T~iseases_B3~4p:<`'b8-bg?_, i9Ei5 Other support.= T--lational Insi:;u*:e -of-AIlergy - and Inf ectious Siseases, National Institute -_ for Cancer Rtsearch,ha*.iona: Instit-otes-of Health, and U. S._Depariment of ngricu!t°are.- Froin the- I3epartment= of Pedi3triEs, Microbinlogy and Iramunology, and_ Patl:oiogq, fsaylor College of Medicine, Housto-r;, and- -irana-Farber Cancer -lnst;tute-,- Elarvard Medical School, Boston. antibodies have shown that hyt-.'.,3 and I FA-l surface molecules a-re-assDciated with T= lytnphocyte-mediated killing. - -Somewhat surprisingly, -the Iv:ac=, molecu,e aasociyted with= CR,-functicn,-nnd the I.Fw-i molecule assuc+3ted cvith-'E=1ymphocyte-medisted killing, have been found ta be structurally related.- This suggests that at leas?-with regard receptor and- corttp;ert:ent receptF~: type -one V'CRr) h.ave_-been- cha:acterizea- by - purification and function-i^fiibiting antibodies. Recently; anti-MacEI aniibods was found -to inh.ibiL the complement re*_^ptor type three (CRs),-sugEesting that-the _CRS is identical to-or associated _with the- ure:-iotisly- biQchemically charactcrized- Mae- I molecule: - Studies- on T-- lympaocyte-mediated -imc^unity- with function-blocking MACROPHAGE AND T LYt:IPHCJCYTE-ME-DIATT--aIMMUNIT_Y: SIMILARITIES AT THE LEVEL OF THE KAC-I ANDLI`A-A MOLECULES A number of cell surface moleculFs_ have recently been chara_cterized= which-are important in riac:opliage-anc_=_ymphecyte cell-mediated iriimutiiyn i}~e r~acrophage Fc_ - - 162

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to these mOlecules,_-similar molecular m¢chaniSm= -urederlie_`r_r,ac_rophage and _ '.ymphocyte-med'aated -imrre:ni,y.- The £iridiag_ cn-Mac=1 aez*.I L`F A -3-are reviewed and the ~°ntut:o;tar; in ~licatiers are ~:iscus~d. _ i_n-~ Volkman, A(ed j: kfviron=:er 3r 1hagor_-y.re _ Biology, New :FO k- Marcel Dek keL 1984, _Cpringer. ; . A. pp. 109-i2f3.- = Other support: U. S Public Hea+t : Service hrid- air Rmarican rancer So_ ew 3unior_ aculty Award: - THEIR YyEFICIVNGY lN A NOVEL HERITABLE DISEASE TI-IE-M2c-1; LFA-11 F4MILt OF LEUKttiYTE, AD-IiESIGIV GLil_'t1PIGTET_f+iS, AND - FUNCTIONAL AN_l`ST12UC`1-JRAt, INTERItE1,ATiv;-SElIPS AMONG ertacrcph..?ges. - Specific ee-cognitior, of orsonized microorganisms _is- facilitater_-by CeII surface adherence reactions 3re of central importance in the immune funcfion, -of iyk:phoc-vtes;=-- nicRCcyfes.- -an=d= gran;iC6ckte," 1-y::phcrcyie5° -adhere o an<igett-pieseriting= raaEr:whag_~-, or ~dPndrrtic ce s:^ the induction of -T-lyrrYFocytE---_ irumone responses_-a.nd tfi-target cel7S 9n celi-medeated killing= AdheSive in,eractions are-_ fundamental to a _ wide spectrum of fuflctiorrc -yf granuloc_vtes, n?onocytea, and mediate microlie-cell] adher:oA prior- to- ihe='triggering of cytoskeletal events leading= t;, memCEanz_recepto_rs-for IgG s_r;d for the_third component-of complement is3;. which endocytosis. - Adhesion mediated by = 1g% -(Fc) =receptors can _ aiso trigger this research has led rto t} 4Aef:niiior., wit ; monoclonal antib-~esr o,`-: _riovrl :eritabie disease that msnif_sts itself in defects it, Ieuxccyre adherence anA snoti::i-;.= With the use of FrrAbs a novel d_istase- has been recogn-ized- in wttich- the Mac-1, LFA-I and p150,95 glycoprot4ins are d4fic;cat- Recurrent bact:rial ittfecton. progressive periodor:tjtis,- - persistent leukoeytasis,- and[or delayed ur :bilicai_ cord separ ation have c.Pen- described ',n _ patients whose_ neutrophils se-monstrated _dep.essed phagocytic _f unctioi and deficient (Mac-I). lymphocyte function-associated antigen i (LFPr--1), and p15Q;95. Furthermore. -characteri=_ed orr,eukocyte surfaces that is_ imTortant in ruany- of the -abcve -adhesion higtl=tnolecular~we-iQht glyco<rroteins ivith identicai }4 subunits Aas recently been --_ gnabody-dependettt =killing - of iarge= cells, ir._dependt;r<tev of Pddocycosis. A=famili of teactiohs. -i4onoclo5a'__aniibodies have -been= i:+strumental i,-eiu_idat:ng the structu=_al interrelationships ard-_functidns= of .hese thnEe gtycop_roteins; "rriacropliage= antigen 1 adherence and-che:notaz.is Springe?. T. A_; and Anderson, D. C. Ir; Springer, T. A. (ed',; Hy5 ,`dorn2 T- Y7o.vgy in the Bjostienre_ an Wedici:.e. Chap. 11, New York: Pleau6 'PublisFing Cvporation- 198{,-pp. TCTI~?0$. - -Other support: ttiatidyal-Ittsti;cites of I{eaithm from the LaDr_,ratory of Membrane _;mmunochemistry, Dana-Farber ~Cancer -1nstitute, Department cF.r = -ftticology, Harvard Medical School, -goston; and=- Baylor College of - 2vtedicitte, i,epa*_tme-w of PFdiatrics,-I?oz!stori, TX. 163 ~: -- ~ l

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S'I UI3IFS ON ~.` N:7CzEIESAS.SOCSA-Tt'E13 VViT!-I THE ACTIVA -T_:ON-OF-MLihINF.- b101~fON"~ka..£A 1~_- PH:~C~Y°I'ES e(IIVET!C$ £3`t--' .~cN'-~3 REQJIREfviENTS F-`.)R __ E (LF_A)=1 AI1T10EN- IN INDUCTION OF LYMPHC3C'f TIE-FL.tNLY`ION-A3SOCIATC~ VITRO Ivlacrop?w-ages -activated and primed inv:vo, although not_ residznt or respa,is;vF -- macrophages, express the lymphoeyte function 2ssociated (LFA)-l antiger. By contrast, the biochemically related Niac=' antigen 7s expressed on alt populations of ntacrophagcs. ln the present paper, we studied regulatior, of-the i.FA-i antigen in vitro. _ LFA-I could be induced in--~iFro ~n thioglyc.>liate ('FO)=eiicil-d ~ut not- an proteose peptone (PP)-elicited or zesident macrophages. -_ Specifically, nsacrpphage-activating- factor (MAT),_ irtterferon-r (I='Al-7), or picogram amounts oI endotoxin (LPS) induced LFA-1 on T;r-elicited _ macrcrp:.ages following overnight incf:batior:. Intzrfro n, -a or -~, fucoidin and colony-stiznulating factor were not effective. While some levels of LFA-i could be detected as soon aa 10 hr paai ezpression wa,: obs¢rved after 1$ to ?2 hr of - incubation. Tke inductiori could-be completely abrogated by cyclohezimide, suggesing that protei n syn° ,esis w'2~i required. i nese results inyicatz that :he induction of LFA-l - on mononuclear phagocy:es-is closelly--regulated- and that the _r-_Wuiremerics_=for_ =_ucii_ -- induction -are- distin:t fr0-M °but share certain-sinn;iaritia_s with, induction of cytotoxic functions and expressioo 6f Ia-ant;gen The Jor~rn~f o~ lmm:~no(ogy I35~i);17-l3~, L985: 1From-?ke DeRartment_of P-athology;=Duke-'yniversi:y_:v1dical-Ceeter; Durham, NC, C, and - the La)soratory-ot'ailembrane-immLnoc}cemistry. Dana-zarDer Cancer In'stiut>,-:-iarvard = _ ,~Sedical ~choo!, F3os;cu: _ COMP-LEME N"i'. RECEPTOR TYPE THREE=DEPENDE*!T DI-GRAf?A-T_'-iOPd OF OPSOINIf.:.e5 ERY"ITIROCICTES BY MOUSE MACROPliAC,ES _ Z The role of-the comnplernent receptor type 3(CR3) on thidglycoilate=r~iicited _, peritonea! macr:~phages rt'FO-P1Di) i:. tlse-destri;ctio~ _of opso.n.fzed partic_les was studied. - I We f-ound that sheep red-b:o3d celfs (E) that-were opsonized .vit=i an igt`1 ,rnonoclonal- - irdt (E'=1gr3C) w ld-3y TG4Nih ar:t-orssniag antibody an complemen=ereyse, - wereas -- there_was little Jysis-of =--p:etryated=:vith e3ther`3'ie- antibody or the cqmplernent sou-~zz - a;one__ Furthescaore,-this ] Ssiseould be infiiF i;ed- by anti-CRI monoctonai _ant:bodies, that had previously heen-shown to-inhibit binding of E-IgPv1-f-" to-the CR3. -Kinetic studies __ of phag_ocytosis-and- lysis'-iridicated .hat lysis o:` E--lg;z.-C occurs -aftr phaqncytos:s,-_ suggesting -that lysis -i3--an- intracellular- event. -- Further- findings suggestzd- that intracelrul=ar lysis was pr+,rnvtedby CR3 bound to the phagocytosed target; because -a - monoclonal-anti-CR3 ant:bady,decreased the rate of phagocytosis of E-IgR:-C but not its- magnitode, whereas the rate and extent of lysi;-wearr strikingty inhibited. FurtlrermEZre, _ Tt'i-.^-M that had alreadv iaterna!i:ed aenopsoniied E selectively ly;ed _ E-1glvi-C that were added later. These data- confirm that the in.eraction of- the CR3 with its ligand on E-IgM-C promotes rapid j)iiagocytasis, and they further sugge3t that the CR3 facilitatez _ degradati3n-of_the target-particle-o:iie internalization has occurred. The Journal of i'mrrt_:ro7ogY :'3(4);?2b8=?672, i983. - 164

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Other support: U. S. Public rlealth Service. =a~.oratar, of -lricmbrarie Irciniu-noeheru-sYry,--Da:au-Far-3er Cancer institute, Frdnr the I-(.__*va7d_7E1ed:cal &N-A; Eosron.- - THE-FL1N:.:'I_ONOF L-FA=I IN EELi,=?vI'DIATED KILL-ING-A-tJD:ADHES!ON: _ S"I'LTD3ES ON HERITABLE EF_A:-I, Tiac=1 DEFICIENCY AND ON LYMPHOID- -- CELL SELF-AGcihE:,P_=PION' - step in CTI 1=.-mediate'.1 kiaing, anLL'-s1FggeBt-L-FA-i=f3ncti3n's as-af- adhesion -.9rofe.n.. - I and 2. Killing b; patient 2 -L 3 L was pQoriv ii]iEihit`:d -: en anti-_L?A-1= MAb- was - added- to the- assa: _` and pretreatment cf kiilers- or effector,= gave za,uivical 2r - no blocking. CT:L f*ay patient 1 was- inhibited -hei anii-LFA-I svas- included_ ir._ the -assay. The findings with-=Parrerit--1 and 4 °CTt suggest that LFA=t -on -target _cel;s can also cantribut_i to-the CT:,=targ_tinieracti:rn-.= --Da#a prese^ted here suggest a 3t-rong siimuiacity -between phorbol ester-induced celf'ce:l aggr oation and the LFA-i-dep.nde:rt adhesion &rolil'yraticsr: -bfter srimulation with the -lec-tin -phytohema$ostirrin (P-i3A). PBL - f rorr_- LFA-! deficient patients showed an i*~airecl prolifera*_ive response to PHA. C ontrasting results were found for the ciiantetarively=tiiorp severeiy i.-FA-1 deficient f'TIv of patients Natural kiiiing= was assessed -ofi the R' 5i;2 -eryth-. oleukernia cell line (HLA -negatiye): - All LFA=: deficient itiCiviauais showed lhw-leveis =of NK cell-mcdiated cytolysis co:npaied -td that of farssily'~srnb4rs and unfeiar_e4i isditiduais.=_ FSL were also tested _-for - lits-threatning inI'ei.:ie..s were found to be -deiirieRt in :.FA-I and two other surface ;xolecules which utilize -the sasne-fl -subuhit; hac--l an-~-~ NI30,93:- _ To -aasess ta:3ral killing; per_ipheral hlood lympnocytes (PIfL)-fro:rr LF--A-l- defiFient sndiy_dual4,- their farr:ilie3, and unr_intc_ -contr>>:s-were e;iltured a:one-or _wih JY cells for= sis *~,.avt.= i;mphoryte iuncaon associatec# antigen-i (LF4=i) is-a ceil-surface-glycaprotein identified in mou~ aad- ?_iman by mon-_ional antibod-ies-=which-inhibit cytalyiic T ^pliocrte-(ChL;--~cdi~ted-=ytol,sis: Re;er:t.y= a ourr.he: of_yatient£=with -securring, 7kork K Load3n: 13lenun? Fre`,,-i-9$5, pp 'si I-3112. In: Hcnlart,-P. and=MaT-T, E. (eds:):-MecAanisrns o! Cell-Mediated Eytote_cdci_rv 11, New .)pr:Rb°er,-T. .'I.-el SI. _ - -- - ©tF,-er suYporE: Nat:cnal Ir,stitutes of Realt;_: From-°Dan3=farber_L_.ancer Institute, Harvard ?Aed~mat School, Roston;=-Faylor _iolleze of Medicine, Houslon,-"EX; and- Stanford Lrniversity-, StanfAed,-CA= STRUc' T I.JRAL ORGANIZATION OF -Ii+TERFHAS£ 3°I3 FISROI3LAS 3 STUDIED EIY TOTAL AL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY critical angle was- approached. A- similar result was obtained- when the nuclei -were- -staine.d with Hoechst dye 33392.- From this measured angle a cytopiasmic refractive- index in the-r-ange 1.33-R-1.374 was coanputed. The plasma membrane of-3T3 cells- was-° stained with dil-C'fi-(3)= and 3he cytoplasmic_ _compartment- was - stained with fluoresceiny l-deztran = (FF-1 dextran)= or with carbozyfluorescein_ We have -with the water-so7ulife cationic dye, dil-C,-(3), fluoresced- only as the glass/cytoplasm -- - -- We studied-the lamirrar organization of-3T3 fibroblast celfsgrowing_oa- glass- sli:;es= -- by use of t-otal internal-reflection illumination to eacite f_luoresce-r.ce7 emission _(TIRE) from labeled molecules and stained=cellular compartments that are-very-close- to the cell substrate-contast region. Mitochondria, diitanG from the-contact_regions-and stained

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demons,rared a high degree-o corresp_~fide_r.c; between the low-reflectance zor;es- in the 3ef1_ection interfe=ence-_ii=age of a!ivy ceil=anv the TIRF rmages of ~oth the plasma -memhrane and cytco~smi: compart?;ent, ~ TIRF phorrrcne;fy-~f se.e-c3ed contact regions ` -__of cells provided_ dasta-::om- avhich_the absolute separation of eel: and- su5strate_ waS computed. From a-popul}tion o;-3TS cells microin;ected with fl::orescein-!a3e,ed actin, _ motile and adherent interphase cells -were selected_ for ;tu4y. For adherent ce;ls, ~i=h = displayed f/aoresceef stress fibefs~ t he TiitF image`was composed of intense -patcl:es_a-^.d- less interse-regions that corresponded, respeetively,ia the -foca-1 conta=t-and close-_ontzct-_ :.ones of the eeT7eciion-i=ntrf?rence image. The intense patches corresponded to the _ endpoints of the stress fihvrs. Celis-of motile-rnorphoiog;, which formed some focal contacts and extensive cl:.se=mntact zortes gave=Ai--3ctin TIR F images of relatively even intensity:- Thin larreffia: =reg SAns-of°:he cytoplasra- xere found to-c3ntain concentrations of actis not significan-tly -different from :,ther-close- consact regin- n, of tt:e. ce41= The major-anal,vt-ical problein-of TIRF microsco,Y-is separation of the effects of proaimity to substrate, refractive indeiAnd Fi:rorescent:-pr-0t>e soncerrtratiorfv:: the Iocai hrightness'of" the T'}RF image: From vwresults, it aTpears possible-to -use T1RF microscopy to measure the-prozimity-oI` differentzcmponents of-subs tra;e contnct-regicns of ceilc. f,anni, F.: Waggoner, A. S.; and Taylor, D. L. _- - The Journzf=of Ceil $iolo.t0:IC91=ii02, 1985: rrom the Center for Fluorescence Research in Biomedical -S,:iences-ar.d= the-Bepartr?tent = of Biological Scienses. Carrsegie-Miellon:Uni°:er,sity, Pitts6urgh- - -LIGF_'-T-SC'ATTFRINv C#fAAJC;~`D!IRiA3: CriEM^T?tC::CST'RAU~ATI0N OF HUMAN 1lTsZJTR0P-Iii1.R_fKiFZTICS FOLLOWED BY FLOW C-1'Ta'?M:LTRY - The light-scattering properties o__ human-neutrophils were compared o_n- a cell-by-cell basis -before~ and after stimulation with chemotactic peptid-e usinf f'C- cytomeiry. Between 20 and 180 sec after pept;de addition, side (40`) scatter declined by up to 49Frand forward scat_er-increased up-to 6%. Beiween-3 and 15 rrrin,-side sc_atter increased up°-to 15% and'-forward- scatter aecreased- up to .i%. Ass-c-ciation- of -a fluorescence chei=.dattrastact with neutrop:.ils was n ost rapid during the i,.;t:al phase of -increasi:ng-forward and decreasing side scatter;--a;jd association saturated before the maximum increase in s+'de scatter-. Evidence is prese*ded-that the-o5served- changes-i*: _ scatter- .vere not a co!+senuence of -cnemoattractant-inducPd ce-ll-cell- aahesfo!!_ o? neutrophil- degranulation. _-Rather;- the ea_rly_ phases -of light-scatte:ing changes-are interpreted to_represent men-ibrane ruffling by the stimulated neutrophil;-tFe later phases polarization of the neutrophil morpho'sog-y. - - MicNail,-P. L., Kennedy, A.-., 5rag3oner,-A: S., :aylUr. D: L., and Murphy, R. F. - Cyrometry 6 '- i2, 198 5. Cther-support: National Institutes of Health, National Science F oundation, and -Health-- Kesear,h-and Services Fr;imdat;on.° Frorn the Center 7or Fluorescence Research in Biomedical Sciences a_n_d-th_e Department of Biological Sciences,-Carnegie-_Mlell-on University, Pittsburgh. 166 1 ~ ~J''r

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CGNDI'I'IC?NS REQUIRED FOR EKPRESSIGh! 6F MEMDRA1vE IL I;3N B4CELi,S _-- observation that rnaE_=ophage membranes stimulated prol-iferation of thymocytes and an IL 1-dependent TzeEl line but nat IL 2-dependent T-sells, and this activity v.as,inhibited by a polyclonal goat anti-IL I antibody. Additional biochemical studies r.eed-to be done to unequivocally esab!ish the interreiatitnship3 between the soluble _ IL 1_- and the membrane _form. Nevertheless, t-.vo_ irrportar_t features of the membrane IL I_ were noteworthy first, it was essentiar that Ia-bearr:g, fized macrophages express it to activate antigen--specific T cel] lines, and second, its expression could be dissociated from the secretion of IL 9.= BRcaute B cells are known to function as-ar.tigen-present,ng cells, we investigated whether these cells express trrembrane-IL f_ The authors have eeported that raacrophagcs-bea*a rriitogcr:ic activ t- for T cells in -thes'r Qlastna m-embme that-we have=tsrated 'rFierrbrane-interleuici n i'. Mer~tbrane IL- F_ was studied by -using' eitY':er paraformaldehyde-fiaed°rr:acrophags or isolated macrophage membranes. The reasom for ascribing-this membrane activity to-IL I was based on the Kurt-Jones, E.'A., I'.4aly, J. Ivi: and (in:emue, E. R. -The Journal oj_ImmLnology !35f?):1545=50, 1985. Other suppori, Nat-icnat IE_stitutes of Health= From the Department'of Pathology, i-!arvard-Medical School, Boston. -_ IMM-iISNE C(?M?LEX'EFFECT (;N I!SLrBINE MACeUPI-IAc-sES 1. IMMUNE COMPLEXES SUPPRESS INTERFERCN-=r INI;rTCTIGN OF Ia EXPRESSION ` We have studie-d-the effects of im:riune comple?ces on the expression of macrophage surface proteins-in r(t: u. -Increased expression of the H-2-atolecuhs I-A, I-E, and K on -the macrophage menibrane- was induced by i:.itro culture with crude lymphokine or -interfero n-y:_ -£xpressiorr of ait three of the-molecu!es was additienal i;icreased by stimulating the cultu?es wi3h -heat-kailed Listeria monocytogenes. Addition cf- soluble immune complexes to-the cultt:rerdid not have-any effect on-macrophage expression of` these proteins. However, significant inhibition of lyer-phcltine or inteFferon- ; ind-u_ctioa = of I-A. I-E, and-H=tK was observed when macrophages were culturea or- p!ates_ to which immune compleaes had beerr bounE. This inhibition was dose dependent, required an immunog!obu!ir_ (Ig) molecule with an intact-Fc portion, did not require the presence of T cells, and occurred in the presence-of indor.:ethacin. Complexes containing [gG:, IgG2a, IgG2b, and IgE, but-not t_gM or IgA, ansibodies mediated-the inhibitory effect. Virgin. H. W. IV, Wittenberg, G.-F.,-and f%nanse, E. R.= -_ The Journal of 7mm%no7ogy 135(b):3735-3-743, 1985. Other styport-- Natio>iai-Institutes of Health and-National Institutcof General Medical Sciences: - From the I-Iarvard Meaiicyl=3choo!, l;epartmeret uf Pathology. Boston.

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IMMUNE COMPLEX EFFEC3'S ON MURINE Ia1AC_R_OP-IfAG£S.- II. Iivll=.diINE-CGF:SFL';= EFFECTS ON ACT'IlrA'I'EL_` MACRCI'IiAi:Es C2"IUTOXICM, ME4$.~'~AN IL-I AFtiC-.kN-I7GE'IEf I'RESEN1`:4T_:`7N We i4Y"$ti],tated the efect° of i'T-i$Z'yS' Ei'ilh'yle7te_ on rpak_.rop4:arSe ft-luctionc =1R --_--- rt'tro. Immune complexes brihiSit IymphokE<<'snds?tlon r,f both I-Ak expre' esf and cytotox;c activity by fetal _cel se:um eiisited ~-acrophyses dlFin ' g 6ni-terni ~"f dauy, cuiture. In-addition, induction of >nteDen Presentation wa° sign=fi~ntl~ inhibite3 by immune_c4mrle>iese xprevsioil of membrane nt°t,et.xin I-(IL-I, y me:e:brane-,,,1.jnd- ` form o?' tfie I -cell mltcasrr rerui ed for antigt:n= prr~entatio n _by fixed c Ls" _~as minimally inhibited by mtn1ine co-rkiptPxeY.. "I"ti?aefote, inhibition of ant grn fiF sentation was primarily due to efcect- on Ia ~piessian ra.he_r thar m mb ane IL-1 _ exp:ession. The inbibitor- effect ^f immune complexes was not found dunnj skors tFrm ulture-(4 _ _ to 48 hr) whe_L activated ~;iaga 1 bearing high levels of iv~ fro:n- r;.icc i~.fe~:e~ - - with L:siPrla rrion6cydogr:.e; wer° C..a_ eia2ed- Immune a.2t:1p1ex'°c,, R+a1rnaineC,- or - even increased ieveHs of -both ._.~° and cl.oto=icit; acti.ared nt-croph ses.- The impEicatittts of these .''inx1= ~&s fo: imsnun e complex n,odul:aion of the immune respon se -- are discussed. Virgin, H. W. IV, Kurt-Jones, E. A., Wittenberg, G:F. and . R. _ [ !!e oJl ral CJ [.'iFntdn p(flgy -m3mi j .4 r44 S 74y, W). ~- -- L3rher- su per°; Nat ona irisutes_of HeattF- U._S. °ub, ic Health Service; National_ Institute of Get:e-:aI Medical Scis^;.es. ~ !- From Harvard h,ed.cal SchoYl- I<epartm t of t arhology pos°on MA {-_ ~ SUPPRESSION OF MitVIUN°'- RESPONSE TO c.i_.? ia rnoncrylogenes; MECHANISM(S) OF IMMUNE COs4PLEX SUrPRESSICINU - - ~ We I?avt_ investrgvked po ffkle m c_hanisms undar ng ;.n2 .:unY cSi,Tp'::x-s°ppressio;f of resista.nce to-Lisre la rne . niqy-ogenes-Irh;bltlon of resistance was ounc; when Immu_r we-Q forrre d ri v yo in aE.~3~re mlce--A in . ~ni n;,,Fe f:ce complexes adoptiteij --_ transfer;ed, with specific a-_? body_ Suppress! - a- w_a.; also foynd- whzn_ not!iff-mmune mice were .njected-wi-th irr,nipne comQlexas preformed _i~ s'_rro_ iVe nYezaegated the rrze- of_ compfetaent- by deccmu=gm,.-Ating mice - with-- cobra veaoim ` factor purified - 6y _- high-pressure-liyuid cliroresaeOgrapfiy. Chcnplete a`epEe,ion of serunr C3 did not e:ir7ina-te immun -= complex s ppre~si~n vf' resic,3nss to f.= -urr3cy o6er_<s_ suga st',ng 'tha. complement actiyation _is` not requirPd.- for itimune- complex j;tppression. Infection=indnced_-cha!tges E _n the c~rface pherlotyar.d= tunctionel prepe t=Y: of macrophages frorn norrnal And irnn:urie c(impiez-suppressed tn:; e=Ners also invastigated. -Macrophage zxpressieu of both JI-2K and Ia molecules increased during the response of _ normal mice to L. rna r~yicg:rrt. Hewever, these d~sa ngs were not found in immune cov±pleA-sbpp_essgd m"ice. In contrast,,necr,brane interie_Em I expression was ittsrea-_d i;n macrophages from supprYssect niYe campared with_ macrop-hages =`rorfi normai mice. MacrophLges_from i: rnono°y:oger.es-=ef?cled normal an-I _ immuie _eon plex-suppressfd- - mice expressed cytotoxicity- abeinst tumor c- /9s in -vitro. - We` cinceude :ha_r -jb±nre complexes-do not inhibit resistance-to L. rrohecytogesos by activation of compierFnt or- - decreasing m2crophag? cytoxic activity. Rather defects-in la expression by roacrophages from suppressed mice _ might be one component responsible for immune complex suppression of resistance-of L. monocytogenes. Virgin, H. W. IV, Wittenberg, G. F'.; Bancroft, G. J., and Unanue, E. R. - 168

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Injection-rvtd Im>nunity-S0(2):34-3-353, z985. _ Other_;upQo.-t.- tJ.~.-Public-Heai:7 Serv'_cF, National_ Institutes of Health, and Nationa[ Institute of General-Medical Sciences. From_the Department of Patnology, Harvard Mecticai Schoo:,-L+oston. _ RFLATIONSkIIP Oi- MAi,ROPIiAGE 127 AND -IvIEMBRANE-i_I, I- E){PR'£aSIOI~f T O ANTIGBN~ PRESFNTATIOt.C prese-ntation. Using this system, xe have demonstrated that quantitative variation in these t3vo-pararaeters-was associated with changes in the magnitude of the T cell response. t<urt-lonei, E. A., Virgin, It° W.-IV, and G`nanue. E. R.-_ The Journal of fmmrusologyi35(6)3652-3654:-1985. Other support: {Vationat--tnstitutes oLl3ealth. _ Fro m the Department rf Pathology, Ha, vard Medical School, Boston. membrane IL I expression on maci=9phages in culture. By then fixing the cells: we are _ able to preserve constant levels of these molecules _rluring the assay for afitirsen_ Membrar e-earfessioc of Ia mTlecules by antigen-presen,ing cells is critical_ for the induction of T cell-response3-tF2-forcign protein an[igens. In addition, antigen-specific T cell proliferation ha;=been thought to be dependent on interleukin I(Ii. I) secretion by antig_en-presenting cells.- Iteceatly, xe have described a$ovel_ membrane-bound form of IL- 1 that is required for -the presentation of- antigen by antigen-pulsed, fixed macrophages. _ Membrane IL I is a mitogenic protein found on the macrophage membrane that, like`soluble IL -1, stimulates thymocytes and-iL i-dependent T cells but no'- IL 2-dependgtit=T cell- lines. This activity is ir:hibited by a-polyclonai anti-fL 1 antibody. -Membrane-IL I is an integral membrane protein-and does not represent soluble IL I nonspecifically bound or tized to the macrophage membrane. Additional biochemical-studi=_s are reqcvred-to defit-e -the possible interrelationships- between soluble and membrane IL 1. We have now develoned a method= for independently varyin,g tfie levels of Ia and-- ACU T E LUNG :NHURY IN RAT CAUSED USE.B BY IMMUNO~`iLOBUL-I*4 A IMMUNE COMPLE?CES- Mouse IgG andr.gA, wit1a reactivity to dinitrotrhenol conjugated to carrier-proteir., have been isolated rom-tnyeloma proteins- by means of a variety of affinity -techniques. The IgA was preptn:nantfy in the-dimeric form. The in vitro and in vivo biological --- aEtiVitie4 of .gA-kontaining immune co~lp:ex_es we-rz` assessed in the-rat-. IgA-cortair.i ng° -Immune- cocnplexes- were cesnonsttyte#, in - a dose-dependent manner in vitro, to activate neutrophils and to generate= (D:_i* In additiorr, these imm+me - complexes showed evidence of conp!ement -activation in vitro, by the use of -immunofizatioa techniques. When IgA was instilled into the airways-of rats and antigen was injected-intravenously, acute lung injury occurred, as reflected by increases in lung - permeability and morphological changes consisting- of- bieb6ing- cf - endothelial cells, :ntra-atvgo(ar _hemorshage; and f ibrin -deaosition. -- i'~e !ung changes - were directly proportiona.} tn- the~ amount of IgA- instillecd into the airways and failed to occur if intravenous injeetien of antigen was om9tt:e~. Lung i*ijury did not occur in animals thae received an in+:avenaus iniection of antigen in the absence of-an airway-'tnjection of

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IgA. _ LLng--irt;ury related.. to=:gf+=eont3inirig- immune cosrsplexes_ was complement dependent but neutrophil independent. In _ompanios studies with mouse Igi: -~contaimng immune consplexes, -aeute lung injury=-aiso :occcurred and-had morphologicai feat_res-==- ; ost associated wit:: lgU -assa=iated-iz neune=cornplex-indtt_ed-acute lung injury. --_-- ° have lung-damaging proper-ties rrd-that=t;ie pathogenic -n3echarisit are different -_frorn _ "I'hese- studies -indic-ate -for the-first tirrie that immune ec:rsplexes=- containing- - tgA, _-- bovirie seerorn aibufain; alon3produced-t-A4ence_oflurtginjury. injury in the--rat.- N-either=antibody alone nor antigPn-(phosphoEylcholih¢ linked to_ - to phosphorylcholine also demonstrated the ability of Ig4 antibody to cause acute lung by the use of-gorphornetry tecntrioues = - Studies with another moddcional Igr a ntibody=contairring, antigen-binding aetivitv and tgh-associated -acute immune compiex- induced injury of rat lung were reinforced complement and neutrophit dependent. T he similarities and differences between igi- -_ immune complex-induced damagy; neutrophils sverv--+?ore evidenf. Rc~rt£ lung induoed by--Igt:rcrntaiciog=imnu-ne-ccritplexs; whetheriof inouse or rabbit origin, was -similar to those associa,ed=-vAth 1gA-induced lu:eg injury except that, in the case of lgG --_ Iohnson,_K. it., Wilson, B. S.: _Till,,G. O:, and Ward, P~ A:-, __--- Ja4rrral of triinical-:nvesagw;ons'?4.33g-369, 1SS3_ -- : rom the Depart_mentof-I'zt:nology, i-lnivtrsity~f ?~eiehigan_.~izdica_-~hool, Ann ~ri~r, -- v:htr_st:,pporl: Na?ional_ Institut¢s of Healths - THF DN/4 SYN 1`HETIL' RESPONSE OF NORMAL AND ABNORMAL -i-=EJ\4k_N -- LYM E'l-+_OCYFEs Tt:l i`iiiVA L_C'iNIC`. ACiLr THI~aDLE OF CiR kN-*vLOCYT.'ES AS AHELPE-%PC>PtjLATIOv'=,. _ - and the regulation of cellular DNA synthesis and mitosis. - observations-add to-our knowledge of the relationship betv-een-mevalonate metabolism - -_- DNA briskly in response to rnevalonis- aciy in=the absence of=-neutrophii help.= -These -° -- granul-hytes- in_ contrast= tv=H ce l-chro¢ic _ ly!np;-,oqytie ieu4er*tia ,°lFs that synt!=esiz° - - mevaloiiaie respofi.se-of normal E=rosette=nezativeeelis is eahanced_by the -presence o;-- lymphocytes-faia to respoad--to mevalona-te,-whsreas-P rosettv-negative-celis do. _`I'he -- neutrophil_= are responsible for t he help -neutropfiils -provide:= Normal E roset: -positive _ neither neutropleii lysosomal *enzymes nor reactive- oxygen species _ generzted by lymphocyte DNfe-synt;3esu-tn the absence of mevaFonate.- Our experiments suggest that - 6ranulocyt_s preexpdsed-- =T =8sevalonate do- not -Acyuire _the = ability= to- stimulate - extracelsular -mevaionatF-derived growth factor that- in turn stimulates lymphocytes. ___ the absence of tvFvalchic-acidFfurther increases the tympnocyte response. He have- beea unable to demonstrate the +rodustion by_-gra_r;ut-ocytes of either an intracel7ular or - numbgr of neutrophils: Peizcuba: -o,:-of lymph-ocyiss wit . ne-titophi?s for 2q-h.r, vsen in - to mesalonic_ acid, but thei-r response can be significantly e nha,-ced-bj one-half _ their ceEis each freeof-rros:-ootttaaiaati-o;, by ttiother.=-Purified lymph5cytes respond pooely Idordzr, t9 investiga.te the role of rteuiropf.ils- in`-the DNA s_r_r,thetie- response -of human peripheral blood lymphocytes to nievalonic acid,-we ol:tainedyreparations of-both - -Laron, R. A., Kluskens, L_ E, andYachnin.S~. - - Journaf of Allfrgy and Clinical !mmanology 74:2$0-291,-E9g-.5~.- l70 -

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Other support: 3!ational-Instit4te of Arthrii3, Metabolism and_D~igestive-Biseases and -Naico Cattcer_Rm arch-Futta: _ - _- - - - - From She -Departments oE-Medicine and -?aholcgy--and=ih-r_ vommittee- on_-Itnr :unoiogy , The U nivtrsity of_ Z$icagrz Sc ?ool oF_ Medicine.- OX_YY.r'iENATED CHOLES T EROLS SYNERGISTICALLY IMMOBILIZE ACYL CHAINS AND-ENHANCE PROTEIN HELICAL STRU_ CT{JRE IN HUMAN ERYTf-IROCYTE MEMBRANES Fourier -transform= _infrared -spectrescopy --revealed --that - insertion of 20or=hydroxychol-esteiol into hurs an erythrocytz membranes (1096 -of total membrane sterol) immobilized-_ the lipid acyl_ chains to- a degree eq-ciialent to--enrichir,g- total membrane cholesterol by 50%. Raman spectroscopy showed that the amount of acyl chain rotartters was--rsot si¢nific2nt;y aBered by the presence of-20a-hydrdxythotesterol, indicating that acyl chain immobilization was limited to an inhibition of iateral rtTion: The presence of 20a-hydroxychol`sterol may - synergistically enhance - the =cyL-chain-immobilizing behavior of rembrane cholesterol. .n addition, protein helical structure was not - altered by -20o-hytfr2xychol-esterol. The insertion of- 7a-hydroxyc:?olesterol into ;sythrucyte membranes -resulted in-an increase in -protein- helical structure which was_ comparable to that-_observed for erythrocyte membranes enriched- with _pure cholesterol by 50%. - Ho»ever,- both acyl chain mcbiiity_and conformation cvere=-unchanged. TheSe resalts suggest a-synerQistic treha_vior between - oxysieroisand cholesterol :n-modifying erythrocyte-tnembrane packing. Rooney, M. 1E'., Yaz,-}srir,, S.;Eucuk, 0., Lis, L. I_, a, Ka-uffman, s: W. Biochimica et_ 8:ophusica .ict3 830:'33-33, 19,85. _ Other support: Northwestern University Research and U.S. Public Health-Service. _ -Frorn- the BiomediFai-Engineerin& Division, Northwestern University, Technological Institute, Evanston, -iL;-the-Departrment of Medicine, _the iritzker -School of- Medic:ne; Universi:y of-Chicago; the Department of Med:cine, the Chicago Medical School; and the -Department of Physics and-t.he Liquid-Crystal-insti ute,-K;nt Stare-Urri.ersiy, OFi. 171 r,. r. .e y:

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pathway--(:ep?rotriencs-C apd E?} - As was _ ss-n, _[l _ resa ft-, oF= t;ese studies suggesr =_ in -the studies reported here.- tissue expiaats from different leve: of rodent airways, from trachea=to bronchioles as=srttall as 2f3a -ntierons in diatne er_ were exposed that could inhibit mucin secretion {PCE;-pr stirp;;lal.ory prcd:icy-of the lipoxygenase - _ affectsct-, t-his could lead to- zacreased_synthesis of products of the cyeloox;!genas cascade -_- metzbolitm of arachidoois acid it_;`sese- -cells.- Depending =on theL enaymEtic .atlaways_ the exposed epithelial cells. =-They hyRothesized that exposure to Fristobatite coe:id affect-- authors-were_-ir;terestesi-ia_fhe,effects-of cristobalite, a knovvn toxic and-fibrir:<-genic -particulate, upon mucin secretion and production o` p.flstaglandi;<s and leukotrienes by to nontoxic concentrations of-^ristotiatite parricles-of ay-ize less -than 20 mirons,- T'r,e-- intrapulrnonary a_irways apd-into _thz small- bronchi~oles, slight stimulstion -of szcretion-- _ tissue from-differettt leie~~=of ;he-respiratory tree - As one progr4sses-deeper- into the - crlsto5al;te -has a-d;fferentit-l_effect r.n -n1-Jcin secretion -by explants -tif--rodei.t a'a'w.ay -- _ EXPOSURE OF-SFviALL ALO.WAl'S-TO CRISTOBALITE IN VITRO changes to-significant inhit;ition. r-n. NA T n Asd- S,ries, 'rol. t°:3,- 2eek, £. G.-and B:gson, -_. (eds:r_ #: F%i7ro E}jects_ of == kfinera%Dust. ,Beriir3-Heidelberg. Spr;nger-J-erlag;!9$,. Other ;! pPortY National.l:stitla:es of 3-Iealt!:. From the Departments of - Path,alog;~ Civil F ng:neerin g and Pl-~ysiologylBiophy;ics, University of V ermont, Burling*.on. CVTOCHALFkSIN D-iNTJstCED INCREASE I~'S'-ACTIIC SYNTIiESIS- A:VD CONTENT IN A VARIETY OF CELL TYPES - Treatment= of a variety of mesenchymal cells (normal and tran_formed- rat fibrobiasts,- bovine aort-ic _endothelial celis, rabbit smooth m;,ecle cells) exhibiting different rytoskQletal organizations and derived from several species with- doses of cytocha3asin v(CD, 2-6 -p>Va for 2D -h) sufficient to induce cytoskeletal rearrangement and 3ltered cellular rrrorphology result, in an increase in-the relative conterit-and rate of synthesis of actin. -These data extend our previous findings for-klEp-2 cells to other cell types and provide further evidence-for-our hypothesis that the CD-induced cytoskele-ta' reorganization triggers stimulation of actinsynthesis and the-resulting increase in actin content. Brett, 3.-Ci.,-Tannenbuams-J.-and vodman. G. C. Cell BioloBy-Internaiional Reports 9(3):?23-F-1L',19o5. Other support: National Science Foiendstion and Nationat Institutes of-Health.- - From the the DeDartment of Pashology.-C.oilege-of Physicians tA Surgeons of Columbia University, New York. - - 172 ~ f~ - ~

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IE4dk4BRANE-CYC:f._t+I', AND k9AC?tDYAF_I;CiLATION=uN'DESc THE I_NNFLijENCE OF [_ .'1'OCH!,LASIM KINETIC S.'.'DIM3Rf'HnPMrT :IL STUDIES - _ I I -Fibrobtasu exposea-t-a hig"F_c.+ases af_cytoshalasiff _accu-F_u:az-vcry-hig;-disc-¢t_, enddgtaFmic vacuoles. the merncrare- of ---:ck is lerives by interna?intioa g f- _pfasmaiEmma. Y]o rpitame_try co^fir_ms tha-._=the amoti9i of st:rf ace intericfrized,i3 equa1-to the difference=oetv,e-r the-csrig.ral ce.: ~lrfact area (hefore_CD) a~:h the reduced surface -area measurable after ~~-induced rounding. -Co:resp3ndiagly, there is a-aeariy two-fohi increass= in the ac*.ivity of _ tl:P- ectoenayme- 5'-nucieotidas4 (a marker for- plasma me7brane%interna;l_, within !he cytoplasm, after treat3-en; with t:r.=_- Macrovacuoiation increasesce:i vr~Iume-hy - 3p%.- Surface ne~sbraLe is ernali;cc as s.acropinocytoti- vsicies _ar a rate mtasurabte by-the= acc::^ruia 2gn -^f horseraditr _peroxidbs =(--I4RP), a marker of_!'uid=Pflase pinccytoss-._-3J_ptak----of HRP is shown-io,~be- enhanced at a:' times during exposure io_t:;TD and *M balanced ty-acceBerated exocytic ecrcling-of_ membrane ezcept during_ a phase ( - 4-8- irr; in- which _pin acytic _uts.ake, -,azceeds_ exocytosis. Vesicular memtisra_:e- accumulated -_intracei:at`~arly _is tar:s--_period .ia_--etained in- the _ . ettdopjasm - and by =33tCce5si_ve - fi15ioni fornts= Vac11fNe5 in close __ approximation to microulame ni-agg:ggates=. - Dnce estab:is:.ed -thi_s ne:v ma:.~Qvac io.ar tr_wthrane -cotnpartmeit is in -dynan'.ic ectuil'+hrium with ,he-celf surface and is membrane is cycled like the p[asma- membrane, in a mutua' exchange of pinosornes between :_he- several vacuoles and :he cp=1 surfa: e. In drug-free medium.-vac;;oie membrane -apparer;tly- rfvert;- to the surface by pi:iocy-totic recycling,--and the cells- =recover_ --n_orriai characteristics s-5 rr after=withara.val of cytoc`±alasin. _ Brett;j. i1. and Gotft'?Ln.-v. C. 4 lssrie & Cell E6(31):3?5-33r, leg4: Other suppprt:_Natiora;_Institutes of Heaar;, From- the Tiepartmest of Pathology, College of Physiciar:s & Surgeons of Cofum hia - ilniversity; I:ew York. disorder.- -HavyryerE ir-liver h:opsits _firo:..=a patient navl:.g -ne5natai°onset Ai.D, - hepatoceiiular pero;i_~taes were greatly reduced-in site ano- number, and sedimentab:e catat-ase was n:arked6t-ct.itninished. "1'he presence of increa.sed: co :centratior_s of erun* pipscvlic.aeid and the hi:e-acid--in~rmed+ate; tsihydrozycorrostanic-,cid,_ i^ the neqnaai- -A.! D patient are associated with a genf-aiize_d diminution of peroxisoma-Lacti,itiss that -°- was-noi observed-in the patient vsith X-;inked A: D. _-- Cald J`is; her. 3, et nl_ :-- _ F-E'Fc-OXISOiv1A?, DEFFz?v IN NFG_ NAT_AL=ONgL-T-A__4D-.`.-Ll-F.;'_ED- - Ad32-cENt7LbUfCQDYSTRGP-i-IfES = Accumulation ot - very long -chaiz fatFr_ acids in X fi~ked and neor~ata6 f orrta of adrenoieukodystrophy._(Ai,D) appears to he a_consenuence of de-Cicient-oxidation of-very long chain fatty acid,_ a function that t,as- been attributed_to peroxisomes. tero*isorees were readily -iCentiiied- in_ liver 9iopsies- taker._ from a patient having the,-- X--linked - - acsente 227:67-70, h.~g5. Other support: National 3n3titutes-of Health, National Science Fouadation ar,d Gail 1. Zuckerman Foundation. - 175 I

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From the Uepastmen> of ~aahclogy, Pediatr~s; and i+iE~~ro:ogy -an~ the i,iver fcesearch -Center,- ISlbert- Einstein Colkge _tsf: Mes.:cine,-'ilre= R:onx, 14i=; fise -=Departments of Pathology ar;Pediatr:; ;~euroldgy; C:.ildren's Hospital of ':ichiga t,_-and -Wayne_-Statv F=- John F.=Kem,ed}# Insti:ute ; the-3dhns=-1#3~k~ns F"sniversity_'S:.hodi of i~3edicls~; B"a}G n:nfe;= MD; and aockefeller University, F]ew York. ~-_ UL'I'RASTItLIC'i'LIRAL-Ati'D L1`-FOC:3F.MICAL DP'Olt#STI?;A'I'ION OF _ I PLROXlSQA%ri.1_I_iv CULTUR£D FIBROBLASTS FROM PATIFcNTS _ ~-_ - ' ' I- - WI T a PEItOXIS9MA-~ .- -,* iErCIENC_Y---DISOIRDERS:z= - - _ -- (pla;trSalugec.1 occ~liar.j i~perl?5tisosTies-~- ~brotl@~i3tiFe?1al =3yri4~roT~ _- The oAidation_ of verg-Iosg ehain= fatty acids and-synihssis =o!` e_ther-glvem:ip:ds -- University_S<.hopl-of Msdit~ne_-Detroit;--tlse Departments of Ce1t-Biolog3 and Medicine and Kaplan.~Qancer eente=f_ Ne-w York-E3nive.sity School of-MPdicirie,=Ne:v York; t-ha _ !CHR3` -i=_ a ra=e, inher ited nietabolis disease- cbsracteiized by- an apparent a135ence of per9xisoa.es, -an -azcuatulation=-of -eery For:g' ckai& fatty= acids; an d a dE <rrease -of plasmaloge_ns in tissuet-an-1 cultured fibroblasts from these pat?ent§ As peroxisomes -are _ ubiquitous i., macnin_l-an pe:ls,we-sxa:niwd r.:rrnal aria t;1?R-S--c.lt ze:; f;broeFas for ~- ° their-presence,_usirrg an elec=ron_microscopic h utochernica; prrr--edure fg- ti;e=subce::ulaF _ ~ localization af catalases a_p&-roxisorna= :~rker= enzyme =Sn:a1l (0 08=G 20 µni) round or - . __ ! s.t ~gh- tly ovai perpxisames, wlerf seen in=both=nornsal and Cl-fR3 iibro`±lasts The number . ~_- -- of _r+erexisornes was aza ;zed-.t:,oryno:r,etricaby-and ;ound to bsignificaFtly_ recuced- in -mI!_CIiRz cell iines:=_-Thes"4zsu:ts- are discus3ed-in relation to the u ndeclying des?cz in _Ve-rozis`mal funstioa and biegenesi3 i_ this disease. Anas. 3. A., Moser, A. B., and Gold'jrsctier herL. __ - Tne=journuo of Cell B clogv-1!' `7g9-1792 495,95 . _ ~ _ ©iher sup;~ri =1~iatiorea! listit:..tes-o` P.ealth. = 1 -_ -- - 1 f £i lb i A i a 53 N F f M: ='Iho a I ~ eri ns r_--g-eune, e ._ ronx,e jOj kizs rom thR A o o e nd has l Y; School of Medir.ine,Baltimorz, MD. _ l= iviti tid e ~ lf th h d i ~ l f ? as gct tse . ver pap es o -t e vasctsler e e past severa years, as3ects of # ~e metabolism of kinins and -ARiG- I by pulmonary endothelinm have -been defiseed. Th_ e ~_ - 174 harnogenates,-and many-enzynses.'-One of the most critica! deerErinaRts-of-tsre selec-:vity~ of metabolism of pertides is=t~e ~ocation-of theis in~cti~:-ating en7yrnec. '*'1-,u3 at i:~e = remarkable feature_cd=tAe lung is-the selectivitp-isf ihp processing, espec'rally=x:ien one _- considers-that mosl peptides are sndis,4amr,ately degrade} `oy=bloo` en_ynes. tissiie = quality o`-biologically activj! .peptides allowed to enter= the systemir circulation. The - Ti:a pulmonar;_ vascular be4ran be-considered af a staiiee gate =for cots_trolFing- the - PTct;tvESSING0F_gNcFIOT_sl*lSlis7 AND OTHER -3FI'TID-1;-S H-'ITIIF L4INCS = -- tSe lungs. Bradykinin a~d angiotensin (Ahiv1 are the m3st studied of ihe pi:ysiplogicalV s<eli_biology 3hai_ts s_great-extetlt determines the=access-of pepts'des to the-ir-metaf-uAi..~ing- fnzynfes. _ThE -psxseyt-chap:ea:-is devoted -tir the=piocessing of polypeptide hormones by in portant pept de=_ rtor.-.-.ania=prt~~~e5 b3~ the t+nV.. In the discession-sestion- of this= baper, i*.-is noted that :-he biological a.tivities-of t-he kiains Fn-d ANG II- disappear, whereas t§o activity of Al3G-7 is enhanced during circulation through various vascliiar - beds. To a large-deg:ez_the changes in acti-vities of-ihe-vas_,active poly[eptide: are -independent o. -eotymes -and=:eliular elements of-bls,od buc are ctosely related= tn

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-- -_:c3cus=has_ been or:--a-ce!a not prgviously knnwn tc-ezist untal the advent of electron 7nizr9scopy. S:doF h4~a! cels ;etecltveLY--piocess -a encs Gf v535activc substances, cell surface.. - - -=ano re,.Iated Feptsdea c'ya;4y_ =how _that the cr.ticrl, :n,::a rvT-tao~ oCcuI a'-- ., cea surface - tra o~ _:nct~}Qtiz en~ytnes, zceptyrs aq; the r$sease of grdd,c-s. A cr-a!!enge for the t'us :L -es-to - eyam to h-r_'s ti gecg ra-h, Ef_th_ p11.-: vn ~ry endothelial incfuding :4~ I a~i bradz Kin-ir.= Stidues of the pul :~oraary processing of ang;ctensins-- . Ryan. U. S. In: Fishman, A. P.-a;,d Fisher, A. B. (eds.): Handbook of -Ftiysro(ogy - : he Res-pirotory System 1. Bethesda, Nta- - :'he_ Ain_e ica_,- _r-h}ssot-..gI c _ 3,ciety,-;9g5,.--Chap. fv,- pp. 351-364. _ t)th¢r -suyport: -Na*.icny! Ixistitutes of I-Iealt :. From the Department of Mc1 ci=_e, .,: z e.sity of Miami a: ;oo7 of nt=di ene-; Miami, F.- - THE PIII_MOTv ARY _ ENPr.^THRIaAI. SZiRRACE -re'Xnt _yLa:s. = Endot=hc!iaf ce'!s_ z_rs;=- pctssm hormones, dr';gs; add many oioc_-borne -° substances- by n7ea as of_en'syntea;-and: transj>,rt ©rocesses. -in-tUt - some Tori'?o'.ev, i<!oE'td ,'he undLrstan diTg of endothelial ,,e-ta~;ic ~E~ ~~r ti es3as ~: yrsased e~am~ticbl;v in andathe!:al sur.`ace,_ s=5 jc'_L_We-?:at -s i se;f compIcx. ECs t?ssess e^do be!iaf_pr o ;ect:oss - antiaggrexatQry'- agent (yG12J._ Many Qf-these c3ClpFeR e7leta`-i~iic- reactions occur at the =- the=rt!tase vf,anct#:er va.sodilato., 1'GI2, aYd can stiMu!`te the Tetease-of a rower rtceptors and stirt~;iaZe tha-rviease of vrDsiacyci nTh;:s. nraevkir_in can amplify converted to angiotense5 Il.-_ BradyKinan not thus- negraded can act on -cndo*.hefia! --_ zinias and--angioter~in3 by EG=_ Bradykinir is inastivatya xhereas_ angiotensin I is - reincorporated 'ar3t:acellz9ar!y in tts n4eotid^e. Equally coinp'_ex=is the rtetabo!isrn of the -- -- surface. -Fu =c on a_I -ccr?ea!tx ity-+s -cxernN!ified--by ine n e;aho;isns r,f the adenine nucleotijes.- A?'Fr; ADP, ahd-A*hP are metabolized :,y-enzyrnes of- the =ndJthe!iaI sur#`aca-tc release -a:;enesi^.c.-whic!r tr:ay be irrnnediete-!y-taken up into endoth.ium and - ;,a, an~-se!lu;ar ~r:~~ucts in>eract a;th ECs vi~ spec:fic-receptoss -on ~he t~t~na! g!ycocaiyk_are_;iot weli-un3arstoQds Vui t'ze glycocalyt: ca?:no-be-:~isua!iz~d; it may act a.;- a--tno!ecular- sieve and +rovid? a substratum for-ths i±:itiatica- and- p=ogr=ssion lzf -and caveolae- a.,° GP!1-a.: a fuzay-coa_-, or a!ycoca!y{. ru=,ceions of the endwt!>r!ial- -= irr:mune;ogic reactions. _ ` -- - -=Ryarr._U. S., Ryan,=J:._w: an!-Crutcniey, Ey: I.- - Federation froteEvings.a4:00 B-4'3LY,-!4g5: , ,,~ q Other art Na;i?aa-, Jns±itute= of 3-Feai ~z~ and-the Resear1: Division, Miami Heart-Institute ldiin:_ Be-acn: FL. - From tl-:-eDtSartmcnt of ivledi,:ne; _L'nivzrsity_af >v3ia:ki S-csoe; of b#~~-inv,=?vliami, FL, -

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PULMONARY ENDOTHELIUM AND PROCESSING ORt'LA_S_M_%+_JO_i U-t_ES:- 1~~Nf '~h STI3i.TCTURE AND .-, WhiL- trme: lna9-V uf"_a,e consiaered exc u,vvlY sn ?er~ts of gas e=cha_nge,- they ;ave aada:icru func ona nak-xes ssartiy re.ated to such ezcnang :_ dtta srch act vitY _ is_ _ the eb;la*.y _to, prtcts -selsct_ .ively--hc,rts,ones_,= k rrrur.e =~f~~sr?c~ s an_~- Wher:._-°=Eatory-=`- _-_ substance: as thPY= pass thi=c_ug:: the lungs via the blcodstres:n. iiss the Iungs cdnver*t venous blood to arterial blood, they also regulate the-ertry of hormonal substances -into- t.he systemic 3r*_erial circulation. Through this selective processing. products of specific _ reactions of the lungs can influence specific 2ct2ons of tiss'?££ aF:d (Pr&ans at ad'_st3E'.ce. -_ - The first -level-oi.-con+par; mientation in the lungs_ is the separation of t14e_ blood suplilv -- - from th6-air-supply. Quits_clearly, circnlating-s;:bstances are u. 1ikely to havr,_acr;zss to enzymes beyo?d the first ceLisiar 1?.yer lining the -vesse s s the erkot :e:iun°a. °= he present chapter describes fenctios.- I anc structurai as;,ects of- the ir.ter_ction of solutes and colloids cf_-plascia v+Fth puit;,ona.y Fndnffir}i4i-cel:s. First the basic architecture and env;rollR''enf :.e the lungs a"t? £i)n'si(ir[cd, The atruetiAre and situaii'JR=oI the leJngs with-In the circutato:y sy,te m-wvhich make_ =;hzas-t_we,l ,s!i:_#-for gas_ erch_e nae -m3y_ weti =gxpiain h;.w the- l~ngs are sr efficient in processing sr,me hormones. 1aR :eft- level of compartmer_tation is within t;e -pulmonaro circulation aAd -is- governes by th@ -flow characteristics-and -relative surfaes areas of t::e vesseis. lrigures ar= giver_ in :ha sect-on which emphasize the overw:__Imirig sig:;ificnnce -of endothelial cells of the pulmonary- -_ bic:olorascula;ion in -praxiding_a surface for int=-racti9n- with blcc=u-forrae 3i;bstrtes, In following s'e~ctiors o~ :his- ~per, Iri?munocytochem,trt; End~~:ceiial iel:ult;ire- and 35irt?cr- ~yrccial~[.4~iG~- -`~E`_.~s3t;i°-:~ =-Fr=Jj?r?~n3, --=CiY~'©1ae, c.#~i£E-.-. -~sryt~ai7~a= $e`e-= _ _ ~ _ ~ - di;cusse- ~. It=w ~11 be imI"'--ru=~t,nr tLture-studis to cxa:ine to what ~~- _te nt ~:.~ul;tion - ° ° and -t-rampor{ -r!s_1?gYies ~ affeect- t21c oR_eral'. E u35£tiors-~-`--Rg, lil eI!dothFliiFfi.-a-c-ti~"."_e arid of endcthet,ial- surface stiuyturt---projections.-caveo1_ae, g15~~=a1~ ,-.^.zyme _ re.apt:.rs_,_- - the overall _fvnctionng nf t:ha iungs Fn.-n:aintaihing the quai:ty af _t.he internal m,liab: -Pyan. U. 5. ard Frp-k;2er-:erst n--, I:- 1_ _- ,_ - - c-= -ln: Said. i. Ie fed.): The Pu,rmar ar, CdrnYlnti9+t c. d .fcute Lung ;hijrry, AIou^t. R=sco,= r Futufa-Publi3hing C c Inc.,_I-9Sa,W . 37-5(). 1= - ir Danee suBnort:-Natiaaal Institutes o_-Eiealih: - -F'rom the Llnivesity of rO iam- -:S.#sc~o1=of_Ivfedic ne .inniar,.i, EVIDENCE FOR A ROLE 0: _t-:YL3RC,'-VY RADIC4:.. I1M;vtiJNi;-COMPLEX-i1+1DUCFU 4ASCULITISS Previously -it was ,hcwa tha*.- tissue injury o^curring_ aclte immunt-cnmplex-induced v a_;.ulitis= svlrieh- is cosnplemeni= and neutrophii-denendent, es _ signifi^antly attenuated by the presence of' cEtaiase, wuvgesti_r4 the nathogenic role of- --11,20g gy nrra3ed flo3nm activated -neiPi'p3rlTils. - WF fit_y:Y show that -sigai mSJnt -proteytion is= -'-so afforded by =pretrartmtrzE o= -aniiwais- with apolac_cferr.n, a naturally occurring- - ; ehelator of- _-iroa. _ Iroa-saturaiei _ iactoffrrin - is- devoid- -o" -~rosective: effects. -- ~_ - - :2eferoxa8n:fe mesyla;e,=2 syri=E£ 1'' ir(i c,elmal°i: }tas pFCte't t£T--i`{eSt.S~ Infils!on cf_- Anic-3ron, especially F6I:-t1, peA e^tiatex tfL& tssue I;l;ury. S=an;fscant-protectiotl ficm tissue injury-is also-produccd-by_treatment of rats with dimethyl si;lfoxic:e, a potent y y ger, l~orphoiogical':_y, animals treated with ?hese protective_ ~- h drox 1 radical scaven _ interventions s7iow the irfiux cf natrophis into s:cs of immune complex deposition, but there is n:a.rkedlyattefluatyd edema, littls or nohemorrrhagp, and littie -e-videncebf ! s

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. endoth~Eiai Ce}1-S~t2Fr'~_~=iti ct~i---'_-~~_traa`z '1-the fi:Ad:ngr in aanprttec.'.ed-a niiniEi.- inase _ data _S l:p'oTt ih'd S6 gge8t1!?t: t5at ]nS3rttin e°c()rr S3ee7.-1^~ei;.eti EnJury-~a? ~ linked to -geiierateo:. of HaOa from actiLat-d_neutropniEs and th. subsequent .onse;3ion of-peroxiae_ta ;he ' _ - -- - - - - liydroay-+- rasi,...at. = -- FliRivl, S. E. Gj., 3t`arri. P. A:. 3orason, K:=J.,and Till, Cr. 0. _ - Antrieaer $otrnFU c! P a.lho#og3~ t;5.3%S-3-g3; 1984. G1her surgar: b:aional Institutes cf=d?eajth. ' From tht-Department of Pathciogy; the t.Tni~rersiry-o"'Wreh'sgan- h°edica? -Schooi, Ann Arbor: _ klEpHR('jT~'J Xisr.-'NE?;r?R `z ; iu - -EVdL'-LE::.E Fu: -THE ROI t OF OXYGEN R AD:CAi:,S ai': A C.7TE infusion of sheep antibody t^ glomerular b-sernent membrane anti-tsBM ha-2tigEornerular ==_ basement meinbrzne). The aa iglon3erufar baeme.a -mxmt ane art:br~y has been Ac:a.e gfiom>ml~2: irjory _njhe -rat has been= inducgd-by [he ~,rarenal ins:a.:rte~ial end^'4n i}al o, el~T:Eis- cel s. _, ese dnt~ 3ugge3t that ac~?t- gto~ ru:ar -irr;ury peod :ced _ ~ ul3r _ - rats o:--o ev;dea1 of . ;tcy of -ertle: oiGwer protective iu-the ;nti-GEIsS mcdil. klorpho!ogica,iy. g;oaer_oa= from cataiase-prote:ted _$gai~t -en~otheiia=-ce!t- !alj;Iry following SyStemiic aCtevation of complement, was not The hydroxY: r~e`oa:=~~vei ge;; di:neth; 3_Efox:de; wh:ch has beea sh-o:vn- te proteet -- priatective_ effect of superrede dis:Lutae was not found to tse statistically - significant. 'Preatment of anima_s uriEE=i superozide dimr!utasP caused a-s=..aEL red=rction in the-~egrey = o, -g1bnneruia: ;njttry; again assessed by- a reduction_- in proteinur ia. However, this 33%-=protestior-ag~~+s' ZEomer jA: i,:~ury; as a=s-tiaed by Eeductirn in the yroteinr-nia. Treatment of' anintaie with cata'ase prey~ed-; ;n a dose=depe;~3n-t Eri9nrtr. As-nT.uch as --=inductittz of acute prcteic> Fia, wh=ch iseakz d~~ing the-first 24 hours. Following in;_EtiC?n of te,eAit!ir~o(~y--,-ar:'4e. in:etksr, .-gio:ilgrUl'ar _n "sl~ resulie~i-;-w3.~ _t'~e denFuis`Tg of gioraeruEar v4sc_;ar ba;emcnz=-mmbrane asso<.iated si,5 eat;ns:ve---dataage or - -?#e.E-n r'.^t:on SSf ~gl;~ ~E-~r PGdotFe-iea`_ veEi, and s.ozl SS- epitE:Eaiat-Ce'. =36t prC-_°35?S:- verified_=to _be of the var iety :hat is toa3-pEement and _noutsophi' iPvendent for the by aatiglomeruiar bas emeisi mzrst"Ireg areE3ted to H-0. productio=R fro~ activated ; - _ neutrophiEs. Rehan, A., 3oFrtson,X._ J., 'W;ggins, R- C,_Krnkel, R. G., and Ward, -!', A - LatiornF:ory In3esrioe:id5 5[(4):.',?0-40112, 19$4. methionyi-EeucyE-phenyt-aianine (FEviLP) and particu:3te-agents (iramune co:spEeEes- . _ - Actiyaaion ;def ined as Eysoso5-aE ebzyenc secretion- and generatiou of_ OiA of rat I neutrophils- has -beeP measured with the-use of varying doses of 3olubEe- s:irnuEi- (phorbol E atyristate acetate (PRitA); -calciu:t ionophore A23E$i; and N-formy_I- I Oihz7 aLpport_ blatUona7-Ins;5t4tes of Health_ - Frora the Deparimr&rE of }ote_:na: Meoieine and Pvt% I o:o3y, eh> t3ai ervi,y of Michigan - ~iedica; ~'n~f,l.nt~=~?rbc~E;= ~A:-II;:UT.`¢E#,-ACII'lAtIC~s:~iD EirEfZ't:F LY?C7iYGNASE - AND r'YC1 OO]CYGEhE-ASi; EEVE#:BsTORS - z77

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anc3 zymosan particlex)r _With-eitl?er- t:.e-csl_iutra :onopftore ~r the cner.totactic i secretion. =Wher cell s=1^6ti'd6fDn `?J_:ti_ IR57-sl9)b}i-p-Sti :uii-(i_CP-.mun$ Soii7pleF.es -c_-° zy7no>ari -. small. Cytoc: a!asin- f? -grea4 e:.'iasced-.t,a_ en~~me ~e:ease rezco ~se to- a:~e =-c4emotactic_= peptide tsu?=ttas: fiit'ee,fecF= -an ttehtroy'r.if responses to o;he:- so!uule air=rli. Tl:e cell resaonse- to PMA resulted-i : the gea*est production- t;f GrL wit : signiCisan: -I;;r.ya-W ;FMh.fs), Euhstantial enay~awrelea.s.,~ joccu.e:{-_ it.a ;ha arno"t Rf -%L producev very- fFar"s":lesf =was Studled; sign3'rmca:;£ a'no33nts of enzy:ie release occurred in-p3ralldn with - the generation vf_ substantial=-apsour.ts-oC 0,-_ The presence of cytoctalasir B-enhancRd the Cell responses to IryilEAne .ori pleiec but had an iitf=_il`iitoc`r eife-ct on %yrnh:Fan=-inCi Jce(: _ responses. _-As--eape-c%ed; t_h?'amount cf° lysoryu_te secreted by stimuate!` rat neutro,hils- tended to exceed the an:ount o: 8-glucuronidase released froT, the san:e cells. Neutroflhif__zesiorfses-_wgre__invest',gated in thr:_ presence cf-arugs that_ were demonstrated -in the rat reut-efhil to iihihit either :.`,e ~~ oRygenase oi the cyclooFyganase pathways. tnhihitors of the cyclooaygenase pa_hway (indomethscin, piroxicam. ibllurofet ,B4y733i}; with few ewceptions, consistently ~-thanced the enzyme secrc.tlo7 .`ea'txlrs~,- wn~c '~t~ig ts otib /Z 3ener33i53n were less _ca~a_?-cEr_ vut'ter!. ed _ to F'_ predomi:!antly #nh-i~i~i;y. L~r~~g-s_ ith-inhihitory et'fects on the °°noaygenasc p: - " , ai _way Unordihyd;,oguaiaretic acid ar,d uaCazat-rorr.i had si&nificant .nhibitory- effects on btl: enzyme secretioa as well as - -enerairon ;~f- r',=. _=Thecy-dzta=sa38est-thai--act:vation---=- respz;nses (enzyme -secretioE -and- (°is`--generation) of =: et t.eutroph;ls- may be- dissociated - ;i.e., one not=aaw+ays acompanying-the-other)r _gtrrther_;-it-agpea*s aha: neutrophil _- activation, as defined-hy enzyme secretion,--is enhaFce& by products of the fipozyge-nase= release and t3a'-proiuction hy=zcf waied mt-_nauirofir;i-ls may ;u_ under z_parate_coni s.. pathway izns=suppressed by-~jT_?od+ictS.of-tf7f; cytlooAygena.s:-,^,".it_l:wa-ye l5viPr3tion-of .{=.+ is r.::t 4f:ecied=in such a-i.fea:-°i,et.,-riaFLue_. Tal:eg--tosr-tlRe.}d:e -dat`r,-sagge3t_-[hat -'sFlzyen_ e_- - = -f3'ard r- A:.=Salav:kt l' C., and :oh.: s iK j Arnerica.n lortrne! of r'aarcfogy 1!b_223-s3Tr I--fi4. Other suppora: Nationaf-Institutes of Health: -- __ ---- Frarm the £epartae wof f'&I'r_ ilogY,-Ah t_nwe;sity of- Michigan -= Medical -1-;~oof;-Ann -fS.fFFEPENTs-ATfOf±I OF t! '-._$i:K:PN LlrvXE'r`s:A CEi.i. i.INE A'.n.'D-EXf;RESSdi1N GFC`JLLA£sE:^IASE INHf-B:iLl: A hu ,~tancoltagen..se inh bitoe=(Cit of M, 2J,-c?jo has been e.=_tensive y har; terized i!t sk;n fibroblasts_and Ide_ntifiec in a variety -of con:ertzfe -,-issues- Ryciuse Ru-_- a.veoiar -rriacropl?a~es -Fy-rtisesize -and s_eereee= hsith_ a_ coh~gaiase and Li that a- immuno!ogicalls~ and- functionally identical to their-crunt°~aarts in- fibroh!asts,= we studied the prpd;ic3i2: of_sizc.h.-proteins ayan icr!m2tu.e3tua}an cv.<- line(Hi60)_ that_ can _ Se induced to-stifferen.ia.e along ,sono,-ytic or granulocytic pathwxys. The cells f2le_= to synthesize collagRriase-t;nder ony culture conditio : tested.- Howe-vei, upon exposure to -l,25-dihydro--y+ita.-_tin-L?s or phor!+o:-esters (P-n:A), both of whirh prom-ate_i.,or:ocyiic differen tiation of -4l~6v; these =c=l"Is xyn;},zsi-zed and -released-_Cf, in a 4ose-deper-dent -manner. Furtherrore. the ex_tnt= of C:`- expression was para=-le;ed by the-acqnisition by such cef's of the monocytic marker _o3D3, indicating that inhibitor production and -different-iation are closefy__corn lated. Th?s Cl=w3s isrl.muaniagically-and furlctionally - -- -

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m I& 5 ® ~ .- _ ~ - - 3deuE.at_tc that ~r du~~d_ oy h~t~crokhages= ~a ku~a skin t:b vvi~?_. The qua_r_..[~ of f sy _:; e4~o -by _ °t~u et r-u a=ed ,ls .o~ ~ t ~ : ~id ~~iac:. : :9n yrOi3u:Cd E j/- , 31_i ~ _<.oin'3- 6?=, PTp& Ve 106 ~ "sa3 pe.= day. -- Iu C;~ntrAS-t- andiffereatiarPd it:.6t~ pPl.s-bcooseed 'it1r-!;r ~~ -cEtecta~.e ~~ ~ i ~-?~ g ~e _ 'i?~ ~ lls ~0 i_..ce 8o_ '31MI1u:cCytw Der_ yay;, tm er«. rigly,_ wtie __Hl MI_ cails~ ere st:m '~e ditfereniiatio!I by S'irlle3}l, i~-su!f6xi(3e 3£ T 3r3ic a^.d, ty ay B:Sie produced the --'H.F:+2CV!!i - - ' -' . -_-' -°- --- _ ---" - - -- - - - -. ~_ - _- -- -_--~ . - _ . -_ShaY!t, Z. u.. WQeg1$_ H~C. es~.flE- Prc?ce9dfrg-, o' the Nnfidrtal r--l+e.r, -y. S=9nnes oi _ ehe VRrtQd St;ates -o; - Am?ra 3f:ZY80; ±a4. ~083. ~ct~e! su~ ~re' SIe~~~a~i - n ?!zut 6 +ealth : rror the Gi ;S;on oS ?'1 Bi,logy, Wash,2f0r ~,.;iversity_~h x,~ _ of~ed!.,.ne; ~ivis;un v? Decr:atoiogs, Lz?~ar:n;erf or ed:cine, ,, The ~ewuh-Hcsp=?al at Wa~hin__ bn U aiversit;, ~~e~ica1 Ctn,er, St, iot.!; and Divisi~c of-s~e r.atdiog=.s re~r_oeent cf Medicine, Vis- -M_dicai CF.;ter/Uni3? srof Tqr._nes-aoe Cpfite for the aith Sc,;ncc-ey; Mee°:r? :2. i;: ®

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FP:IEiILI,3.t,-=A: GTiECATI->N iNCI=RQS'i_C-(?BZTR1JC, c~'.;pt7LV.O" ARY DiSEASE: USE .f'i'Tis i66I INERIt isj~Ea TU A'NAI YZE _I's IERtiEt%IATE Et+I`l;RarslVTrtEidTAI1 AND GENETIC .~~'ISK EAv ;-QRS exa~- ~--- T ine-th nt ic k7 t ~ f- i ! n t l ; ili t f i- -69 spo+rses of 10:t noV:pu:-rvnriary patient controls for the T,evaler+,:a of two clinic,a: . O r J lo t O ynv e CO ro it en a iani Fene ~CtOr to :F r:s arn a .-s-aggregation in chroniC obstructive ; ?trlrnonari= disease (COPD), -125 first-degree (1d)-- relatives and ~ 56 5pou_ -Of-1 `s_ "~.34E3 ;sat.r3ts 'r'er.°~ ei'par°d . _:}? 222 1.4 re,Q.i 'e3 end- ---- vita, capaciiy? :n^ z) cttroris bronet+:tis rLB° ca~igh ahd-sn,tu., `or ~--r*~.:nths ~r yzr - - outcor:e- _1) aiSways vbstructson iA'~; : s°c Torced=ex,~irat;..r~ volume s 6g~e of forced - - - . ~_ _ _°~ - - for =; years). The loglinear--molei_ was used to study airect_-and indirect (f.e., those - i r t r e ir,ed ated_-by -c?ser isk factora; ow-ppneh_ts o tam_ ia agg _ yaucn:,_ _T1.1ret aisk *c=;s ~ " were foc:n;idn be iQCepens#ieas>ociaied witls av~ a:dio: ?~r~ ~ ntitry -niaf: ~fie;ency-= I ~-_ !EiZI ai?eie), _ per-s3r:a cigazeta aaeakino; and- -p-re-n;al _ gar; t:e _:r.oiingo lae -a~;e_ d ~ relatives ef~CSI"D patients -were m ore-li&-ely to have 4?'si allele, be k>;av~y smokers - -- -~i- - _pa&~s per day_). and be e.~.~f~e~-tS parEF tal sithAir7q than ia:adv- of ~o1=rC~, $he~ -- - 9-~_ -- three factors also OZnstiYut£d indirect c,~l`i~nePt`- of 3i~iiid: Eg~5r~4`3':OFi. However, ~ after contrc!1'sao_for-tFe three-=factor,-7d relative, oa CC?D patients_kvere more 'i=:R1y to F= have At'!-ana CII3 haa td re-laEi-es e:-controis (airect co:rtpor:en;,. ;hiss -direcq c?-nt.monen` } sight have a genetic b-as:s=:reca>se no-such asso. ;tis;n was soundl- :vhen spouses instead #- - ' of le re =_atives were comparea. Thus, both s: ared cnviro nmenta3 factors (,-ersonai -sni passi_ve smokirg) and s-barecl -genetic factors (oi-ant:-tfypsin-and a poFsible airect ee=re:ic _= c43IF:ptin~c:) i.fint?ibiltc to fam.;.i3e a _ggrGoatii3 n-In-i.l7pD= Ti.E=!O~'il58af 2 useful t04l fs?r an=lyg3fg-faOLl;al _gg:E,'rati-on in d+.S«es of 3n_;iii:?-?oTwtetIDo;Os~:- -_ h.hry,-IN. J.>~e~ty, T. H., Ioclnran, ~+`_ S.TS1*, S. G,, and Co:wn. B.-Fi. Genetic Epidemiology E:155-I6u, 19a3. - Other Su,~gort: Lebanese ~atiEnal Cou~c-if ic "earch and !+lat=~a, Institutes of tiealt=.- - s~-rism -tbe-Departmsnt3 of E??fdenEioiOay, B;c,t?tisticS-a_.l- Enviroec:.i~5'Etai -`-:f=itla-;cien4es, The .0h1s Hopkins University School of Hygiene and ~ -- - - Eet+a aYi_ Public #iea:.h, Fa't' Qre. HEALT:i-PEL_A_°i'=cD-1;YCHDSOti:AL Cfiti.aELATES O~FfdEURi3TICISSM. A STUDY --OF ADULT MAi.E T tll?tiS IN FINLAND 8ome- 1-iCal?h related psychosocial lc.rt=:ates 'o the Eyse ~~k neuro'icisn: _sca'.- w2re examined in a quPytiornaire stydv n3 15550"_-!nonozygoti_ (Tdz)-end-3?55 dizygotic (LZ) raale_ twin pairs represr.nt;" the edult male_ txin- poDzlatinn in Finltr.d- in -an_:yses of- the individuals,_ 34% -o : the variance -en. neurwtici;:n 'was associated with: psycholr_,gica: -variables (siress -_of=da:ly- a,:,ivities, life satisfac?ioF, quality of sleep, arcd extroversion-tlre-eFplanatory rate of this variable set was "s0%), psychotropic drugs- (5%); alcohol - (4~z,), and s:~o-ing (~~1_. Fvnroticisn= was also assOCiated wit __ sOCia:, :ii~ -ciiadge, and medical variables. - In pairwise ana3yse:.,-the heritability estieaate (h=-) was 0.54 for pairs-living together and 0.39 for pairs living- Ep:rt. It seeatsthat treritability_ _ 180

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i M baclc-ground variables showe,rs similzr intrapair _difi'erencez as between individuals ir, the - -- es:;t:,ateF are con.oundeo by the closer intrapair r=latioteship between memb2rs of -hsZ. than -DZ-pairs. In =pairwise analyses, 221% of the intrapair dif_ersnce oi reurotic-istri-ir_ MZ pairs was assoc"sated vrith intrapair differences in the aforeatentioned variables. - The - following explananatory_rates were-fo'a nd: -psyolJgicafi variables, 21%; prychotropi_ dregs; 2%; alcohol4se, 2%;-and ;m-oicing ,-1%.- -.Neurot-i:.-s-m_m z'- pairs discordant for - following- variables- service- vs. farming- work, use -of alcohol, use of antacids, hypertension,= heavy physical work, quality of sleet;,- chang@s of workplace f or - negative reasons, smoking, ai<d uEe of tranquillizers. It appears that in Finland, environmental factors explain at least E1% of the variability in-neuroticisan, and that factors determinir:g deuroticisra are also associated -with health rclated 1- ehaviur such as smoking, use of alcoi.oi and psychotr_opic drugs. Koskensuai, M., Langir_tvainiR_ 1-l., Ka;,rio, '-, and Sar'a, S. dcta GeReticaeJHea:=.^.e-et Ge.-:=_e!lo:o;iae33=307-'s20, 198d. -Frorn-Ehe Dep4rir:Ynt-p£' evbliertlealth Science, University of f:elsinki, Helsinki, Finland. iiIdNISIiT1r1NS 31 AItE-ABAl" 11 `r'nLlr.?A T;4N1nr_-.' fvf:Si-i`r; - _ irN:%.R^NlvlrNTA4..F~iSSih3ILARfTY-A;J-D-lk=EIr,l-IT A H£a1H.T - Within the FinPasl: Twin-Golto.- t-tf iice=sexed aduft twin pa`sr_s,a suhgioup of pairs=_ - separated at an early age Iras been -;aefltified. -;a-1O _pairs; both icot:viRs _ responded to questionairys in 1975 _ aid 1979. _ An ecvironmentai dissi:niiz_rity score---.vas fdrined :vhich i ---°- corsesrs ot items on Kt.etnef iAy rw;ns=naG uvea ar[er ser~arat~latr;SYn~ same -camtm:nityr -. atte_ndesd t~~e sa~ scn'i3., were in the ~~me grade- at-schooi; fiow-_ten tney -met, how one pair-changey from DZ to-hiZ. The following intrar.iass edrrelateons- for heigh3 and weigkr were found contacted-for blood sampling ;1' b'oodgrot:ps). Of 15 pairs with no zygosety d`iagnosis, 10 responded (I no address, 2 abroad, 2 refused). Six--pairs ryereclassified JM? and 4-DZ. It:- i2 MZ and 8DZ pairs ;ndergoing bEoidgreu3-determirsation, the i:assificatiart-of - only -0f ten they met cptnrnor- friendr and relatives, and whether they ~ttended the same clubs or n9t,.etcr To-validate dhe-Tzygosity diagnosis obtaiaed~y question :aire ;n 1915, those_ _ palrs whose Zygosity-Wag l:nrtnown-a?d those Wtt hthe lek5t cont-act- after se-paratiL'n-were Age at - = No Cases kY¢ight= --- Heig4t separatiun- MZ _ DZ =_Mz= DZ- MZ DZ 0-5 _ 18 - 61 L.88--0;1 0.8A -0.70- 0-'0 -30 95 _ 0.87-030 0.92 0.70 =_ LanBinvait:io, H., Koskcs:vk,y,=M., Kaprio, :., and Sis.onen.-°. ,4ttgGenet:cue.N_edica° etierrtetloi;;biae-33.21 -Z58,i98cs.--°- _- ()ther suppart: YrjcaJahnsson Feun.d<tion. - From the Depa: tment of PubJic Health Science, :3niversity o" Helsinki, and finnish Red - Cross-Blood Transfusion Srrvice, Helsinki, Finland. 181 - 1 r.;'' ~ ~ ~ - C>ot

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°FINNISH-;'Yv`-I?VS REA'cED-APART=a'I: PtIM::AL["1'Y?ACTO?S : his att3dy-is based on-A-atz -_fso%n°:6;-'adult'twiz-pa'us separate,lnt ILryears of `age - individua; data was studiesl.=_ViR overall e-ayl-anatory=rates were iow (2.1 --4.496'i. ':'he 0.83). The effect-o%- sepflration on ¢erso7a:ity- factbrs--by- aHlalysis-of variance -9f-- `_ 7-30 pglr-ta) measuring the environmental dissimilarities after yet:arat'soY-(reliabii:ty in 19;3 Later in 14?§ a'QlystionpaiFe sent to the twirs reared apart yiekleda scale-;ra»gr - obtained as part-of the quet: oRrra;zt-ityd'y-carr+td out ir-t.e entireYinnic:y` Twir=-Cohort scale short fCrS]), iife=,at;:fGuie- (!'S.V(~Aiia=d:! at.::-st?.e3Sot ~ai:~+4c°, sv;.ie_~- (SDA1=tas-=- Cr les3.-InfCrfrla-=-:7r, on Crr$or3a!iTy :3csor's: e°.tr2d@ra9 aPf (-E) -a3Zil Ce2%oiiciam -('Y) (s~.-pt = =_----"_ defi nitive si::dy=groyp wasf,srned by ;viec;ing those pairs with a- dissimiiarity score greater-thfln-13, TISe-f3-1Qwitlg-.ititr3`-'as"aCCrreiatiotls 5:'sre `vgta-aed_: - - - - Age_a - `:c.nf cace3_ F - - N - - - ' S SDa_ separatiue= - MZ I3Z,= Mj -- IYZ - FItZ- DZ = -- MZ~ DZ ?~Z DZ 0.5 W -- 61= 0.40 =O.!T - _rs.34=- "7- _- 0.22 --0.18- 0=04 0.`st.~- 0-10 30 - 95 0.3&_ 0.1-2- 0.25 - 3.!V 0.40 0.19 (Y.06 0.l2- ~ar.gitrvainio~ H., If~prioti s., Xcsker r~. ~`:, and i c..nqvi,t, J. Aeta ?ieretlcae--Medicae et 6gy+tellnl,;gaae-33:25-9-25A; ld. _- Ot1,er support: Yr;it ;annssarr. FouTdatioir From -the I'iepaz.'ment, of Public Heal~ Science s:.4-Psycrriatry, University of rsie;sinki.- -= i'S i CF-II A;'RIC HOSf'I -t-AL.IZAT~~r: iid Tk'3NS -_- were-con:pared for and-alcohviisrr- Tecora-tinkrtge- oi -hosp_ pta7 .ecords_aF; dea.h-certificates==or=t:e=years :9Y2-1979 waacarrivd ottt forpersons ia ehe-- _ -Ei>=nish Twis"-chor2=(18Mr '_-ike-semed twin =.,airs). T,e--ratio of tne number of ~= observed 3s. that of 3x_pectea' 4oncardanz pai:s aid tme -ratic of concoryafic- rates betwe@r, MZ and DZ- pairs w_re greater~ -an?ong ma!es- than `ferna1es,-2ndl 5reates-_ amont,- young (40 years old cr-less) th30 ane;ng older pairs. T he highest difference ivas found in- ~_ schizop'±renia and the lo.ye3t 3n nPISrC es. Pairwise concordance rates for sc:rizooh-eniy- _ (1!.0% for :'fiZ and `1.5*- for DZ)=sEem ta° indicate grtat-environmental influence ;i.gl: ~= proportiarn of-discord=nE -pa:,s}_ with apparent genetic liabi!ity _(u.1=fo!d ratio in concordance -between- MA °-and--I33 .airs}. ° In -neurotic disorders, the difference of pairwise concordance ratesbmkweel MZ-;nd-DZ pairs (6-.i<% es= 4.0%) was eeate :ow, not strongly supgcrt:" a genetic--hy~otl~esia;--Gr the M 1Z= pai~s=;,onc~arlaF`-for- psy~hiatric --hospitaliz.ation, 47% had iived t3gethe-, for their -,whole iifF tie:?: oi those disccrdaat 16% lived toge`her. The correspoadirg figures for ; Z pai<s_w_re !?Sk apd=i5%. T,'-,e effect of - ~ ° - intrapai-r relation ships in disease-con_ordant pa_=s should be e taken- in:o fl,cou:rt -whern evaluating the ef-iect-of-genetic and en-sirorurentai-fattorsinpsychiatric diso-:ders.- _----_ _ Kos:err.:o. A'., Langinvai?tio; e-i:. isapirio, J., Lonayvisi. i.; and Tienari;-P. --- At'.ta freneticae ti:edicae et Gene_Ilologiae 33:321-332, 1984. 182 - Hospitaiization rates_ of =mdnnaygot'ec=(?~t'_) and difygoliv =(DZ? twir pairs in _Firl land = _

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epartWe:~=of F~-biic 'riealth~ie .c~ a~d ar~artmof Ps;chiatr;, tJniversity _- Froti the C or F~eLi n~i.~i. sr:o=i~epart- of FsycFiaMy, ?'n:ygrsety-!_^uI-, i3iu, :;anr~ __ SNORING AS 41:iMK -Fr_!_C!"O°_ F.;h-_HLFcRTEii`Si_^~Ld AND :%.xyGINn= Y.-C"-t'OR;3 _ _ - -The assxiaticd ~ir!or;-ng-as;tlr ~ypetezl.oo and isch er4ic_ ` ar' d~easP _~I:i"#~) was tested by postal qsestioenai= ia apepoiation of 3847- men a nd 3e`ti4_wo *tera -aged 40-69 years: IiY~rte~siuo assocta.e~ h gniv s,gntf,_artie t~itii s-,oEing tr;_ e:a:i- e risk (RR) of hypertension betwge , habitual snorers an_u-nevrseorg s E iflg 1.94 . .n men and -3.t¢ in - - -wo:T:e4. T}iSs a2soc'_atioT waA a;io found Y'hc`:e adjusting for ?t'S$y~FiLaSs iridetE. A -- significant association between angina pectoris-aid hab.tiEa_"_sno-ri:lS was observed i=7'nen iRI? 32:221. Ik-_svomen the relative rislc-was rtot sig:ificent. An association between ha-b-itua'_ snorin#- ;nc-.angina- peCtcria= in men w--ss= a.3c, frauaR: aft-e_r al,usiing for hypertensio: gnd bo-:vy-L ass ~rde-x .°R- - 2.01, _P=!].0i).- The zeiztivF- risks -for _ n yocarc;al :4farrtton_ aj;_ hospital _2a7lss~~ f~;7'_iliD _ for I!ab;rt_ai aflo~rs _3v~e _ - nvn-siS32if1ca_'It. -- -'_- - _ -- - -- - - _- -- ° - _ _ _ _ _- - - _ KiisKfAFz_~. M_Bt-t'ci. - The Lasrcet ;I1893-F96f i4&5=- _= OtFtgr--srrppcrt' -'I'he-_Medi_a: ResearCh iou_n,ie, A-cade_Tw of F;naani:, and th<.--9ravo-- - Nur..:i Founda,ion: ~,_---, From tb hs^_ISaranen:3 =f-rilb'-le r'ealtiz'.sctence and _Neurr_: oy- nevgrs -, of -S-Ielsl3tk3, - Fimand. CAitCgl'e_STJDIE3_1N TW'IizS AND FAMILIES OF TWINS -- The study_of cyv.^(u_ sa ce for canccr nzwin. Qa)rscaz be uEed ao eexa,m- e~the fois~ of heritaht : `acto= i.: carce=- ~ 5 :nRish ? v in ~~`rt - - it iiKe-s°zed a u)s mt , -pairs = 4ora-aefDre 1958 Iin Finland vi h bo?h co-twins alivt in :~6'. !n - _ i<,_'+5' o :s) --_- was - - - - linked to the F,4n1F -.-y'ncar. {k.c Str-3 dat to , yield _ Inc1.1°tka ~d3e- f __ancp- .i5 to= 1rJ8 i. __ Zygosic_v-was deteTr>Zi; eu-bv_tbe yati#*d Gni'sEionnaire me:hod in 19E-3.- rE -total o: 1,112 cas;e o: cancer was=ro =nd in t}e tw4n- series uo to-Iz8, among : u68 iwias.- Ivlu!tipie orimary catzcer=uaszound amo .g- 4D.persons-~~ 3a6),--T1-ere were : 33 MIZ gairs-_ discordant _fcr ca-r._ce.--,ar:d 18 -_pai_s !n- 'vhicn byth ;*sarnbers= had- cancer (Frybandwi:~e cor:~odyr:ce r,reF13.496' . :n tke_I3%pa;rs +`ere were4-7~ c.tscorda;,t pa.:~__ - and= 34 ro.-.~r~ar: ~-~ (pr .ba^dwi e-~!reedanatg--_ i'.~5} '`he c set-c!' ;_w = malignancy were excluded (basaYcell=earcinot::a,-:n situ uterixte cervix _carcin osna, urina_; t:ad~r pa;,il[roasa, ~of,s_y?hcm~a e*~=aki maelofbrnsa) -iitaligr,e ror t.~7- bairs were I2_~concordaFt an3_2(ii Aiscor,;Q. t -(raie 3~ -and fo: DZ pairs 23 cn_ ncofda"t_=asid 413 _ -._ di"scOrda_nt (raie-t0.Si%J- -- _ _ - -_ - _ = -- - --_ - _ -_-- - - -_ .-. - _. -'1'he-an3iysiscttaFgrdancg,itiieai';t hat oaerall-hered,inry -(2ctc?r3 d~ r,ot-S,ontribuEe = tr wch to=the igcider.c° .f ca =ce* .: ,_ in accordance .+-ith theTpid nx:ogtc r.rd~ncE fo:= -- The primary r~~ie o-f er,~~.:2.^T?rt-tar?acta"s -y ?he-?sIQ1Gg; Of FF:4a c`dlrCtzi .i. _ Kaprio, J Koskgm_a:o. rt! ,?'eRao, , dangi:;:?i-io, >z. Fukkala, 1 Ri?a,- z, and Sarna,-S:_ _ - 1B3 M_ 11 IM a ® I E r..e _ ~'- ~

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Tn: Fam:Ira1 Cmr,°er lsi In3eM atiortas ;3esa r .=^en''e}en c, B_= 1V"== 92 ;9 - (Ka_rgee_ Basel, 1585). Other support: Sigrid iusc:ius-kou ndation. Fror the Finnish Twin Co-±ort Studyi5epartm-_ent_of Pub:ic Pcat*.h Scienca,- Un:v=rsiey- O: H@Yinlei, and Finnish CS`cer ReQistry. Helsinki, e'in='s7fd. 184

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A`°TIVE PitOjR:~"LS- Follr.wing is a?is} of the prineioal investigators, or = in"stitUtions,-of 6rOiects under way or acytivateti in t_i e,^.^,iod _ . sins:, the p~eyinur. Refs~t; ;ogether with th reseprctiv2 pro;e~ t141es. i,ouiple*8d projects a'g listed 3 i alatrsect;oic:-- -- ------ 3RI?CCIgAI. ]IF:V?''-1-Ta3AT_i3R PROJECT TITLE OR INSTd'YTU i IJh' - ROBERT H, ABELEES, PFt.Li. - Development of F33stase inhibitors --Pru;e-ssor of pioc=;:cmistry. Brandeis -UII;Versit'+, Waltham, IaA_ LEO G. ABO'u0, Pt.D. - - Pr6je.ts~^-' o/..Rrui~c Research L'ied-- Bao_v''..°.7:istry,-Cente' for Brain Rysear-ch-, Lt~-iversity of Rochester wledical ^enter, ;iozhester, Nl`_ DOLPH O_ ADAMS, M.D., i'tt.D. Pro;essor oJfath.^.ogy. C3 ske_ University .:e=dica: Oent-e-; E,urhaat:,- hJ0_ h:icotime t:ansf=*-d _positcon-iA ;iver cells _ Role and regulatiort of protein phosphorylation du: ng ar icr ;phage - ac tivation ~ EF7r9 F 3'°~ A~~t_iR,iR, ~.0. _ - - Assist¢rlt Professor of Patkology, L.F: ;varsity ol be.rG?oa:; College of __ Medicine, -Bur-iir,gton. ;OIIN J: ALBERS, P:qZ-. Pgsearch Assvcciat9 Professof of Hed__sine, University of `n3sh:ngt.on- vchoel of Mesh,cine, teattle=-= HARRY N. ANUON-LA_^,ES, Pa.li. Professor of &:oche-mistry. Harvard 7lniversity Sc:iaol-oz Public tiea.ch,_ Boston, MA. ~.r.- Assi-sta.a: Pro;Fssor-of Pathology, Thie_ Jewish Hospital of St. iou5s, MO. IRIT AVIRAM, ~"r<L3: Depertmznt-vf 9i--sheR;rstry, The Facult; of-l_.3fe Sci= rmes, Tel Aviv Uairersity, Israel. - A:r-ay muritr secretion: Gfferts :;i=.~£nwcts- trom-h.act~ria associ_ated wiih chrclnc,~ _ -- t7r)Slchltls High deasry Iipoprotein quantita:ioi - Brosynhes~F at:dpsox_ssing_of I'DGF-iike - polypept:des in human rhalio zrt ceils in _ culture Giolsgy-of the lymp hol-ine; soisb:e i_rnmune :esponse 3ukpress" (SiaS} isterferon-activationo!' s,!p:esso: T cell pathways - Iso la*.ion, propertits and physioloaica! 4uncti_rsa of :Ieuttophil s'3cx: roTe o- _ BI;RNARD M. BAB?DR, M.B. Pg.O. Studies on the enechanisr:3 of actiYatirn of - Professor o; :yfedicine, New England the resoiratory burst in neutrophils Medical Center FIo3p:ta!,-Bsston, :4A. SA aMULL BALK, M.0., -Irh.iX- Serum r-nito$en ho*?ros~es, ions, viral Pathciogst. New England Deaconess- tra-r_sf:oraning genes and tumor reversal in E-_IQSpital. Boston, ?+~.~-i. spV-ro5rlnte aIISd ai_stoP-oneovis ir°itiatio ro_s --cel_l repEication _ 185 0

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-PitIN;'3PAL ?YVEi.'ICATOR OR I:_VSTiT',TT36k - BEe-INE-! i -;M.s JOEL S . , . _; . Assosia!e P.rc 3esscr of ,:;ediririe, - Hosp9ta'_ o-t the Z1E:'?ver5ity.,0f_- ; Fvn7lsyivania, Phiiadelphiz. __ - "; _ _ - - !~!Cf:A~iv ~_ )ti':NCi,-.1s:.1`~i. - Projessor of bfedicrxe ; em.eriwJ, University of Southertt Califoe nia ~ School of Me3iC-ine, i.o-- Angeles; lisiiir<g_hssori!!~, f'alifasia Institute of Technology; Director 0! E:prin±eh:o' Cgr4iology and 3csea,iJi., 1)eve:ay.-,ei:. Htint-ngton Medical _ Research-institotes, Passs;en3,=S=A. - PHYLLIS B. BLAIR, t'H.D. - Pro;esso: of .rrimai:ology Un-iv:~itY of Califwnia; Reskele=y: -- - - "i'HOMAS R.-BROK.ER, F--H;D. _ l,SSOC7Qe=e P?ff,;e8sor_oL Biv,:l:;MisiPys -Univeesi:y of Rochaster S>hooe Qf = ac_e3izatiob-cJ the pla#ele.t fatri nog-ea ~eceptor Coronar s spasm; cerchaI rnicrocirculation Eegulation of $atu=al-Ecille{ ce l activiti Ce!lulay transforma,_o_r._ hy-papilEoma ;i-es rec Cl:?blnartts v?ed=cir:e, Rochestw ,1# a DC3[tO'THy L_ BI-UC3-IFLAs :EN_ _Y:r-;i. - Oncogeive-ezpression :1 =etal mouse lung - Heparan-suifatt proteoglycana and hlood= Senior S-cientisi=nnd_Dep:rt=, Gdrect6r.-W; homjtos.yti-c _rnec`anisms Alton Jones Cell-Scin'~ s~~ter, IrF.,_ - - - - --=-_ --_ - _ _ - - - ---- -- -- - - - Lakr ?'__2cid, N Y _ - Ass%Ftr711t P>_ofev]'or.-Sate UnYver3itj'-of -- New York, Downstate Mea-ical Cen:er, 8roolfry ,, NY _ = ssocrfae eaCa~ .~C1J., Red Cross, Rochester DiYi~~n;- Ftochester; NY JOHN 5:. FIJYtCH. !vi D.` _`= Centrol-of aT^ch=des±y acid ~~genatio, ib= _ A ~ -- DAViD L. HL?SBEE, P"st."_ Ey~~nuclear aromatic hydrptarbon zra;tsport °ro7essor o13`oxicol0-gy. Tezass A3iM by serum lipoprotemE Univ_fsity College of Vete Fn3ry- Medicine, Coti?ge S-atio n. EDWARD_i. CAIvl-FBEL-L, INI.D. - --A.EststanF Prvjfe35QT of 'vPdici7feK W3shington U-ivers :y ~~- of MedicEne,=St. Louis, LN.t_? LAN BO CHEN, PH.D. Aasociate Professor of Pa?hology: _ Dana-Farber-Cancer i:,3'titate, Boston,= MA. YUAN-TSONG CHENyM.D., Pp.D. - Assistant Pro,Iesaor-oj Pediatrics, Duke Un:vrsitr-&:edica! Centec, Darharn; NC. Modulators of-infla.-rrnaio;y, celi p_rot?o_ly_t4c== -activiiy Sti;dirs on human oat cell carcinomas keconrroinant DNA al>9 roaches to assess _ risk for lung cancer - - I _

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Ph;NCIP-AI. IN'i£STIs:fi T GR _ Li'Z 's1V_%"I'iTEI-T1Q1 -- _ - W. L L IA-Csi 9~t. =C=si-l L hN,-P'ri. C=. =-AsSdslfaRi e?es~+~rch --&rieE:list, Cardio- vascular Center, Llriveaitv of Ir?wa Collegeo.` ivled-iciire_A®v--a City. - DOUGLAS EROCK--CINES, M.D. ProJessoroj hfediiin_gr!oykital of the University of Pena3elvania9 _ Philadelphia._ CU;?'S 1. CHvIN =Iv1.D=`= .tssisto~l rr;~j,essor=o! Ltr~:'-o~;> an~ -Pedtat-i: s. Tr.?-7ohR- ~;otki^s ° Gacology Ceater 1?aitu:.or= MD. GARY- A_ C>.AWS. N, M.r'S., Rx.U. - =Assis:aw. Professor. _Llizive.sity-oi- - - - iailfor nia, $a ~ Fa~?!cK~.?. - - B 1'.'_.AI1=L:=CL1=V-1 =TG_FR.-F%.D_ - Asastaw science. Washing:~n_IJ:.:verity oF _ Dental Medicine, S.. Louis,-MO. CHARLES G.- ACt;i:HRANF, h:.D. Mem.&r. Jtpertnrer!:-of -fsra;nano~- po;hofogy, Scr-ipps-Cl'anic and-Re-search - i-oundasioa,-1« Rol1_, CA. -RG3ER-i W.-t;'OI,M-.A-4, 7M.^.- Professor of b;edi=i7e. `::1`,lP = -- = University Sc#ior,l-of-1<icdicine, Fhiladelphia,]?;,. - - - EVA BROWN CR.-'~.iv3~I?, -PhX-;. ~3soFief~ ~ra~e.~s4t yj ~xLicrny cr.d --_ Cell BioZopv_ Downstate 3vt_edical Centef, R-roblclyn, NY. _ -PROJ£C-i _TS'iL£ -?a.hoph}siulYOy;_o: t_he sore.na.;-;icro_= circulaiion immune ir.;ury -of huraan endothelial cells -EiocheTiser v and ft:nstior of humarr graau:cpoietis =ntig?ns Nuclea; NTPase and selective RNA splicing/tra n=_p^rt- -R-oiAof =3 segm.enil in Vsegi?tgnt-_exdre=,aon Riediatiu:~ sy=e[~s_in-irv.iamn:a:o_ rv iung ` disease initiation of-pIasrna coagu:?tioct_End kinin ferttiing. systettts_in ~ rna . - _ s:uuies cf iRiiarsria_rr ,si °.g ar_ ih -oiirl; = r.:o_;~e1= _ CARL E. CRUETZ, ft-_.Fs. Role of protein phospho=ylatioz in Assist9nt Professor ~-F`narrosojogy: ."scatsnz:-iaduca3ratech,_ia:nine ~lease _ University of _ Vii ginin. School of Me,d-;eine; Cha:lot-tesvil.e GIDilNC'ZAN--1, mw.: Ptt.):. _, Role o: rnet5_ns on sYpero-ide-itnl Professor of P!!ys'r_°o{ Cirenmisery. The Vitamin C.o..ic-t; in hin.lhgicaL,vsterms - --Hebrew-Universitj~ --J-erusalPm, Isr?el. - _ ® IVAN IJ'Aivt"ffRNOY, M=is., 1=l---.D. _ Deveiip,~entally pluripotent human lung f'rojessor_ o~IF~helogy. I:aanema:i:. cancer stean cells U nlvErsi.tj! S5.hfiG1 Ot: l'{e~e-lne, - - °_ - - - - - Phil2delpm3_ PA. , = Ai,9ER'I!F. =I3E3SSLIZC,'TH, --ILd.G., I'tt.I).== -_ Study o} 3ltere # aiphs_glebin genes in Professor of Medrri:iF, _ V_ete_;arls lgukemia-and solid +_Tors- wd:niaistratior:ivle~::3i_Center, Sari Francisco, CA. 1$7 - _ fz "I --to - - t

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ffRI14a:.IPAL-IzWyFr-Ss IGA T 0E-- OR lNsTFT-;JTIO!+i-- _ - - P T E1t'H.----aiUFS'tiERiT, Prof_sSar of ,iFolecr c.= v.ology, . - UnivSLslty _oI_{`.a:itQrn!a- tSrPeiev- = - HAROL-D F. DVL`RAK, ]v',::_ - !'i:aef , L`ep?rtrneni-oJ Pa0w4egy: Beth Is ael 13os; ilal, osson, MH= --' --. V. GER[E ERWIN, Px,D. _ - Profe4-sor of Pharmacology; 1ear- Vnlver3lt;_of Gcolc=,aGt School of Pharmacy, Beulde; ;-- - - PRO.i-I;G T --T_FFLE- - _ --.ansforrtiag ge.,es off two _;,ute lEUkem-4_ Pathogenesis of tumor desl:oviwia - Effects of nicotine or ne;:rooertiie secretioa by intact at::usg briziq, a i har_.==foge- . t7cstudy F- ALVAN R.iINS-I_E:.r-3; k'_:l).- Smoking, detection bias and primary lun = - _ - _ ~ - Profs'3sor of ttiedrrirls' fR3 P!1e° - _ cancer meology.= 7 a1e-Ur.ivers.ty S,.^hool ~of- kiedisineN'i?re-Fvec, T-I-1=.^,MAS}f: Fi15LA`:- Prb. Structti-e,-oroperties and reoulatinr of Asscciate P.clessor_of jhstet:ic,;-anii fnouse=pia=ma protease inr::m i vv± `r`ux I~3ie7s'iV i,y.re-klcgv t- _ I= Med-icai Cemr,_N wYork' JPAa.,' B. FLKr'.R3 P`~rD Che ~ -~ _yii ,yaea1 vir31 int rat.Dt ` ' 7 s ~ i ~ - crllor - es r1rc ! n S3JCa- t, - ep Fsler[ trans Si[lat-il_ r of Microbro.ogy._ ,Colurn`:a :3niversiiy .:'olleg4 o1'_Yitys ciae+s ~4 ~ar$c4~ ~ ~ _ _ ~York. - -- t- - ~ _ JOSePE1=A. F'OAITANA; _M.D-, Pa.D. Glycasyltra:sferases_apr ~:yco~+roteir, -4ssistanf f-o,'es.to , Med,c~ -antireihess.-iP diffrrea+.atian induced - ~ ~i~chPmrst~r lres tiirg.niz~Lfriversity phenotypic reversal of -nya ignanby Medical Center, biorgan_- retir_cic acid cy iic vucieoti;es and ciher ~ -_ agenrs JiJDITti._ANN FO?TLR, -IPD. - L.vc;vi;rn_ent of e asfin fiber- in 1uRg_ -iseaz%_e- t'ro%sso: and Chairpersnmm [1e}ar:men' _ o' 3ae:ogy. S;r~_ s use=Ulniversit.; Syracuse, NY. M *i E'OV RIGFIARE i3 -- s ._ , , - - Assisr9Jf: FrJFeSSor-.r[f =Pe63jG:L12s, Chitdten`s- Hospitai Cotpo?ation, - E;ston, F~=!~~_ _ IRWIAtF?IT--C` IOFt Ps.ti.. - Frofessor of Diochemtcr-r, Duke =. __ University Pv?edicaiCen*,er, Duisam~, NC~ ERI2OL-O. F7tIF.-DaE_RG..M.D. _ -_ Asso-_`iafe eTtjfesSGrvf 1-afhnlogy, Stanford University, StanforrJ; CA. Role of giyco;amiriogiy-c-atts -it=_-1utg-edew-a=-- ---- C6ntrnl ef_ti:e $ios3FitFies of saperozsde_= ~ s "utasc,_ Complementing ht:man c~1=s aiti"lo.^-g~ - yeast DNA repair genes - -- lgis - w-

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-=PTtINCi1PAi.IIVVESTIiATfsEI gltt33ECI TITLE - - fi3R I?:3TI T i'TIO?7 =-KJEL.L=EEJnE_hf_D. _- Ni~-otine, ,~atecFolans-ines, and ` =Proiessar~J fisto~o~3~: 'Ihc_I~~rol:r~sko ~Y rroeolocri~se n ~e~_5ne Institute, S?ockzoim; Swede ; S?r~kifig, dypa~rane :~europeiad_es and models of Parkinson's disease _ JA?:iESS W.lvAI1HA T7, PH.D_ Direct demonstration of high-affini:y -l,s srstnni Professor of Bcycre3nisiry. drug=DNA interaLf-iisnsby :esir:ctior._ Untversaty °ot South Alaba-na, IvlooEle:="` enzyme-maDP;rig, JACK ::XULf3IE_ g-s3.D. - The mast-cell in i?ttersta:ai psen.onay° Professor of Pntt<ofoey. hicMa..^3er filiros=_s iJaiversity: Harni_tor., Ontario, -Canad3, J. BEi-tNACtD=-L. -GEF, ivl.H.-- T+ssue m?irix and phagocyte ^;ury _ " =Pro, ~ssor-oJ ~Nedi~:znr Yaie University _ - relative contrihutions~.-` proteases and Schooi of h4edi~i^e, Ne~ I:a~en, CT oxidants CHOU ZEN (°rIEAJ, Ftt.D. Imn:i:nogloi;utin enhancer elements in _PostdoCL^-rGl -_F-IlioMt.--I~~.S3Qnal in3titute<. di:.=iFa sp;ci-fic-ge=:c °Spir3zslon _ - - -- -- of-Health .1ethus?a, MD. JACQLIES~E. GIELEN, T~i.D.- 'I'pwar=Js_a molecul2r-urderstanding-o#' - Assoc:ote FroJessor- Ldboratory of meno-oxygenase regulatory snechanisms in -- - nfedrealCrartsl-ry. Toxrcotogy and _ ar-_mats-a^t#man - Jlyaiene lrstituie-oi= Pathotogy, University of Liege, iaege; jelgium. GORDON NELSON ('i?LL,?efi.D. Epidermal growth faceot receptor gene in Professor of M edicine, Univers:ty of _ episumoid carcinoma - California, San -D"iego, i.Y=Jolta. - CABAIRL C. GODMAN, M:D. - Cytoskeietal organization of-the endothelial _ Professor o, Psth;riogy.-Colurnbia tell=in regulation o#' shape ~onts-5etility and University College of PFrysii<ians & -= zur?ace-r:.overrient- - Surg_eons, New Yrsrk i%A3ZREN_1<-I. OOLD, IS1.D. Effect of ozone on airway mast sells- Projessor of Medicine, C3rdiovascu{ar -Itesearch Ins*.itute,-University of California, San Francisco: 4LFRED L. uOLDB-ER--PH.D. _Seiective degradation of-darnag;,j cellular 9iojessvr of _Physiolcgy. Hat vard _ -proteins ruTeins -_ -IVIealical-Scheol, =Bostof,}.~tA. SIDNEY (;OLD-FI3f'lIfi-R, I...D. -- = Extraceliular matrix-cytocliernis?ry and Professor o# Pamelogy. Albert Einstein ultrastructute College of h:edicine, The-BFonx; Iv -. WILLIAM E.-GOLD;vIAN; Fa.D._ zJ'ordetella [ertussis-trachea;-cy2otos-in Ass_'stant PrvJessrOr of :!Jifcr o8iotzgy dnd /rnvnensoEogy. Washington University - ScFool of ivledicine,-St. I.oui{, MO. G - _ F

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FRINCSPAI:-INV>t;`: LICA I-OR PROJE! T 'f'-I T L - OR INSTITUTION CHARLES S.?1sREEI<<RERG,_M.D: _ Trat~sgl_utr:3inates snd a:herosclerosis = Assistant ProJesssroJ Medicine. Duke University Medical-Center,- Durh3m, NC. MARK !. GREENE, M.D., PH.D. Suppressor ceiis is: syngeneic tumor Director of Immunobrologv,_'Jnivarsir_y immunity - of Pennsylvania, P-hilad-elphia. . NOSUYOSHI MAvIN{), M.D., FN.D.= Professor oj.In4tomy; University of - Texas Health Science Cer*.er, San Antonio. - CAROLINE-R. HALL, M.D. - Associate Professor of Pediot*ics and - Medici, e. University of Rochesler School of-Medicine, Rochester, NY. _ Nicotinic receptors of LHFSH axon- - tertain3ls i:, the nscdiarn eminence Nicotine on prolactin secretion in development - Interrelationship c:` ina'zctious lower respirato_ry tr°act-disease in infancy, and host and CHvlrorlment3e factors to later- development of chronic-lung disease LINDA-M: HAI.L; Ftt.D: Genetic differences in nicotine sensiti°: ity Associate Pra; essor of Ceneics and -in Drosophi:r, -:efancgnsterstrains Ivewoscience. =A?ber. Einstein Col=iege - of MedicaAe<,f-Yesniva-Uriv¢rsij;_T't:e = 9ronx. NY.- PAUL- HAM(3SH,--M.D. = Cigarette smoke-and tipoprotein remodeling - -Associose Professor of rk~ysiology and by th-e lung 8ir; phys:rs, an:t ': edizine, =vcorgeiown-- -University Schools of Medicine and - - hentistry=_~zr; iagts,-T~c:, -- - RON,-.L 13-G.-FiARVEY,-I'tt-:D-,- - - Novel a7cicarsinogenis co'!marirts riti _- ProJessor of Orgsnrc-:'.'Femrstry, ihe_-_ flaven e=_= . = Universi[y of.c::hic jo, [:,: - ROBERT M. HOFFMAN, Er.D. - Methionine dependence, triethviation and - eissistoat=Proje3s6r of PdFatries i ~ organic trattsfcrmatic*rt- Residence.=Lniversit.yof_4~nlifor;ia Scho.;l-of Medicine,_La Jcrila. - -Regulatiote of cellular uncogenes- N AYNE HOSS, rH.D-- St'3iie; of -nisotine irterar_tiE,n-wiin blood Asso£isate Pr_ofessdr.-C.enter-for-Brain -.- cells Research, University of Rochester : _ 7viedical Center, R .c':Pste.-,, i+fY.- -- -RIC'rIARD L. HUGANIR, Pt.i:.= 'I'he nicotine acetylcholine receptor. _ Assistar: tt Professor. The Rockefeller regulat;or.-by protein p^osphofylalisn Uttiversity, New York.-= _ HAROLD P. rON: S, P-d.D. Calcium-de-pet:.dent regulatory proteins and -- Assis%ant Professor of fiine?semisrry. °- nPuCrophi-l activatii n- Universi-ty r,f-"south Alabania, Iv3obile:-- M^vRRIS !, KAIthOY§K4', M.B., B.C ti. The molecular Basiso: ru':mon ary= yhastuck-ProJessor-ol Po=sisologi£6: _- surfacta^t secretiorr by :ype II - Anatomy, Harvard `:3e_dical Sczh')l, pneumocytes: studies in ir:tact_celt> a nd a Boston, MA. cell-free system _ ;9d A ,`~~.~- 'a[ i/,

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RRiNCPA[, 1iV=fi,IG_!!_'EOR_ - - -_= PROJECT `I`fIL~' - = f%R_IF:S~`=f i~.i~~"i~llai Th°-r=::e-of_pEatele-is in ^_uEect cell-metas_ases- Prof"sor of _zdrdicine,-New-Yo-R- Medical CentEr, New GrEea:+s,'C.A. University -Med:cml-t=e^ter, Nei: Yorx. R.4BERT W. KARR, M.n. Development and differentiation o-t normal Assistane 1'rvje~srjr of ~fecticine, and-"teukemic :^~-no;Yte3 _ ~lniversit]*ot :?~wa~_~o~~ ~~ty. _ i}t1iRt,EY L. KAL'hMA?!_ ?a.D- Oncogene_ in chemical carcinoge:esis :. tri~J4ssor fij~ Pi2dhoi,".gi, Sa.a.-ia-~:',s'~!~rsity-= - -,- ~--_ _ - -_ _ - - - - of New York, Dow:, tate_Msdical - - _ --Center, $rook~ yniN11- iN' GEC'iE)E`cD-M. KE.iTi-1= FH,D. Assistant Projessnr of Anutnrry, University of Wisconsin School of - -_ Veterinary lyiedici:te; _ Tia;:isoa.- -- - ° -Institute; i3sffalo, -N-Y. _ - ~m~et:,ioi~g_r. Fccswe=[ Fa;k I+~~snor2 = - HEf1tZ-K~-0 -.}f1=ER, M.D.=,=EH.D, Multi-ta_rgeting vaith_hybridos;jas on tu..~:~to~ J~irer~o~ Derarate~t of A~^?~~i ~r - cells - _ : i:ng_riettroeriai.cfine ccll Director. New YorkS=_are Research Institute-for N`euri;c:emisrj znd-i:rug - Addiction, New York. _ _ MQRKKL?~'siSKENYJC.,_IE+l.D_= = The Einnish 7wi,_ Cohort Eollow-u; Study =Yr,ifEssor ard -,`,'I:zir.mnr. t3eparFrnend ei = Public Health 3'cie:,fc. University of -#el3e.ai:i' -Helsimki, 1=i.:,and: AEE:, LATEH,4;=PH.1=: [:eneric basis fsi nicotine :espor__se: VALEi'cIE-iC. LLw"llfyRtfl, PH.D. -- V rai and cellular factors co:Rtrcllir. gg Gues:°ftesearr#er. 2d&tional Concer - papillemaviius transcripts Inctitute, Eet-l:eseI2; MD. _ College of Me=sicin P, _Itouston : % -bron.hial-ly=;,p;a=yte3 A3sararrl ?roj'e ssUr~i;~ _~`edieine: payior- ia[nuncg6? ,1=n #rT.o~uction=.~,-y 7=uFra 3- E._trLlN~/Ti LAWK-E.NWZE,-M,''i3, Eff?cts o? ei~arez~ s:c~~g en --_ _ Sciertist. The ~Veizrr;aaf Ins_tiata of - Science, Refio-ot, Issael3OSE?H D% LGCKER,-Fv:.D.; P-y.L;. DNA meti.; lation in neupe asi;_ Assisiont?rofissar-of-Pathology= a:rd - -BfffChc'm?siry. :Jnlv_e4s :y of -1'itl;tlurgh -Scl2ool of Medicine,-i ittsburgn, Pb.. _ iYI I i f,M _ F' 3.^. -Mecha^:isrn_of S.t).:i. _es:or-pri~Me, re; ai3- Professa.-snd Ho_ad. D?Dart.-:err of c';ernic-Al carcinogens RONALD B. LL+FI'iG, -PH.D. _- int~ract:oas hA.~e~n 1<id~ ;:r_;es tnd= - Microbiology and irmmurmiogy,'~`

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PftINCI-PA'e. INVESTIGATOR -PRvjE_C_ T T-i5'i.t<=. - OR t!vSTI-UTION=-_ RONA;,D J_-LUKAS, of-n-icotine or: nPuro:,a1==-__ L3irocter, Laloratorv of . TTe:oo- ezpressiort of_acetylcrioline receptors ckemis-rry, St. Jose>n's H=y~p ita! astdMed3cal Center, Fhoeni=, AZ.: JAN Ra._ LUNDBERG, 3i-D. __ Senspry neuzu-pept;des attd-s rnke indyced Ass3alanf PrcJessor_o,f c}armacology, irritation of t_ha respiratory-tract Karoli.ska Ihstitute, Si~k~±cicn, Sweden. HENRY T. LYNC)!, it' I3__ Genefic and 5ioanarKer s udics of cancefs Professor and `'h.aima:.=4'tey5art :-let of -=rf the resriratory tract, pancreas anci PreYent%ve=Medzci~u:J'-Puls!:c - urinary v!a; de: Mea!th. L:fpightcn University School of Medicine, (:i__ah;, NE $RUCc A. MACHER, PjLD~--_--,- - Che.,is3ry and-hiology-o~cocnp:ez Assistan. Pro,%e3sor-pJ R~Carmac,-i,,ica! carbhydra2es Ch-o.rEisrry, Univers:ty of Ca!fort:iy, - Sart Francisco. -- - Moan t Sinas School of ;E3ed.i,_:ne,_New disease York, - - A=sociale Professor =; -%`Fa:,,!Agy, -- azd ir: a^ animal :,edel of Patkinson's HOWARD S: MAKt V cr1.D. Nicotine action on brain n-e:,rotransrtritie_s=- - Jerysaien:, Israel. r_IRID MURAD, Fx_i. Mechanism of rAtric oxide activatio^ af _ -PioJessor of Medirine -and Piar_m.a- guanyiate cvL!Qse cology, Stanford University, and Cnie; of Me.r.icine, Patc Aita :'.A.1-!ospet.al= Role of cyclic GMP ir. smooth muscle Stanford, CA. - - - relazation CHRISTOPHER MUJ3LAS~, M.D. lectromechanicas proper ties of airwa; ,tssislant. Professor of Medicine, Uni= muscle - _ versity of Cincinnati, OH. JAY A. NADEL, M.D. Mechanisrrts of airway hyperreactivity PrQJessor of Medicine, Physiology and Radiology, Cardiovascular Research - institute, Lmive,sity of L'aEifornia, Saa Frarcisco. RICHARD A. MARK-HAM; Ivi.D.- - T cel!-mediatedd immunity in PsFr4omc_=nas Assistant Pro;essorvJ M:dicine and of aeraginosa -MicroSiology and %mman6!ogy, T he Jewish Hospital of St. Louis, MC= Endocrine control of huTi~r, er.do_ the!iat_ WALLAC'E L: McK-ELH4N, Pt!.D. cell re-generatiorr Ser:ior SciFnt:sr W. Alt_r,n Jo.^.es Cell -_ Science_Center, Inc., Lake Placid,-NY. ALAN C. McLiAU1;Ili.INa_ °H.D Lecturer in HiochYmistry and Bio== - I~ eractian of divalent cations .vith=mode_l ph_ysr`cs. U niversity of Pennsylvania- and biological memGranes School of Medicine, Philadelphia. ST>=-LLA MI-ThiAN-f-ItOSENEAUM-,-P_i:.D. Molecular analysis of-!wrnanaenita! _- Professor of Virology, Hebrew - pa_pilloma viFus -University=Hadassah Medical School, - -_ 192 -

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® _ F1tINCI'AI. INVESTIGAT-3°'. PROJECT TITLE OR-IkSTITUis9lu:-=- -_ S:1SA NWf+?'LADK,=P-A:D-_ I~o~.~cu:ar sad ~seiis a~lysis e-`-s~a3r cbil- ~ssocl~te ~~ ~e~s~~~I rY~ cv t;*g--ancer. _- -Ger..elics: , Tts [34iv.ersity of Tesas- = - - .0f}f Scie 4c! LS;~ra ?tywE_ -=_ _ -_ _ --_ --.- - - -_ - ----- ' F -ep DONALD J. KcL..C:-, CaimoduliQ interactions with c?rget - .issociWe Prafessm= of L'iserristry. Ciark ,-- proteins and sy napZic _vesi_c::es University, Worcerter,-lvrA.- STEFAN NIEWIAROWSKI. lyi-D., PH:D, Platelet interaction with tikriaogen and its - - -- = P'rojfss'7F 3f Pi`f-ys'Cfo.g3: Shr2Y :bX~3is _ -,--=;gF'-i ir-;E3_3iC°- in !-!.eEF7oat3-s_:'s-._ R~arch. [ eater: I emple L;tiv~rs;.u - = SchoE:l--oi-_ Mx9isiue,.Fhiladeiphi3, rA.-°- :4IrE i M. OiIV£R_ 1H.D. RRoLlation of the membrane oxidase of Professor o-J Paatho`ogy. i?rtiversit,ii of human po_v-h><rph,uclea-z eul:ocy;e-s Nm -faexico Schu:; oE Medicine, = A}buauero,Ate. _ F_ A!ILLMM ORR. M.D- --Role of local factor3_ i§_ pulmonary Atsorlwe Professor of f cisiogy_ 1Jui-- =_ tnetast_Qsis- _ -`l+ersirj/ of 'r.Eani~tabra,-I=`i95i-rie& - . -_ -- =- - ~ - - - -- - 1sftnitoba. Canada_ _ - Y _ ~.~`r'OtA1~A, :~Y ~-rcmsiase=inhibifo;~ ia cagare:;e~ ~o~e _ Wead.Dzp.,: d..c;i of in:oer::ne and toha~.~ = - - - _ -Biaherr:is.ry; "Niedieal_ Foundation of &uif$1o~ Buf£a'-_;M N4 -' -- `_ _ - IiCRIS bi._ P£'z'.~.`[?',,i_1rA~.L3~ -_ Biology and=~noEecular biology of the-- assWaw ProJes3os of Medicine. Section - _,di1'ferertia=.ion-olf _ _a_humau mit::ocytoid cell of Rh~tumatologv-Ctan.cat=ssar,uttoiow_ line -,__ - _ ',JSi~y=of C aii~Drai3-~hFo: of IVIe!'iicifle, Sar._ F`_r?a:=ois_Q. - - - DENNIS R. F'i~'I ~SI~S y._- iR;_7sTi:ei_t'-..`'iE9n of t#in '_-so `vE.a peerYu3~d - - -Pr5_lessorc; PFarmacolo6y, Univer rt;= - li-..o to study niyon ne metabolise:; and -_ of Colorado Schoo of Pharmacy, _ ateiaboi';c;rterections Bo4i',ier £DGAlt fICK;-M.6j F'0.1). 32:e biochemical `?asis-of e:hdnc;ed oxygen - _ - Nofes3o. Afinunolog=y. Tel Av_iv- _ rasiLa9-productien_hy Iyreepho;;ir:e'_ a7n iversity, Tel AEiv,'uraO. acti+3ted°macrop:ages_ - ® a-s?-rs~laceoa and cellular s*eYay-alisr_; SAVVA-iCR£ Y FaZZO_-~1.D., PP.D- --1?rote es;o~ oj 9siwlrg~ viuke sssocdu:e Pro :?niieisity Medica:-yeraler, D;:rhasu, - NC. _ _ -Boyd Professor of_tivh-rrnistry, Louisiana -.State-URtverty, Baton Rouge. ~I IA 3~= YC3I is=3Cz F~.Sti.,-Iv1:I~ Iu=Fsr-igai;~~=~{-th°--o~ y-igai;~~=~{-th°--o~y o_~:-a_a:;-_ Ser:eofi.ec:uFe.-_=irc-F.'iscopqtro:ogy, DeptidPa :n human lung disease Royal hostgraduate -MedicalSchool,_ I-laa;mersmith I-F_osvtta!; ioadon Englard. BViL-LIAFi-A_ PRYKII ,-Pli.-i3--=- Free -~ica che_~is3r; of_cigarettesan_,1~t ® 0 I -~P

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PRINt:I_PAd,_ tN`VTSTEOXTOfd --PRO IECT "I'ETi:g OR I1FST# s id'!':ON - F~I~~.~EI 4oal;-ss of_s~yuECS: ra~;airsd for bayicie F.ese ~rch dc;eslis~, ~~~ ~~ry=~' pap:it0%j2 -v=r ~s ::anS:ormaii:,~ and - PaiFwa(ogu; anton eaous pIasmi; repiiyation - - Bet7esdaMD_ RAMIT RAHA13}Alt7FF-,=M.r-,- _ -_ Projesscr of P_hysioldgy,l1ebrew - University=Had--assah-?r1edi<i'Sehool, Jerusalem, ?sraeE. T:%v?C: E 1~~ i.`RFiAN., MA Proiessor of Msdici:-c - D%se-.=1sr. io>;-oJ--Ccrs.'isaseu-!4F. Disea.re,, College of biedi-=ise aed- I`rtotistrs 3> New j-rseyf?,etv'Je:sey- hiedical ScAook, Newark: 3CDHN ;,. REPIINT; Iv::dJ. - - _ :Iss7slf'!F4 D:ree-ir-.. Wb.k`.-~`~::'1? s.L~4a _ lns7ilfstrt:=Associale P:ole,scr oj_ M_edici"._ " rniL'ersisv e=_Coin" ado -_lealtb Scie11ce3--_renter'-DavF~ RUFiER-~RE3Ad'sCY_, M.D. - - --Ass:'.z'if3ie --Projessoi-of, !,?i?0rC'd:ecffiYe -_ - MediciP.e,-U-3Liy~r$it-y=l'~~~atir Medical Cen ter, San Oieg_a~: - - -Flumora; ef£ects ot sin-aff°cea carcir;oma- of i7iE lusg _0_n e~vromi:scnflar ir-ans!revioa --_=-Catechgh-min.~-. C1?fiab3fism iG chronic -- s3mo1Fing a.:iYZ'_aly _ Basic mcenanisl*-s-of I: ng injtzr y-from - _?fi`Ea'_e~v+1#a~t~ - --- - = - Ine ei'Sect=u` IOicvtirtE ofl 33te_ri.:E_-an -_ ca.rdirvas-ct1lar :-e vad;'Larnict PETER M. ROSS~ PPA): 0NF damage and sel?ctivY snain-tsr aice- of VIRGINIA L> ftiCHi`%tOAID. P-t!,H. _ - Resensc-h sti Fsociale, PGC_=Ffig Tsor.4:vert Researct, Fbusdatiorr, Seactt-e; E': w= R~aex%eh .~~se~ru_e:~ee-R~ t_e~ellEr aaimai genes i.-New-1'sr'-,c. _ ;!'A -~~. RYA I+I, Pn.F3 =- = Ynteract¢~ ZI-" _h?rrofies_Wi-eh eef13 cs? ie- Resea-ch Pro3essor-aj 3fedesrne< ?I,.i= palmooarrv~-usc vialt 9ersitti of Miami Sc; oo' of _Medxi in, Miami, FL. _ BRAHMI P.=SAW=, PA.D_ -' =- = s`elenium-biadin6 pcoseins' _ - !fead.-Paora?n Blc{he!ntstr.:-Sout:;ern - Research i~stig~te, ~irmi~gl~.~, A~. ~.Oi~A A~~;~l'!"F'LiA~ P~:U.-= _ ~EeLLpmei:.: o. m nc~Iona~~ntihz~ies *~s .;issocieit Professor of Yeditirse and= ~ - carci:.ogen-DNA adducts ' -- - - E n9irnn/13en1Q[ f{lelh:es. C(}iLmSblc - - University, New_York. _ PrrJ_>,r.;or-oj Phd.-acoiog;._y2i=ida_r}il, of aar=na acids i:~r~~:dica: art:r;es arI{ _ University School of Medir . qc; _ veins - EY. RAMA SA`Sf'RY;=6~~.Pt=.Lr- Mater.-_a3 srsekh 3~ and b;oW concentrations rdashv_i11e,-717h. _ - =alL ece o: nicotine on the re:ease of - ace-i1y_.cbol:Fe in the humar ptPcetita and its - ;in0;.ationson ehe fgta' gsow:b

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M ® ® PRINCIPAL INVESTIGATOR PItO.:E-Q 'i TITLE - OR-If:'s"IFTE7TION ~IiALES -t::-SCOGG5N_ MI.L. :`ne somatic -celtZeneflics-oe' Psstsg_cancee ;-3et'd.-D-7;i,iion As°..ociat_ -Profe3soc_-Yf-Mcffiiins. Qzi-- _--_-_ v'crsFty of Colorado Health SF:encgs Center, Denver. - ROBERT ~Q °I ¢=1~5.~ roo~~itroent f=fn::o3=a±~i ca£cinogs:~e,:s ;n frvlfrssar of.Prunoiog_r; I~ayo Qlinic nor.;.al, greFeop_ast'sc an_= y_iignant human aas#=Founc:abon; Rochester. Iti5N. etrthelial cei13 Es=NRY,SERS-IE Y:,-P-E:O.- ~vc;r~~rit d£ ~a ~r~i~r~i ~o4ze c_f ReserErch SeuntiYi _lv, Neurochemistry_ =-a ° I~~.l i is3u sescase _--_-.-_-I's'ivis?OIf; Il3ili3f3-S. K-1;P.E ~35tSiElt~, `- ----_ - ---` - - WaF3'3 ISi3Il ~, AFer~_l~r-]c.< ISAlA~fU :;IiiEQEiTUt, Pa.Fi. Effect o£-thiols anid disuIC:_' Ies 4oc Senior Z~ec.wrer in Bsochernss-ry: -Ihe- c,holest"Di meta_bolis>±i- Gco_-ge S. Wise FaL:ti;ty £az L,i£e Sci_Gnces, T'el` A_-9;.-=l11_iversltys Te!_ Aviv,-Israei.: ROIS£RT 1: S--d.LAR,:--WT PiT.11. ~'cesesrci3 -.fssocio:e Professo:r of ClytGk?l"c::i;a C_ 31ete:2?p-;;iid 3libpopil'eati,: r'y ParhoEos, New York :Jn:- ersi:y - _ Rewarch ;;rvice =QoIsiwate-= Memorial _ -liospitai.-Roo3evvlt WaW. N s. KENDALL A. SMU=I<; M.D. Dissection of-tke s'ukaryotic DNA ProJessor-oJ _st*edi~.~zns. Dartmouth- repi:carirn vaAhwav -__ Medical echc;I. Fi~xo~`~, Ni~. EN S. $ZrITlff_ F-H.I2. _ - `;TEVE SeteCtivity u£ -DNA -r±ethr'_at_o:~ :e nr=rma'-_ - Assistan9 Research 5--rFrtisa. Beckman ° =and-oecog_~aicaa; r ~,~sl'or~o~ celfs Re;ea:ch Inst~t~1P of=t:,e C,t.y of ?~dy_ es_- _--_uazxe. 6A___ _ TsMO-.=d Y -A. -SPRI?JQERF P%.O. gtas:,ies ~ mae~v~F~ag~-ss~pc~u'at~oits =^d - _--AssdSJli.4t Pr?fe_sS>+_r-Eif PCa`ioiog,r`Csit_;:-_:- sif£ei'ari:E3tiESTrvsirtg a^~--oFv:-lo,;at antibodies Labrratorv of Membrane ,lrrsmikno--- =chemis:ry. I:a_a-F3_ ~r Cancer - Institute, Bostu~, MA.- ERsCi. STA1`[i.BRIDG'-E, PH.I). - T~as£tr o: s~c:£ic :a~iv~ysa~ h~ »a~ _ 7fssrci:.tF Prvlessor :f M:crn=- th:orr,osyifles to rcipl~nt cells bifFogy. University of Cal£_raia, tavins - IvQltMAN C. STAL~sqv4.?j. _ AI=+zelas-aia`ai=horFie: ~r~a~iia~3~ to Professor of Phvs[ocoGy. Cardao-vasc;:;a: en4d mac:emodes-uies an dog a-a# - Research Institute, Qniversityo£ _ sheet) lung i:aai£ornia,-Ssn Fraxrcisco. DANIEL STEINBERG, M:rJr_-, P?::Q. Professor oY R3ed_rc,tn§.: Hea,4 Divisio:r- _ Arterial degradation of low density _ -gflMelQb3tic !`3l3et~E, T11-~-iJ,is'°rsity o!'-- :'_p::?r?.'tE-iFiv in 9i7o Qa1i: o: oia_ at San INFgo. :,a Jolla. 195 .E

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- PI3INCLPAL- ililiYES"I'Ea:.; -B = - - _ _ PPMJEC' TTI'LE _ OR dNS-. I'F?_r"FiON? . - ~'I'1O1lAASP ~'' I . ~ . .;-y.; _ sa.chuse:s General Hospital. Bgsfon.. - FLEUR L. STRAND, fq!.):'i- -- Projssor of BiodTgy~ New York __ vLf.vfs3_ty. New York. - _ ~ D: -LAMSING-'fAY].,gR._ P3,L'., ~ ar~=~ie ~felio~ f= - - Proiessor nr Bioro~v -C . 1 U .iverait_Y. :'itts3urvl~, PA - - ?,.OR, N:u. - JOSEPH -c,HA?LES T,: Associa:-e ltesearcR &7re.srsf:-Cfsy of Hope I;-Esear_s`..`d-I33iiis~Y, JQHIV .A, TS-ic?MP'aO:',- -"-~,4- As sr;.iy:e=yr=o-,'essor -of Ph.ar;haceuti ca: _ Clrtsfis.ry. Uriverss'4 of Colitrado- 5chooio*_`_Th3rL3'v,.B4V~&er,-__ - -- - - ___ ~ ~ --_ - - 1-- , V ~ W_Ijj~ ff&=s1 FFo*g,uu, 1 .oyfde~°-y_Mx-_:_ - . _ _ EMIL 1;-_ i?4AtJU-E;':4a.D. -_- t<'J~air~san_ancr=Profe =or.-Liey o.f ~= Patho:ogY, wash=sgcors Uoive°s .tJ - ~ ScFso'-of Medicine, S. Louis. MC. _ F=° ea ~ _ , ~ -- - -_-~ - ° ' ElfrRS1i..D E. -_--- -1Functit'irt"~_'F ai-1atvrEfy of `el3e Idfng rtaCro-L`lia°@ - Prenatal a ni-p3str~Ral effects of nicotine and ACTIR peptides on s,euromuyr;xlar--°= -- kS7-eioFfi="-s .: and s3f0.or -bebwd-ri.F', F?t` -~_-. rhmotasis of macrorhages - 4Ceritcplasmig-a-Daor--~ial:tr in =heonir obstructive jfuldiiC?:-.p_r37 yis°a,iE iChrdrYa>ngra-phic sezaratiofr and ca-wi; :reiive met3L-olism 4f d- ac-ri- - 1-9icoiSle - -- tsea effects CUrenal fune=isn or dr , -e= m:satxi:is-r3 Physionathelugy of ncrfnat a:fd-,c+iv_ted Macro_nlagev- Functional arigssis ef-elt o~ os ~r~z~~s =_ -_- ac-tiva.ted du_riFg vi[ro=be?esis serr:r~f,la$;. 13niversivf oI"L;al fyrnia,- San Fiancis;;v. - HF:.E7+? VrLN V.:1NAKiS, Pt,i _Pra,essor of i?in-rnem , ~y. braa.~,es i: nivenitY;'~a!t :zm, i~A.`_ -- - `cBLGT:-IyV1+W .~LASZE-11C,-Pil D. = F'Se.Fisi:vr'rs£_isTidHSt, Ohio s_st5te-: _ - Pu ifi=ation and <ropeties- of a-go1ur1e:= NA.134P$ glycohydrolase isolate3 from_tiig _- gpeoESgr-; M. prollitera Potential for luf:g-c_ncer greventionby :Iltili:et~:1~ de-glu4S:rf_)p' 1-.r-atlon. EVELvW-WALPSiEIN, Irk=--.b- - Seri3_r-i,ect~-Pr.-Dw.partm¢nt of_ _- _ Biochemistiy, Tel Aviv CTniversity, Tel Aviv, Israei. PI:TE:R H.-WAISh, -P;a.D. ')`er;:ple-- ' Medicire Proj-sor o , r _Uaiversity School of_M?ds6ae,= - _ Repair activities for 0a-ffeetnyl8uaeute D1F A aoo3=ct3 in hu:-nats i;, :rhoeyte, o: emokers rs: 9-',lns3:okers Inieraciion of glateletz wit!i-coagulation fa;.tors_1X and X- P: iladelpla+, PE'e'ER A.-WALD,-Pi1.L). -oxygefr=derivd free racical-, iMmune =_ -Proj_essor and Chairrnan. I;epaFirr:err oj ;,omp Ieties snd tissue is;Ijry - = -Peiirc!^gy. ihe University of '=1ch!&an, - Ann Arbor. 196 r@=

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PR[NC'PAL INVESTIGATOR PROJECT i iTLE CiR :NSTiT'--MaN - - t,EORG; Frc3NBAiJhi. `F.D, saeioien pep=iCe rnetfE:orine sulfoxide - - i3Sil:-'_~'i QpWrm ar'-_ f.i. A mS'3ytfa+-e°an2il_^=-l~~-n ~S=-A ~."35s?-,^-1~ . - &-f.PdfmCri _vf-,WGf-^_d?7e, T11e_GraYuate - dt?erue agal n3 protein oxHe'a'-t.on -Q1Q :`l-aJ'tin aPgryaai=_Gn in smoice's SAMUEL E-WJ[SS. PH.1;. _Sequence=madificatibny ic -virai DNA by t? v~ess5r ~~ e?i~hd ~is:r; be?~;~~yren? ~~~iiies _ V1b?lGg} li:e University of C5%cAg%t.i.3capo. - - i-i:0V-`A3e G. ~Q`~~~tS ~~ ~± rn`c ._mcr,~ }ag~ co's_agena e anu - a> _ ~iS~ISe~y7C~°iale3`Sr.r.~r .''r-~.6_"~s, Jf~-1-E± =i:3!~gE~:-a~" @~"@ iF.`=_:~itC. wo;rita! at Wajl:ing?;a Uni.ersity - R:ecicai Center Sr L>t=lass, 'r0. =MW-HAVI. 1_-tiE:~ :.-i.J. _ ~ec~.a^is.ng cc~.:trol:ing ion tra.s~Ttin Asrstvrst frcJessor v; :E'ediclne, Urn- - -airway epithelia -versay.of laws=wllegE of Me_d;:ffe_ __ IGwacity - A'ZE-_l`.°_yNFIM.'-,LN3; -M_D `~eCCiirF_as tT:Tlbit=_` if BCOst<3`x5d:r. __ Prsvlessvr %yd Char'rm~_!;e; _=:men. of = fo,niat : ~ :oclo~` :is~ ierj;i:~itarY_- _ ~ni+er5it-9f _~ =-3t~w aAd-c:[2raCt2fFz3t!n-fi-Gf ; :e- - .raG.itenbllr& TAt5yS3~'~=ig,C~e-r1es1.= _- _rE:c~iGi~ -Zl?~7E:.d*_~.~~---- ~AUL V. wOOClr = !=1, i~ ~. Effects of chemica; ya:ci noger-, jpoa-gene -Prc!essof c,+-Aredicine fse;d ?hvrma- -__ --=k4.i ~n the-pancreas 3vlvzy. r=eorgeor::m Lt=i:~ersi.q -,Vedi~'a3 Cea+_-ter,=Was:±ington= STANI.EY-YACF-IV:?_, M.I3.- Models for the=pathager:ess ai PrvJes~vr-of M, ~ieipie m!i i;kz£;' _- '= ° b,herosElerosi==A; bio:aoical-effez.ts of -- - Scerivn v{ ~ermat~cg~/~crcvc~g_t; V;t-- oxygenated =steFel te,ipo,ds, s)-cneva:ofl c Univetsry of CFj_--cygrc_ k4ed3;ai %eAtar; _ acici and-LholAsteral biosynt7esK_anu the -- -_ - cticago. bi+syut: css nd eg :aaon of cLll growth. ~eFl -- DOIN A L D A. Yt?YN G, M.D : Pa~,iilari~v:rus Yedtc ns -and brvfes.;vrv;-wfedicrRe. i3nivyrsityir2nsform2ticn - Ro_fies>_er;_Roehester, Nf.-

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CrJMPLE;FD PROJECTS = Follaniimg-ir-a li.;tof'th_e principal inves-tig-ts;rs, or i:.sti_s.i-vli-s, ~ of projects that have been completed -grio: to-tFe-peried covered in= _ this Report. Sev;ral cf the indiv;duals na:ned- are deceased. The titles and -affiliations -iiatee were- those effect at- the time the wors; wass in progres5, : i ay__ l~ra;e.aer ~, taiegiv, _,~- r~rversi y of ~*~i~ersity ?~'Y~ical ~c:ie>;e£ rlc~ York, -- - - 'f`ezas - Syst_em= Cancer- -C-enteF; 1~T1 N Y. _ _ -},:lde:son_-i4e-SF_WI and Tv_-nor=Ins-:tia- -- }iouston. R.-FR EDEP.;CK EELRER, ta-.D: _ Associate -: EofTsso,r ef_ -ArtraJ)fiy =3a.1- - _- DOlwflNCsi3 M: A~ViAD(?, .%?_''.- Dire.oi_. - -L.aSCraiOry of -~er:,ata'-_ Professor oj=?isFrmecoloer, University Srience Duke ::niversity- MeQicat of -Per,asclvaais School of ~led',cine. = Cesje,, Gurs,-n, N!'.-- Fhii.Ae_ h:a= _ ~ MARIO D. Af;ETO,-Pw?:' STEPHEN _'~': AVRES-, M°U= _- tsse-c'awr pro;essFr - of -P.w tr:Jr 'og.; ° Director,_ -Card opeilmonsry : ~~iornt~ry, - - Medic~ Coifrse of ~~~ir,a;: ~ia = : ~r - - ~:nt .ncen['s Hospiti, New York. NY. -Comraonwedlt` Univers;ty, P ic;_i^ont. _ L.ILSL=I:--B>.El? _ M.,. CLARENCE Irt, r?GR Sr,=MDAssoctatPofessm- ` s; .ssociatt-Cfirricoi Profeisof of Medicine, Cou nea` Un sv= Ca`13-RE _ of ifai°:ersity-tt C-a-Iifoinia ;iedica:-tenLo:- ri,y `,:an_ & rt.geers, New ferl=_ N-Y. Los Angelel: Hastrrr;s = ~ ~e# =:nP=- _-gi °° AJed`icr~e;" =° - f 1- Director ~r= L-..r.barawri2T; -Tee Burke -~ Universi:vc, Sruthcrn zali:'orhia Schoel- Rehabilitatic~n -Centee=-V:'hiftJzjgins, N: -- - a% ~leaiciaP, Los Aneeies. OS~~;R ; EAyCl~~;A;; sH.D. _ Urii~~r='ti-Aiban" - n 1= *~ r~= ~: - - iJ;ii.ersi., ~~~- e~ x vg.e_w aa.,'~ }3 : I3ealiFz aliin z6re- mD ins A-! Garqf Mf;d :F-a -'-~ ~e ge -ca '_ .Jn_ ?_ fia tls-0 :ujgY; JoA=1s`= Hop ki ANTHONY P.=AM.ARCSE,-Ptt.D. -FREDEERIK4. BANv,-S~~?:DI:-_-_ -- !rs -=ciar in ~C4Jst9tT } {iErr'~7Gy:,Zc['e,4, i h - -~-- __ __ _ -r9f~_ f2 ~7"i= ~lTrrdt `E ' _ E. %. -ANGEL k_K0S,'r...., 'F!.DFrofetr3r crj_ -1116t -1~9v Bo°:ea n...::..iF}~R~ BARGER, IM.D. ; L'niversit°,+ a 11 sct3~k--£citoul- vf B RC7DA G BA-RNEy; '3 QtI.D D: MURRAY - A*itrEViNE, MI.1? _ MA. - Ph;siolo8y, _ H?svard 'Me-ddital School, _= = Boston, MA. _ rJniv4rs:iv sCfiool ; rWk.~ier ~-6, ~,niffer ~-ojzss}-- of Meiicin¢ Mad:sos ~Fc3e_..Or- rstilifiatei of ' =~FssiotoA~, _ - 'Jnivrsi:v-ilczavl of Medicine, New ,~ssociaa Pro±~ss,~r _~' :,iedtca?se, =T---_-`+~ ' Professor = _ 01 Medici ce, rulana- rs~DE.R `K W. LARNFS, tt, JOSE.P!: C. AR(--DS,- i3.Sc. _ t ol~r~ ie ~~t~La ~e stt F~F:~oz,-~2s: _ _ Johns a3px:ns University sc_n x --ALAH ~._AR~4*'I'A~E FII:~, - Me~ieinerB1ltas,~e, F~l : i- Research Gi.ec,rr, H_rzleton Laso .T. C. BARNES, Y:Sc = £urape-=Flasrogate, -I:c:E. __ -Ydrkshim, --R9seorck- Sc+entis, "hi:adeiohia State ~ -England. E°iespital; p::iYaae-lpnia, ?A: ~- _ ~_ NLARz'i..Yl4 -'S_ E:RNa:1~i= (R°"SCO), PH.D. 4 :R~ ~.-BEi,x E?~ :~_TJ:_ F` -Fssistam Biele ist - -~_d= .£ssis:or ~ s:or~ , B , t g ~tsso;io~§ Professor a' ~,~rolei~y. ^_~r-cll U+

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Y t .- R?li.PH S. BECICER,-rF`.ii °-_ _-_ - - - - - - if.'-EAHa _K. HIS`:;!Fc3, P`a.D., D.S.:. ~3 PrQjrssor of C~Sema.=i ~ izEvers v a: Hatestc:3-Hcittstaa,'LX _ l, u<ajt~ ProfeszvP o; Ora: :i~auo~y`, -a.aluxatory- o, Pha+mbkt',og°. Harvard Fi;dBAINifi4--l$i i I!4 D_ aci-f Des al ~,edici-ttC Bosto., MIA: ~; t i~trectc: Esr.er~c:,t, -~ya >'.at=v Aging IkA >if b'L C.iF. :vi.ir.= i St::dy_ Veter a~ _- = Adrri=.-i;tfafi~a -Pioje~.~c=: -a,.dCnrej, ,-°r-ivisis3 of j ~ ~'¢ati~fl.'Iinfc;9-n'~tt lri'.9: I~tvcl~~me~tal ~Iexrcli~g~ ~0~~.6~_ s;t d:cai ~aes N-r York ~i e tinive , SAMUEL BELLE'TMD. , Y e s e N~ : Drfctcr Dir_stan_ of t ra_rdi:ngy = Philadelphia = _ G-reera .__Hc,spitsiI FRED G. BOCK, ~: ghii23elph,a I'A A3so` rte Cs _cCr RE e: rck Scie~tr=?, Biolcg,c-stat.anRase?ei; -parl; t ~N? IiRRV7 >rEN,:CEARAF,, 3vt D. _ a+srf f Frcje or ; :Paam- &lemor,ai Inshtute,=ap°agville. NY. _ _ ;~ ~ _ ~ , DeWnmie+ M, d,F~. ~c _~_ as.~~, ~dA_ ~ s rc ~ur ;~rcles~ ~~ ~ -_- = 1_ss s?ozt ->Flrreto>-. ~~erie~ .- c iinicai WI:LIA_31i r BENEDICT, MI) _ P esesr a, sreuer,-''dmp!e Univers:t, I~ As is# 7 i~~j~ or--pedaa:>:c NGa fl: tiaencsCenier, Ptitade:p", P.A. JI; !,riveraety of Sa,.tfiem -Ca,farpia_ Schoo = r ® BOENIGs -Pa_Dl. = aE- -Iv5e3;ciae. Divisioff~E of Hleimataagy_ }I;E;R?vcMT V_ _ ana- Mle~-ca:- - GeF:Htis~~, tihilti-et:"s Ileast ~~r n e~=~~*~y --and _~c~~l 3C =~~ ~e~~.=~~ng~ie5== B's~.hcta:try, _ _NpindieAp Ice_searcF _ -_ `- -- - -- - - _ - - - _ - -C-e_ 5 -er, 3.CR=43gt37_ KS - - _ -- - __ -~ . RRgBARe : --an der S~rRG. W.D. I. - - _ -_ . -- - I R¢sea,~h Pe~~}u,ct r~ essc I:~iE3~ r"~01-:N_R, :H.JJzi= - ~ ys Bfc,s.a,r.>4?cs; spav ~is: Y rT- t;.alifc r,ia Pro;e.,sor &r =~r` ~zl~?~rr:a Institute _ - w! . Senoo1 of piik'ic sealta;l>akland- of ?s hsalagh lsas-adcta- _ - ~ a ~ lv3II~~: EE~AN, ~.Tr._ . VAL:EF- M. BOO~ERkx. F. Projestor -j P`el.r~-sflc-~?ogY, :;1e:vars±s;s- grfl Fso: -and tx'ead, De_ pa m-n: 'fXVi1c fl m Schao. -or:edecine, ioa T's,arinacoeogy_ Hawarti E'$!ver t B~PhEV BI#AGA-I f~ ; M_~:V4C3s MP~:SE ex= katr:-- Pro;ei-w 0 -P-hyue:csn SairT Se::~or - I:xest~o- ~:c^se: I~ee e Js,ve:Nt, Sca~. of Iv~~.c 3 auis, ~es arG: _Foan3atice, Chic-a_ g5 IL. CESARE-_BIa_ENC;r°IGRi, -M.:~.-- _ tIR"Orvr'} BOSSE, ; -assoraafe-- rJirectvr, Normative Agisg_ I D4v'~ion f c'anccr its~-ar~~,. U_miveraty gtvdy, Veterans Adm,nst,at;nn -_ofperilg3a, ger4.2g.a, ItA!} Ciii-z.+S, B."35MI:, MA.... FIV..F=:~ A_BICi:IF`cMANIR-i 2._ T_om 4.,srst:rx3 a rojessv6 oj ~rted7c;ne ar:d R~seaek _Proje ;o~ Pv ic de Resi1ae- ~ - AL:.~N ~- ~otr~lt.:~t -~a~ra.esr~ 1~~tk - ~ .ar:3ltna-_ S~te _ t l ity _ C a~~gR _;2ale Sh ~ hysiciomns ~- Surgeun= Xo.iege of P ~- _ ~ -_- - - ~ ~idwa:Ft=__ 3~~?_s~al -==I_osyita.+,_=- ,_e~ =i, NT¢.i:_~sRATa,._.s ..I:R, It;.~ 1 = Y~re;_~_,1~3': Assrsi~r.- -Proessor- -oj- F#,acr,arcioBY: .13rtheasterir lhia Unive_rsities- C?Il-ege_- -HIO-FeESEARCHCGNSULxAN^S, INC, -ocf-4iedjcit:e, Rav"aava. - ^ b M C..afi rla e`, A. E!t; P Pt; EAR1 I3 - . = . 3- z: . - ~ ~ ig_IG-ItFSEAa><-: INS `I':"tiT'EYZ_NlC. C= -F:idge; :~ A. Frofessor_a,fri Chairrnar.,_rs,`s ~r rFr~t-p=- Eiccher:iistry, _ The ilaiversity of =-_ -_ l - Ve=aan::vallege zi_Iyte^icir_e, Barl=ngton _ I ® : n . - °--°- - -_ ---- - _ __- -----~_ -_-_~_~'---

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GEOFFREY L.-EgiNK&fAN; 5-#:D. _ ELROY T. CANTREL..Li PE.r3r As.,~lote-Profess_or-of=Mel-icine,=Wayne_- -Ckairssan,_Degartinent-of PhariFacoi46y, aais I_Jnhisrsity SchooI cf Medicine, _`I'exac : o:lege of Gsteo,,ath:c *3ei'sriee,=- - Detroit, 3-EI. d:4ortll ~-~24 State T._T ;-lYcrS_ty,-De=atOn- _ _ _ RQBERT E. BROOKS, PaD: _ WfLLI4h~ H, CARNES, M.D. Associate Prpfessor_ oj Pai}o,rc~gy,- University oi T~ah Collegg Yf--~dedt~en~ - i~niverrity of -Ciregon=-~edical- School, Wt Lake City-- - Portiand. MARCUS N. CARROLL, JR., PH.u BARBARA is.-BROWN, Pa.D. Cnief, Divis;on of P; rrn3cc_og`y, 'I"re CFigf, Ew~riment~~ iSZc~:iatry, Yetgra!fs- B,vOKdaie- Hospital -Ce3ter_ -Pirookl'yn, - Administration H,spital, Sep;aveda, C A: N :'. - RA :'I44OND R. BROWN. -PR.D.- _ WILF IA-M A: CAIL?'ER, M.r.. Professor of _ Cini:el C>ncology. _ Professor of -Hen:atology and Med:cat - LTtfiversity of Wis,,onsin Medical achr,ol, - Oncology, _Hahnenfanci- Medicat College, Mgdiso;3.- PiilaJelprtiz, PA. JOSEF BROZEK,4N.D. _ _ -WILLIAIvf ALVIN CAR:ER,-M713. - - ~ e ~ - - - '~ : - _3ychuz:;gY, ,..ehr#n-_ -i>n,mersay,- _ Mcr-rnbrc;togy, - ?he_ = Johns Hwfins-- Eethlehe^, PA: University - Sc+ecl= - tf I~leuicif~, ---rofess!., Cra ~ :asr~:~,, ~';~srtcn~iic!=f _~s.srsiTepe @j_ t~~-irrCdrte -,.rsd--_ - ~_ _ i- - - _Ajizz;a.iQ:: f-r-'~'~le3--..`or r1_ 5'3yCr_na '. :.an !~--`~SF.t i:SF.ti: i:rf_iaFi:Ea-s_rI.13. ~ is H -g^ errctorrarenc:,e- Research - -- _ ~. _ - -_ -_ - ---- -- __- - -= - - -- =Labora-to?y', Pn-fe3"3t'rr 0 i P3t7-of_-g"i- !fn7d--- SLIz - r- i;_C I.NGHANf, MU M' ~rE;.i nr, trri>egs~~ of '~i_h ~r x ~i or A3t. ?tLEtS: c:4fessoE'-`=- of= Jf o(o.:,-:cs, It4d Colum4ia -Un-bersity_ - L'otiexe - ^t Phvii=st~s & Sr;r geon3.SNew i u-n- NY. I I p~ i I~~ ^~R: * ~'H ,~. _ ~cfessor of F_ror'seFr. .ry o +id fJwiiit~n. A. SON?A RiJISi, MM. U3rve=-sity--cf Puerto _ Rico Scho;:--of ~ ~ ate Projessar c ~ Medici e _a>:d =Med-e rz, S,..~ Jasr.. zi;rstoa~p, Iloi"~rsity e i u,~gor ~ea'.Ih sCier:cec ?entEr, POY`~1-d: 7AtK yti.AlON, MILD:= - ~- I$i-L£MEei` _ : IIA OF LiS- - Ge nErai Hospisat,-Los A,,geil-- -~_.A, B£I=JA%F.I~ Pt1R`rt~W~, ~ ~ Ne- Ytrz -Univris!-v_- h;edics. Cen wr, A3va°rae Prr,'ssfe' of '~e;:cisre, New yo rk. - University o: Chicago, Chic_ag6s iL. ---- --_ ° -- __-_ _-__ -- --- _ - kRANCM-C.-C#HAO, M.L ; Et?.U.- _ E.1NM.1 S,zrr'r.r -!nves-tg,cuv , L;enter for _ n]ood Chre; Pat3:3ioe ist; LAs Angrles Cou. - Re:esrch; bc:o-r,,-G.A _- -- _- - - -- - `--- _ -- - - ~_-- --__ _ -_`- _-°- - =-afSz_^-:e--gr-1 tSs;' _ '- _-,4nes --=3iF=ls7~"i: itIr'FIAR=I2 U. RYtg`RUMPqt~b- ° ANGELES :AN F. C_iii e.isOWSy.is PH-ii. SIS[±R M._EMfLYiA-HF,.-L,-PIA.3. _ -Assi3tent=-_ Profe~ _aj . Biecre,i3ir;=; -Cl.-a:rri!an, Depa-cnier= -of Che_t_istry- - Sis_uiEal solieg"Yirginis, R; ;hcr%3nd. -_ ?cegis Coaege; Westan; MA. - - SANFORD _%.t2ODOSI-I, K--- D. BRUCE F_ t:r`3ME?O'`7£ NCD., P-n.1Y. Assista>:' P.rojnsst-ir af -Mer,'-icin; _ `3'e,f>s __ Ffok3rd Hughes irrstiiu'e; Jnivz_sity o:_=_ -t:Inivtrsity-Schoee e:-Medi;=ne, Poston, Prr;jt.sor= of Cleemivr~,_ Rr.;chigan= StatP - -Loz _Ange`?3, C A:- _ Universiy, East La nsi hg = E- -Miar:aa Sc'saoot of Medicine -lvlitiFni, FL. MA_== - f -f f , ~

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NArFE P, M. JC__qO?_X, Da D._ PAUL "~ _ COsT& JR ., paxL s5, '-rstri. :za;ar sarota.v ~ssoerote k.o_jessar of ,Psycno.mv, Hgrisu?liraI 3iiC - TechFisa_ .'.atdte-_V«IYef3I:Fj. o:=:Maa8a'.`!Iuse "s °.-at- DQs'On, _ --"SIn!yerr:r_y, GreEPwb=::o. 13C=Ci:eSCe'. -- - u'reffr,r ?s_3!chGp}tarf`~. 2~.G;~to ,~GesearCF- DfOfeSS~: - of A'a.h g-; tY sity O` ` ns Ad rin_siraiicn Vermont inilege nf R~edic,ne; p~Ir1i: gtcr.. Labo.aG,ry, _ Hesyito'. sepsiive,y, CA. ROBERT : : Cc;~IRIs Pt:D: _ .isafst.. ~? -- -krr~jcssor -- o; _ Soziology, IiPI~~ T. ~I ~°~f, D.Sc. Frvfessor-r, B,Gc?-vnrstry, CcWr- b- ia== I:ueversiiy ~.~ Cnica_go ; L hic3go, t1L - a- - _-- - _ - - - _ _ - _ University £,Glibgr-_ --7 ?hy36tiiarS -curgeotts- New York,=H=` - _iRVING?_ ORAWFOR;;'.:1'._ _ = Prof.essor and vx,ar~ -t, t~~ait .~r; Jw_ YD. CC__FFAAN.M'':- Micrnyio:ogs~-. =-U*L.e,si_ty af ,;,~ ~e;;Fic=l Heo=. Iz~r~~here' ~ss'iege~.~edisir:e, Iaw_~its. Depart- _ rrren ; - i-niversity= Hospitai; SS#or, Mi.- _ - T. iifvSL"iTHY-I<RO~ICI=A`4:II -_ - -=-_ -_ -- --- - -- -- - - - - - - - .- ALLEN B, C(DI-iEi7, ..-*f~_.Ii t::.lk- __ ---,- C'atlidrnia, i.slaeare of?Ae(fic;rl_-, irytn e. Asscc~~e- J?rsiess w of - YEdicine: Chief, }iealt5 E..:e=es c.,e-aivr. :', iLdelgh-ia.-PA-..1ssor:ai_ _fes :.r t :~dciRe rsi~ - ~ - - ~i -~': ~t-rric~ary= Sectien-_ Ty~t ~sta~¢I~ :° =C;~>i :OfL E -_C?m; ~a m~n- ~h~sr~lo~ i ; ~ar_ect~.r, arr_ oT ~, i ~ - ~p T l .ff ;I--s-.,,°-° -t -stT. = ry ~onnv fed#3> = t seaarq..,'Pene.4rc = Epidemi^'.oVy . = 4w Iess:;>_ _ --:~= d;ire2c~:, ~I ~a;~ ~a#ifa:nia=Scn~o;~fMedic:ne;_Da-it-` --praQrarr!~ -r-e~ i-'~~--~.f. -HiQkxn3=t1E?1yEfs?t~ -- _ - - - _ _ _ - ,,.= C Sch%o4 _-o-f-_Hygl2t=w s;'ti_Fgblic Heai,tl;, R_searck r7epartryter.:,- 5t.- _ Joserm'= _ valtimare,-`.!O= I:os;fita1, Il,rtiank, CA. _ -CiA_NIL 0O_ PtN_,=D.Y.M., M.P.- ±. D_AII_-ri__51-_£;RtJMF-Ai.-~ER_: i'x_M.-= _ - - A_sEsF:~3tt - ? -iesa"v, of . Ve-ridfir'f' ==-PrOfe3soa -Qnd ° 0.0irir{an, DeDJa"[_me.^.! of E -Etisireymantai, Pop-Oa,ion -aM cgy,- `_tr3:versi - wf i3niymi t ; =-,6 f Pen~yhaa.ia_ _C~ncrei iuE O:is:it-Ic - -11iol fe,eri-buy ~Ledici n~~Pi;ilatieip:ia,PA. Ooiorada% s;;u:;ie-.. ALA_N_O. COLLINS, :`v.D: A_Lg-: r-ID3 MvIK_ ri.D, PH D; _ - ` ;Ji - - -,~hthG`9vt3~og y,' - ltE-3c'u-~i'~ - S-~yc4~te- -P.'F}r~°23o: = n=._ ~'iL~ ' s7=}if3l7gj_`_-- Lect~er--- lJ9i -- ~l s' ,~ 'ensi:it~~ for ise,eavioia:- _Genetics-- - Associ te -i~ -_ Nedicot -,tnth>apolory, an-iversity of ~~lcr°a:o, Buui d`r.-= Peai oav =-IVFusFun: = Harvard- LJni ersity; - IULIvS,I-I. ~.`,~tr~r, = ~ eardi=a?tular _ k?searc-h __ =-TYOP&AS RDA"WH;= R_ -r: . D1_- . - - = --- InstiIII_'-g;-=_ cIn-i`remiES =---.~.-i _-catifwm4'd-_ ==~~`c7r'c_:fZlz Fri}.'-t'-;-sar of -fY_mli':r.e;=;='sr`.."tt7tl __ [idi~ra.sit-y_ kfiat;: of _l4ed:,..:._e, .~,-cst:>ri, _?1e;_icai C_enter, S_aMFatici;ce _. ~A. IJE A.3' it~ Cl??.TI:~'-R-Sr?'LO_ dssoc:aie _ -rJirec=o=, -De-parta~enr- ot_ R, F, =O,~MZA0N, f`E:M. ~a~orar__ _- 6~~dicise~ - it. _ ~taf_,'i ___ rfe:~Ur _= o,i = Bot~n~ ~_ ~ ' 4,-.tsia -IIespita?rlvtad_san. W=r - - 13:ider:,ity. Ntsy York: - - - _ _ -- J;JH; P_ sEi A??SY, M.D1.; Ps.rS. _ -_ _ = -!."ls=n~l ~'rojr s3- -of '~`~rgery ~ - ~~ssacrve Pi'ojgssor_of- 3i!rgef i~recto}; Surgical ~ahdratozy of -Cee?I~r __ -'3tri~saty-wr_-Pvlb~.~--+.:sota, 3~firnecpolis. ert -~=,fctejh -CoFIP = ~ C.- ' ' 41k ~I* s'Olt -, i a - - - 'y r- - $Y, ~~ _.~ }. ,,, ~T~'wtr r - -~ - - - ~LF R =^~ * ~ - - - . s : ` - c.` `~_ 4E-,i:rine _of- }~~Ii>a -~i1t:.a~r<i3y;=ih;ef ~u~ =c t ter ,.e yet¢r-ns: ExechEixe:= Psy_`o ~?e_er~. -~1,e Age a g. ~. ~° , - -Ceaer oi'-Td~ Ergtarsd-, i~:c:, Rost_n, Au nini~ra.ianosp~ah~ The rJ-o;,~, ~~i3-

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>yD?i<ARJ '--_,i~uv[II3~, Fi;.~: ~Oti N R: ~t r ~t~-~';=i t~. p rof<Fso:= t,=-~~rrs~ce ~rs U4iy..ity- A'_setiat_=-°-r o,fe;;nr= 3j Fal.,E;icgy '. _` " of L. , :aire SE'ey ?S: T SalL'3@Y o~ ---_ ~ Gf ~t ch_ip3n. 4nfl Ab6P. ~.~ -- -- ---_ _ - -' - __ .~ - - - _ - -- . RALPH L. DORFMAN, T.P, EireE,or - 0- -!-q&rn~.r- '_WorceE>er Ht_R_11E 7EVAN-S_ M-15y: Foundation for - - uer3fim~s= Roio~. _ " D:r ecco-t _ U-1-sartrse=u- -c-f-' P cd iatr:ecs, ,- - RgSe.7r£!!= - C ry-3rp;l I i!um`rF+- - aits trtu tC T kri_°.-,--m._---=- -. - - Center at Daf17s; Ca_r;3'ovasEui~f~ ~ Psychiatry, vn =FFrsit" of :,x`-:,S~ii?f; - - 1E-._ _ _ _ Un°vE-r5ity -= o: T'.Fam eif th aci2:'~.e = Pr?fk acr - -O,`-- ps y=holGr:F-- lnsti;ute, ~` As3i-SIi!.ilt =-PrOf£3Sv3 - =V~VRhYSdofoaY, HAi» `.-EYS; i''i.K, "-.~.D-,--u S-I. -'= - H. FRED DO ?:? i'e EY, S'Fs-: U. _ ShrswsSUry;-tvlA: __ deeri.h Hospital and Medical -=Centtc - of _ _,---_- Esrookl-_rn, i;rooiiiTn; NY. INe.Fcyist Hnsp:?al of BeiFarrnine ['h@'cge; L-ouisE`iile, KY. lCa'iCernia= Sc-hxl-= of- -Medicine, Los i-iA:vS La_ FALK. -Fa=D. JAMES 1. DYAR,-PaZ-. = Adjmv -Prvfe,si;- rf -A3saVint irtfestot g?_ --~saio~Y:`- Dr~koi:~dYs t'nivers sj of ~u?Ler=r _ F= Rnh :~s._ _ - _ -- ` Chtrf, -Pw.nanery Fur£t;OL~aCu rstorr DANA rARNSS'i)R'`-t -'M.Ls. - assr x^s P - q. FeS O> f 34! ea Hr ,v K _ D i-is or _o- ; Eiv n= ~ - Unrsit,-os ~t;icagh "h=_ a - crt:f ~rr~fa:,~ Uni e-rsiEy i~e~;h =vic~ ~` 3 L Hrvard?nivecs.,y,-~roh =- ~ - ,ZOBERT - [rHT, >'t:.6 rrofetsoi 3~ ~c,ir ;-Y; Ii ~3nat e ~GAT) _}E1NT ~TEK~:_.ii. - ~-__ _ - -=UniVimity. Ea..t -1...a:._Unt = - - _ - --_ __ _ --=yetio-' =i.et;:FS?£`--sb7 ~-i:oce`fcP.??;isy, The _ = reafge s. *is ~_ei?e. o wite S ierce JO"iIV IW-.-Es^J&LSTi INfur.D:=- Ygi-r~.v_v t-ni-v_,~.;A ~-- 4viv_1`rae!. _- Assis:amt YsT:9sse; -of ~ r~. ~s _Afi ~r-%pe. _ Stas_ LJn-iversity of to_ va t oF_ge = of ?CSE-P:D. E: D MAN; M.13: ls4edicine, Iowa Cit_y._ fm:nUnot'a.'FG! 2rsE; Scr!pM°-'Cli,c-= ayd ridrecsdr, =bnstitute oa-~ - ---.^. f_ c' ;_=- ~ ~F-.'~-''v~:~. Q - - '-~- - -- -- .~t~7_75~ti- 1 `~< 9~ __d~.: i~ ]r~ i _ - i1_D. - ~ -~ - ~ B~~F_ c-iif.'s ii~"t:HEir :T -n-:"S: _ - -`_ _$aearc - Ca.deusi3,- 1a; sj _ =SF_ .-_ T~ ~- - - - ~_ -_ -_ - - R_Se's s;:. :Z~:£~ t=. ~- y ._-.~ `i8n-=- i?~~` o~i= -Mssachus-ets ~h-oat =of .:L-;L^m-e,- + ~ _ W=ez er: : H.'MAR_EFhG£iBEit-G,M-D.= .trterriang Fh--s-i£iar, Cedz af I*_3anon -FkANi`= C.=t:-'ERGUSONI;t: i-:ospitals Los Ang2'"CA Cha:p-na,-r, Denar[n?erEr- o` Pliafmaso:ogy :r.e_ A56aisy Medical Coliege -cf -'~,~naon ( Al.tL-E-fF`..N-Ir--ERIC1GSON'. ::-I3. University, r~7Dsnv. ?v"f. - - _ F 6'es.._r_ af ~'h3r...~ ,$;'. ?t _unev1'rSit?l fi: - -?imi-~ - co;,e~ E --Phi'Mcy '-~- 6ESSin . ~ -_ . .. . ~_ - C..~=£ort~.°~ _ ?-:°=e.~r --York_ (. .v_e:s. q ___-- s- MeCi:.a4 Ce :ter New :';;rk. - = - HE$+RY :. LS-ERY PID.- - _ - -- ~__ -_ _ -' -_ . - - - - ~ : eseah:h f»amc-?tr©,£. Mason Research ':°I-1EODO1;E i`d._ }aNtri<Y M.y_= ~ insiitute, Worc_esty=-_-MA. -__ -_ _-- _ _-P.dr.=i_nr;- =- Pulmonary )3esez-rsi = 3.aboratorpr_ IAoe:ett- Zio.- Hvspital, San Professor = of ~ __Q=~)'lC~:7~: -_ ` >nnd C°!i'sf -af- E__3_,:J?m:qfo- '`hiF?,ga--Bo?_d __ -- B3ch~m:szryi Queec-_ iro+i_g_?f the C;tv-_ of i~e-alt?~, ChiE-ago, II ~f URsvi3ityoI i`ew V::rk= ~ tiingj4Y--- -'*`A:1,T"-R 3-- 1:u~-AwT: T fD;' 3)<IA-M-1. FISHf?iahi; lvm.D. ` Francisco, CA. _ Z...i - - ~ ~z -

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EDWIN R. FISIJLR, f-4_P. -_ rpE((:r =_ 3~_-=-=_~abG~a`~SQr`ie3 _ Sh&JY.i ::y !#aigCq--_ Df: rctGi; --~77ait?'~~t _ of = - - _-_i _- .ndsiu.a_ -_: ~1rss;~-_ oi_=--_s`7E`aioc~'{ -= PlE~7S?i.=-__`ivir~?d- =a_°.ES°ary=re_--=e``a1'ser EI~i ~? VIZEC-_- r. nE r e c arg h t A ~ 'E2rii,'YesltJ'-- /3i _eitl~F scho~i; c.r- . --~ +,.rf4ati~.n '~esEC~c;i l~isti,.~te, _r'skia.:f2,- _ j r x, d i a ~ F c.v:isu r~r n f calf ~oieve4r ns Ir crk - ~~ Adm-r..saionM=dica C~, er _ Say = / i ~ ~ ! s9C<afr rcSOo! !_ -_-.t:,r .-N oc re Nra` -"yI -. Jx'K Wr2flNfaE:_ F;t3. Yrk LiF vers-iy ~_'.,,,ia! Czter, RE,w I " ~. I~ S xktolm, Swde MOl:;Tf'N trALr3STO+_ Mr HYS cr<2, ~e_ Kkra9itlskv i95f,ii~ -- - ' - - 3IR.=.=A_ - FLGD-R1j_.-MYRR.':ED, Drrrcds>, .G;tr:ute of -a..~~e'_t;' BL tv.$Y. UQiY?r5i'ey _ of y3~- -sn`-a5~_iSa_ii - AssrstG._?_ Professor- tf E-rEv:rv,,-ssWal _. Fra-m-kco, C_A.; ~=' IrFL sc r ItS £ranciscoProjz1 c of Bare oJuand rVt,,.,L-aiM R_. -NFI?o`Ati PHMrrrrrmatrrsSJr gyLr v? s,tv ta!-,i.?:iA, P es,dent z a Jo ia Car.cer Re3FarL h $erkp_Iey ~ 164editi^e E'alii'ers;a :i.-4ac-aI - ;rer??s= = - -f3QiYE:$Itj of M~rtia:r~ 3..~rj_i: (i:-_ _..=7~'i~ji'is2 A:~"u~:Clm6. -U':~S3er~it}- of __ A ~ -E - -Net,~-.-113 Y -CA tDN~ ii D _- I Associate _PT..EesS^r-.- ~rf '~~:~:ffc`'~'~ ~ B_1. FREFD=A DsRM-I . - ze r,aty of e..~ he,: i st+for: ia- Schr--,_; Dlireierr o -~-~.ocer ReIr~ ;-W3]C; Z{C` -- s)f _4Efe r3-c_ Los i"ge°eS. ~a~ytta a, d .Medca-I. ._ Ce ,-tp-rSa2 _ 'II ,~ rca : ~o, ;~ M iCPyEL r - -. GEi~Kr~S, l~i: s, ~'~t.L._ r .s~Fzs3't?$ en- - _ Vwfhqlir;r?rzrr,- _~ A:.LA-N P. ; ~ED.'.:a3~, ~:.~'s. - =l~~a :~tEn - of .Medaci~, Usnrers~y of ~ s ~ ~n f31n ,,. Ca rcorna _ B rnecHc_4~ ~_ AARO`r-t.. Fi3EEMANPK L. ~ = i ` i~ a ,. , 3; - YQii~1°3-hi , rA_ ~Ie_Fa,7cn°= Heailr P es ar~ ir i . _ Ro iva1! P =ssisldC = =_-PrOfgssc. MEdreer--e; - %~1i''4rnia St°L-: t` Pf_rlav is. -r+hnetrain _ Med r T'h ; ~A:vt~IV~ ~ c Lr`t~e c ~EY~ ~ a r ~; ~ ezecrr rg; ~ l a .~r_., 13rr c F ti ~ ~? ~ ~ r= ? }_.~ C y c andE: ni ~ -- - .r±:_m - -_~ - - - -of - -Suri_rY. ''yF- JI^%tris `ra_~~~Sans-- --_V ~e ~a K 10-2 = r}- P-.z.~- iag P- £ .ssz3t y er t -- t2sPa.cF, - R~4c ~.=---Zo~:~r,~'taI anh LIniYersaK of _ ~Ed~;in=; s D_- _ Nedi°a-I ficQter, Sa~ _r*an~~:sc;;,^A. _ ~a tirsoae R KONA-LD-46! GIL ; ~TE = L2 recrer, JJasic SaiencE Research Uait, J,tFPEC?!i , M r'__ __ _ ! a:eRr "e~,e- c.-P J-r3a:caii . r.Tniersa ;. s~ . _ Atsisianf -Profiess7r _ of parrri c?toa~ ~awai a[-MrCo"cnoiulu. iviedi aioI'_ege Oei.ia, Cciclr:ncrd_ _ _t`PiZA~~:. LL-:LIC4, i E.L'-. _ lu~Ei'tc}."e, -eSear~ i -_- -_ ~! -_- _ - i ! I , . _ ~~y= Maro- Medicam scholil_ RoOne5ter. MN. Ic =and =f oti Pfa Praisessor -and Chat: s:!?rr= Depar~mAm-o Cunun&aaF;rossor -_o,, TI;e ~~r~lin ka ~tt=rnc:t -.~r,ricine - and ImmuraoJrigy, - ~QViren~r n~1 ~ g~E ' ~.a ~cp:ory - or -'-~!; = FJ;IB ~CP~ y , ..-- ' ~ .J`HOtVCA.S M. GOCKE, M.D. Assac:at-e°-- Professor of PrevenfJye -~ _ -GE-iBER=-i-t.-F__-3E-D~~1-_, v3`aX° _ Yedic;,e-and Cr.rar'rhni- ~#:eaL.-k_51kettin- - -o1 -r'ath--` £t:- = vince3re'.- =.4a;1, Cv::ege of =_:Aelisiy., a.-1 Dentistry, HosPital, Worcastr MA _ = -- - ;Ersey Ciix IkJ: C.,

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U __ i _ - _.- . 1 - D_AalIL7T'S.-GOLLENByRl~,--O-J..-_ t dsscc dte=- Prc~_ss.tr oi Paehor ry, dttgrr f..g . !~ c a_ t° gi_r: rrE Lr, -- ~ 1 = Te~nple = CtisiYersity ~~aItb -Ssie:..ez= M:crvblo+og,=.haboratgries ;;3cg ~ _ Center, Phitade:phia 1eW s5 ~='lsiee -N~eii~al ( en:_e?; Queens A Fi t ~ i t - s on a;tta a t.5s~ eLt r tt ?AVTL C-GLIs lRt~~ a. ~ s Associate P o;esso c,` Per._~nrr hfo;y, H ODAA Hasvart School of ^cr,t ':iediciF<, A ss=jca:a= Fro,e sc; Comr;u ty ar?u -' 'S3 y of IOn Ik Fr dlrorr=~:.ertltTi ~~ edrL=t;e lj1 :A - B , .-- _- - -.-_- - `_.-_ . _-- ~ _+ 13 - LEONIDE GOFDS°IEl-N, E3Sc. _ 3s,oc,~e =- Pro,f9sso yi -=Psysh;a:ry. h R GCitT"_f3.V,M, M V .Sc , -InstiYute for- MPntal 1']eait';- S=iences, -PH-D. dolledo y of ME ediane & l$3eW istrJ ~i ..eW- === dssociate Carlcer== °s'tz sea._h-:; Sc,emti:u' -- jzrsPV Rutgc%s Meu7-a! - Sc_:r.,l Ee,a,-im l;t- - - r` =tpcr. i?n:~ .- P=seataara}°. Ti:cmrvey.__3, r'ncwwe11 , ark ?`cn-i.r.ri3. =lalSil[lite-~ Fliitfalo, NY - Lef-tkzer_- :39 - B! : h ..- Cc.I!ege, E3nive-r=;ty of :-r~scian, I.ond;r-a i'`-A. HADDYM.z=, rjT; Englafld- _ = Prr:IesSOr= ~nr~ r pa+r~arr- i3C±3rt'nI<t o: _ Pkrsiology, =:;^iversitv--'^iversitv-- r' i:~-I2:bSfY~= - ~0 I3iN-W7 t~09 P OD.j -C : --- - - ~ `%ey~ o~-vSr+e'in'.a, ~sGF :Tti7n'". Un:ve-Tsity -Sckooi of Medicir,$;-Crief of - Profesvor b;" ;°,+'o-molo8y;_North Carolina =.atlo.'r`tr) - -Sertit_ce, _-- -= i%etera.+S=~-- -State College, F.aleigl3. Adrsraniit#atiar~ :~t~s~ita;;_ ~'est= ~ o~vpry ProR`clia~-..- ' _fessrr ° uyT-.a.=mtca(og;, = Peofes,or of Patitor'3gy, goston FRANK E.-~IT=f-i~~iE;-Pn:i~: _- IRA GORr~M,I1 %rEi<TRUDEF IT'. GG.""iISC tlAL i; Psi:if. ---Medica~ C-er~"tar. -r-i:f_ahor.ia Citsr, ~ssists*- ~rofessor- of _. ~tlbt~e~~.trY, _ _ - !' ...- }Kole~t~. - ~hooLof "~edicii,e, P ;iladeiphia. i .~irector, /ns.rtute- J`°-- Physicians di Surgeons, Ne- York. Dirertvr, CaFd'_opulmerary Unii, T be L873,4,en2u - __ Uo3pital; -= dssocJat-_ - in _ - Colyrr.bia- :ifti°=er..E-i-`._y Colleg-e_ G? 10SEP41 FA :S>;iI-I_.:~,-M.D. ,- i7raiversity 1"r- -Pennsyl4a:+i~ I M_a_ E-vREE*?, M.U.-- - hledicine Virology, - St._ Louis Unlverzty_-Me_dicat ~- Cen er, St. Icrbis, MO. 'viAsctsIT HAMOSH, PH.D. ; = i?zseareh Assostata, - DeparLneFt of - -.:. CLARK C;°IFFIN; :1t.I:). = Pbysiolygy and Biophysics, Georgetown Head, DeParimeni_ of Biocize: Eistry, M,D. University Scti<_-tils s? Medicine ar.d ~er:io. Biocremi,t, a:uthwest Ite--sarc :_ _BICHAIZD, }IA-V>tt. M:D. ~- Institute, San Antonio, TX, - Assistar: Professor of Medicine, LT ~1^[cTGAi I. GK7~~!vtAN, '`£.D,, P: .l?. r.i?~ecsit;_ o'_Califo=nia -+yledica3-Ce;,_ier, I- Saan trancis;;o. i- _ Associate C7inicai Professor--oJ hfeaicine; _ ~ '.3niyer?it} of California Medical-Center, I3ERBEAT R. HAWTHORNE, M,D.- 1 L3s An,geles Chairman, -- Departrnent - of Surgerv; T;niversity- oi ren: sylvania_ i,radvate , -CARL C. i,RvtY_;ZIT,_M.D., PIi.D. School of h4edi.ine, 21:iladeiphia. ~ Assrsreate - :,:- - PFysio'^iy- u'~d ~ 1~_ Pharmacologybj _ OI['~ A. II4~ E~, M,D. ,:' `. rtversi+.p aduate - cchxl of Associate Pathoioyist, -Mallory institutz - lvaria G I Penns I ~I~TH ; RL_ GR %~, i `~- ~tata U ,iversity,=":ort~r idge. ~ I Andersoa I-Iospita: ar:n_-T umor ,nstitu;e, -- Dentsstr=}°, Washington, y£', Jniversiti -of ieza< Medical center, i Houston. BERNARD HANES, PK.D, - I- Professor o-f Fr"ealth-ycience Califo r y ._ Medicine, Phiiadeiphia. of Pat'r.olo8Y,_- BJSton- ~ ity Ilospital, - - ..oston- INTP=.

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inc.,Sethesdr, MD~ _ iERR~' k~tSRT Jxd^~ ~lt ~r~r~- ~ER~~`Fe ~ x ?~~ ` IiEF?~cI t'. ~~ L~ - ~ re = r ri _~'~r! p y~~~;~ - I~ Associate ~'r~essc= of Axer ~bFoi.4p ~ve and Ear -+ ~ ~'i°oig~.r.d~x~ Jn Yest,v Schp'Ols Of _ -- k _Md-.=ine yn_d -DeCfls_trr_:rasha;~vor,~DIC . -l;._:..' I US i=;AL_R~fi' ' '" - -- _ = ---it l= . :.,~. Assezl.:.e ` Pra;~sso- a - u Xe~ - and L~i@Rc[~CE_ L ?IEiyR, J~. ~..,I . Drrec~r of ~.-giC f -_ .ke~s r,-F m-i<«ass ar<d L.uiiege of Souta£anaa:r~, ChaTiPstor = ~ E sB E = CURT 3~rr~= Ar' -P~,3. ~rr~~e £~ l~>thz a~. dicvi~~ ~i Protessnr _arrd CarrF.~EV is~n a v¢~4e~_3tztg ~~^,ver~i,~ of ~'c~ ©rIE ~I 1 _ _ Res~rcfT `Aed c_l-CoIeje o_ SIo ~Y rook, Sy:,ro3z, ~;°ch at%ic B tun ` - ~ virg.Aia v= +r-ced - ~ciC~Icr': E J4~Vlri P r ~ z 3tROME !.°iiBri~AR i~p.~7 ~.a.;~~i~te Frcif~s c° p-, rrnsc~:~~y: '~'{ - - AmSrf E Irsi-.It c[i3 eg~_ ~cences_ ~~v~f~_- ~~__~?_~~a zaxe_ ~sF:=} Tfiaa~i;. eRrat~~ilV___ - - L3esi/fB-A . .{©~II~3ON. PifA^-- -" ? V- -- t~ t--OLMA=NT, ?=:.B .f sarqnu $rajss .* c,~= ~och ~ is~r~, East ~ - - Lo;;s,att4 S4ate Vrtvers;iy ~Scaoa' ~. ` 'Cerir_essee Seae UJnivitf.it3' Cc,_;iege_ of - - _ irRdtce:x New~riea-ts = I-^ine 'nhatson "t-x. ~ f)3F+AID (?' E A. f~rJ TER ~Ai~N,'~t 'R- ~}f~~ ?. ab r~ r+tl~esiiaspiI,~=1~w Ysr=:. Raec~-,?T ~_e~Fk;' srhu^~ ' _ ,_ yT~tiy4rsit; r N¢-.-e Da_-me =Nstre-B i- _ REBDY ~~pNt RG"RB ,lin ePJiarrrionetics- M. E t i atr~Y Nti~ar~ ; ii,m_r ~a p>t*4_ r ~s7sFderti ~d i e_ o F ~, .,~~stat~_, -~.if#' ii~Y` - 9i~,, -- ANBY4r` ,4 Kd~1CF;~C~t ~k L T Rt.BER W_.?#ULLPt.D~ r, a7~~ F,ar Ra-bs, ~ts e U'nwersity-TarsaFtz.seA, Rr-J`NA' il n~-.=. Y,u IT_'HfNSsN , PkXAMNG-LB R. 1CA'd.AN, Psi-1i. - - ~~ear~;: Birrr~r.~ =~~.:nd= ticr for -=-1`,irecipr, ~-a~ef~Fa ~o%-~i~~~®!-~~+et:cs ~2~i~sa? ;?.n-rcti a 6~__~; ~4.:- =_-~!iaakisiilu.Ye- - amd= i - :-lespit_!.-Cie~°:a~d - _ = 'S5'z5 = C1.a~~clk_ W_~IE::~$i~,U~.~ ~I9 R) SCA:~CH i~+s.Ti~1:>, t lt:c=$3, I_e. - - _Y7eje3 Gr = of A3Ed:~iiFe --iiiFd DF>C ° ~ - -_ - __ - - ~ -. _ - ~ _- - ~~ - f'.s'.-3-=_- 9ni6c354K = i=.LP''IO NSx JINiE ~ a!-~, Y~' 9 F ~rrR"r %va kaSt i.aro~i.,"a~ Univs,si y~ G _ NORM4N W. PEUMSTRA. ?-q.D. _ - tv!edi_=ne. G:eer~: J'rv)~~or _ of P,yckslery. i7irecznr, Jiumait F~actyrs L':vers-tyY i-[ARR;~ f Sta Bax a vehi:.t <ndi~ PLt~~~~~yt, ' ~nok ocu,r,c .g~. tio5RS, l ~nrr~i Prosoei i YAUTiN: iEI= Er .°_D. n.ie5olear. C5E..:ae`2a UlF+ ver3:t= College II .. ~=-2e-s2arek: .-Asec.:le - in C~ve^fagy-c;' nf P'.~.yE:cia:r~ S-yerr: ti~~' York. _ - Cy_-'Fithe`r<'S'ry, S~ ~ !7rsnf'l3co s_:Cita -- JA V-ed:-,-al 1-seer es, _Sah_ F ra.._:c o, C.k- , GEOFCr JACFJ'St~-11il M; .-_ _- '-` .- --- _-_- --- -_- - -~ ` '_ - - __ Pnif£S3G a5d =JWS:i, icp-_!eIIer.p TJS -LAROr_J-. Raiiphg, t,;nit'y u: `-So,ther: Bepaa:~-n: u: - E=pe_,^en E~ - "Ca;:'~a~3r, ef Me"ciref-os_

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A`1iAL...AH_KAP?AS: Ax.D.- ---- ~ cL'vjt~it![=_'.~3d S23' o-_ s ~}aif:°,dft =- ~n_? _- .slsst~l_ ?ro.'Qs.i.'f, :a'-.'aSlv'a - - Sli- _ t= RowKafelleF iIaiverfq}, NewYcrk ti~sro er$Lfy,- Naiv_;e-szv ~t~t~ -L'oi?eg2 ` t: ~Aedi -ne, ;efsejI-ity w~ - j~Al~A DJ k .:ysistau-= _ _-D.recr,~ -= --:;erdLovascy?,r SiIiy ICUUL.ANDi;R, 1ki.D. _ - - LabofntcryInstiuctor -. rl- ':edicisee, - -'rcjesao- an ~ hafrrr- spa-r:r:^ :+ of -Raht1Ei7%ara!- ~ lvle&--of -e GI'cge-_ .-an~d rtirt::'S::s aT.'t i jile<o]Gb ~Imi- rrSie; 't;ostiitai, Philadelp5 a P-'-~. of LGnc Lutid§wedE-_.'1 DON LA:~E~~rf~..~i. E~'t.D, - -Pr; ~ H KN~arP r. t? Assa.cr: ' - ~?ro;Eis~~ a~ - ~' . ~ ~_ - - eser'v~: _s=_`.3f_ 'e`~'or _ a: r~~ Bi~siiFa =UGt:vQrsi'-.. _ o?' __~Jers<C:_1---_ ---_- rvle@z ~ tin;sersity School -of =~~iedie=nA; L~stLr,, ~ievit i;E, ~i rl:ng!on: MA. PAG'~ ~ L.~R~'~R, -r~i.~ F._ fasag ~ rf~s o~ ~? er~a . n -ftOB£?t l ev. RRU i,1 -K,?F':=-.C'r - _H15p 11alaWorLes.er, 1.~'?. - Trolassor -o,: Ckemazr-y,- University of - Rc;iieste_=!Ko<:nestef_ NY __- - JOSEPH M. L.AiIWLFdYI:S, Wa-PH.L°. =L [:iLI KATZ. PH.D.-- -- - LAWRENCE _i,= KUPPER, P'i!. :. __ r ~ f -` F - ° ~i - iGS.E e o = G --, _ _ , . U,1 .~ssot ue=ro' -roieSSO.' 3-- - _ ,~ssr-~ia2e S ~- UeriersiTy of f -iLr4o-_:.`F;iGZoo, IL. aJa=='e.'a=iy- o[ N6rth `s:ert7iiRa .~;Cr.t.c;3 Gt - ` - - - - _ -- _ _ . - _ ~~_ _ _ - - -_ P:34`.c r' a.t >..^a~_C31{ i .- = i l ` Y L:aL3FFMa .- s m. - , D S11:RLL ~_ r~! _pa2t~t£ss-~V-.sF3 . ~'!i vFf .tv- MARVIN K'isC`-' 1`R _ + of New York D4w_ r3 ,at a.ecsical ~-eztLr. _ adea° 'x k __U~ ~e-sit; Iv<ed:4x':- t Brookiy_. ~, hY.= - New Yo k - - ~t - = ~° _ - - - ~ AhLLL K )rYS, Pv.DCHARLriS-W. L.ARELL£- Pu D- s D rec!+_a, L_ a~racty _af' Physiullcgica~ Assistara- Proy=soT nl -nvircvi ~:ai -- L t_ k,~'_et!E: e ot~E~. s 5t i~='..rtres_a_ ~Fiov: hygiene e,f.e sa .- i~+fPSS k,: ollegg; ~-~ - -3= of P!s~-`._i#'e°lth,-t~iR~' ;;F,C iS- _ _, - -ii 1.3~i .p.s, 3'!~. y _- ~ _f^~cP".~B.~!.-SNF~ ~~ ~S'. L'~~ -4f9]es3C-z -_of _ -mec.alq _,- t1-.IvP;'3 tf- ~ P.~j2 t? rY.fd 'm,=v"'j7~.~=~~3E31~;.5 j irDdA-R^ 1-K1.AI5E-R, h=.D. ~-_ ~e~o- Scies£isl s?L siar v tEr LAtRFy3!. M.M. - ~~ '- , _ ^ - _.` - ~'hi~3gi:-Cni of ~e~it°:~, : r#C;~:. 1-'a._._~ -~r:~;C .y~ - - i~i`:3rE -!ty_ G: - ~E~_-_ . ~_ °?.texici Sci oo~ fics- datloniqr Exp3r. eritaa BigEcg°-; Pro/essuof - -~atilr~: nY Rio-t`wes!e* ° - -_ - ~ Inc., Jh:ewat<_,y, M& _ t31vers sf ba_Lv La! Sdaol, ChP-a~D l~ D. ~ ]EROME _i4LFiFIERMrN-W6. MICHAEL E. LqiSA M Deea=tis;eF, v_ r' ojezsmx o0 pa:h??l'o-g} xe4 yo.K ~ :*tologyHov,.t Sicas_sfie ; _ of Uave ylr.m ~ Nled~F l_s, . . [{ aiaversi!y os ''a_h-..~rsr School of .`E , '- ..Te~7.e ~' _ I i ~-_ - - ~'_Ii.`E, vrR K = K£l~ ts E i ~s P.~" ?v~i ~ ~~~,i. I3- a P;ehrns dL a C:IIe eof Vir1~31 t 3° c~t:el _m~- .1 s- s1n3 r^u^;y _ - - - - A L 71'~'. ~. K a~JJ]aa,-~_ b7 ¢ s ~rate Pojessor of ~~. ...;:ry_ New iG_ rA. - - ~ , - - - -_- ~----- 5 or'.' Ls-~vEg3sty. "'JEoi~ E G ~: - - --- - =G'arrSI e~ vM -- t= -- _ - - _- _- -- - - ` -- - --- - v~arJ ~ 6r. = nf-4~.cI'£~ 7c.-=- iIll'-2~° L -p tJi e3.42! _ :Ird.lcr3i_ G33r.1 _ C~l4:P`_~,a. i dCL..Ua L K:JL ~]`iL~ ~~ F Departmerl-I -_ of = Pa3~ ~logy asid _ Po/essor and Cna ~an, i?epartm,.at of > _-~ _ il>~~1~:2. !vt; ~osc pis r~Ha!Gr.~ f~ - - - ~-. -- ~ 'i; - - - - - Pa - .iay y- `- - RosE-Presb,terta et.= -Ltk~s ~*':iai s te Les-ve~, ~ euv~ n' Beig irs. a 0 iGCatry, u _ se._ ~ ...ggE vi 1 i-_ pa_ie1~;7arv - - ~~ ~a~_r~t:,r;' a _ - .!?:aaE$- ~1C3 -YGus'- iFi.~~i~-al -_ i t.S?.i=2bE, sX ~- HisT =d~G G s. ti - ~sa~xe~;n.ve;sitri i~ o -~Eptef, Ch!CagG~ .L:~-- - ` `_ - - ,. -_ --_- - -- -- - - -- - - - `-- - i

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jAMF.& = L££ r" . P. LONG, PH.2_~ --- __ Asr"s:aFi,~Pr6fes=Of-=- v!_ B1~,-,c..m":lsyj ~-prof___or c~'=- Rherm ,---:crlogY.--- StaF-- Sit~`i- f -oi~i3 =~FCZ!t1 Sf ~_ fe_d?-_ et, _ s ~ ~r ve rr~ [ 1 lwya ~ Ql,e- _ ~ _ _arm, - -- -- -= -_ - -` _ _ ~-~w-~ ~ifs~. -- _ - _ - - - x'[3WE,R.7 i_£r-iER, FIWD,_D."i',_. . isShNe! L..LONi.i=ENEK=-R== ~s~;~ c T, ineKss~ L~i~ rs,% ~~ ~ssx-~_= PsJ~~sos.f__P,~p~w^!v - ' <;, slaoya,~ o,` m -nnE~c E- ~3e~~~ ~ v~trers, y of ~ Pi-3tLZP ~?. 1,E Qst--_-.-.S114T , I'::.D ., D.Sc. P.vjesso. vr- Chema_rr, Noriheaster E y^LAYTr?N C-. ~SI IKD.- i'::=O ` QiEemtt Pos*om, MA- _kLSt ng- _ siolsav v _ _ Wdt ine _ cnq ~etnarvv;~, :,c~iye n!t _ c` __ ~;athT ~ n Rr_3lrRdt A. iE RKNERj 1.- t, Calra.r:3 - ~jcnc_ul- tr= Fr.ed:c,ne, Jos I"1 . 4'syoc- t~Ee b7e= 3beF i'd'pQS Clt.n.9c- and A. n_Q _ ,°s. Resear .k_ Foundar,Qt:jLa TQ:1Q,_C.A. - r'';F.fa -9 ~_ LOU:dA.M.::--- = I CCIL~ _ £U~`~~IRFRGER, t~ dr: - ds r-or f _ . `_rr,,,.e, s:,r .._-.'. - ~ ~~n~ers±y #~edi; CoT:Rge; ~:ew V?:~ ~ crlf ~; ~rt?,en Tf c'v~ ~ emist-y ,. Svvjm Insru;l:te_io: Experiment?z ~C-3=-*- - 1 = -Resea-_h, Laus2t,ne "voi!ae-!a14. XN-NL'Tt+ - M"OFtiL --LirNC_tL =-WD._ -- - = - _- , - - -- _ _ Sc'..L.,_ L.=.,.D: = rAi( A: LEV$','NtD_ are, <s,,v- Err°ertEa..t c/ -pr,rr,c!cgy vnd- r e ior, med-t?a Cc'.t?v O. _ So1 A..orecc.e- -P;--_Ie., s-r o; - M_rh_rn , y, a __ ~epa;chAssa, - ancs- Rmswha - ' ~; k1sdicine. Sm3Ftam-=,acO-_ -- - =_. - --_ -_ =_ --- - _ ~t~~ s:aE _= r-F' .lo:~'. oj-= 3Fsy. F!±_L!?= D , vb°'»-'IS Gsto~ia <~ iv5r~i;y C:i1fte of u- 3'er,Jvr Lee-.urec -,n rsn7vrctnvla2 v , R~~ai F!sic ~~ So,ve ~r; f4x~,rk. ._ ~~~ :s,et~ h~-t~ ~nzlon- t:ng.ar3 IGR~~ F x!P~I4 t~I L3 ~,II ti~ LS tP~ F,_o fesay~ vnd D~_rcrf3eparment a= ~ _ ` `.A. Lt,=,B-~-i~,s-i.i. T-nttroisgyand: ijm! ~i~gnis,_ New AVER!c.- ~ - ,e,Dek ~_: W Painio y js =e; - Cv gip Meuicine anz -. ~II~ePS l`3S*' - .S a?De5ltiirtiery -- W.- _ - _ -_t'~e~ti l4Y'1-a. le.. _MANN% PI D, =D } ~ V_i~-£ dtEl~LR, ~.G. Ascvs,t3~_ ~`rv; ess ~F ar -of Ph a*m-a-c! ic°eY- ~r~Te ~ of ~~~ierrF-a, ~~I^ e e;y of TEmp+e aitv+e*si;yS h:~ c. Fk9rmis~_ t Minii~?~ ~~ N ,, - -- -FfA~s.+elphra, PA= ESTEN 0. LtNDSE?F, I+-L'-, P-H-:9. S'-_ _- ][~s?~?t=~ ~ -_~_=£:,Spi`a!- ..2Sea!_c~i -_ A35 al'sai= ~~i~3svr 'ip_ .~~ -~ r_nLL- -~9n -rs' Nl' iabofcivey St. Cyr,err,!?8yss- -hosp:w, -~v =, Ange:~ Ce:,n.`;LU r vesi;y vt-~. tr.cFr: _ _ Rv~ Rr I-f.i..1NINEVL, P.q3B. fJr+ria =10 ie:gx i~Raje1Je .__ 61--- -P rE~-s3" 6'f C o -~%i _f1cCz!+Fp --- r n y ay ~-~e ,CH,~ - :A?atJ,-,~3.D. = instmxFor in 'V-x?:ca3 and Rc.lerio!srY; _ £~B!Ai~3~ ~..inF~~T""t =~ r ?Gfet c~ r 1,e3e- f ~~ C_ar~'~= ~ P. oiezsooj 5i't ~~ ~r,astvy e cinP B tinivers,~ScFs~ of M o s!cn y_= _ A~7s: CFMISTJ.~£R-`.!. !~IRI~~at~: -M4.~. =_-- - Assistant Frv,cssvr if--Msl;c:ael a?!d-_ _ ~c_ .,` ~. I ^.:r3,W~3:. ~i.t'.3f: _- ~?ertv ; ~?iEisic,:_:c>f- Infectious = Diseases, Ajff!.~e= : sncer r=e,earc:i Ancti~qe. --=Setc:F Y_:aii Cvliw _of- Medicens.- !er:ey _ _ t~tnc, 7V4A. - -- - _-- - - ° - -~ -- - - - -- - -- -. _ - - -~i~~ _- --- -- - -. - -- __ - _ - - - - _ - - - - ` ° - - - - --- ---_ _ ``~ _ -_ - - -- -- I_ i _ -- - 1 Fr ° j~ i~

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LoViSisn--Siat-=Ur5-1 2 s ,v --~siW = CL --1t33GriC!!~ P r~-f 3tNr L'-f= ~ el ~ahult=~ _ l ;~- ~~~~i! SJ - I:eriStv - ot - M~lrw_=s43_VCb.°._ i£8gd~- C _ HE rtiR `!`W Mbi`L~TOSni£M.D Fvjessar tr~= -~ I~'i~:=f ~" P_;isr~- - - - _ E °__- __ -- . --_ _ ° _ _ __ __ i gr~,0v~uia: `_- la~; Duke =,issrst~4r .grc7-'sstr r7- Pir~_Rr_~i~tr~ nn : VT'-ivorslE"-.,'feliC3i ~en:?.-, i31~:}sSi'S, Silrgery.__U~illvefS3[y_-3Y. iaa_L4rRl&=jChE)bl, __ _---- - _ - -_-.-- -- _: . __- _ - - -- ~-_ Of MELi.Cirle,.--Sai. I'.an;.14r(e` -FOL{DEfi.-1470v'ER.=M.Lr~.- - _- _ - == -- - - "-__ -.--' - _ _ - - _-_ -___ _ a?5r tlf _~~vicbqg .7ics'IUBit'LQ_CLCAASS SO{R E- ik:ESiicae °Co'lege = -3f- -Sutn f4r :4na, iN`--, I3E'r-esda, +:.*: - X_RilSSVLLM..A:7JN;M.D.= HEPHE4. MaKElsNNiS. J-_=:,_?st.i~_ ~ ~ Pr IF_s?r ot-~o~t^:r~. ~`z+ftr;3tia'*Gy P,Ps.uir _ . =1c _ v:5gy-- XCune~1= Tsia-~r.~~ - IsF M:ed9s1 le,- fs:gdicEm ~.~'-.oti3-`_4n ,=°trx. -- - LVi:anif_ ktGJN4LDI_G: M¢SSc?i5, VICTOR-A. M-K',';SIC- P~oj?sfcr ~d CA~irhw =~f~r.r~~~; ,` -= f o;~~r c= ?rr~~-~:ne, _ 7F~ roa~la - Pa-r_hologY, "UrsrvErs:t; of Utah Ceilege of _ Hopm lurivm-v s;.hocT of _Mad3.1__ ~ Medicine, Salt :.$ke C"ty._ _ - Rai:imOrE, X. ~3A~14~"cIC_EF_ ~-t~. -- BERNARD _ =Prejesror- u. - - .icting C°_e3::nza,n, ~ 'stvnt ~~~~~ar ~ ~~ v~}, . _ _ . ~rrEa~~.:,2i~ : Medical _ jzr`i~: s~r.~ i1.~PC ~sal 1'~ ~2, ;'hs13i~ . ip,~l-:~~ C'Dt:E~v _: ~utir Cara3;r,a, Char,°ston =- -PA.= l~_~.L Y-T: i~C~E~., I~.-i= ~.~. iAN~~G , ~I[Ll.:E[?..~'-_s.,-°.~_~F.~: -associate- Pro;-essor as Medr:ins, -Mej ltal,9;ojessr,r :v~~=Psych rirl- and _Psychtc __gy. roiLege=of Sau.hC'ato_lin .=ffia;iestbe. _D;-ertsi,_ Men:a-L rzeQ,ti ResearcFi Ni c itl7C° - :~3!: ~ _=RrL'ie:.

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JERAL-I` A. MITCHE_.L, F;.D= c4ii3ER: Fi. Ni:;:InlE ki.L3. A3socrade Praf:ysoi=of _-znwomy- Wayne = Qioj:ss'.r o{- Efedrerne, Drew State University S-0hoo- 4f k'_¢diciae,- - Eus:graduate __ Riedicz;_ S;tinst- ane I2t~jt; ?~`, ~lni,eecity of ~u: er?~ crer._ .c;, E,iiSlctary Dise%s:e -_ -~_ctScni, - CHAK..ES Mi7 s MAN-, M.D. _ LA-iier King Flnsp:ra, Les Amonge'es = -.9-Fretmr, -1i¢DdF$rsi . m U : - :_: Liseases, Ci'y 0- ~atii na: m-Ei,;~= ~.?~~ R;<~-~_i~A ~~A_ i..A_;3~A'~C_R_ -CenE~~,=DuarT- CA. 3r~3'.~ nR Rge. TN. HUGH 1vOtvTCOMRR i;_M.i3. FRANZ OE.4t_r. EP.D, - ' -.__=i7z4Gici3te 9^ie3,.,.- Gf Szs FClllEi. .'~r3fFSSQr of f = -Phis% G4~,^,~~ He"d, f ~ - = us~;verciiy o Pe: sy!va.aa x°,ho.._ -s - Secti,n - ;n =LSSohc :ic a! -i?ka: i _ r !.,y,, ~ fJ _iv.r_iEy of Maina :.nz, West ~ ~ a-rip __ Ge.~ia::y, E -_. G SS_0NTG0N:ZRIJ 'j<. L=ri.D- Texas SouthNyst-Mteica: 4 __^!_ D--ag.-_ __ Psoi£ss _ oi- pJkv*WvgYa.dLI;>ect, - I M_st:m y -.or Ce;:uia: Rese`-,^h; GE:,aZCE R:M-rx e-E; NtAl;_Fz.i2. __ ;;niversity of ivebra_sk3, Lincoln. -Ditecior, _ R cs*01- Park - ~mcr~:_= ;~titi_ts E3~ia!o, -d~°. _ BEVER. Y p2,iGE,N, ?"!?.__ £atwe.r- Ftaear k =SrFnist _V, -Roswp! - REMCr'Ei:_M. MOS_kgrs:D_ Park M rwiriai I: :*itvte. _ _ A SS °a±: Pfo;cYs -W :r£t ne -,=q = C-~rgeeav it~i,br__«= gr~'c~=:~huo:,==~EW~SE;HAs;E~~ ~f~_- _ {Eas!ungtdn I3_c`.=, -EarEcw--=,_ Uk -artnien= of ful;riecu!ar 9io:±~gy,-:iva!& Re:eaith'_ ij' -Vosavei! 1-3L?RLE3'=L>r Mn_T:.E-i' W0. °x!k =vi'dis::.~'' Ei_.!`2`y--" _ ~ --~ro1esTor- I- Medr.~ni?---and_D. f .,?or. Ca:deo=ResF raeors - L a'aoxa.a:y. ALBERT Es.J,qLMR _- tiniversir_y yi S5i<i~ein ~a1i4U,r5:3 Sc2oo.ssisttns - r°--'rJ-fesci=- .=a1hmtogv- -_ o- Loa Uraven:ly a#` T"o ecl,., rclerf-, C::-:. _° EDMOND _ AN'Ii'3tz"'.,'-.1'.-31;RP-Y=- _- M.U_-M=i.- -- =SGX-_ :-- .- - °- - ----- ~SSis.t.aTj ~;:_:i~ uL~ - - ~ --- _ -- x"- - - _Es u-Jo:r-P^/essor af Eios;otis:res- und __ Leztarer- -DeY rFre_nt ei -F_o-J -Scir:cie MEd?ft.:v, The J37nu-4fur: ?1ftiYCSS3~= TQc~h:^.3.~&-_ $i:~-r V!IY-- c:_ _ -_ +uf=ki~Oicir3,Bai[ir:roFp.'~;~;. ~aii=orr~iti Mavis: - - WiLLiA vi=S _MURRAy J^ D1E.rtN vi~ F:zsKr R, IVe.D_ - SertG-s '~;fn i/ =_ ~vs 2a"as3= AssGc :Gt°.`<?r Pl _.r - ----~-SEr. --n.~_~. Prcsaz _ of-.amcrgy, LaUaratory Ba _.- ior; =iE LTaive si,_-o+_S_ r...'_ _ C aliforr.:a Sr:n;c~3 --- - -o4"h4edi ne, L Ang¢iet R1CrI..RD L. NAE .°~; itt L`. Prsqessor:ond- =DeFa.t^a nt -)f AiARt`S'`E~iE?~SRSI~ ~4i;Pii.~ _ P2ie=mU~1, K siii~i+L 3.i° 3tai_ i1 -UY [Y_ - !si rcTes`'Z):- ofu A.ea:)_ rnj in = Cf'.-irge of fa E ~iclr'~,- E~iclr'~, , [crSk7EF. _ -_ - - ' - _ _ O3sielrr'cs=an; {,'3:n_.e :.~ i,.. .el :.~i, C:,-iuafiaa = - d ~sTi `es 5't]/ _~~1 ce? ~ F~;sds:'sr:s $f tiE Oy G B. i+`UM A` H s -li.L. S~rge_e_s, ~ ew -~o:i:.= -_ ~tic. cnr~: 3 ir~! ! r,ficr_atvry- Ham _u~_g,- lN_gst4Germarty =. - - ~ ~ - - - - 9 N(Di='3-"'" U : nAi;:i7 1! G1-: - _ HriROYuH? EiVEA rLAi,L. Assoca P. t'rof~sb... ~t PG heiog ~'ct~~~i _ Assoeiale-_ProJess . -of - bft,iic;na S;,e- pMspyter~ai ~: L.kemc ~_c .. C_eri* ~ aZh-i'_3 --`e~c~eti.;- -i;i:vF;Eiiy :r>'hCiES!=, _4i = C

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EDWARD W. PRL:K~-.*!; M.D. -WAL iE'R REDISC-H, ks.D. __- _Cccirrr~?, and_ Lx~iT~ntal ~her~~:;;t_~~, -Bost3n= ` New YoFk'= =iJriit~rsi,y -_ ~.c,ho~`s =ct:_ iraivxrstty s.°hoC1-o: Med iine, Aou=:on, Mea:cine,= .nd ~IYTJ =Re~eafcl~ ~evi~ ~ htA. ,Cldu+a r Mecno=.iat I-i:sp?al;-id4_v-Yo- t C ARL W. PIP_ RC CF; M.3., i'Iz.Y: F'rito=L-tAM REGL:,SOt., *n,3. Professor, De;`a=tments Ci` Pathology and- -=Pro;frssor and=c;lrairman, :)apartment-of= Microeiology-lnmitirro:oT;: -- WasFington Medical Oneo[ogy-,- Medical Ooileg_ O. Uni~vere_ity School oE-[4iedic: ny_-Director -Eirginia Rich:noRd. = Pathology attd- L3bor=torn Tw edici ne, The Jewish Hospitlil-of St L-ruis,- St. Lou:s, LY1'dNL-M. PU0,-1ei.D.- - - ht0. - _ i'rojbtc - P'roro33or of PutFof: gy,- rrarva?d Medical Schoo ;_ = Hos*oa;- --yFairn:an, - 1vl:ALrOyM=n. PIKE, 'tt.0.- Department of PathC[?gy, ; hil3ren's Pru~-~jvssot ol- tomr:aunif; =#fdic::;e a:n:~= Y-:i=sv=it>t Medical Center, -~osten_,'yiA. _ Pe3itatric.t. Univers:tY Qf - S~-utl ern Lalifornia -Scho,o1 o`= -Ncedici;3e;- Lds - HOBART A. =t2EIDA,A;4-N, M.D. = i Angeles._ .-Pr1Jessor- _ cj- - ?ser;:rine, = I#a'nnear~_ _ Tvied.ca;- -Oollege = end- - :-iospitat ~ OTAKAR ::?'.Ol<ti.AK MD.r?*tt .D._ _ PhrtaCelp=ia,-P... Exec><,iyf= iDirector, D-wsvse Med _at Resear-h ^e-ter, lnc-, Dovee, DE. EILEEN RL"MCLDO'iONNELL, .Pt?:D:~ °- Principa% Res-earch- Assn!s.`gte: l1[ar;-arei F -1_;3O)c-R.Si1'Ol_i.Q l-ct;, ;31 Medical Schcol ry-ve 3i zuo, Center :or ~ rJ:. ~_._. ,- lr;~_nd L=`~ora.~ary; ~nive-s ty g.~, Reseafc n, P~stor~, NiA. _ C:-~1!4trE -- _ 1:ERRER:' Y: RE~'!vOi'-'J-S, M.D. - - ~ - C. iK Pc ::3E~a'L- Ps t` Pa;rs,oF of ~i!ra ae.` ~e~._ P~trno.~3Ty Direct~,r ~~ ' 3fo'ogico = Research. = uection. Yale nr.e_rsity - Sn. cm_ _ of ' ~ ' or= Id°dicins_h ewflav~n, OT -._, _ oodatic1 -asnun - Medical Padza ;' f= Research, Pas adAaa, CA, Associate -=Pro;ess~ --=o~ = Pa~hoi:rgY; PFc~tiF~4 ~ - -tNS~I'acU?iEhiis - t,fniversit~ 61'-Soythern-Ca~iforn:a School r = = KOLLANL3 v:-REYivcaLOS, hi it. - S, N. PRADHAN, NS.B., :a.' Assistant Projessor oj -Pa?hologY,_ f f_ ieXa6 -= Scuth-w-_estern-- Pro!essr~s ` of PhvrriC;~i~Bi; H.ow&-d - = 13nive-ryiiy-=- o 2:~r`E~2: Unive'sity~ College _ of tl3edicirY `dedic~: ~;hoo:~r'~r`E~ Washington, DC VICTOR RICHARDS, M.U.- H. R. PRqT'>"=":'HOlul.S, -T..D:_ _ _ C:ie; ofSr<:aer_y, 13retoyier;ay ;Aedical Professor -oJ- PatiOlogj - cnd Dean, Ceater, -Sat~-=ra ncssco, CA. tviedical C_oeiegz-_ of -&;ith--Oarohna, Charleston. -AR:11S RICEiiERS, P:.^. OORPOR371Od of-Medicine, Los Angefes, RroFkl t°n, NY. _ 0A RTtN V P'r T a1Pr r-~i~T W7LL"e; H.=RIES'rN, , y : ,- _ - - - 1' - Chief, - Depaf=meat of Oral - Pathology,- -~':or _ _ Eeochem.s:.= _ _ Lrre - b_crences iy-~:- ';'i.mt~.n t~ lie; a:ch 1r; itute, .-~se~ga°--- iF~l P:zc ~x -NuwarkOit rios; !__--- RO'VAL ! E. RASls'_U_SSLtd,-PH.t . ~ _ Associate <djiehct - Pro,ressor, Departments ofComun F' and -0A`d3EL BRir tiN, f'H.I3 - ° Environmental fAeacEn_a +rni `_rsity of - Assi3far Projes of ~ ne-nicot Biology, r California College of Nied c ne-. !_rviae, The Ro..k °eller L,nivers;t-NeW York. - i - ~=- i - 210

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8_ Ei. R-IGDONt,-M.D: - -- - SVA FNE L. RYAN, PH.D. Pxofessor-_;,f _ Patfi.c'ogyr -isti-vers:ry os Professor of Brochzrr~istr UniversFty-ot -- Texas: Medieal B.ar<a, . tox= Nehra.ka c4lege ot ;M-ed~i-nF, ©ma}s._ _ SYDi`'E:'-C RITTEN-._--£Fc t, Jiii.rs. PETER _,F._SA~ arojessor of Bacter;olog;=, University or - s-read, 'nter.sive Treatment Ce.nrar, Sair,t- Sizuthern Caiiforniae Los A,Egelea- - Joseph Y-_;,spita+, Burbank, CA. - EUGENE 1;GBEIi;TS, ITH.D. - PAUL UL SALTMAN. r^H.D. Research Scientist, C_i.y of Hope ~cesearcn_ Assistant Professor of Biochemistry and Institute, D-narte, CA.=°. - Nutrition, -sJniversity - of Soutnern California School of Medicine, Los BENSON B. itf}Ea M.D. Ange:c~. _ A ssot~we Projessor -oj .Surgery; i%hi Cardias Surgery, Uttiversity- of ULRICH H. SCIIAEPI?I. M.D. California Sc :oo? of Medicine, San - Director of Nearorhar:rtarology, Mason - Francisco. - _- I?esearch_Institrte, Worcestsr. MA. JOSEPH H. ROGERS, M.D. Jt3P.GEF.-U. SCHLEGEL, M.D.YPft.D. Ha:3 Name of Jes±.<.,°r Hospital, Gadsden, _-Projessor and Chairman, Department of AL. -Surgery, Tulane ilniversity-Schoo: -of _ Medicine, New-Orleans, LA. - AOBEp'_ C. ROSAN, M.D. -As;oc_iate gro,fes_sv:=- c.t Pathology and-ALVI,. It: K(i#r?IDT, PtE.D.= -_ Pediatr:cs, St Lr-uis University Schooi of Director -of Couns?ling-, Tufts U3ive7sity, _ Medicine, Associate Pathologis:, Cardinal =- M_ed?`ord_= MA. - Gientton Men:oriaF Itospi;dl -_-for - Children, St. Louis, MG. JAKOB SE:IiMID T, M.D., PH:D. Assistant Projessor o1= Biochemistry, CHARLES L. RO5E; F-tt.D: Division _of= Fiological--S4ienees, Sia,z -_ Clir,i_- Director; Direuor, _No::.,ativb =-_ Universiiy-of N3w YSrk-at Stony-B_ook, Aging Study,- Veteratrs Administration_ Stony =Brook. Outpatient Clinic, Boston, MA. = ISAAC SCHGUR, D.D.S,, Ph.D., DSc. JOHN A. RCDSECkANS, Pti.D.- _---_ Dean, ';Jniversi.v of Llinois_ College of As.sociate Pro/;=ssor o;- -PhaFr>racology, Dentistry, Ctsicag_o-- Meci.=al Coi}eg4 o` -Virginsx. Virginia ~ Com:r~-onsveaal3 UUni~rsit;, isiYhmon d. -- SCRIPsS CLi;?IC -AND R_ E'SEAILCfi FCSU?r'DA '_ ION, - JOHN R. ROWLANDS, P-H.D. = La Jolla, CA. Staff -ScieRtist, Southwest- Research -ISstitute, San An_to nia,, TX._ MAURICE-S. SEGAL, M.D. _ Clinical Professor o; - Medicine, Tufts BENJAMIN A. F-UBiN, Fa.D: - - University School of Viedici-ne, Director, - t,sista.k Professor of Public -Hzal.n,'= Deeartment of Inhaiation- Tlseraov. Baylor University College -oL Medicine, _ Boston City Hospital. Boston, MA: HodSstoa,TX. _ RONALD E ;;UBW, T-H.:;. CARL C. SELTZER, PH:D:-- ProJessor of _ Pharm4co-_lcgY,- Medical Nororary Rrsearch- Asso_^iate. Peabody Col'rege_of Virginia, l'ticnmond_ Mu=_eurn. Harvyrl- University, Cambricye, MA. H£N>~Y I_ IfJSSEA,-I~~.D. _ fresident. 'I-he Russek Foundation, Inc., LUCIO SEVERI, M.D. Staten Island; NY. -Director and Dewc, institute of Anatomy W. C. RUSSELL, ht:D: and Pathology, __ DivisiJn of_ Cancer University -of _Texas _ Medical Center, 3tesearcl-, University of Perugia, Houston. Perugia, Italy. 211 -

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CHARLES R. SHAW, M.D:= - LUCILE SMITH, P3t.D_ Chief, sactio~_of-P~e3iea'- ven:ties, FSi.D:- Professor of Biochemistry, Dartmouth Andersoe Ho,;aital - and T umor _ Me::it:al Schoo_l; Hanover, NH. Institute; Pro;essor of = B;-_!ogy. -The University of-Texas aLidaascrir:;1-douiston. LOUIS A. SOLOL"F, 1vr.D. Blanche P. L.evy=_ Dfsiinguis}ea' Service CHARLES E. SHERWOOD, M.D. ProfessQr; Professor -of tiJedicine; Assista.ht Professor _ of - Radiology, Director, Research Lipid- Laboratory, University -of Rochester School of - Temple Llr_iversity- -Heal-tfi Sciences -Medicine and Dentistry; Rochester, NY. - - Center, Philadelphia, PA. SHOJI SHIBATA., M.D., P14-D., - SHELDON C. SOR'IMERS, M.D. Professor of Pharmacology, University - Director of Labora:ories, Lenoz Hill of Hawaii $chdol of iv[edecine,-tlonolule. Hospital; Clinical - ProJPssor of _ _ Pathology, Columbia Universits College GERALD SHKLAR, D.D.S. of Physicians & Surgeons, New Y orK. Charles A. -Brackett Professor of Oral _ Pathology: Head, >;epartment -oI' Oral ERNEST SONDHEIMER, Pt?.D. AssocrJte=_Prrrjessor- o/- Biocnemi.4try, - Mpii~i i ar,d Oral= P{#Foiogy, Harvard School of Dental M-edieina, Boston, M4. -Co:iebe-of- Forestry, State_ Universit; of New York Syracuse. DAVID-L-:_SIMO_ SIMON, M.D. Ilrstructor in - Internal Medicine, T. M. SONNEL+O C-N, PN.D. Cincinnati General-+]-Iospital,-Cincinn4ti, Distingaisheit _ Servk~e PTosar -oj OH. zootagy: - Indiana University, Elopm:ftgton. ERIK SKINHOJ, M.D. - Ch:F;, Dpa:tmy5t 0. ' ~eu afogy, SAM 55-01-10OF, ft D - 4= I3ispebjerg Hospital, Copnnhagea. _ Pead, Dep-artint=-v-'= Macromolecular ~ Chemistry, -The Institute for Cancer ~_ NATHAN H. S7.,OANE; Px.^. _ R_esearct , Thilade;phia; PA. ' Professor oj Bioch_ernisrry, = The University of Tennessee Center for t; e SOUTHWES"I' RESEARCH INSTLTUTE, Health Sciences, Memphis,- San Antonio, TX. t HANCk 1J SL3R;-P9.D.-= DAVID ]vi: SPAiti, M.D. i Associate Professor of filluman Grnetres, Director, Department-_of Patholcly, The_ ; Sackler -Scttool of Medicine, Tel A:av -)rrookd3le Hospital - C3 ~teF, P.ookly. ,- _ Ursiversity~-Tel Aviv, Israel° NY. THEODORE A. SLOTKIN, Pn.D. _- ALEXANDER SPOCK, tvLI?. Assistaat ProJessor- oj Pharmacology, Assistant- Professor _r.j- Pediatricsr Duk- Dzke University Mpdica_ Centes,_- IJni:ersity Medical-College, Durham, NC. -Dur ham, NC. O£OKfc-W.ShSETTRS,-KD= -=-'rREDERIdt: J._STARk, M.D.= - ;ssocia:e in PatRCfJBy> Northwestern --Prt3ressot -- -oL_ N!ariti%r,- - Hsrvar(i -_ University ?.edical 3chool:_f Fr:cago_,=IL. LTniYrsit?; Sch-of- Public =:-Iee':th, DENNIS Ivi. SMITH, I`:z.D. Boston, MA. i Ass:3a:nt ¢rvjessor ef B:clvgica! C. STEFFEE, M.D. GENE M. SMITH, P$.D. Assistant ProJessor- of --Psychology, JACK P. STRONG, Ivf.^.- ~ Associate Professor - of Pathology, - Harvard- kledica_ I_I Schooi. Mcssachus~s j General Hospital, Boston. - Louisiana State- Laniversity -Schoo-I--of - I- Sciences, _ Wetlesle_y College, N'eIlesley, Director oJ -Faboratorres, -Method'rst ~ = MA. 'rlospital, Memphis, TN. _ hTedl:c,ne, New Cxlearu.

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-IS+LSRdQN_P.__SLILi$£RGEf"C ?v1:n _ jGEk.~~.LD:M--T-. r.Ti:.'No. Iy:-D: *;aivtnit9-B~4l=vue 'ieCeea: ~n?er~ 14~': ~~rge , Nexw''~ork. rr oje~s_sor attd--Gf;a;rRs-ar;, Department of -= Professor =- of Medicine, Coiurnti, Dermatolog-~}-~d S=,-vHisc ~3y, t`~e:~-York=- _= rtni+~er,ty _ E.o~e$e --E!' i-hys;c=aris-- & - _Adrnittistratior:,_ - Harvar~ University, Europ2, Ltd., iiairogate, ';'or-kshire, `c3'oston,-Pv1ri Eng=and,_- Associare-= Profssr-F=- of r'ryck3affgy, iiead, De~ar?s~ + of_ Dr:~® _Wiet no.isrn -c,ra.~uate---=- _S:.hco, of _- :rs°usiness_ aetd-~io_.err<istay_,-YazTet.^ Laboratories RENATG TACi..iRl;?ti.ia. Q, ki.-TUf:NER,_P::.-e'. Arizona-Hesltn-Sciences Tucson. :,os Angeles., CA. -MARC D. THAMES. ,:L.D. STEPHEN_F. VATNER, hi.i} Senior !?csParch : plsow, Mayo_ Clinic and_ Associate Professor of Medicine. Harvard--_ E'orrdatian, Rochest '~.?>_ ?+iea_eal Schooi, -NPw England _2eg=una'- Unaers:?y -of Co!Fsada iv:ed:cal Cente_:_.=- De.'var. UNIVERSITY _Jr-SAhi EP.ANCISiO - iaf-rrant:scu; CA. LYNN MN. -T AUSSG, ia.D .-_ - _ Associ$ e- Professor ana as ociate - U[~'iV~ERS[Ti a SOUTHERN Cacsrmzr, -De gart ~ent of Pediat i s, _`Al`-IFt3Ri~lA; D_ AVi-^W. 3';:L Iv?AGb,_M.Li -- UNION CARB:DE CORPORATION ;7irector, Wcbb-Warir_g =Lung `ristituier _ Nuclear uiv~isio :, Jal< Sidge, T;l: _ rriina.ie Research Center, Sua=t!:horo, - - CAROLINE BEDELL THOMAS, M.U-- -MA; AssoeiatG in k:e5icinv_, Pete_r_ Benr - Prrzfessor=and Ct i.»tar D?pa~:yeri*, of -_Iviedic_ i nF, Ba?t3rr,~re, MD_ :- Fi i I^ T S. VESELL, lvl.M; _- .io!a;s- ao3kins -Uciverei;- Sr: o`- o, _- Professor E=eritus of -'v!leaicine, -':: e- Br:g; artt Hospital, Boston. MA. _lJn:ve-rsay of _L'aliaorcia,=Berkeley. =--S. Hershey :1iedibal Center, Hershe;. JEROME '=. T'aOAdAS. P,?=D. --= Pharmacologys - Penrtsy~.~ani9 " State Professor of -Sanitcry_ -_&no-ineeriro, *,;mive:siry College of Medicinc, Iv',i.ton JA;.iS E. P. TO`MA.~~'- P-d.D. = BRAtd:SLA :' V-1DiC, =Projessor and Ci:airm<n,-:Deepart:r-_eni of- = ProJes.sor _o!- Anatomy, FeorgfAewn -?harmacology, Chifago_ Medical= Schoot,- Unive yicy_- Schools- ar Medicine - and Ins=tit-:te- for Pyfedica' ixe~e9rc-h, _-^-: ic,ago,_- =-L"atistry- Washinvts,-s, J-.= ROMEO A._ VIDONE, N'.D. Pres:den.- a:ra P!irecor - of < Research, _ University =School of Medicine. -New - - ANDREW M. 'Y`UiLiETSIC-0, Ptt.D. .issaciate - Professor of -Pathology, Yale -Litron-Laboratorie-s_ `1.tti.; RQchester,- Fzi s. - Haver. - C~T - .- - !?esFarcn` _Assxiatie -irt Pathalc,gy, Yale - -= Pro; essor of Pat3rolog-y, State iioivtrsity University School = of REedici:ne, _ l;le-wdf Iowa College e.` Mediciste,fo-wa-tr'ity. Haven, CT. -1 ~larrs7atirb$)'.--_ Corne!l_ =~iaii'eEsSty-.= _ ~rz'?Shingto n -,"hool .^.f _1'le.`'.7cE:'e,'e, Seattle. Medncal Coilegej New s'ork. - _ IRENE Y. WANG, Pa.D. JAMES TRAV]S, FK.D: Assistant_ Pro%essor= of fsasac 3ru: vfrr:rcaf ProJessor of = Biochesuistry, The immuio'ogy a:~d Mirro8iotogy, Medical University of veorgia, A?hes: University oi Sout3'Carclina, Charleston. LIE SHA TSA:, Pti.D.= _ E, D, WARNER, M.D., 3e4tdcT.TRAVE.L,-M.D_ - PETER-K. VS')G, Ptt.1D. _ ~css~isste =_ ~rcf~ssnr oV- - C:ir;~l -_ P.~fess!~r of Mir=obiarogy, Un versity .~f 213

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SHIELDS WARREN, M.D. A. STANLEY WELTMAN, PH.D. Director oj = LaCor_atories, _-Cans:ez Rssocia:e-Pry-jessor 4 j Phurrr:ecoiegy and - Research-- in-stitute,- Iaiec4 - England Researci:, Brookiyn College of Phsrmacy, - Deaconess Hospital, Boston, MN. B,ooheyn, NY. YASUSHI tyATA" zBE, N.D. -- SIMON li. WENDER, P:I.D. _ Associate-bfem3er; The= Wistar Institute-_ Research - -Projessor- of 8icchemrstry, of Anatomy and Biology, Phiadeiphia, University of Okahoma,-Norman. PA. DUANE ci. WENZEL, Pzi.D. BARBARA K. WATSON, Ttt.*-~'. Professor and Chairman, Department of Assistant B~rteriologist; -- ?vlassaL:R,uetts Pharmacology and ToziCoogy, The C.Enererai-Haspita'. Research-Asa:,=iate i., University o:`- -Ifat:>as S_-ho-3i of Bacteriology and 1mmw7ology, Harvard -= P harm_ acy, La-wrence: Medical School, Boston, MA. THOMAS C. WESTFALL. Px.D.- LEE W. Wf:I"I"ENBERCc, M.D. _ Projessor -oj r?harntacology, University -Projr`ssor of Pathology, University of of Virginia School _ of Medicine, Minnesota Medical School, Minneapolis. Charlottesville. JOHN S_ WAUGH. PH-D. _ -FF:EDERICK E.-WHISKIN, A?.D:C.M.- Pro,/essor - of Chemistry, -Massachusetts- = Di<<ctor,_ Divisioo - of He.ath and Institute of Technology, C'amaridge.- _ Personality Equ;:_,brium, The Age=Ce_ tee of New England, Inc:, Boston, MA. THOMAS E. WEBB, PEi.D. Professor of f'hysrvlogirs~ Chemistry, ;AMES A: WILL' D.V.A4.. 1'~.D. Ohio State University College of_ Projessor and Chc~irman,- Deparment of Medicine, Columbus. Veterinary Science, University of Wisconsin, Madison. _ _ RI:,HARD_L. WECHSEE~;-M.11. Cliaical PF.ysiologist: = Montefiore RCGER-J. WILLIAMS, M.D- Hoipital Institut_e -_of Research. Professor of Chemistry: - Director, Pittsburgh. PA. Clayton Foundation Biochemical Institute, The_ L'aiversity-_ of=- Texas, -JaHN_ V. WEIL,- M.D: - Austin. .dssistr.nt _ Professor =oj Medicine, University of Colorado Medical=CeFter; JOHi;~ T. WILSON,Pn:D:= Cell and Assistant Professor of Denver. 1. BERNARD WEINSTEIN, M.i:. Molecllar Biology, ; E;,dica_ Coliege - of Georgia, Augysxa. -Projessor of Medicine-and Environmental Sciences, Columbia University, New _ALV=N L. WIN-TERS, Px.D. Yor-k.' Assistant Professor of Microbiology and Riochemistry, -The University of- A. WEINSTOCK, Pti:D. Alabama, University. Researc4 _ B-dochernis!, L`fe Sciences Division, lIT Reyearch -- institute, DANIEL H.-`.r`lISE-MAN, M.D. Chicago. IL. Assistant - Projessor- of Pediatrics; University of Southern Caifornia; _ SIirM[1-hD A. 5kEI'I'ZMAi:' M.D. Chirlren's Division, t.os Angeles County Assistant Professor of - Medicine, General Hospital, Los Angeles, CA.- Hematology-Oncoogy Unit, Massachu_setts Ceneral-Hospital, Boston. BR.UCE ,A. WODA; ;4.D.- .Issoci:ae - Professor of Pati oiogy, RUSSELL W. WELLER, M.,.-- University of Massachusetts, Worcester. Patholozis-t, Memorial :I^spita- of - Chester Count -, West Chester, PA. 214 W -

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GEORGE ]YCDLF;=D.PytL. - _ _ Professor of Physio:'ogicai - Che.nislr-y, Department t of Nutrition - anu Food - Science, Massachusetts Institt;te -of -?echnclogy. Cambridge.- - J. EDWIN iVC3OD,-;h.D. - Instructor in Medicine, Boston 'University _ _ School of Medicine, Boston, MA. SUMNER WOOD. Ja., M.D. Assistant Professor of- Pathology, 'I'he_ - Johns Hopkins _ Unire rsi ty _ Scfiool of - _ Medicine, Ba?tiraorg,- MD. JC2FiN .-~', WY" 'I'I'; M.D. J:rofessor _ of_- .Patho:ogy, St. Louis University-Scrto-G.1 of_MeGicine, St, i.ouiv,-- Mr. ° '.C3JI YOSI-IINAUr,, FP.D.- CebteT for -, opulation Research, NIc_'FID, lyatiotra! Ipe:itutes-of--fi#ealth, -32thesEv,_.. MD. 315 0

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