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NYSA CTR 1

1995 Report

Date: No date
Length: 154 pages

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nysa_ctr1 60040920-60041073

Fields

Named Organization
Albany Medical College
American Cancer Society
American Heart Association (Voluntary health organization that focuses on cardiac health)
Voluntary health organization that focuses on cardiac health and stroke. AHA occasionally teams with tobacco retailers to engage in promotions/fund-raisers (see http://www.smokefree.net/doc-alert/messages/247136.html and http://www.rawbw.com/~jpk/stand/Pictures.html).
American Red Cross
Archives (National Archives and Records Administration)
Army
Boston University
Boston University School of Medicine
Bowman Gray School of Medicine
Brown University
California Institute of Technology
*California State University (several, specify city)
Cancer Center, Philadelphia
Chapel Hill
City of Hope
City University
City University of New York (CUNY)
Cold Spring Harbor Laboratory
Columbia University
Council for Tobacco Research - USA (CTR) (Formerly Tobacco Industry Research Committee (TIRC))
Originally organized as the Tobacco Industry Research Committe(TIRC) in 1954, and renamed Council for Tobacco Research - USA, Inc. (CTR) in 1964.
*Department of Energy (use United States Department of Energy)
*Department of Health and Human Services
Department of Veterans Affairs (VA)
Downstate Medical Center (in Brooklyn)
Duke University
Eli Lilly (pharmaceutical company)
Fox Chase Cancer Center
Georgetown University
Hadassah Medical School (Jerusalem)
Harvard Medical School
Harvard University
Hebrew University
Howard University
Hunter College
Illinois College
Indiana University (Located in Bloomington, Indiana)
International Union Against Cancer
Jefferson Medical College
Johnson and Johnson
Johnson Foundation
Louisiana State University
Lovelace (Biomedical Research Foundation)
Loyola University (Montreal) (Became Concordia University (Montreal))
Merged with Sir George Williams University to form Concordia University.
March of Dimes (Voluntary health organzation concerned with birth defects)
Mayo Clinic (Located in Rochester, Minnesota)
Has a nicotine dependence center; runs the smoking cessation program at the Mayo Clinic
Medical College of South Carolina
Medical College of Virginia
Medical University of South Carolina
Michigan Cancer Foundation
Microbiological Associates (Research lab in Bethesda, MD)
Research lab in Bethesda, MD. CTR contracted with this lab to do the world's largest inhalation study, involving more than 10,000 mice in 1973-1982.
Monsanto
Montana State University
National Academy of Sciences
National Eye Institute (part of NIH)
National Heart Lung and Blood Institute
National Institute of Allergy and Infectious Diseases
National Institute on Drug Abuse (An addiction research center in Baltimore, MD)
An addiction research center located in Baltimore, MD
National Institutes of Health
National Institutes of Health (NIH)
National Science Foundation
New England Deaconess Hospital
New York Academy of Sciences
New York University
New York University Medical Center
North Carolina State University
Northwestern University
Oak Ridge National Laboratory
Omega Alpha (Honorary medical society at Univ. of Pittsburgh)
Phi Beta Kappa
Plenum Press
Red Cross
Research Council
Rockefeller University
Roswell Park Cancer Institute
Rush Medical College
SAD
*Scientific Advisory Board (SAB) (Only use SAB with name of specific org.)
Scripps Clinic and Research Foundation (Located in La Jolla, California)
Scripps Research Institute
State University of New York
Tel Aviv University
Texas College
Thomas Jefferson University
Trinity College
Tufts University
Tulane University
United Brotherhood of Carpenters and Joiners of America (UBC)
Univac
University Medical Center
University of Alabama
*University of California (use specific branch)
*University of California Berkeley (use University of California at Berkeley)
University of Chicago
University of Colorado
University of Florida
University of Illinois (at Champaign-Urbana)
University of Iowa College of Medicine
University of Kentucky
University of Massachusetts
University of Nebraska
University of New Mexico
University of Pennsylvania
University of Pittsburgh School of Medicine
University of Rochester
University of Texas
University of Texas at Austin
University of Utah
University of Vermont
University of Virginia
Upjohn
US Army
VA Medical Center (Located in Minneapolis, Minnesota)
Vanderbilt University
Veterans Administration
W Alton Jones Cell Science Center
Washington University in St. Louis
Wayne State University
Welch Foundation (Supports chemical research in Texas)
Yale University
Yeshiva University
Named Person
Abate, Cory T.
Abood, Leo G.
Ace, Mario D.
Adelman, Burt
Adler, Kenneth
Alber, John J.
Armitage, Alan K.
Austin, Austin
Axe, David
Barber, Frances C.
Barclay, Laurie
Barnes, David W.
Befus, A. Dean
Bellet, Samuel, M.D. (CTR Grantee, Philadelphia General Hospital)
Studied the correlation between cigarette smoking and the ingestion of nicotine and serum fatty acids with Dr. Alred Kershbaum.
Berg, Leslie J.
Berkeley, Lawrence
Bevan, John A.
Black, Ira B.
Blalock, Edwin
Blum, Mariann
Bobak, David A.
Bock, Fred O.
Bockman, Dale E.
Bone, Gene During
Bonner, James F.
Booker, Walter M., Ph.D. (CTR Special Projects; Pharmacologist, Howard U)
Defense
Bowden, Drummond Hyde (CTR SAB Pulmonary Pathologist, U. of Manitoba)
Defense
Bowery, Tom O.
Brooks, Robert E.
Brown, Barbara B., Ph.D. (CTR grantee, Psychiatrist, Veterans Admin. Sepulveda CA)
Industry Consultant, CTR grantee and a CTR Special Project recipient.
Brown, Raymond R.
Bryson, Rebecca
Buonassisi, Vincenzo
Burrows, Benjamin
Burstein, David F.
Cabot, Myles
Cameron, Bruce F.
Cantrell, Elroy T.
Capra, J. Donald
Carne, William H.
Carroll, Ann M.
Cart, William Alvin
Carter, John R., M.D. (CTR Special Projects, Pathologist, Case Western U)
Funded by CTR, Director of Inst. of Pathology
Carter, William A.
Castro, Albert
Chan, James C.
Chao, Francis C.
Chiu, Robert H.
Clark, William G.
Colman, Robert W.
*Comroe, Julius H., Jr. (use Comroe, Julius Hiram Jr., M.D.) (CTR SAB, Grantee, Pulmonologist, U of CA, San Francisco)
Defense
Connor, Basil O.
Connor, Dean M.
Cooper, Philip
Corley, Ronald B.
Cramer, Eva Brown
Croce, Carlo M., M.D. (CTR SAB, Physician, Kimmel Cancer Institute, Director)
Dr. Croce is Editor-in-chief of Cancer Research Journal. He is a member of the National Academy of Sciences. He is currently Director the Human Cancer Genetics Program at Ohio State.
Cross, Carroll E.
Dallas, Dallas
Delaney, Olin P.
Domino, Edward F.
Downey, Fred
Duarte, Hope
Dvorak, Harold F.
Dyke, W. Van
Earle, Richard H.
Eichel, Bertram
Einstein, Albert
Eisenberg, Arthur D., Ph.D. (CTR Assoc. Research Director 1991, Asst. Secretary 1997)
Defense
Enget, Hyman
Erick, Carlton K.
Erikson, Raymond L. (CTR SAB 1992)
Defense
Falk, Hans L.
Farnsworth, Dana L.
Feldman, Joseph D., M.D. (CTR SAB)
CTR SAB
Feldman, Joseph David
Fenton, Richard
Ford, Donald H., Ph.D. (CTR Assistant Research Director)
Found, F. Welch
Franc, Paris
Francisco, Sun
Frankel, Jack W.
Freeman, Aaron E.
Gill, Gordon N., M.D. (CTR SAB)
Defense
Gleich, Gerald J.
Gold, Barry
Gold, Warren M.
Goldberg, Ira J.
Goldman, William E.
Good, Albert
Gore, Ira
Green, Maurice
Griep, Mark A.
Hall, Caroline B.
Hall, Linda M.
Han, Min
Helson, Donald J.
Henry, Carol L.
Heywood, Paul O.
Ho, Deborah K.
Hoover, Herbert, Jr.
Hoss, Wayne
Hughes, Howard
Hull, Robert W.
Jackson, Barbara J.
Jacobson, George
Janoff, Aaron, Ph.D. (Received funding from CTR)
Jefferson, Thomas (3rd president of United States, 1801-1809)
Jone, W. Alton
Jones, Harold P.
Kaplan, Arnold R.
Karr, Robert W.
Kenter, Amy
Klaiber, Edward L.
Kline, Nathan S.
Kmiec, Eric H.
Koch, Tad H.
Kunkel, Steven L.
Kupiec, Martin
Kupper, Lawrence L.
Lai, Eric
Larson, Roger K.
Laurie, Gordon W.
Lee, Edward
Lee, Eva
Lee, Wen Hwa
Leff, Stuart E.
Lennon, Vanda A.
Lerner, Richard A.
Leung, King
Levin, Eugene G.
Levy, Blanche P.
Levy, Jay A.
Levy, Stuart B.
Lewis, Kim K.
Lewis, Paul D.
Li, Edward D.
Lion, Fabian L.
Loosli, Clayton G., M.D., Ph.D. (CTR SAB)
Lotan, Reuben
Lowell, Lowell
Luka, Ronald J.
Lundberg, Ian M.
Ly, Chang
Lynne, Susan
Mandi, Ines
Mann, David E.
Markey, Lucille
Markey, Lucille P.
Markey, Lucille W.
Markham, Richard A.
Martin, Billy R.
Martin, R. Russell
Mason, Robert W.
Mckee, Edward
Mckee, Kelly T.
Meyer, Edwin M.
Meyer, Julia
Miller, A., M.D. (Asbestos researcher)
Mitrani, Stella
Moore, George F.
Moser, Kenneth M.
Motley, Lee
Murphey, Marcy
Nadel, Jay A.
Nahm, Moon H.
Naylor, Susan
Olson, Eric N.
Pac, Donald M.
Palmer, Albert B.
Parker, John W.
Pfeiffer, Louise
Pierce, Barry
Pierce, G. Barry
Pierce, O. Barry
Pike, Malcolm C.
Pizzo, Salvatore V.
Planck, Max
Poster, Ann
Pratt, Virginia
Reed, John C.
Reese, Michael
Reidy, Michael A.
Repine, Olin E.
Reynolds, Rolland C.
Richards, Victor
Richmond, Virginia L.
Rifkin, Daniel B.
Rinker, Carrie
River, Pearl
Robert, Eugene
Robinson, John M.
Rogers, Joseph H.
Rosan, Robert C.
Ross, Peter M.
Rowlands, John R.
Ryan, Una S.
Sabatini, David D.
Sanborn, Barbara M.
Sato, Gordon H., Ph.D. (CTR SAB)
CTR SAB
Schmidt, Alvin R.
Sell, Stewart
Semenza, Gregg L.
Ser, Henry
Severi, Lucio
Shaw, Charles R.
Shore, Heath
Simmons, Harold C.
Simon, David L.
Sloan, Nathan H.
Smith, M. L. (RJR)
Spain, David M., M.D. (Pathologist, NY St. Down-State Med. Ctr., Anti-Tobacco Exper)
Director, Pathology Dept.,
Springer, Timothy A.
Stare, Frederick I.
Stearns, Mary
Stile, Wayne
Strong, Jack P.
Sunday, Mary F.
Sussman, Raquel
Taus, Lynn M.
Thomas R. M., Ph.D. (Conducted Gallup poll on smoking prevalence and attitudes wi)
Thompson, John A.
Tseng, Ben Y.
Turk, John
Val, Al
Van, Barbara J.
Villa, Lydia
Vogt, Peter K., M.D.
Wagner, Robert R.
Wai, Peter N.
Wang, Irene Y.
Wang, Xiao Fan
Welch, Robert F.
Wilusz, Jeffrey
Winters, Alvin L.
Wiseman, Daniel H.
Wolf, George (Prof. of Physiological Chemistry, MIT; CTR grantee)
Professor of physiological chemistry at Massachusetts Institute of Technology, and a Council for Tobacco Research Grantee.
Wong, Dean F.
Wood, Edwin
Wooley, Paul V.
Wright, Richard M.
Wright, Thomas C.
Wu, Reen
Wyatt, John P., M.D. (CTR Scientific Advisory Board)
John P. Wyatt served on the CTR Scientific Advisory Board.
Wykle, Robert L.
Date Loaded
11 Jan 2006
Box
0329

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1995 REPORT of Tile COUNCIl, FOR TOBACCO RESEARCll-U.S.A., INf'. TIlE COUNCIl, FOR TOIIACCO RESEARCII-U.S.A., INC. 900 Third Avenue, New York, N.Y. 10022
Page 3: 60040922
SCIENTIFIC ADVISORY BOARD to The Council for Tobacco Re.arch- U.S.A.. Inc. as of Deccmher 31. 1995 G. BARRY PIERCE. M.D., Chairman Dislingulshed Professor of Pathology University of Colorado Health Sciences Cemer Denver. Colorado LEO G. ABOOD, Ph.D. Professor of Pharmacology and Biochemistry Depa~menfof Pharmacology University of Rochester Medical Center Rochester, New York BARRY (3. ARNASON, M.D. Professor and Chairman, Department of Neurology University of Chicago Chicago. Illlnois DRUMMOND H. BOWDEN. M.D. Professor (Emeritus), Faculty of Medicine Department of Pathology Univcrsily of Manitoba Winnipeg, Canada MICIIAEL J. BRENNAN, M.D. D|rcctor Familial Cancer Prevention Clinic Haq~cr Hospital President and Medical Director (Emeritus) Michigan Cancer Foundation Professor of Medicine (Emeritus) Wayne State University School of Medicine Detroil, Michisan CARLO M. CROCE, M.D. Director Jefferson Cancer Institute and Cancer Center Philadelphia, Pennsylvania RAYMOND L ERIKSON, Ph.D. Professor of Cellular and Developmental Biology Biological Laboratories Harvard University Cambridge. Massachusetts GORDON N. GILL. M.D. Chxllrman, Faculty of Basic Biomedical Sciences Unlver~ity of California, Snn Diego School of Medicine La Jolla. California W. K. JOKLIKo D. Pint.. Department of Microbiology Duke University Medical Center Dudmm. Nrslh Carolina llENRY T. LYNCIi, M.D. Professor and Chairman Director, Creighton Cancer Cenler Department of Prevenlive Medicine and Public Health Professor of Medicine Prcsldent. Hereditary Cancer lnstitule Creighton Univen:ity School of Medicine Omaha, Nebraska HARMON C McALLISTER. JR.. PlI.D. Scientific Director The Council for Tobacco Re,arch- U.S.A., Inc. New York. New York DAVID D. SABATINI, M.D., Ph.D. The Frederick L. Ehrmnn Pmi'essor and Chairman, Dcp:mment of Cell Biology New York University Medical Cenler New York, New York JUDITII L. SWAIN, M.D. Herbert C. Rarer Professor of Medical Sciences and Genetics Chief. Cardiovascular Division University of Pennsylvania Medical Center Philadelphia. Pennsylvania PI.~'ER K. VOGT, Pff.D. Mcmbcr The Scripps Research ]nstilutc La Jolla, California Scientific SlaffofThe Council HARMOI~ C. MCALLISTER, JR., P~;.D. Scientific Director ARTHUR D. EISENBERG, Ph.D. (3EORGE A. HASHIM, Associate Research Director Associate R~. catch Director DONALD H. FORD, PII.D. A,c, mci:~te Research Dircclor
Page 4: 60040923
C) CONTENTS Introduction .................................................................................................... 5 Abstract Titles and Authors ........................................................................... l I Abstracts of Reports ....................................................................................... 25 L Cancer-Related Studies .......................................................... IL Cardiova~ular System ........................................................... 4 IIL Cell Biology ........................................................................... 4"/ IV. Developmental Biology ......................................................... 145 V. Epidernio~ogy ......................................................................... 163 VI, Gonelics .................................................................................. 164 VII. Immunology and Adaptive Mechani.mns ............................... Viii. Neuro.c.clcnce .......................................................................... I111 IX. Pharmacology ....................................................................... 190 X. Pulmonary and Respiratory Systcm~ ..................................... 212 XI. Radicals .................................................................................. XII. Virology ................................................................................. 227 Xlll. Miscellaneous ...................................................................... 241 Active P~ojeCls ............................................................................................... 247 Completed Pmjecls .............................................. .: ........................................ 271 Index of I'rinc~pal Investigators ..................................................................... 304 Introduction Researeh funding of The Council for Tobx¢o Re,arch by its spon~or comp~. nits continued at a significant level in 1995. "rim total amount of funds awarded re.arch 8rants amounted to $19.~;50.000 in 1995, bringing to mare than $262 lion the Council's suplmrt for its researeh program since it began opcvations in 195.1. The Council rcmalns among the lcadlng Wivate funding ogeneles in the nation. supporting various types of b~¶i¢ biomedical investigation: cancer, cardiova'.cul.~r di~a~s..cell biology, developmental biology, epidemiology, genetics, immunology. neure~ienee, pbarm.'gology, pulmonary di~a~s, radicals and virolo.ey. New original re.~areh proj~.cts approved and fimded in 1995 to|al~l f,7. while many more continuing anti renewal stndies were also funded. Through 1995. Council has suppc~ml n cola! nf 1.491] original investigative projects and meelings .'~ part of the Council's m~arch program. The tee;plants have bc~n mc~r~ than I.(I(X) independent scicnlists a! more than 3110 m~ical ~h0ol~. ht~pitols re,arch cenlc~. During 1~5. ~'~mtces p~blL~hed .t26 r~po~s on their investigations ~apponcd by ~ Co~neil for Tobacco Re~afcf. Grantees are encouraged to publL~h the ~'~tdts of all of their investigutloes. AbstraCls of Ihe papers publ[sh~ dudng 199.~ ~re |nclud,~! in this report. Over the past 41 yea~, there have been more the1 ~.T00 sc~tifi¢ carious by investigators suPlx~ed by The Co~nctt for Tob.~co Re,~areh. in Ihe summer of 1995. we w~ s.~ddcl~-d ['~ the sudan and t~ne~l~*cted of the Cfairman of" the ~ounc|l's Sc|enfific Advi~ry Board, Joseph D. Fcldm:~n, M.D., of L~ Jolla, Catlfomia. Tfls annual report is a n~mo~al to Dr. l:'eldm~; many distinguished contributions to biomedical science arc acknowledged in t~e dedication appearing el~where, Dr. F¢|dman has hecn succe~ed as Chalrm:~l of Scientific Advisory Board by ~. Barry Pierce, M.D., Professor and Chairm~n ~meritus. Department of Pathology. University of Cotorado School of M~dicine. Denver. Gordon N. Gill, M.D., Chief of EndocrinologyJMet-'q~oltsm, Unlve~ity of' Caliromla-S0n Diego. has been elected to the post of Vice C1~airman of the SAD. Manfrcd L. Kamov~ky. M.Sc,. Ph.D. of Harvard University retired from the Scientific Advisory Board in April 1995, having given ten years of dedicated and leadership in the process of ghent applieutlon reviews and fending, of re.arch, Dr. Kamovksy. a native of South Africa, came to the United Slotes research fellow and became a naturalized citizen in 1956. He spent mo~t of his career at Ilarvard M~lienl ,School ~s ti~e llarold T. White P~fe~.~or ~nd Ch:finn~n the Deparlmtnt of Biological ChemLqry. He has been the recipient of many and awards and has held memberships and offices in the most prominent societies In his specialty. He is the author of some 300 papers, chapters and books. Successors to Dr. Kamovsky and Dr. Feldman on the Scientific Advi~j Board have not yet been elected. In 1995. we welcomed to the Scientific Advisory Board a distinguished new member, David D. Saballnl. M.D.. Ph.D.. of New York University School of Medicine. Dr. Sabatini. • native of Argentina. trained in that country and at Rockefeller University, New York. He h~ ~rvcd as F-gederick L. Ehrman P~ofes~or and Chairman of the Department of Cell Biology at New York University for than 20 years. His awards, .vi~itlng professorships, lectufeshlp% editorial appoint. meats and hono~ are extensive, and he is recognized a~ a leader in the field of mern. lwane and protein research.
Page 5: 60040924
0 0 IN MEMORIAM Joseph David Feldman 1916-1995 Joseph D. Feldman was a native or Hartford, Connecticut. He received the Flnchelor of Arts degree from Yale Un;verslty in 1937 nnd wax nv,~rd~l the of Medicine degree from Long Island College oT Medicin~ in 1941. From 1942 to 1946 he served as a Major in the United Slates Army Medical Corps. sen, in~ ~,~ Betallico Aid Station Surgeon. After the war. he hogan his illu,xtrtous career in p~tholo,ey, scrvin$ firq n,~ dent at Ihe New England Deaconess Hospital. Boston. Subsequent appointments were at Yale University School of Medicine. Flad0te.sah Medical School in and the University of Pittsburgh School of Medicine where he rose to the rank or' Professor of Pathology. In 1961. Dr. Feldman moved to L~ Jolla. Callroml~, whcr~ he held eoncurronl appointments in the University of Callfomla-San Diego and Scripps Clinic and Research Foundation. In 1974. he was named Chairman of th~ Depanrncnt of Immunopathology at Scripps. and he continued active affiliation with the Scripps Re~,camh Foundation even after his relircn',cnl. Dr. Feldman w~s an editor of • number of prominent medical and scientific joumals, and served as the Edilor-in.chler of th~ Journal of ImmunoTogy. lie also • member of the pathology study sections or the National Institutes of Health and a coosultant to • number of b|omedical research prob~mms. His scholar~hlp and ability were acknowledged by his election to Phi Beta Kappa, the Society of Xi. and Alph• Omega Alpha Honor Medical Society. His leadership and expertis~ in ~mmunopalhelogy were recognized and acclaimed inlc~natiotmlly. Dr. Feldman joined the Scientific Advisory Board oT The Council for Tobacco Research In 1974. In 1991 he became the Chairman of the Scientific Advi,xory Board. • po~t which he held until hie unexpected death in the summer of 1995. His contributions to the promotion of basic biomedical research ~e Bratefull,y ~cknowl- edged by his friends and colleagues on the Scientific Advisory Board. pxxt and l:~e- sent. The Council for Tobacco Research--U.S.A., Inc. is honored to dedicate this annual report to Dr. Feldman's memory.
Page 6: 60040925
0 Joseph D. Feldman: A Personal Remembrance G. Barry Pierce. M.D. Distinguished Ccntennlal Profess~ of Pathology University of Colorado ltealth Sciences Center, Denver I am pleac, ed to have Iho opporlunity to remini~e about .l'oe because it allows me to express my Brat|rude for ,,11 he did for me years ago. I know I am not alone in my indebtedness to him because he w-~,,~ a giving man. I had a great affeclion for him which was reciprocated: rcciproeatod in the sense o1" an .bier Ixt)ther cemstanlly guiding and tcaching a younger (me. I|c wasted no words; be said what he thoughl, not ncces~rily what you wanted to hear. As a rcsul[, you always knew where you stood. Fortunately. he had a good sense of humor which we both needed upon occasion. We met al the Department of Palh~dogy al Ihe Universily (if Pittsburgh in .lune of 1955: he an Associate Professor of Pathology. recently returned from Israel. me a newly appointed and lowly fellow. Rank, race, creed, none of there mattered to Joe. Whal mattered was how the game was played. At the end of each exerei.~, wbether it be squash, correcting a manucfipt, or writing a grant application, toe's clo~ing comment was always the same: "All right." But it w&s the fleeting smile or lack thereof that told you if yo~r performance met with his approval. In reflecting on our friendship, it was puzzling to have known a man so well and so long and to owe so much to him. yet to know so little of his life. We knew he suffered from autoimmune disez.~, but he never complained about his health, .iust ignored it..ice was n private man. proud and loving of his family, extremely effec- live in his profession and loyal to his creed, friends and values. I:ut you only knew this by aberration, by the example he set. .Woe was patient; you learn a lot about a man who goes over your manu~rlpt with you. i~e taught me that no~zns arc not supposed to modify noun.¢, split infinitives are a s|n. and hyperbole is intolerable. | was told Io read poetry to learn economical u~;e of weeds. ! was fond of bmencks, hut apparently they duln t count. At that ttmc dldn't know ~oe was u Yale graduate in English. and a Phi Beta Kappa at that. To almost split an infinitive. To Joe was highly definitive Of laser men. Those lacking ken. The near.deed was prohibitive. "All right." Joe was a .~cbolzr. He loved scholarship. It was manifest in all of his activities. As an example, he organized the sophomore course in pathology so student.q learned about disease by studying carefully edited ca~ summaries m a ~mlnar format. Then they did the autopsy on beautifully dissected specimens to ch.cc.k their de~du..ctioos and diagnoses. Hc put the onus on the students to learn not cozy t.he tac!s ot.o.t .s~s~. but [he ability to use those facts, using professors as referees non coaches. He nao deep c¢~nccm for the students. He understood their needs and ambitions, but his efforts on their behalf were often unrecognized because of his low waffle. Oedit was not important to him but students were. This course was a thing of beauty and it was exciting to be pan of it. He wa_~ generou,¢ with his time and taught me to u~c the electron microscope. He led me through the frustrations of lea .ruing to ~_e.ak.gla~ knives .and obtain able sections free of "schmutz" for examination wzm toe electron microscope. then there woe the thrill of photographlng and knowing .something impcxlant that for a moment nobody el,~c knew. The only rule was do it elegantly and finish it with r.cholarship. R Joe wa.~; fun to he around, and so was Natal. Donna and I recall with p~ea~re receiving an invitation to n party at the Feldman~. in addition to g,,ood fun. lb,*re would be real food. It began snowing heavily the morning of the party and by e~ening the city was paralyzed by it. To recently at, rlved Canadians the city wa~ rionsly quiet, the old trees and hon~s were covered by snow. which crunched und~:r, foot. We walked the mile or so to the Feldmans and were warmly received ~nd ate and ate and ale. We were the only guests and there was food for about 5t). P~r Nomi. all that hard work. Io he opprecleted only by people who liked to hear snow crunch. There w~s love in that family and it showed that night. Our p~ths diverged in 1961: Joe went to Scripps Institute. but we always met at the .¢prlng meetings and kept np with family off0in¢ and science, lie was ¢o proud of Nomi nnd talked n hil or Ih~ girls nnd tl~ir doings. Years later I~ would tdwa.,.'.~ ah~tl his granddaughters. l)uring his tenure at Scripps. Joe nsxttmed the editorship or the Jot~nnl of Immunology. Over a I 6 year peritxl il not surprisingly became the weemircnt pul~li. cation in th;d field, largely I'll:arise the editor xvas fair. considerate, ohli~in,~,*, ,and .~hohtrship was his traderaark. I lc tralm,'d many young g|entists at .~.'rlpl~. any one of whom could reiterate tales comparable to Iho~ of the Pierces. His effcctivcnc5 Chairman of the Scientific Advisory Board of CTR is well known to z~ll of u~. We will mi~¢ him. In his retirement there w~,~ :~n0ther activity of which I wax unaware, lie friend G~med the Ilebrew Free Loan Association of San Diego. It~ puq~ose wa~ provide loans to newly arrived .~ewlsh immigrants. He was head of the loan ¢ommlt- tee which to date has made 150 loans. Think of the impact this made in the llvcs people starling a new life in a new strange countw. What a remarkable man. We rejoice in having known him.
Page 7: 60040926
ABSTRACT TITLES AND AUTHORS !. Cancer-Rdal~d Studies COM ~.t~'~. R ~'q~T~"tJT~N OF Ct~UUGA'rlC~ AND ~.~A ................................................................................ ~IR~AL MA~ ~ ~ ~1~ ~ FAMILY A ~1~ A~ KI~ ~ARI~ ~ ~~ III I~N l~lll~l~l~, I~. ~, I, ~. ~ M. I~ ~t ~. I~RI.Y ~ ~ K~ ~11~ ~ ~~R~AL~ ~V~.hK ................. . ..................... ..... ......... MA~I~I~-~ ~S ~'~ • I ~MAN ~ ~ ~M.~ ................................................................................... II
Page 8: 60040927
II. Cardio~a~ular Sydem IlL Cel~ Dlnh~o' 12 ~ UR~-M~A~ ~.~ PA~IWAY: kIF~l~ ~ A~ AND ~ R~ ~V~ Ak11~ ~. C4.~ ~ AND ~ARAG~ 1~7. ~ ~1~ ~ ~ff.~M. RA~ A~IA~RIMIDI~ ~~ (~A~ ~,L~e.~ I~. ......................................... . .................................... ~F~L ........ . ......... , ......... • ............ ........,...,... .............................. ~FA~= ¢~ ~ ].W~.T.~ .............................................................. 13
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HY~AR~ RE~ H~I.~R TRANSI.rK'A~ ~JN ~ ARYI. AND BklP ~ ~(~I I:R A(~ IV~y IN ~IMARY I~AI. RAT I~1)~ L ~ ~/*MJ~Jllm, M .................................. 14 I~t. ~ I~. T. ~ ~ ~, T, ~ .............................................................. AL~A~S ~ ~A~ AND EEI~V~ ~.A,~,D.W .......................................................................... 15
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0 0 ~R~ ...................................................................................... 16 TIE WW lX~MAIN (~ YI~.AS~:)CI&TED Fit OTF~N BINDS A I~O~IPI~.RICI I L~IAPt O ~RA~ ~ ~IE MAMMAl JAN YAPI~*~A~ ~N) IV. l)eve+opmenln! 111nlc~v 17
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4~ C) 0 "4 ~.~ GLY~lR fib ~ ~X~F.~SK~ k~ A ~Hg], ~ MI~;AKAR MY~.~ ~T~Y FA~S. ~S~ ~IEIR R~ AND REfi~T~ V. Epldemlo~ogy VL Genetics $|'f~'Dt~£~"I~DMUTANT~OF'IltR{~$UBI~tffOFpRql~fNK~ASE(~2IN,~AlI{~IHIM~TA~ lg VIi. Immunology & Adapflv~ Mechan[,~ms RI~AL VAL~A~ ~ L~A~ MIM~Y IY A~-RE~ A~R~: ~A~ AND 19
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VIII. Neurc~¢lence ~I[I~L I~£~1~ ~, ~. [ ..................................................................... II} ~AL SffA~AL F~K~ IN: ~" {11~1~. ~.Yff~ IN RA~ IIII~K'A~PAL IX. rharmacolog.y ACUIT~ Lrl~,q~ EXFO~IE SUff'RES~ TILE. RF2AIR ~ ~-M~ I~.~IIANINK ~ 1,~31~ ~ ~W.~) ..................... , ................................................................. ~lE ~X~ ~ ~Y~ (~ ~ IIUMAN AND ANIMAl. ~J~ ~ ~L~E AT ~~S USED ~ A~V~AL ~A~. ~, D.T. ~ ~ P, H ......................... 191 ~DY, ~B,I.~.~B~hH ..................................................... ~A~ ~ C~UM ~UX FA~ ~M ~t~ IImKAT TJ.YMm ~.~ ~Y 2O X. Pulmonary & Respl~Ifory Systems 21
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0 0 XI. R,d;¢sls Xll. Virolog.v 22 XIlI. Miscetb~ou5 23
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C) 0 0 Abstracts of Reports Followi.$ ~,~ al~tmcts of teix~s ~ new ~s~h ~t C~il ~at have t~ in ~i~tirw ~l~ si~e ~blicafi~ of R~. ~ nt~ of the g~t ~ip~nl is in ~Id ~e abslm~s ~ g~d u~ ~ ~in~: I. C~.Rel~ed Smdi~. Cardiovascular 5yslem, III. Cell Biology, IV. DevelopmenlM Biolegy, Epidemiololy, VL O~etics, VII. Immunology and Adaptive Mechanisms, VIII. Neuroseien~e, IX. ~ol~y, X. ~I~ XL Radials, XII. Virology, XIII. I. Cancer-Related Studies COMPLETE RECONSTITUTION OF CONJUGATION AND SUBSEQUENT DEGRADATION OF THE TUMOR SUPPRESSOR PROTEIN I)53 BY PURIFIED COMPONENT~ OFTHE UBIQUITIN PROTEOLYTIC SYSTEM The wild-type tumor suppressor pmteln p53 is a short-lived protein that impatient roles in regulation of cell cycle, diffe~nfiation and survival Mutation; that inetctivate or alter the tumor suppressor activ;ty of the protein seem to l:e the most common genetic change in human cancer and m-e frequently assoc[ated with changes in its stability. The ubiquitin system has been implicated in the degradation of p53 lx~h in vi~,o and M P[tro. A mutant cell line that hmhers a thermolahile ub]q. uitin-activating enzyme. El. fails to degrade psi at the nonperm|sslve tem~ratu~e. Studks in cell-free extracts have shown that covalent attachment of ublqu~tL~ to the protein requlms the three conjug~ing enzymes: El. a novel spec|es of tier protein (ub]quitln-conjugating enzym~ UBC).E2-Fi. trod an ub|qu|fin.proteln llgase. E3. Recognitioct of p53 by the llgnse is facilitated by formation of a complex between the protein and the human papillomnvirus (HPV) oncoproteln Therefore. the ligase has been d~signated E6-assoeiated lWOte]n (E6-AP). However. these in vitro studies have not demonstrated that the conjugates serve as intermediates in the proteo]ydc process. In fact, in many cases, co~jugation of u~iq- uitin to the target protein does not signal its degradation. Thus. it is essential to demonstrate that p53.ubkluitM adducts serve as essentlM proleolytlc and ~re recognized and degraded by the 26~ pretense complex, the proteotytic arm of the ubiqultln pathway, in thls study, we demonstme that conjugates of pS~ gen~r. ated in the presence of puriGcd El. E2. E6-AP. E6. ub]qultln and ATP. axe spectfi. eally recognlzed.by the 26S protease complex and degraded. ]n contxast umconjt~gat- ed p53 remains stable. The ability to reconstltule the system from purified comi>o. heats will enable detailed analysis of the recognltlo~ process and the motifs involved in targeting the protein for degradation. 25
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C) 0 0 Shkedy, D.o Gonen. H.. Bercovich. B.. end C|echanover, A. FEB$ Letters 348:! 26-130, 1904. (~her support: United States-lsraal Binatio~al Science Foundation. Germ.',n-lsrael; FognCzlion for Scientific Research and Development. Israeli Ac.-~Jemy of Sclcnc~ and Hummities, Monsanto. Inc.. Foundation ro¢ Promotion of Re~atch in the Techn~on • Re~zrch Fund administered hy the vice president nf Ihc Techninn for Research. From the Department of Biochemistry, Rapp-',pnrt Institute for Research in the Medical Sciences, Tcchnion-lsracl Institute of Technology, Ilalfa, Israel. THE ROLES OFTUMOR SUPPRESSORS pRB AND p53 IN CELL PROLIFERATION AND CANCER Cell numbers age normally controlled via a balance between cell proliferation, differentiation, and apop¢o~L¢ in a mufficellular (,'ganism. A disturbance in this bal- ance may lead to an uncontrollable cell proliferation and result in malignancy. Fai[uro to wlthdrsw from the cell cycle and terminally differentiate may cause organ dysfunction and developmental abnormality. Moreover the genom¢ of each cell is carefully monitored to maintain its intcgrity. Genomlc lesions in a cell need to he repaired before DNA replication and cell division occur. A cell conloining unro- pairable germmic lesions should not be allowed to proliferate and must be orderly disposed by programmed cell dealh apoptosis, to prevent, oncogenesls. Each of the cell fates is mediated by sequentially organized biological pathways at the molecular level. The control of cell fates is a well-c~mrdlnaled parr(romance starred by key players in cell cycle regulation, in recent years, important advances have been achieved in the d~seetion of cell cycle control, differenti.~tlon, and apopto~is regula- llon. Increasing evidence points to the involvement of both the retlnoblastoma gone product (pRb) and p$3. two well-characlcrized tumor suppre¢*.ors, in mulliple path- ways to coordinate these esscntlal processes. Sang, N., Baldi, A., and Giordano, A. Molecular and CeJlular D~fferentiallon 3( I): 1-29. 1995. Odor support: National Inslitutes of Health and the W. W. Smith Ch.:,ritable Tarot. From the Institute for Cancer Research and Molecular Medicine, Jefferson Cancer Institute, end Depmments of Microbiology/Immunology and Pathology, Thomas ~'efferson University, Philadelphia, PA. CHROMOSOMAL, MAPPING OF MEMBERS OFTllE CDC2 FAMILY OF PROTEIN KINASES, CDK3, CDK6, PISSLRE, AND PITALRE, AND A CDK INHIBITOR, P27~¢', TO REGIONS INVOLVED IN tiUMAN CANCER Orderly progross|on through Ihe cell cycle requires sequential aclivalion and inactivation of cyclin-dependent kin&~s (calks). This is achieved in part through the 26 association of cdks with positive regulators called cy~lins and inactivation or cyclin- edk complexes by u rnpidly growing number of cyclin.,cdk inhibltor~. Recently. Ihe role of" cell cycle control proteins both as primary effcctors and a~ medi.~lor.~ of tumorigenesis has become a sub~ct of increased interest. Hero we report the chin. mo~omal mapping of two cdks. cdk3 and cdk6 two putative edks, PI~SLRE and PlTALRE, and one cyclin-dependent kinase inhibilo¢, p27, to chromo~om;d re~ion~ which may he altered in human tomato ~md examine their po~.~le im'olvcmcnt in some ofthe~ malignancies. In particular, two of the klnases cdk3 and P1SSLRE and PITALRE, the edc2.related kinases recently cloned by us, map Io regions previously shown to exhibit lo~s or beterozygo~ity in breast and o¢her tumors. Built;cA, F.. M~cL~chlan. T. K.. Sang. N., Druck, T., Veronese, M. L., Allen, S. L., C'~iorazzl, N.. KofT, A., Heutmer, K., Croce, C. M., and Giordano, A. Cancer R~carch 5.';:I 199-1205, March 15, 1995. Other support: Tile Sociely of The Memorial Sloan-~etterln~ Cancer Center. National Cancer Institute. and the W. W. Smith Charitable Trout. From the Jefferson Cancer Institute nod Department of Microbiolo~.y and Immunology, Thomas Jefferson University. Philadelphia, FA, North Shore University Hospital Cornell Universily Medical College, Manhasset, NY, and Program in Molecular Biology. Sloan-Kelterlng Institute, Memorial $1o~n-Ketterln~ Cnnccr Cooler, New York, NY. A STRUCTURAL AND KINETIC COMPARISON OF PROTO-ONCOGENIC AND ONCOGENIC NEU HOLO-RECEFFORS EXPRESSED IN INSECT CELLG The proto-oncogenlc and oncogenlc forms of" the rat neu receptors were expressed in the baculovims system to characterize their structural and enzymatic differences. The epitopes of their extraceliular domains, lheir molecu]:u" weight~, and kina~e activities were similar to mt neu receptors expressed in fibroHa~ts. The Iors were pa~iaily purified using a phospho-agarose column and were analyzed to compare kinetic parameters using ATP as a subslrate. The oncogenlc form ot" the receptor showed a significant increase in V., (56%) over the proto-oncogenic form. Structural analysis of these pro~elns using sucrose gradients showed the o~co£enic recep(ms to have a 62,6% incr~ in aggRgated receivers when comp:u-ed to prolo-oncogoni¢ reeeplors. These studies ~re the first to link enzymatic activation and the physical form of the receptor using isolated Rceptor species. LeVca, C. M., Myers, J. N,, Dougall, W. C., Qi,'m, X,, and Greene, M. !, Receplor 3:293-309, 19~3, Olbnr mzppon: National Institutes of Nealth end the Lucille Markey Charitable From the Dcp~rtmcms of Pathology, Center for Receplor Biology, and Biochemi~try and Biophysics, und Johnson Foundation, University of Pennsylvania, 27
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O~ 4~ 0 0 0 0 INTERMOLECULAR ASSOCIATION AND TRANS-PiIOSPIIORYLATION OF DIFFERENT N£U.KINASE FORMS PERMIT SH2-DEPENDENT SIGNALING AND ONCOGENIC TRANSFORMATION The neat oncogene encodes a 185 kDa receptor tyrosine kin•st. A single point emft~c~_o_n..( _V~I~___5~--. >G.!u) w.hhin the p!_8.5`~. transmerebrane region results in higher .;~¢.cy oi receptor otm~Jzaton, constitutive activity of tyrosine klnase and cellu- lar transformation. The oncogenlc potential of this mutated form of p185"(lermed Tneu) can be inactivated by slat.directed alteration of a lys~ne gcsldue in the con- served catalylic domain. In this report, we have utilized the i~ysical and functional inter•ellen of a full-length klna'~e-deficient neu protein (T757) and Iguncated kinase. active Tneu fames to delermine critical protein dom.~ins for Tneu ollgomeriT.'~tion and the resultant biological consequences. Analysis of v~rious truncated Tneu reuta~t~ ¢onErmed teal the transmcmhrane region w'ds crucial for pl85 dlmcr~zJ~llnn. Receptor as~x:iatlnn facilitates intcrmolccular pho~ph~gylatinn of kin;tse..de~clent reulant "1757 by truncated kin•st.•clive pl85 pru~eins, and tho trans-phosplx)rylated kln•se.~eficient T757 was able to associate in ~,itrn wilh proteins cohlainlng SH2 domains. Receptor.receptor inleractions resulted in enh:,nced signal I~nsduction potential and transformation of cell-linc.s co-cspr~xing different ncukln~tse These studies erepha.,~ize a novel f~ture of protein-protcln interacllon and the func- tional significance of p!85 dimerization, intermolccular pho~phorylation and signal- ing which may result in cellular transformation. Qian, X., Dougall, W. C., Fel, 7... and f~reene~ ~.|. |, Oncogene 10:211-219, 1995. Other suppo~: Lucille Markey Charitable T~st. From the Center for Receptor Biology, Department of Pathology and IJbornto~ Medicine, Department of Physiology. University of Pennsylvania, School of I~edlcine, Philadelphia, PA. PURIFICATION AND CHARACTERIZATION OF THE BCL-2 PROTEIN The oncogene product bel-2 functions as a represser of programmed cell death and is • 26-kDa protein with • single predicted transmerebrane segment located at the cadcoxyl terminus. 'The bcl-2 protein seems to function in different subeellular cornpaxtmenls, as evidenced by several biochemical and ultrastructural studies, The wesent study w~ performed to purify bcl-2 woteln in significant quantities neces- sary for strUclural and funclionai studies. For this purpose, the bcl-2 gent was over- expressed in either baculovirus systero or lymphocyies. Initially, attempts were undertaken to purify Eel.2 protein using conventional melhods such as ion exchange or gel fittrallOn chromatography. During Ibese purification attempts we delermlned thai hal-2 protein is highly hydrophobic and prone to uggregation as might be expected for an integral memlxane protein. By ion exchange and gel filtration chm- 2g malography, this protein could he partially purifi~. In order to purify bcl-2 to eat homogeneily ~d avoid t~ aggmgati~ ~, ~ p~p~d immu~affin~ty ~lumm using I m~l~=l untidy ~vel~d against a ~t~tlc ~ide cho~zn either coupled to CNBr-activated Sepharose 4B or cross-linked into protein A~epha~ by dimethylp~melimMate dihyd~h~de, Cellul~ extol equivalent columns. A~ximately 5~ pg pu~fi~ ~1-2 ~eln ~ld ~ ~ove~d mated ~ silver staining and immunoblottln~ Fuehrer. purified ~1-2 w~ el~t~ into ~-B lymp~yt~ which do nm eg~ss thi~ ~tein in ~¢knt quantity to delay I~ ~set of gluc~o~icoid-lnduc~ a~osls. el~tlon of h~og~sly ~m ~1.2 ~e~n. the ~lls we~ f~nd ~11 suwi~l in Rs~ to glu~o~icolds. llaldar~ S,~ Jean, N., DnBois, G. C,, Teknyeme. S,. Reed, ~. C., Fu. C~e, C. M. A~hives of Bi~bemlstW and Biophysics 315(2):4S3~88, 1~4, ~her snp~: Neat;enid Cancer Inst;tnt~. F~ t~ ~nt of Mi~blology and Immunology. ]eWe~on Cancer ~nt of ~a~y, ~as ]eKe~n Unive~iW. ~il~dclphia. ~ ]olla Ca~ Rehash Foundation, ~ Jo~l~, CA. BCL-2 RELIEVES DEOXYADENYLATE STRESS AND SUPPRESSES APOPTOSIS IN PRE-B LEUKEMIA CELLS The influence of bel-2 activity on 2°-deoxyadenosi~e.indueed apop~o~is was investigated in 697 hurean pre.B leukemia ceils stably t=nsr~ed with plasreid pHeBo-BCL-2et (697/BCL2 cells). Apoptosis was induced by the 2'- deoxyadeno~ine analogue,2-chloro-2°-deoxyadenoslne (CI.dA), with the concentra- lion for apopto~is in one.hell of the cells at 24 hour~ (LD~ being 10 p.M for cells and 120 p.M for 6971 Bcl 2 cells. There was • strong positive correlation between CI-dATP levels and apop¢oClc index (coefi~clent of determination, r~ = P = 0.027). When 697 cell and 697/Bel 2 cell lines were treated with 5 p.M CI.dA. CI.dATP did not significantly accumulate in the latter. 'The CI-dATP/dATP ratio 0.03 in CI-dA treated 697/Bcl 2 cells but nearly 6 in treated 697 cells. Bcl 2 ovetT'~O- ductlon also suppressed the accumulation of dAMP. dADP and dATP in cells exposed to 2'-deoxyadenoslne in the presence of pentostatln to abrogate th~ pro- nonnced inversion of A'I'P/dATP pools associated with 2~-deoxyadenosine expo'cure. These results suggest that one consequence of Ix:l-2 activity is suppression of 2'- deoxyadenoslne phosphowlatlon and elevation in the ai~optotlc target cells. Relief from dcoxyedenylate stre~ imbalances ireplies a novel upstream site of !~cl.2 activity. 29
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0 0 Gun, X., Knud~n. T. B., lbrahim, M. M., and llaldar,,q, Cell Death lad Difl'erentilfion 2:69-71~, Saouary 199.S. Other support: National Institute or Child Heallh and Iluman Development. From the Departmen! of Pathology, An-'qomy ,,nd Cell Biology. Jefferson Medical College, Philadelphia. PA. and Deportment o1" Microbiology and Immunology, Jeffersc~ Cancer Institute, Pfiiladelphla, PA. LYMPHOMAGENESIS IN THE SCID-HU MOUSE INVOLVES ABUNDANT PRODUCTION OF HUMAN INTERLEUKIN-IO Both human (hu) and viral (v) intedeukin.lO (IL-IO) a~pcar to he important ¢o~'actors in the survivll and growlh of lymphehlastnld cell lin~'s infecled wilh Epsteln.Barr virus (EBV~. When mice with s©vcrc combined iromun,- derlcicney (SCID) are in.~ccted wilh human peripheral blood lymphncytes {PBL) fn~n nom~n! individuals who are ~rrc~os||ive rot EBV, lho majority of hn-I'BL-SCID mice will develop In EBV-assoclatcd lymphoprolirerafive disezz~e (EBV.I,PD) nf hamnn B. cell origin, not unllkc .,mroe ca;es or EBV-LPI) lent ~zra seen in immunocompro. mi~d individuals. The role of hulL.1O or vlL-10 in Ibis chim=ric mou~ n~xlel or EBV-LPD is unknown. In the pre~nt sludy, we .~huw that hu-lqfl.-SCID mice theft develop EBV.LPD have slgniricanl elevation of serum hulL.IO levels compared with mice that do no( develop EBV.LPD {P = .005). vlL-IO was undcteClabl¢ in all ~imals. T~e EBV turno¢ samples express tran~ript fo~ hulL-lO ~nd hulL-IO recep- tor, express huIL-IO protein by immunohistochemical staining, and show specl['¢ binding or recombinant (r) hulL- I0. In ~tro analysis or the Functional cons~q~nces or rhulL-10 binding to IL-10 receptors on t'r~h EBV" tumor ceils shows Ihat 10 can prevent programmed ¢cll death &s well as promo(e prolit'erztlon and can do so It eoecenzradons or hulL-10 found in ~h.o. Thus, hulL.lO production by EBV° tumor cells rely contribute directly to their roallgnant outgrowth in the hu-PBL- SCID meuse by two autocrine mechanisms: prevention of p~rammed cell death ~d proliferation. 'The implications of such findings with rogard to EBV-LPD in humerus is discussed. Ba|occhi, R. A., Ross, M, E., Tin, J. C, Chou, C.-C.. Sullivan, L., l-l~.ldar, Monne, M., Selden, M. V.o Nlrula, S. K., Sklar. J., Crcx:e. C. M., and Caligiuri. M. A. Blood g5{4):I0~3-1074, Febn~a~ 15, 1995. Olh~r support," National Institutes of Health. National C~ncer Institule, American Canc~ Society, ~nd the Coleman Lcukerola Re.~.arch Fund. From the Departments of Medicine, Molecular Mcdiclnc. Moiccul.',r Immunology, ~nd Physiology, Roswell Park Cancer Institute, Buffalo, NY, Scherlng Plough Research Institute, Kenilwo~h, N J, Depmlment of Microbiology and Immunology, .fefferson Cancer Institute, Philadelphia, PA ind the Department of Pathnlogy, Brigham & Women's Hosphd0 lllP~anl Medical School, Boston, MA. 3O A GERMLINE INSERTION IN Tile TUBEROUS SCLEROSIS (T~r2) GENE GIVES RISE TO THE EKER RAT MODEL OF DOMINANTLY INIIERITED CANCER The Eker rat heredhnry rennl c~ncinnroa (RC) is nn excellent example of ,~ mendelion dominnnt prcdisl~illon to a specific c~neer in an experimem=! animal. We have previously c~tahlished a new conse~ed linkage group o~ rat chromo.-.om~ 10q and human ch~>rno~ome 16p13.3, ~nd shown that the Eker mut,~tion i5 tl/~htly linked In the luberons .~lcrosls (Tic2) gene. We now describe a Bermline mulalton in Ihe gene encoding T.rr2 tensed by Ihe insertion of nn nl~roximately 3 kilob~.~e DNA fragment in the Eker rat, rcsulling in aherran! RNA expression from the mutant allele, The phenotype o!" tuherous sclerosis in humans dlfi'ers rrom that ot Ihe Eker rat, except rot the occurence of rena! turnouts. The Et:er rat may ther~rore pmvld~ insighls into species-specific differences in ~umouri~enesis and/or ph~nolype- specific mul,~tions. Kobayashi, T., Hiray-~mn, Y., Ko~ay~shl, E., Kubo, Y., ~nd tllno, O, Nalure C, cncllcs 9:70-75. Janu,~ry 199.~i. Other support: Ministry or ~Ine~tloe, Science, nnd Cullum or~npan, ~nd the Vehtcle Rzcin~ CommemoraHve Foundation or j~p,-m. From tl~e DeFtr~ment or Ex perlmental Patl~olo~:y, Cnneer Ins!irate, Tokyo, ]~n. THE EKER RAT, A MODEL OF DOMINANTLY INHERITED CANCER SYNDROME A class Of cancer Genes, called the tumor suppressor ~enes or ~l~oncogenes, ha~ been revealed by the study of hereditary human cancers. Although these gem-s are rece~ive in oncogenesis, they render heterozygous can~ers highly suSCel~ible to particular cancers and so apl~ar in pedigrees as doroina~tly inherited dlso~c~s. Such a domln~ndy inherited predisposition w~s described in rats by Eker. llino, O., Kohayashi, T., Mttani, H., Kobayashl, E., Kubo, Y., Tsuchiya, H,, Kikuchi, Y., Nishizaw,,, M., and Hirayama, Y. Tr~n~la~ation Proceedings 27(2):1529-1531, April 1995. Other support: Ministry of Edueatlon, Sclcncc and Culture or.~apan. From the DelX~rtment or Exporlm~,mal Palhology, Cnncer Institute, Tokyo, 3apan. TIlE PREDISPOSING GENE OF THE EKER RAT INHERITED CANCER SYNDROME IS TIGHTLY LINKED TO THE TUBEROUS SCLEROSIS (TSC2) GENE The 5"ker rat is • promisln~ ~n~mal model or c~cer predisposition s)~nd~mes. 31
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O 0 In thk ~tudy, using 129 hackcro~s animal~, we have established a new con~rved linkage group on ~t IOq and hum~ !~[~.3 whereby th~ ~ker mul=li~ w~; found ~o ~ ti~hZly link~ Zo !~ tu~r~s ~ler~is (TS~) Bent. ~is will ~ the fi~t slap tow~ I~ ~sili~al cloning and i~nti~Ration of the ~dis~ing Ek~ mulali~. ]!;~, O, Kobz~shi. T., Tsuchiya, H., Kikuchi. Y.. Kobay~hi. E.. Miznni, ll., :nd Himyama. Y. Biochemical and Biophysical Research Communicelions 203(2):!302-130~. Scptcm~r 15, 1~4. Other sups: Minis~W of Education. Science, and Cuhure F~m the ~ment o~ Ex~rlmcn~al Pathology, Cancer lnslilute, Tokyo./ap~n. EARLY DETECTION OF KNUDSON'S TWO-lilTS IN PRENEOPLASTIC RENAL CELLS OF'lifE EKER RAT MODF.I. BY TIlE LASER MICRODISSECTION PROCEDURE llereditary renal cell caminom=.¢ invariably develop hy the age of 1 yc~r in Ekcr mrs. At Ih~ histological level, renal cell carcinomas develop through multiple stages from e~zly preocoplastlc lesions (e.g., phenmypically ahemd tubules) to ndcnom~. We previously reported that ionizing mdiadon induces additional tumors (large ado- n~mas and carcinomas) in a linear dose-respon~ relationship and Ihat loss of het- erozygosity (LOH) a! chromosome I0, where Ihe predisposing tuhemus sclerosis tTsc2) gent is localized, was found in Ibu renal cell carcinomas which developed from hybrid FI rats carrying the Eker mutation, indicating that in heterozygotes two events (one inherited, o~e somatic) are necessary to produce at least large adenomas and carcinomas. This study was designed to examine LOH in the earliest preneoplas- tic lesions, using a laser microdissectlon procedure. We could accurately dissect single altered renal tubules o~t of freeze-dried sections and clearly detected LOH in 4 of 19 altered tubules (21%). This is the first demonstration of LOH in single renal tubuges. Our p~r~nt results support the theory of a s~co~d, somatic mutation (second hh) xs rate-limltlng step of renal carcinogenesis in the Eker rat model of dominantly inherited cancer and the tumor suppressor nature of'the T.w'2 gent function. Kubu. Y.. Kllmck, F., Kikuchl. Y., Baana~h. P.. and Illnu, Cancer Re.~arch $5:959-~. March I Other support: International Union Against Cancer and Ministry of FJlucation, Science, and Culture of Japan. From the Department of Experlmcntal Pathology. Cancer Institute. Tokyo, Japan, and Department of Cell Pathology, G~Tnan Cancer R~;earch Center, Heidelherg, Germany. 32 NAD(P)II:QUINONE OXIDOREDUCTASE, (DT-DIAPHORASE): EXPRF~$1ON, REGULATION. AND ROLE IN CANCER NAD(P)H:quinonc oxidoreductzsc, (DT-diaphorase or NQO,) is a that promote.~ obligatory two-clectrou rcduetlon of qu~norr~s, pmwntln~ their patton in redox cycling, oxidative stress, and neoplasia, NQO, is ubiquitously expressed. However, a large amount of variation in NQO, gene expression wa~ noitc.ed among various human tk~es. NQO, gent is upregulated in livers of carcinoma patient.% trod its expression is induced in response to a vm-iety of com- pounds, including plan.~r aromatic hydrocnd~n.% phenolic teeters, tumor promoters, and hydrogen peroxide. De|etiOrl mutagenesis in the gent promoter.ldemified several cis.elements including antloxidznt re.~pon~e ale. meat (ARE), xenoblotlc response element, and AP2 element, which regulate the expression and indzg:tlon of the NQO, gent. Among the~ DNA elements, ARE is the most important tax.clement required for high basal expression of the r4(~, gent in tumor ti.c, mes, as compared to the normal tissues of the s~mc origin, and for its induction in response to xeno~iotics and zmtioxldants. Nu¢lcozlde .'~'(tucnce of the ARE tndicaled pRseueo of three API/API.likc elcrnents nnd n (;CA Mut~lioual analysis indicted a reqtdrew~nt of two API/APl-like elen~nt~ as inver~ rel',cats at the interval of three b:z.,:e palr~ for the ARE activity. Th~ GCA box in the ARE was requlr~ for Ol~imum ba~l and induced expre.,~ion. ARE is a novel tax-clement hecau~ a slnglc APIIAPI-llke element did not stlm,d~te ~.cne expression in response to xenobintics end antioxldants, Band shift and ~up~hlfl a.~.~'~ys identified Jun, Fos, nnd novel proteins in the hARE-nuclear protcln com- plexes lhM mediate regulatiou Of the NQO, gent exp~P..,;iofl. A hylX~hetical n'~P~el to sludy the mechanism of signal transduelion f'mm xenohiotlcs nod anlioxicL'mts to the ARE resulting into activation of lhe NQO~ and olher phase I! enzyme genes is descrihed. Evidence is presented to demonstrate the role of the NQO, in preventing, the bind;ng of P450 reducta~.uetiv-',ted benzo(a)~yrene qulnones (semiqulnones) to the DNA, presumably by eliminating their formation. Joseph, P., Xie, T., Xu. Y., and Jaiswal, A, K, Oncology Research 6( ! 0/I !):~25.~32, 1994. Other support: Nat lena! Institutes of Health. From the Department of Pharmscology, Fox Clause Cancer Center, Phila~elphla, SUSCEPTIBILITY TO TUMORS INDUCED IN MICE BY ETHYLNITRO$OUREA IS INDEPENDENT OF RETINOBLASTOMA GENE DOSAGE The relinobla~;toma gent (RB) is a classic1 tumor suppRssc, r. Severs! have shown thai RB dosage is important in determining biological effects. To explore the effect of RB dosage on susceptibility to cancer, three groups of C$TBIJ6 mice. each of which expres.~s a different amount of Rb protein from two. or Ihrce ullelcs, were treated at postnatal day 12 with a single 60.m~.~g 33
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C) weight i.p. do~ of the DNA-alkylaling agent N-ethyl./V:nilrosourca (ENU). Mice ~emzy~s ~or Ihe RB ge~ dcvelo~ cha~clerisdc piluila~ tumo~ wJlh n~rly c~p~ele ~nelmnce. whether ~ not they were t~ated with ENU. Tumm in;fluted earlier or progres~d mo~ rapidly, however, in ENU-treated mice. Funhe~nre. although mice t~ated with ENU had a higher incidence or scvc~! n~pituhary tu~s com~d with unt~ated c~t~ls, no significant diffcmaces in the incide~e of the~ lumors were found ~tween wigd-ty~ mice (mR~'). mice ca~ing only ~e n~al R~ alle~e and defici~t in Rb protein exp~ssion (mRB"). ~nd m~e over- ex~ss;n~ gb WnteJn from two nn~al murlne R8 alleges ;rod a hnm:m RB tmns~ (~". hRS" ), ~e~ studies u~e~ore Ihc tissue a~ m~hani~tic ;~ificity of tumor W~;s~sition caused by an in~fited ~0% ~daction in RR ~age a~ indi- cate that mo~ ENU-induced tumors occur independent of RR inactivation. H~ethe~e~% t~y suggest that cenain ~inl mugation~ induced hy ENU m;ty Ft~ici- pate in t~ ~uence of mol~ular steps involved in progr~sion of tu~r-pr~e. RB- ~ficient cells to t~ fully malignant state. Riley, 0. ]., ~t, C..C., ~ang, C-Y.. Jones. D.. l.~, F. Y..ll. P, and Lee, W..ll. Caner Re,each ~4:~7-6101, ~em~ I. !~4. Other supra: Hatio~l Institutes of Heal~h. Fr~ t~ l~titute of B;otechno~y. Center f~ Molecular M~cine. Unive~ity of Texas Health ~icncc Center. San Ant~io, TX. THE ONCOGENE qin CODR..q FOR A TRANSCRIPTIONAL REPRESSER The ~tmvJral oncogene via c~ for a ~eln lh~! ~longs to the winged helix fam}ly of t~n~Hpl~nal ~gula~. ~e Qin prmein is I~ali~d in 1~ nucleus ~d binds [o lhe ,~ DNA c~nsus s~ue~e z~ ral brain fac[~ I (BF-I). Cellular O;n sh~s 8~ater srfinily to DNA ih~ ~s vi~l Qin. Alone or rus~ [o the DNA-b;nd- in8 domain of the yeast GAL4 prolein, ~lh Qin prolcins ac[ as/ranscri~i~al ~s~. ~ mawr [~H~i~al ~sion domain m~ !o lhe mgi~ or ami~ ~Ms 2S2-39~ or vt~l Q~. L[, J., ~ang. H. W., ~i. ~, Parker, E. ~.. end Vogl. P. K. Olh~r supra: U.S. Hcahb 5c~ice. From lhe Deparlmen[ o~ Molecular and Ex~rimcnlal Medicine. ~o Scripps Rehash lnsliluic. La Julia. CA, Mcmerlal S[oan-Kel/ering Cancer Center. New York. NY. and Dcpartmcnl or Cell and Molecular ~inlogy. I]niver~lly or ~1. A~m~. SI. Andrews. UK. 34 OPPOSING ACTIONS OF C-ETS/PU.I AND C-MYB PROTOONCOGENE PRODUCTS IN REGULATING THE MACROPHAGE-SPECIFIC PROMOTER5 OFTIIE IIUMAN AND MOUSE COLONY-STIMULATING FACTOR- I RECEFrOR (C.FM$) GENES The receptor for macrophage colony stlmul~Iting f,'Iclor (CSF-I), the c.fm.~; product, is a key determinant in the differentiation of monocytic pha.t, ocylr.,t. Dissection of the human and mouse e..fms proximal promoters reveaTed roles for nuclear protooncogenos in the tmn~rlptlonul regulation of this ['.enc. On one h.'md. ¢'.et,.f.r.et.¢.2. :rod the macroph.~ge.sp~cific f.'sc[or PU.I, ~tt not the, factor PEA3. Irtm.r-~tivalcd tlm e./m.r proximal promoter. On the other hand repres.~ed proximal promoter octlvity in macrophages and blocked the attica of r- er.r.I .'tnd c-el.r-2. Ba.~'d e-fm.r promoter t~clivity was almost undetect~hle in the Icukaon|la line. which cxpres.~d high levels of (,-m.vb. httl w~s ~ctlva]cd ~s cell~ fercnliatcd in r~ponse to leukemia inhihilory f~ctor and expressed ('.fm.~r The represser functlon of'c.myh depended on lhe COOH-terminal d~m~in of the Icin. Wc pmpo.~ lha! cts-foclors arc ncccs~P] for the lis.~ue-r~tHeled expr~i0n of ¢-./m.r and lhal c.nLs'b acls Io ensure corrccl temporal exprc.~ion of r-fi~es durin~ myeloid differentiation. Ruddy, M. A.. Yeas, B.-S., Yueo X., Barnett, C. J. K., Ross. I. L., Sweet. hl. flume, D. A.0 antl Oslrowskl, M. C. Joumal of Expcrimcnlal Mcdlclnc I~0:2309.2319, D~cem~r 1994. O~l~cr soplx~: Nal|onnl lleallh antl Medical Re~ereh Co~encll of Au::tmlla ~,~I th~ Queensland Cancer Fond. From lhc Dcp.'~rlmcnl or Microhlnlopy, DuLe Unlversily Medical Center, Durham, NCo end Center for Molecular Biology and Riotechnolosy, Unlvcrsily of Queensl-'mdo Que~n~l-',nd, Australia. INSULIN-LIKE GROWTH FACTOR l! REGULATION OF GENE EXPRESSION IN RAT AND HUMAN HEPATOMAS Insulin.like growth factor II (IGF !I) regulated tls~ue-sp~cltle g~r~ expression in hepatoma cell lines, but had no effect oo expression or li~ue.sp~ific genes in pri. mary cultures of El4 and newborn rat liver cells depleted of erythrotd cell~. change was observed in these primary cultures with respect to c~-fetoprotein (c~.FP~. albumin, c~j1oker~tin 19 (CK 19). "/-glutamyllmnspeptid,'~e (GGT), tots. Two well-differenliated hepatomas. HepG2 and FTO-2B. lia!ed hepatoma, H,A.C~. did not ~hew incr~o~d p~o~ifor~tion in lh yet showed gent expression changes in respon~ In IGF I1. In HepC;2 cell% IGF I! incma.~d nlht,mln mRNA levels and resulted in ~ ,.;hlft fm~n chl~ler~ of ccll,~ po~ili~'c to I(XH(~ of the cells expmsslng immunohistochem~cally detectable albumin. The Iranscrlption factor IlNF-31~ mRNA ond protein levels of the bile duct m~rLer~. CKI9 nml GGT. wcr~ also incr~s~t in the pn..'scnce of were ~tol nlTecle¢l, i|Koluding ~l-anlil~l~:in. nnd Iwo livcr-swcific Iran~crlpti~m 35
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0 0 tots. HNF-4 and HNF-3cz. in FTO-2B cells. IOF II increased the expmsslon of albu- min. CKIg. and GGT. without accompanying changes in albumin and GOT mRNAs, In H,A,C., cells, lOF i! reduced CKI9 and OC.3 protein levels and GOT. Irznsferdn, and HNF.313 mRNAs. The elTe~s of [OF II on H.A,C, cells were not blocked in the presence of an anti,ral lOF l! recer~or anlihod¥. We conclude thal IGF i[ affcc[$ tissue-specific gone expression of hepatomas and qoaliladve and quan- titative z~pecls of its influence on the hepatomas is dercndenl on their degree of dif- ferentialion. Zvibel0 l., Brill, $.. and Reid, L. M. Journal of Cellular Physiology 162:36-43. J99.';. Other support: American Cancer $ociely. National lnstilute~ of [[ealth. nnd The Richard Molto Foundation. From the Albert Einstein College of Mediclnc. Dcparlmcnls nf Molecular Phawnacology and Micmhiology and Immunology. Bronx. NY. THE PROTO-ONCOGENE/TRANSLATION FACTOR elF4E: A SURVEY OF ITS EXPRESSION IN BREAST CARCINOMAS The eukaryot|c Iranslation iniriat[on fac[or ¢IF-4E binds [o the cap structure of" mRNAs as one component of the elf4 translation inilialion complex, which medi. ales lee recruitment of mRNA to lhe ribosomes. Ovcrexpresslon or elF4E can result in onco~nlc lransformalion and unconlrolled growth of mammalian cells, prcsum. ably by facilitaling the expression of growlh-conlml genc produCls which am nor. really translalionally repres~d. Whereas the mechanism of clF4E.medialed lransror- malion is bcing actively pursued, clinical investigations into Ihe expression uf eif- 4E in prcva|enl human cancers are lacking. We have recently initialed a semen of breast carcinomas by probing wilh elF4E anti,rum. Using Weslem blots, we have analyzed lee level of elF4E in 3g caminom=e, 7 normal samples and 3 fibroadeno- mzs. We found |hal elF-4E was elevaled 3- to IO-fold in virtually all the carcinomas we analyzed, but not in fibrosdenomas. This analysis was also confirmed by immunohistologlcal staining in xlm, showing Ih-'q overexpression of elF4E can be readily identified al lee slngle-cell level. Our results suggest that an elevation of" elF4E may be an esseotial component in the development or breasl cancer. Kerekalle, V., Smiley, K., Hu, B., Smith, A., Gelder, F., and De Benedclti. A. (Rhoads. R. E.) [nlemational Journal or Ca.cer 04:27-~ I. 1995. Other support: Louisiana Educallon Quality Support Fund. From the Department of Biochem|slry and Molecular Biology, Dcpartmenl of Patholo[y and Department of Surgery, Louisiana State Universily Medical Center, Shreveprsl. LA. 36 HERITABLE. POPULATION-WIDE DAMAGE TO CELL5 AS THE DRIVING FORCE OF NEOPLASTIC TRANSFORMATION Prolonged incubation of NIH 3T3 cells under the growth ¢onslrainl of cOnflu- ence results in the death of some ceils in a manner suggestive of upoplo~is. Suecexsive rounds or prolonged incubation at confluence Of the surviving cells duce increasing neopIastle ~ransfon~alion in the form of increments in d~sily and tmnsfonned focus formallon. Cells from the poslconfluen! cullur~,; arc given a recovery period of various lengths to remove the direct inhibilory effect confluence before tbelr growth properties are sludled. It is found thai with e~ch round of confluence the exponential growth rate of the cells at low densities gets lower and the size of isolated culon[es of the same cells shows a similar reduction. The decrea,,md growth rate of cells from the lhlrd round of confluence sists fur >f-AI generallcms of growth a! low density. The pmpo~ion of cohmlc,~ tainlng ghml cells is m,ch higher al'lcr a 2-day recovery from confluence th:~n after a 7-day recovery, Retardation of growth at low dens~y -~nd incre~Lxcd ,"~turatlen dcn~hy appear to bu Iwo shies of Ibo ~me coin: both occur in the entin: l~pul~tion of and precede the formation of tmnsl'omv.~d fact. We propose that the stowd~wn growth nnd the fi~,alion of giant cells resttll from herilnble damage to the o:11~. which in turn drives their tr,'msformation. Similar results have been reported for survivors of x-lrmdlafion and of treatment with chemical careinogen.~ and arc tied with the aging process in animals. We suggest that these chan~es result free radical damage to membrane lip|ds with particular damage to lysosomex. Pro~ea.,.es and nuclcases would then be released to progressively modify the growth behavior and genetic arability of the cells toward autonomous proliferation. Rubin. H., Yno. A.. and Chow. M. Proceedings of the National Academy of Sciences USA 92:4943.Ag47, May 1095. From the Department of Molecular and Cell Biology and Virus Laboratvry, University of Caliromla. Berkeley. CA. NEOPLASTIC DEVELOPMENT: PARADOXICAL RELATION BETWEEN IMPAIRED CELL GROWTH AT LOW POPULATION DENSITY AND F, XCESSIVE GROWTH AT HIGH DENSITY Tho role of hcritablco populat[on-w|de cell d~mage in neoplastic development was studied in lee 2g L sublinc of NIH 3'r3 eclls. These cells differ from t~ 17~ subline used previously for such sludies in Iheir lower frequency of "sp~zlan_,~us transformation at high population density and their greater capacity to produce brae, dome transrm'med fact. Three cullures of the 28 L sublin¢ of NIH 3T3 cells wcr~ held under the constraint of confluence for ~ wE (5 wk I° assay) and th.,'n twice in succession (2° and 3° as~ys) for transformed fact and ~lumlion d~nsity. After the 2° assay, the cells were also passaged at low densily to determine Ih~ir exponential growth rates and cloned to determine the size and morphological lea- lures o[ the colonlcs. Concurrenl me~suremonls were ramie in each ca~e wilh con~x~l 37
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cells that had Ix'on kept only in frequent low-denslty pas.~ges and cells that hod been ke~ at confluence for only 2 wk (2 wk I°). Two of. the three cultures transfcn'cd from the 2" assay of the 5 wk l° cultures produced light transfomled f.oci, and the Iffird produced dense rnci. The light fncus-fo~m;ng cultures grew to twice the control saturatlon density in thelr 2" ashy and 6-g times the control density in the 3`" saturation dons;ties for the den~ fo,:~s formers were abe, el !0 t;mcs the control val- ues in ~oth assays. All three of the cultures transferred From the 2" ns~ay o~" the .q wk I° cultures multiplied at lower rates than controls at low densities, but the dense focus formers muhlplled f.a~tcr than the light f.ocu~; fonncr~. The reduced rate.,; multiplication of'the light focus formers persisted For >50 ~onerations of. exponential multiplication at low densities. Isolated colonies f.ormed f.rom single cells of the light focus formers were of a lower population density than controls: colonies Fanned by the den.~ focus formers were slightly denser Ihan the controls hut occupied only half. the area. A much higher proportion of" the colonies from the $ wk I" cultures than the controls consi~ed of' giant cells or mixtures or giant and normal-appearing cells, The results reinforce the previous conclusion that the early increases in ~turation density and light focus formation are associated with, and perhaps c~used by, herita- ble, population.wide damage to oc]ls that is e~sentially epigenctlc in nature. The rn~re aclvanced transformation characterized by large incre~s in .~turat;zm density and dense focus f"ormatlon conld have originated f.rom rare genclic changes, such as chromosome rearrangements, known to occur at an elevated Frequency in cells desta. bilizcd by zntcccclent cellular damage. Rubin, I!,~ Yao, A., and Chow, M. Procc~dlngs of. the Natlc~al Academy of Sciences USA 92:7734.773g, August 1995. From the Department or Molecular and Cell Biology and Virus Labor,rotary, University of Czlil'omla, Berkeley, CA. SPHINGOSINE-I-PHOSPHATE, A NOVEL SECOND MESSENGER INVOLVED IN CELL GROWTH REGULATION AND SIGNAL TRANSDUCI~ON, AF'F~C]'S GROWTH AND INVASIVENESS OF llUMAN BREAST CAI~'CER CELLS This review will focus on the role of sphlnBoslne and its phosphurylated derlva. live sphingosine-l.p~osphate (SPP) in cell growth regulation and sisnal tran~uc- llon. We will show Ihll rainy Of. the elTccts attrihured to sphlngosine in quiescent Swiss 3T3 flbroblasts arc mediated via ils conversion to SPP. We ~ Ihzl SPP has |pp~ate properties to f.unclion as an inlracellular second me.~senger he.,,~ on the f.ollowing: it elicils dlvers~ cellulm' responses: it is rapidly produced from sphin- goslne by • specific kln,,,,e and rapidly degraded hy I specific ly,se= its cnncenlra- tlon is low in quiescent cells hut increases rapidly and Iransicnlly in response to the growth fat-lot,s, fetal call" seam (FC:S) ~nd pl~,lelel derived growlh factor (PDGF); it releases Ca~ from internal sources in an InaPt-independent manner;, and finally, it may llnk sphlngolipld signaling pathways to cellular Fax.mediated signaling palh- w~ys by elevating phosphztldlc acid levels. The effeClS of'this novel ~cond messes- 38 STUDIF.S OF TI IE STRUCTURE OF TI I E METASTASIS-ASSOCIATED 67 kDa LAMININ BINDING PROTEIN: FATTY ACID ACYLATION AND EVIDENCE SUPPORTING DIMERIZATION OF THE 32 kDa OENE PRODUCT TO FORM THE MATURE PROTEIN The level of' expression of' the 67 kDa hlgh-n~nily lnmln~n h|mlin~ pruz~ln (LBP) conclatcs with the rnosression of many sol;d tumors. The eDNA clone r(,4, th~ 67 kDa LBP is sufTicient to encode a polypeptide or only 32 kDa. and there |~ no rendily identifiable mechanism for mcmhmn~ assrciatlon. We have overe,'~pres~ccl Ihe tr=nsf.ecled 67 k~a humstcr LBP in q.unfities Ihat have ena~led us to unalyz~ the memhrane.huund form of. the pmte|n. Treatment of' the purified LBP with methyl transesrerification reagents, followed by GC-MS. identified the ¢ovalently bound ratty acids lulmltate, stearate, nnd oleate. The f.atty acid modification m',y provide mechanism f.or membrane assoclatico. Molecular mz.~ deform|nation by MALDI. TOF MS demonstrated the true molecular mass of.the pn~ein to be 66.7 kDa, com- patlble with the SDS-PAGE ol~er~allon of' 67 kDa. Treatn~nt or the LBP with neu. raminida.¢. O-~lycanase. or Endo-F glycos|dase has no detectable e~f.ect on apparent molecular mass or the pmteln, and the MALD~-TO]~ MS did ~o~ show evi- dence of mass heterogeneitics typically observed with glyonsylated proteins. Reduction with dithiolhreitol or/~-mercal~oeth~n01 had no ©ffccl on the apparent molecular mass on SDS-PAGE or o~ the relative quantities of. molecular species on MALDI.TOF MS. The experlm~ntally detem~incd amino acid compo~i. lion. however, was f.ound to be consistent with Ihe 67 kDa form 1~ing • homo~imcr of' the 32 kD,, precursor. Prellminnry experiments a~o sus~.est the! the hi~h-affiniW I~minin binding characteristic of'the protein may be mod~l'~ted by ml. zzs yel. unic~.~n. lifted memhr'.me acces.,~nj molecule. L'mdowskl. T. I!., Dralz, E. A.. and Rtarkey, J. R. Biochemistry 34(35):11276-11297, Other support: Natimml Science Foundation. From the Department ol" Microbiology nnd Department of Chemistry ,~nd Binchemislry, Montann State University, FJozcmnn. MT. 39
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CONTROL PATHWAYS OFTHE67 kDa LAMININ BINDING PROTEIN: SURFACE EXPRES$1ON AND ACTIVITY OFA NEW LIGAND BINDING DOMAIN A number of" papers have been published on Ihe clinical corrclalion of the expre,~sien or the 67 kDa lamlnin binding prolcln ([.BP) with Ihe metastatic potential of ~olid ~mors. Both mRNA and protein expres_sion levels have been reporled, hut hath lee relationship between them and Ihc molecular nature of the 67 kDa surl'ace prcxJuct remain unclear. We have utili;'.ed a homnlyp~ .verexpres.,;inn Syslcm to invesligate the cell surface pre~ontalkvn of Ihc ~7 k|)a 1.131' and Ihe cnnlrJhullnn of this proeein t(D the invasive phen(~ype hi" cultured cell line,~. We mporl hem that the cellular mRNA ~evels do not directly relleel the levels of the 67 kDa LBP oh~n~ed on the cell surf*co in this overexpression system. Melhnlrexnte amplification of tran~fccted pla~mid~ expre,~,,ing the 67 kDa LBP lead,; t. :m inili,I clevalion of Imzh the LBP m~NA and surface pn~ein levels. 'llds it acconzpanied hy an allercd, more fla,ened, cell morphology. Later. apparent adaplatlnn of the cells to melhotrcxare is ~ccompanied by | downregulation of the surfucc expre.~si~m of th~ proteln, mRNA levels, however, remain elevated. A nine amino acid .sequence. CDPGYIGSR (pep. tide 1 I). within the ~3 chain of laminin I has been idcmif'~--d as a probaMe hlnding domain for the 67 kDa LBP. Previous studies have identified a region of the 6"7 kDa LBP which may be involved in lamlnin interaction, although not necessarily via the pepllde I I domain. We have identified a second site within the amino acid coding sequence of the 67 kDa LBP which al~ shows biological activity both in vitro and in rim. A peptlde with this ~quence. LBP residues 205-229. hinds I~ninin-I in a peplldc ! I inhibhahle manner. Thc receptor.derlved peptide modulates invasion of ha~rnent membrane matrix in vitro and inhibits esperlmenlal lung colony formalion when in.~.,.cted zffong with BI6BL6 mouse melanoma cells. Ilowevor, pretmamzent of the melanoma cells with the peptidc enhances lung cnlnny formation. Thus. the interaction of Iho 67 kDa LBP with basement n~mhrane malrlx appears Io involve a complex series of'event,; includln~ multiple adhesive silos and tight rcgulatlon of cell surface expression. Landowski. T. H.. Uthayakumar. $.. and Slarkey, J. R. Clinical & Experlmcntal Mclastasls 13(~):357-372. FY95. Other suR:xxt: National Science Foundation and National lnslilutes of Health. From the Department of MicrobiOlogy. Montana State University. Bozeman. MT. COMPREHENSIVE ALLELOTYPING OF llUMAN RENAL CELL CARCINOMAS USING MICROSATELLITE DNA PROBES The vo~ llippcI-Lindau locus on chromo,rome 3p is a tumor suppressor ~ene known to be involved in nonpapillary renal cell carcinoma. A prcvlous los.,; of h~. eroaygosity (LOll} study aimed at determining Ihe allelotype of kidney tumms has indicated thai in addhlon to 3p, chromosome arms 5q, 6q. IOq. I Iq, 17p, and 10p may also harlx'a" tumor supprc,;sor genes. Ilowcvcr, cytogcnetic sludles reveal thai 40 chromosomes 3p, 6q. 8p. 91xl. end 14q most frequeady undergo kzxyotyp]c chznE~s in renal tumors. To resolve these differences, z collection or mlcrosat~llite DNA prohes has b,~n used to'scan for LOH so that 90% of individual lumor genom~z were rendered informative for allele less. The ashy is capable of detecting quantita. live genomlc alterations in tumor ceils as well. We find that LOH ;s most f~"quent for chromosome orm 3p. llowever, in no tumor is 3p ex¢lu.~ively alTeczcd. LOll 6¢ 8p, 91R, -',nd 14q is ~]~ distinclIy elevated for both nonpapillao, as well as lary tumors and suggest Ihat many of the tumor ~ppressm" loci involved may be common to the etiology of ho~h forms of kidney cancer, Thrash-gingham, C. A., Greonber~, R. E.o I Inward, S., Bmzel, A., Brcmer, M., A., Sal~,~r, t1., FRed, J. J.. and Tas'lof, K, D. Proceedings o~'the National ^cz~lemy of Sciences USA 92:--.8.r~4-295,~, M.~rch I Other support: Bets Foond~don, Lucille W. Markey Foundation, U.$. Pu51ic Hcahh Service, and an approprladnn from the Commonwealth of Penn,~ylvanla. From the Institute for Cancer Research and Division of Medical Science, Fox Cancer Camera Pfiil~elphla, PA. II. Cardiovascular System INDUCIBLE NITRIC OXIDE SYNTHASE ACTIVrrY IN MYOCARDIUM AFTER MYOCARDIAL INFARCTION IN RABBIT Tbe aclivity of nltric oxide synlhas¢ (NOS) in infaxcted and no'infarcted myoc,'~dium was ~lcrmined. NOS OCliVily. as meas~ztd by conversion ©f ["Claret- nine to I"ClcimJIline. was significanlly higher in the inf~,-cted area of myocard~um (22.7 ± 3? final/rag as compared to 7.67 - 1.0 in noninfarcted area). NOS ~ctivlty within the area of risk remained on control level. Increased inducible NOS activity was observed on the first postoperative day and persisted for at least 14 declined 3 weeks after infarction. Citmlllne formation was inhibited by nltro-L-arginlnc and N-omcga-monomcthyl-L.-arginine. Tho locallzafio~, of NOS .by monnelonal anti-NOS antibody indicates mononuclear eelWmlcropn~ge~ as likely smucc of the e~yme. The concentrations of tumor necmsls faclor,.alpha and intcrlcukin-I hera wcm not inerea, v.'d in peripheral blood or mynea~dium. Dudek. R. R.. Wildhlrh S.. Confor~o, A., Pinto, V., Suzuki. H., Winder. S,. end B|ng, R.,L Biochemical nml Biophysical Research Communications 205(3):1671-16~0. Decemher 3{I, I~)94. 41
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Other supgx)~t: Margaret W. and iferhert Iloover. Jr.. Foundation. Pasadena. CA, Gustavus and Louise Pfeiffer Foundatinn. John and Lilli Lauh Trust and the Lluella Marcy Murphey Foundation. Pasadena. CA. From the Department of Experimental Cardiology. Huntington Medical Re,arch Institutes. Pa.~dc~a. CA. and Depar~men! of Environmental Engineering. Califomla lnsdlute of Technology. Pa.~a~cna. CA. DEXAMETIIASONE INIIlBITS TI IE EXPRP, SSION OF AN INDUCIBLE NITRIC OXIDE SYNTIIASE IN INFARCTED RABBIT MYOCARDIUM Infarcted areas of rabbit myouardium shrew relatively hiL~her inducible nitric oxide syntha~ activity, measured by the conversion of L-|"Cl:trginine In L-I"Clcit- rulllne. The principal finding in this study is that dexmnethu.qme (2 m '~1]) prevenls the induction of inducible nitric oxide syntha~ in heart muscle when given before, or even 3 hr after coronary mlezy ligatkm. Additionally cyclic GMP levels remain unchanged following treatment wilh dexamelhasonc. It is Ix)ssihle that the enhanced production nf nitric oxide by inducible nitric oxide synthase accounts, at lear,! in part, for the depression of myocardial contractility seen in myocardial infarclion and in olher clinical conditions. Dudek, R. R., Wildh/rt, $., Pinto, V.. GJcslcr, G.. and Bing, R. J. Biochemical and Biophysical Re,arch Communications 202(2):1120-I 126. July 29, 1994. Other support: Guslavus and Louise PfeilTer Research Foondalion. Redlands. CA. Mart.user W. and Herbert Hoover, Jr., Foundation. Pasadena. CA, and the Pasadena Foundation. Pa.~adena. CA. From the Department of Experimental Cardiology, Huntington Medical Research lnslilutes. Pasadena. CA. INHIBITION OF ENDOTHELIAL NITRIC OXIDE SYNTHASE BY CYTOCHROME P-450 REDUCTASE INHIBITORS Nitric oxide synlhase (NO$) shows sim;lar;lieS to cytnehmme P.450 reducta.~e. The Iwo enzymes catalyze the oxidalion of N.oPhydrnxy-L-arginine by NADPII and oxygen to nitric oxide (NO) and citrulllne. Nitric oxide synlha~ ~clivity i~ inhlbiled by L.arginine analogs like N-c~-nitro..Loarginine, which does nol affect cylochmme P-450 reductase. Dihydrocrgolaminc. mlconazole, and Iroleandomycin are classical inhibitors of cylochrome. The present study shows the concentration-dependent 42 inhibitory effect of the~ compounds end of L- but not D.N.~0.nitro.:~rf.ini~ on activity of constilulive nitric oxide synlhase from bovine nortic endothelial cells. Activily of nitric oxide synthase was estimated by measuremenl of conversion of [~Hlarglnlne to [~lt]cltntlline. The tested cytochrome P.450 inhib|tors are likely interfere wilh hcmc of nitric oxide synthase. The data confirms a similarity as well as funcllonal differences b~tweco the enzymes. Dndek, R. R.. Con lotto. A.. Pinto. V.. Wildhirt. S.. and Suzuki. H. (Bin~, P.S.E.B.M. 209:6,0.(~t. 1995. Other support: Margaret W. and lterbert llonver. Jr.. Foundation. Pasadena. CA. From the Dep~rtment of Experimental Cerdlology. Huntington Medical Re~ea~ch Institutes, P~sadena. CA. and Department of Environmental Engineerinf, C~Iifomi,~ Institute c;f Tcchnnltey. Pa.~'~dena, CA. IMMUNOtlISTOCHEMISTRY IN TIlE IDENTIFICATION OF NITRIC OXIDE SYNTHASE ISOENZYMES IN MYOCARDIAL INFARCTION Inducible nitric oxide synlhase (iNO$) acliv;ty, as measured by conve~;~n of L-'*C-argln;ne to L-'*C-citmlline is significantly increased in infarcled rabbit myocardium as compared to healthy myncardium 48 h after comn:~ry occlusion. Th~ aim of this study was to Iocalise the nitric oxide syntha~e (NOS) isofurms in norm~! myocardium and compare these findings with NOS activity in oclls of the inf~rctcd region. Methods: Activities of HOD isororms were cnzymatically a~zy~ in normal and infarcted myocardium. Localisalion of NOS was perfumed on identi~l sections using specific monoclonal lgG antibodies against endolhelia constitulive (eNoS) and macrophuge inducible tiNeS) nitric oxide synthasc. In addition, mae~ofha~cs were identife d using fluorcsccin co41jugatcd CEromPure rabbit IgG, Fc fragment. Resulls: iNOS activity increa.~d significantly in infarcted as comp.~red to normal myocardium (O.42(SEM 0.03) v 0.85(0.0~) fmol-pg'~.min4, P--0.02, re~pectivelyl. However, eNOS did not increase significanlly in infarcled regions 10.18(0.04) 0.24(0.06) fmot.p.g'*.min ', P=0.16, respectively], eNOS was immunohl~tochemically Iocalised in endolhelial and endocardial cells in norton! and Infarcted tissues. presence of iNOS ucfivily in m.'~ages in infarcted myocardium wa,~ immunohistochemicully. Cardiomyocyles .~nd neutrophils did not label Wilh the anti. lxxlics to eNOS and INOS. Conclusions: (I) Infiltrating macropha~es are th~ m:~]n site of incrca.,~'d iNOS activity in infurcted rabhlt myocardium, (2) eNOS ucfivlty not significanlly inere.'tu.-d in infapcled tlr...~ucs us compared to nnnnal myocarditzm. (3) Neulrophils aml c.'wdlomyneytes do not express NOS imunoreacllvhy in and normal rabbll myocardium. Wiidhlrt, S. M., Duckk. R. R.. Suzuki. H.. Pinto, V., Nar~yan, K. S., and Blng, R. J. Cardiovascular Research 29:526.-531, 1995. 43
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C~ C:) 0 0 LO Other suppo~l: Marsarel W. and llcrberl lloovcr. Jr.. Foundalion. Pasadena. CA. OUSlavus and Louise Pfelffer Foundation, Redlands, CA, and Ihe Pasadena Foundation, Pasadena. CA. From lhe I~parlm~nl of Experimenl;d C:~rdinlogy, I lunl;.gton Mcdlcal Research ln,,titutc% P~",~ena, CA. CARDIOVASCULAR DISEASE AND NUTRIENT ANTIOXIDANTS: ROLE OF LOW-DENSITY LIPOPROTEIN OXIDATION Increasing evidence indicates lhal oxidative modification of Iow-denshy llpoprotein (LDL) is cau~lly related to athero~lerosis, Oxidatively modified LDL (oxLDL). in cofllrlsl Io nalive LDL. is lakcn up avidly hy macmphnge.s, leading to form•lion of llpld-l~.fen foam cells. Foam cells are F~lhognomn~ic of Ihe alhen'~cle- rolic fatty streak. Modified LDL may Mr, o promote ntherosclerosis by many other mechanisms, such as recruitment and retention of mon~x:yte-mncrophages. T-lym- phocytes, .',nd smon~h mur, cle cells in the arleri.-,I intima, and eytotoxicity tnw-',rd endothelial cells and macrophage..derived fozun cells. Tbo "oxldalion hypolh,.,~is of atherosclerosis" is supporled by a numher ofln ~'iv. Ending,.,. such ~s Ihe Ix'e.~nce of oxLDL in atherosclerollc lesions, and increa~d 6ter~ of =utoantibodles against mod- ified LDL in patients with atherosclerosis, As • corollary of lhe oxidation hypolhesis of uthcro~clcmsi~, anlloxidants Ihnt can inhibil LDL oxidation may ~¢! -s antiatherogens. Th'is concelXion is supporled by animal studies showing that antioxidents such .~s probocol, butyl•ted hydroxy. toluene, and a-tocopherol can slow Ih¢ progression of *therosclerosls. Epldemio~ogica! ~ clinical data indicate a protecllve role of dleta~ antioxidants against cardiovascular disease, including vitamin E. 13-carotene, and vilnmin C. Likewise, basic ros~arc6 studies on LDL oxidation h',ve demonstrated a protecllvc role for ~ntioxidanls, present either in lhe aqueous environment of LDL or a¢~ociatod with the lipoproteln it.ll. More studies ~re needed to c~tahlish the elTectlvonc.,~ and determine the required doses of specit~c anlioxidonts to prevent and possibly Ireal cardiovascular disea.~e. Frei, B. Crilic~! Revlewz in F~xl Science and Nutrition .15f Other support: American llearl Association. Mass-',chusetts Affiliale, inc. and Hational ln~lilules of llealth. From Ihe lion,on University School of Mcllicine. Whilakcr C;trdlovnscul:lr ln~lil,fc. Bu~lon, MA. 44 REDUCTION OF COPPER;BUT NOT IRON. BY HUMAN LOW DENSITY LIPOPROTEIN (LDL)-- IMPLICATIONS FOR METAl, ION-DEIq~NDENT OXIDATIVE MODIFICATION OF LDI. Cell-mediated oxidative modification of humnn low density Iipopwtein (LULl. mosl likely an import.'ml early step in atherosclero~is, requires rednx oct•re ions such as copper or i~xm. We have previously shown that imn-dL-VeMcnt, in con- trial to copper-dependent, oxidative mndific.~liou of LDL requires supero.clt~. i~yslnlngical red,el•hi. In Ihc presenl sludy, we sought to explain thc~ results. LDL w:t~ inctlh.'lted at 3?oC with Cu-" (10 p.M) and bathos,pro•no {11C, 3f,~l p.M}. an indicator molecule which specifically complexes Cu*. bul not Cu". In time- and concentratlon-dependent manner. LDL reduced Cu:" to Cu'. An LDL cou~ con!ration as low as (10 p.M) of proteln/ml (about 20 nu) reduced •haul 7 p.M Cu:' within I h of incubation. Complexutlon of the Cu' fanned under the~ condltlou'~ with BC significantly inhibited oxidative modification of LDL. as asse,~red by • garose gel elec~ropheresis. Proincubation of LDL with N-ethylmafelmlde had no effect on the rate and extent of Cu~" reduclion nor LDL oxi~tion, indlc*tln.e !hat sulf~ydryl groups associated with apolipoprotein B are not involved. Ad~lh~tm of either superoxide dismutase o¢ eatata~ or increasing the a-tocoph~rol content of LDL from I 1.8 -- 3.0 to 24.4 _* 2.8 nmol/mg of protein also had no significant effect on the kinetics of Cu~' reduction by LDL. In contra.,~t, incubation of LDL Fee'-el!rate (10 p.u) and the indicator batbophenanthroline (BP. 3f,~O ttM) t~ulted in ~o significant Fe~' formallon, even at LDL concentrations as high a5 200 Itg of pro. tein/ml. However. incubation of LDL with Fe~'.cltmte und un en~.ymatle ~urce of supemxide led to rapid fan•alton of Fc"" nod cooscqucnt oxidative modification of LDL. Addition of BP inhibited iron-mediated LDL oxidation under thc~e condhtc~n.~. Our re.~ulls indicate that reduced metal ions are imporlant medlato~ of LDL tlon. and that LDL specifically reduces Co:'. but hal Fc". These data. tl~refore, h~Ip explain why copper, in addition to being chemically more re•clive, is more potent than iron at mediating LDL oxidation. Lynch. S. M. and Frei. B. The .l'oumal of Biological Chemistry 270(10):5158-5163. March 10. 1995. Other support: National Institutes of Health. From the Whitaker Cardiovascular Institute. Boston University School of Medicine. Boston. HUMAN SUCTION BLISTER INTERSTITIAL FLUID PREVENTS METAl., ION-DEPENDENT OXIDATION OF LOW DENSITY LIPOPROTEIN BY MACROPl IAGES AND IN CELL-FREE SYSTEMS LDL in the cimulatlon is well pmtecled against oxidation by the highly efficient antloxidant defense mechanisms of human plasma. LUL oxidation contributin~ to ntherou:lcrosls, therefore, h.~ been hypolheslzed to lake Elate in the Intcr~tit|.'~! tl~,~|~l of Ihe arlcriul wall. We investigalod Ihc antloxklant compo~itlon trod the cap,~chy ICl 4.';
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0 0 '0 inhibit LDL oxldalion of human suction blister intersliti*l fluid ($BIF). a so;table representative of inlerslitial fluid. We found Ihal Ihe concenlratimts in SBIF of the aqueous small.molecule antloxidants a,~corbate and orate were, reslx:clively, slgnlft- ca;lily higher (P < 0.05) and identical to plasma concentmtk~ns. In contrast, lipopro. tein-a,isociated llplds and lipld-soluble antioxldams (~-tocopherol. ubiquinol-10, lycopene, and 13-carotene) were present at only g-23% nf the concentrations in plasma. No lipid hydmpemxlcfes could be detected (< $ aM) in either fluid. The capacity of serum and SBIF to pro~ect LDL from oxidallon was investigated in three metal ion-dependenl systems: copper, iron. and routine macrophoges in llam's F-It'l medium. In all Ihree systems, adclitinn of ~ fi~, (vnl/wfl) of either serum or SBIF inhibited LDL oxidation by > 90%. The concentration that inhibtred macrophage- mediated LDL oxidation by 50% was as low as 0.3% serum and 0,7% SBIF. The enzymatic or physical removal of av2orbate of orate and other low molecular weight ¢nmi~ments did not affecl Ihe abilily or eilhcr ftuld In prevenl I.DL oxld:lli(m..~nd the high molecular weight fraclion was Its pmlccllvc ;is whole scram ¢~r SBII.'. These data demonstrate that bolh serum and SBIF very cffeclivcly protect LDL from racial ion.dependent oxidation, most probably because nf a enmulaliv¢ mctal-binding effect of several proteins. Our data suggest Ihal I.DI. in the intcrs~itlal fluid of Ih¢ a~rlal wall is very unlikely to gel n~lifted hy racial i,m-mediated oxi¢lali~xt. Dabbagh. A. J. and Feel, B. Journal of Clinical Investigation 96:1958-1966. October 1995. Other supperl: American Heart Association. From the Whltaker Cardiovascular Institute. Boston University School of Medicine, Boston. MA. DIFFERENTIAL EXPRESSION OF VOLTAGE-GATED K' CHANNEL SUBUNITS IN ADULT RAT HEART -- aELA~ON TO FUNCTIONAl. K° CIIANNEI.S? Polyclonai antibodies against each of the K" channel subunits (Kv 1.2. Kvl.4. Kvl.5. Kv2.1. tad Kv~.2) shown previously to be expressed in adult rat he.an at the mRNA level were used to examine the distributions of these K' channel subtmlts in adult mt atrial and ventricular membranes, lmmunohistochemistry on isolated adult rat ventricular myocytes revealed strong labeling with Ihe anti.Kv4.2 and anti-Kv 1.2 antibodies. Although somewhat wealcer (than with anli-Kvl.2 or anli-Kv4.2), posl- live staining was also observed with the anti.Kvl.5 and anti-Kv2.1 antibodies. Ventrku~tr myocy~es exposed to the anti.Kvl A antibody, in contrast, did hal appear s|gnificantly different from backgroond. Qualitatively similar results were oblained c~ isolaled adult rat atrial myocyles. Western bint, t of atrial and ventricular mem- brane proteins confirmed Ihe presence of Kvl.2 Kvl.5, Kv2.1, and Kv4.2 and revealed differences in the relative abundances of these subunlts in Ihe two morn- bmne IXtparaliens. Kv4.2, for example, is more abundant in venlrieular than in atrial membranes, whereas Kvl.2 and Kv2.1 are higher in atrial membranes. Kvl.5 levels are comparable in the Iwo preparations, In contrast to tbese results, nothing was 46 deleeted in Western blots of atrial or ventricular membrane prolelns with Ih~ Kv1.4 antibody at concentrations that revealed intense labeling ofa 97-kD protein in adult mt brain membranes. A very faint b:~nd was detected at 97-kD in the atrial ventrieular preparations when the anti-Kvl.4 antibody was used at a 5- to I~-fe]d higher concentration. The simplest inleq~retatlon of these results is thai Kvl.I an abundant pr~ein in ndull rat atrial or ventrlcular myocyles. Therefore, il unlikely that Kv 1.4 plays an important ro~e in the formation of functional lion.acllvared K' channels in these cells. The rehttion(s) between the (other four) K" channel ~lxtnils ~nd the delmlarla~'~tlon.aclivnled K" channels identlfled electrophy~. inlogically in ~dull ral nlri;d nml ventricnl.'~r myecyles is discussed in the sludy. Barry, D. M,, Trimmer, J. S., Muffle, J. P., and Nerbonne, J. M. Circulation Rc.~:,rch 77(2):3fil-369. Anl~usl Other support: Monsonlo-Searle/Wnshinglon University Biomedical Rc~earch Program, nnd the N~linnal inslitnles of Health. From Ihe Depnrtmenl of Molcenlur fllology :tad Phnnnncolo!~yo W~;hln~tnn Unlversily Scl,ml of Medicine. SL Louis, Me. and tbe Dei~rlmen! of BIt~:h~'mlstry and Cell Biology, Slale Unive~ity of New York ~t $1onybrouk. III. Cell Biology EXPRESSION OF AN EPIDERMAL KERATIN PROTEIN IN LIVER OF TRANSGENIC MICE CAUSES STRUCTURAL AND FUNCTIONAL ABNORMALITIES To examine the role of keratin Intermediate filament protelns in cell structure and function, Imnsgenic mice were isolated that express a modified form of Ih~ human I{14 keratin prolein in liver hepatocyles. A medified KI4 eDNA sequence was linked downstream of Ihe mou~ Iransthyretln (~i~} gene p~m~ter and enhancer elements to achieve targeted exl~-~inn in hepatncyle~. expre~ng high levels of the transgene were found to have abnom~al keratin fil~ment networks as dcqected by indirect immunofluo~esecnce using an antibody sp~:|ftc for Ihe Iransgene prnduet. Lighl lind electron mi~'o~-'opk: level histological analysis of isolated liver tissue showed in many eases degenerative changes thal included inflammatory infiltration, ballooning degeneralion, an increase in fat containing va~. ogles, and glycogen accumulation. These changes were most evident in older mice over four months of age. No indication of typical Mallory body structures were idea. lifted at either the light ~ electron mlcroscople level. To evaluate ~'~ecretc~a, fan~tion in transgcnlc llve~, bile acid seerelion r~les were me,~sured in isolated perfumed liver 47
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C) 0 and t'ouod to be approximately Iwofold lower than aged-matched controls. These findings indicate that expression of an abnormal keratin in liver epithelial ceils in Ih~ in r/ro setting can alter Ihe structure and function nfa tissue and suggest a role of the keratin network in cellular .~ecretion. Albers, K. M., D'Jvis. F. E., Perrone. T. H., Lec. E. Y.. l.lu. Y.. and Yore. M. The Journal of" Cell Biology 12g( ! &2): 157-169, January ! 995. Other support: Nstlonal In,~titute~ of lfeahh. From the Departments of Pathology and Pharmacology, University of Kcnlucky College of ~.'~ed[clne, L~xington, K Y. CLONING AND EXPRESSION OF GENES CODING FOR PROTEIN KINASE CK2 o AND p SUBUNITS IN ZEBRAFISil (DANIO RERIO) eDNA clonc~ coding for Ihe a and/~ subunil,; of protein kimme 2 {CK2) in zebrafish (Duni~ rerio) have ~n i~olaled. Sequencing of the eDNA clone~ has demonstrated that one contains Ihe complele coding ~qucncc for the p sul~Jn|l of CK2 while the ~. clone is Iruncated and lacks 183 nucieotldes of the 5' coding region. Comparison of the deduced amino acid sequences shows an extremely high dej~ree of evolmiona~ sequence conserv-lion of these ~wo proteins. Northern analy- sis of the mRNAs coding for the a subunit indicates thai this me.~nger is present in I h embryos as a 3.6 Kb and a 1.9 Kb species, both of which decrease in 24-h ¢rnlx)-os. In the case of ,go the major mRNA species of approximately h7 Kb main- lains its level during the period of embryogenesis studied. In sam hybridization of early embcyos, using antisense RNAs against ~ and/~ mRNAs demonstrates tempo- nd and tissue specific expression patterns. The ar mRNA decree,s al'ter Mm;tUlao ~ben it is evenly diseribo~ed. The/3 mRNA is maintained at high levels between 4 and 24 b of development0 showing in Ig h embryos a higher concentration in deve~oplng neural tube and in the embryonic optic and otlc vesicle. Danio(li. J. L.. Altonde. M. L, Welnberg. E. S., and Allende, J. F. Cellular and MoIeculm" Biology Research 40(5/6):431-439. 1~94. Other supporl: lnlemational Centre of Genetic Engineering and Biotechnology (|CGEB). FONDECYT-Chile0 and the David M;~hemcy In~lilptc of Ihc Universily of Pennsylvania. From the Depar~amenlo de Bioqulmica0 Facultad de Medicine. Univcrsidad de Chile. C~silla, Santiago. Chile, and Department of Biology. University of Pennsylvania. Philadelphia. PA. 48 PROTEIN KINASE CK2: AN ENZYME WITH MULTIPLE SUBSTRATF.Y, AND A PUZZLING REGULATION Protein kina~ CK2 (also known 0s casein kinase !1) is ~z ubiquitous euka~j~tic ser/lhr protein kinn~ Ira:sent in the nuclc,s end cytoplasm. CK2 i.~ kn~,.'.'n to phi';. pherylate more than I00 sub.~traies, many of which n~c involved in Ihc contro! ol'cell division and in signal Iransduclion. ~ review cefllers on the structure and fun:tinn ofCK2 a and B subunits and on the regulation of its ~cliviIy. a topic Ih'~t r~main~ tp be clue|dated. An ~nalogy is drawn betwc'~n CK2 ~nd lhe cyclin-de~nd¢nt kindles (edks); lx~lh types or proleln kinases share many sul~tretes nnd are aclivz~ted by reg- ulatory subunils.~Allende. J. E., Allende. C. C. Protein kin~se CK2: on en;,ym~ wilh mulllple subsidies and a puzzlin~ regulalJOn. Allende, J. I~. and Allende. C. C. FASEB .loumal 9:313-323. 1995. Olher support: ICGEB-Trieste and FONDECYT-ChIIe. From Ibe Deparlamenlo de Bioqulmlca. Facultad de Medlcina and Dep:zrlam~n~o d~ Biologia. F~cullnd de Cienclas. Unlversldad de Chile. Sent|aLto. Chl]e. DNA INHIBITS THE CATALYTIC ACTIVITY OF THE e SUBUNIT OF PROTEIN KINASE CK2 The recombinant a subunit of protein kinase CK2 (casein kina~e 2) from Xennpu$ Ioe~'i$ inhihiled by the addition of single stmn~d or double stranded This inhibition is compel|live with the casein substrata, h~v;ng an app3z¢~! Ki o~" nM for an 86 bp DNA fragment. Assays with a fragme.t containing the putative pro- mater of the human CK2/~ gent indicated thal the affinity of CK2 for this fr~,m~nI w~ not greater Ihan that of other unrelated DNA. The inhibitory capacity of toward Ih¢ I~olein phosphorylating activity of CK2~x is greatly reduced by th~ pres- ence of the .8 subunit which can completely reverse II~ inhibition. The inze~cli¢,n of CK2~ wilh DNA can ul~ be as.,~yed by the nltroeeliulo~e filter binding assay. assay demonstrates that Ihe interaction of CK2a with the lasted DHAs is not sequence specific and that the ~ subunit can also greatly diminish the binding of CK2e to DNA, Casein al substrale concentrations also is inhibitory to CK2¢~ binding. Likewise, polynnionic inhibilors of Ihe CK2 catalytic activity, such es bep~fin, poly(U)o and copoly(Glu:Tyr) polypeplides, can compete for and inhibit binding of DNA to CK2a. However, quartet|n, which also inhibits CK2 pho~ph~ry- latlon activity, and ATP do hal uffecl DNA binding. A muranl CK2~ in ~hlch t~mlc ueids rcplucc Iwo lysinc resld~s in positions 75'; ~nd 76 of Ih~ ¢¢ pepti~ chain is less su~epfihle to DNA inhibition, and|curing that this basic re,alan of the cult is involved in its interacl;on with DNA. GaliCa, M,. Jacob. G.. Allende, C. C., and Alleode, .I. ~ Biochemistry .'t4(I):122-127, 1995. 49
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0 0 Other 5upporl: International Centre f.or Genetic En~incerin~ and Bi~echnolo~y cod FONDECYT.Chtle. From I~ ~pa~meoto de Bi~uimica, Facullad d~ Medicina, and ~pa~am~lo de Biologia. Facuhad de Ciencia% Unive~ided de ~ile, Castile, ~nllago. ~ile. IDENTIFICATION OF A STARI.E RNA "ENCODED BY TIlE II-STRAND OF TIlE MOUSE MITOCtlONDRIAL D-LOOP REGION AND A CONSERVED SEOUENCE MOTIF IMMEDIATELY UPSTREAM OF i"13 POLYADENYLATION SITE By using a combination of Northern blot hybridizalion with strand-specific DNA probes. SI nuclease protection, and sequencing of oligo-dT-primed eDNA clones, we have identified s 0.8 kb polyfA).conlaining RNA encoded by the ll- strand of the mouse mitochondrial D-loup region. The 5' end of" the RNA maps to nu.c~e~, ide. 1.54.17..a region complementary to Ibe .~tar~ or IRNA"- gone and the 3' Polyanenylateo eno maps to nucleotfde 16295 of"lhe rename, immedintcl u lream oFIRNATM gent. The H-strand P-loup region encoded Iranscrlpls of" s~milYar sP~e am al~ detccled in od~er verlebrale systems. In Ihe mouse, ral. and human systems, the 3' ends of the D.loup encoded RNA are preceded by conserved sequences AAU.AAA..AAU.U.A .A. or AACUAA. thai resemble the polyadenylation signal. TIc steaoy-sl~te level el the RNA is generally low in dividing or in vitro cullured cells. and markedly higher in differenliated lixsues like liver, kidney, heart, aml brain. Furthermore. an over 10-reid incrca~ in the level of Ibis RNA is o~.erved during the induced differenliation of C2CI2 mouse myoblast cells into myotuhes. These results su~gesl thai the D.loop II.strand encoded RNA may have ycl unknown blo- Ioglcal ronclions. A 20 base pair DNA sequence from Ihe 3' terminal rej~ion conlain- in~ the conserved sequence motif binds to a protein from lhe mituchoudrial exlracts in a sequence-speclfic manner. The binding .,¢<clflchy of ~his pro~eln is distinctly dilTeren! from the previously charactcrixed H-strand DNA terminalion sequence in Ihe D.|oop or the H-strand transcripllou terminator immediately downslroam or tbe 16S rRNA gent. Thus. we have charecterized a novel poly(A)-contalning RNA encoded by the tl-strand of the mltnchondrial D-loop rcglon and .',lso idenlified the pulatlve ultimate termination site rot Ihe H-strand Iranscription. Vi]ayasarathy. C.. Zheng. Y.-M.. Mulllck, L Basu, A., and Avadhant. N. G. Gent Exp~.~siou 4:125-141. 1995. Other suppo¢l: National Institutes oF ltealth. F~om the Lalx~ratorics of. Biochemistry, Deparlmonl of. Animal Biology. School Veterinary Medicine. University of Penn~ylvanla. Philadelphia. 5O CYTOSKELETAL DOMAINS IN THE ACTIVATED PLATELET Platelel~ circulate in the blood as discoid cells which, when activated, chan~e shape by polymerizing ~tin into various structures, such ns filoi~dia and fibers. In order to understand this process, it is necess.'~ry to determine how m~ny other protein.~ arc involved. As n first step in defining the full complement of" actin- bindin~ proleins in plalclets, fil~mentmes (FI.eetln .affinity chrom,ato~raphy used. This nppronch identified >..~0 different proteins from ADP..~¢tlvated hunL~n blood platelets which represented 4% of soluble protein. AIIhough a number o1" prolcins nra previously identified platelet ,'~ctin-binding prolelns, many other.~ -'~ppc:,red Io he mweh Fot~rtcen dlfrcren! polychm~l anlil~sdie.~ wcr~ r~i~cd these apparently novel proteins -',nd used to sort them into nine categories h~scd on their molecular welghls .and on their location in the sarcomere or striated mu~;cle, in fihrohlasts and in spreading phtelels. Ninely-thrce percenl of these protein¢ (I~ of 14 proteins lesled} wcrc fonlxl Io be nssncinlcd wilh nctln.rlch slru~'ltliv.¢/~l Font dlslincl nctin filamcn! slrncltzres were round Io femn durin~ Ihc in[li~I 15 mln of activation on glass: filopodia, bmellipodia. ~t conlraclile rin~, cn¢irclln~ degr~nul:lting gramlles, nnd Ihlek bundles of I'ilamcnls rcsem~lin~ stres~ .Aclin.hlndlng prolelns not localize! in lh~ di,,,cold cell bc.'camc highly concentra~£d in oue or ~nolhcr of these .'tclin.bzlsed slniclt~l~S during spreading, suc:h Ih:~! slrUclure conlalns a different complement of proteins. These resulls pre~nl crucial information about the complexity or Ihe platele/eytosl{elelon, demonslr~ling four differenl aclin.be.sed slrnclures form during Ihe firsl 15 mln or surface lion. and Ihet Ihero remain many us yet unchameterlzed protein awaiting furlh~,r invesligalion Ihat are differentially involved in Ihls process. Bes~, F. L, Cell Motility and the Cylo~keleton 30:50-(~, 1995. Other SUPlX)rl: Nalional lnslitUleS of Heallb, From the Division of Biology and Medicine, Brown Univ¢~ily. Provldcnec. Rh CAENORiiABDrri$ ELEGAN$ CYCLIN A- AND B-TYPE GENES: A CYCL1N A MULTIGENE FAMILY. AN ANCESTRAL CYCLIN B3 AND DIFFERENTIAL GERMLINE EXPRESSION We have cloned cDNAs for Cnenorlmhdids elel~ans c.vcllns At, B and 03, While cycllns A! and B are mo~t closely related to either A- or B.type cycllns of other species, cyclin B3 is less related to these cyclins. However, this cyclln is mo~l similar to the recently identified chicken cyctin B3, Our identification or a Ceenorhohditls homolog demonstrates that O¢lin B3 has been con.~ervod in evol~. tlon. c~lin AI is a member ot an A-typa muhigene family: however the cyclin cDNA only recognizes • single band on nol~hem blot~. A single-sized RNA is observed for the cyc#n B3 eDNA. In contrasl, Ihree different Inmscripls me observed ror the O~t'/in B eDNA. Ba.~-d on our analy.r.es using RNAs from gennllne-defCCllve mutnnls and from populations eorlched for males, one r,vt'fi, B lran.~cg;pt i.~ specil'¢ 51
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to the paternal gennline. "The two olher c.~lin B Iranscripls, as well as the c.vrlia AI ~d c)~tin B~ Iran~cfi~s. 0m mosl obund~! in lbe malemal p~li~ 0nd are ~nl al lOW levels in olher fiss~es. Mn~ovcr. !~ 3' unl~n~lat~ ~glon~ o~ e~h C~nnrho~lls c~]in eDNA ~s~ess ~v~l copies oF ~enfinl I~n~lali~ol control elements shown in Xent~e.~ and Drnxnphita maternal cyclin mRNAx to foncli~ durin[ ~[e~sls and ~arly emb~ogenesis. Kreutzer, M, A,, Richzrds, ~. P., Oc Silva-Udawarto, M, N., Temenak. J. K~btich. ~, A., ~hner, ~. ~., aM Rennell, K, J~sl of Cell Science 10g:241.%2424, Other sop~n: Basil O'Connor Sta~er Award from the March of Dimes, and National Sci~c~ Foundati~. ~rom the ~paflm~nt of Molecular M~hiology and Immunology, Unlvcrsit7 of Missouri-Columbla, Columbia, MO, ~riedHch-Micscher-La~nitor~um dcr Max- Pla~k-Oe~il~harf, Tubingcn, ~dcral Re.bile or Ge~any. LIPOPOLYSACCHARIDE SIGNALS ACTIVATION OFTUMOR NECROSIS FACTOR BTOSYNTHESIS THROUGH THE RAS/RAF-I/MEK/MAPK PATIIWAY B~,ck~round: Lipopoly~accharide (LPS) is known to activale macrophages, causing the release of toxic cytokines that may provoke inflammation and shock. One at" the most important led best studied of these cytokines is tumor necrosis fac- tor (TNF), Details of the signaling pathway leading to TNF biosynthesis remain unclear, 11ze pathway is branched in Ihe sense that TNF gene tran~ription and TNF mRNA translaliOn are bnth strongly slimulaled by LPS. Recent evidence has indi- cated that MAP kinase homologs bocome phosphotylated in LPS-sdmulated cells, su~,gestlng their po.~siMe involvement in signal tron~uction. Wc sought to test this hypothesis. Materials end Methods: Measurements of LPS-induced MEK and ERK2 activity were undertaken in LPS.~nsitive and LPS-inscn.~itivc cells. Transreedon studies, in which domtnsn! inhibitom of ros end roJ'.! were u~d to b~ock signaling to the level of MAP kina~, were carried out in order to judge whether the THF gcnc tran~rlp~ion and TNF mRNA translation sre mndulaled through this rmlhway. Results: In RAW 264.7 mouse m-,crophages, both ERK2 and MEKI nclivity are induced by LPS trealment. ~n the ~me cell line, dominant negative inhibitms of ros end roJ'.l block LPS.induced activation of the TNF promoter, os well as deft. presslon of the translational blockade normally imp~-'d by the TNF 3'-untranslated region. A constitutively acllve form or ral'.l (roJ'.gXl:~) was found to augment, but hal replace, lhe LPS signal. In LPS.inscnsifive cells (RAW 264.'/× Nlli 3T3 fusion hybrid cells and primary macrophages derived from CaH/Ilc~ mice), ERR2 ~ctlvity was found to I~ r~fraczory to induction by LPS. Conclusions: The rm/roJ'.IlMEKIMAPK pathway is chiefly responsible for trznsduction or Ihe LPS silent] to the level of the TNF gent and mRNA. roJ'and rof-! ~2 lie upstream trows (or actually represent) the physical l~'aochpoints of the tmn,,:rip. tlonal and translation activation signals generated by l`P~. 'The lesion~ that prevent LPS signaling in macfophages from C3H/HeJ mice. or in RAW 264.7 × Hill fusion hybrid cells, occupy a proximal position in the si~natinE~ pathway. Geppert, To D., ~hltehar~, C. E., Thompson, P., and Beuller, B. Molceul,r M~icine I ( i ):93-103, November 1994. Other suplmrl: National Institutes or Health and the Texas Department Auxiliary Velerans of" Foreign From the Department of Internal Medicine, University of" Texas Southwestern Medical Center at Dallas, Dallas, "iX, The Harold C, Simmons Arthritis Center. Dallas, TX, and The Itowarcl Hughes Medical institute, Dallas, T,'~. CONSTITUTIVE ACTIVITY OF THE TUMOR NECROSIS FACTOR PROMOTER IS CANCELED BY TIlE 3' UNTRANSLATED REGION IN NONMACROPHAGE CELl. LINES~ A TRANS.DOMINANT FACTOR OVERCOMES THIS SUPPRESSIVE EFFECT The role of the mou~e tumor necmsls factor (TNF) promntcr, 5' region (UTR). and 3' UTR in TNF gene expression has bcen examln~d in thz~'e macrophage cell lines (HeLa. NIH 3T3. and L-929). The TNF promc~ter is m~cropha~e-specific. On the contrary, it consfltmively drives reporter ~ene sion in all three cell lines. Not only the full.length ]~x)mnter but al,co tronca~d sions or the promoter, 10ckin~ NF-~B binding motifs, are active in each tyl~ el" cell. The THF 3' UTR effectively cancels reporter I~ene expression in IlcLa cells ar~ in NIH ~T3 cells but fails to block expression in L-929 cells. I..-929 ceils eentein tar Ih~t ovcrcomcs the inhibilory influence of Ihe TNF 3' UTR. lls .~lofl upon the presence of'.',.cqucnc~ found in the TNF $' UTR. Cell.fu.~lon e~rirn~n~s reveal thal this activalor is trans-dominnnt. These ~tudies hi,hitch! the c~cnt~al played by the TNF 3' u'rR, which silences the TNF germ in cells that mi~.ht wise expre.~,~ THF. They nlso reveal Ihe existence of~ escape mc, ch.'~nirm inappropriate synthesis of TNF might occur. Kruys, V,, Kemmer, K., Shod:hay, A,, Jon/~encel0 V,, and Beutler, B. Proccedlngs of'the N~tion,~! Acndemy or Sciences D~A 99:G?3~TT..lanuary lrY~2. Other support: National lnstilutes of Health. From the Howard Hughes Medical lnslilute, Dallas, ~ and l`udwi~ ]nslilute for Cancer Re,arch, Lausanne Brunch, Epalinges, Swilzerl.~nd. 53
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A REPORTER TRANSGENE INDICATES RENAL-SPECIFIC INDUCTION OF TUMOR NECROSIS FACI'OR (TNF) BY $111GA-LIKE TOXIN--PO.~smI.F. tNVOt.VE~Jn' OF T~F ,N ttE~mU, nlc U~E~C s~'~t~zo~. We have examined the hypothesis that TNF may play a pathogenetlcMly imporo tent role In the hemolytic uremic syndrome. ~pccil'ically. we con,.;idcted th~ possibil- ity that shigatoxin, which eventuates this syndrome, millhl induce TNF hlo~ynthesis, and/or that T~F and shlgatoxin might sensitize anlmals, each to the toxic effects of lhe o~her agent. Shigalonln was round to ~nsitiz~ mice to Ihe lclhal cfrec~ of I.!~ and to Ih¢ lethal ef~'¢c~ of' TNF. On Ihe other h-',mlo pretreafn~nt (if" ;mh,al,~ willl either TNF or LPS did not noticeably sc,sitize mice to the Icth-',! effect or ~;~toxln. lntrapeHtoneal injections or shlgawxin did adz induce the production of detectable quantities of T~F in the plasma of" mice. When ~igatnxln w.',,; injected into Irons. gothic mice bearing a ehloramphenlcol acelyllronsf'er-,;e (CAI"~ re~o~cr gene th-',t indicates TNF synthesis. CAT activity was induced within the k~dncy, bu~ not in o(h~' llssu~s. We therefore conclude Ihal shigatoxin acts to induce TNF synthesis wilhin the kidney, and a! the same time increases renal s.cnsilivily to Ihe Ioxlc effects el" TNF, While this mouse model does not reproduce the hemolytic uremic syndrome Is [t occurs in humans, it does suggest thst local synthcsi.s of" TNF within the kidney may contribute to Rnal injupd induced by shigatoxin, Hm'el, Y., Silva, M., Giroir, B., Wcinberg, A., Clear'j, T, B., and Beuller, B. Xoumal of'Clinical lnvestlgztion 92:2 ! 10-21 ! 6. November 1993. Other suppo~: Nation~,! Inslitutes of Health. From the Departments of Pediatrics. Pathology, and Internal Medicine, llow~rd Hughes Medical Institute, University of"Texas Southwestern Medlcz~ (enter, Dallas, IX, and l~partment of' Pedlalrics. Unlvc'rshy ol" Texas Medical School, Houston, TX. THE SYNAP'rIc VESICLE PROTEIN SYNAPTOTAGMIN PROMOTES • FORMATION OF FILOPODIA IN FIBROBLASTS Nr~catO~L filo~ia are acdn.dch c~oplasmic extensions that am involved in motility and reo~niti0~ in growth cones ~nd maturln~ Ixonal endings. A detailed undcrstandin~ of neuronal growth will depend on clarification of the membrane fusion events occuwing during filolx)dial exlension. The synoptic v~sicle protein syn,p¢otagmln r~ems to lx~ intimately involved in e~ocytoti~ membrane fusion. Hem we show that Gbroblisl cell lines tronsf'~cted wilh synspto~agmln fore, long, highly ~anched, cello.rich filopodial IXocesr~s, with the expres.~d syn-',ptmagmin helng incorporated into the plasma membrane. In contrast, ~11 linc~ expressing either or" Iwo other synalx;¢ vesicle protclns, SV2 or synnp~ophysin, generate noly ~udlmentary processes, and. llke neur0~s, sort SV2 and synaptophysin Io small intracellular vesi- cles. As presYnal~iC calcium entry regulates synapllc vesk:le f'usion, nut re.~uh.~ indi- eate thst syn-i~o~agmin might link neuronal aclivlty with synaplie growth. Feany. M. B. add Buckley, K, M, Nature 364:537-~0, Augu.~t $, 1~93. From the Dep~lm~t of Neuroblology, Harvmd Medlcal School, Breton, MA. TIIE SYNAPTIC VESICLE PROTEINS SV2, SYNAPTOTA~MIN AND SYNAFTOP! IYSIN ARE SORTED TO SEPARATE CELLULAR COMPARTMENTS IN CliO FIBROBLASTS ss~ the synoptic vesicle proteins SV2. synaptolagmln, and We cxpre.. " • - 'n information contalncd by phys|n in CHO Rbm~lasts to investigate the largch ~ pro/tin. All thr~ prol©ins entered different cellular ¢omparlmcnl~. Synaptota~mln , h la~n~ membrane. Both SV2 ~nd sy~a~ophysln were sorted to was found on I ¢ P • . .--,---:- -^s,,,,alizcd w" eadv en~o.~omal • " ul s na w - a.. ,.~,,,.,. lib . • . small mtrac¢llular veszctCSo ~_._Y P.._P,~_= .......~...~;a ~ have Ihe ~am~ ma,~c~, while SVZ did not. ~.v~om~,,,..~ ..._=,.-::;: ~.~, ;'~h t=.sr=ted ~v~ mcnlnlion ch,,tr~terbti¢.~ ~ autl'~nts¢ synnpuc v~. =¢,~=, m endoson~! m~rkers. V/e also created ectI lines expres~in~ both 5V2 w&s sorted fro ................ ~-~sin ;rod lin~s e~p~5~|ng ~nd s~".~-°~?-~..-s~ile .,,~ci,s ~, =ll ca~. ,he ~tel., "- ...... ,'~tk~ns ware not found in the .~ame ermine.lie.. ...... syr~piic vcstcle-hlce stPJCtmes i z-,,~c ~,~mt,~ • ....... t .......mt.IR of syn~lic vesicle blue.sis. F~ny, M. B., Y~. A. G., ~lvy, M. L., ~nd Buckley, K, M. ~ J~d of Cell Biolo~ 123(3}:~7~-~g4. Novent~r 1~3, ~her ~p~: Nati~nl In~thute~ of He~hh. A DISCRETE SUBPOPULATION OF HUMAN MONOCYI'ES EXPRESSE~; A NEUTROPHIL-LIKE PRO1NFLAMMATORY (P) PHENOTYPE v emonstra~ed that n discrete and nalurolly occurring sublet1~ul~ti~ of mtm~ 10s c~mst,l.lc Zo-.~tr, p o. ,,,,- ...... ~.~.,, ~,--..-~, {:0~, lev~'l cell-~urfac'¢ exprc~. by I) ~id ~v.lh~rence to extmcellular mat.,- .-,~"~ ..... ~" - " " ' . and -integrins; 2) high capacity to produce re,clive oxygen sion of o~-.. P,, .. ~3~ ...... '-~-s -nd o~ .omit,nose inhiblton and 4) pro- Ic¢~lylic ;tcliVily alga, list a ~OI(~I~IC p~plmC ,u . " "
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0 c.n. : |'|lJefastin. and solid-phase fib~onectln, even in the presence of protelna~ inhibltor~. However, P mon~les expm~s littl~ or ~ cell-surface H~.DR antigen. that they are unable In ~icipale in s~ci~c immune resents. In contrast, the mmaind~ of cimul,ling mo~, have a low proin~amma~o~ ~lenHal bu~ c~- ta~n ~he ~lation o~ ~e; wilh bi~h-]e~l ex~ssion of IILA-DR anliB~. P m~)~es can ~ad~fy ~ ~pzrazed from the ~maindcr of m~ytes on the basis of D their cap~ily Io a~e~ to fi~in: and 21 z~ir o~nl ex~s~i~ ef ltLA-D~ :nti~cn when ~nw cylnmct~ ~ immunomag~tic ~ad~ a~ used. Our ~ata that, ~'ben ~mited to Aite~ o£ ~nflammatlon, P mnm~tc; ~n either ~mnto futinn o£ inflammzti~ or c~t~te to ti~ injury, American/oumal o£ ~i0to~y 257( ] ]) :L775-I.7XA, Other ~uppo~: National Heart, Lung, and Bh)nd Institute and Department Vete~n~ Affai~. r ~tom Ihe Division of Respbato~. Crilic~l C~re. and ~cupatlonal Pulmnna~ Medicine. ~anmenl of Medici,. Unlvemily of Ulah !/enlth ~cien~ Cenler. Lake Veterans Affairs Medical Center. Sail Lake City. UT. and Lung [mmunobi~mical Rese~h ~to~. Unlve~i[y of B~fnghom. Bi~ingham. UK. MONOCYTES RECRUITED TO SITES OF INFLAMMATION EXPRESS A DISTINCTIVE PROINFLAMMATORY (P) PHENOTYPE Only a re|nor proportion of monncytes responds to ch~moallmclanlSo To test the possibility that chemoaltraclant-responsive monocytes have dislinclivo I'unctionnl characteristics, we enriched or depleted monocylc W~paratlons for cells having a ~o;nllammatory (P) phenolype and tested their responses In biologically relevanl chemoattraclznl,;. We prepared monocyte suhlx)l)ulatlons hy one of three indepen. dent tcehnlqoes to minimize the chances ofartlfacts: I) dcptellon of P monneyzes hy adherence to flbronectin: 2) c~richment for P monocytes by negative ~leclio~ for NLA-DR antigen: and 3) flow cytomelric sorting. We measured responsiveness of monocyte sub]x)]xtlalions to N-formyI-Mct.Leu-Phe. CSa, zymo.~an-activated ~mm, and monzx:~e chemoatlraclant prolein.! by three parameters: I) polarization, 2) aclin polymerizallon, ~Uld 3) directed migration. With each chemoallractanl and each pars. meter, there was • striking direct re|ationsbip between the responsiveness of the monoovle preparations ~nd their content of P monocyles. Our data indicat© that lhe capacily of monocytes Io be recruited rapidly from the vasculzture inlo sites of inflammation is • property of • subpopulat|on of monoc]nes with a distinctive, ncu- Irophil.llke prolnflammatory phenolype. Owen, C. A., Campl~]l, M. A,, Boukedcs. S. S.. and Campbell, F'. J. American 3oumal or Physlolo~y 267(I I):LTg~.L796, 1994. 56 Other support: National Heart, Lung, and Blood Institute and Department of Veterans Affairs. From the Division of Respiratory. Critical Care, and Occupational Pulmonary Medicine, Department of Medicine, Unlversily of Utah Health Sciences Center. SaI! Lake City. UT. and Salt Lake City Veterans Affair Medical Center, Salt Lake City. LIT. CELL SURFAC~-BOUND ELASTASE AND CATHEIX31N (3 ON HUMAN NEUTROPIIILS: A NOVEL, NON-OXIDATIVE MECHANISM BY WHICH NEUTROPI IIl~ FO~S AND PRESERVE CATALYTIC ACTIVITY Off SERINE PROTEINASES Ser;ne l~Olclnases of human polymorphonuclear neutrophils play an iml~n~t role in neutmphil-medlated pmteolytic events: however, the non-oxidative m-'z.cha. nisms by which the cells can degrade extmcellular matrix in the pre~ence of pro- teinase inhibhors have not been elucidated. Herein, we provide the fir~t rep~ that human ncutmpbils express persistently active cell surface.bound human leukocyte elasta~ and colhepsin G on their cell surface. Unstlmulated neutmphil~ have mlni- real cell surface expression of these enzymes: however, phorbol ester induce~ a 3{)- fold increase. While exposure of neulrophils to chernoattractams (fMLP and stimulates modest (two- to threefold) inc~ases in cell surface express|on of r.eHne proteinases, priming with concenlrations of lipopolys~eeharlde a~ law as I(X) leads to striking (up tol0-fold) increase in chemoattractant-lndueed cell surface expression, even in the preface or serum proteins. LPS-prlmed and fMLP-stlmulat. ed ncalrophils have ~I00 ng of cell surface human leukocyte elastz_~ actlvity per I0' cells. Cell surface-bound human leukoc~e elastase is calalytlcally active, yet is remarkably ms,slant to mhththon hy nalur'ally occumng pmteznase tnhtbdors. data indicate that binding of serine proteinases to the cell surface focu~s and serves their catalytic activily, even in the presence of protelnase inhibltors. Upregulated espres.qon of persistently nclive cell surface-bound serine on activated nemrophils provldes a novel mechanism to facililaze their egress from the va~ulature, penetration of tissue barriers, and recruitment into sites of inflaming. llon. Dysregulation of the cell surface expression of these enzymes has the to cau~ tissue destrnctlon during Inflammation. Owen, C. A., Campbell. M. A., Sanncs. P. L., Boukedes. S. S., and Campbel]~ E.J. The 3auras! orCell Biology [31(3):7"/5.'/89. November 1995. Other suppo~: U.S. Public Health Servlce and the Department of Veterans Affairs. From lhe Division of Respiratory. Critical Care and Occupational Pulmonary Medicine, Department of Medicine, Un[verslty of Utah Health Sciences Center, Sall Lake City. UT. Salt l.'~ke VAMC. Salt Lake City, UT. •nd Dep~rtment Physlolog|cal Sciences and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC. 57
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i COLLAGENASE EXPRESSION IN TRANSGENIC MOUSR SKIN CAUSES HYPERKERATOSI$ AND ACANTllOSIS AND INCREASE,C; SUSCEPTIBILITY TO TUMORIGENESIS In I ~rles of Iransgenlc mlc¢. the human tlss~e collagenase gent w&,; ¢xprensed in the suprablsal layer of lhe skin epidermis. Via.ally. the mice h~l d~ and ~caly skin which ulxm histological analysis revealed acanlhosis, hyix~eratosis. and epi. del~ll hype~lasia, At the ullrastNelurll level, intercellular gran.lar materials were ab.~'~! in Ihe lransgenle skin epidermis buI canine! wa~ maintained Ihrough Ihe intacl desmosomes, De;pile a diversity of underlying etiologies, similar mo~holog;cal hyperproliterative chan~es in Ihe epldennis --,re el)served i. the human skin di.~'ascs of lamellar ichthyosis, alopic dermatitis, and psoriasis. Subsequent experiments demonslrate that when the Iransgenic mouse .,;kin was lrealed once with an initiator (7ol2.d;melhyl-heoz|o|=nthracene) and Ihen Iwice weekly with a I~moter (120- telradecano),lphorhol-13-aCClatC), there was a marked increase in lamer incidence among tran~enlc mice compared with that among ¢onlml lltlermates. The~ exl~ri- meats demonstrate Ihat by overexpresslng the highly specific proleolylic enzyme collagenase, I ca~adc of evenls leading to profound moc~hulogical changes which aug:meal the sensilivily of Ihe skin Inwards carcinogenesis is inili-',lcd in the epidermis. D'Armienlo. J.. DiColandrea, T., Dalai, S. S., Okada, Y., Huang. M.-T., Conney. A. !!.. and Chitin, K. Molecular and Cellular Biology 15(10):$732-5737. Ocl~her 1(~)5. Other support: U.S. Public Heallh Service. From lee DeFartmenl or Biochem|stW. Universily of Medicine and Denlistry el" New JeP;cy-Roberl Wood Johann Medical Schrml. and Lsboralory for Cancer Research, Coileg¢ of Pharmacy. Rulgers-The Slate Unlversily o1" New Jersey. Piscalaway. NJ. Departmenl of Medicine, Columbia University College of Physicians and Surgeons. New York, NY, nnd Department of Molecular Immunology and Palhology School. Cancer Research Inslilule, Kanazawe Universily. lshikawa, Japan. NERVE GROWTH FACTOR STIMULATES TYRO$1NE PHOSPIIORYLATION AND ACTIVATION OF SRC IlOMOLOGY-CONTAINING PROTEIN- TYROSINE PHOSPHATASE I IN PCI2 CELLS Rat PCI2 cells respond to extracellular peptide growlh factors in at least two distinct ways, When Ireated wilh nerve growth factor {NGF) PCI2 cells exit the cell cycle and differentiate to a neuronal phenotype, whereas when teemed with epider- mal growth factor, they pmflferate. We examined the potential role of Sin homology 2 (Slt2~.¢ontalnlng protein tyroslne phosphatases (PTPs) in the difi'erentlation process. PCI2 cells express substantial amounts of both SH-PTPI and 2. SH-PTPI. but not Stl-PTP2. becomes tyroslne phospho~laled following NGF. but not epider- mal growth factor trealment. The enzymatic activity of Sll-PTPI toward an exoge- nous substrat¢ following: NGF treatment is increa,,cd 2-fold. We found that SII-PTPI 58 hinds to the NGF receptor Trk^ ~n ~Ytro end that unti-TrkA immunoprccipitates h,~ve PTP ~tlvily. ~¢se rcsulls s~w that SII-~I is diRc~ntially ~tivat~ by NGF in ~!2 ceils and sngg~t that this anllwli~ may p]ay NGF-ind~ differentially. Vam~tas. V.. gopTan. D. R.. Sell~ M. A.. and ~emoW~ ~e Jonmat of Bin]oglca] ~mlstW 27~4~):25629-2553~. ~to~ Other sup~d: Nation] Institutes of Health a~ the L~illc P. M~ey Trust. From the Fox Chase Cancer Center. Philadelphia. PA, Eukaryotic Signal T~n~l~t~n Group. Melee.tar Mech~sms of Ca~ino~nesis La~. Basic R~arch ~gmm. and Nmi~=l Cancer Tnstitut=-FR~dck Ca~er end ~vel~ent Center, F~e~ck. MD. THE UBIQUITIN-PROTEASOME PROTEOLYT1C PATHWAY Mammalian cells contain two distinct protcol~ic pathways that arc involved in different a.,~pects of pmteln breakdown. Proteins that enter the cell from the tsars- cellular milieu (such as receptor.medioted endocytosed proteins) are degraded in lyso~mes. Ly.~osomal degradation of intracellular proteins occurs mostly under stressed conditions. Nonlysosomal mechani~ns ~re responsible for the highly ~lcc. tire turnover or intracellular proteins thai occura under basal metabolic conditi~s. bat also for some aspects of degradation of intmceilulnr proteins under stre~. important nonlysosomal protenlyllc pathway is the ubiquilin system in which pro- teins are degraded by u 26S wotea~ complex following conjugal;on by multiple molecules of uhlquitin. The "catalytic co~" o1" the complex is a 20~; prolca~ cc~m- Plex also known ~ the Woteasome. Ciechanover, A. C¢11 '79:.!3.21. Octobur 7. 1994. Other support: United Slates-Israel Binatlenal Seience Fou,dation. German.l~r~ll Foundation for Sc;entiflc Research and Development, Israeli .Academy of Humanities and Sciences. Israel Cancer Society. Foundation for Promotion of Research at Technlon. a~d a grant admlni~e~d by the vice pros;dent of research at Technion. From the Department el" Biochemistry, Rappaport Institute for Research in the Medical ~iences. Technlon-lsrae! Institute of Technology. Hairs.
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0 ~0 0 PROTEIN SYNTHESIS ELONGATION FACTOR EF.le= IS ESSENTIAL FOR UBIQUITIN-DEPENDENT DEGRADATION OF CERTAIN N'-ACErY'LATED PROTEINS AND MAY BE SUBSTITUTED FOR BY 11~E BACTERIAL ELONGATION FACTOR EF,Tu Tirgnling or different cellular proteins for conjugation and sub~eqacnl degrade- lion via Ihe ubtquilin pathway involves diverse rcc,gnili,m signal.,; and di.~linct easy. malta faclOCS. A few proteins arc recognized via their N-terminal amino acid residue and conjugated by • uhlqultin-pmtein ligase that recogni?es this residue. Most sub. strain, includ~nl] the N'.acetylated prnt¢in,~ Ihal con,,lilUre Ihe WL~I mn.~Eily o(cellu- lar protein's, am targelcd by different signals and arc rccogni~,ed by ycl unknown lag. a~es. We have previously sho~ that degradation or N-terminally blocked promins requires • specific factor, designated FH, and that the factor acre along with the 26S i~'otea~e complex to degrade ubiqUilin-conjugatcd pmlelns, llere, we demonstrate that FH is the protein synthesis elongation factor EF-Io~. tu) P;zrlial sequence sis reveals 100% identity to EF.le. t'h) Like "------------------EF-Io~, FIi hinds to immobilized GTP (or GDP) and can be purified in one step using the corresponding nucleolldc for elu- tion. to) Guanine nuclcotidcs thai bind to EF-I¢~ prelect Ih¢ uhlquilln .,¢ystem-rclated activily of FH from heat inaclivatlce, and nuclcotidcs thin do not hind do m~t exert this effect. (d) EF-Tu, Ih~ homologous b~cterial elongation fuclor, can suhstilule for FH/EP.la in the proteolytic system. This last findung is of partlcul~r interns1 since the ubiquitln system has not been idealized in pmkaryotes. The activities of both EF-Io¢ and EF-Tu are strongly and specifically inhibited hy uhiquilin:zhlehydc, a specific inhibitor or ubiqultin ixopeplidascs. It appear% IhCrcfor¢. Ihal El:- I(z ulay involved in releasing ubiquldn from multiuhlqultin chalm¢, thus rendering the conju- gates sur, cclxibIe to the action of the 26S pro~caxc complex. Goncn, H., Smilh, C. E., Sicga~ N. R.. Kahana, C., Merrick, W. C., Chakmhunly, K., Schwartx, A. L., and ~k'channver~ A. Procecdingt oflhe Nariooal Acetic'my of Sciences USA 91:7Mg-7652, August 1994. O~her suplx~rt: United States-Israel Binalional Science Fo~ndatinn, ecrman.kraeli Foundation for Scientific Research and Development. Israeli Ac~cmy of Scicncex and Humanities, Monsanto, Rappaporl Foundation, Foundation for Promolion of Re,arch in the Technion, and the Albert Good.~rein Research Fund adminlstcred by the vice presidcm of the Technlo~ for Re,arch. From the Department of Biochemistry, Rappeport Instltmc for Research in the M~ical Sciences, T¢chnion-lsr=¢l Institute of Technology, Hail'a, l.,a'ael, Monsanto, Inc,, SI, Louis, MO, Department of Molecular Virology sod Genetics, The Wcizmann Institute for Scimce, Rehovot, |sra¢l, Departnlent or Biochemistry, Ca.~e Western Re.~rve Uolverslty School of Medicine, Cleveland, OH. Department of Btochemklry, Mcdica! College of Wisconsin, Milwaukee, WI. Edward Malllncrodl i~zrlmenls of Pediatrics and Pherm=cologyo Divison of Hcmatology-Oncotogyo Children's Hospital and W~hlngto~ University School of M~i¢ine, St. Louis, MO. THE UBIOUITIN-MEDIATED PROTEOLYTIC PATIlWAY: MECItANISMS OF ACTION AND CELLULAR PHYSIOLOGY Uhlqultin modification of m.',ny protein lar~et,~ within cells plays imlx~rtant roles in • variely of biologlcal proces.~es. Among Ihcs¢ are regulation of g~ne alan, regulation of cell cycle and division, involvemenl in the cellular stress ~pon~. modification of cdl sut'f~c¢ recel~0~, DNA ~palr, iml~ort of ~'o~ein~ into mitoclmodria, up(eke of Wecursor~ of neuroffansmittcrs into synapto~om¢~, sis of pcroxtsome.% as.*,¢mbly of ribos~es, and programmed c¢11 death. Tbe mocha, nisn~ that underlie these complex ira:ceases am poorly understood. The t~¢~t modification occurs in Ibe ubiqpiHn-mcdlated pro~¢olytie pathway, Recital mental evidence indicates that the ublqultln system is involved in Ih¢ degr~lation milotle cyclin.% on¢opro~eins and tum~ suppm.~.~_ rs, in the removal.of ~. orm=l o{henvise damaged protein% and in proecssing or antlgen~ m~lrlctcd to cL't~ I MIIC molecules. DcgrMation of • protein via the ublqultln system involve~ two steps. Initially, muhlplo ubiquilin molecules am covalcnll~ lln1(cd in =n A'TP-d~p~n- dcnl mode In the protein substrata. The tlrge/ed protein is then degraded by • specific, energy-dependent nnd high molecular mass protear¢ complex into hie m~ublquhin ~duc=s am also found in t~ c~l], for example, thee invoivlng nuclcos~al hist--. ~xpim th~ consldemble ~gmss Ihat h~ ~n m~c in dating t~ ~e of ~ti~ and mle~ of the ubiquifin s~, many problems ~ma~n unxolv~. F~ example, lltlI¢ ix E~wn ~ I~ signals Ihat largel proteins for ti~. While a few ~elns am t~gelcd f~ ~g~dat~ following ~ognifion or N-In,anal amino ~id msld~, t~ v~t mzj~ty of ~llular ~oteins am t~gcl£d other signals, ~ idcmity of I~ native cellular substmt~ of the system is an0th¢r im~anh yet un~solved pmbZcm: ~ly o few ~oinx have ~n ~ogni~cd ~¢ sub~l~tes of II~ system in vlvo. ~ ~ of Ihls ~vtcw L~ to di~cu~5 I~ aims involv~ in u~quilin ~ivafion, ~l~l~ of su~t~l~ f~ c~j@ali~. degm~li~ of ubiqnilin~jugaled p~elns in I~ mll-f~ syslem. In addit[~, Israeli Academy of Ilumanilio$ and Sciences. Is~li MinislW of H~hh. ~ Rex~h FuM, Ismvl Ca~er S~iew, Found~li~ fvt ~O~livn of in t~ T~hni~. H~s~ F~d f~ M~ical Rcs¢a~h, a~ M~santo. Inc. Fr~ the ~paflment of Bi~mistry, Rappa~fl Instltm~ fo[ Re~earch M~i~! Sci~¢s,T~hni~-lsmel lnstlt=e of T~hnoIogy, Ha=f=, DROSOPIilI.,4 RIBOSOMAL PROTEIN $3 CONTAINS AN ACTIVITY THAT CLEAVES DNA AT APURINIC[APYRIMIDINIC SITES A ~at ¢DNA.¢m:oding Hbosomal pro~eit~ $3 w~s used to clone the S~ homctl0g in Drosophila mt/anogosfer. ~ Dro.¢opldla gone was in turn u.~ed to con*;lmCl fusions belween S.1 and glutnlhione S.lransfernse Ihz~t were overexpre.'~etl in 61
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¢0 0 0 R.~,'heri('hiu ruli. purified on affinity columns and Sllhseqlzcnllv .sod hit anll~xl ; .... .-,,,~y ~cu,c i~ ~.t was ori~inally letted ~o ~t~i~ lhe s,~cllular l~adon of $3 hy W=mcm (immun~h~ot) =nalysi;. A; ex~cled, the 53 anligen wa~ found =~inted wilh purified prepa~ns of ri~ ~es, bu~ notably the protein was aim nh~rv~l in Ihe nucleu~ where ii w~ls ~ound tO ~ lightly ass~iated whh ~he nuclear malrlx. ~is ~suk. cnmblned wilh the facl ~ha~ S~ c~tain~ a nuclear ~o~i~ali~ signal and ~hat th~ pn)lein sha~s so~ ~ol- o~y Io a )'ea~l nucleate sene, sug~e;led ~hal $3 mi~hl ~ssibly have a role in DNA mem~li~m. Tost~ were initially ~rfe~ed to s~ ~r S~ contain~ DN~ acl/vlty, where il was subsequenlly dclc~incd that Ihe protein 5~cifically cleaved DNA containing an apurinic/apyrimldlnlc si~e via a ~-ellminalion rcaclion. ~e DNase ocdvhy was in~livaeed by and~y to 53, indicating Ihol Ihc opurinic/apyrimldlnlc lya~e activity wm ass~ialed wilh Ihe Drmnphi/a 53 protein. ~;~kcn l~elher, Ihc~ re~uhs ~ug~e~t that $3 is um~g a growing class of mafli~u~.lionul pmfeh)s with mle~ in tran~ription/~ra~lalion and DNA repair. Wilsm III, D. M., Dmlsch, W. A, and Kelley, M. R. ~e lt~mal of Binlnglcal ~mist~ 269(41):25359.25~f~, (~lo~r 14, 1~)4. Olher support: Nalional Inslitutes of lleallh and Ihe Louisiana Agricuilural Exffrlm~m SlaHon. From Ihe Molecular Biology Program, Loyola Universily ~eclicnl School, Mayw~, IL. Department of Bi~mislry, Lmisiana State Univcrsily, Balon Rou~e, LA, a~ ~anment of ~diatrics, S~lion of P~lialrlc End~rinolo~y, Wells Center for Pedi~lric Research a~ Deparlmem of Bi~hemislry and Molecular Biology, Indiana Unive~iW ~1 of M~licine, Indiana~is. IN. TIIE ROLE OF TWO CONSERVED AMINO ACIDS, GLUTAMINE (X) AND ASPARAGINE 137, IN O"-MErl l YLGUANINE-DNA MErl IYLTRANSFERASE STABILITY, ACTIVITY AND SUBS'IRATE SPECIFICITY To assess the possibility that two conserved amino acids (glutamlne 90 and asparaglne 137) in O'.methytguanln~.DNA mcthyltr~nsfemse (MGMT) are involved in prozein-substmte conlaCl and/or discrimination between favored and nod-favored ~ubstrales, families of proteins mutant at these two sites were expressed in alkyl- transferase.deficient bacteria and analyzed for stability, ability to repair O'-melhyl- guanine (MG)-containing DNA, and ability to differentially repair a preferred (MG- containing DNA) versus a non-prefened (tree hose MG) suhstrate. All seven pro- teins mutant at glulamine 90 (excel~ a praline mulant) were stable in bacleria and repaired MG-conlaining DNA (>50% or wild.type levels). A repre~otative gluta. mine ~0 mutant protein was nut, however, significantly diffcrenl from the wild-tjq~e protein in the preferential repair of MG-containing DNA versus MG free base. Of eight proteins mutant at asparaglne 137, o~ly glutamine and ~rlne mutants repaired MG-centaining DNA to any degree (g.5% and O.g% of wild-type regpectively) and only Ihe glutamin~ mutant protein was dc~ectable in hacrerial soni~tes by Western 62 blot annlysls. Alnnlne and leucinc roman! alkyt-transfemses, in:~.'tlve and unrtahlc non-fusion proteins, could, however. ~ stably exposed in bacteria as S-Iran~fe~ fusion proteins, allhough she proteins wer~ still inaclive in repair. ~ese resuhs suggest ~hat while glulamlne ~ has no direct role in M~.DNA m~lh~k~nsre~.medlmed ~lr or f~e b~esi~ed DNA subslmle ~a~gi~ t37 is implant in ~th the slabilily and l~ivily of I~ protein and m~y ~lri~te ~o I~ f~ati~ or functi~ of I~ active silc of the ~lein. Pic~r. R. 0.. Morgan. 5. E.. and Kelley. M. R. (~l~h. SV. Ca~i~g~esis I ~(9): I ggs- I ~2, 1~4. ~her sup~: Nati~nl In~tltutes of Health. From {he Division of IlemafolngylOncolo~y, Dcpn~menl or Mcdicine and Program in Molcculur Biutogy, Slritch Sch~! of Medicine, Loyola Univ~rsily Medi~l Cemer. Mayw~l, IL. a~ ~anment of P~ialdcs, S¢~ion of Endocrinology. Wells Center for Pediatric Research and Bi~hemislry Molecular IHology. Indian:= Universily Schtml of Medicine, Riley llo~phal for ~ildmn, lndian;~fis. CLONING OFTIIE MULTIFUNCTIONAL RAT APURINICIAPYRIMIDINIC ENDONUCLEASE (rAPEN)/REDOX FACTOR FROM AN IMMATURE T CELL LINE The cloning of the ral AP endonuclease gene affords us the opportunity to investigate Ihe regulation of n crucial multlfunclionul enzyme h~vin~ both DNA repair and rndox aclivity following v,qrJous p,~mdign~ in an or~anism ll~.~t ha~ h~,n extensively u.~d in research, parllcu|aHy in Ihe areas of aging ~ld hormonal control of gem= expression. Wilson.T, M.,Camey, L P. and Kelley, M. R. (Deul~h, W. Nucleic ~clds Rest, Itch 22(3):530..~i~ I, 1994. (Nher supporl: National Institutes of Health. From the DeparSment of Pediatrics, Section of Pediatric Endoctlnotogy, Wells Center for Pediatric Research, Riley Hospital, Indiana University Medical School, Indianapolis. IN. and Program in Molecular Biology, Loyola Unlve~ity Medico! $chooi, Maywood, IL. CALCIUM PULSES DURING THE ACTIVATION OF A PROTOSTOME EGG Transient increases in free cyloplasmlc Ca-" pass through the c)Io.~ol of a ~.i~ variety of fenilizlng dcutemstome eggs anti do .~n from the sl~nn entry po~nt to [t,~ 6.1
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0 0 .Ix antipode. The~ Ca*" waves occur through the release of stored Ca~'. and they pro- vide most or all of the activating stimulus for these eggs. Bared on several indirect tines of experimental evidence, it was proposed that ptotoslome eggs arc activated by a prolonged uptake or ca~. from the medium due to sperm-indncnd membrane depol~rlzJtion, and th~l this uptake Ihen slarls an activation wave similar Io Ihose in deuterostomes, except that it moves inward from the whole surface ralher than across the egg from pole to pole. To test lhese hypolheses, we microinjeeted Clteetnptertts pergamenloretes oocytes wilh semJsynlhetie recomblnanl aequorins and measured light emission in response to bolh fenili;,.:,don and artificial activation by excess K'. £¢kberg, W. R., Miller. A. L,. Short, L, G.. and Jaffa. L. F. Biolo~ica! Bulletin 1175:289-290. October 1993. Other support: National Science Foundation and the National Institutes of Itealth. From Ihe Marine Biological Laboratory and Department of Biology, Howard University, Washington. De. CONSTITUTIVE AND INI)UCIIII.E ~B BINDIN('} AEI'IVH'IF~ IN "IliF. CYTOSOL OF v-REL.TRANFORMED LYMPIIOID CELLS Constitutive and inducible ~B binding ~clivilies associa[ed wilh v-Rcl and c-Rel in the c)'to~l of v-Re-transformed cells have been idenlified. These aetivilies were resolved by eleclrophoretic mobility shift ~L~ay and chromatographic techniques inlo a hlgh.molc'cular.weight pro~ein-DNA complex designated complex ! containing v- and c-Rel -nd lower-molecular-welghl complexes II. II! and IV which conlained only v-Rel and which were stimulated by nnclcoddes, h~w pll. and detergent. These experiments suggest that interaclion of v.R¢l and ¢-R¢1 decreases the DNA-blnding activity of each. Hndgson, J. and Enricllo, P.J. Journal of Virology 69(3}:1971-1979, March 1995. Other support: National Institutes of Health. From the Depa~ment of Microbiology, State University of New York. Stem)" Brook. NY. DENDRITIC CELL PROGENITOR IS TRANSFORMED BY A CONDITIONAL v-Rel ESTROGEN RECEPTOR FUSION PROTEIN v.RclER A conditional v-Rel estrogen receptor fusion protein, v-RelER, causes estrogen- dependent but otherwise unaltered v-tel-specific transformation of chicken bone marrow cells, tlere, we demonstrate thal such v.rel-transformed cells e~chibit B lymphoid determinants in line with earlier studies on v-relER-tranffonned cell~. However, following inactivation of v-ReiER o~opro~eln ~etivily by admini~ration of an estrogen antagonist, cells dlfl'emntlnte into antigen-im~sentin[~, dendrhi¢ cell~ a~ judged by several morphological and functional criteria. Additionally, under )~et dlf. fcrcnl culture conditions, v-talER cells differentiate into cells res~nbling i~lymm'- phonnclcar neulrophils. Our studies therefore susgest thal the conditional v-RelER, and probably also the authentic v-R©h transfocm a common progenitor rot neu- trophils and dendritic cells. Bcehmell. G.. Madroga, .I., Daffier, P., Brlegel. K., Schwlu-z, H., Ear|ella, P,J.~ ~cl Zenke, M.. Cell g0:341-352. January 27. 1995. Other support: National Institutes of Heallh. From the Institute of Molecular Pathology, Vienna, Austria, Max.Planck.tn.,~titut fur ~alwlcklungshlologie, Tuhlngen, Federal Republic of German),, and Department of Microbiology. Stale University of New York at Stony Brook, Stony Brook. NY. THE ROLE OF TI IE CARBOXY TERMINUS OF v.REL IN TRANSFORMATION AND AC'TIVATION OF ENDOGENOUS OENE EXPRESSION Proteins within the Rel/NF-~B transcription faetm" family can be dlvid~d into two functional domains, a homologous amino terminal ¢e~ion. the Rel Homology Domain. and • divergent ¢arboxy terminal domain. The amino terminal srquence~ specify DNA binding, nuclear localization, and interaction with Ihe hob family of inhibitonj proteins. The cad)oxy tennlnus of each prmein functions ~s a tran~r[~. tlonal ncllvation domain, however, pmclre definition of sequence requlrem~nts hz~ been difficult. 'To further define'tbese sequences, small 100 bp delet[ons were strucled throughout the carboxy terminus of v.Rel, Each resulting mutant . ~.~aynd for DNA binding. Inc-dlzation, protein complex formation, a~ivalion ~f endogenous gent expression and ability to transform Ixmc m~row cells and fibro- blasts. Surprisingly. deletion within the caeboxy terminus had marginal effects on transforming potential. However, three sepaz~te regions were required for full active. lion of germ expression. Taken together, Ihese results suggest thai the eazt~o,xy tenni. ous of v-Rel contains multiple sequenc~ tl~t participate in the ~,etivalion of gent expression. Smardnva. J., Walker, A,. Morrlson. L. E., Kabrun. N., and E~nrletlo, P. J. Oncogenc 10:2017-2026, 199~. Other suPimrt: National Institutes of Health. From the State University of New York at SIon)' Brook, Department of Microblology, Stony Brook.
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0 0 0 0 INVOLVEMENT OF RAL OTP•se IN V-SIn-INDUCED PiIOSPHOUPASE D ACTIVATION An early response to the tyrosine kinase activity of v-Src is an increaLse in phos- pholipz-~e D (PLD) activity, which leads to the generation of biologically active lipid second masse•Bars, including phosphatidic acid, lysophosphatidic acid and diacyl- glycerol We have recenlly demonstrated that v-Sin-induced PLD activity is mediat- ed by Ra% although Ras involvement was indirect, requiring a cytosolic Factor for PLD activation. Ras interacts with and activates RaI-GDS, the exchange factor ~'esponslhic For tee activation of Ral GTPa.ses. Item we mpon that thL~ newly identi- fied RaVRal signalling palhway mediates PLD activation by v.Src, PLD activity could be prc¢ipitaled From v.$rc-transformed cell lysates with immohillxed RalA protein and with an anti-Ral antibody. A mutation to the region nf RalA analogous to the "effeetor domain' of Ras did not reduce Ihe ability of RalA to complex with PLD, allhough delelinn of • Ral.spocific aminn-lerminal region did. Ovcmxprc.~siem or RalA potentiated PLD •clivation by v-Src, nnd expression of dominant nag•live RalA mutants inhibited bolh v-Sre- and v-Ra.s-induced PLD activity. Thus RalA is involved in the tymsine blna~ aclivatiou of PLD through ils unique N terminus, and that PLD is a downstream target of a Ra.VRal OTPase cascade. Jiang. H.. Luo. L-Q.. Urano. T.. Frankcl. P.. Lu. Z.. Foster, I). A., and Fcig, L. A. Nature 378:409-412. November 23. 1995. Other ~uppon: National Institutes or Health and American Cancer Society. From the Inslitut¢ of Biomolocular Structure and Function. Department of Biological Sciences, Hunter College of City University or New York. New York, NY. and Dep.~rtment of Biochemistry, Tufts University School of Medicine, Boston, MA. SELECTIVE ACTIVATION OF PROTEIN KINASE C ISOFORMS BY v.Src Protein kinase C (PKC) is • gene Family consisting of no less than I I di.~linct is•forms. In both routine and rat fibroblasts, we detected expression of four PKC is•forms: Ihe conventional PKC o~, the novel PKCs 8 and ¢, and the atypical PKC ~;. Wilh the convenlional and novel PKC is•Forms, membrane a.~ocialion has been used to show PKC •city•lion. In cells transformed by v-Sre, them was a Ca"-depen- dent increase in membrane association of the ~ isof'orm relative Io lee no•trans- formed parental ceils. The [ is•form had a slightly increa.~'d membrane *as•clarion in marine fibroblasis tmnsron'ned by v-Src but not in rat fibroblasls transformed by v.Src. However. since it is not clear whether cellular distribution of [ is•form carte. lalcs with activation, the data arc inconclusive with regard to ehis is•Form. rnlcre~tiogly, of the CaZ'-indcpondcnt PKC i~Forms 5 and e, only the 6 tsar'ann was preferentially associated with membrane fractions in v-Sre-transformed celia. The lack of PKC e Ictivation was not due to lack ol" responsiveness to diacylglycerol (DG), .~ioce exogenously supplied DG and phorb~l ester were both able to induce membrane association of PKC e. Thus. the differemial activation or the I~ and • is•- foams by v.Src sugge,;ts a more complex mechanism for the aclivaliou or the novel Ca"-independcot PKC ;sol'nnns, involving m~e lh.~n simply elevating DG levels. Since PKC has been implicated in the intracellular signals activa~cd by v.~rc that lead to transformation, the selective activation of PKC ¢~ and ~ su~.ests • ro~e in mat•genesis and transform•don for these PKC is•forms, Zang, O., Frankel, P., and Fosler, D. A. Cell Gmwlh & Differentiation 6:1.'367-1373, Novemt~" 1995. Other support: National Instilutcs of lie•lib. From the Institnle for Biomolecnlar SIIllclure and Fnnction nnd Dep'~rtment of Biological Sciences, lluoter College of The City University of New York, New York, NY. COPPER, ZINC SUPEROXIDE DISMUTASE IN ESCHERIClllA COLt: PERIPLASMlC LOCALIZATION Cu.Zn.~OD Porified from R.whrrlch[a roll bus been nsed to ra;.~c ant;bodles ;n rabbits. The resullnnl anti.~cmm wa.,~ found to reco~niTe n siogl~, h~nd on '~Vc~k'm blots of SDS-polyacrylamidc gel elcclmphemgrams, and that single band coincidcd with the position of Ihe Cu0ZnSOD. Uitrathin sections of fixed E, coli ~vere Ire•ted wilh Ihe antibody followed by protein A bearing 10-am gold particles, Eleetren microcopy revealed that Cu.ZnSOD was largely lo,~lized in the peripl~m in I~lar bays, Barmy. L., Chang, L. Y.. Day. B.. and Fr|dovlch, I. Archives of Biochemistry and Biophysics 319(2):Y, Og-51 I, June I01995. From the Departmenls of PJinehemistry and Medicine. Duke University Medical Center. Durham, NC. A CATIONIC MANOANIC I~ORPRYR1N INHIBITS UPTAKE OF PARAQUAT BY E$CHERICHIA COU A manganlc porphyrin (MnTMPyP), which catalyzed the dlsmuta|ion of O.~, facilitaled the ,-ternblc growth of • .,rndA .~rr~R strain of E.~cherlclda rot[ •ad protected a superoxide dismutase (SOD)-competent parental strain •gainst para~uat. St,rprisingly. the latter effect was more complete than ~he former and the mimic could block the inductions of fumara~ C and of glucose 6-pht~.~phat¢ dehydm~en~,~ hy pamquat, even though SOD could not. An explanatitm for tl~.,~ ~pix~rent ek~xes was fi~md in the ability of MnTMPyP to inhibit Ihe uplake of p-.ln~t|,at by
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O O coll. MnTMP.vP was accumulated by E. coil until its inlracellular conccnlration was 20-reid 8realer than the exlracellular concentration. This happened in a glucose plus salts medium, but oat from a rich LB medium. MnTMPyP was hound Ohio cellulnr macromolecules and was maintained in the reduced state wilhln E. coll. The free form of Ihe reduced MnTMPyP w-',s autoxidizahle, hut the heund form was nnl. Con.~equenlly, the free form could catalyze the oxidation of ascorbate, while Ihe bound form did not. Liochev. S. I. and F'rldo~ich, !, Archives of BiochemisIw and Biophysics 32 ! ( ! ):271-275, Augusl 1995. From Ihe Department of Biochemistry, Duke University Medical Cenler. Durham. NC. STAFIII.17.A'T~ON OF TETRAIIEI.ECAI. DNA BY Till:. OIIAI)RI II'l F DNA I{INDIN(J PRfYI'I:.IN QUAD " " The 57-kDa hepatic n~lear protcin ~Q~/) h{n,ls l{gh[{y ant{ spec{fi~l{y ~ par- alle{ lelrahellcal form of the IgO switch region [)HA (Wuisman-Shumer, p. ;rod Fry, M. (1993) J. Biol. Chrm. 26g. 3306-3312). I lure wc .~m,w Ihat QUAD is a he;it -slahlc proleln, maintaining --gf~, uf its tetv-,helix binding aclivily -'~fler and becoming fully in•tile•ted only after 30 rain at 100°C. To demonstrale Ihal ~UAD protects bound quadruplex DNA, naked anti QUAD.bound tetrahelices were boiled, the protein residue in lhe complex was dlgaslcd with trypsin and qu;~druplex •nd slngle-strand forms of the DNA componenl were resolved by elcctrophnresls. Whereas naked quadruplex DNA became fully denatured after 2 rain at IIX)°C, 55% of Ihe QUAD.buund DNA was conserved as a te|rahellx after 6 mln at 100~C. The~ findings support the proposal that QUAD may aCl in vh,o to slabilize turf•helical DHA. Welsman.Shomer, P. and Fry, M. Biochemical and Bioph~ical Research Communicalions 205(I):.105-31 I, November 30. 1994. Other support: U.S.-Israe! Binationai Science Fund, Israel Science Found•lion, and the Fund for Promotion of Research at Ihe Technion. From the Unil of Biochemistry, The Bruce Rappaporl Faculty of Medicine, Technlon-lsrael InslilUte of Technology, Hail•, Israel. THE CDC2-RELATED KINASE. PISSLRE, IS ESSENTIAL FOR CELL GROWTH AND ACTS IN 0: PHASE OFTHE CELl. CYCLE Mammalian cell cycle progression is regulated by several protein kin.',~s that 6~ are activated by cyclically expressed proteins called cyclins, Thesm cyclin-de~ncl~nt kinases, the prolotype of which is the edc2 mito.~is-promotlng kina~, are kno',vn to phosphorylale substratum the modified status of which is critical for Ihe cell In progress into sequential phases of the cycle. Recently, a new cdc2-related protein kinase has been discovered. PISSLEE, named with respec~ to its homoto+y to the cdc2 PSTAIRE amino acld domain. Here we report Ihal by usln+ bo~h antisense and domlnanl-negalive mulanl ¢onstrocls of PISSLRE when overexpressed in cells, • ~rowlh suppression is found. Furlhermore, Ihe dam;nan! nc'gatlve I'~'~rn~ t+f PISSLRE hall cell cycle progression ;n G,-M. Therefore, PISSLRE is es~nllml for cellular proliFeralion, and its effecl is exerted in GCM. This describes the fi~l eel. dunce since cdc2 of i cdc2 related kinase aclin~ Ihrou~h Li, S., MacLachlan, T. K., De Luca, A., Culudio, P, P., Condorelli, O., and (;,lord-an, A, Cancer Research 5.';:.1992-.1995, September 15, 1995. O~her supf~m1: Sbarro lnstilule for C:meer Research nnd Molecular Medicine, Nelional instilt, tes of I lcnllh, From the Jefferson C, ncer Instilule and Departmenls of Microhlol~,gy nnd Immunology and Palhology, Thomas Jefferson University, Phil~etph;a, NOVEL NFAT SITES THAT MEDIATE ACTIVATION OF THE INTERLEUKIN-2 PROMOTER IN REPONSE TO T-CELL RECEPTOR ST/MULATION The lranscription factors NFAT and AP-I have been shown to b~ es~ntlal for inducible intedoukin.2 (IL.2) expression in activated T cells. NFAT has ~en p~vi. ously reported to bind to two sites in the IL-2 prom~er in o.ssoclatlon with AP-I the distal antigen response element at -280 and at -135. On the basis of D~e I footprinling with recombinant NFAT and AP-I prolelns, gel shift assays, and ruction experiments, we have identified three additional NFAT slits in the IL-2 pro- mo~er. Strikingly, all five NFAT sites am essential for lhe full induction of aclivity In response to T-cell receptor stimulation. Four of Ihe five NFAT sltc~ are pal or compo.~ite elemenls able In hind AP-I in association wilh NFAT. The~ display • diverse range of cooperalivity and interdependency on NFAT and .AP- I proteins for binding, One of Ihe NFAT siles directly overlaps the CD2g.re.~pon~ive element. We presem evi~nce Ihal CD2~ inducibility is confen~-'d by the AP-! ponen! in NFAT-AP-I composite elements. These findings provide further Insight into the mechanism involved in the regul*tlou of the IL-2 promote. Rooney, ,/. W., Sun, Y,-L, Glimcber, L, H,, and Hocy, T, Molecular and Cellular Biology 15( 11 ):6299.6310, November 1995. Other suppo~: Tul~rlk, Inc. 69
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0 0 C) From the Departments of Genetics and Medicine. llarvard Medical School. and De~m'tment of Cancer Biology. Ilarvard School of. Public Health. Boston. MA. and Tularik, Inc., So~Jth San Francisco. CA, THE DESMOSOMAL PLAQUE: ROLE IN ATTACtIMF.NT OF INTERMEDIATE FILAMENTS TO THE CELL SURFACE Cyto~kelelal-membrane attachments play an important role nol only in regulat- ing cell mocilily, adhesion and maintenance of cell shape, but also in tran~nclng sig- nals generated at Ihe cell surface. The~ atlachmcnl.~ are orion mediated hy intercel- lular junctions Ihat compri~ macromo~ecular complexe~ specialized for interaction with a specific filament tRY. The most prominent cell surface attachmenl silo for intermediate filaments (IF) is the dcsmosorne, an intercellular junction Found in abundance in lissues th,,I experience mechanical sirens. A number of rnnleculcs in the desmosomal plaque have been suggested as candidate IF linkers, including desmoplakins, plectln, ~)0 kD IFAP, desmocalmin, band 6 protein, and a 140 kD lam|nlike protein. Of thee. the most abundant and ubiquitous constituents are the desmoplakins (DPs). Desmnplakin (DP} I and !! are Iwo ma~)r related pndclns heated in the inner portion of the desmosomal plaque. The predicled sequonce of DP I suggests it will form a homodlmer comprising a central ct-helical coiled-coil rod and two globular end domains. The smaller spliced form, DP il, apparently has identical end domains but lacks two thirds of Ihe central rod. The C-lem~inus contains three regions wilh significant homology, each of which is made up or # 3g.residue motif also found in two other molecules involved in organi~tion of IF, bullous pemphi&oid antigen and plectin. When polypeptides including the C-terminus of DP ! are ectopically exwesr,~-,'d in cultured cells the proteins align with keratin and vimontin IF, whereas those lacking the C.terminus do not. The last 68 amino acids of DP are required for alignment along keratin but not vimentin IF, and residues 48-68 from the C-lerminal end a~e critical for this interaction. These results suggest that the C-terminus of DP plays • rote in the attachment of IF to the desmosome and Ihat Ihe presence of I spe- cific site is necessary I'of interaction with keratin IF. A sequence at the most N-ter- mlnal end of DP appears to be required for efficient incorporation into the desmoso- mat plaque. Interestingly, this region bas not been reported in the homologous bul- lous pemphlgold •ntigon or plectin molecules and may represent • desmnsomal tar- goring sequence. Green, g. J. and Stappenheck, T. $. In: Citl, S. fed.): Molecular Mechanisms of Epithelial Cell ]unctions: From Development to D;sea.se. R. G. Landes Company. pp. 157-171, 1994. Other support: National Institutes of IIcalth, American Cancer Society, March of Dimes Birth Defects Foundation, and • Focu~d Giving Award ream .Iohnran and .fohnson Co. From the Departments of Pathology and Dermatology. Robert IL Lurie Cancer Center, Northwestern University Medical School, Chicago, IL. 7O STRUCTURE AND FUNCTION OF DESMOSOMAL TRANSMEMBRANE CORE AND PLAQUE MOLECULES Desmosome~ =re intercellular junctions that function in cell<ell adhesion ~n~l attachment of. intermediate l'iloments (IF) to the cell surface. De,cmo~leins desmocollins are the major components at" the tmnsmembmne adhes;nn whereas desmoplaklns (DPs) are the most prominent components of the plaque. Ra~d on .,~-quence similarity, desmogleins and desmocollln~ are related to the c,,lclnm-depondent homophilie adhesion molectates known as cadherins, Like th~ cla¢,dcal cadberins, the desmosornal c~dheHns cont,'~in rour homologous e~trocellul~r domains bearing putative calcium-binding sites, • single transmembr~ne rp',nnin~. domain, and a C-termlnal cytoplasmic tail. MoTecules in the desmoglein contain a unique C-terminal extension within which is found • repeatin~ motif th'~t predicted to form two ~3-strands and two turns. Stable cell lines e,'cpre,:~in~ desmoglcin I have been generated from normally non-~herent L cell fibrobl~t,:. In study the contribution of this cadherin to desmos~mal adhesion. The • '~lUonce of desmnpTakin (DP) I suggests it ,~'ill fon~ homodimers cemprl.;,in~ a con. trnl e-helical coiled-coil rod and two globular end domains, The C-terminu,~ cont31n~ three regions with slgnifie~nt homology° e.'ch of. which is m',de up of a motif also found in two other molecules involved in organizer|on of" IF, butto~ phigoid antigen ~nd plectin. Ectopiealiy expensed polypeptid~ includin~ th: minus of" DP ! spc'cifienlly afi~n with keratin ~nd vimentin IF in cult.red cell~, whereas Iho~c l~king this domain do not align ~vlth IF. The last 69 amino 0clzl.. DP are required I'or allgnmcnl nhmg kcndln Ix~t nt',¢ v;menrin IF. zmd rc~i~h~c~ from the C-tennin;tl end are critical for this intet~ction. Tlesc results su~e~t that tT~' C-len~imss ot" DP plays n rnle in the ,'ttlaohment of IF to the d¢:smo~me ort~ theft specific site is necessary roe ;nten~cl;nn with ker~tln IF, A .~equer¢c at the n~t term|no1 end of DP ~ppeara to be rcq.ircd for efficient i~orporation into th~ soma| plaque, lntereslinglyo Ibis region has not been reported to he proven! in homo~ogoua bullous pemphi¢oid antigen or plcctin molecules and may reproach! cksmosomal lar~e{ing sequence. Kow-~lezyk, A. P., Stappenbecko T. S., PamJ, D. A. D., P-Ika, H. L., Vi=ta, M. A,, Bornslaeger, E. A., Nilles, L. A., and Green, Biophysical Chemistw 50:97-112, 1994. Other support: National Institutes of Health, American Cancer Sociely, March Dimes Birth Defects Foundation, and a Focused Giving Award from Johnron Johnson Co. From Ihe Depmments of Pathology and Dermatology, R. H. Lurle Cancer Center, Northwestern University Medical School, Chicago, IL, and Mass~y University, Department of" Physics and Biophysics, Palmerston North, New Zeal=rid. PIIOSPIIORYLATION OF TIlE DF-.SMOPLAKIN COOII TERMINUS NEGATIVELY REGULATES ITS INTERACTION WITH KEI~ATIN INTERMEDIATE FILAMENT NETWORKS Desmopl.'~kins (DP~) are the most ~humlant Dmte|ns in the innerm~.~t p.~nion of" 71
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0 0 0 the desmo~mal plaque and have been pro/x',sed to play a role in the attachment of intermediate filaments (IF) to cell-cell cont-'.ct sites. Our previous results suggest Ihat Ihe globular end domains of DP perform dual functions: first, to target DP to 1he desmo~me via the NH~ terminus and second, to altaeh IF to the desmosnmal plaque via the CO01t terminus. When ectopieally expressed in most cultured cells, the COOH terminus pile; the rod domain (DP.AN.SerC23) exhibits striking cnalignment with keratin IF networks. However. in certain cell types (e.g. PIK2) or in cells treated wilh fo~kolin to activate protein kina~ A. DP.AN.Se~,C23 exhibits a diffuse cylo- plasmic distribulion. A variant molecule (DP.6N.OlyC231 in which a .~rine located 23 amino acld~; frnm Ihe COOII Icrminus is altercd to a glycine, Ihcruhy dish,piing a protein kina~ A eon,;en,~us pho.,~phoryluti,m site. co-h~:-',lixes with kenllin !!: ncl- wo~ks regm'dless of" cell type or fi)rskolin treatment. Analysis of the phospho~plide mal~ of the~e DP variants and endogenous DP is ccmsistcnt with Ihe phosphorylation of the .,trine 23 residues from the C(X)i! lerminus. 'l'he.~c re~ull~; sng~esl th-'|t I~or~lati~m of a .,,peclflc residue in the DP f.~'X)l I tcrmie),s may ncgalively reg,lale its interaction with keratin IF networks. Stappenbeek, T. S., Lamb, $. A., Corcoran, C. ~., und (~reen, K. 3. The 3oumal or Biological Chemistry 269(47):2Q3~ 1-293~4. Noveml~er 25, I~.~)4. Other support: American Cancer Society, and the March of Dimes Birth Defects Foundation. From the Departments of Palhology and Dermatology. Robert II. Laurie Cancer Center, Northwestern University Medical School, Chicago, IL. INVESTIGATION OF'rilE INFLUENCE OF CYTOSINE METIIYLATION ON DNA FLEXIBILITY' To test Ihe influence of. pydmidine methyl ~roups on DNA ~cxibilily and helix repeat. Iwo ~ts of 14 mixed ~quence DNA molecules, srmnnlng a range of. Ionglhs from 158 to 180 ba~ pairs, were cyclized wilh T4 DNA li&',~. The two ~ls dilT~r~J o~ly in that the Cyl-5 positions of all cytosines (~0-90 cytosine residues per mole- cult) were fully metbylated in the members of one sel. Determination of. the molar cyclizalion factors, persistence lengths, helix repeals, and torsional elastic constants revealed no sitmificant differences between the two sets. The~ results imply that, at least for mixed sequence DNA, the biological con.~'quences of"cytosine methylation are likely to derive from ©itber I~al structural distortions in the helix, which do propagate as altered Iwisl. or from direct prolein.methyl eytoslne interactions. Hodges-G~z~ia. Y. -',nd H~erman, P, J. The ]onmal of Biolo~ical Chemistry 270(I):197-201, January 6, 1995. Other suplx~l: Locille P. Markey C'harltahle Trust, and the Univcr~hy o1' Coh~,v.lo Cancer Cenler. From the Department of Biochemistry, Biophysics and Genetics. University of Colorado Health Sciences Center, Denver, CO. 72 Upon withdrawal of inlerleulcln-Z (11..-3) from human factor-dependent ery- throleukemic cell line "rF-l. 1~/.2 mRNA and prolein levels decrease within ~ to 24 hours, Accompanying this decrease is the o~set or apolxosls as determircd by flow cylomatric analysis of DNA degradation. By ~ to 18 hours of depr;vation upprox;. mutely '70% to 80~ of the cells have entered npoplosis. Downregulation of. I~Oteln Icina.q: (PK) by a 24.hour incubation in 100 nmol/L 12-O-tetradecanoyl-phorbol-13- acelate (TPA) in Ih¢ prc.q:nce o!" IL-3 dramatically reduced hcl-2 mRNA levels, and imhmcd ~poplosis in the presence of IL-~. We have also found that even in the prc~- ence o£ IL-3. two inhibitors of PKC. light-activated calphostin and H-7. sub~tantlally reduced the levels of ~'!-2 mRNA between 8 nnd 24 hours as me-t~ured by a semi- qunnlilative rever~e tnmscripla~/polymentse el'rain reaction ,'ls~y method: ho~ve~'er, the cyclic nocle(~ide-dcpcadc,I PK inhibitor ilA I(X~4. that is n stn~t,r~l an31o~, t,r 117 htlt u poor inhibitor of PKC. did not reduce J~'l-2 levels in the prcxence of IL-3. This decrease in/wl-2 mRNA was accompanied by a declloe |n/wl-2 pmteln levels by R to 24 hours aflcr ztddition of light.~tivuled calpho.~lin. In addilion to interferlnf. wilh the malnlenance of hrl-2 mRNA levels inhibilion of PKC ~vith !1-7 inhibited the induclion of h¢'1-2 mRNA in factor-~prived TF.I cells re;timuleted with IL-3. The cyclic nucleolide-dependem PK inhibitor HA 1004 did not inhibit IL-3-1nd~.tc~ed hcl.2 mRNA. Studies with actinomycin D showed thal transcription plays a major role in maintaining b~'l.2 levels in TF-I cells, and it is therefore likely Ihat IL-3 plays a role in maintaining/x'l.2 m~nscrlption throu[~h acllvatlon of PKC in these cells. Rinaudo, M. S.. Su. K.. Falk. L. A.. Haldur, S.~ and Murson, R. A. Blood 86( I):80-gg. July 1995. Other support: U.5. Public Health Service and National Cancer Institute. From the .llollund Laboralory for Biomedical Science, Amerleon Red Cross, Rockville, MD. and Jefferson Cancer Institute. Thomas Jefferson Medical College, Philadelph|a, PA. RAS FARNESYLTRANSFERASE INHIBITORS SUPPRESS THE PHENOTYPE RESULTING FROM AN ACTIVATED RAS MUTATION IN CAENORIIABD[TIS ELEGANS Attachment of Ras protein to the membrane, which requires famesylatlon at its C terminus, is essential for its biological activity. A promising pharmacological appm.~h of antagonizing oncogenie Ras activity is to develop inhibitors of fornesyl- transferase. We ,se CanmrlmlNlltis ele.eanx vulval differentiation, which is con. trolled by u Ros-mediated signal transducllon p.'zthway, as a model system to test pre. viously identified famesyltransferase inhibitm's. We show here that two famesyl- transfcr~ Inhihilors. manumycin and gllotoxln, suppcss the Mul¢ivulva plzenotype re.suiting from zm ~'tlvnled h't.60 ra.v m,l~tion, hul not the Multh.ulva ph~notype
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0 0 I',,3 C,rl 0 resulling from mutation; in Ihe lin.! gone or the lin.15 gone, which act dowmtream and.upstream of let.60 rex, respectively, in the signaling palhway. These results arc consistent with the idea tlut the suppression of the Multivulva phenotype of ms by the two inhibitors is specific for gas prolein and Ihat the mutant Ras pmiein might be more sensitive than wild-type gas to the f'amesyllransferase inhibitors. The work suggests that C. elegons vulval dcvelopmen! could be rlvo system for evaluation of famesyhransferase inhlbitms agninsl Ras-actlvaled tumors. Haxa, M. and H~,n, M. Rocecdings or the National Academy of Sciences USA 92:.t3.t3.3337, April 1995. Giber ~upport: Naliflnal Institutes of Ilealth and the March nr Dimes Foumlalion. From the I~partment of Molecular, Cellular and E~velopmcnlal Biology, Universily of Colorado, Bo~lder, CO, and Tokyo Research Lalmratori~, Kynwa I lakko Kogyo Co. Ltd., Tokyo, Japan. TIlE ROLE OF THE ARYL I IYDROCARBON RECEPTOR NUCLEAR TRANSLOCATOR PROTEIN IN ARYL HYDROCARBON RECEPTOR ACTION The aryl hydrocarbon (AH, or dioxtn) receplor mediates carcinogenesis by a wide v~icly or compounds, it aCls as • iigand-dependent tran~ripffon faclor. Many investigators expected Ihal the All receptor would prove to be a member of the steroldlthyroldlrefinolc acid receptor superfamily of proteins. However, recent cloning of the two subonils of the DNA-blnding rr4~n or Ihe All receplor has shown that this is no~ the case• The~ subunits, the All receplor nuclear translocator prmein (ARNT) and the lig~nd.binding All recepior monomer, do not contain line finger DNA-binding domains, nor do tbey have any olher ~quence similarities with mere. hers ef Ibe above family. Instead, tbey bolh contain basle helix-lcop-belix (bHLll) motifs and also another segment of .'~-'quenc'e simil-',rity, the "PA$" region, btlLI! motifs in o~her transcription factors are known to function as dimeri?.~llon and DNA- binding domains. Present experiments are directed tow~J under~tandlng the mocha- nkms of acliofl and the ro~es of the two subonlt~ H•nkinson~ O. Trends in Endocrinology and Metabolism ~6):240-244, 1994. O~her suppor~: N~donal Cancer Institute, Department of Energy, and Tohacco. Related Disea~ Research Program of the University of California. From the Deparlment of Pathology and Laboratory Medicine, and Labor•for7 of SmJclural BioloBy and Molecular Medicine, Univcrsily of Califomla, Lo~ Angeles. CA. 74 RECOMBINANT BONE MORPHOGENETIC PROTEIN 2 ACCELERATE~ BONE CELL DIFFERENTIATION AND STIMULATES BMP-2 mRNA EXPRESSION AND 9MP-2 PROMOTER ACTIVITY IN PRIMARY FETAL RAT CALVARIAL OSTEOBLAST CULTURES Recombinant bone morphogenetlc protein 2 (IfiBMP-2) stimulates ectopic fom~ation in vit,n. Using primary c~ltures of felal mt ¢=lvcwlal cells that differentiate to fonn bone nodules in ~tro, we demouslmte Ihat rhBMP-2 c=n acceler.',te this dif- feremtation process as measured by acceleratod mineralized bone nodule ferrnali~n. rhBMP-2 alex) ~¢¢elemtes mullilayering of these osteobla5! cultures. We ~]~o demon- strafe that rhBMP-2 increases and accelerates the appearance of alkaline mRNA cad osieocalcln mRNA. In addition, rhBMP-2 increa~s the nmplilude expression of endogenous BMP-2 mRNA and accelera£¢s the appe:~nc'e of mRNA exwession. We timber demonstrate using transient tmnsfcctkm a~..ays with the BMP-2 I~omolcr and luclferase reI~lcr gone constructs that ~hFIMP.2 I~';illvely affects BMP-2 tmnseriplion. The BMP2. response region was mapl~d ~.'twecn and -g76 of tim 5' nanking region of the BMP-2 gone, The resulb; SUgg~,q th~t .~l least F,'~rt of Ibe mechanism for d~BMP-2 action orl bone cells is ~.'ltvati~n of endngenrat,~ BMP-2 gone. Thi.~ could lend to amplicatkm of the BMP-2 signal in bolh a paracrlne anti m,tocvine fashion during I~ne cell differemiatlon. Harris, So E., Fens, L Q., Ilarrls, M. A., Ghosh-Cboud-C'houdhut7, N., M. R., Wozncy. !.o and Mundy, G. R. Molecular and Cellular Differentiation 3(2): 1:37.155, 1995. Other supporl: National In~tltutes of He•lib. From Ihe University of Texas He•lib Science Center, Department of Medldn~, Division of Endocrinology, San Antonio, TX, and Oenellcs Institute, MA. EXPRESSION OF THE BMP 2 GENE DURING BONE CELL DIFFERENTIATION none mnrphogenelic prol¢in 2 (BMP 2) ~nd lransforming growlh facler p (I"GFI]) =re aclively involved in bone fmmalion Imd ~tmdding, TGFI~. a powerful slimulml in the early stage of bone cell ~owth ~ed matri~ fownatlon, inhibits differ. enlialion and in vilro mineralized nodule fond•lion in primary felal rat calv;u'ial ostcobl~sl system. TGFI~ al~o ne~alively regulates BMP 2 expression al Ihe lran. scriplioe•l level. BMP 2 gcne expreJsion is ¢lxltmlled by • ballc~ of tmn~rll'~io~al fac~OrSo ~ knowe and some yet IO be i~emfficd. Immortalized o~tcobla~t celt I~n~s ~eneraled from • Iransgenic mouse canyln¢ BMP 2 promoter-driven SV40 13rgc T anllgon Iransgene are described as powerful lno|s for s~udying regulation of BMP 2 ~ene expres.~ion ~nd bone cell diffcrcnlialion nt Ihe molecul,~r level. Gbosh-Choudhury, N., llan'k. M. A., Feng, ~. Q.. Muncly. 0. R.. and llarrls, ,q. I~,, 75
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0 0 0 Oitical Reviews in Eukaryotic Gene Expression 4(2&3):345.355. 1994. Other support: National Institutes of llealth. Frmn the Univershy of Texas ffeahh Science Center at San Antonio. D~parlment of Mcdicln~. Division of End~rinology and Meta~)li~m, San Antonio, TX. ESTABLISIIMENT OF PURE NEURONAl. AND MUSCLE PRECURSOR CELl. CULTURES FROM I)£O.TOPIllLA EARLY (;ASTRLILA STAGE EMBRYOS ~]ma~ cultu~s of Dro.e~hiln goslrula stage emh~ic cells will divide te~inally diffe~nllate into mo~h~agically r~ogni~ahle ~u~s a~ mu~le~. p~n~ypicslly mixed nature of Ihis prima~ cullum syslem has mm~ it difficull off, lively ~lyze va6~s pa~mete~ of cell growth and diffc~iafion h~r indivhl. ual cell ~y~s. We rein here a simple and economic melh~ io separme early emb~i~ precurso~ for different cell ryes. using a shallow linear ronrlentln~ Rcoll gradient ~1 unil gr~vily. ~e se~raled c~lls we~ collecl~ into rmcli~, cul- tured, and ~nalyzed for their ~rowlh and differenlialion pallems. ~)e In~er and denser cells of lhe flrsl fractions differenfialed to yield pure ~uronal cullnres. judged by mo~hologic, immunolo~i~, and hi.heroical crileria. C~lls in the fmcli~ diffc~nliated line ~ p~om~n~dy mu~le-enrlclred cell ~)~lali(m. which also conlai~d a ve~ small ~enlage of neurons morp~dogically disli~l from those in I~ pu~ neuronal fractions. A~ximately 3S~ of I~ e~flv gm~la slate emb~onlc cells differenllale tale neural ~]ls. and ~S% of t~ non-neu~nal lln- ease cells laler devel~ ~nlo ~lomlnanlly mu~le ~dal~nn. ~)e melh~! is highly ~pr~uclble. can pr~ess 3 x 10) cells ~r ~u~. a~ Ihe ~ove~ is >(~ the in~l cells. ~e so,ruled ~lls are suilabic for cell biological analy~s as well for hi.heroical and ~lecular studies of n~ron and music p~curs~. H~yashi) L ~nd Pe~z-Magall~s. M. In Vitro Cellular & ~vel~mental Biology 30A:202-20g. April I F)om the ~panmenl of Mof~ular Oe~llcs. Bcckmon Rehash institute of {he Cit~ of Ho~, ~ane. CA. 3~11, A SEQUI-~NCE ISOI.ATED AS A Iq(O'l'l..'lN KINASI.'. C IIINDIN(; PROTEIN, !$ A NOVEL MEMBER OF TIlE ADDUCIN FAMILY We ~ccenlly cloned a partial cDNA (3511) h~r a pn,tein kina~c C (PKC) binding r~)lcln fn~m a rat kidney eDNA library aml dcmcm.'dnlted thal il it a I'K(" sith~Inde in ritrn (Chapline, C.. Ramsay, K., Kl-',uck, 3:, und Jaken, S. (I'~)3) J. Bhd. Chem. 76 268, 6855-6961). Additional libra~ screeninB and ~' rapid amplification of eDNA ends were used to obtain the complete open reading frame. Amino acid analysis, DNA sequence analysis, and Norlhem analysis indicate that 3.~11 unlqtm eDNA related to a- and ~3-ndduclns. Antiserm prepared to the 35tl b~teri.~l fusinn protein recognized two polypeplides of g0 and ~0 kDa on immuaohlots kidney homogenates and cultured renal pmslmal tul-,ulc epithelial cell extract¢. The. 35H-related prolelns were similar to a-and fl-addudns in Ihat they were prcfercn. tially reeo~ered in the Triton X-100-insoluble (cytoskelctal. CSK) fractk~n o[ cell extracls and wc'~e predomin;mtly local&ed to c¢11 Imrdcrs. Phorbo! cxter~ stlmtllatcd plmsphnrylntion of CSK 3511 proteins, titus emphasizing that sequences ~-cordlng to PKC binding activity in vitro are also PKC sub~lrates in ~.ivo. The phorylated forms of the 35H proteins were preferentially recovered in Ihc fraelion, thus demonstrating Ihat phosphoryladon regulates their C'$K a~c, Ctalir~n and. thereby, their function in regulating eytoskeleml as~ntblies. We h.~ve an~lber PKC binding protein parlial eDNA (clone d$) from a r, tt fibr~blart libr.~ry with substantial homology to e~-adduein. Antisera raised against this expre,c,~¢d sequence recognized a protein of 120 kDa. the reporled size of n.addocln. immunohlmx of renal pmxlmal tubule ephhelial cell extracts. A 120.kDa pmte|n that cross.reacts with die clone 45 (a-addue;n) antisern copreclphated with .I~!1 immunecomplexes, indicating that e~-adducin associates with 35H prate;as in Taken to, ether, these results indicate that 35H is a new. widely expres~d form of ndducin capable of Forming heterodime~s with c~.addu~in. We pro~x~.~e naming this adduein honmlogue ~-mldu¢in. Dang, L., C'hapline, C., Mousseau, B., Fowler, L., Ramsay, K., Sreven~, L L. and Jukeno S. The Journal of Biological Chemistry 270(43):255.~4-2.d~540, October 27. 1995. Other suppom National Institutes of Health and American Concer Society. From the W. Alton .lones Cell Science Center, Inc., Lake Placid, GENERALITY OF THE SHARED ACTIVE SITE AMONG YEAST FAMILY SITE-SPECIFIC RECOMBINASES~TIt£ R Sn'E-S~aF~C ~rCO~mN^S~. rOLLOW~ Mutations or the ;nvarianl Int family letrnd msidues, the RHR tri,ad, and th~ active she lyrosine, wilhin the Zy,qosarcl~arom3~ex rm,.ril slte-spcclfic rccornb]na~e R caes~ Ihe same "step.arrest" phenotypes as they do in the Flp recomhin~e of $nccAaramyct$ rertt.i.¢iat. In "half-slte" recomb|nalions, the R recombina~e exhlbhs catalytic completnentation between an RIlR triml mutant and an ~¢tive site mul;mh Stnnxl ¢utlillg by R ftdlows Ihe "tmn~" I)NA cleavage ride. 'rh~:se r,.~uh.~ ~w,., best explalned by the assembly of a furgtional active site from partial active hadx)red by the R monomers. Complementntion tests using single and double step- arrest R mutants verify crilical prediclions of the "shared active site" n~l,:l. A wihl type inomm~er I~dred with nu gl Ig tril~l-TyP" d¢~dde mutual is n catalytk'ally 77
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live combination, Pairwise combinations of a single or a double RltR mutant with R(Y358F) yield comparable levels of catalytic complement•lion. These results strongly imply conservation of the mechanism of active site as~mhly and the mode of substrate cleavage within the yeast family site-specific recombina.,,es, and perhaps within Ihe I~ger lot family me•robin•sea. Yan,~, S. H. and Jay•ram, The Journal of Biologlcal Chemist|y 267(17):127X9-12796. April 29, 1994. Other supporl: National In';litUte,; of lie•lib. From the Department air Microbiology, Unlver~ily of Texa.~ at Austin, Austin, "iX. ACTIVE-SITE ASSEMBLY AND MODE OF DNA CLEAVAGE BY Flp RECOMBINASE DURING FULL-SITE RECOMBINATION A combination or half-site subslrates and step arrest mufants of Flp, a alto-spe- cific recomblnase of the integrase family, h~l e~riier revealed the following features o1" the half-alto ~'combinatlon reaction. (i) The Flp active site is assembled by shar- ing of catalytic residues from at least two monomers of the protein. (it) A monomer does no~ cleave the hs~f site l• which it is hound (DNA cleavage in rather, i! cleaves a I~alr silo bound by a second i~p monomer (DNA cleavage in Irons). For Ihe X intcgrase (Ira pro~ein), the prmotype member of the ]~n! family, cat- slyliC complemenlalion belween Iwo acdve.site mutants has been n~;crved in roan- lions with a suicide alfL substrale. By analogy with Flp, this observation is strongly suggestive of t shared active site and of trues DNA cleava/;e. However, reactions with linear suicide o,,t/~ substrates and synthetic Holliday.junctlons are more compal. ib~e will) c/s Ihan wilh fron$ DNA cleavage. The~ lot re.,~ults either argue ~ain.~l n common mode of •clive.site asscmbly within the lot family or cbellen/~e the validity of Flp half shcs as mimics of the normal full-alto substrates. We devised a strste~y to assay c-talj~ic comp~emenlatlon between Rp monomers in full sites, We found that lhe full-alto re~ct~on follows the sh~red |calve-alto paradigm and the rran~ mode of DNA cleavage. These results su~&est that within the lot f=,mily, a unJlary chemicul mech~nlsm ol" recombinatlon is achieved by more than one mode of physical inter•co l~on am•oK the recombinase monomers. Whanl~, !., I._-e, .l., and J,,y~r~m, ~, Molecular and Cellula~ Biology 14(I !):7492-7498, November 1994. O~hcr support: National Institutes of Health and the Robert F. Welch Foundation. From lhe Depm~ment of Microbiology, University of'Texas at Austin, Austin, TX. 7~ DIRECTED PROTEIN REPLACEMENT IN RECOMBINATION FULL SITES REVEALS TRAN$-HORIZONTAI. DNA CLEAVAGE BY lqp RECOMBINASE One round of site-specific recombination belweon Iwo DNA pa~n~rs mediated by Ihe FIp recombinase require.~ the tweak•go and reformation of four pho~ph~i. ester bonds. The reaction is accomplished by the combined action or four monomers. Within the recombin,'ttlon complex, what is the n:la|ive disposition nf o FIp monomer with respect l• the tm'~:et diester thai it cleaves? To oddress thi,~ q~c~- tion, we have devised a strategy for the targeted orientation o]" Flp mnnnmcr~ v,.i|hln full-alto recombination s||b~trates. Our experimental design is not depentlcnt 'altered hindin~ specificity" of the rccombinase. Analysis of the pattern of DNA cleavage by this method reveals no evidence for DNA cleavage in ci,~. A monomer hound to its recn~nltion element within the full slte does nnt cl~::|vc s¢i~silo phosphodiester hand adjacent Io il. Oar rest|Its are most consi,~tent with 'tran.,~-hm'i~,.nnt||l cleavage'. Cleavage by FIp ecct|rs m the seissil~ pho.~phr~lic~t~,r distal In it, hot within the same full site. The ~:eneral experimental design cmpl~)'cd here will he or wide.,:pread utility in mechmdstic annly~s at" m|clcic ~id Ira||.~ucli~n~ involving multimeric DNA--iwotein assemblies. Lee, J., Whan/;. I., Lee. J.. and J,,y~,r,,m, M. The EMBO .~oumal 13(22):$34~...'~354, 1994. Other supporl: National lnstltutcs of Health and the. Roberl F. Welch Foundation. From the Department of Microblolo~y, University or Tex0.,~ at Austin, Au~tln, T~. MECHANISM OF SITE-SPECIFiC RECOMBINATION: THE Flp PARADIGM This chapter has oulined Ihe mechanism of strand breakaE, e and Irc.io~nln~ dnrin~ Flp recombination. The Rp system provides • paradigm tror the strategic assembly a mulli-component biological machine designed to produce a lo~ieal chemic•! oul- come. The chemical simplicity nnd mechanistic pnrslmr~ny employed b],' Rp mediate an apparenlly compllcnled .-,el or phosphoryl transfers between IWO DNA molecules are likely to have parallels in Syslcms Ihat carry Oul similar chemical transactions in oucl©ic acids, for example, DNA lmnspositlon .',nd RNA splicing. The success in analyzing Flp recomhlnadon has resulted from Ihe design of ~-'combln~llon halEsite, Ihe construction of step-:~nesl I~p variants, and the of the reacllon into pafllal macllons. ,Icy•ram, M. In: Ecksteln, F. and Liliey, D. M. J. (Eds.): Nucleic Acids and Molecular BinloDy, Vol. ~, Springer-Verlag. Berlin, Heidelberg, pp. 265-286, 1994. Other SUPlmfl: Natirmal Institutes of Health. From the Departmenl of Mierobioloi~y, University ot'Tex,'~ at Austin, Au.~lin, 79
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ROLB OF PARTNER ItOMOLOGY IN DNA RECOMBINATION ~ CO}41~N~.NT^R¥ |^$E PAIRING NIE~ ~IE ~'-llYI~OXYI. I~ ~A~ ~h~olule ~molo~ ~lw¢~ ~ner ~uhslr~fes w]thln Ihe ~l~md exchan~u ~li~ Js In as~ial ~i~! f~ ~nati~ ~ial~ ~ the yea~ CJEC ~na~ Rp. Us~ ~b~ of ~inlly de~ half- and full-~ila FIp s~l~tcs, we ~m~strate that the s~nnd joinln¢~ep of ~comblnal~n exquisilely s~nsillve to spear homology. At e~h ex~nn~ point, 2-~ spacer n~d~ ~j~cnt to Ihe nick within I~ ~leavnd s~ of~ su~l~le mu~t ~ir wJlh t~ c~s~din~ ~ment of t~ us-nicked ~t~ r~ t~ ~ su~ slate r~ e~c+l stand ~in~ in l~ ~m~nanl ~e. In ~ wilh lhe "ci~vali~mnsmucl~hilic atl~k" m~el r~ e~h or ;he two tl~ +te~ ~ Rp ~,mbin~li~ (stra~ cleavage a~ stm~ ~ining), ~ ~ that • e llmit~ stand ~iri~ ~mlS the DNA-nucleophile (~'-hydmxyl) r~ allxk on i~ llr~l dio;ter (3'-p~+~otymsyl-Rp). ~rin~ one mu~ +f ~+mbinati~, 4+ ~al ~m ~im or l~ s~ (2-3 ~ ~im at e~b s~ cml) mu~ un~. Pol- lowln¢ ~mnd cl~va+e, within = DNA submm~ a~ ~ir ~th l~ ~dm+ ~ ~ stm~ uni~, In shim ~I. the extent o~ ~ mi~mli~ or I~ ~valmlly cl~ lloll~ay ;ntc~iale is llmil~ IO lhe ~nlral corn or the s~+¢r, ~ lea- plated ~;it;~inl or m~mive n~leic acid ~ (which is astral In the ~y ~ ulili~ by m~r ~binal~ my+terns aml by RNA ~l~in¢ ~ ~. ~d +a~mm, M, ~ f~l or B iol~ml ~mlmtw 2~R):4(~2-4052, Fc~aW 24, I(Y)~, Ol~r su~: Nali~l Institutes ~ lleall~Nali(mal ~ Fonndalkm, mad llm Ro~m F. Wd~ ~t~. ~ ~ ~mment ~ Mi~bi~y, U~vemity or Texas at Amain. A~i,, ~. FUNCTIONAL ROLES OF INDIVIDUAL RECOMBINASE MONOMERS IN STRAND BREAKAGE AND STRAND UNION DURING SITE-SPECIRC DNA RI~'OMBINATION ~ The slte-Sl~cific recomblna~ FIp tom ,~h~'ht~rom~.~ ~'r~vi, da~" accomplishes recombination between two tlrget DNA sites by executing a pair of strand exchanges at either end ot' the strand exchen~e region. One round of recombination rmluire.~ the cooperative aclion of tour recomblna.~e mo~mer~. We demonslr:llC hem thll, in the pre~mee of the appropriate nucle~llniles, a ,~ingle FIp mmlt~t)cr :lsst¢i- ated with ill bindin¢ element can mediate strand cleavaiJe and ~l,ran~d joining, at .the exehlm|¢ site pllospha~e Idjlccnt to ;I. Our mulls support a mo(vc~ m recommn~hOn in ~ich pairs Of FIp monomers re,eric catalytic roles In mediate the first and scc. end ~tS of ilrand breaka~PJunio~ reaction~. They (li~ravor a rnodcl Ih,'st involves a relay of ~combina.~c monomers between binding clcmenL~ to as~mblc sep~role active sit~s ro¢ ~ra~d cleavage and strand joining. Oor data ~ consblent wllh lx=~ka~e a~l joining vc~ctlons ~in& cacrlcd out by a sln&l¢ composite actlv= site In which ~ resldu~s ¢ontrihete ~o bo~h reactions while o~her~ ¢0~tril~t¢ te ~r4 or the two Lee. J. and .y,,~-~m, M. TI~ Joumat o1" 131olo¢'¢u| Chem~J 2"/(X39):23203-232t I. Se~mNr 29, 199~. Other suppml: Nutionsl Institutes or llealth, Natioeal ~¢ic~c~ Poundatlon, sad the Ro~ F. Welch Fou~ati~. From the Depc, lment or M~croblology. Un;~ers;ty of'Texas. Austin. TX. JUNCTION MOBILITY AND RESOLUTTON OF HOLLIDA¥ ~TRUCTURES BY R.P SITE.SPECI~C RECOMBINASE ~ 1Y.~tNo ~,^~'rNuR coMpA'rtsn.n~ KMJaI~ IU',~'OM |H NAll(M~ At~olute homoloey between parln~r sublimates within the strand rc~ion (spoeer) is art essential requlr~t for recx)mbinatio~ m~distmJ site.specific rec~inuse Rp. Reeem expeflm~ms suggest th~,t 3-late Fair adjacent to the points ot'exeb~n~e at each end orthe spacer is utilized in plementadt),Jepcnd~.s!randjob.tng .r~cliO~...H~___.o~'_~_d.t~l~v~o of the specer is also threat, but how boe~olol3 ~s t~.~o m mm~ poeltlons FIp recombi~tiou by assaying strand cleava.~e_~n.o resolut .Km.. m a Holllday junctions in which the I:~¢anch poinl .~.~R~.. ly m pmml~ mo~l~. space', or is imroobillzed ~t e~¢k position wkmn me spacer o~ i,. ^ i. ,he Ixanch I~int is located just outside the spacer qal Ine jum~o, u~ ,.~ ,-,~, v,, frozen ~nmcdiatcty ad,~¢ent to this po~tttou within the specer. KeSO cases il most olden mediated by exchange of the s¢|ss~le branch ~olnt or I~'~ximal to it, m~d rarely by exchange o~ the sclm~ile dislal I~ il. In lilhl ol'lhe~e rand l~VlOU~ R~II~, we di~cu~s pomlbl~ ltslinI l~a, vlvtr compalihilil~ durin~ Flp l.ee, J.. I~. J.. a~l Jwyaram0 M. ~ .Iournlvl of" Biolo~icnl Chtmlst~ 2"/0(32):19086-I~I092, Aulu~ I I, 199~. Odor ~npl'~1: N~llonul Science Foundnllon. Nalonnl Inslilules or ll~llh. Rnhev1.F. Welch Found~lion. From Ii¢ D~pnrlrnem oP Microblology. Unh,¢rsily oF Text. Auslln. TXo
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0 0 MAPPING SITF~ OF INTERACTION OF p47-PItOX AND FLAVOCYTOCHROME b WITH RANDOM-SEQUENCE PEPTIDE PHAGE DISPLAY LIBRARIES During assembly or the phagocyte NADPll oxidase, cylosnlic p47ophox Iran..Iocate,( In Ihe plasma memlx~ne and hinds In fluvocymchrome h, and bindinC dam|ins fro. p47-phox have been identil'~,d on the C-lerminal lails of both flavocy- Iochmme h subonits. In the pre~enl report, wc further examine the inleracliou of the~,e Iwo oxldase componenl,,; by u~;ing random-sequence peps;de phage display librar)" Imldy~i,~. Screening pIT-phox wilh Ihe peplide lihr;.-ies i(icntlricd five IX)len. tial ~,ite~, of interaction Wilh flavcx:ylochrome b. inclmlini: three pruviou.,;ly regi0n~; oF interacllon and two addilional re,ions of imeraclion or p47-phox wilh $pgl-phox and p22-phos. The mid;lineal sil~ were mapped tn a domain on Ihe predicled eylo~dic Im~p of gpgl-phox encornpa,;.~ng residues .~"'q'R V~RQL" aml ¢o a domain flear the eytosoliu C-tcrmioal ~all of ~pgl-pbox encomr~.,;sinj: re.'~idues F°"EW.FADLL''. The mapping also confirmed a previously reported binding .d.m~..ain on gpgl-phox (E~"SOPRGVHFIF~) and putative Sre bomolof, y ~ domain binding sites on p22-phos (P'~PRPP" and G'"GPPGGP"). To demonslrate Iha! additional regions identified were biologically sienificant, peplldes mimicking ~pgl-phe, x SmlUenCeS F"LRO$SACCSTRVRRQL" and E"WFADLLQLI.F,';;Q" were synll~$ized and assayed fro. their abilily to inhibit NADPH oxidase aclivity. The~¢ peplides had EC,, values of ! P.M and 230 p.M. respecliv¢ly, and inhibilnd aclivation ~ mick~d prim" m as.~mbly but did no( affect aclivily or the prca~,;em- bled o~i&~e. Our data demonst|'ale the u~rulness of phage display lihraD, analysis for the identification of hiok~/,jcally reicvam sites of prex~in.pr~ein imeraclion and ~how thl! the binding oF pl7-phox to fiavoc)'lochrome h involves mukiple binding skes along the C-terminal tails o!" both I~pgl- and p22-phox and olher regions of gp9 I-phox nearer Io the N lermlnus. DeLeo, F. R., Yu. L.. Bun'ill. J. B., Loellede, L. R., I]ond. C. W.. Jesallis, A. and Ouinn. M. T. Proccedin~ oflhe Nalional AcMemy of Sclences USA 92:711(~-7114, July 1995. Olher supporl: National Institutes of Heallh, Arlhritis Foundation Biomedical ~Sclenc~ Gram. and Ihe Nadon~l Science Foundation. From Ihe Depmtmenls of Velerioary Molecular Biology and Microbiolo~y, Montana Slate Universily, Bozeman. MT. ' TOPOLOO{CAL MAPPING OF NEUTROPHIL CYTOCltROME b EPITOPES WITH PHAGE-DISPLAY LIBRARIES Cyloclceme b of human ncutrc~ils is the central cornlx)ncnt of the microbicl- dll NADPH-oxidlse system. However. Ihe folding topology of this integral mem- bran~ prolein remains undetermined. Two random-sequence hac~c~ioplmge pel~ide Iibrarlea wece e~d In map stru~ural features of cylnch~mlC h hy (lelcnnioin~ the eplto~es of n~moukmal antihodk;s (mAbs) 44.1 sed ~4.1. slx:cif'¢ f~" the p22"~ and ~2 l~P91'~" cylochrome h chains, respectively. The unique I~Plides at phale ~lected by m.Ab a~nily purification were .deduced tram the phage DNA sequence|. Phyla ~electe~ by mAb 44.1 displayed the consensus I~lXid¢ sequence OOPQVXPL which is nearly idcnllc~l Io '"GGPQVNPI" of` p22". Phage ~lec~ed by mAh .q4.1 dis. played Ihe consen~ sequence PKXAVDGP. which re'~mblcs '~PKIAVDGP" of" gp91'~-. Wexlem hlotlin~ clemonstratcd specific bindln¢ oF el~h mAh live cylochmme h subunil end ~lected phage peplides, In flow cytomctrle analy'~I,~. mAh 44.1 bound only I~nne,qhili~d neutrophils, while 54.1 did no~ bind pem~eahilbcd cell~, llowever, mAh ,'~4.1 immarm~dlmenled detergcnl.~mluhili~! cyhx:hr~nc h ill sucr(~e llm(llenls. The~ r~sulls suBgest the "GGPQVNPI" rncnl of p22"' is accesslhle ~m its intracellular surface, but the v~PKIAVDOP" region on ~pgl,~' is hal ,'K'ces~ihfe to anl;hody, and probably not on the pmtel~ I'~m, ilt. J. B.. Quinn. M. T.. Jutila, M. A.. Bond. C W., and ,let, ail~',. The inure,! of Biological Chemisl~y 270(2R):16974-169gO. July 14. 1995. OIher support: U.S. Puhllc Heahh ~vlce, Nallonal ~clence R:)undallon. AnhHttl Fonndalion Biomedical Science Gram. and Hatioual |mtilutes or Heallh. From the Detriments of Microbiology and Veterinmy Molecular BIOlow, Mnntan~ Slate Univer~ily..Baseman. Mr. ASSEMBLY OF REGULARLY SPACED NUCLEOSOME ARRAYS BY DRO,~OPIIIL~ CHROMATIN ASSEMBLY FACTOR I AND A .q6.KDa IllS'ION-E-BINDING PROTEIN To ascerlnio the mechanism by which nucleo~omes are asaembled by factorl derived from Drosophila cmb~7o~, two proleins termed Dr~ophila ehromatin assembly faclo~ (CAFs) I and 4 (dCAF-I and dCAF~4) were fra~lon~d and purl. fled from a Drosopl~ila embryo extract. The assembly of chromatin by dCAF-I, dCAF-4, purified hlstones. ATP, and DNA is a process that gtmCrlll¢| spaced nuclcosomal arrays whk a repca~ length that I'e~m~l¢l; thll at bulk nativ~ Dro.vopllila chromatin and is hal obligatorily coupled to DNA replication, The a~Jtcmbly of chromalin by dCAF-! and dCAF.4 is nearly complete wkhin 10 rote. The dCAF-I activity coporified wilk Ihe Drmophila version of chromatin a~Rmbly factor-I (CAF-I). a factor that has b~n found In be requiRd for the assembly of` chromatin during large tumm. (T) anti~cn.mcdlaled, simian viru~ 40 (SV40) origin. dependent DNA replication. The dCAF.4 activity copurlf'~zd with a $6.kDa ten©.hinding Ixotein Ih~tt was podl'~! Io >90r~ Ilomog*~l~ily. Bulgar. M.. llo.'r., Kamakaka. R. T.. and KadonaR~, J.T. Proccedlngs of the Nallonal Ac,qd~my of Sciences USA 92:11726.11730, l:N~emt~r Ig95.
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Other ~upport: National Institutes o1" l{ealth and Natkmal Sclcncc Foundation. From the Department of" Biology, and Center for Motecular (iefletic,% University of Calil'0~nia. San Diel~o, La ]ofla, CA. DIFI~RENTIAL ACTIVATION OF ERK AND JNK MITOGEN.ACTtVATF,D PROTEIN KINAgF_~ BY Raf-I AND MEKK Growth factor~ activate mlt~en-aclivated protein kina,~e,~ (MAPKs), includini~ ¢xtraccllufar sil~nal-rc~ulazed kina,~s IERK~) anti .fun kin:l,,~s (JNKs), ^ltlxxs~h Ihe sil~nllin~ claude lrmm growth factor recq}tor~ It1 h'RKx ix relatively well under- stood, the plthway leadln¢ to JNK activati(m is more oh.~cure. Activutkm ofJNK hy ~1'~idermal Mowlh factor (EOF) or n~rv~ growth factor (NGF) w;~ del~r~ent ma li- Ras activation, wherea.,, ]NK activation hy tumor nccn~is I'g~tor ~ (I'NF<O Ras.independ~nt. Ras aclivates two protein kina.-,cs. Ral'-I and MEK (MAPK, or ERK. ~|nss¢) kinaze (MEKK). Raf-I contributes dimclly to ERK activation hot not to JNK a~ivation, whereas MEKK parlicir~tcd in JNK acti~,atlon but caus~! ERK aclivmlion only after overexprcssioe. "rhe~e z~-sulls demonstrate the existence of'two dl'~tlnct Ras-dependont MAPK ea.,~cades -- on~ initiated by Rpr-1 leadln~ Io ERK activallon, and the other inkiated by MEK K leading to JNK activation. MirKfen, A.. l.in. A., McMahon, M., Langc-Carser. C., D~rljard. B.. Davis. R. J., Johnson. G. L. and Karln. M. $cler¢~ 2(~:1719-1723. Dcc~mt~r 9. 1994. Other suppo~l: National Institutes of Health and the American Cancer Sociely. Fronl the l~partment of" Pharmaco|ogy. Prelim in I~iomedlcal Sclenc¢~. School Medicine. University or Cellfomia a~ San Di~co, l.a `iolla. CA. DNAX Institute of Molecular and Cellular ]]iology. Pale Alto. CA. Division of Basic Sciences. National ,fewish Center for Immunology and Respiratory Medicine, Denver, CO. and Howard Hu¢hes Medical Institute. University of Massachusetts Medial School, Worc~mr. cJUH N.TI~,RMINAL PHOSPHORYLATION CORRELATES WITil AC'TIVATION OF THE ,INK SUBGROUP BUT NOT TI IF. ERK gUFlGROUP OF MITOGEN.ACTIVATED PROTEIN KINAgF_.~ ¢.,Iwz transcriptional activity is stimulated by phospho~,lstion at two N-terminal sites." ger.~3 and -73. Phospho~jlatioo of thc~ sites is enhanced in respon,~ to s variety o4" extfacellular stimuli, including growth factor, cytnkines, and UV irradia. $4 finn. New n~mF~rs el" th~ mlto~n-activatcd protein (MAP) kimse Iroup of sipnal- tr~sducin~ cnzy~s, tc~cd JNKI, 5i~ to t~ ~ivatlo~ domain of c.Jun and ~i~¢slly ~t¢ t~ sit~. Ho~veh t~ N-t~i~l sit~ of c-Jun ~R al~ su~t~ ~ ~ ~p~lst~ ~ t~ ~ MAP kin~s. ERK! ~ ERK2. ~te t~ ~s. ~ ~ that unlike t~ ~N~ ERKI ~ ~K2 do ~ ~- ph~late t~ N-te~inal sites o~ cJun i~ vitro: i~teed they phosph~late an inhihlt~ C-le~inal ske. ~, t~ ~1~ of e-Jan l~ ~v~ at t~ N-te~i~t ~ites ~s wkh ~tlvati~ ~ t~ JN~ ~1 ~ ~ ERKs. ~e ERKs t~ sti~bt~ or AP.I ~tivky. ~r stay ~m t5~ t~ diffeRnl ~ of t~ MAP k~a~ 8m~ a~ involv~ in I~ st~uiet~n of AP-I ~tlvlty thigh t~ di~c~nt m~nisms. M~, A., Lin, A.. S~l. T.. ~d~a~. B,, ~h~ M., Davis. R., a~ Kadn. M. Mol~utar a~ Cellular BloWy 14(t0):~.~. ~t~r I~. ~r supS: Nat;~al Institutes of ll~lth ~ I~ A~ricnn ~r ~olla, CA, ~ment o~ ~#~Io~, $~k~ M~#I Center, Unlv~ky of Texa~, Dallas, TX, and Howa~ Hughes Medical Institute and Program in Mo~ul~ M~ici~. Unive~ity o£ Msss~h~ttn M~lcd Sc~1, W~ter, MA. JNK2 CONTAINS A SPECIFICIW-DL:rl'ERMINING REGION RBSPON$1BLB FOR E~CIEN? cJun BINDING AND PHOS~ORY~TION ~¢ Imn~pt~l ~tivity or cqun is ~¢~nt~ t~lh ~p~lal~ ¢ t~ sit~ ~ a c.Jun ~i~l~i~t k~a~ UNK). All ~lls cx~ two disll~ JNK ~ivit~s, 45 a~ 55 kD in si~, It is ~ cl~ which or I~m is the m~ t~lnt ¢- Jun kina~ ~ ~ I~y ~iflcalty ~ c-Jm. ~ 4~ED ¢o~ o£ JNK was idenlifi~ as a new mem~r of t~ MAP klnaR ~mup of ~iBnal-t~nsducin~ en~ ;NKh He~ we ~ I~ ml~ier cl~i~ or I~ ~kD ~ of iNK. iNK2, ~i~ exhi~ls ~ i~ntily z~'slmilzr Rplat;~ m }NKI. ~phe thin c~ si~laHty, l~ t~ JN~ d;~er g~t~ in t~ir a~lky zo Im~ ~th ~Jun. INK2 bi~ cJun --25 ti~s ~ e~¢~lly I~n ~NK I. ~ ~ a ~11 hu I b~r ~ t~aM ~m than ~KI. ~ ~luml ~is f~ ~iz diKe~ w~ i~lipl~ a~ I~ m a mall F-st~-like ~ m~ I~ ~lalyl~ ~ ~ !~ e~me. M~ling ~s I~! this Rgi~ b ~l~m ax~ I~ I~ ts likely to ~e ~ ~lts explain ~ two cl~ly ~lat~ MAP ki~s can di~ in t~ir Ibllily m ~i~ s~ific s~l~les S~ I~R~ elicit di~e~t biol~l RI~L Kaltunki. ~., ~. B., ~i~lny, L, Sluex. H. K., ~. g.. M~. O., Dnvls. R.,
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0 !'0 0"1 Genes & Development 8:29%.3~7, I(~4. Other suR)orl: National Institutes or H~lth and t~ A~rkan Ca~r S~icty. F~m lhe ~punmenls of ~a~aoolo~ and Chemistry. Center ~or Molecular Qe~llc~ ~rlm in Biomedical Sciences. Sch~! o~ M~dicinc. Unlvcr~ily or Mol~ular Medici,. and ~parlment nf ~;~hemlslry and Molecular ~inln~y. ¢-Fo~ TRANSCRIPTIONAL ACTIvrrY STIMULATED BY II Rag-ACTIVATED PROTEIN KINASE DISTINCT FROM ;NK AND ERK R~s prmeins exert their mltot~nie a~cl on¢ogeni¢ downstream protein kinms. An imporlanl question is hmv Ras-generaled signab reach the nucleus to uclivate downstream tur~el plex of ,lu~ aml Fos proteins, which activates mito~en-indu~ibic ~es. is • n~r nucl~u" tu~et o£ Rus. R~s can slfmulule AP-I activity by inducing c~J~ Iram.crip- liO~. l .pl'ooes~ which is pmbaMy mediated by the ERK! and -2 milosen-acllvated prolein (MAP) kina.~cs, which phosphorylate the Iran~cripdoo lacier EIk-I/TCF. Belidel/ndu¢in| transcriplion f.rom J't~ •nd jen genes, mltcll~ns and Ras prolelns ~nlm~ AP-! ~otivity Ihro~h p~Islioe ot" ©-.tun. l~pho~lali~ of. Ihc c- ,lun aetivalion dorn,,in leads to ¢-je~ ind~'tioe through an •utoreSulalory loop. Ras- and uht/vlolel-responsive prolein kinsses (hat phosphorylate c-Jun on serinc m=ieuet II I~ilions 63 ~d 73 and slimulule ils tra~acripdo~al aclivlty h•ve been kl~ntit'lecl. ~ pmline,.dire~ed kirmses, lem,.ed JNKs. are novel MAP kin•sea, it is nol ele=r, however, wheiher ¢-Jun is the only recipie,l and .INK the only transducer oC the Rus sisnal ~o AP.I proleins. A sho~ seq~n~e surmundin8 the m~l~ ,l~.riK ~•tinn site o1" ¢.]un is ~s~rved in c~Fos and is purl of" its ucti. vatmn ~om•in. sul~eslin8 Iha! c-l~ m,y he slmil•rly r~gulated, Hem we show Ih~! RII do~ ind~d augment the trunactitxional ucllvlty ol" c-Fee lhrou~h phospho~jla- liC~ ~1Thr 232. Ihe bomolo~ne of" ~r 73 of" c-.lun. However. Ihis is mediated by • ~wl RII- ~ mito~,n.~po~siw" pmllne-dim~ed prol~in kinase Ihal is ditTemnt rein ]NKs and ERKs. Thercf'oce, ~! least three types o1" proline-directed kinascs Immmit ~ and mitolen-~-ra~J signals Io the tran~qcril~io~l m~hin~. H•lu~ 371:171-175. ~q~emb~r 8. 1994. Other lupport: N|lionll lnslilut~s o1" lie•lib. Arneri~•n Cancer SoCiely. Tob~- Rel•led ~ Reselu~h Prelim. and Ihe Leukemia Seclety oC Amer~•. ~ I~ Depmmem oT Phermaoolo~y. Cemer I'o¢ Molecular 13~ics. Unive~slty et'C~lif.omb •¢ $~n DieBo School of Medicine. L~ ]o11•. CA. $6 IDENTIFICATION OF A DUAL SPECIFICrrY KINASE THAT ACTIVATi~ THE JUN KINASES AND p38-Mpk2 On~ Ras-dcp~ndent prolein kinase cn~e leadin8 from I~Owlh factor to the ERK (cxtfaccllular signnl.regulnted kinn~z) su~.l~roup of mltollen.acl|v~lled protein k(na~'x (MAPKs) is ek~nd~nt on the IX~ein kin.'~c I~r.I. ,~.h the MEK (MAPK. or ERK ki,axe) dunl ~p~ificity kinalex. A ~.¢~d I~O,¢ln eaxclde ieadin¢ to acliVlZlinn el" the Jun k|na~l (,INKs) is d~pendenl t,m (MEK kln~Be), A dual.,~pecificlty kinase II~t acllv~le~ iNK. n~med JNKK. wzB klen- lil'¢d that funcllo~s helwe~,-,n MEKK ,rid JNK. ~NKK nelly•led Ihe JNKz Ixzl ac~iv•te the ERK~ and w•s un~poosive to Rat'-I in Irallsfect~.',(! HeLa eellq..tNKK • lr~o re:siZzled anolher MAP~. p.~A (Mpk2; the mammalian homolol~ of HOt31 yeast), whose activity is rcgulaled slmilarly to that of the .INKs. Lin, A., Miedcn. A., M=rlinelto. H., Claret, F.-X., Lan~e-Carler, C., Meeetwlo, F.. J~n.~on. (3. L.. and Kzrin, M. Scicece 2~8:2it6.2900 April 14, 1995. Other supporl: Natlon.ql Irmitutes or Health. Rom Ihe Del-,a~Iment of" Pl~armacolot,,y. Pmeram in Biomedlc•l ~¢ienee~. University of" C•lil'omia-San Diego School of Medicine. La Jell•. CA. Divilion of Science. Nnlio~al 3cwi~h C~ter for Immunology & Rezpiraton/Medicine. D~ver. CO. and Signal Phermm:eulical~, San Die~ CA. INTERACTIO~ OFTIIE p53-REGULATED PROTEIN OADD,I.'i WITH PROLIFERATING CELL NUCLF, AR ANTIGEN GADD4.~ i.'¢ n ubiquitoo~ly expr~t.ed mnmmalian ~ne thai i~ induced by DNA d•m•g¢ and ¢¢rl•in olher slre=se$. Like •nOlher p53-rcgulated geol. p21"~'".who~ prod~cl binds to eyctin-depemL-nl kin~;es (C~'I) Ind pmllt'mllinI cell nucl.r •nligen (PCNA). GADD43 has ~ asxcx:ialed wtlh ~rOwlh tuPl~len. Gzdd4$ w•~ found to bind to PCNA. • normal ~n! or Cdl~ complex~ and a pro~in involv~cl in DNA replication and repair. Gzdd45 at/ms,lied DNA excieten repair i~t vitro •nd tnhibiled entry of" cells into $ phase. Title results ISl|blilh GADD45 as a link hetw~n the p.'~3-dcpendent c¢11 ¢yc!¢ eheekpoin! and DHA mr, air, Smith. M. L. Clan. L-T.. Zhnn, Q.. Bae. I.. Chen. C.-Y.. Oilmer, T. M., Ka~lzn, M. B.. O'Connor. P. M.. md Fornace. ^. J.. Jr. S¢i©nce 266:1.176.13~t0. Novemher 25. 19~4. Other ~tplx'~t: Natiooal lnstilutCs of Health and lhe Leukemia Society or Amerk~ From Ihe Lahornlory of Moieeular Pharmaooloey. Developmental Therapeutics ProM•re. Nalio~al Cueeer ln.~lilute. Berber.d=. MD. Johns Hopkit~ OnooloBy Center. Baltimore. MD. and Glnxo Resem'ch Institute. Rc.~¢arch Tr~nBle Pa~k. NC. It?
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PROTEIN PIIOSPHATAS~ IN PROKARYOTES: REFLB~--'TIONS OF ~E PAST. WIN~WS ~ THE ~r ~wt~ of t~ i~ntili~ a~ m~e ~ l~ ~ph~teins. pmte~n ~d ~ein ph~l#~ in ~k~ organisms la~ su~lnnli.lly ~hin(I with ml~ m euk~ic m~ni~, llowcvcr. (me ,hin~ ha~ ~come appa~nt in ~m ~ca~l~ wall lh~ ~s dirked Ih¢ ~ntcin pl,~ylafi,m nelwt)~ks of ~¢s f~ t~ ¢uka~oles a~a~ to ~ on ~ ve~ fi~l Ca~ually of this ~iemiflc Jer~ i~ Ihe ~aning and valldi~y ~a~e-#~ euka~ote-~ific ~ein kln=~s ~J p~c~n p~a~a~s. Another likely casually i~ the ~p~ that pmlcln pho~phorylalinn ~m ~dhl~ ~o the ceWs ar~nal of ~gulal~ m~hani~. (~ junior ~o all~er- i~ a~ gene ~gulation. ~ wides~ad natu~ o~ l~ ~rging ~mllc]s ~ka~ic and euka~ic ~i~ sug~st that II~ ~fiMin~ hl(~k~ ~in ~lati~ ~tw~wk~ a~ in f~ ~filc a~iela in nat~. ~d m~ have ~ o~ti~ as ~ulal~ in t~ Wimltlve ~lg~ ext~t ~G~ e~ dive~e of~r ~nt ~e~t~ domain. Ir~ix ig iNo. even to = limited extcnl, t~n the alkn" ~ein ~lal~ ~tw~s or ~a~ic o~i~m~ may ~ as wlmk~w~ inlo as yet undJgove~d ~ions of the signaling ~two~s oF t~ir euka~ ~n. Ti~ will tell K~H~, P. J. ~ ~t~. M. ~ su~fl: Nlt~ll ln~ilulcs ~ Health a~ Nal~al ~k~ F~dat~. ~m the ~pa~ment of Bi~hemlstW and Anaerobic Mic~iolngy. Virginia ~l~hn~ in~titu~ a~ State ~ni~hy, Bl~ks~rg, VA. ISOLATION AND CLONING OF A PROI"EIN-SERINFJ'lllREONINE PHOSPIIATASE FROM AN ARaiAEON A div~ttont metal ion-stlmulated prolein.scfine/threoninc pho~phalase. PPI-arch. w~s purified approximately I.O00-fo~ from the extreme acldothermoph~lic trche~n St,(fWo~c,s tolfamric~ (ATCC 35091). Purified prepami~..s conta~.nea.40 ~.o~ ~O~.or to~al pmzeln as PPI4rch, zs determined by ass~y-ing so~mm m:~ecy~ su||atepo|y- aerylamk~ ~els for pr~ein pho~pfimase activity. The first 25 amino acld~ of the pro- teJn'a g, quence were identified, as well a~ an internal .,~quencc spanning some 20 amino ~gid~. Using this information, we cloned the ge~e for PPI4rch via the aPlgi- c~tk~ of ~ and conventional cloning te~hnklues. Tbe genc for PPl-arch predicted a prolein of 293 amino acids that bore striking resembl.~n~e to the members of the E~Om, tim PPI/2A/2B superfam,ly. ~ core ol the .protein, sp~.nmn.g gemotw~s~_, .m 275. Ix~es~'d 29 to 31q6 identity with t~csc cucaryat prmein pheeqmat~es. Uf me • 2 re~idue~ found to be at~olulely conserved among the krmwn eocar~al memhen~ of tbe PPIt2PQ2B supeWsmily. 33 were pre,~ent in PPl-arch. If highly crmservatlv© sub- stltulions are included, this trial reached 37. The great degree of .~lucncc con~va- tion between molecules from two distinct phylogenetic domains imp1~s that the mem~',~rs of thls e~.y~ supelTamily had evolved as ~ialt~. ~l~l~ ~¢ln ~ta~s wi~ to I~ diver~e of ~m~ of ~ A~ ~ Eu~ f~ ~g. J.. ~me~. A. 3. M.. Buckel. S.. nnd g~y, P. J. J~mal of B~teriol~y 177(22):~ 10-6317. N~em~r I ~3. F~m the ~pa~menl~ of Ri~hemi~lry and Anaerobic Microbiology. ~lrlinla Polytechnic Instil,It ~nd S!a¢¢ Unive~ily, Black,burg, VA. end Pittm~n-M~ ~m~ny. T~ llaute. IN. DNA STRAND EXCIIANGE IN T~ IE ABSENCE OF HOMOLOOOU$ PAIRINO The str~ed exchange reaction that i~ wMely u~-d for In vitro studl~g on go~.s pairing step. With the ~ of a stflglC.,~tg~'~fl ellggl~t PNA ~ • ~ ~-~" ~ f~ that ~ ~nt~ of ~myle~ ll~z ~ mn ~u ~ ..... t1~ of ~e~uplex ~kt a~ ~ e~h~ W~n t~ duplex f~$m~t ~ily imply a p~Jng ~ ~idng stc~ F~ t~ Jcff~ ~r Insetted. ~as Je~ea~ Unlve~ity. ~ll~¢l~Im, Pk. and !~ ~pa~m~t of Mi~biology. Comell Untidily M~ical Y~, NY. TELOMERIC PROTEIN-DNA POINT COHTA~i~ IDENTIFIED BY PHOTO- CRO99.LINKING USING $.BROMODEOXYURIDINE ~9
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0 prompted this photo-cross-linking: stud),. Multiple pr,~ein.DNA photo-cross.links Ire formed upon UV imK~iation of Oxytri~'hn telrm~ere~ rec(m,~tltuzet] with a synthetic olisouucleotide terminating in 5'-T,.T,,T.T,,O~G,,G.G,T,T,T.T,G,(],G~G,-3'. Size. speclr'K: sub~tltution OF ctrtaln nucleotldes with $.hromndcoxyuridlne (RrdU) greotl), increz.~ the photo-cro.-J~-linkln~ yield, each substitution favoring a specific pr~cin- DNA cross.link. For example, substitution o1" BrclU for T, rcsulled in 25% cross. linking: of the bound DNA, a lO.l'old increase over the unsubstituted DNA. subonits of 1he telomere protein cross-link to, and are therefore near. the DNA. Three point contacts within this nucieoprotein complex, involving: the a subunit, weee eslabli.,hed usin~ BrdU sub,,~litutinn: Tyr239, Tyr142, and I1is292 cross.link (3,. T,u and T,, respectively. One phmo.cro.~s-link, l"yr2.tg-G,, occurs amid a shorl acidic stretch of the a subunil, counter to expeclalioos for amino acids thai approach Ihe polyanionic DNA. The lwo remaining cross-links are to umino acids in hydrnphohic rex:ions of the primary pnlypeplide sequence, cnnsisrent with the hypotbesi,; that hydrophobia interacti(m~ account los the s,lt re,~i.~taocc (>2 M NaCI) of thl~ I~oteln-DHA complex. The~e Iwo ~holo-cro~.llnks surest that the telrencre prolcin may bind t¢lnmeric sing:lc-stranded DNA by inlercalatlon of nromalin ~sldues into I nucleotidc lattice. Hicke. B. J.. Willis. M. C. Koch, T. IL, and Cech. T. R. Biocbemisl~ 33(I I):336,1-~373, Other sul~port: Natlon~! In,aliases of ltealth. Prom the DCl~rnen! of Molecular. Cellular and Development~d i~iolo~,.V, ltoward Hushes Medical Institute, and Del~trtmen! of Chemistry and Biochemistry, UniversiW of Colorado, Boulder, CO. ~ VITRO ASSOCIATION BETWEEN THE JUN PROTEIN FAMILY AND TIIE OENERAL TRAN$CRIFrlON FACTORS, TBP AND TFIIB Transcriptional activator proZeln~c interact with Ihe 8eneral transcription ~'actnrs TATk-bindin¢ pr~eln (TBP), TFIIB and/or other TBP.at~ociazed faclors (TAFs). Using alYinily chrom~osraphy we demonstrate thal n~mbers of lhe ,l~n family of tr~e~pdonel activators inter~ with Ixxh TF~P and TRIB in ~tro. TBP binds Io both the N.terminal acllvation domain and C-terminal bZIP reg:ioos of c-.lun, wham~ TPIIB binds to only the c-Jun bZIP domain. This interaction requires Ibe dlmeriZZlion o!" Ihe .fun protein, The ability ot" the N-terminal •city.ion domains of ¢.Jzm, .lunB, JunD and v-Jun to interact with TBP in ~,itra cortelat~ with their tnm- acripdomd aclivily ~k z,/l~o. Domain mlpping experiments indicate that c-.lun inter- acts with the conserved C-lenninus of TBP. Studies using a set of TIqlB inframe deletion mutuals cicmoeslralc thai C-terminal amino acids 17~-201 and 238-316 play an important role in modulating Ihe interaction between TFIIB and cJun. Although phe~phozTlallOn of the cJgn H.4crrninzl activation domain stimulates c-Jun Iran- scril~ional activizy in z,iz~, it has no effecl on the lbility of c-Jun Io interact wizh either TBP or TFIIB in z,/tro. These d~a su~Best that the Jun family of aclivalor pro- zelos may ~cdvate lrwnscvipdon by interacting wlzh the Saner•! tru~rilXion ~P ~d I~IB. F~Elin. C. C.. Mc~ll~h, A. V.. a~ Kr~, A. S. Bi~h~m~al ~mo~ ~S:%7-974, I~. ~her su~: A~r~an Ca~er ~ty. From the Division n£ ilacmatolo~y and Oncolog~, University of Alabama Bimdn8ham, ! IETEROGENEITY AND MICROTUBULE INTERACTION OP THB ClIO ! ANTIGEN. A MITOSIS-SPECIRC KINESIN.LIKE PROTEIN SUBDOMAINS EXI~F3SF.D IN INSBCT ~ ~ The CilOI antigen is a mitosis-~pecif'¢ klnesin-likc motto" Iocaled at the Inter. :,.o~al reSion of the spindle. The human cDNA codln¢ tot the antil~n contains a domain with r.equence similarity Io the motor domain of klnesin.lihe protein (NIslow et el.. I~:mre 359. 543, 1992). Here we cloned cDNAa cncodin8 Iha CHOI antlllen by immuno~reenln¢ of a CHO Uni-Zap expression library. which the oriBinul monoclonal antibody was raised, eDNAI of CHO 953 amino acid polypcplkic wilh I calculated molecular ma~ of 10P kDa. 'The 1'4. zennlnal 73~ of the antiJ~en was S?~ identical to the human clone, whereas rcmainlng: 27% of the ©odinB fabian Ihowed only 48% homolol,y, infected with haculovirus cant•inane the full-lenSth insert pr~xlu~d 105 and 95 polypcptides. Ihe same doublet identified as !he ortBinal an!|len in CHO cells. Truncated polypeptides con'cspoadir~ to the N,zerminsl motor and C.lm'mlnal tall produced • 56 :rod 54 kDa polypeptide in $f9 cells, respcetl.vely..llull .and. N.4.erm.tnal proteins co-~edimenzed with. and eauged bondllng of. brain micr~uout~ whereas the C.te~mMat polypeptklc did hal. Cells expres~in8 the N terminus formed one or m~e cylopP,~mic processes, lmmunol'luorcsccncc us well as electron mlero- scopIc observ~lons revealed the ~ of th;ck Ix~ndle of mitre(ninnies, whleh were cln~ely p,'¢ked, fon~|n8 a marginal ring: .just benealh the cell rn,m~brane and a core in the procer, ses. The di~uslon coel]~clent a~l ~Jimentalion coefl~ient wer~ dczermlncd for the native CHOI aat;~ by ~1 filtration and S~ d~naity eat ccntrifu~ztio~. SeSl¢cliv¢ly. The rmtivc molecular mass of' ov~rinduced prolein in 519 cells was calculated am 219 kDa. sugeestin¢ thai the anti,,tin exists M a dlm4r. Intrinsic CHOI antigen in oullured mammalian cells forms a larBer native (native molecular mass. 362 kDa). which may suZ'jes! the presence of additional molecule(s) associating with the CHOI motor molecule. Kurl~rma, it, Drugas-Gramoic, $., Mack•we, T., Vassilev. A.. Khodjalmv. A., and Kobayashi, H. Journal of Cell $clcnce 107:~115.3499. 1994. 91
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0 U'I CHARACTERIZATION OF A MINUS END-DIRECTED K1NESIN-LIKE MOTOR PROTEIN FROM CULTURED MAMMAL/AN CELLS U~ing the CH02 monoclonal ~nlibody raised ~zgain;I CliO spindles (Sellitto. C., M. Kimble. and R. Kuriyama. 1992, Cell Mocil. Cyrtxdeleum. 22:7-24) we identified a 66.kD protein located at the interpha~ centn~ aml milo(ie spindle, lsolatctl eDNA• for the anligen encode • 622-amino acid I~dypeplidc. Sequence •nalysis revelled the pre'~,once of 340-•mlno acid homologous to the mo¢or domain comcrvnd urmmg o~her members of Ihe kin~sln superfamtly. The I'~cin i,~ composed of • cenlral ,z-hellc,I p~mkm wilh globular demains at bo~h NH~ and COOil termini, and the ~itul~ to the n~moclonul anlitxxly resides in the ~-mtal ¢~-hellcal stalk. A serle~ of~ledon ¢on~rucls w~m created foe im v/t~v~ analysis or micrmubule interactions. While the miermuhule binding and bundling activities require bod~ Ihe presence of'the COO11 terminus and Ihe e~-heli- cal domain, the NH~-terminal half of the antigen lacked the ahilily Io in~oracl with micfo~bules. 1"he full-length as well as deleled proteins cor~isling or ihe COOH- lewnintl moWr ~ Ihe central a-hellcal stalk suppmlnd micrmuhele ~liding| wHh veloeiW ranging from 1.0 Io 8.4 Fm/trnlnute. The speed of mlcrozubule movement ¢kepel~d with decreasing kngths of" 1he central stalk ,filched ro the COOH-termi- r~l moloe. The microlubules moved with their plus end lending, indicating that the ami,~en is • minus e~d.dlmcted motor. T~e CHO2 ~mluen~e shows 1(6% identity to llSK'r, a gone located at the cemromeric end or the human MHC region in chromo- some 6 (Ando, A., Y. Y. Kikuti, H. Kawata. N. Ok•auto. T. lmai, T. Eki. K. Yokoy~m•, E. Sceda. 1". ikemura, K. Abe, and H. Inoko. 1994. Immunogened¢$. 39:194-200). indicating Ihet HSET might represent a human homoingu¢ of the CHO2 amlgen. KiIr~ama. R., Kofron, M.. E,~snor. R.. Karo. T.. Dr•gas-Granule. S,, Omoto. C. K.. and Khedjakov. A. Th~ Journal or Cell ~i~IoIw 12~4):1o49. I(}59. O~her s~l~x~: Nat|onal Institutes of Healm ~nd the National Science Foundation. From the Dep~r~men( of Cell 8|oinsy and Neuroan~torny, University or Mioneso~,. Minn~-ap0;is, MN, and Depe,~ment o£ Genedcs and Cell Biology, Washinglon St~e University, Pullman, ~A. 92 IDENTIFICATION OF INTRINSIC DIMER AND OVEREXPRI~SBD MONOMERIC FORMS OF y-TUBULIN IN SP9 CELLS INI~d.CrED WITH BACULOVIRUS CONTAINING THE CIILAMYDOMONA$ ~.TUBULIN Sl~UENCE A new member of the tuhulin supeffamily, y-tubuiin, is Ioealb.ed at mica. tubule-organlzing centers (MTOC~) in a variely or orpn|sms. Chhm~.~bmlnnox cDNA coding for the full-length ~'quence of y-lubulin was expres,,~'d In in~ect ova'. tan 5t9 cells using the buculovlrus expression system. Approximately half of induced 52 kDa "y-tubulin was recovered in Ihe Impematanl •tier centdful~tlon SF) cell lysates al 18,000 ~ for 15 minutes. When the cell supernal•hi w~ analyzed by FPL,C on a Superdex 200 si~ing column, Chlamydomonax 'y-rubella into two major peaks. The lagging peak contained a monomerle from of "plubulin with a sedimentation coefficient of 2.5 S, which interacted with the Sul~rdex umn in a salt-dependent manner. The leading peak, with an app~ren! molecule' mass of 900 kDa. corr~lx~Kfed to a molecular ehsporonln complex, lad TCPI ~elea~d folded 'y-tubulin polypeptlde from the complex in the pRs~nce of MsATP. The released 'y-tubulln mo~,omers were ca~ahle of binding to microtubules in and hinchemlcal qu,'mtltles of'actlve ~ were further purlfled usin~ ~ comhi. n~tion of s|ze.cxcluslon and io~l-exchenge column chromatography. The endo~er~'~u,z SI9 cell y-lubulin migrated t'a~e~ Ihnn Chlamydnmon~x -p.tubulin with an appeN, nl molecular ma~s or 49 kDa on ~els. Annlyw~x o~ gel filtration and sucrose density gmdlent centdirugatiun showed the. while overexprelsed Chlael.w~m~m~ts y..lubulln was pse~-nl in a mo~omerlc form, endo~-,nous 7-tuhulln from $19 and llaLa cell~ extols •~ • dlraer. ~ r~sulls m=y sug~xt the possibility that "plubulin ¢ouhl foal a hete~dlme¢ with hilhe~1o unknown Va~ilev, A., Kimble. M.. Silflow, C. D., I~Voie, M., and Kurlylma, R. ./oumal orCell Setonce 108:101~3.1092, 1995. O~her suppofl: Nallonai Institutes of Health. From the Depa~men( of C¢11 Biology and Neuroanatomy, Univerdty of Minn~o~, Minneapolis, MN, Deparlmen! of Genetics and Cell Btololy, Univer¢lty of Minnesota, St. Paul. MN, and Depa,mem of Plant Biolo|y. Univer~ity of Minaes~xa. S~. P~ul. MN. FACTORS DETERMINING SPI~CIFICITY OF SIGNAL TRANSDUCTION BY G-PROTEIN-COUPL~D RECEPTORS: ~u~ RECEI'TO~ TO Among suhfamilics of G-proteln-coupled r~l~ors, ag~|sts initiate several ~II signulin~ eve•L• dCl',ending on tl~e z'~'el~oe subtype (R) and the lYi~ of G-pat,,in (~i) or effcetoe molecule (E) expressed in a p~tieulas cell. Determinants of stln~llng specificity/efT¢iency may operale al ~ R-G interface, where events are influ~n¢,ed by cell architeclure o¢ ,~ccessoey p~zein~ round in the Rcq~or's mlero~nvirt~mem, This issue was addressed by characterizing signal eransf¢¢ from R to O rollowln$ 93
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0 sla~¢ expression of the ~ ndmncrgi¢ receptor in two diff~nl ~m~ne envlr~- ~in8 to ~s G-~cins in ~h ~11 ly~ ~s elimin~led by ~nussi~ toxin ~l{~t I~ R~ signal t~nsFer ~o~d by ~litul~ flf cell mum- latin of G-~ein= in the two different envlr~ment~. In this siBnal ~y. ~isl-i~ ~tivati~ of G w~; 3-9.r~d 8mater in ~-12 w~h NIH-3T3 ~=~dm~rBic mce~or Iran~eclanl~. ~ ~ll-s~iFm diffemn~s tr=t~. ~ =uB~nl~ si¢n=l transfer in 1~-12 ~rx~,x NIII.~T3 tr=nsfeclams ~ ~s~¢e = 2-3-fold ~r level of ~pt(~ ¢xistinB in t~ R-G~pl~ ~i~h a~nity. 8u~y~.~'.yl im~i~phal~nsilive a~ist Nmlin~), sug~sting ~ G-~e~ in I~ t~ ~11 ty~. ~ler@nt exl~l~ or ~-12 ~t ~ NIH. 1~!~ =u~si~ 5'-O-(thi~rip~te) blndin~ In ~in G-~ein in = ~nl~. I~~ m~n~. ~ data i~i~ that t~ transfer or sibyl f~ R to G ~is~ ~ivali~ orG. Sz~ M., Km~kz, R., Dingus, J.. Wil~x. M.. ! lilde~. J. D.. a~ ~n~r, ~ M. ~ ;~1 of Biblical ~mi~ 27~25): 15269-1 ~276. ]u~ 23. I ~ ~: N~i~al lnsdluze~ o~ l~ahh. ~ ~ ~pz~nt Of ~oloEy. Medical Unlve~ily ~£ ~onth REGULATION OF %-ADRF.NERGIC RECEPTOR EXPRESSION AND $~GNALING IN PANCREATIC 13-CELI~ Activation of %-adrener~ic receptor~ (%-AR) in pancreatic 13-cells inhibits inmlln seC~lion in re~lxmte to vxdons slimuli, and acule or long-term r~ulslion of e=-AR receptor.mediated effects may influence the tissue response to glucose dlshorneo~i~ As xn initial aplXOach to this issue, we delermined the m't'¢¢1 of vuri- mac metabolic and hormonal treatments on %-AR expression and ¢onplir~ in the plncmati¢ 19.cell lines Hl'r-Tl$ and RIN-SAH. Radiollpnd binding studies ([~HIRX.$21002) md RNA blot analysis indlclte that both procreate I~-¢ell lir~'~ expm= ff~e ===-AR subtype [for HIT-TI$ the maximum bindin~ (B,,) = 113 -+ 28; tot RPH.SAH Bi~ ,= 93 ± lg £molt~nB ot'cellulzr prmelnl. Tre~lmem oFHrr-Tl$ or RIN-$AH ¢¢!1s with ~lucoc~llcolds Idexamelhasone, hydrocorti~me, or prnd. to,~_10~¢ (I p,M)] increased (%.AR mRNA level and rcceplor pm~cln density three. flvofold, The ~ueoc~Jco,.indu~l in~rease in receplor density in HFr.TI$ cells m sssoei~d with I) m incr~.~ i, the amount of mc~p(or~ co, p1nd to G p~ei, ~ .d.ete.rmined by analysis of hlgh.affinity 5'-guanylyl imidodiphosphalc-.,.cnsitive bindl~ of PHIUK-14304, n s~l~tive %-AR ~onisl and 2) a grcalcr inhibition or 94
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From the l~plrlmcnt or Cell Biology and N~uroanatnmy, Univer~ky of Minnelpolis. D~partment of Bioch,,mi~try. Univer~hy or Wisconsin, M~lison. WI, Immur~dcs. Cambridge. MA. and I~rlrrcnt of Bioto~y, University of" Virginia, Charlottesville. VA. REGULATORY MECIIANISMS THAT COORDINATE SKELETAL MUSCLE DIFFERENTIATION AND CELL CYCLE WITIIDRAWAL Skeletal muscle differentiation entails the coupling or mu~cle.slx:cific g~fle exxon to lermlnd withdr~wal rna'n the cell cycle..(;cvcrM mnd~ls have re.ally been pro~ which attempt to explain how regulate,,d CXlXCSsion and funcli~m or myoBenf¢ transcription fzctocs e~sums Ihel pmlircrati(ffiand diffcccnli;slion of skele- tal mu~le cells are mutually exclusive processes. l..a~tzr, A. B.t Skzp~Ic. $. X.. and Novitch. B. Current Opinion in Cell Biology 6:78~?94, 1994. Other support: National Sc|ence Foundation. Lucille P. Marhey Charltobl¢ Trust. Muzcu/ar Dystrophy Association, and the March or Dimes Birth DefeClS Foundation. From Ih© Department or Biological Chemistry and MoTccular Pharmacology, Harvard Medical School. Boston, MA. INHIBITION OF MYOGENIC DIFFERENTIATION IN PROLIFERATING M¥OBLA$'F$ BY' CYCLIN DI-DEPENDENT KINASE Although Ih¢ myogenic regulator MyoD is exprc.~cd in proliferating myohlasts, differentiation or ghost cells is limited to Ihe Go phase of the cell cycle. Forced exprecsion of cyclin DI, but n¢4 cyclins A, B, or E, inhibited Ihe ability or MyoD to transactivate muscle-specif'¢ genes and cowclated with pho~phorylatloo of MyoD. ']'ranlrcctlo~ or myoblasls with cyclin-dcpcndant kinas¢ (Cdk} inhibitors i)21 and p16 ~l~mentcd muscle-slX¢il'K: gent expression in cells mzinzained in high conccn. trttions or ~erum. suggesting that an active cyclln-Cdk comtdex suplx~s.¢s MyoD ruf~tlo~ in proliferating SEll, ok. $. X.. Rh~. J., Splcer. D. B., and La.c-.ar, A. B. $¢|e~ 267:IO22-1024. February 17. 1995. Other support: National Science Foundation, l..uctlle P. Mad~,y Charhabl~ Tn~u. Muscular Dystro~y Association, and the March of Dimes Birth Defects Foundation. From the Department of Biological Chemistry and Molecular Pharmaeolosy, Harvard Medical School, gozto~, MA, and Department of Pndiatrk: Hematole|p. Oncology, C'hildren's Hospital and Dana Fadoer Cancer Institute. Botlo¢l, MA, CORRELATION OF TERMINAL CELt, CYCLE ARREST OF SKELErrAt, MUSCLE WITI! INDUCTION OF p21 BY MyoD Skelel.I muscle dilTercnti~;on ~ntails the coordination of muscle.specific eslwe~slon and terminal wldxtmwal fn~n the cell cy¢1¢, This cell cycle an'~s! in the (3. ptms~ requires the relinohlastorna tumor suppressor proRin (Rb). ~ fu~¢tlnn of Rh is neptlvely regulated by cy¢lin-dcp¢~dent kinaces (Cdks), which are coasted hy Cdk inhihltors. Expression of MyoD, I skeletal mu~clHpeciflc transcriptional relPdalor, activated the expression of the Cdk i~ibltor p21 durin| differentlazl~ murioc myocytcs and in nonmyo~enle cells. MyoD-mediatcd induction or p~l did not require the tumor suplym~sor protein p53 and correlated with cell Cycle with. drawal. Thus, MyoD may indtR'e tom,gnat cell cycle ,,rmst during ~kelatal differentiMion by increaslnB Ihe expression of p2 I. Halcvy, O.. Novltcb, B. G.. Sptccr, D. B., Skapek, S. X., Rhee, .I., Harmon. O. ~., Beach, D., and Lassar A. B. Science 267:1015-1021, February 17, 1995. Other support: National Science Foundation. Lucille P. ~larkey Charitable Trust, Muscular Dystrophy Asr.oclat|on. and the March of Dimes Birth Defects P~ndadon. From the Department of Biological Chemislry and Molecular Pharmacolo|y. Harvard Medical School. Boston, MA, Department of Pediatric HamatolosY- Oncology, Childrcn'a Hcepital and Dana Father C'anccr Institute, Bceton. MA. and Howard Hughes Medical Instilute, Cold Spring Harbor Laboratory, Cold SpHn| Harb0¢, DEREGULATED EXPR~,S$1ON OF E2F-I INDUCES $-PHA$p- ENTRY AND LEADS 1'O APOPTOSIS ~F-I, the fi~l ge~ ~t idcmlfi~ ~g a family or ~F fact~, is thou~h~ to plW • ~itical role in G~ pmeRlsion of the cell ~d~i~! ~tivki~ ~ E2F am m~ul~ d~ng the ~I1 ~le, mainly ~ t~ f~ati~ of c~cxes ~n ~Fa~ ~m[ key R~ulat~ ~11 ¢~e s~
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the rctinoblastoma protein and mlared prole;n.~. To ~unher unde~tend the roles o£ E2F in t~ ~11 cycle ~g~si~, wc have ove~xpres~d exo~s ~F-I ~ using a t~c~li~-~t~ll~ ex~ion system. We ~ve f(~ that the i~u~d ex~s- si~ o~ E2F-I in R~-2 fi~asts ~m~cs S.pha~ ent~ and su~ntly le~d~ to ~i~, ~ ~osis ~ in an ~F.I do~-de~ndeot manner. Cell; ~i~lnnt ~o lhe I~tion o~ ~l~i; ~ve !~ the ahilily Io exp~ss ex~e~s E2F-I. Cell~ ~winI in low serum ~ mo~ sensilive to Ihe E2F-l-medla/ed cell de•lb. x~i~ of ~,F-! mulants Ihai implir DNA hi~ing ~ l~ns~livalinn d~s nm ~llcr cell c~ic ~ss~ or indu~ =~plosis. ~cse ~sulls de~ a ~vel ~lhway to ~ptmi~ and de~t~te that ~matum S-phn~e em~ is ~soci~ted with a~ ~ll ~ath. M~ul~r a~ ~llular Biology 14~ 12):~ I~-glT~, ~m~r 1~4. ~r supS: Nat~al Institutes o~ lie•lib. ~ t~ Center F~ Mol~ular M~i~nstitute of Biot~f~y, Unive~ity or Tex~ Health ~ ~nter at San Antoni~ San Anl~io, TX. DISSOCIATION OF P34:'~ C'OMPI.ffX FORMATION itROM PHOSPHORY~TION AND HIS~NE HI KINASE A~IVI~ ~ ~ll ¢~ic inhibit~ m~;~ was u~d m exami~ t~ ~tivat~ o~ t~ ~ ~cin kin~ ~ S ~a~ or t~ ~11 c~cle. Additi~ or mlm~ine to cyclin~ e#t~lial ~lls halt~ ¢~11 c~c t~ve~e in 5 phil, coi~ident with an inhiblti~ o~ ~ his~ HI ki~ ~ivlt~. Mi~i~ tmatm~t did ~ alter ~4--' synthesis ~ tu~v~ howe~r, ~11 ~p~i~ o~ p~ was d~ to ~ar u~c- t~oMe l¢~ls, Althou~ •ctivity o~ p34~ was inhibitS, I~ abilit~ or the p~ein to f~ hiih ~ul~ w~t ~p~x.. a ~o~n~ nss~ia~d with kin~ ~i- v~ti~ in ~4~, ~ ~ a~. ~ ~ts ~dlcate that p~'~ ~p~x f~ati~ ~n ~r in t~ a~ or ~lal~ ~d that p~lati~ of p3~ is I~n ~ui~ [o ~ivatc the~ ~r~d ~exes. ~, ~. T., Fault, M. P, E~tt=, ~ Z., S~r, M.. and ~r, ~. B. 98 DIFFERENTIAL REGULATION OF P34~'~ AND P33" BY TRANSPORMINO GROWTH FACTOR.13, IN MURINE MAMMARY EPITHELIAL Cyclin.dependent klnas~s (cdks) arc • family of ~mclielna ~ I'unctionpl•y~l a critical role in cell cycle traverse. Tronsl'orming growth factor-p, ('I'13F.p,) i,i • potent growth inhibitor of epithelial c~lls. Since edks have been sulic,,ited as I~lssl- hie hioehemlcal markers for T13F-III growth inbihttlon, we inv~liti|uled tha efT~l of TGF.p~ on cdc2 and cdk2 in il normal mouse mammilzy epithelial c~ll line (MMH) and a 'TCIF.II-reslsianl MMIZ cell lin~ (BGIli.2). TGF.I3~ declea~lell newly aymhe. siT.ed ¢dc2 ix'.leln levels wtlhkl 6 h alter ,'lddilllm. Coln~klent with IhN d~rcn,ze in newly synthesized cdc2 proleln was a marked rc~hlctlon in its ahilhy to rlho,ipl.lr~, l•t¢ 5islone H I. This decrear~ in kinase activity is not du~ to a eho.n~e in ~liee(ly,~ltate levels ofc~2 protein, since retINA end total r~otein levels ~1" ,.'etc. arc f~t reduced until 12 h after T13F-ll, addilkm, This suggesls that the kinase iletivity dependent on newly syntllesii,,ed c'dc2 protein. Moreover, the protein another cyelln-dependent kina.~, c'dk2, is nol elTected by "T~F-B~ addition, hut its kinase activity is subxieniially reduced. Thus, it IIT~arl Ihiil "l~F-tl kln,'lse aciivlt)' or lx~ih ede2 and edk2 !~, distinct mechanisms. Faul~lch. M. P., Ehleri, 5. '!'.. Anders, R. A.. Burnelie. R. J.. and Ixor, !r ,loumnl or Cellular Biochemistry ~$:S 17-~26. I Other SUplmZl: U.$. Public Health Service, National Cenel~r institute, and lee Amerleen Omcer Society. From the Thoracic Diseases Rese•rch Unit, Department of Biochemtst~ and Mole~ulai" Biolol~r. Mxiyo Clink:. Rocheler. MN, end Deperlmtrll or Cill Vanderl~ll UNiversity, Nushvltle, TH. CONDITIONAL BINDING TO AND CELL, CYCLE-REGUI.I, TED INHIBITION OF CYCLIN-DEPENDENT KINASB COMPLEXES BY P27I~' Mammalian cultures prlmarily regulate cell c~lc traverse during (3,. For lresslon through G, and commitment to DNA synthesis, the •etlvlty or a I'•rnily or proteins, the c~x:lln.deperl<knt klnises (edks). is rcquimcl. Them am two primly fell" ulatory portions of 13,: (,x) Ihe 13.-G, Irensilion. which allows entry into (I1: the 13,-S lransillon, promotinl entry io DNA synthesis ind commitment Io ctll divl. siou. In the pr~r, ent menuscripl, we provide avldene~ for o'oss-lllk htlween Ihllil Iwo cell c~jcle lrsnsillons. Exlrlcls prepared from quleicent moutle mlmmll~ ephhe. lial cells •re shown lo acl in • domlnlnt m~nnor to specifically Inhibit the hislone HI klnase uclivlly of pr~fomled/active cdk2, ~1c4, cyclin A. or c~lin E oumptexis from 13,-5 cell exlracts. The in~ibliory llotlvily iilses as ec41s enler q.leacillei end decreases once c-llure~i are si|mul~ied to begin 13, trllvenle and endosenliua edk Iclivliy becomes evidcnl. This llCllvily is asslx:ialt~l with the rc|ul•ted blndlnll or cdk inhibilor p27'~ to eyclln A#edk2 kinl~c complexes upon mixiolc of till exllll~ll. Renioval of p~-?" from the quk:sccni cell extract spt~il'l~llly abolishes Iht tnhibt. lol~, effcch 111~ inhibilory nelivily umt p27~'~' hilldlng in v#ni deptnd on incubuikln
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0 0 D'I of the extracts at physiological temperature or the presem.'c of • rcduclng agorot. ~ _RSUhS wl~gest an interplay between the acquisition of quiescence, edk activity and U. lraver~. ' Eblen. S. T.. Faut~ch. M. P.. Antra. R. A.. and l,eor, R. R. Cell Growth ,$ Diff~rentiatlon 6:915-925. Augu,~t 1995. Other support: U.S. Public Health Service, Nalional Cancer Inslilute and Amerk~ Cancer Society. From the Deparlment ot Cell Biology. Vanderblh Unlvcrslty, Nashville0 TN, and Thoracic Dilates Rer.e~rch Unit, and D~parlmcnt of Biocbemislry and Molecular BioloD', Mayo Clinic. Roche~ler° MN. FUNCTIONAL COUPLING OF GLYCOSYL TRANSR~.R STEPS FOR SYNTHESIS OF GANGLIOSIDES IN GOLGI MEMBRANES FROM NEURAL RETINA CELLS The synthesis of Ihe oli~saccharide or g~ngllo~idcs is carried out in the Golgi complex by successive suW Iransfe~ to proper $1yco/ilfid acce~ms. To examine how the Ixoduct or one |lyeo~lation slq~ coupl~ with the next transfer step. the endogenous gaoglio~icl~s or Gol$i memlx~es from 14-day-old chick ¢mh~o reline were lll~ld from CMP-I'lllNeuAc or UDP-I'IlIGalNAc or UDP-|ltIGal in c~m¢ll- liona which do not allow vcsic,jlar inlcrCOml~rlmenlal I~nspotl. After s~lumli(m or ihe endo~neus acceFor capscily, labeling w~s mo~tly in the immedlale acc~Fors or lhe c'o~RsFonding lalxded sugars. However. some bl-,olcd interm~liatcs !o more ~lyoe~yl•led l~mgiio~iclca iflh~ membranes were incubated in a second step in the lxe~n~e of the necessary unlahekd sugar nucleotides. This wu I~nicularly evident in the care of memlxl~S incubaled with UDP-I'HIGal. in which meal oflhe rHlOel,ldmted lecto~ylceramkle aynlhesi;,cd in the firsl slep was convened to GM.1 m~! QD3, or to GM2 or to GDII in • ~ incoNlion slep ~ Ih~ IX~S~nce of u~. l•bded CMP-NeuAc alone, or Io~l~q" wilh UDP-GalNA¢. or together with UDP. Gel plus UDP-GalNAc. respectively. Conversion wa.,~ lime depenclonl and dilulion. indqx~dml. ~ince prior R?ons usinB Ixdeldin & indlcat~lhal Imn.,d'cr steps cal- Ily¢ed by GIINAc.T. Gel.T2. and Sial.T4 localize in the trans-Gol¢i network (TGN). ~r resulls lead !o Ihe followinB major conclusions: (a) Iransfcr sleps cat- Ily'~l¢t by G|INAc-T. Gel,T2. and S|al-T4 colonali~,¢ and m'~ funclionally coupled in lhe TON: (hJ proximal Golgl GaI.TI. SlaI.Tl. and SlaI-T2. and their correslx~din¢ Blyc, olipkl ~¢eFors. ¢Xlond Ihelr pccs~nc~ Io Ihe TGN. and (c). GalNAc-T and Sial- T2 coml~.e for • common ix>el ol'acccpIor GM3 in Ihe synlbesis of GM2 and GD3. Msxzud. M, K.. Danlolll. J. L.. and Maecioni. !I. J. F. The Journal of R|oloBical C'bemktty 27(X.'H):20207-20214. August 2.~. 1995. WHEN CELLS STOP MAKING SENSE: EFFECTS OF NONSENSE CODON$ ON RNA MITrABOUSM IN VERTEBRATE CELLS It al~Cars thai n~ orgnnkm i~ immune to the el~'cts or iton~cnse ¢~n~Imts t~n mRNA abun~nce. The study orhow non,.nee c~lo~s alter RNA m~abollsm II still at an early stage, and our current undentanding derives mor~ from Incidental vlgn~les lhan from exl~rimental underlaklngs thai addresi molecular n'~chlmhms. Ch•llengcs for the future include idontil'ylng the pne pmducla end RNA sccluenc~s Ihet function in nonsen.~ m~i,,td RNA Ires. r~.olvlng the cause and ¢onsequen~s ~ Ihcr¢ apl~rently heing mo~ thin o~ cellulm" aile and r~,ch~nlsm for no~onsc~ncdblc~! RNA loss. ~md undentandinl~ how Ihele tiles and mecha- nisms ~ Plated to both ~x~slitotiv~ 8nd spedalized I~thw~rys easing m~! mRNA decay. Maqu•t, L. F. Olher ~uppml: National ln~ilut~ o1" He•lib. From the Del~mem of Ilum~n C~nedc~, Ro~well Park Cam, er lrBlltule, Buffalo. NY. LACK OF AN EFFECr OF THE BFRCIENCY OF RNA 3'-END POR MATION ON ~E E~CY OF REMOVAL OF ~ER THE ~NAL OR ~g ~NUL~MA~ I~ON IN INTA~ ~ Ev~c~ exists f~ stud,s using intact ~lls t~t int~ ~m~al can ~ influ. • ~ ~nylati~ can ~ influenzal ~ I~ reactivity of u~tmsm tpli~ d~. ~e~ ~stdls i~icnlc that ~s within 3'4~i~1 int~s ~ fu~t~ In I~ ~moval of upsl~am int~s ~ ~11 as the f~ati~ or RNA 3" ends. Bvl~ 101
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removal Is lacking. We reporl I~ thal mutations within polyadcnylotion sequences that eJl~ ~c~ ~ inc~ 1~ e~y o£ R~A 3"-enO ~o~a(ion ~ve no ~th~ ~ ~ t~ri~ ~ ~R w~ u~! £~ I~MO i~ (~). ~1 ~mRNA h~ six inl~ I~l sti~flvely ~ by ~. ~lztive ~ n~l ~vels, ~h ot the del~i~s was £~nd IO ~du~ the n~zr a~ c~loplzsmic levels of n~clelr level o~ unp~es~d RNA 3" ~ds, and d~ase ~d RNA 3' ends. In ~lrast, Ihe su~til~ion usin~ I~eg~ o[ t~ m~l~ ~-zlobln [cne wa~ £ou~ to inc~a~ ~ inc~l~ t~ n~ar lavcl of ~e~ Y e~s. ~e t~ dill i~ales Ih~ (;) t~ ~te or 3'~n@ £~l~ ~alfy limbs l!¢ ampul. ~mRNA ~ ~d {it) t~ ~letions ~a~ a~ I~ e~Je~y or RNA 3'~ r~al~, ~ill ~h ~ the dclel~x ~ ~l~ ~X~ RNA~ in ~ c~ wM I~ an a~o~l ~tio of int~-~ntalnJn~ to ~t~ RNA £~ citer i~. ~[~, at least f~ RNA )'~/~atl~ ~ s~ing my Rfl~t I~ su~limal xt~nGths o£ I~ c~- s~i~ m8ulst~ ~ a~ rosy ¢.~t~ to ~ that ~. fl ~ t~ t~ i~~ of ~ RNA ~in~ ~ Y~ ~ati~ may ~ ~: ~.5. ~ Health ~i~ a~ Nali~al 1nMitutcs o£ I fealih. ~ ~ ~m or H~ ~l~. R~il Pa~ C~r l~6lu/¢. Buffalo, NY. INTEGRIN ~ CYTOPLASMIC DOMAIN DOMINANT NEGATIVE EFFEC'q'S REVEALED BY LY$OPHOSPHATIDIC ACID TREATMENT lnle~r;n reccplora Iocalix~ Io focal ct'mlaCl silc.~ and interact with Ihe cylrw.kclc- toa vii the/~1 cyloplasmic domain. To study the role of. this domain in wJhesion, wc have Cxp~',m~ in NIH 3T3 ~lls a eDNA temlsting of lhe interleukin 2 reoep~or a subuni! exlraocflular and transmemhrane domains, cr~oected Io the integrin fll cyto- plalmle dmnain (P-~R-#31). Since Ihe extracellular domain of. the chimeric pcmeln has no role in edhcsion, this protein could uncouple adhesion f.mm intracellular 102 events. As expected, in a cell line expressing IL2R-18I, Ihls chln~m was dirtied !o focal confa~ shez. Unexpeoledly, Ihe tells exhlbhed rtormal a~hes|oo lo (FN). Hoverer. when a rapid reors~n;zat;on of th~ eytoskelclOn wax induced uqlnl l:ym~ho~phmidic acid (LPA), IL2R.~I cells d~tacl.~d from FN in coeh,~! to wild- lype ccllr.. ~ delachmenl in resl~m~e Io I..PA coekl be prevenled with cylO~hebllin D. an inhibitor of" actin polym~izallon. ~ results imply dill I ~1 domain, which is uncoupled from edheslon, can eoml~e with the e),lopl•lmk domain of nallvc intcgrln ~1 for c~loskclelal I~leins. As I ~mS~lUl~ce. the 1131 IXolein acts as a dominant negative cKC¢lOr of edl~io, by dlm'upllng Ihe Iml. gfin-cytoskek:lo~ cut, cellOS, Smilenov. L.. Brte~wil~,. R.. and Mar~•nlonle, F, E. Molecular Bioingy of the Cell 5:1215-I223. Novcml~r 1994. Oll~r SUplx~: National In~ilule ol'G~n~ral Medical Sciences. From Ibe l:)cl=nmenls of. Pathology and Analomy and Cell Biololw, Colle|¢ of l~ysiciaoc and Surgeons, Columbia Unlvcrsily. Hew Yol'k, NY. and lnslilult of. Tt!11 ROLl1 OF 1"111! I DOMAIN IN LIOAND BINDING OFT RE HUMAN INTEGRIN We report here the an~lysi~ of polenlial ligand binding domsin~ wilhln the human inlegrin (x, sulmnit, t known eollale~lamlnin mceplor. This inlelrln is eft~. lively Mocked by the mouee menoclonal antibody I B3.I. A tinselled verllon Of II~ et subunil lacking the NH,.lerm|nal half of Ihe exlratellulm" demaln is sol rec~. sized by me~ocle~al Inlllxxly IB3.1. Pmlhermme, we have iaollted a eDNA lathing the ! domain fm~ chicken a, bearing significant homology to Ihe hum~ led ral e, seqeer,~l. Replacing the httmm ! domain wilh |Is chkkml the surfate exp~sslon of t funclin~al betemdlmer wlth endoleneus mouse ~, en NIH 3T3 cells. However, IB3.1 does not bind to the chickar,~uman chimera, demonstrating that the human ca, ! domain is required for epilope reeo|nilion, Mutatlon of Asp~ within Ihe I dooutln to alanioe resulted in surface expmtsle41 of an e/I heterodimer recognized by i B3,1 bel with markedly mdtk-'ed bindlnI to IV or lamlnin. Since • ptevieusly reported mutation of • homelol~us Asp in the Mac.! ] domain has similar e~Xl~"es, these results sugBest it etmrel rol~ for the I domain in li~mxl rocogni~ion for Ill inlet.rln e subenlts co, raisinI this domain. Kern, A., BriesewJtT.. R., ]Bank, I.. and Marcanlonio, !~ !~ The Journal or Biological Chemistry 269(36):22S t 1-225 t 6, September 9, 1994. Other SUl~i~l: Natk.,nal Institutes of Health. From the Departments of Pathology and Anatomy and Cell Biology, Colle~I or i~yslcians and Surgeons, U"ohJmb;.a Unlvcrsity, New York, ~Y, and I)epartrtwnt of Medk:ino. Cha|rn Sheb;i Medical Center, Tel Hashemer, Israel. 103
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0 AN INTERNALIZATION MOTIF IS CREATED IN TIlE CYTOPLASMIC DOMAIN OFTHE TRANSFERRIN RECEPTOR BY SUBSTITUTION OF A TYRO~INE AT THE FIRST POSITION OF A PREDICTED TIGI IT TURN Receplors are intemali~d from lhe plasma membrane at -I0 llm~s the rate of bulk membrane. Th~ predominant model for the molif that promotes r~pid int~mal- izati~ ~ a requircmcnt rot a lyro~ifl¢ located in the first position of a tight tame In this repmt wc show Ihat an intcmaliz.atlon motlf can he created de nol~ by substituting a tyrosinc for the first or last residues o1" s tetrapeptlc/c GDNS {rcsidue~ 31.34) I~ll ix I'~'edleted to form a light turn within Ihe cyt<~plahm;c domain of the human transterrin receptor. There substitutions restore wild-lypc levels o1" inlemal- izat|o, to Ir~nxtcrTin race, ors that are poorly internalized due to mis.,.cnre mutations in the native internalization modf. The imrt~Juction of a tyrosinc at Ihe first or fast pr~|tion of the GDNS letrupep(ide in a transt'errln receptor coal,tiling an u~il'ied wild.type |nlemalizali(m mollf signlflc'amly incma~s Ihe inlemaliT~lkm lute that o1" the wild.type reccplor. Our rc~lls indlcalc lh~! a fum:li~mal n.vcl inlernaliz.'~. lion moltl" can he creeled hy placin~ ,~¢cit~c ammallc amlnn ~ids wilhin the overall sideline o1" an existing I~-Ium in a cytoplasmic dumaln uf a roccl~or. i'ytowdd. B., Juclge, T, W., and MeG, raw, T. R. The ]o~mei of Biological Chemlsl~j 270( 16):9067.9(Yt~. April 21, ! 995. Other suppoe: American Item Associalion. New York City a~llate. From the Department of Pathology, Columbia Univer~ily College of Phy:;iclans and Sc,3eous, New York. NY. HUMAN 11..-3 RECEPTOR SIGNALING: RAPID INDUCTI~3N OF PHOSPHATIDYLCHOLINE HYDROLYSIS IS INDEPENDENT OF PROTEIN KINASE C BUT DEPENDENT ON TYROSINE PIlOSPIIORYLATION IN TRANSFECi~D NIH 3T3 CELLS Although tyrosine kinares are cleady activaled allot li£and enga~emenl ot" lee humm iI~3R in both hcmatopoiclle and nonhematopoiclic cytoplasmic environ- me, his, a role for phosphollpid hydrolysis Imd prolcin kinare C in IL-3R signal Irans- duction is eme~ing. We I~ve used NIH 3T3 cells transiently transfected with human II.,-3R ~- and I~-suhueits to study phosphatidylchuline hydrolysis in responre to human IL-3. We have found that NIH 3T3 cells expressing the ¢omplele human IU.3R respoed td human IL-3 with a rapid and suminecl increase in glycerol. Aceompenyin~ this w~s a rapid inc,~ea~e in intracellulm" levels of" phospho- ~jleholl~e. The protein kinase C inhibilo~ H.?, however, was not effective in inhibit- ing ph0~phatldylcholine hydrolysis in response In human IL-3 in NIH 3T3 cells expres,.in$ tl~ human receptor. Thus Ihe human II.-.'IR induces a rapid klnmm.C-independenl hydrolysis that ix in conlrasl to the slaw protcln-klnase-C- dependent increase in phosphetldylcholine hydmlysls induced by the murlnc recap. Ior. Simultaneous wilh lhe increase in diacylBlyccrol levels w[~ un ingrate in mem- brane-bound protein kinare C en~.ymc sultrily. Immunoblntllng wilh isol'orm- !04 spaCil'¢ Abs a~tn~t pmlein kinase C showed Ih~l, whereas the E-iserorm b ,:e~litu- lively membrane bound. Ihe e.lsofonn of protein kinase C is Irandocate~ w the membrane in response !o IU.3. Activalion of phosphatidylcholine hydrelyill pm~eln kinase C activa|ion requimcl lx~h c,- and 19-receptor subunlts. To Ihe r~ladon~hip of tyrc~ine phoepho~latlon IO the ~ctlvallon of Id~phelidyld~llne hydrolysis and pro(ein kinase C Iranslocalion, we used lhe I~fk: and unrelaled lymslne klnase inhibilors genisiein and hc~oimycin. Both iuhihilors clue- lively blocked human IL-3-induced lyrosine pho~phorylalion. In addition, both inhibhors I~ncked phosphafidylchollnc hydrolysis and I~O~ein klnase C Iran~loca- lion. 'i~er~ dale. comhlned with our previous Rport showing th~ r.jun |nduclion by IL-3 ix del~ndent on protein kinare C. suggest thai. in hematopol¢il¢ and non. hemal(q)OlClic celb expressing the human ILo3R. pho~phalldylcholine kydroly~is and protein klnase C are downstream eft'actors of 13~osine: phot~plx~,lalion in the IL- 3 signal lran.,~luctioe cescnde resulting in immedial¢ early responre ~ene induction. Ran. P.. Kitamura, T.. Miyajlma, A., end M~tllon, IL A. The Jr~mal of Immunology I.~1:1664-1674. 1995. Olher support: U.S. Public Health Service. From the llolland Lalx)ratory for BioMedic-I Science. American Rid Rockville, MD. and DNAX Research lnstltule. PUle Alto. CA. A MEMBRANE PROXIMAL DOMAIN OF THE HUMAN INTERLEUKIN-3 • RECEPTOR I~ SUBUNITTNAT SIGNALS DNA SYNTHESIS IN NIH 3T3 CELLS SPECIFICALLY BINDS A COMPLEX OFSRC AND JANUS FAMILY TYROSINE KINASES AND PHOSPHATIDYLINO$1TOL 3-KINASE The hi~,h affinity human iniedeukin-3 receiver is a helerodin~dc protein ing or an c~ and 13, sulxmit. The I~ subonil is reslx~nsible for rece~or lilnal tramdu¢- lion. We have shovm that a membrane proximal domain of Ihe eyloplasmk~ Ltll of the human 13, subuni! (amino acids 451-51"/} ix minimally requir~l for human IL-~ ai~n,q DNA synlhesis in quies~nl lransreeted NIH 3T~ cell~, Glulutht~a ferase (GST) fusi0~ Ixotei~s of Ihis 45 I-517 regio~ and another regicxt 45 I-~62 that includes an acidic domain IX~ViOUsly shown in o~her rm:el~ors lo bind Sr~ finally kin~es m conslre~ted. Purified Lyn and Ldc Iclnare. but not Fes. could pho~pho- ~lale tyrosines in bulb domains. Adsorption with lysatea Crorn the human 11.-3- dependent hemelopo~le cell lirm CrF-I) or 313 cells and in v/~ro phosM~>rylulon showed that bolh th~e domainz we~ intm~ely phoq~ho~laled. Phceph04mlno acid analysis, however, revealed Ihat the ma~)rity of pbusphoqlatk)n was on f~lna thRoqine mher th~n tyro~ine. Ad~mq~ion of these domaim wlth 3T3 or 17-1 cell ly,lc~, follow¢~l by immunoblott~ng, showed that c:doplasm|c lyTos|ne klrm~a Lyn. Fes. and JAK-2 could aim alably associate with both domains; howevm'. Sre family k;nuses -',re more strongly recognized by both regio~s than JAK-2 klnn~e. In 8ddJ. lion. FIm~phatldyl;ncsitol .'t-klna~c from cell lysates wax nile round sL~bly with there domains, hut GTI~t~¢ aetivalini r,~olein. Yav. $o~1. o¢ (3r~ w~-te i
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0 0 0 RI~ P. and Mutton. R. A. The Journal of Binlo~ical Cbemlsm~ 270(12):6986-6893. Mamh 24. 1995. Other support: Naliunll Institutes of" tlealth. ~' From the Holland Laboratory for BioMedical Science, American Red Cross, Rockville, MD. ET~ PROTEINS: NEW FACT'ORS TI IAT REGUI.ATE IMMUNOGLOBULIN HEAVY.CHAIN GENE EXPRESSION We used a DNA-pmtein interaction screening melh~l to L-,nlate a cDNA. Erg.3. who"~ pmducl hinds to • site, designated 'a', presell in Ihe immuno~l¢~dln (ig) hsevpehain gent enhancer. F.,rg-3 is m alternatively spliced product of the err Bane and eOnllins •n Ell DNA-blndin~ domain. Ri-! and PU, I, relsled Bs proteins, alse bind to the same site. In addilion, PU.I bir, ds to • ~:cnnd site, designated pR, in the Ii heavy-chain enh~lcer. We demonstrate that the .n" hlndlnB site is crucial f~r lg holvy.chzin ~me enha~ce~ function. In ~lditicm, we show IhIt ERR-3 and FILl, but not PU.I, can lCliVlle I relx)rler ¢onstrucl eonlainin¢ a mullimer of ixnteln.blnding .sEes, s~ically with helix-loop-helix protein El2. We discuss how comhinator. zal interaczmns between members of the helix-loop-helix and Ets families may account for Ihe tissue specificity of these proteins. Riv~l, R. R., $1ulver, M. H., Steenbur~en, R., and Murre, C. Molecular aM Ceilubr Biology 13:7163.7169, November 1993. Other support: Natio,sl Institutes of Health and the Searie Community TrusL From the l:)cTwlmenl Or BiolofM, University of Califomla. Sin Dic~o, La Jolll, CA. E'2A PROTEINS ARE REQUIRED PeR PROPER B CFJJ. DEVELOPMENT AND INITIATION OF IMMUNOGLOBULIN GENE RF.ARRANGEMENTS El2 and E47 arc two helix-loophellx transcription faclo~ lhat arise by altern-- live spllein¢ of the E2A Cent. 13olh hive heel implicated in Ihc regulation of intrnuno~lc~)ulin ~ expression. We have now generated E2A (-/-) mice hy geuc targeting. E2A.null motsnt mice fail to generate mature B cells. The anc.,.t of B cell development occurs at •n eddy stile, since no immunoclobulin DJ rcarran~ments can be detected in homozy~ous mutant mice. While immunoglobulin germline I, RAGol. mb.I, CDIg, and X5 transcripts are dramatically reduced in fetal livers of E2A (.../-) mice. 1329 and p.* transcripts are present, bul It lower levels. In ~dition, 106 we sbuw that P-t-5 transcripts am si¢nil'tcantly reduced in f~.al livers of II~A (.-/-) mice. The.~ dIl• iul[P~est a crucial role fo~ E2A products IS central reSUlStO~ In early B cell differentialion. Ball, O., M~ndag, E. C. R., IT.on, D. J., Amsen, D., Kmisbedc, A. M., W~intrauh. B. C., Krop, !., SchlL, csel, M. $., Fceney, A. J., van Roo~, M., van der Valk, M., te Riele, H. P..l., Berus, A., and Murre, C. Cell 79:!?85-g92, Dcecmher 2, 1994. Other supporl: Haliunal lnstltutos of Heullh, Searle Community TnJst, Dutch C'lncer Soclcty, •nd the He~berllnds OrBanlz|tiun for Pure Re~e.arch. From the Delx~dment of Billet, v, Univers;ty of California, 9an O|elo, La/ella, CA. DClmrlment o~ Mntec,dar Genetics, Depmlment of lmmunnh~W. The Nelherland~ Cancer Institute, ~m.~terdam, The Netherlands, Department of Immunology, The Scripps Research Institute, La Jolla. CA, and Depaftments of MediCine and Mnlccular Bink~.y ,~nd Oenetics, The .Tohns HopklnA Universily School of Medicine. Baltimore, MD. ACTIVATION OFA TRANSPECTED FGFR-I RECEFrOR IN MADIN-DARIBY EPITHELIAL CELLS RESULTS IN A REVERSIBLE LOSS OF EPITHELIAl` PROPERTIES Besic fitm}blIst growth factor (bFGF) is a potent mito~en for • wide v~rlety or tell types derived lrrom me~xlenn arK! neureeetodecm. The ~llvit), of hi'OF is m~ll- lied by several lyp~ or closely rcb4ed receptors bulonBinI Io th~ 13~'eti~*~ki~L~e family of Rceptors. We have f'o~nd thet Madin-Dadoy qdthellal celll (MDC"eO do nol seem to produce bFGF or bFGF receptors. Hi~,h level eXlXeSslon of humm bPOF eDNA in these cells did not produce any mlto~enlc or morpholo$1eIl effu¢ls. Exprel~ion of the mome-derlved eDNA encodin¢ R3F mceptm'-! (POPR-I) in MDCK cells sesulled in the •equ|s|llo~ of • fibrobl•s~-Iike morphololy when the lransrected cells were cultured at low density in the Ixcseuce of 0,6~ ~elul ellr serum and 20 nghnl bFGF. Acidic fibroblast |rowth racier (aPOF) tile indueld tkese mOrl~olo~ical chan~es but nol keralinneylc ~j~wlh factor. TI~ moqg~o$1¢al effect was nol accoml~tnied by increased hFGF..induced cell l~otifemlio~ lind did nol rcsu|t in the loss of epithelial cell mlrkers ~uch as cyloken~lins, However, lha morpbuTogical tran~iliun was uc¢Oml~nied by chun~es in the intracellular dlstdbu- lion of aclin. In spite of the~ chanEe~ the Iransfected cells l'onned monolaytrs evt~ in the presence of hFGF, Ccexprcsstoo of bFGF and I~PRol in the MDCK tells resulted in similsr moq~hologlcul effect~ that were not dependent upon eso~Amous hFGF. Thexe morldmlr~ie~l effects were mimlcEed by exlx~ure of MDCK cells to either orlhovamKlate or phorbol ester. Parental rand PGFR-I expresslnI MDCK cells formed monol•yers that displayed hieh electrical resistance. Incubation of monolIyers of FGFR-I.transfecled cells with bFGF resulted in Ihe loll of trans. epithelial reslst-',nce. Monolayers or porentll MDCK cells did not Io~ their Items. epithelial resistance in response to bPGF, •lthou&h exposure Io phorbol tiler did I07
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O f~Sutt in the Io~i of'their trans.ep~theliat resistance, indicating that the effects on the I~lial I~sistance are rnadladed by prmcJn kina~ C activdtinn. |nterestioGiy. orthovinadile did no~ ¢~use a loss oF tfansephhetial resistance, soGCextin$ that the lOSs of tranr,-ep~thelial resistance is separable from the morpha,otoi,~ica! transltinn. MIBdal, M., Soker, S.. Yardcm, Y., sod ~Teureld, .toumal of'Cellular Physlofo~ 162:2~6.276. From the Dcpmlm~nt of BioloGy. Techelon. I,~a~! Inslitule of" TechnoloL,.y. Israel. and D~partmcnt o1" Chemical ImmunoloGy. Weixmann Institute of" Science. Rehovot. israel. TIlE BCL,-2 FAMILY OF PROTEINS: REGULATORS OF CEt.I. DEATil AND SURVIVAL The B¢1-2 protein inhibits alx)ixo~is induced hy • variety of signals, in a range of C~II types and in diver~,e orsani~mr,, ,,nd it i~ implicated in Ixah normal ck:vclop. meat and oneoGenesl,;. Deq~;te thi~ centr-I role, the mechanism of aclion of Ocl-2 is no~ ~ ek:ar. Recent studies have uncovered a numher of Bcl-2-rela~'d Cene pred- uct.~ Ihat reSu|ate apoptosis either ne&ativcly or Im~itively, and Bcl-2 forms het- erodimcr~ with at I~LSt ooc oF ttK~c prot¢ios, I~-x. "l~is article disuse, ca the rote or the Bc1-2 family of proteins in the litht ofthe~ findings. 1'rends in Cell Biology 4( 11 ):399-403, November 1994. Other support: National Institutes of Health, American Cancer Society, and AASTROM Bio~:ier, ces. Rrom the ~menta of Pa~helo~v and imemal Medicine, University of Michi~n Medk:al SdK)ol, Am Arbor, MI. ALTERATIONS IN DIFFERENTIATION AND BEHAVIOR OF MONOCYTIC PHAOOCYTES IN TRANSGENIC MICE TI.~AT EXPRF.~$ DOMINANT SUPPRF.~OR.~ OF RAS S|GNAI.ING • r~ address the role or r~ signaling in monocytlc ph-,gocyte~ in vivo, the exl}r¢~in/s of .two dominant suptxcssor~ of in ~,/lrn rax signaling pathways, the ear- hexyl.termlnd reBion of the GTPa.-,e-activatin~ pmlein (GAP-C) and the DNA hind- in| domain o1" the mm.~riplion factra" eL~-2, were ta~Betnd to this cell c(xnpartment. A $-k5 po~inn or the human ¢./ms proximal promoter w~.,; ~own to direct expres. ,,ion of the thmsBenes to the mooucyt~c ilnea~. At a result of the GAP-C expes~in~, ~s.-GTP I~vels were reduced in mstur~ lab-ritual macn:~hil~s by The tef~inal diff~mlali~ or ~yt. was alter. ~ evi~ ~ t~ ~umula. ti~ or atyp~t ~ ~llx in t~ b~. Mature ~d~l ~~ it~ chanBes in colony~tlmulatlnS r~lor I-dc~dent survival sad ~r, 9x~ssi~ of t~ cot~y-stimulatioG f~t~ I-sttmulat~ ~e~ umkine~ that ms ~li~ is critical in m~ic ~lls after t~ ~lls ha~ I~ t~ ~in. D. L, ~a~, 5. B., R~dy, M. A.. Schenkm=n. D,, and ~t~wsk~ M. C, Milk,at ~ Cellular B~lo~ I~(2):6~-703, Fe~ I~. Ot~ ~p~: N~i~al ~nslitutes or Health. From the Department of Microbiology and Dep~rtment or Pathalol~, Duke Unive~il~ M~icat ~nler, Du~. NC QUANTIFICATION OF DNA-PROTEIN INTERACTION BY UV CROSSLINKINO Measurement of the affinity of a protein I'or a promoter s~quen~ is ~rhleal when assessing its potential to r~Bulate transeriptlon. Here we n~or~ that the DNA prolein cro~slinkioG (DPC) assay can I~ u~d to meaa~tve aiTinlt:v, amount stud mole. cufar weiBht or DNA blndin~ proteins to specit'E: and non-specific DHA ~,qucne~. B), appl},ioG a theoretical anal),sls to evaluate the bindin~ data, it ~ shown that tll~ aft;nit)' constants or. two pro(elns (named DPC'80 and DI~IO?) to the M'r3 re|Ion of the moose th),m|dine klnase promoter were 2 X 10" M. whtch is lip times hllher than to non.spceil'K: DNA. Similar affinEy comtants were round when the p~urifled pro~elns corresponding to DPCS0 and DPCI0? imtead of nuclear extracts were uted to aue.ss the reliahilit)' of the DP~ assay. A value for cfo~linkE'L$ efficiency was detonn;ned as 0.07, howev~, it is no~ needed for computat;en of the DHA.pmtein alTinit),, but with it the •hind•nee of a h;nd;n$ pm~ein can he estimated. In sum- mary. the DPC am), is u.,ml'ul for quantifyinl~ DNA binding pmtelns and thereby jedBin$ their inllucnce on transcription. Molnar, G,, O'Lenry, N., Par(lee, A. I~., nnd Bradl~-~, D. Nucicin ^cids Rex~•n:h 2.'t(l~):.'1.'lllt..'t~26, 199.';. Other support: N~ional Institutes of llealth. From Ihe Danu-Farher Cancer tnslitute, Division of Cell Growth and ReBut•finn. BoslOO, MA, and BioMolecular Assays, Inc., Wot,,m,
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0 UI IDENTIPICAT~ON OF THE IN VITRO PHO~PHORYLATION SITES ON G~. MEDIATFJ:) BY PI~O'' Overexpressio~ of pp6(Y" in mouse fibroblasts potentiates both agonist. induced Ilpllliq thcough/~.adrene~i¢ recep(o~ and cyclic AMP accumulation in re.spOn~ to cholera toxin |Bad,man, Wil.,~,l, Luttrcll. Moy~rs and Pa:~ (1990) Pro¢. Natl. Acid. Sci. U.S.A. |7o 7462-746~, Moyers. Bouton and Parson~ (1993) Mol. C~il. Biol. 13, 2391-2400]. In reconstitutlon ¢xperiment.~n vltr~. tlo~ oT G,~ by immune.complexed p~ - resulted in enh•ncc(I rates of reccp{or- modi•ted iPJan~sin~ 5'-|7-thioltripho~phatc (GTPISI) hindin¢ aml GTP hydrolysis Illau~k~tY. Pitch~r. Lulcrell. Li~J~r, Kur~.e, Parsons, Canto and L~owitz Prec. Natl. Acad. Sci. U.S.A. 89, 5720-5?24]. These Rsults suggest Ihat one mecha- nism by which plxhO"~" affects si~nalllnB through the/~.adrencrBic receptor is hy ~phoryl•tion m~d tuncfi(mal altcratffm o1" the G protein. To clucklalc how phoP/lition of G, mlcht affect its function, wc xubjccled pl~phorylul,~l, rccomhi- nlnt G. to t~pti¢ phosPhol:cpdde malysis. Phospl~rylxlc lX:l~i~s were purified. by h.p.Lc, and analysed by Edman d~radatlon to determine the cycle numbers at which nldiolshell~d phospho{yro~ine wax released. Candidate pcptldcs that con- ~llnnd Tyr Rsidt~_ at the ¢orrespondin¢ po.~itions were synthesized, pho~phorylatcd In v/t~ by P~', lad their migrations in two-dimensional eleclrophomsi.VLI.c. were ¢orl~ with tho~e of tnJl~i¢ pho~phopcptid,~s from intact G,. We relx)rt here lhlt G,, k pho~phorylated on two residues by pp60 ", namely. Tyr-3? and Tyr-377. Tyr-3?lles near the site of I]Y binding in the N.terminus. within • regio~ postulated le modulate GDP dissociation and aclivation by GTP IJohn.-.o~, Dhanssekaran. OUl~•, Lowndes, Vaillaneou~ and Ruoho (1991) J. Cell Biochem. 47, 136-146h whi~e 'P~ is k)ca~ed in the extreme C.lerminus, wilhln a region of G. import•hi for re0el~or interaction [Sullivan, Miller, Masters. Beiderman, Held•man and Bmcne (19~'/) Nature (London) 3~1, ?12-?151. The location ol" these R~idu~ sug- ~l~s that pholi>ho~latio¢l may affect the funclion of bolh of lhe~e regulatory Moyer~, J. S.. Lindcr, M. E,, Shannon. J. D., and Parso~.~, S. J. Biochemie,a $ounm1305:41 !-417,1995. Other sul:~x~l: National Institu{~ of Health and the University of" Virginia Pratt From the Oel~lm~n| or Microbiolo~, Molecular Biology Institute and Cancer Center, ¢Mivcc~ity of" Virginia Health Sciences Center, Cherlol~csville, VA, and DepaRme~t of Cell Biology and Physiology. Washington University School of Midicin~, ~1. Louis. MO. , USE OF MONOC1,ONAL ANTI-LIGIIT SUBUNIT ANTIBODIF.~ TO STUDY THE ~'rRucTuRE AND FUNCI'ION OF THE EIVTAMOEBA III~TOLYTICA GAIJGALHAC ADHERENCE LEC~N lal~ctote (Oal) lad N-ac~ylgalacto~mln¢ (GalNAc).spccific surl'acc Icctln. The II0 la:lin is a heter~imeri¢ Ixo~in comlx~d of ~vy ( su~nils link~ ~ disulfide ~ds. ~l~l~al #ud m~l~al lnli~l~ (mAb) ~i~ against a lig~ ~it-glutathi~-l~nsfe~ ~s~ ~ein ~ its stmci~ and fusion. F~r li~l su~nit-~iY¢ mAb which Rcognized dislincl epilo~ on five differenl light Immu~b~s with t~ mAh ~stm~ ~miBmti~ ~n ~ ~he ~teins ~ anuly~ by I~ su~nits do ~ exist f~ of l~ ~te~i~r in ~avy su~nh .nti~l~s h~l ~vi~y ~ s~n to alter ~l~m~. and.lisht dmle mcngnh~ domain, McC~. J. L. W~ver. A. M.. ~ Peld, W. A., Jr. Gl~m~u~le Jonm=l I 1:4~2~76. I~4. ~r such: Nali~al Insdlulc~ o~ ll~hh, From lhe ~parlmenls of M~icine. Microbiology and P~lholo~y. Unlve~ky Vir¢ini~ ~h~l or M~, ~d~ville. VA, CllARACTERI?ATION OF REGULATORY ELEMENTS IN TllR 5% FLANKING RF_~ION OFTHE RATGPIlB GENE BY 5TUDIF.~ IN A PRIMARY RAT MARROW CULTURE SYS'II~M Gly,~pm{¢in (GP)III~II•, an int¢~rln comples R~nd on the awf~"e or is a receipt tot" fibrino~en aM ocher tipnds, and is invoh~d in plat•let Beca_~F. GPIlb is ~FectYcally expeetsnd in mepk~jo¢~-s, we hey• ~ndl~l the ~ - flanking teglon of the mt (r) OPllb lcne as • medal of a mepkemjoc)~te.~etfk: gene. The sludges presented hem used a rat marrow expr~sslo, syltem, which the study of prim•n/cells und~goln~ ks'mlnal diffc~cotiation inlo n~Alulry¢~e~. 'The delerminallon of me~k~n~ocy~=-specil'¢ expmssio~ of DNA constructs w~ possiMe by immunomagneti¢llly sel~lrating n~pk•~o¢~el from to~•l bone mw cells. Transient exprcsslb~ co~stn~ts, ¢omeinin¢ v•~jin| lenldhs of Ihe ~'. na~in~ region from -:39 to -a12 ~ Ice•lized a regul~.u~ e!ement t~)ween .-4_~. and -439 bp up~cam of the trenacfiptlonal mrs site. Th~s Rg~0~ ee~tai~s a O^ conscmus binding element betwecn -457 and -4M (GATA~,). Purther conetruets ckmo~str~¢d the~ this GAT^ binding el~nenl wu indeed e~sentla! for exprmeio~. A 2~.bp substitution, covering the region -4S0 to -426 immediately downstream of the G.ATP,~, demoostndnd that this region was es~ntial for full expr~sion, wbieh sugfcsts thai Ibis regio~ may inlerac~ with the GATA~. site in pmmotlng hil[h-lcvel linepce-specific expre~slcm. To define regulalory elements between the QATAr,, and the tmmcfiplionat start site further, we letted additional constfu,=ta dental horn 15¢ originM -912 co~n.~ct~ each of whi~ omtalned the GATA~, but had different 50. bp del~tlon~ fro~ -4.9) ~o the start sil¢. Virtually all or the~ cons~'ucts c~tinued to twice the kvcJ of expt'c~Jo~ of Ihe full-Icn~th wild-tyr~ construct: tl~r~fol~, thil III
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0 re|ion may cam•in • negative regulatory element. Comparison of our.data with expression sludles i~rformed with lhe 5'-region of" the hum.',n OP|lh gent using HE].. cells. • cell lir~ wilh sorr~ megakaryocytic proi~rties, demonstrates significant dilTcmnces, which may reflect our ur~ of primary rat Ix~ne marrow celia. In parllcu- tar. our study points to Ihc importance of" the GATA,, for high levels of GPIIh expr~ui~ in developing mepkaryocytes. Block. K. L.. Ravid, K., Phung. Q. H.. and Poncx, M. Blood g4(lO):3393-3393, Hovember 15. 1994. Other support: National Institutes of" Health and The Schulmun Foundation. Prom the l~p~mn~nts of Pediatrics ~ Phxrm~cology. Univemlty nf Pennsylvania School ol" Medi¢in~ and Childr~°s Hospip, I of Phil'ad~lphia. Philadelphia. PA. and I~,parlr~'m of Biochemistry. Bostrm Universily S~h~ml or Medicine. rhythm. MA. STRUCTURAL REQUIREMENTS OF PLATELET CIIEMOKINES FOR NP-UTROPHIL ACTIVATION Using mcomblnantly exp~ssed proteins and synlheli~ peplides, we examined lh~ cm~.-'tur•~'functionzl features of the plalclct ¢hemokir~s. neulrophil-mctivalin~ p~pllde-2 (NAP2) end plat•fez factor 4 (PF4). thai wcrc im]x~"tant in their activation of" neolrophib. Previoos studies with the chemokir~ inter]~ukin-g (lUg) had shown that Ihe N-t~'mlnal region preceding the first ¢ystein~ residu~ was critical in defining neutrophil-activetin~ Ixopcrties, We examined whether NAP-2 and PF4 had similar structural mquirem~ms. In the Al•-glu-leu4rg (AELR) N-termlnus of NAP-2, sub- ~itutton of Ear R abolished Ca~" m~bilization and elasta.,~ s~cretio~. Unlike the par- eat m01ecule PF4, AELR/PF4, the hybrid.formed by replacing the N-terminal sequence of" PF4 hat"ore the first c~tein~ residue with the homologous .,~qucnc~ NAP-2. stimulated Ca~" mobilization and elastase ~cretlon. Furthermore. the effect of ~ino ~id substitutkms in the ELR motif differed from tho.~ seen with NAP-2 in that oomerved substitutions of E or R in NAP-2 abolished activity, hut only reduced negtrophH aczivedon in the hyl~d, These studies show that just as with IL-8, the N- termini of NAP-2 and PF4 am critical for high-level ncutrophil.activating function. D~enddxation studies provided information ~ receptor binding. NAP-2, which binds almost exclusively to the type 2 11~.8 receptor (IL,-gR), did not de,,~nsitize ncu- trophils to activation by IL-$ I~cau~ IL-g (:ould bind to.and activ~t.e via h~._h t.ype stud 2 IL,.gR. AEL~JI~4 q~pezrs to bind to both typ~s o~ receptors cecause ~;tlznd .eutrophils to NAP-2 w,..tivetion, but wa.,; not desensitized by NAP-2. be(~u~e it desensitized to ~nd w~s desensitized by ItJ. Thu~, although NAP-2 and AELR/I~4 s~zr~ ~ amino ~cid homology, they have different ~-cep~or afllnities. Studies were performed to define the role of the C-termini of the~ pl•telet ,.-hemlines in recap(or binding. Hcpm'in aud • mono¢lorml antibody specific for the ~p~,~m.binding domain of PPI bosh inhibited Ca~" mobilization and elx~ta..,c furlher stq~ting that the C-terminus of thes~ chemok" .meg is im__l~, ant in.r~c_~or binding. Synthetic NAP2.., failed to mobillxe Ca~', whereas PF4.,.,. aria !12 induced Ca~' mobilization and ~cretlon of' el~tase at high ~o~.'~nlmttons. i~tus~is toxin inhlbH~ ~t~il mtivzzi~ by 4~ to ~, ~biishinB z role for tein<~ ~ s~ us t~ l~Rs in ~tival;~ ~ t~ P~ C-t~ln~ li~s. Calcium mo~li~Jti~ studies using H~ ~llz ~ly zx~z~ I~ 2 l~gR s~w t~l at ]east ~ oft~ ~iv~ ~ I~ ~ C-~l~l ~d,ng !~ tim eyste~ ~idue ~ I~ C~i~s of I~ c~in~ influ- ~ their ~ul~il ~llvali~ Yam Z.. ~zng, ~., Hol~. ~, C.. Stewart. G. J.. Niew~wski. ~., n~ ~n~ BI~ 84(7):23~2339. ~zo~r !, I~. Olh~ su~: Nal~! Inslilul~ o~ Heallh. From the ~pnrlm~l o£ Physiology and zhe Sol S~y ~mm~sis Cemer. Temple Univenily Sc~I of M~ici~. ~il~lphll. PA. Rh~kn~ ~ r Bi~cchnolo~y Inc.. King ~f ~ssla. PA, ~ponm~lz or ~ialrl¢~ and I~ogy. Unive~ly ~ ~nnsyl~ania ~ of M~ki~. ~iI~1~11. a~ A CELL CYCLE-REGULATED INHIBITOR OF CYCLIN-DEPF.~DENT' KINASES Cyclln.dcpcndent kinases (Cdks) pmvlously hive b~en shown to drive ma~or cell cycle transitions in eukaryo~ic ocpnisms ranging from yell Io humans. We report hcrc the identific•tio~ of • 21t-kDa Froleln. p2g~' (inhibitor of ey~lln- dependent kina~), that binds to and inhibits the kin•so activity Cdk/cyclin c~nplexes from human cells, p2g inhibitory activity fluctuates dudr,~ cell cycle with maximal levels in G, and accumulates in G,o and G.-ar~sted ~lls. Thex¢ ~'~lts sugg~t th•t eomml ol" the G,/S Imnsilk~n may be influenced by a f~nlly of'Cdk inhibiting that inched• p2g~ and the re,:emly descrit~,d inhibltom and p16". Flcngst. L.. l~lic. V., Sllng~rland, ,~. M., Lees, E., and Reed. ~L L Proceedings of" the National Academy of Sciences USA 91:~291-~,295. Jun~ Olh~r supporl: N~lional Institutes of Health. From the Department of' Mol~uiar Biology, The Scripl~ R~areh Institute, Jolts, CA, and Laheratot7 ~ Moiceular O~cology. Massachusetts G~neral ~mcer Cemer, CharTestovm, MA. 113
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A NOVEL INHIBITOR OF CYCLIN-CDK ACTIVITY DETEC'/~D IN TRANSFORMING GROWTH FACTOR [3-ARRESTED EPITilELIAL CELLS 'rrensformin~ lrowth lictor 13 (TOF.p) it a polent inhibitor of epithelial cell lrowlh. Cyclins E and A in assoc|afion wilh C'dk2 have been .~hmvn In play a role in the O,-to*$ phase transition in mammalian cells. We have studied the effects or TGF-~,mediated lrowth in, st on G./S cyclins E and A. fnhihltion of'cyclln called kinlse by TGF.J3 it primarily due to a ~ecrease in ©yclin A mRNA and pro- lain. By ~ontrasl, while TGF.I3 inhibits icoumul~tion orcyclin E mRNA. the reduc- tion in eyclin II prolein is minimal. In•reid. w¢ find Ihal the activation or cyclin aszocl~ kinase that normally accompanies the G.-to-$ phare transition is inbibiled. A no~,el inhibitor of eyclln-Cdk complex~ was detected in TGF.F.treated cell lysates. Inhibition it mediated by a heat,stable protein Ihat tarsals both Cdk2 and Cdc2 Unaser,. In Oearresled cells, • similar inhibitor of Cdk2 klnare was detected. The.~ dala sul~est the existence of" an inhibitor of cyclin-depen~ent kin•re• induced unckr diffemm conditions to mediate antiprolirerative responses. $1in!ledand. ,l. M.. Hen$st. L.. Pan. C.-H.. Alexander, D.. StarapFer. M. R.. and Molecular and Cellular Biolocy 14(6):3683-3594. J'une 1994. Other supper1: U.$. Public Health Service. National Institutes o£ Health. and the U.$. Department of' Energy. From the Del~rlment of Molecular R|nfogy., The .~crlpps Research fnstitule. LI ]o11•, CA, end Lawrence Berkeley Lzboratonj. Life Science~z Division. University of. Calitomia. Berkeley. CA. 0 0 01 I'0 RECEPTOR TO NUCLEUS SIGNALING VIA TYROSINE PHO~JPHORYLATION OF THE Pgl TRANSCRIPTION FACTOR Rocent atndics on signal Iran~fuctlon stlmu|at~d by interrerone have defirP.d p~hw~yz th~ link cell and'ace ncoptors to Iw~ez ~.nes in the ngcleus. After blndln~, nonr~eptor lymsine kin•sea are activated that phospho~late components o£ latent DNA-bindln~ facto~ in the cytoplasm. These pill•ted fictoes form multim0fi¢ ¢ooIplexes thai Illm$1ocate to the ~J¢leu,~ and bind to sl:~ciflc DHA seque~ in the promoters of induced $~,es. A 91-kD factor (pgl or StatPl) is acti- vated by tnt~ftrerona and serves as • subunit pmrtncr in the ~mpO~il~n of dlv~se Defy, C aM Rei~., N. C, TEM ~{4):I.~-IM. 1994. Other supl~o~t: Nalioual Institutes of" Prom the l:~rtmem of" Pathology'. Stele University oF New York, Stony Brook. PHOSPHORYLATION OF EUKARYOTIC PROTEIN SYNTHFJI$ INITIAllON FACTOR 4E AT SER-209 Inili,qllO~ f'ictor 4E (elF.4B) binds to the m~'TP-conlainin| cap of. mRNA and facilitates the em~ ormRNA into the inilisllo~ cycle of I~oleln si~ clF-4E is • phosp~cm, and the pho~pho~lat~.d term binds to mRNA 3-4.fetal moee ti¢hlly th~n Ihe noflpho~phepjlated form. A prevlotm ~tudy indk'a~d Ihal the meier pho~phorylalion ,ilia was Ser-~3 (Rycbilk. W., Rus~. M. A.. and RheaS. R. F_,. (19R7) J./~it~/. C/~tm, 262.10434-10437). In the prew:nl study, we Ihe~i~.ed Ihe pho~phopeptide expceled Io result from tzTl~ie dille~tion of elF-4F, O, pl~oserylly~ine. Surprisingly. the Iry~ic and synthetic phoephopq~ld~ did not ,.c~ni¢~te e_.k~lrophoratically. AoeordinBly. we redelonnlned the pho,~pherylalion ~]te..by zsolalm~ • chymotzTl~ic phosphopelxide on reverse phase hlllh hquzd chromatography. The peplide was sequenced by Bdmmz d~|rldado~ and responded to '~$HADTATKSGSTTKNRP". The size of pheiphorylatlon de~ermlned In ~ •or-209 by four m~thods: the increase in the ratio of dehydroel~. nine Io .rerine derivatives durin¢ Edman de~radatlon, the relea~ of" ~2P, Ihe Nrther digestion of. the ebymo(n,/ptic phozl~plid~ walk trypsin. Glu-C. and Aq~.N, and she-directed mul~enesis of. elF-4E eDNA. "i'he 5209A variant was not pbo~phory. Ioted in -~ ~hhil mllculneyte lyt, lle syslem, wheRas the wild-t,yp¢. ~,q3A. and vadanl~ were. Thi~ site f-',lis within Ihe con,,angus z~quenee for pho~pborylatten by protein lain•re C .In,hi. !~.. Cal. A.-L.. Kciper. R. D., M|nieh, W. B.o Mcndex. R,, Beach. C. Stepiztski, J., Slol~r~ki. R.. I)zt~ynkiewic~,, E.. ztr~! Rhoads. R. It. The Journal of" Biolo~ic'al Chemi~lry 27(X24): 14597-1460~. June 160 1995. Olher support: N,,llonal ~nstltute of General Medical Sciences. U.S.A. and the CommillCe for Scientific Research. From the Dei~rtment of. ]31ochernistry and Molecular Bioloty, Loulatana $llte University Medical Center. Shreveporl. LA0 Macromoleculer Struetur~ Anelyziz Facility. University of" Kentucky Medical Center, Lexlnlton0 KY. and the Del~rlments of Biophysics and Cheml$1r~. Univac•by of W~rl•W, War, w, Polled. RECEPTOR-SPECIFIC INDUCTION OF INDIVIDUAL AP-I COMPONEN'I~J IN B LYMPHOCYTF_~ The induc~le oucicar transc~'|pt;on ricer co~plex. AP-I° typlcall), eoml,,ts or hetcrndimcf~ hetw,cc~ 3un and R~ pe:~eins. Although eomponen~a are drawn f~ families orR~ ~ul~ I~tle is k~ a~t t~ ~ ~ul~ti~ or~m. ~ividual family ~m~ ts ~ul~-s~ir¢ ~ w~t~ tM ~ sipalinI ~. wa~ ~ ~stbl¢ ~ ~t~ of atl su~flits, ~ ¢1~ t~ iu~s, AP- I ~- ~ms m ex~i~ ~otfow~ ~ivali~ of ~m~ B lymr~es thigh ~o ~fale ~¢~o~, the surf~ IS A¢ ~or, a~ t~ C~ ~pt~ tot T ~ll ~fl~. ~h ~s ~ ~imutati~ i~ to ex~ss~ of ]unB ~ Juno mRNA
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0 0 U'I I',3 U1 ~d proleln: hnwever, incJuclion of./unB mcdlaled by ami-l~ Ah was ~ein C (PKC).~n~m. w~ i~l~ ~di:led ~ C~O !i8~ was ~si~:m m ~C ~. ~ t~ f~s ~ stim~al~ diver~ even rufl~ w~h ms~t to ~ ~.~. AII~ ~h stimuli I~u~d c-~. ex~i~ of ~R was slim,- I~s s~ifie It ~th the mRNA a~ ~eln levels, in i~ by snli-lg ~l ~ by C~O li~. Sllmula~ cx~cs~im w~ in a~l c~¢ PKC-~em. ~ ~ul~ ~vi~ evi~n~ ~m ~e~m-s~- ~ tri~e~d ~ two su~a~ ~ Ih~h ~ral¢ ~tl~ways thai differ in PKC de~e~. ~e ]~mal of lmmu~y ! 54: 3~.~. 1~5. ~ ~: Nat~al Institutes ~ Ilethh. ~nl ~ ~in~ Re~a~h. ~ Unive~ity M~al ~ler. ~. MA. 115 stably expres.q the DER prote~n exhibit reduced ~t~ i~o t~ S ~u~ ~ I~ ~11 ~ ~ qu~t ~Hs ~ ~m ~;muta~ in l~ u~ of est--. ~h tnln~ IM role of AP! In this ~s. ~ ]~mnl ~ ~i~;~I ~mls~ 2~ 19):11 ~-11513. M~y 12. Mye~ ~ol~y Divisi~. F~ I~ Fox ~l~ C~er ~m~. ~il~l~i¢. PA. CAP-INDEI~NDENT TRAN~I,AT~ON AND IN'T~RNAL INITIATION OP ~A~ IN EUKARY~C ~R m~A MO~L~ inI cell l~wlh, Alli~. lenllkllly ml~i~llllbie lyee~ Ivlh II yllll led ~ ~ioi~al ml~ fm ~ ~ ~tnl~ exist. 117
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0 0 INITIATION OF PROTEIN SYNTIIF~IS BY TIIE EUKARYOTIC TRANSLATIONAL APPARATUS ON CIRCULAR RNAs The n'bo~ scanning rondel predicts that eukaryotic ribosomal 4&V subunil.~; onl~r all mes~nler RNAs at their 5" ends. Here, it is reporled that eukaryotle somes can initiate translation On circular RNAs. l~t only if tim RNAs comain inlet- nil ribosome entry sile elemcnls. Long-repeating polyl.~pllde chains were sy, lhe. a|lrad from RNA ¢ireks with continuous ~ rcadin~ frames. These results indicate lhat ribosc,~ can translate such RNA circles for multiple ¢ot~ecutive rounds and lhat the free 5' end of' II messenltor RNA is not necessarily the entry point for subunits. C'hen. C. md Sarnow, P. • ¢-ience 2~:415-417. April 21. 1995. Other support: National [nslitutes of ltelllth and the Lucilfe P. M+',flcey Charitable TRmt. Prom the Molocullr Biology Program. Dcpatment or Binchcmistry. Biophyslc~ and C~ncl~es, Dyde Lalx)ratoryo Onivcr~;ty or Colorado I feaith ~.'icnct...~ Center. Denver. CO. MSXI lNHIBrr~ MyoD EXPRESSfON IN FIBROBLAST x IOT~CELL HYBRIO$ 'Pransf'er or human chromosome I l. which contains the m.y~) locus, from i~- nm mcam human chromosome ! I and addttmal human chromok.,,mes fllil [o acfi- vel~ mj~D. We show lhat human chromosome 4 inhlbils m.lP~D aclivation, m~-~D . . _adu,'~lxo,~er re_porter c<msa,J~ show that repmssion is at the transcriptienal ~_v~/+..~..'~om~. +.` rrll .n.~m..-.¢on.lllinin¢ hybrids Io¢111z¢ the r¢lx~sing aclivily to me relic>n o14p mat onotalns me l~rncobox Benc MSXI. MSX| is exlx~:s~J in prl- m~'y human fl~ and in 101"~ cells containing human chromosom~ 4. while imrenlal IOT~ cells do not express Msxl. Forced expression of Msxl repasses m.yoD enhaneer activity. Msx! protein binds to the m+yoD enhllncer and likely rex, ,.,.,. ,~. we txmchxk tim MSXI t~ibits tmsc~p~ion of m.y~ ~ that J-j~D is a m'101 for hormobox Icm re&ulat;on. Woloehln. P.. ~. K., l)e~nin, C.. Kil~an~. ^. M.. Goldhemer. D. J.. Sassoon, D., Cell $2:611.620. Aueus125. 1995. + Other support: Hatlonii lrmitutcs or Health. !18 CHARAC]'ERIZATION OF THE SUBTYPE OF MUSCARINIC Rg, C~PTOR COUPLED TO THE ,~TIMULATION OF PHC~PHO1NOS1TIDE HYDROLYSIS IN 132.-INI IIUM^N A,~r'ROCYTOMA CELL.S Stimulnt;on or muscar;n;c rccCpIoP, I incrcasCS phospholno~illde (PI) hydrolysis in 1~2-1NI human aslroc)'mmu mils. To evaluate the suM~pe of re.piers which m,,~liate PI h)'dmlysis in 132-1NI cells the elT~cls of: a) the nonxelectlve M1 nist. carh~chol: h) Ihe sclcclive MI ~onist. 4.hydmxy.2-hutynyl.lrln~thylommo. nium chloride-m-chlorocarbinilllt¢ (MEN-343); ¢) the nonteleet|ve anla$onists. atropine and scopolamine; d) the relatively selective MI antlllonist, piRm~epin¢. the rellltiv¢ly ~l¢clive M2 llntll¢onim. AF-DX 116 {I piperidinyl~cetyl-5. I l.dihydro.GX-pyrido,2.3.b-l,4.henzodlazeplne.6.ene) melhoctrzmine llnd f) the relatively ~leclive M3 anta&oni~1, hexahydrceil~-dife~ldol (HHSiD) on PI hydrolysis in 132-1NI cell,, were studied. The cell pools of Inodtol- phospholipids were prelahelled by incubatin¢ 132-1N! cell¢ in I low anon|tel tulnlnB medium (CMRL-1066) supplemented with ['lt|ino~ttol (2 IxCI/ml) for 20.24 heur~ at 37"C. The cells were washed and resuspended ia a i~ydolollcel tlon. and Pi hydrolysis wus mcesurcd by accumulation of' I'tllinozitol-l-pl~zphate (IP) in the pre~ence of' 10 mM LiCI. C'adoachol produ~,d time and conc~tminn delx:ndenl PI h).dmty=is (EC30. 37 p.M). McN.A.'44~ did not cause silmifleam hydrolysls of PI i~ I ~2- ! N I cells indicatin¢ that the z~ceptor was not of M I the above mu~cmlnlc zmtag~:m|st~ caused a ¢oncentr~io~ dependent d~reaas in the level or IP in rcspon~ to ©arlxx:hol (100 IzM). The rank ~ of their idTinldes val~s) was: atropine (&~) > HHSID (7.6) > pi~zcidn¢ (6.8) • ~lhoetrm~ine (6,0) • ^F-DX 116 (5.~). This rank order suplx~s the concept thai M3 (ozher M2p. ¢lendular M2) mcelXmS am linked to PI hydmlyais in 132-1NI celia. HH$iD. which is sele¢llve for M3 reeep~or~ or Ihe smooth mul¢le ha: hilher affinity for n'~:arinic reeqxors in 132-1NI ~lls than AF-DX 116 whk:h lz selective f'or MZ Rcel~ors in ¢llrdia¢ tissue. If Ihe'meepfor in 132-1NI cells had been M2. IXUl of'the rank order for llffintties wo~ld have been medzo¢l~mine > AF.DX ! 16 > HH$1D > pircnzepine. From III1 of these obzervation~. ~ mt~l¢|dn~c receptor for P! hydroly- sis in 132-1NI cells is tentlltively ehllracterized as ot" M3 type. $1ephan, C C llnd Saslry, B. tl. R. Cellulllr and Molecular Biology .3t~(7):701-712. ! 992. 119
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Other support: The Study Cenler for Aneslhesla Toxicology of Vanderhilt Unive~sity. National Institute of Child Heallh and Iluman Development. and the Nationll Institute of General Medical Sciences. ~ the Deparlments or Pharmucoh~y and Anesthesiol(~y. Vanderhift University School or Medicine. Nashville. 'IN. EF1=I~T OF FIBROBLAST GROWTH FACTOR-I ON TIlE TIIREE- DIMENSIONAl- GROWTH AND MORPI IOGENESIS OF liUMAN SALIVARY GLAND EPITHELIAL CELLS EMBEDDED IN COLLAGEN GELS ~ resu|L~ repocled here ixovide evidence that human salivary gl*nd epilhclial e~lls were capable of ~cons~uctlng epkhelial ductlike ~ructures similar to develop- |n~ saliva~ glmxIs when ¢¢11a were embedded in a thr~-dimensional collagen gel under serum.fr~ culture conditions, in panlculur, we ot~erved tlmt FGF.I ruvocd proliferation and duct formation by H,~E cells. A similarity with salivary gland ~tro~tUreS ~ a|no Rinfore~d b~ th~ fact that secretory componen~ af~J I~"~ofen~n were identified in ductal }iSGE ~ells in collagen gels, Myoken,'Y,, Myoken, Y., @kamo~o, T., .~alo,,L D,, end Takada, K, In Vitro Cellular & Develnpn~nlal Binh)~y .11 :X4-X6. February ! ~}5. O~her ~uppm1: Jal~nese Ministry of Education, Science. and Culture. and Nulional In~itute~ of ll~lth. From the Deparlmen! of Oral & Maxillol'acial Surgery !. School of Dentistry. Hiros~ima University, Minami.ku. Hiroshima. Japan, and W. Alton Jones Cell Science Center, Lake Placid. NY. STRUCTURE AND FI..INCTION OF TRANSCRIPTION-REPAIR COUPLING FACTOR ~ ~. SmUCtU~A~. DOMAIN~ AND BINDING, PROPERTIES ~ ! 30-EDa mid |one product is required rot crmpllng transcription to repair in E~,heHcJ~ie ~e/, Mid displeoes E. (~/i RNA polymerase (Poi) s~alfed at a lesion, binds to Ihe damage recognition protein UvrA, and increases the template strand repair rate during transcription. Here, the interactions of Mfd (tran~crilxlon-repalr ¢onpl|ng factor, TREE) with DNA, RNA Pol, and UvrA wero investigated. TRCF boor~ Itonspecif~cully to double slranded DNA: blmlin~ to DNA ix~(h,ced ulternal- ins DNas¢ l-pro~ucled and -hyp~nmnsifive regions, su~ge.~tin~ im~sible wrapping or the DIqA ,,round the enzyme. Weaker binding to single stranded DI~/A and no bind- ing to single stranded RNA were observed. DNA binding required ATP, and hydrolysis of ATP promoted dissociation. Removal o~r a stalled RNA Pol also requ|rcs ATP hydrolysis. Apparently, TRCF recognizes a stalled elongation complex 120 by directly interacting with RNA Pol. since bindh~g 1o a synthetic tr~n~'ir41on hub. b~ w¢~ ~ s~ t~n bi~in~ to d~e st~ DNA, aM ~idlnI to ~ RNA Pol ~nzy~ a~ to in~iat~ a~ el~t~ ~plex~ in t~ ~ of si~ 5'-O-(th~dp~phnte) w~ o~. St~tu~ru~ ~tysi~ s~ t~ ~sid~ 379-571 am in~lv~ in bi~iog ~ ~ ~d~ RNAP. ~ ~1~ ~if~ ~¢i~, residues 571~1. bi~s to A~ ~d duplex ~lyn~i~ (DNA:DNA or DNA:RNA). Dissolution of Ih¢ lcmaw c~plex u~n hydmlysls e[ ATP ~o ~ulms I~ ca~xy~ ~in~ of ~CR ~n~lly. Rs~ues !-~?~ bi~ Io UvrA ~d ~liver I~ damage ~nilion c~nt ~ I~ exeisi~ nuck~ Io I~ Selhy~ C P. ~nd ~, A. Other supporl: National Institutes of Health ~nd the Human Frontier Science Pm~m. F~ ~ ~n~m o~ Bi~isl~ ~ B~yslcs, Univ~ity of H0~h ~lin~ ~h~! of M~i~, ~1 Hill, NC INIlIBITION OFDRO.~OPlllI.4 EGF RECEPTOR ACTIVATION ~Y 'THE SECRETED PROTEIN ARGOS T~e Drosophila bomologue of the mammalian epklennal ~rowlh factor (EOF) Rcepmr (DER) is a race,or tyrocine kinase involved in mamy tta~s o1" fly d~velop meet including photoreceptor determination, and wing-vale t'ocmation. Its pdma~y aclivating ligand is the Spitz Ixo4ein, which is similar !o mam~mallan TOle..~ (ref. 12). Argos is u sum'uteri prolein that, like Sphz, conlair~ a single EOF modf. II I$ a represser of cell determination in the eye. and acts in o~her titluel, including Ihe wing. Because ArSos hue the oppmlte effects to DER in the eye (Ihe femur bleeka phomgecep~o¢ detenninatlon, the latter prorates it) we have tesged whether I! ~ts by blackleg the DER pathway. We show that Argn~ does in&.ed tepm~ thi,, I~hw~y i~ ~t~ and find that. l, ~tro. Argo~ Ixoteln can inhlbil the aetlvat|on o1' D~R by ~plt,¢ Thus the determination of" cells by the DER pathway is regulated by a balance hetwcen extracellular activating and inhibiting signals. This is th~ flrsl in vim earn. pie of an extracellular inhibit~ ~ a roceplo~ lyrosine kinase. Schweitzer, R., Hewer, R.. Smith, R., SEiI~ B.-7.~ and Freeman, M. Nature 376:699-702, August 24. 1995. Olhcr m~ppod: Hallond Institutes or Health. Israel Academy of ~c|enc¢. and the Medical Research Council. From the Department of Molecular Genetics and Virology. Welzmann Institule of Science. Rehovol. Israel. and MRC L~boratory or Molecular Biolngy. Camlxldl~. UK. 12t
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122 SPIilNGOSINE AND SPHINGOSINE L-PIlC~PHATE IN CELLULAR PROLIFERATION: RELATIONSHIP WITH PROTEIN KINASE C AND PIIOSPIIATIDIC ACID Previous studies from our laboratory have shown thai sphln~o~inc (ZJ~ang ¢1 al. (1990) ~. Biol. Chum. 265° 76-~1) and sphingosine I-ph~ph~ate (7.,han~ ,el (1991) .I Cell Biol 114, 155-167), metabolite~ of membrane sphln$olipids, stimulate release o1" calcium from internal so~rces and increase proliferation of qules~nt Swiss 3T3 fil~oblasts acting in a fundamentally difl~rem~ p¢o~¢in kirtle C-ind~p~- dent pathway. The mitosenic effect or sphingo~ine was aceomlmnied by an Ir~maac in the levels of phosphatldlc acid (PA), a potent mltogen for a variety of cell types. that may function as an intracellular ~:coud rnessen~s- (Zhang at at. (1990) .1. Biol. Clsem.. 265. 21,309-21316). ~phingaslne also induced emty increases in sphir~odne l-phosphate (SPP) levels that preceded the increase in PA (De~mi et af. (1992) .1. Biol. Chem. 267. 23122-23128)..SPP itself prnduc~! • more rapid inc~a~ in PA. thus suggesting that it may mediate the effects of sphinge~ine on PA accumulation. The concentration dependence foe the formation of PA induced by SPP ear.fated with its effect on DNA synthesis. Similar to sphln~osine, also stimulated the activity or phospholipm~e D. "atheugh a slgnifieam effect wa~ obaceved at a much lower con. cemratkm. Ilowever, in contrast to previous relp~ts with sphin~0dne. SPP did inhlhlt the PA ph~ydmlase nctlvky in cell homo~enatce. 21m~, in addition to its effect on mobilization of calcinm. SPP can increase the level of PA, mo~t likely via activation of phosphollpase D. We smggest that SPP medlmca the effect of ~phln. go~ioc on PA accumulation in Swiss 3T3 fihrohl~ls and may r~gutate cellular pro- lifemtiou by affecting multiple tmnsm~m~'sne signaling pmhwnys. 123
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JOUrnal of L,|tdd Mediators 8:!69.17~. 199.t. O~her support: Natio~al Institutes of Health. From the Department of Biochemistry and Molecular Biology, (;eorsetown Unive~ily Medical Center, Washinctofl, DC. J~PI I|NGO$1NF.-I-PIIOSPIIATIZ. AS SF.CONI) MF.~SI-2N(';F.R IN CFJ.L PROLIFERATION INDUCED BY PDGF ANt) FCS MITOGENS Orowlh ~i|nallin¢ n~twodcx that u.~ gtycefopho~;pholipid meta~xdites al second me~nll~x have been well c4~rac~eri~d, but less ix kla)wn of the sec-nd me.~n- ~ derived l.rom sphin~,olipids, another major clas.~; el" memhmn~ lipids. A tantuliz- Ini link between xphlnsollpids and cellular prnlil'e~llofl has emerged l.rom the dis- covery that Ibe sphingolipid rnetabe~ites ~phingosine and sphiugoslnc-l-pho~phatc ¢tmulate ~¢owth o1" qule~u::ent Swlr~ .~T3 fibroblaxtx by a pathway Ihat is indcpcn- &mr o~" proleiu kinas¢ C. Sphln&o~in¢- I.pho~ph~e is rapidly ixoduced From sphin- l~0~ine and may medlale ks bloloclcal effects. Furdcrmore, sphlngosine-l-pho~T4~te IdS~era the dual $1¢nal transo'uetiofl p~thwa~ el. calium mobili~tiofl and activation nt I~lip¢~ D, prominent evemx in the control or cellular I~olil'eralion. Ilefl~ we repofl that activation el. sphln~,x~ine k.ina~ and the formation el. phe~ate are imporlant in the $iCnal tranr, duction pathways ac~ivmed by the peseta milo~ma plalclet.dedved growth lacier (PIX3F) and I'etal call'senna (F~$). Olivera, A. and Spiegel, $. Nature 365:557-560, Octoher 1993. O~her xupix~: Notional In¢itute, or Health. From the Department el. Biochemistry and Molecular Biology; Georgetown Unive~ky Medical Cen~er. Washln~lon, DC. SPHINOOLIPID$ METABOLITES: A NEW CLASS O~$ECOND M~ENOERS IN THE REGULATION OF CELL GROWTH "l'he inleractlon o1" &rowd~ l.actofs with specific cell xurl.~ce recep(ofs trig~erx mulliple imracellular signaling pathways that culminate in DNA synthesis and cell division. Orowth signaling netwo~c.,; in which glx=rophnspholi~d meta.he,~it, es, I~ diacylglycerol, inosltol 1.4.$-trisphosl~ate (In.~P,), pho~pheti(dc xio, ann nrncm. donee acid, serve as .,monad mccscn~r~ have been well churacteri~.ed. Much le~s is 124 known or the ~econd mermen&era derived from amolher major elba or memtw~ne lipidx, the sphin~olipidx. All sphin~oliplds, includin~ cetamlde, aphiu|omyelln, ce~ebrosides, gangliosldes, and tall'irides, ¢onlain (I) a le~$,.~haln xphln$old lhalr b~:kbon¢, of which sphir~oslne is the most prominent. (3) an amk~c.llnked r~tx ~-~, ~d O) • ~o~r he,d s,~p (h~,~,l ro~ ~-~,m~. !~...r,~1.d~lh~ r~ R~in~myelln, and carbohydrate resldues of varying ¢ompicxlly ~or IlY~O.li~hi .~. ~oliplds). These ublquilotzs cellular components have Ion$ beeft know~ to play all impoflant, yet und~fi~, role in cell Srowlh R~ul~ion. Spinel, S. In: Hu0 V. W. (F,d.): The Cell Cycle: Regulators. 'l'ar~ts, and CIin~al i~num I~re~s. New Yod:.pp. lit-119. 1994. Other ~ui~: National Inxtltutcs of Health. From Ihe D~partment of Bi~hemlatry anti Molecular 1~ioloBy. ~niversity Medical C~ntcr. Wa~hinBt(m. DC SPHINGOSINF_,-I-PHOSPHATE, A PUTATIVE SBCOND ME$$1~NOER, MOBILIZES CALCIUM FROM INTERNAL STORES VIA AN INO$1TOL TRISPHOSPH&'rE-INDEPENDENT PATHWAY Sphin~o~ine-l-pho~phate, a metabolite ot" ~phin~olip|de which has pfevi~dy ~ shown to stimulate DNA synthesis and cell division in quiuacent cuttufet ol. Swiss 3T3 fibrobtasls (Zhan~, H.. Dcsal. N. N.. Otivera. A.. Scki. T., Brookcr. O.. and Sple~el, $. (1991) J'. Cell Btol. 114. 155-1~7). indu,.~l a tear, slant in~reo~ in intraceltular free calcium independent of extfawellula~ calcium. The increale calcium ,was completely abolid~d when iulmcellular calcium p0ola ws~ ckpk~d with thepdr, ar¢in, an inhibitor of'the endoplasmic ~ticutum C~'.ATIhu~, The RSlX)nSe for csdeium.Rlease induced by slddns~ine-I-~.. • with the Con~ntretion recluirl~l ~ siimutallon otDHA s~il~sii, l~ nltlmtu@l lhe ¢ilchim I~Sl~ d~led with Ilive ehellollI, Illhoulh Xl~iulodnl- I. I}llo~lphlle did nol lllfnUale Ihe relltli I~ eilhei 5rldlkintn or ton, onll¢in. C0fltly, pdor slhnulalion cX dl~ cells I I:~'ad~klnln had no tlTect on lhl aphin- losirl~-i-~hosphlie-lnduced ell¢i.m .il.ll. ^ldloulh. incl~ased inollltol (l,4.S)-iri~l~l~e l~v~la, complcle mnlbilion of I to ~ ~hlle Rm, nlilon by pretr~ wlih ll-~.l~lrld~ln~ll~l~-i~lit did Ii~'d cells, I~in. an mcedol (l,4~S)-Ifalhlt Inlalonill. blocked ~i re i induced ~I i~osilol (I,4,~)-i|4Xl)hO~l~lie, I~l did r~ xittflclnlly alicf Ihi {I ult Ibe rcle.',l (if anichid(mlc acid. anolher iiinalinl mollie known !o ICa"l, witheut ino~ilol Ii~id lumoicl or calcium irifl~x. Dor i~ll llillilt mnl s~hin. tl~ilnc.l.i~l~li~ie l~ilil,:s @ll~ from inlemal alorcs Ixtmaitll lhroul5 a mt~hl- nL~l indcl'lCndenl of in~ilol Iipkl hydroly:ds and ilhldonic lld lltal Ind Ihll
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O Matt|e, M., Brooker, O., and Sp|el~el, ,~, The ./oumal of Biological Chain||try 26P(~):31gl.3181~, February 4, 1994. Od~¢ suppo~: N~ional |nat;lutes of Health. I~,om the Department of Biochemistry and Molecular Riofo~y, Georgetown University Medie~1 C'efller. Washhlgton, SPHINGOSINE I-PHOSPHAT~ A NOVEL SIGNALING MOLECULE. STIMULATES DNA BINDIN(3 ACTIVITY OF AP-I IN QUIESCENT SWISS 3T3 FIBROBLASTS Sphingo~ine and sphlngoslne I-pho~phat¢, met•halites at sl~;ngoliplds. •lima- !ate cell pro.liferatioo in quiescent Swiss 3T3 fibroblasts and induce transient tncms~$ in mtmcellular free calcium (Zhan$, H.. De•at. N. N.. Olivera, A., Sekl, T., Brooke¢. O.. and Spiegel. S. (Iggl)J. Cell Biol. 114. 155-167). However. lltllc is yet I~own of the nuclem" events that follow the early responses induced by sphlngolip~d met•hollies. Using • gel retardation assay, we found that specific DNA bindin8 a~tivil7 oTactiv•tor prolcin-I (AP-I) wa~ markedly inere~ed at~¢r trcMmcnt orqui. e".~nt Swiss 3T3 fibroblasts with sphingoslne I-phosphate and sphingosin¢. The DNA binding specificity ot" AP-! was confinnnd with competing probes containing eormen~us sequences or AP-I, AP-2, AP-3, SP-I, and NFI/CTF. The c-J'o~ ~ wig detccled in the AP.I complex using anti-c.Fos anlibody. The do~ m~ponse for atlmulstkx~ ~ DNA binding ~ctivlty or AP-1 by sphlngosi~e I-pho~- ~ co,related do~ly with ks ctTec/on DNA sy~ibesie. ~rtbermore, an inhiblto~ of sphingosine kin•st. Dt,-tk~'o-dihydrosphlngosine. which inhibits sphlngosice- indt,~d DNA symh~b and the formation or sphlngos;ne I-phosphate, abo inhibited epldngm~ne.mtmulaled AP-I DNA binding activity. This result t'unher supporls I~oP0~sslre~u.tha.I spbingo~.ine I-pho~l~. ate mediates the mitngcnlc effect of tm indicate that sphingosme l-phesphate-induced DNA synthesis and cell division may result tforn activation aT AP-I protein, linking signal transduclion by sphingc~lipkl me~abelltes to ~e exist-salon. ~u, Y., Rosenthal. D., Smut•on, M., and Spiegel, S. The Jmm~al or Biological Chemistry 269f23):16.T12.16517. rune IO, 1994. ~ suppo¢l: Nat|~al lnsdmles or Health. From the Deplrtment at Biochemistry and Molecular Biology. Georgetown Unive~ity Medical O~nte~. Wmhln~on. DC. + INVOLVEMENT OF A PERTUSSIS TOXIN-SENSITIVE (3 PROTEIN IN TIlE MITOGENIC SI(3NALING PATHWAYS OF SPH1NOO~INB I-PHO~PHATE Sphingoslne I-phosphale. = sphlngolipld mclabolitco was previ~udy reposed to increase DNA symbesis ifl quiescent Swiss ~ flbreblasts and to induce inere|~..s in intrncellulnr free calcium (Zhnng. H., De~i. N. N.. Olivem. A.. ~eki, T.. Brooker, G.. and Spiegel, S. (| ~ I ) J'. Cell Biol. ! 14, ! 55-167). In Ihe i~l~ent Ixetrestmem of Swiss 31"3 fibroblasts wire pennia toxin reduced sphi~|o~t~ l- phosphate-induced DNA synthesis. Sphingosine I-phosphate decreased cell,let cAMP levels -~nd aim cau~.'d • dremic decrease in iso~olerenOf and ftm,kolln-~tim. uloted cAMP accumulation. P~rtu~tx toxin Ire•lineal IX~Rmed the i~htblm~y tf~! or sphlngoslne I-phosphate on cAMP accumulation, suggesting that a toxin.~enshlve (3, or G,-like pmleln m~y be involved in sphtnlodn~ l-~ts. mediated inhibition ot cAMP aecumuladon. Mitogenlc co~cenm,tlo~.~ or sphinllo- sine I.phosphate stimulaled pmductlon of inosltol Phmpludes which w~ inhibited by pc~lussis toxin, white the re~,x~n~c to br~dykin~ wa~ no~ alTected. Furthering•, c~icium release induced by sphin~oslne t-pho~pl~c, lxtt hal by bradyldn|n, wn~ stlenunted by perltissix toxin trcatmenh However. xphin,eoxine l.pho~phate-indlx~l phn.~ph~kllc n~id nce.'unt,httio~ w~s unalTe¢led by I~-~ltmsb~ toxin. The Inc~n~a,.~ in specifie DNA binding ~¢tivity or nelly•tar prolein-I, which was induced by meat or quiescent Swiss 3T3 filxoblasts with sphinsogine I-phoeph,~te. was also inhibited by pentBsls toxin. "l'hese resnha suggest th~ some o~" th~ sphingoslnt I- phesphate.imhR-ed signaling pathways are medlmed by (3 proteins that me nut,Irate• I'or pcflut,.,~ix toxin. Oondemote, K. A., Maltie, M. E., Borer. A., and Spiegel, $, The .To, real or Biolnglcal Chem~ry 2"/0(1"7): 10272-10277, Aixil 2g, I995. Other supix~: National Institutes or Health. From the Department of Biochemistry and Molecular Biology, G¢orlletown University Medical Center, Washington, ,STEREOSPECII~CITY OF SPHINGOSINE-INDUCED INTRACELLUIAR CALCIUM MOBILIZATION AND CELLULAR PROLIFERATION SphinBosine is a positive regulator or cell IrOwth in Swiss 3T3 (Zlmeg, H., B~,ckley. N. E., Gibson, K., md Splclel. S. (1990) J. Biol. Chem. 2~, ?6.fit). The pro•era •lady inveglg~ed the slereo~pe¢ificity aT cell Im)lWeratlon and its mlto~mie signal Iransduclkm mec~niam~. Stereolsomers (c/$ and irons) xtlmulaled DNA synthesis, whem~ neither thr~o-sphlngo~inc (cls or irons) n~ Ix.-threo-dibydrosphlnge~|ne had any Pmvio~slyo we lmve ~hown tha/~phin~sine- I-phoR~e may mediale the mltogeal¢ effect of sphingo~ice (Zhan~. H.. l)essi. N. N., Oliver•. A., ~eki, T.. Brooker, O.. end Spiegel, S. (1991} J. Cell Biol. i14. 155-167). Howcv~, no n~ t27
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0 0 were found in the formation of D.(+)-erythrn and z.-(-)-thren-sphingosin~.l.phos. plm¢ derived from the respective sphlnsosine isomers in int~:t cells. Thus. the slemo- Ipacif'~ity or ihe m.~'~onse Io sphinsosine may reside it the level o1" specific inlracc|- lubr ter~'ts for sphingosine.l-phm, phate. Sphingosino-l-phasphate triggers dual st|hal tnm~lu¢tion pathways or ~clivatlon or pho~pholipzse D iezdlng In inere~s in tl~ kvels of" phosph|tidic ~id and mo~ilixatlon of calcium f.rom internal sines. Bah ~.F)-er~vt/~rn. and t.-(-)-t/tren-sphin~mlne i.~ncrx inch~'ed similar inerea.,,e~ in phosphatidic ~id concomitant with identical decrea~ in plmsphatklyleholine levels, In contrast, only the o-(+)*erTIkro-stc~-oi~ner~ (cix and trunx) were efre¢. live iu Pete,.sing calcium from inlrscellular stores. Our re.~uhs suggest Ih~l the re, ha. lion of'.p~..'phzlidic acid is no~ sulTmiem In mediate .~l~ingosine-sfimulated DNA syntheszs. However, the stereo~pecificity of the sphin~osffze-induced mobilization of ~l¢ium From internal stores seems In correlate wilh the induction of DNA synthesis by sphin~o~ine slereoisomers. Ollvem, A., Zhzn~, t!., C~rlson, R. O., Mettle, M. E., Schmidt, R. R., and Spiel~¢l, ~ TI~/oumal of Bioloalcal Chemlslr7 269(2?):17924.17930, .1uly ~, 1994. Other suppo~: National Institutes of" llealth and The Mini.~lry of. PJlucalinn ~nd Science, Spain. From the Department of. Biochemistry and Molecular Biology, Georgetown University Medical Center, Washln~ton, DC, zml the Depar~ment ol" Chemistry, University of Kon~tanx, Kwnstanx, Germany. SPHINGO$1NE I-PHOSPllATE RAPIDLY ACTIVATES TIlE MITOGEN- ACTIVATED PROTEIN KINASE PATHWAY BY A G PROTEIN-DEPENDEHT MECHANISM Addkion of sphin~osine I-phosphate induces proliferation or quiescent Swiss 3T3 flbroblzzls I~ unknown mech~flisms. To identify Ih~ palhways involv~l, the z~oilhy 04" ~phingosine l-pho~phate to activale milo~en-aclivaled protein (MAP) klnt~ ~ studied. Sphin~osine I.phosphme rapidly aclivated the I~f/MAP kinz~e kinlz~ (MKK)/MAP klnese palhway, end the concentration dependence for MAP kEqzl~ aclivalion eorrelaled wilh lhzl for induclion or DNA ~jnlhe~is. Both MKKI and MKK2 ~ zctiv~led by ~hln~:~ine l-phosph~le, ~sessed by specific immune compl~x kin~e zsr~ys. I~tor tre|irn~.mt o1" Ihe Swiss 3T3 cells with I~lussiz Ioxin inhibited ?0-~0% or the zphln~slne I-phozphate-stimul~ed MAP klnzre ~:llvily. Thus, ~ or ihe direct or indirect lar~ls o1" exosenoas~phlngoslne l--pho~phate Wu..I.. ,~pi~, ,%.~ and ~lurgill. T. W. The ]~umal or Biological Chemistry 270(19):114~4-114R~. May 12. CHber support: Howard Hughes Medical lmlilUle, Nalional ln,~tltule~ or Heallh. and American ~ncer Smiezy. From the Howard Hughes Medical Institute, l~parlmenla of Medicine and P~arma¢ology. Markey Center for Cell 5ign~.ling. Univeraity of Vt~.$1nl.z Health. ~es Cenler, ~adollesvillc. VA. and t~ ~pa~menl or Bi~nzmtst~ ano Mol~ul~ Riol~. ~l~ Uni~Hy M~I ~. W~in~ ~. SPHINGOUPID METABOLITES: MEMBERS OF A NEW CLASS OF LIPID SECOND MF_~SENGERS Abur~Innl evidence has he~n z~-umulelcd to stmr~ly su~t that sp~ln~lipid melalx~lltes m-~y be e new class or |nlracellular second mc~,efl~er~. Howev~', many questions remain to be answe~d. Are sphln~:~ine, aPP, SPC, and e~r~m|de ~ only ~v~ive sphlnBoliplds or are Ihere still others remainin~ to he dizeovernd? AN clTccls of there enoqx'mnds the same or aimilar in all ~lls or ~u~ Ih~lt effacls Iimiled to specific cells and tissues? More complete dezcHpzlons of" the tm~hznim~ latlon of cell growlh and oth~" cellular pressmen should ~mable us 1o Ih~;e q~stions. ,~plet~el~ S, zmd Milstlen, $. The .~oumal or Mcmbran~ Biolo~ 146:22~.2.17, 1995, O~her support: NMional Instlzulea ol" Fleal~. Prom Ihe Deparlmenl or Biochemistry nnd Molecular Biolol~y0 Oeor~ltown Univer~ily Medical (~nter. Washington, D~. I~! I.Aboralo~ of Neurocbemi~lry, Nallo~al Institute or Mental Health, Belhesda, MD. THE ROLE OF CALCIUM INFLUX IN CELLULAR PROMF~RA'rlON INDUCED BY INTERACTION OF ENDOGENOUS OANGUO~IDE OM I WITH THE B SUBUNIT OF CHOLERA TOXIN The B au~unit of cholera toxin, whleh binds speciflr~lly to patlio~lde OMI Is miloeenk: for quies~-nt Swi= 3T3 fllx~huds, It was ix~vioully shown that the dkl c=u~e an increase in the influx of c~kmm I'rom extr~C~llUilr =rod Ihmaliompnolo~. C. (19~) Exp. C¢1! Re~. 1~7. 414-427). In =orion o!" known growth I'netoet. the B smbunit induced ai|nifl¢=~tt. DN.A after only a !-3 h tre=tny:nt. W¢ utilized.thls unkl.ue ~1. y the increase in calciom influx plays • ro,e in u su~n|t-i.r~ .t~. m|!o~m.~r/. were l~'icny Ire=ted with the B subun~t In l~ ~.,en~...~ followed hy remo,~al of the ~ceke~ and fueber iacut~llon m Is for Ihe remaining time required to measure DNA $).nd-~is. When 129
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only I~Sem during the first 3 h incubation. DNA synthesis induced by either the subgnil o¢ felal Ix~vine senm~ was completely abolished. However. bulh nickel (I aM] aM th~ L-type volla~.'~.galed calcium channel inhibitor nicardipin (I0 inhibiled B ~ubunit.induced cell prolifemlion wlthou~ abram•ling the response to fetal bovine serum. Using a gel retardstion assay, we found that the B suhunit m•rkedly stimulated specific DNA-hinding activity of the tranxcriplio~ factor, acti. ValOr prmein.I (AP-I) which functions L~ • ma.ior convergence point coupling early evOnts ~lduced ~ a variety or milo~ens to long fenn growth ~spno~,s. P~exeflce of ' 7 a~my uzmg c.v~ poeye~l ~mtd~ly. C(~aft. which markedly inhibited ~,ubunil-indueed DNA synthesis also compl~ely ubolish~ AP.I DNA-blndlng activ- ity slimulsted by the B subunil. In sharp co~trasl. Co~'4tll ~ no effect o~1 DNA-hind- in~ activity of AP-I induced by the tumor prom~eer. 12W~-tclr.'~lc~anoyfphorl~l 13- actinic. Oar resulls suggest Ihat calcium influx is n key ekmenl for I~ah DNA.hlnd. ing actlvily of AP,,I and ecll prolil'erullon induced by binding of the B subunh or cholera toxin to cell ~ffaee ganglieside GM I. Ruckiey, N. E,, Su. ¥., Milsticn. S.. and Sp~gel, ,~ Biochimlca el Biophysics Act• 1256:275.283. 1995. Other support: National Institutes of Health. Prom the Department of Biochemistry and Molecular Biology, Georgetown University Medical Center. Waxhingt~, DC, and L~boratory of Neurocl~mistry. National institute OF Menial Ilcalth, Bethesda, TIlE WW DOMAIN OF YES-ASSOCIATED PROTEIN BINI~ A PROLINE. RICH LIOAND THAT DIFFERS FROM THE CONSENSUS ESTABLISItED FOR gre HOMOLOGY 3-BINDING MODULES The IArw domain has i~eviously been de~rihed as • motif of 3S semico~ residues found in seemingly unrelaled pro~elns, such ~s dys~rophin Ycs4zsoci~ed i~ein (YAP), z~! two I .r~sc~il~ionzl regulators, Rq~-$ and FE~. The molmla~ ion of the WW donna has ~ unknown until this time. Using a functional ~ OF a eDNA expression library, we have identified two putative li~nd~ OF the WW ~ln of YAP, which we nam~J WBP.I and WBP-2. Peplide sequence pmtmn ~ween the two partial clones revealed a h(xn~logons region consisting of a proline.fich domain followed by • tyro~ine msidoe (with the shar~ s~ucnce PPPPY), which we shall call Iha PY mo6£ Biedln~ ass,wj~and site-specific mula~c- ne~is hlwe shown that Ihe PY molif binds with relatively hish a~nity ned specificity to the WW domain of YAP. with the preliminary con~nsus XPPXY helng cri6cal for binding, Herein, we have implicated the WW domain with • role in mediating proleln.prmein interactions, as a variant of the paradigm ~et by Src homology 3 domains and their praline-rich ilpnds. 130 Clan. H. I. a~ Sudol. M. Proceedings oftba National Ac~my ~ USA 92:7819.7~3, Aunt I~ ~ ~u~: Nati~l C~ f~itute, ltu~n ~t~ ~i~ ~mm. ~d the Nati~l l~itutes ~ Health. ~m I~ ~t~ OF Mol~ular O~1~y. R~kereller Unive~lly, New Y~. NY. CHARACTERIZATION OFTHE MAMMALIAN YAP (YE$-A$$OCIATIRD PROTEIN) GENE AND ITS ROLE IN DEFINING A NOVEL PROIM|N MODULE, THE WW DOMAIN We report cDNA cloning and characterization oF the human and mouse ortbologs of the chicken YAP (~cs-associated lZrotein) ~cne which ~ a novel pro{tin I~"ll hinds to the SH3 (Src homology 3) domain or the Yes pmto-encoim'~ product. Sequence camp•than belwecn mouse, human, and chicken YAP proteins showed an inu~tcd sequcucc in the mouse YAP that rClX~ented an Imp~feet r~ptst of an ul~tre•m sequcnce. Further analysis of this sequence revealed • putative pro- tein module that is found in various structural, regulatory, and si~nal~g mol~:ules In yeast, nematode, and mammals iuclnding human dystmphin, lkeau~ one of prominent features of this sequence motif is two tryptol~ans (W). we nam~t It tl~ WW domain (Bark. P.. and Sudol. M. (1994) Trend~ Bi~rm. $¢t. 19. 531-.'t3.~). Since Hs dellnestlm, rrmce proteins have been ~own to contain thk domain, a~d ~ here ou the wi~k~sprcat! distribution of the WW mndule nnd l~'mnt a discus. sion of its Ix~sible fonction. We have al~o ahown that the human YAP pm la w~ll con..fred among higher eekmyotes, but it may not he com~ved in yeast. Its expm,~- skin at the RNA level in adult human tissues is nearly ubiquitoua, beinlt relatively high in place•t•, Ixordate. ev•ry, •nd testis, but is not detent•bin in periph~nd blend leukecy~es. Using fleorescenee i. ~/m hybridization on human metal~a~ ehmme- ma and by analyzing rodent-human hyb¢ids by Southern blot hybridization Ix~lymerme chain reaction amplification, we mal~ the human YAP ~ to chro- mosome hand l lq13, st region to which the multiple codocrine neoplasla typ~ I has been Serial, Me Bark, P., Einbeed, A., Kastury. K., Dmck, T., Nesrird, M., Huebner, K., and Lehman. D. The Journal of Bioleglc•l Cl~mlstry 270(24):14733-14741. June 16. 1995. Other sup~rt: National Institutes of Health. the Annenherg Foundation. Hebrew Tcclmical lnstitnte, N•tional Cancer Insfitule. Human Fmmier Science l~mm. the Klingenstcln Award in the I.tl
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I':rom the Lal~)ratnry nf Molecular On,~dogy. Rockefeller University. New York. NY. ~pean Molecular Biology Lair•tory. lleidelberg, Germany. and the ~plfl~nt o[ Mi~bi~o~y and lmmu~ow, Je~e~ Caner Institute. ]effcrs~ M~al ~lle¢e. ~ila~l~ia. PA. CtlARACTERIZATION OF A NOVEL PROTEIN-BINDING MODUL~ THE WW DOMAIN We have idefllifi~d, characlerixcd and cl~l human, mo~s~ and chicken eDNA or I rl~v~l pro~eln that binds to the Sic ~m()ln~y do~t~ 3 (All]) or the Ye~ pmto- onco~ene p~uct. We subsequently named it YAP for Ycs-as~iat~ ~lein. A~is or t~ YAP ~e~ R~al~ a ~cin mmlule that w¢~ ~ml~ in ~d it ~ WW ~maln. Usi~ a tu~al ~n of a eDNA e~ssi~ li~, we ha~ id~fiR~ !~ ~lallve ligands ~ Ihe WW d~ain ot YAP which we ~l~ a ~l~s ~-fi~ ~¢i~. Bi~i~ a~ys and xile4~ir~ sis have ~own thai I~ ~line-r~h ~ir bi~s wilh ~lali~ly high =~nily and ~l~¢ily m t~ WW ~n ~ YAP. wkh a ~limina~ ~nsuA t~! k diE~l f~ t~ gH~-~in~ PXXP m~if. ~is su¢¢e~ls th~ the WW d~in he~ a ~¢ in m~tatlnl ~in.~lein Int~ti~s vb ~li~-~b ~i~s. similar ~t di~i~t f~ ~ ~y 3 (SH~) d~ain~. Ba~ ~ this find~=, ~ h~xi~¢ Ih~ Mdil~l ~ein m~ules exist a~ Ihat I~y ~ild ~ i~ated using pmline-rlch Sudd, M~ ~n, H. !., B~¢c~h C. Ein~¢ A., and Bor~. R Ol~r sup~: National Cancer Institute, Klingenxtcin Award, Human Fronlier $¢i¢~ ~, ~d I~ Nalimal ln~ilutes or Health. F~ I~ ~to~ of Mol~ular Oncolo~. RockeFeller Unlvc~ily, New York. NY, EMBL, Heidd~r¢, and Msx-~llbruck-Ccntcr For Molecular M~icine. B~in.Bu~, O~any. MEASUREMENT AND MANIPULATION OF CYTOSKELETAL DYNAMICS IN LIVING CELLS A new era in c¢11 biology is at hand with the devdopm~m or tools fer imaging molecular furctions in living cells and tissues. Specific chemical and molecular 132 events can now be me~uicd ~d manipula(ed in cells in order to explora th~ nlsms or ~11 f~i~s. In ~ular. ~t~elelal ~sR~ sR ~in ~lly a~ s~ially in single ~lls r~ lo~ euk~otes, pfan~s. li~t-ba~ Ra~Is a~ el~ic light m~y. Oiulia~. K. A. a~ ~aylor D. ~ ~nt ~iol~ in Cell ~iol~ 7:4-1~ ~her su~: Nati~d Instilut~ of He•lib. Nali~al ~ic~ F~ati~, ~len~ a~ T~h~lo~y ~t~, a~ I~ ~tts~r~ Ca~ Institute. F~ t~ Univc~ity of Pittx~h a~ ~¢ie ~11~ Univ¢~]ty. SPECIFIC CLEAVAGE OF MODEL RECOMBINATION AND REPAIR INTERMEDIATES BY THE YEAST RADI-RADIO DNA ENDONUCLBAgB The RADI m~! RADIO ~nes o¢ $ac~.lm~s c~'r~ri.dae '~m required for br<h nuclcolid~ excision r~pair ~d certain mitmlc Iccomblnatkm e~'enls. Ilcre, mo,,kl Iccomhlr~ion and icpeir intermediates were u~cd Io d~ow th,~ RadI-R~ll0.medlated cleavage occurs at duplcx.sincl~strand junctions. MoRover. ¢leavll~e occurs only on th~ strand containing the 3' single-stranded tail. Thus. both biochemical and L.en~tic evidence indicate • role for the Rad l-Radl0 compl©x in the cluvate of Jpe- cifi¢ recombination inlermediates. Furthen'nore, these data luCl~s111181 RIdI-R~dlO enclonuclease incises DNA 5' to d•maCed buts during nncleotide excision r~palr. Bsrdwello A. J,, Bardwell, L. 'romkinson, A. E., and I~edherg, E. C. Science 265:2082-2085, September 30, 1994. Other supper1: U.S. Public Health Service. From the L~boratory of Molecular PatholoW, Unive~shy or Texas Southwestern Medical Center at Dallas. Dall~, 'IX, and Institute fo¢ Biolechnolo~,~y, Center for Molecular Medicine. Unive~;ty of Texas Health ~clence Center at Sm Anloflic~ $~m An(nolo. TX. RFJ.~ ,F-~Bp~B STORY Discovery or NF.KB transcrilxlOn factors gencrlted • new con~pI rot gent activations: the rcgulat;o~ by cyK|plasmic inhibitor molecules. 'This system is ubklul- tous and cvolutior~lly well ¢Onser','ed. A wi~¢ variety of exc~K~s and endo~nout ~¢nts generate a muffitude of r~gul~tory casc,~des which ¢onver~ on this eytcq~s- complex, resulting in the release of the K~ive Ira~oril~i~ I~or. 133
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0 0 De~pffe ~J~at interest, the s~saling pathways are t~nslati~M ~ification is involved. Clearly, o~ of the ra{a.llmitln8 steps ~Is ts l~ id~tir~ati~ of kln~s thai daftly ~p~rylate ~e~ sy~tem(~) involv~ in l~ (~Br~ali~ in viw~. 1~ inic~diate ~lhway~ i~lu~ ~s, oxygen ~dical~, tyr~i~ kisses, and/c~ the Ras-Ra£ system. ~ ~kx~ ~l~tjvely ~d? ~ an~ will ~f~ ~ example, in dexi~in$ ~ ~r/els or the NF-,B sys~m exl~ to ~ne~l F~ti~s m~t. Unveilin~ h~ a ubiqut~s f~t~ differentially ~ulates key ~llular ~. I~ns ~ui~s fulu~ inveslipti~s, ld~ti~cal~ oF target ~e~s involved in ~ll c~ ~ ~e~ti~i~ rosy ~v~ ~ ~w way to u~ersta~ ~w this system ~ I~ to lym~id and ~lymp~id ~. ~her ~: Hati~sl Institutes or Health, A~rican ~d ~ C. ~r ~t~. Fr~ the Mol~ufar Biology and Virology ~r~t~, ~e ~alk Inslltute, San DIe~ CA. AUTOREOULATION OF I~Bt~ ACTIVITY ~tiv~ ~N~KB ~ins is ~ula~ by IKB ~eins. We ~ that i~ei~ or I~B~, a ~ ~ t~ ]KB fami~ ~i~ ~ ~ ~ ~i~tim ~ NF-~B in t~ ~ ~ t~ N~B ~x. ~at~ of !~ ~ein ~s ~i. inhib~ ~-~B ~v~y. it t~ ~i~ d Ixg¢ is ~ventd. t~ ;~ of ~ exi~ ~ m ~u~l~ ~ ~ lxB= R~tes t~ ~ivily of ~ ~ NF.~B, ~kh in ~m R~ulales ~ I~ ~ P. J.. Mi~o. S., ~ V~a, I, M. ~!~ ~ Naiad AtOmy ~ie~ USA 91:2~.32, ]anu~ 1~4. ~m lee Mol~ul~r Biology and Vi~lo~y La~ainry, ~e Salk lnslilule, San ~, CA. 134 ENIIANCED IxB~ DEGRADATION IS RESPONSIBLE FOR CONS'TITIJTIVB NF-xB ACTIVITY IN MATURE MURINE m-CELL UNE3 Nuclear f~:tm xB (NF-xB) is u ubiquitous tran~ctipllon factm which birds to (lecameric DNA ~cqt~nces (KB site'.O aeKI regulll~ tr~'l~plkm of mullil~la The acllvlty of NF-xB is rc$.lnlcd ~ an inhibitor l~-~ln. IKB. which NF-xB in I!~ cylopla~m. Rel¢~ of I~B ~ ~ul~lU~nt nud¢~r Ir|msle~lliou NF-K~ ~m~'~lly t~l~ire ~.liv~tin~ si~nuls. However, in n~tu~ mudne B DNA-bimlln~ ~¢livily ~1" NF-~II is ~lilutively nud~1~r ~ a~llvule~ Ihe 1~ u m,~d~¢r fnr m~ltm~ B cells. "1~ ||ml~mand Ihe I~.~is ~r Ihe ¢~n~ltl~|tive NIZ-~iB i~1|. ration, w~ esamlned Ihe ~ni~ of NF~B I~ I~B in ~h ~B ~ml mmu~ B ~11~. I~ Rgulat~ :~ ~stitullve st~te~, ~s~ti~ly. We f~d thai ~ 1~ a~ plOh, ~m~ or I~ IKB family, a~ ~1, a ~m~r or i~ NF.KR ~amily. i~ augmented in malt~ B ~lls. Both IKB~ and pl~ ~ as~ia~d ~'hh N~KB ~e~s a~ ~4~ ~t or t~ ~¢ins ~ t~ cy~m or mstm Iran~l~ali~ o~ a small ffacli~ ~ N~KB ~tcins, which R~nt Ihe ~nathu. lively mtlve NF.Kfl in ~lum B ~lls. We cxtlmatc t~ t~ ~ca~c ~ivity Icaxl 35-to~ $~er i~ matu~ B ~lls than in ~-B cell~. Rapid dc~atlon i~ daftly involv~ in ~stilutive N~KB ~tivali~l. ~au~ st~bili~ti~ ~ I~ by a ~c~ inhibit~ cauls I~ of NF-KB a~ivity in mal~ B ~lb. ~e~ R~ull~ ~vidc ¢vid¢~¢ that ¢~linuous ~rd ~pid ~¢~ti~ ~ IKB~ in t~ n~ of ¢xlcmal ~limuli is causally involved in t~ ~nxlilutiv¢ aclivati~ of NF-KB mal,~ mtlrin¢ fl cclb. Miyam~ S., ~la~ P. J., aM Verm, L M. Mol~ular a~ Cellular Biol~$y 14(5):327~3~2. M~ I~. Ol~r xu~: N~tt~nal lnslilutes of II~llh and I~ H. N. e~ Fm~s C. ~m Ihe Molecular Biology ard Vifol~y ~rato~, ~ Sulk In~lllut¢, San D~$~ CA. QUALITATIVE CI IANGES IN TIlE SUflUNrr COMPOSITION OF mINDING COMPLEXES DURINO MURINE B-CELl. Dll=pERBNTIATION 'We repmt here that the major KB.hinding complex in murin~ malure comlx~,¢~l of a F.SO-Rcl heleroditner, wher¢~.~ the ne0or indocible fore: in I',ee-R cells is u p~)-p65 hClefodimer. Treatment of u pfC-fl cell llne with Ilpopolym~charide 135
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chan/,es the subunll composition of ~B-hinding complexes from ph(t-p~ tn phO-Rel. This ©hlmge is preceded by Ihe enhanced Rel expression and corrclates wilh the exp~ssk:m o~ the gene [or the immunoglobin ~ light chain. The hetcrodlmerlc p.~0- Rel eo~ptex binds to the inlronlc enhancer.KB site in Ihe immunoglnt~lin ~c light ~hain ~ M lead 20-fold more stibly than does the p~O.p65 dlmer.'These data sup- por~ a model i~ which ausmentation or Rel expressio~ during Ihe differentiation of pt~l~ ~e~Is to mature B c~lls I©~ds to an exchange o1" t(B.~inding subunils resulting in the l~In~edpdofml ~clivatinfl eX Ihe g:enc for Ihe ;mmun~lohulin ~ light chain. Miy~no~o. 5.. Schmltt. M. J.. and Verma, I. M. Proceeding,~ of the National Academy of Sciences USA 91:5(h~5OCff), Mly 1994. O=her support: National In~titutm of Heallh. Arncrclan Cancer $ociely, and Ihe ! !. N. and Frances C. BarBer Foundation. From the Mo|¢cular Biology and Virolosy Lab, ratory, The Saik Institute. San Die=o. CA. TUMOR NECROSIS FACTOR a-INDUCED PIIOSPiIORYLATION OF IKRc~ IS A $|GNAL FOR ITS D~RADA~ON ~UT N~ DISS~IATION ~OM NF-~B ~l~ of t~ ~ ~lysi~ It ~s ~ ~ulal~'lhat ~falkm ~ l~O~ le~ IO its dis~t~ ~ ~-~B, a~ f~ I~Ba i~ ta~ctcd f~ ~pid ~at~. II~ver, this ~~i~.~iat~ di~ial~ ~s o~ I~ Nmi~l Ac~my of Scie~s USA 91:1274~ ! 27~. ~r ~ N~: N~al lnslk~cs of Heallh. A~can ~r ~ly. ~ I~ H. N. ~m t~ Mo]ecut~r Bi~o=y and Virology ~rat(~y, ~¢ Salk lnstilulc, San Die~ ~. ~ ~=t~ ~ Hum=, Tumm Viruses. In~ilu~e ~m Vires R~mh. K~ Univ~ly. Ky=o, 136 ,,~ r~ ~~iff~,,.,nF ~'~. ~" ~ ~i"s '~ "u~. RNA ~ly~ I! ix a su~lmt¢ f~ I~ Abl ~t ~ t~ Sm ty~i~ kinl~. ~i~ s~ifi¢ity is ¢~fc~ ~n pa~ ~ t~ SH2 d~=in. ~ Abl ~H2 d~in bind= the exchanging it wilh thM of S~, wmc, ~s ~ ~ t~ ~r).~ ~ u. =~ ~ F~ I~ ~t of Bbl~ a~ ~nt~ f~ M~l~ul~ ~n¢~, Unl~lty of EXPRES$1ON OF TIIE B2Pl TRANSCRIPTION FACTOR OV~RCOMP.~ I~ TRANSFORMING GROWTI! FACTOR-MEDIATED GROWTH SUPPRESSION in m~d-G, and ~ associated with dect~sed G, cyenm-dep~, t~_.~ Ktna~ mlinlcnl~ce Of the Rti~K~b|lsto~a tumor suppl~ssOr ~p~ocein KO In IOl that disr, ociale E2F from Rb and Rb.mlated po=ypelxmnes- po~csibillty that control of E2F may he a downstream event or TGF-~ ,:on,i,,=, wi,h ,ha, ,ihl,i, i, ,he ,,,,, tally reduced in l"GF-/~-Ircatcd celts. We have alSO = conlalnlng the human E2FI gcn¢ to overexpres= the E2FI product epithelial ~ells thai were growth anestnd with TGF-~. We find that I.'17
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0 0 !',3 of E2FI can overcome the TGF-0-medialed effect as inca,cured by the activation of cellular DNA synthesis. Thece recults suggest that a likely doWnslrcam target for cyclin.dependent kinases, which are controlled by Tf31:-~. is the activation of E2F. $chwarz. J. K.. Ba~ing. C. H.. Kovesd;. I.. Dattn. M. B.. Blazing, M., George, $.. Wang. X.-F.. and Nevins..f.R. Proeecdlngs of the National Academy of Sciences USA 92:4~.487. Januaz7 1995. Other support: National Institutes of Health and the lloward tlughes Medical ln,~ritule. From the Departments of Genetics and Pharmacology, Division of" Cardiology. De,pertinent of Medicine. Howard Hughes Medical InsritUle. Duke University Medical Center. Durham. NC. and C, enVec. Inc.. Rnckville. MD. TRANSR')RMING GROWTII FACTOR I'~ INDUCF,~ TIlE CYCI,IN- DEPENDENT KINASE INIIlBITf.)R P21 TIIKOUGII A P53,1NI)I-'PF, NDENT MECHANISM The transforming growth factor Jgs (TGF-~s) are a group of mulllrUnctional growth I'actofs which inhibit cell cycle progression in many cell types. The TGF-/3 induced cefi cycle arrest has Ixzen partially atlrih~ted In the regulatory eft'ccts of TGF-/~ on both the levels and the activities of the G~ cycllns and their kina~ pan- net's. The aclivitics of these kiP.uses arc negatively rcgu|ated hy a oumher of small proteins., p21 (WAFI. Cipl). p27'~. p16, and pl5~'=. that physically associate with eyclins" cyclin-clepandent kinases, or cyclin-Cdk complexes, p21 has ~ previous- ly showa le be transcriptionally induced by DNA damage through p53 as a mediator. We demonstrate that TGF-~ also causes a rapid transcriptional induction of p21. su~Be~lin~ that p21 can respond to bo~5 intracellular and extrxcellul=r signab= for cell cycle arrezt. In contra~t In DNA damage, however, induction of p21 by TGF-/~ is no~ depe~k'ot on wild,type p53. The cell line studied in thcr, e experiments. HaCaT, con- taint tw~ mutam alleles o( phi, which are unalde to =ctivaxe transcription from the p21 premoter when overexpmzsed. In addition. TGF-/~ and p.~ act through distinct elements in the p21 prom~er. Taken to, ether, the..~ findings su~st that TGF-/~ can indoce p21 through a ph~-indcpondem palhway. Previous findings have implic~ed I~?"~ =~d pl~~=' IB effecto~ mcdiatlng the TGF-/~ growth inhibitory effect. These resulls dentonltrat¢ that a sin¢le extra~llular antiprolif~-ative signal,'l"GF-~, cm act throt~h multiple signaling pathways to ¢licil a growth arrest rcspong. Dana. M. B.. Li. Y.. Panu~. J. F.. Howe. D. J., Xiong, Y.. and Wang, Proceedings orthe Natlorml Academy of Sclences USA 92:5545-5549. ]un¢ 1995. Other sul~: National Inatitutcs aT l-~alth, the Program in Molecular Biolocy Eiol¢~'hneloffy. Bx! the ~ncl~'~er Comprchenslve Cancer Center of the University of Califomia ~ Chapel Itill. 138 Fro~ the Dep,qrtment of Pharmacology. Duke Unlverxlty Medical Center. Dudtam. NC. and Department of Biochemistry and Biophysics. Prosram in Molecular Biology and BioCechnolo~y. Lincherger Comprehensive Cancer Center, University of" Norlh Carolina, Chapel Hill. NC. TRANSFORMING GROWTH FAC]'OR/~ ACTIVATFJ THE PROMOTER OF CYCIJN-DEPENDENT KINASE INIIIBITOR PI5''~'= THROUGH AN ~pl CONSENSUS SITE Transforming growth f~ctor/~ (TGF-/3) causes ~m~lh m in the O, pi'm~e In many cell types. One pro~ahle pathway for this growth inhibition is thren~ the TGF./~ mediated up-regularion of the eyolin-dependent kinaR (CDK) Inhibitor p l.'PTM, which apccifically inhibits Ihe enzymatic activities of CDg4. and CDK~. active cyclin D.CDK4/6 complex is required for pRb pho~phorylatlon to allow cell cycle to progre~,~ from G~ to $ pha.,~. To ~ludy the molecular mechanism of pl5t~'~ induction by TGF-/~. we |saluted • 7804~tse pair Womotcr sequence of the hem,'m pl$ jp:ne and in.,.erted this fragment upstream o1" a lu¢iferase r~porter When this construct was transiently Iransfe¢led inlo HaCaT cells, lucil'era~m activity was induced mine than lO-fold upon "I'GF-~ treatment, indicatit~ that the Ioduetiou of pl$~'~ expression by TGF-~ is partly exerted at the transcdptien level. Promoter delelion analysis revealed Ihat the sequence from -I 10 to -40 Rlative to the tran- scriptiou staJl site is capable of eenferdng the IO-fold induction by TOF.~. Within this region there are three $pl consensus sites. Mutation of one of thea¢ thus, GGGGCGGAG, subsramlally reduced both the induction hy "I"GF./~ and tfte promoter activity, whereas mutations in the other two Spl sites and the spacer sequences had tittle effect. In addition, gel rnohilily shift assay indicates that the transcription factors Spa and $p3 hind to this SpI alia. Taken tolether, thes~ suggest lhal I specil'lc $pl consensus site is involved in the mediation of induction as well as the b,-eml ixomoter activity of the pit gene and that $pl and $p3 transcrlptloo factors might he involved in this regulation. LI, L-M., Nichols. M. A.. Chandra~ekheran. $., Xiong, Y. and Wang, X,-i~. ~ Journal of Biological Chemistry 270(4~).'26750.26"/$:~. Novemher 10, Other suppml: National Institutes of Health and the l..ucili~ P. Ma~kw Foundation. From the Department of i~urmacology, Duke Unive~slty Modical Center, bJrh0m, NC. and the Curriculum in Genetics and Molecular Biolosy, Department o1" Biochemistry and Biophysics, Program in Molecular Biology and Lineherger Comprehensive Cancer Center, Univeralty of Noah Carolina, Chopal Hill, NC. t39
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0 0 FUNCTIONAL ANALYSIS OF THE TRANSFORMING GROWTH FACTOR RESPONSIVE ELEMENTS iN THE WAFI/CIPI/~21 PROMOTER The tr~nsforrnlng growth r~ctor/~ (TGF-~) ar~ a gnmp or mullir.ncliooal growth f~ct~s tha! inhibit cell c~cle progressiou in many cell types. The TGF-B- indu,~ c~|l cycle arrest hen hcen partially attributed to the regulat~'y effect,~ TOF./3 or~ b~h the levels and activities of the G, c~Iins and their cyclin.dcl~mlen! kif~se r~rtner~, The ability or "r~F./~ to inhibit Ihc acfivily .f these klnase com. plexe'~ derives in pan from its regul~m'y elTccls rm the cyclin,.delx~mJent kina~e inhihitor~, p2I/WAFI/Ciplo p27~' and pl.S, U[xm trcatmenl or cells wilh TGF./3, the~ three iflhibltors 5i~ to ~d ~k the ~ivitie~ ~ ~ci~ cyclln-cyclin.~. ~t kinl~ ~exes to cau~ ~ll c~ a~st. Utile ~ k~wn. ~wever. ~i~ th~h ~i~ ~F.~ ~ivzt~ t~ c~]in-~nt kina~ inhibitor. In the else o£ p21, ~-~ t~stment le~s to an increase in p2f mRNA. Thi~ i~ in p21 mRNA is ~nly d~ to t~H~i~al ~tivat~ ~ t~ p21 ~ter ~ ~ To ~u~r ~fi~ the si~alin~ ~thwa~ thigh ~h ~.~ induc~ p2t, ~ hl~ ~ a ~tail~ fu~ti~al analysis on t~ p21 ~m~. ~mu~h ~h ~let~ a~ mutat~ anat~is o~ t~ p21 p~er. we have defi~ ~ir ~ that is ~ui~ for t~ ~tiv~i~ or t~ p2t ~ter by ~P-~ In ~iti~, ~i~ ~ is ~nt to drive ~-~-m~iated transc~pti~ f~ a ~vi~y ~n~sive ~er, ~iimi~ ~! shi~ as~ys ~strale thll ~ ~sive el~t ~i~s ~i~ally to ~l ~cin~ ~ ~ins a~ t~ t~n~H~ r~t~ S~I and Sp-3. ~ M~ies ~scnt the initial ~ ~wa~ defining the signaling ~lhwa~ involved in "l~F-//.mmliafcd Oatt~ M. B., Yu, Y,, and Wun~, ~ J~! o£ Bio~iczl ~mist~ 27~4g):~623-28628, ~em~r I, I~. Ot~r ~u~: Nat~al [n~ilule~ ~ I [eahh. F~ t~ ~n[ or ~z~lo~y. Duke Unlve~iW M~llcal Center. Du~am, NC, TRANSCRIPTION-DEPENDENT REDISTRIBUI3ON OF THE LARGE SUBUNIT OF RNA POLYMERASE II TO DISCRETE NUCLEAR DOMAINS A ~bpopulation or the largest subunit of RNA polymer~se l! (Pal II LS) is |oc~ted in 2050 discrete subnuclexr domains that are clo~ly linked to speckle domaink wh~-~ slore splicing prmeins. The speckle-associated fraction or Pal II LS is hyp~ho~pho~lated on the COOl[.termlnal domain (CTD), and it is highly rests- lain to extmcl|on hy ck=lerg~nts. A diffu~ oucfeoplasmic rr~ctkm or I~1 II I.~ is rcl~ lively :h)'lx~phmph~_ Isled on the CTD. and it is easily exlracle~! by dclergcnls. In tranzcrlptlonelly ~ctwe nuclei, speckle bound hyper~latcd Pal l! LS mole- eul~z ~ di~Hb~ted in irr~ulady Shel~l sr~cklc domains, which appear to he intcr- eonneclnd via • reticular n~twol~. When transcripti(m is inhibited, hyperphr~plmry- fated Pal If L~ and splicing protein SC3~ ~wnulate in speckle (k~nains, which are 140 transformed into enlarged, dot-llke structures lacking inlerconn~-ttons. ~len ~ ~[~ f~ t~H~i~at inhibitS, ~1110 ~ ~35 i~c~t~ s~k~ ~t~m Of t~i~ally ~ti~ ~Ils. ~ ~td~li~ or ~1 l! a~ 5C35 is sy~h~s, ~v~tble. a~ lem~mu~ ~n~nt. Ii~ ~.~im~ to di~le su~k~r ~ains t~ hs tmn~p~nl ~tivH~ ~rin~ states oF tmn~ri~i~al inhibiti~, h~pho~lat~ ~ with t~ ~ sites £~ spl~i~ ~ ~ ~'~ tein~ ~istH~e sim,lm~ly ~inG to t~ ~lf t~ ~ic~. Ot~r such: Ma~h of Dimes Birth ~fect~ Foundati~, Donashu~ Medical Re~h ~u~zti~. ~ l~ NzI~zl [nstitmes or H~llh. ~om the ~pa~nls or ~tholo~ and Genetics. Yale Onivc~sil~ ~i~, New II~. ~. AGONIST-INDUCED EXPRESSION OF TISSUE INHIBITOR OF METAIJ.OPROTEINASES AND METALLOPROTEINASES BY IIUMAN MACROPIIAGES IS REGULATED BY ENDOGENOUS PROSTAGLANDIN E~ SYNTIIF,,SIS The regulslop/effect of endogenously synthezl~,d elco~moid m~tabolltes the expression or tissue inhibitor or metnllopr~ein~s ('rIMP), Int~tkio! nasa, and 92.k[)z ~elstinase by hurrah macropha~,,s was exlmlned, T[MP ~ mcl- allopro(¢inase production were stimulated with three ~onists that produ~ disti~'t pmterm or eicosznoid s3mth~is: li[x~olyszecharide (10 p~Jml), ~atur~,d (10 I~g/ml), or zymase (l rag/m1}. Indomclhacin (3 Ix~nl) or MKl~86 (3 p.M). • specific inhibitor or ~.! ipoxyg~nasc, was used to cxamin~ th~ role of" end~'n~s m~aholi~s of m~chldonic acid. Rc~clles~ or the agonist use~, TIMP pe0du~lion m~opheg~ wz~ inhibited 65% by indomethEin, synth~is or intcnzlit|•l nine wu reduced 70~, and expression of 92-k1~ plalinaze wm deerez~,d 40~, In conlrast, inhibition of' I~uk~rlon~ synthesis h~d no elTecl on metatloproleinsse or TIMP pe0ductk~. The agonlst-~timufated income in TIMP mad eollagena~ lion w~s directly ce~clated to the eumulative ~$1andin E~ level induced by the agonist used. ~o if'~espon~ to an ~goni~ wa~ poor° the exo~n~us addition of" pro~tnglnndin E~ could hal inerea~ "]'IMP or eoil~ennse I~Xlu~lims more then Iworohl, indle~ling ms impml~nt permissive effecl ~l'the z~oni~ e~ the reSulmion o{' each protein's expre~ion, The ~ni~n of indmnelhEin Inhibition ~" TIMP uelfagenz~e production wit studied by l~heting the cells with [~$].~nethtoni~ ~nd perl'onnlng |mmunoprecipttatkm using specific mt~'mm, lndomelha~in mmkedly inhlhlted the Iil~'~olysacch~rldo-indueed blo~ynthes|a of bmh T1MP end 141
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0 U'I ate. Northern analysis revealed parallel suppression or TIMP and collagens.so stemJ)'.mate mRNA levels by i~h~in, indieatin& ~ranslat~nal ~t~. ~ ~l~ti~ ~ infl~mmat~-~ll TIMP a~ int~tltiM ~ll~enase expression by prosts&landln E~ suB~est; that therapy inhlbilin& the cellular response to ~st~Elmdins may ~ u~l in ~t~us ~ systemic disuse states involving ~ t~ Divisi~ of ~t~ogy, W~hingl~ Unive~ity ~h~l of M~i~, a~ the Divisi~s of Respira1~ and ~iliczl Care and ~atolngy. Washington Uni~ity Sch~! ~ M~ici~ at Jewi~ I I(~tal. 51. b~is. MO. COMPLET1E DEGRADATION OF TYPE X COLLAGEN REQUIRES THE COMBINED ACTION OF INTERSTITIAl., COLLAGENASE AND OST~X'LAST-DERIVED CATHEPSIN-B W~ have studied the degredat~on o1' type X collagen by mctalloprotclnases, eathepain B. and O~l¢oclast.der~ved lysales. We had pre~ously shown (Welgus, IL O., C. |. Rials', J. L. Seltzer, T. M. Schmid, and J. J. Jeffrey. 1990. J. Bhd. Chem. 26~:1~521-13527) that int~rstltial collaL, enas¢ rapidly altacks the nalive 59-kD type X molecule at two sites, ren~ri~ a final product of 32 kD. This 32-kD fragmem. howover, ha~ a T, of 43~C due to a v~ high amino acid ~emcnto and thus remains helical at physiologic core temperature. We now repot1 Ihat the 32-kD product reslal~ my further ~ta~k by several matrix metallopmleina~es including inte~Slitial onll~a~e, 92-ED ~elatlnase, and matrilysln. However, this eollagenase-genetmed fra~menl ~n be reedily degraded to ~ompletion by cathel~in B at 37"C and pH 4.4. lnter~tingly, even under acidic conditions, cathep~in B cannot effectively attack the. whole ~-kD type X molecule at 37~2, but only the 32-kD collascna~-generated reer~l m~lt. Mo~t impotently, the 32-kD fr~menl was also de~rad~ at acid p}! by l~es |~olaled from maine ostencl~m. Degradation of the 32-ED ~en fr~menl by o~leoclast ly~ates exhibited the following pro~rtles: (o) cleavable occurred only at acidic pH (4.4) and not at neutral pN: (h) the cyslelne pr~ein~,e inhPo~ors B64 and leupe~in comidetely Mocked degradation; and (¢) specil'¢ anti. body Io cathei~|n B was able to inhibit much of the lysate-derlved activity. Based ~o~ these data, we po~ulale that during in ~qm endochemhal bone formation Ih~.~cn is first de, grated at neutral pH by interstitial collagens.st secreted by n~cbtng eanilaE~--derived cells. The re~uhlng 32-kD fragment is perature and further degradation requires osteocla.~t-dcrived cathepsin B supplied by invading bone. 142 Sires. U. L, $chmid, T. M,, Ffisxar. C ]., Wang, 2'.-0., Gluck, $. L. and 11.~, Journal of Clinical Invesd~tioe 95:2089-2095, May 1995. Other suplx~: U.S. Public Health Service. National Institutes of Health, and Edward Mallinckmdt Department of Pediatrics. From the Dermatology and Renal Divisions, Oep~mem of Medicine, W~hlnl~en Unive~ity School of Medicine at The Jewish Hospital, St. IAmis, biG, of Pedimrics, St. Louis Children's Hospital, St. Louis, MO, and Department of Biochemistry, Rush Presbyterian-St. Luke's Medical Cenler, Chicaito, IL PRENYLATION OF RAB5 IS DEPENDENT ON GUANINE NUCU~)TIDB BINDING Rah$ is u small molecular weight GTP-I~ndlng protein Ih~ fl~'lions In endo. cylic vesicle ImlT~c. Like o~har Rss.reisted prolein~, Rab5 is IX'enylted on C-t~rml. nal cysleln~ residues, although it lacks Ihe typical C.termlnll ~ remit (wham is any aliphafic amino ucid and X is rely amino acid) Io d]m~ this IX:ll.tranllalional modification. We have inve~i~ated slruetural Rqu|remenls for the In v~'Po ~eranylatlon or Rah$. Rab5""~, a point mutant Ihal has impaired abilily Io bind or GDP, undergoes modil'~+otlon to a limited exlenl and at a ~evanely reduced n~te when compared to cognate Rab& A second poim nmt~m, R~~'-. can be Ixoce~d Io apl~oximmely the sarn~ extenl as wild.type albell at e redlx'ed rate. $|1~1 Ibl lif- ter mntation resulls in defccllvc GTPase acllvity, the~¢ combined ot'~erVallons Ir.,.Jt- talc IhM guani~ nucl¢otide binding plws an impoltmt role In tht lion reaclion and sugl.esl thai the GDiX.boond foem of Rat~ is the preNrved marion for inleraclion with Rib prenyltnmsfen~e. Thi~ idea Is tupported by lha find- ing thai non.hydmlyzable GI"P analogs inhibit Rubs prenylatlen, while i~ ~qlrn pm- cesslng of both H-r&,~ and the 'y2 subunfl of regulatonj, O Ix~in# is unalTeeted concentrations of guanoslne ~'-O-(thio~riphosphate) (OTPI~) up to 400 p.M. Mmeover. a truncation mutant Ileklng the C.t~,minal cysteines, Rab5'~', mrv~ an inhibilor of RabP' genmylgeranylallon when lisanded wilh ODP bul no~ Thus, Ihe reeognillon of R~b$ II a sut~rsle by Rab prenyhransl'erase Involve~ Mruc. tural elements exclusive Ol" the C lerminus and de~ndenl upon Ihe ODP.bindinI conl'own-~ion of the IXotein, Sanl'onl, J. C, Pan, ¥., and Wesding-R~nlck, M, The Journal ol'Bio]ogical C'~misl~ 26~(~2}:23TI3-23776, November I$, 19~3, O~her suppo~l: Anm'ican Cancer Society and lhe Nalicnal Inslilules or Health. From the Deparlment of Nulrillo~, Hazard $cheol of Publlc He¢flh, Bo~Ion, MA0 143
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O O "DIE O-RICH AUXILIAR DOWNSTREAM ELEMENT lIA,~ DISTINCT SEQUENCE AND POSITION REQUIREMENTS AND MEDIATES EFFICIENT 3' END PRE-mRNA PROCESSING THROUGtl A TRAN.~;.ACTING FACTOR A dowr-,d~,~m G-~ch ~qucnce (GRS}, CKK",GGAGGUGUGGG, has hecn ~o viou~ly shown to influence Ihe efficiency of 3' end pr~v.:essing or the SV4I! late polyadenylation signal. We have now defined several imptm,,nt paramete~ tar ORS. m~diatod polyadenylatlon. The ahilily of the ORS to influLm~ .~' end pfo~.csing elTmkney wa~ sensitive to individual and multiple Ix)ant mutations within the merit, as well as tl~ po~|tion of el~ elem~! in tl~ down,~tre.'mt t"egion. Competltk'~ Itm|ysts indicated that the ORS functioned Ihrough a titralable trues.actlng r~o~. The ORS-specific DSEF-I protein was fmmd to be bound to the ~me population of RNAs as Ihe 64 kl~ protein of the general polyndenylatiqp faclm" CalF, indicating that DSEF-! is associated with RNA subxtrates undergoing 3' end proce~¢ing. Punbermoce, an as, mciatlon was o~alned between the relative strength of DSEF-t protein binding to ORS variants and the relative ability of the GRS variants to medi- ate elTgicnt cleavage in v#m. Finally. mutations in the ORS affected th~ efficiency of ©ro~.linking of the 64 kDa protein of CalF. The.ca dat:t define a novel class of aug|liary downxtrcem element and suggesl an important role for DSEF-I in .'3' end Bqta, P. ~., Ford, L. P., C'hen. F., and Wilu.~., J. Nut|tie Acids Re.arch 23(9):162,5-1631. 1995. Other xuppo~: National In,~titutcs of I Icalth. From the UMD-New Jersey Medical School, Duparlment o! Microbltllogy aml V&l RNA BINDING PgOTEIN MEDIATES THE ASSOCIATION OF Vgl RNA WITH MICROTUBULES IN XF_.NOPUS OOCYTES Lo~al|zed RNA$ are foend in a variety of somatic and developin~ ~11 types. In many cues, microlubules have been implicated ~s playing a role in facilitating trans- po~ of the~e RNAs. Here ~e mpmt tha¢ Vgl RNA. which is Ioca~i~-d to the cortex of Xotopu; latvia t~x:)'tes, iS as~ceialed with micgotub*J~es M viva. Because th~ ubiqukeex nature o1" tuhalin, the assncltt|oe of ~ecil'g RNAs with mlcrotubule~ is likely I~ involve faotors that recognize both RNA and mlcrotub~les. VSI RNA bindMg plebe|It (Vgl RBP). Ix~vin~sly shown Io bind with high affinity to the vega. tel Iocall~tion site in Vgl RHA. appem to function in this capuchy. Vgl RBP ix ~r, oalated with microtubulm: it is enriched in micmcul~le extracts ngtx~cyte~ also co.preci~ltetnd by helerolo~, polymerized tubulin, ffanhern~e. Vgl RBP b~nd~,~.ty |,,~u~-~ rathe sp~ir,~ ,~=|,tio. or._vgL.e~A to in vit,'~. ~ data suggest a general model for how specmc Kp~,s can tg to partlcular xit.,.~ via common eytoskeletel elements. Elixha, Z.. Hav;n. L... R;ngel. i.. and Ylsraelt, J. K. 1,14 The EMBO Journal 14(20):5109-$114o 1995. From the Department of Anatomy and Embryology and Department of Phawnacology, Hebrew University Medi~d gcho~,.l~n~lem, Itmel. CELL DEATH: PROGRAMMED. APOPTOSIS. NECROSIS. OR Them are at least two mawr types of active or physiological eell death. The ~t ~ll.known form, apolXOSis or Typ~ I, involv~ early hue .k.~'. collq~¢m, sat|on of chrom~in, genera|on of nucleo.so~, xl IMders, and.ca, try. me~..tat .~n.v" m little ~ no e~fly altemio~ of lytm, om~. It t| .mint cornm .on!y see.n .m. ce..s from highly mitotic lin~, and the cells are pnagocyto~co uy net|hi.'mS c~.!l, or infiltrating macmphages. In m~amo~o~i_ng, or see.re.tory ~ll~. and u .nc~. g where the majority of cells die. the l~lk agree eytoptalm la cona~n'~a of the lysosom.'d system well berate nuclear collapse |4 man|felt. Th|a form ~f tell death has ~ ten'p~d Type It cell death, and we r~. ¢.rt W_t_his te..r.min.~...o~y. requ|rement for protein xynthesi$ is marc eharsetenxt,~ o! Ty~.. !1 ¢~11 developmental situatkms than it ix for Type ! cell death, The vl~atio~ mn force t reaxf~--ss~nl of Ih~ aspeets Of phpiol~i~l ~11 (:~ath t~l ~ t~|y the I}iO~G,~l al~l ~)t~hotogy O[ o~.nt ceils Will llcIp eimiy Ine nmlecular and bi~chcm|cId findings. • Zlll~eri, ~ Burseh. W.. Tenn|swoed, M,, lrld L,ockshin, R. A. Cell ~ath and Differemlatlon 2:g7-96. April 1995. Other support: Nat;onal Institute on A$1n~. P.S.C. CUNY. Medical Council of C~lnldll. and ~atior, lll Cancer institule and Human Frontkra of Sellncl Prognlm. From the Depenment or BioloLy, Queens Collele Ind Orlduite Canter or CUNY. Flushing. NY. initiiut fur Tmnorbiolog~e und Kr,~.4~s. f_org.h.U ,n.l:..,Univirlitat AusWia. and W. Alton Jo~es Cell ~cknce (~enter. t.age t~acto. IV. Developmental Biolog~ EMBRYOGENESI$ IN V/TRO: STUDY OF DIF'~'RENTIATION Of EMBRYONIC STEM CELLS Embl~enesls is the fimdamentet process of dllTerenthiion of all dlall¢l 145
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0 0 0 later differentiate to all cell types. Recently. lines of embryonic stem cells have been ettablithed in culture from blastocym. The cells am pluripotent and can differentiate in vhv~ tO all lineages. Interestingly, differentiation oF these cells can also be induced in ,,fl~. Morphological and molecular evont, t that are characteristic of the develop- ment of the embryo can be: mimicked in t,irro by growing the cells under contmllnd conditions. Furthermore. the ~ of earl), develof~nent can now be studied and manipulated in ~,/wo and specific lineages of stem cells can he ob~alned and grown in culture. Du~bnlk-Levinton, M. and Renven~y, N. Biology of lhe Neonate 67:77.$3, 1995. Other support: United States-lsrael B|nalional Science Fonndalion. From Ih~ Deparlmem of Genelies, lnstilute of Life Sciences, The Hebrew Univerthy, Je~ue~lem, Israel. MEK 2, A CAENORilABDITIS F£EGAN5 MAP KINASE KINASE, FUNCTIONS IN RAS-MEDIATED VULVAL INDUCTION AND OTHER DEVELOPMENTAL EVENTS Activated Ras initiates a cascade of sequential phosl~lallon events inclnding the pl~ein kinases Rat, MEK and MAP kinase. The Let-60 ]~as mediated signal mmsduerkm pathway controls vulval induction in Caenorbabdit~ de,fans. Both Lin. 45 Raf and Sur-! MAP kina~ have been determined to be es~ntial factors derln8 vulval indootion~ however the C. cleans reek gene ha~ not been idemif'~d. In this .pa.~..r we have cloned a C. ele&anl reek gene, reek.2, and demonstrated that the MEK.2 p~eln possesses the biochemical properlles of MAP k|nase klnases: The C. ele&anl MEK-2 protein can phospho~ylate and activate a human MAP klnase (ERKI) t~d MBK-2 |tself can be phosphocylated and activated by immunoprecipi- tared mammalian Raf. The reek.2 gene plays a key role in the let-60 ms.med|a~ed vulval indeotlon pmhway as loss-of-function m~ions in the gene Obdi4 and sif~if':~mtly reduce the signal tran|mitted throegh Ras. mek.2(k~l]4) completely soppremed the Mtddvulva (Muv) pbenotype of a hyperactive/et-60 ms mutation and animals hommy~us for mek-~(t~ll4~ also d~#ayed a p~ial larval le*hal pBeno- type. Antmala homonyms rot met,2(h294) exhibited a highly pen~rant steele and Velvaless p~meo~e. Mk'reloJe~;ou of a gain-of-function reek-2 m~-dm resulted in Mev a~d e~he~ mutam I~,m~y~, whereas miero~njectlm of a deminamme~a- tiv¢ re.rot.firm pot onl3~ anl~e,~d the Muv phen~y~ or an activated let.60 mutttioa Ira! al~o craned an egg-laying defective phenotype ia o~e~wise wBd lype ank~als. O~f remits demon~lme tha! reek.2 ac~s between lin-45 tar and sm'-l/mpk.l in a signll iran~uedon palhway used in the control of vulval differentiation and oth~ deve~opm4m~l events. Genet & Development 9:742-7:55, 1995. 146 Other support: U.$. Public Health Sc~vlee and the American Cancer Society, From the Department of Molecular, Cellular, and Developmental glololty. University or Colorado at Boulder, Boulder, CO, and Department of Chemistry and the Institute of Geronotology, University of Mkhigan Medical School. Ann Arbor. Ml. ,r;UR.2, A NOVEL GENF_., FUNCTIONS LATE 7 3 IN THE LET-60 MEDIATED SIGNALING PATHWAY DURING CAENORH/IBDITI$ ELEGAN$ " VULVAL INDUCTION We de~erihe here a new gene ac~ing downst~um of let.nO r~ in the vulval tag- haling pathway or Coenorl~lS~rts eleRons. The sur-2 ~ o~ I~IS) ~ is defined by eight mutations Identified in a genetic ~creen for aupl~et~ol~ of the Muhivulva phenotype of ler.60(n1046), an UeliVaeed let.60 m mtllatlon. $I~r-2 lions result in pleiotropic, incompletely penetrant phenotypea that Include a Vulvaless phen~ype in hermaphredi~es, defects in development of the mala grma(lal almornmlltle~, and larval lelhMity, indicating a role fro" d~ ~rur-2 ~ uet in multiple developmental events. Genellc epislasls malyses au~le~t thal ~r.2 Is required late in lhe vulval signaling palhway, downmman of let-60 Rm. and Is likely to ac~ down.qream of the Raf/MAP Kina~e cascade. We cloned the a~r.2 DNA..medla(ed Imnafoemntion. and have shown IbM it etN:x)d~ a novel pt'~eln. We also show that a sur-2::lacZ tran~me i~ expressed in lhe vulval pmmr~or e~lla at the time of' vulval deten, inatlon. $1ngh. N. and |Inn. M. Genes & Development 9:2251-2265. 1995. O~her support: U.S. Publlc Health Service and d~e Lucille P. Markey Poundatkm. From the Department of Molecular. Cellular. and Developmental Biology. University of Colorado at Boulder. Boulder. CO. THE DROSOPHILA EXTRA SEX COMBS PROTEIN CONTAINS V/I) ESSENTIAL FOR ITS FUNCTION .,t..S A REPRES$OR OF HOM~J3trlc OBNI~3 £~tra sex cmd,.s is a member of the Pt~,~h Group ~ne~. whole preducts required rot stable long term transcrlptienal ~'ixessio~ of the homeotie ;ene~ of the Bilhorax and Anteenapedia complexet. 'The Pc-O proteim are _.r~._tred to the .,~tially restricted domains of homeoti~ ~me exlxe~io~ emblithed by tho am. siently expre~tsed repressom, e.g hunchback, but are nol required for the funetlonlnjt 147
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O O of these e~y represr.~rs. This implies |we distinct mocks of'represskm ,,rid rains the c[~.~tiCxS how does the transition from eady transient repressio~ to stable Pc-G-medi- ated repressim occur?. W~ile other Pc-C proteins ~e required continuously through. OUt developo~t, the t.sc UP. is only present transiently in early emSryns, suggest- ~ that t~' may play a ro|e in mediating this transilion to stable long term Pc.G- mediated repression. The predicted esc protein contains mulljPle copies oF the WD mot|f, fouad hi G-protein/~ subenits as well as non-G prote'Fns involved in diverse ~tluhu' fenctkms includin~ transcriptiouzl repression. The sequence aherationa of a numbtT of t.xt" mutations cause LqlirIo acid sub.~titutlons within the WD repeats. k~entif~j'in~ them as essential for the function of' the exc protein a~t a represser or homeotlc ~e expression. Other WD Ix~telns are components of" reve~ible macro-. molecular a,emblles and the WD motif has recently been directly implicated in rnediatlni[ inleractio~s with other i*d'oteins in ~ch co¢~plexe.% We ~ that the tsc proton is similarly invotved in the initlal recTultment of ~.G repre.~.e.or~ to the homeotic ~enes to estahli..t5 their stahfc long term represx;o,. Sathe. $. S. and llarle. P. ]. Mechanisms 0£ Development 52:?7-8?. 1995. From the Department of Genetics. School of Medicine. Caxe W©.~tem Re.~rve University, Cleveland, OH. THE EX~A gF.X ~TMB$ PROTEIN IS HIGHLY CONSERVED BETWEEN DROSOPNILA WRIU$ AND DRO~OPltlLA MELANOGAfrER F.xtea ~ex combs (esc) it me ef the Poly~mb Group genes, wh~e p~lucls am required for king term mainlenmee of ~ t~ally ~sl~ d~ins or ~k ~ e~i~ ~itially e~biis~ ~ t~ ~ucts ~ t~ ~entsli~ ~ We ~lly S~ th~ ~ e~ ~ein ~ fl~ ~s ~t~ WD ~if[ whi~ in ~ ~nS ~ ~ di~iy t~ in ~ein~ein ~nter~ti~s. ~utat~s ~ ~ a ~ of ~ ~ ~l. We ~ thin l~y may mediate ~t~J~ ~W~ t~ ~ ~r Pol~ G~ ~eins. ~iting t~ m t~ir S~ ~ k~k. ~o £u~r in~sti~zte the £uncti~zl im~zn~ or the WD ~ifs ~ ~ti~ ~ f~ally SmUt mgi~s of t~f~ ~ein, ~ ~ave D~fM ffri~f, a diu~tly ~laI~ Dr~ila ~ies. We show I~ I~ esc ~ h~ ~t~ of ~atim, ~kul~y m ~ttl~ which a~.not ~ m ~ WD ~nses ~v~ from align~I or all known WD m~its, suggests I~t ~n 14g ization has been highly conserved dunng evotutkm. ItS ntlnry cnm~o exhibits the greatest diver~nce, bet even the~ dllre~ences are conservative of hs predicted physical properties. These observations sugpst that the e:c protein is fu~ctloually compecl, nem4y e~ery residee m~in| an imlx:xtaot eonldl~ltkm t(t its function. Sathe, $. S. and Ha~e, !'. J. Mechanisms of Development 52:225-232. 1995. From the Deplrtment of Genelica, SchoOl of Medicine, Clse Weltem University, Cleveland, OH. IDEN~FICAT~ON OF FAT-CELL ENHANCER ACTIVITY IN DROSOPHILA MELANOGASTER USING P.ELEMENT ENHANCER TRAP~ To Identify Series important in fiat.cell metaT~lism and developmerd, we have screened l~ila atocks crating an e~gineefed transpel~ble ekm~nt that can reveal the presence of nearby enhancer elements. We have identified IhOH ,,.-" ,hat speclr~ enhancer ek~ments. We Inli¢lplte that I1~ l~el as~tatea will i,ro,,,,..k., n, ,,ing ~ for stndying fat.~ell q~e~ille ~ exlxe~n, rmu~'m~. ;,t ~hwv~'.h'~o lines ~"live in the developing fat carl ahould lxovKl~ In Garry point and Kr, a zlne-I'mBer protein, am ix'e~nl m the fat I~O~y, Bole lletorl fioally, in a $in11¢ ilne transgenc activity ia F, tesenl in the peopnttor celia or e~ic fat 5ody. The lanes a.ttociated with there enSam'er-lfap Ilmr~ mary be invol~ed in fatell development. lloshizaki, P. K., Lunz, R., Glx~h, M., and ~nson. W. Ge~ome 38(3):49"/-50& 199& Other support: American Cancer Society, llllnoi$ Division. American Heart Assocls~icn. and the Campgs Resem'ch Erom the University of lllin0lI (::ollele or M~dicine at Chicalo, l:~l~rtment or Biochemistry. Chicalo. 11.. and Uaiver, lty Coll¢le of' Medicine at Chlcale, l~rne~ or lmmunotosy and Mk~obiolos~. CSicalo. IL. 149
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0 I~OT~IN KINASE C ISOZYMES IN PROGRESSIVELY TRANSFORMED RAT IIMBRYO FIBROBLAST~ ~ role of individual prolein kinlse C {PKC) i~form~ in pmgrer.~ive transfor- mati<~ of n ret embcyo fibmblas~ oell line (REF52 cells) has been evaluated. Normal ce. line. KI~P iS ce.s are moqmowgically transformed but have o~ly limited ¢ap~i~v fro' grov~h in mh agar. Meszage and prmein levels f~r PKC~ and PKC.~ ~ ~imilar in REF A and B cells, indicating that expresslod'of SV40 large T did ~ diru~y influence the amounts of PKC#. However, PKC~ localization was influ. m~d. PK(~ ~ associated with focal contacts of REF A but not REF B cells, h~dicmflng Ihat ehanges in luestion rather than comenl are an early event in REF52 orll transfocmatio¢l. Clones of REF B cells were selected for growth in soft agar (REF C orlls). Leveis of PKC-8, but not PKC-a or e, were increased in ~veral of these clones, su~esting that increased PKC.~ cow,tent may facilitate anchovage.inde- ~ind nd~t ~rowth. in o~he¢ stvdiex, we have determined that PK(~ interact with their inn I~oteim/~ubstrates through their regulatory domains (RD: L. Lira et oi.. mnlmt .PK.C |lg~aling by competing with endogenous wild-type |)KC~ for binding pl~eln/~ul~trate interactiens. Overexpresdo~ of th~ RD of PKC-8 inhibited growlh in sof~ ~ of one repre~tative REY C clo~e, whereas overexpcession of the RD of PK~-¢~ procr~ed growth i.n sof~ apr. These results suggest that RD expres.do~ may I~ a u~ful aplxoach for aeminmm negative PKC inhibito~ with po~emlal isozyme Lisa. L,. Ramsay. K., and Jakeu, S. Oell Growth & Diffem~tiati~ $:1185-I 194, November J994. Other ,upp~: National in~itutes of Health. From the W. Alto~ Joc~es Cell Scienc~ (X'nter, Lake Placid, NY. HMO 17 [$ A CHROMATIN-gPEC1FIC TRANSCRIPTIONAL COACTIVATOR THAT IN~E~ THE EFFICIENCY OF TRANSCRIPTION INITIATION W..e hive examined the effect of HMO 17 on mmsc~ptio~ by RNA polymer~e H by u~ ~bly and mmlysis of HMGl7.ccmtalnlng chromatM templates comisl- in| M r~Ma~ly ~ nuel~momal an'a~ys. Structural analysis of the clu~matin indi. ~! ~ HMGI7 :s incoqxnted into chromatln in a physlolngieal manner with the full eomp|em~t of core histones. The transc~'iptionsl studies revealed that HMGI7 sdmulmes tmmm~ion in conjtmct/on with the seqnenee-specific activator GAL4. VPI6. This ~Muc~ w-,s observed with ~hromatin. but not with nen-nucleosomal tern. plat .~/and required ~he presence of HMGI7 during chromatin assembly. The incor- Foranon of HMGI7 into c~romatin resulted in a 7- to 40-fold stimulation or GALA. . .VPl6,~ctivmnd U'amz:rfptkm to levels that were eomp,~able to those observed with histone-froe DNA templates. In contrast, transcriixion from HMGlT-containing chromal~ ~ not detectable in the absence of GALA-VPI6 m" with a GALA deriva- 150 live IGALe(I-14?)I lacklnl lhe VPI6 acllvelion domain. Rnullyo the Ino~pomlon of HMOI7 ~o ~#lin was f~M Io i~m m~P~ tmn~i~al ~iv~ thai ~ ~ RNA ~ly~ ]L ~. S. M.. Km~. A.. ~ K~ J. T. ~s & ~vel~nt 9:1~8-1~1.1~5. ~ su~: Nmi~al institutes of ~Ith F~ the ~nt of Bi~y, ~ Bi~ical ~s. Unlv~ity of ~lif~i~ llutcbls~ C~ Cent~. geattle. ~A. FOUR-JOINTED i$ REQUIRED FOR INTERMEDIATE GROWTH IN TH~ PROX|MAL-DISTA~ AXIS IN DROSOPHILA Genes capable of lmnslating po~itlonal information into relulated Smwlh ll~ st Ihe heart of moqxhogenesls, ~ few g~ors whh ~hls I~+-llon have been Id~ntill~d. Mulants in lbC Drosnphil~ four.jointed ~) gene show reduced ISowth and ~hered diffcrenti,qion only within r~trlcled ~ of the lXOXln~l-dlmd (PD) axis in leg and wing. thus .0 is a candidate for m gene with Ibis comdlnatlon C~mi~lenl ,wilh a pmilio~sensillve role, we show lhit.0 is exposed in a pmlem in the developing leg, win~ eye and e~lie lobe. The.0 tern encodes a nev¢l lype !1 membrane glycoprolein. When lhe cDNA is trenslaled in an M ~qlPo liO~ syslem in the Ixe~ence of exol~mOue mlct~omal membrar,~, the potion of ~ome of ihe molecules is ¢le~ved, yielding a ~'x~¢ed C.eatmln-t We pmpm~ that.0 enendes a secreted signal thai functions as a pmiliv¢ mlulalo¢ of regiomal gmmh and dlfferendstio~ akmg the PD axis of ~he imaltnd diet. Villano, ]. L. and Kalz~ F. N. Developmenl 121:2767-2TI7, 1995. From the Dep~ment of Biochemisl~, University of Texas Southwestern Mldlcll I~enler. Dallas, TX. WIRING DIAGRAMS: REGULATORY CIRCUITS AND THE CONTROL OF SKELETAL MYOGENESlS During the p~t year, targeted mutage~sis in mice has begun to clm't~y th, ml~s of individual memhenc of Ihe MyoD family of myogenic reeulatm'l In VOllebl~te 151
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development. In this review, we discuss these studies both in the context of. tissue interectleez necessaw to induce skde~zl muscle precursor cells during cmb~7o~cne- sis ~nd the molecular cireuitr~ that regulates the terminal differentiation of these cells. l~s.~r. A. and Munstcrberg. A. Current Opinion in Cell l~iolo~y K:432-442. !~4. arbor suitors: National Science Foundation. LueiUe P. Marcy C*haritahie Trust. Muscular ~stropby Ar, r~iation, and the March of Dimes P~irth Dcf.cel.~ From the I~pzrtment of" Biological Chemistr~ and Molecular Pharmacology. Hm'vard Medical Scbooi, Boston, MA. C~OMBINATORIAL SIGNALS FROM THE NEURAL TUBE. FLOOR PLATE AND NOTO~IORD iNDUCE MYOOENIC bIlLll OENE EXPRF.~SION IN THE SEMITE The neural luhe. floor plate and notochord are axial tissues in the vertebrate embryo which have been demonstrated to phty z ale in semite morphogcncsis. Using i~f ~tro oo-cuiture of" tissue cxplanL% we have re(tailored inductive inter'actlons of"these axial tissues with the adj,-ant semitic mcsedcrm in c~ick embryos. We hove found that sign, s from the neural tube and l"ieor piatc/no4oEhord are nece.~pj for expt'ession or Ih~ myopnic bHLH regulators MyoD. Myf'5 and myogcnin in the ~mite. Eventtmlly semitic expression of. tl~ myo~nic bHLH ~cnes is maintained in the it~n~e of th~ •xi|l tissues. In o~.,an cu!ture, at early dcvcloprnental sta~es (tIH I I-)~ lndecti~n of" myogenesis in the thece most recently formed somitos can be mediatnd by the neural tube to~etber with the floor platc/notechord, while in more festal somltes (st~es iV.iX) the neural tuhe without the flo~" plate/notochord is sufficient. ~y recomblnin~ somiles and neural tubes from different uial levels of'the ~nl~j~ we I~ve farad that z secoud signal is necessar~ to promote competence or the ~omile to respo~ to inducin~ signals from the ncur~! tube. Thus. we prelate that st I~LSl tWO signals from axial tissues work in combination to induce myogenic bHLH ~eue expression: one sigr~l derives from the floor plate/notochord and tbe other stgn|l derives from regions or the neural lube other than th~ floor plate. F, funnmt~Z. A. E. and Lair. A. B. Development 121:65i-~0. I~$. Other sW~port: Natio~al Science Foundation. LucJllc P. Mnrkcy ChariUthlc Trust. Muscul~ [~troplty Association. and the March of" Dimes llirth Defects Foondnlion. From the Dep~rtment of. Biological Chemistry and Molecular Pharmacology, Hsrvmd Mediell School, Bocton. MA. 152 BCL-X~ IS THE MAJOR BCL-X mRNA FORM EXPRESSF..D DURINO MURIN~ DEVELOPMENT AND ITS FRODUCT LOCALIZES TO MITOCHONDR|A Most exampTez of"cell dumb in an;male L~e controlled by Iz ~encti¢ program that is activated within the dying celt. The apoptotlc proces~ is further r~al.st.ed by ~.~t of ~enes ttmt act as relxe~sc~ of celt d~ath. Of these, ..t~. -2. is .e ~x~ .sl~...In, zz v~nety Of" eml~'yonic and pmtnattl ti.~suea w~.~.h s.u~pstS 8 cr!.tzes!.r~, le I~ ~'./.~. m ~enesls and tissue honlc~tasts. S.urpns|n,~ty0 mutmlt .mice .w!th t .ml~..t~..otsru~to~. o/ 1~1.2 appem" normal at birth she compzele rrmturat~on at tympz~olo tissues oetor~ succumbing to fulmi~nt lymphopenia m~d polyey~ic reflal disease by 2.$ w~4~s of ~c. This sugp, csts that there may be |enos other t..hm &rl-~.~at c~n.rel~uist,~ sis during development. To ~gln to investigate this poaslbilityo v~ have czon~a characterized the murlne ~cl-x ~ene, whose human counterp~t divines |tdkin$ homology to brl-2. The prediet~ mmin~/x't.x~. ~e product cxhiblts z high lavel el" ~rulno acid identity (~7%) to its human countcrp~./ust like Bcl2. the murine withdrawal. In addition, the bulk of" the bcloX~ pro~uet lueat|zes to tl~.pen~r~ mltoch~dHa as assessed by s ~x'~-x.-tag cxwcssioa system..s~st, .~.tnm. ~. r~cl-2 and/~'~-x,, j~'otelns prevent c¢i! dc~th by | similar rn~l~msm, r~'~..rs tsmo most a~ndant/x'/-x mRNA sp~ics expressed in eml:~ic and .~ult tl~..on~. ~ levels or I~l.x~ mRNA al~ear higher than those or/x,t-2 .d.uri.ng .e~. ,I)~jo~al~.~ve ment m~d in several ~dult o~ns in¢!udihg beoe nmm~w, main, ~_x~... ~o.tnym.t~. results from an unsplicnd/x'l-x tranlzcrtF4, b~/-x~ mKr~A.se exit. seed.In embryonic and posmatal ti~tues. Smprisiogly.~ the.e.x.pre.ss _t _o~ at" ../~r. ,Xs.~. n~.;tve regulator or programmed cell death) was undetecmme t~ • ~smve assay and polymemse chain re~ctloa ~'ml~sls or mouse tissues. Zlst~d on its timm and developmental patterns or exwe--lon, it sppcm that t~-x m~ pray m~ impm,. tzmt role in th~ ~gulatloo of"cell death during d~velopment and tism~ bom~ostasls. Gonzalcz-Garcla. M., Perez.Ballestero. R., Ding. L.. Duan. L.. Boise. L.H.. Thompson. C. B.. and Nones, G. Development 120:.Z033.3042. 1994. Other support: U.S. Public Health Service. American Cen~r Society. and Sandoz Feendatle~ for Gerontological Rescm, ch. From the Department of" Pathology, University el" Michigan Medical $cboo]. Ann Arbor. MI. and Howard Hughes Medical Institute. Departments el" Molecu~m" ~eneties and Cell Biology. University ot'Chlcazo. Cldeqo. IL. PLATELET OLYCOPROTEIN l|B OENE EXPRESSION AS A MODI~ OF MEGAKARYOCYTE-SPEClFIC EXFRESSION Olycopmtcln (OP)llb/111a i~ ~ integ~ complex nornudly restz4ctcd in its expr~-
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0 0 U1 gl~ion. The rece~or (~m~ex is expcessed a( high ~s du~ng m~tin ~. GPllb is only expms~ ~ m~turing ~g~k~tcs and ~ ~ ~ ~ hu~ ~er a~ ~ st~s using ~gak~ ~11 li~s. i~ a GATA~ S~¢ (4~ ~;~ u~m of ~ l~Ficeal stad site t~t in~lves ~ d~ ~ tim ~ ~ ~ a ~il~ eff~. Wh;~ t~ way. ~ ~ ~udies of t~ ~t 5'-fl~king ~g;~ using a ex~si~ ~xim~y fi~f~d, ~ile mu~at~ at ~ GATA~ ~v~ ~lfili~ t~ ~!1 I~ ~ f~t of f~u~ Sl~ will ~ to fully ~y~ tis~iF¢ ex~. BI~. K. L~ ~ M. ~ ~u~: N~i~I Instltm~ of H~IIh a~ ~ ~hulmen ~ t~ ~~ of ~ial~ a~ ~gy. Unive~ity ~ ~nsyl~nla $chml of Medicine, and lhe Division of Hematology. ~ild~n's Hospital of 154 MATURATION.DEPENDENT CHANGE~ IN THE IU~ULATIO~ OF LIVER- SPECIFIC GENE EXPRESSION IN EMBRYONAL VERSUS ADULT PRIMARY LIVER CULTURES During rat liver development, which stms on day tO of embr~l~m|~ and emil EI$, all pemnchymal cells are thought to be a hecnoleneees popell~lon of blpetentinl progenitors, able to give rise Io both hepSecytet and bile de~ cells, We established primary liver CUllURS from embq~enle Ilvm at varies devel- opmemal stages, from El4 to neo~tes, as well as ad~.lt rats. ~ ex.~!~ regulation by three known differentiating agents, heparin, oimetflyisuamxloe (DMSO). and sodium butyrate, were examined in these primary cultures. Alpha- fetoproteln (a-FP), albumin, gamma.glutamyltranspeptldate (GOT). and gtu- tathione-$4nmsfer~e-P (Yp) were CXlXCssed by cultured liver cells through fetal development, vChe~as insulin-llk¢ growth factor-il (IGF I1) recep~e¢. fetal parenchymal cells, w¢,t not IWe~mt in cultun~! neonatal cells. Ilepnrin |nerea~d ~-I~ levels in fetal liver ~ells, but not in cells obtained after bitch. The expreslkv~ of GGT and Yp wa~ eu:~dlnatety vegetated..The t.wo ~ wove .u.p.t~gulat .~. by.sodium. betyrate and down-regulated by DMSO m cutture~ t:ver ccl~s tram a, ages tested. However, the mgulatlon of these two genes by sodi_u~ buty.~t¢ and DMSO was not apparent in neonatal and adult liver cultures. ~m|ium eulyntle increased a-FP and albumin mRNA expression in El4 and El5 cells, but n¢~ in El6. neonatal of adult cultures, and its luhdition caused !~...~. Sex ..IXe~;iOnlof al .l~i~- mifl, We ~lude that the regulation of gene ex .ix'~s~ in ixlm .sly ilve.r the three agents tested is altered after birth. In fetal cel~, ~xl.ium t|u.tyratu se.rv~s 81 It strms general differentiation s.g..em, tnefe~..'mg_exlxess~x~ e_l.m.ts_ot Iones isde for lxXh helm0cy~e~ and bile duet ceils. By cmtrmt: OM.~U._I~p~I,., ~tl O goner expretsed in bi~45~,2 cells, e.g. GGT, Yp. and cytokeratm tV (~th. Drill, S.o 7rebel. !.. and Reid, I., M. Different|alien 59.'95-102, 1995. Other support: American Cancer $oclety, National Institutes of Health. and The Richard Molls Foendatkm. From the Cav~e~ and Liver Omtera, Albert Einstein College of MedkJne, and Microbiology md Immuno!o~y, Albert Eh~teta umJqe of Meotcim, PRIMARY CULTURE OF DROSOPHILA EMBRYO CELLS Pr|m~ry cultuRs of Drosophila embvjron|c cells off~ a ~lq~ system to sl~y the t~nsltims ~ u~iffemntin~ ~lls ~to a v~e~ of~ll I~ ~p~ wi~ ~wer ~ Drns~hila class;cal ~ ~l~ul~ g~t~. t~ ~iy~.th~ ~ ~lble in ~ using diff~miafing em~o un~n~anding of ~velo~nt. ~pt~tion of prima~ em~o ~11 ~velo~ i~e~dy in t~ latices of S~ and his ~lle~l (~r 155
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U1 and Us•seen 1968) and Shields and Sang (1970). The procedures for preparing cut- turns ate te~hnieally undemanding sad can he adapted to .q)lve • wide range of logical problems, Important variations include the culture, pr single emb~.os (Cross and gaft~¢, 19/8), as well a~ the developmenl of techniques for lad•ling me prceur- ze¢~ of differem cell types soch a,; neurons and museic cells (Furst and Mahowald. 19f,5). ~dvaterra, P. M. and Hayashi, l. Cell Biology: A Labor~ory Handbook. Academic Pre~;. pp. 3R3-388. 1994. O~her tUpl~Ct: National Inst;tute~ of Health. From II~ Department or Molecular Genetics, Rcekman Research Institute of the Cily of Hope. Duarte. CA. MYOGENIC REGULATORY FACTORS: DISSECTING TIIEIR ROLE AND REGULATION DURING VERTEBRATE EMBRYOGENESI$ The elucidatio~ of the myogeflle regulatory factors (MRFs) has resu|ted in a |urge of r~sesreh aclivity that promises to bridge molecular and embryological tppt0tchea In muscle research. In this review, we consider the arguments thai would plane MRPt in a key determination role for the skeletal mu~l~ lineage.in tl~. vefz~ brate embryo. Amphibian. avian, and marine syatems are compareo an~ recem developmems in the molecular embryology of myogene~is are reviewed. A model is pfopoled In aecoent for the apparent di~:~cpancles in conclusions drawn from stud- let ualng ¢~ltured cells that indicate • role for the MRFs in determination, sad .~tu~- tel of MRF md mu.cle sane expression in vitw Ihat indicate a role in diffeRntiatlon but no* in lineage determination. ~t.c~on, DoA. Devele~e~tal Biology 156:11-23o 1993. ~ Prom the Department of Biochemistry, Boston University School of Medicine, Benton, MA. DIGIT TIP REGENERATION CORRELATES WITH REGIONS OF MSX! fHOX ?) EXPRES$1ON IN FETAL AND NEWBORN MICE We report that during monse fetal development transerlpts of Msxl nnd Msx2 the Mx~r~ ~xpre,ion domain i~ alway~ more mslal ,tan M$xI. P.[ mnn onto max and Max2 are expresr~,d in cells or the nail bed and hair follicle, We have reend that the regencralive ability of moo~e dig~ til~ is reslrlclnd to levels i~ whleh the ampu- tation plane is within the region of Mxvl, bul not Max2, expression in early felll digits and to levels where bolh Mxxl and M.tr2 are ¢xpresf~d in late fetal md neon-. tal d!g!t~..Fetal digit tip regeneration is.rapid lind completed b~f blab. wheren~ seem. tel denis tip Rgeneratio~ requires 4 weeks and is somelimes Imperf¢¢L In b~h fetal and neonatal digits, we find that both Ms, vl and Mgx2 are exprez~d during Rlenem. tlon. but not during wound healing associated with proximal impel•lions whtre no rel~ter~ive response is observed. ~ data support d~e hypelhl~ia that Iha exptl~ sins of M.r~" genes are impotent for digit cells to initiate and pe~icl~te in a repntr. atlve response. Reglnclli. A. D.. Wang. Y.-Q.. Swetoon, D., and Muneoka. K. Development 121:1065-1076, 1995, From Iha Depallment of Cell and Molecular Biology, and the Molecular and Cellular Biology Program. Tulane University. New Orleans, Bo~ton Unlvenity ~cheel of Medicine. Breton. MA. RESTRICTED EXPRESSION OF TYPE-Ii TGF/~ RECEPTOR IN MURINE EMBRYONIC DEVELOPMENT SUGGF~"TS A CENTRAL ROLE IN TISSUE MODELING AND CN$ PATTERNING The type-lI TOF~ rnc~or n~-diates many of the biological responles to TO P~. An examination of the expression of the type-ll TOF/~ recept~" durinl; mouse .embryngen~is Iherefore provides specific information •bum the role of'rGlz,~ dut. mg emlxTogencsis than has been available In date. We have imlated the gee>role marine homologu¢ of Ih¢ hemm type-ll TGF,8 receFo¢ ending Io eaon 2. The marine and human sequences show a high degree of homology. Using routine pro~ we found that type-ll 'I'GF,8 reeeptor expreaion is rel~ulated in bo~h e spatial and • temlXXal faxhioa by using t~ ;ira hybddixation and ribomtcleem lion a~ays. Type-ll TGF/~ receplo¢ expeestlon ia localized to the mezenehyme dur- ing ¢ritleai interactions with adjeccm epithelium such as developing hair follklel, wh;sl~er follicles and tooth anlaBe. !~ the eenlral nervous system, type..ll reeep(or expreszlo~z is highly restricted to the floor plain. ~lmng exprt~lon is deteeled in migrating neural cre~ cells, meninges and chorold pin,u,, gpeclrl¢ enchymal locally, allen or Iype-ll'TGF,8 receptor is al~.o ob~rved i, lung. kktney. intestine, stomach, and bladder. The rellficted cxp~lJon of type.If TGF/! ree~plor tn rnc~enehymal cells at sites of epithelial-mesenehymal interactionl auglezta type.II TGF/~ receplor plays a maim" role in mediating the e~tablidmnenl ef embP/. ocic r~an systems. The highly restricted expr~,ion of type-II TOP# ~,el~Or In developing CN$ sngge~ts nn important role for a u~"dne/thR'ontne kin•In In pattern- ing of Ihe nervous system. Wang. Y.-Q.0 Sizeland. A., Wang. X.-R. end Sa~Been, D. • Mechanisms of Development $2:275-299. 1995. 157
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0 From the Deportment of' Biochemistry. Boston University School of Medicine. Bmle~. MA, Whitehead Institute for Biomedical Re.~arch. Cambridge, MA. and Dcpailmenl o1" Pharmacology. Duke University Medical C~l~r. Dmham. NC. ECTODI~M.MESENCHYME AND MESENCllYMF.-MF.~ENCI IYME I~ACTIONS REGULATE MSX-I EXPRESSION AND CELLULAR DII~'~REN~A'TION IN THe- MURINE LIMB BUD 'The ~oi~|| eeto~.rmal rld~e (AER) is a specialized thi¢l~ing or the distal limb m~se~hyme ~at h~ been demonstr-ted to suppoa llmb omgrowth and proof llmb dev¢10~morlt. The homoo~ox gone. Msx-I. is associated with the distal limb m~s- ¢~chym¢ (l~o~ss zoo) and its expression depends upon the presence o1" the AER in chick limit. We demonstrate hem that the expression of Max-! is del~ndeot upon the limb ectoderm in the mouse, but that the inductive capacity of. murlnc llmb ecto- derm is hal restricted to the AER. Msx-I can also he maintained in limb mes- e~chyme by the ~JbGtitution of PGF 4 fo~ the ectoderm: however. ~ see that local cell-cell interactloos me required for high levels of expression. Disruption of cell- cell lnteractiom in Ihe limb mesenchyme resuit~ in a dramatic decrease in Msx-I levels and a precocious expr~sioo of. MyoDI, su~J~esting that the limb environment ~a dlll"~ntlation and promotes cell proliferation durin8 ezdy development. BMP 4 and i~GF 2 can also maintain Mss-I expmssio~ in limb mesenehyme as well as reticule acid which is usually associated with polarizing activity in the early llmb. Max-2 expression do~s not apl~ar to be dependent ulxm c~ll-¢cll interactiom as m~asured in these experiments. Taken toselh~r, our data suCeL-st that the eXlX~Ssin~ of MIx-I. but not MIx-2. no~ only ~luims factors From the limb ectoderm, but also mlic~ ttpon cues f.mm focal cell interactions and thai the spoli~! distribution of indue- live eal~cltics io limb ectoderm differs belw~n the avian a~J mu~in~ systems. WSml. Y. and ~w~s~ D. Developmental Biology 168:374-382. 1995. Other suptxxt: Natior~l Institutes of Health. From the Department of. Biochemistry. Boston University School of Medicine. • o~lon. MA. SECRETED SPITZ'TRIGGERS THE DER SIGNALING PA~IWAY AND IS A LIMITING COMPONENT IN P.,MBRYONIC VENTRAL ECFODERM DETERMINATION The ~ zone enoodir~ a TGF.a homolog, has been shown Io affect t subr, cl or developmental process~ that am similar to tho~ resulaled by DF.R. the Dnxw~pkila EGF receptor homolo~. This wot'k demo~mes that Spitz trivets the DER ing cascade. Addition of. a secreted, I~t mx the membrane.mao¢itled ro~m to $2 l~o~hila ceit~ exp~ssin| DER gives dec to • rapid tymsine a~tophosphoPp Inlion or DER. Following autophosphorybtioo. DER as~ociales with the ~ nd~pler protein. Coma~uemly. s£qivation of" MAP kiaaee iz ohae~ed. The p¢orlle of MAP kirtle activation provides a qutmhatlve as¢~y f.or DER scflvadon. A dine hetw,.-~n Ihe levels of. Spkz and MAP klnnse activity w~ observed. The secreted Spitz pmleln was expms~l in emhnjo~ to a.,u~.~ its biological a~tlvlty. An in cell f'~t~ w~ nh-..ef~ed in tl~ ventral ectod~fm, such thai lateral cells ac~dml the ventral-mini f','~les. 'The result indicates that Bm4ed actlvatlo~ of" the DER may normally give rise to a mi~rtoire of did'crete celt ralce in the ventr'at e~.loderm. Sl~tinlly restricted processing of Spitz may he rcsponslbl¢ for this Eroded aetivedlmt. The Rhomboid (Rho) and Slur Iwotein~ weR su~ested, on the halts o1" genetic int0r- ~ctlo~L% to act as rmxhdato~ oF DER signaling, No alteration in DBR autoph~pho- ~lation or the pattern o1" MAP kina~e ectivalion by secreted Spitz waa when the Rhe and Star proteins were ¢OeXl~essnd with DER in $2 cells. In mutant for rh~ or Star the ventraltzlng effect of secreted Sphz is epist|tic, that Rhe and Star may normally t'acilitatc Fmcessing of.the Spitz p~'ecursor. Schwcitzer, R., Sheheral~ny. M., Sager. R.. a~d Shilo, Genes & Development ?:1518-1529, 1995. Other xupporl: Nulional Institutes or Health. German Cancer Research Canter, Wol f..,~l Fund, anti Ihe Minerva Foundallon, From the Dcparlments or Molecular Genetics and Virology and Membrane Research tad Biophysics. Wcizmann lnstltule or sclcnc¢. Rchovnt. israel. RF.GULATION OF TIlE XENOPU$ LABML HOMEODOMAIN OI~IB~, HoxA I AND HoxDl: ACTIVATION BY REFINOIDS AND PEPTIDE GROWTH FACTORS Vertebrate homolo~ues or Dro~pkila labial am likely to play key ~oles in anlempo~terior axis formation. How©vu. little is known aboot Ihe relulatlon of these genes during vertebrate development. Hc¢c we examine the exix~.,,llon and ~¢ulatlon of.the Xem)p~ labial Imateodon~in ~enes, HozA! m~d Hox[)l. l,{oxAI • was espressed around the dorsovcntral circumference, of" the mink in neurula cmbryce, with later expression in spinal cord. midbraln, hindbrein, and endolym. phatlc duct. By mid gastruls. HoxDI was predom|namly expressnd in dortolatend and ventral .ectedefm, with a pp in eXl~eSston at the dorsal midline. By ~eu~ls. ventral expression h~l d~lined with mo~t exp~mtsion rextrlcled to de~ohte~ memo. dem~ and eclnd~rm. Retinoic ecid r, lro~Bly induced HoxAI and H~x~! throughout the ectoderm and mesendnderm or gsstrula sta~s, while h~ old~- emSr~ retinotds induced ectoplc eXlXeSslon or these ~nes in more limited re$1oos. Induction by mtlnolds was inde~ndcot of pmteln synthesis. Suq~t.. singly. HoxA! w~ Ixpr~J~ed 159
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0 0 at h|lh levels in i~olated animal ¢sps in the ab~nce of ~wth f~ b~F a~ ~tivln A singly ind~d ex~i~ of }loxDl, AI. in an;real cap~: however. RNA s~umulated ~ly many hour~ a~er ~l~tlm of t~ f~. ~e~x~im of thymld ho~ ~ceplor (c~A) ~venl~ i~u~im of HoxDI by ~ti~ic ~id in animal c~. c-erbA also ablal~ e~i~ of HoxDI in ~te em~. su~e~ting a m~ f~ ~d~s retards i~ t~ ~gelatim of HoxD! ex~im. ~ant interf~ing ~ivln t~ ~nt~ ex~im ~ lloxDI i~ t~t~. ~plkating i~ of HoxDI. Our ~ta i~ic~e that inducl~ of legal-like h~omaln ~t is ~plex a~ may ~ui~ many f~. K~m, P. J. ~ S~e, H. U ~l~ntal B~logy 167:3~9. I~5. ~ ~ ~ite~ad lnstit~e f~ Bi~ical Rehash Manhunts Inuitute fm ~nol~, ~m~klge, MA. EFFICIENT HORMONE-INDUCIBLE PROTEIN FUNCTION IN XRNOPU$ A major problem in analyzing gene function during Xenopt~s development has been the inability to induce gene expre~don in a temlxmally controlled manner. We have attempted to ~olve this problem with a system of hormone-actlvated protein function, using the myogenic gene MynD as a paradigm. We show that microinjec- lion of RNA for MyoD fused so Ihe llgand-blndlng domain of either the estrogen o¢ glucocortlcold receptor results in hormone-dependent activation of MyoD function, u sssaynd by eCtOplc i~duction of muu:le-spceific ~lin mRS..A, ledu.c, lion is.lightly regulated in both imlated mimal cap~ and intact cmbcyos. Wlffi ¢etop:c muscle-~e- clfic actln cxpre~ion inducible after 2 hr of Imrmon¢ trealmenl. Higher levels of MyoDoreceptor fusion pro~ci~ that native MyoD protein are preach! in embryos, appmently a result of ioereL~d fusion protein stability. This is the tint demonma- tion that homtone-inducible fiction proteins can work effectively in a complex KoCh, P. and Siva, !!. L. Developmental Biology 171:267-272, 1995. P-me Ibe Whitehead Instkute for Biomedical Research and Depmmenl of Biology, Mt~ttcfiu~ts levitate of Technology. Cambridge. MA. 160 A RHES~JS MONKEY MODEL TO CHARACTERIZE THE ROLE OF GASTRIN-RELEASING PEPTIDE (ORP) IN LUNG DEVELOPMENTm EVIIX~HCF. FOR ~TIMULATION OF AIRWAY OROWTll Gastfin.rele~ing peptlde (GRP) is developmentally exl:xe~ted in leman fetal lung and i~ a growth factor for normal and neopl~tl¢ lung but it~ ro~e in normal lung development has yet to he clearly defined. In Ih|S sludy we have chal~tcted~ expre~slo~ Of GRP and its nce1~or in fetal rfieaus monkey lung and dttermiood the effects of bumbesin on fetal lung development in ~itro. By RNA blot ~alya~ ORP mRNA wt~ finl detectable in fetal monkey lung at 63 dayl geftttion, mched hq~h- eat levels at I(O days gestation, and then declined to neat adult levels by 120 days geslatk~: a I~ttero clmely poralleling GRP exprettlon in hemm fetal lung. As in human lung. in situ hybridization localized GRP mRNA to neuroendoertna telh though during the canalicular pha~e of develol~,nem (belwten 63Jr0 days GRP mRNA was pre~ent no( enty in cl~k pulmonary neumend(~'in~ tells, bm ulso in cells of b~dding ait'way~, lmmuno~hlod~emiatry ~ that bemb~|wlike |mmunme.nctlvily w~ lwetenl in neume~d0¢rk~ tells, bet not In lxtdd~$ altway~. sug~est~g that In buddiog akwlys either the GRP mRNA is ~ot translated, is npldly ~reted, or n relaled, but dlfferenl RNA is p~ent. RNau: protection a~alytis using a prol~ Io Ihe monkey GRP receptor demonstrated Ihat the tlm~ ct'~r~ of R~tpt~" RNA exprer~io~ closely pantlleled Ihe llme coarse of GRP RNA e~pr~,Jim. In ~ttu bylxldlzatlon ~howed Ihat GRP reCelXO~ wee primarily eXl:~es~'d in epithelial ~lls of the developing ai~rays. Thus, GRP would appear Io be teCreled from neumen- docrine cells Io act o~ larget tells in developing airways. Thin hypothe~i~ w~ firmed by organ culture of fetal monkey lung in Ihe pre~nte of bomboain and buml~sin antagoeisls. Bombesln U'tetmcnt at I and I0 nM sigaifkamly In¢~tlm.d DNA synthesis in airway epilhelial cells and sif, nifica~dy ir~'el~0d ~ numblr size of airways in ©allured fetal lung. In fact, culturing 60 d fetal lu~g fog $ d wllh I0 nM bombesin increa.,mt airway si~e and number nearly to that ebtt~ved in 80 d fetal lung. The cffecls of bombesln could be blocked by specific ORP re~pior antegonisL~. Thus this sludy demo~f4rate~ ihlt GRP ~ t~ exi~ll*ed o~ air. way epithelial cells in developlr~ fetal lung and that the interacdo~ OfORP whh the GRP receptor sllmulale~ airway development. Li. K.. Nagalln, $. R.0 and Splndel~ E. R. Journal of Clinical Inve~tigalioft 94:1605-1615, Oclober 1994. O~hef ~uppotl: National ln.~titute$ of Health and Natkmal lmlitute of Child lleallh and Human.Development Population Resea~:h Ccnt~ gram. From Ihe Diviaion of Neuro~icncc. Oregon Regional P/imate Re,J~areh Center, Beaverton. OR. DEVELOPING AND REGENBRATING SYSTEMS AS MODEl,3 FOR TROFHIC EFFECTS OF ACITI NEUROPEPTIDES Neurons undergoing dr~ic metabolk: cbengcs, such u pedl~ral edng from nerve traumn, cemml neonms in tissue celtm, nilrmtrbtal m-~e~i for 16t
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0 0 162 From the Delmrlment Of Biology, Carlelon College, Norlhfleld. MN, I.R.C.M.. Monlreal, Quebec. Canada. and Deparlmenl of Oenelics, Unlverllly or IIllr~il Colle~le o1" Medicine, C'hicago, IL. V. Epldemiology POLYMORPHISM OF GLUTATHIONE $-TRANSFERASE M I AND LUNO CANCER RISK AMONG AFRICAN.AMERICANS AND CAUCASIANS 1N LOS ANGELES COUNTY. CAUFORNIA Bockl~rmmd: Glut:lthione S.t~n~l'era~e MI (G~i'MI) is mlve in the tio~ of a numher ofcal~lnogens, includin$ poly,,tomatlc h~dmea~s, such I thoR' present in cigarette smoke. In ahl~l ~ -55~n Of individuals, dependinI On the nk: grmep, there is a virlual al~ence or OS~l enzyme a¢livlty due to deltllt~l of holh coplc~ ol" Ihe GSTMI Bene (GSTMI null genolyp¢). Thl~ geneli¢ phlsm of the GSTMI gene locu,n has heen IXOlX~d as a ri~k fll¢lor foe lun$ eanem'. However. results ~ Sludies are in~,o~sislenl. P~rp~e: We conducted a lrol sludy OI" imlienl,'c wilh incident lung cancer and polmlatkm c~trol mhJec~ to
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O examine the association belween homozygous deletion of` t~ ~S~ f Cc~ a~l lung C~ ~ a~ Af~c~-A~c~ ~ Ca~aslans. Mell~.~: ~t 35 h~tals ~ ~les ~, Calif~il. ~ ~li~ ~tients with a fi~st diaB~sh of lung ~r ~n ~cm~r I. I~, a~ Janua~ 6. 19~. Oft~ 8~9 ~tentlally eli- Bible et~ palients, 207 h~ died by t~ time the;r phy~ician~ had feceiv~ our ~o~t f~ ~ssi~ to ~t~t t~m. We enmfl~ 3~6 elf;hie ca~e ~tie~ (167 can-A~ncam~ and 1~9 Ca~asians) a~ 731 eligible control Aub~ts (2~ African-Americans and 473 Caucasians, all ~sidcnts or ~ Angeles County). S~pl~ of ~[le M~ ~li DNA su~nl f~ d~al~ ~ t~ GSTM I ty~ by a ~1~merase chain ~t~-~x~ a~ay we~ ohtainc(I ram 342 case ~tlents ~ 716 ~lml sub.s. ~ ~ds mti~ (ORs) ~d P~% ~n~dcn~ inter- vals (Cls} f~ lung cancer ass~iat~ with homozyBou~ deletion o£ the GSTMI lent, i. I~al a~ a~cr Sl~tificat;~ ~ a humor of ~levant char~cristics. ~timat~ by Io~ixt~ ~g~i~ analysis. Rr.~utLs: F~ ~tients with .11 lung ca~e~ ~, the GSTMI null ~n(~y~ was as~;ated will~ an OR of 1,29 (9~% CI ~-1.77~ ~ OR was similar am~8 Afric~-Amcricans (OR I.~); 9~ ~7~.~) ~ Caucas~ns (~ - I.~7; 95% ~ = 0.91-2~). ~e ass~iati~ wax st~st f~ Squamous ~ll cantata (OR - I.~7: 95~ CI = 0.93-2.63). ~ an ~ ~ I.~ (9~ ~ ~ I.I 1-2,~2} ~ the GS~ ! null ~ in t~ ~o I~ c~ d~ a~g s~m o~ le~ than 40 ~k-~. ~t ~ ~;~. t~ ~ ~v~r ~,~ (OR - 0.~ 95~ CI = O.~-l~). C~,si~n~: ~r G~l ~ a~ IM ~k of lung .~r o~11 in this ~fati~. H~ver. ~r ~ ~ suBB~ an el~at~ Hsk rm I;8~ s~km with this ge~y~. Impli~lions: B~u~ t~ ~r of~ analy~s with;n stria ~ lifeti~ smkin8 kls~ wa~ lira- it~, I~er st~. will ~ ~ to ~fi~ t~ ~inss. ~. S, ]., ~ly, A. K., ~, J., Hav~i, W. C., C~ntcr. C. L, ~ M~ J. R. O~er ~n: ~a~ or Calir~ia Toby.Related D;~a~ Re~amh ~m. California Public Ilealtb Foundsti~. National Caner lnslilute, and lntli~mes of Halt5. F~ the Divis;~ of ~cupatlonal and Envimn~ntal Medicine, Division or Sch~l of Medicine, Los Angeles, CA. and ~parlment of ~armacoloBtcal ~s. Uni~ity ~ Hc~tlc M~ical ~l. UK. VI. Genetics SIT~DIRt~-'TED MUTANTS OF THE/~ SUBUNIT OF PROTEIN KINASE CK2 D~'xMONSTRATE THE IMPORTANT ROLE OF PRO-hi] Th~ re|lowing amino acids of the Xe~opats laerix .B subunit of protein kinase CK2 (casein kina~ 2) w¢~ changed to alanlnc: Pro.hX (/~ .,); Asp-59 and Glu-60 164 and Olu 61 (j~.~); His 151-153 (/?~,.~). The last 37 amino acids oflJ~ ~ "~xyl ~ ~ ~ (~,~.,). ~i~ti~ ~ CK2~ ~tal~l¢ lu~n~ ~ivlty w~ mansard with casein as suMtrate and t~ followinl Rlative activities were ~ults i~icat¢ that ~ ~-58 and t~ s~;~ ~d~ cluster pta~ ales In int~t~ with CK2~. ~BS ~ttc~ 3~:211-214, ~er su~: Intemati~at CcotR f~ Ge~tic Engi~ng a~ g~teehnology ~ ~CYT-~ile. F~ !~ ~to de Bi~olmlca, ~llad de M~ina, ~d ~memo de ASCARI$ AND CAENORHABDITI$: COMPLEMENTARY OROANISM$ FOR STUDYING OERMLINE DET~.RMINATION As am approach to undemanding how the/A.n~ll~ is dot~n~Ined in nomatodll, we have cloned and have begun Io characterlse two families of' 1~ in the name. lade. the glA and c~clln genes. 'l~esc 8e~es are potential components of the gcnnlinc-specil'¢ P-granules. A role fo¢ glh proteins in ~ee~nline speeif`ieaticm I~¢ome more likely with our n~cnl finding, using antibodies to $1h. that ~ I¢in(s) segregate with the ¢emdine peeem cells in the C. ¢ltl:~ embryo. We Idle expect tim the 3' UTRs of eack genc in the~e families, which appes¢ to have muhl- ple regulatory motifs, may post.lranserip~iomally coordinate their expr~lon In devetopmem. The availability of unlimited stag~- and tlssue-speeif`l~ RNAI from A.car/~ could allow us to i~olate other RNAs yet to have been identified thai m~y he Imund by glh zinc I~gerl in the P.granule "lreasure chest." Roussell. D. L. Gruldl, M. E., Kreutzcr, M. A.. Richa~ls, J. P., sad IBennelk In: Boothroyd, J. C, and Komuniecki, R. (Eds.}: Molecular Approlchas to Pm'ashology, Wiley-Lisa, Inc,, pp. 321-340. 1995. Oti~-r ~oa: Natloeal Science From the Department or Molecular Microblology and Immunology. $¢hool or Medicine, Univ~ily o1" Missouri. Columbia, Me.
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0 0 EPITOPE-T~g VECTORS FOR EUKARYOTIC PROTEIN PRODUCTION We report on the constmclioe end use of tw~ eukaryolic expre.~slon vectors ~hhi~ add we.ll-eharaetedzed epilope tags to ihe N lemdg.i nf prozein~. The millay of ese voctot~ Is demonstmed for detecting the expressiorl~f a variety of proteins, As the addition of these epltope lags elm in some c~,,es ohvlatc the need to generate spe- c|fle antisera to each iodividuel protein, these vectors provide a facile means both to moet~or peolein expression and to pedfy such expres.,,ed pro~clns. ~ells. M. k. and Chernolr, Jo Gene 152:197-159. 1995. Other suppo,l: National Institutes of Health. Prom the ~ox Chase Ca,k-'er Center. Philadelphia, PA. MURINE CIIROMOSOMAL t,IX:ATION OF EIGIIT MEMBERS OF 1"! IE HEPATOCYTE NUCLEAR FACTOR 3/FORK HEAD WINGED HEUX FAMILY OF TRANSCRIPTION FACTORS ' A lO04mino4cld DNA-blnding motif, known ~ the winged helix, was first ideatil]ed in d~e mammalian hepatecyle nuclear facto~-3 (HNF-3) and Drnjopkilo fork head family of t~mcdplioa factor. Sub~luently. more than 40 diffeRnt ~s 0us! ¢or~in the wln,god helig motif bays been identif'~J. In the studies described have. we have determined the merino chromosomal Iocatio~ of eigM members el'this I~ne f-mnily. HI~-I. HI~I-3. HIll4. HI~I-$. HFH-6. HFHog. BF-I. end BF-2. by in~Ifie hecbm~s analysis. These gnne~, designated HNF-3 fo~k head homolog I (]~1), J~jTdo HiM. HiltS, Hjq~. HJltgo H~9, and HJhlO. respectively, mapped to 6 dif"re~ent mottle auto$omes and ere Ihu$ well dispersed throughout the mouse geaome. Ba~d on thi- mapping information, we pn~lk, t jhe chromusoma! toe, ion of these geae~ in hemam and discuss the po~emial of Ih~e gene~ as candidate~ for tmclonad mm~e mu~iom. • Avrlk~m, IL B., lqetcher. C.. Overdier, D. G., Clevidence, D. F_. Lai, E.. Cce~, It. !!., Jonidns. N. A.. and Copeland, N. G. Genomlcs 25:388-393. 1995. Other support: Halional Canner Institute. American Cancer Society. Hationsl Institete~ of Health and N~ional Imtitute of General Medical Sciences. ~ the Mammalian Genotice Laboratory. ABL-Basic Re~earch Program, NCI- l~rederkk ,U'ae~ Re,arch and Development Center. l~rededck. MD, Deparlment of Biochemistry. College of Medicine. The Univemity of Illinois. Chlcago. IL. and Div|sim of BndocHnology and Cell Biology and Genetics Program. Memorial $1oan-lf, e~ering Clmeer Ce~ef. New York. NY. 166 GENETIC ANALYSIS OF GROWTH INHIBITION BY GAUI.IKB.~ IN .~;vlCCIIAROMI'CE$ CEREVISME hcB proleins bled to and Rgulnle Rel/NF-tcB tranu:riplion factorl. ~Ve showed i~vkm~ly thz~ • fu~;io~ prolein (GAL,I.I)40) coelaining the DNA-binding domain of GAL4 and sequences of chicken IKB-a(p40) inhibits |rowth in the yeast Socckornmyces tertvi#nt. We mow fJlOW thai p40 must he bound Io DNA to lnh|btt ye:m growth, p40 IXO~elns, bound Io DNA either as GAL4 or LEXA fusion inhibit yeast growth. In contrast, p40 pro(tins that eanno¢ bind Io DNA, such al full- length p40, a GAL4-1KB fusion pro(ale eonlaining a mutant GAL4 DNA-bindlng domain, and a fusion prolein (GAD-p40) containing the tran~eHpllnoal acllvail~n domain of GAL4 fused to p40, each failed to inhibit cell powlh. As with GAL4- VPI6, GAL4-p40 needs a functional cellular ADA2 gnne Io exert ill IroWth- inhibitory'effect in S. mt,/$iae. Using I high-copy supfme~io~ slmtely, we have isolated three S. ceretqsiae genes that restore normal growth to yeaSl expres~lnlt GAL4-p40 or LEXA-p40. We have termed Ihe~e re~culng genes collt~ltvely as genes, for "Suppre.~,,o~ of IKB." Expression of the $1K ~encs ~pecifically supprt~t~e~ the growth.lnhibitory activily of GAL4.p40 and LEXA-p40 Ix, cairns $IK ~ene expression cannas Neck GAL4.VPl6-mcdisled Fowth inhtbilion in $. $1KI encodes a novel i~oteln that co~ain~ e COOll4ennlnal repeal thal ha~ been found in many microtuhole..binding proteins. $1K2 encodes HH)-terminel ae~lyl- tramferase, and $1K3 encodes Ihe yeast rilx~,omal ~1 protein. None of Lt~ $1K reins b;ods directly to pl0 seqoeaces in w'tro, suggesting thet the SIK pro(tins likely to acl dm~m.tream or ihe direct polnl of grmvth inhibilkm by GAL4.rAO, Onr resells may he useful for devising stralngie~ for klanflfylng vemhrale J~hlbitorz t~f IKB prolein~ and of other proleins that inhibit growth in $. Modn, P. J., Dowrs, J. A., Snnd~rass, A. M., and Gilmore, T. D. Cell Growlh & Differentiation 6:789-798. July 1995. Olher support: Massachusells Division of the Amerken C'a~eer $oClely, and the Children's Leukemia Research Assocltdon, lne. From the Depmlment of Biology, Boslon Unlverz;ty, Bo~It~, MA. ISOLATION OF A eDNA ENCODING A NOVEL CHICKEN CHBMOKINB HOMOLOGOUS TO MAMMALIAN MACROPHAOE INFLAMMATORY PROTEIN- J p A eDNA encoding e novel chicken chemokine homologeus to m|mmllan chemokinc macrophage ;nflammalecy p¢o~ein !~ (MIP-II~) was hoisted and cher~. Icrlsed. ~ eDNA encodes a protein which is 7.~80% homelt~oul Io human ~ mouse MIP-II~. All conserved truing acids charscleristi¢ of the mammalian chemokino family have been evelufimadly preserved in chtchen M1P.I~, suSPst. ing similar prolein folding pzliems and funclional prostates. Pewanko, O., lachenko, 1.. and Enrletlo, P. J. 167
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0 c.n Omer suppo~: ~atlona! rnsdtutcs ol'~ealth. From Ole Department of Minrohiology, Slate Univ©rshy of" New Yo~ at Stony Brook. Sloe). Brook, NY. ISOLATION OF MICRODI$$ECTION CLONES FROM RAT CHROMOSOME I0 rr ~We,~...~ted an R~OI0 specific m;crolibrary and imlated DNA marker~ c ~.rlr IP we I1~ currefltl - . • of .. r..~ . ,y eoetmumg tO |~ale as many additional markers from [nis mictollOrlry as Po~siote. slncc it cixt|d be useful in ge~tlc ma in . no~ f°r the ~r ral ~one. but a|so oibcl.diseas~ ~nes located on rat Ow 'I ~PI~. 8- y l~oba~vashl, T, Kaw~u~hl. T., Kishlno, T., Matsumo~o. N.. Nilkawa. N., Mori. ~an, G., Kflnp-Levan. K., ~md ll|no, O. Mammalian Oenome 6:216.:218. 1995. O~her suppom Minlst~ of Education° ~cicnce. and C'ullure ol'~apan. From the Del~tments or Expedmem~! Pathology and Palhology. Cance~ ~nsdt T.o_k,y,~/Jepan, Deparlment or Human Genetics ~, ............. • I-I[i,~IKI UnlVellity ~0110Ol O! M~(;,cme, Nalasaki, Japan. lnstltu~ or Laboratory Animals, Facuit of _Kyot.o University, Kyoto, 3span, and De-m ........... Y-- ." O0¢eborg, C, meborg, Sweden, ~- ,,,~,,, ,,. ,.,=.e.cs, unlvemty or V/L lmmunoloEy and Adaptive Mechanisms 168 retmviral vector encoding/~n abo resulted in an ek~vmed level ~ kin~ ~tlvt~, aTthough t~ increase was not as dramatic as that ob~v~ with ~ or kln~ ~tivity was ~ in ~stimulat~ ~n~m~ ~lls ~ 1~3 sdmule- of ~ in 32~13 d~ ~ result in I~ ~1~ ~Ivm~ ew ~a~. I~ s~i~i~ f~, bck. a~ ~n. ~i~ t~ ~x~i~ of~, kc~, m ~n In ~M~, it did ~ mmkr I~ ~lb ~ ~;fi~ ~ I~3. ~ ~s cant role ~ si~d ~n~t~ ~ ~i~ ~e J~al of lmmu~1o~ 15~:!~ 1670. Ot~ su~: New Y~ ~munity T~st and t~ NMi~al C~r Institute. ~nver. ~. COORDINATE A~D COOPERATIVE ROLE~ ]=OR NF-AT AND AP-I IN 'THE REGULATION OFTHE MURINE IL-4 GENE The tran~ription rector NF-AT plays ~n e~m;~l role ifl the induclMe tmn. sc.dplion or several c~tokine ge~s dudf~ T ~11 activalinn. The distd NIt,AT site of the marine IL-2 ixomote~ binds both NF-AT and AP-I proteins, and.thus m1~reaants a composite regulato~ site that integrat~ ~'- and PKC-d~ndent si~na|i~ I~lho wa~ in T ¢~11 astivatio~, However, the tndlvto'ual ¢ofltdbutionl of the NIl.A1' AP-I components to promoter activity via this ¢ompolitl site hurl resolved, owing Io Ihe ab~.mce eWe ¢le~ly defined AP-! biedin~ li~, which, wh~ mutated abolishel AP-I binding. We de~'ib= here ~m q~p~dy ~lolou$ NI ATIAP-I composite sl~ ;n the tam'the IL,-4 promolef, which can be mumed to t|vely block the recmltment of eaca~ eompo~nt. We show ~It lhe eooplratlv~ coordinate iovo~vement ot bo{h NF..AT and AP- I is n¢~:x~Juary llpr Ibll acflvlty eW the NF.AT/'AP- I composite site, and, utdma/ety, the emlm If.,-4 Roo~ey. J. W., Hoey, T,, and Glhw.'her~ [,. H, immunity 2:4"/3-483, May 1995. Olher support: Tuladk, tncoqx~ded, From the Depa,lments of Genetics and Medic|he, Harvard M~llca| ~¢hool, TUladk, l~'~J. South San l~n~i~-'o, CA, ~md Dq~nment or Caneer Ha~vml School oi" Public Heakh, Bo~to~, MA, 169
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0 0 U1 U1 THE PROXIMAI. PROMOTER OF THE IL~4 GENE IS COMPOSED OF MULTIPLE ~$ENTIAI. REGULATORY SITES THAT BIND AT LEAST TWO DISTINCT FACTORS Immune r~sponses to l~thosens o~en Iced to Ihe 8eneratloo or polarized T helper ~ul~els designated Thl and Th2. Thl cells, characteri~.~l by the IXnducllon of IL-2 and IP~*-y. stlmulste cell.lar immune re.~pon~es important for prmcetlon ~aimuns~ intrac~llul~r pethogens. In commt, Th2 cells, which produce IL.4, are point lalors or B cells ~ml stimulmc prolect;on against cxtracelluiar p~ho~ns. 1l..4 h~s abe ~mcrl~! a~ • key cylol~inc in Tcell dil~erent|~ion si~:~ it h~ been ~ Io direct the development of r~i~e T cells toward a Th2 pheno(ype. Recent slndles have Ixovkl~d |n~ights inlo the tr.n|cril~ional rcguiatio~ or IL,4. including the iden- tiflcafio~ of multiple binding sites for a ~b~nit of the IL-2 ~ranscril~ion factor AT. In this study ~ de.,cdhe the characlerirJ|tJon of an essential rcginn ~t" the IL.4 promoter located immediately ui~'~tream of the TATA clement. Iligh-~cs~lution mut~en~sis of this 33-~ re~io~ Rvealed multiple sites indispen.~able for inducible IL~I transcription. Included in this region sre overlapping bindin~ sites t'or the cycloc, parln A..~.ndtive t'ac~or NF-A'rp and • no~el ¢oestitut|vcly exi~,'es.~d f.actor designated PCC. An additional sequence adjacent to the TATA clement is sho~n to be critical I'or IL-4 tran~'fipl|on in Th2 cells. H~I~e, bt. R.. Roooey. ]. W., and GlJmcher, L !t. The Journal of immunolngy !~4..6397-6405, 1995. From the D,~par~ment of Cancer Biolo~, Harv,,rd School of Public llealth snd Depmmem ot" C<nctlcs and Medicine. H-,r~ard Medical School. !~o~o., M^. RBCOMBINAN'r SOLUBLE CDI4 PREVENTS MORTALITY IN MICE TREATED WITH ENDOTOXIN (LIPOPOLYSACCHARIDE) Endotoxic shock is a lifc-threslcnlng ¢ondilion mediated by cytoklnes telczsed aher exposure to bacterial LPS/endoloxin. Activation of monocytes and ncutrol~ils by the binding of LP$ to the membrane revisor. CDld. plays s key ro~e in Ibis re~lX~le, Pm1~. • soluble I'o~n of the CDI 4 tecqxo¢ eahences the enclolhellal cell re~oonze to LI~. Wc show hem that despke the z,,*onist clTccts ot soluble CDI4 on the endodlelial cell z~slx~R le LP~. teoombinant soluble CDI4 is able to ixolect mice ~ IJ~dnduced I~hetity. This lx~xcction ~ to he associated with the inhibitiml or TNF.ez rel~se. These fesulls sngge~ thai the soluble CDI4 ~ccptor rely fe~ a new form or ~ for endoloxic shock in humans. HuJol. A., Rong. t3. W.. Lin. X.-Y.. Silver. L. and Goyerl, S. M. TI~ ]omnal or Immunology 154:6529-6532.1995. ~, Other suppotl: National Institutes of. Health and the Arncrlcsn l{cart Association. New Yod~ Slalo AITili|le. ~ the Division ot" M~:ular M~llcinc. North Shore Univer~ily Flo~pltal/Cornell Unive~ky Medical Colle~c. Manhassct. NY. 170 CDId-INDEPENDENT R~SPONSE~ TO LPS REQUIRE A SERUM FACTOR 'rXAT IS ABSENT FROM NEONATR_S Mom¢)'t~hnacropheges and nentrol~ils can retlx~d to e~lotoxtn vb ~z high- affinity n~,l~or (CDI4). requiring low levels of LPS (<1 n~l) ~ wldl st Ihl~Jlh anmher pmhway(s) that require~ high levels of LPS (>10 n/Jml), lmh plthway~ _R. ~.lt in tiT. s~"r,~ioe of high k'v¢ls of cy~i.ce, s.ch ~ ~.~!~, and I~Oe of v|rme.~ other efr~:tm mol~'ul.. To rueher define the aet|vallon of etlls by LPS via -, pmhway that does nel im~olve CDI4. we haw mind an expmlm~nt|! m0d~l Ihe~ disti.guixhex CDI4-dependent from CDl4-|ndepen~nl t~ponm usinll scum. ing amoums ut an anti-CDI4 Ab !o block tl¢ CDl4-dependem r~sporne. Al~lysis of the ability of varkms individuals to reSlxmd to LI~ via both Ihe C'Dl4<kpende.! and CDI4-i~I pathways shows Ih|t ndult~ can rcspoed via both plllhway~; furlhenno~, in Ihe pr¢~encc of 100 ng of LPS/ml. the primary ~spon~e is CI)14 indcpendcut. In contra~t to the re~pon~ by ~dults0 neonates can oely r~poed via the CDI4 dependent pathway. Further analysis has shown Ihat the CDI4-k~t~¢.~ I~thway requires a noe-CDl4 plasma prutein present in ndull plam~a thet mi~ing or nonfunctional in neoeate (cord) Cohen, L., Hazlot. A.. Sh~, D. R.. Lin. X.. Sis. C.. Harper. R.. Silver. ~., aM Goyorl. S. M. The J'oumal of Immuno~ngy 155:~,337-$342, 1 Olher suppotl: ~ational ~nstltutes of Health ~nd American Hc~.~t Association. New York Sts~c Affiliate. From Ihe Divisions or Neonmolngy. Depmln~nl of I~liald¢l. Molecular Medicine. Deparlment of Medicine° and D¢l~rtm~t of" OB/GYN. Heath Shore University Hosplzal/Cornell Univerdty Medical College0 Menhaden. FUNC'rIONAI. VAUDATXON OF IJOAND MIMICRY BY ANTI-REC~FPOR ANTIBODIES: STRUCTURAL AND THERAPEUTIC IMPLICATIONS Sever~! anti-m:q~lor mono¢lenal autilxxlies have ~ de~cdhad thlt ~mpele wilh the ligand foe rceeplor binding. The po~ibility that sln~'~,res within the varilbi~ or complemenlarily-d¢le~mlnlng mglons (CDR) of ~ antlb~db| mtlht displW slmilarily with Ih~ r¢cel~Or ligand Im hecn documemed. Ihar~by this imepunologic approach !o explor~ lipnd-n:¢eptot interacti~es, Taub. R. and Greene, M. I. B|ochemblry 31:7431-7435. 19~2. 171
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O Giber support: National Institutes or Health. National Eye Institute. American Cancer Society. and Mackay Charitable Trust. l~rom the Department of Genetics and Howard Hughes Medical Institute and Department of PatholoBy. University of Pennsylvania School of Medicine. Philadelphil. PA. INVI~TIOATION OF NEUTROPItIL SIGNAl. TRANSDUCTION USING A SPECIIRC INIIIBITOR OP PIK),SPIIA'III)YIJN()srI'OI. 3.KINASK Neutrals cabala • molt~npo~nt NADPH oxida~ sy~em th~t is involved in the production or mlcrol~idal oxidanL~. Srimul-don of human neutrophils with the peptJde FMLP activates this resplmtory burst ~ to produce sup~roxidc and abo has been shown to result in activation of pho~phetidylino~itol (Pldlns) 3-klna.,~. "rr~tmem of human r~mrophils with 2-(4-morpl~inyl)-8-phenyl-4H.I.Mnzowran. 4-o~e (LY294002). a Ixxent and sFccit'¢ i~hibitor or P~dlns 3-kina~e, complete ioldbition or Pldlm 3-kin~ activity as well as in inhibition ~ superoxide pmductio~ in FMLP.treated neutrophils in suspeudon= FMLP-stimulaled oxidanl production in Idhe~t cells was also abolished. T~atment of human nculrephils with PMA ~sultnd in production or superoxlde wttho~ actlValion of Pldlns 3- kin~e: LY294002 did no~ block SUl~,roxide production in neutropbiLq exposed to PMA. In addition, LY294002 did no¢ inhibit eellfree NADPH oxid~e activation, CD! lb-~l~nd~nt adhesion, actln polymedzatlon in R~xmse to FMLP, or FMLP. ied~ calcium flux. 'These results subtlest Ihat the Id~esl ~'an.~PJction pathway or the FMEJ~.roceplor involves activallon oF Rdlr~ 3-kles~, which is required for sub- leClUent sBperoxide production induced by the chcmotaclic peptlde. Furthermore, Pldlns 3-~in~ may be Iocaled dim,clly upszream or pro(ein kiflase C o~ o~her protein klm~es, which in t~n activate d~c NADPtl oxida~ Vlahe~, C. ,T., MIl~er. W. F., Brown, R. Pro~nit¢, E. R., Ye, R. D., M~'der, P., Schdm. J. A., Ro~hruss. K. J., Serlin, B. S., and Simpson, P. J. The3oumd oTlmmuno~, 154:2413-2422,1995. O~her ~I~o~I: Nalinnal [n~itm'es of Health. Prom the Cardiovascular Reseamh. Lilly Research Laboratorlcs. Eli Lilly and Compcn¥, |ndlanapolis. IN. I~Nnlment of Medicine. University or C~liromia a¢ San M~~I C~ter. ~n Diego, CA. Dqxmm~nts of Molecular and Experimental inn and Immunology. The Scripps Research Institute. La Julia. CA. and Deplrtmen! of BioloBical Sclence~.. D~Pauw Univer~y. Greene•sale. IN. 172 B LYMPHOCYTE RECOGNITION OF CYTOCItROME C: HIGHER FREQUENCY OF CELLS SPI~CIFIC FOR SELF VERSrJ$ FOREIGN ANTIGEN EARLY IN THE IMMUNE RI~SPONSE AND V OENE USAGI~ IN THE RESPONSE TO SELF ANTIGEN 1"o study immunoglobulln ~ene usa~ in the a~tlbody response of mlc~ te Ihe stir ami~n (A~) me~R ~tochrom~ e (c,~), g cell hybddom~ were ~ frem splenk: g cells of immunized BALB/¢ mice prio¢ Io the o~t~t el" ~e~atle Le. 3 days aller in.jectin[ ovalhumin (OVA)-pdm~ mk~ with mou~ ¢yl eeupltd Io OVA. Monoclo~BI anlilx~dies (mAb) t'm~ all or the ~ve~ primary hybddem~ we oT~alned war ~sitive to a sin,,~4e amino ~id sub~it~don from ~rde acid le flu. tamlc acid at po~itio~ 62 in mouse eye, This is the tpeeif'¢ity or the v~ misty or it calls rc~ing to moose eye a~ d~crmlncd from a~ay,, of It c~lla activated tn splenic fragment cullums. Six or the mAb derive from the 19.1.2 ]$.~ V~ ~.ene which is al~o u~ in Ih¢ R~ponse Io ~ (!-'6) dexlmn and thr~ or these mAb I'rmn the R9 V. ~ne, u member of" the V, Ox.! family. ~ other mAb de~ve dlslinct, nllhou~h similar, V. ~. Atlempts to ol~ain hybrido~B ~c~tinl~ I~mary (unmut~ed) mAh slx~lfic lot- cy~ ~orel~n to mice have been Mmpe~,d by Ihe much Io~ fRq~-~cy of It calls req~oedlnB ~ly to fmelgn c~t in eomr~d~ to the ~lf Ag. This ~sls ihal. ~1~ to ex~ali~ of I~ ~n[~ BAL~ m~ B lym~y[~ s~ific rm a sln¢le epi~ ~ ~lr c~ ~ ~t in bi¢~'r~y than B I~!~ ~ific rm dmil~ ¢~ ~ ~iln of I~ ~ni~lar ~binati~ of V le~ ~i~ ~R ¢yt~ific mAb or Io ~ilive ~l~i~ of~vel~ini B I~ ~ ~s ~. Min~h, J. M., M~lkr, C. M., Bu~, S., ~ ~em~, R. ~r s~: National $cle~ Foundalion, U.S. Public Health ~ewice, and N~al lmtilul~ ~ Hedlh. F~ t~ ~t d M~olo~, Unive~it~ ~ Minima M~i¢aI ~!, M~is, MN. A TH I CEL~ LINE OE9.1) FROM RF~ISTANT A/J MICE INHIBIT~ INDUCTION OF MACROPHAOE PROCOAGULANT ACTIVITY IN VITRO AND PROTECT~ AOAIN$1" MHV=3 MORTALITY'IN WVO Tnduction of imrm, m coqulants has heen implicated in the routine hepttltis virus strain 3 (MHV.3)-induccd fulmimmt hepatic neere~tl. P~vious ~ from our llbont¢or~ hss shewa that the induelion or activi~ (I'CA) con~lales wire the r~islmce/susceixibility to recombi~lnt inbn~d (RI) straine of m|ce. Macrophs~s f3rom resislant, suains or mice exprm~ increased levels ot PCA in response Io MI~.3 stimulation. 1" lymphecytes, however, had u marked reBulatory role In the tlnal expresslo~ or n~cro~u~e'PCA. CD.I" CD4" CD~" l~mphoc~tes 173
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0 0 01 p~lible lulelplible mice were able to inslmct maemphzgcx from su.~cp~iblc mice to ~ -~-~mmum~ ~sm~t m~ ~m a~e Io su~ss ind~i~ o~ ~A. In Ibis ~s~! study. T~II IJ~s ~m ~rived ~ drainin~ ~lit~l ~m~ ~ ~ ~si~mt ~J m~. ~h ~ ~n immuni~ with MIIV-3. All T-~II li~s show~ m~ ~lff~at~ to MllV-3 and MHV-]HM which w~s m~ hi~libility ~x (MHC) ~s~cl~. All cell li~ ~m CD~'. f~r oft~ ~ C~" md ~ wa~ ~'. All ~ t~ ~" ~II li~ ~uc~ IL-2 and two ~ i~e~ (I~ff), ~sis~nt with t~ ~I cyt~i~ ~fiin. ~ ~II I~ O~.I} w~ ~e to ~hi~t the i~ti~ of m~ ~A thigh ~ ~suhz de~u~tc that t~ exis~ o~ s ~I su~lati~ or ~ with s regu[s- ~ e~ ~ m~s~ ~A i~im in MHV-3-inf~t~ m~ ~t~tes to t~ ~sist~ ~t~ ~] s~in ofm~ to MHV-3 inf~li~. ~un8, S., Gomz~ski, R., ~ B., ~in~mtc. R., Ska~ne, E., Perlman. S.. ~l~w~ J~ ~ng, L., ~o~ M., and ~vy, G. Imm~ ~:]~3-~I. I~. ~ ~h~ s~: Mcdi~I Rc~a~ ~il of C~nsd~ American H~R Aspirin, ~d Nsti~al Insthutcs ~ H~Ith. P~ the Unlve~ity of To~t~ Tomato, ~nsds, Montre~l General IIospitM. M~t~l, ~e~, Ca~s, Univ~ity or ~ows, [ow~ Cily, IA, a~ Unive~ity of Te~ H~Ith ~s ~ten H~st~, TX. ALTERING THE ANTIBODY REPERTOIRE VIA TRANSGENE HOMOLOOOU$ RECOMBINATION: EVIDENCE FOR GLOBAL AND CLONE-AUTONOMOUS REOULATION OF ANTIOEN-DRIVEN B CELL DI~q~RF.NTIATION Amibody V. wanq, en~ containing roll mnon~ts of natural 5' and 3' f~.king DNA undexl~ non~eiproeu] hon~ ~ceo~binalion with the eodo~-nous lXk aW~.lC.flm.l, we. .tin .v~. ~sed .lfliS reco~Dnnlhon l~thway to introduce a somatically mulaeee vmable iv) regnon with an unusually hiBh affinily for the haplen p- sm.ay or this ~z~a. ~r, pmssmn ~ the lrans~ene-encnded V re~ion did no~ affect many sspoc~ of sr~J~.driven g cell diffemmia(icn, tnclndlnB somatic hypennutation, in ei~er Ars-apcelrJc trans~cne- or ~s V ~ene-expressing clo~e~. Thus. the mgulMiOfl Or these processes upsets to op~lte ~n a "~)hel" fl.~hin~, in th~l the n~xll~isrn| involved ~re impcrcepllve or the relative affinities for -n/igen of Ihe ~mlibodies expresl~ by B cell clones p~ic~l~lln[ in the imnyJrle response. In c~- 174 trust, the selection of V region mutants leading Io affinity m~umflon a~! cell forr~tion was found ¢o be slrongly influenced by the Wunsvnic V r~gi~, only in clo~¢s cxpr~sing this V region. Hybddom~s derlv~d fv~n Irlnsp~l¢. and endogenous V re|ion-expr~sin~ memo~ cells ~ iso~ed st slmilur frequencies from individual lmmgenic mice. The V regions exprelled by hy~donul~ Ihese groups h~l 2- to 30-fold Ireller af.finity for Ant din their unmut~ltd sm~. desDile the fact that Ihe tmnsgene-enended precl~ors h~ 100.fold hl|b~l' affinily Ih~n their endo~m(~s counte~l~, "[l~se ~sults show I1~ the crlteric~t for entry inlo Ihe ~ ¢O~llplr~men! is e~tabllshed 11ol b~ Ihe Ifflillly ol' I I~ etll°~ V rel~ion relative Io all ether V ~egio~s cxpl~sed durinI lhe relponsl, but by lht Iffinlty or Ibis V l~iolt l~llllve Ioils unmul~ed im~ufler. ThUl, the of. B cell memory is ~e~ullled in 1 "{lo~e-lmonomc~" filhion. Vow, K. A. and Mnnser, T. Journal ot'Exp~inl~nlal Mcdlclne 1~h271-281, ]~uary 199.% Other supp(~: National lnslitutes of Heallh. From the Deparlmen~ or Microblolow and Immtmolo~ ~d The Ieff.er~on Institute, Themes ]elTew.(m Medlcal College, Phih,delphi~, P^. EFFECFS OF ANTi-CD4 ANTIBODY: ENItANCEMENT OF LYMPH NOD[ PPD-MEMORY T CELL RESPONSE In vttro inc~b.'llions of CD4" T cells with anti-L"D4 n~necle~al anlPoody (Mab) demoostrated satumt;o~ binding and inhib|ted lx~h PPl~stimutated p¢otifiemtle¢l ~ delayed.type hypersensitivity (DTH) after p~ssive transfer of' Ihe~e celh into naive rats. In COml~ri.'me, in vi~o adm]nlstmlion or unli.CD4 Mab inlo rala, 2 weeks CF^ immunization (PPD priming), r~turated C04 molecul~ ex~ on T lym- phocytes snd depl~ed signir, c~nt percem~,e~ or CD4" T cells fn~n blood, and lymph nodes, yet railed to ~use significant inhlbltin~ of PPD4timubted DTH ear swellln~. Only repea/ed ~ vlw~ admlnislradon of anti-CD4 Mabz duHn| CFA/PPD anligen prlmin~ e~u~ed silniflcunl decma.~s 06 "" $%) in PPD-tlimu- laled DTH era- swelling. T cells isolated from Ihe blood ~d ~leen d~ved in~ilnlll- cant relative decreasce in PPD~|mulmd pmllf.or~ion after ~Ii-CD4 tm~n~mts. In conemsz, PPD-sdmulated pml|f.emtiun or T cells bohlcd I'r~m 1he dminln| lymph nodes al~er an~i.CD4 treatments showed drern~lic incre~es in PPD msetlvl(y. PPD sllmel~icn or T cells izolalnd fn~n the draining lymph nod~ er zmti-C'D4 MM>4mt- ed rats produced rel~live phenolyplc ~ in CD4 ccexp~zzicnz ~ CD4$RC (inverse memory). CD49" (inflammatory adlp:sion inleSria VLA.4}, and OX40 (CD4" M~), representative of mcn~7 CD4' T cell bl~sls with z~-dueed eap~lty endothelial infillmion. These data demonstrate thz~ depletion of" COJ' T eells by Imli-CD4 Mab injcclions, administered either dudn~ or ,,tier antigen priminB, tivcly enhanced memo~ re~pome~ in T lymphocTtez in Ihe dmtnin~ lymph M~rriso~ W. J., Kennedy. N. L Offner. H.. zmd Vandent~rk. A. A.
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CNIlular Immunology 163:106-112. 19~5. O~her support: Depanrnent o~" Veterans Affairs. the Neumimmunolo~y Research Laboratory. Veterans Administration Medical .C.e.nte.r; .Portia.hal, OR; and_the Depart..me_ms of Pharmacology, Neurology and M~r~mlo~'/Immunolo~,y, ure~on Heallh $cien~e.~ University, Po~land. OR. ' BCL-2 AND BCL-X: RF.OUI.AI~RY SWITCI IF.~ FOR LYMPIIOID DEATII AND ~URVIVAI. un~ a~ outlive I~yf~, a~ well ~ to limit f~ ;~u~ ~. ~ ~ival or ~lis is ~ufal~ by a ~t or ~s fell ~ ~ ~ or I~ ~11 ~th ~F~ism. 0£ t~, ~-2 a~ ~f-x exhi~t a st~kln$ ~t~ o~ • ~ ~w ~nt ~yel~fx in t~ field. uf~y F~si~ ~ ~ ~e of ~ Bc]-2 a~ Bcl.x ~eins in ~uf~i~ lym~ ~th ~ su~ivd. Nine~ G, M~ R.. G~Ilol, D.. ~ G~Iez-Ga~;a. M~ ~r sups: Nation~ ln~litutca or Health. American Caner S~iety. and ~e S~o= ~iety r~ Ge~lofo~al Re~=h. ~ ~ ~nt o~ ~1~!o~. ~ Ualve~ily o~ M;ch;~n. Ann Afar. MI. |NDUCT~ON OF THE TRANSCRIPTION FACTORS NF-KB. AP- f AND N~AT DURINO B ~L $~MU~ ~ROUOH ~E ~0 RE~R To ~ ef~s z~ mi~t u~ly ~ C~O ~iat~ siplin~ ~=~,~ ~w ~. o/t~lvatm~ ~zvaW wh~h ~a~ aflai~ similar 176 signaling pathways utilized were apparent in that pro~in ~cinase C (PKC) dupl~llon did not affect CD40 mediated induction of NF-KI3 ~on as induction by antt-II was abolished. The~ results sugsest that a 'final common pathway' 0t ¢onv~,'~ef~'e of transcription factor induction may exist for two distinct receptors, each of whl~ Is individually capable of" triggering cell cycle progression, despite the use of intracellular signaling pathways that differ at the level of' PKC. Although t~rip- lion Factor induction was similar for CD40L and anli-lg eaHy on, su~l~ in expre~.,.ed NF-KB arid AP-I nucleoprozeln complexes were appm'em at 24 h. Such dilTerencex n~y play a role in de~ermlning the variant effects on B celts or stimula. tlon through the~e two receptors. Francis. D. A., Karras, J. G., Ke. X.. Sen. R.. and Rothstein, T. L, Inlematlonal Immm~logy 7(2):1.S!-161, 199.% O~her SUl~O.: American C'.nc~ Society and the U.~. Public I leallh Servi~. From the I~p~lmonts or Pathelo~v. Me~icifle ~ Miero~iolo~. Eves Memed.zl De, raiment or clini¢~! Rcsem~h. ~o~lon University Mndieal C~nt~. ~o~le~ M^. and Depaflmont of" Biok~y. R~.nsliel C'emer. Bnmdula Unlvet~hy. Wallham. MA. REGULATION OFCD14 EXPRESSION DURING MONO4~'~iC DIFFERF.~ITIATION INDUCED WITH la,2$.D1HYDROXYVITAMIN CDI4. a monceyle/~na~ophage ~'~or for the complex of LP~ and I~P$ bind- ing prmein, is a differemlatkm mzrker for the monocyte/n~ Iin~. We have •nal~.nd the regulation or CDI4 exlxeSslon during I(:,2S.dihydmxyvltl~ln D3 (VitDj)..induced rnonocyllc dilTerentlalion. Using FACS. N~xlhem blottinlk nuclelr am-on analyses, we demonslrale~thll the up-regulation ~.f .CDI4 during monoc),fic cell matur=tlon is regulated mainly at the level ot ~ene tlon, and tit new pro~eln synlhesls is n~quir~d for CDI4 indu¢li~. We hive re~ntly cloned Ihe CDI4 $' upstream sequence and ~ml ils ti~u~-q~e¢ifl¢ xcllvlty. Using sluble Ir=nsfcclion ~ the mono¢~old U937 cell line with a ~ el" delc~iou mulants of the CDI4:5' upstrelm sequence (~x~pled to • i~(xler str.~, we show d~at bp -128 Io -70 is the critical region rot the indu~km ef CDla exlxess;on This region contains two binding siles for Ihe Spl ~plkm f~.~. A 3-bp mul~llon ~ ~he d|stal Spl-blndin~ site not only elimin~es $pl Imm~don, mobil;ly shill analysis does not dulcet a direct inter~fion mine ,.uz,~ om~ ~pz- ~ dala demon~rMe thiI VitD~ indues CDI4 ino~reelly !h11~ ary raclor, and suggcs( a critical role tot Spl in this process. 2:h~,~ng, DEE., Helherin~o~, C. J., Gonzalez. D. A., Chert, H.-M., and The Journal ol' Immunotngy I $3:.1276-32PA, 1994. 177
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Other suppo,l: U.S. Public Hcahh Service. ~tsl an~ Hsr~srd Medical School. B~ton. MA. MONOCYTE CHEMOAITRACTANT PROTEIN.I GENE IS EXPRESSED IN ACTIVATED NEUTROPHIL$ AND RETINOIC ACID-INDUCED HUMAN MYELOID CELL LINES . We have u~-d differential display polymera~e chain reaction Io tale•lily genes Ifl•t are upreCuleted after retinolc acid (RA) treatment of human myclobl~tic ILL-60 celia, Three of the eDNA• cloned hybridized to RA-inducible transcripls on Nor.them blots, one of which was shown Io encode sequences for monocylc cbcmoattraclanl pm~eln. ! {MCP.I). • recently ¢lc~cril-~.! cy~okine that is chemo~actk~ for monocytes b~ no( for neutrephils. Nuclear run.on analysis indicated that the upregulat|on of the MCP-I ge~e oecur~ at the transcripli{mal level in ILL-60 cells MCP-! tranzcrip~ levels •l~o increased after RA treatment of the NB4 acute promy- elecyll¢ ceil line. MCP-I transcripts were undctcct•blc in freshly isolated ncn- trophils by Norlhem analyst,s or reverse tran~crlption-polymcrase chain reaction ban were readily delectable in ncutro~ils •ft~r incubation in media st 37"C fo¢ 20 helms. zugpating th|t an aetivalion event can lead to MCP-! expre~.,tlon in neelro~ifs. o, rein in . iv.ted ... ~111~ ,_~.ta ~ .~..r~ t,,.~ ~.~ MCP.! Se.~ ,a.RA-respo~ive in mye~d ~ ,,.e. •on ,s expresse~ m .et~lroph.s. MCP-I exl~-~,on by aclivated neu~rophils may p~ay a~ imporlant ~ in |ttracfing monocylea !o the si~e of ti~;~e damage or infection. Burn. T. C.. P~rov|ck. M. S.. Hoheus, S.. Roliins, B. 3., and Tenen, D. ft. Blood S4($):2776-278). O¢loher 15, 1994. Other support: Netlon•! Institutes of Health. Hallo.a| C•ncer Institute, and lhe Leukemia S~:iety of America, From the Hcm•tolocy/Oncolocy Division. Beth Isr•el Hospit•l, Depar~menl of Medicine. and Prooam in Cell and Developmenl Biolot#. Harvard Medic•l School. md the l~parlm~m of Medicine. D•na-Fa, her Cancer lnstifl~e. Boron. MA. ~TRUCTURE, ASSEMBLY AND INTRACELLULAR TRANSPORT OF 'DIE T CELt, RECEPTOR PeR ANTIGEN ~ T cell receiver for antigen (TCR) is responsible for the ~cognition of anti- |en associated wilh Ihe major histocompatibility complex (MHC). The TCR expr~,ed on the surface of T cells is •¢~o¢ialed wilh an invsriant slrUeture, CD3. 175 CD3 is ~ ,,m~d to be respomlble for iotrat~llelar s;|nalin$ fol~l ~ t~ ~ ~ h~. ~ ~D3 ~plex ~sisls of six dlK~ ~ R~ls a un~ly ~ex mult~u~nil s~mMy ~m f~ t~ ~ll. ~11 ~s wilh Ih~ ~km ~ ~lafing I~ iot~llul~ ~bly of ~ Within t~ e~ pla~m~ ~iculum, ~ ~.ly-s~:i~ ~ainx ~m~e line i~f~s of t~ ~e~ ~volving cliff.natal u~ of ~ ~ ~te~l~r (~.~ y-8), [-family ~r. a~ ~ 7 ~ 8 ~i~ ~ aR ~umMy Ilfl~ te dlf. feRnS ~ fu~ims. A~m~y of I~ ~D3 ~kx ~ with ~1~ ~ ~ ~le signals m indiv~ull chains w~h ~t~i~ ~ldnI and bly ~ de~dalim. ~ T cell is thvt ab~ lo sel~ a ~k~ely ~m~ fe~i~l ~ efdlsti~ ~3 ~pkxes f~ ex~t~i~ at the ~11 ~y, M.. Terh~. C, a~ Wilem~. T. ~mina~ in IMMUHO~V 3:283-2~, I~1. F~m the La~tory of Molecular lmmunolo~. Dana ~r~r ~n~r In,Irate. ~m~t of ~thn~. tla~-a~ M~tkal Sc~I. B~tm, MA. MURINE B-CELL SUBSEI'S DEFINED BY CD23 The B-cell ¢omparlmenz is ¢ornpo~d of • c~x~plex mixlure of dlattnct Th~ec anatomic sites that are p~liculady rich in B-cell subpopuletkms a~ Ih~ bent marrow, qdgen, and pertloueal cavity. Our laboatmy has found CD23, d~ ity lf, EFc Rce~or (FceR|l). Io be ef value in dlsc~iminztlng • number of mudnt I~. cell subsets, especially when esed in combination with osier B-cell n'~ken. In bone marrow, the site of matt•alien. Ibe Ixetence of CD23 spe¢|flcally dellnlat~l mature reci~cutsting B cells, wire•as its absence dea~ea the maturing B.¢ell tim•. In the spleen. CD23 is constitutively expt~essed m the follionlar ~' me~tle cells md is ab~m on the m,,rgina! ~ and immmu~ B ceib. These latter two lallo~ can he distinguished by diff'edn¢ levels of the heat stable t~til~n (HSA). In the peritmeal cavity. B2 {conve~tiond) B cells display tile CD23 •ntll~n wh~rms BI (CD$+/sister) subset does not. A~ditie~al studies examin|ng the exp~lsslon of CD43 (Icukmlalin) have found this anllgen Io save as at i'ecilXOCal re•deer to CLY23 In many cases, in the ~ re•now, a pan a~ti-CD43 anlibody deices• tho~ B celts that are CD23 net, clive, and in the,spleen and Ix, d( ,oueal cavi!y, d~ majority o.f CD~.~.. mive B! ceJIs are CD43 ix~tillve. To••the*', Ibese •ladles demon•Irate tl~ utttay CD23. CD43, HSA, and other marker~ Io discriminate betw~n B-cell tut',~ets and allow for mine I~,clse purification and analysis of these pope|aliens. fleet. C. G.. Kemp. J. D.. W•Id~hmidto T. J. METIIODS: A Compan|on to Methods in Enzymok~,y $:3-10. 1995. Other suppo~: National Institutes of Health. From the Department of Pathology and lmmtmology Graduate Program. Uniwnky of Iowa College of Mcdlciue. low• Ci~y. IA. 179
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THE EWFEC~ OF IN VIVO I!.-7 DEPRIVATION ON T CELlo MATURATION A number of prevlou~ studies have su88cstcd a key sole for imedeukin 7 (ikT) in the maturation of T lymphocyles. To better a~sess the funet;ou of IL-7 in lym- phOpoi¢~ls, we have deprived mice of ]L-7 in vizw hy long.tcm~ ndm;ni~trntlon ors neulfalizing anti-lL.? anllhmly. In z previous rep,rl (Grab.stein. K. I1.. T. J. Waldschmidl. F. D. Finkelman0 B. W. Iles~, A. R. Alpert. N. E. Bolani. A. E. Namen. and P. ]. Mocri.c~y. 1993. J. £~rp. Mid. I'7g:257-2M). we used this system to demonstrate lhe critical role of IL.7 in B cell mazuration. After a brief Period of Imli-II..-7 treatmenlo mo~t of the p¢~-B cells and all of the pr¢-B and immature 13 cells were depleted from the bone marrow. In the weans mgx~. we have injected anti.lL- 7 antibody for periods otr up to 12 wk to determine the effect or ht r/m IL.-7 depriva- tion on the thymus. The results demon~rat¢ a >99~ reduction in thymic cellularity after extended periods ot antibody administrmion. Exandnatlon of thymlc CD4- arKI CD8- deRned subr.ets revealed thai. on a propcmionaI basis. Ihe CD4+. CI~+ suEm, I was mosl depleted, the CD4 and CD~ sinsle positive cells remained essembl- ly unc~, and Ihe CD4-, CDg- coml=rlment aclually increa.,md Io --.~ or Ihe thymus. F,jrther examination of the d(mbie negative thymocyzes demonst~l~d that IL,-7 delxlvafion did, inc~,d, depieze lhe CD3-, CD4-, CD~- precursors, with expendon or this ~ubset being interupIcd at the CD44+, CD25+ sty. The propor- tional incRa~e in the CD4-, CDg- compa|lmenz was found to be due zo an ~ceumu- latlon of CD3+, T cell re~c, lor e,~ + dr~hlc ncg~allve T cell.~ Additional analysis reve~l¢~! dmt ami-lL-7 lre~lmont suplxe¢,~-d the auditiord~iec~ir~ process oI'T celk, as shown by a si~nlricanl reduclinn of single IX~ilive ceils cxpre.c¢ing CD(~) and beat atalde antigen. Finally, Ihe effec|s of IU7 deprivation on the thymus were round io b~ rcverslble, with a no~al pallcm zion of Ir~lm~nt, The pre~r,I results thus indicate a central mie for IU7 in lee malu- radon of Ihymie-derived T cells. Bhatla, S. K., Tygrett, L T., Grabslein, K. H., and Wald~chmldt, J'oumal cff'Experimemal Medicine 181:1399-1409, April 1995. Otl~n" su~x~: National ]nstitmes of Health. From tee Depenmem of Patholocy, University of Iowa College of Medicine, Iowa City, IA. and Immunex Coqxxation, Seattle, WA. IMMOBILIZED ANTI-CD3 ANTIBODY ACTIVATES T CELL CLONES TO INDUCE THE PRODUCTION OF INTERSTITIAL COLLAGENASF-,, BUT NOT TI~UI~ INHIBITOR OF METALLOPROTEINASES. IN MONOCYTIC TIt P-! CELLS AND DERMAL FiBROBLASTS |n tEis ~udy we have inve~igated whether direc! cell~lo cell eont~t Ic~ivlted parlformaldehyde.figed T cell clones obtained from synovial tissue of petiem$ with omooarthrltls (CA) or rEeumatold arlhritls and target monocytic cells or dermal flbrob~lsts irlflncnced the balance between |nter~tltlal ©ollagenas~ and it~ ¢iflc inhlbilor tissue inhibltm of meta|loproceina~s (TAMP) produced hy the lazier 180 cell types. PHA/PMA-activated fixed T cell ¢lo~es or their membranes alrcmllly induced the production ofcellagena~e lx>th in monnc~k: THP-! coils and in dermal fibrobllsta. In ¢OOlraM, only low levels of TIMP were Induced in THP-I calla ~nd no cEeng~ of" TaMP exWession was olw~,~ed in filxoblasts ~ I f~suh of with PIIA~MA-acliv~ted T ¢¢11s or T cell memtwaee~ Anti.CD,t-aetivatld T ¢~II clones stimulated the woduction of collagenase Eolh in TllP.I c~lls and flbeoblam. whe~as TIMP levels were nol influenced. Colla~mase production in THP-I e~lla induced by anti.CD3 ucllvated T cell clone~ was !) dependont on the de~l of anti. CD3 used to stimulate the T cells. 2) initiated only Wen CD3 wax crabs.linked. 3) inhibited when cyclosporin A was included during T cell activation. Our dell col- lectively indicate tha¢ activated T cells in conlact wilh monocytic ceils or may alter the helance between interstltia! collagenal¢ and its speeifle inhibitor TAMP. This seteetlve induction of a mediator Wofiie repregnlltiv¢ of m0tdx br~ek. down as a result or target cell interaction with Ictiveted T eelgs may be an important lictor in lhe local Wocess of tissue destruction that ¢haracterites o~t~l/thdlil and ~neumatoid arthritis. Miltenburg. A. M. M.. L.ncraz. S.. Wetgus. IL G.. and Dayer. J.-M. Journal oflmmunology 154:2(~55.2667. 1995. Other supporl: Swiss National Science Foundation. and the National In~titull~t of" Health. From lhe Division of Immunology and Allefgy. Hans Wilsdorf Lahoralcwy. Department of Medicine. University Hospital. Geneva. Switzerland. and tee Division of D~matoloBy. Washington University ~choot or Medicine at Hospilal. St. Lo~is. MO. VIII, Neuroscience ATTENUATION OF AGONIST-INDUCED DESENSITIZATION OFTHE RAT SUBSTANCE P RP.~"EPTOR BY PROGRES$1VB TRUNCATION OP THB C-TERMINUS We have invcstlgated the C-terminal tall or the rat substance P t~teeptor (SPR) as a domain e~enllal foe agonlst.induced desensitization. Four pmffeaiv¢ly shorter mutants, using premature lerminallon in the C-termlnus. were comtmeted and pared with the unaltered SPR using octopi© expres~iou of wild-type and mullm receipt in Xenopus oocyles. "l'hete mutants were designated .DI6,_D47. D7~..~d D96 with 16. 4"/. 70 and 96 amino acids residues deieled from the tall. raSpeCttvoly. Wild type SPR. DI6 and D47 exhibited normal ourr~t responses when eh~ltenjtd with sut~ance P, hut IT/0 and D96 h~d redu~d maximal cuwent t~po¢l~ and 5% of wild type SIR. respectively). DT0. however, exhibited lutmanttal tBt
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0 0 UmC¢ to subctax~e P.indoced 6esensitiz.afion in thai 55%. versus 8% for wild typ~ SPR, ~f zl~ ~ cum..m of the tim ~ ~ ~ ~ ~! chellc~c ~th ~ P. ~. s domain r~ ~sldoes 3~R to 3~ or t~ ~t sPR. t~h ~ ~ f~ I~ fu~al Klivily ~ I~ ~. ~ays an cssentlal P~ t~ ~ of B~l~i~l ~mist~, Sc~l ~ Medicine, Unive~ity or TRAN$IEN']" ISCHEMIA STIMULATES GLIAL FIBRILLARY ACID PROTEIN AND VIMENTIN OENE EXPRESSION IN THE GERBIL NEOCORTEX. ~TRIATUM AND HIPPOCAMPUS Aetroe71i~ ~tlvatino plays a major role in homeostatic maintenance or the can- tral ~rvous system in feslx)rise to neuronal damzse. To assess the reactivity of astro- c~les In trlmslont cerelx~l ischemla of the ~erbil. we studied the levels ofglial fibril- lary ~idie protein (GFAP) ~ its mRNA. GF^P mRNA increased by 4 h aAcr carotid artery occlusion, re~ched peek levels by 72 h with a 12.fold increase over control ~ then staled declining ms early as 96 h po~tischemi~. An examInation of the sj~ciflc re¢ions or the brain revealed mn increase in GFAP mRNA as.,mciated with the fore1~In, midbmin, hippocampas and striatum. GPAP mRNA in the non- Ischemle cerebellum however, remained expressed a! constitutively low levels. Imm~ecd}lot mn|lysis with anIi-GFAP ~fibod~ demonstrated a 2- to 3-fold iner~ in the Iwo~In afle¢ 24 and 48 h of fcperfusion. F'relreatrnent with Ix~tob~rbital and I-($'.oxohex~yl)-3-methyl-'/-pml~JI xantSif~ {HWA 28S), the drugs that have been showsl IO pllXect against bchemle clmnage, prevented the increase in GFAP mRNA in the cortex following iachemlc in,~zry. Forebrain ischemia also induced vimcntin mRHA ~nd Ixoteln quanlltlcs by 12 5 of feperfusloe in the c(xtcx, The levels of c-,f'os and proproonkephalln mRNA increased rapidly within I h after isch~nic if~ury. (lemonm~|n~ a t~l diffo~c~ in mRNA chzn~ followin~ ischemla. ~ resuhe ind~t~ ~ m inc~ in GFAP and vin~.tin. ~he ~wo ~lial'in(m~dia~ fib aunent Fcmeins in the a~a ot" the ischemle lesion may be assoclatad with • glial Kindy, M, S~ BSat, A, N. and Bhat, N. R. Moleculm" ]Bfaln Rcscamh 13:1~9-~06, 199Z Prom the Department of Biochemistry, Laboratory of Cellular and Molecular Neu~obio]ot~, Center of Ex~ellenoe on Stroke of the Sanders-Brown Center on Agin|. Cbll~le¢ Medical Center, Univ~xlty of Kentucky. Lexington, KY. 182 TYROSINE PHOSPHORYLAT~ON OF MICROTUBUL~A$$O~IATISD PROTEIN KINASE AFrER TRANSIENT ISCI-IEMIA IN ~ tymsi~ ~ph~lati~ o£ m~ubule.~la~ ~ein,(MA~kl~q wax ~xamincd in the =crhll brain after trsn;iont ~lati~ ~ MAP kin~ was max~l within I ~ min ~ i~ia a~ mtu~ ~ ~tml I~els a~ early ~test i~a~ in MAP klna~ p~l~i~ ~ ~t~ in I~ hi~, with mi~ i~a~s in ~ i~ ~ ~ t~ .~1 ~F~usi~ inju~ ~ly ~t ty~ ~all~ i~a~ in t~inc ph~p~ylMi~ w~ ~nt~ cyan~?.nit~l.lnox.li~-2,~i~e, wa~ i~ff~tive. ~a~nt of ~e~ll~ with calcium channel 5l~ke~ also p~nt~ I~ ly~;~ phosphor~lati~ of MAP kina~ in t~ I~¢~m~ ~in. AIt~, t~ ~1~ im~y mate ~epto~ and calcium d~n$ the t~msine p~lMi~ ~ MAP kin~, T~ine ~pho~lat;on w~ al~ ~ve~ w~ i~ia ~du~cd u~cr h~ic ~iti~s. which ~ ~ma~. ~ fi~i~s impt~atc a talc ~ MAP k~ m ins rr~ i~hemia and ~ffusi~, Cam~G~z~lez, R. and Kind~, M. ~ F~ I~ ~mcnts of Sur~e~ a~ Bi~mlst~. ~ C~tcr. ~ ~tc~ ~ A~in~ a~ Stake, UnlvmtW or Ke~ky M~ical ~. ~xin;t~. KY. INHIBITION OF TYRO$1NE PHO~PHORYLATION PREVENT~ DELAYED NEURONAL DEATH FOLLOWING CEREBRAL I$CHEMIA Prc~e;n tyrosir~ pho~pherTlation plays an important role in the regulatkm oi" neufonal funclinn. Wa examined the effecls of inhlb~t|on of tyro¢|n~ pFic~pl~latlon on ischcmic neuronal dzmage in the CA, mglon or the h|pp0eampus, in the pd}il hippocampus, genlslein and lavendustin A, lymsine klnzsc inhlb~tors, were admird~- t~,ed 30 rain before initiation of ~-min ischemia and z~'~rruslon. Both pnisttin ~ lavendustin A Mocked tymslne phosphot~Jlztlon and prevented ddayed neuronal de~h (DND). However. Beni~tin. an inaclive analogue of pni~lein, did ne4 bloek DND. G~nistein was dine.dependent in the inhibition of DND after iaehemht and rel~ffuslon. Adm|n~ratlo~ of ~ist~in $ to 10 rain afler tachem|- and ret~rl'uslen was ineffective in Mocking DND in the CA, region of t.h~. hill.am .1~.. ~ ty~. sine klnas~ |nhi1~tors s~lecllvety blocked the pho~hmylatmo oF miem~ut}ul~-aa~ect- ated protein (MAP)-2 kinase following i~hemia and reperl'asion injury. These results s.ggcst that'tyro~ine pho~phm71atinn in the ischemic l~in is imlx'al~nt for 183
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neuronal injury and that MAP-2 k}na~e may play z role |n the oor.et of'delayed neu- ronal death. • Kindy. M. S. ]uemal ofCerelxal B|ood Flow and Metabolkm 13:372..177. 1993. From the Del'~mcnt or Biothem|slry. Stroke Fr(~ram of the ~anders-Brown Center O~ ABing, Uni~'rsity of Kemutky. Lexlngto~. KY. NMDA RECEPTOR INHIBITION USING ANTISENSE OLIGONUCLEOTIDES PREVENTS DELAYED NEURONAL DEATH IN GERBIl. IlIPPOCAMPUS FOLLOWING CEREBRAL I~"HEMIA Olu~m~e plays a key pt~icity of the neurord ~s w~l as n~Jmnel cell de~b. Tbe~ proc~,:s ~re rnedlat~d through the I¢fivation of N-methyI-D*asl~rl~e (NMDA) ~ecel~Or~ and the resultin~ ildlux of CI~'. We exlndned Ihe elTe~ of inhibition of the NMDA receptors at the II~lecullr level o~ ischemic neuro~|l injury in the CA, r~glo~ ot Ihe hippoeampus. In the ~rbil hipixcampus, amisens~ olisenucleoddes (ODNs) dlmcted against the NMDA mCCT, eOr (NMDARI) nxluced the level or NMDA ¢cel~Or mRNA and lipnd bindir~ in the CA, rngio~. Sew,~ o¢ nsense c~igonuclenlld~s h~l no effect upon the NMDA receptor. Administration of antiscnse NMDARI ODNs prevented delay~ ncmonal death fDND) following 5 mln of ceret~al ischemia, whereas, sen~ ODNs did not allot the eft'eels of ischemia. These rc~ult~ suggcs~ Ihat the NMDA r~ceptm" ia involved in the ixocess of DND during cerebral ischemia. Kindy, M. S. .N~roec~enc~ R~reh Comm~nlemlo~s ! 40): 175-11~3. I ~94. Olher support: National lnslitulce of Health. " ~ lhe Di~rlmem of Biochemistry, St~oka Pmgr~n o1" the Sander,.Brown Crater e~ Aging0 Unive~ity of Kentucky. Lexlng~on. KY. BLOCKADE OF ORNITHINE DECARBOXYLASE ENZYME PROTECTS AGAINST I~IEMIC BRAIN DAMAGE PolyamJnea are derived from omitbin~ by the actions ot om;thinc d¢ca~boxylase (oDe). which is the rate-limltin~ step in this I~hway. Po|yamincs play a role in ceil sJcml ¢efC~tll ~K"~cmi• ~vc's i~ IO incre, as~d ODe mKI~IA Imo ¢fl~.,ym~ aCllVlty Ifl the gerbit 5raln. OD~ and pelyamines a~ tho~M to be importam in the Sen~ratle~ of edema and the neuroaal celt loss associated with ,~'~oral isehemla. To test this theory, wa examined the O[X: activity, putrescine l,~ela, and I~e~ro~! density in abe CA, rngi0~ of the hippoeam~us following ischemia and repeffuslon i~Ju~y in the absence m~d preRnee ot" an inhibito¢ or ODC aclivity, a.dlfluomm~hytm~hhine (DFMO). Pmlreatmem or anlmds with DI~IO R~mlled in attenuatk~ or the ode acfiv|ty following S mln of |schemia and 4 h of re~-rfllsicn. In addltk~ O~10 vented the increase in polyamine kvel¢, as determined by mealu~n~nt in the ischemic brx|n. 'Tl~se ahemio~s were no( due to c~ang~l in ode mRNA level. Furlhe¢ ~nalysis revealed that DFMO I~.atm~nl block~l lhe delayed n~romd cell death in the" CA~ region of Ihe hippoeampus thal ac~'npln|es i$¢bemla and reped'usion injury. Adminbtr~ion of DFMO msulled in a do~dei~ndent I~Nfl¢ia! effect upon ~'uronal tell survival. The~ reaul~ so,eat thel ODe efl~y1111 ~,~lvtty and th~ IXnduction of I~ly~mlnes play • signifl,~mt role in the ~lxmle of the brain to i~chemic injury. KindT, M. ~., Ho, Y., and Demp-~-'y, R. J. ~onmal of Ccr~ral Blood Flow and Metaboli~ 14:1040-1045,1994. O~her support: Nmion£ Instflme~ of Heaflh. From the Stroke Program. Sanders-Brown Center on Aging. Deparlment Biochemistry and Division or Neurosurge~, Unlvenily of Kentucky Cenler, Lexington. KY. REOULATION OF ALPHA~-ADRENEROIC RECEPTOR S~GNALLI~O All~.~dr~crglc rece1~o~ (%-AR) medlate • number or ¢~r~iov*.Jeufar and metabol~c effects or eatecholamlnes and ale memhess of a ~1~ fluni[y of which tr~mduce their cell signals Ih¢ough het~.o~imedc lulfline nt:cleolide lxo(clns. Utilizil~ the e~t.ldrenergk: tteep(or Sylt~m a~ a l~.l~itadvo specificity. (~ objective is4o define the micm~vim~ .m~..nt of c~-~. ~m~r~le tots in dilTerent ¢~1 ph,~otypea ~d det©rmine l~w d,ffe~ m mils mtere. environmema ~ cell •mbiteeture co~tributa to (:ell type apeeil~ allmdl~ by The s(rategy entails the use of c~-AR sul~ypes atably eXlX~Ssed i~ NIH.~3 filx~last~ and Ihe Fheonhromocy~om• ee|l line PC-12, two eel| tyFel wit~ dlst|~'t infraatmctum •nd in which the Rcepto~ couple to dilTemnt aigm||l|n$ AhhouBh R, G •nd E ~re the ml~ emltics involved in si~l| ~tt~, and "k~llzed s,gnel transduct~o~ or n~leeules that re, orate events at 185
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G-E Interface. A more complete undemanding of cellular plsstlclty with re~pect to signal transducdon may lead to a new level of therapeutic specificity and the devel- opmem of therapeutic approaches that perhaps target the R-G or G-E interface as ~ to the receplor's hormone binding site. Lankr, S. M. PhermucoloD, Communications 6{I-3):133-137, 1995. Other suplx~: National Institutes of Health. From the Department of Pharmacology. Medical University of South Carolina. Chadestm, $C. HETEROGENEITY AND REGULATION OF NICOTINIC ACETYLEHOLINE R~"EPTORS $~ce 19~6 there has been a remarkable evolution in our conceptual understand- lag of the nicotinic acetylcholine receptor system. It is now clear that them is genelty amens memheta of an extended nAChR family m gene, pt'o~eiu, and funo. alumni levels. This heterngeneity poses challenges to a clear undentandlng of feneticn of these Important molecules. It is also bueoming dear that diversity in me~anisms involved in the regulation of nAChR expt~tsiun and ftmctkm build on ~ diversity of the nAChR family. Continued. exciting advances in these fields will unck~ulxedly e~ur under a deluge of applicatkms of skills of a hum.her of i .n~e~.tlg~.- to~ wed~ing In a variety of diseii~ines who will benefit from reflection m the principles of neurutransmltter-recelxor biology revealed by past studies of the nAChR aystem and from a heightened anticipation that Ipany conve.ntiontl.and Ix~vlnclal views of nAChR biology and regulation will require alteration or amtn- donment by 1996. Lulms, it. J. and lkncher~f. M. In: Inlemational Review of Neorobiology Vol. 34, Academic Press, Inc., FP. 25-131. 1992, COWl suppot~: National Institutes of Health. Prmn the Divislon of lqeumblology. Barrow lqeurological Institute. l~uenix, AZ. AOINO4NDUCED CHANGES IN "HIE AUTORF.OUI.ATION OF AC'E'rYLC~IOLINE RELEASE IN THE RAT BRAIN In ~e~iiu F'isehm" male ntis (33-months-old), the spomaueons and evolced releax- e~ of ACh from eerelx'um were depressed. The defe~ in the vesicular storage and t'ele~e of ACh is the mo~ important parameter which may c~ttrihe~e to the cholln= ergic deficit in the cerebrum of 33-month-old rats. The fz~lback systems fur mstoregelation of the rek'ase of ACh are ol~'~tk~eal in aging tats. ~ duer~tt~ in ACh mle~.e triggers the pos;live-feedl~tck system whkh increases the rate of rdsuM of SP during nging. There is no riced for the neptive feedbe~ system. "l~tmfem. MEK release was depressed. The increase in the rate of SP release dorln| opens a new avenue fm drug developmenl. Nm-peptide SP-antagonisls may b~ ful in the trealment of senile symptoms of the aged. Saslry, B. V. R., Janson, V. E.. and Tayeb~ O. S. In: Hamiu, I. et al., (eds.): Alzhelmer's a~d Parkinson's Diseases. Plenum New ¥o~k. pp. 275-282. 1995. Other support: U.S. Public Health Service and Th~ Study ~enter for Toxicology of Vanderbilt University. From the Dep~meuts of Anesthesiology and phatmacology, Vanderbill Univmlty Medical Cent~. Nashville. ENDOGENOUS GANOLIOSIDE GMI MODULATF~ U.TYPE CALCIUM CHANNEL ACTIVITY IN NIg NEUROBLASTOMA CELI~ Digital imaging flumescence mlero~:~y was mud to investilate the effect of the B suSunit of cholera toxin no calcium homeostasis in t~lutoma NI~ cells. The B subuntt, which binds specifically to pnglimlde OM! in I~ outer leaflet of the cell memb~ne, was found to induce a suslaiued increase ef tnttacellulm' calcium concentration ([CW'~,). The incfease in [C~], was no~ obs~'vod in the able .n~ of estracellular calcium. ~ in Ihe presence of Ihe calcium chelator EGTA. anu was blncked by nickel. Th~ B subunil was also t'~nd !o induce an influx ef ions. as indicated by a quench of the iulr~'ellular furu-2 fluorescence. Tha~ dub suggest that the B suhunlt induces an increase in calcium influx in N15 putassium-lnduced depolarization also stimulated manpuese influx: however, Ihe onset of depdariza|ion.lnduc~l influx, the B subunit had no ~urther elTe~l. This occlusion suggests i.n.voivement .of volt~e. ~ .-d~.'.e.e.e.e.e.e. ' ~n~.~. n~ calcium ctm~..Is. Tmam~. t with BayKg64~, t d,hydrop~dme agontst sdect,ve for L-ty~ .~. lc,u.m induced manganese influx that was no¢ altered by the B subonst an~ apparently blocked the effect of the B subunlt itself. Purthennme. the dihyd~dine channel antagonists nlguldlplne or nlcardipine completely inhibited • subunit- induced manganese influx. Thus. the B subunit-induced mangenesu influx it likely due to activation of an tetypa voltage-dependent calcium channel Spontamous influx of man ..ge~. iuns was ~l.m inhibited by nkasdlplne or hi|aid|pine ~ by exngenoes genglk~kles. Oangl*estde OM! .w~ mo~_ .potem t~ .an.GM3.. but side had no significant effect, The modutatton of L-tyl~ Ca~ctum channels endogenous ganglkxtlde OM! has impmtaut Jmpllcaticns for its role in r~und duvel- ppment, dlfferemla6noz and reW,meration and also for its potential function in electrical excitabillty o~ ueurons. (~rlson. R. O.. Masco, D.. Btoetcr. O.. and ~piczel~ S. 187
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The Journal ol'Ncumsclence t4(4):2272-22gt. April 1994. Other ~pport: Natlo~al Institutes or Health. From the Department or Biochemistry and Molecular Biology, Georgetown Universit~ Medical Center, Wa~hin|ton, D~. DIFFERENTIAL SPATIOTEMPORAL EXPRESSION OF K' CHANNEL POLY~DES IN RAT HIPPOCAMPAL NEURONS DEVELOPING IN $1TtJ AND IN VITRO Nt~ocampal neurons ¢-e highly plasllc in their exckable pro~lies, both dur- ing development and in the adult Ixaln. As voltage.senshive K" channels are ma~or d~0rminants oF mcmhcan~ ex~tabillty, erie mechanism fc~ 8en~rating plasticity is Ihrough regu/atkm o~r K" channel a~tivity. 'To gain insights taro the regulation of K" channels in Ihe hiPlxx:ampus, we have analyz~l th~ sl~timemfxx'al expression pat- lems or five K" chaan~! i~lyl~lXldes ~n nt hiplx~ampal neurons develof~ng in aim and in vitro. Delayed rectlfler-tyl~ channels (Kvl.5. Kv2.1, end Kv2.2) are expressed on all n~umnal somata and Ixoximal dendrites, while A.lyl~ channels (KvlA Incl Kv4.2) ar~ IX'c~nt distally on distinct suhfx~ulsl~ns of n~uro~. The ckvelopm~nt ot" th¢,~ ]~ttcms in .ttu is mort<Sonic; that is. while the time and development viries among the channels0 each K" channc~ suFXyl~ inillally ap1~ars in its adult i~tt~n, sugaring that the mechanisms underlying s~atlal I~,¢ming ate thrcml~h development, lmmunoMols c~nl','m the dilTerenti-I tcmp~'al exr~csslo, OF K" ehennels in the devel~ing hilqxx:ampus, and dcmon~ratc devci~ntally regulated changes in the microheterogeneity of some K' channel ~ly~ptide six,lea. Temix~xl expression I~tlems OF all five K" channels o5~rved in s#u am ~talned in vL~o, while ©e~tain as~ts OF cellular and sulx:¢llular kx;alization am altered for some of. the K" ~annel polyixq~ides studied. Similarities in K" channel nisms are c~x~tmlling s~dmemlxxal I~tteming in bblh shuations. Howevef. dilTcr- enees between levels of exl~eSslon for all sub~,~s stadi~ except Kv2.1 indicate ~klitiona] mechanisms o~rating in ~it~ Ix~l absent in vitro that me important in de~mining poly~-lXide abundance. Mdetic-Savmic. M.. L~nn, N. J.. and Trimmer, J. S. ~ Jm~nal o£Neuro~icnce 15(5):3940-:3g$1. May 1995. ~ suplx~t-' National Institutes oFHealth. Prom the Depadments of Neurology. and Biechemistry and Cell Biology. Stale University orNew York. Stony Brook. NY. ASSOCIATION AND COLOCALIZATION OF K" CHANNEL e- AND I~-SUBUNIT POLYPEFF1DES IN RAT BRAIN Recent cloning Of auxilJaa~ subunks associated with voltage.pied ion chatlnels and their aubsequent ¢oexlxe~ion with the channel formk~ e-ltlbuntta hal that the exlxession level, gating and conductance i~ot~k~ or t~ expmlmd e'--nun. nels can he profoundly affected by the presence of an auxilla~'y subunh In the IxeS~l study, we Ixized antilxxlies ~,zlnzt the p-sul~nfl as~illlt~ with th~ hovlne dendm¢oxin sensitive K'-channel complex and used them zntllx~dlt~ I~ char. xczcrlz~ the related p.~,u~mlt potypepli~s in r~t br~in. The antl.~-sub~nlt w~tlhedles displayed a ~pecil'¢ reactkm on immunoblols of' Zlt brain membranes with a 38 kDa po~jpep~ide, and a minor 41 k]~ pol~¢,pllde, which ~n~spon~ ©l~ly to the predicted sizes or the Kvp2 and Kvpl p-aubunit polypel~ides, resl'~tivdy, recenlly cloned From fat brain. Rc~ipr~al colmmunopreeipilati~,m CXl~fiments ~evealed that the j~.su~unit i~lypcpticles m ~izled with Kvt,2 Ind Kvl,4, not Kv2,l, e.subunlta. Immunohistochemical zlaining revealed that the I~.subunlt polypep~idea were widely dis~'il~ted in adult ~ Ixzin. lvloreover, the e~tlal~" dlsld- butlon OF I~-subunlt immu~re~ivity co~esix~ded closely with tmm~nme~,tlvlty fo~ Kvl~., and to a lesser extem KvIA, Ix~ nm with Kv2,1, ~ ~esuits that neuron~! mechanisms mgy exist 1o direct the selecllve interleliO~ or K" e~ and 13-subunit I~Yi~Fidez, z~ Ihz I~ pr~erties ol'K" channels in zl~eiflc sub- eel]olaf domain~ n~y he regulated by the t~0nnm]on of halemmuffim~rl~ K" ehlmn~l cm~plexez conlalnlng specit'¢ ~mblnz~ions ores. and [B~ub~mits. Rhodes, K. J., Keill~ugh, S. A., Bzrrczueta, N. X,, LOlX:Z, K. L., and Trimmer, 'Th~/oumal of Neuro~ience !~'7):~3~0-$371, July 1995, Other ~u]~m1: LedeHe Lz~x~zorles, the Center for gk~lech~'~logy at ~to~y Br~k, and New Yc~k St~e ~clen~e and 'I'e~hno~o&y From the Dcp~rlment of CNS Biological Research, Medical Research Division, Lcderle Laboratories. American Cynnamid Company, Pearl River, NY, and D~pmlment of" Biochemistry and Cell Biology, St~e Univerelly OF New YoH~, Stony Brook, NY. IMPAIRED R~ON BY ANOIOTEN$1N I! MEDIATED BY ~E AT, We demonstrated Ixeviously that hi~q~ocanq~l d~nlate Lyrm n~lmm were sen- ~i~ ~ m~in ll (AH) ~ ~ntly di~ ~ All a~l~ di~y to I~ ~te g~s ~hi~ ~ule ~11 I~ ~ ~tkt~n i~ti~ ~ Ih~ t~ AT, Mt~ ~ ~ of ~ ~ ~ w~ ~ e~ ~ e~ o~ All ex~ts ~ ~. ~ ~ ~iti~ ~ ~ ng All ~lnlm~ 189
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the~e expcrlme~al conditions and that the impairment can he efTcutively p~vented by pretreatment with 20 mg/kg of Ioszrtan IP. Lee. E. H. Y., Ms. Y. L. Wa.Tner, M..~., and Annstron~, D. L Peptk~ 16(6):I0~9-1071, 1995. ~ support: Institute of" Biomedical Sciences. Academia $inica. Taiwan. Republic orChinL From the Institute of" Biomed|cal Sciences, Academi~.$inica, Taipei, Taiwan. Republic of China. and Division of" Life SciEnce;. Umversity o1" Texas a! S~ Antonio. St. Antoeio~'IX. DO~E AND TIME DEPENDENCY OF ANGIOTEN$1N l! INIIIBITION OF HIPPOCAMPAL LONG-TERM POTENTIAT]ON We prev;onsly reporled that injectkm of" 1.0 pd or 4.7g p~ an~iotens;n II (All) shove the hippocampus in r~s inhibits long term polontiatio~ (L'J'P) imluclion in UCtc4 m urethanc~anc~thet,zed Spra~ue-Dawle), rats. LTP was meaxured in ~ o4" ~ relatJYe change in s|o~e of the ~'~ha~on ~I.~P compared to ~L~line. EfSx~S of 0AS, 0,956. 1.195. 2,39. and 4'/8 ~ All and lime delays of 30, ~O, 90 a~l !~0 man with the 4.78 F-q dcee were detennlned. Resuhs were significant and demons~ale ~mt All inhibilio, of' LTP in dentate Iranule cells is lxah dose and time __~/~. ^._ _ncp.nz sl .ow~.y zner ! h and is complete over the .exl ~0 rain, eOnlinUes rot" another 30 ram0 ~ then full), pecovevs by the end of the next 30 man. This time ~could be due to the imemalizatlon of' the All, interaction with • cytoso- Wa,/'ner, M.3., Polm-Cunain, J., and Armstrong, D. L. Peplkks 16(6):1079-10~2, 1995. Other mzppxt: National ~nztitute~ o1" HeMth, From the Division of t~re Scic.ces, University of" Texas ut San Antonio, San IX. PhannacoIo~ ACUT~ ETHANOL EXPOSURE SUPPRESSES THE REPAIR OF ME3"~Yt~UANrN~ D~IA L~l~S IN CAnArD ADULT M~ ~l ~ ~c~y ~ ~;at~ with an i~ ofc~ in nu~s ti~ gas. lmym~, phm'~nx, end mouth. Aicohel ak)ne has n~ ~ s~ ~ with af~! c~ts ~g f~ 4 to 5~ (~r ~ wl~ ~I~) (whiskey), ~t~st in ~t~inln~ t~ ~ism(s) ~ibte ~ t~ eff~s of c~ at~ cx~um ~ m ~s of DNA mpMr. have ~gon to a~ss t~ eW~ of a~te ~ %tn~" tlc~! ex~ on mare. ~llm DNA ~;r. Tow~ this ~. ~ ~ ~ i~ibiti~ or DHA methyhransferase (MGMT) by a single int~iton.] injection of 30% ~an~ in ~ult male c~ ~. ~is ~bibit~ I~ ~ ~ le~t 24 hr. tmt~ nts. ~ fi~i~ or ethel's eff~t ~ MO~ ~ivity i~t ~ts imp]ks a ~al ~t of MG~ DNA ~ir m~. ~s ~]y ~en all~ to in ~t ~a~h. Wil~ III. D. M.. Tentier, 3. $.. ~y. I. P.. Wit~. T. M.. and Kelley. M. Alcoholism: Clinical and Experimental Research ~r su~: Hnti~l Inxtitute~ of Health. ~om I~ Dew.mona of ~dlatr;cs, Section of ~iatrie ~nd~rinolo~. We]l~ Molecular B~l~y. l~iana Univeniw M~ica) Sch~l,/ndlana~lle. IN. and Mol~ul~ Bi~ogy ~m and ~t of Bi~mit~. ~ola M~al ~. Mayw~. THE TOXICITY OF AZIDOTHYMIDINE (AZT) ON HUMAN AND ANIMAL CELLS IN CULTURE AT CONCENTRATIONS USED FOR ANTIV|I~.AL THERAPY AZT. • chain lerminator of DNA synthesis originally developed for oh•moOr. al~, is new pre~hed as ~ an~muman immu._n~x~erieie~ v~ (HIV) ~ .a ~0 to 1500 m~person/day, which corresponds tO ~0 tO 60 p.M A,~'. ~ l~uman ~ is ba.,~d on a study by the manufacturer of the drug and their collaborolot~t, whlch • •potted in 19S6 that the inhibitmy do~ for HI',/t~epticatlon was 0.0~ to 0.$ A~" and that for human T.cellt was 2(X)0 to 20,000 times basher, i.e. 1000 ,A~T. This suggested that HIV ceoM be safely inhibited in humans st 20 to ~0 A~I". However. after the llcen~ing of AZT as an anti-HtY drag. aeveral studies gapEr, ted 20 to 1000-fuld lower |nhib~tm7 do~'s of A~'T for hu~m and real cells than did the manufacturer's study, ranging fr~ I to ~0 p,~. In with this. life threatening toxic effects were reported in hurmms txe~tod with ~ at 20 to ~0 I~M. "rhe~fure. ,we have re-examined the growth inhibitor7 do~s of ~ for the human CEM T-cell llne and sever•| other human augl animal cells. It was f91
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0 roond that at I0 ~M and 25 p,M AZT. all cells are inhibited at least ~ afl~ 6 to 12 ~l, ~ ~tw~n 20~ l~ •fief 38 to48 da~. U~x~tedly, v~i~ts of all A~, at tM ~p ~ as ~ anti-HIV dm~, is highly toxic to human c~lis, ~iu, D, T, z~ ~e~rg, P. !!, ~ su~: ~vlte ~ti~s. F~m the ~pu~ment of Mol~ufar and Cell Biology, ~niversit~ of California, Be~e~. CA. H[V AS A SURROGATE MARKER FOR DRUG USE: A RE-ANALYSIS OF THE SAN FRANCISCO MEN'S HEALTH STUDY Our analysis ofdn~ use and mmbldlty dilil from a cohort or 1034 men yields the following to•ells: I) HIV iofecilen is a slro~g indicator of drag use--HIV.positive respondents reix~nd an avera~ lifetime dose of recreational dmgr~ (excluding marb jut~0 2.3 limes higher tha~ HlV-ucgntlve re.qxmdents. 2) llomosexuallty is indic•lot of drag eso--hommexual respondents relmcted an average lifetime dose 2.0 tim~ h~q' lhan hetero~extud rests. 3) The incidence ¢d" AIDS-definlng dis- ~ w~ not limited to re•farad•his infected wah HIV. but was almost cornpl,,~ely limited (~5) to respondents who refx~ted usir~ drugs. We also addrcss • IXeViOUS report (A~cher e~ ~!.. 1993} that was bused on the ~me datab~ and ixa'lx~ed to show that HIV alone oofselales with the deveinfxnefll of AIDS. Specif'ically, we show Ihel the ~laticmship between HIV infection and CIM+ T Cell fo~,t is weaker Ihm relXWted by A~' a eL., md provides little evkle~e for support the hylxxhas|s that Iong.lerm. habitual drag use can cause Ihe condillOnS lu~wfl as AIDS {indei~'Menl of the pretence of HIV), and refute the hytxahesis that HIV irene came• these enndidons independent of drug use. Ellison. IL J., Downey. A. Genetka 95:165-171. 1995. Other suplxm: IA'ivate Donations. Prom the Department of Molecular and Cell Biology, University of California, Berkeley, CA and EECS Computer Science Division, University of California. Berkeky, CA. ATTBNUATION OF AGONIST-INDUCED DESENSITIZATION OF THE RAT SUIB,,TrANC~ P RECEPTOR BY MICROINJEC'rlON OF INOSITOL PENTAKI$- AND H~XAKISPHOSPHATE$ IN XENOPUS LAEVIS OOCYTES R~ently. inosltol hex~isphosphate {phyiic Icid) was shown to blnd to photore- 192 • eplor iweltln Ind bl~k its intelllll with dldllltn, lleh In inllillllen lalihl ~t~d_, act that. tnmk.ol pol~l~ld~es could Ilt~" O I~otein<x~l}led s'~4or dinilii- tiff.-IO inveltlgile the pelslble miel of higher lenltlol lot desemlilzitlon, we have expresand the tat su1~tmc:e P oocyies. 'rhe fumllonxl expmuion of sutllancu P receFlor wal monite~d by yogi. age-clamp recording of sub•lance P.induced C'/l,l-dependem CI- eummtl. When control oucyles were silmulaled wilh sulltlllce P (30 hilt), •tier 10 rain er w~tns the second responses if sub•lance P were Ippmxtmalely 15% or Iha tint alp•ales. Cyiosolic injeclien of. ino~ilol peniaklsphosphaic (100 lllvl) or Inotttol htxakisiitio~. phule (100 llM) Inhibiled the I'eduction of the ~ substance P.indueed curt•hi responses, maim•thing the second r~spenses if 57-58% of the inili•l reilpensei. '1'he protective elTecls of inosilol pentakisphosphale and inesilol hexaki|phelphale agalnsl a$oilisl.lnduced desensitization were cone•nit•lien and time lllptndtni and structurally specific, tn thai inellioi hexlsulf•ie lind lnolllol Iril- lnd tetriklallhnl- pinlle isomers were Inactive. Microln.iectien of. Inositol hexikispholphilte change Ihe polen~y of. sub•since P ol" Ihe ~'nsltivlty of" the exlnialed Itlbatanel P receplor if substance P, (h) Inhtbii 12-O-lelradecinoylphoibol-13-•etlali.indtic, id loss of" suhslance P-induced current responses, or (c) aller the curt•his ill•lied by micml.n.~'l, ion or inositol.!.4.5-1rlsphosphnle. These I~sulls luggelll Ihal Inelitol penlaklsphc~phile and Inos~Iol hexxklspholphate have speclrle Inhtbllory Ifllell en the agonisl.i_.nduced loss or rpspo~., slven~ss .of" Ihe rl ~ul~l .~c~ these pmtecllve efTecis or inos~iol hexaklsphosphai¢ ilainli clelenslitzltion wen • hlo ohserved wilh Ihe enclolenoua iysopho~phalldlc ncld/pholphatldl~ a~ld rililpior, indieallng that lhis mechanism is not specific to ecloplc recepiori. "rheie rtaulta Sail. lesl that inosilol Fenlaklsl~hosphale and inosilol hexllkispho~h-il may he novel I~rm~olellkal tools for she tedy of atonisl-lnduecd delensitilalion. ~lkawa, ri., A-fleas. i. E., Sherlf", M., and llenlly, M. R. Other luppml: Nalional lnllilUlei of Heullh. I~m the Depa~lmeot of Biological Chemislry, Seheol or Medicine, Univinlly of Callfomil ii Davls, Diti~. ~A. EVALUAT/ON OF CALCIUM INFLUX FACTORS F~ROM ~IMULAT~D JURKATT-LYMPH~ BY MICROINJECTION INTO XF.dVOPU$ Acid extracts of" Ihepsigargin-acfiva~ed lurkat cells have be~n shewn to hays lair•cellular activity in inducing a dose-dependent rapid chloride mlcrolnjectlen in Xenopus taevis focus. The exzr~cls act by eiev~zion or thro,r.h cuicium ¢,w/. The f~or~) res~siblo rer this ~iv~y h~ve cMclum influx factor (CIF) and have been fomgl tO he small, t~lativoly I~l mel~- culcs (< 1000 dallcms) Whose activity i~ abolished by alkaline pbuephatale t~at-manl and IX~enliated by co-lnjecllon of ~adaic acid (I laeteln phmphata~e Inh~blie0. 193
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is produced in • lime-clapt•deal m~nner following thopsigargin trestment of JuAst cells, beln$ first elevated •bore Ixe~l kvels by 2 rain. lmr~ellul~ CIF Icllvi~y is completely ab~m horn HG115-4011- neuronal cells, which I~ck. c~pacitative ent~. On Ih|$ b~is. il aPlxmrs that Ju~at cells. ~dvmed by sdmu]i Ih~ ~plele inlemal c~l¢ium sto~s, p~e one or m~ CIF ~c~ivilies Klln~ imr~llulxrly, end X~ ~ may ~ s ~1 t~! to ~fy and c~efi~ ~Fs. ~m~. D. ~ Healey, M. R. ~ Jo~al of Biolo~cM ~emi~t~ 27~!2):~29-~32. Ma~h 24. 1~5. ~r su~ Nstim~ Instilules of Hesllh, F~ t~ ~nment of B~logi~l ~emi~t~. Unive~ily of Callf~ia ~h~] of MKki~. ~vis. CHROMATOGRAPHIC RESOLUTION OF AN INTRACELLULAR CALCIUM INFLUX FACTOR FROM THAI~IGARGIN-ACFIVATED |URKAT CELLS: |VID~N¢I P¢~ MULTIPLE AC~IVtTIES INFI.UF.NCING CALCIUM ELEVA'I]O~q IN XF2COPU$ Acid extracts of thapsig~rgln4timulsted Jurkat cells revealed holh intracellulm" and extniodlular activities slimulating C~'-dependent (21- oust, ms on Xenopus lacvis ~y~es. Chromatographic frx~km~inn of thai• exit•eta on gel filtration se~ two ~ttve fractions of M, approximately 6(30 and 400. Mm~over. the M, 6UO fra~im • xhibited both imracellelar and exit•cellular activities, llowevef, the lair•cellular letivily was ab~n! from exlracls of onstknel•tcd $u~at cells, sogge~ing if• imxluo. lion wts stimulated by lhalYsigatgin. The further purificatio~ of this fraction by high peff~an~ d~in |ayef chromatography reso|ved • st•fie fraction wh~h w~ let|~e only on mk:rolnjection and whkh required calcium entry for activation of cuvrem mponse~ These msuhs sugge~K thin • single 8mhemi¢ c~kiem influx ~ can be res~ved by pudl'tca~ from confounding activities detected in crude acid extract.*,. Kim. H. Y~. Thomas, D., md Hsnley, M. R. The Journal of Biologicul Chemlsu~ 27~17):970~-970~. AlXfl 28. 1995. O~bor zul~x~: Nmleml Institutes cA" Health. ~ the Del~ment of Biological Chemistry. School of M~d~cine. Unlver6ty of C~lifomi•. Davit. CA. PHENCYCLIDINE INHIBITS EPINEPHRINE.STIMULATED PLATELET AGORBOATION INDEPENDEHTLY OF HiGH AFFINITY N.METHYL.D- ASPARTA'TE (NMDA).TYPE GLUTAMATERECEPTOR$ The p~ycho~omimetic analgesic phencyclkline (P~P), which binds to • high 194 affinity site on the neuronal N-methyl-D-s~panate (NMDA).sensltiv• ~lutamate recepl~, has ~vi~sly ~n f~ tO b~ to plat•lets with high Mflmty a~ to s~ally ~1~ t~ ~ of ~t~imul~ ~el~ a~ el el. (1~2) Bi~m. Y. ~5, 35-39). We h~ ~ ~ ~t I~k ~ ef ~i~ aWmil~ of 14 s~l~ic ~P snails al ~ high ~n~y bindin~ ~lt¢ m ~s, ~ ~at !~ M~h a~nily ~ bi~ip~ site in ~el~s Is dl~t ~ I~ ~tl ~MDA ~or. ~ ~r of ~y of six of I~ aaMof ~ delaying f~ on~et of epi~r~-stimulated pla~ ~atim al~ did n~ ~!- ~I I~ nnk ~r of I~ir ~nd~g a~nlt~s f~ ~met~ ~ ~in bl~g tiles. ~ d~a i~i~*e that t~ a~l;ly of ~P ~[ogs ~ inhibi~ e~m.stimel8~ ~. gmlm is ~ ~lat~ to their •Silty to bi~ m t~ hi~ a~nity plat•let ~ ~1~ site. Fu~. (+)MK-~I. whi~ bin~ to t~ n~ h[~ #~nlty ~iog ~tte in n~s as d~s ~P, fail~l Io inhi~l epi~hHn~timulat~ plelelel ~ti~n. fu~ su~sli~g Ih~[ I~ silt M which ~ ~ls in plme{~s i~ ~ ~la[~l m ~ NMDA-Iy~ glul0mate ~pt~. ~nher stoics show~ that 5-~ ~pt~ ~d cff~s ~ platel~ ~lim a~ ~ Involv~ in ~-~d inhibiti~ of epl~ ~ri~-~duced plat•let ~t~ of M~inal ~emltl~. Nail•el l~ilule or via~ ~ DiFsd~ ~d K~y D~ ~a, MD. 3'-AZIUOTHYMIDINE (ZIDOVUDINE) INHIBITS OLYCOSYLATIOH AND DRAMATICALLY ALTERS OLYCOSPHINGOL1PID SYN'DI~lS IN WHOLB ~T~J.LS AT CLJNICALLY RELEVANT CONCF_3~TRATIONS Re~.m in t~tro work with Ge~gi-eru~hed membranes •bowed that 3'.4~klethymt- dine.$'-m~phme (AZTMP), the prlmmy intracellulat" mel•bollte dothymldlne (A2T). is • potent inhibiter of glye0eylttkm reaetkms (Hall tq' .7. B/~. C~m. 269. 143.~-1435g) and Ixndk:u~d dm AZ'T treatment of wlmk cells should ~ sb~ilm" inhibition, in thil n~x~, we vori~ this predictinn by slmwi~g that treatmcnt of K562 cell• with AZT inhibits lipid and lxoteln glyco~ylation, lag pngliceide synthesis at clink:ally ~,levant co•carat•dons ( I-5 I~). and suppmsml demenslrate that these ch~x~s do net result from nonsp~:ifk: effects on either the scereWry apparatus or prmein synthesis. On t~e othcr h~f. stud{e• using isolator nuclei as • model system fer clmxnmomal DHA p~icatk~ show that ~ Is a very weak inhibitor of DNA s3mthesls. ~ obscrvMk)ns stronl~ly su~/~tt thlit the myelo~uppressive effects of AZT' in ~'~.o m dua to inhibitk~ of IX~in and or IVpld glycol•don and n~ to efrcets on chmmosema! DNA replk~i~. ?'an. $.-P.. llsley. D. D.. Frohflck, C.. Steel. R.. Hail. E. T.. Kuchf•, R. D. and Melanc~n. P. 195
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0 On The Journal of Biological Chemistry 270(39):22836-22~4 I. Sci~cmh~r 29. 1995. Other supf~)r~: National Instltutes of Health ~nd American Cancer Society. From the Oep~nment or Chemistry and Biochemistry. University of Colorado. NICOTINE EFFECTS ON PRF_~YNAPTIC RECEPTOR INTERACTIONS i~'esyneptic regulation or neurotransmitter release in the brain inHucnces the ~ount 0f ~urotransmilt~" released. This may m may not del~nd on mrve activity. ~ c~n ~ modulated by other f~etors that influence the membrane polential or the nerve ending. The neurotr~nsm~or itselr can inhibit its own release by ~ctlng on p~e~|plk: ~torecep~ors. Othc~r transmitters can also ~ct presynaptically to mndu- I~e Imnsmiltor outflow. The ten,laxity ~nd diversity of such modul~{~'y mecha- nisms of r~le~se here received n~ch attention. We were particularly interc~ed in recep~of~ceceplor interactions influenzal by Ixtrticip~tion of nicotine in the ceotrel actions of ni,~line involving pre~y~alxlc effects. "the mec:l~nlsms of these reCel~¢W-recep~¢: inlur~,'tlons haw hecn di~u~ced reeemfy. In receptor heteroregulation, neuro~ransmitters or neuron~dulator~, hy bindln8 ~o reeq~xs on the he,real membrane, may he d~le ~o resulste the terist|es ~nd function of the ~cosnition si~es of another transmitter or modulator recei~or. Whemss autoreceptm meeSanlsms ~egufate the sensitivity of a receptor and are modulated by the levels of its tigand 15rough positive or n0~ative feedback heleroreceptor mechanisms in~lud~ direct or iedimcl interact|o,s Ix:lweon dill"cant receptors ~nd transmitters, involvin$ the plasma membrane, intr~cytop1asm*tic Ioo1~. or inter~io,s m the nuclear level. Sershen. H.. 'roth. ~... L~tha. A.+ and Vixi. E, $. Anmils of the New York Ac~dem~ or Sciences ?~?:23~-244. May I0. 199~, ~ the N.S. Kllne Institute for Psychiatric Reseamh. Center for Heurochemlst~. N~/ROCIIEMICAL EVIDENCE OF HETEROOENEITY OF PRESYNAPTIC AND SOMATODENDRITIC NICOTINIC ACETYLCIlOLINE REC~.PTORS We de~,ribe su.,dles of the elTec~s oi" different nAChR ~,onlsts on the release of dilTeren~ tr~mm~er~ ~nd ~he modul~ ale of presym~ic nicotinic n~eptms in ehemk~! signal ~rmsmissicm. In ~ldkion. using difTemnt nAChR ~,onlm and ant~- 196 onlsts we attempted to eharacterlze the presyna~ic nAChRs locatod or~ the noradren. ergic axo~ terminals in the hi~x~csmpus and yes deferens and the nAC'hRs located on the myemeric plexus. The advantage or relea~ ~tudles is that one c~n he certain whe~ the t~nsmJttor comes from, ~nd thus elm 1o¢8¢@ the Ilt¢ or ~¢tioo of diHerent nAChR ~onists lind ~mt~cmlsts, m~klng the ehlrac~tk~n the rC~l~ors mher Vi~'|, E, S,o Sershen, ||,. Balll, A,, ~i~e, A** Windisch, K., Jur~y|, /~jtha, A. Annals of the New York Ac~emy of Settees 757:~4-99, M~, lO, 1995. Other sul~: Hun~m Rese~.-h Fund (OTKA). From the Department of Pharmacology, institute or Experlmental Msd|elne. Hungarian Academy or Sclcnccs, Buda~st, llunpr?, ~nd ~he Nath|~ $, Institute for ~Tch|at~:: Res~ch, (~en~cr for ~Koch~nis~o Orl~l~t~n~, SUBTYPe.SPECIFICITY OF THE P~ESYNAFT~C afADRBNOC~TORg MODUI.ATINO I IIPf~OCAMPAI. NOREPINEPI tRINE RELI~$B IN RAT In ~vn Ix~in microdlalysls ~nd hi~h-I~'fmn~nce Iklu~d ¢hrom~toSrephy with ele~ttrochemlcal detectio~ were used to study the effect of different ~lt~tive af mtt~onlsts on hilqxx~ml~! norepln~n~ (NE) release in fl~ely morin~ aw~k¢ Symmic ~dmlnistretion (0.5 m~ |.p.) of either ~ a~,-~e~i~ BRL &M0~ er the a~o-~tagonist ARC 239 did not si~niflc~otly ehan~ Ihe I~1 release of NI~. At • higher dose {5 mg/kg |.p.) ARC 239 w~s still ineffective. ~ BRL 4440~ caused a significant increase of the extr~cllul~" level of NE. $imil~ ~ults were o1~ained from in vitro pcrfu~ion exFerim~ts. R~ hips~c=mpni slices were k~ded with [~H]NE and the elcctrlcal stimulation.evoked reka~e of |~i|NI~ wu doter. mi~ed. ~ ~.~nt~,onists were a~lied in a eo~centmion ren~e of 10" to 10"* M. ARC 239 w~s ineffective, where~s BR[. 4440~ si~nlf'~n~ly ir~rel~d the electrle~. ly induced rele~e of ['H]NE. In ~ with ~e dma of n~x~lialy~is ~ per~. slon experiments. BR~ 4440~ dlspl~'ed [)HlYohimblne from hlppoc~mpel c~l membranes of r~t br~in with hi&h affinity whemu ARC 2~9 was The pK, values of eight dill"urine w~dnmer~ie comFoueds showed a very ~ ear. relatio~ (r = 0.98, slope ,, I.I I P < 0.0001) in h~p~Impus ~d ~n~tl the c~-~drcmoc~ors have ~ ctm'~,r~d ~ e~ubeype. Our hippocampal NE ~h',~se in m is reBel•led by c~4drc~x:~ors, a tpeci~ vadatkm of the human ~z~t-subzype. Kiss, L P., Z~illa, O., Mike. A.. 2elias, T., Tolh, E., LaJtlm, A., and Vi~i. Braln'Resea~ch 674:238-244, 199~. Other suppozl: Hungarian Reseaxch Fund and from OMFB. Front the Depmlmen! of Pharmacology, Institute of Experimenlal Med[elt~, Heagarian Academy o~' Selenees, Budapest, Hungary, and Center for Neumehemlstt% N.$. Kline In~tltele for Psyehlatr~ Rezemh. Om~f, elm~. 19"/
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DETERMINANTS OF %oADRENERGIC RECEPTOR ACTIVATION OF G PROTEINS: EVIDENCE FOR A PRECOUPLED RECEPTOR/G PROTEIN STATE w The abil.it.y of a~. ist-OC~, upi~_ e%-adrenergic rece~ors to activate G preteins -~, "t Immm~n~-;~ "¢z-t.~l"~pnin}ffipnosphate (i"SlGTI~S) binding as an endpolnt. Epinephrine (EPI) stimulated I"$1GTP'y$ binding in a Mg"-dcpendent manner. ~howing Ixxh mictomolar and millimolar cation affinities, Prior trcalment of cells with pertusds toxin ¢oraplmely eliminated the EPI stimulation. The ixe~,nce of GDP ~ bm! I"~IOI"PIS binding and intrepid the Ixoponion or EPl-slimulated e.ec( on l~f'i*stlmumted binding, such that the agoni~t re~pon.~ w~B !lone!ely greater at higher Na" levels. In saturation binding studies with me a(~'~¢ ol ~ a h~$h a~nlty ¢o1111xx1¢~ was also present, indicating a Na'regu- I.ited I'~¢¢1!~¢~G pr~eln interaction. EPI induced high a~nky |"S}G1"~$ h;ndlng in ~ l~ence of Ha* Ind i~e¢~l the a~ni~y of the high affinity component under N|'-f~ Olmd{li~s, ~ Iclecllve am-edrener~ic ant~on{~t rauwof~ine produced rightward shifts of EP! dose-~esponse curves and decreased the basal level of [~SJGTP~ binding ~¢ro~ the sane r~ge or ¢o~-emratlom. ~ extent or ¢lecreasc w,a~ de~,ndent upon the e~.~-ndfe~ergic receptor expretdon level, i~lic~[ing that ~m reee~o~ ¢omrib~e Io b~at G lxetein acdvation in the alsace of hist. The abiltly of raewolscine Io decrease b~ll |~$1~ binding was dimin- ished u the level of Na" was iacreasnd, suggesting that bo0t agems act to reduce mgeplor/G protein interacllon, by distinedve mechanisms. ~x,-Adrenergic recepto¢ anullonittt reduond basal G preteln agdvatlon with a rank o~er ror maximal effec. dvel~l~ that wu dlf~rent from their recepto~ binding af~niti~s. These r~-~ults port ~e exi~tenee of precoupllng I~Jw¢on ~-a~rer~r~ic rc~plor~ and G pmtclnz: coup mg tin I~ diminished by both Na" and antz~oo|sls" whereas agonists increase fhe elTzcigw~ of re~lxor]G prozeln coupling. Tiin, W.-N. Duzic, E, M,, Lanler~ S. M. and D~th, R, C. Molecular Phan~acofogy 45:$24-$3 !, 1994. Other lupl~a: U.S. Public Health Service. From the Dqxtrtment of Phtrmaceutkel $ciefges. No~heastem University. Boston. MA. and D~erlmont of Ce~l Molecular Phetmacolngy. Medi¢a! University of Sooth Carolina. Chadeslm, $C. USE OF !ii(}!4 API:INITY. RADIOIODINATED PROBES FOR IDENTI~CA TION OF IMIDAZ(X,INE/GU^NIDINIUM RECEPTIVE SITES 'Vm'le~ ldmmra¢olnglcally active compounds with In imidm,.ollne or guInidin- lure nl~i~y a~e m0ngni~ed by membrane bound Ix~eins that app~r strtgturally and finetionllly dL..llnct From konvm hormone recelxogs. Such entitic~ are termed imida- soline binding sites, I reap!ors, or imidlzoline/guanidinium receptive sites (IGR$). t9g To facilitate the aden!if!cat;on and sm.~'to~l ana!ysis of IGR$, we develel~d dowillzed molecular prohe~ exhibiting high |ffinily and lelectivity for I(31[$. The molecular IXO~s a~ structurally related to eirazolk~, an imida~line that high affinity for IGRS in bo(h central and peripheral tissues. The t~tmnt moleeu~a 2- [3-aminophenoxy]mcthyl imidaxoline was radioindinatnd to yield 2.[34mlno-4. |*~|| iedophenoxy~thyl imklaxollne ('~I-AMIPl), which was used to identify IGR$ in brain and peripheral listens. '~'I.AMlP! was co~verled to the photminah|v~ aryla~d~ derivative ("I-AMlPl) and n~d Io ideally Ihe M, or the liBand subunlt of IGR$ in various tissues including lxaln, pancreas, kidney, and liver. The results of these studies indicate that them are muhlpl¢ binding pmtelns for them molecules that dafter in Ihelr apparent molecular weight, ti~ue diatdbut|ofh intratb. stm Ioc0tlm and li~nd z~ccof~ition l.anler, ~. M. t.~nicr. B., Bakthavachalam. V., McGrl~h. C. R.. and Ngamey~r. J. Annal~ of the New York Academy of Sciences 763:10~ l I I. July 12. I Other smppo~: National |nstitulcs of Health and the Medical Un|~ers|ty of Carolina. From the Deplrtment of Phannaeolngy. Medical University of ~outh Cmollna, Chadeslon. $C. and Reseagh glochemicals International. Nadck. MA. NICOTINE EFFECTS AND WORKING MEMORY PERFORMANCE Nicotine and other nicotinic d~up may have a variely of therapeutic uses. Cognitive enhancemenl is one effe~! of .leo!inn which show~ promise for the mcnt of disease slates" Studie~ from our laboratory" and e4~rs have shown that fine !alcoves cognitive performance. Improvements in tests of attemiv~mas Ind memory in humans, monkeys and tats hevn been found in many but not all We hsve co~tducted a scrlcs of" experiments examining nic~ine and nicotinic nlst effects on rats in working memory task in the redial4nn ma~e and T..maz& Chn~lc subcutaneous nico(ine inl'usi0o cons!sternly ira.paves choice a~uragy of normal adull rats in the win-shift woAing memory verston of the radlal4tm task. This chronic nlcc4iue exposure also at(eneat~ the monm~y h~hrlclts in am~ maze choice accuracy eaured by lesion~ to ixojecdons of the ppecampue and the basal fmebrain, two areas alTecled in Alzheimer's dbea~. The nk~xln~tnduc~d improvement was robust in the face or bmh a~onisl md antagonist ehelkn~ge with doptmlnerg~ o'mp, In contrast the nine chm~ic nic~ine adminlstmleo dkl ~ nifcantly alter choice aceuracy in n T.m|ze spatkal altematkm wefklng task. This lank of efl'¢~"y of n'~ine in Ihe T.mase t~'k may have been due to the greater Imlx~ance of prooclive interference inherent in this lask. Nkotine or ether nigetini¢ agoni~ls may he userul treatm~ll$ for Alzheimeds dlteaute and ether syn- dfomes chafacteriT~d by cognitive iml~drment. Levin, ~ O. 199
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0 0 In: Rc~,~l Advances in Tobac'~o Science, Volume 20. Proceedings of a Symposium Presenled at the 48th M~ti~ of the Tobacco ~emists' Resea~h C~fe~e, O~s~, NC, ~r 2~-~. I~. ~ ~: R. ]. Re~ds. Alz~imer's F~n~ati~. ~d the Nat~nl F~ t~ N~um~havi~l R~a~h ~1o~. ~pmmont nr Psychlat~, Uni~nlty M~i~l ~nter, ~am. NC. WORKING MEMORY PERFORMANCE AND CIIOLINERGIC EFFECTS IN TIlE VENTRAL TEGMENTAL AREA AND SUBSTANTIA NIGRA The nlcollnic anlagrmist me~amylamlne has been h~und to im~ir working memor,j perFormanc:e i, the radial.ann maxe (RAM) after s~. or i.c.v. ~dmini.~m. ticm. Mecamylumine has important interactions with dopaminergic (DA) systems. Meo~mylamin¢-induced memo~j dehcits in the RAM are potentated by the D~ amlBoeist raclolxldc and reversed by the D~ aBonist quinp;m~e. The eico~inic ~ nist nicotine Im.s been found to improve wodcin~ memm), pcrh~rnance in the RAM |her I.e. or i.e.v. •clminislr~tion. Nicotine-induced rnemor~ improvement in the P~M is polentiated by Ihe D~ a~onist quinpirolc. The midhrain DA nuclei. Ihe sub- rand• ,il~ra ($N) and Ihe ventral telmentul area (VTA) hive relatively dense con. oentmion~ of nicotinic receptors which may ~ erid~•l si~es or action for mecamp famine and nk~ine. In the ©unbent stud),, Ihe elTects or mecamylamine (I, 3.3 and I0 p.B/~ide) infusions into the SN (~ = 12) •nd VTA (~ = 13) on working memo~ in the radial.ann maze were examined in adult female Spr~ue-Duwley rats. The IO- p.~/dde dose el" mectmylamine siBnifi¢•ntly impaired radlal.arm mu.e workinB memo~ peal'romance when infused imo either the SN or VTA. No sisnil'|e,'mt effecL,~ OF mecamylamine on resFonse l,,tency were seen. The nk:ocinic ,,~'mlsts cytisine (O.I, 0.33 ~ |.0 p.~de) and nic~ine (0.3. I.O and 3.3 F~ide) were admlnislen~l in • o0umerb~lanced ordm'. The hash dose of cTtislne (I p.W~ide) nearly caused • sicnillc•m d©~¢it in choice ~racy. Nic~ine slightly depres~d choice uccu~cy but not si~i~¢md~ in this s~ndy. The interaction of nicofir~ and mecamylamlne was the~ studied. A dose of 1.0 is.~/side of nicotine ¢•u~d a si~nil'~ant decrease in ehoi~ aeotaracy. Inte~estir~ly, Ibis was si~nif~amly ~versed by • 3.3-1x~ai~ dose ~r m~unylumine. Studies of the musc•rinic •nt~onist scopolamine (!, 3.3 and IO p~'skb) and {he mus~,ink: agonlst piloc~ne (3, IO and 30~c~ide) did no~ detect sf,~ai~atu citers cm RAM cheice accuracy. These data sapporl the involvement of nicodnk: Inne~ition of the midbrain DA n~clei in memory Function. [~v|n, ~,. D., B~i~, $. J.. Chrlslopher, N. C.. and Auman, ]. T. Brain Rebirth 657:165-170, 1994. Other suppo~: N•tional Science Foundalion. From the Neumbeh,vioral Re~e•reh L.~h~ratory. Oep',rlment of Psych;atnd, Duke Univerli~ Medical Center. Durham, NC. 2O0 NICOTINE EFFECTS ON MEMORY PERFORMANC~ In: Clnrke. P,, ~uik, M,. Biol~al Systems If. Advn~ Oth~ su~: R, ~. Reynolds, Alz~imcr's A~iat~, ~d t~ Foundati~. ~ t~ ~m~ts o£ P~chiat~ a~ ~ol~y, ~ke Unlvo~lty M~I~I ~, ~am, NC, ACUTE AND CHRONIC NICOTINIC INTERACTIONS WXTH DOPAMINB SYSTEMS AND WORKING MEMORY PERFORMANCE Nicotine h~s I~cn found to improve memo~ rats. monkeys, and humans. I~tc~ctiees of nicotinic sy~cm~ with depemine (DA) system my ~ im~ant f~ this eff~t. We ~ m ~s ~ st~s ~ nl~ tin~ aghast a~ animist ~t~ with DA s~t~s ~i~ m~ ~ x win4hi~ ~ing ~ task in t~ plmfc. N~i~ also h~ ~i~ ~ ~ ~ ~ ~ial-~ ~ ~ DA ~t ~1~ d~ not by itself imp~ve r~iai-a~ ma~ mcmo~ ~rfo~ance, but when 8iven t~et~ with ni~i~ it ~ an elevat~ d~~ i~ in c~i~ ~y. N~i~ w~ e~i~ nk~ine, t~ D~s ~lst quinpimlc im~v~ RAM ~utc n~inic mani~fatlo~ c5~ic ni~tine ~ministrati~ cxamp~ or the diff~nt ~uml su~tmtes that und~lie the mcm~ im~vemont ~u~ ~ ~utc a~ ch~ic nixie. 201
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0 Annals of the New York Academy of Sciences 757:24:5.252, May It). 1995. From the Department of Psychiatry. Duke University Medical Center Research Service. and VA Medical Center. Durham. NC. EFFECTS OF RETINAL LE$1ONS UPON THE DISTRIBUTION OF NICOTINIC ACWI'YLCHOLINE RECEPTOR SUBUNITS IN THE CHICK VISUAL SYSTEM lmmunohistocbemistry was used in this study to evaluate the effects or retinal ksions upon the distribution or neuronal nicotinic: acetyicholine receptor subunits in the chick visual system. Following unilateral retinal lesions, the neuropil staining with an antibody against the p2 receptor subunil, a major cnmpnnent Of ct-bungam. toxin-insensitive nicotinic receptors, was dramatically rcducnd or completely elimi- nated in all of the ¢motralateral retinoreclpient stmClUres. On the o~her hand. neuropil miniog wilh antibodies against two a-hengarotoxin-sensillve receptor subunils. a~ ¢t~. wa..g only slightly affecled after retinal icxions. I~'crea..~cd neumpll for (ot?.lik¢ immunoreactivlty ~ only observed in the nucleus or the basal optic ~¢and layers 2.4 ..and_ 7 of the optic lecture. I~ erR-like immunnreactivlty, slight re:lion of neuropd staining could be observnd in Ihe ventral genieulate complex. srtseum lncti, nucleus lateralis anterim', nocleu~ lentffonnls mesencephali, layers 4 and 7 or ihe lecture, and nucleus supracbiasmatk:uz. Taken tngelher with data on the localiution of nicotinic rocepto~ in the retina, the pre~n¢ resuhs indi. cole that the ~2 subunit is transported £mm retinal ganglion cells to their centad tar- gels. whereas the el? and at~ subunil immuonreaclivlty appears to have a central era. Sin. The source of" Ih~e immunoreactivilic~ could be. at leaSl in pa~. Ihe .qalned pedkerya that were observed te contain el7 and ag sobunils in all relinorecipient crees. In agreemenl with Ihls hypothesis, the 1~2 subunit of" the nicotinic acetyl- choline receptors was no¢ frequently found in perikarya of the same areas. Britto, L. R. G., Torrao, A. $.. Hamzssaki-Brhto. D. E.. Mpo~ozls. J.. Key~r, K. T., Lindstrom, J. M., and Karten. H. J. The Journal of Comparative Neurology 3:50:473484. 1994. Other support: National Institutes of" Health, Council for Smokeless Tobacco Rese~'~h, Muscular Dystrophy Association, California "~pbacco-Related Disease Rcsea~'h Program. FAPE~P and CNPq (Brazil). and Fon~ecyt (Chile). " Prom L~c D~pwlrncnts of Physiology and Biof~hysics, Department or llistology and Embryology, Institute of Biomedical Sciences. University of Sao Paulo, Sao Paulo SP. Brazil, l~'patlment of Biolog3r. College of Sciences, University of Chile, Santiago, Chile, I~partm~t of Neurosciencea. Unive~ity of California San D~..go, ~a ./olla, CA, and Oep~meot of Neuroscience, University of Pennsylvania, Philadelphia. PA. NEURONAL NICOTINIC RECEPTOR STRUCTURE AND FUNCTION T~ best characteriznd suhtyp~ from the branch ofthc neuronal nicotinie reecp- 202 tot" (nAChR) gene family which does not bind et.bunprotoxio has the se~nlt stoi. chinmotry (a4)~(p2), and accounts for >90~ of the high affinity nieotin~ blndln$ in mammalian brains. Clwonic treatment of cells ttansfect~! with a4j12 ACh~ an increase in the amount or AChR wldl phermacologie~d cher~stl~z and a time cour*..e that parallel the nlco¢ine-indu©ed increase in I~aln a41~2 AChRs, Thi~ is the result of a decrease in turnover of. ~ffaee AChRs. Many of these surfac~ AChRs permanently functionally inactive. The wedomlnant sut~ype of the t~eneh of. the neuronal AChR gene family which binds a.bungaxolo~dn (a-BTX) corollas a7 sub. units, cO AChRs predominate in brain, while ag ACHRs predominate in retina, and aTctg AChRs are a minor component in both tissues, aT and ag homomert have similar cbannel properties, but aS homomers and native ag AC[IRs haw affinity for a-BTX and higher affinity for small chollnergic ltpnds than do home,nets and a7 AChR.s. The high Ca~ permeability and rapid d¢~¢nsitization of a7 and a8 AChRs may enable them to participate in unusual syneptin mechanisms. Lindstrom..L. Anand. R.. Pens. X.. and Gew.anich. V. In: Effects or Nicotine on Biological Systems !I. Advances in Pharmacological Sclcnccs. Birkh.'mr~.r VeHag. Basel. Swit~.~and. pp. 4:5-~2. 1995. Olher support: National Institutes of Ileatth. MuscuTtr Dystrophy Asgglatlon. and Smokeless ~ Reseorch Council. Inc. From the Departmen¢ of Ncurmclence. Univen~ity of Pennsylvania Medical Phlfmlclphla. PA. cY'rOCHALASIN MODULATION OF NICOTINIC CHOLINERGIC RECEPTOR E.XPRE.~SION AND MUSCARINIC RECT:4~'OR FUNCr/ON IN HUMAN TE671/RD CEILS: A POSSIBLE FUNCTIONAL ROLE OF THE CYTOSKELETON Previous studies have shown that ecfls of the T~671/~D human clonal line express musele-type nioulinh: aectyleholtnc receptors (nAChR) and m3-typa earinlc ~cetyleholine receptors (mAChR) whose numbers and f.unetien am by agonlst treatment and second messenger modulat~n. Hem we show tba~ cyt~:ha- lasln treatment, which cau.,~..s disruption of' aotln n~two~ks, indues marked changa~ in the numbers and distribution of" nAChR, but not mAChR. Moreover, whereon cytochnlastn treatment fails to alter nAChR funclie~ slgntfieamly, i! ~u~ly ales mAChR-mediated phospbulnositlde hydrolysis. Tz~-~zment of 'rE67 I/RD eel,s with different cytnchalasln nnalopes (rank order efficacy at S Ixlhnl is.H C ~ D > A ,,, E) produce~ a two- to fourfold increa~ in ,umher~ of memtwane-t-,oune nAChR (!1... in units or ~peclf',c '~'l.laheled ~.hun~,~mtogin binding per milligra .m of membrane protein), nAChR up-regulation is evlden! after 1.2 days of eytoebal~,,m exposure, is max;real after 3.6 days of drug Ireatment. and is dominated hy an approximately 10-fold increase (per cell) in an intraeellul|r nAChR pool. CytochMasin-lnd,ced nAChR up-regulation is similar in magnltud~ to. ~x~t no( |dril- l;re with. up.regulation of nAChR follewlng chronic exposure to nicotine or ester. Norlhern blot analysis s~ows a four* to five.foM coordinate incr~ in kyats of mRNA that cncnde nAChR (t. ~1, T. or ~ subun~ts in cytochalasin-lreated cells. 2O3
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0 0 su~$esfing that nAChR up-re.~ulatlon has a possible tran~ripfional h~sis. Studies ~ usi~ a "Rb" e~ux &~ay indicate that cy~halx~J~nl h~ ~ si~ifi~nt e~ ~ nA~R r~ti~. By c~t~t, cyt~helasin Ireat~ hoe no ef~ ~ t~ num~ o~ mAWR as as~ss~ by ~nding studie~ with the fadi~ntagonlst fa~l~ qu~nuclidinyl ~n~late, ~t it ind~e~ ma~ked enha~em~! ~ stlmufat~, but not ba~1, ph~pho~iti~ hydrolysis. ~e ~tt~fie~ stt~t that p~um~ m~ulatlnn by cyh~hala~in treatment oF cytoskefetal mlcrofil~ment oF t~ ~la~t~c ~ptnr xu~amily) and nA~R {a pmtmy~ oF t~ li$~. pt~ ~ ~u~amlfy). ~c ~ult~ al~ su~$e~t ~iMe new mte~ ~r su~: Men's ~ Wo~n's ~s o~ ~a~ow Neumlo$ical and ~pi-Hab Ph~nlx. Inc., Ari~na Disease Conlrol Resea~h Commissar, Alcohol, Dm$ Abuse and Mental Health Adminlxtrati~. and t~ ~mokeless • 0~ Rc~h ~il. ~ the Divi~ oF He~olo~y. ~ H~mlo~ieal ln~itme. ~nlx. D|VER:~|TY AND PA~r~RN.~ OF RFJ3ULATION OF NICOtINiC RECEPTOR In ~ ~s we ~xpl~ ~ ~ivc oF nA~R ~ i~al ~ts fm m~ulm~ influx, i~l~in~ ~ ~im~ vii #c~ ~sible ~h~ ~ ~ (I) ~ n~in~ li~ (2) ~ ~ ~e~. (3) ~ ~w~h ~ (4) ~ ~ t~ t~ n~ ~ (S) ~ ~ ~in~ ~ ~mkel~ ~lul~ S~li~ ~isms that f~ nA~R dive~ity an~ mgulato~ pla~lkhy in syn=~ re~llng ~ in play im~l ~1~ in t~ ~Buimi~ or~ ;~1~ fu~t~. Annz~s ofl~ New Yo~ Ac~cmy of ~ie~e~ 757:1~3.1fi~. May I0. I~. 204 Other suppc,l: National Institute on l~g Abuse. National Instttut~ Disorders and Stroke. Smokelesl To~cco Resem~h C~flcil. Arizona C~tml Re~mh C~mls~i~. ~d Ihe A~m ~i~ Di~ ~ the Divis~ of N~bi~y. ~w N~o~al Imtitute, ~nix, 11101 ! AFFINITY BINDING OF TIlE ENTAMOEBA lllSTOLYTICA LI~'I'IN TO POLYVALENT No^CETY LGALACI'OSA MINIDF~ Entnmn~/~x hi.~tnlytlca tmphozoitcs initiate pathogenic colonization by adher- ence to host glycoconjupte.~ via an amebic ~urface I~ctin which binds to IllaCtOl¢ (Oil) and/~,acetylgalactosam;ne (GalNAe] resido~s. Monovafan! ~md muftival~m carbchydr~tc ligands were screened tot inhibition or £./vlsro/.wlee lectin.n~dtsted humzm red nell hemagglutination, revealing that: (1) the synthetic multlvMent v~qly- copro(ein GalNAc,RSA (having sn average 0/'39 OIINAc residues link~cl to bovine ~,rum albumin) was 140.O00.fold more potent m inhlbi~or than nmnovMent and ~00.000.t'old mote potent thnn monovalm Oal; and (il) small symlmi¢ rmalztvs- le.~ li~snds which bind wlth high affnity to d~ mammalian ~l~ti¢ lectln do no4 bind vflh high affinity to the £. Ahlo/~i~o lccti~ Rzdiolill~d ztudie~ re~aled smumble hlndln¢ of '"i-Ga~A¢,,BSA to £. Iii#t~t~tic¢ (K~. :~ I0 ~-- .~ riM. B,~ = 0.9 ----. O.Og pmoVm¢ membrane protein). Mc, dmal required the presence ol'¢Meit~m chloride (~0 p.M) ot ~mdium chloride (~0 aM). I~d h~ • bro~d pH maximum (pH ~). GMNAe~BSA wns 200,O00-foid morn than monova~ent GalNA¢ in bleckin~ radbfiBand hindus. Among synth~k~ ~'he- dde-derivallzed linear polymers, the (3alNAcll and GMNAco3Ga|~ dedvaltves the most potent, with OaINAc~ and Oa|NAcr~3(Fuc(x2)Gal~ derivatives much weaker. The dam suppo~ • mode! i, whi~ a ~ique pattern # ~ multiple GaINAc ~s;dues am the hlghesl affinity ta~e~s fm the E. bi~ro~wlen leetin. Adler. P. Wood. S. J.. Lee. Y. C.. Lee. R. T.. Pelrh W. A., Jr., and The Journal of Biological Chemismj' 270(|0):$1M-$171. Ml~--h 10. 1995. I~l~q" supIx>r1: U.~. Army ~em|csl Ind glologic~! Defmse Comm~md. Research Dmvelopment and Engineering Center. and the National Institutes o1" lteallh. ~ the DepOses of Phmnacolo~y a~d Moleeulm" Sc~e, The .lohns Hopkins Sclmol of Medicine. Baltlm~c. MD. U.S. Army (~emicM t~s+,rch C~mmand, Aberdeen MD, Department of Biology, ;ohns Hopklns Unlverdty. Baltlm, MD, and lhe Departments of Medicine, l~theloly and Unlvers;ty or'Vlr$1nia P~hoc! of Medielne. Chmlmmsvillc, VA.
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the pm'~si~ •! [he molecular levc! h~e ~ limJt~ by t~ l~k or x DNA I~nsr~- t~n syst~. R~endy. met~s for Irxns~nt Iransf~li~ of E. kls[~v~i~ ~!~. We m~ h~ I~ ~vclo~l ~ a stab~ tran~ti~ systQm lame ~er, ~ 1R st c~mmi~s ~ 0-~ ~ ml" was ~ in ~al dil~ a~ ~;~ in ~!-5)3 ~;um. Aher 72 h ~ ~h a~ 37~, ~lg ~n- ~t~s ~r ~ ~ ml"~ kill~ I~ ~ l~ a~. We ~l~d that E. is qui~e ~itive Io G4I~ a~ tha~ ~o ~ ~ u~ ~ a ~l~eable turks. Vi~. R. R.. ~y. J, E.. R~la~. B. D.. Slm~l~. J.. M~n. ft. J,. a~ Pelri, W, A., Jh ~ su~: ]effre~ Mcm~ial T~st ~ t~ Nati~l Inxtit,t~ or f lcalth. VJ~J~J~ ~svJlle. VA. ~ ~nt o~ ~a~ If~lth. lf~a~ ~ o~1~ H~I~. B~. MA. PHY$1OLOOICAI- DIVERSITY OF NICOTINIC ACETYLCIIOLINE RECEPTORS EXPRESSED BY VERTEBRATE NEURONS The last duc~cle ~ ~vezled ~n aswundiRII degree of physiological and s~ruc- rural diver~iW in neumtransmilter-agtivated receptors, in l~rdculzr, molecular stud- ies Of the acetylol~line-pted receplors in vertel~ate muscle ~lls and Tnrpe~n elec. t~'yles lave birth to a new era of research on st related subramily of nicotinic recep. tefs (nAChRs) exprelsed by central and peripheral neurons. This review focu,~s the nAChR lubly~z expressed by neurons, viewed mainly from a physiological per- Iplotlve, h is intruded to ~omplemont several excellent recent reviews o~ muKlc- type AChRs and on the anattemkal dtst~butlon, blochemlstW, and molecular biology The wlx~ slm~m~xed in this review has helped answer • long-standing question: How can the well.known behavicxll, cognlti~e, and add~ive effects of nicctine he reaoe~ed with ~e ]mueity of evkkme for C~S s~mapses wh~e the tmnsmisslon is medilted by nleotlni¢ reeelxo~? ~ identification or" • large family of neuronal nAChR subunk Becks and an anmj of functlonally distinct nAChRs has given a bmis for the dive~ effects of n~x~ine. Pedmps mo~ impe~ant is emergent evidence for hmctlonsl roles Of We-, as well as IXmSynapllc. CNS nAChRs thai contribute to ext~io~ modulal~ Innsmitl~r ~lear~. and therein effesl s~mal~ fonnaiinn. We Orll Sml1~Z~ the ml~ar ~ thll( ha.~ ¢alaly;,.l~! th~ franstf|ol~ the eUr~om "Status nioolinic~s." next dlsc~ss the dlvccxlty in suhonil con~ition, ion~ Ix~m~,abillW, Iocslization, ~ modulaiion or neuronal nAChRI, and Tin•fly oool|(l~l' ~ltysiologi~"~ reasons for thc exlent oJ" n~ChR channel diversity ~nd pro- IX~ ~ rme~ch IV~3eh~, D. S. ~nd Role. L, W. 2O6 Annual Review or Physiology 57:521-546. 1995. Other support: National Institutes of Health and the McKni|ht and Hl~chl From the l~pmlment of Cell Biology and Atmt~ny in the Center for Ne~'obiololty and Behavior. Columhla Universily. College or Ptzysieians md SuW.eons. N~v Yorl~. NY. 2O7
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male Spr~t, ue-Dawley rats under nembutal anesthesia. At different times ($-60 mln) efMr .niootir~ administration, n~ce4ine and its metabol;teo cotinine, were determined by F|PL~ in plasma, liver, kidney, heart and brain. Within 5.10 men after administra. lion, nicotine Co~ntrations reached peak values Jn plasma (2.1NJpmol/rnl) and the orpns analy=ed. The plasma level of nicotine deereased h7 5(F£ within 20 mln (half.dine) after its intrav0floos administration. The hair-time of nicntinc in the hgain was about ~0 m;n. Th~ hatf.ttme~ of" n;cn¢ine f~f the echo" {wgan5 were sin. ~ mJ~m" metabolite, eotinlne, accumulated in plasma, and by the ooncemrat|ons of n~cotine and cot;nine in plasma were about equal pmo~ml). While ¢ntinine accumulated in plasma, nicotine wa.,~ eliminated by the kMggy. While the nJcotin~ concentrations decreased with time in all mgan~, cotinlne eon0entrations remained constant. These observations indicate that n;cotine is tenuity elimin~d ~ metatx)lized to eotinine while cotinin~ exhibits a long retention tame and accumulates in plasma. This plasma accumulatinn may ,:ontrlbute to activation of PAF turnover rate and to thromtx);is. Saistr~', B. V. R., Chance. M. B.. $1ngh. O.. Hum, J. L.. ]an~on. ~'. E. P~tMf1~co|og,y 50:|28-|:~6, 19~;. OOter support: U.$. Department of Heahh and Human Sen~iceg-Natlonat lnxtitute on Drag Abuse. and the Study Cefaer for Anesthesia Toxicology. From the Depmlments of Pharmacology and .Aue~dhesiology. Vunderbilt Univer~;ty School of M0di¢ine. Nashville. "IN. SMOKING. PLACENTAL FUNCTION. AND FETAL GROWT!! Scientific literature published during the past century indicates that cigarette smoking dudng pregnancy his si|nil'gan! adverse effects on the development or the fetus and the health and development of the newtx)ro ~by. TI~ haztrdons effect~ of smoking dudng pregnancy inolude po~siMe six)neon, fetal death, fetal growth retar- dation and short, or Iong,.tonn developmental deficits in the infants. Ckagera shoot the exposure of ]xe~nant women to toad',co dust and gnoke factOrks in Eurofg. There was also a high rate or mmtahty among the mrants us femdot in tobacco-reMt~d occup-~ions. In !035. Somag and Wallace o~,erved that ¢fgaf~te smoking by pregnant women increa,,ged fetal heart gate. and they q~cculated that th~s was pro~51y due to trampf.~,~'~t|l tgamfer o..f ni.c~ue ,into fetal Camp~ll ol~a,'vnd that a woman wan smoked heavily oaring ,pregnan~ was tO h~v~ mole difficulty duging the coarse of pregnan¢.y, p~_.uritl,on. ~ lactation, than a no~smoker. In 1940, Em~anberg and onllahe~toes revert|gated the influence ot n~pt|n~ and tohee0o un~e on wegnam albi,o ms. 'T~ey round that the young of nicotino,,treated aa well as tobacco-smoke<expend rats were umkrwcight compared to tho~ of control untreated rats. These studies rated imp~tsnt questions al~)ut the ¢ff©,,'ts of tobacco smoke and nicotine on intrauterine fetal growth retardation (IU(3R). In 1957. Simpson reported that babies of women smokers had signifieamly lower bisth weight than those of nonsmokers. Simp~on's stud;as were eo~f'm~ed by ~v~'a| ~¢hers involving mof~ than half a million births. These studies have been dis- ham w~n s~ a~ ~ g lighter th~ ~blcs ~m to m~ of p~al fu~t~ in IUGR ~ve ~ ~ R. ~. R. ~ Jm.V.~ In: Sn~t~. ~. V. R. (~.): M~tal ~. ~a~=r Other ~p~: Stay Center fm A~st~si~ Tox;c~o~y. V~d~Ut Uni~lly. Nsxhville. TN. U.S. ~t OF ~jl(h M~ical Center, Nashville. ~. OPIOID ADDICTION. PLACENTAL FUNCTION. AND FETAL GROWTH Ill;cat u~ of opinld drugs is a mqjor and Iong-::tanding ixoblem in the Uni~! tiO~ inC|t~CS • signJf'mmnt perc~ta~ of wm~n. ~ ~.p¢~.~. g~..~ .v~nun p~K - pating in drug treatment programs suppmled by t~ reationat that|lute on L~ru| ^bu~ reached a level or30~ in the 19g0s. A ma~xit7 of~-~ mn ~ heating age, In the United States, an cat;mated 10,000. children annually have lx)m to women usins ot~d drugs, mainly mo~. i~.a~t .M~. "n.. the Oplold use during pregnancy has Ip~ mv~si|pted (Nslng p~t 35 with t~gaed to its effects.on the wo~n, the fet~ ~ the child. Its use m~lical, obstetflcah and management pr0btems0 inclndmg the di.r~_, lay in ing the pregnant heroin ed(llct~ f~t distress due to matcma| aosunen~ tram me drug, which is even rno~ harmful to the fetus than pmive deF~donce: and utatlng withdrawal symptoms in the fet,s and infant. A COml,mhensive metha¢~ma hence treatmeflt (MI~) with app~Me ~ do~go le'~els can possibly t~t~ the hl¢|(~n~ of obstetrical and fetal eomphcaltton$. In: Sastry, 8. V. g. ted.): Placental Toxicology. Chapter 4. CRC Press, pp. 1905. Olher suppogt: 5tttdy Center for Anesthes|a Toxleotngy. Yamkrbilt Nashville. TN. U.$. Department of Health and Human Services. and the Ntt|~al instilule on Drug Abut. From the Departments or Anesthesiology and Phtrmtcolng~'. Vande~oilt Unlverslty Medk:al Center, Nashville. TN.
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0 0 01 -%! COCAINE ADDICTION, PLACENTAL FUNCTION, AND FETAL GROWTI! One of the serious and growine problems which will have an impact 06 the heahh and well.being or IXegnant women is cocaine/crack atmse. It is estimated that I I~E of the 13. $. population regularly use cocaine; 2 In 3% are hellcvcd Io use cocaine during prognancy. A survey in 16 states ~nd the District of Columbia invoh,- lag .'~OJ)O0 people indicates that women of childhearing a~e fig to 34 years) censti- lute I~ql, or all regular user~ of cocaine. The human fel.~ is dependent upon the maternal environment for its safety, health, gmwlh, and development. Cocaine and olher abused dmg.~ ingested by I~egnant women may damage the develop~nent orthc fetus by many mechankma. Cocaine and its mclabol;tes may interfere with normal physiological processes or pregnancy, resuhln~ in fetal I~,~, prematurity, and oh;tet- ric|l compliealion,~ threatening to both maternal and infant health. Co~alnc anti/at its metabolltcs may enter th~ placenta and interfere with placental translmrt of nutrients to tM fetus. Thus, Ihey cut the maternal supply line to the developing fctus. Cocaine a~(~tor its metabolites cross the placenta and inlerfcre wilh ~:rowth and develolm~nt, both physical and mental, causing reduced birth weight, birth dcl'ccts, learning and l~havt0ral disorders, and newborn distres.,c The immature enzyme systems in the developing fetus cannes datoxlry dru~. ThereR)re. cx)caine use may cause complica- tions due to its effects on the mother, placenta, ~tus sad newborn infant, S~stry, EL Y. R. In: SastQ', EL V. R, fed. ): Placental Toxicology. Chapter G. CRC Press, pp. 1995, Other support: Study Center for Anesthesia Toxicology. Vanderbilt University, Nashville, TN. U.S. Department of Health and Human ~ervlces, and National Institute on Drug Abuse. From the Depa~ments of Anesthesiology and Pharmacology. Vandcrhilt University Medical Center. Nashville. 'IN. STUDIES ON THE DIVERSITY OF MUSCARINIC RECEPTORS IN THE AUTOREOULATION OF ACETYLCHOLINE RELEASE IN THE RODENT CEREBRUM USING FURAN ANALOGS OF MUSCARINE Prev~o~s studies hive indicated that two t'eedl~ack mechanisms, one positive and the olher nesalive, regulate the rate of auatylcholine (A~h) release. The positive feedback system has at least three COmlxments: a muscarlnic receptor (Ms), of subsran~e P ($P), and influx of extracellular Car'. If the amount of ACh released from the presynapt;c ne~e lermin~l is low, the released ACh activates Ms, which. stimulates the rel©a,;e of SP. Substance P increases Car. influx, resulting in the rclea,~ of fulther quantities of ACh for effoctlve chollnerglc transmission. Similarly, th~ comlx~cnts arc present in the negative feedhack system: a ixe~ynaptic mus- c~inic: reocptor (Mi). relea.~c of mcthionine enkephalin (MEK), and inhibition of Cs~" influx. If the ACh in the synaI~ic gap is b|gh. it sctlvxte.~ Mi. resulting in the relea'~ of MEK. Methionine enkcphalin decreaxc~,~ Ca~" influx which (k:creaxcs the rate of release of ACh from the cholinergic nerve terminal. 11~c critical first stc~ in 210 bo~h ol'these fecdlx~k systems is activation of p~%maptlc mu~arinic racu~o~ Ms and MS. No ~elective s~onists have Ix, an described far presynsplic mu~earlnle Iors. Therefore, we studied the effects of $-methylfurfuryhdmethylammonlum ($-MFr) and 5.hydmxyfurfurllrimethylammonium ($.HMFT) on the simultan~s release of ACh, SP. and MEK from the suI~,rfu~d mouse ~heal ~ll~s In our search for selective a~:t'mi~t~ for Ms and Mi reC¢l~O~o $.MFT and $.HMPT w~m selected for thk Ix~rtx~e hecause they have dlve~xe effects in t5¢ per~'d~l nervou,~ system. .~a~try, It, V. R., Tnyeh, O. $., end Jai,~wal, N. Annals of the New York Academy of Sciences 75"/:!94-196. May I 0, Other supf~rt: U.$. Dei~rtment of Health and ilum~n Services, Hafional Institute on Drag .Alxtsc, anti The Sandy Center for Anesthesia Toxlcolo.r.y. From the Departments of Anegtheglology and Phan~aonlogy, Vandef~llt Unlverdty Medical Center, Nadwille. NEUROPHARMACOLOGY OF NICOTINE: EFFECTS ON THE AUTOREGULATION OF ACETYLCHOLINE RELEASE BY SUBSTANCE P AND METIIIONINE ENKEPIIALIN IN RODENT CEREItRAL SLICES AND TOXICOLOGICAL IMPLICATIONS I. The neunmal release of acetylchollne (ACh) and iu~ anlO~lUlation by neuro. modulalor~, ~ub~taoce P (SP) and rnethionlne enlcephatin (MBK), have ~ studied using superfused rodent carets'el slices. Nicotine exerts si|nificant affects on autore~ulation or ACh release, which may hive to~i¢oloBical implicatluns. 2. Positive and n~,ealive feedback systems have b~n postulllt0d fee au~megula. llon of ACh rele.'u~. The components of the positive fcedtmck ayltem Include a carlnlc (Ms) reccl~oro SP, and nCllVatin~ of C*a~' influx. Low levels of ACh In the blophase of the cbolincrgk: synapfic Cap may IrigW the positive feedb0¢k and high levels of ACh may triter Ihe n,.,lative f0edback sy~un. 3. "]'here are also neuronal pathways foe direct reciprocal R~ulatlons of SP and MEK. 4. Low coneentration# of nicotine tri~.~rs the Rlea~e of ACh followed by MEK and SP. Release of SP causes neurocenlc in~nm~ion. $. Nicotine and its metabolite, cofinine, actlva~ platelet aclivadn| f~lo¢ (PAP>. hydrola~ and thereby enhance the lumover rate of PAF. This effect may to Iobo¢co-induced arterial thrombosis in peripheral and central nervous systems. Sadry, It. V. R. Cllnical and Expcrlrn~ntal Pharm,,¢olocy and Physiolofff 22:2~g.290, 1995. O~1~" supporl: U.S, Deparlment of Health and Run)an Scrvlces. National In~thut~ on Drug Abut, and The Study Center for Anesthesia Toxicology. From the Departments or Pharmacology and An~sth~iology. Vanderldlt Unlv~lly M~xllcal Center. N~dwille. TN. 2It
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0 0 "xl EVALUATION OF TEll=- NATURE OF RAT RETINAL ACETYLCHOL|NESTERASE USING A SPECIFIC SUBSTRATE AND A ~PBCIFIC INHIBITOR 'rite occurrene~ o~" cholinesterascs (ChE) has hcen dem~lslrated in retinLs or ~ev~fal mlmmlllan species. Usil~ BW2~4C~l and iso-OMPA as selective inh|bitors or zeetylcholinesterase (ACHE) ~nd buty~lcholinesterase (BChE). mspcetivcly, it hz~ been demo~trated that the rat Rltnal ChE is predominantly ACHE, Therefore d~e kir~|e nature of inhibition or the rat ~inal AChE by BW284C51 was studied ustnl ~tyl-(l-mmhyhbiocheline (AMTCh) as az r, elcetive ~ra~e of ACHE. AChE activity or the ral r~tinal sonk:ates was assayed using AMTCh as the sub.trite in the prc~nc~ or 5.5.dlshiobls-2-nitro1~-nzoate and yellow $-tbio.2.nitmhenzoie anion Was mna~rud by the ab~)q~ion at 412 ml~. using z spectropholomeler. The sul~trate (AMI~) was vlHe(I hetwcen 0.1 and 0.5 raM. The inhibitor concenlratlon~ wer~ 2.1 and 4.2 aM. DooMs-reciprocal plots I~lw~n su~trate concentrations and Ihe v=lOCJties for the cn=ymlti~ hydrolysis of" AMTCh in the r~,~nce and abscnce of inhibitor ~ constructed. This study Save the f'ollowir~ rcs.lts: BW2R4C$1 was a potent inhibitor of the hydrolysis of AMI"Ch by rat retinal AChE (IC50..S.2 nM). 1~¢ nature of the inhibbion w~s found to he ¢ompelilive as the double reciprocal plols with and wilhoul the inhibitor eros~d on she ordinate. Saslr¥, I~ V. It., Singh. G., Lon. P., and Jan.son. V. E. J0~m~l or C~'ular Ph~mnacolof, ff and Therapculics I I (3).~I01-409. 1995. Othor ~plx)rl: The $1udy Cenler for Anesthesia Toxicology and lhe Smokeless ~ Research Council. ~ lhe l~l~Imems or Ancsthesinlorh' and Phannacoingy. Vanderb|It Unlv~slty M~lical Center. Nashville. TN. X. Pulmonar~ and Respirator~ Systems D~..~LIZ.KTION OF Till=. SUPERIOR CERVICAL GANGLIA INIIIBITS MA~T CELL MEDIATED TNFe~-DEPENDENT CYTOTOXICITY. 1. POT~ITIAL ROLl=' OF SALIVARY GLANDS De~entreli~tion or pn|lioneclomy of" the super;or cervical ganglia (SCG) redueei pulmOmU), inflammation, as well as chemolaxis and activation of circulating negtrol~ils, However, lhe pro(ectlve ¢11"ecl or dectntralizaltbn was abulishe(I when combined wtlh Rmoval of Ihe sol)mandibular $1ands (sialndcncetomy) in the sam~ InlmlTs. Thus. it has been postulated thai the submandibular glands (SMG) an Imli-lnflammatory factor(s) that is controlled by ccrvical sympethelic nerves, Deee~ralization OrSCG did no( modify in vitro histamine release or in vivo levels or rat ml~! cell prolease It, but it reduced mast cell (MC)-mediated tumor ncerosis tor ez (TNFe).dependent cyto(oxieity. Combined decen!ra]iza!ion/sialad.ence.[o.my abro!ated the inhibition or MC eytolox.ic ecfiv.lty0 as we nave s.n~. w.n pr~woos,y, pulmona~ inflammation a~! neolrophd furlc[zons, However, smmocmectom)" 212 inbibhed MC.mediate~ ~'NF(~-depeudent cyto(ox|cily, m ~mMi~ ~{¢h Wl. ~z that SMO ~ a r~s) I~t ~ ~ti~e MC c~xk ~lvi~. or t~ errors or SMO-derived r~1ors, s~h as epical $mwlh ~1~, ~e ¢~h f~. ~ I~f~inB ~wth r~ ~ ~F~), ~ t~t ~ly ~. ~ ~ MCs with ~F~ IO' ~1 inhiffi~ M~ ~~ ~. cal sy~t~ in~ati~ ~d 5MG b ~ ~ dish~ ~ ~ ~ ~l~aW infl~ati~ ~ ~ut~bil ~i~s ~fi~ Base,re, E. Y., Mathis~ R., C~er, L, ~vi~, J. S., ~d Be~ A. ~ Bmln. Be~vim. a~ ]m~ni~y 7:~3-3~ 1~3. ~r su~: AI~ R~a~e F~ndat~ r~ M~al R~h. From the Deparlmenl of Medicine, University or Alberla, Edmonton, ~parlmenl or Medical Physiology, Universily of Calea~, Calp~, Can~a. TEMPORAL ANALYSIS OFTHE ANTi-INFLAMMATORY EP1~CT'3 OF DECENTRAUZATION OF THE RAT SUPERIOR CERVICAL GANGLIA Bilateral decem~allzatio~ o1" Ihe supsHor ,cervical pn$1il (SC(]) mJuced imlmonary inflammation Ihat develops 4.~ h after iudUClion ol" anapbylasil in NippoxtrongTt.s braxiltensis-senaltlzed r~ls. Hi~lamlne levels in pedlom~l lavllle fluid mid rut mast cell Im~ease type |! in serum were ;ncmased IO eornparubk levels in sham-opiated and d~cen(ralzed rats. fn v/tro stimulation or Idv0~ll~r rml¢~ (ALM) with llpopolysacchaHda (LPS) provoked tumor nec'~lis I'l~er-cc {TNP-a) release d~at was two Io three times Irea(~" with onchellen~d clu~,nmdbu~d ra~s with ~d~m..o~rated ra~. However. ahor allefl~ ¢heller~e Ll~-itlmulated rdease from ALM of sham.oporated ratt i~eased threefold md lamd at l~124 h. whereas with d~.~llix~! ms mle~e of thi~ eytok|ne K.tual~ deem~l at 4 m4 $ h. The irv.'~ase in the pl~x:yto~is alnd respiratory Ix.'sl ord~cuhllinl he.ll, a( 4 and 8 h ,,tier alle~en eha~nle in ~ham oper~ed rats was ~edu¢~l by decenlrali~atlon. These results sugEe|z that the ,~lenuatlon of induced pulmo~a~ inflamnltion tl~t ~ with de~-f~ralizatk~ of the SCO is nifty asso¢ialed wlm dow~eeuladon or neu~ophll a~d mac~ Mathison, R., Carler, L, Mowat, C., Bissonnelte, E., Oavisoll. J, ~, and Bel~lk ,4,, D. American Journal of Physinlogy 266(35):R 1537-R!543, 1994. From tl~ Depmlmenls of Medical Physlole~y, Faculty of Medtcinu, Unlverally or Cal~a~, Calr~lry, Alberlao Chnlda, and Pulmonary and Cell Biolo$~ .Rlzalreh Group, Heritage Medicid ResoL.ch Centre, University o1" Alherta, F.dm~nton, Allots, Can~z. 213
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PRETRF..ATMENT WITH AN ANTIBODY TO INTERLEUKIN-5 PREVENTS LO~$ OF IMJLMONARY M, MUSCARINIC RECEPTOR FUNCTION IN ANTiGEN-CHALLENGED GUINEA PIGS Inhalational challenge w;lh antigen decreases the function or inhihilo~ M~ mus- elflni¢ autorl:ceptors on pirlsymllhetic nerves in the lung. ilx:r~sing the release of leetiyleholine from the vagus nerves and l~tiating vaplly induced hrouchncon. strictlon. It is Ix~sible that eosinophiis cau~ M~ rec'cplor dxsrunct;on, p~rlups relet!iin| positively charged IXOleins that m'e M~ receplor antagonists. O~cause of the prohlble role of intefleukin-$ in initiating and maintahiing the eosinophil infillratlon. we tested die function of neuronal M~ recepiot's in antigen-challenged guinea pip after pretreatment with a monoclonal antibody to inteflenkin-$ (TRFK-$). Ovalbumin wits liven intliperitoncldly to sensitize the Imimals. Three weeks later. the animals were injected inlriperitoueally with either TRFK-$ (240 ix~kg i.p.) saline. Beginning thre~ da)'s later, they were challenged with an ovalhumin aerosol fo( 5 mln on each of four consecutive days. M, r~eptm function was tested 24 h al~er the last antigen challenge. Electrical stimulation of hoih vagi caused bron. choconstriction and bilcl)'cirdia. ]n control animals, piloclllpine atienuat~d, and llmine polantiated, va~tll)' induo.-d blx)nchoou,strlction by stimulalin~ and ~lnckin| nouronll Mz mitsclrinie re~ptors, reSl~.Ctively. In challenged animals that did not receive TRFK-$ these effects were mlrkedly reduced, confirming Ill. receplor £unetion. In TRFK-$-treated guinea pigs. the effects of helh pilocsrpine and gal- himlue were Ihe same li those in control animals, demonstrlting normal Mz receiver function. Pretreatment with TRFK-$ selectively inhihlted the millration of" eoslouphils into the lunts its measured 5)' lung favaBe. Thus the function of ~ mu~ clrln;c receptors in antigen-challenged gulrlca pills can ~ protected h)' ;nhz~t;ng eosinophil influx into the hags. Elbon. C. L.. Jaeoh)'. D. B.. ~d Fr)'er. A. D. AIrle~an J~lrnal ot'Re~pbatory Cell and Molecular Bioloty 12:320-32R. I~)$. Other support: National Heart. Lung. and Blood Institute. Ameri¢an Lun~ Association. Id the American Heail Associalion. From the Department or Environmental H~fth Sciences. Scl~xll. of.Flygier~..and Public ltealth, and Division of Pulmonar# and Crltlcal Care Meeicine. JOnnS Hclltins Asthma and Allergy Center. Joims Hopkins Universit)'. Baltimore. C~GA~ SMOKE-INDUCED ERONCHOCONSTRICTION AND RELEASE OF TACTIYKININS IN GUINEA PIG LUNGS Two series orexperlments were taxied out to determine whether the reic=se or tachyklnins is involved in the bronchouonstriction induced by inhalati .on of cigarette smoke in guinea pigs. In the first se~s. cigarette s..moke co~s. is.ten!ly i.nduce.d, lxon; choeon~ction (&IU. - +203~, and ACdyn ~-46~) zn ancsthetzzeo gumea psgs, anti the ~pOnse w~s only plni~dly blocked by bil~eral cervi~l vagotomy. However. the smo]:e-lndueed bronchial constriction was ¢omplete~ a~x)lished in animals 214 re~iving a systemic Cal~aiein IWetrectment to de~tm# the IKbykini~liniM fi~ affe~. In t~ ~ ~Hes, was i~t~ed ~ a~g. th~ times of t~hykinins afl~ I~ ~lea~. ~v~. t~ ~h~t ~~ ~ to sm~e w~ ~ni~ hv an me~ow a~ cafcil~in ge~.mlat~ ~ide *L! in s~w~ t~ ciga~tte sm~e trigge~ t~ plays an im~a~ ~le in the ~ke-i~d ~st~live eW~ pigs. ~. L.-Y.. ~. Y.-P.. Hong. L-L. Other su~: Swedish Medial S~dish National ~vi~n~ntal ~i~ B~. ~ati~al Institutes of Health. a~ I~ Unive~ity ~ Kent~ky ~ ~ Health R~h In,irate. ~ t~ ~an~t of ~a~ol~. Ka~in~z Inslilute. St~kholm. Swan. ~ ~m~t of ~ys~lo~. ~nive~ity or Kentucky. ~xingl~. KY. XI. Radicals TIlE PRODUCTION OF NITRIC OXIDE IN ENDOTHEUAL CELI.,~ BY AMPHIPHILES Lysopho~phatidylcholinc. an endogenous detergent is an endoChelium-depan- dent smooth muscle relaxant, which acts through the releate of nitric O~ide. It iS known to activate a number of memlmlnc-bound cnz3~mes. B~se or the r~l~k~- ship ~tw~n d~e~en~ ~cfion. rel-x~ti~ of end~halium-intm r~it ~ml~ and the rele~ ~ nitric oxide, w~ ~k~a~d the Ix~sibili~y that o~her m~phi~ll~ al~/x~duc~ nitric oxide lrmm md~helial c~lis. We thcreflx~ inwsdpted th~ of digltonin on relax~ion of p~©ontr~cted nitric oxick from fr~hly hervested bo~ino enc~heHa! cells as del~rmin~ ~ chemi- luminesc~nee. We found th~ bo~h di~itcx~ln md LI~ rele~ n~lc oxide ~ ~hat ~his process is inhlbit~ by the NO syntha~ F.~ler (L-NNAME). Life Sci~s ~(t6): t 143-1153. I~. ~hw =up~: ~ Ma~mt W. and H~ H~r. 215
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From the California Institute of Technology, Deparlmcnt of Environmental En|ineering, Pasadena, CA, and Department or" Experimental Cardiology, Huntington Medical Research 7nstitule.~. Pasadena. CA. INVOLVEMENT OF INDUCIBLE NITRIC OXIDE SYNTIIASE IN THE INFLAMMATORY PROCESS OF MYOCARDIAL INFARCTION Inducible nitric oxide synlha.se (iNO$), which cataly;,.e~; the reuction ot" t.-argl- nine m t,-citmlline and nitric oxide (NO). ldays an ;mllo~lant sole in immune-medi-',ted cardiac disorders. The present repot1 summarizes and discusses findings on the induct|on of NO$ in myocardial infarction of rabbits. INOS was slgnific,,mly inc~ in infarcted myocardium 4g h after cot(mary artery llgation. The effect perti~ted I'o¢ 14 days and declined thereufter, lmmunohistochemlcal locallzallnn revell~l macrophages as a major" source of iNOS expression: INOS cxprcsslon w.',.s al~o present in infaxctcd human myocardium. Increased iNOS a~ivily appeared to Ix: related to the iMnction of apoplosis in infiltrating macropha¢cs and cardiomyocytes. Moreover, preferential inhibition of iNO$ by $.mcthyllsoChiourea sulfate ($MT) resulled in algnlf'¢ant improvement or left ventricular pet/~nnance and increased regional myocardial blood flow. These findings soggest that selecliv¢ inhibition of iNO$ activity may provide • therapeutic strategy in cardiac disorders such as myocardial infarction. Wildhirt, 5. M., Dudel:, R. R., Suxuki, H., and Bing, It. J. 7ntemational ]ournal of Cardiology 50:253-261, 1995. Other support: Gustavu~ and Louit.e Pfeiffer Research F~Jndntlnn, Redlands, CA. Margaret W. and Iierhert Iloover, Jr. R)undation, and the I.luclla Morey Murl~ey Foundation, P~aden,,, CA. From the Department of Experlmenlal Cardiology. ||unllngton Medical Rer, e.'trch |nstitutcs, Pasadena, CA. THE REACTION OF NO' WITH O;" AND HO;: A PULSE RADIOLYSIS STUDY ~ reactions or No" with O;" and with HO; were studied t~ing the pulse redl- olyds ~¢huique under pseudo first older condtliom where (|O;" 1, + | HO;I,) > |NO'|, at pH ~.3-10.0. The rate ¢onstan~ of the reaction of NO" with O;" was determlrcd bo~h by n~nilortng the decay of O~'" at 250 nm and the formation of ONOO" at nm Io be (4.5 - 0.5) x tOe M"s", independent of ionie strength and pt! in the age 216 ot"6,1-10,0. The rate co, slant of the reaction of NO" with HO;" wu d~,nn|n~l by following the decay of HO; at 250 nm to be (3.2 • 0..3) X 10" Goidstein. S. and C~'Jl~ski, G. Free Radical Biology k Medicine 19(d)-.50~,-.~ ! 0. 1995. Other support: l~rael Academy of Sc;euces. From the Department of Physical ChemLctry, The HeMew Univerdty of .~emsalem, Israel. NITRIC OXIDE RF.GUI.ATION OF SUPEROXIDE AND PEP.OXYNITRIT~ DEPENDENT L| PID PEROXIDATION :'~39--I'O~M^'nO~ O~ ~ Superoxide (O'~). nitric oxide ('NO). and tEelr reaction product peroxynitrile (OONOO') have all been shown to independently exert toxic tWl~l molacule lions. Because these reactive species are often generated in ex~l dwlnl dlvane inftammatoW a~J ocher pathologic clrcumstan~:s, we ae~,ased the infltme~ of "NO on membrane lipid peroxid|tion induced by O~, H~O~. lad "OH derivld front thine (XO) and ONOO'. Experlmental conditions in lipid oxidetlon ~t~__s ad.iusted to yield different rates of delivery of "NO relative to mtea ofO ~ and ~O~ generation, by infusion or either "NO or via "NO released from oe.nltroso.N• acetylpenlcillamlne $.nitro~oglutathione. Peroxldation of pho~phatldyleholi~ IipcP somex wag ~ssessed hy fom~allon of thiol~rhlturi¢ acid.reactive products and by llq.ld chromntogr~phy-mass spectrometry. Liposonles exposed to XO,,~It4v0d live species in the presence of'NO exhibited both stimulation an0 Inhibiti~ of lipid peroxldatio~, depending on the ratio of the rac~ of reactive, oxy.~ I..l~. e# .p~. u¢- tio~ and "NO introduction into reacllon systems, Nitric oxme al~ did nee lipid pemxidntion. Llnolenlc acid emulsions peroxidlzed by XO.dsriv~d reactive species showed similar do-,.e-dependent regulation of lipid peroxidation by Mass spectral analysis of oxidation products showed formation el" nitdto-. nltrosoperoxo-, and/'m" nitrated lipid oxidation adducta, demonstrating that 'NO resonance (ESR) analysis of incubation mixtures ptovmeo no cv|(amce for t~ton of paramagnetic imn.lipld-nilri¢ oxide complexes i~ reaction .R/~IeMI...l~.xynlt~. ~l- mechanisms, was also inhibiled by "NO. Pamxymmte-meo,ated tides was pertially inhibiled by "NO, inf¢ff _~ reaction between "NO and ONI~IH. It is conclnded that "NO can bo(h ,flmulate O=~Tl,OJ'OH-induced lipid oxi¢~tlcm and mediate oxidant-wolecfive ~eacl;ons in membraecx at higher ratel of "NO lion, with Ihe Wooxidant t, er$~r antloxldant outcome criticall~' dependent on ralattve ¢o~,,entrations ot individual reacllve specks. Prooxiden! reactionl el' 1¢O will after O:~ reacl;on wlth "NO to yield IXXem ~meond~j oxidants such as ONOO" and 2t?
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the ~ntioxidlult effects of'NO a co~:F~nce of di~c~ ~ct~n with ~Ikoxyl ~ ~r- oxyl ~icM inte~iatcs durinI li~d ~roxidati~, thus le~in~fin~ lipid chain ~i~ Ru~ H.. R~i. R.. ~ill~ M.. TeHeri. R.. K~n~mm~n. B.. ~m~. S.. Ki~k. M., ~ F~n, ~. A. Olher sup~rt: FuHbri8ht Foundation. National Instilmes of lle~hh, and the Univ~i~d de Is Republica ~d ~jo de InvestiB~io~ C~nlifica~ y T~n~cas. F~ the ~nments of A~hesiol~y. Bi~hemi~l~ and Mol~ular Genetics. Pediatrics. and PharmaeoloBy and ~he Mass Spectrometry Shard Facility. ~m~h~ive ~r ~nter. Unive~i~y of Ala~ma It Bi~i~h,m. Bi~in~am. A~ ~parlmenlo de Bioquimic~. Facullad de Medicina. Universidad de Re~bUca. Montev~eo. U~guay. a~ ~he Bi~hysic~ Re~reh Inxfilule. ~'TAILE Mn(lll) PORPHYRINS MIMIC SUPEROXIDR DISMUTASE IN VITRO AND SUBSTITUTE FOR IT IN VIVO ~everal m~nsanic porphyrins, wilh sut~t~uenls on the mcthlne Ix~dge carlxms. were prcpllred and examined for stability, redox behavior, catalysis of the dismata- lion of Sulmroxlde radical (O;). and the ability to protect a superoxlde dismutase (SOD).~ull atraln of E. coil against dissolved oxygen and a SOD-competent strain agai~t plmquat. All of the compounds te$1ed exhibited reversible redox belmvior and wgr~ ltabk to EI~A in both the oxidized and redtged stat~-% and several were able to ¢ltllyzo the dismutation of O; with the rate constants olP -IW M" s". The milked Wolective effects of some of the~e compounds exceeded that which could he ant|eipmted oo the tmsis or such rate constants. The letrak~ ft-mcthyl-4-pyridyl) compound ~ reduc~ en~maticatty a¢ the expense of NADPH and nonenzymati- ~ltlly by (~tH and was gop¢ in the reduced slate within E. coil Since the rate constant for reoxklation of the red~ form by O; is 4 x |0" M" s'% it appears that this pound acts in Wvo as an NADPH/X~SH:O; oxidore~ucta~ rather than as an SOD mimic. It+s ability to facilitate aerobic growth of the SOD-null strain can be explained o~ this basis. Faulknef. K. M.. L|ochev. S. I.. and Frldovich, I. The Journal of Biological Chemistry 269(38):23471-23476. Sepl<~mber 2.3. 1994. Other support: Johnson and Johnson Foct.q+ed Giving Program and Eukarion Inc. From the l~partment of Biochemist~. Duke University Medical Center. Durham, NC. 21g ESCIIERICHIA COLt EXPRESSES A COI~ER- AND ZINC~AINI~ SU~ROXIDE DISMUTASE A mut~t of F~he~c~ia ~li. u~le ~ ~ m~- ~ ]~tatnlnl su~mxi~ dismutag (S~). was f~ to ~ta~ ~st k~Is ~ w~s j~ to ~ a ~r- #~ zi~tzintng SOD ~ t~ ~s ~ tnh~ cyani~ a~ in~tiv~i~ by either H~O~ ~ dle~yldhhi~af¢. M~er. dlethyZdithi~a~mate-in~tlv~t~ enzy~ c~M ~ ~tivat~ with ~(II). ~c~stltuted enzyme. Hke the n~tive enzy~, w~ una~ by ~A inhlbil~ by c~n~. ~is enz~e was, funhe~ ~e]y ~]e~ sh~k, in k~ing with a ~fiplasmlc I~alizati~. a~ it was st~ly t~ ~blc g~th. ~is enzyme wa~ al~o ~s~! in the ~D~t~t ~nt~ Fail~ to det~t it p~viously can ~ attfi~ted to igs ~plazm]c l~ti~tl~, real lability, sen~itlvity to pll, a~ to its ~latlve ~iW. It wilt ~ ~ i~tlnl to expl~ the ~yplc ~q~ im~ ~y t~ a~e of this ~OD, Bevy, L. T. ~ Friao~ l, ~ J~mal of Bi~lnglcal ~mist~ 2~(41 ):~31 ~25~ ! 4, ~lo~r 14, Ol~r ~up~: John~ and ]o~ F~zsed Giving ~gmm ~ ~Z~I~. i~. F~ the ~nmem of Bi~mlstW, ~ke Unlv~ity Medical ~r. NC. SUPEROXIDE AND PEROXYNITRITE IN'ACTIVATE ACONITA~E~. Bur NITRIC OXIDE DOES NOT The ~¢cheHc~;a coli ~I m~xx~binaot human cyto~olic ~'on|taset ~ inactlv~md by 0~:, with a rate constant of -3 x 10' ~" $": the c, orrezpondin| value for the porcine mitochondrial a¢onitase is -O.g x I0' w' r'. Nitric oxide, which ia repmled to inactivate Eonitase, did not do ~o at z pen:eptlble rate, while incut~len with per- oxynitril¢ led to a rapid loss of aconitase aetlvity. We ~ th~.t !he m[~rtad tivalion of aconita~ by nilrtc oxide in t~w is acttmlly meomted thrOUgh poroxyf'll- trite, the product of the reaction ~twcen O~: and NO'. Hausladen. A. and griduvicb. I. The Journal of Biological ChemlstW 269(47):29405-2940g, November 25, 1994, Other supporl: Johnson and Johnson Focused Giving Program and ]~klrlon, From the Department of BiochemlstW. Duke Unive~s|ty Medlca! C~nter, Durham, NC. 219
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SUPEROXIDE DISMUTASE PROTECT,*; AGAINST AEROBrC FIEAT SIIOCK IN ESCtfERICIIIA COU Exposure of e superoxide alia.inulase.null (soda .vodR) strain of IO Iambic he~ stress (45 tO 48:C) cau~.,d a profound Io~a or viability, whereas the same heat slR-,,s applied tnaerobk:ally had a negligible effect. A auperoxide dismu. ~-eompcte~t paternal strain was re~ist|n! Io the lethal effect ot the ~mblc It f.ollou,,s thai aerobic hea~in~ imr,~,~s an Oxi'*-':-- ~- ...... Inltor ©(~111l¢me~rd "r~.:. -it .... ' .... ,.,m~v~: ut~rocn OI wn|crl ~emperalur¢, d~ I~ability of vkal cell com~e~ ove~;~s su~xi~ ~ica/s. - F~ t~ ~mcnt o[ 81~h~ist~, Ou~c Univenky ~al ~cntcr, Ourh~m, A $'UPEROXIDE DISMUTASE M~M~C PROTECTS Soda Soda F-.fCltERICIilA COU AGAINST *AEROBIC HEATING AND STaTIONARY-PliSSE DEATH ~.~.~ ~. zn,s..e~_.lusm% .are the t.ollowin~, ob~mtvalkm~: (a) Stallon~y.pha~ .., w~ aR~wmm m me ~A ~dR, eel ~ m Ihe supem~tlde dismu(a~e (SOl)). su~xide, p~led lho s~A ~9 s~raln a~insf ~lali~a~-pha~ dealh; (d) HeltinE .~ ~A ~B st~in to 42~ ClUed a I(~s oF via~lJly $OD-~mp~onl parental ~train and prevenlaMc by the man, ante ~rpbyrin. ~ in ~ ~Rn~ st~in ~ I~ mmg~ ~h~n ~nl~ that J~l~. ~. i~zz~ l~s ~gizy m su~dmzc £~ SOD in ~k~iu ~i. ~hiv~ of Ei~mis~ ~ Bi~i~ 322(!):291,294, ~cm~r 10, ~ I~ ~n~nt of Bi~isz~, ~ke Unlvmhy M~al C~ler, ~. NAPHTHALENESULPHONAMIDES BLOCK NEUTROPHIL SUPEROXIDE PRODUCrIoN BY INTACT CELLS AND IN A CELL-FREE SYSTEM: MYOSIN LIGHT CHAIN KINASE RESPONSIBLE FOR THESE EFFECTS? ~ekct|ve ama~J~ts of' myosin llgbZ chain Einsse (MLCK) |e.~. ML-7; iedonaphlhalene.l-~elphonyl).Ut.hexahydro.l,4.diazcpine hydmchloridel were 220 REMODELING OF' TIlE PLASMA MEMBRANE APTER STIMULATION OF NEUTROPHILS WITH F-MET-L.~U.PHE AND DIHYDROCY'fOCHALA$IN B: IDENTIFICATION OF MI~MBRANE SUBDOMAIN$ CONTAINING NADPH OXIDASE ACTIVITY Supcmxlde (OZ') production by neutrophlls stimulated with .ehemoluctic tid¢~ [e.g., fonnyfmeth;onylfeecyf.l)heaylalal~ine (fMLP)I is mmtm b~ ln~mmm in rate and duration after pretreatment of the cells with dihydrocytechalasin (dhCB), suggesting a possible ml~ far the plasma memMane ~! m~mdmu~ skeleton in the ~ulatlon of the Oz" germating system. Analysis ~fplm membram imlated from the~e cells by bepycnlc lucrme density Iredlent ~ed|menlaliOn showed them were no significmt vatiatio~ In th~ distdb~t;on of plasma membrane hetween coetml and dhCB-ffea~ed cells, whereas a a[gnir~mt redis~dl~k>n ~'pillml membrane maAe~ was ol~erved in dhCB + fMLP-trealed ceils. Instead of taunting to 31-35% sucrose, as in the foamer two gmul~, plasma mambramt mtrkm were Ixoedly distributed over 25-50% aucro~ in Ihe dhCB + fMLP.4mated ~lht. In addition, -R0% degnmuladon was achieved in Iheae cells, whereat little release (<5%) was observed in control and dhCB.trea~ed cells. Anal.v~is of the eat fractions for membrane steletal (aetln and fod~in) and NADPH oxidasa 221
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0 0 (¢y~ochrome b and pt?.ptwx) components in dhCB + ~LP-l~at~ cell~ ~n- • e ~ disl~bufi~ or plasma membrane mmtker~, wilh an overall ei~hffold n~ ~ ~ -~ I~. ~ ~sults ~l~o that ~ ~~ sur- ~ ~S ~ m I~ ~w m~a~. w;Ih ~l7 a ;uhfr~li~ o~;~ ~ga- ni~ plume ~m~ ~t~inin~ ~11 or I~ c~nts n~es.~ fm ~tivc Of ~i~. ~r ~sults su~ s role ~ plasma m~a~ la~l organi~tim and ~i~li~ of t~ ~a~ skelet~ in the ~gulat;~ of Ihe O~ ge~raling tern. ~ supS: NaI~zl In~.ilules of lfeaith, ~ I~ ~m~t of M~mbiology, M~t~z IMMUNOCYTO~HEMICA~ DETF.CrION OF lIPID PEROXIDATION IN PHAOOSOMF~ OF HUM~ NE~HI~: ~RE~TION WI~ PROTON OF ~V~HROME 8 with a num~ of tiss~ ~ets to f~ tox~ mcta~lites s~h ~ 4-h~mxy~nal (4.HN~ 4-HN8 la a lip~ ~mx~ti~ ~t get.ted ~ f~ ~d{~l alt~k ~ or ~ils ~ ~ ~ ale ~z with ~o~s X~nk~. flz~- ~ his ~! ~r. S~i£~ ~l~l~al znt]~l~ ~o~nixing 4-HN~ein immu~yt~miczl]y ~ ~ ~tigens in c~fix~, mol~lar distillali~. ~, si~i~{ 4-HH~ein M~ fs~lins w~ o~, ~ma~ly to t~ ~~HI ~ ~ ~m~ ~D c~ ~ 71~ 4-HN~ein tlv~ ~ ~6~ B~,l-~x-~itive. while ~lla from t~ ~I fal~r f~ ~'~ a~ ~'s ~Ib, ~g~ively, ~ d~mted thai 4-HN~ 222 SUPPRESSORS OF OXYGEN M~TABOLITES FAIL TO REDUCE VEIN GRAFT INTIMAL HYPERPLASTA Objective:. 'To evatuale the rok~ of reactive oxsen metabolites h~ the Initiation of int;ma{ hypcrptasia of vein sxafls insetled into the arledal circulation. 223
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.~etttng: University pathologic re~earch laboratory. Animals: Spfague-Dawley rats. |ntervention.. Animals were randomized to receive either the xanthine oxlda~ inhibitor allopurinol, the iron chelating agent stsreh-e~jugated dcremxam;ne, nr the I~ee-fadi¢~! scavenger 2 l.aminosteroM U743gPG. Control animals were included for each Src~. Tho epigastrlc vein was inserted into the right common femoral artery. Vein ~lt'ls were harvested 30 dlys po~toperatlvely. The degree or intimal hyperpla. Sis al ~ two |nastomo~cs as well as al the mldgrarl was calculated. Main Oul©ome Measures." The vein gratis were divided into thr~ sections designated proximal an~omo~is, mid~,'afl, and dislal anislomosis. Intimal and medial are,,s were determined in ~n ol~'~ver-blind fishion and eXlXe.~.~ed &~ intimal Bruits: Pretre/Rrn~t of the animals with any ol'lhese agent.~ resulled in no nificant reduction in the degree of intimal h3~-~rplasla in any treated groups parod with the eonlrol animals 34) clays postoperatively. Conclusions: Anerbl reconslruction often involves inlerlx~ilion of vein menls into the allertal ¢ireulalion. These veins are ~hjccl !o i.~ehemla and relX:d'u. llon. with Ihe ix~ential for generation of re•clive oxygen reel•hollies and vein I~aft injury, resulting in intimal hyperplisia. We h¥1x)lhc~b, ed Ihel live phan'nacolog;c intervention either to ~aven~o or to reduce Ibe pr~uctlon o1" t~..ctive oxy~,n met•boll:ca would ,qtenu~e the inillal vein graft injury and Ihus limit the subsequent developm~t of inllmal hyl~q)lisia. Tl~ese data create doubt is to lhe iofluen~ of reacliv© oxygen m~ta~x)liles in Ihe initlalkm of inlimnl hyp,.'rpla- si• in the vein graft. G•~es. ]. D., Hir~ch, G. M., and K•rnow;ky0 M. J. Arohives ol'$ur~ry 130.'97~-0R0. Selxember 1995. Other supp~l: The Upjohn Co.. Kalamazoo. MI. From the Depanm~ma or Surge:T and Pathology, Harvard Medical School, Brigham and Womon°s Hospital, and Massachusetls General iIospil:d, I~o~ton. THE KINETICS OF THE OXIDATION OF L*A$CORBIC ACID BY PEROXYNITR ITE iamoxynltdre [•=NO•-, oxoperoxonitrale(l.)] is a strong oxidant thai may be formed i~ t'h,o by the reaction of O~ and NO'. Oxoperoxonitfate(I-) reacts wilh molecul~ |n aqueous acidic ~olutions vll pathways that involve the highly reactive ~ti~ve q~oxopcfoxonitfare either as •n intermediate in • firgt.order reaction or as a nt in • simple ~¢~gl.order reaction. ESR experlmonts show that hydro- g~tl oxoperoxonilrate oxidizes monohydrogon c.|scorbate by one electron: when mixed It pH ca. $ and pa~¢! through • flow cell within 0.1 !, the Iwo-line ESR slg- nil of the ascorbyl radical anion (a~ = 01R T. g -- 2,00~) is ol~-rved. The overall slolch|ometW of the reaction wis I sol ota~orbate oxidized per sol of oxoperox- • nitrate(I-) added. The kin•lies or lhe reaction were studied over the pH range 224 4.0-7.5 by stoPl~ed.l'low spectrometw. Hydrogen oxoperoxonimtte oblcr~d ~00 and 350 nm, ~md the oxoperoxonilfare(l*} anion at 302 rim, di~ppl~ tatar predicted for the first.order isomerlzation to NO;. The rate incRa~s ~ pH 4 to $.8, and then decreises with increasing pH. The fate veal•don Ix&pats a him•locals•' reaction either hetwzen the oxot~roxonkfate(I.) anion and aseor~ acid or be:worn hydrogen oxoparoxonitrate and the mo,~ydrogen ascorb|to anion. Although the two pathways arc klnctically indistinguishable. Ihe pK, vaJues or aacorbic acid and hydrogen ox~roxonitmte strongly sng~es~ that tl~ reactin~ speclcs are oxoperoxonitratc and rnonehydro~n iscor~te. The second-ordor ra~ constant for this reaction is 235 -- 4 M"s" at 2YC TEe enthalpy and entropy of activation All* = 9.3 - 0.5 Iccal/~mol end ~$* ,= -16 ¢ 2 ¢al/(mol K), t~l~etlvely. Bmlett, D., Church, D. F., Bounds, P. L., and Koppenol, W, Fr~ Radical Biology & Medicine ! ~( ! ):85-92, ! 99.% Other suppml: Na¢ional Institutes of Health. From the 'Depanmems of Chemls,y and Binchemit~'y and Bl~ynlmi~l |nlthut•, Louisiana State University, Bstor~ Rouge, LA, and l~pmm~nt of Chlmlstt~ tad Physics, 5ontheistem Louisiana State Un|vers|tyo Hammond, LA. OXIDATIVE STRF_.S5 BY MENADIONE AF'PECT~ CELLULAR COPIER AND IRON HOMEOSTASIS Menadione produce~ DNA strand breaks (DNA sh) in cultured Chinet~ fibroblnsts which are+ Io • greet extent, mediated by OH radJeM. A I~l|onable hypothesis is that H~O~, | prod•el of menadione metabolism, rca¢ls with nt}elw Iforl and produces OH r~dical in sil~. C~onslstent with that, l,lO.~enlnth~dlne prevents menldione-Jnduced DNA 15 at low (< 200 I~M) eo~l¢~ltfatlofll of 11io chelator. However, at higher PHEN concentrations, the •IT•el is revcrl~l a~ld l~i enhancement o1" DNA s5 ~ oil',•fred, The PHEN-]no~ce~ elthan¢osl~ of DNA becomes mor~ evident at high (> 60 p,M) men•all•n• concentrations prevented hy he•Cupid•lee (NEO), an ef~cient copper chelator. How~v~t'o olTers only • slight protectio~ apt•st DNA s5 caused by mcrmdlo~ •l~le, '111o results are consistent with the followlng events: (i| the pre~uets metabolism causes copper ion release from some ~ellutis ¢o~pmlme~ (11) in presence of PHEN, • Cu(I~IEN)~ comldex is forme~, (iii) the Cu(PHI~. compline known to be ve~j claslogenle, inducing DNA damage in • n~luei~ mvltom~nt, Evidence is also presented thai memldione metabolism e'autes an tort•cellular ¢hebtahte iroo: in the presence of | ~stamt 2,3°-dilP~ tlon. the DNA sb Weduced by i~ing COOC~Wat~OllS Of men~Klion41 gressively less susceptible to inhibition by the chela¢or. Therefore the DNA dmnage m4ginated from menadione metabolism s~ms to cauw.d by I.wo con,~ugated and synefgist|e e,v.enls, vix., the pt'eduetlon of.m.antlv~ oxygen spec,es and the release of ec~p~ and iron from a cellulw stora~ s|t• Into • 'free' from pool, capable of catatyziog DNA damaging reactions. 225
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0 0 0 HYDROGEN PEROXIDE AND DNA DAMAGE Very dearly F~ ~ play m imlXXlmt role in DNA dam~ ~~ ~ ~.l~s.it ~. ~ w;. ~,l) ~s .~ ~ .. O~ ~,. ~i~ ~e~ DNA tests may ~nlt[ate m~i~ ~ ea~;~n~ ~ ~n s ~xt it is ~c~y th~ epi~miologi~! st~ve ~w~ ~y i~ st~s ~ ~ or a~r (Stevens e~ establis~ why and ~w Fe(ll) ions ~h t~ DNA. ~s to ~.h~dled ve~ ¢~fuily ~ w~M ~ ex~ su~ a vital ~. In: A~, O. T. a~ ~alli~ll, B.. (~s.): D~A and F~ Urn., ~, ~ 83-93, ~h~r su~: FAP~P F~ lh~ Institute o~ ~hemist~. ~pa~ment o~ ~i~hem~t~. Unive~ity of Sao CELLULAR DNA DAMAGE BY HYDROGEN PEROXIDE IS A1"FENUATED BY HYPOTONICITY Chln~¢ hamster filxobllsLs (line V?9) withsm~d well exposu~ for 30 rain to h~x~lic medium. ~ing to 259~ physiological phospbate-~ff~,~l sallce (PBS). Under these conditions, the cells become resistant Io two eff~ls or H,Oz: DNA dam~e and inhibition of cell clone formation. "l'Ke nom~l s~nzilivily Io lhc DNA..d~m~|nI attica of H+O+ is r~tored if. after exposure to hypolmic PB$, th~ oel~ ar~ |ncld~lled in isotonic cell, culture medium. However. f~t0~tion ol's~siliv- hy is mx obl~n~ed on incubation in botonic PB$. The normal ~ensitivity !o H~O+ is IlSO RsloR~ it one of Ihe tollowlng reducing -gents is added to hypotonic PB$: 226 XII. Virology
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Chow. L. T. ~md Broker, T. R. Ime~irology 37:150-t~8. 1994. O~hcr s,ppon: U.S. P, blk: Health Service. F~n the Dep~rtmcnt of Biochemistry and Molecular Genetics, Schools of Medicine ~ Dentistry, University of Alabam* at Bim~ingham. Birmingham, AL. REGULATION OF IIEPATITIS B VIRUS ENI ENIIANCER ACTIVITY BY IIEPATOCYT~ENRICllED TRAN.~CRIPTION FACTOR IINF3 Heights B virus (IlBV) EN! enhancer can ~c~ivaee the expre~ioe or IIBV non-tlBV ~ in n liver-specific m~nner. Fly performing the etec~rof~m~elic mobil. it.v~hif~ a~ays0 w~ demo~raled that the three relaled, Ever-enriched, fl,c~r~, HNF3e~, HNF'31~, ~ HNF3'y ~xdd all hind to the 2e site or the HBV enhancer. Mutations introduced in the 2~ site to abolish the binding by HNF3 red.~! the enhancer ~ivity approximmcl.y 15.I'o1£ Moreover, exixe~on or HNF3 antilew S~lUenc~s Io ~ Ihe exprl~lsloe or HNF3 in lluh.? he~tom~ cells led to reduction of ~he ENI enhancer ~clivlty. These results iedic,~e th~ HNF3 po~ivel). regulates th~ ENI enhancer ~:tivlt)" and this regulation is mos~ llkel¥ medlate..d thn~.~h Ihe 2c site. The requlremen! o~" IJNF3 1'or the ENI enharmer saivity could cxi~am the liver spectralW of this enh,,n~r clement C~en, M. Hieng, S., Qian, X., Costa, R., ~nd Ou, J.-H. Viro1~y 20~:127-132, 1994. O~h~" supporl: Nslionsl Inslitutes o1" From the Dep-nmem or Microbiology, Univer~iW of Southern C,llromia, Los Anleles, CA, and Dep~rlment of Biochemklnd, University ot" Illinois College or Medicine, Chic~,o, IL. AID~: VIRUS OR DRUG-INDUCED? We have seen above thut the Drug-AIDS hypothesis correctly predicts ~ o£Amcrlc~ AII~. On the otha- h~u~! the HIV- hylx~hesis h~s no~ pmdletod AIDS toc~ctly, me~e impo~ant, it his f,,iled to produce any decrease in mo|ldlty, cure. m a vaccine. AlthouBh pro~ble, the D~-AIDS h~othes|~, until expeHmon~l~ proven, remains an intelle~.~l ~'~stmet. Because o1" the number or I~tients sufferin~ from AIDS. the imp}i~tlons o1. this hylmlhesis In Public tlcallh. 225 ~nd the potential for prevent;on of new AIDS cas~, testing t.~ drug hyp~h~is should have a high priority. Becsuse this hypothesis m~es verifiable I:~t'edi~llons. exp~r~m~.mt,,I testlnl! is regime and Ixxctical. ~ug toxicity tin be tested exp~rlmentally in animals, ~d In human cells in tissue cultme. In ~dition, drag toxidty can b~ les~d eptdemlololi- ~tly in heroins who are ~ddic~ed to reeremioeal drugs, te~s ~n ~ c~d~ at a mall £~i~ of t~ e~ ~ in ~ HIV h~s~ If t~ d~ hy~sls ~cs to ~ c~l. AI~ ~Id ~mab~: [~m~. that ~ ~ c~~ of ~zu[~ c~n. ~ th~h Impel ~ AI~ di~ f~ I~ir ~h milers. Ka~i s .~ with ~l with ~ nul~l~. ~ th~ t~t e~h of ~ ~ll~ill~ A~. ~ ill,it drop. ~ to ~fi~ ~ to its ~e ~r s.~: ~vzte ~zt~z. F~ the ~pa~ment or Mo1~ular and Cell Biology. Unlve.tty or Be~oley. CA. FOREIGN.PROTEIN-MEDIATED IMMUNODEFIC~ENCY IN HEMOPHIMAC~ WITH AND W]~tO~ HIV ~tein.AlDS h~sis t~ I~ Hum~ lmmun~r~ic~y V~i c~me~ul clotting ~tor VIII cau~ tmmu~u~si~. ~e f~n~tei~ ~l~m, ~ in t~ !~, ext~ t~W llv~ ~ simultmtly d~m~ s~in, ~My ~m~ ~ w~ t~m with t~ c~k mi-HIV ~l t~ hiJ~ 5~5 ~ud risk ~ i~m~ ~g u~ ~ male ~z~ ~ disi~ drag u~~ ~nsruCm ~ f~ign ~elnt ~ !~ imm~p~t- 229
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0 0 g'l r'0 slve than rec~,eational drug use. 3) The age bias or hemophilla-AIDS, i.e. th*t the annua~ AIDS risk increased 2-fold for each 10-year increase in age~becausc immuno~uppreraiee is u function of the lifetime dose of" foreign proteins received from tr~sfusJons. 4) The restriction of. hemophilia.AIDS to immunndeficleney di.~- elses--heeanse foreign I~o~e|rm cannot ~use non-immunodeficicncy AIDS dis- el~s, like Kapc~i's sarcoma. 5) The abson~ of AIDS diseases above their normal background in sexual par[nets or hemophillucs~bocause transf.uslon.medlated hemophdtucs~because fo~.gn pnxelns, nm HIV. suppress their immune sys. tern. 7) Stabiliution. even regeneration, of" immunity of. HIV-positive hemol~hiliJcs by leng4erm armament with pur~ f"uctor Vlll. This shows that neither HIV nor f"uctm VHI pi~ H|V are immunosupsx~sslve by themselves. Therefore. AIDS cannot be Ix~ve~lod by elimi~tion of HIV from the blood sul~y and cannot be rationally treated with ganotoxic untiviral drugs, like AZT. Insleud. hemophilia-AIDS can he IX'eVemed and hu even been reve~cd by treatment w~th pure factor Vlll. D,,~berg, P. H. Ge~tk~ 9S:$1-70. 199S. Other support: Private Donatlom. I~rom the Department of Molecular and Cell Biology. University of Callfomla at Bedcctcy. Berkeley, CA. DNA RI~OMBINAT~ON IS SUFFICIENT FOR RETROVIRAL TRANSDUCTiON On~enlc rotrovlruses can? coding sequences that am transduced from ¢¢llulm" Ixotoom~c~-s. Natural transduction involves two nonhemoIogous recombinations and is thus extremely r~e. Since transduction has nevm" been rep~d~ced experimen- t~lly, i~ mechanism has been studied in terms oF two hypotheses: fi) the DNA mod~l, which postulates two DNA recombinatkx~s, and (ii) the RNA mockl, which puatulates u $' DNA mcocnbinat|on and a 3' RNA recombination eccun'in~g during mvc~se tmu¢l~io~ of" vbal and prot~ RNA. Hem we use two viral DNA oonatz~u~ to test the prediction of the DNA n~lel that the 3' DNA recombination is achieved by e~nvmlional integratien of a rotmviral DNA 3" of the chromosomal Frotoonee~ene ~ding rogim~. Fm the DNA model to be viable, such recombinant viruses must be infeeticm without the puqxxtedly essential polyporine tract (plx) ~r~the 3' long termirml repeat (LTR) of all rctroviruses. Our constructs coding re,cane horn ll&vey samoma vires which is na[uraly linked at the $' end to a ref~vkal L,TR ~ ~ificiatly linked at the 3' end either directly (con- sU~'t NdN) ~" by • eollolar sequem~ (eontu'u¢~ SU) to the ~' LTR of a retrovirus. • xh ~ lack the ppt. and the LTR of NdN even lucks 30 nu¢leotides at the $' end. ~ oomtn~s proved to he inf.ec~ious, ixoducJng viruses at tilers of. 10~ focus- formln~ units per mL Sequence analysis proved that ~ viruses were colinear with input DNAs and that NdN virus lucked • ppt and the $' 30 rmcleotid~ of the LTR. 230 "The results indicate that DNA recombination is su~cicm rot ren'ovtral ~ that ~ither t~ ~ ~ L~ c~plete L~ Is ~tiaf f~ ~vlms ~l~l~, DNA ~mbinat~n explains the rollow;fls o~e~i~s by ~if~ with t~ RNA m~el: (i)ex~fimcntal ~c~ging e~c~y or vi~! RNA. a~ (ii/ex~lal ~s with ~s~t ~o ~. ~ ~ f~ ~dlngs oft~ Nzti~al Ac~emy o~ Sci~ces USA ~:24~2~ M~ I~. ~m I~ ~pz~men~ o£ Molecular and Cell Biolo~. Univ~izy o~ ~e~y. CA. CHIMERIC PROTEINS COMPOSED OFJUN AND CREB DEFINE DOMAINS REQUIRED FOR INTERACTION WITH THE HUMAN T-CELL LEUKEMIA VIRUS TYPE I TAX PROTEIN The ~gulation of" human T-cell leukemia vbus t.)~e l.(IT11,V.,.l) k~g ten'nln~l r~eat gcne cx~s,~ion is dependent on thm~ ¢i~-uchng cl~11£rlts IUIOW~ •~ 21-bp repems and the Iransuctivatm" protein "r~x. Muttgencsis hat demonstrated SC~lUenCeS in e~ch ol" the 21-I~ repeats can be divldcd into three dcm~*~ns d~sllnated A. B. ~ C. ~x stimuhtes t~ bi~i~ of~EB to t~ B ~uin. whi~ ~ e~li~ ~ ~x ~iv~i~ o~ HTLV-! ge~ ex~si~. In ~is sl~y, ~ de--re Tax will stimubte I~ binding ~ CREB to I~ ~V-I 21.~ m~ls stimulate CREB binding to the c~sus ~lic AMP ~ e~nt (~) ~t ~ in ~ ~mat~t~tin ~er. H~v~, ~x stimul~ CREB a ~sus ~E in l~ ~text o~ t~ 21-~ ~ts. i~atlng ~ I~ s~u~s in stimulating CRE8 binding. ~ d~i~ t~ m~hanism by wh~ ~x stimulates CREB bind~g t~ ~i~ ~ti~ inte~l~t ~x ~ ~EB. we ~ t~ mtmmal~ t~hy~d s~ in ~ with ~tro bi~ing ~ gd ~ta~li~ ~ys. ~h~ ~ J~ic~ ti~ in either the ~sic m leuci~ zi~ ~gi~ oF ~EB ~vem~ in--liOnS with Tax. Sin~ ~veml stud~ have ~t~ th~ T~ will ~I~ bi~ing of u va~ely o~ diffem~ ~s~ mg~.le~J~ zi~r ~ins to t~lr ~i~ f~ int~ti~s with ~x, ~ ~tes ~m ~SOle ~U~ in ~ ~ in ~1~ interims with Tax ~ tn~a~ b~ing ~E~ to t~ .I- ~ ~ts in ~s~ to Tax. ~ ~ ~fi~ t~ ~ins ~ ~h i, ~ a~ in ~.it~ inle~i~ ~ t~ ~V-I Tax ~in. Yin. M. ].. Puul~n. E.. ~ich L. ~ ~nor, R. ~ 231
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0 [,.q Journal of Virology 59(IO):~20~.6218. October I~. ~ ~: Hati~al Institutes of Health, From the ~pa~mcnts or Internal Medicine and ~croh;ology, Divisions or Mol~l~ Vimlo~ ~ H~tolo~-~lo~. Univ~sity or Texas S~thwestem M~! ~mer, ~lfas, ~. PROTEIN DOMAINS INVOLVED IN BOTIi IN WVO AND IN VITRO INTERACrlONS BETWEEN HUMAN T-CELL LEUKEMIA VIRUS TYPE I TAX AND CREB Gcn4~ ¢xpre~ion f.rom the human T-cell leukemia vires ty~ l (HTLV-I) IonB terminal repeat (LTR) is mediated by tlu~e dx.acting regulatory elements known as 2|-bp sepcats and the transactivator pro(ein 'lax. 'T~ 21-bp repeats can be subdi- vided into thre~ motifs known as A, B. arid C, each of which is imlxxlant for maxi- mal ~eSle expression in response to Tax. The B motif.contains nucleo4id¢ sequences known as a cyclic AMP response element (CRE) or t~x-response element which binds members of. the ATF/CREB family of transcription Fatima. Though mutalinns of' this element in the Frf.LV-! LTR eliminate tox activation. Tax will not activate most other Ixomoters eontainin~ abase CRE sites, ira this study, we invc~tlpted thu mechanism by which Tax actlv-tes gcn¢ ©xpresslcm in co..iuncti~m with mcmh,~s of the ATF/CREB family. We f.ound that Tax c~hancnd the binding of ono mcmher of • the ATF/CREB family, CREB I. to each orthe tl~ HTLV-I LTR 21-5p repels but oct another" member designated CRE-BPI or CREB2. 'Pax enhanced the binding of CRI[BI to nonpafindromic CRE binding sites such as those found in the HTLV-I LTR, but Tax did nm enhance the binding of CREBI to palindromic CRE binding sites such as round in the somatostatin IXOmoter, This findin$ may help explain the failure of. Tax to activate promoters containing consensus CRL .,dies. Thc-,,e studies were extended by use of. the mammafiam two-hybrid system. Tax was demonstrated to interact dim:tly with C~EB I t~ no( with otber bZIP ~roteins, including CREB2 and .Tun. Sit,:.directed muta~p-~-sis of both Tax ~ CREB! demoustrated that t~ amino terminus of.Tax ~cl bod~ the basic and the Iouclne zipl~r reSicgs of. CREBI were required for direct interactions between these pro(~ins bo~h i. ~t~ and [n ~(tro. This interaction o0corrnd in v/vo and thus did nm require the presence of.the HTLV-! 21-bp ~:ats, as previously suggested. These results d~finc the domains required for inter~ction between Tax and CREB that are likely critical for the activation of HTLV-! geu¢ expression. Yin, M.-J., Paufssen, E. L, Sueter, L S., and Gnynor, R. B. Journal of" Virology 69(6):3420-3432, June 1995. R'0m Ihe Divlslon of" Molecutar Viroh'~y. Dq~arlrnenL,~ of Internal Medicine and Microbiology, University of'T~xas Southwestern Medical Center. Della,q. TX. 232 HUMAN IMMUNODEFTCIENCY VIRUSES R~OULATED BY ALT~R~ATI%'Ig TRAN$-ACTIVATOR$: GENETIC EVIDENCE FOR A NOVBL NON- TRANSCRIIYrlONAL FUNCTION OFTAT IN VIRION IN'PECTIvrI"Y Thirteen genetically sllered HIV.! proviruses we~ creatnd. Thesa various gcnon~s can be Rgregatnd in(o three grou~: (i) i let of tot(-) vL,'l~m ~ hew a functional HTLV.I 'PaX inserted in ~. (It) ase(Of.tat(-) virules with 131}4 bindin$ s~s ;nsefled in U3 and a Gal4--VPI6 eDNA inserted in m,~ and (ill) a ~t FiiV geuomes that are S' and 3' TAR(-) and are GaM-binding-slta(+) in U3 GaI4--VPI6(+) in m,/. We found that elm,s in ~oupc (i) ~md (II). although were fully .co~. ple.m~, nted foe .~ira! gene exi~ession bm, ed c~ quantltalive rn~utu~ merits of v~al ffotem synthes,s and on the visca|izallon by ¢lactron mtcnxu~opy the proper ar~embly of' morphologically co~Rct virlons, lntersstln[:ly, group (I) and (it) virions were defecllve in a al~ding eytopathic infection when aslaysd in T- lymphouytes. Group (ill) vlmses, although capable of ixoducin| imact Tat a~o could no~ use Tat fmt rany¢filxio~-,ne expression because el" the TAR(-) gano- type. Ilowever. this class of viral ~enomes produced vlm~ that were hi, My inf,:. flout and eytopsthic in primary and in ee~tlnuously prepared T-lympheeyles. These three gronl~ o1" viro~ are all tran~¢r~tlonally Tat--TAR indep~nde~. Thel~ distinct differences in infe~tivity/Cylopathlcity pex~id¢ ~enetic eviderK~ that 'Pat vi~es a lraw,.,crtpflonally iz~,~ fon¢lion in detemzining infaetivlty ~ eyto- puthiclty in the setting ol'a alxcading viral infection. Gives that all HIV virions n~,. ma~y contain fou~ intact copies Of TAR RNA, out" findtnp suggest a I~.examtn|tion of whether Tat could he a vi|~(m,.TAR.assoclated pro(sin md the po~sibla Imptica- lions of this for virus inf'ectlvity/cylopethlclty. Huang. L.. Jo~i. A.. Willey. R.. Ore~stein. L. and ,/eunf,. K.-T. The EMBO Journal 13(12):2~,~-2~96. 1994. Other st~xl: National Institutes of Health. Fsom the Molecular Virology Section, Laboratory of' Moleeuhw Microbiology, National Institute of" Allergy and Infectious Diseases, ]Bathulda, MD, and Department of" Padmlo~,, C, eor~ Washinltou Univm;t~, Washl.~to.. IX:. GENE THERAPY FOR HUMAN IMMUNODEF~CTENCY VIRUS GENE~IC ANTIVIRAL STRATEGIES AND TAROET~ FOR INTERVEN'IION Oeee therapeutic strategies for the Irea~ment of hum~ immunodefl¢len~y type I (HIV- I) infection have received inctessed ~emion due to lac~c orc~moth~r- apeutic drags o¢ vaccines that show l~.te~n efficacy !~ v~vo, .~m ¢n~rlinI rofefTnd to ~ u "genetic antivirals," is reviewed, Oem41c antivira]a DNA or RNA elements that are tfa~si'ennd into cells amd affect t~4r tarots either dlr~:tly, or after exp~c~ioo as RNA or ixoteins. 1'~ in¢1~le a~nthen~ oligonuclcotides, ribozymes, RNA decoys, transdominant mutants, toxins, immunoeens. Tbey olTer the po~ibility to .tsr~ simultln~ousl.y m~lti.p.i~ sit~. in IIlV gccome, thereby minimizing the produclzon of" a~istant vmt~, we review
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molecular mechanisms of genetic antlvirais, their HIV mo;ecular targets, and di~uss issues concerning their application as anti-HlV agents. Dmpulic. B. and Jeang, K,-T. Hurowl (~,ne Therapy 5.~27-939. Aught 1994. Other suppo~: National Institutes of Health. From the Molecular Virology S~cllou. Laborarory or Molecular Microbiology, Nadouz] ]mtitutes of Allergy and Infectious Di.,~as~. Natlonsl Institutes of Health, Bethe~la, MD. F.XPRE~$ION CLONING OF GENF~ ENCODING RNA-BINDINO PROTEINS RNA-binding proteins are important regulatory factors in gene expression. Studies on the structure and function of these proteins are complicated by limitations in the technology for their isolation. One approach is to pmify the pro~in, to per- form micrm~uencing of amino acids, to ¢o~slnct decenerate oli¢onuelco~ide~, and then to zemon libr~es. An ahemative to this is to isolale exlxe~ion cDNAs direcdy. Here, we deu:ribe a method to isolate cDNAa encodi~ RNA-binding Ix~eine, using a direct screening assay for RNA-prmein.binding interactions. For this purpor, e, we modiC,~ the proc~ commonly tm~d in isolating cDNAs encoding sequenee.spe- clflc DNA-hlndln~ proteins. Us~g this ixotocof, we have isolated • 1.5-kh eDNA thlt co~s ~1" a humln protein th~ hinds to the human immunoderK:icncy type I (HIV-I) T~-roq~onse element (TAR) RNA. Modifications lind derails of our tcch- nlque are presented below. G-tiBnol, A. and Jea,~, K.-T. In: Methods in Molecular Genetics, Volume 4. Academic Press. Inc., pp. fg-28, !~)4. Other support: National lnstltutcs of Health. Prom the Molecular Virology Section, Laboratory of Molecular Microbiology, Natlen~! ~nstltutes of Aller¢y and Infectious Diseases, National Institutes of Health, Bethe~a, COMPARISON OF RBGULATORY FEATURES AMONG PRIMATE LENTIVIRUSES This review hi~hlights the general similarities and some specific differeoeez in th~ regulatoP/mechanisms u.,cd by the palmate lentivimses. From the perspective or 234 the regulatory proteins Tat and Rev and their respective RNA tarVtl. TAR aed RRE. one can conclude that much is c~ ~ HIV-! f~t that n~-~i~al effe~s have ~n ~ ~inf~s t~ J~a that t~m ~main un~ ~n~s i~ivid~i~ to s~i~. ~ cle~ a~e~ of the ~c~ ~ ~ in t~ ~V-~W s~p it ~istent with this. Having stn~d t~ n~ve, it is ¢le~ th~ ~IV, wilh I~ a~iltt~ to inf~t ~" cells ~nd to cause AIDS in m~k~s. fm in ~ivo HIV-! ~tho~esis. ~ ~nt el~idat~ of dete~inants for SIV (DEw.uR~ et aL I~; K~ ez al. I~lt 0~ ¢t ~1. 1~2~ ~ et hi. 1~3) ~ould ~o ~ I~g way tow~ u~tanding of HIV- 1. Jel~R~ K~T. ~ O~tlgn~. A. B~in.Hei~rg. ~. 123-1~, 1~4. ~r ~p~: N~li~al ln~timten of H~lth. ~ lhe ~Io~ ~ Mol~.lm Micmhlol~. Inr~t~s Di~s. N~ti~l Insti~m~ of llcallh. BelUga. MD. DEFINITION OF A MINIMAL ACTIVATION DOMAIN IN HUMAN T-CELL LEUKEMIA VIRUS TYPE ! TAX Fou~.cn mutants were used to delineate a minimal activation domain in the Tax p,~el, or hem,. T-~ll I~.kpi, ,~ t~, ~. ~,..~, ,~x..usi,s • o,z~.-.'~.. fusion protein and a respemwe promoter onmammg ~x~ consemus mnola8 tlt~. we found that activation was "squelched" by coex .i.x~. ~lou of wild.type ~ in Irons. When Tax mutants were tested ~ squelching, many..c~.~ed eruptiVely against GaiTs. However, those @raining changes in amino acids 9~9 to 32 l'alted to inhibit activity. In particular, three mutants that were expsls~l}d stably, with changes at amino acids 289, 2~6, and :320 respectlvety, did not squelch GaITs activl. ty. On the other hand, mutants with indlvlduat changes at amino acid 3, 9, 29. 4t, 2"73, and 33"/ef~¢ientiy inhibited Gal'i'x function. ~ other mt~tlmts failed to stably expressed. In separate exix.'~|ments, when fused alone to the DNA-blndin$ do~aln of GAS4, amino acids 289 to 322 of Tax coof'crred fr~nx activation ability. This fusion It~oteln was able to activate a core pmmo{er. These t'mdi~gs su~s~ that amino aelde 299 to 322 define a fegh~e that cordacts am essential tnm~cdp41oe and that this reg|on is • modular activat|on domain. Semmes. O. L and Jeartg, K,,,T. Journal of Virology 690):1$27-1833, March 1995. 233
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0 0 Other support: N~li~al Institutes of Health. From the Molecular Virology Section. Laboratory of Molecular Microbiology, Nadonal Institute of Allergy and Infectious Diseases. P.ether, da. MD. GENETIC MAPP/NG IN HUMAN AND MOUSE OF THE LOCUS ENCODING TRBP. A PROTEIN THAT BINDS THE TAR REGION OF THE HUMAN IMMUNODEFICIENCY VIRUS (HIV- I) Productive infection with HIV.I, the virus responsible for AIDS, requires the involvement or host cell factors for completion of the repl~catlve cycle, but the/den- tif~tion of these factors and elucldalion of their specific funcllons has been dill- cult. A human eDNA, TRBP, was recently cloned and characteriTed as a podtlve rellufator of gone expression thai binds to the TAR region of the HIV-I 8cnome. Here we demonstrate that this factor is enc'ud~ by a gcoc0 TARBP2, that maps to human ©bromosome 12 and mouse chrommome 15, and we also ider~ity end map one human pseudogcne (TARBP2P) and two mouse TRBP-related sequences (Terl~.rsi, Taebp2.r;2). The m~p lecatio¢l of the expressed gone identifies it ~.,~ a cmdfd~le fo¢ the previously identified faclor encoded on human chromoecome 12 that has t~en shown Io he imp~l~t for expression of HIV.I genes. Western hlatting iP4iCateS that d~pife h~h s~u~nc~ coOsel"Valk~l in human and rm~se, f/~ TARRP2 protein differs ~ apparent size in primate and rodent cells. KocIE, C. A., Oazignol. A.. Graham. K. Jcung, K.-T.. and McBHde, O. W. Genomios 25:(~-72. 1995. Other support: National lnstlzutes of Health. Prom lee Labocztory of Molecular Microbiology. Natkmal Institutes of Allergy and [nfeclious Diseases. and Lalx~atory of Biochemistry, National Cancer Institute, National Institutes of Health. Bcthesda, MD, WHAT REASSORT~ WHEN REOVIRUS GENOME SF.G~fENTS REASSORT? Reovlms ~ermme segment reasso~tmcnt is cloudy a complex phenomenon. On the one hand, vastly different pnomc segments are readily steepled, like the ST2 SI genome segment into the ST3 ~cnnme: it and the ST~ 51 gcnome ~gmcnt have diverged ~t9% towel randomness in their r, eennd Ease cndon p~id~ms (8). Them is. howev~', a requirement for the exchange to he effecled: and feat is two mutations in the ST'] $4 pnome segment, These s~me two mutations am required for lee acccr~ umee of|ll ST2 gonome segments introduclions imo the ST3 gcnomc, including that of the $T2 M2 genome segment, the second Ease codon position of which has diverged to randomness from that of the ST3 M2 ~enome segment by only 2% (8). 236 AN RNA.PROTEIN CONTACT DETERMINED BY 5.BROMOURIDINE SUBSTITUI~ON, PHOTOCROSSLINKING AND S~UENCINO An analogue or the replkaL,~ traeshdionM operator of boctedophlle RI7, thai ~tains a $-bromoorldlne ut position -$ (RNA I), ¢omplexe~ with a dlmer el" tM coat protein and ph~ocrosstinks to the coat protehl in hif, h yield upon excitation 308 nm with • xenon chlork~ excitant las~'. Tryptic digestion of th~ nocleopruteln complex followed by Edmla degradation of the u3q~ic fralment bear. ing the RNA indicates crosslinklng to tymdne 85 of the coat protein. A control experiment with • Tyr fI5 to Sue 85 varimt ceut protein showed binding but no c~ossllnkln~ at saluratlng pm~in ce~entrltlon. This is consistent wkh the tlon from model compound stymies of prefeRntlal pholocro~llnk~nl¢ of BrU to the electro rich aromatic amino acids tP/l~ophan, lymsine, and hlsddine with 30fl nm exc;tal~, Willi~, M, C, LeCuyer, K. A,, MeiRnheimer0 K. M., Uhlenb¢ck, O. C., and Nucleic Acids Rcseapch 22(Z1):4947--4952, 1994, Other support: Natlenal Institute ot'Genefal Medical From the Department or Chemistry and Biochemistry, University of Colorado, Boulder. CO. 237
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0 0 A~IATION OF MOUSE FIBRINOGEN.UKE PROTEIN WITH MURINE HEPATITIS VIRUS-INDUa~D PROTHROMBINASE ACTIVITY Previously. we clemonstr~led induction of u unique macmphage prothrembinzse during infection of BALB/~J mice by mour.e heNtitis virus strain 3 (MHV-3). By immunologic screening. • clone representing Ibis p~lhro~hinasc was isolated frmn • cDN.A libr~y and S~ZlUenead. TI~ .,~qucnce identified this circe a.~ repm~nting pa~t al| lene. m~.-~/Vp. Ih~t ,.n~ndes • fibrlnn~cn.like protein. Six ~'f(fitional cl~s were isc~ed, and one clone, pl I-3-1. encompass~l the entire ¢~Jing region //trip. Murine macroph•~cs did not constilutivel), cxpres~ ~.q'/AIp hut, when inf~ed wilh MHV-~, synthesized m~#~.q~clf~¢ mRNA. rmt¢/~/p mRNA induc- llo~ was euriler and si~nifican0y ~reater in B~/~] than ~/J m~cmpha~es. ~h~bln~ ~dvky w~ ~m~slrat~ w~n musfiblp wax ex~s~ f~ 3-1 In RAW ~.? cells. ~e~ d~m su~st that m~.~ enc~s ~ MHV-I~u~d ~h~bln~. P~. R. ~. ~ng. ~.. Rc~r. ].. M~e~-M~. N.. ~i~wit~ J. ~ ~ ~y. G. Other sup~: Medical Rese~h Council o~ Caned~ and Nalion~! lns~hutes of ~ the ~p~ment of Put,fogy and ~ato~ Medici~ a~ ~pa~ment of Microbiology and Molecular Genetics. Unive~ity of Texas M~ieal Sch~l H~sl~, H~. ~. ~ ~a~ment of M~ine. To, to llmpltal. Unive~ity of~z~ T~to. C~a~. PA~ OF DISEASE AFTER MURINE HEPATITIS VIRUS STRAIN 3 INFI~"TION CORRELATE~ WiTH MACROPHAOE ACTIVATION AND NOT VIRAL P~PLICATION Murine hepatitis vires strain ;3 (MHV-3) produces • stmln-depandent pattern or dil~e ~ieh has been used as at model for fulminant viral hepmitis. This study was underU~en to examine whether there was z conclusion between macml~ge activa. ~ton ~nd •usc~ptibility or resistance to MHV-3 infection. Peritoneal macrol~zges we~ bolaled from resistant A/J and susceptible BALB/c] mice and. foSowlng stim- ulation with MHV-3 or lipopolysuccharide (LP$). analyzed for transcription of mRNA md prodUCliOn of interleukin.I (]L-I). tumor necrosis factor alph• (TNF~). transforming ~ewth factor p (TGF-p). mouse fibrinogen-like protein (musfiblp). UsSue fa0tor (T~. leukouiene B,. and prostaglandin E~ (PGE~). Ma©rophag~ from BALWC] mioe Ixoduoed Creater amount• of IL-I, TNF-~, TGF-13. leukotricn~ and nmsfiblp following MHV.3 infectim than macrophages from resistant A/~ mice. whereas in response to LP$, ec]ulvz~ent amounts of IL*I, ~['NF-ez, TGF-F, and TF were pt, eduoed by macsophages frorn both slra~ns of mice. L~vels of mRNA or IL* I, TNF.(x, and musfiblp were greater and more per~istenl in BALBIcJ Iban in All ~, whereas the level• and kinetics of IL-I, TNF-a, and TF mRNA lowing LPS stimululion were identical in macrophages from both strains of mice. 238 Levels of production of PG~ by MHV-3-stimulated macmphlps from r, lsttnt and su~ib~ mice were e~ivalent: ho~er, the ti~ ~ fm indu¢i~ of ~ diff,, but t~ total q~tity of ~ ~ w~ i~uffiei~t ~ ~hlffit i~u¢i~ of musfiblp, a ~mgulmt kn~ to ~Im with ~ve~l of Nlml- ~nt ~t~ ~i~ in ~u~i~e mice. ~ ~lts ~e m~ di~. ~ in ~i~ of inflammat~ ~iat~ ~ MHV. 3 ~f~t~ In m~h~s f~ ~slstant A/J a~ sox~i~e BALBIc$ m~. ~i~ may explain the m~ ~pmlc ~i~ a~ fi~n ~ili~ ~ ~unt fm t~ lethallW of MHV.3 In lu~ ~;ble m~. Po~. M.. Rotstein. O.. Cole. E., Sinclair, S.. Pan. R.. ~z. g.. Finlemtt, R.. ~ung, S.. G~zynski. R.. Fnng. U. ~l~wil~ &,Rao. Y. ~.. ~ ~vy, O. J~mal ~ Virolo~ ~9):5252-5~, SeFem~r I~5. Other sup~d: Medical Rexea~h Cou~il o£ Canada and National Instltutel of Health. Fr~ t~ ~pm~0ts of Su~e~ ~d M~ici~, T~to Hmpit~, Univmlty of Toronto, T~Io, ~t~o. Canada, and ~t of ~t~l~y, Unl~nlty ef Texas Health Scie~es ~nter, H~st~. TX. KEY ROLE OF A CCAAT ELEMF.NT IN REGULATING HEPATITIS B VIRUS SURFACE PROTEIN EXPRESSION Two sel~rate promoters, the upctrcam p~$1 and the downst~am $ p~ccnoterx, give fi~e to transcdpls en~Jing tl.ee forms or Ibe hepatitis B virus surface i~eln. Overproduction of large surface Ix~.eln Ixp~auze of incremed pr~l eanscdpts Imds to a block in sec~tlon of •!I forms of the surface pro~c~n and of virion partklcs. We show here that • CCAAT ©kment in the $ prommcr not only tneretlcs the Imount ef $ transcrlp~s, bet also deceases the amount ef preS! txanscrlpts by up to fl~feld. Consequently, mutations in this element cattse intracellulm" aceemulatte~ of itlrflet proteins becaose of the secretory block. 1'he, fore. this CCAAT elcme~ ~ te be critical for mainl|inlng the high ralio ofS ve£sus preSI trtntedpt! that il nl~** sary for the viral llfe cycle. Lu. C.*C.. Chen, M., Ou, J..ll., and Yen. T. S. g. Virology 206:1 !$5-115~. 1995. Other suppod: National Institutes of Health. From the Department of Pathology, Univenlty of C~lifocnia. San Franelu:o, CA, Department of Microbiology, University of S.~thcm. C~..llf.om_i•, Lmjt.nel,z..CA, and Anstomk: Pathology Service. V=tmns ^,airs MmtCll Cruller. 3In HanClleO. CA. 239
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0 0 PHOSPHORYLATION AND NUCLEAR LOCALIZATION OFTHE I[EPATITIS B VIRUS CORE PROTEIN: SIGNIFICANCE OF SERINE IN THE THREE REPEATED SPRRR MOTIFS Hepstlds B virus core protein (antigen) is an important rerulogic m~rker of heF~ilis B virus infection. 'This protein is found in Ihe cyloplasm or the nuclei, or I~h, of infecled hep~oc~es. A nuclear localization si[nal hes pmvlously been iden. lifted in the core protein sequence. This signal overlai~ three repeated 5PRRR motifs. In this report, we demenstmte ~hat substitution of all of the rerine residues in thee th¢~ SPRRR motifs with alanlne can pmvem alums entirely the I~spho~'la- lion of the ~ protein in Huh-7 hep~oma cells, enhance nuclear Iocali~afio~ of the core protein in both Huh.7 ~ nonhep~lic cells, and alx~lish cell cycle regulatiou of n~lem- loc~lizaden of the core protein. Since the three co~ prmein mut,,nts which retained only one serin¢ reddue or e~ch of [he thRe SPRRR motifs could he pbos. pho~lmed to similar de~'e~ Ihere three reriue residues likely could ser~e ~ the a~el~or s|tes for pho~phorylstien with equal efl'~ieney. These results, together with the observation that the three SPRRR n~clfs overlap the nuclear localization signal of the.co~. ~ prol.ein, .~.ise ~.he pomib.ilky that nucleJr lucalimtion orthe cue protein is nept|ve|y regumted by phc~pho~y~atlon of the refine residues in the SPRRR mutiFs. I.J~o. W. md O~ J.-H. Journal of V|rulogy 69(2):1025.102@, ~chruanl 1995. oth~ suplx~1: N~tienal Institutes of H~slth. • * From the Dqx~ulmen! of Moleculsr Microb~olo~' aM Immunology and Del~rlment of Phwrn~utical Science, University of Southern C~llfomla, Los Angeles, CA. MULTIPLE SUPERINFECTIONS FAIL TO ACTIVATE DEFECTIVE HUMAN ~MMUNODEF~CIENCY VIRUS-I (HIV-I) INFECTION OF RABBITS ~upeHnfection of human immunodeficlency ~in~s (HIV)-l-infected r~bbhs with Treponema pallid~ra. Mycohocterlum avi~m, herpes simplex CandMo sibilant. MycWJlan~ t~(,ognitu$, and malignant catarrhal fever vin~s as well as irradiation corsica trettm~nt, falls to activate production of infectious virus. For up to 6 mon~ ~ter infectio~ of r~bbits with HlV-inrected cells or fre~ vires. [here ~e nei- ther elMJ~al s3mq~'n$ nor po~tive lalxx~tory tests for d~ctlon of HW. Howevor. afar ~per~fec~ien with o~her ~ts, HIV sequences may be transiently found in i~ ~lcod mononucle~ celts by polymeme chin reaction (PCR), ~ multi- I~ antilxx~|e~ to HIV ~migen~ may be detect! by Western bfo~tlng. Bo~h the PCR po~hivh~ ~xl Western blot reaedvi~ become neg~dve with time a~r superlnfce. lion. Other th~n delayed he~lln~ of the skin lesions predoced by T. pallld, m and c~ni| in HIV-infected mbblts, them is no evidence of any immune abnormality. Af~er death, ,~g s~qucnces am detectable in the splenoc~es of ess~ntial|y ever~ NIV. infecto¢l rehbi[, ~xl the splenocyt~s of elght or2~ infected r~bblts mspouded by pro- lithr~fo~ to HIV pepsins. In ~ddition, ~e~ SC~lu~nces are ~ctectal~ in rabbits thet ~re ink"ted with lymphoid cells from HlV.infected r~bbits. Howevu. after mult~p~ 240 testing of both pe~pheml blood or living ral~its ~m kill~ (~l~n. ~;n. lymph n~es. liv~. v;ms Ms ever ~n c~vi~in~y d~t~ In ~t~st to ~ ~r ~blis~ ~s. I~ ~ta ~ate ~at ~V-I ~f~l~ ~ it ~y ~ u~gul as a ~1 to stay t~ h~an ~gat~ to de~nt J~al of Acqui~ Immune ~ficieney 9:211-226. H~ Health ~k~ ~teg. H~st~. XIlI. Miscellaneous USING INHIBITORS OF METALLOPROTEINASF.S TO TRF.,AT ARTHRI'rI~-- t,J~s~B SAm ~t^N tX~ Arthritic diseases in general, and rheumatoid atthritls in particular. ~Wssent a complicaled muttifaceted set of clinical di~dor~. The clinical ~,~nptoms and logic features result from a ClaUde of biologic psthways ttmt involve a~l~tl and chmele inflammatiou, the immuce response, ~nd metallc~oteina~ ~t therapies are often b~red on the concept of "ix~ .yl~n~:. y",in.which ~ drags are administered slmult=~o~dy, with the hope that eeeh will be Satiate4 at inlenup¢ing one or another of tbese p~lhways. H_m~.e~.r. ~ .the~.=p~$ ~ alw=~s specir~ ,.d they o~en ~l. As know .l~d~ o~_me t~)c ing these pethways cominues to meret~, w~ ,h~ .to [onnu.la~ "cl~ pier. that arc targeted at specific moleeulur mecnamsms aria are e,ectlve m wvo. Successful inhibition of metallopre¢¢inases is sure to be among them. Vineenti, M. P., C'la~, I. M., and grinekerhoW~ C, Arthritis & Rheun~ism 37(g):! 115-1126, August 1994. Other support: Nstlonal Institutes of Health and the RGK ~cendadon (Austin, 'IX), From the Dartmouth Medical ~0ol, Hanover, 1¢I4.
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KINETICS OF PEPTIDE SYNTHESIS STUDIED BY FLUORESCENCE OF I~.UOROPHENYL ESTERS TI~ kln:tlcs of the ~actlon or Boc-alanine-lrifl~,rophenyl. Boc-alanlna.tetr~. fluorol~yl, Boc-alanine-~mellnorophenyl, and Boc-alanio~-p-chloe~etralluoeo. i~enyl ~ {BocAtaOTrl', ~ocAlaOTrp. BocAliOPf'p, and BocAhITf'c. I~espcclive. Iv) with leucin~ amide and with valine mathyl ester have I~n m~asured usin~ chsn~s in flsx)rophenyi chromnphora em|ss~n ~ 375 nm. The kir~ic ~tl cannel he w~li I~ with a simpl~ sacond<xc~ inaction schem~. M~l~urcm~nl$ of. Iho raac- liOn kinetics at diff'erent ocmcontrat;ons of the reagems showed that the exlxession for the ~eaction rate is V,,kC~.~"~. i. which k is the reaction rate comtant. C. is the concent~ion of either LeuNH, or V~OCH,. and C,~ is the c~:,m'emmion orthe fluo- rophenyl ester. This reaction equation indicates • complL~x, probably eha|n-like, f~ctioe mechanism, The order of reactivity foe these active esters with ValOCH, is BocAlaOTfc>BocAlaOPf.F>BocAlaOTf.p>BocAlaTd'. The apl~rent rate constant, k, for the ~ction with LeuNH~ is higher than that foe the react;on with ValOCHs. Permyalmv, E. A.. Medvedkin, V. N., Klimenko./.. Y. Mitin. Y. V.. and Permyakov, $. E., Jr., {KrelsinRer, R.) lnterr~k~nal Journal of Pep(ida & Protein Research 44'.472-476, 1994. From the "l~orcfical ~ Exp:rim~nt~! Biophysics. Russ;an Academy or Sciences. Pushchino. Moscow. Russia. and lnstitutc of Ps~c;n Re,arch. Ru...~isn Academy of Science's. Pushehino. Moscow. Ru,ia. p.CHLORO'~ETRAFT.UOROPHENYL ESTERS OF N.PROTECTED AMINO ACID~ p-Chlorolelrafluoeophenyl (Tfc) esters of protected amino acids and pcp¢ides are more reactive than are the well known p~ntafluoeophenyl (Pfp) esters. Two re~,enlS, p-cMoro~etrafluorophenyltrifluoroacctatc (Tfc-OTFa) and di-(p-chlomletra. tluorof~m~l)em'oonat¢ (di-lTc-c~xma~c). can be used foe their sT.tl~es, themb~ avekli~ me ~ the alle~i~ dlc~lohex]~lc~'bodiimide. This is ea~x~ially impoetant f'or bulk prep~ions. M~ny Fmoo- and Boc-~mlno acid-OTTc ~ers have been syn- th~izmd and ch~,acledzod. The hexadecam~'io tandem repeat H-(AlaAlaLysi~o),- OH was a~jnth~sised using di.Tfc.r.attx~te f'or the I~l~ration of. lTc-cstm. Motived[kin, V. N., Klinl~tlr~, L. N,, Mitin, ¥. V., Kr~ldnger, R. H., $heb~Kn~itz. lntem~di0nzl )ournal of P~ptide & P~e|n Research 44:4T/-4N, IP~4, From the Ir~titet¢ of" Protehl Research. Russian Academy of Science, Pushchlno, Moseow Region, Russia, Department of" Biology, University of" Virginia, Charlottesville, VA, Department o1" Chemistry, University of" Virginia, Chetlottesvilk~, VA, and Penn Bzlneh of" the Ins4itute of Applied Chemistr,/, Penn, Russil, and Biochemical Finn 'Biolar'o Olaioe, L~vla. 2~2 DEVELOPMENT OF A HIGH.AFFINITY RADIOIODINATED LIOAND FOR IDENTIFICATION OF IMIDAZOLINFJOUANIDINIUM RECEFI'IVE $1"f~ (IGRS): INTRATISSUE DISTRIBUTION OF IGRS IN LIVER. FORBBRAIN, AND KIDNEY rmidazolinelgu,.mklinimn receptive sites (IGR$) am memt~ene ptolelns that exhibit hll~h amnity for various compounds with an imldazollne o¢ lu~idlnhm'l mo~y. TI~ ~tn~me of these hlndi~ sile~ and their strniffcar,~ in d~ macolo~ical action of" such li~nd~ m~ unclear, To i&lm~s thil be,~, wl selective high M'rioily componn~ that conld he radioiodinal~l and u~d a~nity foe IGR$ in rabbit kldncy membrane, as d~ennined in competition bladln studies with l'Hlic~zoxan (K, - 12.$ ± 7.$ riM). and was t~dioiodina~ed by ehk~, ramln~-T oxidation to yield [~I]AMIPI. The blm~inE FmF~llea of ["~I]AMIP! determined in m~m~anes ~u~d from IWO rqxet~t,,tive li~ues, ra.~.l~. codex and rat liver. $p~:ir|c bindlnE of ['I]AMIPI w.- saturable and of" hi~n amnt. ty. ~ determined by Scatch~u~ an~ly~s of ~uration ~iodln~ ;solherms (rabbit _kid- .ey. K~- 2.0 ± 0.9 n~. B.,. = 5~4 ± 201 f'mol/m~, five exp~n~nt$: rat liver. ,,[~ ,, 2.6 ± !.3 riM, B.. = ?3 ± 10 rmo~n~. Ihme experiments). |':'i]AMII~ bi~t~nl~ In bit kld.e), m~n~r~.es was inhib;t~ by va~ous im~olioes ~ |~anidini~m Ix~nds. with the foll~wln~ ~nk oe~r of ~otenc~: drazolino (K,., 3.(S id~,~oxan (hi,h-affinity sile K, ,- 0.2 ± 0.1 nM a~ low-affinity site K, ,~ ?6 ~uarmbenz (hiEh-affinity site K, ,. I.? ± O.M nM ~ k)w,~ffinity site K, ,~ 201 ± 72? nM)> amik~ide (K~., 625 ± I~O riM) > e~o.idi~e (K, ,, 22~X) ± 200 gM) > oclo~idin~ (K~ = ~422 ± 172 n~), I~'I]AMIP! bindin~ was no~ inhihlted by ~e e~- |drene~ie ~cep~m antagonist rauwoh~ne or endo~mous agonists for mltt,~' Rce~ors (epioephrln~, histamine+ ~.efe¢onin, and dof~mtna), The rank oeder of ¢o~petinS li~nds is conslstont with the ~f'mition e( the ['~i]AMIPI 5indiol~ as an IORS. Re~.ptor autmadiosraphy was used to deten~ina the intratissne diatribe. lion of" IORS in rat liver, rat forelxein, and canine kidw.'y. Autoradio|rams indicated homogeneous spccil'¢ hindin~ of" [mI]AMIP[ in tl~er. Renal ["I]AMIP! 5tndio| was ol~crv~! as discrete co01ieal ra~s, Autor~io~orams of rat forcta'a|n tissue s~tio~s the subfomieal oee.an I"I]AMIPI blndins was irmlb;te~ I)y tx~n Lmi~axa.ne an~ colo~icd and molecular c~cnzatmn mm~s cn y. lvkovic. B.. Bakthavachalam. V.. Zheng. ~.. Perini. A.. Dis. D.. Bosch. $.. Ncumeym', J. L., and ! ~.nler, ~, M. Moleculnr Pharmacology 46: I ~-23, 1994. O~her suplx~1: National Institutes of" Health, Medical University or $omh and Contrat de Rech~che Esteme lnstitu~ National de Is S~n~ el de Is Recherche Medicsle. 243
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0 From the DeI~rtment of Pharmacology, Medical University of South Carolina, Charles{o~. $C. Research Biochemicals International. Nati~k. MA. Pharmacologic Mo~ecuIaire et PhyslopatholoBie Rename. Instilu! Louis Bugnard. Toulouse. France. a~l Hypeftc, flslO~l C~nler, Division of Surfer?. Bowman-Gray School of" Medicine. Winst~n.Salem, NC. COMI~INED IMMUNOCYTOCHEMISTRY AND ENZYME CYTOCHEMISTRY ON ULTRA.THIN CRYOSE~I'IONS: A NEW METIIOD We combined immunneytochemistr~ and enzyme cytochcmistry to locality, two d~ffc'cc~t proteins on the same ultra.thin cn~)section. In thLn m~thod the immunocy- toch©mical ~x:alizatinn is vlsualized with colloidal Sold prol~s and the enzyme cyto- chemical detection is achieved with cerium as the capture a~nt. The immunocyto- chemistry is ¢xpoducted first so that any potential adverse elTcets of the enzyme cylo- chemical pro0edtw¢ will no( aker the antibody bindln~ propezlies of'the cryoscctio~s. TzZ:iz~wa, T. and Robinson, J. M. The Journal of Hiztochemistry and Cytochernislry 41(I I):1635-1639, 1993. Other support: Hallo Found~tlon. From the Depwlmcnt of Cell Biology, Neurobiology, and Anatomy, Ohio State Unlverdty, Columbus. OH. COMPOSITION OF TIIE TRANSFER MEDIUM IS CRUCIAL FOR HIGH-RESOLU~ON IMMUNOCYTOCHEMISTRY OF ~Y~ONED HUMAN NE~O~I~ ~ ~ t~ ~ ~ti~ st~ ~i~ w~ c~t~i~ o~ .ltm-thin ~ ~i-thin ~s f~ im~~mist~ is t~ ~qfer of ~i~ to EM g~ ~ ~v~i~. ~ a ~y of ~ ~ul~ of h~ ~ls. ~ have f~ that T~i~w~ T. ~ ~n, J. M. ~ J~l of Hist~mlst~ a~ ~mls~ 42(8):I 157- 1159, I~. ~ ~ ~menl o~ Cell B~Iogy, Neurob~logy, and Anatomy, Ohio Slate Unive~i~, ~m~s. OH. 244 USE OF IA-nm IMMUNOGOLD PARTICLF~ FOR IMMUNOCYTOCIIEMISTRY ON ULTRA.THIN CRYOSECTION$ We present a new application for the uae of small immunogold pmtldes (,,,IJI- nm diameter) for ultrastmclural immtmocytocbemistry. These mall ~old pafti6l~ have been u.~ed en ultra.thln cfyo~ections in co~juncti~a with a silver enhoncew~flt procedure that does hal degrade ultrsstmctural detail. We have treed the human tmphil as a model system, in which known protein marker~ or two dtlT~,mnt plasmic Fanule~ were localized, in the development of this pmcedpxe. The 1.4-nm immunosold p~rlltfes coupled with silver enhancement yield inten~ Inhell~t for kx:alizatio~ of lactofen'in, a marker !'o¢ the sp¢cif'¢ eranuks, and myelot>eroxida~e, a markef for the azurophil granules. Double labeling io which one anti~m w~s visual. 17¢d with IAmm ~old and silver enhancement and a se¢oad anti~en wan deteet~d with colloidal gohl-ll~G oa the same ultra-thin ewosectioa was sueceasfully achieved. We also show that IA-nm diameter immtmo~old por~Id~s penet~ cryosectioned neutrophils to a greater extent than 5-nm or 10-am immunolold probes. These results show that small immunogold particles, alan| with silver enhancement, are a u~el'ul addition to the immtmolabeling methods available for u.~ with ultra-thin c~jox¢clio~s. Takizawa, T. and Robinson, J. M. The Journal of Histochemist~ and C~tochemlstfy 42(I 2): 1615- ! 62:3. 1994. Other support: American Heart Association, Ohio Affiliate and lhe Naito Foundation. From the Department ot" Cell BiotoBy. Neurobiology. and Anatomy, Ohio State Uaiverxity, Columbus, OH. A(~R./F.,OUS TWO.PHASE PROTEIN APPINITY PARTITIONINO A LABORATORY DEMONSTRATION OF A BIOTECHNOLOGICAL PROC"~$ 'The lechnklue described hem is one sOlUliOn to the cc~lex Ix'Oblem or fractiona{ion. Its facility is dgc in pail to the runt that.the compon~11ts el" the two. phase system ~re also household products. Polyvinylpy~olidone (PVP) can he round in many types of lotions and hair care IXOdUets while mahodextrirm aR u~ed ta many hoped thM it begins tO demystify at lea~t one asl~"Cl o! mo~m form of this experiment will mos~ I~ety fit into the pm~eln ehemistr~ section of Ih~ blocbemistr~ laboratory curriculum for undergraduates or possibly advanced ~,¢. onda~]-level sta(k~nls. "
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~ogmal of C'hem~:al ~docatlon 71(7):$90-591. July 1994. Oth~ support: Nitlo~zll ~,c|ence Foundation Scia~:e and Technology Center. I~Yom l~e University or l~ttsburgh and C~megie Mellon University. Pittsburgh. PA. INDIRECT USE OF IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY FOR ISOLATION AND CHARAC~TE~IZATION OF P~OTEIN PARTNERS rmnx~li~d mere M'~nJty chromatogril~y in c~junctlon with modem return. blnw~ DNA technology, allows emciem immobilization of a tagged protcln to • matrix with a consistent ~nd defined sl~tiM oriontatlon. The zmlquc nature of this immobilizstio~ method makes il immune to most hiochemlcM .rc~e_ nts. The small slee of the oligopelXtde t~. typically six contigoo~s~stidine r~id0es, while suffl. cie~t to Mlow stron~ end Ipecific interaction with a el]Elate! reef•l, offers minimum lnteffcrm~e with mOSt protein functions, both enzymatic and physical. Together. the~e characteristics define a system that allows tl~ L~..,cmhl), or t~nd prmeln com- plexes, their f~ife puril'zc~lion in • native ~ functionally active state, and a means to d|z~:t the interactions I~min. Tn the fz~um, blodztmical similes involving in~er- ~tlons between polypepiides ~ml o~her mw:romolccules will most likely see IMAC playin~ :m inerezllngly importer role. Szwadogo, M. ~nd Vxn D~ke. M. W. in: Sallow, J. ]~. ted.): Ocnellc Engineering. VoL !'7. Plenum Press, Hew York, pp. $3-65, 1995. Other lupp0~: National Institutes Of Health and the Robert A. We~ch Foundallo~. From the Department of Molecular Genetics. Department of Tumor Biology• University of Texas, M.D, An~rson Cancer Center. Houston. TX. 246 Active Projects Following is a li~ of the principel investigators, or iP~itut~. ~ ~]~s ~ .~ w~v ~ ~m ~tivat~t in I~ ~1~ si~ t~ ~vi~ P~, t~t~ wl£n l~''~ive ~t 1i1~. ~ltt~ ~ ~ lt~t~ ~ z I~ ~1~. PRINCIPAl° INVFJffl~sATOR ~()R I@TUTII)N CORY T. ABATE, I~.D. Medicine. University or Medina ~ ti~'lry. Rrd~ll Wm-,d Johnson Me~zicaz Sclmol. pi~-'at~vay. NJ. . SYF~D $. AiIMAD, M.D~ P,c~x'ch A~|ut~ pml'e~'o Temple PA~ M. A~H. ]ORGE E. ALL~D~ P~e~r or B~isl~. Univc~ily As~iatc Professor. Unive~sily of Col. ~ ~ver. ~. HARRY ~. A~HIADF~, ~n;~ sezfr Investigator. ~Id Sprin~ I!~, NY. SUSAN A. A~IN. ~.D. Scnlor Memhex. lnstllgle for NARAYAN G. AVA~ANI, ~hD. ~NA~AH M. BA~ER. M.D. Ass~ant ~fe~. AI~ Eins~in PROJP.CI" TITLP~ Immune effeclor meeh|nl~ms In ulhero. ~'kr~is SIznsl ,ransductlon vl_a l~o~eln klna~,*s and Sil:nzl trattsduction by the Inlerlsukln-) emttve d'~xdm ADe~idon ~'e,m~" in ~ rm.lm ~1 ~t;dylinmtlol 3'.ktnase and Inaulln 2~
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0 0 PRINCIPAl, IN~FLRTIf;ATOR (JR I.%,Wi'ITUTION SRIUATA BAGCI !I. PII.D. AI~51Inl Prol'e,+~lr. Center rot, Mnlecular 3iol~ly o{ (.hll Dl,tea~% C~lle~e d IX, n. lit~y,Univeaily of Illinoi% C~ica~. IL ST~V~N BALK. M.D.. P,.D. VYTA$ A. BANKAITI$. l~l.D. Universily of hlablmi al B|rmin~ham. Birmln~h~m. AL DAVID W. BARNES. P,.D. A~tocille Professor of" Biochemis~ryl- Bioph~sie~, Oregon $1|le Uoiver~ily. Corgalli~, icine. The Johns IJo~kins U. niver,tily ~ cd" Medk, ine. I~ahirnore. RLAINE L. DEARER. M.D.. Pit.D. Au~tanl Pmfe~uw of P=lh~y and rt~m/Medicine, tlr~vn Unive~i¢y, l~wi- demi~, RI. WILMAM R BENRDICT, M.D. ~ fe=sor of BioCechnolucy. Baylm Col- of Medicine, The W~Jlands, KAREN t. BENNETT, Assluam Pmfe~or, Unive~W of M|,mud, Colum~=. MO. NI~|M BENVENISTY, M.D., L~.D. S~nlot Lecturer, Life Scier~es Inslilule, Helxew Universily, Jerusalem, Israel. DARWIN K. I~ERG. Profe~,m' ~m:l Chairman. Deparlmenl of Eiology. Universily of Caliromis. San Dielo. L~ ]0~la. CA. LESLIE J. BERG. PN.D. Assistant Profet,.or. Harvard Unive,|~y. C~mlx~le. MA. SHELLEY L BE, RGER. P,.D. Alt, islan! Professor. The Wislar Insfilule. I~iL=kl~is. PA. ]uDrD! BERMAN. I'~D. A~oc~ate Pmf'¢sso¢0 Univernlty of Min. msol=. SI. Paul MN. BRUC~ IIEUTt.ER. M.D. A~eehtte Professor of Internal Ua|ver~y of Texas Soethwe~cm M~lical C~. Dalla~ TX. I'ROJECT TITI,F: B|(¢h~mical fun¢linn,¢ or Ihe human marlin++ ,~t~Wt~eins I'~ aml E7 Re.gulaliem of CDI Itcrm expresslo~ during uman T lympht+cyle devek+q~r~nl M~spholipid Iransfer pmleins and Oncogenle Iran,d'mmatlon in ~etum-rree Rel:ulal~o~ of Ihe DNA melhyla.se geae ~1 v~. tracer <:ells Xe~p~a ~el/&w~al honml~uen and Ihe ~ role of Ih¢ rel;m~a.~toma gem in lung po~ranule components of Ihe germllne cell .hesse Ir, olstkm and ~=l,'a~tefi?adon of laf~el g~ few myc gel ca+-,¢ade~ in r,¢um~s Fumlio+t and imer-~llo~ of the T cell cif¢ tyro,dnc kina~¢. TSK Transcriptlf,~al activallOn by Ihe lum~ sup pre.t,~ 1,53 "fh¢ role of hnRNF~ in lelomem ~mclure arxl Mol¢ollar eh'mlng of the #.ps eDNA 24R PRINCIPAl, INVI~TIRATOR OR INSTITUTION RICIIARD J. BING. M.D. Pmf¢~c~ of M~llclne (Emeritus), Unlvef- ally t'ff .~elhem California ScUd of Medl. ¢{ne. Uo= Ar~des: Visltin= A~.~a~e, Calf Ex~rlmemal Ca~iology a~ Sciemifi¢ Developmenl. lluminglon Medical Rehash Insl;(e~. ~na. C& BARBARA K,~IRSI~. ~.D. c;~. ~ B~x. NY. GAIL A. BISllOP. RhD. Al~Itlanl Pmf~ of Ml~ob~ Internal M~ici~. Unive~ily ~f Iowa, I~a ~ty. IA. DAVID A. BOBAK. M.D. As~iale Professor. University ~f Vir. gin;& ~d~tesvil~. VA. BRU~ M. BOMAN. M.D, m Univmity, ~ha. NIL MEREDm I BOND. A~itte SlaW. The Cleveland HENRY R, B~ Rt.D. • ~fes~. Unive~ily of ~xas al A~in. Austin. TX. ~G~S DA~D BOYD, R~D, Assistant ~res~. Univenily of Texas M.D. A~ Ca~r Cemer. TX. R~ ~ BRE~ART. R,.D. B~m. MA. ~V~ J. BRI~ R LD. " ~hy of New ~my. ~awny. NJ. ~N~AN~ ~ BRI~HO~. c~mist~. O~rtm~th Medical I I.~. Nil. ~MAS R. BR~E~ ~.D. ~f~ of Bi~mi~. Unive.~y Of Ala~mk ~l ~ M~ki~ a~ Analysis of the ~egle~ I=e~e cluster immune m~ce S~r~l~ nd fumti~e dd~ family Of 01'F, APC I~e prod,el.binding pm~elna An~hlde~ie mid a~d myefltwllhw pmtela Mulallo~s in the C.lermlnus of v.rel and Role of ufoki~s~e and IIs rl~plor In eelon exn~" i~vadon Genetic analysis or ~lk~llo~ pmleln A in llumzn F~ldll0mavlrtut Rplleatim a~ I#r,e exlxm~ i~ pdma~ I~mm heml~my~ 249
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0 0 I"RI~C|PAL, INVI~qTI~ATOR OR I~STITUT|ON .~EANNE'rTE C EULI~SKh ~.D. ~ Surgeons, Columbia University. New Y~ NY YURI BUSIIKIN, ~I.D, ~h In~lku/e. New Yo~. NY. KA~RYN P~e~r. Columbia Unive~iW, Ileahh ANNE ~ ~AI~F. ~ARD J. CAMPBEL~ ty ~ ~h. ~. R~. !~. AsSmtlle Profels~. Duke Univerqily M~al ~. ~. MARIA ~ C~DENA~ A~n~i~le ~f~m. Duke U~ive~ily PA~ICK L ~ASEY. ~.D. As~ilI+ P~fe+sor, ~ke Univershy AN~ a~y, N J, P~fessor, Dartm~h Medical ~ch~, I!~, Nil. PROJRCT ~ITI.R lnterac¢|~ of micrnluhulcs with cell cycle etTcclor~ Profmties r4 the ALL-I Ixt~ein assrgluled wi~ h~nm acme Cell ~ienali~ inlcraclkms in l~ll~ o~I f~- Immunnphilln,calci~urin c~mptexes wilh and witl~ in~n~mm~"~sr, m~s Struclure a~l functinn of pf~e;n famesyl. Tolerance aM/O~imm~nlty Io inll~n~ IIA m a lram~-'elc ~lf milan Role of the g~bB3 pmlein in growth lacier- coupled signaling Exlractllular n~rix &gradalk~ in I~ lung Neuroimmunc inlerscllon'~: mechanisms of ~-~elamine effects on lym~e rune- Mapf~ng Ih~ sleml l'~ndlng sites of ACAT - a key enzyme in mhenmclen~ig 250 PRINCIPAL INVI~TI~ATOR OR IN.~rlTUTION ]FAN.PIERRE CHANGEUX. I~t.D. Profe.c.~of, lfl~litul Plst~r. ~s, ~. JONA~ IAN CI IRR~, M.D., ~t.D. la~lilule for Cm~er Re,each, I~il~el- ~i,. I'A. KA~II.~N R. CLIO. M.D. A~islanl Ad~l CIlF~G-MING ~UONG. AAR~ J.O~IA~V~. M.D.. Professor or Bi~hemillfy. PAU~ 8, ~RKF~ ~.~ As~ble Pmfc~. McGill Unlvmhy. W. ~ ~VE~ND. ~D. Ih~al C~. ~ Y~. NY. M~.ANI~ I!. ~BB. ~.D. A~bte ~. ~tlh~em M~al Cenle6 I~!1~. TX. RR~ II. ~11RAH. ]E~EY P. ~HN. ~.D. Ass~iale Prnressor. Emery Atlanta. G~ ~UD~ L ~. D.V.M.. ~. . I~.~. ~f~ of Bi~mist~. Unlv~i~ of Mi~ St. ~1, MN. ROBF~T !!. ~A. R~.D. ~s~l~t~nt ~fes~ Pcofe~r, The Ilebrew Unlver~ily of PROJRCr TITLR Funcl~o~ and ealx~ss~on of I~ ~ n~lnk Genetic alterations in ~lcal and vulvar ~ ~I ~ f~ ~S ~t~x ~ nicm;~ l~tl~ml~ of RN~ of I~ MAP kint~ pathway in ~11 ~ysio~o~ ~l~l of m~ltipll 251
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0 I~RINCIFAI~ |NVF~RT|~ATOR OR I~TITUTION A~t*nl Professor, State Univerd~y or N~ Y~. ~y B~, NY. ~RICK C DE B~R, M.D. ~ ~ M~i~, ~ily o~ Ken- WAL~R A. DE~H, ~*.D. ROBOT C. IHCKSGN. ~.D. ~ of BinOmials. Uni~hy of Km~ky. ~xinBt~. KY. M~. H~. TX. A~ ~. Uni~hy ~ M~i- c~ ~ ~is~ e~ New levy. R~ W~ ~s~M~ca! Sc~t. Piscm. ~R H. D~B~G. Unl~y ~ ~iF~ ~. ~y. CA. Wi~IAM R. ~K~RG. W~n~. D.C. E~NE A. ELION. Asslslaal Pm~essor. H~vl~ Medical ~RY ~. ELAN. ~.~ ~ A~. Bi~i~m. AL (~ ~mh a~ ~atio,. lee.. Palo A~ CA. ~y ~ New Y~. ~ B~ ~. 2.~2 PROJ RCI" TIT1.1.: r'r~n ~ning to the k, ot~i coml~ex Fcmctim~ of ~rum ~my~old A pm(ein (SAA) Oxidmive ~re~ and the eukanjo~;c ~cl~r of X-nxoqumin~ l~atkm of th~ human R.'~a~e ~ne p.S3.deficient mlc~ Mechanism and fate of r~Iroviral-mediated Ira~mckctlon otcelfutar .,equencex Molecu|m- m~cb~sms d' aluminum. and nickel mxicky R~rovira! @he L, enes and ~llular prom gcn¢~ Ac.vat~'m ~ M pha~e by l~,r~! es~ Role of' endolhelin in the pathngenes|s of lnlerscllons medlaled by glutamate uptake tran.,qx)ncn= ~¢¢wcen ocurons and I:lia Mnrtalily lrend,( m~nn¢ smokers and I*RINCIPA~ INVES~TIGATOR OR INSTITUTION A~I~HAM FA|NSOD. Leclurcr, HeMew Univemily-Hudass*h Mdell ~1. ~. I~1. ~S V. FALLER. M.D. ~e.D. Mri~ t~ Palholoey, B~l~ Ufllv~lily. B~. MA. ~OMAS ~ FANNING. lit ~s~l(~. ll~a,I Medical ~1. ~. MA. ~ ~ ~RD. M.D. DAV~ A. ~. ~U~i~ity or New Y~. N~w . Y~. BR~ A. ~EEMAN. ~.D. ms. B~in~bsm. AL. ~e ~f~ ~ M~lici~ aM B~- M~AEL ~Y. R~D. T~y, Haifl. !~. A~N b. ~YER. ~ . . . ~. MD. XIN.YU~ ~. ~. ~w II~v~. ~. ~A GAL~HOUS~ N.D. Res~h Inst~t~, N~aslem U~v~i~ ~1~ ~ ~i~, ~011. Molecular mechanlsm¢ COnlrolllnl bloc, d flow i~ the lu~ Function of LINE.! retmlr~nsposons In httmm breast c~nc~r Im ch~ in DNA n~,lr C,a~ i'a l~,chemhml uul~ts or ~lh~¢ orpniz~dm Vascular end0~clial blndtn~ ¢f xmddne ml- date
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PR|NC~PAL |NVF',RTIf~ATOR OR INSTITUTION PAUl,. ~. G~ER. ~.D. Assis~t ~fe~. Unlv~sity o~ Tex~s An~ TX. RI~ARD B. GAY~R. M.D. Ass~ia~e Professor of Medicine xnd ~. Re~. ~!. B~t~hnology ~nd Medicine. A~iate ~es~ of ~t~ Univ~- ~il~I~ PA. Ro~ P. Imwm ~fesI~ of Biol~. li~, Ueiv~it~ of Olif~ia Sc~I of Assistant ~fessor, Harvard Medical ~. ~. MA. I~ MA. A~O G~A~. M.D. A~istan{ ~ofessor, Jefferson Cancer lnstitu~. ~as )effer~n Uni~rsity, DA~OI~ ~,~ ~, R~ !~, Pmfe~ or C~mkt~, Ilumer N~w Y~ NY. PROJRCT TITI,E Regulation or neu~oesl nicotinic ACh recq~ tor expmsd~n Character~ation of the cellular lran~rlplion The molecular basis for the effe~ of cell adh~ion on mslignan! t~nsfmmadon oom~o(ein¢ Charac~eHr, mion ~" the mdm2 e~,ene and ils in~ua~ion wlth p.S3 U3 ~mRNA and dlxy~r~ll RNA I~¢~ing Receptor Factors as~clalcd with ~n F, IA.likc prmcln from Novel ~ctivities as~xcialed with tel *'inhihi- pl30/Rb2 a novel target rot the AdS oncop~dns ~ The figmd billing s~ ~ I~F rcccplm~ ~ a target ~or growth modulalo~J~ Regulatlon of the infedcuk;n-4 ~ Rellulslion ~mRNA translsticm 2~ PRINCIPAl., INVERTIGATOR OR INSTITUTION SANNA M. GOYERT. Nmlh She~ Univcrshy Ilml~tah Man~ms. ~el. NY. SRRGEI A. GRANDO. M.D~ P~.D. A~,to~te Pr~'ess(~. Unive~ity of Minne- ~a. Minneq~i~ MN. JFJ:FREY A. GRAY. P,.D. Pro4"e~m an4 Ileal, ~menl d ~y- ANN M. G~YB~ f~ilule o~T~h~, Ca~d~, MA. KA~II.~N J. GRF~N. Asxi~l~l Pmfes~r ~ Pm~logy. Noflh- weslern Univer~ily Medical MARK L GR~F~ University o~ I~nn~ylvxnia S~I CAR~ W. GRRIDER. MARK A. GRIEP. slay ~ ~ka. ~n. N~ SIDNEY ~ GROSS~RRG. M.D. ~ ~ Wi~i~ M~wauk~. Wl. Y~GRU~BAUM. ~.D. J~. I~!. PA~ J. HAG~MAN. M.D. ph~ics ~ Ge~t~s. Unlve~ily of ~1. SUBRATA HA~DAR. ~g.D. f¢~ Unive~ily. 3effe~ Cn~r ~. ~il~iz. PA. PROJRCTTITLE M~lold diITe~mi~io~ le~: COI4 Kmdno~e chol~r~ic Mcdlatlofl b~, cal©cholam|ne systems clT=ct~ or n~-~ine on b~h~vlor Molecular erfecl~ of' n|cofln¢ In sldstal Rwird syslem Epilhelial difIr©renliation and nenplaslI." Recer<o¢ intc~cllons in cancer THomenm= in cellular DNA mlfl~c~oe kimtlcs Clmmclcd~allon of the humm JHK vires Studks of m~k~" activities in a cell-f~e Role or pmleln t _yr_odne Id~mphor~ladon In The role ofDNA mc~hylmioo in I~e ~lX~- Mol¢cular i~d fu~tllonll ¢hlracledudon of 2~
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0 0 f..rl 0'I PRINCIPAL INVESTI(|ATOI~ OR I~IT~ON MIN HAN. ~.D. A~t~lan~ ~c~r. Unive~t~y of Colo. MI~IA~ R. HANLEY. ~.D. slay M~if~, Davi~, CA. . ~RRY W. HARDY, ~e.D. As~islanl ~fessor of Oe~dcs. Wesl~ R~e Unive~hy. ~1 ~. ~. OIL N~w YoA Sch~l of M~icine, Slony B~, NY. PAUL O. HEYWOOd, hi.D. rule. ~ ;~1~ CA. Mem~r and Chief. ~nccr D~O~H K. H~HI~KI. ~.~ Assimant ~fe~ of B~:ical ~m- isl~. Unive~ily of Illi~is ul Chfca~o. AN~ ~. JAI~W~ ~.~ ~. ~il~ PA. MAKKUH} {AYA~M. ~.D. A~i~ ~. Univ~{~ of Tex~ PR()JE('T TIT[.E I%-~igning ,n RNA-bind;ng Ro~ .as of ~ur.! and olher ;cnc~ in ras-n~diat- cell signalling during an;n~! develop. meal and in cancer Rcgula~ ,)~ of I~ C~m~ini of multi~e ~MP h~mx~thyl:~ mof~ular basis o~ stable Ion~ Re~l~ of ~tit~ D v~s ~ ex~ Furcllon o( surrogate light chains in B cell Role oT NAD(P}IP. qul~mne o~ido~educ~ase I (N0Ol) in csrcin~'n mid drug mctal~o. .~lep.arre~t vat{ants o~ Flp PRINCIPAl, INVF~ITK{ATOR OR IN,CiTITUTION KUAN-TEH JEA~G. M.D., ~.D. ~ ~ M~I~ Biol~y. ~t~al I~ilm~ ~ I le~tth, ~a, MD. WI~ED JE~RI~. D,~ R~ ~ ~ ~is~. Assimm ~r~. Un~e~ffy of As~ate ~. Unive~i~ of Te~ ~~ K. JOK~K. O.~ ~. ~. ~. J. O. J~lll. ~e.~ JAM~ T. ~AGA. ~.D. ~CMif~ ~ ~ ~ I~ ]oth~A.~ C . ~AYA KA~EIM, ~ ~. Ik~ Univ~ily ~ Jerusstem. Illd~ssaE-Mcdical Schwa, WI~IAM H. KAN~ M.D,. Assistant Professor of Medicine grid ~3~CA. a~cs. J~ns Hopki~ Unive~gy, JOHN F. K~Y, M.~ ~ ~ M~. ~. MA. PROJECT TITLE Role of Tst Imd ~llu1~r fi~ors in HW cx- The role 2 in lowering cell surface expr~ssmn--,ofo I1LA nmlecuf~ Mokc~far tar~ir~ ~ ~ ~ ~i- Ccllubr lliclill of Illlllin hldritli
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PRINCIPAL INVF,,ST|(';ATOR OR IN.~TITUTION AMY KENTER. Assislan! Professor of" Microbiology and Immunolofy, University or IIlino,~;. ]UDITI! KIMBLE. Professor. U~ivershy of Wiscomin. Madio ~o MICIIAEL P. KING. I~l.D. New York, NY. IIANNAll I. KLF.IN. P..r). As,~cia~ Pm(~". New Y0d¢ Unlver~ily Medical C'enler. New Yod¢. NY. ERIC H. KMIEC, PII.D. Assn¢i~le Pr0fcs.~r. Thomaq Jefferson Unlv4~ty. I~iladdl~ia. PA. TAD H. KOCH. P~.D. hofcs~or of Chemic,/. Unive~y or Col. THOMAS J. KOOADEK. As~)ciMe Professor. Univer~;i)' of Texa% A~ TX. HEINZ KOllLER. PH.D. Proresl~r o( Microbiology,. University ot" Ke~lucky Medical Center. Lesinl:l(m, KY. WI~ H. KOPPENOL. I~. A~ Pml~o~. Louisi-na Stale Uni- ANDREW S. ](RAFT. M.D. Prof©ssor o| Medicine. University of Alatmma. BinnlnfJ~m. AL. ROBI[RT H. F.RI[TSII~K3ER. F~tD. Pi~fessor I~.ta. Chadou~vi,e. V~. SKAIDRIT[ KRISAI~S. P,.D. ROIISRT D. KI~ITA, F~tO. Ass|a~ael Professor. University of Col- FI$iNOJII~I KUNG. I~.D. ~. ~ We~em Reserve Us|vet. $ity. Cleveland. ON. RYOKO KURIYAMA. P..D. Associate Prof~sso~ of Cell B|olc~y and ~urounalomy. University of Minnesma. Mir~eapoli~. PR~JRCT TITI.R PRINCIPAL INVF3"TI(;ATOR OR INSTITUTION ABE[. LAJTlfA. Director, Center fo¢ Neumchemi~?, 'The Nathan S. Kline Institute rm PsyehialrJ¢ Re.arch. Orang. NY STEPIIEN LANIER. P..D. ical Universily of ~;outh Carolina. ANDREW LASSAR, P.,D. Assistant Prnfes.,,or. Ilarvard Medical .~,¢lm~d. Flmmn. MA PAUL J. LAYflOURN. P,.D. veery, l"-~t Ccdll.s, CO. MLIRIF.Io I.EDF, RMAN, Asslstanl I'mfcssm of Illok~gy. Virl~inll Po~ic in~allule xr, d Stele Unlve~ity. EVA LEE. Assnclal¢ I~rofcs,-~'~'. |nstit~e or fllo~ech. C~I~.. U~t,-~"~ky or Tex~ Ikahh Science WEN.HWA LEE, P..D. Professor an~ Director. Institute of chemisln/. University of Texls Health Sci. enc¢ Center. ~an Ante.lo. TX. JULIAN L. LEIBOWi'I~. M.D.. Aux)ci~e Pm(esr~ or Pathology ~nd oratmy Medicine. Unlve~ity of Texas { {ealth .~ien~ Cemer. lissom. TX. EDWARD B. LFX)F. l~tD. C~nsa~um and Ass~'i~e Pmfex,,~w. Mayo Fntmgati(~. Rochcsles.. MN. HEINRICll LEONtlARDT. Inxlructor in Pediatrics. Fran~ Volhard Klinik, B~lht. EDWARD D. LI~VINE. ,A~ixlant Mcd;cal Research Prorcsso¢ or hychla(O,. ~ U,ivers~, Mcdicat ter. Dud~m. NC. ~ON D. LEVINF, M.D.. Pml'eaSm'. Univmity of CalifornTs. ~n Fru~i~co. S~m Pr~c|~:o. CA. KIM K LEWIS. Am~ci~e Pmt'es~o~. Unlve~sity of land ~h~l of Medici,. fla~im~e. MD. PROJR~T TITLR G1utama~er~ effects ot' nlemlne
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PROJF.CT TITI Fl|nclhmal an~ly~i~ 4~" lhe ,~,'ogene tlln aml il~ chicken ,:ulhil;,r Selective a%,,~mbly of K ch;mnc/s, dcfcrm[na. li~m c)f the rook'cut:it Lymphoid VDJ recombination in human $CID Molecular ~.ix~cl~ of DNA Neunmal n~tlnlc rece~nr-.lmclure Molecular i~nellc~ of cant~ in I INI~?C 260 PRINCIPAL INVESTIGATOR OR IN,~rlTUTION ROflERT G. McKINNE~ ~I.D. Pmfes~ or Gc~llcs ad Cell Blol~y. MARK K. MFRS. ~.D. A~si~lafll Profeslor. Ilatvlrd Medical ~. ~. MA. Pmfcssor of Molecvlar Biology and Pha~logy. Washlngwn Unive~iey. ~ ~ MO. O~D MEYUIIA~ Ilehrew Univenily.lladassah Medical Ass~ ~I l~lal, ~ MA, ~. ~I~ OR. J~ D. MINNA, ~m, 5~ ~ C~, ~v~- si~y ~ ~x~ ~h~ M~1 ~lla~.~. ~LLIAM J, MORRISON, ~ Health I~. O~ KARL M~G~, Assls~am Pm~essor. Harvard Medical ~. ~RN~IS MURR~ ~,~ ]AV~ NAVARR~ ~fes~ of ~s~y a~ B~ysi~. University of Texts Medical Branch. ~ A. N~. G~ANAN H~VE~ M.~ A~i~l* B~
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0 0 PRINCIPA/. INYKSTI~A'rOR OR ~O~ Senior ~clu~er. ~=r.llen Univer~ily. R~ ~ K.~D~, ~.D. ~ l~ ~. Uni~ily DAVID N. OASR;~ N~ M.D. ~Y O~-~R. As~ate ~. ~e~d ln~tltule A~iII~ ~mm~o~, D~¢ Unlvcr/ity ~dical ROBOT ~ ~N~ ~D. reeky, ~ of Ve~n~ M~ici~, !~. ~ P~ER, ~.D. Assist ~es~. Waksman In, flute. Ru~e~ - ~ State Unive~ily of New RO~ P~, ~.~ B~.TAO P~ a~r ~-~, unlve~ity ~ Kem~ky. ~x~ KY. ~o~e.er ~.Di~cal ~m~sf~ and ~ f~, ~ ~A. PROJ R~-Ti" Tm.R Clo~ing and l'un~tkmal ~ns~,y~;.~ of The MMTV long terminal ~al: ~ene Putificstlnn of I~ yc=q rec~mhlnant C~II Pr~r chromosome ~e~atinn and the dr~ila ~ ~eln O~etic snalysls o~ ras-si~nat t~n~uct~ ~hways in tmm~n~ m~ ~lsr ~tsm of Mol~ular analysis of hepatitis B vies X ~ly ~i~in ~a~km hy am~x. ~F-~la-likc ~ Rex~iv~lio~ o£ isolxcd mito~ xpl~rat~s t.O~gee~ ~e ms pro~n and cell cycle No~d gcr.es cxpre~ed in the cell cycle 262 PRINCIPAl, INV~ITI~ATOR OR IN~rlTUTIilN SARAll L PAR~N$. l~l.D. ~il7 ~ Vl~la. ]AMF.~ O. PA~N. ~.~ xlty. Na~hvilk. flKN~I~ ~r U. PAULI. sity. College of Veterinary Ith~. Asx~iate ~f~ of Internal ~d~ a~ M~mb~gy, Untve~ily ~ V~tnla, MOR~M~ ~N~ As~iale ~es~ of ~ialri~ VIVEK M. RANGN~KAR. ~.D. MARK M. RASF~ICK. ~.D. II. AHARON RAZIN. ~VEN I. R~ED. ~.~. NA~ C. R~II. ~.~, R~ALD R. ~IOIE~ ~.~. A~isllflI ~fe~, Unive~ity ~ M. REID. ~.D. ~fes~. Uni~ily ~1 lflll. ~. Ilexd. ~menl of v~ily. Sh~ve~. I.A. CARRIE RINKER~OIAE~R. ~I.D. Assislunl Pm~es~r. Unlversily t~r Chl. rROJRCT TITL~ ln~er~i©m or m',O'" w~h tt~ R¢l~uladoe of f,~'l .tml~,~In q~lk-inll Tiute-qx'cifle tum~" ¢fllA, nd~lh~llal c~ll Idheskm in veflcbral ~ nmlmlasls +,I' I M¢clmnls .ms of ~tamt~. hkl~k~ mh- Regulation ot'pl~lelet protein expt'~slo~
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PRINCIPAL INVP.qTI(|ATOR OR L~.~ITUTIO,~ ~ W. ROGERS. ~.D. A~iltsm ~. University o~ Ulsh, ~HA W. R~ h~.D. A~aflt ~ of A~tomy ~ Cell B~I~. C~um~a Unive~ity~ll~ of ~y~ian~ ~ Su~q. New Y~k. ~. G~A~ M. R~. ~.D. BY. ueive~ity of Maryland Sch~l nf ~ssJst~nt ~fe~sor o~ Bi~hemlstry. ~IOMAS ~ R~I~IN, M.D, A~¢ociate Professor o~ ~ediciae and nler, u~m, M~ HARRY R~IN. ~.~,, D.Y.~. ~ ~ M~t~ B~, ~i. K~T W. RUNG~ tJ~ Re~h J~ ~c~l~. OIL ~PNEN ). RUOS~ M.D. FABIO P. RU~, ~I.D. A~ista~ ~fes~. 3ohn~ lh~ins Unl- ve~hy ~1 uf Medici,. ~ahi~e. M~ AN~ K. R~I. M.D. Atds~m ~ o~ M~tcim. K~N R~. ~.D. As~in~ MemO. in, flute ~m ~ne~ R~ ~il~l~a. PA~ ~. gA~VA~RRA. ~.D. Rehash ~misl. Beckman Rehash Instate. ~y ~ H~. ~a~. CA. H~ K. ~A~ ~.D. Assi~l~nt Pro~esso~. C~se Western ~e ~i~nily, ~vd~. OIL PROJECt Tm.R The reBut.ilion ~ neuronal nicotinic receplm Modulation of Central nicntine-reeeptor chann~l,~ F~ec rxdk~! en~diated cyfotnxiei|y Molecular mechanlxm5 of mu,ccle flher diversification in tran,~mic mice TR .I~AP.I DNA bimlin.~ pr~ein~ in !! lyre- 0vamita|;ve .~.d;cs or.,~.poman~us tntnsro~. marion in cell culture T©lomere function in chmmmeme stability u~J nicker .~ru~tum Pmlci~ation of" mast ~ell t~ta,,,e in airv~ys inflammation Agrin, nlcntinic neuronal acetylchnline recel~f~ (';~,e repfllon a~d prr~in functlrm hkntific~ion of the gcnc targets of on:o- Molecular and biological aspects of a mirophic f~c,~ in Drlllr~iln Regulatkm el" allemztive p~mRNA, sl~iclng I~j rmf, a Dm~of~ila snRNP prolem Initiation or tramladon by internal dho~m~ blnd~g 2~4 PRINCIPAL INVP.qTI(;ATOR OR INSTITUTION DAVID A, SASSOON. I~t,O. As~i~lanl Professm. Mr, tat Sinai Medical Center. New YoCk, NY. J. DENR¥ SATO. hi.D, Researeh S~ientist, W. Alton Jinxes Cell Science CLm~r, tadcc rl~cld. RORERT Ii, SC! IIESTL, l~t,D, Assislam Pmfesso~ of Toxicology, vai~ Sch~l of Public Ileallh, Ooxton, ~IIRISTIAN W. SCIIINDLER, M,D., I~LD, As~sfant Pml'ex,~mr of Mc~licin~, Colum. flRU~ J, ~IHA~, ~LD. AXl~Jate Profes~, tiltyard ~cdtcal R~ fnst~, University o~ Caml~t, ~ Hill. ~. GIN~ SERRERO, R~mh A~iatc ~, Univcn;~ ~ Mu~lu~, ~h~, MD. JA~F~INE SllARON. ~.D. AtlJsllnl ~fe~r. Boston University ~ ~ M~i~. B~t~, MA. BEN~ION SHI~. A~ia~ ~f~. ~ Weizmann Investigator and Assi~tl~l Pfofess~, Jml~ ~a~s ~nier, Ifma~ ~. ~, MA. ANN.BIN $11~. Assi~ttm ~f~, Univ~ily of ~xas ~l ~1 at H~ ~. ~ sity d Calif~i~ ~n ~ DAVID A. SIRBASKU. ~I.D. ~cs~ ~ B~mist~ a~ ~lar Bi~. Unlvm~y ~ Texas ~ul~ Auislmt ~f~. ~umh~ Unlv~ty. New Y~. PROJECT'TITLE Molecu~r ~sis of llmb develep~m finn ~ ~ Signal tran~ti~ in t~ IL.3 family of c~i~s M¢~lir ~ ~ fulfil B~IIs or Molecular analysis o£ ~cH~l~.repalr ~pli~ ~ hum~ Factors thlt control T-zell-speel.fle lane expression during lymphoe.jt¢ merit
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PRINC*IPM.. INVF~TI(;ATOR OR INSTITUTION I Uh?J~l. L. sirE. A$~ociIte Member, Whitehead In~tilule for Biemedieal Reseltch. MA. IIAROLD C. SMITII. FIt.D, Associate Profe,~sor. Unlver,dly of. ~er. Roche~er, THOktA$ E, SMrrlIGALL. Asst~am Professor. El~ley Institute for P.cs~amh in Cancer an~ Allied Di~em Unlversi~y ot" Nebraska Medical Cuter. LAUR~Jq ~PAYRA~, ~,~ A$~o~iat~ Professor, University ~ ,TANUSZ M. ~OWADSKI, As~on~a$c l|©,~ca~cb Biolog|~ Un~ve~ity Anm, i~on! ~of.e~,~. University ~f Ken- DAVID L. $~R. g©n&~ Staff lovegi~atOro ~d S~ing llarho~ Lalx~atory. Cold Sprin~ llarhor. SARAll SPIEGEL. i~t.D. AssistInt Prof.e~sor of Biochemlstr)" and Mole~uhr Biology0 Geor. l:e~own Unlver. sit)' ~cal Cuter, Washmgt~a. D.C. ELJOT IL SPINDb'I~ M.D.. $¢.lentiat. Oregon Regional Primate Ree, eamh Ce~ler. Bemvmo~. OR. HIKOLAU$ SPOEREL. PH.D, AS$1U|al Professor. University of ER|C #. ~'rANBR I DOF~ Ase,~iate Prefer.or of Micrab~ogy. Uni- verity o~ Calit'omla. lrvlne. CA. JEAN R. ~TARKIEY. P~hD. Assoclale Professor of" Mictob|ology. Montana Sate University. Ih,xemmn. MT. JANET N. ~TAVNEZER. P~t.D. Professor. University of" Medical School. Woecesler. MA. KARl $'~'~¢PANS~N. M.D. Profesa~¢ o( Neumlofy. Beth [s~*1 ptt|l. Boston, MA. PROJF, L~r TITI.R The ~.le ot" re|inoid~ d~wing antem~oqterio~ Occurrence or almlilmpm~ein R mRNA ing factors in huma~ liver Regulation of" I'as aignalin~ by the e-lea pmloemco~ne tyrmm¢ kinas¢ Genes invaded in $V40 pOSllr~nslal~l h~i~ ~e in tr~gen~ m~e $~ial a~ lem~l orpnD~l~ of DNA ~mtkm Mol~uhr mechanisms of t~ ~lulmti~ by gangli~i~ GM I Potential roles of novel and mutated ~in m~ in human ~ m ~ su~tly in D. ~la~a~ su~ ~ in ~xn ~r Adhesive ~cepmr:llgsnd inler~ction~ in Regulation of transcription of germtin¢ imm~l~lin ~ Hex~hi~ (Tena~in): an PRINCIPAl. I NVF.~"I~.AT~R OR IN,qTII~J'rloN DAVID M, ~fERN, M.D. Colle~ of l~y~i~ k ~r~. New Y~. NY. MARK Y. ~LF, M.D. A~i~ ~e~ of M~ici~, ~II Unlversily Medical CoI~. New Y~. ~UR L ~AND, ~l.O. ~ B~l~y. New Y~ New Y~, NY. DAVID ~. ~AYER, M.D~ IIEIDI ~UIH.MANN. A~i~l~l l~e~. Mmt~ Sinai SC~I ~ M~i~, New Yo~, NY. ~EN I. S~RLEY, Asslslanl Ptores~. tn~titule of Human Nulrlli~m. C~flmnhin Un~versily. New Y~. NY. MARIUS SU~I~ A~i~tanl ~'~, ~ R~kefeller Uni. ~ily. Hew Y~, NY. KA~IY A. SU~A~. ~.D. Asslstan~ ~rcs~. Un;v~;ly of K~ DANIEL ~. ~U~gMAN. ~.D. ~RGNY 5V~S~N. ~.~ P~es~or. K~linsks In,titular. IRA A. ~A~A~ M.D.. ~.D. NAOKO TAN~ ~.D. ~RIC M~-SHO~ TANG. ~.D. A~b~ ~e~. Unlv~ity of M.D. A~ C~er C~. I~st~. K~N~ D, TAR~F. Senior Member. Institute ~or C~neer Re~. ~il~t~ia. PA. PROJRC'r TITLR Aging. diat~ete~ ~t va~lar Cytokine ~ repula~lon: coati of mRNA ~ti~al m~ly~i~ or ~t-3 du~n~ m~. nmtian Delermlnams of secretion or human Endogenou~ inhibllors of mlorotuhule t~ ~thway An ex~al Function o( the foam ~11 Ii~inla) F~#II~ ~ ~Ir ~ ~#~I~n.DNA
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0 IPIIL~'IPAL INVI~qTIGA1X)R OR INSTITUTION D. LANSIN~ TAYLOR. Pr0fe~ of Biologlc~l Science. Carnc~ie- Mellm Univmt~. F~tt*.bur+h, PA. CORNF+LI$ P. Tt~R I IOR.~". Asso¢||le l~'ofe+e, or o! Pnlholo~y. Beth l,.rlel lk~,ph~l. Boslo~. MA. ~EPIIEN R. TtlOM. M.O.. P..D. Ass|stanl Prol'e~,~or. University or Penn. DONALD]. TIPPER. Pl'ofea~or. Uoiversily of Mas,~achusett,+ /v~dicl! Ceolef. Wnec~-~ler. MA. ALAN E. TOMKINSON, P..D. Astisl|nlt Plol'e+~sor. Unive~ity of Texas Heallh P~|ee+ee Cenler at san Anlonlo. ~m~ Amonla, TX. NICIfOLAS TONK$. P,.D. ~e~ior ~IIIT lnvealigator. Cold Sprlnl~ Ilarhor l.Jl~ralmy. Cokl .~1nlt Ilarh~. JAMES ~'OTTTRIMMER. PH.D. Ass|+llnl l~fes~or or gi~hcmistry and Yo~ al Stony llnm, k. $~ony Ilre<~. NY. PIIILIP hl, TSICItLI$. M.D. Seolm" M~mber. Fox Ch,,me Cancer Cen- let. PhHMelFhla. PA. MITCIIEI.J, $. TURKER. PH.D. A,+~oclate Pml'es~r. Unlverslly or Ken- It~ky. L4minltlo~ KY. INDER M. VERMA. I~I.D. Pmfe~or. The ~Ik lnslllUle. San CA. H~RB~RTW. VIRGIN. M.D~ PH.D. A~|slI~ Prot+e-or, Washinllee Uni~er- slt.V. ~. L~is+ MO. THOMA$,I. WA~MllYr. PH.D. ~ ~ I~l. l~s ~ly. IA. Asti~ ~e~. N+mhwettem Uni~r- ~e~ ~ B~y. Univ~y ~ Cati- ~. ~ D+~ ~ J+dlm CA. MAP kin~e pho~hal-~ MKP-I and c~- Iml orcell pmllferalkm G~e~iC i~dali<m and recomlilutlno of l~aln polassium channel camplexex Role ol'a novel Imkyrin re~al Ix'olein in c~!1 c~le ~gubthm Pmmoler ~gi~ ~lali(m a~ ge~ tn~- tival~ ~gulal~ of ~F-~ ~ I~B: ~iat~ early ~ fu~i~ T~ role of Fc.R-B cells in n~al and aut~mm~ i~li~ ~ anti~n Tymslne p~phnryludno or RNA ~ly- PRINCIPAL INVF.~'rI~ATOR OR I N,~'~TUTION XIAO-FAN WANG, Assistnn! Prol'exsor or Pharmacology, l~ke Univ~nlly f.k,d~cal Center. Durham. STF+PI IF.N L. WARRF.N.M.D. As~ixlanl Profesaor0 Ynle Universily ~ of M~dlclne. New Haven. MA'I"rlIr..w ]. WAYNL~R, Pmfcssm'. University or Texax st San A~k). San Anwnk~. TX. MICI IAF.L A. WFJ$S, M.D. Ct,IARLES ]. W~'t'Z, M.D,. PtI.D. As+ixllnl Professor. Ilarvard Medi¢ll Schmd. P, mlon. IlOWARD G. WEI.GUS. M.D, Pmfe.~or of Mcdlcin~, ]ewlsh tfmphah Washlnglon University Medical Cenler. SI. louis. MARIANNE WF_~SI.INP,-RF.qNICK. Assislanl Profes.mr of Nulrilionnl Bio- che.m.islry. Ilnrvard ,~chonl of Public Ilen,h. Plmttm. ALI~'~ANDffR STI~.N WI IrT~J IF.AD, Pml'e~sor of Medical (.;enelicm. Trinity College. Ouh3in. KA~II~RINK WII,~)N. I~.D. Assi~IInl Prl~re.~sm'. The )~hna II~pk|ns WILLI^M 13. WOO0.1~.0. Pr~exxr~, Univet~ity of Cotm~lo. Brad- der, CO. LAWRFJqCE ~+. WY~I. ~lnff R¢lm~4~"t~'. Nalloonl .~ewlsh Denver. CO. TIMO~IY 'YEN. A,,~o~|ate Member. ~n'q|tu~e for Cancer Res~rch. Phil~ellgs|a, PA. CtlO-YAU YEUNG. A~|~l~mt Pmfcsnm, Unive~ily ol" IIIin~s. JORI+ K. YISRAEL1. L.eel,,rer. ltelx, ew Universily-ll~as~nh Medical ,~tm~l, .Icrusalcm. PROJRC'r TITLR Study ef the TGF.Seta ~ilmHnI palhway(q) in srm~th inhlhlllon m~Aic ty~ine k|n,~s. ¢~loeellln and Nicol|fle enhancer memory: Inll, rs~ltoo~ wtlh anI,Jm~nsl. II Caster,related tran~ripl~n fa~toPs: stria- lure ,,nd mcchan|+trns Ge~e mlolatkm in entminmznl or t~ mare. malian ein:~i~n clock TKe role or cell-cdl c~muct in metnllapm- leimme Ind~llo~ The ale orP,& sln~4ure in ve~ hl~k~ty C~mml of exl~vSmim+ el" Ihe laMammato~ Molecular analysis or ehmmalln.hlndlnl A~I~ ~ k~ ~ ~ Trans.~gulati~ ~ family ~rs or a Mar ~ni~s a~ ~lh f~l~ ~a. nitml~ 269
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0 0 i',3 PRINCIPAL INVI~qTIGATOR OR R(STITUTION BERNARD ZAL~, M.D., D.So. Director of Research. Hopltal de la fmmu~oly, univc~ity d Kentucky, 2"/0 Grantees of Completed Projects Following i.~ a list of the fflneipal Investiptors. i~iv~als n~ ~ ~. ~ lilies a~ a~li~ lisl~ ~ ti~ t~ w~k was ~ ROBERT It. AB F~F3, P~fesrax of Bioch~mhclry, R~IC Un|- versify. Walthnm, MA. LEO G, ABOOD. ~ofcsscx of Brain Re,earth and B~hcm- kl~, Cemer fm Brain Re~--'arch, Univer- sity of Rmhester Medical Cemer. Rochcs- ter. NY. MARIO D. ACE-TO. P,.D. A.oc;ale Professor of Pharmacology. Medical College of Virginia, Virginia Cm~monweallh University. Richmond. VA. DO~PI! O. ADAMS, M.D., P.,D. Pml'essm of P'ad~doD.. D~ke Unlv~sily Medical C~n~cr. Durham. NC. tAN Y. R. ADAMSON. P,.D. Pmfes~m" or I~th~c~y. Un;versity d" Man- iloh. WE'mi~eg. M;mi¢n~. C',nada. BURT ADELMAN. M.D. Assislaml Pmfgsf, or of Medicine. Medical College r,l" VisTUla, R~mw.d. VA. KENNETH & ADLER. P..D. Assimnt Pale*Jet of Pafhdoff, Univer- sity of Vermont College of Medicine. Budinpnn, VT. CLARENCE M. AGRES$. M.D. Aswndale Clinical Pmfeum of" Medlcin¢, University of Calirnrnia Medical Cente¢, los Angde~, CA. ANTIIONY A. ALBANESE, Din~or of t.~hor=tocE~. The Bud¢e Reha- hilil¢ion Cen~er. WhEe Rains. NY. JOHN J. ALBER$. P..D. Associ=le Dire¢lor, Northwest Lipid Research Center, llarhervlew Medical C~fllgr. ~lle, WA. AN'rTIONY P. AMARO~E,, PH,D. lnslruCm" in Obmldes and Gym~o~7. The Albany Medical College of Union Unlvemtly, Albany, NY, T, ANGELAKO~ M.D.. I~.D. Professor of Physiology. |oslon Uni. realty. ~ch(~l of Medicine. ~ml*n. MA. MURRAY ANGEVINB. M,D. l.Jnivem{ly of Witcm~in ~ch~d of lvkdl. dee. Medbon. WI. JOsEPH C. ARCO~ D3~. 1~rofea~ d" Medicine. Tulams Unlv~ily School ot' Mcdieine, New Orlwn~, LA. ALAN K. ARMITAGE. R,#~h Director. Hukion Eufol~. Harm~te. Nenh Ym~:hlm, hal 271
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0 0 ALAN D. ATT~E. I~.D. KAR~N J. AUBORN. ~.D. M~I C~, New Hyd~ ~. NY. ~AS M. A~E. ~.D. ~1~0 M. AVIAn. M.D. fear of ~a~o~ogy. Pennsylvania Sch~l of Medicine. IR~ AV~RAM. ~D. ~i~% ~el AvivUni~i~. DAVID ~ AXE~OD, ~.~ Ass~tate Professor oF Microbiology. Waksmzn Insdml¢. Rul~c~ Univer~zly. ~atzwzy. HL ~l~ M. AYR~. M.D. ~i~. ~u]mnary La~ramry. 3slot ~'s I~pilM. N~ Y~. NY. B~NARD M. BABIOR. M.D.. ~.D. H~d. Divisi~ of alchemistS. CA. L~LIE EAER. M.D. A~i~e ~es~ or Medicine. Colum- ns Univcrsily College of Physicians SAMU~ BALK, M.D.. ~.D. pId. B~. MA. ~DERIK 8. B~G. M.D. ht~. ~ J~ns ll~im U~ e~ MO. 272 RORERT L. I~ARBIERI. M.D. A~si,;tant Protector or Ohstetrics and Gynccoh)gy. llarvard Medical School. Oo,ct~. MA. LAURIE BARCLAY. M.D. Uniformity ~t" Snmh Fi(~i~. Taml~. FL. BRODA O. BARNF.(;. M.D.. PlhD. Prol'e~r.(w fAffili.~le) of PhysioloBy. C~d- Slate Univcr~iW. r'.urt Collins. CO. FREDERICK W. flARNFJ;, Je.. M.D. Assm:iale Prol'e~nor of Medicine. The Jc~m~; Ik)pki~ University .e~h~4 of M~I;- cir. ~hin~. MIX T. C. BARNFJ;. D..~k". R~ea~ch Sciefl~.~l, Phil~lelphia Slate Ih~- pit-.I. Philadelphia. PA. CARLG. flECKER. M.D. Asseclal¢ Proton, mr o1" I~llholo~y. Cornell Univer~hy Medical ColleBe. Ncw Yo~k, NY. FREDF~ICK F. BECKER. M.D. Prnressor of Pathology. Universily of Texas M. D. Anderson Cancer Cenler, Ileusle~. TX. R. FREI~.R~CK RECKER, i~LD. Associate Professor or Analomy and Director. Labor~c~ of Pe~atal Scler~e, Duke Univer~y Medlc~ C~lcr. J3mham. NC. RALPtt S. BECKER. P..D. Professor or Chemislry, Universily I !~t~. Ito~o~. TX. A. DEAN BEFUS. ~l.D. Professor of Medicine. University or Alhen~. Edmnn~m. Canada. BEN.IAMIN BELL. M.D. Director EMeritus. Normative A£in~ ~t.~d~. VeK, r~r~ A(fmini~lralin~ ()intellect Clime. ~. MA. SAMUEL BELLET. M.D. Dirtier. Divide~ of Can~i~o~y. Philedel- ph;a General llmp;lal. I'hilad¢l~h;a. PA. BARUJ BENACERRAF. M.D. Fabya~ Professor and Chain.an, DUpa~. menl of Palholo~y. llarvard Medical ~t. ~. MA. JOEL S. flENNETT. M.D. As~c~e l~nfessm # Medic;he. Hosphal or the Unlverslly of Pennsylvania. I~il~l~lphis, PA, MICIIAFJ, FIENNRTT. M.D. Professor of Palholocy. Universily or Texas .%q~hwe~em M~dical C~nler. Dul- I~ TX. BARBARA J. VAN ~ BERG. M.D. R~ch Pediatrician. Adjunct Prol'cssor in Bio~lalislicx. Universit)~ of C'~lifomla Scl~x~! o1" PuMic Ilealth, Oakland. CA. ANDRE BERNARDS, P~.D. Ax.~b;lan! Profcs,,,nr of Meal;clan (Genet- ics), The Cancer Center, Ma~r~¢husells Gener~.l I l~ifal. ~csm~. MA. ERNEST BFJJTLF_,R. M.D. F, xpedme~al Medcme, ~enpps Re~.~teh Insdlele. L~ Jolly. CA. JOHN A. BEVAN. M.D. Professor of Phan'~scotogy. Un;verslty of California Sch(ml of Medicine. Los Angel~x. CA. BUDIIDEV BIIAGAT. I~l.D. r-mfes~m" of PhysiC. St. L~u;s Univtr- sily School of Medicine, St. L~is, MO. CESARE BIANCIFIORI, M.D. Division of Cam'er Re,ear',h, Unluckily of Perugla, Pen~la. Ilaly, IIYLAN A. B1CKERMAN. M.D. Assistant Professor of Medlelnu. and ALVAN L. BARACH. M.D.. Con~ultanl in Medicine. Columbia Univemlly COlI~ or ~slcbns & ~ Ookl.mr M~- modal Ilospltal. New Yod~. NY. ~IO.RE~EARCH CONSU~TANT~ INC. C~m1~d~e. MA. BIO-RESEARCll IN~TUT~ I~C. C~mlm~l|c. MA. SUBAt. BISNAYEE. PtI.D. Associate Profuse(. C"od~li In~lltut¢ for Medical Re~ch. C1md~, NJ. DEBAJIT K. BISWA$. FILD.. D..~. A~.~c;ate Pmfc~0~ of Oral Bkgoly. Ll~e. Dental Med~Ine. ~e~, MA. IRA B. BLACK. M.D. ~ and Cldef. l~vlslon ef Duvel~p. menial NeuroloLy. Cernell Univertltl~ Medi.~! Colle~.. New Ymk. NY. PllYLLIS B. BLAIR, I~.D. Profe,o~ ot" lmmu~lora'. University of Califm~ta. l~-rkeleT. CA. EDWIN BLALOCK. PH.D. Professor. Univcr~ky of Alablma. Ingham. A~. MARIANN BLUM. I~t.D. Assistant Profcs~er. Fishier| ReNet~h Center for Neu~obleloly, Mount Sinai Sc~ ~M~I~. ~ Y~. ~. FRED O. BOCK. A~xt~e Io~|cal Slalk~. Relw~ll Pad~ Imlltul~. ~lT~lo. NY. DALE E. BOCKMAN. Proressm" snd Chairman. Dupsrtment or Anatomy, Medk:al C'ollep or O~or$1a. Aupm. GA.
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O O IIERMAN V. BOENIO. P~LD. Ile~ld. Dep~Rmem of Chemistry and Bin. chemistry. Spindlemp Re,earth Center. Leain~o,~ KY. CONSTANTIN A` BONA, M.D. Profestor of Microbiology. Mount Sinai ~choul of M~ici~. Hew York, NY. JAMES F. BONNER. P,.D. ~ofc.or or Biology. Califoraia ln~itute of Teck~lo~y, Pa~dena, CA. WALTER M. BOOKER, Pit.D. I~ofessot aml |1~. E~r~rtm~tt of Phar. maeo~.,~y. Ilow|rd University. Washing- loft. i.p~, FRANI~IS M. BOOYSE, I~I.D. Senior Investigator, Michael Reese Re~,amh F0~fldalioe. Chiea¢o. IL. RAYMOND BO~$1~ P,.D. Associate Direclor. Normative Aging SItKly. Vel~a~ Admint~r~in~ ~t ~{e. ~. MA. TOM O. BOWERY. I~t.D. R~mh Pndes~ot, Pssdeide Residue Lab- oratory. North Carolina State College. Rale~h, NC. It. L~ON BRADLOW. P..D. l~sthme fo¢ lion, one Re,earth. New Ynd~. NY. $COTTT. BRADY. PI,.D. Asso=iate Professor or Cell Biology and N~mcden~. Uni~ity of 'rex,,. South- western Medical Center. Dallas, TX. J. MARK BRAUGHLER, P~.D. Assistanl Professor of Pharmacology. N~rll~$*e,m Ob~ Univerr, ilics ~ of EDWARD BRESNICK. PIED. Professor a~d Chairman. Department of Bin~..,hemi~ry. The Ueiverxily ol'.Verm~t C'~dlel~ of Medicine. Buriin|t,m, Yr. GARY R. ERIGHT. Pit.D. Asd.~Jm I~of~,m~. Cede W~tem Reserve University. Cleveland, Olf. 274 GEOFPREY L. BRINKMAN. M.D. Associate Professor of Medicine, Wayne State University School of Medicine. De- troll. MI. ROBERT E. BROOKS, Ph.D. Assneiate Profes,~o¢ of Pslholo~y, Univer- sky of ~ Medial ~l. ~lan,l. OR. BARBARA B. BROWN. lhcD. Chief. Experimental P~ychia~ry. Veterans Admlni~radou Ih~l,~ah ~¢pulvc~a. CA. MELIS.C~'t A. BROWN. Pit.D, Re,earth Aqsislanl Pmfes.m¢ of Medicine. Microbiology and Immunology, Ore¢oe I{eahh ~iences University. Pnnlaed, OR. RAYMOND R. BROWN. P,.D. Pm1"e~or of Clinlcal O~logy. U~vet~ily of Wi~coosln Medical School. Medim. WL REBECCA BRYSON. Pit.D. Associate Pmf~sne of Psychology. San Diego Stale Universily. San Diego, CA. NANCY L. R. BUCIIBR. M.D. Rer.earch Prot"c.~.or of Pathology. Boalon University School or Medicine. Boston, MA. DOROTIIY L. BUCIIlIAG -F.~/, PiI.O. Assislam Professor. Stale Unive~sity of New Yo~k. Downstate Medical Cenler. BrooElyn. NY. SUE BUCKINOIIAM, M.D. Assistant Pm~essor of Pediatrics, Cniumhia University College ot" Physicians A Sur- geons. New Yod~. NY. KATIll,E, EN M. BUCKLEY. Pit.D. Assi,aam Pmfessm of N~Jmbiology. lbr- vl~l Medical SdNn,l. Bmkm. MA. VINCENZO BUONASSISI. M.D. ~m{m' .e~cienli~ and I~ty Di~ctor. W. Alton Jone~ Cell Science Center. Lake Placid. NY. JOIIN W. FIURCit. M.D, A~,~x:ial¢ Medlenl Din~r,r. American Red Cro~,~. Roehe.~tet Divi,~ion. Rochester. NY. BENJAMIN BURROWS. M.D. As~m~iate Pmfc~mr of M~dicine. Univer~i- ly or ChignOn. Chlc~g~. IL DAVID L RUSBEF~ Pmfesqm of Toxlcolo~y, Texas AAM vershy Cnllege of Veterinary Medicine. Collese Stalkm. TX. DAVID F. BURSTEIN, M.D. Assistant Professor of Patlmlo~y. Mount Sinai School of Medicine. New Y~rk. NY. F_ M. BUTT. M.D. Chief Pstholo|ist. Los Angeles County Genotl Iimpital. ~ Angeles. CA, RIC11ARD 13. RYF.RRUM. Proressm of Chmislry. Michigan ~ate Univeodty. FJe~ I.amslr~. ML MYLES CABOT. ~enlor ,%ienti~. W. AIIon Jone~ Cell .~i- once Center. L.~ke Placid. NY. SISTER M, EMILY CAtlILL. P..D. Chairman, Deparlment of Chemistry. Reels Cnile~e. Sexton. MA. EOWARD J. CAMPBELL. M.D. Ass~am Pmfesmr of Medicine. Washin~- ton University School of Medicine. St. Louis. MO. BRUCE F. CAMERON. M.D., P~.D. Howar~ Hughes Institute. Univ.e~.~ity or Miami ~ool of Medicine. Miamt. t-~. ELROY T~ CANTRELL, l~.O. Chairman. Department of PharmK'ofocy. Texas College of Ostee~lhi~ Medicine. N~h Tex~ ~ale Un;~ily. ~. TX. J. DONALD CAPRA. M.D. Professor of Microbiology and Internal Medicine. Univer~ky ~" ~exax ~ulhwest- em Medlcnl ¢..'eflle¢. t~lla~.. TX, 275 WILLIAM H. CARNE$. M.D. University of Utah College of M~dieine. Salt Lake City. UT. ANN M. CARROLl., I~t.D. Assislant Professor or Mlerobkflo|yl Immu~olngy. Albany Medical C011¢11¢. Al~ony, NY. MARCUS N. CARROI.t.. Jn.. Chief. Division of Pharmaeotolw. The Bm~k~le Ilmpilal Cemer. Brooklyn. NY. DENNIS A. CAR~ON, M.D. Univenity of C~lifm~da. San Dl~|e, !~ Jolla.CA. WILLIAM A. CARTER, M.D. Professor of ll©matoloLv and Msdieal Ontology. Halmema~ Medical Odle~,. PhileddpMa. PA. WILLIAM ALVIN CART~. M,D. Assistant ~ofessor of Medicine end Microbiology. The Jc~m~ H~klns U~lv~. sky ~chool of Medkine. Behime~. MD. JOHN L CAgTEM, OT, Jn,. Assneiale P~ofealor. Tuflt University, Breton. MAo ALBERT CASTRO, Pit.D. Dire¢lm. Honeone Re~eareh L~t'¢4~t~)': Profes~w of ~dmlo~y and M~dl¢Im, Uni. ve~it.y of Miami $i:hool of Mndlelne. Miami, Ft. FRANCO CELADA, M.D.. i~.D. Senior lmmunolol~lsl and Professor of Immunology. Iloq~tal foe Joint Dbe~t, O~ho~edic Irmitule. New Yeck. NY. LEOPOLD R. Profess" of Blnehemi|u'y and Nulfltlrm. Uni.~rsity_ of ~ Rk~-Seho~ of Medi. dee. ~n Jute. JACK CHAt.ON. M.D. Associate Professor of Anesthesiology. New York University Medical Canter, New Ymk. NY. JAMES C. CHAN. I%D, Asso~b~e Profesm'. Unlversky of Texa~ M.D. Aeder~e Caner Center, I1~o~. TX.
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FRANCIS C. CHAO. M.D.. I~t.O, $enlm" Investigator. Cenler for BIn~d Re~amh. B~t~ MA. SUSAN LYNNE CIIURC! I, M.D. Ass~am Proressm of Pediatrics. Washing. ton Unive~ily. ~. ~ix. MO. IIAROLD A. CllAPMAN. M.D. Associel¢ Pmr~,~N" of Med/cine. Br~Bham aM Wom~'s llmpi~al, Hc~t(m. MA. DONNA CHAPRONIERE. ~Iran~eway~. Re~earch Lah~rsmry. ('am. hr~d~e. En|bml. YUAN-TSONG CIIEN, M.D., PII.D. Assistant Professor of Pediatrics. Duke Universfly Medical Cemer. Oudmm. NC. MORDECIIA! C1 IEVION. P~t.D. Chairman. Institute of Biochemistry, Ilebmw Uni~city of Jem,~alem.llxda,~,ah Med~11 Sctx~. Jeruulem, I,~rsel. IX)UGI.A$ BROCK CINES. M.D. Prof~,~w of Medicine. I In,~ta| of I~ Unl- ver~ity of Penn~ylvaflia. I~ilml~lf~ia. PA. CURT I. CIVIN. M.D. Assi~m~ Pmf~w td" C'J~--ol~v and I~di- atrlc% The Jollth~ IIopkins (]m'oh~y Cen- ter. It~ffin~h'e. MIX ROIII':RI' A. CI.ARK. M.D. I~mh.'.~P,r of Mmlk:in~. I Inive~ity of It~wn. Iowa Cily. IA. WILLIAM G. CLARK, r~,.D. Director. Psychophurmscolncy Re.'~arch L~lx~atmy. Veterans A~inlslm~ If~- ~1. ~1~, C~. CHILDREN'S I IOSPffAI, OF IJ~ ANGELF~, Lm Angele% CA. WILUAM M. CIIIIJAN. I'~.D. Avd~tam Pmfcs~s,. Tesa~ A&M Universi- ty. C~Mle~e Station, TX. ROBERT H. CHIU. P~.D, As~,ir,~nt Pmfes',or in Re.knee. Unlvet- shy d Qlif(~'~i~, Lo~ Angeles. CA. JAN F. CI.ILEBOWSKI. R~.D. Asstmm ~ of Biochemist. Med. i¢~1 Co11¢~ ef Vi~inla. Richmm~. VA. SANFORD CHOOOSlt, M.D. Assistant Professor of Medicine, Tufts Universily School of Medicine, Eo~ton. MA. SUSAN CLASTER. M,D. Assistant Research Igemslofogi~t. Chit- dren's llnspital. Oaklaml Re.arch Inset. lute, Oakb~l. A~iare BRIAN L a.EVINGER. ~.D, W~s~init~ Uni~ilySc~ ~. CilAR~ G. ~BANE M.D. Mem~r. t~, ~ JOfll. JAY D. ~AN. M.D. ~t~ H~, ~t V~.f~r ~. ~. U~v~ IfmpimL ~t~. MA. Astarte Professor ! [eahh 2?6 F BERNICE H. COtlEN. PI~.D. Pfofessce of Eptdemiolo~y. The Johns I.l'o1~ins Universily. Bahimme. MD. DANIEL COItEH, D.V.M., M.P.H. olosy and Public Ilealth, Umversity or ¢i~. ~il~ia. PA. MYRON S, COIIEN. M.D. Assi~tam PmfessO~ of Medicine. Microld. ology and Immunology, University of No~hCamllna. Chic! IJiJl, NC. MARY ,~UE COLEMAN. l~t,D. Professor o1" Biochemistry., Associate Pmvo~t aM Dean of Reseamh, University ot" Nocth Carolina. Chapel Hill, NC. ALLAN C. COf.LIN~, ~.D. Associate Professm of Pharmacology. lr~titute for Beh~viorsl Gene~lcs. Unlver- sity of Colorado. Boulder. CO. ROBERT W. COLMAN. M.D. Pm(essor of Medicine. Temr~e University Seh~x~ of Medicine. Phlladdphia. PA. JULIUS H. COMROE, Ja.. M.D. Director, Cardiovl~cular ReseaR'k lnsti. lute. Univcrd~.y of California Medical C~. t~. San Francisco, CA. ROBERT L. CONHAIM. A.,~nei~e .~-'~misl. Unlve~ily of Wisco~- ~ifl, M~di~o~. Wl. DEAN M. CONNOR$, M.D. A.~,~,i~le landor. _Dq~n~e~ or Lj~x~ra. to~ Mcdlci~¢. SL Ma~'s Ho~itl~. Madi. 1lOG. WIG PHILIP COOPER. M.D. ClinlcJI Pml'~ of $ur~e~' and Su~BicaI L~lx~ .o~J of Cellull.r..Ph~siof ogy. AIb¢ll Ek~slem Colleee ol Mealcir~ of Yeshiva University: ChieT, Surreal ~r- vice. Veterans Administration Hospital. The Bronx. NY. RONALD B. CORLEY. P,.D. A~o~i~e Prot'essm or Imm,o~.3'. Uni.,~'~ity Medic~l Cen~er. Dm1~. GERALD R. CRABTRFJE. M.D. A~,e¢iale 1~o1'~4~' of Palhololly. StJnre~d University. Slanf'onL CA. JOIIN P-CRAIGIIF~D. M.D. Pmres~r~ oi" P~l,,~k'~.. Univmlty of Vef. mnnt Cnllelle of M~di¢ineo I~urliflllmn. v'r. ROBERT L. CRAtN. F~t.D. Au|stmt Profcss~'~ ~:blo~y, Unlvml. ty of Chk'q|o, C'~alo~ IL EVA BROWN CRAMER. ~.O. Asse¢ia~e l*~ore~w Of Analomx and Cell Biology. Downstate Medical Center. Bl~x)klyn. NY. IRVING P. CRAWR3RD. M.D. Pmresso¢ and C~ainnan, I~panmot of Micmblolocy. Uni~ity of lowt C'ellepe of M~dic~, Iowa City, IA. I". TIMOTltY CROCKER. M.D. Pmfes~m of bledi¢in~ Unlvemily ~1' Cell- foods Cdlel~ of hk.dicior, Irwin. CA. CARROLL E. CROSS. M.D. Associale Ptofe.~mr of Medicine and Human I'hysiok~.v: Dif~tor. ~lle~ Of Pulmonary Mndle~ne, Unl~y of ~ll- f~ia ~ ~i~, ~vil. C~ CROSS 3urt~k. CA. CARL ~. CRUET'Z. PH.D. Auismm Pm~e~sor or Phlnn ~l~ol~y. Unl. ve~sity of Vir|inia School of Medicine. Chado.esville.Vk. 2"/7
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Cn cn ~ 01" MierobJology. l.m,m*mclo~y School or Medicine, St, louis. MO. GIDONCZAPSKI. M3c.. PH.D. Professor of Physical Chemistry. The Hebrew University or Jeru.~nlem. AL~F,,RT DAMON. M.D.. I~t.D. Lecture~ ee Anthropology: Rcsesrch Asso- ei.t~ |O Medical Anthl~lQl_ nay, P~s~y MUl~tlt~. |lir~I~ Unive~dty, C'mMid~c. LARRY W. D~NIEL. Pd.D. AIIOCiIIC Professor. Bovnnan ..Gray ~1 o( M~iCi~e. Woke F~st Ua:vcrslty. W~.~-~em. NO. THOMA~ R. DAWBER. M.D. AmX~'~me Pn)re~ of MKltcinc, Boron Univer~ty ~chool of Medicine. MA. A.IJIUI~I' It.. IJGIS~ROTH, M.D.. Profe.or o( Medicine. V~lermr~ Admln- istr~t~ M~ical Comer. Sun Francisco. ]OLIN P. DELANEY. M.D., P..D. ofMimteso~. MinaealXd|s, MN. • ~F.ROME A. D~I~EVo PH.D. F~,~x' of Pm~entive Medletne, Univer. si~ of W|sc~m~n. Mediso~. WI. EVAHS S. DEHERTS. I~.D. ~ ProA:ssor. C'nse Western Rese~e U~i~s~. O~v~ind. OH. 2~ ANDREW S. DIRNER, P,.D. Exccative. Psycho.Research. The Age Center of New ~nBland. Inc.. Er~m. MA. EDWARD F. DOMINO. M.D. MPr~. ~ .e~l~o~ of Phsm~s~logy, Unive~ily o~ ~hi~ln. Ann A~. ML RALPii L. [X)RFMAN. P/I.D. Dircc~o~ of" L~lx)ramrle~. Wmce~e~ Form. dalion In4' FJq~rlmenlsl Bi~ogy. Shrcws- ~tm/. MA. I!. FRED DOWNEY. A.s~istml PnW~snr ~f Physlolo~y, Univer- s|ty of Texu I ktllh S¢i~n¢¢ Comer a( Oaf Ins; Dire~qur of Cardiovascular Research. Cardiopulm(~m~ |r~imle. Mclhodir.! rdtal o(Dallax. Dall&%'lX. HAROLD F. DVORAK. M.D. Chief, Del?artmcnl of Pathology. Beth bra~! Hoq~lM. Boston. MA. ]AMES J. DYAR. P~f.D. Assimm Pm~o~ of B;o~., BelL~ml.e COllege. Lo~sville. KY. RICHARD H. EARLE. M.D. Cffief, I~lmoaary Pnactioa La.l.~.ratory; As~ ~ ~ ~, un~- ROBERT ~CHT. PH.D. Professor of Anatomy. Michigan Stile Us~.ersity. Estt Lassie. MI. ]OHN W. ECKSTEIN, M,D. Assisltnt Profes~o¢ of Imemsl Med;¢ir, c, Stale Uni~..y ~N" Iowa Cdk~e d" M~i- ,~ne, Iow~ ~ty. IA. BERTRAM EICHEL. D.D.S. Director. lagt|tutn of SIomatolo¢ieal Rera~h. 5~ience Re~s F~, War,own. MA. RFJJBEN EISENSTEIN, M.D. Professo¢ of Pathology, Mo~nl Sinai Med- ical C~lcr. Milwaukee.WL. HYMAN ENGEt~ERG. M.D. Attending Physician. Cedars of Lch=ee~ I io~ldtM. Lm Aegele.¢. CA. VICTOR !!. ENGELIIARD. I~t.D. Profexgm' of Mi~obiotc~y. University of Virginia. Chado~tcsville. VA. ELLIS F.NGLESBERG. I~t,D. Pmfcs',o~ of Mictobk~k~'. University or C~lifomb. Sama R',rl~va, CA. CARLTON K. ERICK,qON. I~t.D. Pmfesmr of Phan~-olo~y.'/be University of Texas Cbllcgc of Pharmacy. A.slln.TX.T,. V. OF.~R ERWIN, Pin.mr of I~m~;~'~..v: Dean, Univ,- sity of Colorad~ ,~chool of I'harmacy, Boulder. CO. HENRY L ESRER. P..D. Re.~rch lmmu~oto=|~t, M~o~ ~r~Itule, WnrC0tler. MA. JOHN R. E.TrERLY. M.D. Assmi=te Professo¢ of Pathology. Univer- sily of Chiea|n Prilzker Scheo| of Medi- cine. Chi,:ap). II.. tlUGH E, EVANS, M.D. Director, Del~mem of Pedialri.c~ kh lios~tal and Medical Ce~er ol nmo~* lyn. Bro~lyn. NY. HANS J. EYSEHCK. Pst.D.. D.SC. chiatry. University of HANS L. FALK, P~r,D. Addict A.~.ocbte Prorcu.or or Patho~oF, y, University of So~lhem California School or Medicine. Los An¢clc~. CA, DANA L. FARNSWORTH, M.D. Hcn~J IG Olive' Pmre~o~ of Hy~ie~ Direcloro Univcrslty Health Services. vsrd University, C~mbrid~e, MA. ALVAN R, I~TNSTEIN, M.D. PrefOrm" of Medicine and Y,IC U~va~tty Scho01 of Med~ine, Have~. GAD FEINSTEIN, I~.D. Senior Lecturer in Bioehemislry. Gentle S, Wi~e Ccnler Tel Aviv Universily. RICHARD FENTON, P~D. Instructor in Physiology. University of Massachusetts School of Medicine, Wo~-¢stcr. MA, JAMF~ R. FERAMISCO. ?mressm of Medic;no. Unlverdy ~" CMI- f~nla, San DkI~, CA. F~ANK C, FI~ROU~ON, J~ M.D, ChMmmt. D~p~ttm~t of The Albnt~y Medical Cot|ego of Union Univerdty. AI~. NY. £ 5~L~IIEN RNK. M.O.. P~,D. Assimm Pmt'e~m~ of Newololy. M=~- chusetts General Ilospltnl. Cherleslown. MA, TII~ODORE N. FINLEY. M.D. Oi~zctof. Putmmmry Rese~eh CA. WILLIAM I. IqSHBE1N. M.D. Ilcahh, Ch;cago, It,. EDWARD A. RSHER0 M.D., PH.D. Assistant ProfessOr of Bkx:hem|~try. Th~ Medi¢II ColleI~ or l~nn~ylvanl*, l~lla- d¢I#~. PA, 279
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Rt.tS-~I.l. $. I'I~IIF, R, M.D. Uoiv©r~ily of Maryland School of Medi- cine, l+ah|mcxe. WILLIAM II. PI.~IlMAN. I~'~kk~l, L~ Jolla Ca~'~r Re~,tr~h Fern- dlllo~, l.J Jolla. CA. BIRGITrA FI, ODERUS-MYRRIW.D, P,.D. A+,sislanl Prof¢~o¢ or b%vironmemM Ily. |ieoe. The Karolinska Inxlilute, SlOCk. JUDITI! ANN POSTER. I~l,D. I'~l~¢~sor amml Chairpcr~m. Dcpatlmenl or Biololy. Syracuse Univor~lly. Syracuse. R/CHARD B. FOX, M.D. ~'t I~' Pulmon~ Divisloe. Minnc~polis JACK W. FRANKEL, PIt.D. ~llinl i~ Mndk:al Re~eamh. Veler~s Admin~mlon Medk:al Ce,lo¢. Bay Pin~-s. II. L FREEDLANDER. M.D. DiroClOr of Can~:r R~amh. M~m Zion 11041~ital and Mcdk:al Center..%n Fran. el~eo+ CA. AARON E. FREEMAN. P..D. Staff .%k~tbl. Qlifomia Biomedical qea~th Fmmdal~n. La/ella. CA. MICHAEL C. GEOKAS. M.D.. Pit.D, Professor and Vice-Chairman, Depart- m+nt of Medicine. University of Califor- nia School of Mc~+¢in¢. Davk. CA. MICtlAEL D. GER$11ON. M.D. Proresmor of An=lomy and Cell Columbia University Colle|e of Pfiysi- cians& Surl~on~. New York. NY. GERALD J. GLEICH. M.D. lory f~ All+Ii¢ Oi~llel, M~o Olnle iclne and Imm~nololy. Mayo Meal. GABRII~ C. GODMAN, M.D. I'mt'¢~x of Pmhok~y, C~lumlda Unlv~f, lily Collet~e of Phyl]¢lanl & Surle~nl, N~v Yod~. NY. BARRY GOLD. I~D. Associate Professor, Unlverslly of Na. brawl Medical Cen~er. Eppley ]nslitete. ~zh~ N~ WARREN M. GOLD. M.D. Professor of Medicine. Cardiovasevllr Research Inlllt~e. Univently of ~llfm. ~ S~ ~ C~ ALFRED L. GOLDBERO. 1%O. ~heoL Bollmt. MA. " IRA J. GOLDBERG, M.D. /mimm Pmlt==x ef Medici,, Columbia U, iverlily Cellele (d" I~ysl¢i~l & I~m. ?kw Yak. NY. SIDNEY GOLDFI~CIIER. M.D. Prol'estmr of Plthololy, AIb~'l Ilinllein Colkce or M~ine +t Yed~lva Unl~+. sl~, 11~ Borax. NY,
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0 0 0 PAUL GGLDIfABER. D.D.S. Associate Professor of Periodomology. Hmm~d School or ~ M~i~, ~ M~. WILLIAM E. GOLDMAN. I~t.D. Aslialant Pr~feseor of Microbiol~gy and Immunology. Wa~hln$1on Universily Sckeol or Medici.. ~. ~i~. M~ IRA GORE. M.D. I~ofesset ~ Pathology, Boston Unlversily S~ol of Medicine: F~.le.t of La .~. o9, JOItN W. GORROD. D.C.C. Lecluret in Btol~.armlcy, Chelsea Col- luhd. MAURICE GREEN. M.D. Ditc~lor. Institute for Molecular Virof ~o~Z. $1. Louis Univcrsily Medical Cer~er. CHARU~ S. GREENBERG. M.D. Assistant P~ofessor of Medicine. Duke Uflive~ity M~dical Cenler. Durham. NC. AU,,AN D. GRINNELL, P,.D. Professor of l~iolol~y. Univer~ily or" C~I- ifomfa. ~ An~l~ CA. ARTIfUR L ORO~S. Seni~ Biochemist, Southwest Re~earch In~kul¢, San Anlonio. TX. MORTON I. GR~SMAN, M.D.. P..D. Associale Cllmcal Profcsso¢ of M~ici~. Unive~ily of California M~ical CoMer. ~ A~e~es. CA. CARL C. GRUNZIT. M.D.. A~iate in ~ysiology a~ Pha~aeol- ~y. Univmily of ~nns~v~ia G~Uale ~1 ~ M~ici~, ~il~l~b. PA. JOSEPII J. GUARNERI, ~.D. Attending Mierohiolo~isl: Director, M~roh~lo~ ~alones, ~g l~la~ A~iale I~ofcs~or n¢ Commu~h~ and ~vim~memal Medici., Univer~dy IIIRA ~ GUR~, D,V.M., M.V.~ ~LI). NY. ~NK ~ G~IRIE. RhD. H. B. H~G. M.D. k~ ~ Vi~inia. F. J. HAZY. M.D.. Pm~es~ a~ Chaiman, ~partment of ~ys~. Unlve~ily of ~la~a Direclor, ClNiopulmonary Unit. The ~nkenau llo~pilah A~iale d~, Uni~ily NOBUY~III I~AGIHO. M~D.. ~f~ ~ Anal~. U~v~ky of Texa~ Ileallh Science Cenler. San Anto- KATHERINE A. IIAHAR. M,D. As¢isl~l Pmfe~ ~ial~cs ~ Meal;- New Y(~. NY. CAROLINE B. HALL M~. A~b~ ~fe~ of ~ia~ ~ Med- iei~, Unive~ily of R~sler ~! or M~i~. R~Rr. NY. LINDA M. HALl.,. PN.D. As*ociale Pmfes~,o¢ of C.~tellcs rex5 Ncu- I~¢lenee. Albert Ei~st~n College of" Med. icine of Yeshiva University. Tic Brenx. NY. MARGIT HAMOSN, I~LD. R~.earch Auocia~e. Depmlmcn! of Physl. otogy and Biophysics, Georgetown Uni- versity Schools of Medicine aml Dentistry. Washington. DC. PATE. llAMOSII. M.D. AnsnClale Professor of Physiology and ~i~hy~ic~, and M~licine. Geor~elown ~ive~ily ~s oF Mcdici~ ~ I~n- fi~, w~i~. ~. lllDRSABURO I IANAFUSA. Professor. Th~ R(~ckel'ellcr Universily. New Yol~k. NY. BERNARD llANES, l~hD. Prore~o~ of Ileatlh Science. California State University, l~hddge, CA. OUVER HANKINSON, Ptt.D. Ac~neiale Pm(e~so~ o¢ Patholocy, Unlver- sity or" Califo~ia, Lm Aoceles. CA. ULLA M. NANSEN. P,.D. Chief, Ij~tmy oi" E~k=~ic Trap~cr~p llon. I)~a-Far~r Cancer In~lilule, ik~teo, MA. PRTER C. IIARPEL. M.D. Pmfc~w of Medicine. Mo~nl Sinai School ~ Medicine, New Yo~, NY. RONALD O. llARVEY, I~hD. Pmr~em~ of Org,~c Ch~mi~lr~. TI~ Unl- v~sity ol'Chica¢o, Chicago. IL. RICHARD ]. HAVFJ.. M.D. Assisom! Pmfess~w or Medicine, Univer- sily of C|lif.orni| Medi¢~! Comer, Sso Framiseo, C~. ISUMI IIAYASHI. P..D. Assislant Research Seicntisl. Beckman Resesrek Inslliute, City ~" Hel~. Dulrt¢. CA. ~ IN A. IIAYF-% M.D. Associate Pa~holoStM, Mallow_ l~llllute ef MA. PAULWE HE17~'~, Rc~mk Associa~ in Cytolo~_y and ch,~ni~,. San Franei~ Imthm~. or Med. ical Sciences, ~n I~m~isc~. CA. CAROL L HENRY, I'~D. Di~ew. Del~nmem (d" EXl~'lm~mtml 0~. eoloCy. Microhtolo¢ieal Auneiales. Inc.. Bethesdl. MD. HERBERT It, HER~"OWI'I~ PH.D. Associate P.~'.of.esso.¢. o~_ Mie.robIpJ.ol~, ~n umvenl~. ~f Mm- C~ a~ ~nli~. ~. ~. AVRAM IIERSHKO. M.D.. I~I.D. In~kme eTTecknology. Half'a. Iraqi. HENRY D. NOBERMAN. cine o~ ye,~hivm Univm~y,'Pne i~nx. NY. 2R3
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0 EBBE CURTIS HOP'F, MD,, Pmfes~r and Chairman. Division of cbialri¢ Rc,,~-arc, h, Medical College or Vir. |inia, RickmowJ. ROBERT M. HOI~MAN, Alumci|le P~'ev, o¢ of Pedialrlc% Univer- sity of Caliromla at San L14e~, LI Jolfa, CA. JAMES C. IIOGG. M.D., P,.D. Prof(~ssor of Palholol~y. University of Brilish Columbia, Vancouver. British Columbia. Canada. JEROME L IIOJNACKI, Assistlnt Professor of Biological 5el- em'e~, Universily of Lowell, Lowell, MA. RUSSELL. L. tlOLMAN. M.D. Louisiana ~ Univetsky Sclmol of" Med. OIJ~ A. IIOI.TERMANN, M.D. Redneck Scienlist. Lobtmd Laboratory, Univcrsky of Nmre Dame, Nmm Dame. FRBDDY IlOMBURGER. M.D. Presi~nl and Director. l|io-Reseal¢t~ In- itiate, Inc.. CamMidBe. MA. JONATHAN M. HOROWrl7., I~.D. Asa|sl~t Pmf.esm¢ of" Microbiology and Immunology. Duke University Medical Center, Detham, NC. DEBORAH K. HO~HIZAKI. PH.D. Assi~nl Professor of Biological Chem. Islry, UniversHy e( Illinois, Chicago. It. WAYNE HOSS, PH.D. A~ocilte Pro(~tor. Comer for B~in Re. ~eareh, University of Rochester Medical Center, Roche~r, NY. RICIIARD L. liUGANIR. P,.D. Av, oCil~e Profess, De. ~ Johns Ih~kin,; Unive~ity ~rK'l'~xd o1" Medicine. l~allirm~e. MD. ROBERT W. HULL. I~.D. Pmfe~or or Eic~o|i,:M Science% 5~10 Univershy, Tallaha~ee. MIEN-CIlIF. flUNG. A,,~i~tanl Pmfe~,~or. Univer,|ity of Texa~ M.D. Antrum Cancer Cooler. IInnslcm, TX. RONALD R. IIUTCI IIN.q~N. I~.D. Re,~.m~ l)~re¢lor, I;m~mlatkm h~¢ ~chav. i~.al Rc'~s~:h.~ul~u.dn. MI. lit RESEARCH Ch~ca~. IL KYOGO ITOI I, M.D. Assistanl Pmferaot' of Surgery. University of Texa~ M.D. Anderso~ Cancer Center, I Inusmn, TX. BARBARA J. JACKSON. PII.D. Staff Scientist. tin,ton Bimnedic=l Re- seamh In~lgule, Baton, MA. GEORGE JACOBSON, M.D. Professor ~ad I1~1. l)q~tm~m of Radi- ology, Uolve~si~ of ,~mlhem CalWomia School of Medicine. I.m Angeics, CA. JF.RRY IIART JACOb'.ON. M.D. Direclor, Divi.~i~m of I'~ectmphysinlogy, New Yolk Eye and FJr Infirmary. New Y~k. NY. JULIUS II. JACOIISON Ih M.D. A+moeilte Pmfe+'~'w of. Surgery" an.d ~- ~lm of SU¢~=I Re~lmh, Umv~tly ~ Vermont College of Medicine, liar. I~lem, VT. 2R4 SUSAN JAKEN. PH.D. Senk~ ,~:iemi~. W. AItnn .k~es C¢1t Sci. er, ce Center, Lske Pf~cld. NY. GRAIIAM A. JAMIF.SON, Ph,D. Senior Scientist, American Red Cro~s. Rockville. MD. AARON JANOFF, Protester of Pathology. lteallh $ciencc~ Ccnler, Slme Univ~i~y or New S~my g~, NY. RONAI.D JF.MMF.RSON, A~t~e Pm0:um ~ Mk-n~l~,¢y, veery d Minmso~a, Mi~al~lis, MN. JOLYON JF.STY. PH.D. Associate Prof"e~c~ of Mcdlcine0 State University of New Yak at ~ony NY. DAVID A. JOiINSON, PH.D. tL,.dmnt Profe~or of Biochcmbtry. East Tennessee Slate Universily College of Medicine, |~tson City, TN. ~OIIN P. JOllN~ON. M.D. A~oclme Dir~or iM Cllni~aJ C, en¢i~isl. Childrm's Ho~pil~l Medical Center, Oak- lad. CA. HAROLD P. JONES, RI.D. A~islar~ I>rof¢~tu~ of Biochemi~nJ, I.~t. verdly of S(~Ik AlaMm~t. Mobile, At. OSWALD R. JONF.~, M.D. SI. L~ke'a IIm~tal, New York. NY. WIIJ.IAM $. JUSKO. l~t.D. Assocism Pmfemm¢ of I'harmKemics: D;. ~lor. Clinical Pharm~.okine~k:~ IJl~ora- taT, Millard Fillnmre l/o~pilal. Buffalo, NY. MAR$11AL~ E. KADIN, M.D. A.~..oclate PH:d'ener of Patholet_v. Medi¢|l School, Beth IsraelHo~pilal. Boom, MA. ANOREW A. KANDUT~tCH. I~.D. Staff Sck:n|i~l, TI~ Jaek,t0q Laboratory, Bar llm~', ME. FA-TEN KAO, PH.D. Profes,mr, Eleax~ Rnmevell In~lhale ft~ Cancer Re~,arch. De~ver. CO. ARNOLD R. KAPLAN, PH, D. Cl,~el~d Psychiatric I~titute at~d Hmpl. let. Cleveland. OIL LEE M. KAPI~N. M.D.. PN,D. Asalmnt Pmf.es~,or of Medicine, Haf~lrd Medical School. Massachusetts Oeneral Ih~p;lul. Baits. MA. ATT'ALIJ.H KAPPAS, M.D. Professor and Senior Physlei|n. Ib.,d~efeller University. New Yodt. NY, MORRIS 3. KARNOVSKY, M.B.. B.C~. ~ka|t~k I~.el~tor of .Pa.lh?l~al ~Ay,, Harvard Medical ;chc~l. SIMON KARPATKIN. M.D. ~ e~ Medicine, New Yed~ sity M~ka ~, New Y~, NY. ROBERT W. KARR. M.~ shy ~ I~ l~u C~y. llRA~I K~PARIAN. M.D. ~lory;.Instr~el~ in Medicine. Hahne. ~ M~il ~k~ s~ lintel. ~11. ~+pbim. PA. YONA KASSIR. ~I.D.
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0 0 Assimm P~ofe~or or Bicchemislry. Uni. versity of" Texas Southwestern Medical Cemer, Dallas. "IX, $1111~EY L. KAUFFMAN. M.D. ~ of Pmholoiy, Stale University of New York Downxtate Medical Center. Brooklyn. NY. MARK S. KINDY. PH.D. Assist~m Pcofesscx or Bloc:hero,n/. Uni- vecsity o1" Kentucky. Lexi~L~on. KY. DONNA KINO. PH.D. Mol~ular B~I~. Unlv~ty of lleahh ~ ~a~ M~I ~. N~h ~3~lg~H B. KIRSNER. M.D. Professor of Medicine, Universily or Chi. m~o ~hool or M~di~ine, Chlca~o. IL EDWARD L KLAIBER. M.D. Senior Seiemist. The Wo~-esler Founda- tion for Experimental Biology. Inc.. ~hmw~bury. MA. k l'mte.or or _h .y~. iau~._Bostoo ily ~clmoI of M~dicine. no, ton. VICTORIA P. KNUT~ON. PH.D. A~Is~am Pmfe-~or of Pi~mucolo~y. Uni- versity or Tex~ Medical School. Iloml~, TX. HEII¢~ KOIILER, M.D.. P..D. Adjuncz Professor or Pslhology, Univer- sity or California. San Diego. La ,lolls, CA. WILLEM KOPI~.NOlo, Pu.D. Associal{ Pmfevuw. L~Ji~iana Stale Uni- versity. P.aloo R,ui~e. I.A. ALVIN I. KCJ~AK. Pit.l). A~ociatc I~fe,~w of Chemktry. New Yak UniveBity, New Yak, NY, ROBERT W, KREIIJCK, Pu.D Professor of Chemislry, University of Reghest~r. Rod~er. NY. ROBERT H. KRET~INGER, P~I.D. Profesm" of Biology. Univcrsily of" Vir- ginia, Clmdmtesvgle, VA. KLAUS E. KURI"/'NER. Professor aed Chairman. Depar~mem of Btockemisl~. ~u~ College o¢ Ileallh en~s and Rush Medical College. Rush- Presbyterian-~L Luke's Medical Center, Chicilo, IL. ROBERT A. KUIIN. M.D. Associate Profesmr. Division of Ncuro- surgery. New Jerr, ey Stale College or Mad. icine. Jcr~y City, NL ARUN P. KULKARNL I~t.D. As~o¢iale ProFeslor. Univereity of South STIG KULLANDER. M.D. Pml'essor and Ckairman. Deparlm~nt of Obsletdcs and Gynecology. Unive~sity o( Land. L.nd. Sv~de~. STEVEN L KUNKEL PH.D. As~oc;aze Pn~'e~m~ of' P~hofog~,, Univer- Sily of Michigan Medical School. Ann Ad~or. ML MARTIN KUPIEC, P,.D. Assi~tanl Pmfess~', Tel Aviv Unlvers;ty. "!"_,,! Arty, h~ael. LAWRENCE L KUPPER, Pit,D, Assoeiale Professo~ or Biostalislics. Uni- versil.y or Norlh C~0Una School of Public ttesllh, Chapel Hill, NC. GORDON W. LAURIE, PH, D. Assistant Proresso~ of Analomy and Cell Biology, University of Virginia. Chef Io~lcnille. VA. JAMES T. KURNICK. M.D. Asnoctate PUt _lFtlogi~, Masslchl~e~ts Oen. eral Ilospkal. R~o,. MA. MARVIN KU~CIINER. M,D. New York University Medical Center. New Yolk, NY. CIIARI.I-.~ W. LAI~FJ.I.F. A~i,~l:ml Prnre~,--,o¢ of l:.nvl~menlsl giene. Jefferson Medical College, P~ifa- delphia, PA. AARON J. LADMAN. I~l.D. Profemo¢ a.d Chairman. Depmlmenl of Anatomy. University of New Mexico Seheol (~ Medicine. AIb~quentue, NM. ERIC LAI. PH.D. Amlslanl Pm~ or I~armacology, Uni- reeky or Nmlh Carolina. Chief Hill. NC. TIIOMAS C. LAIPPLY, M.D. Pm{essm el" PUL _lpk~. Northwemm Unl- versify Medical Sch~l. Chieal:O. IL. MICIIAEI. E. LAMM. M.D, Professm" of Pulhology. New York Uni- versity Medic.~l Cenl~. New Yod~. NY. TIIOMAS A. LANGAN, lh f.D. P~ofcssot" of l%mnlcololy, University or Colorado School or Medicine. Denver. CO. DON LAPENAS. M.D. Assistant Professor of PUIhology. Univer- sity of Vermont College of Medicine, Burlin$1on, VT. PAUL S. [,ARSON. PH,D. Haa~ Pnd'cs~m of Pt~m~'olo~. Medical Coll~ge or Vi~inia. RIc~moM, VA. ROGER K. LARSON, M.D, Chief of Mgdlcine, F~ Ct~nly tal. F~s~o, CA, GUSTAVE A. LAURENTJ, M.D. C'hlef ~ Medk:ifle. ~. vl.cem's Wt ~.I~', MA. A~s;slanl Pmre~sor of Medk:l~, Coil~ dr M¢lki~e. I l~u~,;'~, TX. PAUL LEBOWrlz, M.D. Scala Rescamh g~imli~. Yale Univm'd~. New I la~. EDWARD LEE'rE. PH.D.. D.~C. P~f~aor or Chemi~y, U~ive~i~' of MI~- nesola, Mimtespoli~ MN. STUART E. LEFF, fad Univmi~, Stanfl,md. CA, JOSEPfl LEIGIITON. M.D. Pmfeuef of Pathololy. IVledk~l Collate of Pennsylvanis. P~iladelphia. PA. VANDA A. LENNON. M.D., PH.D. Professo~ or Imn~nolo~y ~nd Ncnmlngy. Mayo Clinic, Roch~lef,~ kiN. PIlILIP M. L~ OUF-3~E, PH.D., D.~'. Pmf~" of Cl~mluP/. Nonl~l~m Unl. versky. Boslo~. MA. RICHARD A. LERNER, M.D. Aa~-ta~e Member, ~s Clin~: and Re. search I=mmd~ic~ IA Jolla. CA. LAWRENCE LIT-KING LEUNG. M.D. Unlveed~y. ~m, rool. CA,
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0 cr, I~-ZION LEVI, i~t.D. S~nk~ L~eluRr, Techoio~.Israel lnslilule of Technology, Haift krael. EUGENE G. LEVIN. P,.D. Assistant Member, Scripps Cllnlc and Re~'~rch Foundation. La JoHk CA. JAY A, LEVY. M.D. Associate Professor or Med;clnc: Re- se~'~ A.ocle. Cancer Rexea~ch Insti. lute, ~niversily of Califmnia ~h~l of M~i~. hn F~ CA. STUART B. LEVY. M.D. Pmf~ of Mdicin~. New F.ngl.d M~d- teal Center. Tuff., Univer,.ily Medicine. l~ltm, MA. PAUL D. LEWIS, M.D. I~nlor L~urec in Ilkmpad~dnff. Rnpl PoVlFaduale Medical Sclv~d. Ilaml~r- emile I |o.~iLd. Lond~m. I~gS~. RANDOLPll V. LEWIS. I~.D. Associate Pr~e,or and Ilcad. De.part- mere of Molecular Bio~y. Univers,y ~,'~w~. Lmm~. wY. MTCHAI¢ W. LIEBERMAN. M.D., P,.D. W.L. M_ondy. Jr.. Professor ad Chai~nan of I~holo~. Baylor Collq¢ of Mdiclnc. H~ultek lX. AVERIL.L A. UEBOW. M.D. Chlirm~. Dep~lmen~ or Pathology. Yale University School of Medicine. New Haven. CT. IRVIN IL LIENF~R. P,.D. Pml'e,o~ of Biochemistry. Unive~ily or VALF..RIE K. UNDGREN. l~.O. Ou~t Resea~ckr. National Cancer In~d- t~¢, B~he~, MD. JOSEPll S. LIP~ICK. M.D.. P,.D. As~..ciste Pro/'es.~or of Microbiology. Side University or New York. Stony Brook. NY. 7.V[ LIVNEII. PH.D. $ci~ntkt. The Weilmann tnstitult of Scl- ence. Relww.~. kr~..l. RICARD() V. IJ.OYD. M.D.. PH.D. Assi.~t~l Pmte~..mr of Pmhology. Univer- ally of Michi~. Ann Arhm. MI. CLAYTON G. LOOSLI. M.D.. I~i.D. * Ilaxtings Professor of" Medicine and PatholoLv. Univc~ty of Sc~hcm Califor- nia Sch~xd of Medici,. I.m Anplet CA. REUBEN LOTAN. I~t.D. Profe~m~ and Deputy Challman. Univer. lily Of Texas M.D. Anderson Cancer ~n~r. I~, TX. F3EN O. UNDSETII. M.D. Rt.D. Sl. Jo)ei~'a Hospital Re.arch Labora- Io~. ~. Paul. MN. ROBERT H. IJNNELL. PIED. At~cle pmtr¢,o~ of Chorally. Unlver- sky of Vmt, ~udin~lo~. v'r. FABIAN L LION'~'I'rl. PH.D. Prol',~or of Bilxk-mid~. Bo~ton Uni- ,r~k¥ ~ of Mdiclne. Breton. MA. DONALD B. IX)URIA. M.D. As'.ocla¢ hofe,O~ of Mdici,~. Com¢ll Universily Medical Collate. New York. NY. RONALD J. LUKA$. PH.D. Di~-ctor. Laboratoq of Neuroehemistry. St. Joteph's Ito~pit..I and Medical Center. Phc~lix. AZ. RICHARD A. MARKHAM. M.D, Assistant Pmressor of Mdh:lne and of Mtembio~ ~nd lmmufl~l~gff, TM J~- IAN M. LUNDBERG. M.D. A~iale Profe,or of Pharmacology. Kamllr, ska Ir~lilule. Sloekholm. Swdcn. KENNITrI| MI'~RRII.L LYNCll. M.D.. Sc'.O.. IJ.D. tellm'. Mdkll College of Soeh Carolina. Chd~to~. SC. [IRUCE A. MACIII!R. Bt.D, Assistant Plofessor of Pharmaeeulical Chemlsl~'. Univenily of Califoenla. San Frar¢i~o. CA. HOWARD $. MAKER. M.D. A.~o¢iate Profess~ of Neurolqy. Mou~ Sinai ~ ~ Mcd~. New Y~. NY. BILLY R. MARTIN. P,.D. A,e¢iall P~fe~w, Mallei1 Collage of Viiglnll. Vllllnla Comnillllihh Uni- venily. Richmond. VA. CIIRI~TOHIER M. MARTIN. M.D. A,istam Prate,or el" Mdlcini ~d DI. lariat. Division Of lnfKIbw! kto~ Hall College of Medklnt O. ~'I~VE MARTIN. PH.D. Pl~eslei. Untvmtty el" Cldifonlll, Icy. CA. R. RUSSELL MARTIN. M.I~ Prate,Go of Medklno. and INES MANDI. P,.D. REGINALD G. MAION. M.D,, !~.1). A.,udstant Profeuor of Biochemlsl~. Co- Professor and Chlirmln. I~ftmlf~l of lumbla Universily Colic~ of Physicians & Patholoff. Univmtly of Utlh Cdlege of Surg~m. New york. NY.Medk'ine. Silt Lie Cily, UZ JOHN H. MANHOLD. 1~.. D.M.D. horessor and Director, Department of Pathology and Orll Diaposls. New Jev,,tey Colle~ or M~ i/~llsl~, lely City. NJ. DAVID E. MANN. I~t.D. A;csl~tate Professor of Pharmacology. Temple Unive~ily Sebnol of Pharmacy. Pkildel~it PA. FRANK ARTilUR MANNING, M.D. Assistant Profe.o~ of Obdetri¢l and Gynecology. Women's Hospital, Los Angeles Cotm~/Univerllty of Southern Califomla Mcdi¢al Center. Los Anplcs. CA. HAIM MANOR, l~t.D. le of Tecbnol~¢y. 15ira. Im¢l. JOllN F, MANO~, M.D. lnslmclor in Viroloff ~ Baeteridc~y. Medical College of South Carolina. MASON RESEARCH INffrruT~ Wo~:esler, MA. ROBERT W. MASON. ~.O. VA. ~NALD i. MASSAR~ M.D. As~ill W~iqm ~ARL~ McAR~UR. ~D. H~ U~. lily O¢
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On ItFJqRY C, Mc'GILL. l'~t., M,D. Acliall lt0td, .D~,parlmem or Palhology, LoulsiMa Stile uniterdty School of Med- icine, New Drleans, LA. llENRY D. MclNI¢~ll. M,D. Piofello¢ of Medicine ~ DiRilor. Car- diov|scular Lahomtory, Duke Unlver.dty Medical Oentc¢. Durham. NC. I~RD~ A. M¢IVER, M.D. A.oliale Prates,los of Pitho~oiy. Medical Cll41¢le of ~mnh Camlhra. Chillelloe, SC. EDWARD MCKEE, M,D. Ilroft,~loi" a~d A¢llng Chalrmm. Del',l~i- meet of Palholo_ly, Medical Collese or Souih Carolina. Cilarlcslon. KELLY T. McKEE., M.D. A.ociate I~ofesr~r of Medicine, Medical Cellel~ of Scmh Camlloa. Charleltm. SC. WALLACE L.. McKEEIfAN, I'N.D. ~nior SCICmt~, W. Allo~ ~ Call ~ Cl~l~. Lake Plicld, NY, VICTOR A. McKU$1CK. M.D. Profciloi of Mtxfi¢ine. ~ Johns Hopkinl Univeility School of Medicine. Balti- mor~. ME. RO.~ L MclJAN. M.D. As~e|ale R, ofas~or of Medicine. Emo~ Univ~tty ~k, hool of Medicine. Atlanta, GA. WILLIAM F. M,~'VARY. JR.. P~e.D. I.Miversily ~4:h~oI Of Medi<hit, Boslon. MA. NEAL McNIVEN, TI~ W~l~ l~.mdallo~ fm til Biology. She~vsbu~. MA. PAUL]. MEB~IAN. I~,.D. Alllttlll ~ of lliololkil Science.% N~,lhtm IIli~ds Unlvershy.~DIKalb, IL ! IAN.~ MEIER. D.V.M. Senior Staff Scienlkt, The Jaekmn Labo. ratory. Rat II,,rhor. ME. ROGERIO MENBGI IINI. P~t.D. Professor. Unlver~ity of S|o Plulo, $1o Paulo, Brazil. EDOAR F. MEYrdt, Je., Associate Prof¢-~o¢. Texa~ A&M Unlver- $i¢y. Coflese ~lstlon.TX. EDWIN M. MEYER. Assocle Piofl~lor. University of Florida College of M~di¢ine. C/ainesville, JULIA MEYER. PlI, D. Ar.se¢iate Professor of Oral Pathology. Unlv~ri|ly of IIIinoi.q Colle~je of De~ist~, DOV MICHAEU, Assistiu! Professm of Biochemistry and Sur~y. Unive~zity of CMifomia School of Mediator, San F~i~c~, CA. MICROBIOLOGICAL ASSOCIATES. INC. B~tl~.~a, ME. BERNARD ]. MILLER, M,D. Aslimm Pmfes,t~ of Anatomy, Medical Coflege. Philldetphll. PA. JAME~ O. MILLER. M.D.. F~I.D. Profeuo¢ of Psychla~ry ard Psychology: Director. Men~-I Health Resea~h inslitule. U~ive~ly of Michlian. Ane A~or. MI. JERALD. A. MffOIELL R~.D. A~,o¢le Professor of Anatomy, Wayne Stile Unlver~;ly School of Mediclne. D~- IrOil. MI. STELLA MITRANI.RO~ENRAUM. I~.D. Pmf©.,emr of Vimloly. Ilelwew llada#;lah Medical School. Jeruxalem, Imd. CHARLES MITrMAN. M,D. Ear.for. Deplmnenl of Respiratory Dil~ ea.,,~. City of 1lope NiliOtlll ler. Duarte. CA. HUGH MOWrGOMERY. M.D. Asse~iate Prore~or of Medicine. Univer- ~ll~l~ PA. GEORGE F. MOORE. M.D.. I~.D. Dileclor. Restart I~, Memodll taitit~e, Buffalo. NY. DAVID A. MOSCATELLI. P~tD. Researck Assistim Pl'ol'essor, New York U,ile~ity Medical Career.New Yod~. NY. KENNETH M. MOSER. M.D. Allimnl Proressor o~ Medicine. O¢orle- m*n t~imky Medical School. WiihlnS- toe, DCo HURLEY LEE MOTLEY. M.D. Pml'¢lior of Medicine and Director. Car- Soulhern Cllitornia ~CnOOl ol Meo:clne. ~ A~lele~, CA. BARBARA M~OCZKOWSKI, Stuff .~-.iemi~t. C11ifomla Institute log~al Research. La Jolla, CA. R. ALLAN MI.IP"~)N, P,.D, lie Lahomo~. Rockville. I~RID MURAl). M.D.. I~.D. Slunfmd UnlvelsltT, Stanford,, li~ ~al ~. ~o Aim. CA. CIIRI~TOPHER MURLAS, M..D. -- . man. I~nmem or Med*cme. Ruse unt. ~|ly, Chkllo. IL 291 WILl JAM ~. MUR~Jt, Y, ~*.D. Staff ki~tbl. 11~ Bar Ililbor. Mill. JAY A. NADEL, M.D. Professor of M~t¢ine, ltliloloU aM ledk~.oi#, Cardk~e i-l~ll, RICHARD L NAEY~ M.D. Cd~ge of Med~lo~, litany, MOON H. NAHM. M.D. in,Ion Univ~rsky, ~ll. L,'~I~, MO. SUSAN NAYLOR. PtI.D. Associ~e Pmteilor oF Human O*md~,. The Universily of Texas Health Cenlor. Sm Antoelo."lX. DONALD J. HELSON, PH,D. Asse¢lale Profe~Kx of Chemistry. Clad~ Univmky. Woreeilor. MA. GERA NEUFELD, PN.D, Senior I.~tum'. 'l~ilon.luttl hillull of TIlhnelol~. HM~, field, OEORO B. NBURATH, I~I.D. NAROLD H. NEWBAtJ.,, M.D. ,k~..~lm Uahirlily ~¢hool (if Medl- ainu. BMImmm, MD.
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JOHN E. NIEDERHI~ER. M.D. Prores~r of SurBery/On~olo~y, Stanford Untve~hy, $~lnford. CA. ~"r~FAN NIEWIAROWSKi. M.D. I~I.D. I~f~or of I~j~siology. l"l~o~l~Is Re- m~h Cemor, Temple Uni~'~|ty School of Medlci~e. Philadelphia, OAK RIDGE NATIONAL LABORATORY O¢c R~, PRAN~ OESCH. PH.D. Professor of Pimrmscoloc7: Head, Section on Biochemical Phsrms,,*oloty, Univcr- shy of Mains, Mainz. Germany. SF.-KYUt~G OH, PH.D. Associate Professor or Microbiology. B~ston University School of Medicine, Rmtm, IANET M. OUVER. PH.D. Profc-.sm" of Patholc~#, Univmky of Mexico ~¢hool or Medicine. Atbuqverque, NM. ERIC N. OLSON, I~.D. Acsociate Pmfesso¢ end Assoclale Bin. chemist in Biochemistry and Monocular Ilicle~ly. Uni,~sil~ of Tcxu M.iX Amber- SUZANNE OPARII~ M,D. Professor of Medicine. University of Afab~m~ Birmingham, WILLIAM OIUC A~x'ial¢ F~feuor of Padx~cy, Univer- sity ~" Manitoba, Winnipeg, Manitoba, DONALD M. PAC...~ P,.D. Pmfesso¢ of Physiology and Direclor. rn. sllmte for Cellular Research. Ufliversky of Nebraska. Li~mln, BEVERLY PAIGEN. P~I.D. The Jack,Do L~boca~n/. Bar Harbor. ME. ALBERT B. PALMER. P,.D. Ass|slam Profes~or of Pathology. Univer- day of Toledo. Toledo. OI I. JOHN W. PARKER. M.D. Assoc|ate Professor of PatSol0¢y. Uni~- sl~ ~ ~ ~lir~la ~1 of M~. ~. ~ Ange~ CA. MARY STEARNS PAR~HLEY. Ass~am Pm~e~or of Aa~omy ~s a~ O~y. ~lumMa Uni~ si~ ~lkge nf I~yx~iuns New Y~ NY. BENDICIIT U. PAUIJ. D.V.M. Associate Professor of F~hology. Rush. Pre~byterinn.St. Luke's Medka/Cem~', ISRAI~, l~cirr, PH.D. Pm~es~¢. The Weiz~mmn lnsd(u~e e~¢~. Reborn. br~l. EDWARD W. PI~IKAN, M.D. C~airman. Department of Pham~a.cotogy end Experimental University School of Medicine, I~OSlOn, MA. MARY D, OSBAKKEN. M.D. Assi~Jnt Professor of Anesth~sla sad Bio- chembl~j/Biophyslcs. Uoive~hy of Paon- sylv~nil, Philadelphia. PA. ARTItUR PENN, P~t.D. Research Pmremmr of Envlmnmemal M~d- iclne, New York University, New York, N¥. 292 DENNIS R. I~'TERSEN, PH.D. I~OfCs~Or of Phlm~acolo~y. Unlveuily of Colorado School of Pfisrmacy. Boulder, CO. ~ W. PETERSON. lht.D. Associaee Professo¢ of Biology. A~ansas Colle/~e, Batesvllle. AR. DAVID E. P~TTI30HN. PH.D. Proressor of Biochemislry/Biophysies. Universi~y of Co~ado. Denver. CO. PICK, M.D., PH.D. Profe~s~ of lmmu~lo~y. Tel Aviv Uni- reality, Tel Aria, Israel. O. BARRY PIERCE. ~ M.D. Distinguished Centennial Professor of PathoJocy. U~lve~ky of Colom~ Health MALCOLM C. PIKE, Px.D. Professor of Communily Medicine and Padbt~s, Universi~, of So,item C~ifor- ~ia ScI~Ol of lV~icl~e. Lm A~lele~ CA. JAME~ M. PI~AS. Ph.D. Asslstsnt Professor. Uoiversily of Piffl- RAY C. P1TTMAN. PH.D. Reseamh Biochcmist. University of Calf fomia, Sm Diego. t,~ JoHa. CA. SALVATORE V. PIZZO, PH.D. Associate Professor of Pathology, Dulls University Medical Ce~er, D~rSsm. N.C. CHRIS D, PL~T~OUC~. PH.D, Prof,~ssor o.f ChllraMI, university O1TIIII JUUA M. POLAK, I:~C, M.D. ~-nlor Lecturer i~l Hlllo~llh0~y, PoltMIdaate Medkll l'~hocl. OTAKAR L PO(,LAK. M.D.. I~I.D. Bxecutive Director. Dover Medlcsl Re. seIRk (~er, i~c.. D~wer, OB, C. M. POMERAT. PH.D. Di~c~ of Biok~ieal ~ ~ Foundation for Medial Rehash. Pain. LOU~S PONCZ, Seeinr Re~eamh A~ect~. Ca~ Wemm ~ University. Ck~ela~d. OH. S. N. Iqt~DHAN, MJ&,. PH.D. Pm~e~or of PHarml¢ol~3,, Howard Unl. H. ~. PI~'rl'.'iltOMA~. M.D. Colk~ of Scuth O~llra. Ch~ll~lO~ DAVID M, PREP'Old, PH,D. Distin~ulshecl l~roresso¢. Unlverslly of PROCEt~ & INSTRUMENT~ CORPORA- TION Bmokl)m, NY. EDWARD V. PROCROWNHc. M.D, A,echte Pml"e~or of I~Mbtde¢ Univ,. sW of Mi~ipe, Am Ad~or. CATHERINE PRODY. PH.D. Sclenti~. The I~,l,-~_ror Skk Children. MARTIN S. PgO1ZEt~ Chief, Dcpn.~. me.at of Oral Pitholo~, New~ ~y Hm~ Ncw~. NJ.
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MICHAEL A. REIDY. P,.D. Research Associate Profes.~¢. University of Washingto~ Seallle, WA. HOBART A. REIMANN. M.D. Prol'essm oi" Medicine. tlahnemann Medi- cal College and Ilmpital. Philedelpifia. PA. MICHAEL S. RABSON, M.D, EILEEN REMOLD-O'DONNEU.,, P,.D. R¢l,e2.l~h Scitmtiu. Laboratory _or Pathol- Medical School: Investigator, Center for ~ Natiooal Cancer Institute. vethesda, Principal Research Associate, Ilarvard • Blood Research. Boston. MA. RAMI RAHAMIMOPF. M.D. F~fcs~/t of ~hysiolo£y. tlebrew Univer- Stly-Hadassah Medical School Jcmsaicm. Israel. ]OLIN E. REPINE, M.D. Assistant Director. Wehb-Waring; Lvng Institute; As~oclate Professor of Medi. cane. University of Colorado Ilealth Sci. ences Cent'. Denver, CO. ROBERT RE, SNICK. M,D, Associale Professor of Reprodu~ive Med- icine, University of California Medical Cem~. Sm DIC~ CA. PH.D. JOHN C. REED, M.D.. P,.D. LI )olle Cancer Research Po~edatio~, La Jelll, CA. ROLLAND C. REYNOLDS. M.D. Assis~t l~ofesto¢ or PmholaLv. Unive~- sJty of Texas Southwestern Medical School. Dallas, TX. SOLON I. RIfODE, llr, M.D., PH. D. Profe¢,,o¢. University of Nebraska Medical Ceme~. Omaha. NE.. TIMOTHY L IU~AN, M.D. ~ and l~mc~or, Division of o~,¢y, Univer~.~y of ~edi.~i~e ~ WIZJJAM REGELSON. M.D. Pmt~te¢ a~d Chairman. Department of Medical ~ .0~ loly, Medical College of Virginia, Rietm~ VA. LYN?~ M. RFJD. M,D. Wolb~ ~ or Pa~hologT, Hazard htl~. Children s llospital Medical ~. ~um. MA. WARD RICHAPJ~RICE. M.D., Ass;stem PmfesJo¢. Univm|ly or C~nc;o- neff. Ci~'innad. OIL VICTOR RICHARDS. M.D. Chief of Surgery_, PRsbytedan Med|czl Center, San Franci~:o. CA. VIRGINIA L. RICHMOND. PH.D. Research Associate. Pacific Northwest Res~,ch Foundadoe, Seattle, WA. ARNI$ RICHTERS. P~.D. A~JMe Proras~or or Pathol~o Univcr. Slt.y Of Soulhem Califom|a Sch(k~l or Meal- lone. Los An~les, CA. HEIMO RIEDEL. Investigator, ,foslin Diabetes Center, Boston. MA. WILLIS !1. RIESEN. P~I.D. Senior Biochemist. Life Sciences Divi- sion, lit Rese~ tnslilule, Chica~o, DANIEL B. RIFKIN, I~cD. A.~htant Prores,.or of Chemical Biology. The Rockefeller University. New York. NY. R. It. RIGDON, M.D. Professor of Pathology. Universily or Texas Medical Branch. Galveston. TX. SYDNEY C RITTENBERG, PH.D. Profes.,mr of llacterioloRy, University or Soulhem Califomla, Los Angeles, CA. A. ANGE R1ZZINO, P.O. Ass~x:iaCe Member, Unlversit~. of Nebraska Medical Ce~. Eppley lnstilute, EUGENE ROBERT~. PH.D. Reseach Scicmist. City of Ho~e Ream'oh ln¢lllule, D~ane. CA. JOHN M. ROBINSON. PH,D. Astatine Pmfe*Jo¢. Ohio ~me Unl~'rshy Colicle ot' Medicine. Columb~. OH. RRNSON B. ROE. M.D. Associate Professor of Surge~; Chief. Cardiac Sur~c~, Uni~'~;ty o( Callromla School of ~i~. ~ F~i~ CA. JOSEPH H. ROGERS. M.D. Holy lqame of ;ears Hospital. Gadsden, AL. D~NA RON. PH.D. Asslstam Professo¢. T¢¢hnion-lsrael Insti- tute of Te¢tmology. Hairs. Ixmh ROBERT C, ROSAN, M.D, Associate Professor of Pathology and Pediatdcs. St. Louis Univgnity School of Medicine; Associate Patholo~|st. Ca~linal Ok:nn~ Memo¢ial lf~,pital fro" Cidldr~n, SL Louis. MO. CHARLES !. ROSE. Clinic Director; Director, Normative Aging Study. Veterans Admlnl,.rallon Ou~patlent Clink:, Boron. MA. JOHN A. ROhECRAN~, Px.D. Assoelale Professor of Pharmacology, Medical Cotlelc of Virginia. Virginia Commonwealth Univerahy, Rlchmofld, VA. PETER M. ROSS. PH.D. Re-.each Asteciatc. TI~ Rocket'clltr Unl- ve~ky, New Yodc. NY. JOHN R. ROWLANDS. FIt.D. Staff Scientist. $~lhwest Rl~tan:h lnstl. tu~e, San Antmio. TX. RENJAMIN A. RUBIN, Aasistam Pmfe~m e~ Publle Neef~, Bay- l(x" Uolvt'~ity COI~ ~ M~d~ir~, RONALD P. RUBtW. I~.D. Pmfetto~ of PHarmt0ol¢~y-. Medical C~I. le~ of Virgini~ Rtd~meed. VA. HENRY l. RU~E~. M,D. PRsidem. The Rws~k Fnendatl~n, Inc.. Sttaen Islam1. NY. W. O. RUS,S'EU... M.D. Unive~Ity of Tex~ UNA S. RYAN. PH.D. Research Pmfessm of Medicine. Unlvef. lily of Miami School of M~diclne. Miami, WAYNE f,~ RYAN, Pt~D. Profe-,.sor of Biechamlsl~. University Of Nebraska College of Madleine, Omaha, NE, JEFFREY D. SAPI:I~R. PH.D. Ass(x:tate $1aff Sclanllst. The mto~,, Ba~ Had~or. ME. PETE~ R SALISBURY, M.O,, I~.D. Heed, Intensive Tteam~ant C~nt~r. Saint Joseph Hospital, B~'~. CA.
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0 0 PAUL SAL'TMAN, PH.D. Assis~nt Professor or Biochemistry and Ffulrkio~, Unlversky of Southern CMifor. hie Sehoo~ of Medk:ia~, Lm AeL'ete~ CA. BARBARA M. SANBORN. P..D. Profesmr University of Texas Mcdlcsl ALVIN R, SCHMIDT. P..D. Dirccto¢ of Counceling, Tufl,c University. MedfoM, MA. IAKOB $CHMIDT, M.D. P~hD. ~q~d~tan( Pmfcs,.,or of Binchcmist~. Div;- of Bio~o~.icsl Science-,,, 9ste -Univer- sity of New York ~ Slony Flrr,~, NY. AZIZ SANCAR, M.D,, PH.D, A~le Pml'es~0¢ OF Biechem;~'y. Uel. ve~il~ of Noah C~olin~. Cl~pel Hill. NC. REGINA M. 5ANTELLA. PH.D. Ass0clete Professo~ of Medicln¢ and Envl- ronmental Sciences. Columbia Univer- st~, ~ York, NY. I!. WII.~JAM SCIINAI~R. M,D. Assistant Professor of Pediatrics. Wash- .intton U~iversity School of Medicine, St. Louis, MO. ISAAC SCHOUR, D.D.S., P,.D., D.SC. Dean, University of Illinois Colleee of Dentistry, CMctgo. ft., BR~HMI P. $ANI, I~.D. II~, Prolein Biachemistry, Soulhem scc, ch Ins~ilu~ Btmdnghnm, AL. LUCIA ~"HIJGER, M.D. Mallory Pathology Associates. Boston. MA. B.V. RAMA .~ASTRY, PH.D. Professor or Phxrmacology, Vanderhill Unlve~ily. N~u, hville, TN, GORDON H. SATO. PH.D. Director. W. Alton Jone; Cell Science Ce~. L~e Plaid. NY. WALTER SAUERBIER, PH.D. l~fessor of Mkrebiololy, institnte or llu~m Gc~c~k:s. University of Mim~'so~, Assis~ant l'mfessor. University of Col oracle Health Scknces Center. Denver, DAVID W. Ptofes~m" of" Immunology, University of" Rochcsler, Rochc~er, NY. ROBERTF.. SCOTT, M.D. Professor OF Pmhology, University ,f Ten- nessce. Mem~is.'lN. SCRIPPS CLINIC AND RESEARCH FOUNDATION La Jo~. CA. SID~EY A. ~-~JDDER. M.D. o~y. Uelvefs|ty m ~.'miJmnia. uavts. C~. ~ D. BCHER, M.D. ~ el' Pedfauics and Human ic~ Jceq~ S~ Research lmtkute. C'~il. dre~s Heq);~al of Ffiilacklphie, PA. JOROEN U. SCHLEOF.J.,. M.D.. PH.D. Professor m~! Chairman, Department of Surgery. Tultne University School of M~dlcine, New Ode~s, t.A. STEWART SELL, M.D. Professor. Unlvers;ly of Tex~s llesleh 5¢i- e~ce Con(or. Ilou~, TX. CARL C SEL'rTJ~. PH.D. Honorary Resesrch Asso~iste, Peabody Museum, Ilsrvard University, Cam- ~, MA. GREGG L. SEMENZA. M.D.. P~D. Assistsnl Pmfessm. Johns Hopkins Unl- versily School of Medicine. Baltlmore, MD. HENRY SER$)IEN, ~.D. Research Scientist IV, Neutochemisl~ Divi~k~. Nad~n S. KIks¢ Rescm¢'h m~, Wt~'s Ida'. NY. LUCIO SEVERI, M.D. Director and Deun. Institute of Anttomy end Pathology. Division of C~neer Re- search. University of Perugia. Perugis, linty. CHARLES R. SHAW. M.D. C~ief. S~ctk~ of IV~dical C, enede~, M.D. Ander~ I Iospitel a~ Tumor Institute: Pmre~ of Biology. ~ Unlversi~ OF Texa~ It I I~tl~. I I~, ~. JERRY W. SIlAY. l~hD. Ass~iate Pml'es~nr. Univerxtty OF Tesss II~tllh Science Comer, Dallas. TX, ISAIAHU SHECHTER, PH.D.. Senior Fellow. Eleanor Remevelt Inslilul¢ fee Cmeer Research, Desv~. CO. CIIARLI~ E. SHERWOOD. M.D. At~istaal l~,ofessor of Rndiotegy, Unlve~ sity ot" Roch~ler ~ of M~klne md Demi~, ~, NY. SltOfl SHI~ATA. M.D., PH.D. l'mressor of Pt~m~)gy. Univml~y of Hawaii School of Medlci~e, Henold~, HI. GERALD SHKLAR, D.D.S. Chides A. grsck~t Profess~ of ~ ~- c~ ~ ~1 ~. -~ ~ ~ ~m~l ~. ~. MA. PAMEL~ A. SILVER. PH,D. As~o¢lme Prore~Jo~ or Biologiea| Chem- istry and Molecular PMnn~olo|y. DaM F~'C~nc~ lnsti~e, Bneto~ MA. DAVID L. SIMON. M.D. lmtrm-~r in lnlem~l Iv~dkla~. Claelrmtl Gene~l Hmphal, Ciaeln~ll. OH. MICHAEL SINENSKY. PH.D. Hend, Lipid ~nd ~l~eks Divisi~ ~ ER1K SKIttle;. M.D. NATHAN H. SLOAN~, PH.D. Of 'renn~s~ Clltltf for till. I|ealtlt ~el- HANOC~ SLOP,. I~.D. Aslecble l~ofesso¢ of H~mLq Oonetlel. $~ckkr School of Medicine, Tel Arty Unt. versily, Tel Aviv. lutel. THI~DORE A. SLOTKIN', I~D. Ass|st.a,nt Professor or Phstmaeo|~|y. Duke university Medical Center, GEORGE[W. SM~'I'T~ M.D. Associate ks I'~. N~d~v~ttm Uni. 1~JtffiS M. SMITH. I'~.D. Assistant PmfeJamr of BJolotluel S¢i. FR~NC~S L $MrrH, PH.D. Shriver C~nter for Me~te! KENDALL A. sMrnl. M.D. Pmf~ of Medl¢tm, Dmm~th School, H~over. NH. LUCIL~ SMITH, Professor of Biochemistry. Osrtmouth Medied School. Hueover.
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0 STEVP~ & SMITH. PH.D. NASSismnt Reled~ch ~ientist, City of Hope ad<mzl M~ieal Cenlcr, D~ar~e. CA. LOUIS A. ~OLOFF, M,D. Blanche P. Levy DIsdnlnished Service ProNsle~1 ~fe~ of Medicine: Di~- ~or~ R~h Lipid La~I~, Temple unlv~l~ l~hh ~s Cenler, ~da- SHEI.DON C $OMMER~. M.D. D&ec4or of L+beratodes. Leonx llill llos. ¢~bi~; Cl+ics' l~of.mx of Pa,.~. Co- Unive~ily College of Pfiysm:mns & Surln~m. New Yod~. NY. ERNEST SONDHPJMER. PH.D. Yo~. Symcu~. NYo T. M. SO't'INEBO~N, I~LD. D~stla~vtshed Sorvic¢ Professor of Zoo. IOl~. Fadla~u I.hdver~+ly. Bloomtr~loe, IN. ASHWAN! K. ~X)D. PH.D. Canclr Research Scicnlist Ill, Boswell Park M~-,oHM Iw+tilute, guffak~ MOIIAN ~OPORI. PH.D. Scienllst l,l, Lovelace Medical Fozmda. li0~. Albuq~e~. NM. OKOROE D. SORF.NSON. M.D. PtOrel~or of Pathololy. Durlmomh teal $¢'hool, H~nover.~H. SAM $OlU~R H_+Idt l~p~_rlm~.nt of Macro_molecular Cbemtsl~r~, The lnslitllle foe I..il~er Re- touch, Ft~illckll~il, PA. ~ RI~EARCH ~NSTITIJ'~ ~n An~qdo, TX. DAVID M. SPAIN, M.D. Dlmclo¢, +l~eparlmenl of' Pathololy, Brookdlle H~lal Ce~. Brooklyn, NY. SPARKS. PH, D. ~¢ientls~. Univeraity o£ Roche~ter. Rod~e~. NY. ALEXANDER SPOCK. M.D. Assistant Professor of Pedlatrics. Duke University M~ical Cemer, Durham. NC. TIMOTHY A. SPRINGER, I~.D. Senior Investigator and Vice President. TIe Center foe Blood Research, BO~lO~. FREDERICK I. STARE. M.D. Professor ¢4' Nul~io~ ll~,~ard University School of i~bllc Ilcallh. Bosm~. MA. DANWL s'r~NB~RG. M.D., PH.D. Pm£¢+sor of Mediek~e, Univerddy of CMI- fore}a Sehn+l ol Med~:ine. San I~e¢o, La CA. NEWMAN L STEPIIENS. M,D., F.R.CP. Protesr, nr of Physlololy. Univer~cily of M+milolm. Win~, Manhole. Can~fa. TIIOMAS P. ,~ROSSF,/.,. M,D. Ilead. Diviskm o1" P.xper;memal Mcdlclne. Brigham and W~men's Ifosl~lal. Bosfon, MA. NANCY E. STREET. PH.D. Azsislam Profesaor of Microbiology. Uni- versily of Texu Southwestern Medical ~:hoel, Dallas, TX. JACK P. STRONG, M.D. As~¢~ Prot"es~or ~ S~e Un~ky ~:bool oT Med~'i~e, New Odeans, ~. DONNY STROSBERG. PH.D. Chaid~rol~zor o( Bkeheml~' and Immu- nology. Pasl~w l~lilul¢, Paris. Franc~. 29R SURESH SUBRAMANI, PH.D. Associu~e Professor of BIo~osy, Unlve~l~ of Calirorai* at Sac Diego, IJ Joll~, CA. MARION B. SULZBERGER, M.D. Professor and Chairman, Deparm~nt of l)umatolocy and Sy~hilolof#. New Universily.llellev,e Medical Center. r~ew York, NY. MARY F. SUNDAY. M.D. PH.D, Assblam ~ ~ ~l~y. JOHN P. SUNDBF.RG, D.V.M., Ph.D. SmlT Sclend~, The Jack,on Labor~tory, Ba~ Ha~ee¢. ME. RAQUEL SUSSMAN. I~t.D. Assa¢iate Scientist. M~zine Biological ~, Wo~dl ltole, MA, TORGNY !1. SVI~,KON, M.D. Pmfcssm, Karotlnsl~a Institute. Stock- holm. Sweden. LORRAINE S. SYMINGTON. A~,,+istanl Pml'emmr. Coh~m~ia UnlversilT, New York, NY. MAKOTO TAKETO. M,D.. PH.D. Associale Investigator. The McLaughlin Ren:~--h Imfimle. GRa~ Fails. MT. LYNN M, TAUS.qlO. M.D. AxP, ociate I~of©s~or and Aug.,elate Cha~ m~, ~nt or ~i~r~s. AliBI lled~ ~s ~l~, ~, ~ KENN'E'T~ M, TAYLOR, M.D. Profe~or aad Chief of Cardla¢ Royal I'~lSradual~ Medical DANIEL G. "I~NEN. M,D, Medical School. leek laml Hospital. Rmme, MA, VICTOR P. T~IUt.ANOVA. A~t~¢bl¢ Pr~*~'~. Columld| Unberdly. New Yerk. NY. MARC D, TtlAM~ M.D. ~ R¢-.eaR~ Fdlew. M~j~ Clink Four, be. Rmhe~er. CAROLINE BEDEL~'I1qOMA~. M.O. Hopkins University School of Mtdi- ck~e. Baltlmo~, ~EROME F. THOMAS. PH.D. Professor of Sanitary Engle~tlnlt. Unt- MATrlIEW ~. TIIOMA$. PH.D. Ar~iM*m ~ of PalMlely, MicroN. vemty $¢hool of M~ir, e. St, L~III, MO. JOHN A. THOMPSON, P~D. Associate Professor of P~arma~utieal Cl~im/. UMvmlty ~" Colm~ ~ JAMIES tL P.'rOMAN. I~.D. Pro(cure" sad Chairman. lnstltv~e for Medk~ll Research, It.. ANDR~PI~/M,'rOMlZTIKO, PH,D, LMx'~alod~. LM.* Re¢,hester. NY. ANNAMARIA'rORRIANI.GORINIo I~.D. Pml'cmor o4" Biololy. Mamdmsem Inml. t~ of "rec~nology~ ~ambddl~, MA.
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0 JAN]aT TR~VELL. M.D. Asf,0cilte Plofes$or of Clinical Pfiasms. eol0gy, Comell University Medical Col- lep.-Ncw Ymk. NY. IAMI~S TRAVIS. I~.D. of Geo~a. A~f~ns. GA. WAYNE M. TRE,BBIN. M.D. A¢$oclale _Program Director of Internal Medicine Residency. Salem Hospital. Salem. MA. LIE SIIA '~JAL Re,earth Associate in Pllhnlogy. Yale University School of Medicine. New Itav~n. BEN Y. TSENG. I~LD. Associate Ad~ut, ct hof~.x. Unive~lly of Callrocnia. San Diego. La Jolla. CA. PHILIP N. TSICIILI$, M.D. $e~ior Member, Fox Ch~e ~ Cemer, Pt~l~dd~. PA. PIIILIP W. TUCKER, I~.D. ~ofe~o¢ of Microidolo~y. Unlvers|ty of Texts $omlmee~em Medical Cemer, Dal. |~So 'IX. GEP, ALD M. TURINO, M.D. Profcs~o¢ of Medicine. Columbia Univer- lily Collep or Physicians & SurBeons, New Yo~k.NY. JOHN TURK. M.D. A~ Profe~mr of Mndk.i~e and P~ho~. o~y, W~dd~m Ueivem~, ~. Lo~is. MO. ANGEU, L TYNER. I~I.D. Alslsfanl ~ of Glandes. University of lllir~/m. Colle~: of" Medicine. (~icago, IL JONATHAN W. UHR. M.D. Pmfcs~x' and Ch~rlman of Mi~idology, Pre/~mr of lnlemM Medlclne. Unlversip/ ¢dr Texas Southwestern Medical Center. D~lla~. TX. EMIL R. UNANUE. M.D. Chairman and Pml'essor, Department of Patholo~t. Washington University School of Medicine. St. Louis. MO. UNION CARB|DI~ CORPORATION Nuclear DirkS. Oak Rid~. TN. UNIVERSrT'Y OF SAN FRANCISCO San Fr~cisco. CA. UNWF.RSrFY OFSOUTTIF.RN CATJi'~RNTA Angeles. CA. MICllAEL W. VAN DYKE. PII.D. A~,ci,~lanl Pmre~mr of Ili.h*gy. Unive~ity of q'exzx M.I). Amlerson Cancer Cemer. II~.TX. ltELEN VAN VUNAKIS, PII.D. Professor of Biochemkln/. flrandei.~ Uni- versily. Waltham, MA. HAROLD F- VARMUS. M.D. Pmfettor of Microbiology and ogy. University or Callfomia. San Fran- cisco, CA. STEPIIEN F. VATNER, M.D. Assoclme Prof¢~x' of ~ic~. ll~a~ M~al SC~. New E~la~ Reg~l ~m~e R~h ~r. ~th~ MA: As~ia~ ~ M~. ~er ~ BH~ Ill,Ill, ~t~, MA. E~I~ S. V~L M.D. ~fes~ a~ ~ai~an. ~pn~mem o¢ ~. ~nsylva~a Sta~ sky ~lle~ or Medici,. Milt~ S. I~r- ~OM~ W. VICKROY. ~I.D. A~s~ ~. Univ~ky of ~ BRANIS~V VIDe, ~fcs~r of Antony, ~rg¢own ~ni- ve=k~ ~s or M~i~ a~ Washmp~. ~. ROM~ A. VI~NE. M.D. A~i~le Pmtcssc~ of Pathology. Yale Univeraily School o[ Mcdiclne. New H~n. ~. LYDIA VILLA.KOMAROFF, P~.D. Asse¢ille Proresso¢. Children's BOS~o PETER K. VOGT. PX.D. Professor and Chairman. Depar~me~! of M _k'fVidol~y. Uni~e~dty of So.hem Cal- Worn|a, Los Angeles, CA. ROBERT R. WAGNER. M.D. Profes~m of Microbiology, U~iversity of Virg;nia. Charlo~esvill¢. VA. ZBIGNIEW WALASZEK. R4.D. Retearch ~c~mist. Ohio Stale Univ~;ty. Columbus. OI!. EVELYN WALDSTEIN. P,.D. Seninr Lecturer. |)epimmenl or !airy; Tel Aviv University, Tel Aviv, PETER N. WAI.SH. I~.D. Pmfe-Jor of M~dici~e. Temple School of Medidne, Phih~Jel~b, PA. IRENE Y. WANG. P..D. A~isla~ lq~r~x, of Basic s~! Clinical lmmu~olo~ a~ Mi~ ~edical LEE W. WATI~JqBERO. M.D. Pmf~mr of hdmlo~y, I~l~nity of Mk~. ~ Medi¢¢ ~1, Mi~p~llk MN. JOHN $. WAUOR. I~.D. mi~e of Technd~y, C~mbrld~, MA. THOMA~ E. W~BB. I~.D. Professor of PhysioloBi~sl Chem|tCry, Ohio Stale University Collie of M~di. clan. Columbus. OH. JOtlN V. WEI~ M.D. Ass~ant Pr~'esser of Mudkh~. ~ ~.of Calor~lo MMka! f.~nler. Drawer. LU-HA! WANG, P,.D. Associ~e Professor. Movnt Sinai School o1' Medicine, New Ymk, LAWRENCE J. WAt~fGIl. P..D. A~o¢iale Frofes~o~ of Bielogy. Brandeis Univmily. WM~ham. MA. I~TER A. WARD. M.D. Professor and Chain~an, Deparlment Of' Pathology. The University of Michi||n, Ann ArF.6r. MI, D. WARNE~ M.D. Pmre~o~ of P~hoio~y, St.~e U~ivecdty of Iowa Cotte¢c or Med-~¢ine. lows C~ty. IA. SHIELDS WARR~. M.D. DiRelo~ o¢ Labor~od~, Cancer P.e=eard~ lnsllm~e. New England Dcaco~ss Hospi- tal, Boston. MA. YASUSH! WATANAB~ P,.D. As~clale Memt~r. The Wist~r 1n~l|l~e of A~ammy ~md Rie~y. Phibdclpbil. PA. DAVID B. W~NBR, Direct" of Bio~ecknole~y, Wistar latll. m~e. l'kil~ddplda. PA. I, BERNARD W~INSTEIN. M.D. ~:kme~, (:elm,lda U, Iv~ndrt. Collele el" Pt~ileS & S~ NewYel~ NY. A. WEINSTOCK. I~.D Resca~h Biocbemis4. Life $~r~l DIvi, skin, lit P.cse~ch lnsdmte. Chicaso. II. SAMUEL B. W~IS$. PH.D. JUDTTH WETSZ. M.D, P~fesso¢ of Obsl~tdcs and MilKm S. He,.hey Medical C~m'. s0,1van~l Stall University, H~d'~y. PA, 301
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0 0 SIOMUND A. WEr~.~IAN, M.D. HOWARD O. ~U~ M.~ ~. ~is. MO. N~ K. ~R, ~.~ Assist~t Pro~essor o~ Mcdiclnc. Western Rescue University, Cleveland, RUSS~ W. W~LER, M.D. ~W~I ~ter. Oixb~ ~m~ of S~e~. WashiflZtm Mi~A~ L W~H. M.D. ~t~ of lows Ca1~c~ or ~edJ~. lows O~l~ NY. SIM~ H. ~. ~.~ ~i~ ~ N~ OK. ~ ~~. M.D. ~ ~ ~alman, ~mcnt or ~~ ~d ~xi~o~. ~ Uni. vc~i~ of K~nsas ~h~l of ~tcy. ~ ~ ~f~. Un;~i~ ~ ~. VA. ~ ~ ~, MA. AI.EXAHDER S. WIIlTEllEAD. Assistant Professor of Pediatrics. Chit- drcn's Hospital. Bo~wn. MA. JAMES A. WILI~ D.V.M.. Pv~fCsso¢ and Chairman, Department of ~n_.~/Science. University of Wis¢o~. sin. M~iso~ Vii. ROGER ,h WILLIAMS. M.D. Professor of Chemistry; D|~tor, Clayton Foundat!on Bi~hemicM Institute. The Uni~ily of Texas. Au~lln. ~. JOIIN T. WILSON. I~f.D. AsSist~A Pcoi'as~o¢ of ~.ell ~ M~l~r Ololo~. Medical Callek, e of C~-or~il. Au- gum. OA. JEFFREY WILUSZ, P~LD. AsSislcnl P~oJ'csso¢ or Micro~nlo~y snd Molecular Ge~ll~. Unlverxky of-Medi- cine t~ ~ntist~ of New J~y. New ~ M~al ~. NewJ~. ALVIN L. WINTERS. P..D. Assistlml II~'o~e~or of Micmbinlo~y Bt~hemistry7 The University of Ala- ~. Bi~m. A~ DANIEL H. WISEMAN. M.D. Assi~a~¢ Pmfusor of pediatrics. Univer- vision. Lo~ A~e;es pital, Los Anleles. CA. BRUCE A. WODA. M.D. • Associate Professor of I~thok)~/. Univer- ally of Msss~hns~t~s. Viorcester. MA. GEORGE WOLF. ~me~ of Nutrition aml Fond ~e~, Mass~h~tts Institute of Technology, ~. MA. DEAN F. WONG, M.D. Asnistsnt Peofcsso~. ;ohns Ik~klns Ilmpi- tsl. Baltimore, MD. L EDWIN WOOD, M.D. l~¢tor in Medicine. Ilost~t Unlver~hy Sdtool ~ Medlcln¢. BroWn. MA. SUMNER WOOD. Jx., M.D. Asslstam Proresmr of Patholpgy. The ~hns ll.~Id~s University Sclmol cine. Baltimore. MD. PAUL V. WOOLEY. Ill, M.D. l~so~ of Medicin~ ~d Pt~m~ololy. Oeor~t0wfl University Medical Center, Wash,oaten. DC. RICHARD M. WRIGHT. PH.D. Senior tnstructor. Webb.Warin~ Leo| Institute. University of Color~o~ Denver. CO. THOMAS C. WRIGHT. ;~., M.D. Axsistsn! Prol~sor of Pathology. Colum- bia University College of Physlci|ns & Smyrna. New York. ;OSEI~! M. WU. P..D. Professor of Biochemistry. New York Medical C~IK~. Valhalla. REEN WU, PtI.D. Assocl|te Adjunct Profes~.r..CMi[0mia" PfimMo Research Cooler. Umverstly ot Callftw~a. D~vis, CA. JOHN P, WYATT, M.D. Prol'essm of ~oU, S~, L~uls Univer- sity School of Medicine. Sh Louis, MO. ROBERT L WYKLE. Professor of Biochemiswy. Bow~. Gr~y ~hool of Medicine. Wake Freest U~iver- ~tty, Wlnst~e-Satem. NC. STANLEY YACHNIN. M.D. Professor of Medicine ~nd Chief. Sccdo~ of HemstoSo~,/Oncok)Zy. ~ Univ..ratty of Chlc~lo M~dicst C~'ntcr. Chictlo, GARY VELLE~. I~.D. Associate Professor or Neuroblololy, Massachusetts Gcncrn! Has~ital, Charleslo~. MA. ANDgEW YEN, P~.D. As~e¢i~e Professor. New York ~t~te CrA- G1' Vetedmmy lvlcdklne. Comell Uni. versts, llhKe. NY. "rIEN.S~,E R. YEN. M.D., Flt,O, A.istam Pmt'c~or of l~thotnlY, Unive~- sky of Coliromia. Sm Frmci~o. CA. DONALD A, YOUNO, M,D. Profeaso¢ of Medicine. University Roches~e¢. R~chester, NY, ZAHRA ZAI~F~I, Px.D. Assistant Profess~" of BloloILY. Q~ee~ Cotte|e of ~he City Ue|vera|iy ef New Yod~Hmhi~. NY. MAURIZ]O ZANffTTI. M,D.~ Assoei~te Pmfe~e~' of M~dlci~. Univer- si~ of Calirm~ia ,~I~I of M~II¢I~, $~n D~eso, t~ Joffs. CA. MAD~ICE ZA~IOER~. PHJ), A~ l'm~r o~ Onedol~, Univer- sity of Roche~, Roel~ster. NY.
Page 154: 60041073
0 0 INDEX OF PRINCIPAl, INVESTIGATORS Alber~ K. M.. Al|ende. J. E.. 48.49, 164 Anderson. $. M.. 16~ Avadl~i, N. G., Bcru~ A. D.. 212.213 Be~tl, K. L.. 51.163 B~ntsty. N.. 14~ Bi~. R. J.. 4 !~3. 215-216 B~ke~oK. ~. E.. B~ker. T. R., 227 Buck~y. K. M.. M.S~ ~. J.. ~. C~h~. A., 25. ~9~ ~t*. R. H. !~. 228 C~i. O.. 216 ~. W. A.. 61-63.~ ~ ~. R H., 191-1~, 228-2~ ~k~. W. R.. 63 ~ R ;., M-~. 167 ~. ~. B.,217 ~h. h. 67, 2~ R-2~ ~. M..M ~,A.. 2~4 G*y~. R.. 231-232 Gil~. T. D.. G~. A.. 2~ Gli~r. ~ H.. 69. ~69-170 ~m, S. M., ~. K.. ~-71 ~. M.. 27-28. 171 H~e~, R J.. 72 H~. ~, 2S-~. 73 Hm, M., ~3, H~in~. O.. 74 H~, M. R.. lSl, I~.!~ H~s. S. E., 75 H~y~hi. L. 76. Hey~. R G., 172. Hi~ O., 31-32. 16g H~ht~i. D. K.. 149 1d~e. J. R., I~ Sai~wal. A. !(.. 33 ]aken. $.. 76. 150 ]amler, on. G. A.. I~ ~ay~ram. M.. 77-R! Jeang. K, T. 233-2~ Jerome. R.. 173 Je~itk, A. ].. ~2. 221-223 Joklik, W. K., 236 Karin. M.. ~4.~7 Kam~y. M. J., 223 Kaslan. M. B., ~7 Ka~ F. N.. ~h, ~ II., ~, 237 K~I, W. H., 224 ~h,A.~.,~ K~sin~, R. H., ~2 K~h~, R. D., 19~ Ku~yxml, R.. 91-93, ~lh~. A.. 1~!97 19g. ~3 1~1-1~2 ~, ~ Y.-H. R, 33 ~i~l~ J. L, 173, ~vin, ~ D., 1~-201 ~iMs~. ~. M.. ~2 Luke, R J, 186, ~, ~. M., 214 M~r, T.. 174 M~u~, L. E., 1~1 M~lonio, ~ E., 1~103 Ntufeld. G.. I~ Nu~, G., I~, 1~3, 176 ~. J.-ll., 22R. Parsons, $. ]., IlO Petri, W. A.. Jr.. 110.205 Poflcz, M., i11-112, 153 Re~. ~. C., 2~ Re~. ~. I.. 113.114 Reich. N. C.. 114 ~ei,l, ~ M.. 3~, I~ ~h~ds. R. ~. ~. !!~ Robinson. ~. M.. 244-245 Ruhin. I!.. 37 Ryd~, K.. ! 16 1~6-1~R S~si~. B. V. R. 119. 1~6~ ~7-212 ~to. ~. D.. I~ Shil~ B.-7~. 121. I~ Si~. H. ~. I~-I~ Sp~l. S.. 3g. 122-1~. IX7 S~I. ~ R~ 161 S~rk~. J. R.. ~0 ~. R ~. 161 ~no~, K. D.. 40 ~, C.. 17R T~ki~. A. ~. i~ ~. ~. ~.. 46. l~g-I~ ~.A. ~. 162 Yankee. M. W.. 246 163 ~hmldt. T. ~., 179-I R0 ~ng. J. Y. J.. ~37 W~y~r. M. ~.. ! ~ ! ~ W~l~s. !t. G.. 141-142. 180 W~ssling-Re~ick. M., M3 Wilu~ J.. 144 ~ll. J. K~ I~

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