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Report the Council for 20 Iresearch-." U_s.a., Inc

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Named Organization
American Cancer Society
American Red Cross
Archives (National Archives and Records Administration)
ATF (Bureau of Alcohol, Tobacco and Firearms)
Bureau of Alcohol, Tobacco and Firearms
Basic Research
Bristol-Myers (In Connecticut)
Brown University
California Institute of Technology
City University
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.
Duke University
Fox Chase Cancer Center
Georgetown University
Harvard Medical School
Illinois College
Johnson and Johnson
Lederle
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 Wisconsin
National Academy of Sciences
National Institutes of Health
National Institutes of Health (NIH)
National Science Foundation
New York University
Northwestern University
Plenum Press
Preventive Medicine (periodical)
R.J. Reynolds Corporation (second tier subsidiary of RJR Industries)
Red Cross
Research Council
Schering-Plough Corp.
*Scientific Advisory Board (SAB) (Only use SAB with name of specific org.)
Sigma Xi
STAT (Stop Teenage Addiction to Tobacco)
Stop Teenage Addiction to Tobacco - anti tobacco group started by Joe Tye.
State University of New York
Thomas Jefferson University
United Brotherhood of Carpenters and Joiners of America (UBC)
University Medical Center
University of Alabama
*University of California (use specific branch)
*University of California Berkeley (use University of California at Berkeley)
University of California San Francisco
University of Colorado
University of Massachusetts
University of North Carolina
University of Pennsylvania
University of Texas
University of Texas Medical School
University of Virginia
US Army
VA Medical Center (Located in Minneapolis, Minnesota)
Washington University in St. Louis
Western Reserve (Medical School)
Named Person
Abood, Leo G.
Austin, Austin
Berger, Frances C.
Boyd, David
Busi, Yuri
Cai, Anne L.
Cam, Israel
Campbel, Edward J.
Canto, Israel
Chuong, Ming
Dallas, Dallas
Davis, Davis
Feldman, Joseph D., M.D. (CTR SAB)
CTR SAB
Feldman, Joseph O.
Found, C. Berger
Gill, Gordon N., M.D. (CTR SAB)
Defense
Houston, Houston
Hughes, Howard
Israel, Beth
Jefferies, Wilfred
Jones, W. Alton
Kent, Amy
Kmiec, Eric B.
Lira, Ellis Horwood
Ly, Chang
Markey, Lucille
Markey, Lucille P.
Medico, Howard Hughes
Orf, Mark I.
Pierce, G. Barry
River, Pearl
Rose, Nadia
Ryder, Kevin
Sabatini, David D.
Sherry, Sol
Simmons, Harold C.
Smith, M. L. (RJR)
Washington, George
Date Loaded
11 Jan 2006
Box
0422

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Page 1: 60111634
REPORT THE COUNCIL FOR 20 IRESEARCH-." u~S.A., " Inc. 1995
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199S REPORT ,f 'I'llF, COUNCIl, FOR T{}IIACCO RESF, ARCll- U.S.A.. INC. Till,: C(IUNCII, I:OR "I'(IliACC(! RF..GI,'.ARCI I -U.S.A.. INC. 900 Third Avenue, New York, N.Y. 10022
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SCIENTIFIC ADVISORY BOARD to The Council for Tobacco Rc.r, carch-U.S.A.. Inc. as of Oncembcr 3 I, 1095 G. BARRY PIERCE, M.D.. Chairman Distingui.~hed Profex~r of Pathology Universily of Colorado I l~llh Denver. Colorado LEO G. ABOOD. Ph.D. Professor or Pha~acology ~nd Bi~hcmi~(ry Dcpaamcnf of Pha~acology Univ~ily or R~h~s~cr M~ical Cen~er R~hes~er. Hew Ymk BARRY G. ARHASON, M.D. Professor and Chai~an. Unive~ily of Chicago. DRUMMOND II. DOWDEN. M.D. Professor (Emeritus), F~ul~y or Medicine ~panmcnt of ~thology Unive~ily of Mani~a Winning. Canada MI~IAEL J. BR~NNAN. M.D. Dircclor Familial Cancer Prevention ~inic Presi~nl and Medical Oirccmr (~er~lux) Michigan Csncu F~ndalio, Professor of M=dicine (Emerilus) Wayne Stale Unive~ity Sch~l of Medici~ D~lroi~. Michigan CARLO M. CR~E, M.D. D;rccl~ Jc[[cnon ~ancer Insdm~e and ~anccr ~cnlcr ~ilad=lphia. Pcnnsylvania RAYMOHD ~ ER~KSON, Ph.D. Pmfe~f of Cellular and Devdopm~l~l Biology Biological H*~ Unive~ily Cambridge. M~chusetls GORDON N. GILL. M.D. Chairman. Facully of Basic fiiomcdicnl Sciences Univcrsily of Califomi.. San Diego Schr~t of Mcdlcit~c La ]olin. Califomia W. K. IOKLIK. D. ~ttt.. ~paflmcnt of Micmhiology Duke Univcrsily Mcdical Ccmer Dmham. Nnrlh Camllna IlF~RY T. LYNCII. M.D. Prorc~or and ~ni~an Di~ctm. C~clghl~ Cancer C~ter Dcpn~mcnt of P~vcnlivc Medicine ~ Public llcallh Professor of Mcdici~ P~idcnl. I lc~dila~ Cancer Inslilulc C~clght~n Unlvcrsily Sch~l of Mcdlclnc Omaha. Nebraska HARMON C. McALLI~ER, Sclcntific Dirccfor ~ Con~il f~ Tobacco R~a~h-U~.A.. Inc. New Ymk. New York DAVID D. SABATINI. M.D., P..D. ~c Frederick L. ~mlan Pmfc~qor and Chaim~no, ~pam~cnt of Cell B~logy Hew York Unive~h~ Ncw Y~k. New Y~ JUDITI! ~ SWAIN. M.D. Ilcr~n C. Rotor Prorcs~r orMcd~al Scicnces sad Genetics Chief. Cnnliovn;cular Division Univc~ity of Pcnnsyivnni= Medical Center Philadclphia. Pcnn~lvan;a P~ER K. V~T. PtLD. Mcm~r ~ Scripps Rcsc~b Institut~ ~ Joiln. Caligula SclcnliBc Staffer.The Council I fARMON C. McALLISTER, JR.. I~I.D. Scientific Director ARTIIUR D, EISENBERG. PII.D. GEORGE A. IIASHIM. PII.D, Assncialc Rcxcnrch Direclor A.~mciale Rer~a~h DONALD !1. FORD. PILD. Ax.~ocinte Rc~arch Dirtclor
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CONTENTS Introduclion .................................................................................................... Ah$1ract Tilles and Authnrs .......................................................................... Abstracts of Reports ....................................................................................... 25 !. Cancer-Related Sludies .......................................................... II. Cardiovascular Syslem ........................................................... 4 I IlL Cell Bin}ngy ........................................................................... IV. Developmenlal Biology ......................................................... 14~ V. Epidemiology ....................................................................... 163 VI. Gentiles ................................................................................. VII. Immunology and Adaplive M~hanL~ms ............................... 16~ VHI. Neumscience ......................................................................... IX. Pha~acology ....................................................................... X. Pul~na~ and R~iramry ~y~lems ..................................... 212 XI. Radicals .................................................................................. XII. Virology ................................................................................. 227 XIII. Miscella~ous ........................................................................ 24 I Aclive Proj~ls .............................................................................................. 247 Compleled Projecls ....................................................................................... 271 Index o~ Principal Invesligamrs ..................................................................... Introduction Research fundinl~ of TI~ Council for Tobacco R~s~a~h by i|s s~'msor compa. nits continued at a significanl level in 1995. ~e t~al ~munt of funds ~eseatch ~tan~s ammmted ~n $19~$50,~ in 1995, brin~in~ to ~te than S26~ roll. litm ~he Cnoncil's snp~ for ils ~sea~h pm~mm ti~e il ~n o~ratiom in 19~. 33~¢ Cmmcll remains tenet Ihc lcadln~ pfivale [un&n~ agents in supporting varies ty~s nr h~ic hi~cdi~l invalidity: cancer, c~iov~ular discascs,,ccl/hiolngy, tlcvelo~enlnl ~ol~y, epidemiol~y. ~e~llcs. immu~nBy. neuro~ience, phann;~ology. ~tlmona~ disc~s. ~di~ls aml ~imlogy. New nHgin:d re~nrch pn~jccls approved and funded in I~.$ It~aled 67. while m~y mo~e cnnfinuin~ and renewal sludics were also funded. ~t~ouSh 1995, the Cn.ncil has s.p~ncd a total ~ 1.49E miginnl invesdBadve pmj~ls mccllnss as part of the Conncil's tesen~h program. ~e ~clpie~s Shah I,l~ll imlc~ndcnl ~icnllsls al mt~ Ihan 3(~ mt~lical ~h~l~ Durlu~ I(~)~. ~ntees ~thli~ed 326 ~l~S ~ tl~i~ invesli~at~s tu~l by ~lc Council f~ T(d~co Rc~a~b. G~s ~ c~$~ Io ~lblish all of th=ir invcstlgafi~s. Ab~s of I~ ~ ~blish~ dufint 1~3 ~ i~l~d in this ~. Over I~ past 41 yca~. t~ ~ve ~ ~m Ih~ 5.~ ~icnlir~ ~MI. cal~s hy invesligalo~ suppled by ~ C~il rm To~ R~h. In Ihe snmmer or 1~5. we we~ ~ndd~ed hy I~ sudan and ~ex~led ~olh or Ihc ~lalrman or Ihe Council's Sclenlifi~ Advlso~ Board. Joseph O. Feldman. M.D., o[ ~ Joll~ Calir~ia. ~is a~ual ~ is ~ memorial to ~r. Folding: his mnny dislinguish~ c~lriholi~s Io b~mcdlcal ~i~e ~e ack~wl~d~cd in dedlcallon ap~nfing elsew~. Or. FeMma, '.,~s ~en su~ed~ Scienlific Advisory Board hy G. Oar~ I'~ rce. M.D.. Pro~ess~ and Chul~an Emcrilus. ~pa~menl o~ Pal~logy. Oniv~.~:hy or Colo~ Sch~l ot MediCine. Denver. ~¢1~ N. Gill. M.D,. ~ic[ or ~d~ri~lo~ela~llsm. Uniw~i~ California-San Diegn. h~s ~en elecled In Ih. ~:,)s[ or Vice ~ai~an of Ihe SAD. M~nr~ed [,. Kamovsky. M.5c.. ~.O. ,I lla~u~ Onive~ity ~c~¢nlific Aclviscwy Iloanl in April I'P)~, havi:~ ~ivcn Ion ~cu~ of ¢~cliculcd and I~¢rdfip in the pros of g~=nt a~lir:z;on ~vlew= and r~mdlnp of research. Dr, ~amovksy. a n~llve o~ S~lh Arri~. ~e ~o Ih¢ Unil~l SI=~ =s a rcsea~h fellow and became a nalurallzed -;dzen in 19.~6. lie ~nl mosl of his t/~C ~p;trlm~nl of flk~logJcal ~,cmist~. lie ;ms ~cn I~ ~ip~nl O~ many ~nd awards and has held m~m~rshi~ ~ o~s in the most p~i~nt s~iet~s in his s~ci~lly, lie is lhc ate~ or s~ze 3~ ~, ch~plem ond Successo~ In Dr. Karnovsky and Dr. Fcldman on the ScleMific Advlso~ ~ have nol ycl ~n ~leclcd, In I~5. ~ ~lcomtd ~o I~ Sci~liV~ Advi~ Boa~ member, David D. Saballni. M.D., Ph.D., or New York Univ~rsilx School or Medicine. Dr. Sa~tini. a native or Argenlinz, Imlned in thal R~krfcll~ Unive~ily, New Yozk. l[c [ms ~d as ~er~k ~ and ~aJ~an or lhe De~dm~l or Cell Biology al New Y~k Onlvr~[zy for mo~ lhan 20 yca~. Ills awards, vishln~ professorships, l~lu~ships, cdil~al mcnls ~nd honors are cxlcnsJve, and he [s ~copnized ~s a le~er in lhc ~eld or mere. hrnne msd pmlein research.
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C) IN MEMORIAM Joseph Dav|d Feldman 1916-1995 Joseph D. Feldman was a nalive of Hartford, Connecticut. lie received the rl~helor of Arts ~k:grce From Yale Univeraity in 19.17 and was nwanlctl the D~tclor of Mcdiclne dcgrce from Long Island College'of Medicine in 194 I. From 1942 1946 b¢ served as a Major in Ihe Uniled Slain| Amly Medical Coq)s. serving as a Balallion Aid $1otinn Surl:¢on. den! al Ihe Nc'w England Deaconess }los'pitnl. Bosloe. Subsequent were at Yale UnNct'shy School of Medicine. Hadassah Medk:al School in and the University of Piltsburgh School or Medicine where be rose Io the rank of Professor of Falhology. in 1961, Dr. Feld,;'.:.'. moved Io IJ 3ella, Callfoml=, when~ h~ held coocurrenl :*ppoinlments in Ihe Uni',,?r;ity of C, allromla.San D]¢¢o and Ihe Scripps Clinic and Re.search Found~,lJcm. I~: 19"/4. he was named C'balrm,n o1" Ihe Dcpartmcm! of lmmunopathology at Scripps. and he ¢(mlinucd Klivc al'filiallmt wilh ~h¢ Scripps Rc.~earch Foundallon even aflet hi.,; Rti~mcnl. Dr. Feldman was an ¢dilor or a number of prominent medical and scicnllfic jo,,rn~lso and se~ve,I as the Editor-in-Chief or the .~oumal of Immunology. I|e was al.~o a memtcr of ~hn Patholo3y study sections or the Nallorml Institul¢~ of Health and a ¢onst*lt.'m! Io a number or biomedi¢=l Rsearch programs. His schola~hlp and ability were acknnwlcdged by his election to Phi Eel,, Kappa, the $oci~y of Sigma Xi. and Alpha emma Alpha llonm" Medical Society, His ic~der~hlp and expcrli$© in immunopalhology were recognized and Ic¢lalmcd inl.'malionally. Dr. F©ldman joined Ihe Scientific Adviso~ Board of TE¢ Council for Tobacco Research in 1974. In 1991 be became Ihe Chairman of Ihe Sclcnlif;c .nard. a post which he held until his uncxpecl~ deatk in I~e summe~' Of 1995. Ilia contributions to the p¢omollon of basic biomedical scsca~-h aJe gratel'vlly a~knowl. edged by his fri~mds and cotle~gue$ on the Scientific Advlsm7 Board. past and seal. Thn Council for Tobacco Research-U.S.A., Inc. is horned to dedicate Ihls annual rcpo~ (o Dr. Feldman's memory.
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Joseph D. Feldman: A Personal Remembrance G. Barry Pierce. M.D. Dislingu|shcd Cenlennial Professor of Palholngy University of Colorado ! Icalth Sciences Center. Denver I am plca~cd to have the opponunily Io reminisce almut Joe because it me to express my gratilude for all he did for me years ago. I knnw I am nnl ah)n~ in my indebtedness In him ~causc he was a giving man. I hml a grcal nffecli~m fiw him guiding and leachin$ a ~mnger (m~ I le wasled m~ wnrd~; he said whal I~ Ihnughl. nnl necessarily whal ~u wznled Io hear. As a ~sull. y,u always knew wbere sl(~. Forlunalely. he had a g~d sense of hunmr which wc both needed n~n Wc met at tl~ ~panment of Palhnh~kT at tl~ Univcrsi~y .f Piltxl~urgh of 1955: he an Ass~iate Profe~or ~ Pathology. ¢ecently rctu~O fgom l~el. me a ~wly op~intcd and lowly fellow. Rank. race, creed, nt~e of these maltcfcd Io ~. What mallcrcd was how the game w~s plaid. At Ih¢ e~ of each ~xc~ise, whether h M squash. ¢or~cling a m~uscripI, or writing a g~anl applicalinn. ]~ cl~ing cornmeal was always Iho sa~: "All right." Bul il was Ihc fleeting senile or lack Ihcrcof Ihal Iold y~ i~ ~ffo~a~e met wilh his approval. In ~flccting ~ ~ fric~shlp, it w~ puz~ing to Imvc known e ~n so well aM so long ~d In owe so much In him, y¢l ~o know so lillle of his life, We k~w hc suffered from auloimmunc di~e. ~1 ~ ~ver complained a~ul his heallh, jusl igno~d h. J~ was a ~ivale men. ~oud and loving of his family, exlre~ly live in hi, profession and loyal ~o his ~cd, friends and values. ~1 you only knew this by o~allon, by fl~e ex~ple he seh J~ WLS palicnl; you I~m a lot a~ut a m~ who g~x over your manuscripl wJlh you. lie I=ught me Ihal nouns a~ nm ~p~scd Io mnd;~y nnnns, spill infinilivcs arc a sin, and hy~r~le is intolerable, l was IoM In ~=d ~try In learn ~onnmicnl u~c or word;. I was r~d of limerick, ~1 =~afcntly Ihcy didn't cmlnt, ~l Ihal limc didn't know J~ wa~ a Yale gnduate in ~gli~. and a ~i IIcta Kappa =t Ihat. To almost spill an infinitive, To J~ ~as highly O[ lesser men. ~]lo~ I~king ken, ~e neapdaed wax prohi~[ive. "kll righl." J~ was a sc~l~r. I{c Io~d ~]a.hip. II was manifest in all of hi~ a~ivhi¢s. As an example, he o~anlz~ Ihe sophom~c cou~c in palhnlogy so slu~nls learned a~ul disease by sludyln~ ¢~fully ediled case summaries in o ~min0r fo~aL ~¢n ~ did Ihc am~sy ~ ~amifully dissecl~ s~cime~ Io check Iheir deductions and dia[nose~ Ilc put Ihe onus ~ I~ stu~nls Io learn ~ only Ihe foals of disease bul ;he abilily !o use I~sc f~ts. using ~esso~ as reruees and ~ac~s. deep ¢o~em for Ihe slode.ls, lie undc~(~ Ihcir needs and ambid~s, bm his ¢ff~s on t~eir ~half were ~ten umc~gni~ ~usc of his low profile. Credit was nol im~flam Io him bul ~enls were, ~is ¢~ ~ a thing ef ~auty was cxching Io ~ pa~ or il. lie ms gc~mus wJlh his I;mc and laughl me Io ~ Ihe clcclron m]cro~o~. lie led me Ih~gh I~ [~slrili~s o~ Icaming In ~eak gl~s~ [nlvex and ~la;n able sect~ns fRe of "~hmutz" fo~ ¢xaminalion with the clcctr~ mic~osc~. then Ihcre wa~ Ihe [hrill of ~n[og~ing and knnwing ~cthing im~flanl Ihai a ~cnl ~y cl~c knew. ~c only role was d. i[ clcgamly and finish it wilh scholarship. ]oc wn.s fun to be an~nd, and so w~ Nomi. Donna nnd I recall with pleasure receiving an invilation to a p;irly al Ihe Fold:hans. In addilion Io good run. Iherc wouhl be real fnnd. It began snowing hen~ il7 the morning of Ihe party ,~nd by evening Ihe oily w~s paralyzed h)' il. To rcccl, tly .'uTived C..mndians the city was l!lo- ¢iously qnlel, the old trees and braises were co-=red by snow. which crunched under. fool, We walked the mile or so In Ihe Feldr..:,.,s and were warmly received and and ate and ate. ~V¢ were the only guests m~l there w~ food for at~ut 50. Nomi. all dial hard work. m be appreclaied unly by people who liked Io hear snow crunch. "i]lcre was Iovc in that family and il showed that nigln. Our palhs diverged in 1961: ]oc wt:M Io Scripps Inslitute. h~t we always reel :1 lice spring meetings and kept np with family affair~ and sclcnce, lie was s. pn~l Nmni ;rod Kl|kCd n hlt of 1he girls mid II~ir d~ings. Yent~ Inter he would nh'~a)'s a~ml his grandda.ghlcrs. I).ring his tenure Ill Scripps. J~ nssnmed the edilo~hip of file /ounal of Immunnlogy. Over n I~ year ~ri~l il nol su~risingly ~amc Ihc ~¢eml~nl puhli. carinn in Ihal field, largely ~ml~e Ihe ¢dilnr was fair. co~iderale, obliging ~nd of whom ~)uhl rcil~lc lairs tempo[able Io Ihose or lhe Pierces. Ilis effeclivr~ss ~ Chni~an of tl~ Scientific Advlsoff ~oard of ~ is well ~own Io ~1 or u~. will miss him. I. hlx rclime~cn~ Ihcre wa~ nnoiher ~livily of which I was nnawa~, lie and &lend fi~m;~l Ihc I lehrcw P~ I~an A~ialion of San Diego, IIs pu~sc was provkle Ioanx to newly a~ivcd Jewish immigrar, l~ He was head 0f Ihe I~ c~mil- lee which to date h~ made I~ loans. ~;nk of tee impel this made in the lives ~ ~oplc s~aning a new life in ~ new slmng¢ Whal ~ remarkable man. We rejoice in having known him.
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AI|SI"RACL~T TITLES AND AUTIIORS I. Cencer-Rebt,.¢l Sludles II
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hhW. JC ~l ................................................................................ 12 13
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14 t~
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16 I?
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Vo Rpldem|olo~y Yl. (;enelics VII. Immunnln~y & Adnplive Mechanisms 19
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VIII. Neur~inc|ence IX. Pharmacology X. l~ulmonnr? & Resplrnlor7 STslems 21
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XL Radials Xll. Virolal~y 22 XIII. Miscelluneous ~,~ lI.Ot (n~'~4 ~-1 .uolmel ~ F.3T~S OF,V.~IiO1T.C~D AMINO ACIDS. ~t,~,~;.. v. H. Xl;m.4*~ L. N. M;d~ Y. ¥. K~mk*l~. It. I |~ S~**~./. ?at~kmA;tl4 ¥. Ir~ m4 re#lgmws, H.N. .......................... D~V~I J3PMFJ~" Of A IIIGII.~J~JNr~'~ IUkOIOK3OIHATEO Li~ANO ~ IO(~F~T~FIC'A'F~ 0ir A NP~W MKllKXI. T~IU~a.T. *,,4 It*l~ J. ~4 .......................................................... 23
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Abstracts of Reports Following are abstracts of reports on new research acknowledging supl~o,1 from The Council that have appeared in scientific journals since publication of the 1994 Report. The name of the gt'anl recipient is in bold type. The abstracts are grouped under Ihese headings: I. Cancer-Related $1udles, II. Cardiovascular System, !11. Cell Biology. IV. Developmental Biology, V. Epidemiology, VI. Genetics, VIi. Immunology and Adaptive Mech•nism~, VIII. Neuro.'tcience. IX. Pharmacology, X. I~lmon~,/tad ResplraloP/Systems. XI. Radicals. XII. Virology. XIII. Miscellaneous. I. Cancer-Reln:ed Studies COMPLETE R F.CONSTITU~ON OF CONJUGAl'ION AND SUBSEQUENT DEGRADATION OF T! IE TUMOR SUPPRESSOR PROTEIN p53 BY PURIFIED COMPONENTS OF TIlE UBIQUITIN PROTEOLYTIC SYSTEM The wild-type In•or suppresso¢ prolein p$3 is • short.llved protein the! plays import•n! roles in regulation of eel[ cycle, differentiation and survival. Mutations Ihat inactivate or alter the tumor suppressor activity of Ih¢ protein seem Io be the most common genetic change in human cancer ~d are frequently associated with changes in its s~ability. The ublqt:itin system has been implicated in the degradation of p53 both in t.h,o and ~n vitrn. A mutant cell llne that ha~tx)rs • thermal•bile ubiq- uitin-activating enzyme. El, fails to degrade p53 st the ~o~permissive temperature. $1udies in cell-free cxtracls have shown that covalent attachment of ub|quhin to the pfoteln requires the three conjugallng enzymes: El, • novel species of ubiqultin.ear. rler protein (oblquitin-conjupling enzyme; UBC),E2.FI, and an ubiquitin-pmlcln llgas¢. E3. Recognition of p53 by Ihc llpse is facilitated by form•lion of,, complex between the protein and the human pap-'-]lomavlru$ (HPV) oncoprolein Therefore, the llgase has been designated E6-associaled pmtclo (F_J~-AP). Ilowever0 these in t'#ro studies have not demonstraled that Ihe conjugates serve as essenlial intermediates in the pmlcolytlc process. In fact, in many cases, conjegallon or ub;q. uitln to the larger protein does hal signal its degradation. Thus, il is esscnlial Io demonstrate that p53-ubiqnitin addUClS serve ,,s essential proteolydc intermediates and ate fecognlzed and degraded by the 265 ,-,rote•st complex, the pmleolyllc arm of the ublqultin pathway. In this sludy, we de:.-.onslrate thai conjugates of p$3 goner. sled in the presence of purified El, E2, E6-I,P, E6, ubiquitin and ATP, a~ specifi. cally focognized by the 265 protease complex and degraded. In con•tat ed p53 remains stable. The ability to reconstitute Ihe system from purified coml~- nears will enable detailed analysis of Ihe recognition pro~ess and the structural motifs involved in targeting the protein for degradaxloo. 25
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C) Shkedy. D.. Gon~n. ll.. Bercovich. B.. and Ciechanover, A. FEBS Letters 348:!26-130, IC~4. Other supporl: Uniled States.Israel I];national Science Foundation. German-Israeli Foundmloo fo~ Scientif'¢ R~'~rch and Devchvmcnl, Israeli Ac~idemy or Sclcnces and Ilumanit|es, Mo~samo. Inc., Fo~mdatinn for I~'omoti~m of ReJ;earch in the Techni~m end a Research Fund administered hy Ihe vice prcsidcn! of the Tcchnion for Re.,,can:h. From Ihe Dope,lineal of Biochcmislry. Rappaporl Iw;litule far Research in Medical Sciences, Technlon-lsracl In,~titulc uf Technology. I I;tira, Israel. TIlE ROLES OF TUMOR SUPPRESSORS pRl~ AND p53 IN CELL PROLIFERATION AND CANCER Cell natal:ors are normally commllcd via a holancc bclween cell prolil'cralion, diffcrenfialion, and apoplnsis in a multicellular organism. A disturbance in thL.; bal- ance may lead to an uncontrollable cell proliferation and rcsull in malignancy. Failure to wilhdraw from the cell cycle and term;nelly differentiate may cause organ dysfunction and developmental abnormality. Moreover the rename of each cell is carefully monitored to mainlaln ils inlcgrily. Gcnnmic lest,as in a cell need let ~ repaired before DNA rcplicalinn and cell division uccur. A c¢11 c¢)nlalning nnre- pairable genomi¢ Ieskms should not be --,llnwed m proliferate and must ~c orderly disposed by programmed cell death apOplo~is. Io prcvenl, oncogenesis. Each of the cell fates is mediated by sequentially organized biol~¢ical pathways at the mo|cculnr level. The conlml of cell fates is a well-com'dinaled performance starred by key players in cell cycle regulalion. In rectal years, importanl advances have been achieved in the dissection orccll cycle conlrol, diffcrendadon, and apopto~is regula- llon. lncreas|ng evidence patois Io Ihe involvcmcnl of bolh the retinoblastoma gent product (pRb) and p53, two well-characl©rized lumnr suppressors, in multiple path. ways to coordinate these essential processes, Sang. N.. Baldi. A., and Giordano. A. Molceu|ar and Cellular Differentiation 3(I):1-29, 1995. O~her supporU N|lional Instilutes of Health and the W. W. Smilh Ch~trilablc Trust. From Ihe Institute fo¢ Cancer Research and Molecular Medicine. Jefferson Cancer Inslilute. and Departments of Microbiology/Immunology and Pathology. Thomas $efferso~ Unlvenily. Philadelphia. PA. CIIROMOSOMAL MAPPING OF MEMI~ERS OF'rilE CDC2 FAMILY OF PROTEIN KINASES. CDK3, CDK6. PISSLRE. AND FITALRE. AND A CDK INIllBITOR, P27TM. TO REGIONS INVOLVED IN IIUMAN CANCER OrdeTly progmssio~ through the cell c~cle requires scqucntia| actlvalion and inactivation of cyclin-depcndent kinascs (cdks). ThLs is achieved in purl through Ihc 26 nssceiatlon of cdks with positive regulalon called cy~:llns and inaclivalicm of cdk complexes by a ~pidly growing num~r of cyclin<dk ;nhib;Io~. Recently. the role of cell cycle comrol proleins ~lh as ~ima~ effeclors and as medialtws of tumo~gcncsis has ~comc a ~bject ~ inc~sed inle~sl. II~ ~ ~n tl~ chin. niosomal mapping of Iwn cd~. ~k3 and cdk6 I~ putative cdks. PISSLRE and PITALR~ and o~ cyclin-dc~ndem kin~ i~ibil0r, p27. to ¢hm~somal which may ~ altered in human lumo~ a~ exami~ their ~¢~iMe involvem~l in some of these mali~nancles. In panlcular, two o~ I~e klnas~ cdk3 a~ PISSLRE ~d PITALRU. tl~e cdc2-relaled kin~s ~ntly cloned by el. map Io mgi~s p~vi~sly sh~n Io cxhibil loss or heler~ygosily in ~e~ a~ other tumor. Bullrlch. F.. Mac~chlan. T. K.. Sang. N.. D~ck. T.. Vemncse. M. L. Allen. S. L. ~iorazzi. N.. KoK. A.. limber. K~ ~. C. M.. and Giordano, Cancer Research 55:11 ~- 1205. Ma~h 15. I~'~. Olhcr suppori: The Soclely of ~e Mem,-e~.d Sioan-Ketleflng Cancer Center. From the ~efferson Cancer Instltule anx; Depavlmen~ of Mictobiolo~y and Immunology, Thomas Jefferson University, Philadelphia, PA, Noglh Shore Unlveraity Ilospilal Comell University Medical College. Manhass¢l, NY, Program in Mole~l~r Biology, SloawK~leri,g I~filule. Mem~al C~mcer Center. New York, A STRUCTURAL AND KINETIC COMPARISON OF PROTO-ONCOGENIC AND ONCOGFJqlC NF.U ! IOI.O-RECEPTORS EXPRESSED IN INSECT CELLS The proto-oncogenic and oncogenlc forms of the rat oe,, receptors welt expressed in Ihe baculovims system IO chararlerize Ihclr slnJflural and enzymatic differences. Tl~c cphopes of their extracellular dumains, lheir molecular weights, and icin~se ~clivilies Were similar to rat neu receptors exp~Jsed in fibmblasts. The recep. tars were parHnlly purified using a phospho-altarose column and wee analyzed Io compare kinetic parameters using ATP as • ~lbslrate. The oncoganic form of Rcepeor showed a significant increase ;n V,, (56%) over the proto-oncogcnk: from. Slructaral analysis of these proteins using sucrose gradients showed the oncogcnle mcel~ors to have • 62.6% increase in aggregated receptors when ¢omF:med to the pmto.oncogcnlc receptors. These studies arc Ihe f;rs~ to link enzymatic :ctivalion and Ihc physical form of the receplO¢ using isol;~ted reccplo¢ species. LeVee, C. M.. Myers. J. N., Dongall. W. C, Qian. X., and Greene, M. L Receptor .'1:293-309. 1993. Other s~,ppor~: Natlonnl lnstilulcs of II~hh and the Luclile Mnrkey C~aritnble From Ih¢ Departments of Pathology, Center for Receptor Biology, and Biuchemlstrj and Biophysics. and ]nhnsnn Fmtndatlon, University of Pcnnsylv0nia. 27
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C) INTERMOLECULAR ASSOCIATION AND TRANS-PI IOSPIIORYLATION OF DIFFERENTNEU.KINASE FORMS PERMIT SI I2-DEPENDENT SIGNALING AND ONCOGENIC TRANSFORMATION ~.The nr. oncogene encodes a 185 kDa receplor tyrosine kina~. A single point mutation (VII664--.>GIo) within the pigs~ transmumhrane region rcSUllS in higher efficiency of receptor dimerizalon, constitutive activity of tyrosine kinase and cellu- lar transformation. The oucogonle polenlial of Ibis mulalcd form of" Tneu) tan be inaclivated by alia.directed alteratlon of a lysin¢ residue in Ihe con- served Calalyfle domain. In Ih|s report, we have utilixed the physical and functional interaction of a full-length klnase-deficiun! ncu prolcln (T757) and lruncalcd kinar.e- active Tncu ~'orms Io delen~in¢ crilical proleln domains for Tnuu oligomerization and the resoltanl biological consequences. Analysis o1" various truncalcd Tncu mutant= ¢oflfirmed thai lhe Iran.~wcmhrane region was crucial for p lg.'i directly.arian. Rcccplor associaliorl facililales inlermolccular phnsph.ryl;,li.n of kin~¢-def~clenl mulanl "1"7~7 by Iruncalcd kinase.aClive plX.(; Pr(l|eins. und the Imns-plu~phorylaled kinasc-defic|cnl T757 we.,; able Io associate in t.iln) with proleins COhlaining SII2 domains. R¢ceptor.receplor iotcr,,¢lions resulted in enhanced signal tranxduclion polenllal and Iransrormalion or cell-It.ca co-exprcs.~ing diffcrem =mukinn.~¢ I'orms. l~ese studies ¢mpha.si~,.e u novel feature of proteln-protcin inleracllon and lira J'unc. lionel si~nificanc¢ of p 18:~ dimerization, inlemmlecular phospborylatlon and signal- in= which may result in cellular Iransrormatinn. Qian. X.. Dnu~all. W. C.. Fui. Z.. and (;eeene. M. !. Oncogene 10:211-219. 1995. Other support: Lucille Markey Charilahl¢ Trust. From the Ccnler for Receptor Biology, Department or Pathology and Laboratory Medicine. Department or Physiology, Universlty or Pennsylvania. School of Mndicine. Philadelphia. PURIFICATION AND CIIARACTERIZATION OF TIlE BCL-2 PROTEIN The oncogcne product hal-2 functions as a repres.,~or or programmed cell death and is a 26-kDa protcln with a single prcdiclcd Iransmemhmne .S~men! Iocaled al the carboxyl terminus. The bcl.2 protein ~ems to function in different subeellular ¢omparlmems, as evidenced by scv~al biochemical and uhraslmctural studies. The present sludy was ~o~ed to pu~il'y bd-2 protein in signil~canl q=,anlides neces- sar~ for sllllclural and I'anelionll Sludies. Foe this purpose. Ihe b¢1-2 gent was over- " expressed in either baculovirus system or lymphncytes. Initially, allcmpls were undertaken to purify bcl-2 proleln using ¢nnvcnlional enethnds such as ion exchange or gel fihralion chmmalography. During these puriflcalinn attempls we determined that b¢1o2 protein is highly bydrophobic and prone In aggregation as mighl h¢ expected for an inlegral membrane Ira/gin. FJy ion exchange and gel Iihr~tion chm- 28 malography, this protein could he partially I~Jri~,:cl. In order Io purify Ix:l-2 IO eat homogeneity and avoid the aggregation p=oblcm, wc p~cpa~d immu~o=ffinlty columns using a monoclonal antibody dcvelop~l against a synthetic peplld¢ chosen from residues 61o76 of the amino acid sequela of human Ix:l-2. The Inlibody was either coupled In CNBr-activaled Scpharose 4B or ¢~oss-link©d into protein A-Sephamse by dimcthylp;mel|midate dihydroeblofide. Cellul~ ¢xtracl cquivalcnl In 10' hel-2-ovemxpre,~slng insect celts or lymphoeyles was apptled Io immunoelfinity columns. Approxim~lely ~00 IJ.= purified b:l.2 pn~ein could be recovered as esli- mated b)" silver slaining and immun0blollin8. FurthermoR. purified Ix:l-2 protein was el¢clmpo~Ind into Pre-B lymphatics whk:b do nol express Ihls Fro~eln in suf- ficienl qo,'mlily Io delay Ihe on~;el of ¢lu¢o¢orti¢oid.induced apoplosis. Following ¢lcctmpo~tlon of homogeneously pea Ix:l-2 Protein. the cells were found to cell sun~ival in respon~ to glucocorlleoids. Ilnld~r, $., ]cna, N.. DuBols, G. C.. Tnkuyamn. $.. Reed, J. C., Fo. S. S.. and Croce.C.M. Archives or Biochcmist~ and Biophysics ~15(2):483488o 1994. Other sup~mrl: Nnlionnl C.nccr lnslilulc. 'From the Dcpanmcnl or Microblology sad Immunology, JelTer~on Cancer lnslilUle, Dep.',rlme~ll of Ph0rmacology, Thomas ]efrerson Universily, F'hlh'Melphla. I..= folio Cancer Research Fmmdalion. I.~ Julia. BCL,-2 RELIEVES OEOXYADENYLATE STRESS AND SUPPRESSES APOPTOSIS IN PRE-B LEUKEMIA CELLS The inl]ucnce o1" bclo2 aclivily on 2'-d¢oxyadconsine-induccd apop(asis was investigated in 69"/human pre*B leukemia cells stably tfans|ectcd with exp~ssion plasmid pIIcBo-BCL-2¢z (697/BCL2 cells). Apoplosis was induced by the 2% deoxyadcnosinc analogue,2-chloro-~.'-deoxyadenosin¢ (CI-dA). with the con~'enlra. llon for apop{asis in one-half or Ibe cells at 24 hours (LD,.) being I0 p.M rot 697 cells and 120 p.M for 697/Be] 2 cells. There was u strong positive con, elation I~ween CI-dATP levels and apoplollc index (coefflcicm or determination, r~ == 0.95; P = 0.027). When 697 cell and 697Rcl 2 cell lines wcm ImaZcd with 5 p.M Cl-dA. CI-dATP did not significandy accumulate in the loller. The CI-dATP/dATP main wa~ 0.03 in ClodA treated 697/Bcl 2 cells bul nearly 6 in Ire,tied 697 cell'=. Bcl 2 oveq~ro. doction also suppressed the accumulation or dAMP. dADP and dATP in cells exposed to 2'-deoxyadcooslne in the presence o1" pemostatln to abrogate the pro- haunted inve~slon oF ATPIdATP pools associated with 2'.dcoxyndenosine exposure. T~cse' results suggest thai one consequence of Ix:l-2 activity is suppression of 2'- dcoxyadenosine phospho~lation and elevation in the apuplollc tercel ceils. Relier from d,'oxyadenylale stress imbalances implies a novel upstream site of bcl-2 29
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O Gan. X,, Knurls(n, T. B.. lhrah;m. M. Cell Death ~d Diffcrenfialion 2:69-7R. ]amlar~ Other su~: National fnst ilule of Child I Icalth and I hlman ~vclnpmcnt. From Ihe ~panm~nl or Palhology. Analnmy and Cell ~iology. ]cffcrs.n M~ic~l Colla~a. Philadelphia. PA. and ~pa~mcm oF ~icroh]ology and Immunology, Jefferson Ca~cr lnsfi~ule. ~;l~dclphia. LYMPIIOMAGENESIS IN THE SCID-I IU MOUSE INVOLVES ABUNDANT PRODUCTION OF I IUMAN INTERLEUKIN- I(I Both human (hu) and viral (v) interleukin-1O (IL-10) =ppcar In he iml~nan! cofaclors in the survival and erowth or lymphohlasB:id cell line-.~ infected with Epsleln-Ban' vires (EBVJ. When mice wilh severn combined immune dcficlency (SCID) ~ injected with human p~:fiphcral hhn~l lymphncyt¢~ (I'BIJ from m~ml i~ividuals w~ are scro~silivc h~r EIIV. Ihc majurhy of hu-I'llL-SC~l) mica will develop an EBV-ass~ialcd lymp~proli~cralive discmc (EIIV-I.PD) of human ~11 origin, not unlik~ snmc ~cs .f EflV.IJ'I) Iha( a( sccn in raised individuals. ~e role of hull.-I0 ~x vlL-I0 I, Ihis chh~ric ,m.sc m~lcl ~BV-I.I'D is unknown. In the pre~¢nl slmly, wc ~how Ihal hu-I'lll.-~C~D mice dcvcl~ EBV-LPD have signi~¢anl elcvalinn of ~tum hulL.H) levels compaRd wi~h mi~ Ihal do n~ dev¢l~ EBV-LPD (P - .~5). vlL-IO wig undeleclnhle ~imals. ~e EBV tumor ,mplm cx~ t~nscripl for hulL-IO and hulL-H) Reap- Int. cx~ss hulL-IO prolcin by immunohtsl~hemical slulnin¢, and show binding of ~b~anl (r) hul~10. In dtro analysis of I~ funclional c~scqucncm of~ulL-IO ~in~ Io IL-10 rc~pl~ ~ fresh EBV" lumor cells ~ows Ihat 10 can prcvenl ffog~mm~ cell dealh ~ well as pmmole proli~cralion and ~n do at conccm~tlons of ~IL.IO found in viva. ~us. hulL-In pr~ucli~ by lumor cells mW conld~le dirtily to I~ir mallgnanl ~lgrowth in I~ hu-PBL- S~D mouse by Iwo lul~rioe m~hanisms: p~vcnlion of prog~mmed ¢¢11 dcalh ~d prolife~don. ~c implioa(;o~ of such findings wilh regard to EBV-LPD hum~s is di~ussed. Bli~chl. R. A.. Ross. M. ~. Tan. J. C. ~ou. C.-C. Sullivan. L. llaldar. Mmn¢. M.. Selden. M. V.. N~la. S. K.. Skier. L. C~. C M.. and C~ligiud. M. Other sup~: Hati~l lnsdlulcs 0[ Hcahh. National Cancer lnSlilule. Amcrican Canc~ ~icly. and ~he Coleman ~ukcmia Rcs~rch Fund. Fr~ Ihc Depanmenls of Medici,. Molecular Mcdicin¢. Molecular Imnmnology. and Physiolo~y. Rosw¢ll Park Cancer Inslilule. Buffalo. NY. Schering Plough R~ch lmtimtc. Kenit~nh. NJ. ~mcnt of M~mhlology a~ Jefferson Cancer lnstilulc. ~iladelphia. PA and Ihc Dcparlmcnt of Pathology. B~b~m & Women's II~ild. Ila~rd M~ical Sch~l. Doslon. 3O A GERMLINE INSERTION IN TIIETUBEROUS SCLEROSIS (T.~r2) GENE GIVES RISE TO TIlE EK F.R RAT MODEL OF DOMINANTLY INIIERITED CANCER The Ek~r rat mendelian dmninnnl predispc~ilion Io a specific cancer in -n experlmcnl,d animal. W'e have previously established a new conserved I|nk--,ge group on rat chromosome 10q and human chromosome 16pI.'!.3, and shown Ihat Ih© Eker mutation is lightly linked In the tuhemns sclerosis (Tsr2) geu¢. W¢ now dcscdbe a ~ennline mulallon in Ih~ gone ¢ncodlng T.Tr2 caused by Ihe ins~'lin~ of ~n apl~oxlmately 5 ~ilobar~ DNA fragmenl in Ihc Ekcr rat. rc~ullin8 in abe~anl RNA expression from Ihe mulant allele. The phenolype of luberous sclerosis in humans differs from that of Ihe Eker rat. except for the occurenc¢ of renal turnouts. The Eker rat may ther~'for~" I~ovld¢ insights into species-specific differences in tumourigenesis and/or phenotypc- specific Kob.~yashl. T.. N'~lure Genetics 9:'/0.75. January 1995. Olhcr suplmn: Minlslry of ~lncadon. Science. nnd Cult,m of Jnpan.'and Ihe Vehicle Racing Commemnraliv¢ Foundation of Jap.'m. From the Dcp::muent of Experimenlal Palholngy. Cnncar Institute. Tokyo. Japan. TI IE EKER RAT. A MODEL OF DOMINANTLY INHERCFED CANCER SYNDROME A class Of cancer ~cnes. called the lumor suppresso¢ Befits o¢ -'.nlioncogcncs. h~ ~cn ~vealed ~ the s~udy of h~cdila~ human ~ AIIh~¢h t~sa • m recessive in ~co~enesis. I~y tenor hcl~mzy~ou~ ca~ hilly su~plibla to pa~icular ¢ance~ a~ so ~p~ar in ~dig~es ~s ~mioanlly ~ri~cd diso~ Such a dominantly inhedled p~dls~dt~n w~ d~d ~ ~rs by ~er. Iileo. O.. Kobayashi. T.. Mhanl. H.. Kobayashi. ~. ~u~. Y.. Tsuchlya. H.. Kikuchl. Y.. Nishi~wa. M.. a~ Ili~yoma. Y. T~nsplan~ali~ P~eedings 27(2):1529-1531. April OIh~ sup~: Minisl~ at ~ucation. Science and Cullu~ From Ihe ~pa~n~ o~ Ex~ri~nlol Palhnlogy. ~ncer Insdlule. Tokyo. ]ep~. TIlE PREDISPOSING GENE OFTIIE EKER RAT INIIERITED CANCER SYNDROME IS TIGI ITLY LINKED TO THE TUBEROUS SCLEROSIS (TSC2) GENE The Ekcr ral is a promlsing -'.nimal model of" cancer predisposition syndromes. 31
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In thi~ study, using 129 backcms.~ animals, wc have e.~tnbli.~hcd a new conserved linkage 8mup on ral 10q ~ human 1~13.3 wh~ehy ~he I~kcr mul~lion was Io ~ d~hlly linked Io Ihe tu~rous sclerosis ~) ~coe. ~ds will ~ the fi~l step ~ow~ ~he ~sklan~ cloning ~d idemifi~li~ o~ Ihc pmdis~ing Ekcr mu~ion. ilino. ~.. K~ba~ashi. T.. Tsuchiya. II.. Kikuchi. Y.. Knhayash;. ~.. Mhani. II.. and ! li~yama. Y. Biochemical and Biophysical Research Communic~lions 20~(2):1392- Scplcm~r 15. 19~4. Olher supeR: Minls~ o~ Educnlion. Scic~c, end C, Ilu~ o~ From Ih¢ Dc~mant o~ Ex~dm~m=l P~tholo~y. C~nce~ In~t~l.lc. Tokyo. ~npon. -- EARLY DETECTION OF KNUDSON'$ TWO-IIIT.~ IN I'IH:.NEOPI.A.~TIC RENAL CELLS OF TIlE EKER RAT MODI[L I|Y TIlE MICRODISSECTION PR~EDURE ~(s. At die histolo¢iol level. ~cod cell ca~in~as deveh~ d~mu[h muhlple stages From e~ly preneoplaslic I~ions (t.R., p~nntypically :hcrcd tubule) to ~cnomas. ~e p~cviously ~ned Ih~ ion;zin~ ~i:ti~ induc~ additional tumors (lace eric- names and cattiness) in a linc~ dose.resist Rlationship ~nd that loss oF ¢rozy~hy (LOll) at chromoso~ 10. where the p~is~sing tu~rous sclerosis {Tar2) ~cne is I~allzed, was found in ~he Rnal cell carcinomas which devel~d from hy~d FI eels cl~ing ~hc Ekcr mutation, indicltin~ tha~ in hctcrox.y~s two evcms (one inhcHlcd. ~c s~atic) ~ ~ccs~a~ m pr~ucc at I~sl large ~d carci~mas. ~is SlUdy w~ designed Io examine LOll in (~ earlicsl lic lesions, using a laser mtcrodisseclion W~cdure. We could ~cura~cly single altered ~nal Inhales ~t o~ frcc~-dried scclions and clearly dclccled LOI 4 of 19 ~hered tubules (21%). ~is is t~ fi~t dcmnn~t~lion of (.OH in single tubules. Our ~¢~nl Rsulls s~ Ihe Iheo~ of a second, somallc mmafi~ hil} as ~le-limiling si~p or Rnal ca~inn~enesi¢ in Ihe ~¢r rat m~el or ~ominenlly inhcriled cancer and Ihe lumor su~m¢~r ~lu~ o~ Ih¢ ~r2 gent ~unclJon. Ku~, Y.. Kli~k. R. Kiku~;. Y...~nn;~h, P.. end Illno. O. C~nc~ Research 55:9~9-9~. Ma~h I. t995. Olher suppo~: Inlcrnalion:l Uninn Against Cancer and Minlslry or Science, and Cuhu~ of Japan. From Ihc Department o~ E~rimcm~l ~holo~y, Cancer Inslimt¢. Tokyo. ~ Dcp~nl or Cell Pal~loey. G~en Cancer Re~h Conic. Ge~ny. 32 NAO(P|I I:QU INONE OXIDOREOUCTASE, (DT.DIAPilORASE): EXPRESSION, REGULATION. AND ROLE IN CANCER NAD(P)ll'.quinone oxidm'eductasc, (DT-diaphomse or NQO,) is a thai promotes obl;~aton] two-eleclron ¢tduction ofquinones, preventing their partici- Pation in rcdox cycling, as|dative slress, and ncoplasla. NQO, is ublquilously expressed. Ilowever. a large amount or varlal~OO in NQO, gent exprcsslo~ was noliccd among various human tissues. NQO, 8ene is upregulalcd in livers or hepato- carcinoma patients, and its rxpr~sinn is induced in RslXmse to a varlet7 of com- pounds, including plan,mr aromatic hydrocarbon.~ pl~enollc ~m|oxidanl~chembpm- I~clors. lumor promolcrs, and hydrogen peroxide. Dclcl;on mulagenesis in Ihe gent promoter, idcmlflcd several cis.elements including andoxidant r~sponsc tie. mere (ARE), xenohio~ic response element, and AP2 clement. ~hich regulate the cxprcsslnn and indoction of the NQO pent. Amo~ these DNA ©tcmcnts. ARE |s the most mlmrtan! (-ix-element required for high h;~al expression of the N(~K~ |one in lumor ¢i~suc.~. as compared to the normal lissucs of Ih¢ some o~igin, and for inrhiclinn in reslXmSc Io xcnohlntics and ~nlioxidaMs. Nucleolidc sequence .r ,he ARI~. hnlienled presence of three API/API.llke elemenls and n GCA Mutatlno;d a~:dysis indic;~tcd a ~.'tpdr~ncnt of two APtlAPl-llke elements as inverse rcl~at~ .'. the imcrvnl of three base pairs rot. ihe ARE i¢liVily. The GCA box in the ARE was ~equlred for optimum ba.~l and induced expression. ARE is a novel rLr-elcmenl bcca.sc a sinplc APIIAPI-likc ¢lemem did not sllmolnte ~ene express;on in rc.-,~p~n.,ce tO aenobintics end nnl|oxident~. ~Jand shlh and suporshlrt a~says identified Jan. Fa~c, .~nd novel proteins in the hARE-oucle-',r protein com. pleats Ilml medialc regulation o~lh¢ NQO, gent expresslrm. A hyfmthetlcal model to study Ifi¢ mech,'mism of" signal Iransdu~tion f"rom xenohiolics end anfioxidams to Ihe ARE res.hing into acllvation or Ihe NQO, and mher phase II enzyme |¢ncs is dcscritcd. Evidence is presented to dcmonslmtc tl~e role of the NQO, in preventing tile binding of P45(I rcduc~ase-~cdvat~ henzo(o)py~¢~¢ qulnoncs (scmiquioones} to Ihe DNA. presumably by cllminating their fo~nalion. Joseph. P.. Xie. T.. Xu, Y,, and Jalswal, A. K. Oncology Research 6(10/I I):525-$32, 1994. Other support: Natinnal Inslltules of i|eahh. From Ihc Dcparlmem of" Pharmacology. Fox Ch,,s¢ Cancer Cenlcr. Philaddphiao PA. SUSCEI'q'IBIUTY TO TUMORS INDUCED IN MIC~ BY ETIIYLNITROSOUREA t$ INDEPENDENt" OF RETINOBLASTOMA GENE DOSAGE The Rtinohla,~mm~ gen¢ (RB) is a classical tumor suppressor. Sevml slndks have shown th~t RB dosage is |mporlanl in determining biological effecls. To explo~'e the effect of RB dosage on suscep(ibilit7 to cancer, Ihm¢ groups C57BU6 mice. each of which ¢xpscsscs a differem amount of Rb I~otein from one. Iwo. or three alleles, were treated al pOSthOle| day 12 with a sln~le 60-m~/kg body
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weight i.p. do~ or the DNA-alkylaling agent N-ethyl-N'-nltromurca (ENU}. Mice heterozygous For Ihe RB [enc developed cl'mracteristic pituitary tumors with nearly complete penelrancc, whether or hal they wcrc Ireatcd with ENU. Tumors initiated earlier or progressed more rapidly, however, in ENU-lreatcd mice. Furlhermorc, ahhough mice Ircated with ENU had a higher incidence of several nonpiluitary tumors compared with untreated controls, no signific--,m differences in the incidence of these tumors were I'ound between wild-type mice (mR/]'). mice carrying only one normal RR allele and deficient in Rb protein expression (mRB"), nnd mice over. expressing Rh prulcln from two nnn~al murine RII alleles ;anti a human RII Iransgcne ImRU", h~lJ" ). 1hose studies undew, core Ihc ti.~suc and mcchanktic speclflcily oF tumor predisposition caused by an ieherlted 50% reduction in RB dosage -',nd indi- cate that most ENU-htduced tumors occur independent of RB inaclivulinn. Nonetheless, they SUl~[CSl that certain imint mutatinns induced hy ENU may p:mici- pale in Ihe sequence of molecular steps involved in progression of tumor-prone, deficient cells to the tully ma)i~nam state. Riley. D. L, Lai, C.-C., Chan~. (~..Y., ]on~s, 0.. Lee, F. Y.-ll. P., and Lee, W..ll. Cancer Research 54:60~7-6101, Decemlx:r I. 19~4. Other suppml: National Institutes or | leahh. From the Institute or Biolenh~ology. Center for Molecular Medieinc. University of Texas Ileahh Science Center. San Antonio. TX. THE ONCOGENE qin CODES FOR A TRANSCRIFFIONAL REPRF, SSOR The relrovjral oncogene qin codes for a prmein what belongs to the winged hcllx family or tmnscrlptional regulators. The Qin prolein is localized in the nucleus and hinds to the same DNA consensus sequence zs rat brain factor I (BF-I). Cellular OJn shows greater affinity Io ONA then docs viral Qin. Alone or fused to the ONA-hind- in~ domain of the yeast GAL4 prolein, bush Qin proteins act as transcriptional rcpressors. The m'~jor tr~nseriptlonal repression domain maps to the region of amino acids 252-~95 of viral Qin, L|, J., C:hao~, II. W., Lzi, E., Parker, E. J.. and Yogi. P. K. Cancer Research 5S:$$40-~544. December I, 1995. Other xupl~rl: U.$. Ilcalth $crvlcc. From the Departmenl nf Molecular and Expcrlmcntal Medicine. The Scripps Research Instltule. L~ Julia. CA. Memorial Slonn-Kcltcring Cancer Center. New York. NY, and Dcparlmenl oF Cell and M.leeular Biolopy. IJnivershy ~f St. Andrews. St, Andrews. UK. 34 OPPOSING A C~'IONS OF C-ETS/PU.I AND C-MI'B PROTOONCOGENE PRODUCTS IN REGULATING THE MACROPIIAOE-SPECIF1C PROMOTERS OFTIIE ilUMAN AND MOUSE COLONY-STIMULATING FACTOR-I RECEIrI'OR (C.FM.T~ GENES The receptor for macrophage colony stimulating faclor (CSF-I). the r-finn product, is n kcy dclcrminant in the differentiation of monocyllc phai~ocyles. Dissection of lhe human and manse ¢.Jm~ pmximal pmmolcrs rcve,,led Ol~O~in= roles for n,clear prnlnonco~enes in Ihe Imoscripllonal rc;ulat|mt of Ihil [~ene. On the noc haml, t'.r~x.l, r-~t.r.2, attd Ihe m~roplmgc.specifi¢ I'aclO¢ PU.I, h¢=l teal Ilu., fucl,r I'1.~^.1. o'rmx-acllvaled Ihe r-~nx proximal promoter. On Ih© other hand repre.~d proximal promoler acllvily in macroph=ges and blocked the ~ellon of et.r-I aml r-e#s-2, llnsal r-J~ns pmmot©r ;~.:'.i'~'ily was -',lmma undclcclable in the leukacmla line. which expressed hish levels of ¢.-m.t'b. but W~LS activaled as cells dif- fcrcnlialcd io rcslmnse Io leukemia inhibitory f=clor and expressed c.~mx mRNA. The reprcssor I'nnclion of c-m.vh depended on Ihe CO011.1enninal domain of Ihe pro- rein. Wo propose Ihal cls-f.'tclor~ are o~.'¢s.~ary for Ihe lissee.l~slrieled expression of c-J~,s and that e-myh acls to on,sure correct temporal expression or e.~n~ duriu| myeloid di ffcrenllaliOn. Rcddy. M. A.. 'Yang. B,.-S.. Yu~. X,, Bnmetl. C. ,L K., Rots, I. l, Sweet, hi. IhJmc. D. A., anti Ostrowskl. M. C. .louma| uf F.xperhnental Medicine 180:.2.'!0q-~..~ 19, t~:ccmber 1~4, Olher ~UplUtrl: Nalitmal I leallh sad Motile;:! Rescurch Council or At~tmlia nile| (~ucem;land C,'mcer Fund, From Ihe Dcpnamcnl tff Micmbiology. ~,~:.~c Un;versily Mmlical Center. Durham, NC, and Center for Molecular Biology and Biotcchnolotzy, University of Qtmen.~lnud. Queensland. Auslralla. INSULIN-LIKE GROWTII FACTOR !1 REGULATION OFGENB EXPRESSION IN RAT AND IIUMAN HEPATOM^S Insulin-like growth factor II (IGF I1) regulated llssoe-specil'm gene eXl~ssion in hepatoma cell lines, but had no effect on eapress~on orlissue.specif'¢ genes in pri- mary cullures o1" El4 and newborn ral liv:'¢ cells depleted or erylhmid cells. No ch~mge w~ observed in these primary cultures with ~spect to ~,-felol~'oleln (e-F'P). albumin, cylokeralin 19 (CKIg)o ~pBlulamyllranspeplidase (GOT). and IGF II recap tars. Two well-differcntlatcd hep-~tomas, Hel~32 and FTO-2B, end a poorly dilTcren. lialed hcpnlnma. I I.A,C~. did nnl show inerc~.'~d pmllfcmtlon in Ihe I~enoe or IGF II. yet ~hnwed gent espr¢.~ion changes in ~esponse tn IGF IL In Hei, A32 tells. IGF II incre~ed Mhumtn mRNA levels end resulted in a shift team ¢lu~ler~ of c¢11~ po~ilive to I00% of Ihe ccll~ expressing immunohlsloehemtcally dele¢lahle albumin. The Iranxcriptlon Factor IINF-3p mRNA and prntelu levels o1' Ihe bide ducl marl~rs. CKI9 and GOT. ~.,re nl.~ increased in Ihc t,msence of IGF II. Other' l~cn~ levied wt:t~ trot all'coted, including ,t I.m~llttypttln. mtd two liver-specific tmn~crlplltm [uc-
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tars. II~F.4 and HNF-3a, In FTO.21~ cells. IGF II increased Ihe expression of albu- m~n. CK 19. and GGT. wilhout accompanying changes in albumin nml ~GT mRNAs. In II,A,C~ cells. IGF II ~,ccd CKI9 and OC.3 pn)Icin levels aud GGT. Iransrt~in. and IINF-3~ mRNAs. ~ effccls o~ IGF II on II.A C~ c~lls were bl~ked in ~hc prtsence or an and-tel IGF II rcceplor anli~,ly. ~c cnnclu(~ lhal IGF II affccls llssuc.s~ci~c ~cnc cxpmssion or bepalnmas and qualilalivc and quan- d~aE~c a~cls of ~ts in~u~nc~ on lhc h~p0tomns is dc~,dcnl on lhcir dcg~cc of f~renfialion. Olhcr supp~: American Cancer ~iclx. Nalion~l In~lh,re; o~ licallh, and Richa~ ~olin F~ndafi~. From Ih~ Albafl ~insl~in CollcGc of Mcdicino. D~parimcnls of Mnlccnl~r Pha~acoln~ and ~icroh;olo~ and Immunol,~. Omnx. TIlE PROTO-ONCOGENE/TRANSLATION FACTOR clF4E: A SURVEY OF ITS EXPRF-_SSION IN BREASTCARCINOMAS The eukaryolic Iranslalion initlallon faclnr elF-4E binds to the cap sl~cturc oF mRNAs as one componenl of Ibe c|F4 Iran,.lalion initiation complex, which mcdi- .ales Ihe rccn~ilmenl of mRNA Io Ibe ribosomes. Overexpressi,m of clF4E can rcsuh m oncogenic irans,rnrmali0~ and uncontrolled growlh or mammalian cells, presum- ably by facililallnl~ Ibe expression of growlh-cemlrol gcnc pr~lucts which ore nor- mally Iranslalionally reixessed. Whereas Ihe mechanism of clF4 E-mediated Iransfor- malign is bcing aclively pursued, clinical invesligalions ;nlo Ihc cxprcssinn ¢'d" eiF- 4E in pccvalcnt human cancers are lacking. We have rectally inillalc¢l a screen of b~easl carcinomas by probing wilh elF4E anliseru~n. Using Wcslcm blain, we have analyzed Ihe level or clF4E in 38 carcinomas. 7 nomlal samples and 3 fibroadenn- mas, We f.o~nd Ihat elF-4E was elevated 3- Io 10-fold in vlnually all Ihe carcinom,x~ wc analyzed, but hal in fibrosdcnomas. Thi,~ analysis was also cnnrlrmed by immunohislolo~ical slaining in zitu. showing Ihal nvcrcxpression of clF4E can be readily ;demified al Ihe single-cell levcl. Our rcsulls suggcSl Ihal an clevalion or elF4E may be an cssenllal componen{ in Ihe dcvclopmeul of hrca,;I cancer. Kerekalle. V,. Smiley. K,. Hu. B.. Smith. A.. Gclder. F.. and Dc I]cncdcll;. A. (Rhoads, R. E.) lnlemalional Journal of Cancer 64:27-31, 1995. Giber support: Loui,,iana Education Qualily ~upFnl Fu.d. From the Dcparlmenl of Biocbemislry and Molecular Biology. Departmenl of Palholo~y and Depallmatlt of Surgery, Louisiana Stale Un|vcrsily Medical Center. Shrcvclx~, I.A. 36 I IERITABLE. POPULATION-WIDE DAMAGE TO CELLS AS TI IE DRIVING FORCE OF NEOPLASTIC TRANSFORMATION Prolonged incubation of NIH 31"3 cells under the growth constraint of co~llu- cncc results in the death of. some cells in a manner suggestive of apoptosis. Successive rounds of prolonged incubation at c6nfluence or the surviving cells pro- duct ~ncrcasing ncoplasllc transformation in the form of increments in saturation densily nnd Iransfnrmed locus formation. Cells rrom Ibe poslco~nuenl cullure$ are given a recov©ry pcdt~J of" vnrimJs Icn¢lhs to remove the direct inhibilnry el'fccl of confluence before Iheir growth properties ore st,died. II is rmmd that with each round of confluence Ibe esponenlial growlh tale of the cells al low dcnsilies gels lower and Ihe size of isolaled colonies or the same cells shows a similar progressive rcdoc6nn. The dccrca.,tcd growth rate of cells from the third round or c(ml]uenee (~r- sisls for >(X) gcncralinn~ of [,.mwlh nl low densily. The propmllon or ¢ohm|c, lathing giant cells is m.ch higher aflcr a 2-day recovery from conllueoce Ihan slier 7-day recovery. Relardadon of growlh el low densily and incpcased saturation apicar In be IWO sides of Ihe s:~nle chili: boll) ~cur in Ihe el)tire Popol:illnn of ~lls -~nd precede lhe f.nnalim) nf IransGmned feel, We propose that Ibe slowdown growlh ,nd Ih¢ fonlhlllnll of ~illllt cells flag|It| frill1 herilnble Illtnla~C tO life cell~. which in turn drives dteir tz,~nsl'ormatlon. Similar ~csulls have Ix~o c~po~:d for the sa~ivors of x-inadlalinn and of"lrcalmcnl wilh chemical carcinogens and are ated wilh Ihe aging process in on)reals. We suggest thai Ihesc changes resull ram free radical damage to membrane lipida with particular Jamage In lysosomes. Pmlcascs and nuclcases would then be rele.',,~ed to progRsslvely modify Ihe growth ~havlor and gcnclic stability of the cells toward azztonomous proliferation. Rubin, IL, Yao, A., and Chow, M. Proceedings of"the Nalional Academy ol'Selences USA P2:4843-4~t47. May 199.~. From Ihe Dcparlment of" Molecular and Cell Biology and Virus Laboratory. Universily of" Califomla. Rcrkeley. CA. NEOPLASTIC DEVELOPMENT: PARADOXICAL RELATION BETWEEN IMPAIRED CELL GROWTI! AT LOW POPULATION DI~NSITY AND F.XCESSIVE GROW'i'll AT HIGH DENSITY The role of hcrilable, population-wlde cell damage in neoplaslic development was sludicd in the 28 L subline of Nil! 3T3 cells. These cells dirfer from Ihc sublln~ used previously for such slud|cs in lh~'ir lower f~equency of "spomaneoes'" transformation at high Population density and their grealer capacity to produce large. dense transformed loci. Three cultures of Ihc 28 L sublinc of Hill 3T.1 cells wcrc hchl umlcr the conslralnt of confluence foe $ wk (5 wk I" assay) and Ihen assayed twice in succession (2" nnd 3" assays) for transformed focl and saturn|ion densily. After the 2" assay. Ihc cells were also passagcd at low density to dclennine Ih~ir exponential growlh rates and tinned Io dctcmfine the sir.c and morpholog|cal feao tarcx or Ihc colonicg. Cemcun'¢ol ioeasnrcmenls were m,,uic in c;a:h case will) conlml 37
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cells Ihal had b~n k©pl only in I'requ~nl low-densEly p-~ss.~ges and cells Ihat h,~d kc~l al confluence ~or only 2 wk (2 wk le). Two ~ t~ Ih~ cu~lurc~ tran~cd ~rom th= 2" assay nf Iha ~ wk I" cuhu~¢s pr~uccd li~hl Ir:ln;~nm~d ~i. ~(I third pr~uc~d den;~ r~i. ~ light f,~us-fo~ing cullurcs grew In twice th~ cnnlml salu~lion dcnshy in Ih~ir 2" a~y a~ ~-R I;m~s Iho cnntm~ dcn~ily in ~ho 3~ satu~lJon dcnsilics rot Ihc dcn~ P~us ~n~ w~e a~tll ID limes Ihc conlrnl ucs in ~lh ~+~ays, All Ihrcc o~1~ cultures ImnsPc~d Prom Ih¢ 2" assay ~ the ~ wk I" ~llurcs mulliplicd II lower rains Ih~n conlrolm al low dcnmilJcs, hul Ihc dense (~us fo~rs mullipli~ fast= than the light f~ns G~. ~o roduced rains of mullJplicallon of Ih~ llghl ~us ~o~ ~i51e(I for >50 gc~rallnn; o~ multiplication a1 ~w den;iti~. Isolalcd coi~s fo~d ham single cells of Ibe light ~u5 ~o~ers wc~ or a lower ~pulal~n d~sily Ihan conlmls: colonies fo~d by Ihc ~n~ r~us fo~¢~ w~ slighdy d~nser Ihan Ih= comml; hul ~upicd only h~l( Ihc ace. A much higher pro~nion or ~ colonic~ Gas Ihc ~ wk I" cnhurc~ Ihc naturals consisled ~r ¢i~1 cells or mixlurcs o~ gianl and natural-opiating cells. densily and lichl r~us f~ali~ arc ~ioled wkh. =nd ~rhaps ~uscd hy. heril~- b}e, ~pulalion-wJdc ~ama~e to cells Ihal is css~lially cpi~cnclic in hal.re. marc advanced Iransr~Jon characlcrixed by I~r[c/ncrea~e~ tn ~;dundi(m and dense r~uS ~O~lli~ could have ori~in~led ~mm ram ~enellc changes, such ch~osome r~n~emenls, known to ~cur zt on elevated f~quen~ in cells Robin, IL, Yao. A., nod ~ow. M. ~dings or Ihc Nal;noal A~d~my or ~icncc~ USA 92:7734-773~. Augu~i From the Dep=rlmenl of Molecular and Cell ~iolo~y and Virus L:hnr~lory. Univ~i~y or ~lit~ia. Berkeley. CA. SPHINGOSINE. I.PHOSP}IATE, A NOVEL SECOND MESSENGER INVOLVED IN CELL GROWTH REGULATION AND SIGNAL "I'R,~NSDUCTION. AFFEC]'S GROWT~I AND INVASIVENESS OF IIUMAN BREAST CANCER CELLS This ~vlew will focus on the role or sphingoslno and its phospberylated derive- live sphingosine-I.phosl~z•le ($Pp) in :cell growlh regulaliOn and signal Imnsduc. lion. We will show Ihzl many or ibe effects altribuled In sphingosine in quicsccnl Swiss 3T'~ fibroblasts are mediated via ils conversion to SPP. We propose Ihat SPP has approl:~at¢ prope~l|¢s Io function as an intracellular ~cond messenger based on the following: it elicits div©~ ccll,,l~ responds; i~ ix rapidly produced tram sphin- ¢osine by a specific kinzsc and rapidly degraded by • specific lya.~; ils ¢onccnlra- lion is low in quiescent cells bul increases rapidly and Inmsicmly in mslxmse In Ihc g~owlh I'~tors, fetal ceil'scram (FCS) and plalelCt derived growlh factor (PDGF); il releases Ca: from internal sources in an InsP,-indepcndenl manner; and finally, may link sphingolipid signaling pathways to cellular r<~.,¢-medlat©d siy.nallng ways by clcvallng phosphatldic acid Icvcls. The elT¢cts of Ibis novel second get on growth, diffcrentialion and invasion of human brcasl cancer cells will be discussed. Spiegel, $., Ol[vem. ^.o 7.han~, !1., Thoml~on, Drcas! Cahccr Research and Trealmcnt Other supporl: Nati~nal Im;liltll~ or Ileahh ~nd The Minislry of Edncalion and Science tSp:dn). From the Dep~rtmcnt of [~iochemislry •.;d Molecular Bioloi~y and Vincent T. Lombnrdi Cnnccr Research. Cooler, Georgelown University Medical Ccnlcr0 Wasbinglon, STUDIF_~ OF TI IE STRUCTURE OF TIlE kDa L.6,MININ BINDING PROTEIN: FAT: .'~CID ACYLATION AND EVIOENCE SUPPORTING DIMERIZATI( ,: ; ~)F THE 32 kD• GENE PRODUC]" TO FORM TIlE MATIIRE PR(; I~IN The level or expressinn o(. Ihe 67 (LBP) correlates will( rise prol~sslon aT many solid tumors. TEe eDNA clone for the 67 kDa LllP is s'd'ficicnl Io encmle a polypeptld¢ o1" o~ly 32 I~Da. and Ihe¢ is no readily idcntifiat~lc mechnnism for mem~z~ne =ssocl•tion. We ha~e overexprts~'tJ the ln,nsl'ccfed 67 kDa h~m~.~ler LflP in quantilicS Ih,ll have enabled us IO ~n•lyze the membrane-bound form o~" die protein. Tr~tmcnl oir lee purified LBP with m¢lhyl trans~--,leriGc•lion read.eats. I'ollowed by GC-MS, idcntil~cd the co~alently boood [ally acids palmilalc, slcaratc, nnd alexia. Th~ laity acid modific•tion may t~ov~lc a mechanism for memhrnnc association. Molecular mass detenninallon by MALDI- TOF MS demonstrated Ihc truc molecular mass oflbe Ixolein Io be 66.7 k[Pa. com- patlble wilh the SDS-PAGE observatino o1"67 kDa. Trealmcnt orthe LBP wilh ncu- ramin;dase. O-glycanase, or Endo-F ~lycos.:.aasc has no delecl•ble el'feel on the apparcnl molecular mass or ibe prolein, and Ih,. ivlALDI.TOF MS did hal show evi- dence of mass hctcrogcncitles typically n' .rved wilk glycosylatcd proteins. Reduclion wilh dilhiolhrcflol or ,8-mcrcalXo.':'~anol hod no effccl on Ihe molecular mass on SDS-PAGE or on Ihe r .t silva quanlilles of molecular mass Sl~cics on MALDI-TOF MS. The cxpcrimer'*z;y determined amino a¢|d compost- lion. however, was I'ound Io be eonslstenl wit;. :h-" 6"/kDa I'orm being • homodimcr of Ihc 32 kDa precursor. Prellmin.qry experiw.,:.,|s also suggest Ihel Ihe hich.al'finlty laminin binding charm:lerisl;c ol" the prolein (pP, y be mndulaled by no. as ycl. un|den. dftnd membrane accessory molecule. l,,'mdow.~kl. T. I I.. I')ratz. E. A,, ~nd Slarkey~ ,~. fli(~:hc:nislry 34(351: I 127(':- I 1287. Olher s.pport: Nalional Science Found•linn. From Ihe Deparlment of Microhiolocy and Depnrlmenl of Chemislry nnd fli~hcmlst~, Mo.tnna Sl~le University, flo~'.cman, MT. 39
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CONTROL PATIIWAYS OF TIlE 67 kOa LAMININ BINDING PROTEIN: SURFACE EXPRESSION AND ACTIVITY OF A NEW LIGAND BINDING DOMAIN A number of p-',p~rs have bccn puhllshed on the clinical corrclallon or lh¢ expression ol" Ihe 67 kDa laminin binding protein {I.hP) whh Ihe m¢lnst;itlc [x~tcnllal of solid tumors. Both mRNA and protein exprcsshm levels have t~en reporlcd, hot product remain unclear. Wc have udllzed a Immotypic ovcrcxprcssion system inveslip=ale the cell surl'aee prcscnlalion nr ihe 67 kl)a I.IIP aml the c~,urlhulinn of this prntcln In (he inv=sive p~n(~y~ orcollorcd cell lines. W¢ ¢c~rl hem Ihal cellular mRNA levels do n~ dirtily refill Ihc levels or dm 67 kl);~ LIIP nhscrvcd on Ihe cell su~uce in Ibis ov~re~pressiem system. Mcdmlrc~a~e ampli~calion gatten¢~, cell m~h~logy. ~ler. apparent adaptuli~ of the cells Io mclhot~cxat¢ ~ccomp=nied ~y • downregut~tion of the su~ac¢ ¢~p~ession or the protein, mRNA levels. ~wevcr, remain elevated. A nine =mim~ ~id scqt~cncc. CDI'GYIGSR li~c I I), ~ilhln Ih~ ~ thoin o~ laminln I has ~ idenlific(I as ~ ~mhahlc hlndin~ dom~in for lira 67 kD~ L~P. Previous sludi~ have idenlifiml ~ rtginn nrlhe 67 L~P which may ~ involved in laminin ~ltr~Cl~, ullh~h ~ml ne~essarily via Ih~ ~ptide I I domain. We h~v~ ~enli~d • s~cond si~e wilhin Ihe ~mi~ acid in t~rn. A ~plid¢ whh Ihis sequent. LBP ~siducs ~)5-229. hinds huninlo- ~plide I I inhlbituhle manner. ~c ~cc~,)r-dcriv¢d ~plidc mmhdatcs lay=shin nr when injcct~ alung with BI6BL6 merest mchmoma c~lls. I lowevcr, p¢cWealmcn( the melanoma cells with the ~plide enhances lun~ colony formation. Thus, ~hc interne[ion or the 67 kDa LOP wilh ha~nmnl membrane malHx ap~nrs m involve complex scr~s or cvenls including muhlpl¢ adhesiv~ sties aml [ighl rcgolalion orce[l su~ace expression. ~ndo~ki. T. eL. Ulh~y~kumar. S.. end Sttrkey, ]. R. ~inlc=l & Ex~i~nl=l Mclaslasls 13(5):3S7-372, 199~, O[~r supeR: Nad~l Scie~e Found~llon ~nd Natlrmal [nsli¢ot~ or l [cal~h. From the ~p=~menl of Microb~logy. Mnnmna St~c Universily, Bozeman. MT. COMPREilENSIVE ALLELOTYPiNG OF I IUMAN RENA L CELl. CARCINOMAS USING M[CROSATEI.LITE DNA PRolrlI-:S The yon llippel-Lindau locus on chrnmosomc .'Ip is a tumor suppressor l~cne Enown [o b¢ involved in nonpapillaw renal ¢c[I carcinoma. A pmvlous loss of het- ¢rozygosity (LOll) study aimed at delennining Ihe allclolypC of khlncy Inmors has indicated Ihal in addilion to 3p. chromosome arms 5q. (Xl. I(kl. I Iq. 17p. and 19p may al~o h=r~r lumor suppress~w genes. Ilowcver. cylngencllc sludtcs reveal Ihot 4O chromosomes 3p, 6¢ Sp, 9pq, and 14q most frequently undergo k~,olypic ch~¢es in ~'nal tumors. To resolve these d~fferences. • collection or microsatelllte DNA pro~es has been used to'scan I'or LOll so ~h-~l 90~ or individual tumor gcnomes wcm rendered inform=live for allele loss. The assay is capable or detecllng quanllta- llve gennmic alice=lions in tumor cells as well. We find thai LOll Is most for chromosome ann 3p. Ilnwever. in no lumor is 3p exclusively aff~ted, LOll 6q. ~p. 9pq. and 14q is also dlslinclly clcvalcd for bolh nonpapillar7 as well as papil- lary lumurs and snggcsl Ih.ll many of Ihe lumO¢ supp~essor loci involved may b¢ common Io Ihc elinlogy of lmlh forms of kldn¢7 cancer. Thra~h-Binglmm. C. A.. Greenber~. R. F.0 IIo*" ;d. S.. Bmzel. A.. [rl~mer. El.. Gel1. A.. SalaT~r. II.. Freed. J. L. and "r--srlol'. K. proceedings el" Ihe Nation.'d Academy of Sclences USA 92:2/I.$4-2~.'~. M.'~h Iq95. Olhcr supimn: Rclz Foundntlcm. Lncille W. Markey Fmmdatlon. U.S. P~lblic I[e~dth Service. and an approprialinn from Ihc Cnmmonweahh of Pennsylvania. From th~ Iostitule for Cancer Re,arch .'rod Division of Medical Science. Fox Chase C~ncer Ccnlcr. Phil-~d¢lphi~. PA. II. Cardiovascrdar $2slern INDUCIBI.E NITRIC OXIDE SYNTIIASE ACTIVITY IN MYOCARDIUM AFTER MYOCARDIAL INFARCTION IN RABBIT The nclivily or nitric oxide synlhase (NOS) in [nt'a~ctc~ and nol;inrarclcd rabbll myoc~lrdinm was determined. NOS activity, as m¢,,sured by conversion of I"Clargi- nine to I"CIcltmllin¢, was siRnificanlly higher in lira inracled are or myo~ardium (22./~" 33 fmnllm~ ~s compared to 7.67 ¢ 1.0 in ~infareled =uea). NOS ~clivily within the area or risk remained on control I©v¢l. Increased inducible NOS =¢tivily was observed on Ihe firsl postopc~allve day and pcrsisled rot. al leasl 14 days; it dccllned 3 weeks after ini'a~cllon. Cilrullln¢ ('nan=floe was inhibited by N.omega- nltro-L-arglnine a~! N.omega.monomcthyI-L-arginlnc. The localization of NOS by monoclonal anli-NOS antibody indic=loS mononuelear ccllstmac~ophages as the • likely so,~rcc or ihc enzyme. The conccnlraliOnS or tumor ncemsls fnc[os-dpb~ and inlerleukin-I h¢la were nol incrcascd in peripheral blood ~r myocardium. Dssdck. R. R,, Wildhlrl, S., Conforto, A., Plain, V., Suzuki. I1.. Winder, S.o and lilac, R. J. 131ochemlcnl trod lliophysic~l Research Communications Dcccm~r 3it. 1994, 41
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(D DEXAMETIIASONE INI IIBITS TI IE EXPRF.~;SIf)N f}F AN INDUCIFILE NITRIC OXII)[:- SYNTI [ASE IN INFARC'I'ED RA[IIHT MYOCARDIUM Infarcled areas of rabbll myocardium show rclalivcly higher imh, cilde nitric oxide synlhasc aclivily, measured by Ihc c~mvcrsiem of L-I"C[;¢gininc Io L-["Clclt. rulllnc. The principal [inding in this slody is Ih;ll ¢lca~unclh~lsonc (2 mgjltg) prcvcellS Ihe induclion of inducible nilrlc oxide synlhase in henri muscle when given before, or even 3 hr after cmonary artery llgation. Additionally cyclic GMP levels renmiu unchanged h)ll,)wlng treatmenl with dexamethasonc. |1 is I'xsssiblc Ih;d the c,h;mced production of nitric oxide by inducible nitric oxide syntltase accounts, at least in part. for the depression of myocardial Conlractility seen in myocardial infnrcti(m ~nd in o~hcr clinical condi6ons. Dudck. R. R.. Wihlhirt. S.. Pinto. V.. Gieslcr. G.. and llinlI, R. J. Biochemical and Biophyslcal Research Communications 2D2(2): 1120-I 126. Suly 29. 1994. Other support: Oustavus and Louise Pfclffer Research Foundalion. Redlands. CA. Marg~'cl W. and Ilcrhet~ Hoover. Jr.. Foundatinn. Pa.~adcna. CA. and lee Pasadena Foundation, Pasadena, CA. From the Departmenl of Expcrlmental Cardiology. t[unlinglon Medical Research |nsdlutes, Pasadena, CA. INHIBITION OF ENDOTIIELIAL NITRIC OXIDE SYNTHASE BY CYTOCIIROMF- P..450 REDUCTASE INIIIEITORS Nitric oxide synlha,,e eNOS) shows similarities to cylochromc P-450 rcd~cta~e. The Iwo enzymes calalyze Ihe oxidati~m of N-t*~-hydfoxy-I,-argininc by NADPI I and oxygen m nitric oxide (NO) and citrulline. Nilric oxhle synthase aclivily is inhibited by L-argininc analogs like N-~u-nkm-L-arginine. which does not affect cytochrome P-450 ~educlasc. Dihydrocrgotamin¢, mico~azole, and tmleandomycin are classical inhibhors of cylochfomc. The present study shows the concc~lrallon-dcpendent 42 IMMUNOIIISTt3CI IEMISTRY IN TIlE IDENTIFICATION OF NITRIC OXIDE SYNTIIASE ISOENZYM -ES IN MYOCARDI,'tL INFARCI'ION Inducible nitric oxide synthas¢ (iNOS) aclivlty, as mensued by eonve~ioo of L-'C-arginlne to L-'C-citrulline is significantly increased in infarcted rabbit myocardlum as compared to healthy myocardium 48 h arler corona.,y occluslo~. The aim of this sludy was In Iocallse the nitric oxide synthase eNOS) isofonns in nownal myocardiom and compare these findings with NOS acllvity in cells orth© inraxcled Rgion. Melhods: Activities of NOD isoforms were ¢nzymatically a~sayed in normal and infarcted myocardium. Localisatlon of NO$ was performed on identical sections using specific monoclonal IgG ~libodies against endolhelia constilutlve (eNoS) and macrophage inducible {INOS) nilrlc oxide synlh~se. In addition, macmphage$ were identified using Iluoresceln conjugated (2~mmPure rabbh IgG. Fc fragment. Resulls: iNOS activity increased significantly in Infmcted as compared to nowna| myocard|um [0.42(SEM 0.03) s, 0.85(O.01t) fmol.p£".m~n". P=O.02. respeclivelyl. Ilowcvcr. eNOS did not increase significantly in infarcted regions 10.18(O.IM} t' 0.24(0.06) final.rig'.rain '. P=O, 16. respectlvelyl, eNOS was lmmunohistochemically Iocaliscd in endolhelial and cndocardial cells in natural and Infmc'ted tissues. The presence Of INOS aclivily in macmphages in infarcled myocardlum was ide~lll'ied immunohlslOd~emically. Cardiomyocyles and n~lmphils did hal label w[Ih Ihe anti. bodies to eNOS and INOS. Conclusions: el) Infiltrating maet'opheges at~ the main site of increased INOS acllvlty in infarcted rahbh myoc~rdinm. (2) eNOS aclivily is rmt significanlly increased in infap:tml tissues as ¢omp,'trcd to ~ownal myoeanlitmt. (3) NeOlmphils and cardiomyocytcs do hal express NOS inmuo~cactivily in infuriated and normal rabbit tnyncardium. Wildhlrt. 5. M.. Dudck. R. R.. Suzuki. H.. Pinto. V.o Nan)an, K. S.. and BIn¢. R. J: Cacdiov,~sculttr Research 29:526-53 I. 1995, 43
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C> O~her supporl: Margarel W. and llcrberl lira)vet. GUSl/VUS and Louise P~ei~(er Foundalilm. Rcdlands. C~. and the l'a~adcna Foundation. Pa--dcna. From Ihc ~panmcnl o[ ~x~imcm~l ~n~lltulc% Pa~a(Icua. C~. CARDIOVASCULAR DIS -F_~SE AND NUTRIENT AN'rlOXll)ANTS: ROI.F. OF LOW-DENSITY LIPOPROTEIN OXIDATI~)N Increusin8 evidence indicates Ihal oxhlalive mc~dil'icalion or Iow-densily li~prolcln (LDL) is causally ~¢lulod ~o mhcm~clcm~i~. Oxidalivcly mmli~od !.~1. (o~LDI.). in c~l~l Io nal;vc I.DL. i~ 1okon /n~al/on or lipid-I~lcn ro~ cells. Foam ccll~ a~ palh~tgn~mmlc o~ Ihe alhcr~cle- relic ~ally slrcak. M~ified LDL may Mso promolc mhcm~clcrosis hy many olhcr mechanisms, such as r~ilmenl on(I mlenlion ur nmn*~ylc-m~crophages. T-lym- ph~ylCs, and sm~lh muscle cells in Ihc arlcrlal inlim~, and cyt¢~t()x~ily toward endtHlmlial cells and macrophage-dc~ivcd adtcfosclemsis" is sup~n~ by a humor oxLDL in al~losclcrofic lesions, and incre~d lilers of,uloanli~lics againsl m(~- tried LDL in ~denis with al~m~lerosls. AS a c~o]la~ o~ t~ o~idal;~ by.thesis nr alhcrnsclerosls, anlinxi(hulls Ihal by an;real sludies showin~ Ileal anlioxidams s~h as pmb.col, hulylalod hydroxy- (oluene. and a-locophe¢ol c~n slow the progression of alhero¢clerosis. Epidemiolo¢ical a~ ¢lln~al dale i~icalc a prmeclivc role or dicla~ umioxi~nls u~ainll ca~iovascular disease, includin¢ vil~mi~ E. ~-cam~cn¢. and vitnmln C. Likewise. ~sic rcsca~h sludies ~ LDL oxld;.i~ have dcmnn~lraled a prolcclivc role for anl~xid~ls. WC~nl ~lher in the oqucous envlmnmonl with I~ li~rmein il~lr. Mine m~ies am n~dcd d¢ic~ine I~ required d~cs o~ ~cific anlioxldanls Io prcvem and W~ihly Ircal ¢ardi~ascular disea~. Frei. B. Crili~l Reviews in F¢~ Scic~c aml Nu ri hm Nalional IhSl/lUI~s n[ I Icahh. Ih~lon, 44 REDUCTION OF COPPER,'I]UT NOT IRON. BY HUMAN LOW DENSITY LIPOPROTEIN (LDL) -- IMPI-ICATION$ FOR METAl. ION.DEPF.NDENT OXIDATIVE MODIFICATION OF Cell-mcdialcd nx|dal;vc mml~ficalion of human Imv den~ily |lpopmte|n tLDLI. mosl likely an imlmdunl cnrly slop in nlhcmsclerosls. ~¢quircs r~los or'live ions such os copp<:r or iron. We I~va Ixcviously' shown thai i¢on-dqcndcnl, in con. trasl Io coppcr-dcl~mlenl, oxidative modification o1" LDL requires supcroaklc, a physiological rcd.clanl. In the prc~:nl study0 wc r, ough! Io explain II~sc di~.'n.'pnnt rcsulls. IJ)h was i~c.hatcd :~1.17eC with Cu~" (10 p.MI n.d balhocopml,~' (11C'. H.M). -~n indicator Inoiccille which spcciflcally comple~es Cu'. bul ~ol Cu llme- anti conccnlr.~lion-dcpcndenl manner. LDL reduced Cu~" m Cu'. An LDL con. conlralion as I.w as (10 p.M) of p~otcilVml (.'1heal 20 hill) Icduccd abOtll ? p.~! Cu~' wid~in I b of incubation. Comptexatlmt of |I~e Cu" I'on~ed under Ihe~¢ ¢ondillt~s with DC signiricontly inhibilcd oxidolive m~dil'icadon or LDL, as assessed by agarose gel elcctrophorcsis. Prcincuhation or LDL. with N.~thylmaleimlde had no cffecl on Ihe rate ~nd ex~cnl of Cu~° reduction nor LDL oxld~t|no, ind~calin~ Ihal sull]~d~y! ~roup.~ ~sr, o(:i,,led wilh npolilmproleln B ere nol involved. Addhi~m o1' ©ithcr supcroxlde dlsmulase or ¢atalasc or incR~sin¢ Ih¢ ¢Hocophcrol ¢Onlcnl or LD[, f~nm I I.g ± 3.0 to 24.4 + 2.8 nmol/m8 or proletn also ha~ no significunl elTect on Ihc kinetics oF Cu~° rcducllon by LDL. In conlraSlo incuhallon or LDL with Fc"-cilralc (10 ~.M) oral Ihe inlfi¢,'ttor h~lhophonanlhmllne (BP, .~(~0 pM) resu|lcd no significant I'-e~" formation, even at LDL concentrations us h|~h ~s 200 i~¢ of Ic;n/ml. Ih~wevcr. ;nc.halion or LDI. wilh Fc"-cilrale and ~n enz)'mat;¢ sm~¢¢c of supemxidc led to r~pld foemalion or Fe~" and consequent ca|dative modification or LDL. Addilion o1" BP inhihilcd iron-m,-dialcd LDL oxidation under Ihes~ condillons. O.r r~.h.,~ indlcale Ihnl reduced meln| ions ~r¢ impo~lanl medJalo~ o1" LDL oxide. lion. aml Ih.~t LDL specifically reduces Cu', ?Ul nol Fe". '!1¢s¢ dale, Ihe~eror~, help explain why copier, in ~ddilion Io being chemically mo~e reactive, is mor~ Ihan iron nt medialin; LDL Lynch. S. M. -~nd Frel. I~. The Journal of Biological Cl~emisl~ 27(X10):51.'i8-5163. March I0. 1995. Olher sol-,pan: N=llonal |nslilulcs o1" I lcallh. From the Whil-~cr C.~rdlov:scolar lnstliute. Tioston Unlve~hy School o1" Medlclnc, Boslon, MA. IIUMAN SUCTION RIJSTER INTERSTITIAL. FLUID PREVENT~ METAL ION-liP.PENDENT OXIDATION OF LOW DF.NSITY LIPOPROTF.IN BY LDL. in the circulalion i~ well prelected against oxidalio~ by the hichly c(l'¢|cnl ~ntioxidnn! dei'cnse mcchanlsms Of hum.'m plasnm. LDL. oxidation contribulinF to nll~:rosclero.~is, Ihemrore, h;l~ hccn hypnlhcsizcd to Inke pl~'c in Ihe inlcn~tillal fluid or" the urtcrlel v,~ll. We Invcstil~ut~,'d Ihe untinzddm~l COml~|tion m~cl tl~ ceF~ity IB
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inhibi! LDL oxidation of humaa suction hlister interstitial Iluid {Sl~ll~. ~cp~esemadve of inle~stilial fluid. We found that Ihc c(mccnlralion~ in SBIF o~ ~hc aqu~us small.mol¢cul~ ~nlioxid~s ~c~lc ~nd u~le ~ere. c~lly hi~her (P < 0.05) ~ idcnlic~l Io pl~m~ c~ccnlralinns. In tcin-ass~i~itd lipids and lipld-solublc antloxidam~ (~-I~nph~rol. ubiqulnol-l(~. lycopcnc, and ~-carot~nc) were present It only 8-23% nr ihc conccnlrulinns in plasma. No lipid ~ydm~/oxidcs could ~ ~clccled (< ~ nM) in eilhcr fluid. ~c ca~acJly o~semm a~ SBIF Io protc¢l LDL ~mm oxidali~ w~ invcsligalcd in Ihrec racial ion-de~nd~nl systems: cop~r~ ir~. ~d murh~ in~mphngc; ~n I lam's F-IO medium. In all Ihrco syslcm~, a(Mhi(m n~ ~ ~% (wd/vnl) o~ cill~ ~[m. nr XlIIF inhlbhcd LDL oxidallon by > ~. ~c conccmradon Ihal inhihhod macropha~c- mcdi81~ LDL oxid:li~ by ~ w. ~ low 0~ 0.3~ strum and enzymatic or ph~i~l removal ~r ~lC nr utile =n(I ~r IhC high mol~ular wcighl (r~li~ ~ ~ pmleclivc .~ wh.lc dala dcmonsl~ale Ihal ~lh ~c~m and SBIF vc~ cff~clivcly pmlccl LDL (ram racial ;nn.Oc~nd=nl oxldaliou, moil probably ~cunxc or a cmnulalivc mclal-h;mling cffccl or ~vcral pmlc~nx. ~]r dala su~csl thai I.I)1. in Ihc h~l~rslhinl Ruhl .r Ihc a~cri~l wall is very unlitcly Io ~cl mmlificd hy .~lal hm-m~dh~l~d Oabba~h. A. J. a~ Frti. B. ;oumal or Oinic;I Inv¢~li~=llon 96:19~8-19~, ~io~r ~ ~. Olher sup~: Amcri~n llean Ass~ialion. From Ih~ Whli~er C~diovascular lnslilUlC. Boslon Univ~rsily Sch~l o~ Medicine. Boslon. DIFFERENTIAL EXPRESSION OF VOLTAGE-GATED K" CI IANNEL SUBUNIT$ IN ADULT RAT IIEART m REL~nON'rO FI]Nf~IONAL K' ~I|ANNF.I ¢? POlyclonal antibodies aR•insl c•ch or Ihe K" channel subunils (Kvl.2. Kvl.4. l(vl.~. K~'2.1. and Kv4.2) shown previously Io b¢ expressed in adult ral hcan mRNA level were used Io examine Ihe dislribulions of Ihese K" channel subunils in • ,dull ral •lrial •~! vcntdcubr mcmlx~n~s. Immunohislocbemist~ on isolated adull r~t vemricul-r myo~yles revealed strong labeling with Ihe •nli-Kv4.2 and anti-Kv 1.2 =nlilxxlies. Although somewhat wr~ker (Ihan wilh anti-Kvl.2 or anli-Kv4.2), posl- live staining w~ •lso observed wilh Ihc •mioKvl.$ and anti-Kv2.1 antibodies. Venldcul~ myocyles exposed Io the inli.Kv 1.4 anlilxxly, in contrast, did nol appear sl;nificanlly dirfcmm from background. Qualitatively similar resuhs were obtaln,'d on isolal~l adult ~t alrlal myoc,j~,. Wcslcm blols or atrial and vemricular memo brahe proteins confirmed Ihc presence of Kvl.2 Kvl.~. Kv2.1. and Kv4.2 and revealed differences in Ih¢ relative •l~ndances of Ihese subunits in the Iwo m~m° ~ l~cp~ations. Kv4.2. for example, is m~e abundant in venlricular than in memlx~nes, where~s Kvl.2 ~ Kv2.1 ~re higher in atrial membrane. Kvl.5 levels • re cumpar•blc in Ihc two preF~rations. In cooer•st to these result;, nuthing 46 delecled in Western blols of atrial or.venlri~ul~r membl~ne proleins with the anti- Kvl.4 anlilxxly al ¢oncenl~liofls Ihoi ~waled inlen~ la~lin~ o~a 97-kD ~nleln ~dull ~1 brain memb~n~s. A ve~ ~ainl ba~ was dclccl~d al 9T-kD in the al~ial ~nd vcnlricular preparations when Ihc ~li-Kvl.4 anti~y wzs used at a ~- In I~old highcr conccnlrallon. ~e simplesl inlcq~elati~ O[ I~C ~SUIIs iS ~al Kvl.I is nm on ~bundanl proleln in udnll ~l alrial or venl~cul~ my~yles. ~m[o~. il unlikely IhDI ~vl.4 plays on im~anl role in tl~ ro~lion dnn.a~iv~led K" channels in Ihc~ cells. ~ mimic(s) ~lw~n the channel suhunils ~nd Ihe de~l~ri~lion-u~ivaled K" ch~nels ~enli~ ekclrophyl. iologi~dly in ml,h rm ~ldnl mid vonlriculnr Circul;~li(m R~ca~h 77(21:~h1-369. Au~ Ogher s,pporl: Mamsnnlo-Se=rle/Woshln~;lan Universily ~iomedic~l Rese=rch Program. aml Ihe N~li~al Inslil,les o~ and Call ~ioln~y, Sl~le Univ~ily of Now Yol~ at Sl~yb~. NY. lll. Cell Biology EXPRESSION OFAN EPIDERMAL KERATIN PROT1~lN IN LIVER OF TRANSGENIC MICE CAUSES STRUCTURAL AND FUNCTIONAL ABNORMALm "F_.S To examine the role Of kcralin inlermcdi~l¢ fil~mcnl I~oleins ;n cell stmClUre end [unclion, Ir'ansgcnic mice were isolated thai express • modified I'om~ of Ihe human KI4 kcral|n prole;u in liver hel~tOCyles. A modified KI4 eDNA (KI4,P} s~iuenee was linked downslre~m of Ibc mouse Imnslhyrelln (I"TR) ¢¢1~ pmmolcr zmd enhancer clemenls to achieve I~r~elcd express;on in hepatocytes. Ilepalocyles ¢xpresxing high levels of tl~ tr~nsgcne were found Io have •lx~mmal kezllln fil~menl networks as detected by indirect immunofluorescence using ~n antibody specific fur the Iransgenc prodnc¢. Light and eleclro~ micmz¢oplc level hislologic•l analysis of isolaled liver lissue showed in many cases degenerative chanses I1~1 included uo~es, and ~lyco~en ~ccumulatlon. Th~sc chan~¢s were most ©videm in older mice over four m~tb$ or age. No indicMIoo of lyplcll MIIIo~ body slmclurc$ were id6no llfied al ©hher Ihe lishl or eleclron mi~os¢op'~ level. To cv•luatc ~¢r~lnr~ rUnCl~On 47
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C~ and found In he a~proxim.~tcly Iwnfnld lower Ihan aged-matched conlmls. findings indlcale that ~xprcssion o~n ~h~nnal kcr;~tin in t'it'o selling can alter Ihc Sl~lure and funclinn of a li~sue a.d ~ttggcst a role of the kcralin nel~rk in cellular Arbors, K. M.. Davi~, F. E.. Pcrrnne. T. ~tc Journal o f Cell Biology 12R( I &2): 15~- I OtJ. J an.a ~y 1995. Other sup~n: Nalinnal Insl/lut~ of I lcahh. From the Dcparlmcn~ of P=lholofy and Pharm: cology, Univcr~ily ~f Kentucky College of Medicine. I~ing~. KY. CLONING AND EXPRF.~iSION OF GENF.¢; Cf)DlrqG Ff)R PRf)TE[N KINASE CK2 ~ AND fi SUBUNITS IN ZRBRAFISI I (D~NIO eDNA clones c~ing for Ihe a and ~ suhunil~ of pr.~cin kin;txc 2 (CK2) dcmonst~led Ihal o~ cnntaln~ Ih¢ compete tilling ~eqncncc for Ihe CK2 while the a c~one is I~ncaled and I~cks IX3 nucleolid¢~ of the 5' coding resion. Comparison of I~ deduced amino acid scqucnce~ ~how~ an exlremely high ~gree of evolulion~ seque~e tin.walton of Ihe~c Iwo prolcin~, N~hem analy- sis oflhe mRNAs c~ing for ~he ~ suhunil indicalcs thal this messenger i~ pre~nl in I h omens as a 3.6 Kb and a 1.9 Kb species, ~lh of which decrease in 24-h omens. In Ihe case of ~. Ihe maim mRNA s~clcs of appmxima(ely loins ils level during I~ ~6~ of em~oge~sis ~icd. I~ xlm hybridiT~lion of early em~os, using anti~n~ RNAs against ~ and ~ mRHAs demonstrates tem~- ral and lissue s~ci~c e~p~sslon pallems. ~e a mRNA decreases afler when il is evenly dist~bated. ~e ~ mRNA is maintained m high levels ~lwcen 4 ~d 24 h of development, showing in 18 h emb~os a higher concenlralion in d~vel~ing noun1 tu~ and in the em~onk o~ic ~nd ode vesicles. Daniolti. L U. Al[endc. M. U, Wein~rg. F. S.. and Allende, J. F. Cellular and Mol~ular Biol~y R~areh 4~5/6):431-439. 1994. O~hcr sup~: Inlernalional Cenlfe of Genetic Engineering and IlCGEB). FONDECYT-~ile, and Ihc David M:,hn,~y In~lihlle Itf Ihc ~nnsylvanla. Fr~ Ihe De~aa~nlo de Ri~ulmica. Fa~llad de Mcdlcln~ Unlversldad de ~ile. Casilla, S~miago. ~ilc, and Depa~mem of Biolo~. Unive,~ily of ~lila~lphia, PA. 48 PROTEIN KINASE CK2: AN ENZYME WITtl MULTIPLE SUBSTRATF.S AND A PUZZLING REGULATION Protein kina,~e CK2 (,'tl.~o known a.~ casein kinnse II) is a uhlquito~s euka~nlic pho~ylalc more Iha~l I{~ ~u~lr;tle~, many of which ~re involved in Ihe ¢~ml of ¢cll or CK2 a and ~ sub,nits ~nd ~ the ~gtdalion of ils ~llvily, a Iopic thai ~mains ~ elucid:~tcd. An ~n~dt~gy is drawn ~tween CK2 and t~ cyctin~c~nt kin~ses (cdks); ~th eyes of protein kln~ses ~ham m~y subsidies and am acllvaled by ulato~y subu.lts.--A~londc. L E., Allende. C. C. Pmleln kinase CK2: with muhipIc s.bxlrale~ and a puzzling ~gulalJon, Allende, J. ~. and Allendc. C. C FASEB Journal 9:313-323, 1995. ~he~ sup~: ICG~B-Trlestc ~nd FONDEC~-~dle. From the ~aan~n~o de Bi~imi~. F~ultad ~ Medicin~ and ~aam~m de Biologla. F~.h~d de CIcnclas. Unive~id~d de ~ile. Sanliago. DNA INI IIF~ITS TIlE CATALYTIC ACTIVITY OF TIlE ~ SUBUNIT OF PROTEIN KINASE CK2 The recomhinant ~z subunll of protein kinasc CK2 (casein klnase 2) from Xe~opt~ Io.~'i~ inhibited by the addition of single slranded or double stranded DNA. This inhibition i.~ competitive with the casein subttrate, having an appa:enl Ki of 160 nM for an 86 bp DNA frngmenl. Assays with • f~gmant conlaining the putative pro- motet of the human CK2~ gone indicated that the affinity of CK2 for this wa~ nol greater than thai of other unrelaled DNA. "lrlte ~nhlbJlory capacity of DNA toward the protein phosphmylaling activity of CK2o is g~catly ~educed by the pRs. cnce of the/~ subunlt which can completely reverse Ihc inhlbilion. The inlcractlo~ of CK2a with DNA can also t¢ assayed by the nilrocdiutote fiber binding assay. This assay demonstrates that the inleraction of CK2er with Ih¢ tested DNAI is nol sequence specific and thai the ~ subunlt c~n also greatly dlm|nish Ihe binding o| CK2a In DNA. Cabin at subs*rule concentmtlms al~o is inhlbilory to CK2a DNA blnd;ng. Likewise. polyaninnlc |nhibilors of Ihe CK2 calalytle nctiv|ty, seth as heparin, poly(U), and copoly{Glu:Tyr) polypeplldes, can compcle for and inhlbll binding of DNA to CK2m Ilnwever. quercelin, which abo inhibils CK2 phosphoP/- latinn activity, and ATP dn not affecl DNA binding. A mulanl CK2a' in which I.~mic ncids replace Iwo ly,~ine resid.cx in positions 7.~ aml 76 of the ¢r i~plitfe chain l.~ le.~ susccpllhlc In DNA lnhihilio~, indicating Ihal this basic minion of Ihe mole. cule is involved in its Interaction wilh DNA. Galica. M., .~neoh. G.. A|lcnde. C. C., and Allende, J. E. rliochcmisttV .14(I ): 122-127, 1995, 49
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O O~ O~ 0 O~her support: imemalional Comte f'or Gcnelic F.ngincerin~ and I~iolechnology and FONDECYT-Chi~e. Frnm the Depart•menlo de Bi~cluimica. Facullad de Medicin.~. and Deparlamenlo de Biolo~ia. Facuhad de C'icncias, Univer~id~d de Chi|c. C.~.~illa, S,~nti-~go. Chile. IDF.NT|I'~CATIOIV OF A STAllLE RNA ENCODF.D BY TIIF. II-STRAN~) OF TI IF, MOUSF. MIT(')CI IONI)RIAL D.I.OOP REGION ANI) A CONSF.R V|~I) SI:.(,)UF.NCF. MOTIF IMMEDIA1/~.I.y UPSTRF.AM OF ITS POLYADENYLATION SITE By using a combination of Northern blol hybridization with strand-speciGc DNA probes. SI nuclease protection, and sequcneing or olil~n-dT-primed eDNA clnnes, we have ideal|tied a 0.g kb imly(A)-containing RNA enc~led hy Ih¢ I!. strand or the mou.~ mimchondri.~l D-loop reSion. The ~' end nr d~e RNA maps nuclcodde 15417. a region complemenlar,/Io the stun of IRNA'- gene .~.d polyadenylated end maps to nuclear|de 16295 of the genmne immediately or IRNA~" gene. Tile II-str=nd D-loop region encoded trans©~i~ls or simdar s)~ are also delCcled in other vencbrale Syslems. In the mouse, ral, and human syslcms, .1" ends of Ihe D-loop encoded RNA are preceded by conserved seq.ences AAUAAA. AAUUAA. or AACUAA. Ihal rexcmhle Ihe I~dyadenyl.',linn .'d~nal. The steady.slale level at the RNA is generally low in dividing ~r in vireo cuhured cells. and markedly higher in differemi:lcd lis;ues llkc liver, kidney, I,c~rl. and brain. Furthermore. an aver 10-fold increase in the level or Ihis I~NA is uhservcd during the induced dilTeremiadon or C2CI2 mou.~e mynhlasl cells into myoluhcs. These results sul,'~¢st that the D.Ioop II-slrand eneod,'d RNA may have yet unknown hio- !ogieal runcllons. A 20 base pair DNA sequence I'rnm Ihe 3' terminal ~e8iou contain. m~ Ihe conserw~d sequence modf binds !o a pmlein I'mm the milochondrlal exlra~ls in • sequence.specific manner. The bindin£ spee;ficily or this pmlein is distinedy dirrcrem ream the I~'evioosly characterized H-slrand DNA ten~nination sequence in the D-loop or the H-stored Iranscriptkm l¢nninalor immediately downslre-~m of the 16S rRHA g:ne. "~hos, we have char-clef|zeal • novel poly(A).conlaining encoded by the II-strand ol'lhe mi~ochondrlal D.Ioop region and al~ identified the puladve nhim-~le tc~Tninalion site I'of the H-sir•rid Iranscriplion. Vijayas.~rathy, C., Zh~ns, Y.-M.. Mullick. ]., Basu. A.. lad AvadhanI. N. ~. Gone Expre.~slon 4:125.14 I, 1995. Olher support: NmioMI Inslitulcs of Ileahh. From the Laboratories or Rionhemislry. Depanmee! of Animal Biology. Sclmol or Vete~ina~ Medicine, Unlvershy nr Penu~ylvania, I~ilndelpl,ia, 3O CYTOSKELETAL DOMAINS IN THE ACTIVATED PLATELET Plalele~s circulale ia the blood as discoid tells which, when ~:liv~led, shale by polymcrizing -~ctin inlo various struclures, such us filopodla ,rod fibe~. In artier 1o umlem,,nd this p'ocess, it is ncte$.~'~ry to dcltn~in¢ how m.'my other pmlcins ore involved. As n firsl slq) in defining Ihe full compk:men~ or Kiln. hi,~ding prn~eins in pI;~telels, filam~nlo,s (F)-'~clin affinily chromatography was n~d. This approach ideal|fled >'30 diffcnml p'oteins from ADP.,,cllValed h~ntan blood pla~clel.~ which represented 4% of sol.Me I~meln. AIIhough a number of Ihese proteins arc previm,.~ly identified plneelel nclin-bimlin~z peele|us, many appeared to I)c nnvcI, I:t~lccn difl'erenl imlyctnnnl nntibndi~ ~vcn~ miscd Ihese ;t~parenlly n~vel protcins .rid uscd to so¢l Ihem into nine calcgorics I~d on their molccolar weighl.~ ~ml on thci~ I~llen in the ~ome~ of slri~ music. fihrohlasls and in ~preadin~ plalelels. Ninety-lh~ ~l of I~se p~s (13 l:m~r (li~lin~1 nclin filrmcnl slmcm~s w~ rmmd Io rl~ dufin~ I~ iohlal mln o~ aclivnlion on ~la~: filo~ia, lamclli~la, a oonl~lile fin¢ dc~nulalin~ ~ran,les. nnd lhick bomlles of ~inmenls ~semhlln¢ iI~II ~. Aclin-hh~lln~ prnlclns eel l~aliz~ in lhe dL~id ¢cli ~n~ hl~hly Sl~Clure conlains a di~e~nl ~mplemenl of p~eins. ~ tcsulls ~nl inf~ali~ ¢~ul the complaxily of I~ plmele~ ~yl~kelelon. ~l~ling lh¢l foot diff~renl ~lin-~d ~Im~lu~s fo~ du~n~ the r~l 15 rain of s~ llon. and dial Ihcre r~ain many is y¢l unchamcl~ ~lein awlillni [unhcr inv=sligalion Ihal a~ di((c~nli=lly involved in Ibis llear~, F- !. Olhcr sup~n: N~li~al Inslilulc~ or Ilcahh. From Ihc Di~i~i~ o~ Biology nnd Medicine, Brown Univ¢~ily. ~id¢~, RI, CAENORIMBDtTI~ ELEGANS CYCIJN A- AND B-TYPE GENES: A CYCUN A MULTIGENE FAMILY, AN ANCESTRAL CYCLIN B3 AND DIFFERENTIAL GERMLINE EXPRESSION We have cloned cDNAs tar Caennrhohdili$ ele&on~ ey~lin$ AI. g ~d gJ. While cyclins A ! and B ,,re runs! closely re|ah:d Io either A- m" B.lype cyclins or other species, eycfin gJ' is teSs tel•lad Io thes,- ¢/,¢llns. However. Ibis ¢ycl|n is mo~t similnr to Ihc reeenlly tdendl'ied ehinkc~ r.y¢'fin BJ. Our idcnliflenlion of a Cne~nrlmhditi~ homolog dcmonslrates thai ~'. h~ B.t has heen conserved in evolu. lion. cy('ll, At l~ a a~:mb~" of an A-~ypc ntu.'~il~ene fam|ly; however the cyclln AI eDNA only rccognlzes I single band on norlhern blots. A single-sized RNA is also observed for the ryclin B3 eDNA. In ¢onll~Sl. lhre¢ different ira~.ct4p~s are obs~ved- for Ihe c).('lin B eDNA. Ba~ed on our unllys~ usin¢ RNA~ from ~:n~line<f¢l'ective mulants ,~nd I'rom populnlions cnrichnd I'ar males, o~ q~lin B IranscripI i~l ~pecil'l¢ 51
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ca) LIPOPOLYSACCIIARII)F. SIGNALS ACTIVATION O1: TtJMOI( NF.CROSIS FACTOR BIOSYNTi IESIS TIIROUGII TIlE RAS/RAF-I/MI;K/MAI,K PATI IWA Y • Dack~round: Lipopolysaccharide (LPS) is known Io activale macrophagcs, causing Ihe release of Ioxic cytokines Ihat may provoke inllammallon and shock. One of Ihe mosl imporlan[ and besl studied of IheSe cytokines is Inmor nccrosls I'ac. lee (TNF). Details o1" Ihc signaling palhway leading to TNF biosynthesis remain unclca~. The palhway is branched in the sen~ thai TNF gcne transcription and TNF mRNA Iranslation are bolh strongly stimulated by LPS. Recent evidencc h;,s indi- cated thai MAP klnase homologs become phosphnryla~ed in LPS-sdmulatcd cells. suggesting Ihcir possible involvemcnl in signal Iransduction. We sough! Io trs! Ibis hypolhcsis. hlalerials and Melhods: MeasuremenlS of I.PS-induccd MEK and ERK2 activiiy were undertaken in [.PS.sensitive and I.P.~;-inscnsilive ceils. Tr:,nsfcclion studies, in which dominanl inhibitors of rex and r~.! were nscd Io block signaling ¢o the level o1" MAP kina~e, were carried ou[ in order Io.i,dge whether ~he TN F Rene Iran.',crlptlon and TNF mRNA Iranslalion are modulaled Ihro,gh Ihls i~lbway. Eesulls: In RAW' 264.7 mouse macrnphages, hell, ERK2 and MIlK I aclivily are induced by LPS [rcatmcnL In the same cell line, domin.'mt nag:~liv¢ inhlhilors or ra~ and roJ'-I block LPS-induced activation el" the TNF promoler, as well as derc- presslon of Ih¢ tran~;lational bin(shade normally impo,~cd hy d~c ']'NF 3°-unlrnnsl,~tcd region. A constitutively aclive ronn o1" ral'-I |rq[.RXII) was fiml~d nol replace. Ihe LPS signal. In LPS-ins~nsitive cells {RAW 2(,4.7 X NIII 3T'3 I'p,;ino hybrid cells and primary macrophagas derived rrom C311/IIeJ mice). ERK2 actlvi~y was found Io be refractory Io induction by LPS. Conclusions: The r~/roJ'.I/MEKIMAPK pathway is chiefly responsible for transduclion o1" the LPS sit.nal to the level or the TNF gone aml mRNA. r~nnd r~J'-I $2 lie upslream from (or actually rep~scnO Ihe physical bmnchpolnts or Ihc tmnseHp. lional and Iranslalion ~livaiion signals gcncralcd by LPS. "~nc ieslon$ Ihol Ixcvrnl LPS signaling in macrophagcs rmm C31t/l~.eJ mice, or in RAW 264.7 x NIII 3T.1 fusion hybrid cells, nccopy a proximal posit!on in Ihe signaling palhway. Geppen, T, D., .Whitehursl, C. F., Thompson, P~, ~nd lleuller, I~. Molecular Medicine 1(I):93-103, November 1994. Olhcr support: National Inslilules of Health 0nd Ihc Texas Drparlmcnl Leldies ^uxilia~ Veterans or Foreign Wnrs, From Ihe Deparlmen! o1" Inlernal Medicine, University o~ Texas Souihwcslem Medical Center at Dallas, Dallas, TX, The Harold C, Simmons Aflhritil Research Center, Dall~s, TX, nnd The I Iow~rd II,ghes Medical Inslitute, Dallas, TX. CONSTITUTIVE ACTIVITY OFTIIETUMOR NECROSIS FACTOR PROMOTER IS CANCELED BY TIlE 3' UNTRANSLATED REGION IN NONMACROPIIAGE CELL LINES: A TR.~NS-DOMINANT FACTOR OVERCOMES TI IIS SUPPRESSIVE Tim role of Ihe mouse tumor necrosis I'nclor (TNF) promoter, 5' unir'a~slaled region (UTR), and 3' UTR in TNF gone expression has been examined in ihRe non- macrophage cell lines (lleLa. NIll 3T'J, and L.929). The TNF promoler is hal macrophage-specific. On the. contrary, |! constitutively drives reporl~ L::Coc expres- sion in all three cell llncs. No[ only Ihe full-lcngrh promoler but also Inmcal~d ver. simms or the pmmneer, I~klng NF-xB bindlnc mot;Is, are active in each lyp¢ of c~ll. Tim TNF 3" UTR effectively cancels reporler gone expression in IIdJ cells and NIII 3T3 cells hut I'ails Io block expression in L-929 cells. L-929 cells contain n Inr thai overcomes Ihe inhibitory inl]ucacc of lee TNF 3' UTR. IIs acllon depends upon the presence or ~quenccs round in Ihc TNF 5' UTR. Cell-fusion experim~nlS reveal Ihat Ibis aclivalo¢ is trans-dominnnl. These slndi~ highlighl Ihe essential played hy the TNF 3,' UTR, which silences the TNF g~e in cells thai mi£Jl! olher- wise exprcss TNF. They nlso reveal the exislence of on escape moth:mitre whereby in-',ppropriaee synlbesis ofTNF migh! occur. K~uys, V., Kcmrncr, K., Shnkhov, A., Jongenccl, V.. and Ileuller, I]. Proceedings or the Nnlional Acedemy of Sciences USA 89:673-677, January 1992. O~her supporl: National lnstilules of Heahh. From the ltoward llughes Medical lnslilute, Dallas, "IX and Ludwig Inslltute Cancer Research, Lausanne Branch, Epallnges, Swltzcdand. ~3
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O A REPORTER TRANSGENE INDICATES RENAL-SPECIFIC INDUCTION OF TUMOR NECROSIS FACTOR (~NF) BY SIIIGA.LIKE TOXIN~I~)SSlBLF. |NVOLVEMENT OF TNF IN IIEMOLYTIC UREMIC SYNOROMF. We have examined Ihe hypothesis thai TNF ma I- , |a " '" • -y real Snll~a[oxln wh;rh .~..,....__., • • • .~ ,~,,).. wc c¢l(ISl¢lerc(i ....... ~.mu~u;~ ires s~fnorol~e mi " . . Ix , .I and/or tl,-',[ TNF and shi~atoxin mi,,~ ...... :-" ., .gh[ ,n.ducc TNI. bmsymhes~s, Ihe .L __. o. e~"' ~--J,~mze anmlalS, each ll) IIl~ toxic Omer agent. ~hl;a[oxln was found Io scns;llzc mice Io Ih~ lell~l cfl'ccl ed' I I'S and Io Ihe Iclhal c|l'CCl (d" TNF. On Ihe oilier hired, prelre~lmcnl ¢)1" u.inl;~ls w;ih ¢ilher TNF or LPS did no| noliccably s~nsillze mice to 1he retinal effect o/" shi~atoxln. ]mrap~Hlonea| in, co|ions or shiga~Oxin did no| induce Ihe pr~luclinn or d~teclnhle quanlilles or TNF in |he plasma of mice. When shig..,Ioxin was injected iron Irons- genie mice bearing a chloramphenicol aCelyhr'-Jnsrer..,se (CAT) ~c~)ner indicates TNF symhesis. CA'/" activity was induced wiehin |he k~lney, but not in o[her llssues. Wc |betel'arc conclude Iha! sh|galoxin acls Io induce TNF Synllk~,;is wilhin Ihe kidney, and at I]~ same llme increases renal sensilivily to Ihc Iosic el'/'ccls of'INF. Whi|e |his mouse me(hal do~s hal ~'e~oducc tim I • a.. Icmo yt~c uremic sy,clrome ~ il occurs in humans, il do~ su~es! Ihal local synlhesis ofTNF Wilhin Ihc kidney may contribu!e to renal inju~ ind.ced by shigaloxin. lintel, y.. Silva. M.. G;rolr. B. Weinbcr£. A., C'lea~),. T. B.. and Ileullcr. Journal or Clinlcal InveSllgallon 92:2110-2116. November 1093. Olher supporl: National |ns|Jiulcs or llcallh. From the Depa~lm=nls or Ped;alrics, Patholo~,v. and Inlernal Medicine Iluward |lu~h~ Medical/ns|itute. Unlvcrsily orTexas ~thwc~!cm M~ical Cem~r. Dall~s. TTXX. and [:)cpanment or Pediatrics. University or T©xas Medical School. Ilousmn. THE SYNAPTIC VESICLE PROTEIN SYNAPTOTAGMIN PROMOTES FORMATION OF FILOPODIA IN FIBROBLASTS .,.N.EI~C~. At. filOlx~dia are aciin-rich cyloplasmic exlensions Ihal are involved in molmly ~o ~co~niliOo in growth cones and maturing axonal endings. A detailed undcrslandin~ or neuronal growlh will depend On Ciaril~catiou o~ !he membrane [usion evcals occun~n~ during ~ilopodial ex[ension. The synoptic vesicle protein synaplolagmin Seems Io lx~ imlmately involved in we show thal.l~b.roblaSl cell lines [ransr¢clcd with s na Io~a mln • ----... Iwo oiler synapli¢ vesicle pro!tins SV2 or synaplophysin choral processes and like neurons -^- ~'~,-* - . .-'.. g ~ only n~Jimcnla • _ " • - , ~,, ,a~ ann synaptol~ysm c~es. As prcsynapl~c calcium enl~j, rcgul-,es synaplic vesicle fusio cole Ihal s n • • . . . . • n. our rc.~ull~ Indi- Y aplOlai;mm might link neuronal acl,v~ly welh syw-,pl~c growlh. 54 F~'~ny, M. B, and Buckley, K. M. Natui'c 364:537-540. Augus| 5, 1993. From the Depallmcn! of N~mhiology. Harvard Medical School, Boslon, MA. Tile SYNAPTIC VESICLE PROTEINS SV2, SYNAPTOTAGMIN AND SYNAPTOI'IIYSIN ARE SORTED TO SEPARATE CELLULAR COMPARTM~NT.~ IN CI IO FIRRO[IL/tSTS Wc cxprcsscd Ihc syn~piic vesicle proteins SV2. syrmplolagmin, lad synapIn. physin in CliO [ihr~blnsls Io invesligal¢ Ihe tnr~eltn~ informal!on conlai~d by each prolein. All Ihree peele!as entered dilfcrenl cellular comparlmenls. S),naplola¢min w.~ fouml on the plasma memb~ne. I~olh SV2 and s),naptopltysln welt sorlcd Io small imracellular vesicles, bul synaplophysin colocalizcd wilh early endosomal markers, while SV2 did noC SV2-conlaining vc~iclc$ did nol have the ~rn~ rcdi- mcnlafion character!sites as aulhcnlic synapl!:: vcslcles, ~ven Ihough Iw~sfecled SV2 was snncd from endo:~omal markers. Wc alxu cl~'.oled cell lines cxFcsskll~ bolh and synap~.~lal~min, holh syn,~plolagmin and synaplophysin, and lines exp~ssinB RII Ihrcc ~ynaplic vesicle peele!as. In all cases, Ihe proleins milton!ned !heir dlsllncl comp,'~rhnenlaliT-'qions, were not Found in the same organelle, and did hal synaplic vesicle-like stmclures. These Rsulls have impmlanl implicallOOs roe of synapllc vehicle biogenesis. FeRny, M. 13.. Yee, A. G., Dairy. M. L.. end l~uekley, K. M. The Jmlm:d el" Cell Biology 12.1(3):~7.~-~1M. i,l,wemhcr 199.'1. Od~cr s,ppo~: National |nsli!¢ll~.l: or Ilcnlth. From Ihe D~parlmen! or Neurobiology. Ilarv.~:d Medical school. Boston, MA. A DISCRETE SUBPOPULAT1ON OF HUMAN MON(X~YTES EXPRESSES A NEUTROPIIIL-LIKE PROINFLAMMATOkY (P) PHENOTYPE Wc have demonslra~ed Ihnl a disciple ~..J nal~all~ nccur~n~ subl~ol~la!~ human mnnncylcs expresses n aemrophil.;ike prn|nn,~mntory (P) pheno|ype. P nmm~:ylcs conslilule 2l)..'111~/,~ or the cin:ulallnp mo~ocyle pool nml a~ clmn~¢leri~¢d by I) avid mlhcrcncc Ii) ex!racellular malr|R Ihr~gh hi|h-level cell.surface sion o| ~-.. I],-.. and I~-intcgrins: 21 high cap;Icily Io produce R,,clivc oRy|~n spc~'iP-~. 31 hip.J~ conical or serine p¢c~einascs ~nd =,.prole|n:se inhlhho~, and 41 tcolylic nctivily agninsl n sohlble pepl|de Imtnm! leukocyte cl'.lsllSe I~uhslrnle.
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I'l ! |clastin, and solid-phase I~hroneetln. even in the presence or pro~einase inhibitors. Jlowcver, p monocyles express liltle or no cell-surface II~-DR antigen, su~estin~ Ihol thc~ arc unable In Pa~icipale in s~ci~c immune rcs~m~cs. In contrnsl, the rcmaindcr o£ clreulating mon~ylcs have a law proinllammal(~ry ~lcnliM ~1 con- tain the ~pulsdon of mon~ytcs whh hlgb.level exprcs~o, of IILA-DR ~dgcn. P mon~yzcs can ~adily ~ ~paralcd £mm I;zc remain(~r ~;[ monocylcs on Ilzc ba~is or I) their capacity In adhe~ Io fibron~lln; m~l 2) Iheir ub;enl expression or II~-[)R antigen when flow cylOnlcl~ ~r immunml~z~nclic ~;.I,. are n~c~. Our {Ezt;I indic~le Ihal. when recruited to slice o[ inflammation. P nmm~)te~ elm citllcr pmmmc re~o- luli(m or in~ulnm~tlon or c~trihulc to Owcn. C, A., Camp~ll, M. L., Boukcdes, S. S.. ~mcrican Journal o£ ~ysiolo~y 2~7( I I ) :L77~-1.7~, 1994, Olhcr ~upporl: National IlCarl. Lung, and I]h~nd In~litutc and I)cp:zrtmcn{ ~zf Vctcra~ Affairs, From Ibe Division at Rcspiralory, C~ilical Care. and Occupational Pulmonary Medicine, Dcpanmcnl o[ Mcd~in¢. Un~versily L~ke Vetcran~ A~fairs Medical Center. Sail Lake Cily. UT. and Lung ~mmunobi~hcmlca~ Research La~ralow. Univcrsily o[ I]im~i~gham, Birmingham, UK. MONOCYTES RECRUITED TO SITES OF INFLAMMATION RXPRF~S A I)ISTINCTIVE PROINFLAMMATORY IP) PIII:.N()TYPE Only a minor proportion of. moaocytcs reSlxmds In chcmo:lllraclanls. To ICsl Ihe Ixs.'..',ihillty that chcmoattractanl.re~rmn,,ivc momK'ylcs have di,~linclivc runclimlal characteristics, we enriched or depIcled monncytc prepara¢ions For cells having Ix'ninl]ammalnry (P) phcnoeyp¢ and tested thclr rcslmnscs m biologically relevant chemoatlraclanls. Wc prepared monocylc suhpnpulalinns hy ~mc e)F tlzrce indepen- dent Icchniques Io minimize Ibe c.~mnces o1" arliFa~t.s: !1 dcpIcti~m of P mnnncytes by adherence to fibroncctin; 2) enrichment f.m" P monoeytes by negallvc selection rot IlIA-DR anliBen; and 3~ flow cytomclric so~in~. W¢ measured rc.'~onslvcnc~s of. monocyze suhpoj~lalians Io N-I'ormyI-Mcl.Leu-Pbe, On. zymo.~n-aclivalcd serum. a.~d monocyte chemoallraclanl pro~cin-I by Ihree parameters: I1 polarizalion. 2) aclin polymcrlzation, and 3) directed migration. With e~h cbemoatlmcionl and e~ch para- meter, there was n strikln~ direct relationship between the responsiveness of monocyle preparalions and Iheir corneal of. P monocyt¢~. Our dala indicate Ihal capacity of monocy~es Io be recruited rapidly f.rom Ihe vasculatnre into silos of. inl]ammalion i$ a propeny or a snbpopulalion of monocylc~ whh a distinctive, ncu- trophil-like proinrlammato~j phcnotype. Owcn. C. A.. Campbell, M. A., FJoukcdcs. S. S., and Campbell, F~. J. American/oumal of.Physlology 26"/(I I):l.TR6.LT96. I r~')4. 56 Other support: National lleart, Lung. and Blood Insthot¢ and Department of. Veterans From the Division of Rexpiralory, Critical Care, and Occupational Pulmonary Medicine, Dcpanmcm of Medicine. University of. Uta~ HeMIh Sciences Center. Lake City, UT. and Salt Lake City Veterans/,flair Medical Center, Sail L.~ke City. UT. CELl. SURFACE-ROUND ELASTASE AND CATIIEPSIN O ON IIUMAN NEUTROPIIII.S: ^ NOVEL. NON-OXIDATIVE MECHANISM BY WIIICH NEUTROPI IlLS FOCUS ,AND PRESERVE CATALY'TIC ACTIVITY OF SERINIE PROTEINASES Striae prutcinascs of human polymorphunuclear nculrophils play an imponanl role in neutrophil-mediatcd prolcotylic cvenls: |lOWeVCr, the non-oxldat|ve n~-cha. nlsms by which the cells can deg~dc ¢xlrac.;llu|ac matrix in Ihe presence or pro- teinase inhihitors have not been elucidaled, llerein, we provide Ihe first report that human oculrophils express persistenlly active cell surface-bound hum~ leul:ncyte claslur.e and cathepsin G on their cell surface. Unslimolated ncutrophils have mini- ma) eel) surface cxprcsslon of these enzymes: however, phorbol ester induces a 31). f.old increase. While exposure o1" neulruphils to chemoattractants (~,ILP and stimul-tes modest (two- to Ihreef.old) increases in cell surface expresslo~ or prnteinoses, priming with conccnlralions or lipopolysm:ch~rld¢ as low as 100 leads to striking (up tol0-1"old) increase in chcmoattraclant-induced cell surface cxpresslon, even in the presence of r, cmm prm.';ns. LP$-pr~mcd ~nd IML~-stlmolat- ed neutrophils have --100 ng of. cell surface human |¢ukucyte elastas¢ aclivily Par I(P cells. Cell suK~e.hound humnn leukocyte elasla.,.e ls ~tnlylically aclivc, ycl is remarkably resi.~la,I to [,hlh;tlon hy naturally ~.:cunir~g pmtelnase |nhibimr~. These data indicate that binding or scr;n¢ pro~e;nar, es to the cell surface Focuses a~NJ serves their catalytic activity, even in the presence of. proteinns¢ inblb|tora. Upregulated cxpressinn of pe~islently ~cllw: cell surf~cc-lx'~und scrlne pe01elnases on actlvnled nculrnphils p'ovides n novel mcci~anism Io I'acililale Ibelr egress I'mm Ihe vasculature, pen¢lmtlen o1" tissue ban'|era, m~d recrtl|lment into alice o~ ioflamma- elan. Dysrc~ulalion of. Ihe cell surface expression of. these enzymes has Ihe polenllal tO c~use li.~suc dcsln~ctlon during ;nflammatio~l. C)wen, C. A., Campbell, M. A., Sennas, P. L, Boukedes, S. S.. and Campbell, F... J. The Journal of. Cell Biology I ~31 (3):775-?89, November 1995. Other support: U.S. Public: Ilealth Se~icc end Ihc Dcparlmcnt or Veterans Affairs. From the Division of. Respiratory. Critical Care and Occupational Pulmonary Medicine. Depaelment of" Medicine. Un.lve~sily o1" Utah I{eallh Sciences Cenler, Sail I.ak.- City, UT, Sail L,'~kc VAMC, Salt Lake Cily. UT, nnd DepannRnl ot" Anatomy, Phys.'nlogicnl Sciences and Radiology, College of" Vcterlnary Medicine, Ninth Car~i ~.'t State Unlvcrsily, R~leigh. NC. 57
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C~ C") COLLAOENASE EXPRESSION IN TRANSGENIC MOUSE SKIN CAUSES IIYPERKERATOSIS AND ACANTI IOSIS AND INCR I':ASF.~ S[ISCEPTIIllLITY TO TUMORIGENF~IS In a ~rics of ¢~nsgenic mice, Ihe human I;~ coll~gcnasc g¢~ w~ cxprcs~cd in Ihc suprabx;al lair of !~ skin ~pi~e~is. Visually. lhe mice had dry and scaly skin which u~n histolog;~l ~alysis ~vcaled acanthosis, hy~keralnsis, and epi- decal hy~l~;ia. A~ t~ ult~astmctural level, inte~ellulur absent in the (~nsgenic skin cpld~;s b~ c~lact w~; maintained dtmugh the inlet d~smosomes. Despile a divcrSily o~ underlying edolngles, similar mo~hologi~l hy~ro~ite~tive changes in the epid~is arc observed in the human skin disca~s of lamellar ichthyosis, aloplc dermatitis, and psoriasis. Subsequent experiments demonstrate that when Ihe I~nsg~ic ~o~ skin was t~alcd o~c wilh an initiator (?.12-dimethyl-~n~laJanlhracene) a~ then twit= w~kly whh a promoter (12-~. t~tra~anoylph0r~l-13.acdatc), them was a ma~ked increase in ~u~r ;nci~ce amnng transgenic m~e c~parcd wilh that among ~lrol litte~ates. ~e~ cx~ci- meats demonstrate that by overexpr~ing the highly s~cific prolenlytic enzyme collagcnase, a cx~ade of events leading to profound mn~ho~ogical chang~ which augment the sensitivity of the skin Iowards carcinogenesis is i,hiated in the cpide~i~ D'A~iento, J., OiColand~, T., Dalai, S. S.. Ok~da. Y., Hung, M.-T.. Con~y. A. I I.. aM ~hada, K. M~l=cula~ and Cellular Binlogy Olhet sup~: U.S. Public Ileahh From Ihe ~p~m¢~l of Bi~mi~, Colle[e of Pharaoh, Rulge~-~e ~panmtnl of Medlcin~. Columbb ffnive~il~ College of PhT~kian~ and Surgeons. New Y~, NY, and D~anmtn¢ of Molecular Immunology and Patholog~ Sch~l. Cancer Research Inslilute. Kan~awa University. hhikawa. Japan. NE~VE GROWTI I FACTOR STIMULATES TYROSINE PIIOSPi IORYLATION AND ACTIVATION OF SRC I IOMOU)GY-CONTAINING I)ROTFJN. TYROSINE PilOSPIIATASE I IN PCI2 CELLS Rat PCI2 cells reqmnd In extracellular peplide growth factors in at Ica.~t two dislincl ways. When treated wilh nerve gmwlh faclnr (NGF) PCI2 cells cxll the cell cycle and diffcrenliate Io a neuronal phcnolypc, whcre-',.~ when Irealed wilh epider- mal growth factor. Ihey prolife~'ale. We examined II~ potential role ofSrc hnmulogy 2 (Sl|2).eontainlng protein lyrosine phosphalascs (Frl'~) i,, the difl'erenliatlon process. PCI2 cells expr~ substantial mrmunls or bolh SII-PTPI and 2. SII-PTPI, hut hal SII-PTP2. hecomcs tyrosine phosplmrylated I'ollowing NGF. bul not cpidcr. mal growth I'aclor Irealmcnl. The cnzymatic activity of SIl-i-rPi toward an exoge- nous substrate following HGF trcatmcnl is increased 2-raid. Wc round that SI I-PTPI binds to the NGF receptor TrkA in v[tro and that anli-TrkA immunoprccipitatcs have FI'P activily. These resulls slmw Ihat SII-IXI'PI is dlffcrenlially phnspIm~).lated and acllvatcd by NGF in PCI2 cells and suggest that this activatlo~ may play a role in NGF-imluced dill'ere.It;alton. Vambutas, V., Kaplan, D. R., Sells. M. A., an~; ChernolT, J. The Journal of Biological Chemistry 27lX43):2.~629-256..~. Ocl0her 27, 19~5. Other support: National Institutes of Health ~d Ihe Lucille P. Markey Ch:~rhable Trusl. From the Fox Chase Cancer Ccntero Philadelphia. PA, Eukaryollc Signal Transduclion Gm.p. Molecular Mcchanis,ns of Carclnogencsis I~boralon]. ABL- Basic Research Program~ and Nallonal Caucer Instilut¢-F~dcrlck Cancer Res~an:h and l)cvclopo~ul Center, Frederick. MD. TIlE UBIQUITIN-I'ROTEASOME PROTE,";. ~'TIC PATHWAY Mammalian cells conlsin two dlstincl ~'o,:ol)1i¢ pathways thai m¢ involved in difleren! aspects of prolcin l~'cakdown. Protclns thai enter Ih¢ c¢11 from Ihc exlr~ cellular milieu (such as fcccptor-medlated endocyloscd proteins) a~c degraded in lysosomes. Ly.~osomal degradallon of inlr~ccl|ular prolcins occurs mostly under slrcssed condilions. Nonlysnsomal m~chanis,.~s am responsible I'm Ihe highly live lumovcr of inlracellular pmtelns that occ~,~ under basal metabolic cm~ditlons, hul also I'or some ~spccts of" dc[,'radatlom of ~mr~cellulnr pmtclns under sl~css. An imporlanl nonlysnsom~l proleolytic pathway is Ihe ublquitin system in whlck pro. Ictus are degraded by a 269 protease compl. :" rollowln¢ conjug~tlon by mulliple molecules or uhlquitln. The "calalylic cam". d,e complex is u 205 pm¢ca~ com. plcx al~ known a~ the prolc.'~some. Ciech~,nover. A. Cell 79:1~-21. October?. 1994. OIhcr support: Unltcd Slnt,',~-Ismcl Biuatlon~l ,Rcicnce Foumlalion. Occma~.lsraeli Foundation I'or Scientific Research and Devclopmcnl, Israeli Academy of Ilumanillcs and Sciences. Israel Cam:©r S,ci©ty. Foundation for Promotion of Research at Tcchnlon. and a grant admlnislemd by the vice Ixesi~knt of Rsearch Tcchnion. From the Departmenl oir Biochemislry. Rappapocl lnstitule rot Research in Medical S,cicnccs. Tcchnion-lsracl |nstitulc ofTcchnolou. IJaira. Israel. "59
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O PROTEIN SYNTIIESIS ELONGATION FACTOR EF-Ir~ IS ES.~ENTIAL FOR UBIQUITIN-DEPENDENT DEGRADATION OF CERTAIN N'-ACETYIJ~TF.D PROTEINS AND MAY BE SUBSTITUTED FOR llY TI I1"~ FIACTERIAL ELONGATION FACTOR EF-Tu Tar£.cling or different cellular proteins For c~mj.gali~m aml suh~qucnt lion via Ihc ubiquilin palhw-ay inv(~lvcs diverse rcc~gnilifm xign:ds and (li.~lincl cn/.y- made f-',ctors. A few proteins arc recognized via Iheir N-Icm~inal m,irm acid rc.~id,c and conjugated by a ubiquilin-pmZein liga.~e thai recognizes Ihi~; re~i(lue. Most snh- sleaze% including Ihe N'-acclylalcd pr~ein~ Ihal con.~lilule Ihe vnsl m:zj~rily or cellu- lar protcin~. ~rc targeted hy dil'fcrcnt slgn;ml~ and ;arc ruc(~g,i/cd hy ycl uukn.wn lig- uses. Wc have prcviuu.~ly shown Ihal degradation of N-tcrmin.'dly blocked prnlcios requires • specific faclor, designated FII. and Ihal Ihc I'aclor acts along wilh p~otea,;e complex to degrade uhiquitln.canjugalcd prolcin~. Ilcre. we dcm(mslralc Ihal FII is the prolein synlhcsJs e ongalion l'-',ctor EF-h~. (u~ I';~nlal sequence analy- sis re~'aals 100~ identily to EF-I~,. fh) Like EF-I~. FII binds to imumhilized GTP {or GDP) and can be purified in one slap using Ihe concsponding mml¢olidc I'or clu- lion. (c~ Guanine nucleolides Ihal hind to EF-la pr(~ccl d~e uhlquilin syslcm-rcl;~lcd aclivity of Fil lrrom heal in•city•don, and nuclculhlcs Ih;~t d. n.I hlml du m~t cxcd Ibis effcch (d~ EF-Tu. Ihc homuk)~ous haclcrial chmgaliuu f-',cl.r, can suhstilute I'[~r FII/~F-h~ in the protcOlyliC syslcm. This lasl fimlung is of panic|,lar inlerer4 since Ihc ubiquilin syslem has no( been identified in prnkarynles. The activilics EF-lu and EF-Tu are strongly and specifically inhihilcd by ubiquilinahlchydc, a specific inhi~tor of ubiquilin i.'~lpeplida~es. I! altars, Ihcrcf.re. th.'~ I~l:- I ~ may bc involved in rclcasing ublquilln from mulliuhiquilin chains, Ihus rendering Ihe conin- g•to,; su,;ccptihlc to the •allan of the 26S wore•so c.mpIcs. Guncn. I1.. Smith, C. E., Siegel. N. R.. Kahana. C.. Mere|ok. W. C., Ch-'|kmbo.lty. K.. fichwartx. A. L.. and Ciechannver. A. Procccdlngs nf Ihc National Academy or Sc|eoccs USA 91:704g.76~2. Au~:u.,;t I (Y)4. Olhcr support: Unltcd States-Israel Binaliunal Sclencc Rmmlati.n. Gcrm:.n-I.;r;~cli Found•lion for Scientific Re.arch and [)evel,)potcnt. I~racll Ac;ulemy of Sciences and Ilumanities. Monsanlo, Rappapo~l Foumlalinn. Fmmdnlinn rue Pro,n.llnn Research in Ihe Technion. and Ihc Alhert Gmxh, tein Research Fund [~dminislcrcd by Ihe vlcc president of the Technion for Research. From the Department of Biochemislry, Rappaporl Inslitutc for Research in the Medical Sciences. Tcchnion-lsrael Institute of Technology, I IoiFa. israel. Monsunlo. Inc.. St. Louis, MO. Dcparlmcnl or Molecular Virology and Genetics. The Welamann lnsdlulc for Science. Rchovol. Isr~l. Departmenl of rJloc'fiemism/. Case Wcslem Reserve Universily School of Medicine. Cleveland, Oil. Departmcnl or Biochemistry. Medical College of Wisconsin. Milwaukee. WI. Edward Mnllincrodt Dcpa~menls of" Pcdialrlcs and Pharmacology. Divisoo of Ilcmatology-Oncology. Children's llospilal and Washington University School nf Medicine. Sl. Louis. MO. TIlE UI]IQUITIN.MEDIATED FROTEOLYTIC PATIIWAY: MECIIANISMS OF ACTION AND CELLULAR PIIYSIOLOGY Ubiquilin modifiealiun or many pn'Jlcln' larEel~ wilhin cells ph~ys impurlunl rol~ in a variety of biological processes. Amm~¢ Ihese am maul•lion orgeoc cspms- stun, regulation of c¢11 cy¢1¢ .,nd division, involvcmcnl in the cellular stress r~pon~, modification of cell surl'~e reCel~orx, DNA rel~ir, import of pfoleins inlo mitochondria, uptake of precursors o1" neurolransmitlcrs into synaptosomes, htoFne- sis of pcmx~somes, assemhly o1" ribosomes, and pro~r~mm~ cell death. The mocha. nism.s thn! underlie these complex Fracases are poorly under, load. The besl sludie~ modificalion occurs in the ubiqultin-med;-'zted pr.olcol),tic pathway. R~:enl experl- menial evidence indicalcs Ihal the ubiquilln s)'~tern is involved in Ihc del:n~lalion of milolic cyclins, ononprotcins and tumor supp~.ssars, in Ih¢ removal or abnormal and otherwise damaged pmleins, and in proccssin. ,r nnligens mstdcled to class I MIIC mnlcculas. Degradation o1" a protein via Ihe u:.~uitin system involves Iwo dibble steps. Initially. multiple uhlquiltn mole•ales =P. covnlemly linked in an ATP.depen. dent mmle Io the protein subslrate. The largeled prolein is then de~raded by • specific, cncrgy-dependenl and high molecul~ ~n;.ss protcas~ complex inlo pelf;des and flee amino acids, and I'rcc and reulilizablc ~::,iquilin is Rleas~d. In addhion, sla- ble mono-ubiquitin adducts are also round in '.1~ cell. for example. Ihose involving nuclcosomal hisloncs, D~pile Iho considerable progress Ihat has been mad(: in elucl- doling Ihe mode oi" aclinn and roles of the uh|qullln system, many probk:ms remain unsolved, l'-~r example, liltlc is known on the ~;l~n~ls Ihal t,~r~cl Fml¢ins rot de~rnda. lion. While a raw proteins arc largctad for deg~adatino rollowin¢ recognition o1' Ihelr N-tarminal amino acid residue, tbo vast majorily o1" cellular pt'olcins are Im'geled by olher signals. The idenlity o1" Ihc nalivc cellular subslrales or ihc syslem is anOlhCr Impmlant, ycl t,nmr.olved problem: only ¢ few l~'olei.s have t~.'en as suhslrate.~ at tim syslem it~ t'iw:. The scope o! Ihls review is Io di~uss the mocha. nisms inv,lved in ubiquilin activation, selecti.~n~of,~ubstrales fo~ con]uB:tino, and degmdallon n~ uhlquilin.cnojngaled pro~clns in Ihe cell.l'me system. In nddillOn, we shall st,mmarize whal is cur~cnlly known uf Ihe physiologlc:d roles or uhiquitln- mediated pmteolysis h~ Cieehaunvero A. Biological Chem L~lry I IopIX:-Scyler .175 :.';6.~..~ ~ I. Scplem~r 1994. Olher support: German-Israel Foundation for Scientific Research and ID~velopm~nl. Israeli Academy ol" Ilumanilles nnd Sclenec.s. Israeli Ministr# or Ilcnhho Cancer Research Fund. Israel Canto Soclely. I ,-~:ndallon f'(x PYomol|no of Research in the Techoion. Ilcdson Fund I'or Medical Resc;;rch. and Monsonlo. Inc. From the Department of' Biochemistry, RaFj.,-.~rt Insthulc /'or Research in the Medical Sciences. Tcchn[:)n.lsrael Institute ol"£ccl:nology, llaira. Israel. DRO,~;OPIIILA RIBOSOMAL PROTEIN $3 C'.~.:,"TAINS AN ACTIVITY TllAT CLEAVES DNA AT APURINIC/APYRIMID. • ;C SITES A ral cDNA-cncndlng ribosomal protein $3 was used Io clone the $3 baronial in Drosophila melannl~as~er. The Drosophila Bane was in lure used to conslrucl fusions belween $3 and ~:lulnlhionc $-tran*f~.rase that were overexpresscd in" 61
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O TIlE ROLE OF TWO CONSERVED AMINO ACIDS. GI.UTAMINE 9ll AND ASPARAGINE 137. IN O'-METIIYLGUANINE.DNA MI~'I'IIYLTRAN,~FFjtA.,;E STABILITY, ACTIVITY AND SUBSTRATE SPF.CIFICITY To assess Ih¢ possibilily Ihat two conserved amino acids (glutamlne 90 an asparaginc 137) in O'-mclhylguanine-DNA methyhr~)sfer~.~e (MGMT) are invnlve~ in protein-subslrate cam•el mid/at disc~mination bClwccn favored and non-favored sub$1rales, families of i~.ole|ns mulanl at Ih~'.~ two silos were ex~'essed in transrerase-deBcient bacleria and analy-~.ed for sl,,bilily, ahillty to r©l~ir O'-mclhyl. guanine (MG).conlaining DNA. a~d abilily Io differentially repair a pre~'en'ed (MG. conl-"inln8 DNA) versus • no~-prcfcrred [free h~se MG) subslr~t¢. All seven pro- teins mutant al glut•mine 90 (¢xcep~ a F, rollne mul-',nl) were stable in h.'cleria and repaired MG-comalning DNA [>50~ of wild.lype levels). A reprc~ntatlve glut•. mine 90 mmanl prol¢in was not, however, significantly differen! from Ihc wild.tvl~ prolein in the pr,'fcrcmial repair of MG-conlaini,g DNA versus MG free base.'~)f eight I~'Oleins mul,,nt al aspatagine 137, only glulamlne aml serine mulanls repaired Me.cam•thin8 DNA ~o any dc~rce (g.;~% ,,nd 0.8% of wild-type respectively) and only Ihe glulamine mulant proleln was delectable in b;~clcrlal sonlcate~ hy Weslern 62 Nol analysis. Alanine and let~cine molanl alkyl.l~nsfemses, in,clive and un~cr.,Me as nnn-fusion protein.,;, could, however, be slably ,'xprcssed in bacleri', ~s ¢lmathion0 S-Irnnsl'erase I'usion pmlcins, allhough li¢ proteins we~© still in-',cl|ve in These results suggesl Iha! while elm•mine r.,~ h,,s no ditto! role in MG-DNA melhyhransl'er~se-medlaled repair or free b,'~c/lesioned DNA subslr~te spcci~clty. ~p,,ragine 13'7 is imperlan! in hath Ihe stahilily ~nd acllvily or Ihe prattle ~d may conlribule Io Ihe fom~alion or function of Ihc ac~,ive site of the IXo~cin. PiepCro R. O.. Morgan. $. E., and Kelley. M. R. ,l~tulsch, W. C~rcinngr'nesi~ I.~(9):11~95-1902, 1994. Olhcr support: Nalional Inslilules of lie•lib. From Ihe Divisiou or Ilcm:mflngy/Oncology, Depnrlmen! of Medicine and Ihe Progr.~m in Molecular I~iology. $1ritch Sch:),nl of Mndicine. Loyola Unlver~ity Medical Cooler. Maywmxl. IL..~ml Department of Pediatrics. Seclion or Pedialric F.ndncrinnlogy, Wells Center for Pediatric Rese-",rch and Biochemislry and Molecular I~i~flngy. h,dinna U,fivcr~ily Schm~,l of Medicine. Riley IG~:~phal for Childru~. In,li.'p~=p~diso IN. CLONING OF TIlE MULTIFUNCTIONAL R,~ I" APURINICIAPYRIMIDINIC ENDONUCI.EASF. [r^PEN)/REDOX FACT~ ~:'. FROM AN IMMATURE T CELL LINE The cloning of Ihe tel AP endonuclear~..~ene affords us the o~perlunily IO invcsligate the Rgulallon of a crucial mullif;,,:ciional enzyme having bolh DNA rcpalr nnd index aclivily followi~g v.'winus p~n',dlgms in on o¢~..'mism Ih.'tl h=~ hcfn cxtcnsivcly used in research, parlicularly in I1~. areas or acing and hormonal conlrol of gcnc expression. Wilson. T. M.. Carney, ]. P. ~nd Kelley. M. R. (~)-.ulsch, ~Y. A.) Nucleic Acids Research 22(3):530-531. 1994. O, her soppon: Nationnl lnslhules of II~hh. From Ihc Department of Pedialries. Section or Pedialrle Endt~crinolog~. Wells Center Ior Pedialric Research. Riley Hospital. Indiana Universky Medical School. Indianapolis. IN. and Program in Molecular ~iology. Loyola Univer;ily Medical School. Maywood. IL CALCIUM PULSE.S DURING THE ACTIVA .-~.ON OF A PROTOSTOME EGG Transient incrcn.~s in free cytopl~mlc Ca~" p~s through the eytosol or • wide ver~ely t~f fcrlilizing dcutcroslome eggs .nd do so I'ro~l the spenn efllry poinl 63
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C) C~ anlipode. The~ CaJ" waves occur through the release nf slorcd Ca", and they pro- vide mos! or all of the ,,etivating stimulus roe these eggs. l]asetl on several indirect llnc,~ of experimental evidence, it was i~lmsed teal protostomc eggs a~c ~cl~vnted by a prolonged upl=ke ~ff Ca~' from Ihc mcdlmn due I. s~.n-ind.ccd mcmhra.e dc~l~izali~, a~ Ihal Ihis u~ake then sla.s a. uctiv:~linn wave similar to Ilmsc in deulcroslomcs, cxcepl Ih=l it moves inward from Ihc wlmle surr~ce ralher across Ihc egg from pole Io pole. To lesl Ihcse hypnlhc~cs, we mlcminjecled Choelz~pleru$ prrR~mentaz-~s o~yles with semisy~tl~ctlc ~ccnmbinam ~e~luorins and measured lighl emission in res~nse ,o ~lh rerliliz~lion and a~ificial .cdv~lion by exce~s K'. ~kberg, W. R.. Miller, A. ~, S~n. ~ G. ~nd Jaffc. L. F. Biological Bulletin IR3:289-2~. ~to~r 1993. Oihcr sup~: National Science FonndalJon anti the NaHonal Instizotcs of I Icahh. From Ihc Marina Biolnglca1 Laboralory and Department of Biology. Iloward Universily, W~shinglon. CONSTII"UTIVE AND INI)IICIIII.F. xll ilINI)IN(; ^CrlVITII.:S IN TIll~. CYTOSOL OF v-REL-TRANFORMEU LYMI'I IOID CI:'I.L.S Constitutive and inducible KB binding aclivitle.'~ as'~ci;,tcd with v-Rel oral c-Rel in the cylosol of v-Re-t~nd'onned cells have been identified. The.,¢ activities were resolved by elettrophocctic mohility shift ;L';say and chromatographic technltlucs into a high-molecular.weight prolein-DNA complex designated complex I containing v- and c.R¢l and lower-molecular-weight cumplcxes B. III and IV which contained only ~-Rel and which were stimulated by nucleotldes, low pll, and detergent. These experiments suggest thai interaction of v-Rcl and c-R¢l decreases the DNA-hinding activity of each. IIodgson. J. and Enrietlo, P. J. Joum',l of Virology 69{3):1971-1979, March 1995. Other support: National Institutes of Health. From Ihe Depanmenl of Microbiology, State University of New York. Stony Brook. NY. DENDRITIC CELL PROGENITOR IS TRANSFORMED BY A CONDITION^I. v-Rel ESTROGEN RECEPTOR FUSION PROTEIN v-RelER A conditional v-R¢l estr~en recel~or fusion protein, v-RelER, causes estrogen. dependent but otherwise unaltered v.rel.specific transformation of chicken bone marrow cells, llcre, we demonstrate Ihal sn=h v-rehlransfonnrd cells exhlbil B lymphoid determin,~nts in line with earl;or ~iudics on v.r¢lER.l~nsformcd cells. llo~vcr, following inacliva(ion of v-RelFA~ o~ol¢ln ~livily by ~minisl~ion of an c.~lrogcn nnla~onlsl, cells diffcrenliole imo ~ligen-~lin¢ de~filic c¢11~ Is judged by several mo~hologlcd a~ funereal criteria. Addhionally, u~er ~el dlt- fercnl cuhum condilions, v-relKR cells diffc~ial¢ ;too cells Rsemblln~ ~- phonuclcar neulr~hils. O,r slndi~ I~refor¢ suegcst ~al I~ co~ili~al v.R¢IER, and probably also Ihe =ulhenli¢ v-R¢l, I~nsfo~ a comm~ progenil~ for ~u- trophils and ~ndrilic cells. B~h~h, O., Madroga, ~., Do~cr. P.. Br~, ~, K.. ~hwa=, !1., Enr~llo, P. ~, ~d Cell 80:34 I-3~2. Janua~ 27, 1995. Other supeR: National Inslilules of Hcallh. F~ the In~ilulc o~ Mnl¢c,lor Palhnlogy, V~nna, Austria, Max-Pla~k-lnslkul fur Fmlwickl~gshioiogic, Tubingen, Federal Re~blic of Ge~ny, and ~nmem of Microhiolngy, Slnl¢ Universily of New York al Slay Br~k, Slony B~k, NY. TI IE ROLE OF TIlE CARflOXY TERMINUS OF v.REL IN TRANSFORMATION AND ACTIVATION OF ENDOGENOUS GENE EXPRF.-.SSION Proleins wilhin Ibe Rcl/~qF-KB Inmzcri.nllon f.~'tor family can be divided inlo two fonctional domains, a homologous am!~¢,~ Ic~mlnal rcl~lon, the R¢I Homology Domain. and a divergent carboxy Icrminal demain. The amino tennioal s~quences specify DNA binding, nuclear localization, and inlemClion wilh the Ixl] famil7 of inhibitory proteins. "The carboxy terminus of each i~olein functions es ,~ transc~ip. fional activalinn domain, however, i~ecise d~.~nhioe of sequence Rqultemems l~en diffi~ulL To furlher define'thcs~ ~u~nc~, sm|ll 100 b~ ~eletlo~s we~ con- structed throughout Ih¢ carboxy lermlnus ~..~ v-Rel. E~¢h resultin| mut~nl ass.~yed for DNA binding. Io~-~li~fion, p~o,'~in complex fonnallon, ncliv~io~ of endogenous ~ene expressio~ and abilily Io transform ~ m~Tow cells ~ blasls. Suq~rislngly. deletion within Ih~ cerboxy Cerminus hsd m~rBinel effects on Ir*nsformio~ polenllal. |lov~ver, Ihme sepa~te ~loe~ were requJ~'~ fo~ full lion of geoe expression. Taken Io~rJher. the~e rcsuhs su~es! th~ the e~boxy tcmd. nus of voRel contains multipl© sequences Ih:d pnlcipate in the ,,~livatlo~ of exp~ession. Smardova, ,f., Walker. A., MorHson, L. F-. Kab,'~n. N., and Enrlello, P. Oncogcne 10:2017-2026. 1995. Other supporl: National Instilules of lleallh. From the State Unlversily of Now Yo~,. al Slony Brook. D©p|tlmen! Microbiology. Stony Brook, NY. 65
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INVOLVEMENT OF RAL OTPase IN V-Sre-INDUCED PIIOSPI IOIJPASE D ACI'IVATION An ¢"rly response to the lyrosinc klnase activity of v.$rc is an increase in phos- pholipase D (PLD) =clivily. which leads In the 8crier=lion of hloloBieally =clive lipid second mcs``en~ers, including phosphatidic acid. lysophosphatldlc acid and di=eyl- glycerol. We have recently demonstrated Ih,,t v-Src-induccd PLD activily is mediat- ed by Ras. althouRh Ras iovolvemem was indirect, requiring a tyros=lie factor ft~r PLD ,,city=zion. Ras interacts wilh and activates RaI-GI)$. Ih¢ exchange raclor responsible for the =clivalion or Rat Gl"P~scs. I Icrc we feiu)rl Ihat this newly identi- fied Ras/Ral ``ignalllng palhway mcdialcs PLD activation by v-$rc. PI,D acllvily could be precipitated from v-Sre-lra~sfnnned cell lysalcs with immnhilixed RalA protein and with an anl;-Ral antibody, A mutation Io the region of RalA anal=gnus to the "effeclor domain" of Ras did not t'edoc© the -'.hility of ItalA t. complca with PLD, ahhoeRh delelitm of a Ral-spcciGc amloo-lerminal region did. Ovcrexpression of RalA [xllentlated PLD "-,ctivaliun by v-Src..'rod expression of dnmina,t he,attire RatA. mutants inhihlted both v-$r¢- and v-Ras-induccd PLD activity. Thus RalA is involved in Ihe lyrosine binase =city=lion of PLD through its unique N Icmtinus, and that PLD i`` a downstream target of a Ra.VRal GTP'ase cascade. |ian~, I1.o Luo, L-Q., Urano. T., Frankel. P., Lu. Z,, Fusler, D. A., and Fcig, L. A, Nalure 378:409-412. November 23. 199J. Other ~uppocl: Nalilmal Inslilule~ or ! lealth and American Cancer ~oci¢ly. From the Inslitut¢ or Biomol¢cular Structure and Funclion. D¢partmcnl of Diolo¢ical Sciences, Iluntcr Colic@ of City University o1' New York. New York. NY, and Department of Biochemi~lry. Tails Univ¢l'sily School nf Medicine. Boslon. MA. SELECTIVE ACTIVATION OF PROTEIN KINASE C ISOFORM$ BY v-Sr¢ Prolein kinase C (PKC) is • gene family consisting of no less Ihan I I dlslinct is=forms. In both murlne and r=l fibroblasls, we detected expression of four PKC i, sofomts: the convcotio~al PKC ~. the novel PKCs & and (. and the =typical PKC ~. With the ¢onvemlo~al and novel PKC isororms, membrane ass=el=lion has bccn used to shew PKC nelly=ion. In cells transfonned by v.$rc. 1here was = Ca:'-depcn- dent increase in membrane association of Ihe et is=form relative to Ihe nontran~ formed parent=| cell~. The ~ isorom~ had ,, slilIhlty increased membrane association in murine fi~oblasts transformed by v*Src but not in ral fibmMasts If==stormed by v-$rc, llowcvcr, since it is nal clear whether cellul=r dislributlon of ~ isofonn corre- lates with =city|lion, lhc dala are inconclusive with regard to this isoform. Interestintely, of the C=~'-independcnl PKC isornmls E and ~, only the ~ isornrnr was preferentially =~sncialed with membrane rraclions in v-Src.transfonncd cells. The lack or PKC e aclivalinn was not due to lack of resl~msiveness I¢I diacylglyceml (DGI, ",inc¢ cxo=cnou``ly .,.uppllc:d DG and phndxd ester were Ixllh able Io induce membrane -s,,ocialinn nf PKC e. Thus, the dilfmenlial activation of tl~c ~ and ~ iso- f.nn`` by v.Src suggest,, # omr© complex mechanism for Ihc ~tctivati.n of the m~vel Ca"-inde~ndenl PKC Isoroe~.~. involvin~ more than simply elevaling DG levels. Since PKC has been implicated in the ini.'~ccllular signals aclivated by v-S~c that I¢~1 to tfansro~ali~, the sckcllve acllvalion of PKC ~ ~d 5 su~Fsts a talc in m~togtncsis and transfo~lion for t~c ~ng. Q.. F~nkd. P.. a~ F~ler. D. A. C¢11Gfo~h & Olffe~nli~6~ 6:1367-1373. Novem~r 1~5. Other st~p~: Nallonnl I~slilulcs of lie=lib. ~fOII1 llm IflSIiIUlC for Olomolccnlar Sl~clu~ and F, nctlon and Ocparlntenl or Biological Sciences. Ilumcr College of ~c City Univc~ily of New York. New YoA. NY. COPPER. ZINC SUPEROXIDE DISMUTASE IN F_.$CIIERICIIM COt J: PERIPLASMIC LOCALIZATION Cn.7.nSOD porified from R~rlzerirl, " d/hats been u~ed In raise anllbo~ies in rahhils. The rcsultanl anliscrunt was Ion.0 t .iv recognize a single b~ml (~ Western blots of SDS-polyacrylamltle gel elcclrnl'q ;ingrains. and.t.hat s.in..gle b~..nd coincide1! whh tl~c position of the Cu$'.nSOO, Ull¢~th:n scctio~s m .xcoc. ¢oti were Imate. wilh the antilmdy followed by protein A ~'*.aring 10-nm gold parllclcs. El¢clroo microscopy revealed that Cu.ZnSOD was |f.tgely localized in Ihe periplasm in I~tlar t~lys. 13cnnv. U, Chang, L. Y.. Day. I]., and Fr-'.r ~vleh, L Archives of Biochemistry and Biophysics 3 i9(2):.~g-.'; I I, June I. 1995, From the Departments or B|ochcmislry "rod Mcdiclnc. Dtikc University hlcdi~=l Cenlcr. Durham, NC. A CATIONIC MANOANtC PORPIIYRIN INlllBn'S UPTAKE OF P&RAQUAT BY ESCIIERICIIIA COLt A m',nganic porphyrio (MnTMPyP). which catalyzed the distant=lion of f~cilitated the aerobic growth of a s~4A s~lf~ strain or EscAerichta co/( and ~clcd a s,pcroxld¢ dlsmulas¢ ($OD).eompelent parental strain against paraqual. Surprisingly. the latter err©ca was mole • .-.~plcl© than the former and the mimic co.hl block Ihe imh,clinns of fitm~rese C ~,..I ~1" ~lucos¢ 6.phosphate by paraq,nl, even though SOD couhl not. An explanatkm Gw the~¢ ~pptt~¢ot d,scs was finned in I1~ ubilily or MnTMP;tP l= inhlbh the uptake of paraqnn! b~' E.
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('nil MnTMPyP was accumulaled by E. ('rJfi until its; intracellular conccnlrmlon was 20-fold ~rcalcr Ihan Ihe exlraccllular concenlralion. Thi.~ happened in a glucose pl.s salts medium, bul hal tram a rich LB medium: MnTMPyp ~s ~und Ohio cellular macromolecules and was mainlaincd in ~hc rnduccd s~alc wid~in E. earl. ~e ~rce fo~ of Ihe rcduccd MnTMPyp was auloxidiT~ble, hul Ihc ~)und form was hal. Conscquenlly. Ihc f~c form could ~lalyze Ihc nxidalinn ~und ~o~ did hal Limber. S. !. and Fridowich, L Archives o~ Bi~hcmisl~ and Biophys~s 321 f I):271-275. A From Ihc Depa~menl o~ B~hcmJs~. Duke Univcrsily Medical Ccnlcr. Durham, NC. STAItIIolT.ATION OF TETR AI IEI.ICAI. DNA IIY TI II-: f)l fA I)1( I I1'1 .F. I)NA IlINIJINf; I~l((j'l'l.'JN • The $7-kDa hep~lie nuclear prolcin QUAI~ hind*; lighlly ml¢l .';l¢cificallv a par. allel telrahelical I'nnn o~" Ihe IgG switch regiun DNA (W~:i~..m Sf..n~ p -~ml Fry, M.(1993JJ. lli~.Chem. " .... ' "" and ~¢oming [ully ina+liv~led only +fl~r ]0 rain +1 I(~1+~. "1~ ~emon+lralc th,I ~D prolecl+ ~un~ quadmplex DHA. m,k~ and OUAlJ-~,nd I+ln~h~li~++ ~ilcd. Ihe prolei~ residue in die ~mplex w~+ digcsl~ willl I~+in all~ quadrup.ex and sin+lc-slrand fore+ ~[ I~ ~NA ~enl wcrc r~mlv+d hp el~rophnmi~. ~h~r+~s naked quadmplex D~A ~ Fully dcnalurcd aflcr DNA. Weism~n-Shom¢r. p. a~ Fr~. M. BJ~hemicil and Bio~ical Rc~a~h Communlcal;nns 2(l~(I):~5-~ I I. Novcm~r ]0. 1994. Olhcr supeR: U~.-Is~el Binalio~! 5clcncc Fu~, Israel ~;cncc Foundalion. and the Fund f~ ~motion of Resea~h al ~¢ Tc~nion. From Ihc Unil o[ Bi~hcmis~ty. ~¢ B~¢c Rapp;Iporl F=cuhy o~ Mndicinc. Techni~-~sracl Inslhule of T~hno~ogy. I lai~=, THE CDC2*RELATED KINAsE. PISSLRE. IS "ESSENTIAL FOR GROWTI I AND ACTS IN G~ PHASE OF TIlE CFJ.I. CYCLF. [Hammllion cell cycle progression is r~gulated by several prolein kinasc.,; 6~ ~e activaled by cyclically expressed i~oleih-• called cycling. These cyclin-depcndcnl kinase~. Ihc pmloly~ phosphoryla{¢ subsl~ales kinase h~ ~en dis¢ov~d. ~c2 ~TAIRE amino acid domain. I1~ ~minant-nc~afive mulam cells. ~ emwlh suppr~si~ PISSLRE hall ~11 cycle ~r~inn in C. ",. c~llul~r pmli~emlion, ~nd ils c~fc¢l is exc~, :¢ ~n G~-M. ~is dc~ri~ I~ ~m evl. deuce since cdc2 o~ ~ cdc2 ml~led hinge ~¢lir:~ Ihmugh G~-M. Li, S.. MacLachlan. T. K.. De Luc=. A.. C~ludio. P. P., Condorelli. O.. and Giord~no, ~. Can~cr Re~carch OIh~r sup~d: S~ From Ihc Jerker;on Cancer I.~l;I.le and Dcp~rlmenls of Microbloloey and lmmunolo£y =~1 Palholb~y. ~mas Jcr(=.,m Univc.ilx. ~lla~lphla, PA. NOVEL NFAT SITES THAT MEDIATE ACTIVATION OF THE INTERLEUKIN-2 PROMOTER IN REPON.~i~ TO T-CELL RECEPTOR STIMULATION The tran~riplion raclors NFAT a~! AP-I have been show~ Io be eisenlial for inducible imedcukin-2 (IL-2) exprrasio~ in ,~_"tivaled T c~lls. NFAT has been pr~vl. ~msly rcporlcd to biml Io Iwo ~;iles in the IL-2 prom)let in ,,*sociallOn wilh AP-I al Ihc distal anligen rcspou.~ elcmenl al -2~) and ~ - 135. On Ih¢ bests or DNas¢ I ~'oolprinlin~ wilh n~combinanl NFAT ~nd AP-I prmeins, gel shiR aum~. and fection expcrimenls, w¢ have idcn~iF~nd Ih~e nddilional NFAT skea in Ibe IL-2 pr~ motu. Slrlkin~ly, nil five NFAT silos am ¢ -s.~,dlal tar I1~ rail induclio~ of promoqer activity in RspOnse to T-cell Rcq~to~ sllm~dvtlo~, Four or the five NFAT sites purl or composke elements ~ble Io bind AP- I in nssoclalio~ wilh NFAT. Thc~ sties display a diverxe range or cooporalivlty ,,nd interdependency on NFAT and ^P. I proteins for bindinlb O.¢ or ihe NFAT sties dimclly overlaps I1~ CD28-resp<msive element. We pr=ent evidence Iha! CD28 in~,| :bility is co~rcm:d by ,he AP.I eom- pan.at in NFAT-AP-I composite el¢~¢nls. "rl..~¢ findings providc funh<r in;i~h! inlo Ihc mcchani.~m involved in the rcgululio, o1" the IL-2 promoter. Rooney. L Wo. Sun. Y,-L. Gllmehero L. !1..: .,d Hocyo T. Molecular and Celh*lar Biology I ~( I I ):62~9-¢:.~ 10, November 1995. Other supporl: Tularik. Inc,
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O ",,4 0 From the Dcparlments of Gcnclics and Medicine, Ilarvard Medical School. and D~parlmcnl of Cancer Biology. llarvard School or Puhllc Ileallh, Boslon. MA. 'and Tularik. Inc.. South San Francisco. CA. TIlE DESMOSOMAL PLAQUE: ROLE IN ATTACI IMF.NT OF INTERMEDIATE FILAMENTS TO TIlE CEI.L SURFACE Cyloslceletal-mcmlx-ane at~achmenls play an iml~nanl role not only in regulal- ing cell molilily, adhesion and mainlenancc or cell shalx:, hut also in tran.,~lucing sig- nals generated at the cell surface. The~ attachments arc often mediated by intercel- lular ]unclions that comprise macromoleculax complexes .~peclali~'cd for interaction wilh a specific fllamcnl eypc. The most prominent ecll surl'acc attachment site for intermediate I~lamcnts (IF) is the desmosnm¢, an intercellular junclhm I'ound in abundance in lissues Iha! experience mechanical .~lres~;. A nmnber ell" nmlcctdcs in the dcsmosomal plaque have been suggested as candidate I1: linkers, including desmoplakins, picot;n, 300 kD IFAP. d~mocalmln, hand 6 protein, and a 140 kD laminlike proicin, or these. Ihc most ahundanl and uhiquilous con,.llments are d=smoplakins (DPs). Desmoplakin (DP} I and II arc two major related prntcin.~ hx:atcd i. die inner Ix~io. of ~he desmosomal plaque. The predicted sequence of DP I suggests it will form a homodlmer comprising a ccolral a-helical coiled-coil rod and two globular end domains. The smaller spliced fonn. DP I!, apparendy has identical end (hnnains but lacks Iwo thirds oir the central red. The C-Icrminus clmc~ins Ihren reginns with significant homology, each of which is made up of a 3g-residnc motif also found in two olher molecules involved in organization of IF, bullous pcmphigoid antigen and plectln. When polypcptidgs including the C-terminus ol" DP ! are ectopically expressed in cultured cells Ihe proteins align with keratin and vimentin IF, whereas those lacking the C-lerminus do no~. The lasl 6g amino acids ol" DP arc required for alignment along keratin but nol vimemin IF, and residues 4g-~ from Ihc C-termlnal end axe critical roe this interaclion. These r~uhs suggest Ilia! the C-lenninus o1" DP plays a role in Ih¢ atlachment of IF Io Ihe dcsmosome and Ihat th© presence of a cific site is access:u), for interaclion wilh keratin IF. A sequence at the most N-ter- minal end of DP appea~ to be required for clTicicnl incorporation into the desmoso- real plaque. Inlerestingly, Ibis region has not been reported in die homologous bul- Ious pemphigoid antigen or plecdn molecules and may represent a dcs~nosomal tar- geling sequence. Green, K. J. and Stappenbeck, T. S. in: Cili, S. (Ed.): Molecular Mechanisms ol" Epi|hclial Cell .functions: From Development to Disease. R. O, Landes Company, pp. 1:57.171, 1994. Other support: National lnstitulcs or Ilcalth, American Cancer ,~;¢)cicty. M.rch nf Dimes Birth Defects Foundation, and a Focused Giving Award from Johnson and Johnson Co. From Ihe Departments of Palhology and Dermatology, Robert lh I.urie Cancer Cooler, Northwestern University Medical School, Chicago, IL. 7O STRUCTURE AND FUNCTION OF DESMOSOMAL, TRANSMEMBRANE CORE AND PLAQUE MOLECUL.F~ Desm~omcs arc inlcn:cllular junctinns Ihal funcl;on in cell.cell edhesin~ and allachmcol o( inlgrmedialc filamonls ',li-') In Ih¢ cell surface. Dcsmo¢lcins anti dc~mocollin.~ are the major component':- ~f Ibe Inmsmcmbmne atlhcsion cmnplrx,' whcm~s desmopl.'~klns (DPs) are Ihe me. ;womincnl ¢ompenenls or Ihe cywplasmi¢ plaque. Ba~d on seqoence similarity, c~,~.,,mogleins and desmocolli~ m~ r<l~led the calcium-dependent homophillc adhesioll molecules known Is cedherin~. Like die classical cadhcrlns, lira dcsmosomal cedherins cool,in four homolnl~ous eXlmcellular domains hc~ring pulnllve calclum.hlndk~', --|los, a slavic Iransmembl-ane spa~nlng domain, and • C-termlnal ¢yloplasmic lad. Molecules in Ihe desmoglelo subclas~ conlain a unique C-lcrminal extension within which is found a re~allnB mo~|l" Ih,q is predicted to I'orm Iwo 13-strands and two lurns. Slablc cell lines cxprc,~sin¢ der, moglcin I have been generated f~om normMly non-adhe~nl ~ c:ll I'ibcoMasts. In study the contribution or this eadheri~ ~o desmosomal adhesion. The prcdicled sequence of dc.'~moplakin (DP) I so[~ests it will form homodime~s compdslal~ Iml a-helical coiled-call rnd mnd two ~l,'~u:ar end domains. The C.ICrmlnus t'onlalns three reLcimls wilh :~igniRcnnl hmnolo~y, each of w;'~ich is made t,p of a .~.residl.e molil' also I'mmd in two olhcr molecules i'~olvcd in organization at" IF. bullous phigold antigen and ple¢lln. Eclopically r.':.psessed polYl~plid~s in~ludinl~ the C.Icr- minus oI" DP I specifically align wilh k,'ralin and vimenl|n IF in eullure¢l crib. whcrea.s thnc~: I,~klng Ihi.~ thm~ain do no~ ali~n with IF. The last 6A ~ultiml ~¢id~ of DI' hr,.: rcqnin:d fro" ali~.mucol along ker;,¢m lint n~ vhtscnlln IF. mltt l~|ifll~l¢ from lhc C-¢cnninal end arc crilic'-,l foe lhis inlcraclion. These results surliest ih;~l lhc. C-termimm of I)P plnys a talc in lhc allachmcnt o~" IF to lhC dcsmoson~ and lhal s~¢cific si¢c is nc¢cs~'~ry G'~r intereclion wilh I:crelin IF. A sequence= ut Ihc mo~l N- Icmdnal eml ol" DP ,~pl~:arx to be reqnircd ;or erratical incoq~'olion inlo the snmal plaque. Intcrcstln~ly. Ihis region has oul been feporled Io be presenl in homologous hulinus pemphigold anligen or pleclin molecules and n~y reprc~nl • desmusomal larOcllnl~ sequence. Kow-',lczyk. A. P.. Slappenbeck. T. S.. P.' ; t. D. A. D., P,,Ika. Ih L. Virala. M. L. A.. Bomslacger. E. A.. Nilics. L A.. and ¢, ~ ~n. IC. J. Biophysical Chcmislry 50:97.112. 1994. Other supporl: National lnstimles of !!~ ,,,., American Cancer $ocicly. M~rch of Dimes Birlh Defects Foundation. and e Fo,.used Giving Award ITom Johnson and Johnson Co. From the Deparlmenls of Palhology and ['<tmalology. R. H. Lu~e Canctr Center, Northwestern Universily Medical Sch¢.~,. Chic•go. IL. ~nd Masscy Universily, Depanmem or Physics and Biophysics. P~,h...,rston Nmlh. New Zealand. PI IO.~PI IORYLATION OF TI IE D "ESMOPLAKIN COOlI TI~,RMINU$ NEGATIVELY REGULATES ITS INTEPACTION WlTil KERATIN INTERMF.DIATE FILAMENT NETWOP'~$ De-,mlnplakins (DPs) ~re Ihc nmsl nh :.xlanl pr~eins in lee innermos! porlion Of ?I
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C) th," dcsm.osom~.al p)aque and have l~cn Dr(T4~sed tn play inlcnn~iatc lllamenls (IF) Io cell-cell cnnlacl sJl~. Our prcvinus r~ul~ sug~sl lh=l [he ~lobular cod ~maln; o[ DP ~r~ dual Junctions: firsl, dcsmos~e ~Ja lhc NH~ tedious and second, In al~ach IF In lhc dcsmosnmnl pl~uc via lhe COOII Ic~nu~. When ecl~ically cxp[c;~d COOII le~in~ plu~ lhc r~ d~main (DP.~H.~crC23) exhihh~ ~Iriking cnoli~nmcm wilh berlin IF nel~ks, llawcwr. ;n certain cell {y~ (c.~. I~K2) Wilh fo~skolln to aclivate ~tcln k~ase A. ~P, AN.~c~ exhibits a diffu~ cyto- plasmic di~Irlbu~inn. A varlet mnl~ulc (DP~N.GlyC2]} in which a scrine h~lcd 2~ amino acids from I~ COOII Ic~inus is ~ll~d pmlcin kina~ A c~cn~u~ phosph~l~lJ{m ~ilc. c~-h~ali~cs wilh kcralin I1: ncl- works ~c~ardless O~ cell ly~ ~ ~(~kolin Irealmcnl. Analysis o~ Ihe pho~p~plldc maps o~ Ihesc DP varlanls and cmlogcnnu~ ~p is c.nSiSlcnl wilh Ihc phn~ph.rylal;~m phqrylalion of a s~ci~c ~cxidu~ in Ihc I)P COOl I lurmh~.s il~ inlcru~hm wilh keralJn IF nclwmks. Slap~n~ck. T. S.. ~mb. J. A.. Cn~or~m. C. M.. and (;r~Lm, K..I. ~e Journal or Biological ~cmi~lry 26g(47):2()351-2e)3~4, N.vem~r 2~, Olber suppo~; Amcr~n Cancer S~icly. and Ibe Mnreh or Dimes ~;rlh Dc~ccls Foundalion. From the Depanmenlx o[ Palholo~y and Dermatology. Rnhcrl II. I~uric Cancc~ C=nlcr. Nonhwcsl~m U,ivcrsily Medical Sch~l. ~ic;~o. II,. INVESTIGATION OFTIIE INFI.UENCE OF CYTOSINE MRTI IYLATION CJN DNA FLEXIBILITY To rest ihe influence of pyrimidine methyl grnups on DNA flexibility and helix repeal, Iwo scl$ of 14 mixed scqucnc~ DNA molecules, sp:mning a runic or Icn~lhs from 158 Io 18~,1 base pai~s, were eycllzed wilh T4 DNA ligase. The Iwo sets dirrcrcd only in thai the Cyl-5 positions o1" all cytosines (liO.90 cytosine residues per mole- cult} were fully mclhylaled in Ih~ meml~rs o1" one S~l. Dclcrmlnallon or Ihe molar c)'clizallon factocs, l~rsisltnce len¢lhs, helix repealS, and Iorsinoal elastic con.~lanls revealed no si,~nificanl dilTcrenccs bClwccn I1~ two SelS. Thc~ resuhs imply Ihal. least for mixed sequence DNA. the hiololical consecluenc~ of cylosin¢ raethyla~ion are likely to derive rrom either local slruclural distn~llons in Ihe helix, which do no~ propagal¢ as ahcred IWiSl. or from direcl prm~'in-mclhyl cylo~inc inlcraclio~ls. ltodg~s-Gan:ia, y. and lla¢crmun, P. J. The ,louma| or 13iolo~ical Chemistry 2"/0( I ): 197-201, Januory 6, 1995, O~hcr SUpl~n: Lucillc P, Markcy Charilahlc Tru,d, and the IJnivcrsily Cancer Center. From Ihe Dcparlmcnl el" l~inchcmislry. Binphysic.~ and Gcnelics. University of Colorado I leallh Sciences Ccnlcr. Denver. CO. 72 HUMAN INTERLEUKIN-3 RECEPTOR MODULATES BCI~2 mRNA AND PROTEIN LEVELS TIIROUGH PROTEIN KINASE C IN TF-I CELLS Upon wilhdrawal of |nlcrleukino3 (11.~:3) from human flctor-dcpcndCnl cry- lhroleukcmic cell line T[:Olo hcl-2 mRNA and prolcln levels decrease wilhln 8 to 24 hours. Accompanyin~ Ibis decrease is Ihe on~t ol'apoplosis as delermincd by flow cylomclric analysis of DNA dcgradnlion. B)' 8 Io I ~ hours of dcprivalion approxi- malcly "/lYe, to 80~ of the cells have entetecl apopiosis. Downre/ulalion of pmlcin kinasc (PK) by -', 24.hour incuh~lion in 100 n,,~ol/L 12..O-telradecanoyl-pho~bol-1.3- ,-~Cel~lc ('i'PA) in Ihc presence or IL-3 dr~m~ti:..lly Icdnced t~-1-2 mRNA levels, inducc~l alx~phl~i,~ in Ille prc~coc¢ of IL-3. Wc imvc ul~ I'ou.nl Ihnl even in Ih~ prel- once el" IL-3. two lnhibilnr~ or PKC. li~hl-nc|~.-:alcd callgnostin and 11-7. subSh'mllally rcducccl Ihc levels hi" h(.I.2 mRNA between I~ :Mid 24 hours a.~ measured by I semi. quunlilnlivc revcrxc ImnscriplaXC/polymcrasc ':.;,aln ltacllon amy m¢lhi~J: however, 111~ cyclic ilUCle~th|c-¢IcpClldcnl PK iollibilor I;A 1(104. tidal is n ~tn¢lnl~l 117 bnl .., i~mr i,hihhor .1" PKC. dkl ~o! rcd~K •/~'1-2 levels in Iho prcscnc© This ~Iccrc~.¢ in 1~'1.2 mRNA w.'~ ac¢ompan;, .; hy n decline in 1~'1.2 pmicin levels hy I( h~ 24 ho~lrx ,l'lcr .-~hlillon or IiBhl.aCtlVnle~i .~olpIioslin. In mldilio~ In inlcd'¢dn¢ will1 Ihc mainlcomlcc of l~'1.2 mRNA level:. '..hlbillon or PKC with I1.? inhiblled ihe induclkm or 1~.1.2 nIRNA in I'm:lor-dcpri~ ':d TF-I cclb rellinlulnlcd whh IL..I. The cyclic nuclcolldc-dcpendenl PK inhibilor tlA 1004 did nol inhibit IL-3-induccd hcl.2 mRNA. Studies wilh =ctinomycin D s~owed Ihal Iransctiplion pl~,y$ I major role in malnt,'tlnio~ h~'l-2 levels in TF-I ccl,'$, and it tl Iher=l'ol~ llkcly plays ~ role in mainlainlnl~ hrl.2 Iranscrlpl~an "h~onBh aclivallon Rinaudn, M. $., Su, K., Falko L, A., Ileitis, r, ~., ,~! Mufson, R. A. Blood g6(I):~0-g~, July 1995. Olher supped: U.S. Public lleahb Service and N~l|o~al Cancer From Ihe Ilolland Labornlory rnr Biomedical Selene©. Amerlc.'m Red Cro$1. Rockville. MD. and JelTcrson C~ne©r Ioslilulc. "['homes Jerrersml Medlcal Philadelphia. PA. RAS FARNF.~YLTRANSFTcRASE INHIBITO.~.~ SUPPRESS THE PHE~OTYPE RESULTING FROM AN ACTIVATED RAS IV:UTATION IN CAFJ/ORIIABDITI$ ELEGieS Allachmcol o1" RI~ pro{eln Io Ihc n~mb~.:,o, which requires famesyl~llo~ al ils C terminus, is essential lot" its biological ~ct,/=.ty. A promtsin~ pharmacological apWoach o1' anlagonb'.ing once,chic RM aclivil:,- is Io develop inhibitors of I'arnesyl- I~msrcr~t~c. Wc tL~c C~ltlmrh~l~l[l[~ ~lt]~l.$ v :lvnl dilTerenlialion, which is con- Imllcd by a Ras-mcdialcd sisnal Iransdncllon pa:;~way, as a model system Io vioosly idcnlificd I'amesyllransl'erase: inhibilor.;, We show here Ihal Iwo flmesyl- Inmsr¢~,se inhihilors. ~,~xmt~nycin and ~,llnloxi,. ~upl~SS the Muhlvulv,, ~esullin~ I'r.m :in ~cllvaled h.r-(~q r~.r nmlnlinn, hal nol the Muhlvulva phenolylX~ 7:3
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C) rcsull|nC From mulallon; in the Ib~-I genc nr the ft.-13 geno. wldch u~l downstream ond op~lrcom o~ I~1.~ ra~, restively, in Ihc si~nal;ng p~lhw~y. ~cs~ rcsuh; arc consisi~nl wilh the idea thai the suppression or ihc Muhivulva phcnoly~ or I~.~ ~; by the two inhibitors is s~¢ific for Rx~ pmtcln and th~t I]~ mutant R~A pmacin miGbl ~ more sensitive than wild-ly~ z'is'n syslem for cvalualion o~ ramcsyllr~nzrc~sc inhihitnr; n~ainsl Rex-activated tumors. }lore. M. and lien, M, Oilier sup~: Nalional Inslilolas or I lcallh aml the Morch (ff DJm~ Foundal;on. F~om Ih~ D~padm~nt or Moic~lar. Ccllul~r or C.Io~do, 9ouldor. CO. a~ Co. L~d., Tokyo, Japan. TIlE ROLE OF TI IE ARYL I IYDROCARBON RECEPTOR NUCLEAR TRANaLOCATOR PROTEIN IN ARYL IIYDROCARBON RECI~Iq'OR ACTION The aPjl hydrocarbon (All. or dioxio) Rcepmr mediates carcinogcncsis by wide vatiely of compounds. It aces is a ligand-depcnden[ Iranscriplion |.actor. Many inves¢igators expecled Iha! Ihe All rcceplor would prove Iobc steroid/lhyroidlretinoic acid receptor supcr/'anlily or proteins. However. recto[ clonio¢ or the two subunits of the DNA-binding rorrn of Ihe All rcceplor has shown that this is no4 the case. These subo~[s, the All reccl~or nuclear Imnslocator proAein (ARNTI and the ligand-bindinG All receptor ma.ams:r, dn not co~[aln zinc Gngcr DNA-b[nding ~Jomalns. nor do they have any other sequence ximiL'~ritics with mem- bers of the above family, instead, the), both coma|n basic helix-hK~p-hclix (hllLll) mmil's ~d also another segment of. ~qucnc~ similarity, lhe "'PAS°" rc~ion, hll[Jl motil's in other tran~riplion rac[ors arc known to function as dimerization and DNA- binding domains. Present exper[monts isc directed toward undc~tandinR the mccha. nisms of ~ction ~nd the roles ortt~ two sut~n|[s. l|ankinson, O. Trends in ~doc~inoIogy ~d Me{abo|ism S{6):240.244. 19?4. Olher support." National Cancer [nSlilUtc. Dcparlmcnl of" Energy, and Tobacco. Related Disease Rr.s~arch Program orlh~ University of' Califomla. From the Depanmenl of Pathology an~ Laboratory McdiclnCo and I -',horatory of" Sm~clural Biolozey and Molecular Mcdlcinc. Universily of California, Los AnBelcs, CA. 74 RECOMO[NANT BONE MORPIIOGENFI !C PROTEIN 2 ACCELERATES BONE CELL DIFFERENTIATION AND .: ~MULATES BMP-2 mRNA EXPRESSION AND BMP-2 PROMOTER ' .TIVITY IN PRIMARY FETAL RAT CALVARIAL OSTF_OBLAST CULTUR "~ Rccomhinant bone morpho¢cncliC protcln 2 (rhl~MP-2) stimula~cs ¢c[opic bone rormalion ~n ~,i~,o. Using primary cultures of felal rat ca|vax{-',l cells tha~ diffc¢~lla~c IO form bone nodules in vitro, we demonstrate that rhl}MP-2 c~n ,,ccctemte this dif- fcren|ialion process ~s me~sured by accelerated mineralized bone no~ule ,hB MP-2 al~ ~cclcra~cs mullilay~dog of ll,,:s¢ osteoblasl culture. Wc Mso dcmon- slmle Ihn! d~BMP-2 increases and accelcrnle: Ihe Ippcamnce Of ~lk~l|ine phosph~lise mRNA aod ostcoCalcin mRNA. In :~ddil|un, rhEMP-2 increases Ih© ~tplitude of expression or cndo~.ono,s BMPo2 mRNA end ,,K'cclerales the nppearance of UMP.2 mRNA cxprcssion. Wc Gmher ¢|~.monslrate usln~ Iranslont u1tns['ccl|O~, &S~,y$ with the BMP-2 promolcr mid Iucif.crase rcporlcr ~on~ cmlstrucis thai rhP.Ml'-2 p,~ss|t~vely all'eels lIMP-2 Ir;mscriplion. The lIMP2 response ,¢[-'lon w,~ m:~pp:d b~twccn - 196 lens! p;ld eft the nlcch;lnisnl I'or rhl~MP-2 aclion on I~.la cells is .~Iiv.'~in~ of the ClI¢Io~cnous 13MI'-2 gone. "l'bi~ eoohl lc,qcl Its nniplicallOn el" the both a pnracrine n,t(I :lill¢~-~'~ne ~{IshioI1 thsring tmnc cell diffcrcnslatlon. II~rrls, S. E., Fcn~, 1. Q.. II~,xls. M. A.. (~hosh-Choud-Choudhu~, N., Dallas, M. R.. Wozney. J.. and Mundy, G. R. Mol¢cul~r and Cellular DilTercnlialion 3(2):137-155. 1995. Other s.pport: N~ional Institutes of l|eatth. From the Univcrsily of Texas lleahh Sc~" "': Cenler. Department at Mediclne, Division of' Endocrinology. Son Antonio. "~ , and Oen~llCS Institute. CambrldJte, EXPRESSION OFTI IE I~MP 2 GENE DURI~;G BONE CELL DIFFER ENTIATION llonc mmpho~enatlc protein 2 (BMP (TGI~) nm activel), involved in ~ fom~atk~ and renmdclinp. "rGFpo • ~wcd'ol sllmulanl in the earl), staF o('bo~a cell growth and matrix formation, inhibits differ- entialion and in ~'i~ro mineralized nodule fo~mation in primary fetal ~l calwtial oslcohlasl system. TGF~3 also m:gatlvety rcl;U]ae~'s BMP 2 expr¢sslon at the Iron. scripfiona! level. BMP 2 gone cxpRssion is to.trolled by a battery or transcdplio~al rackets, some known and some yet to he idcmificd. Imnmdalized osteoblasl cell lines ~onerated from a t~nsgenic mouse carrying lIMP 2 promolcr*drivon ~V4II large T antiBcn t~nsgene =re described ~,s powmful tools for sludying I~¢.ulallOn of BMP 2 I}ene expression end bone cell di{{'ePmtlalinn Ghnsh-Choudhur)'. N.. I la~is. Me A.o
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C) Crilical Reviews in Eukaryollc Gene Expression 4(2.¢'3):345-355. 1994. OIher supporl: Nalional Instilulcs or ! Icahh. From Ihc UniversJly o!" Tcx;s Ileahh Scicncc Ccnlcr Ul R:,l A,tnnio. Ocpnrlmcnl of Medicine. Division of Endncrinulo~y and Melalxtlistn. S;,i Anln,io. TX. F-STADLISI IMENT OF PURE NEURONAL AND MIISCI.E PRECURSOR CEIJ. CULTURF.S FROM I)RO.TOPIIILA I~RI.Y C;A.TI'IH.II.A .5"I'A(;E EMBRYOS Primary cultures of Druxophiht ~oslrula .siege cmhryonic cells will divide ~nll te~linally difl'crenllate inlo morphologically reco~ni~..ohlc ncnron~ and ml.~cles. The phenotypically mixed nature or this prim:lry cull,re system has re;ale |1 dil'ficnl! In el'fcclively analyze various paramelers of" cell growlJ| .~n(I dill~rcnlialinn for iudivid* ual cell types. We reporl here a simple and ccunomic tnclhod In ~cpar'at¢ early enlhr~'onlc procurers (or diflercnl cell lypcs. ,sing a ~hallow linear reoricnling Ficoll gradient al unit ~ravJly. The scparalcd cells were collccled inlo rraclions, cul- tured, and analy~.ed for their growth anti diffcrcntlal;,n p;dlern~. Tlte I,rgcr an¢l den~.cr cells of Ihe I'ir~l I'rucl;ims differenlialcd h) yield pun: near(real cuh,n:.~c, as judged by morphologic, immunologic, and biochemical erileria. Cells in Ihc lull rracllons difrerenliared inlo a prcdnminanlly mu~;clc-enrid~ed cell p~pulalion, whi~:h al~o conlalned a very small percentage of ncurnns morphologically disli~tct from those in the pure neuronal l'ractions. Approxim~cly .'15% oi" the early p>nslrula S|al~e emb~'onie cells dJffen:nllale inzo neuronal co|Is, :tml 65% or the nnn-ncumnal fin. cage cells later dcvc|op inlo prctlorninantly muscle pq~eh|lion. The nlC11.xl is highly reproducible, can pri~:css 3 x |0~ cells per procedure0 and the recovery is :>~}~ of chc input cells. The ~paralcd c~lls are suilahlc for cell biological an.~fyses as well as l'or biochemical and molecular studies oi' neuron and muscle preco~snrs, llayashl. I. and Perez-Ma~allancs. M. In Vilro Cellubr & Developmental Biology 30A:202-20R. April 1994. From [he Dcpanmcnl or Molecular Genelics. Beckman Research lnslilure of d~c Cily of llol~. Duane. CA. 3511. A SEQUF.NCF. I~OI.ATI':I) AS A I'RO'II~IN KINA.~I'." C IIINI)IN(; PROTEIN. IS A NOVEL MEMBER OF Tlll~ AI)I)t)CIN FAMB.Y We recenlly ©hmed a parlial eDNA 13511} I'or a pr~eein kinasc C (PKC) hinding p~.lcln I'rcm) a r.~t kidney cl)NA library' and (ll:lnollM~liL~¢[ I|1;11 il is U I'K(" s,hslr;Ou in vim~ (Chapline. C.. Ramsay. K.. Kluuck. T.. 76 r'- 268. 6858-6861). Additional libraxy screening and 5' ~pld amplil~cation or cDNA ends were used in oblain the complcle open teadin& r~mc. Amino ~id seq~nce analysis. DNA sequence analysis, and N~hcm analysis indical¢ [hat 3511 is unique eDNA rcblcd Io a- and ~.~uc]~.. Anli~ pepped 1o the 3511 baClcrial fusion pro~cin recognized Iwo ~ly~pli¢~. ~r ~0 and ~ kO~ on inmlunoblols kidney homo¢cnalcs and ¢ullurcd Rnal ptox~ml[ lubul~ cp[l~li~l ¢¢11 ~SH-rclalod prolcins wc~ similar In ~-an~ fl-~ins in Ih~l Ih~y s~ p~r~n- li=lly ~¢¢ovcrcd in I~ Triton X-I~-in~lu~lc (cylos~¢l~al. CSK} fmcllon ¢ltr~l~ nnd wcr~ predominantly I~ali~ In ~11 ~l¢~. ~1 csl~ sllmul~tcd according In PKC binding activity in sV~ro arc ~lso PKC $ubslRl~$ ~ pho~latad forms or the 3511 prate}as wc.e pRfeRnlially recovered fr~clion, shut dcm~stmlin$ Ihat phos~r.1 lallon cgulares their CSK ~s~iat~n anti• thc~by, their fonctlon in mgulnlin¢ cyi~skcklal ~nthl~s. We have iml=l¢~l =nnthcr PKC 5;ndin~ pmlcin p:ni=l eDNA (clone 45) ~mm a ~1 flSmN~l wilh suhxl;mllal homology Io ~-=ddncin. Anliscr= ~iscd a~alnst Ibis expressed sequence rccognizc(I a protcln or 120 k~:. Ihc reported siz~ at n-0(hlucin. immlmobtots of #cnal ~xlnl:l tunic aphl'.-si~l call ~xt~s. A 120-~Oa p~cin thai ¢ross-rC=cls wilh 111o clon¢ 45 (o-addl~c¢ ,) ~nllscri cop~ccipil~led wilh .~SII immun¢compl~x~s, indicalin~ IIiol ~-~dd~cm ass~ialeS wilh 3511 p~eins Taken IOgalhcr. Ih~ r¢sulls i~icala Ihal 35X is a new. w~cly c~pres~d ~o~ or ad~ucln ca~hlc or fo~in~ h~la~ml~m¢~ will1 o-o(~ucin. We pabst ~mln¢ Ihls Juken, ~. ~c Jmlmal of ~io]o~ical ~misl~ 27~d3):2~-~40, ~to~r 27, I~. Olher su~: Hadonal Insfilules oC Heallh u~ ~mc~can ~a~er Prom ~1~ ~. ~hon Jn~s ~ell Sc~ene~ ~ne~. Inc., ~ke Placid. HY. GENERALITY OF Tile SilARED ACTIV~ ~ITE AMONG YEAST FAMILY SITE-SPECIFIC RECOMBINASES--TI~' ~. :1EoS~'ECIFIC R ECOMBINASE Pal.LOWS TIlE FLP ~IARAOIGM Mulalions o1" Ih¢ [nvarlant In[ I'nmil~t t.-l:ad residues, the RIIR Iriacl. a~d I1~¢ active sile [yroslnc. wilhln Ih¢ Z~.¢osnccl~.~.mY~'rs rm¢t'~i sltc-specil~c rtcoulblnase R cause Ihe same "slep-alTesl" phcnolypCs as they do in IhC PIp rccomblnase or Stt~'('ltorontycts rertt,isint. In "half-sile" recoml)inalions. Ihe R rccomhinase exhibils c;~l~l),lic c(m~plcmculnlion belwccn an RIIR Irl..td mnlanl xnd nn .~'live ;lie I.Vnr~inc m||l;u|l..~lrn.d culling by R h~llnws Ihe "lmn.~" 13NA cleavage nile. 'lltc't¢ n,-~||lls arc ICsl explained by Ih¢ assembly o1' a funclinnal ~clive sile from partial |clive sties harbored by the R mahomet's. Complcmem~ilon tests usin¢ s|n~.le and double step° nrrcsl R m.lmlls verily crilic,'ll prediclions of 11~e "slk~d aclivc sile" model. A wild 77
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live combination. Pairwlsc comblnalions of a single nr o d,uhle RI IR mutant with R(Y358F) yield comparable levels of Calalylic complemcntation. These resulls slrongly imply conservalion of Ih¢ mechanism of active site assembly and the mode of subslrale cleavage within Ihe yeast family slte-speclflc recomhinascs, and perhapa within Ihe larger lot family recombinases. Yang, S. II. and Joy-,ram, ~,|. The ]Oum•l o1" Biological Chemisl~ 269( 17): 12"/~t9-12?96, April 29. 1994. Giber supporl: National In~tilute~ at" lleahh. From the Department of Microbiology, Universily of Texa; at Auslin. Austin. TX. ACTIVE-SITE ASSEMBLY AND MODE OF DNA CLEA VAGE I~Y RECOMBINASE DURING FULL-SITE RECOMBINATION A comblnalion of half-site subslrales and slep an'c~l mulanls of Rp. o silo-spe- cific recombinase of Ibe inlc~ra~ family, had earlier revcalcd the followinc of" the half-sale recombinalion reaclion. (i) The Rp aclive site is assembled by shat- in~ of catalylic residues rrnm at least two monomers o1" Ihe protein. [Ji) A monomer dues hal cleave Ihc half site Io which iF is hound tUNA cleavage in ci.~): rather, it cleaves • half sale bound by • second FIp monomer (DNA clcovagc in leans). For d~e ), inlegr•se (Inl protein), Ihc prototype member of Iha Inl family, cat- alylic complcmentadon between Iwo acllvc-silc mutams ha.; hcen observcd in lions wilh • suicide o~tL, substrale. By analogy whh FIp. this ob~rvnlinn is slronciy suggestive of • shared active site und of erans DNA cleavage. Ilowevcr, reaclio~.s wkh llnea" suicide oil/3 substrates Ind synlhelic llollida)',junctlons are more compel- able wilh ca)" Ihan wilh irons DNA cleavage. These Inl resulls cilher argue againsl common mode of•clive-sale assembly wilhin the Inl family or challenge Ihe valldi~y or F1p hair sales ~s mimics or lbe normal full-site substrales. We devised • slrale~y assay calalylie complemcnlilion bclween FIp monomen~ in full sites. We found Ihc full-she reaction follows Ih¢ shar¢d acllve.sile paradigm and Ihe Iron.~ mode of DNA cleavage. These resulls su~Besl Ih•l wilhin Ihe lot family, • unilary chemical mechanism or ~ccomblnalion is achieved by more Ihan one mode ol'physlcal inlerac- lion amon~ Ihe recomblnase monomers. Whang. I.. Le~,,f., and .lay•r-m, M. Molecular and Cellular Biology 14( I I):7492-749~. Novcml~r 1994. Giber suppotl: Nalional Inslilulcs of Hcahh and Ihe Rnberl F. Welch Fmmdnlinn. From Ihe Depirlmcnl of Microbiology, Universily of Texas al Auslln, AuSlln, TX. DIRECTFJ} PROTEIN REPLACEMENT IN RECOMBINATION FULL SITES REVEALS TRAIVS-IlORIZONTAL DNA CLEAVAGE BY lip RECOMBINASE One round of sale-specific recombination I ,:.lween Iwo DNA par~ncr~ medla~cd by the Rp rccombin.-~sa rcquirc~ Iha breakage -.~.d refonnalion of four phosphodi. esler bonds. Thc rcaclion is accomplished '. ? Ibc combined eolian of four FIp monomers. Wilhin the rccombinnlion complc;,, wh-',! is Ihc relative dis~ilion of a Pip monomer with respccl Io Ihc tar¢ct dicsler ,l~,~l" il cleaves? To address Ihis qufs- lion, we have devised a slrale~:y I(~r Iho earl:clad oricnlalion or Flp monomerJ; wilhln full-slte recomhi.allon suhslralcs. Our cxpc~lmenlal deslfn is nol def~n¢lenl on 'abated binding specificily" of the r¢combin--.¢. Analysis of Ih¢ J~llcm of DNA cleavage hy Ihis melhml reveals on eviden¢'~ for DNA cleavage in ci,¢. A FIp mmmmcr h(mml to il.,; rccognilion c~menl wi-,bin Ibe full ~;It¢ do~s roll ¢1¢•vc Ih¢ scb;~;ilc phn.~phndicster trend nd~aeenl Ioil. O¢ir results are nrosl ft'~ns|~qent wl~h 'lnms-lmrixonl.I cleavage'. Cleavage by FIp ueclmc nl I1~ ~cissile pho~phodleSler ,lislal In il. h~ll wilhin Ihe san~: fl,II sale. The general experimemnl deslfm cropIoyed bcrc will I¢ ~f wldcsl~:;nl t,lilily io mcchn.lstlc •nalyses of mlclelc acid Imo¢'K'llons i,lvolving mullimcric DNA--~olcin assemblies. Lee, .I.. Who.g.I.. Lee. L, mid J,.~,arnm, M. The EMBO ~o.mnl 13(22):5.146-3.1.54. 1904. Olhef suppotl: Nalion-',l Inslilules of ! Icallh and Ih¢ Roberl R Welch Foundalon. Fermi Ihe Ocparlment of Microbiology. Univc~ily of Texas al Austin, Au$1il~. TX, MECIIANISM OF SITE-SPECIFIC RECOMBINATION: TIlE Rp PARADIGM This chapter has rallincd Ihe mcchanism ofslnmd b~.--;d;age and R.joinin~ dudn~ FIp rccomhin~lion. The lip syslcm provides a paradigm for the slrategic assembly or a mulG-componcnl biological machine deslgncd Io produce • logical chemical come. Tbc chemical simplicity and mechani~tL" parsimony eroployed hy Pip mcdiolc an apparenlly complicated sol of phosphmyl Iran#rers belwecn fwo DNA molecules are likely Io have parallels in $yslfms Ih$1 can./oul similar chemical tran.~ctions in nucleic acids, roe example, DNA Iransposilio(~ and RNA splicing. The success in analyzing FIp recomhlnalion b~ resulled from Ihe desil:n of recombination hail'site, lhe con~InlCl|On of step-arf~sl lqp variants, and lhe dissection of the rcacllon inlo partial ram:llons. Jnyoram. M. In: fifks~ein. F..~n(l Lilley, D. M. 3. (Eds.): Norlelc Acids and Mol¢cul.',," Biolo~:y, Vol. ~q. Springer-Voting. Berlin, Heidelberg. pp. 2t5!(-256. 1994. Other supfmtl: N;~lionnl Inslilules of llfahh. From tim Dep~rlment of Mifrob|oi~y. Universily of Texas al Austin. Austin, TX, 79
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ROLE OF PARTNER IIOMOLOGY IN DNA RECOMBINATION CPLMPI.EMENI"ARy BASE PAIRING ORII-'.NT~ ~11~ ~'-IIYI]ROXYI. I./)R ~'RANI) J~INING I)t JRING I~.P 511E-~EClFIC RECOMBINATION AhsolUle h~:mology ~lwcen parlour std~lralcs whhin region is an e~Scnlial rcquimmcnl fi)r recomhinali()o mmlialcd by Ihe yeast silc-sF- cific ~combina~ Rp. Using combinalionx of s~cially dcsigned hall,- and fnll-silc FIp subslralex, we demonslral¢ Ihal Ihc slrand joining slop exquisil¢ly senshiv¢ ~o spacer homology. A~ each exchaogc phial, 2-3 spacer nucleolidex adj~¢nl Io Ih¢ nick wilhin I~ cleaved ~trand of nn¢ suhslntle muxl hax¢ pair Wilh Ih¢ co~s~dln$ ~$menl o[ I1~ un-oickcd ~lraml strait for efF~ienl slrand .joining in Ihe recomhinanl mode. In accordance wilh "cis'actlvalionhranx.nuclc~hillc all•ok" mmlel fi=r each o~ lion slops or Rp reck=bin•lion island cleavage and slmnd joiniag), we praise thai Ih¢ limiled slrand pairing art=hiS ~¢ DNA-n~l~hile (S'-h dm ils larg¢l dicslcr (3' -hos-~ ........ , _ , ~ . _ • . I' pgc~ (Z-~ ~e p==rs at c~h ~pacer cndl musl un~ir, rid- Iowln~ ~tr=nd cleavage, wilbin a DNA ~ub~ralc aml pair whh d~c pa~ncr suhslral¢ prior m sl~ union. In Ibi~ m~cl. Ihc extent of ~nch mi~ralinn nr =1~¢ cnvaleml~ clowd llolli~=y immediate i~ limhcd In Ihe ceolral core ,f plalcd ~illonln~ or regctJvc n~lcic acid 8rnu~ (which i~ ccmral may ~ ulili~cd hy other recnmbln=lhm ~¢cm~ =~,1 hy RNA splicin~ ~nc6nn~. ~c. J. and ~=~=r=m, M. ~e lnumal o~ Biological ~emi~l~ 27(~):4(N2-4(1~2. Fcl~ary Roan F. Welch Fou~a6on. From Ih¢ Dc~nmcm of Micmblolo~y. Univcrsily o[ Tc~ =1 Au~=in. Auslin. TX. FUNCTIONAL ROLES OF INDIVIDUAL RECOMBINASE MONOMERS IN STRAND BREAKAGE AND STRAND UNION DURING SITE-SPECIFIC DNA RECOMBINATION ~1~c sltc-.~pccific recombina,~ Rp ~'rrml .~t,'~'l=artmlyc'e.~ rerevi.tiue acca=mpli.~bcs recombination bclwccn Iwo lar~el DNA silos by execuling a pair o~ slrand exchanges al ¢ith~ end or Ihc strand exch==e region. ~c round of recombination require~ the c~ralivc aclion or rn,r rcc~hina~c mnnomcr~. We d~-~n~ffalc here Ihal. in Ih¢ pm~nce nf Ihe ap~=prialc nuclcnphilc~, n ~inglc FIp =led wilh i=s binding ¢lemenl can mediate sl~nd cleavage and slrand jninlng exchange sil¢ ph~phale adj~nl Io il. Our resolls supra = mmlel =~f ~omhinalion in which pairs o[ Rp m~nmer~ revere calal~ic roles In mediate II~e firsl and ~c- ~d ~ls oF slrand breaka[¢/uninn re~li~. ]~cy di~[avor a medici Ihnl inwdv~ ~clay of re¢ombin&~ monomers ~lw¢cn binding elcmenl~ In assemble gN JUNCI'ION MOBILITY AND RESOLUTION OF tlOLUDAY STRUCTURES BY FI.P SITE-SPECIFIC RECOMIlINASE m I"F.STING I~&II'I'NER COMP=&TIBILITY I)|~RING RI.:~I )MIIINATIIIN Ahsolulc hnntolngy helwc©n panner subslrates wilhln Iho Slrlnd exchange region (~i'~tcer) is an esscnlial requiremenl I'o¢ recombination mediated by Ih¢ yell site-specific rccomhinase FIp. Recto| exper.~menls sorest thai .~-hase I~lr homology adjacent In the polnls or cxchang© at each end oftho spacer is ulilizcd in • b~se com- plcmcntarily-dcpcndcnl sir•rid joining macllon. Homolo~), or Ihc cenlral 2 base p=|rs of Ih¢ spacer is also cr|lical, bul how hmtmlogy is lcsted at Ihcs¢ Iwo poshlons Is unknown. Wc have addressed Ih¢ role of homolocy.dcpcndcm branch migr~llOn in Rp recombinalion by =ssoying strand cleavage and resolulioo in • sol of synlhclic Ilolliday junctions in which the branch poi~d is I'rcely or p~lially mobile Ih~ough spacer, or is immobilized ul each posilion v,ilhi~ the spacer or immcdialcly flanking it. A slrong bias in Ihe direction or Hollic"V resolullofl is obsewed ottly when branch point is located jnsl oulsid¢ the spacer (al Ihe junclion or ihe Rp binding mcnl and the spacer). A si~'dficmtlly smaller bias is noticed when Iho I~nch point is frozen immcdlatcly ~j=cenl to this positi~"P whhin the Sl~Cer. ResOlUllOn in cases is most ol'len mcdlnled hy exchange or Ihe scissile phosphodlest¢~ al hrnnch poinl nr Ixoximal In il. anti rarely hT exch-',ngc of Ihe scissil¢ plmsphodlestcrs dislal In il. In llghl or Ihc.sc and pmvions r~ :It. wc discuss possible chcckpolnls testing parlner comp,qibilily during Rp rcc~...~inalion. I.cc, J.. I.cc..I.. and Jn.vnram, M. The Journal of Biolngic=l Chemislr7 27(1(3~h 19086-1909"2, August I I. 1995. Olhcr snppmt: National Science Fonndallou, National IoslilUleS of lie=lib. =d Ih¢ Robed F. Welch Fnundallon. From I~ Deparlmcnl o1" Micmbiolccy, University ol'Tex,,s. Austin. TX.
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C) z MAPPING SITES OF INTERACTION OF p47-PI IOX AND FLAVOCYTOCIIROME h WITII RANDOM-SEQtlENCE P[".PTIDE PIIAGE DISPt.AY LIBRARIES I)uring :s~cmhly ~)f the phagocyle NADPII oxida~0., cylo~;~lic p47-phnx Ifan~h~alc~ I() Ihc plasma membrane and hinds In Ilaw~yi~h~m~c h. and hindhtZ domain~ ~or ~7-ph¢)x have ~¢n idcnliGcd ~m Ihe C-Icnninal lail~ l~hrome h ~u~nJls. In Ihe pr~;cnl [cpnrl. wc furlher examh~c Ih~ Iwo o~i=la~c com~ncnl~ hy u~ing tand==m.~c(lum~cc ~plide phage display library analy~it. ~c~ning ~7-#mx wilh Ihe ~plidc llhnwie~ tial ~*tc~ ur imcracli(m with fluw~yleK'hnm¢ h, including Ihrc~ prcvi~sly rcl~mcd rc~iou~ of in~craclinn and two =ddilional re,ions of interaction ,~f 1~7*phox wilh Epgl-phox and pZ2-phnx. ~le oddJli(mal sties were reaped I, a domain near the cylus¢~lic C-Ic,ninal lail o£ gpgl-ph,x c~nmpussing r~iducs F''EWFADI.L'". The mapping also confirmed a prcvimt~ly ec~fted hlnding domain nn ~pgl-pho~ fE'~SGpRGVIIFI~) and pulalivc Src honmh~y ] domain bindin~ sties on p22-~m¢ (P'?PRPP" ~ G"GI=~;Gp,,'). T~ addilional ~egi~s idealized were biologically signJ~can¢, ~plidcs mimicking ehc ~pgI-pho~ sequences F"LRGSSACCSTRVRROL" and were synlhesizcd and assayed for Iheir ahilily Io inhihll NADI'II o~idas¢ aclivity. ~¢s¢ ~plldcs h~ ~, v=l~ of I FM a~ 230 ~M. rcs~cfivcly, and inhibilcd activation when added prior Io assembly hut did nm affect activily bled oxida~¢. Our data dcmo~trate thc u~futncs~ or ~age display llhra~ analysis for Ihe idenlificalion or hlol~ally rclcvanl sties eft prntcin-p,~ein inlcracli~m and show thai the hindin~ oF ~7-phot In ~=v~yl~hromc h involves mulliple binding silos ahmg Ihe C-l¢~ioal lolls o~ ~lh ~pgl- and p22-~mK and and 0ulnn. M. T. Precedings oF Ih¢ Nalional Academy of Rcicnccs USA 92:711[)-71 Olhcr support: ~tional last;totes nf tlcahh. Arthritis Fnundalhm Biomedical From Ih¢ Dcpa~mcnt~ o£ Yclcrin=~ Molecular Biolo£y and Microbinfogy. Sea=e Universily, Bozcman. MT. TOPOLOGICAL MAPPING OF NEUTROPIIIL CYTOCI [ROME h EPITOPF~C; WITil PIIAGE-DISPI.AY LIBRARIF,~ Cymchrnmc b or human ncutmphils is thc cenlral cnmr~ment (ff Ihc mlcrnhici- dal NADPJl-oxidase system. However. lhc I'oldin~ Inpology nf dds inlegral mem- brane proleln rcmain.~ undetermined. Two random-sequence hactcrioldmge peplidc libraries were u~ed to map .~lruclural fcature.'~ nF ¢yl(v,:hr¢,nc h hy ¢lutcrmining ¢phopes ()f mnnnclonal anlihodics (mAl~) 44. I and ~4. I, specific I'(~ gp91'~ cytochrom¢ h chains, respectively. The unique pcplldcs of p~c selected by mAb affinity purification were deduced from lee pha~e DNA sequences. Phage sclcclcd by mAb 44. I displayed tile consensus pcplide sequence GGPQVXPi. which i.~ nearly identical to "GOPQVNP['" of p22'*'. Phage se|ecled by mAh 54.1 dis. played the conscasns ~quenec PKXAVDGP. which n:semhlcs ~'.'PICIAVDGP" gpglP-. Western hlnttine demonstrated specil'K; binding of each mat) Io Ihe live cytochrnme h suhunil and selected phage I~.pddcs- In flew cytomelric analysis. mAh 44.1 hound nnly pemteahillzcd neulmphilso while 54. I did nol hind iut.qct wnneahili;~cd cells, llowevcr, mAh .~4.1 inmnotoscdlmcnlrd dctes~enl.snh.bilbcd cyl¢~:hron)e h il~ ~tlcri:~e ~ldient~. ~tC~ resnlls Stl~gexl Ihc '"GGI~VNPI"~- mcnl or p22~ is acccsslhlc (m its inlrace~]u]ar ~urf=c¢, but Ihc "~PKIAVDGP~ rc~iem em ~pgl~' i~ nnl acccsslble IO anli~y, ~nd pro~bly hal on Ihe pr~lcin ~c Journal or Riolo~ical O~¢misl~ 27~28);16974-169R0, July 14. OIl=cr x~p~rl: U.S. Public Ilcnllh Service, Notional Sclcncc F~dal]~, R)undalinn lliomcdical ScOnce Gmnl, ~nd N=';nnal Inslilulcs or I leallh. From Ih¢ ~pamnenls of Mi~biolo~ and Vceerina~ Mol~ulor Biolo¢y, State UnJvcrsily. ~oxc~ln. MT. ASSEMBLY OF REGUI.ARLY SPACED ~'JCL.EOSOME ARRAYS BY DRO.~OI'IIII~4 CIIROMATIN ASSEMBLY FACTOR I AND A 56-KDa I IISTONE-IIlNDING PROTEIN To ascc~laln the mechanism by which m:rleosomes arc assembled by factot'~ derived From Drosophila cmhryos. Iwo pro, ;,)s termed Drosophila chromalin as~mhly fnclo~ [CAFs) I and 4 (dCAF-! anC . ~.AF-4) weR fracslonatcd and pu~,- find f~om -', Dro.¢nphil=l emb~do CxlraCl. The assembly of chmm=lln b~ dCAF-I, dCAF-4, purified hislo~s, ATP, and DNA spaced nuclcosom~l a~ with a R~at lent:lh Ihal ~¢mblcs Ihal of ~lk nali~ Dro.tophila ch~omalin and is hal obli~=lori;f as~ntbly of chromalin by dCAF.I a~ dCAF~ is ~rly commie wilhi~ I0 rain. ~e dCAF-I a~ivily c~uri~ wish Ihe ~actor-I (CAF-I), a ~aclor Ihot has ~¢n ~ound chromnlin d,ring I:~¢e lumot (~ nnli~n.mcdi,{cd, simian vims 40 (SVd0) ori~in- dc~ndcnl I)NA replicafi~. ~e (ICAF~ actlvl;3' copurified whh a Innc-himling pmlcln dml was puri~ed Io >~)~ homogeneity. But~cr. M., ha, T., Komakn~a. R. T., and Kodona~=, J. Pr~cc(lings oF the N;tti~al Academy of Sclenc~ USA 92: 117~-117~. ~m~,
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Other support: National In.~titutes or Iteahh and Nati,n;ll Scic,cc R,mdation. From Ilia Department of Biology. and Cooler I'or Molecular (';enctics. Universily Calirom;a. San Diego. La ]ella. DIFFERENTIAL ACTIVATION OF ERK AND JNK MITOGF, N.ACTIVATED PROTEIN KINASE$ BY Rar-I AND MEKK Growlh factors activate mitogen-aclivalcd prolcin k;nm;c,; (MAPKx). incl,lding calraecllular signal.rcEulated kina.~s (ERKx) and Jim klnasz.'~ (.INKs). AIIh(,,gh signaling cascade f.mm growth/'aclor reCeplors t, I'-'RKs is relatively well ,nder- st(v.xl, the pathway leading Io JNK activatinn is more ,bsuurc. Activalion ,I'.INK hy epidermal growlh I'aclor (EGF) or nerve growlh I'xmr (NGF) w;v~ dcl.x:ndent mz li- Ras aelivalinn, whcrea~/NK acllvaliem hy I.m.r necr.sls I':zclnr cz Ras.inde~ndent. Ra~; aclivates Iwo protein kinascs. R;d'-I and MI-2K (MAI'K. ur ERK. klnzse) kinase (MEKK). RaFI contribules dJmclly In ERK aclivati~ hul to JNK activ-',tion, whereas MEKK partlcipaled in iNK aclivaliem but cam;cd ERK aclivation only after overexpressi~n. These reSulls demnnstmlc Ihc exi.~tcnce distinc; Ras-dependent MAPK cascades ~ one initiated hy Raf-I leading m I-~RK activation, and the other initiated by MEKK leading to INK acdvalilm. Minden. A.. Lin. A.. McM--.hon. M.. Lange-Carler. C.. Derijard. 13.. Davis. R../.. Johnson. G. L. and ]tar|n. M. Science 266:1719-1'723. Decemlx~r 9. 1994. Olher supixxl: National Instilules o~" |lcahh and lee American CancEr From the Deparlment of Pharmacology. Program in Biomedical Sciences. School hi" Medicine. Univez~ity of" Calil'omia at San Diego. L~ Julia. CA. l)N^X Inslitule or Molecular and Cellular Biology. Pale AIIo. CA. Division or Flasie SciencEs. Nalional Jewish Center for Immunology and Respir.'ll(wy Medicine. Denver. CO. and Iloward Ilughcs Medical Im.titute. University ,1" Mas~;;zchu~;clls Medical Schmd. Worcesler. c-JUN N-TERMINAL PIIOSPiiORYLATION CORRELAT-ES WITI! ACTIVATION OF TIlE JNK SUI~GROUP I]UT NOT TI IE EKK SUflGROUP OF MITOGEN-ACI]VATED PROTEIN KINASF.S c.]un Iran.~criptional activity is stimulated by phosphon/lalion at IWo N-lerminal sites: Ser.63 and -73. Phosphorylation of these sites i~; cnhancc(I in reslmnse to a variety or extrac¢llul~r stimuli, including growth I'aetnrs. cytukines, and UV irradia- 84 don. New members of Ih-" mitog¢n-activated protein (MAP) kinase 8mup o£ siBnal- t~d.cin~ enzymes. Ic~cd JN~s. bind to lee aclival;on domain o~ c-J.n and s~cificolly pho~ho~latc t~se sites. Hoover. I~ N-le~inal sites ~ c-Jun w~re also sugg~l~ to ~ ~ospho~latcd by Iwo ct~r MAP ki~cs. ERKI Despile these m~s. we fi~ that unlike Ihc )~Ks. ERKI a~ ERKZ ~ not ~- pho~laze the N-retinal sites or c-Jun it, vitro; instead they phosphorylale inhihlm~ C-le~inal site. Fun~om. I~ ~oz~l~li~ o~ cJun in z'h'o zl lee N-tO,thai sties c~clates wile aclival~n ot the JNKs ~ m Ihe ERKL ~e ERKs arc ~bahly involved in I~ ;educl;~ o~ c-~x expmss;~ end I~hy lee stimulation o[ AP-I aclivily. Our sludy su;lesls Ihot Iwo diffe~nl ~hes or lee MAP kindle group am inwlved in the slimul~lion or AP-I ~ivily Ihmu]h Iwo differem mechnnisms. Mindcn. ~.. Lin. A.. Smeal. T.. ~ri~ard. B.. Ct~. M.. Davis. R.. and Molecular end Cellular Biology 14( IO):~R3-6~)RX. ~o~r I~J. Olhcr supra: Nalinnal lnslilules or Ilcallh :r4 ~ A~rican Fr~n Ihc ~pn~,Rnl n~ Phamzacol~y. Pm~r.;..~ in ~ior~li~,l Molecular Gcnclics. Unive~ily or Colifmi... San DieBo. Sch~l Jolla. CA. ~panmen~ o~ Pha~ology. So'~z;:wesl~ Mnd~al Center. Unive~l~y . o[ Texas. Dallas. TX. and Howard Itughc: Medical Inslilulc and Program m Molecular Medicine. Univcrsily or Mass~hu~lls &ledical 5ch~l. Wo~csler. MA. JNK2 CONTAINS A SPECIFICITY.DETER;.|iNING REGION RESPONSIBLE FOR EFFICIENT cJun BINDING AND FHO~PHORYLAT1ON The transcriplional nclivily or c-Jan is auk.retired Ihro~Jgh phosphenjladon at Iwo silos hy a c-Jun amino-lcnninal kinase (JNK). All cells express two dlstlm.1 JNK ~clivitics. 46 and .~5 kD in size. II is nnl ckar which of Ihem is the more impo~lanl c- Jun kinar.e and how Ih~'y specifically recognize c-Jun. TEc 46-kO rorm ol' ,INK was identil'ied as ,'t new member of the MAP kinase group of. siBnal-lransduein]: enzymes. INK I. I lere. we repot1 the molecular elonlnL: or the $$.1cD I'omz or JNK. JNK2. whicE exhibils 83% idenlity and slinkier ~gulat|on to JNKI. Despite this close similarity, the two JNKs differ greatly in Iheir ability !o interacl with c-Jun. JNK2 binds c-Jun -2~ limes more Efficiently Ihzm JNKI. and :s a resull Eas a lower K. toward c-.lun than JHK I. The slructuml b.~.,L: f.m" Ibis dil'l'cmnce was investigaled and traced to a small 13.stmnd-like region ne-~' the catMylic r,~kel of. the enzyme. Modeling ~ggcsts that thL~ reBion is solvent -'.rmsnd and theml'om is likely to serve as a docking sltc that increases the cl'f.ccli~,.~. ~onecnt~tlon of" c-Jun near JNKZ. Th~-se results e~plain how two closely related MAP klnases can differ in their ability to recognize specific subslrates and Ihereby ehe[l dill'cecal biological responses. Kallunki. T.. Su. B.. Tsigelny. I., $1uss. H. K.. D~rljard. B, Moo~. G.. Davis. R.,'. end Karln, M.
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C) Genes & Development 8:2996-3007. 1994. Olher supporl: National Instilulcs of I leahh and Ihe American Cancer Suciety. From the Departments or Pharmacology -~nd Chemistry. Center I'or Mnlccul'.~r Genetics. Program in IHomedical Sciences. Sclmnl ol" Medicine. University or Calil'omia. San Diegu. I.a Jail-% CA. lloward Ilughcs Mcdic-'d In~titme. Prngram in Molecular Medicine. and l)eparlment or lliochemistry ~md M,lec,l~r I~i.logy. University of' Massachusetts Medical Sch~l. Worcester. MA. c-Fos TRANSCRIPTIONAL ACT[VrrY STIMULATED I~Y I I Rat-ACTIVATED PROTEIN KINASE DISTINCT FROM JNK AND ERK Ras proteins exer[ their mi|ogenic and oncogenlc elTects through activation o1" downstream prolein kinases. An imporlanl qucslion in how Ras.gcneralcd signals reach the nucleus In activate downslream largcl genes. AP.I, u hctcrndlmeric cam- plea o1" ,tun and Fos Proleins, which activates mitogen-inducihic genes, in a major nuclear la~gel of Ras. Ras can mimulale AP-I aclivily by inducing c-J'~.r Irans,:rip- don, a process which is probably mediated hy the ERKI and -2 mitogen-aclivated Woteln (MAP) kinases, which phosphorylate the Iranseripllon I'ucror Elk-I/TCI:. Besides inducing transcription tram/as andj~n genes, milop.cns and Rat prolcins enhance AP-I activity through phosphorylation of c-Jun. Phosphorylatlon or the c- ,tun aclivalion domain leads to c.j.n inducllOn through an autorcgulatory Ionp. Ras- and ullraviolet-responsive protcln klnascs thai phosphorylale c-Jun on serinc residues al po$ilions 63 and 73 and stimulate its transcril~iona,I activily have been idcntil~ed. These praline-directed kinas~s, termed ,INKs, are novel MAP kinases, h is not clear, however, whether ¢-Jun is the only reeipieol and JNK the only Iransduocr ol" the Ras signal to AP-I proteins. A shor[ sequence surrounding major JNK phospho~lalion site orcJun is consen,cd in c-Fat and is pa~l or its acti. ValiOO domain, suggesling that c-Fat may be similarly regulated. Ilere we show thai Ras do~s indeed augm~ll Ih¢ Iranscriptiooal aclivily or c.Fos through phosphon/la- llOn at Thr 232. the homologue of Sac 73 or cajun. Ilowever, this is mcdiared by a novel Ran- and mitogen-teponsive praline-directed prolein kinase that is dlffcrent from ,INKs and ERKs. There/ore, at leasl three lyj~s of praline-directed kinzses [tansmil Rata and milogen.generated signals to the transcriptional machinery. Dang, T. and Kzrln, M. Natu re 3,71: J 71 - 1 "/.~. Seplemhcr R. 1994. Olher supporl: National |nslltules of" I|¢allh, American Cancer Society, Tobaccn- R¢lalcd Disease Research Program. and the I.~ukemia Society o1" America. From the Dcp~ulmcnt of' Pharmacology, Center for Molecular Genetics, University o1' Callromla at San Diego School o~ Medicine. La ,iolla. CA. 86 IDENTIFICATION OF A DUAL SPECIFICITY KINASE TIIAT ACTIVA'T~J TIlE JUN KINASF.S AND p.'l~.Mpk2 One Ras<lependenl proleln kinasc ca.~,~c leading: from gmwlh f~lor mecpIom to the I]RK (cxtn~ccll01nr sigual-t'c[-'ulnt~l ~io,'l.~s) suh~|'oop ol' mitolecn.~ctivutcd protein kina.~s (MAPKs) is dcl~mlent on ;:~,, prolcin kinase Rar-I, which ~tlvates the MEK (MAPK, or ERK ki~sc) dtml si~.cifi¢ity kirtles. A second pmteln cascade lending Io ucdvalion or I~ Jun kloases (JNKs) is de~n~l~l on MEKK (MEK ki~). A d,nl-s~i~cily kina~ I!~ nclivales JNK. nard JNKK, w~s li~cd Ihal foncd~s ~lw~n MEKK nnd JN:~. JNKK ~llv~lc~l I~ JNKs ~l did aclivalc Ihc ERKs and was un~s~nsiv¢ to %:[-I in Imns[ccled IIclJ cells. JNKK also activated ~thcr MAPK, p3g (Mpk2: e$ . m~mmali~n homolog o¢ I1~1 from ycasl), whose ac[ivily is regulated similarly :~ ,hal of the JNKs. ~n, A., Mindcn, A., Ma~in~l[o. H., ~a~l. F.-X.. ~n~c-Cadcr, C.. Mceufio, F., Johnson. G. L., and Karin, M. Science 268:2R6-2~, April 14, I~. Olhcr sup~: Hallonal lnslilulcs o~ I leallh. From the Depa~m~l or ~acology, ~uc~m ~ Biom~al Scte~cs, o~ Californla-San Diego School o[ Medicine, ~ Jolla. CA, Division o~ Basic Selene, Nali~al Jewish C~ler rot Immurnl~y & R~sp[~[o~ Medicine. ~, and Signal ~nnn~cnllcals. San Diego. INTERACTIO~/OFTIIE p53-REGULATE,. PROTEIN GADD4~ WITII PROLIFERATING CELL NUCLEAR A~. ":'~ F.N G,4DD45 is a ubiquitously e~presscd ,.,~',~malian gen¢ Ih~l is induced by DNA damage and ccrlain other slresses, i.i;~e ~nother p53-regulated gene, p21""~',who~¢ producl binds Io cyclin4el~:..~dent kinases (Cdk's) and prolif'emdng cell nuclear antigen (PCNA), G,4DD4.T has b.~.n associated with growlh suppression. Gadd4$ w~s found Io bind Io PCNA, a nounal eomponcnl or Cdk co~nlexcs I~d a protein involved in DNA replicalion and r.,'p~ir. Gadd4$ sllmnlaled Ol,lA ©sclslon repair in ~,irro and inhibiled Chip3' or cells inlo S phase. These tcsulIs cslaDlish G,4DD45 as a link belwccn Ih¢ p53-depc~cnl cell cycle eheckpoinl and DNA repair. Smith, M. L.. Cheno L-T., Zhan. Q.. Baeo I., Chert, C.oY.. Gilmer. T. M.o ICaslan, M. ~., O'Connor. P. M., mid Fomace, A. ,i., Jr. Science 266 : 13"76-13R0, Novcmher 2.s. 19')4. Other snpporl: National Institutes or ! lcalth nnd the Lcokcmi~ Society or America. From Ihe Laboratory or Molecular Pharmncoln[~y, Developmental Therapeutics Progn.n. Natinnal C;mccr Institute, Fletl|es~l's. MD. ,iohns Hopkins Oncolopy Ccnler, l]:dlimnre, MD, nnd Glaxo Research lnstilute. Research Triangle Park, NC.
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C) PROTEIN Pl IOSPI IATASE~ IN PROKARYOTF_S: REFLECt'IONS OFTllE PAST. WINDOWS TO TI IE FUTURE? Our knowledge of Ihc ;dentilice and role or ihc ph(~ph.proleins, protein ki~ases, and pmlein phosphatases in prokarynlic organisms laL~; suh~;l:mti:dly behind Ihat wilh regards to eukarynlic organi.,wns. Ilowevcr. one Ihiog h;l~; IIeC¢)llle ap~lrClll in recenl ycars~the wall that has divided lira pr~.eio phosphorylation nclworks or prokaryolcs From die eukaryoles appca~ Iobc ~m the verge or lumhllng down, The first ca,,uahy of this scienlific Jericho i'~ II.c mcanll;g a.d validity or the c(mcepI nr prokuryotc.and eukun.rotc-spcclfic prmein kina,,e.~ aml pr¢)leio pIm,;Idlahp;us. An.lhcr likely ca..uahy i,. Ihu cnocept thai pmlein pho'.ph.ryl:dion rcpre~cut.~ a rclalively recent addillon tn Ihe cell's urinal of" regul.'.lm'y mech:ulislns, imc ~unior lu nllosler- i.~m anti gcnc re~ulalion. The widespread flail|ire ~F Ihe emerging paralh:ls h~lween prnk-',ryotic and cnkaryollc or~-'mi~n.~ suggu~,l th;ll the h.lhlin~ hh~:ks or prexenl day I~n~teio ph.sph~}i~l-',lilm nctwmk.~ are in race ¢inile anci¢ill ida nature an I ill;iv have b~en op~nltive as regulators in the primi ivc ce I.; cxl;int ~CGWC I|le divergence'or our present phylo~enetic dumains. II'lhis is Irue. even IO a limiled cxlcnl. Ihen Ihc alien" prnleio ph~),~phoo, ladon nclwnrks of pmkaryollc organisms may ~rvc as wimk~ws into as yet undiscovered portions of the signaling nelwnrk.~ of their cukaryotic hrethren. Time will Icll. Kennelly. P. J. and Polls. M. Advances in Protein Phosphatases t~:33-68. 1994. Olher support: National |nslitulcs o~ Ileahh aml National Science I"mmd~lion. From the Department of Riochemislry and An~crohic Microbiology. Virginia Polytechnic In..,titule and Slale Universily. l]l:lek,;hurg. VA. ISOLATION AND CLONING OF A PROTEIN-SI~INI.~I'I IREONINE PI IOSPI IATASE FROM AN ~RCllAEON A divalenl metal ;on-slimulaled protcin-scrinc/threonioe phosphatase, PPI-arch. was purified approximately l.O00.Fold tram the exlreme acldnthermnphillc archaeun S.Ij'~,lob~s snlfutor~cu$ (ATCC 33091 ). Purified preparations contained 40 Io 70% o1" Iotaz protein as PPI-amh. as dclam~inod by assay-ing sodium dodccyl sull'atcpoly. acrylamide gels for protein phosphalase aclivity. The firsl 25 amino acids of Ihe pro. tein.*s sequence were identified, as well as an inlemal sequence spanning some 20 amino ~¢ids. Using this inl'onnatio~ we cloned the gone For PPI-arch via Ihe appli. callao or PeR and enovemional cloning techniques. The gent lro¢ PPI-arch predicted a proleln of 29] amino acids Ihal bore slrJklng resemblance Io Ihc members of major Family of I:w0tein-serioc/thmonine phosphatases. From mcmhcrs of Ihe dnmain Era'urea. the PPIr2Ar2B supcrFamily, Tim core or the protein, spanning resid.cs 4 In 27~. possessed 29 Io ]1% ideally wilh IIlese cucaryal proteio phnsphah~.~s. Or Ihe 42 residues Found Io bc absolutely conserved am¢mg the knl)wn cuca~jal mcnthers of Ihe PPI/2A/2B superl'amily, 33 wen: present in PPI-arch. If" highly con.~ervative slilulions are included. Ibis Iolal reached ]7. The greal (I¢~ree of ~equencc cnnscrva- lion helween molecules From two dislin~:l phylogenelic domains implies thai the members of this enzyme superfamily h,¢( evolvod as specialized, dedicated protein phosphatases prior IO Ihe dlvergenc¢ ol" t ",e. 1bees or ihe Archnrn and Enrar.yo I'tom one nnolhcr. Long, J., Cameron. A. J. M.. Buck¢l. $., =~ : ; Kennelly, P. ]o,mal of Bacteriology 177(22):6~10-6517, November From the Ocparlmcnls o1" 13iochcmislr}. and Anaerobic Microbiolo=y, Virginia i'nlytccb.ic Inslilulc mad Stale Universily, Blacksbur~:. VA. nod Pillntan-hhmre Company. Terra I latlle, IN, DNA STRAND EXCIIANGE IN TIlE ,e,.'ISENCE OF I IOMOLOGOUS PAIRING The strand exchange rcactlon thai is w=.dcly used For in t'itro studies on recombi- nation of DNA molecules is generally presumed to rc~lll I'~om •prcceding homolo- gous p~iring step. With Ihe use of a single-s'rended circular DNA and a sho~ homol- ogous linear duplex I'ragmenl as subslral¢, in a model strand exchange Raclion, il wax round thai modest concentrations or ] .,lyelhylene glycol or sail promote I'orma. llon o1" heterodupIcx molecules and sleeted ¢;,change. When the duplex fragment w= parlially rcxcctcd wilh an cxonucleas¢ ¢x~ring a shorl single slmnlled sl~-Ich on Ihe ends. Iha reaclion w¢~ promnlcd by the -~,61lion of comm~seial bovine scnJm albu- min. The transcription Faclor TFIIIA promoled slraod exchange when lira DNA sub.. slrales contained the eognale DNA binJin= sequence recognized by Ihe prolein, The~¢ ohscrvalinn.~ suggest Ihal deteclion or stand exchange ia vitro do~s ~'~rily imply a preceding homologous pairing step. Kmle¢, F. It. and Ilollom~n, W. K. The Journal or Biolngic~l Cbemlsl~7 26~(!,1):1 fi16.1-1016~1. April I, 199.1. From the Jefi'e~JOn Cancer Inslilule. Thomas JelTe~on Unive~ily. P~ilzdclphla. PA. and the Department of Microbiology, Comell Universily Medical College. New York. NY. TELOMERIC PROTEIN.DNA POINT CONTACTS IDENTIFIED BY PI IO1"O. CROSS-LINKING USING 5.BROMODEOXYURIDINE T'n¢ O.~trirhn teiomcre protein sp¢ciF,¢,lly r¢cognlzes single-smmded lelomeric DNA. rnm~ing an extremely salt resistant and klnetically stable nucleo~rol¢in com. plcx. The absence of information ~m how Ihis hctenxlime~c protein hinds Io DNA 89
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C) O) O0 promplcd Ihis photo-cl,oss-llnking study. Multiple protcin-DNA photo-class-links -',re formed upon UV in~dlalion ot" O.~'lH¢'ha lclnmores rcconslilulcd wjlll a oli~onucleofide le~in~lin¢ in 5"-T.T,,T.T.G,~G,,G.O:I~T,T~F,G.(;,O~G,.~ Si:e- s~i~c subslilulJon of certain nucl¢olide; wilh 5-brom~lco~ynridinc (I]rdU) 8really incrca~d Ihe pholo-cro;s-linkin~ yield, each sub~lhullon favoring a ~ci~c pmlcln- I)NA ¢ro~-Iink. For example, substhution t~f I~rdU fear T, resulted in 25% li=tking or the ~und DNA. = 10-fold increase over Ih¢ un~uh~litulcd DNA. subunils of the Iclomcre pmteln cross*link In, and arc Ihcre~or¢ near. Ihe DNA. 3~¢e ~inl conlacls wilhin this nucl~prmein complex, involving the ~ suhunil. were ¢slablishcd using BrdU su~ilUli~: T~239. Tyr142. and Ilis292 crn~-Iink G,. T,,. and T~. re~clively. One ~mlo.cro~.link. 3~r2~9-(;,, ~c'u~ amid a acidic slrclch of Ih¢ ~ subunil, counter Io eX~lallons for amino ~cids Ihal ap~ach d~e polyanionic DNA. The two remaining ernst-links arc In amino acids in hyd~ophohic regions or Ih¢ primary ~lypcplidc scep=cncc, co=lsislcnl wilh hy~dh¢~i~ that hydrnphob~ inler~liems ac¢ouul for die ~dl ~c~i~l=lnc¢ (>2 M NaCI) of IhJs prolein-DN~ complex, ~esc Iwo pholo-crn~s-links sugg~l Ihal Ihe prOlein m=y hind tcl¢=meric singlc-slrnnded DNA hy intcrcah=lhm nf ==rm~=alic rc~idue~ into a nuclcmide latli~. Ilick¢, B. 1,, Willis. M. C. K~h, T, !!., and Cech, T. R. gi~hcmlsl~ 33(I I):33~-3373. 1~4. Other supra: Nallonal In~lilules of Ileahh. Fr~ Ih¢ Depanmen~ of M~ccular, Cellular and ~velopmenlal Biology. I Inward Ilu~hcs Medical Instilule. and Deparlmcnl of Chemistry and Biochemislry. Univcrsily oF Colorado. Boulder, CO. IN VITRo ASSOCIATION BETWEEN TIIE|UN PROTEIN FAMILY AND TIlE GENERAl.. TRANSCRIPTION FACTORS. TBP AND TATA-binding pl,olcin (TBP). TRIB and/or other TI]P-associatcd l'aclots (TAFs). Using affinity chcommog~aphy we dc'monslrale Ihal memhera of the .lun family of tran~Cripllonal aclivalora inleracl with holh TIIP and TFIIII in ~.itrn. TBP hinds Io holh the N-terminal a¢liValion domain and C-torn,lnul h~.ll' regions of c-,lun. whereas TFIIB binds to only Ihe cJun bZIP domain. This inleraclion mquiras dimerizalion of the 3un protein. The ahilily of Ihe N-lenninal ac¢ivalion domains c-|t~n. |unl3. JunD m~.d v-.fun to intel'~ct with TBP i. t'itrn ¢on'clate-,~ with their scriplional activily in ~;'n. Domain mapping expcl'imcnls indical¢ Ihat c-]un inter- acts wilh Ihe conserved C-Icnninus of TBP. $1udies using a sel of TFIlI3 inf~mc deledon mulanls demonslralc Ihal C-~erminal amino acids 17X-201 and 238-316 pl~y an imlx~nant role in modulating Ih¢ interaction between TFIIB and ¢-]un. Allhough phosphorylalion of Ihe ¢-Jun N-lennlnal acdvalinn domain .stlmu|ales c-,lun Iran- scr|plinnal activity in rlw~, il has no elfeel on Ihc ability at" c-Jun to interact wilh either TOP or TFIIB ~n ~tro. These dala suggest that d~e ,lun I'amily of actlva~or 90 telns may aclivale Ironscdplion by inlerac~H~ with Ihe Ezeneral transcription I'actor~ TBP and .]'l:ll I~. Franklin. C. C.. McCullnch. A. V., and Kr=P, l~inchemlcal ~ouma1305:967-974. 1995, Olher SUplX~l: American Cancer From the Division of Ilaemalology and Dinl~ingh:|m. AI. IItTl" "EROGt~.NEITY AND MICROTUBUL~. INTERACTION OF TIlE elIOt AN.]'IGF.N, A MITO$1S-.SPECIFIC KINE.'~T:';-LIKE PROTEIN'~ ~N~,.~'sls oF SlInlKIMAIN$ I:.XI'I~I~$1:.D IN INSF.CT $F9 TIe CIIOI onligcn is a milnsis-spccil'~ ~inesln-like molar Iocaled al Ihe inlet. xonnl rcl~ion nf Ihc spindle. The human c~ NA codln¢ rnr Ihe anllgen ¢onlains d~main wilh sequence similarily Io Ihc mol~" "Jomaln of kinesln.like pcolein (Nislow c~ al.. Nnun't 359, 543, 1992). Ilere we elr'.:d cDNAs encoding Ihe CIlOI by immurm~crccning or a CI IO Uni-Zap ¢',nression library. Ihe sam© species which Ihc original monoclonal anlibody v,~s mi,~d, cDNAs el" ClIO cells 953 amino acid polyp~ptld¢ with a cak:ulaled molecular msss of I09 kDa, The Icrnlinal 73% of the onligcn was RY'~ idc~ltlcal Io Ihe human clone, whereas the remainiug 27% of Ille co(ling region shnwe,! only 4S~, honloloiy. Inse¢l SIV cells inrecled will1 haculovirus cont¢ining Ih¢ ful|-Ienglh insert produced 105 and 95 kDa polypepddes, ;lie same douhlel identified ".,s Ihe original antigen in CHO cells. Tnancalcd polypeplid~; concspondlng In Ihc N-lerminlI meier and C-lenninal lall prnducc~l a .';6 and 54 I~Da polypep1id¢ in SP? ('ells. resptclively. Full and N-Icnnlnal pr(~ein.~ co.sedlmonlcd wilh, ~nd caused I~m,lling of, brain mic~tubules whereas II1," C-lem~inal polypcplide did n~...:'.Is expressing Ihe N lemlinus I'o~11ed on-" or more cy¢of)lasmi¢ prnccs.-.es. Immunu'~-.oresccnce as well as eleclr0n micro- scnpic ohs~rvmions revcoled II1¢ promote o; hick bundles or mlcnxuhules, were cln.~ly.p;~ckcd, funning a nl.',l,glnnl I,i1~ .jqsl hcnemh Ihe cell membrane core in the processes. The diffusion enefficicnl and sedlmenlation ¢oefficicnl wc~e delennined rot ch~ native CHOI anliBen by gel filtration and sucrose densky gradi- enl conlrifugalion, rcsr<¢tively. The nat;re molecular mass or overlnduced prmein in SlV cells w,,s caiculmed as 219 kDa, suggcs'.,,~g that Ihe ond~en exists =s a dlmer. Inlrinsic CIIOI auligen in cullured mammalS.an cells lrorms a larger native complex (nalive molecular mass, 362 kOa), which may su~gesl Ihc presence or additional molecule(s) associating wilh lhe CIIOI meier molecule, Kuriyama, R., Dragas.Granoic, $., Maek.',w',, T., Vassll~v, A., Khodjakov, A., and Kobayashi, II. 3unreal of'Cell Science IO7:.",4115-3499. 1994. 91
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Other suppo~l: Notional ln~lilules of lie•lib and Ihc Japan 5.)ciely for the Promolion of Science. From ~he Dc~nmenl ef Cell Bi~iogy and Ncur~unalnmy. Univcr~ily .f Minnesota. Minneawdi~. ~N. CIIARACTERIZATION OF A MINUS END-DIRECTED KINESIN-I.IKE MOIOR PROTEIN FROM CULTURED MAMMALIAN CI..'I.I.S Using tile Clll}2 monoclonal antibody raised against CIIf) spin.lies {Selliltn, C.. M. Kimhle. and R. Kuriyama. 1992. Cell M.liL Cvtt~.r~clet.n. 22:7-Z4) we identified -', 66.kD pmlein located al Ih¢ inlerpha.,~c ¢enlrcl~¢~me and nl/Iolic spindle. I~nlaled cDNAs f()r Ihe anligcn enc~Jc a 622-aminn acid I~)lyl~plide. Sc(incncc ~nalysi.~ revealed the pre.~cncc o~" ~)4(I-amim~ acid resid,c.~ in the ~)()l I tcnniml~, which hm~ol~,u~ Io Ihc molar dnmuln ~n~od arming nlher mcm~rs ~d Ihe kincsin ~u~Kamily. ~e W~cin i~ composed nr a ccnlntl ~-hclical p~ion wilh ~h~hular dnmaln5 at ~lh NII~ a~ COOII le~inl, and I1~ Cpil.~ I. d~c mom¢lon~d ~nlil~ly rc~idcs in the ce.~l a-belial slalk. A series o~ dclclion C~sl~cls i~ ~'itro •nalysls of micn~lu~le inlcraclions. While Ihc micmluhule hindin~ and bundling ~clivJlics ~quirc ~lh Ihe p~s~ce or Ihe COOI I Icm~inus and Ihe o-heli- cal dam=in, t~ NII.-lc~inal hale o~ Ihe ~li~c. lacked Ihc ahilily to inlcrncl wilh mic;olubulcs. ~c [~ll-lcnglh as well :s delelcd p~cins consisting or Ihc COOII- le~inal molar and Ihc ~nl~l a-~li~l ~l=lk sup~ microlu~lc ¢lidin~ wilh vel~ily ~n~in¢ from 1.0 Io 8.4 ~minule. ~c ~c(I of microlubulc movcmcnl de~elsed wilh decreeing Icn¢lhs or Ihc cenl~l slalk all:ched In hal maim. ~e mic~ubulcs moved wilh Iheir plus end I~in~, indic•ling Ih~l anligc, is a min~ end-direcl~ motor. ~ OIO2 ~quence shows ~6% id~lWy In 115~. a Bent I~:~ed al Ihe cenl~c ~d or~he human MIIC re~i(m in chrom~ som~ 6 (Ando. A., Y. Y. Kikuli, H. Kawala. N. Okamol.. T. Imai. T. Eki, K. Yokoyama. E. S~da. T, Ik~um. K. A~. and Ii. Inoko. 1994. 39:194-2~). ind~alin~ thai IIS~ mi~hl rcprescnl a human homolngue CIIO2 anllgcn. and Kh~kov. A, ~c ]~maf or Cell 9ioh~y 129(4): I(NB- 1(159. May I O~her sup~n: Nadonal ~n~lilulcs o~ II~llh nn(I Ihe N:lional Sciencc Foundation. From Ihe Depa~menl o~ Cell Biology and Ncumanalnmy. Unive~ily or Min~sola. Minnea~lis, MN, and ~pan~n~ o~ Gcnclics and Cell Rinl.~y. W~;hin~lnn Univ~ily. Pullman. 92 IDENTIFICATION OF INTRINSIC DIMER AND OVERF-~CPRESSED MONOMERIC FORMS OF-t-TUBULIN IN SF9 CELLS INFECTED WITII BACULOVIRUS CONTAINING TIlE CIILAMYDOMONAS -pTUI~IULIN SEQUENCE A new member of tile luhulin superl'ami!},. "t-tuhul|n. is localized n! micro- tuhulc-org;~ni~.ing centers (MTOCs) in a v:~:ie:y or eDNA coding for Ihe full-length ~quence or ~,, -:ubulln was expressed in ;n~cc! ovar- ian SI9 cells using the b~culovirus expression s~slcm, Approximately h~lr of the induced 52 kD-', y-tubulin w~s recovered in tile SUlCm=tanl •flee centril'uBallon St9 cell lysatcs at I R.(XlO R I'or 15 minutes. When Ihe cell supern~tant w~ ~malyaed by FPLC on • SuFerdex 200 s|~'ln~ column, Chlamy<fomna~ "y-tubulin inlo two maine peaks. The lagging pea~c comai;~cd a monomeric I'onn or "ptuhulin with • scdimentalion co¢l'ficicnt hi' 2.5 S. w~;' h inlerac~ed wilh Ihe Supcrdcx col- umn in • salt-dcpendcnl manner. The leading peak. wilh a~ apparent molccula~ mass o1"900 kDa, con'espondcd Io u molecular chaFemnin complex, and TCPI chepcnmln rclea.~¢d folded "y4ubulin polypepddc I'rom the -omplex in the pmsencc of M~ATP. The relca~;cd "y-tu~lin monmncrs we~¢ cap.'l~. • of binding Io micmtu~les in ~'~tr~ and hiochcmical q|mnlilies of acllvc monom~:rs .~.erc further puri~',:d us;nlz a ccmlhi- nation of size-exclusion and ion-exchange co;u.'., chromatography. The endop:no~s St9 cell ~.-lubulin migrated faster Ih=n Cldme.~.:~mno$ "f-lub~lin wile an apparent molecular mass o1" 49 kDa on ~.els. Analy~ces .m gel fihmlion and sncro~e denslly go|client c~nl~ifi|g-'.lion showed Ihal. while nve~xp~ssed Cl#hm~.t'd,~m~m~s -ptubuliu was present in n mnonmeric rnrm. cndo~.eno,s -/.lut~din I'rom SF) and IleLa cell~ exlsls ~s n diner. "lllesc resulls n;ny sngees! tile ..x~sihility Ilml "y-lub,lin could a hetermlimer with httherlo unknown molecule(s). Vassilcv. A.. Kimhlc. M.. Silllow. C. D.. LaVoie. M.. and Kurl~.arna. R. Jnunnal or Cell Science 10~:101t.~-1092. 199.~. Olher supporl: N=lional Inslltutes of Health. From the Depanmen¢ ot" Cell Biology and Neuroan~0my. Unlvenlty of Minnesota. Minncapolls. MN, Deparlment of Oenetlc,; and Cell Biology, Univeraily Minncsola. St. Paul, MN, and Deparlmc:d el" Plant Biology, Universily of Minncsol.',, SI. Paul, MN. FACTORS DETERMINING SPECIFICITY UP 31GNAL TRANSDUCT1ON BY G-PROTEIN-COUPLED RECEPTORS: XeGUt.~T~O~ oe SIGNAL 'llt ANSI~'lt FI~OM RECE.Iq'O~ TO G-I'ROTEIN Amon~ snl~familie.~ or G-protein.coulded ..eccpIors. a~oni~lS iniliate ~eveml cell signaling cvcnls dcpcmllng on Ihc rcccplor sn~ype or effector molecule (E) expressed in a p,'trtlm~]ar c¢11. Dclcm~inanls of signaling specificity/efficiency may operate al Ih¢ R-G interface, where evenl] arc influenced by cell architecture or acc~-~sory prolcin.,~ found in the reccplor's micmcnvlronmgnl. This issue was addressed by chnracterizlng signal transfer from R IO G following. 93
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C~ stable expression el" the a~,,,~, adr¢oersle receptor in two cliff aleut membrane envlrou- ments (NIH-3T3 fi~obla$1s and the pheochromocylnma cell line. PC-12). Receptor couplin8 Io endogenous G-proteins in both cell lypes was eliminated by pertussis IosJn prelreatment and R-G signal tz'ansfer restn~e(I hy rcco~'~tilulinn fzl" cell mem- branes with puril~ed bzain G-protein. Thus. the recepl(,r has access Io the same popu- lation o1" G-proteins in Ihe lwo dill'areal cnvjronmenls. In Ibis signal restoration assay, egotist-induced octlvalion of G was 3-9-fo|d are:tier in PC-12 zts cutup;veal with Hlll-3T3 u,.adrenergic rcccplor Iransl'eelant,~. "]lzc cell-slX.'cific dil'l'ercncc:~ in signal Item, far were observed over a range of" receptor dcn'~itics .r G-prntcin corn:en- tre!ion. The augmented signal Iransrcr in PC- 12 v~'r.t'tls NII I-.'~T3 Irans/'eclants occurred despile a 2-~-fold lower level o/'receptors existing i. Ihc R-G-crumpled stale (.hish afflnlly. 8,"nyl-$'-yl im;dod|phn;t, glale.sen.,;itive a~( nisl hin~ing|. Ilzc existence of other membrane rectum Ihal inlh,ence the n.cl~lide binding hehav. ior of" G-proleJn in Ihe Iwo cell lype~. DelerBenl exlraclion of PC-12 hut not NIII- 3T3 membranes yielded a heat-sans|live, macromolecolar entity that increased "S- labeled guznoslne 5"-O-(Ihiotriphosphate) binding to brain G-pr~e|n in a concentrh- lion-dependent manner. Th~r,e dale indicate thai the transl'cr ol" signal I'rnm R to G is resulaled by a cell type.specific, memb~ane-associated protein thai enhances the agonist-lnduced eel|vat|on or G. Solo, M.. Kataoka. R., Dingus, J., Wilcox, M.. ! {ilderbrandt. J. D., and |.artier, S. M. The Journal of Biological Ch©mistry 270(25):!$267.15276. June 23, 1995. Olher support: Hatlonal ]n~titute~ of I|eahh. From the Departmenl or Pharma¢olosy. Medical University ul" South ~arulinn. C~adeslon. REGULATION OFo,-ADRENERGIC RECEPTOR EXPR -ESSION AND SIGNALING IN PANCREATIC I3-CELLS Aclivation of %-adrener~ic receptors (%-AR) in pancreatic ~3.cells inhibils insulin secretion in ~es]',onse In vazious stimuli, and acute or Iong-lcnn re~ulation %-AR ~eceplor-mediated affects may influence the lissue response Io ~ucose dishomeostasis. As an in;tial approach to this issue, we determined Ihe ell'eel of vari- ous metabolic a~J hormonal Irealmenls off a,-AR expt~-ss;on and couplin8 in Ihe pancreallc ~3-cell lines H|T-TIS and RIN-SAII. Rndlol;pnd bindinB slndies ([']|]P.X-821002) and RNA blol analysis indlcale Iha! bolh pancreatic p-cell lines exp~ss the ¢z,,~-AR subtype [for HIT-TI5 th~ maximum binding (B) ~-- 113 ± 2It; [~ RIN-SA|| B~. = 93 _ 18 I'mol/m~ orcell.lar proteln]. Treatment[r lllT-TI3 or RIN.$AH cells with ~lucocorti¢oids |dexamelhasone. hyd~ocorzixone, or predo nisolo~a (I IxM)l incrcasnd (%-AR mRNA level and rcceplnr protein densily Ihrec- Io fivc/'o]d. The 81ucocorlJco.induced increase in recel'~or dcnsily in III'T-TI5 cells was associated ~'idl I| an increase in the amoum or ~ceptms ct~uplcd In G protein as determined by analysis of hish-alT~nity 5°-guanylyl imidndlphnsphatc-sens|tlve bindin~ of |'11|UK.14304. * selective %-AR agonist, and 2) u greater |nhihilion or 94 forsl~olin-lnduced elevation of cellular ade,"~;ne 3'.$',.cyc1|¢ n~nopboapbate after receptor ~tlvati~. Receptor density in HI~-~I~ cells was n~ tltc~d by different ~rowth cen(lit~s, insulin (I FM), photO: ~2-my~istate 13-acetate (I ~M). ~ the sex slcmids Icslnslcmn~ nnd pmgesl~n~ (I FM). ~e~ licoids uprcgul~tc (a~-AR expmss~n =~ si¢.nling in p~atic p~ells, Such ~u. I~ti~ may o~te in n ccll-s~cific manner, :llowln~ discrete m~ulalion or tissue ~s~scs in ~lucosc dishnmcosl;~is. American Journal oF Physiology 269:E 162-[~ l ? I. 1~5. Oilier x.p~: Nazio~l Insl~lule of Neumlo~tc~l Di~e~ From the Dcparlmenl of Cell and Melee.Jar Pharmacology and Ex~rimenlal ~ro~mics, Medical Univc~ily of Soulh Carolina. ~adeslon. MONOCI.ONAL ANT|-DIPEPTIDE ANTIBODIES CROSS-REACT WITH DE'TYROSII'~ATED AND GLUTAMYLATt'.~ FORMS OF TUBULINS Two 'moouckmal =nllhodles. GLU-I and AI.6. ~|sed a~a|nsl ",/-L-ldutamyI-L- 81utam|e act,~. dipcp~i~X¢ (Glu-GIo| and Cog'-dependent ATPa.~ from Por.mrcinm. ~'espcctively. rcco~.ni:~ed the dipeplidc Glu-(;lu sequence. Whereas the unlih~xlies immuuol]..m.~ently slnined very law, it" .~ny, eyl~keletal I'ih~z in cttllt,~tl m-~lian cells, almo.~l all ;nteq~t.'~se as well :~.-. milel|c spindle mlcrOluboles balance visible ~fter Irealmen! of cells with carhoxypeplldas¢ A, Immunoblel an.~lysi| demoesl~tled intense eross-r~a¢lion ot" the ~ntlhodies to the ~x-lubu||n subunil. Tubulin isntyp~ produced ~.~ fusion prolei.ls |n h~lc~a wezc la~eled by both the a.lilx~lics (rely when die pt'oteins did nol czml3in I lylosil¢ Iesiduc at the C len~|. nus. indicating that GLU- I and A 1,6 speci~r=;:.v recognize Ihe dclyrosinaled I'omz o1" a-tubulin. When mlcmtubole prmein put|flee/'ram brain was proud, not only e~. also. to a lesser cxlcnl. ~-Iubulin were zevealcJ by Ih¢ dipepl|de ~tibodic$. A s3m- thelic tripeptide YED coota|nin[ one glu!am); ~,mup linked to Ihe second tesidu~ Ihe peptide via the y position was also mcog.~;.,:d by Ihe antibodies. Since Ihis tide sequence cowespoeds Io Ih¢ amino Kid ~".ucnc¢ o~" polyglutamyated class Iflp isotype at amino ,,aid post!ion 437 to 439. it |~ -.uzgested d~z! GLUol md AI.6 ahlc to recognize the ~ul~'nyllled I'~'m of ~ '.l~lin. These resells indicate Ihaz the C-terminal GluGlu sequence displays strong *~,tigeni¢ily, and Ih¢ antibodies nlzc the sequence present in Ihe C let~inm vf the del)'rosinalnd form Of ..-lubulin and the Blutamyl side chain o1"/~-tubulin. Pad.'c~laxly strong immunoR~clion was deteclnd wilh cili*ry and ~a~ellar mlcmluhules: Ihus. stable a~onema, I mlerotubules -',pp~.~r to h~ rich in po~l-tronslationally modified lub~lln sohenils. K-rlyoma. It., I.cvln. A.. Nelson. D.. Mndl. J., Fr-Jnkfutler. A.. and Ktmbl¢. M. Cell Motility nml I18 Cymskelcton .10:171-182. 1995. Other suplXarl: Nali~mal |n.'qilules of' Ilcahh. "
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From d~e Depanmenl of Ceil Biology and Ne.ro.~nammy. University or Minn~.ota. Minncaff)lis. Depanmenl of Biocl~mislry. Un;vc~ity nf Wisconsin. Madison. Wl. Immunclics. Cambridge. MA. and Oep.qrlmcnl ol" I]i.lugy. Universily of Virginia. Charlottesville. VA. REGULATORY MECIIANISMS TIIAT COORI)INATI.: SKELETAL MUSCLE I)IFFERENTIATION AND CELL CYCI.F. wrI'III)RAWAI. Skclclal muscle dlfl'crcnllalino enlail; Ihe cm, pli.g ¢)1" m.~clc-spccil~c g~nc cxpre~i.n Io Icnnlnal wilhdrawal From Ihc cell cycle. Scvcnll ,m.lcl~ have rcccndy bccn pro/x)sed which allcmpt Io explain myogenic Ir'.m~criplinn facl(~rs en.~ures Ihal prolifcrutlm~ ;m~l dill~:rcmi:~|i...1' ~kcle- lal muscle ccll~ arc mulually exclusive l.aslar, h. !1., SEa.k.S.X.. and Novilch. Cu~cnl Opinion in C~II ~;nl~y 6:7~8-7~. O,her sup~n: National Sci~cc Foundalion. Lucillc P. Muscular Dyslrophy Ass~ialion. ~nd I~ Mnrch nr Dimes Bi.h ~fecl~ Foundalion. F~om Ihc Dcpartmenl or Biological Chemislry and Melccular Pharmacology. I law=rd Medical Sch~l. ~oslon. INIIIBITION OF MYOGENIC DIFFERENTIATION IN PROLIFERATING MYOFILASTS BY CYCLIN DI-~EPENDENT KINASE Ahhough Ihe myogenic rcgulalo~ Myo~) is exp~cs.~cll in l~(difcraling mynhlasls. dilTc~enliation or lhese cells is limiled Io Ihc G,, phase or lhc cell cycle. Forced expression of cyclin D I. but not cyclins A, B. or F~ inhibiled Ihc ahilily of MyoD to Iransaclivatc muscle-specific genes and correlated wilh phosphorylation o1" MyoD. Transrcclion of myoblasls wilh cyclin-dependenl kin*~e (Cdk) inhibilors p21 and p16 augmenled muscle-specific gone expression in cells maintaio:d ~n high cooccn. Italians or seam, su~'gcsling thai an -',clive cyclin.Cdk complex suppresses MyoD function in prolife~=llng cells. Skapck, S. X., Rhcc. J.. Spacer. D. B., a~d lascar, A. il. Science 267:1022-1024. February ! ?. 199S. 96 Other support: Nalional Science Foundation, Lucille P. Markey Charllable Trusl, Muscular Dyslrophy Association, and Ihe h;acch or Dimes Birth Directs .Foundation, From the Department or Biological Ct.:mislry and Molecular Pharmacoloey, llarvard Medical School. Doslon, MA..'.~d Dcparlmeol of PedialriC ilemalolo|y- Oncology. Children's Ilospilal and Dana Frrb~r Cancer lnslhule, Boston, MA. CORRELATION OF'TERMINAL CELL CYCLE ARREST OF SKELETAL MUSCLE WITII INDUCTION OF p21 BY M),oD ,~kclctal muscle dilTcrenti.'~lirm entails Ihe coordination Of nluscle-~l~:ific lethe cxpre.~sinn aml tcm~innl willulr~twnl from Ihe cell cycle. This cell cycle an¢~l in Ihe O. phase rcqulrc~ Ihc rclinnhlasloma lumor suppressor protein (Rb). The funclk~l or Rh is .cgalivcly re,hi:dad hy cyclln-depc~dcm kina~es (Cdks}. which a~ coolrolled hy GIk inhihilors. -Expression or MyoD. a skelel.~i mur, cle-~i~cll'¢ IranscrIptlonal regulator, activalod the expresslon or Ihe Cdk inhihllor p21 during differenllallon or routine myocytcs ~nd in onnmyogenic ceils. MyoD-medlated induclion of p21 did not require Ihe lumnr suppressor IXnleln p~3 and conclaled wilh cell cycle wilh- .drawal. Thus. MyoD may induce Icrmi.-.'d cell cycle arresl during sk¢lnlzd muscle difl'erenlialinn by increasing Ihe exprer~;o*~ or p21. llalevy. O.. Novitch. B. G~ Splcer. D. 3.. Skapek. $. X.. Rhee. J.. Hannah. G. Beach. D.. and Lasslr A. Science 26"7:!018-1021. F'ebn~PJ 17.19~'~. Olher support: National Science Fonnd*tion. Locille P. ~Vlarkey Muscular l)yslrnphy Association. and Ih¢ ;vla~h or Dimes Birth Dcl'ccls Foundation. From Ihe Department of Biological Chemlst~y and Molecular Pharmacology. Ilarvard Medical School. Boston. MA. Deparlmenl of Pndlalrlc Hemaloloiy- Oncology. Childn:n's }lospilal and Dan~ I:a~b~r Cancer lnslilul¢. Iloward II~ghcs Medical Inslilule. Co;'4 Spring Harbor LaboratoP]. Cold Spring II:rbor. NY. DEREGULATED EXPRESSION OF E2F-I INDUCES S.PHASE ENTRY AND LEADS TO APOPTOSIS E2F-I. Ihc firsl gone product idenlified Imong a family, of E2F Ir~nscripllon feelers, is Ihoui;hl Io play a crilical role in G,IS progr~smn of Iha cell cycle. Tr-',nscripdonal aclivilies or E2F are modulazed during the cell cycle, mainly hy fan.arian o1" compIcxe~ between E2F and several key rc|ulalo~ of c¢11 cycle slkh 97
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C) Ihe relinohlasloma protein and relalcd proteins. To further understand the role• of E2F in the cell cycle progressio~, we have ovcrcxpressed exogenous E2F-I by using a tetracycline.controlled expression system. We have fi~und that the induced cxpres. • ion of E2F-I in Ral-2 fibroblasts promote• S-phase entry and subsequently leads to almplosis. The almptnsls occu~ in an E2F- I dex.m-tlcpcndcmt manner. Cells resistant Io Ihc induclion ofapoplnsis have lost the ahility to express exogenous F.2F-I. Cells growing in low serum are more scnsitlve to the 1:2F-I-mcdiared cell doalh. Overexprcssion o[ E2F.I mulanls Iha! impair DNA hinding nr transactivatirm does not a|rer cell cycle progression or induce apoplosis. These resulls define a novel pathway Io apoplosis and demonstrale thai premature .~-pha~;e entry is a.ssoclatcd with apoptmic cell dealh. Shah, B. and Lee, W..|L Molecular and Cellular Binh)gy 14(12):1~166-gl73, December 1994. Other .~uppofl: National lnslitutes of ileahh. From the Center for Molecular Medicinc/Inslitute of Riotcchnnlngy. Univer•ity of Texas Ileallh Science Cooler a! San Antonio. San Antnnin, TX. DISSOCIATION OF 1']4~'~ COMPLEX PORMATION I:ROM PIIOSPIIORYLATION AND HISTONE Ill KINASE ACTIVITY The cell cycle inhibitor mimosine was used In examine Ihc oclivalion of Ihc IO4"= protein kinasc in $ phase of the cell cycle. Addition of mlmosioe to cycling epithelial cell• haired cell cycle trave~e in $ phase, coincident wilh on inn!bidDer of p34"~ histo~e HI kinasc activity. Mimo•ine lreatmenl did no! oiler p'14"*~ syo!hesis or turnover, however, overall phosphorylatlon of p34" was decreased to near unde- I¢clab|e levels. Ahhough aclivity or p34,*~ was inhibited, Ihe abilily or Ihe prolein !o form high moleculm" weight complexes, a pbenomenoo associated with kinasc acli- valion in fifo. was hal affected. These resuh• ind~care thai p~4'~ complex I'onnallnn can occur in the absence of phosphonjlalion and Iha! phnspho~/latlon of p34~" is then required to activate these preformed complexes. Ehlcn. S. T.. Foul•oh. M. P~ Bumette. R. ~. Synd~', M., and LCOI'. E. B. Cancer Research 55:1994-2000. May I, 199~, nines suppml: National lost!lutes of Ilcahh. From Ihe Deparlmen! of Cell Biology, Vanderbil! Univcrshy. Nashville, TN. Thoracic Disea,~..s Re~earcb L!nil. Mayo Clinic, Rocheslcr. MN. and Dcparlment of Ri~a.'hcmislry and Molecular Bi~dngy. Mayo Clinic. R~g:hustcr. MN. 9g DIFFERENTIAL REGULATION OF P.'l,l~a AND P33"" BY TRANSFORMING GROWTII FACTOR-I), IN MURINE MAMMARY EPITHELIAL CELLS Cyclln-dependcot kinases {cdks) arc./'emily of ~xoteins whose I'unctio~ plays • crillcal role in cell cycle traverse, Transforming growlh I'aetor.~]~ (TGF-II~) is a pole,el growth inhibilor of epilhelial cells. $1ace cdks have been SUl~g¢~tcd as ble biochemical mockers fo¢ TGF-~ gro'~'th inhibition, we investi~a(ed tl~ e(f~t TGF-13, on cdc2 and edk2 in a non, el mouse mammary epi!heli.'d cell lioc (MMI~) and a TGF-0-reslst~nt MME cell line (BGIIL2). TGF-I~I dcc~ase• newly synlhe. sized cdc2 pnaein levels within 6 h ahcr mhlilicm. Coiocalenl wilh Ibls ck-cR'a~¢ in newly synlhcsizcd cdc2 proleln was n m:,.'-'cd reduclion in !Is Ibilily Io phosph~r¥. late ht.~ton¢ !! I. This decrease in kinase a..4is'ily is nnl due Io a change in steady.slate levels oft(It2 prolein, sided mRNA and lul:.l protein levels ol'cde2 arc nol red,cod until 12 h after TGF-~, addithm. This stlggests thai the klna•e aclivity of (lcpcodcnl on newly synthesized cdc2 p~otein. Moreover. Ibc protein xynlhexi~ another cyclin-dcFcndcnl kinnse, cdkZ, is no~ effeclcd by TGF-I~, additi~, but its kinase aciivily is subslanlially reduced. "thus. il appears Ih,*! TGF-~ decreases Ih~ kina~c aclivily of Ix~lb cdc2 nnd cdk2 by disllnet mcchan|sms. Faulsch. M. P.. Ehlcn. S. T.. ^oder~. R. A.. Bume~te. R. J., nnd I.eol'. F. II. Journal of Cellular Biochernislp/58:517-526. 1995. nines support: U.S. Public Ileolth Service, National Cancer |n,qilute. tad American Cancer Sr~ciely. From Ihc Thoracic Diseases Research ~'.~il, Departmenl or Biochem|slry and M,dec~dar Binlng)'. Mayo Clinic, Roche.~::~. MN, and Department of Cell Vnndcrbill University. Nashville. TN. CONDITIONAL BINDING TO AND C~i L CYCLE-REGULA'I~D INHIBITION OF CYCLIN-DEPENDENT KI~ASE COL:PLEXE$ BY P27'~' Mammalian cultures primg.ily regula:e cell cycle Iraversc durini Oc. Fro" gross!on through 0, n~d commitmenl to DNA synthesis. Ihe activity o¢ a I'amily or proteins, the cyclin-dependem kinas¢~ (edk.~ ", is required. There ate two ptima~, ulalorS' portieres of O,: fu~ Ihe O,-O, Irans!C'.~., which allows en!~ inlo G I; and the G,-$ transition, promoting ¢nlr~ to DNA synthesis and commilmenl to cell dlvi. slon. In !he presenl manuscri~, we ptov|d.', evidence for cross-talk I~qwecn these two cell cycle teen•ilion•, Exlracl$ prepared f~rn quicsc'¢nt mouse mamma~, opt!he. lial ceils are shown to act in • dominant manner to specil'¢ally inhibit the hi•ton© ill kinase activily of prel'nnned/aclive edk2, odE4, cyclin A, or cyclin E complexex from G,-S cell extracts. The inhibitory m.'t|vi~y ~'i•~ u c~lI• ~les qu~scence decreases once collttrcx arc slimula!ed to ~gin G, .averse and eralo¢coo~s edk cdk inhibitor p27~'~ to cyclin A/cdk2 kinar~ complexes upon mixing of Ihe e~lracls. Removal of p27"' from Ihe quiescent ceil .,'.~tmcl •pec|i'ically abolishes the inhibi. Iory effi..Cl. "lltc inhibilory activily and p27~' binding in vl~'n tk, l~.'nd (m im,'uhat~o~t
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C~ o~" (he extracts al physiological temperature or the prc.,;em:e of a reducing agent. ~e ~, ~bl~n, S. T.. Fau~h. M. P., Ands;. R. A.. and Leor. American Cancer S~icW. From the Ocpa~mem or C~]I O~[ogy. V~n(Icrhill Un~vcrshy. N~shvH[c. "[W. nnd ~io~o~. Ma~o Oin;c. R~h~s~r. MN. FUNCTIONAL COUPLING OF GLYCOSYL TRANSFER STEPS FOR SYNTIIES|S OFGANGLIOSIDES IN GOLG! MEMRRAN .ES FROM NEURAl. RETINA CELLS ']'he synthesis of Ihe oligosaccharide or/:angliosidcs is carried out in thc Golgi complex by successive sugar transfers to proper glycolipid acccptors. To examine how Ihe prodacl of one glycosylalion slop couples willl Ihc acxl transfer slop, lice endogenous gangliosides of Golgi membranes from M-day.old chick cmh~o ;cllnn were labeled from CMP-I'IIINeuAc or UDP-I '11 lions which dn [tot allow vesicular' inlercomparlmcistal Ihe endogenous acceplor capachy, labeling was mnstly in ih¢ ;mmcdi;,~e acccplors the concspondlng labeled sugurs. Iluwever, some lahelcd inlcrmcdi:|les progressed li~ more glycosylaled ganr, liosidcs if ihc membranes were incuhaled in a secured step Ihe presence or the nece~ unlabeled sugar m,clc~lldes. This was par|icplarly evident in the case of membranes incubated with UDP-I'II|G;d, in which most o1" the ['lllGal-lab~led lactosylceramide synlhesized in Ihe Gr~! step was converlc¢l Io GM3 and GD3, or ~o GM2 or to GDla in a second incuhalion slop in the presence of un- labeled CMP-NeuAc alone, or Iogelher wi~h UDP.GalNAc. or to~c~hcr wills UDI'- Gel plus UDP-GalNAc, respectively. Conversi~m was lime dcpcndent and dilution- independenh Since prior repo|1.s usin¢ brcfeldlo A indicate lime tr-',nsfcr stcp~ cat- alyzed by GalNAcoT. Gel-T2, and Sial-T4 localize in ~l~e trans-Gol~i network (TON). oar results lead to the following motor conclusions: (~) transfer slcps cat- alyzed by GalNAc-T, Gel-T2, and S~I.T4 coloc-',li~,¢ ',nd are funclionally coupled in Ihe TON; fb~ proxlm~l Guilt GaI-TI, SiaI-TI, and St,q-T2. and their can'e.,;pondin¢ ¢lycolipid acceptors, extend Iheir pcc~ence Io Ihe TON. and [c), G'.'JNAc.T and Sial- T2 compel¢ [or a coremun I~ml or occel~or GM3 in the sy.lhcsis of GM2 and C, D3. Maxzud, M. K., Daninlli, .1. L., and Mac¢ionl, !!, ,|, Th,~ Jouma! of Biolo~]ical ChemhIw 270(34]:2l}207-20214, A.g.st 25. 1995. Ozher support: Consejo Nacional de lnvecCgaciones Cienliflc~s y Tecnias (CON- ICE'D, Consejo dc Invcsligacio~cs C|enl;.".~.as y Tccnologicas de la Provinci~, de Cordoba (COH[COR), and Sccrelarla dr C~'ocia y Tecnoto~ia de la Unive~ldad Naclonal de Cordoba. From the Ccntro de Invcstlgaciones en Q'-imlca Biologica de Cordoba. CIQUIBIC (UNC-CONICET). Deparlamenlo de Q.imlca Biolo¢ica. Facultad de Cicoclas Quimlcns, Universldad Naclonal de Cordoba. Cordoba, Argon|ins. WIIEN CELLS STOP MAKING SENSE: EFFECTS OF NONSENSE CODONS ON RNA METABOIJSM IN VERTEBRA*';~ CELLS It nppenrs llm! llO orgnnlsm is ~llsntu.,," ;O Ibe elTccla Of Imnscnse ¢(xlons on mRNA abundance. The study of how nons.-'n-~ curious all~ RNA melabolism is slill at an early stage, and our current unde-s,'~adin¢ derives more from incidem,,I vignclleS Ih~n from experimenla! underlakP-.-, Ihol addlcss molccoh'~" mzchanisms. Challenges for Ibe future include i, .:;~l'yln¢ the I:enc prnducts ,,n~ RNA sequences that function in nonsense reed;;...d RN/, loss, resolvin]: Ih¢ cause and consequences of there apparently being mo~ Ihan one cellul',r sile and rncch~ism for nonscnse-medinted RNA loss, nnd un,L%:s|-',nd."n~ bow Ihrse siles and mecha- nisms arc relaled Io hods conslhutive and s:s,:ciallzcd palhways or pre-mRNA proc. essing and mRNA decay. Maquat, L. E. RNA h453-465. 1995. Olher support: Nationnl Inslilules of He-'~hh. From de Department or Ilumun Genelics, Roswell Park Cancer Instilule. Buffalo, NY. LACK OF AN EFFECr OFTHE EFFICIENCY OF RNA 3'.END FORMATION ON TIlE EFFICIENCY OF REMOVAL OF ~ITHER THE F'[NAL OR TIlE PENULTIMATE INTRON IN INTACIr CELLS Evklcnce exisls from sludies using intact ceils lh~I inI~ 1~movaI ~ bc inftu- ¢nccd by Ihe reacliviey of upslmam and do,vnsleam splice siles and Ihal ¢leavag:¢ and polyadcnyladnn can b¢ iaflu~ccd by !~',¢ reactivity of upstream splice sites. These rcsnhs indicate thai scqVenc~ wlfhiP ~'-Ierminal inlrons can runcllon in the removal of upslrenm introns as well as Ih¢ I'omtnlio¢l of RNA .1' crKIs. Evlden~ from studies usin~ intact ceils for an iufluet,ce o1" RNA 3'-end rormutio~ on intmn I01
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C) removal is lacking. Wc rcpml here that roUt•lions whhin l~ly-',d~n),lalinn sequences lhat either decrease or increa~ lhc efficiency of RNA 3'-end form:tlinn have no el'feel on lhe cl'l~ciencics Wilh which either lh¢ 3'-lem)in~l ur lhc l)cn,ltimale intron i~ removed hy splicing. Nn~lhern IRNA) hlul h)~hridi~,.a~inn. RN-',~e m.~pping, a~(l an assay Ihat couples reverse Iran~criplioo and PCR wcrc delclions and a substilution of the polyadenylali,m sequences wilhin I'or Iriosephosphate isomer•st (TPI). TPI prc-mRNA haricots six |.irons that arc con- sthulively removed by splicing. Relative to nnnnal levels, each of the deletions was found to reduce Ihe nuclear and cytnplasmlc levels ur TPI enRNA, increase the nuclear love| of unprocessed RNA processed RNA 3° end,:. In conlrasl, ihe subslltullnn using the I~llyadcnyladon sequences of tl~ mouse la"'-globln genc was found to increase the nuclear and cyloplasmic levels ofTPl mRNA. dccren~ the noclcar level .1" unpr~:e~.~ed 3' ends. and increase Ibe nuclear level o1" lxoces~;ed .'l' ends. The simpIc.,~t inlerprctatlon of" Ihese dale indic•los Ihal (i) the rate or J'-end I'nmlaliou nnrmally limifs the •manta. or mRNA produced and (it) the dclctlons d~crease and the suhstilution increases the efficiency of RNA 3"-end I'ormalion. While eacl, hi" Ihe deleli.ns and tile suhslilntiem allcred Ihe ah~nlule levels of inlron fi-conlaining, inlrrm ~-c-nl;dning. inlrnn 6.free. ' and inlro~l 5-free I~NAs. in no ca.~ was |hen: an abn.rmal raft. of iutrnn-c~mlaining Io |alton-free RNA for eilher |alton. Therefore. al least I'or TPI RNA. while Ihc effi- ciency of removal o1" Ihc 3°.terminal |reran inl'lucnces Ihc cfl~c|c.cy ~r RNA 3'-cml form•lion, Ihe efficiency of RNA .'l'-cnd I'onnaliml eh~:s hill inrlucncc Ihc c[ficic.cy -f rcm~val of ¢ilhcr Ihe 3'-Icrminal ~er pcnullim-',lu inlnm. "Ilia dcpcmlcllcc ~r 'l'Pi RNA 3'.~nd I'onnalinn on splicing may rel]ccl Ihc suh(~plimal .~lren~lhs el" Ihc carte. spending reEulalo~ sequences and may funcll.n In e,lsurc Ihal TPI prc-mRNA is hal released from Ihe chn)malin saints. If so. Ihen Ihe independence o/TPI RNA splicing n. 3'-cod I'~mnalinn may be tel;anal|zeal by Ihe lack or a comparable funclinn. Ncsic, D.. Zhang. J. and Maqual, !, F,,. Molecular and Cellular Biology 15(I):4gg-496, ~anua~y 199:5. Olher supporl: U.S. Public I lealth Service and National Instilutes of I Icnllh. From Ihe Dcpaflmcnl of Iluman G~nefics, Rnswell Park Cancer lnslilulc. Buffalo. NY. INT~GRIN pL CYTOPLASMIC DOMAIN DOMINANT NEGATIVE EFFECTS REV "E.ALED BY LYSOPI IOSPliATIDIC ACID TREATMF.NT Inte~rin" receptors Iocali~..¢ to I'¢~cal crmlacl sil~,~ a.d inlcr~cl wilh the cyl.skclc- Ion via the pl eyl,~plasmic dmnain. To sludy Ihe rule of this d(nnain in adhcsinn, we have expressed in NIII 3"l-J cells acDNA consisllog of the inlerlcukin 2 reccplor subunil exit•cellular and Iransmembrane domain.% ¢nnncclc¢l I. Ihc inlcl~rin/~1 cylo. plasmic domain (IL2R.pl). Since Ih¢ extracellular domain el" the chimeric pmlein has no role in adhesion, this proleln could uncouple adhesi.n from |net•cellular 102 evenls. As expecled, in a cell line expos.sing IL2R-/~I. Ihis chimera was direcled Io r~ol cool•el skew. Un~ly, Ihe ells ¢xhibil~ ~al adhes~n In fibro~lln (~). I lowcvcr, when a mpld Rorganl~lion of I~ cymske~l~ was induced u~Ing lysophmphatidic ~id (LPA). I~R-pl ~11~ del~d f~ ~ in c~lm~l Io wild. ly~ cells. ~c detachment in ~s~n~c In ~A ~M ~ p~vcnlcd wilh D, an inhibil~ of oclin ~cri~lion. ~s¢ Rsuhs imply Ihat a domain, which is uncoupled from adhesion, can com~lc wilh Ihc c~loplasmlc domain or native inle~rin ~1 r~ c~oskelelal p;~leins. As a c~s~ue~, Ihe I~R- pl protein acl~ ~s a d~inant ne~aliv¢ effcc:or or a~sion by dism~in~ I~ inle- grin-~loskcIcz~ connecli~. Smilenov. L.. ~ricscwilz, R.. and ~ar~nlenlo, F. ~ Mol~ular Biology or Ihe ~11 5:1215-122~. Novem~r I~. Olher sued: Nallonnl ]nslil.lc or Gc~ml Medical Sclc~cs. From Ihc Dcparlmenls of Palhology and Anatomy and Cell Biology. Co11~¢ ~zysicians and Surgeons. Columbia Univegshy. New Yo~k. NY. end In~lilulc Biophysics. Rnl~arian Ac~icmy or Sciences, ~ofia. Bulgaria. TIIF. ROI.F. OF TIIF. I DOMAIN IN UGAND BINDING OFTIIE llUMAN INTEGRIN ~,13, We report here the analysis o1' polenllal li¢~d binding domains wilhin human inteGrln co, suhunil, a known colla~en/laminin recqXor. This Jnle~rin is lively blocked by Ihe mouse monoclonal anllhody IBa.I. A lame•ted vc~sio~ or cz, suhunit lacking Ibe Nl|a-lcrminal hall" or ~.h¢ exl~cellular domain is nee recog. nized by monoclonal antilxxJy IBa.I. Funhcmmrc. wc have isolated • eDNA con- lathing the I domain from chicken e~, bear|oR si¢nil~c,,nl homolo&y IO Ihe human mt a, sequences. Replaclog Ihe human I dam=in with its chicken cOUnleq~ml led Io Ibe surface expression of • funclionsl heletadimcr with endogenous mouse ~ on Hill 3T3 cells. Ilowcver. IBa.I does hal b~nd to the chlckcn/human chimera. demonstrating that the human ~, I dam•is, is required [or epilolx: rccognilion. Mutation of Aspr" within Ih¢ I domain to alar,..:e resullcd in suK~Jc ¢xl~'esslon of ~n c~/~ heterodimcr recognized by I Ba.I but witP markedly red~ced bind|hi: Io collagen IV or laminlu. Since • previously Rponed ,r,c!alion of • homologous Asp in Mac-I I domain has similar comequences. Ibes: resulls suggest u central sole I'or I domain in llgand recognition for all inlegrln ~ subunils conlalnlng Ibis domain, Kern. A.. ilrlcscwitz. R,. Bank. I., and M•rc~.nlon|o, E. E. The Journal of Biological Chcmlslry 269{3~):221111-221tl6. Scplcmtcr 9. 1994. Olhcr snplporl: Nallonal Inslilulcs or lleahh. • From Ihe Depnelmenls of Pnlholog)' and At, atomy and Cell BioloW. College of Physicians and Surgenns. Columbia Unlvcrsily. New York. NY. und [)~parlmenl Medicine. Chaim Sheba Medical Center. Tel ;l~homer. Israel. " 103
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AN INTEI~NALIZATION MOTIF IS CREATED IN TIlE CYTOPLASMIC DOMAIN OFTIIETRANSFERRIN RECF.PTOR BY SUBSTITUTION OF ^ TYROSINE AT TIlE FIRST POSITION OF A PREDICTED TI(;IIT TURN Receptors are inlcmalizcd I'rmn Ihe pla.,;ma mcmhr=,nc nl -IO llmc.~ Ibc talc o~" bulk membrane. The prcdnminanl mo<lcl for Ihe mufti Ihat prmnolcs r~pid inlemal- i;'ation proposes a rcquiremanl for a lyrosinc Iocalcd in II,e firsl posilitm o1" a llghl lum. In Ihls leporl we show Ihal an inlem~li;,.01inn molar c~in be creeled dt nnvo hy suh~,lilulin~ I lyro,~in¢ for Ihe firSl or hl',l residues of a Iclr;Ipeplidc GI)NS (residt~cs 31..'14) Ihat is flrndictcd In form a lil~llt turn wilhin the cytoplasmic dmnain of the human Iransf'ClTin reccpIOro These snh.Milulinns resrore wihl-lypc levels or internal- ization Io transfen'in Rceptors that are poorly inlcmalizcd duc Io mis~nsc mutation.,; in Ihc no|lye |nlcmalizazio,~ motif. The inlrnd~,clion or a ~ym~in~ -',t Ih= fir~! or lu.~t po~,ilhm of Ihc GDN$ Iclrapep(idc in a IransfclTin rcccplor c(ml;,ining an mlnl¢~Jificd wild-typo inlcmulixali{m moral" 5ignl/'n:unlly i~cru;~s Ihu inlt:mali;,alhm ra~c above Ih;~! ul Ihc wild-lylX~ r¢ccpl()r. Our rrsull~; indicule I1,-'~1 a I'nnc'li.,al novel inlcmali~a- titan ,T~tir can ~ creeled by placin¢ s/x:ci[ic ammalic an)inn ;laid..; widlio Ihc over:ill " slructurc o1" an cxistln~ ~-~urn in a cylopl.',smlc dumaln ofu rccep~(~r. Pytowski, B., ~ud~c, T. W., and McGraw, T. E. Tile Journal of Biological C11¢mi.~I~ 2/0( 16):9(~67-9(173. April 21. 1995. Other support: American Ilcar~ Associalion. New York City affiliate. From IhC Dcparlmcn! of. Pathology, Columbia Univcr.,,izy C, llcgc or Physicians and Surgeons, New York, NY. IIUMAN IL-] RECEPTOR SIGNALING: RAPID INDUCTION OF PIIOSPIIATIDYLCIIOLINE IIYDROLYSIS IS INDEPF.NDENT OF PROTEIN KINASE C BUT DEPENDENT ON TYROSINE PIIOSPIIORYLATION IN TRANSFECTED Nlll 3"T3 CELLS Ahhough lyrosine kinas~s are clearly activated after ligand engagement of human IL-3R in both hematopoletic and nonhem~topoieti¢ cyloplasmic environ- menls. • role for phospholipid hydrolysis and protein kina¢ C in IL-3R signal Irans- duction is emer¢in~. We have used NIH 3T3 cells transiently Iransfecled with hum~ IL-3R a- and 13-sobunils Io study pbosph~lidylcholine hydrolysis in rrspon~ ¢o human IL-3. We h~v¢ found thai Nlil 31"3 cells expre~slng the complain human. IL-3R ~spoed t6 human IL,-3 wilh a rapid and sUSlained increase in ~lycerol. Accompanyin~ this was a ~pid i~¢re~c in im~'ac©llular Ic'vcls of phospho- rylcholin¢. The Wot,'in klnase C inhlbilm" Ii-?. however, was not ¢/Tccllve in inhlhlt- in~ pho~phatidyl¢holine hydrolysls in rrsixmsa IO hum-~n 11.-3 in NIII 3T3 cells ,:xprcs~ing Ihe human r~cepInr. Thus the human II...1R indu(.'~s a rapid prn~cin- kinas©.C-independem hydrolysis that is in coulmsl to Ihe slaw IXnl¢in-kin=.~¢-C- depondenl increase in phosphalidylcholin¢ hydrolysis iwJuccd hy Ihc murin~ recep- tor. Simulllmcous wi~h the increase in dlacylglycerol levels w~s -',n increase in mcm. brane.hnund prnlcin kinusc C ©nxymc aclivity. Immunohlmlinl; wilh is,h~rnl- IO4 Sl~Cil'ic Ahs againsl protein kinase C showed Ihat, whereas the (~.isororm is consltlu. lively membrane ~und, the ~.isofo~ cr Foleln kinas~ C is Iransl~atcd to mcnlhra~ in rrsp0nsc Io IL-3. Aclival;~ o~ phosphatidylcholi~ hydrolysis and pmleln ~inase C ~ctivafion requi~ed ~lh ~z. and ~-~e~m s,~nhs. To IIic rclallo~hip o~ lymslne p~s~o~l~l~'l In Ih¢ acliv~lion or ph~phalldylc~di~ hydrolysis and pr~ain kinase C I~nsl~a,ion, wc used Ihc s~ci~c a~ sl~clu~lly unrclalad I~rosine klnase Inhibilo~ Banisl¢in .1~ harbim~i~. Bulb inhibilo~ lively hl~kcd human IL-3-induced lyrostn¢ phos~orylali~. I~ Iddilion, inhihil~ bilked p~s~lidylchollne hydrolysis a~ prmdn kin~se C I~l~- finn. ~ dal~, comhi~d wilh ~r ~vio~ ~ ~win~ Iha r-j~a i~KII~ 1[.-3 is de~ndenl o~ prolein kinas¢ C. su~lcsl Ihat, in hcmalo~l¢llc and bcmnlo~icdc cells exprrssin¢ Ihe human I~R. phosph~lidylchollne hydrolysis R~o, P., Kilnmur~. T., Miyajima. ~., and Muf~ ~. A, ~zc ~oam;d o~ Immunology 154:1~-!67~, 1~3, Oih~r sup~n: U.S. Public Ilenllh Strict, From the Ilolland ~bo~tor7 for BioMc~IcM Science. Ame~icmn Red Cross. R~kvillc. MD. and DNAX Rcsca~h Institute. Pain Aho, CA. A MEMBRANE PROXIMAL DOMAIN OFTXE HUMAN INTERLEUKIN-3 RECEPTOR p, SUBUNITTHAT SIGNALS DNA SYNTIIESIS IN NIII 313 CELLS SPECIFICALLY BINDS A COMPi.EX OF SRC AND JANUS FAMILY TYROSINE KINASES AND PIIOSPHATI~:YLINO$1TOL 3-KINASE The hl/~h a~nity hnm:ln intedcukin-3 f.:cepIor is m h¢lcmdim¢l~c protein consist, inl: of an a nnd p, suhunil. T~a p, subonil ix Rsponsibl¢ foe r~pIor sisnal Ir~i~sd~c- lion. We have shown the! n m~mbrL, ne i~:,ximul domain or Ihe ¢yloplasmk: liil or Ihc humon p, suhu, i! (amino acids 45 I-3 I?) is minimally raqoi~d ro~ human 11.-:} to signal DNA Synlhusis in qoiesccnl Iransir¢cied Nltl 3T3 cells. GIm~lhlone $-Irsns- refuse (GST) fusion proteins or this 4S 1.5 IT,e~lon and lllolhc¢ I¢~iol1451-562 that includes an acidic domain previously shc:~, in olher ~ceplors to Mad Icins.~rs were consln~cted. Purified Lyn und Lck kinas¢, but nO~ Fcs, could phospho- ~late tyrosines in both domains. Adsm~i.~,'t with lysates from Ihc human IL-3- depends! hemampolct|c cell line (17-1) o: 3T'3 cells ind in w'~'o phosphmTlallOn show~ Ihat bulb terse domains were inter-~ely phospho~lated, Phos~o,'zmlno acid -~nalysis, however, revealed that the me, mitT of" phosphopjladon wa~ on serlne and Ihreonine rather than tyrosine. Ad.~oq~tion nf these dom~zlns wilh 3T3 o~ TF.I cell lyr.alrs, followed by immunn~lolting, showe4 iha! ©yloplasmie tymslne kinzsea Lyn. Fes. und )AK-2 could also slnbly a~o¢late wilh bolh domains~ however. $~ f~nily kina~s nrc n~rc slrongly Rcognlzed by bolh re|tons Ih~n JAK-2 klnase. In iddi. lion. phosph,qlidyliousiln1.1.klnas¢ I'rom cell lyrics was •lso round stably Willl Ihes¢ dnmuins, hut GTP~s,, m:livalin~ pmlcln. Vav. S~I. or Orb2 wen~
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Ran. P. and Mufson. R. A. T]~¢ |oumal of Biological Chemistry 2T0(12):6~86-6893. March 24. 1995. Other support: Nalinnal |nSlilutes of llcahh. From the Ilolland Laboratory for BioMedical Science. American Red Cross. Rockville. ETS PROTEINS: NEW FACTORS TI IAT REGULATE I MMUNOGLOBULIN I IEAVY-CIIAIN GENE EXPRESSION We used a DNA-prolcin interaction screening me=lull to ix~datc :l eDNA. F~-~. who~¢ prnducl hind; to u ~it¢. (~signalml w. present in the Jnmtu,.~lahulin (Ig) heavy-chain =cn¢ enhancer. Erg-3 is an allc~zivdy spliced prt~luct oF and conlains an Ell ONA-bindin8 domain. Ri- I aml PU. I. relalcd El= proteins, also hind I~) Ih¢ ~am¢ silo. In addition. PU. I hind~ In a sccoml site. desi~nalcd I~ heavy.chain enhancer. Wc ~mon~l~l¢ Ihul Ihc ~ hindin8 silt i~ cr~ial heavy-chela ~¢nc enhancer fu~linn. I, =ddili~m. we show that [~r~-] and Ri.I. bill hal PU. I. Can aclivale = ~ffer co~lmcl conlainin~ a mullimer (~ protcin-h;nding sites, syner~isllcally wish helix-I~helix prolcln El2. We (liscu~t Imw combinator- ial inleraClioos between mcm~rs of Ihe h¢li~-Ioop-ltclix and Els f;tmili~ may ~counl ~or =he li~ue s~cificil~ otiose pro~elns. Rivera. R. R.. Sluiver. M. !i., S~¢¢n~r=cn. R.. and Murre, C. Molecular an~ C¢llvlar Biology I ~:7163-7169, Novcm~r I ~93. ~lher supra: National lnstitules of llcallh and Ihe Searle Conn,,oily T~sl. From the ~panmenl of Biology. Uolve~ity of California. San Diego. !~/Gila. E2A PRO'rEINS ARE REQUIRED FOR PROPER B CEI.L DEVELOPMENT AND INITIATION OF IMMUNOGLOBULIN GI-'-NF. REARRANGEMENTS El2 and E47 are IWO helix-loop-helix transcriptlnn faclors that arise by aherna. live splicing of the E2A gone. Both have been implicaled in the regulation of immunnglobulin gone expressio~. We have nmv gcnen=te(I F.2A (-/-) mice by gcnc targeting. 1~-2A.oull mutant mice fail to generate mature B ccitt. The arrest of B cell development occurs al an early stage, since no immunoglobulin D] rearrangemenls can b~ detecled in homozygous mulanl mice. While immunoelohulin germline In RAG.I. mb.I. CDI9. and J~5 Iranscripts am dramalically reduced in fetal liven of E2A (-/-) mice. B29 and it" Iraoscrlpl= arc present, hut at lower levels. In addilion, we show lhal Par-5 Imnscripts ~m significantly reduced in fclal livers of E2A (~-) curly B cell dlfferendatlan. gain. G.. Maandag. ~ C. R.. Izon. D. L, A n~n. D.. Kmls~e~ A. M~ Wc~l~ub. B. C, Krop. 1.. Schli~scl, M. Riclc, II. P. J.. Bums, A.. and Mu~e, Cell 79:885-892. Deemer 2. 1994. Olhar sups: Nali~al Ioslilules of Ilcsllh. Stifle C~munJly Trash Dulch Ca~er S~i=ly, and lhe Netherla~s From Ih¢ ~pa~menl of Biology. Unive~ily of ~llf~i=. S~ Diego. ~ J~l=, CA. Depa~lmenl or Molecular Cancer Inslilule. Am=tedium. Scripps Rc~ea¢ch In~tltut¢. La Jolla. C~. and Deparlment~ of Mediciae and Mnlcc.lar Biology and Genetics. I]alllnn~m. MD. ACTIVATION OFA TRANSFEC]'ED FG." .'-I RECEPTOR IN MADIN-DARBY EPITItELIAL CELLS RESULTS IN A REVi~RSIBLE LOSS OF EPITIIEUAL PROPERTIES Basic f]hmhlast growth faclor (bFGI~ is a polcnt mitogen for • wide variely of cell lypes derived from mcsoden~ and nemoeclndcrm. The aclivily of bR3f is medi- ated by several lyp~s of closely related rcceplor= I~¢longing to Ihe tymsine-k|nase family of receptors. We have found that Madin-Da~by cpilh¢lial cell= (MDC~) do not seem In produce bFGF or b]:GF rcceplot~, lligh level exp,'talon or human bFOF eDNA in these, cells did not produce any ..'~itogeni¢ or morphological cffccls. Exprcssioo of the mouse*d,,rlved eDNA encoding FOP rcceptor-I (FGFR-I) in MDCK cells resulted in the acquisltloo of a fi~ro~la~t-like morphology when the Iransfected eell.~ were cultored al low density in Ih¢ prep=once of 0.6'i. fetal calf sen=m and 20 ng/ml bFGF. Acidic fibrobl~q gmwlh fa¢lm" (aFGF) al~ induced Ih~e morplmlogieal change= but nOl ke~ali=tncyle gmwlh factor. The m0cphololieal effect was hal accompanied by increased bFGF-induccd cell prcllfcrsllen and did hal rc~.~ull in Ih¢ loss of cpilhcllal cell ma~ .'= ~uch as ¢ylolcetallns. Ilowcvet. mcwphological Iransilion was accmnp.'~nicti by ¢hengcs in Ih¢ inlmcelhdar dlr4rlbu. tion or at=in, In spite nf these changes tl~c tr-',osrected cells farmed mooolaycrs even in the presence of bFGF. Cocxprc~ioa of bFGF =~d FGFR-I in Ih¢ MOCK cells rcsulled in similar morphological effeet~t tea: were not dependent upon exogenous bFGF. These morpholng|cal ¢ffecls w,,re ~;'rlcked by exposu~ of MOCK either orlhovanadnt¢ or phodx~l osier. Parc,=tal and FGFR.I e,prcsslng MDCK cells formed monolayers that displayed hig~ :lecerlcal resistance. Incubation of monolaycr= of FGFR-I-lransfected cells v, it.~ bFGF resulted in the lost of cpithe|ia| resistance. Monolaye~s of parental MDCK cells did ~ol lose th,,ir ¢plth¢llal resisl~nc," in ~sponse to bFGF. although exposure to phorbol ester did
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O result in the loss of Ibelr t~ns-epithclisl resistance, ;ndi~ting that the cffccL,; ,o the Irans-cplthclial r~sist~cc are m~ialed by protein kina~c ~ aclJvatinn. Interestingly. onhovanadatc did nol cause a loss or Iran~pilhcli~ rcsislancc. Mi~dal. M.. So~u, S.. Yarden. Y.. and ~eureld, (;. Journal n[ Cellular Physi,lo~y 162:2~-276. I~racl. and ~pa~mcm o~ Chemi~l Immunnl,gy. WcJ~maan Rchovol. TI IF. BCL-2 FAMILY OF PROTEINS: REGULATORS OF CF.I.I. D~A'll I AND SURVIVAL The Bcl-2 protein inhihils almptnsis induced hy a variety or signals, in a range Of cell types and in divcr.,¢ organisms, and it is implicated in h~th nnnnal develop- meal and oncogene~is. I)csplt¢ this c~lral r,deo the mechanism o1" acfi.n el" llcl-2 is not yel clear. Recent sludies have uncovered a number el" Itel-2-relalcd gene prod- eels Ihat regulate almptosis either negatively or i~sitively, and Rcl-2 fo~rns her. erodlmers wJlh at [casl on~ of Ihese prolein.% Ilax. This article di.-.cus.~cs Ih¢ rnlc of Ihe I3¢1-2 family of proteins in the liahl el'these lindings. Nunez, ~. and Clarke. M~ F. Trends in Cell 13|elegy 4(I I):399-403. Hovember 1994. Other supporl: Nalional InslilUleS or Ileahh. American Cancer Society. and AASTROM Biosciences. From Ihe Departments of Pathology and Internal Medicine° University o1" Michl;an hledieal School. Ann Arbor. MI. ALTERATIONS IN DIFFEREI~TIATION AND FIEIIAVIOR OF MONOCYTIC PilAGOCYTES IN TRANSGENIC MICF. TI IAT EXPRFo~;S DOMINANT 5UPPRE.';SOR$ OF RA.T SIGNAIJN('; To address Ihc role of rns signaling in mnnocylic phagncylCs in viv~. the cxpre~;s|on o1" Iwo dnmlnanl supl~cssors o1" in ~'/Ir~ ru$ sig~laling palhways, the car- boxyl.tenninal re~ion o1" the G'Tl~.~.e-~ctivatio~ Ivy)rein (GAP-el and Ihe DNA bind- ing domain or Ihe Iranscription I'aclm" cls-2, were largcled In thi~ cell A 5-kb po~li0n o1" ,he human c.[ms proximal pront.lcr was shown to direct sion of the tt:nsgcnes to Ihe monocytic Ik.- r~e. As a msuh o1" Ihe GAP.C Iransgene expr~,stoII, ro$.G'TP levels ~ ~u~d i....am¢ ~;t~al m~a~cs ~ 7~. ~c luminal diffcreodali~ o~ m~lcs ~ ~s sliced. ~ cv;~c by I~ lion o~ olypical mon~ylic cells in I~ bill Malu~ ~dloncai m~ha¢cs cxh;~ lied changes in colony-sllmulolin~ ~nclor I~c~ndcnl survival and Sl~Clure. Furlhcr. ¢xpr~ssion of Ihe ¢ol~y-~limulaling f~lor I-slimulaled ~cne u~kin~ plasm;no~cn aclivnior was tnhibhed in ~dloncal mac~ha;~. ~c ~ulls ind~alc Ih:l ra; aclion is crillcal in m~lc ~lls a~ler I~ cells have 1~1 Ihc cagily Io Iravet~ Ihc c¢11 cycle. Jin, D, I.. ~a~. S. B., Reddy, M. A., ~hcnkman. D, and Oslrowskl, M. C Molccul~ a~ Cellular Biology 15(2):693-~03. Fcbma~ O~hcr supS: Nati~l Inslilu~cs of Ilcalth. From Ihe D~parlmcnl or Microbiology 9nd Deparlme~l o~ Palholo¢y. Duke Universily Medical Cooler. Durham, NC. QUANTIFICATION OF DNA-PROTEIN INTERACTION BY UV CROSSLINKING Measuremenl of the affinity of • pro],.i.'l fat • promoter sequence is cHl|cal when as.~ssing ils potential to regulate Ir~nscriplion. Here w¢ ~pml Ihal IhC DNA Ixolein crosslinking (DPC) assay can be use.J to measure affinily, amount a~d mole. cular weight o1" DNA blndinl: peoleins In .s~.: ,Fic and non-slx:cifiu DNA sequences. I]y applying a theoretical analysis IO evalnat.' .he binding data, it was shown that the affinity constants of.two protclns (named DP'~'.~ rio am] DPCI0"/) to Ihe MT~ region or the mouse thy'~idine kin=so promoter were " X 10" M. which is IN limes higher than to non-speciFic DNA. Similar al'finlty conslaals we,~ found when Ihe purified proteins corresponding to DPCa0 and DPCIO7 in,lend or nuclear exlr~.-'~s were u~ed to asses.,; the reliabilily of Ihe DPC assay. A value (or croaslinkin¢ ciT, clency was delermincd as 0.07. however, it is hal need..,d fo¢ computation ol" Ihe DNA.pn'4ein af~inily, b,d with it Ihe abundance o~" -', I~indln~ I~ntein can be estimaled. In sum. m,%-y, the DP~ I~ssay is ~cful for q~antltr)'ing DHA hlndlng ~ins and thc~hy jnd~ing their inl~ucnce on Iranscriplion. Molnar. G.. O'LcaPy0 N., Pardee, A. IL, and i';r~dley. D. W, Nuuleie Ackls R~..~arch 2~(16):.'1.'t 18..1.12(~. 19)5. Olhcr sup[~ort: National lnstilules o~" He:dth. From the l:)an~Farher ~ancer Institute, Divls;oe or Cell Growth and Regubtin?. l]os,on. MA, and BioMolecular As.~ys, Inc., Wobt,m, MA. 109
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IDENTIRCATION OF THE IN VITRO PIIOSPIIORYLATION SITES ON G~. MEDIATED BY Ovcrcxpression or pp60*- in mouse fibroblasts polentlales both agonlsl. induced sisnalling Ihrou¢h/~-adrcncrglc receplor~ and cyclic AMP acconmlalion in response fo cholera toxin iBu,~hman. Wil,mn. Luurell, Moycr,~ aud Par~ons (1990) Prec. Nail. Ac;~J. Sci. U.S.A. 87. 7462-7466; Meyer•. Ilc|lllOl! all(I Parsons (1993) Mol. Cell. Biol. 13. 2391-24{)0|. In reconslilulion cxpcrinlcnls in t,itru, phosplmryl:=- lion or G. by immun~-comptcx¢O pp~?- rcsuhcd in enhanced talcs or rccewor- II Izu~orfE Pilch=r, Lullr¢ll. Lin~. Kuro~¢. P~Mm~. ~aro. at.I l~l~owit~ (1992) Pr~. Nail. Acad. Sci. U.S.A. 89, 5720-~724]. ~e~e re;uhs so~gcst Ihal one mecho- ni=m b~ which p~'" =ff¢cls siBnalling through the ~-adrcncrgic r~cWor pho~o~lalion ~d fu~li(mal all~licm -F Ihc (; pmlcln. "lb clucidalc Ix,w pho~l=tion o~ G. mighl aff~l Jlx ~unclion. wc ~uh~clcd ph,,sph.rylalcd, rccomhi- by h.p.l.c, and analy~d by ~n de~radali~ tn d¢lcnninc II1¢ cycle humors which r~iola~lled p~spholyrosinc was released. Candidal¢ ~Wid¢~ chut leaned Tyr ~;dues at Ihe co~es~din~ ~sil~n~ were synthesized, phosp~laled in vitro by p~" ". and their mi~li~s in Iwo.dimcn~ion=l el¢clrnph.rc~E~Ll.¢. Ihal G. is p~sph~l=lcd on ¢wo resldu= by ~' "..~mcly. ~37 and Tyr-~77. Tyr.37 lies n~ the site or ~ b;nd~n8 in the N-te~inus. wilhln a ~Bi(m ~lulol~ Io m~ulale GDP dlss~ialion and acdvallon by ~P [Johnson. Cupid. Lowndcs. Viill=nooun and Ruo~ (1~1) J. Cell Bh~11¢m. 47. while Tyr-377 is I~=lcd in Ihe exlrcm¢ C-Ic~inus. whhin a ~¢~i¢m of G. for receplor inlcractlon [Sullivan. Miller. Mailers, I~eidcrman, Ileideman and B~m¢ (1987) N~Iul¢ (~ndnn) 334. 712-715l, ~c I~alinn or these r~iducs ~¢sls Ihal phosphowlltlon may =fleet the runclion of both o~ Ihcse d~alns. Moycrs. J. S.. Lander. M. E.. S~nnon, L D.. a~ Parsons, R. J. Bi~hcmical J~ma1305:411~17. Other su~n: Natlon=l Instilutcs of ll~hh and the University or Vir~ini~ Pratt Fu~. From Ihe Depz~ment of Microbiology. Molecular giolo~y Instilulc and Cancer Cenl~r. Unive~sily o~ Virginia Iicallh Sciences Cenler. Charlollcsvill¢. VA, and D=panmcnl of Cell Biology and Physlolo~y. Washln~lon Universily School Medicine. SL ~uls. Me. USE OF MONOCLONAL ANTI-LIGIIT SUBUNIT ANTInf~DIFJ; TO STUDY TIlE STRUC3"URE AND FUNCTION OF TIlE FJv'rAMOERA/i/.~rOLY77CA GAIJGAI..NAC A['qlERENCE LEC3"IN Adh,-rcnce of F.ntomneh~ histol.wicw Imph~oil~. In hnsl cells is medialcd hy a galaclosc (Gel) and N-acelylgalaclremmin¢ (GalNAc}-spccific surface Icctin. "11=c llO iccdn is a hesemdimcrlc protein composed of heavy (170 kDa) and light O5-31 kDa) suhunils linked by disul~de ~nds. P~lon~l I~ mon~lonal andros (mAb) ~aiscd ~ains~ a li~h~ subunil-~lulalhi~e-~4~esfer~e fusion ~leln ~,~ u~d Io probe its SI~ClU~ and funclion. Four li~h~ subunil-s~cifi¢ mAb weR pr~uced which recognized disllncl ¢pilopcs on ~v¢ diCferenl light subunlt i~oromts. Jmmunohlals wilh Ih~c mAh dcm~sl~led co.mi¢~l~ or light ~d ~avy when nnn~duced Imphozaile proteins were .~t~d by SDS-PAG~ i~tin~ heavy s.bu.il aolil~lic~ had ~viou~ly ~¢n ~lown In all~ ml~mn¢¢. =~Ai.li~lll suhuoil ..til.xlics (lid raft. x.ggcstin~ tll~ (iv I~nvy subunli cmitains Ihe t11fl~dty- drain rec,¢nitinn ~0111¢in. Glyc~u.j.~ll¢ ;oumal I 1:432-436, 1~)4. OIImr ~.p~d: N=~tional Insdmlcs or I i~llh. From the Deparlmen(s n~ Medicine. M;cmhioloCy and Palholo~y. University or Virginia Sch~l of M=dicin¢. Omrlmlcsvill¢. CI IARACT~:.R 17.ATION OF REGULATORY ELEMENTS IN TI 16 5'- FLANKING REGION OF TIlE RAT OPIIB CENE BY STUDIES IN A PRIMARY RAT MARROW CULTURE SY.~. '~M Glycnprol¢in (GP)IIW]Ila. •n inlegrin C.'P~:.:cx found on Ih¢ surface of is a rcccplnr for flhrinogen anti olher ligands, • .~J is involved in plalcl¢l aL~¢~lion. Ou:aus.c GPllb is specificity expressed in n1¢;=k~r~tx:yl=, we have studied the flanking re~ion of Ihc rat (r) OPIIb gone a.. • model o( • me¢akaryocyl¢-spccil]c gen,'. The sl,,dies prc~nlcd hcr¢ used • ~11 m~ ..-nw expression syslem, which allows ihc sludy of primary cells undcrgoin= Icrmi:"~: difl'eremiallon Into The dcl~rmin:tllon of me~akaryocyl~-spcci~ .prcssion of DNA ¢onslrlJClr was possible by immunoma~nelic~lly =eparalln= ,~a'karyocylc,= I'rom Im~l Ix~le row cells. Tran.~icnl expressibn cOnSlrucls. ~,t,..aining varying lenl:lhs of Ilanking regio~ from -39 In -912 hp. localize.: • regulatocy elct1~nt I~Iw~n ...460 and ---439 bp upstream oflhe transcrlplional s,.~; site. This region contains a GATA consensus ldndlng ©lemcnl between -.457 a~9 -454 (OATA.,=). Fu=ll~r ¢onslRicts demonsirated lh~t this GATA binding dcmcnl wu indeed mstnlial rm exper~ion. A 2$-bp subsehudon, covering Ihe region -450 Io -426 immediately downsIRam o1" Ihc GATA,,,, demonswaled that this regloe wr.s e~seolial for full expression, which su~esls Ihal this region may iolcmcl with Ihe nATAl, slit ~n'promoling high-level lineagc-,~p~cir~: expression. To define I~¢ulaln~j' elemcms belw~m the G.~TA,., •~l II1¢ Iran~¢criplion.'ll star silo further, w© Icsled additional constricts t~rivc'd from II1¢ original -912 conslm¢l: neck of which conlal.-¢d Ihe GATA,~ bul had dilT¢lcm 50- bp d¢lclions [rom -4.50 Io I~ Slan sile. Virlu~llly Ill of Ih~e Cmlslm¢ls conlinued Io .~how hitdi.lcvcl li.~st,c.~p¢cil]c eapmssion. ~1¢ dele|ed -I.SO In -I(ll ¢~l~lnlct h'~d twice the level of expression of II1¢ fall-len~.lh wild.type ct~tsI~.l~ dwRftW~o this" 111
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re,elan may contain a he,alive r¢~ul~lory element. Comparison hi" our data wi~h expression studies performed with the ~'-~i~ ~ ~hc I~uman GPIIb ~ene I IEL dells, a cell I~nc with ~mc megak~ylic prairies, demonslnll~s d~f[eren~s, which ~y ~flect our use or primaW rat h~e macaw cells. In lar. our sludy ~inls Io Ihc im~ancc or Ihe O~T~,, G)r high levels Dl~k. K. ~. Rav;d. K., Phons. Q. H.. and Pones, M. DI~ 84(10):3~85-339J. Novcm~r I~. 1994. Olher sup~n: Nalional Inslilulcs or I leallh :nd ~c Schulman Foundalion. From the Departments o~ P~ial~cs ~d ~a~c~dogy. Unlversily of School o£ Medicine and ~ild~n's Ilospilal (d Philadelphia. Phil, ddphia. P~. and STRUCTURAL REQUIREMENTS OF PLATE.LET CI IEMOKINF.S FOR NEUTROPIllL ACTIVATION Using rccom~;nantly expressed protein.,; and synthetic peptides, wc examined Ihc SlmClUral/funclional fcalures or the plalelel chcmokines, neutmphil-aetivating peptldc-2 (HAP2) and plastic! factor 4 (PF4), that were imponam in their nelly:don of neutrophils. Previous studies with the chcmokine interlcukln-g ilL-8) had shown that the N-tennlnal region preceding the first cystclne 1esidue was critical in defining neutmphil.activaling pmpeJ1ies. We esamlned whether NAP-2 and PF4 had similar stn~ClmaI reqeiRmenl$. In 1he Ala-gie-lee-arg (AELR| N-tcrmious of NAP-2. sub- stitullon of E or R abolished Ca~" mobilization and elastase .~ccr©tlon. Unlike the p-~r- cot molecule PF4. AELR/PF4. the hybrld.fo~med hy replacing the N-terminal sequence of PF4 before the first cysteine residue wilh Ihe homologous sequence of NAP-2. stimulated Ca" mobilization and clasta.,~ ~cretion. Furthermme. the effect of amino acid substitutions in the ELR motif differed from those seco with HAP-2 in that conse~ed sob~tltutions of E or R in NAP-2 abolL,~hed activity, be! nnly reduced neutropbil activation in the hybrid. There sludies show that jus! as with IL-8, the termini of [qAP-2 and PF4 are critical for high.level neutmphil.ucdvating function. Desensitizallon studies provided informaliun on receptor binding. NAP.2, which binds almost exclusively to Ihe type 2 IL-g receptor (II.-gR). did n(H dcsonsillzc ncu- tmphils to activation by IU.8 because IL-g could bind to and activate via bulb typ~ I and 2 IL,-gR. AELR/PF4 appean *o bind to both types of reeepto~ becaure il desen- sitlzed nculrophil$ to NAP-2 aclivation, but was not desensilizcd by NAP-2. and because it desensilizcd to and was desensitized by IL8. Thus. a|thougb NAP-2 and AELRJPF4 share 60% amino acid homology, they have diffcrcnt rcccl~or afl3oidcs. Studies were performed to define the role of the C-termini of these plaSticS chemokines in receplor binding. Itep:rln and a monoclonal antibody sfc'cific for the heparin-bindln~ domain of PF4 both inhibited Ca" mobilization and elastaxc relea.e.e, further suggesling Ihal the C-termlnus of Ihese chcmokines is imperious in rcccplor binding. Synthelic HAP2,,,. failed Io mobilize Ca:'. whereas PF4o,m and 112 induced Ca:' mobilization and secretion of elauase at high concentmlions. Pertus~is toxin inhibited ncutrophil activation by 40% to 50%, establishing a role for G-prw loin-coupled receptors such as lEe IU-gRs in acl;vxtion by the PF4 C.termlnal pep. lidos. Calcium mobilizallon studies usir~ .t'~60 cells recomhlnantly cxpressinI the lypc 2 IL-AR show thai at least part oft: ~ a¢llvalion by Ihe PF4 C-terminal pepllde i.~ by way Ihlx receptor. Thus. our studia~..,bow that both the N-lenni~s immediately preceding Ihe first cysteinc ~esidue and ti~e C-ten.lout of these chemokines influ. once their neutrophil ~cllvation propeaies. Yan, Z.. 7.hang, J.. lloh, J. C., Stewarl, G. J.. Nicwiamwski, $.. and Poncz. M. Blood 84(7):2329-2339. October I. 1994. Olhcr support: National Institules of Ileahh, From Ihe Dcparlme*tt of Physiology a~.d the Sol Sherry Tlttombosis Research Comer. Temple Universily Schc, ol of M.-Jklnc. Philadelphia, PA. Rarer Itlotcchnology Inc.. King of Prussia. PA. Departments of Pediatrics and Pathology. Universily of Pennsylvani~t ~.'lmol of Medicine. Phil]lelphia. and the Division of I Icmalolngy. Children's I Io;iHrd of Philadelphia. Philadelphia. A CELL CYCLE-REGULATED INHIBIT'.~ OF CYCLIN.DEPENOENT KINAS ",ES Cyclin-dependent kinases (Cdks) pre,~iously have been shown Io drive the m.qjor cell cycle transilit'ms in eukaryotic organisms ranging from yeast Io humans. We rcpo[l here Ihe Idenlificallon of a 28.*:Da prolcin, p2g*'* (inhibitor of cyclln. dcp.'ndcnl klnase). Ihal binds IO and inr,;'*ils Ihe kinase aclivily or preformed Cdk/cyclin complexes from human cells. ; '~ i,~hlbltory acllVily flu¢luales du6ng Ihe cell cycle with maximal levels in G, and .'-~.:umulates in G,- and G.-an~sled cells. The~ cos.Its sogge~f Ibal control of Ih¢ G,/.~ Iramillon nay be In~uenct'd by m family ofCdk inhlbltors that include p2g" and the recenlly described inhlbitor'z p21 and pig"~. Itengst, L.. Dallc, V., $1ingerlnnd. J. M.. Lcc". E., nnd Reed~ S. L Proceedings of the Hatlonal Academy of.~c| :.~cei USA 91:5291 -$295. June 1994. Other suppoR: Nnti~mal Institutes of Hcalti,, From Ihe Departmcnl of Molecular Biolo/;y, The Scripps Research Inslltute, Julia, CA, and Labocglmy of Molecular Cncofogy. Ma$~achuselts General |losp.ilal Cancer Center. Charlestown, MA. 113
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A NOVEL INHIBITOR OF CYCLIN-CDK ACTIVITY DETECTED IN TRANSFORMING GROW'HI FACTOR p-ARRESTED F-PITIIELIAL C-ELL3 Transforming growlh lraclor p (TGF.(3) is a pnlcnt inhlhilor of epithelial ceil growth. C)~clins E and A in a~sncialion with Cdk2 have hccn sh.wn In ph~y a role in Ihe GcIo.S phase transilion in mammalian cells. We have studlcd Ihc cffCClS nf TGF-J~-medialcd growth an'esl on G./S cycllns F. and A. Inhlhiri.n of cyclin A-asso- ciated kinase by TGF.~ it primarily due In .. efecrca~c in cyclin A mRNA .'rod pro- teln. Ely contrast, while TGF-p inhibits accumulali~m o1" cycli, E mRNA. Ihe rcduc- lion in cyclin E prolein is minimal. Inslexd. we find thai Ihe acliv~rion of cyclin E- a~'~oclaled kino...e Ihat norm..lly accoml~nies the G.-Io.S ph.'z..~c Ir:msillon it inhibited. A novel inhibitor of cyclin-Cdk complc~cs was delccted in TGF-~.tre-'.led cell lysales. Inhibition is mediated by a heal.stable pee4eln that targets bolh Cdk2 and Cdc2 kinar.es. In G..-arrestcd cells, a similar inhlbilor of" Cdk2 kinase was dclected. These dale sugges! the existence or an inhibitor of" cyclin-dependent kina.~s induced under different condilions Io mcdiale antip¢oliferalive responses. Slimeerland. |. M.. llensst. L.. Pan. C.-I|.. A|ex=nder. D.. Stampfcr. M. R.. and Reed. S. I. Molecular and Cellular Biology 14(~):3683-~3694. June 1994. Olher supporl: U.S. Public II~llh Service. National Institutes o1" Ilcahh. a~d the U.S. DepaZ'lmgnl of Energy. Frnm the Deparlmenl of" Molecular IZiolngy. The .~ripp~ Rcxnmch Inslilul¢. I~ ~olla. CA. and Lawrence Elerkeley LaboratoP/. Life Sciences Division. University of California. Berkeley. CA. RECEPTOR TO NUCLEUS SIGNALING VIA TYROSINE PHOSPllORYLATION OF TItE P91 TRANSCRIPTION FACTOR Rcccn! studi~s on signal tr~nsduclioll slimulated by interrcrofls have defined pathways Ihat link cell sud'ace recepIo~s to I..r£cl gouts in Ihe nucleus. After inlerfcron binding, nonreceplor lyrosine ki~ascs arc |ctivatcd thai phosphorylate components of latent DNA-binding rectors in ~he cytoplasm. These phosphorylatcd f~clors form muhlmeric complexes thai translocate Io Ihe nucleus and bind Io specific DNA ~quenccs in Ihe promoters of induced genes. A 91-kD lacier {pgl or Slatgl) is acll- valed by imeffero~$ and serves as a subunh parlner in the composilion of" diverse Iranscrlptlon factors. Duly. C. and Reich. N, C TEM 5(4}:1.~;164. 1994. Olher suppeR: Hallonal Inslilules of" I lea!lb. From Ihc Deparlmenl of Palhologyo Slate Univcrsily or New York. Stony Brook. NY. tl4 PIIOSPIIORYLAT/ON OFEUKARYO'P' !'ROTEIN SYNTHESIS INITIATION FACTOR 4E AT SER-209 Initiation I'actor 4E (¢IF-4E) bi~ds !o ';~. m'GTP-co~t~inin~, cap el" euka~jmk: mRNA ;rod I'~ilil;dcs Ihc entry or mRNA iqt:~ Ihe i,iti~ion cycle of protein sis. clF-4F, is a ph~'~phoproteino aml tile phosphorylaled I'omt binds Io mRNA caps .1-4-fetid marc lightly Ihan Ihc nonpbosph~jlaled form. A p~evlous sludy indlc~tcd that the major phosphoryl0tion site was Scr-.~3 (Rychlik. W.. RtL~.% hi. A.. and Rho.'~d~. R. E. (19~?) 3. [li~l. Chem. 252.104.'~4.10437). In Ihe I~ese~! study, we Ihc.~i~.cd Ihc phosphnpaplide expected Io re~.~'tl from Iryp~ie di~eslion of elF-4E. O- Idmspl~oseryltysinc. Surprlsln~ly. the trypl|c and synthelle phosphopcptkJes did comigrale electrophorelically. Accordingly: we rede~ermined the plmsp~orylllion site by i~tating a chymotryl'4ic phosphop~pGd: on zcvc~c phase hlsh pedmm~cc liquid chromatography. The pepllcle was ~o~....need by Edman &gr~Jatlon ~ co~- responded Io "QSHADTATKSGSTTKN.;.;='. The site of phosphowlation determined to Ix: Scr-209 by I'o,r melhods; ~he increase in the ~llo or dehydm~l~. nine to serin¢ deflvativcs during Edm~n drC:ad-"~ion. Ihe release of '~2.P. the further discs!ion of the chymotryplic phosphopept;:., with IrYluin. Glu.C. and AspN. and sile~lirec~ed mulaLzenesis of elF.4E eDNa. "l~e $209A variant was ~0! ph~- laled in a rabbll lCI|CoIucyIe lysnle sy~lem. ,~.p~as the wild-type. SS~A. m~d variants were. This sil¢ fails wilhin the cc" ~.-nsus sequence roe phosi~mylltimz by proleln kina...e C. loshi. El.. Cal. A..L.. Keiper. El. D.. Minie'z. W. El.. Mendez. R.. Bc~ch. C. hi.. Sh:pinski. ~.. Slolar~ki. R.. Dar't.ynklewlcz. !L~ mid Rhmlds. R. K. The Journal of Etiological Chemislry 270(2d;.1157"/- 14~G3. lune 16. Olher supped: National In.-.lilutc of" General b ledlcal Scknces. U.S.A. and Ihe Polish Committee for Scientific Re..u=-mx:lt. From Ihe Doper!men! of Bioehemlslry and Molecular Biology. Louisiana Slale University Medical Cooler. Shreveporl. L."-.. M-'.cromolecular $1~clu~ Analysis F.cilily. University of Kenlucky Medi~....z~ Ccnre'r. Lcxinglon. KY. and Ihe Depart menls of ~i{~physics and Chcmlstry. U.~venily of Warsaw. W:zr~aw. Poland. RECEPTOR-SPECIFIC INDUCTION OF I:.E'. VIDUAL AP- I COMPONENTS IN B LYMPIIOCYTES The inducible nuclear ,anscriptlon I'ach~" complex. AP-I. lypically con$1SlS of' hcferodimcrs b~wcen Jun ~nd Fos proteln~. AIIhough com~menls am drawn from I'amilics o1" related molecules, lillle is known ahem Ihe physio~Lel¢ reg,lalio~l and ~;,r-relnted Gene pruducts. In pnrlicular, it is hal known whether indivldp~l family members is ~limulus-sr.¢¢ific oc wh¢lhcr Ih¢ same sil~nalln ways arc responsible for induction of all sub-nits. To clarify these issues. AP-I com- ponenls wcrc examined I'ollowlnL,. nelly=lion of. p'im.'~y El lymld~Cytes Ihmu~h ~eparatc receplors. Ihe surracc Ig A~ receptur, and the CD40 receptor for T cell inl]ueuces. B~th forms of" sllmulallo~ led Io expresslo~ of JunB and/unD mRN~
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and pro:sin: however, induclion of JunB medi-~ted by anti.lg Ab was protcin kinase C (PKC)-depcndent, whereas induction mediated hy CO40 ligand was resislant PKC depletion. The two forms of stimulation diverged even further with rcsFcct to Fos expression. Ahhonf~h both stimuli induced c-I'-os, expresslnn of Foal] was stimu- lus specific at bulb the mRNA and prntcln levels, in that this onmpunent was induced by anti-lg but not by CD40 ligand. $1imulalcd expression nf c-Fos and Fosl] was in all cascs PKC-independenl. These results provide cvidcnco E~r receptor-spe- cific differences in Ihc expressinn or AP.I compnnents, primarily with rcspcct m FosB. They ah;o indicate Ihal separate inln~celhd.',r F,thw.-,y.~ may he used fnr i,ul.c- tinn of.jun and j~,x gene producls and that Ihe .~.ne tn~n~;eripfinn f;n.'tur (j..ll) may be triggered by two surl'ac~ receptors Ihrough separate pathw~tys d~at dil'l~r in PKC dependcoco. Iluo, L. and Rothsleln, Thc ~0umal of Immunology 1~4: 33(Xl-3.'109, 1995. Other support: National Inslitutes of" I leahh. From the Depa~lmcols of Medicine and Micrnblnlogy a.d tl~e Ev:ms Mcmori;d Department of Clinical Re.~areh. Bosun Universily Medical Cenler. Bn~lnn. MA. ANALYSIS OF TI IE I IORMONE-DF.PENDENT R EGUI.ATION OF A ]unD- ESTROGEN RECEPTOR CI IIMERA The modular ligand.hinding domains of slcrnid rccepl,rs h;we hccn wldcly used to generals protein chimeras thai a~c ligand d¢l~ndenl fur aclivity. In a slmihu- manner, we generaled a series of conditionally aclive ,fund and c-Fos proleins by fusing their earboxyl (COOl I}-terminal ends wilh a COOl I.tcm,in.-d fragment of Ihe human estrogen receptor (ER) that contains the ligand-blndlng domain, funD-ER (DER) and Fos-ER'(FER) chimeras wilh an intact leucine zipper and basic rcglon exhibit hormonc-dcpondent aclivalinn of aclivalor protein-I-dirccled tr~nscriplion in transienl expression assays. One of these fusions, DER, has been examined in delail to delermtue ilS mechanism of uclion. Results from immunopreclpitadon experl- mcms with extracts from D£R and Fos transfc¢led cells demonstr,,~e Ihal FOS arid DER readily form heterodlmer complexes. Surprisingly. the fonnation of Fos:DER helerodimcrs, an,4 possibly DPR Eomodimers. is estrogen-independent. Ilnwever. gel shill assays ¢lcazly demonstrate that DNA binding to API sties by Fos:DER het- erodimcrs or DER homodimers is esuogco.der, eodcot. Moreover. in the absence of eslru~en, the DER prolein is an effective inhihltor .f Fos-mediated transactlvalion. and this effect is reversed by the presence of estrogen. Our ~cst,lts indicate Ihal Ih~ DER protein is a direct, honnone-reversihl© inhibitor ul" F.s and that estrogen con- trois the cm~di|ional positive or dominanl oe~ative '.ctivitics at" DER at the level of DNA binding Io API sites. Accordingly. clonally derived I'ihrnhlas! cell lines that 116 stably express the DER protein exhibit p.~uccd cntry into the $ phasc of the cell cycle when quiescent cells are serum sli~,~. ;.. -.d in the absence or estmgen.'rhis is in contrast to the eslrogcn-treated controls. : .'so results support Ihc hypothesis that APt is impunant f'or cell cycle progressk~:- .':J provide m unique approach roe exant- ining the role of" API In this process. Francis, M. K.. Phioney. D. G.. and R)'dc~'. F... The Jn,mal of Biological Chemistnj, 270(i .n~: I 1502- I I $ I .'t. May 12. 1995. Othcr support: U.$. Public Ileahh Scrvl¢.~. National Cancer Institute. and Bristol Myers Oncology Division. Frnm the Fox Chase Cancer Center, Philnt;clphin, PA, • CAP-INDEPEN DENT TRANSLATION ,'. ~'ID INTERNAl. INITIATION OF TRANSLATION IN EUKARYOTIC gel ?.. :JLAR mRNA MOLECULES Ample ©vidcoce h~s accumu~tcd th:.: t:..ppcd eukaryntic mRNAs can be learn. luted without significant amounts of int., i cap binding protein complex elF.4F. Some of these mRNAs are translated by ~ ~" end-dependent scanning: mcthnnism. while other mRNAs use nn internal inltia~i*.n mechanism.remlniscent or pmkaryotic Ir~nslalional initiation. An intense seamh is '-.InB undertaken for fuctors Ihal bind to IRES elements and mcdlate internal inltlat:=::. This is • fmmldable lask Ihat requlrcs elaborate purification ~chcmc.,~ and fa|thfnl in vitro t~sL'~lizm nssu)'~. The u~ of l gcoelicatly manipulatable system such Is ~,e.ast may help in idemifytn¢ genes who~ products are involved in internal iniliatien. Idcolification of such yeast genes and isolation of their higher eukaryotic homok;.r may help in un~rslanding Ihe molccu. lar mechanism of internal Initiation, AnmT'.:r Imlx~lant qucstlon addresses whclher the cap.indclcndent Iranslalion and interns! ,~nitistiou m¢ch~nisms ape Rgul~ed dur- ing cell growth. Again, genetically mre|;:ulatable syslems such as yeisl and Droznphilo m~y provide useful answer~. "'" "; capindepend~nt Ir•nslation Is |l¢ltly diminished durin~ milosis in msmmalia~ ..;Is mW bo statllng polnl to invesli|ale whelhcr capindependcnt Irgnslltioo is t .','~minatc|y a milotic e~,crll or olher physiological roles; for cap independ. , translslion exisl. llzuka, H., Clan. C., Yank, Q., Johannes, ~'~..ad Sarnowt P. In: Sarnow, P., fed.): Curt'col Topics in I<,.robinlogy and Immunology, Vol. 20.1 CapIndcpendcot Translation. Springer-V.e Hg. Berlin. Germany. pp. 156- I'/?, 1995. Other suppud: U.5. Public Health Servlee. Ram the Department of i]iuchcmisttT, B':ct:, ~[es and Ocnctlcs. and Depamnc~ of Microbiolol:y, Unlversily of Cnlor~do He~;:~, Sciences Center. Denver, CO, It7
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O INITIATION OF PROTEIN SYNTIIESIS BY TI IE EUKARYOTIC TRANSLATIONAL APPARATUS ON CIRCULAR RNAs The ribosome scanning model predicls thai cukaryolic ribosomal 40.; subunits enlcr all messenger RNAs al Iheir 5' cads. Ilere, il is repelled lhat eukaryotie ribo- somes can initiate Iranslation on circular RNAs, but only if" the RNAs contain inter- nal rlbosomc coin/sile elcmenls. Long-repealing polyl~plide chains were synlhe- sized from RNA circles with conlinuous open reading frn~,ms. These res.lls indicale Ihal ribosomes can Ilanslatc such RNA circles for muhiple ©onseculive rounds and Ihal Ihe I'rce 5' end or a messenger RNA is nol ncccs.~fily tile enid] poinl lot" 40.; subunits. Chert. C. and Sarnow, P. Science 268:41 $-417. April 21.199.~. Olher suppo~: Nalional Inslilules of Ileallh and Ihc I.ucille I'. Markcy Charilahle Tmsl. From the Molecular Biology Program. Department of Biochemislry. Biophysics and Gcnelics. Dyde Lahnr--,ln~]. Universily of Col.rad. Ilcallh Science..; Cooler. I)cnver. CO. MSX I INIIIBITS MyoD EXPRESSION IN F/RROBLAST X IOT~ CELL IIYBRIDS Transfer of human chromosome I I. which contains the my~,;D locus, from pri- ma~'y I';broblasts into 10"1~ cells results in aclivatlon of mynlJ. In contraSl, hybrids that re~in human chromosome I I and additional human chromosomes fall Io acli. vale rayeD. Wc show that human chromosome 4 inhibits mynD aclivalion, m.yoD enhancer/promcHcr reporter constnJcts show Ihat repression is at Ihe transeriptlonal level. Chromosome I'lagmcnl-containing hybrids localize the repressing :clivily Io the region or 4p that contains the homcobox gone MSXI. MSXI is expressed in pri. ~ human fiMoblas~s and in 10T'~( cells conlainlng human chromosome 4. while parcnlal 10T'~ cells do hal express Msal. Forced expression or Msll rcpress©s myoD enhancer activily. Msxl prolein binds Io Ihc myoD enhancer and likely rcpz~sses myoO Imscr{ptlon directly. Antlsense MSXI n:lieves repression mediated by chromosome 4. We conclude that MSXI inhibits transcription of myoD and that m.voO iS I larRel rot. homeobox gCl~ ~cgulatlon. Woloshin. P.. So~g, K., Dcgnin, C.. Killary, A. M.. Goldhumer. D. J., SasSoon, and Thayer. M. Cell 82:611-620. Augus125. 199S. Other supix~l: National Institutes of Health. 118 From thc Departments of Molecular and ~Pd!cal Generics. Oregon Ilealth University, P(Irtland, OR, Depaitmcnt of ~ "ochcmisl~, Boston Univc~ity School hi' Medicine. Boston, MA. M.D. Anderson C'..."or Center. Houston. TX. Deperlmant Cell -~nd Developmental Biology, Univet,.~j of Pennsylvania School of Mndici~e, Phil~elphia, PA. and Bmokdule Center fc~ Molecule Biology, Mount Sinai School of Medicine, New York. CIIARACTERIZATION OF TIlE SUBT~ )'i~ OF MUSCARINIC RECEPTOR COUPLFJ) TO Tile S'I"IMULATION OF r iiOSPIIOINOSITIDE HYDROLYSIS IN I ~2- I N I I IUMAN A.CI'ROCYTOMA CCLLS Stimulation of muscarinic mcepIo~ io,:~asc~ phospho|nosilide (PI) hydmlysls in I.'t2-1NI human ostrocytoma cells. To evaluate the subtype or receptors which nlediute PI hydrolysis in 132-1NI cells the effects o1": a) the nonselective MI ni.~l, c;irhachol: h) Ihe selective MI nl~or.i:;h 4.hydroxy.2-~nyoyl-trin~:thylummo- nium chloride-m-chlorocarhinila"le (Mc!~-34]): c) the nonselective unlagonisls, a~ropine and scopolamine; d) Ih¢ rclativee-: ° :;eclive M I anlagonist. FiRnzeplne; e} the relalively ~l.'clive M2 antagonists. A.:-[.)X 116 (I I-2-diethylaminomethyl-I- pipcri(linylacetyl-5.1 I.dlhydro-6ll.pyr;d,~.2.3.b-l.4.bcnzodlazepine.6-onc) and mcthoctramlnc and I') the rclalivcly sclCCli,:© '.1~ anta~onisl, hexahydrosila.di~cnidol (IIIISiD} on PI hydrolysis in I]2-1NI ce:'.~ *,~ slndied. The cell pools of inosilol- phosphollplds were prelahelled by incuL~ • .--. 132-1NI ¢~lls in a low ionsitol con- laining medium (CMRL.-1066) supplemer" .. with I'Hlinositol (2 p.Ci/ml) rot 20.24 hours al 3'7"C. The cells were washed an,.. suspended in a physiolo~ical sail solu- lion. and PI hydmlysls wos measured by ~,,.:.umulation of I'H|inosltol-I-Fhosph"le (IP) in the presence of ID mM LiCI. C~¢";r.hol pro(luccd lime and concentrallon dcpcmlcn! PI hydrolysis (EC$0. 37 p.M~.. MeN-A34.1 did nOl c.use hydrolysis of PI in I]2-1N I cells indicalinE Jmt the receptor was .el of MI lypc. All Ihe above muscarlnic antagonists caused I -oncenlmlion depcnc~nl d~'~se in the level of IP in response to cerbachol (100 p. -";. The rcnk order o(" their ulTmities (p~ values) was: almpine (g.~) > HIISiD (7.6) > p;wnzep;ne (6.8) > m~hoclmmlne (6.0) > AF-DX 116 (5.8). This lank ord~" suW't-.s the concept Ihal M~ (olh~r names. M21]. glandular M2) rcccp,ora arc linked :u ~'1 hydrolysis in 132-INI cells, liH$tD. which is selective rot M~ rcccplors or Ih¢ smoolh muscle his higher affinily for muscarinic receptors in I~2-1NI cells than ,~t~F-DX 116 which is sel¢¢live I'o¢ M2 receptors in cardiac tissue, I1" Ihe'receptor |n 1:)2-1NI cells had been M2, p~'l or the lan~ order for affinhies would have been ~..,uhoctrsmine > AF-DX 116 > HIISID • pirenzepine. From all or these obsesvalim:;..he muscarlnic reccplor for PI hydmly- sis in I ~2- I N I cells is tenlalively characlcr;-,:.l as or M] type. Stephan° C. C. :nd Saslry. B. V. R. Cellular and Molecular Biology ~(?):?0 I-? 12. 1992. 119
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C) Olher support: The Sludy COoler I'nr Aneslhcsia Toxicology or Vanderhih University. Nalioaal Inslilule of" Child I|callh and Ihmlan Developm©nl. and Nalinnal inslitul¢ of"General Medical Sci©nccs. From the Dcpanmcms of" Pharmacol.gy and AaL.~lhcsi.logy Vandcrhil! Univc~ily School or Medicine. Nashville, TN. ' EFFECT OF FIBROBLAST GROWTII FACTOR. I ON TI IE TI IREE- DIMENSIONAL GROWTII AND MORPIIOGENESIS OF llUMAN SALIVARY GL.~ND EPITIIELIAL CELLS EMBEDDED IN COLLAGEN GELS The rcsulls reported hcrc provide evidence Ihal human salivary Bland cpilhcllal cells were capable of rccooslrucling epilhelial ducllikc slrUClUrCS similar to develop ing salivary 81ands when cells wcrc embedded in a Ihr~e-dimensional collagen gel u.der serum.free cuhure condilions. In parlicular, we ohserved Ihal FGF-I Favored prolil'eralion and ducl f.ormalion by IISGE cells. A similarity with salivar~ gland slructures was also reinl'nrced by Ihe fact Ihal sccrclory ¢omponc.I and laclol'crrJn wcrc identified in ducta1115GE cells in collagen gels. M;-'oken. Y.. Myc~kcn, Y. Okam(~m, T., .~;aln, J. I).. ~ad Takada. K. In Vilro Cellular & Deveh)pmcnlal I]|.l.gy .'11 :/N.J(t~. Fchru:try Olher suppo~l: Japanese Minisl~ of Education. ,~cicncc. and Cuhum. and Nation•; Im, litutcs of. I Icallh. From Ihc Department oF Oral & M•xillorocial Sur£cry I. School of. Dentislry, I(iroshima University. Minami.ku. Illroshima. Japan. and W. Alton Jones Cell Science Cooler, Lake Placid, NY. STRUCTURE AND FUNCTION OF TRANSCRIPTION-REPAIR COUPUNG FACTOR ~ I. ~I~I.RTUIIAL DOMAINS ANn nt~mNG, ~of'~;tT~r,~ The I't0.kDa mJ'd gone producl is required for coupling transcrip|ioo to repair in E~chtrichia coil Mf"d displaces E. ~'ofi RNA polymerasc (Pal) stalled at • lesion. binds Io Ihe dam•go recognition protein UvrA. and increases Ihe Icmplate strand repair r.e during Ir•nscripllon. Here. the in~eracllons or Mf.d (Iranscriptinn-Rpair coupling factor. TRCF) wilh DNA. RNA Pal. and UvrA were invc.stigatcd. TRCF bound noospccit;cally to dneble slrandcd DNA: hindlng to I)NA pro(heed •horn•l- ing DNa..,¢ I.Folccted and -hypersensitive real(ms, suggcsling ix)ssihlc wrapping of- the DNA around the enzyme. Weaker binding Io single stranded DNA and no bind- ing ro single stranded RNA wcrc observed. DNA binding required ATP. and hydrolysis o1" ATP promoted dissoci•llo.. Removal or a stalled RNA Pal also requires ATP hydrolysis. Apparendy. TRCF rccognixcs a stalled elongation complex 120 by directly intcraclin~ with RNA Pal. since, b~nding Io • symhctlc transcription bfc w-~s no stronger Ihan binding Io double .~randed DNA. and binding !o r~ RNA Pal holocnzymc and Io initiation and eln(~:ion complexes in Ihe absence or adeno- sine 5'-O-(thinlriphosphalC) wcrc obsen~c.l. S:nRluR.runclioo analpis showed resldpes 3"/9-571 urc involved in hindlni; |~ •siallcd RNAP. The I¢llca~c mo~ll's region, residues 571-0~1. bln(is Io ATP DNA:RNA). Dissociation o1" the leml:~,', o.mplex upon hydrolysis or ^TP •1~ reqnircs Ihc carboxyl Icnoinus of.'TRCF. I'~'-',lly, residues 1-37g bind to UvrA and deliver the damagc rccogn;tinn componenl of" Ihe excision noclease to the Ics|o~. " Selby, C. P. and Saucer. A. The Journal or Biological ChemL~try 270(9):4882-4889. March :3, 1995. Olher support: National lnsdlules of Heshh and the I~uman Frontier Science Program. From the Dcp,'ulmen| of Rinchemist~ and ~.:ophysics. Universlly or Norlh Carolina School of Medicine, Ch,,pcl Ilill, NC INI IIIilTION OF DROSOPIIILA EGF RP.'~ ';',I'TOR ACTIVATION BY THE SECRETED PROTEIN ARGOS The Dro.w~philo hom~logue o1" the m~nmalian epidennal gmwlh factor (EGF) rcccplor (DER) is • receptor tymslnc kin•., • involved in re:n), slaps or Iiy develop meat including photoccceptor (lelorminl|i,;n. and wing-vein I'onnatioo. hs pdmxry activating lig,,nd is the Spilz protein, wl~k;i is similar Io mammalian TGF-a (rcr. 12). Argos is • secreled protein Ihst. like, ji;z. contains • single EGF molir. II is • rcpressor ot' cell detcrmlnalioo in the ey:. end zcls in olher lissues, including wing. Decaus~ Argos has the oplx~sile ell'eels Io DER in Ihe eye (the I'o~ct' blocks photorcccplo~ dctcrm|rml|on. Ihe lalle~ pron'ales Ill we bare leSted whether |l ac~ by blocking the DER palhway. Wc ~how Ih•l ,:~rgos does indeed retm:ss Ihls i~thway vi~ and find Ih-~l. in ~.itro. Argos pmlcin c1~ inhib~l Ihe I¢livalion o1' DER by ~pil~. Thus Ihe delermin•lion o1" cells by Ihe ;,;ER polhway is regulalcd by • balance helween ex|racellulm" aesir•ling •nd inhibi:in¢ signals. Thl~ is Ihe E~I in ~o pie of. an extmccllular inhibitor of • Rcei~c. lyro~ine kin~se. Schweitzer. R.. Ilowcs. R.. Smith. R.. Shill, q.-7,,~ and FR'cman. M. Nalure 376:~99-702. August 24. 1995. Olhcr supporl: Notional |nslltutcs of. l|cr.lth. Israel Academy of Science. ~nd M~lic~l Resc•rch Council. From the Dcparlment of. Molecular Oengtlcs and Virology. Weizmann lns|ttute of Science. Rehovo~. Israel. and MRC Labo~<,ry or Mo~ccular Biology. Cambrld~e. UK. , 121
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C) TIIF. ROLE OF SPI IINGOSINE IN CELl. GROWTII REGULATION AND TRANSMEMDRANE SIGNALING exOgenousWe bsvcsph;ngosiuedCSeribcdinhamSwissour 3T]recenl effnrls Io undcr.~tand Ihe mlto enic f}brohJasls in which we have/d~gn~ld]ed Inducedher or noVelm|logenesls.intr,cellularThls slgnalinginelud~s pathways as Pnlenli~ll medi.~tnrs or s bina nora elu¢idalinu or a porenlial du~l action ~ ¢osmc- me~n~cr role for ~pp. involv~ palk~ys ~lh for ~pid ~hili~cion o~ ~llular lul~ [ree calcium and ~or slimul~U~ of P " ra anulor 5PP. ~clhcr ~uch ~lh~--~~ "~' ~cau~ng to .¢,'case4 levels ~.-~" csscnl/:l for m;I ...... ~ ~s eX~Sl anu to w~ ....... " v~-csls SWill £unher study. ,,., CXlC~[ IIIc~c pafhway~ arc d...:~h~oueh lhc ex~;slcnce o~ a sphlngoli id c I u~m~ c~Cmenl, similar to nhos-k.~:-: ~ p , ~c c which can oct as a mol*--~- r .... -" ...... answered Is s~in~; ...... "'~"g ny~lhcs~s, man a unrve~ll mecha-;.-- -* -, • ~m~ ~ptd moth.hies • . . Ihc cycle ..... - u~ s~l Iran~ucfion ;. ~. ~. , revolved? fs II.s CyCle conscw~ in diffe~nl cell ly~s'~ ..... ,s. ~rc all Ihc chan~ferisi;c~ • • AIIcmpts a! a.~wering Ihc~ q~s(ions are SU~ Io brosden our undersl=ndin~ ormhoBen~sis and likely of ~r Gmda~m=l cellular evems. Spiegel, ~.. Ollvera. A.. and Carlson. R. O. In: Advances in Lipid Resc~h. Vol. 25, Ac~emic ~e~, Inc.. pp. I0.$- 129 199~. Olhcr sup~: Nalional Institmes o~ I l~llk oral ~e C~nccr Rc~earch Foun(l~lion America. From Ibc Dcpa~mcnl ot Ri~hcmi~. G~r~clown Univcrsily Mcdicnl Ccnlcr. Wa~ldnM~. DC. SIGNALING PATHWAYs FOR SPHINGOSYLPIIOSPilORY MEDIATED MITOGENESIS IN SWISS 3T'J FIBROBLASTS LCIIOLINE. Sphlng°sylph°sphmylcholine (SPC), or I~'sophingomy¢lin, a wide-speClmm ~row~h promoling a~enl Ear • varJely o~" cell lypes (D~sal. N. N., and S Spic el 1991. Olr~chem B~c~phyx Res Comm I~1 361 3(x~ ' " g • ol'qui~scer~ $~iss 3T3 ~bro~lasls t~" " : " ). stlmnlales cellular li/'cr~" lops or Ih~n zha , ..... - . . • ;reeler extenl Ih~n o,~-- ~-- p~o phala. SPC r,m..s-,[:.n~l,-~r'~.~T retal¢o mol~'uleSo sphin-osi~ -'-"-~'-~]~. wn growth fac- O-telradeca~'~)~;~'.',~.u_:m:, ~mltogef)~" cJTeCl or an ~-~iva~r ~P__u.l.nRo. s.ine.l.phos._. ,- oxem kln=e C, 12- -v-,---.,-'~,~ ~-)-aCClale. and did not Compele wilh ph,rhol esters binding to protein kln~¢ C in imact Swi,~s ]'r3 flhr.I)lu~l~. IInwcver (InWnregula ~ion or protein kina~c C, by Prolonged trealm • n_o_t cllminazc., the ability or SPC io slimulalc-nl.~,w,i!h phn.rh~,! e.~ler, reiuccd, but ,a, act via ~otb protein k,na,e C.depcndent~';.~,~;cY.~hn(ei~:, SPC induced a rapid rise in inlt~cellular rrec calcium ([Ca"l.) in viable )T'J flbrob- lasts determined with a digilnl imaging system. Although the increases in IC•"l, were obsen, ed even in the absence or calcium in Ihe external medium, no increase in tile levels o1" tnosilol phosphalcs could Ix: (k:,:c~ed in ~cspO~se to malone•it conccn- Iralio~s of" SPC. Furthermore, in conlrosl to ~h,neosine or sphin¢osine- I.phospha|e, Ihe mltogenic errecl or sPc was no~ accomp-,lled by increases in phosphalidic acid Icycls or chan~cs in cAMP levels. SPC, hilt not sphin¢osinc er sphin~:osine.I-phos* phale, slimulalcs IIx: relc;zse or ar~cbldo~lc ~cid. Therefore, Ihe abilily or sPC as on extremely potcnl milnl:cn may he due to tinct from Ihnsc tu;cd by sphincnsinc or sphingosinc. I-phmphate. Desai. N. N., Carlson. R. O., Mallie, M. F_. O]ivera, A.. Buckley, N. E., Sckl. T., Rrookcr. G.. ~.d ,~plel~el, S. T1¢ ]oum;d of" Cell Biology 121 (6): 131~5- I ~9.~,, ~mC 199,1, Olher szIppod: National Insliluics or lleahh. From die Dcparlment o1" Biochemistry and Molncnl•r Biology, GeorLectown Univcrsily Medical Center. Wnshinglcm. DC. SPIIINGOSINE AND SPIIINGOSINE L-PIIOSPIIATE IN CELLUI.A,R PROLIFERATION: RELATIONSIIIP WITH PROTEIN K INASE C AND PI IOSPI IATIDIC ACID Previous studies from our lahorato~ ha;'..- ~own thai sphlngosin¢ (Z~ang el el. (19~O) J. Biol. Chem. 265. 76-81) and sphir-,:.sine I.phosphalate (Zhang el •!, (1991) J Cell Dial 114. I$5-167). metabolites, z membrane splfingolipld$, stimulate fcleasc or calcium from internal sources ~n~ ~r crc-'tsc IXolii'erallon of quiescent Swiss 3"i'x fibrobl-~sts acting in dent palhway. The mJlo~enic effccl in the levels of phosphatidie •cld (PA), • pot.st: r:lilopn for • variety of cell lypes, Ihat may f'unclion as an intraccllulsr second s,,..xsc-ngcr (Zhan| ¢! el. (1990: J. Biol. Chem., 265, 2130~-21316). Spbingosinc also I~.:,J~d cady incenses in spIdn|o~ine I-phosphate (SPP) levels thai preceded Biol. Chem. 267, 23122-23125). SPP il~lr prt~u¢¢d • marc rapid in,eRase in PA, thus sui¢csling thai it may mediale Ihe effecls o£ sph~gosine o~ PA aceumula~km. The concenlralion delx:ndence i'or Ih¢ ro.matic, n or PA induced hy SPP co.tilled wilh iis effect on DNA synl/~sis. Similar to sphin~osine, also slimulaled tl~ aclivily or phosphnlipnse D. ahhough • slgni~cam elT¢ct was observed ala much lower con. centrnllon. I lowevcr, in comr~sl to previous Rpmls wilh sphin~osin¢. SPP did no~ inhihil Ihe PA phosphohy¢lrolasc nclivily in cell homogen.qlcs. Thus. in |ddilloo Io ils cfrecl on mnhili~,:lilm or colcium. SPP can ir, cr~zse the level or PA, mosl likely vln nciivalinn or phmpholipnse D. We surges! Ib(,I SPP medlntcs the efrCCl or sphin. i:osinc o,l PA accumuiat|on in Swiss .'iT,1 l'ibmbl:~.qs nml may Rgulnte cell,1=r pro- • lifer;dism by itlTeclill~ 111ulliplo lr~nsmsmhmne s|~.nalln~ pnlhway:<.
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Spiegel, S. Journal or Lipid Mediators 8:169-175, 1993. Olhcr support: Nalional Instilutcs of Ilcallh. From Ihe Deparlmeot of Biochemistry and Molecular IUoh)gy. Gcorgclown Universily Medical Center. Washington. DC. SFI I INGOSINE- I-P| IOSI'I lATE. AS SECOND M F.~;SI:.N C;I.~R IN CEI.L PROLIFERATION INDUCED llY PDGF AND FCS MI'IXX;ENS Gmwlb signalling ~lwmks mcs~gcrs have ~n well Ltla~clcri~cd. ~c, derived f~ sphingolipids. ~m~ maj~w cla~s of mcm~nnc lipids. A lanlali~- in~ link ~lw~n sphingolJpids a~ cellular proli~¢~llm} ha~ emerged rrnm Ihe dis- COVe~ Ihat lee sphingolipid mcla~lilcs s~in~osinc and sphingozine-I-ph~phal¢ dcnl of pmlcin ~in~se C Sphin~nsi~-I-phos~ale i~ rapidly pr~Kcd Cram xphin- cosine and may m~ialC ilS biolo~ic01 e~fecls. Fu~ha~nrc. SpIlin~osinc-l-pllo~ph~lc Iri~gc~ the dual si¢~l I~nsd~li~ ~lh~ys of calium m~iliT~lion and oclivation o£ p~spholipa~ D. prominent events in lee conlml o~ cellullr ~li~c~lion. I lure w¢ R~n Ihat anlivat~n o[ s~ing~inc kinasc and Ihc ~O~alion o~ sphlngnsine-I- phos~ale z~ im~onl in I~ signal tmsd~li~l palhways ~¢livolcd by Ihc ~lcnt mhogen~ plalelel-dcrived gmw~ f~lnr (P~ and fetal ~lf serum (~S). Oiivua. A. a~ Spinel. ~r sup~: Nali~al In~titules From Ihe Depa~menl of Biochemislry and Molecular Biology. Gcor~clown Univc~izy Medical Center. Wa~hinglon. DC. SPIIINGOLIPIDS METABOLITES: A NEW CLASS OF SECOND MESSENGERS IN TIlE REGULATION OF CEI.L GROWTI I The inlcracllon or ~:rowth factors wilh specific cell surface fcccplors Iriggers mulliplc intraccllular signaling pathway~ Ihnl cehninalc in DNA symhcsis and cell division. Growlh signaling networks in which glyccmpht~phollpld znctal~dites. ~ch as diacylglycerol, inosilol 1.4.5-lrispho~phal¢ (leaP.). phnsphatidlc acid. and a~chl- dante acid. ~;erve am secrmd messengers have been well ch-'.raclcrizcd. Much Ic.~s is 124 known of Ihe second messengers derlve~ ". 'ms anolher major class of membrane llpids, the sphlngollpids. All s~hlngolipi~.., including ceramide, sphingomyelln. ccrcbmsides, gangliosldes, and sulfatides, c.~;~tain (I) a long-chain sphingoid base as Iheir backbone, of which sphingosJn" is lit: most prominent. (2) an amlde.linked folly acid, and (3) a polar head group (hyd~or.yl for ccramide, phosphon/Icholine for sphingomyelin, and carbohydrate residues qf van~|ng complexity for glycosphin- golipids). These ubiquilous cellular compouc~ds have long been known to play all important, yel undefined, role in cell growlh ;egulation. Spiel:el, S. In: Ilu. V. W. fed.): The Cell Cycle: RegulnlnrS. Targets. and Clinical Applicatirms. Plenum Press. Hew York. pp. I I Io 119. 1994. Other suppml: National lnslitutes of I leahh. From Ihe Ocparlmenl of rllneltentislry Itnd Molecular Biology. Georgelown University Medical Center. W,.~hin[~lon. DC. SPI IINGOSINEo I-PI IOSPI lATE. A PUTA"'IVE SECOND MESSENGER. MOBILIZES CALCIUM FROM INTERNAL. STORES VIA AN INOSITOL "iRIS P! IOSP! lATE-IN DEPENDENT PAD iWAY Sphlngoslne-I-phosphate. a melaboli:e of sph|ngollp|ds which ha~ previously been shown to stimulale DNA synthesis ~ - cell division in qulesccol cuhu~'s of Swiss 3T3 I~brohlasls (Zh~ng. I1.. Desai, i' ~'1.. O|ivera, A~ Sckl. T., Breaker. G.. and Spiegel, S. (1991) ./. Cell Biol. 114. 15.~-167), induced a Ir'amicnt increas~ in intracellular free calcium independent of ealmcellular calcium. The in, reuse in calcium ,was completely abolished when in:racellular calcium pools war depleted wilh Ihapslgargin. an inidblto¢ of the endopi~mic micolum Cu-".ATPaR. ~ d0e.e- response for calcium release induced by sp;,:P.goslne-I-phosphale conclated closely wile the concenln~tio~ required fat atimulo:io.-, of D.qA synthesis. The magnitude of Ibe calcium response decreased wile succe: ",.: challenges, allhough .~phingosine.I- phosphate did not attenuate the responses to either bradyklnin or ionomy¢in. Conversely. prior stimulation of the cells wi,.~ bradyki~in had no effect o~ lee aphln- gos|ne-l-phnsphate-lnduced calcium s|g.; d. Ahhoulth sphingoslne-I-phosphate increased inositol (I.4.5)-trlspho~phate lay, I., complete inhibition of inosllol phos- phate formatio~ by pretreament wile 12-f., ;-dccanoylphocbol-13.aceta~e did am block sphingosine-l-phosphate-mediated c.-. ,t.~ n~ponscs. Moreover. in permeabi- llxcd cells, heparin, an inOSilol (I.4.$)-IrlSl,h,,~phale lnlagonisl, blocked Ca~' release induced by inosltol (I.4.5)-tri.~phosphate. ::at did not signiflcanlly slier the Ca~' release ieduccd by ;phlngosine-I.phosphate. Sphingo~:;ne-I-phosphate did not slim- ulatc the rclca.~e of arachidon~c held. another s|gnaling molecule known In elevate ICat'], wlthoul inositol lipid lumnvcr or ca!el" m inl]ux. Our data suggest thai sphin. goslne-l-phosphate mnbillz~ Caz' from in.:~,.sl slates primarily Ihrough a nlsm independent of |oositol lipid hydroly~,;, md amehidonle acid ~elcas¢ and I~at sphingnlipid metabolism may be imlmnam if, calcium home0~l~is.
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C) Matde, M., BrOOkcr. O.. and Spiegel, ~. The Journal of Biological Chcmlsl~ 269(5):31R I-318X. February 4. 1994. Other support: National ln.~itutes of Hcallh. From the Departmenl of Biochemislry and Molecular Riulogy. Georgetown University Medical Center, Washington, ~C. SPIIINGOSINE I-PI IOSPIIATE. A NOVEl, SIGNALING MOLECULE. STIMULATES DNA BINDING ACTIVITY OF AP- I IN QUIF, SCF.NT SWISS 3"13 FIBROBLASTS Sphingosine and sphingosine I-phosphate, mclabolltes of sphingolipids, stimu- late cell proliferalion in quiescent Swiss 3T3 fibroblasls and induce Iransicnt incRases in inlracellular frcc calcium (Zha~g, H.. Des•i, N. N,. Olivcra. A.. Seki. T.. Bronk~. G.. and Spiegel. 5. (1991)J. CellBiof. 114. 155-167). Ilowcver. known of Iha nuclear events thai follow the early responses induced by sphingolipid m¢labolitcs. Using a gel retardalion Iss|y. we found Ihat specific DNA binding activity of ,~clivato~ pro~ein.I tAP.l) was maskedly increased after treatment of qui- escent Swiss 3T3 fibroblasts wilh sphingosinc I-phosphale and sphingosine. The DNA binding specificily or AP-I was confirmed with competing probes comalning consensus sequences of AP-I, AP-2, AP-3. SP-I. and NFIICTF. The c.fos gene producl was det~cled in the AP-I complex using anti.c-Fos antibody. The dose reslx~nsc for stimulation of DNA binding activity or AP-I by sphingosino I-phos. phale coo'elated closely with its cffccl on DNA Synlhcsis, Furlhermore. an inhihilor of sphlngosinc kinasc. DL-thr~'o-d|hydrosphingosinc. which inhibils sphingosine- induced DNA symhcsis and the formation of sphingosin© I.phosphate. also inhibited sphlngosine.sdmulatcd AP-I DNA binding :clivily. This result furlhcr supports our proposal d~at sphingmine I-phosphate mediates d¢ mltogeolc effect of sphingoslne. Our results indicate that s~ingosine I-phosphatc-lnduced DNA symh~is and cell division may lcsull from activalio~ Or AP-I protein, linking signal transduclien by sphlngolipid met•ballets Io g~ne expression. So. Y,.Roscmhal. D.0 Smuls(m. M.. and Splegel, .q. The loumal of Biological Chemistry 209(23): 16512-16517..tune 10. 1994. Other suppoe1: Nalional Instilules of Hcahb. From Ihe Deparlmcnt or Biochemistry and Molccular Biology. Georgetown Univc~ity Medical Center. W.,,~hlngton, DC. 126 INVOLVEMENT OF A PERTUSSIS TOXIN-SENSITIVE O PROTEIN IN TIlE MITOGENIC SIGNALING PATIIWAYS OFSPIIINGOSlNE I-PHOSPIIATE Sphingosinc I.phosphote. • sphingolil,~ melabolite, w~s pRvlously Rponed to incrc.',',c DNA synlhesis in qulcscen~ Sv,; .~ ~T] fit~)blaszs and Io induce Iranslent increases in intraccllular frcc calcium (Zh,,n~.. H.. Dcs~i. N. N.. Oliver•. A.. Seki. T.. 9ranker. G.. and Spiegel. S. (1991)~. C~" P:ol. | 14. l$5-I6"/). In the W©rcnl study, prelrcalmcnt of Swiss ~T~ ~ibroblasts ~. , " pellussis loxin reduced sphingosinc I- phospbalc-induced DNA synlhesis. Sp*..~ ;.,osin¢ I-phosphate decreased cAMP levels ,'rod also caused • drastic dr,:, js~ in iso~0ieeenof lad for~kolin.stim- ulatcd cAMP =¢cumnlnlion. Penussls tox;~ ;~e~lmenl p~venled Ihe inhihil0r] of sphingoslnc I-phosphate on cAMP :,.cumulation. ~uggcsling Ihal • toxin-sensitive G, or G,.llkc protein may ,,: Involved in xphin~osine I.phosphale- mediated inhibition of cAMP accumnlat',m. Mito~enlc concentrations or sine I-phosph.'dc slimulnlcd production p:" ;.~osilol phospbalcs which was inhlbhcd hy penussls toxin, while Ibc r,~pon~ to ~z.tdykinin was not affected. calcium re1~e induced by sphln~oslnc I , :u,.~phale. b~ not by brodykinln, w~s al~ allCnualed hy pcrtussls toxin Ircatmenl. lln.~.ever, sphlngo~ine pbnspbatidic maid uccunmlallmz was tma.Y,. :tel I~y I~.'elnssis to,do. Th~ il¢~ase In specific DNA binding nclivlty of -clivah',r p:otcin-I, which .wa~ |nduccd by Ir,:al. mcnl of quiescenl Swiss 3T3 fibrobl~st,, ..~,h sphinl;osina I.phosphat¢. was also inhibited by I~m~ssis Ioxin. TI~ese resull, . ..$~esl IbM soar of lira sphingosinc I- phosphatc-imhRcd signaling pathways ,'~x'..¢.liated by O proteins thai a~ f~" ~Ru.~sis toxin. Goodemnlc. K. A.. Mettle. M. E.. Bergcr, ~,., .rod Spiegel, S. The Journal of BiologiCal Chemistr/270( :" ~- ;02/2-102"/7. AI~I 2g, 1995. Other support: National Institutes of Ileall~. From the Deparlmcnl of Biochemlslr~ and Mol©cular Biology, Gear|clown University Medical ~entcr. Washington. : .' ,STEREOSPECIF~CITY OF SPHINGOSI;,,.,.~, :,IDUCED INTRACELLUI...~R CALCIUM MOBILIZATION.AND CELI.,.:',,AR PROLIFERATION Sphingosin~ is n pOSilive regulator c,;" cell growlh in Swiss 3T3 I~broblasls (Zhang. !1.. ~uckley. N. F_, Olbso~, K., a~rJ Spiegel, S. (1990)J. B/o/. Chem. 265. 76-81). The present sludy invcstlgatcd the s;ereo¢peci£K:|ty of sphln~osine-indueed cell proliferation and ils milogcnic signal t,'~.nsduclloo mechanisms. $tereoisomcrs (~i$ .~nd Irons) stimulated ::~ .A Synlhesls, whereas ~either threo.sphingosine (cis or I~ons) nor oL-o~;-:o-dihydrOSFhin¢osine had any cffccl. Previously, we have shown thai sphtngosim.-!.phosphate may mediale the milol~enlc effect of sphlngoslne (Zheng, H., Dcsai, N..."1.o Olivem, A., Seki, T., and Splcgcl. S. (1991)J. CtI| BInL 114. 1~;.~-16"/). |towevcr, no major diHeren¢cs 127
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were found in the formation of" D-(+)-er.Whrn and L-(-).d;rro.sphlngosine.l.phos. phate dct'ivcd f.rom Ihe respective sphingozlne ismners in inln¢l cells. Thus. the slereo- spcclfiCil.y of. the response Io sphingosine may reside at Ihe level or specific inlracel. lular lar&els fnr '~phingosine-I-phosphate. Sp|'ingnsine. I-phosph;zlc triggers signal IrausduclJon pathways of antiValitm of ph~sph,lipa~:e D leading Io incrca.~s in the levels of phosphatidic acid and mobilizatinn t)f calcimn frnm intern;d stores. Ilolh D-(+).tryllzro- and t.'(-)-zhrez~sphingosi,e is(~mcrs h,l.ccd slmlhtr in pho~phalJdic acid concomilam with identical dccreasc~ in plmsphatid~lchnline levels. In Cnnlrasl. only Ihc ~(+)-~rythrn.stcreoi~mnc~ (~'ix and Iron,x) wcrc live in releasing calcium fr~ inlracclhdar stores. ~)ur rex,Its surest thai the f{zrma- Ikm of pl~)sphalidlc acid is n(d suf~cicnt Io mcdlutc sphlngusinc-slimulalcd I)NA syndcsis. IIowcvcr. the slereo~cificily of I]re s~zlngmi,e-ind,ced mohilJz~lion calcium from intcmal st~s seem~ to conelote whh the induclinn of DNA synlhe~Js by sphingo~ine stcrcois~c~. Olivcra. ~.. Zhzng. I1.. Carlson. R. O.. Mollie. ~. E.. S~hmidt. K. R.. aml 5pie~el. ~te 2oumol of Biological ~emisz~ 269(27): 17924-17930. ~,1y 8. I Other s,ppom National Institutes of Ifc;dlh :rod TIre Minislry uf Ed,cnlion nntl Science. Spain. From Ihc Dcparzmcnl o£ ~iochcmiszry and Molecular ,iolngy. Ocnr~elown Univcr;ily Medical Ccnlcr, Washinglon. De, aml Ihc ~)cp;Iflmcnl *)r ~c~ni~lry. SPIIINGOSINE I-PIIOSPIIATE RAPIDLY ACTIVATES TIlE MITOGEN- ACTIVATED PROTEIN KINASE PATIIWAY BY A G PROTEIN-DEPENDENT MECI IANISM Addilion of. sphingosinc I-phosphale induces prolif.cralion of quicsccnl Swiss 3T3 I~hroblaslS by unknown mechanisms. To identify Ihc palhways involved, the abilily of. sphinBosine I-phosphate to aclivalc milogen-aclivated protein (MAP) ]:inase was sludied. Sphin~osine I-phosphale ~pidly aclivaled Ihe Rar/~AP kinas¢ kinas¢ (MKK)/MAP klnase palhway, and Ihc concentration depcndcnc~ for MAP kinasc aclivaliOn co~claled with Ihzl for induclion of. DNA symhesis. Both MKK I and MKK2 were -.'rivaled by sphJnBosine I-phnsph'-,le. ;~sscssed by Slx:cil';c immune complex kinasc assays. Prior I~catmenl of" Ihe Swiss 3T3 cells wilh pcnussis Ioxin inhibiled 7O.g0~i, of. Ihe sphin¢osinc I-phosphale.slimulared MAP kinase ~clivily. Thus. one o1" Ihe direcl or indir¢cl tarE¢ls of. cxogcnou~ sphlngosine I-phosphate appears to I~ a O/G. protein. Wu. J., .~plegel, R., and .~lurgill. T. W. The ]numal of Biological Chcmislry 27¢;{ 19): I 14R4 - I 148/(, May 12, I Other suppor~: Iloward Ilughes Medical lnstitule. Nalinnal Inslilutes o1" I lcahh, and American Cancer Society. 12~t From the Iloward Itughes Medlezl l.t~,;llute. Departments of. Medicine and Pharmacology. Marlcey Cenler for Cell .~ignaling. Unive;slty of Virginia Ileallh Science.,; Cenler, Charlollcsville. VA. ~nd Ihe Department of Biochem|sl~ and • Molcc.lar I~iology. Gcorgelown Univcrsl;y Medical Center. Washington. De. SPIIINGOIJPID METABOLITES: MEMBERS OF A NEW CLASS OF LIPID SECOND MESSENGERS Ah.mlanl evidence h~s been ~ccumubled to slrongly suL~ge~l thai sphln~ollpid mcla~i~lilcs may bca new class Of intracrll:Jlar sccorv..~ mcsscngcl~. Ilmvcver, many (I.Csllnns remain in he answered. Are sph;,:goslne. SPP. SPC. and ccmmMa Ihc only ;relive sphi.goliplds or are there still o':..-rs ~malntng Io b¢ dlscovercd? Are Ih¢ cKcclsorlhc~c(~mp~undsll~.~,nmeor,..,, lar in all celts or zzm their e(f.ecIs limited Io sl~cific cells and tissues? Marc camp!.. : descriplions of I1~ mechanisms or ecru. latinn of cell growth and Olher cellular I. ,:sse~ should enable us In answer some o( these qn~lions. .C;pie~el, ,c;. and Milslien, $. The Journal of. Mcmhr~nc Biology 146:? . 2.17. 1995. Off,or support: Nalion~,l Inslilntes of" From the Dcparlmenl of. Biochemists., and Molecular Biolol:y. Ocorg¢lown Universily Medical Cooler. WDshington. JC. and Lnborntof~ of" Neurochemislr~. Nalioual Instilule of" Mental lleahh. Bclhe~..t=. MD. '1"11E ROLE OF CALCIUM INFLUX IN • '"-tJ.ULAR PROLIF~J~.ATION INDUCED BY INTERACTION OF EH.~.;GENOUS GANGLIOSIDE GMI WIT! I TIlE B SUBUNIT OF CIIOLER.* "~ OXIN The R suhonlt o1' cholera toxin, which b~nds Sl~ciflcally Io g~mgl|os;de GMI mitogcnic l'or qpicsccnl Swiss 3T3 fibroSinsts. It was previously sl~wn that the B subunil had no effect on cAMP prolciP .." "'.se C or phosphoinosilid¢ lumovcr. did cause an increase in the influx or ca~¢~.a'.l f.mm extracellular sources (Spiegel S. nnd Pan.'tgimopoulo.% C. (19liE) Exp. ~.'e*~ Res. 17'7. 414-427). In conir~st nclinn or known growth f.~tors0 the i1 s ': .roll induced significant DNA synthesis al'lcr only n I-.~ h trcalmcnt. We ulilizcd .'~:s unique pm~:rty to determine whether Ihe.iocrcase in e~lcium inf.l.s plays n role in B subunil.lnduccd milogenleky. Ceils were briefly ire,ted with the B suhuni! in :1": presence of"calclum ch,'mnel blocEcr~. followed by removal of the blocke~ and .~, ,"or incubatlo~ in B subunlt.rrtc n~dium for the rcm;~inlng tim." required to meas,"-: UNA synthcsls. When I mM 129
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only present during the firs! 3 h i.cuhadon. DNA synthcsis induced by eilhcr the subunil or I'elal bovine serum was campletcly ahollshe(I. Ilnwcver. raM) and th© L-lyl~ vollage.gatcd calcium channel inhibitor nlc~rdipin ( inhlbilcd B Su~nil-induced ~11 proliferation withou~ ~hmgating the res~nse Io ~el~l ~vine serum. Usin~ ~ gel rela~dalion assay, we found Ihal Ihe B s.hunil markedly sllmulaled s~cific DNA-hlnding activity (ff else Inm~ripl~m Junior. unit- valor ~lein- I tAP. I) which fu~lions as a m=jor canvcrgence ~inl coupling early ev~m; induced ~ a variety at milogens In Ioug Ic~ growth releases. Presence c-Fo~ prolcin in Itm AP- I c(~picx was dcm.n~Ir~led as a ~.~shi~l ~nd m(~hilily u~y using c-Fo; ~dyclnnal anl/~.ly. C(~hall. which markedly inhihilcd ~u~nJl-induced UNA ~ynlbe~is also c,m~plcldy a~di~hcd AP- I DNA-I~nding =cliv- ~ly slimula~ hy Ihe B subuniL In ~ha~ conlras~, cavil had no cKecl on mZ oclivi(y ~f AP-I i~luced hy ~h~ lumnr pmnmler. 12.¢~-~eln.l~c~n.ylphorl~d 13- in~ activity of AP-I and cell profi~cratlan imfuccd h~ hiodin~ n~ ch.lera toxin Io cell suKace ~an~l~sidc GMI. ffuckley. N. E., Su. Y.. Miklien. S., ~d %piegrl, ~i~himica el ~k~phys~a AcI~ 12~6:27~-2~.1, 1995. ~)lh~r sup~ff: Noli~al Inslilules o~ ll~hh. From Ihc Deparlmenl o~ Biochemistry ~nd Molecular ffinlogy. Oem~cmwn Universily Medical Cenler. W~shinBion. DC. and i.~hO~l~ry n~ ~our~hcmisl~. Nadnn~l ln~lilule n~ Menial ileallh, llcdm~da. TIIF. WW DOMAIN OF YES-ASSOCIATED PROTEIN FIINDS A PROLINE- RICII LIGAND TIIAT DIFFERS FROM TIlE CONSENSUS F.STAllLISI IF_l) FOR Src IlOMOLOGY 3.131NDING MODULES The WW domain has previously been dcscribed as a mnlifof 311 scmiconscrved residues found in seemingly unrelated proteins, such aL~ dyslmphin Yr.s-as.~ocialed protein (YAP), and two transcriptional rcgulalors. Rap.5 and FE~$. The molecular [unction or Ihc Wt,v domain has been unknown unlil Ihls lime. Using a [unclional screen or a eDNA expression library, we have i~ntified Iwo putative ligands ~.VW domain of YAP. which we named WBP-I and WDP-2. Pep(tale sequence com- parison between d~¢ two pariia! clones revealed a homologous I~glou conslsling of praline-rich domain fallowed by a eyroslne residue (wilh Ihc shared sequence PPPPY). which we shall call the PY moil(. Binding ass,,ys and silc-spccitlc mumge- nc.~is have shown Ihal Ih," PY mnlir hlnds wilh rclutlvuly high ;d'linity aml to Ihe WW domain of YAP. wilh Ihc pr~liminar), cnns,'nsus XI'PXY hcing crilic-'.l for binding. Ilereln, we have implicated the WW domain with a role in mcdimin~ prmein-proteio inleracllnns, as I variant nr Ihe paradigm s¢l hy See Immola~y domain,~ and their prollne.rich llgands. 130 Chen. !!. I. and .~udol. M. Proceeding..~ ol" (he National Academy ~,."Sciences USA 92:7~ 19-7B23. Ausust 1995. Olher support: Nalional Cancer ln.~lilu',e. Ilum~n Fmnlicr $clenc¢ Program. and Ihe Nmimml In.'qii.les of" I|cahh. From Ihc L:~horalory ol" Molecular On¢olo~y. Rockefeller Universily. New Yofl;. NY. CI IARACTI~RIZATION OF 111E MAMMALIAN YAP (YES.ASSOCIATED PROTEIN) GENE AND ITS ROLE IN DEFINING A NOVEL PROTEIN MODUL~. TI IE WW DOMAIN We report eDNA cloning --,rid characterization or the human and mouse onhnlo[~s hi" Ihe chicken YAP C(~-assecialed gmtein) gene which encodes a novel pmlcin Ihnl hinds Io Ihe Sll.'t (Src homc,logy .~) domain o1' Ihe Yes producL Scqnenc¢ comparison between mo~s¢, human, and chickcn YAP pnlleins slmwed an inscrled sequence in the mou~e YAP I1~1 represented an imperrccl refer of an upslr©am sequence. Funker analysis at" this sequence revealed a Imtadvc leln moqlulc tirol is I'ound in various slmcfurul, reL~ulaloe], and sienalin¢ nm/ecules in yeast, nematode, m~d mammals ;nclnd;ng hum'.n dystrophin. FJec,,us¢ one of Ihe pn~nincnl [ealures or this scqucncn nmllr i~¢ two IPjplophan~ (~V). we, nanRd il WW domain (Bark. P.. and Sudol. M. ~"994) Trenffs Bir~'leem. SrL 19. Since ils deline.'tllnn, m~we prol¢ins Imve been shmvn Io cra,.'dn Ihi~ dt~m,~n, ,,nd we rel~ here on Ihe wldespre'ad distribulin;; of Ihe WW module and present -', sion or its pos.,~ible funcllnn. We have a~.so ~own that the human YAP Gene is well con.~crvcd amn.g higher eukar~oles, bu: il may nol be consen, ed in yeast. Its e~pres. sion at Ihc RNA level in -',dnh human ."~',JeS is nearly uhlquitou.,~. 5einl: rtlallvel), high in placc~la, i~'oslafe, ovary, and le , . I~lt is hal deteslablc in Fcriphcral Mood leukucytes. Using Fluorescence in s{rn F., ~,~idization on human melaphasc chromo. somes and by analyzing rodent-human !,yl~ids by Soulhem blot hybridization and polymcrase chain reaclion amplificatio¢z, we ,.~apped Ihe human YAP gen¢ to chro- mosome hand I Iq 13. = re,ion to which ;.~c muItiple cndocrine neoplas|a lype I has hc©n mapped. .Rudol, M., flork. P.. Einhond. A.. Kaslu.',. K.. Drank. T.. Ne~rini. M.. Iluebner. K.. and I.chm.~n. I). ThcJm|roal o~rliolo~icalChemisln/27( ~.l):14733-14~41.Jun¢ 16. 1995. Other s,~pporl: Hntionnl ln.~til,les of/c .;.:h. Ihe Annenber~ Foundalion. Ilcbr~w Tcehnlc;d Inslit, te. Nationnl Cnnccr In.~ "e. Haman Fmmier Science Pm[:mm,. dze Kli.gcnsleln Award in the Ncumsclc.,~;s. 131
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Ram the [.~hmalory of Molecular O/~colngy. Rockefuller U.iversily. New York, NY. European ~'lolecular Diology Laborolnry, llcidclhcrg, (~erlnany, and the Department of Microbiology and Immunology. Jefferson Cancer Inslltme. ]efferson Medical College. Philadelphia. CI IARACTERIZATION OF A NOVEl. PROTEIN-IIINI)INCI MODUI.Em TI {I~. WI, V I)OMAIN Wc have idemifind, characterized and chined human, nmn~ und chicken c~NA of a ~ovel protein Ihal binds Io Ihe SIc hnmnh~y d-main 3 (SII3) ~)r ih~ Yes p~d,- oncogene pro~ucl. We subsequemly namod it YAP f~r Ye~-as~imed pmlCln. Anulysi~ of the YAP ~equcncc ~veale(l a pn~ein nmduie lbal wax [mmd in varinll; sznzclural, ~cgulalc~ and signaling m.iecz~Ic~ llecaus~ ~mc of lhc llmmincm lea- named il lhe WW d~ain. Usin~ a functional sc~:n n( a eDNA expre~si~ li~. we have idcnlified lwo pulazive ligands or lh~ WW (hwnain .[ YAP which ~ n~mcd WBP.I and WBP-2. Peplidc S~luencc cnmparison ~lwccn the lwz) partial clones re- vealed a h{~molngnus proline-rich rcgin,, lllndin~ ossay~ and sJlc-s~Jllc mula~cne- sis have shown thal lhe pr~ine-Hch molJ~ hinds wi~h relatively high a~fini~y nnd s~cJficily Io lhc WW domain or YAP, wilh a ~liminary c~msensus lhal is diffcrenl horn the Sll3-binding PXXP m~i~. ~is suggests lhal lhc WW domain has n talc in medlafin~ ~otein-pmtein inlc~acllons via proline-rich regions, similar bul dislincl ~rom Src homology 3 (SII~) domains. Based on Ibis finding, we hy~lh~ixe lhnl additional pmzeln m~ules exist and that thc~ cmtld ~ isolated using praline-rich ~ptides as Funclional pro~s. Sudol, ~L, Chen, }I, l., Bou~eret. ~.. Ein~d, A., and Bork. P. ~BS ~.crs ]69:67-71. I~. O~her support: National Cancer Institute. Klingenslcin Award, fluman Prnnlicr Sclc~ Pm~mm, and t~ National Instilulcs oF l leahh. From the La~rllo~ n~ Molecular Oncology, R~kc~clier Univer~ily. New York, NY. EMBI., lleidelberg, and Max-~llhruck-Ccnlcr G)r Molecular ~edlclne. Bcrlln-Surch, Oe~any. MEASUREMENT AND MANIPULATION OF CYTOSKEI.R~'AI. DYNAMICS IN LIVING CELLS A new era in cell biology is at hand with Ihe development of louis for imaging molecular funclJons in living cells and lassoes. Specific chemical and molecular ~32 events can now bc mcosurcd and manlpul---:..d in ceils in on~r Io explo~ Ih~ m~ha- nlsms or cell functions. In ~n~ular. ~rally and spalially in single cells hm G,wcr cuka~es, planls, an~imals usln~ lighl-based ~e~gcms and el~Wonic liihl m~:~scopy. Giuliann. K. A. and Ta~lar Cu~enl Opinion in Cell Biology 7:4-12, I~:)~." Other sup~n: Nati~al InsliluleS of ll~lth, National Scie~a Fou~atlon. 5clcncc and Tcchnol~y Cenler, a~ 11~ Pillsburgh C=~e, Inslhule, From lha Unlvarsily o~ Pizlsburgh and Can~gic Mellon Unlve~ily, Pi.s~h, PA. SPECIFIC C1.EAVAGE OF MODF,,I.. RE~ :,MBINATION AND REPAIR iNTERMEDIATES BY TIlE YEAST R/")i-RADI0 DNA ENDONUCLEASE TI~ RADI ~nd RADIO genes or Sm'rl~ ~ ",mFc~'x rcr~'lxlae am i~quln.'d for oneieodde excision repair and ecxlain mi|,st."r, r=eombinatlon evenls. Ilere, model recombination and z~pair inlcrmediales we,...-~'d to show thai R~ I-P-~ cleavage occur~ at duplex-single-strand j,z:.~f~ons. Mo~over, clcava#e occurx only on the strand containing the 3' single-z:,.1.",dcd till. Thus. both biochemical and genetic cvid,'ncc indicate a role for Ihe Ra." !-Radio complex in Ihe ¢lelva]e of" spc. cific ~ecomhination inlcmlcdiates. Furlhe~." edge, Ihese dala su~,e$1 Ihal Rzd I-Rad I 0 endonuclcase incises DNA $' to damaged l-<:(:s durinB nucleo{ide excision repair. Bardwcll. A. J.. Bardwell. L.. Tomkinson~ ,:,. I,. and Friedber~. E. C. Science 263:2092-20g~. Scpicmlxr 30. 1994. Olhcr suplx)r~: U.S. Public Ileahh Service. From lhe L;~l)oralory or Molecular Palho~o~y. Unlversily or Texas $oulhwestem Medical Cenler al Dallas. Dall~. TX, and.. Inslilute roe Bimechnolo,ey. Center h,r Molccul~r Medicine. University of Texes I~-.,lth ScOnce Cenlcr al ~:an Anlmzlo. Snn Antonio. TX. REL~[-KB/]KB STORY Discover), of" NF-KI~ transcriplion f-"-¢lu~s generated • new concep! l'of ~en~ activation: Ihc regular|on by cytoplasmic in:z|bilor molecules. This syslCnl is ubiqul. Ious and evolutionally well eonsen, ed. A wide v:rlely ol'exogenous and endo~:ennus agents generate : mulliludc or rcgulalory c,.scades which converge on Ibis cyloplas. mac complex, resulting in the ~cl~asc of Ih¢ active Iranscrlplion factor. 133
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Ca) Dcspltc grea, interest. ,he signaling pathways are nni yet understood, hm post- translational modification |s |nvolved. Clearly. onc of inhib~tor modjirication by phosphorylatinn and degradatlun. Thus. one of the urgcn, goals is Ihc idcntiricalinn of kJnases thai dircelly i~losphnrylalc b(l| pmlcins aml prntea,;c sy~temfs) involved in IK~ dc~rad~li,m i~ s.iw~. ~lic inl~mled~alc pathways include receptors, oxygen radicals, lyrosine kinases, and/or the Ra~-Raf system. ' • . " ~ help. for example in designing a s~cific inhibitor ~or JIIV ~plicalhm 7~c largels o[ Ihc HF-~B system extend Io genial ~u~inns in cell cycle c~- Irol and s~cific ~un~ions such as Ihe Immune rcs~mse, di[[c~nli:li~, or ~vclop- mcm. Unveilin~ how a ubiqulous lacier differentially regulates key cellular ~unc- ,ions requires ~ulure inv~sfiplions, ldemi~ca6on n[ largc~ genes involved in ccll cycle or differcmJadon may provide a new way Ibis syslcm can lead ~o lymphoid and nonlymphnld Miy~olo, S. a~ Versa, L M. Advances in Cancu R~earch ~:255-292. ~hcr such: Ha6~al Insdlmes o~ HcMIh. ~er~an Cancer ~d Frances C. Bcrgu From Ihe Molecular Biology and ~irolo~y La~ralory. ~e Salk Inslilule. San Diego. AUTOREGULATION OF I~Ba ACTIVITY Tmnserlption flclor HF-0cR regulales Ihe expression of a pie,horn or genes. The ~:¢ivity of NF-~B proteins is regula,ed by IKB proleins. We repot1 that indue, ion or I~Ba. a member or the I~B family of pm,eins, is preceded by activation of NF-xB complex. The promoter of the IKBn, gone contains s KB sill thai is dlrcclly in,lived in ils indue|ion by Ihe NF-KB complex. Degrada,ion of i~Bo~ protein precedes ecti- ,alien of HF-KB DNA binding eclivily, whereas newly synthesized h(13(~ protein inhlbils HF-K]] activity. If the degradation of IxB~z is prevented. Ihe induelion of DNA binding ecl|vily of HF-~(B complex is severely curtailed. These dala suggesl Ihe cxistencc of an amoregula,ory loop whereby |K13~Z regulates the ec,ivity of Iran- scdptlon lector NFoKD. which |n lure regulsl©s the IKRo Chino. P. J.. Miyzmolo0 S.. and Versa, L M. I~oce~dings of Ihe Nallonal Academy of Seicnccs URA 91:28-~2. January O,her supjx)~l: National |nsli,u,es of Ileal,h. American Cancer Socicly. and the I I. N. and Frances C. Bergcr Founda,ion. From Ihe Molecular Biology and Viroloey Lahoralory. "]'he Salk In~;lilulc. San Diego. CA. ENIIANCED IxDc~ DEGRADATION IS RI'."~ONSIBLE FOR CONSTITUTIVE NF-KB ACTIVITY IN MATURE MURINE ~'.CELL LINES NL,clcar lector KLrJ (NF*KB) is a uhiqui:~:s transc~plion feclor which b~nds In decnmeric DNA ~cq,ncnccs 0(B sites) and tea ;latc~ transcription of nmltiple The ;~clivily of HF.~il is regtdztled by nn ;- • ;-lice pmlein, h(13. whk:h NF.KB in ,he cytoplasm. Relea.~e of IKD ; ... uhsequent nuclear tmnslo~atit~ ~d" NF-K~J generally require, activating signals. ;., ,ever. in ma,ure mutir~ B ~lh;. DNA-hlnding activity of NF-K|| is eonslitlu*~ ~.;,v nn¢le,'w and activates Ihe n marker rot malilr~ l| cells. To undcr~Imld ", '~is for lhc cnit~lillll|vc Nl;-x13 v,lion, we e~,mincd the prc~crlics or N:: ~,nd 10(D in l~lh pre-B and ,nalute Lrl cells, the re~ulaled and conslilUliVa St~le.~. , ;pcelivcly. We found lhel expn:sslnn of h(Ik, and p105. mem~:rs of the I~B r: ~ "~:.'. and Rel. • memlxr of llm NF-~R fam|ly, is a|ogmcnled in mnllorc ~ cells. B,... IKB~ and plO$ arc as~ocia,ed HI':-KO pro,tins and SCClUCSlCr most or lhc.~: ,:-o~elns in lhe cytoplasm of maluu~ cells, floweret, rapid l~Bo dissnclnt;on an.; .:gradation lead Io conllnuous nuclear Irnnslncalion of a small fr-~elion of NF-xP ),cans. which reprcsen~ lhe con.,l;lu. ,ively active NF..-KI~ in malurc B c~lls. W~ .~-malc lhal the proleas¢ eclivily is It len.~t ~5-rnld ~rcn,er in ma, ure 13 cells lhnn ;-,. B cells. Rapid degr~alinn is directly involved in constilmive NF-KB .~..." .,~liom. ~cm0s~ sl-',bilizalion of by a protcase inhlh;,or caur~s Io.~ of NF-0(; • °.~.ivily i. malure B cells. Thes~ ressills pro,ida evidence that conlimmus and rapiu ~,,~radalion or IxBa in lit: absence of external stimuli is causally involved in I~'. oonsthullve activation of NF-|(B in mah,re nmrlnc I~ cells. Miyanmto. $.. C'hi,,o. P. J.. and Versa, L Mnlccular and Cellular Biology 14($):3276-.. :~. May 1994. Olhcr supp(wt: Nallonnl ln~qilu,cs of Ileal,., ..~d Ihe H. N. ~md Fmn,~s C. Berg~r Foundation. Frnm ,he Molecul0r Binlogy mzd Vitally:. aboralory. The Salk Inslilul¢. San Diego. CA. QUAI.ITATIVF. CIIANGES IN TIlE SUFIU .T COMPOSITION OF xB- BINDING COMPLEXES DURING MLIRI.'~ -'. ~-CELL DIFFERENTIATION We repro1 here Ih~l Ihe m.~jor xB-bin~.0 ",~ :~nplex In routine malu~ B cells cmnl~.~cd of a pStl-Rcl l,e,emdhncr, whcre~L~ !::.. majnr ind~:ible romt in p~-I~ cells is a pS0-p65 I¢lcmdlmer. Trealmcn, of a pzc-D cell llnc willz lilmpoly~aceharid 135
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changes Ih¢ subunit composilion of xB-binding complexes I'mn} pS(l-p6S to This chan~e is preceded by the enhanced Rel expression and correlales with expression o1" the gene trot the immunnglobin K light chain. "111e helcr(~l;m~-ric Rcl complex bin('s to Ihe intronie enhsnccr.KB site in Ihe immumlglohulin K light chain gent •! least 2l)-rold more st'~bly than do~s the pSll.pt15 direct. "i'hc.~e data sup- port a model in which au~menladon of. Rel expre.~,d0n d.ring tile differe.liadnn or prc-R cells to mature B cells leads to an exchange (11" KI3-hi.ding .~ul~mits resulting in the tran~cripdnnal •clivalion or Ihc gcne tear =he immu.r,glohulio ~ ligh! chain. Miyamolo, S~ Schmitl, M. J.. and Verma, I. M. P='occeding,~ or ~hc National Academy o[ Sclcnccs USA 91 :.';(=.~6.~(IN), May 1994. Olher supporl: Nalional In,~titutc~ of. Ileahh. Amcrcian Cancer S~:icty. and Ihc II. N. and Frances C. I~er~zer Found•lion. From the Molecular Bhdngy and Virnlngy Lahoratury, The .(;:ilk Inslil.le. San Die, n, CA. TUMOR NECROSIS FACTOR ~-INDUCED PIIOSPIIORYLATION OF IKB¢= IS A SIGNAL FOR ITS DEGRADATION BUT NOT DISSOCIATION FROM NF-xB ACtlvalion of Ibe NF-KB/R¢I family o1" trauscriplion I'.~lor~ i~ regulaled hy a cylopla~iC inhihil~. Ix~a. Aclivily of Ixlh= is in lum m¢~l.hdcd by i=ho~ldmryla. lion and prot~lysi~. II has ~ ~tulaledlhal ph.~orylali.n diss~iadon ~mm NF-KB. and [rcc IKBo is largeled for rapid degr~lalion. Ilowcvcr, Ihi; phospho~lalion.medi:~ dlss~ialion ¢vcnl has n.I ~cn dcnmn~fral¢d Wc d~stralc Ihil, ~ntra~ l• lets hy~lhcxis, pho~ph(~rylali(m or Ixlhe induced by lumor nccmsls r~ ~ in Ile~ cells d(ms nnl indnc¢ dissect•It,)•. We pm~¢ a m~el in wh~h (i) i~uc~ ph~lali~ o[ IKB= dcms not rcsull in iion fr~ NF-~B, (ii) phosp~lal~. ~ IKB~ sewes as a signal r~ deg~clalinn. =d (ill) ~radad~ of IxBa ~B while il is 9ill complcxcd widl Miyamo~o, S.. Maki. M.. Schmhl. M. J., I lalanaka. M.. a~ Yerma, L ~¢¢din~s o~ Ihe Nali~al Aca~my of Sclen~s USA 91:12740-12744, ~em~r 1~4. Olher sup~: Nad~al InslHulcs ~ Ileahh. American Cancer ~iely. and and F~nces C. Derger Foundation. From the Molecular Biology and Virology ~ralnry, ~e Snlk In~lil.lc. San Die~o. CA, and ~ralory nf Ilum~ Tumor Viruses, InslilUle [or Vies Kyolo UnlverSily, Kyolo, Japan, 1.16 Src IIOMOLOGY 2 DOMAIN AS A SPE;,",."ICITY DETERMINANT IN TIlE C-ABL-MEDIATED TYROSINE PHOSP:..~'3RYLATION OF THE RNA POLYMERASE II CARBOXYL-TERMIr,~:~L REPEATED DOMAIN The Src-homology (511) 2 domain, r,,~, .' in • varicly of proteins, has • bidding sile for phospholyrosinc-cOnlaining pepli., '~. In adaplor proteins such as G~b2. Ihe S112 domain plays an important role in the .mbly ol'signal transducer complexes. Mnny nonreceplor tyroslne kinases ~ .: g,~ Abl and Src ~ •ls~ contain SII2 domains. Without a functional 5112 dom=.:=}, thet,¢ lye•sloe t:inar.c~ tclain C•lalyli¢ aclivily but lose Ihclr biological hmction, ", :=is resull suggests th,'tl the Sl12 don~in mW be involved in Ih¢ selcc=ion otr bioln~:-'dly relevim subslmles. 't~,'e hive previ- ously shown tim} the carhoxyl-tenninal rel.:~led domain (CTD| of Ibe mammalian RNA polymcr~r.¢ II is • s.bslntle I'or Ihe Abl but no( Iha ~;n: lymsinc I;ina~. This spccificlty is contrerrcd in parl by the SI!2 .•=in. The Abl SIi2 domain binds lyroslnc-phosphorylalcd ITyr(P)I CI'D •r~. ; ~iuiR'd roe Iha pmcessivc •r=l stni- chiomclric plmspImryhllion of Ihe 52 lyros." • i~ the CTD. Mulalion or Ibe Abl $11.! or exchanging i! will} Ih,'tt of. Src. which (: '. no( hind Ihe T).~(P)-CTI). abollsh~J processivi=y and reduced Ihe CTD klna~ • !|vity without any eftr~c! on ~ulophn~- phor)~hltion or the phosplmr~lallnn or no~ :;, ~ |tic subs}t-,Its. These reSldlS demon- strl}le Ihal Ihe S112 domain of" Ih¢ Abl lye•., .inas¢ plays an ~'live mk in catal)'~is and suggcsls thai S112 domain and the lyr~ • kin•s= domain may act in concert tn confer subslrate ~pccificlty. Duysler. J.. Bask•ran. R.. nnd Wanly, ], Y. Proceedings of" =he National Academy or Sc;e.;¢es 92:1555.1559, Fehm~ 1995. Olber support: Nail•hal Cnncer lnslilula. From =he Department or Biology and Cen...r~ M~decul~ Genetics. Unlvet~ily of" Calil'omla. San Diego. L.'t ]oils. CA. EXPRF-~;SION OF TI IE E2FI TRANSCRh. t iON FACTOR OVERCOMES TYPE ~ TRANSFORMING GROWTI I FACTOR ' ~.DIATED GROWTI I SUPPRESSION Inhibilion of" c¢11 growlh by lype/~ lear .,"on~inB growth tractor ('TGF./~) ;n mid-G, and is a.~ocialed with decreased t~, c~lin-dependenl ki~ase a¢livity and maintenance of Ibe roll••blast•ms lumor se:~, tcssor protein Rb in a~ ~laled. growlh-suppresslve state, A vark..; utr recent experimenls suggest Ihal • truncli0nal I=irgel of Rb is the E2F I~nscrlpeTon }racine. In •~dillo~, Ih~ ~¢owlh-sup pressive effects otr TGF.p c~n be overcome : ~, expression ate viral oocogene products that dissociate E2F from Rb and Rb.relalcd p~lypeplldes. These results =u¢¢e~l lee pnssibilily lest control of E2F may be ,~ claw•sir=am ¢vcnl otr TGF-/~ •clio•. Consislenl wilh thai possihilily is Ihe obser~ i:ion thai E2FI RNA levels are dr•sit- tally reduced in TGF-/:~.trcatcd ¢¢11s..We ~.,':;e also used • I~-combinant •denovlms cnnlalning the haman E2FI gtne to ove}e,.press the E2FI prodocl in mink lung epiihelial cells that were growlh are=sled v ""- "i"GF-.B. Wc r~nd Ihat overexpression 1.17
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O 0 i nf E2FI can overcome the TGF-/~-mcdialcd efl'cct as me,xsored by the aellvation or cyclin.dc~n~nl kinases, which am ¢onlmlled by T~I:-~. Schwa~ J. K., Bassing, C. II., Kovcsdi. I., Dalln, M. ~.. fllazing. M.. George, S., Wang, X.-F., and Nevins. ~. R. Precedings or Ihe Nalional Ac~emy or Sciences USA 92:4~-4~7. J~mm~ 199~. O~her support: National Inslilmcs n~ Ileahh and Ihe Iloward ilughcs Medical ]nslilulc. From the Depanmems o~ Genellcs and Pharmacology, Divisi~ of Cardiology, Ocp~lment of Medicine, Howard Hughes Medical Inslilme, D,ke Univer~ily Medical Center, ~hzm, HC. znd GcnV~. Inc.. R~kvillc. TRANSFORMIN~ GROWTII FAL'TOR/~ INDUCF.S TIlE CYCI.IN- Df~P~ND~NT KINA.~I~ INIIIIII'rOR 1'21 "l~lltOI](;ll A 1*~3.1NIJI~I'fiND~NT MECIIANISM growlh raclo~ which ~hibJl cell c~lc ~ss~n in many cell ly~s. ~ ~F-~ induced cell ~¢le a~csl has ~en partially =llri~lcd Io Ihc rcgulal~ TGF.p on ~h I~ levels ~ I~ ~fivilles o[ Ihe G, cyclins a~ d~elr kinase hers. ~ ~liv~ies o[ Ih~ kln~s a~ negalively ~gulalcd by a humor o( ~mall prolelns, p21 (WAFI, Cipl). p27'~. p16. and p15~'. Ih=l physically a~i~e wJlh cyclins, ~clin.de~nd~nl k~a~s. ~ cyclin-Cdk ~plex~ p21 has ~cn pmvlnus- ly shown IO ~ Ir~seri~ionally i~d by DNA domage Ihrough p53 as a mcdialor. We dcmonsl~lc Ihal TGF-~ also ~us~s I ~pid Ir~nscripllnn=l inducli~ su~geMin~ Ihal p21 can ~s~d Io ~h inlrncellular and cxlr;~llular signals rot cell cycle a~l. In co~lras! Io DNA d~c. however, ind~linn o[ p21 by TGF.p de~l on wild-ly~ p~3. ~c cell li~ sludlcd in Ihe~ ex~rimenl~ Ila~T. c~- laths Iwo mulam alleles or p~3, whkh a~ u~ble Io :CliVale I~nScriplion p21 pr~oler when ov~rex~. In ~dil~. ~F-p and p~3 ~1 Ihr~ disli~t clcmems in I~ p21 ~er. Taken ~gel~, I~ ~ndin~s suggest thai ind~ p21 thigh a p~3-i~enl ~lhway. ~vi~s findings have implicaled p2~'" and pl~~ as e([~ ~dialing Ihe ~F-~ gm~h inhihil~ lcsulls demonsl~e Ihal • slogle eXl~cllul~r ~lipmlir~livc si~MI. ~F-~. Ihrough mulliple si~naling palhwa~ Io elicil a Erowlh a~sl Dall~ M. ~.. Li. Y., ~nus. ]. R. Ho~. D. ).. Xiong, Y.. and Wan~. X.-F. Pr~dln&s of the Halional Acad~y of Scicnc~ US~ ~2:~54~.~49. J,nc I ~5. Olh~r sups: Nalional l~SlilMcs o~ H~llh. ~e ~g~m in ~ular Biol~y and Biolecheolo~y. and Ihe Li~ler Com~sive Caner Cenl~ o[ I~ Un~vc~ily o~ Cali~omh al ~z~l Ilill. 135 From the Dcpaflment of Pharmacology, L~ukc Unlvclzily Medical Cooler, NC, and I)eparlmenl oF Binchcmi~lry ~cl Biophysics. Prelim in Molecular Biology aml Binlechn01o~, Line~r~er ~:~.mp~henslve Ca~et Center. Unlvt~ily of Noah Carolina, ~1 Iiill, NC. TRANSFORMING GROWTI I FACTOR t't ACTIVATES TIlE PROMOTER OF CYCI.IN-DF.PF.NI)I3hrI" I¢INASE INIll131T,')R PI.~'~" TIIROUGll AN CONSIINSUS SII'F. 'Tr~nsfonming gmwlh f~ctor 13 (TGF-[. • ~UseS ~rowth algal in the G, ph~ in many cell types. One pmhnhle pmhwa.v : .," Ibis growlh inhih|fi(m is through Ihe T(;F-/3 mcdialed up-reg,dalinn or tire ~. '" :n.dcpenden! I;in;~se (CDK) inhihilt~r pl.~"'.whichspccil~callyinhihilslhecn; , ~ ~llc m'llvilics of CDK4 ~md CDIC6. An active cyclin D.CDK4/6 compk:x is rtq,. ,.'C for pRb phosphorylatlon Io allow cell cycle IO progr¢~.,; from G, IOS ph,'~,. ':'(; slutly Ihe mok'cular mechanism N' plS''" induction hy TC;F-,g. we isolated .~ TEO.bas~ pair promoler sequence or the human p l$ gcne and inserted this fragrm..., upstream of a luciferas¢ rcpo~cr When this conslrUCl was Iransimlly Irar~::-.'led into HaCaT cells, lucifc~s~ nclivily was induced mine Ihan 10-fold upon TC;F.~; tmatmem, indlcalin~ Ihal Ihe induclion of pl$''~'~ ¢xpr~slon by T(;F-,B is partly c~:rled al the Imscdpllon level. Pmmoler ck:lellOn =~alysis ~ve-,Icd Ih,,I Ihe scqucnc-, fn~n -110 Io -40 ~lalive Io Ihe Iron- scription slarl silo is capable of conferring Ihe 10-fold |ndnctlo~ by TGF.~. Within Ibis region Ihere are Ihreo Spl consensus silos. Mulallon of one or Ihese sties. GGGGCGGAG. sohstam|ally reduced bolh Ihe induction by TGF-g and Ihe basal pmmolcr aclivlty, whereas mulalions in I!~ olher IWO SpI silos and Ihe spacer sequences had lillle eff©ch In addition, gel mobility shill assay Indicates that tr~n~rlplien f'aclorS Spl and ~p3 bind to this Spl site. Tal:¢o togelber. Ihese data suggest tha! a specific 5pl con~ens~s si:e is involved in Ihe mcdlatloo of TGF-~I ind,cfinn as well as the I~.nl promoicr ac:ivily of Ihe pit I,.¢uc and lhal Spl and SpJ Ir~scrip~i~n factms might he involved in fl';'~ LI. J.-M.. Nichols. M. A., Chantlmsekhar:.. ,.. ~., Xiong. Y. and Wong. The Journal or Biological Chemistry 270(4.~):26750-26753. November 10, 1995. O~h~r suptmn: Nation,'fl Inslilules of Ileallh and Ihe Lucille P. Marl~ey Found,'~lon. From the Oepanmen! of Ph.',rmacology. l';u:c¢ University Medical CclltCl'. NC. and the Curriculum in Genetics ,',,,~ Molecular Biolo|y. Deparlmenl of Biochemistry and Biophysics. Program |~ iVlol¢cubr Biology and Lineberg¢~ Comprehensive Cancer Cooler, University of North Carolina. Chapel Itill. NC. 139
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O 0 O~ FUN~I'IONAL ANALYSIS OFTIIE TRANSFORMING GROWTII FACTOR RESPONSIVE ELEMENTS IN ~IE ~AFUClPI~31 PROMOTER ~e ~ransfo~inB growlh raclnr p~ (TGF-~) arc a group o( mnllifuncli~al growlh faclo~ Ihal inhlbil cell cycle ~rogressi.n in many cell lyp~s. 1~ TGF~- induced cell cycle a~csl has ~n pa~ially ~rlhuled t~ Ihc regulatory cfFc~ TGF-~ on ~lh tho levels and aclivhi~ of II~ O~ cyclin; and ll~cir cyclin-dc~.denl kina~ prance. ~ ahilhy nr TGF-~ m ;nhlhil the :~clivily ()f lhesc kina~c ploxos derives in pan rr.m i~s rcgulalory ofrccls nn lhc cy~lin-do~ndont kina~c inhibil~s, p21~AFI/OpI, p27"~ aml pl~. U~m Ircam~cm (~r ccll~ witl~ T(3F-fl. th~sc lhreo inhi~Ims bind la ~d bl~k lh= ~livifie~ o~ s~cific c~cll,-cyclln-e~n- dcnl kina~ complox~ Io cau~ ~II ~clc a~csl, l.illlo is known, hnw~cr. ~n mechanism lhmu~h which TGF-~ aclivalcs lhcsc c~lin-~c~n~cnl kinasc inhihhors. In lho case of p~l. TGF-~ Ircs~mem leads Io an increase in p21 mRNA. increase in p21 mRNA i~ partly due Io l~n~c~iplinnal aclivalinn nr lhe p21 promnler by "rGF.~. To ~unher dolinc I~ si~nalin~ ~lhways lhr~h which TGF-~ and,cos p2 I. wc havo ~ffo~ed a delailod runcli~al onaly;b on ~ho p21 premier. ~lh dolclion and mulalion anal~sis o~ lh= p21 prnmolcr, we have defined a pair s~uoncc lhal is mqulrcd ~ I~ aclivafion or lho p21 pmmolor hy TGF-~. In addition. Ibis ~qucncc is surficiom Io drive ~F-fl.medl;,cd Inmscripli~n ~rnm a previously ,onres~nsive ~om~Icr. Prelimina~ ~=I shir~ assays ~cmunsl~l~ lhm ~his TGF-~ ~cs~nsive =Icmcnl bin~ s~cifically In several pmlcJns i, ~.i~r.. Twn lhc;c ~oleins a~ the lr~;~plion r~lo~ sp. i a~ xp-3. 3~ sludics ~cpr~cnl inilial slc~ Inward dofinln~ [ho si~,alin; ~ulhway~ inwdv~d i, T(;F-Jl-mcdin~cd Iran~crlplional aclivalion ~ p2 I. ~alln. ~. ~.. Yu. Y.. aml Wang. X.-F. ~hcr sups: National l~lilul~ nr I leakh. Ram th= D0pa~ment or ~a~acolo~y. ~u~e Univcr~hy Mcdicul Cc, lcr. Durham. NC. TRANSCRIPTION-DEPENDENT REDISTRIBUTION OFTilE LARGE SUBUNIT OF RNA POLYMERASE II TO DISCRETE NUC[. -EAR DOMAINS A subpopulation of the I-',rges! subunlt of RN^ palymerase II (Pal !1 LS) is localcd in 20-50 discrete subnuclear domains lhal are cl~cly linked to speckle domains, which store splicing ixoteins. The speckle-associated fraction of Pal II LS i~ hyperphosphorylalcd on ~he CO011.terminal domain ICTI)). and il is highly tam In exlr~clhm hy deter[enls. A diffu.se uuclc.plasmic I'r~:li(m .f Pul II I.~; i~ rela- livcly hypophosphnnjlalcd on Ihe C'rD. and il is ca~ily extracted hy dclergenls. In Iranscriplionally acliv¢ nuclei, speckle bound hyperphnsphoq, lalcd Pal II LS real©. cules are dislributed in irregularly shaped speckle domains, which appear In I¢ imer. connect©d via a r¢llcular n¢lwm'k, When Iran~crJptltm is inhihiled, hyp~rphn.~plmry. luted Pal II LS and splicin~ prolein SCI5 accumulate in ,~p~ckle dm.~ins, wltich 140 transron~ed into cnlare~ed, dot.like slmctures lackln~ inten:ofmeCllOnl. Wi~n ~lls arc ~¢le~sed ~ t~nscfi~ionol inhibilion. Pal I10 ~ 5~5 ~edls~dbul¢ ~ck {nte~con~cl~ s~cklc ~ttem of t~n~c~i~iondly active celb. Pal II ~d SC]5 is sy~h~us, ~cve~ibtc, ~d Iem~lum de~nd~l. II is c~. clm~d Illal: (~ hy~hosphn~lalion of Po~ II ~'s ~ is I ~llef i1~l~ator or ill It[hi ~iallon In di~c~e subnu¢lear d~lalns Ihan its I~nse#iplional durin~ slalcs or Iran~ripllonal inllibili~, hy~llosp~l=led slated in enlar¢~l s~ckl: domain% which u~der Ihe l;~hl mlc~xc~ colnci~ wilh Ih¢ sl~g¢ xil~ f~ splicln¢ ~lci~; ~d (r) the nucleus. Olher suppo~: Ma~ch oF Dimes Bi~h Dcf:cls FoundalJon, ~na~hue Medical R~ea~ch Foundation, ~nd the N~d~al Instilu~¢s of Hczhh. From Ihc Dcporlmcnls o~ Palholo~y anC Genetics. Yale Univef~ily Sch~l Medicine. New Ilaven, ~. AGONIST.INDUCED EXPRESSION OF ",. ~$UE INIIIBrroR OF METAI.LOPROTEIN^$F.S AND MET^U ~-PROTEINASES BY IIUMAN MACROPIIAGF.~ IS RF.GUL^T~D BY F.Ptf'~JC;ENOUS PROSTAGLANDIN E~ SYNTI I "-----------------~SIS The regulalo~ effect of endogenously synthesized eico~anold mclabolites on Ihe expressinn o1' lissue inhibkor of melollnprolelna.,,es CRIMP). interstitial colla~c. na~. and 9Z-kDa I~elat|na~ by human mac~opha~es was cxlm|ned. TIMP and allopmtcinas¢ producllon were sllmulated with three agonista Ihat produce disllncl patlems or eicosanoid synlhesis: lipopolysat'~harldc (10 I~ml). dcnalured collagen (10 p.g/ml), or zymnsan (I mr,/ml). Indome~.t~cin (:3 ~.g/ml) m" MK~86 [:3 I~M). specific inhibilor or 5-1ipoxygenase. w~s u;,:d to examine Ih¢ role ot cndogenou$ melaholiles of arachidcmic acid. Regardless o." Ihe agonist used. TIMP producllon by macrophagcs was inhibited 65% by Indomc~h=cln. synlhesis of intenlilial collage- I~ase was reduced "~O~. ~md expression of 92.kOx gelalinase was decreased 40%. In conlrast, inhibition of leukolrien¢ synthesi: had no effect on n~tallopmleinas¢ TIMP production. The agonist-stlmulaled ij~:eas~ in TIMP and colla$cnas¢ prodoco lion was directly COVTelated I0 the cumulative Ixostaglandln ~ level induced by the ngonisl used. However. if rerponse Io an a~¢,n:.st was poor. the csogcnous additirm of proslaglamlln R, COtlJd nol increage TIMP o¢ collagennse production marc than Iwofoleh indicating nn im[mrla~)l pennlssivc c,,¢Cl of the ngonisl on Ihe I~¢ulalio~l of e=ch protein's cxpt'csslon. The mechanism o,~ ;ndnmclhac|n Inh|hltlon of TIMP aad collagenase pmd~ctio~ w:]s sludied by la~.:~,;¢ Ihc celia with |"Sl-melhi~nln¢ and perh:m~ing imnmonprecip|lalion tJsio~ xp¢:;ii¢ anlL~,en:m. Indtm~ClhaCln ma~k~lly inhibited the lipplysacchurhl¢-induced hi:~ynthesis or bolh TIMP lad colhl~C. 141
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nasc. Norlhem analysis revealed parallel suppression of T|MP and eollagen,,se sleady-statc mRNA levels by indomelhaciu, indicaling pretranslatiolial cohered. The regulalion of in~amma~o~.coll ~MP and inlerslidal cnllagenasc exprcssion hy prosla~landin Ee su~Besls Ihal therapy inhibiling the cellular response to prosia&landins may ~ useful in cutaneous and systemic disease ~lalcs involving macmphage-m~iated connecllve.tlss~ deslmctlnn. Pemland, A. P.. Shaplm, S. D., and Welgu~ il, (L Journal o~ Iavestigalive De~atolo~y I~:52-~7, 1995. Other sup~: Helical Insdlutes of I lealth. From the Division o~ De~alolo&y, W~hington Unive~ity Sch~l o~ M~ieine, and the Divisions o~ Respiratory and Critical Care uml Dermatology, Washin~lnn University Schmd of Medicine at Jewish I Inspitul. ~t. I.nuis, MO. COMPLETE DEGRADATION OFTYPE X COLLAGEN REQUIRES TIlE COMBINED ACTION OF INTERSTITIAL COLI.AGENASE AND OSTEOCLAST-DERIVED CATI IEPSIN-B We have sludlcd the degradelion of lypo X collagen by m©talloproteinas,'s, eathepsin B, and osteoclast.dedved lysales. We had previously shown (Welgus. II. G.. C. J. Fliszat, J. !,. Seltzer, T. M. $chmid, and J. J. Jeffrey. 1990. J. Died. Chem. 265:13521-13527) Iha! inlerSlilial collagenase rapidly atlacks the native 59-kD lypo X molecule al Iwo sales, rendering a final product of 32 kD. This 32-kD fragmem, however, has a T. of 43"C due to a very high amino acid conical, and tl'u.s remains helical at physio.logic core temperature. We now rclmrl Ihal the 32-kD product resisl$ any fuIlher allack by several matdx metalloprolcin.'~es including inte~titiul collagcnase. 92-ED gclatinase, and matdlydn, ilowevcr, this collagcnase.generated fragment can be readily degraded to completion by calhepsin O at 370C and pll 4.4. lnte~es6ngly, even under acidic conditions, cathepsln O canm~ effcclively allaCk Ihe whole 59.kD |ype X molecule at 37"C. but only the 32-kD collagenase-generat©d fragment. Most impmlanlly, the 32-kD fragmcnl was aim degraded al acid pll by cell lysates isolated from routine osteoclasts. Degradation of the 32-kD type X colla- gen fragmenl by ostcuclast lysates exhibiled the following pmpcrlies: to) cleavage occun~d only at acidic pH (4.4) and not at e.eotral pH; (b) the cystelne la'oteinase |nhibilors E64 and leupeplin completely blocked degradalion; ~d (c) specific anti- body 1o ¢athepsin B was able In inhibit much of the lysate-derlved activity. Ba.~d upon these data, we postulate Ihal during in t,h~ endnchondral bone formation type X collagen is first degraded at oeutr~d pH by interstitial collagenase secreted by ~sorbing cartilage-derived cells. The rrsulling 32-kD fragmcol is stable at core tern- perature and fullher degradallon ~equlrcs osteoela.,~l-derived cathepsin B supplied by invading bone. 142 Sires. U, l., Schmid, T. M.. Fliszar, C. J., '~Vang. 7..Q. Gluck, S. L. and Welgu.-., II.C. Journal of Clinlcal Investigation 75:20~?-209~, M~y I~. O~her ~uppnm U.S, Public Health Se~icc. National lnslhme~ of Ileahh. and Edward Malllnckr~l ~panmenl of Pedi~lfics. Fr~ the Dc~alology and Renal Divis;:.,,. ~me~ of Medicine. W~glon Unive~ity Sc~ml of Medicine ~ ~ It, X{t II~kul. St. ~il, MO. of Pediatrics. SI. ~uis Chihl~n's Ho~p~:~., St. Louis, MO, ~d ~:rlmenl of PRENYLATION OF RAg5 IS DEPENDEI','T ON GUANINE NUCL'EOTIDE BINDING Rah5 is a small molecular weight G'TP.bindlng protein that runcliOn$ in endo- cylic vesicle traffic. Like other Ras-relale(' t~oteins. Rubs is pt~nylated on C-tenni. nal cy~tcinc residues, although it lacks Ihe .'. pious C-Icml|nal CAAX motir (where is any aliphatic amino .'¢id and X is any a:,.i~m ~cid) to dict't this posl-lranslallonal mmlification. W¢ have invesligaled struce., j' requkemcols for the in t*Jl/~tt gera~tyl- gcranylallon of RubS. Rub5"**~. a polnl mt..~nl thai h~ impaired abilily to bind GTP or GDP. undergoes modil]cution IO a limited ealcnl told at a severely reduced i~le when compared IO cognate RubS. A soeond point mulant. Rub5e'~. can be processed to approaimatcly Ihe s~me exlent as wlid-lyp¢ albeil al a t~-duced rate. Since Ibe lab Icr tnulallnn re.~lls in defective ~ aclivlty, these ¢ombloed observations Indl. tale Ihat guanihc nuclcollde binding plays a.t {mpmlanl role in Iho genmylgemnyla, lion reaclinn and sugge~l tltat the GDP.bou,~d form of Rubs is the peefen~J co, for- marian for inleraclion with Rub prcnyllran~f,~rase. This idea is suplxmed by Ihe find. ing that non.hydrolyznble GTP analogs in/..',!l Rub5 Ixenylallnn, while in vitrn Fro. ceasing of both II-nut and Iho "f2 subuni! t,~ eegulalory G [¢oteins is urmffecled at concentrations of guanoslne $'-O-(Ihiot:~,,hospholc) (GTP~$} up to 400 Mocovcr, a tmncalion mutant lacking Ihe C.ten~|nal cyslei~es0 Ribs~4". serves as an inhibilor of RubS" geranylgeranylallon wh.-a'llganded with GDP Ira! not Thus. Ihe Rcognilion of Rubs ~s a subslmte |~ Rub pR.nylmms ferase involves eunl elemems exclusive of the C len~inus and dependenl upon the GDP.blnding co~formation of the protein. Sanford. J. C.. Pan. Y, and Wessllng-Resr ".,, M. The Journal of Biological Chemistry 2~g(3"~).23773-237"/6. blovcmber 15. 199~. Other suppofl: American C.ance~ Society and the Nadon~! Ins¢i~mes of Ilealth. From the Depaflmen! or Nuldlion. Harvard School of Public Heullh. Boston. MA. 143
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TIlE G-RlCll AUXII.IAR DOWNSTREAM ELEMENT I IAS DISTINCT SEOUENCE AND POSITION REQUIREMENTS AND MEDIATES EFFICIENT 3" END PRE-mRNA PROCESSING I"IlROUOI I A TRANR.AC'TING FACTOR A downslreum G-rlch sequence {GRS). GG(;GGAGC;UC;U(;(~G. h;~,~ heen pro. vlou~ly shuwn Io influence Ihe efficiency nf .'V end pmcessi.g .f Ihc SVdI) lale pdyadenylation signal. Wc have now defined several impultam i~lr..tmclers I(~r ORS- mediated polya~enylation. The ability o1" Ihe ORS Io i.fiuc.ce ]" end prnces.,cing efficiency was sensitive Io individual and multiple f~fint mulalinns within Ihe tic- meal, as well as the position of Ihe clcmcm in the down~Ureum rcgion. analysis indicated that the GRS functioned Ihmugh a titralahle/rcn.r-actlng [actor. The GRS-specific DSEF. I protein was Found In I~ bound to the same ~pulatino of RNAs as the 64 "kOa procein of the general polyudcnylation factor CslF, indicating Ihal DSEF.I i~ associated with RNA suhstrates undergoing .1' end processing. Furthermore. an assncialinn was obtained between Ihe r¢lalive strcnglh of DSEF.I protein binding to GRS variants and the relalive ability of the (;RS variants to medi- ate eff~cienl cleavage in vi~rn. Finally, mulallons in che GRS affecled the efficiency of cruss-linking of the 64 kDa protein ,f C.~tF. "ll~c~c data define a n,vcl auxiliary d¢)wnstream clement and suggesl an imponanl ale h~r D.~EF. procc.~ing. Ba88a, P. S., Ford, L. P., Chert, F., and Wihrsx, .l. Nucleic Aci~,~ Research 23(9):162~-16~1. I~.~).~. O~her supp~m: Nallonal Institutes of I le;dth. F~om th~ UMD-New Serscy Medical .~chnol, I)epartmcnl of Microhiol,gy and Molecular Genetics, Newark, N~. Vgl RNA BINDING PROTEIN MEDIATES TI IE ASSOCIATION OF Vg I RNA WITll MICROTUBULE$ IN XENOPU$ OOCYTF.S Localized RNAs arc found in a variety of somatic and developing cell lyi~s. In many cases, microtuheles have been implicated as playing a ale in f;~cililating Irons- pen of these RNAs. Ilere we repe~ thal VII RNA, which is localized Io the vegetal cones of Xe, op~s laeris oncytes, |s as.~ocialcd with microtubules in t,ivo. Because or the ubklultous nalure of tubulin, the =.uneialion of specific RNAs with micrmuhules is likely to involve factors Ihal rccogoize both RNA and miemtubules. Vgl RNA binding protein (VBI RBP), previously shown to hind wilh high alfinily In the vege. tel Iocali~tllon site in Vgl RNA, al~ears to functlon in this capacity. Vgl RBP is ash:luted wilh mlcrmubul~: it is enriched in micrmuhule extmcls of =N~..'ytcs and is also co.Fceipilaled by hctemlogous, polymerizcd tuhulin. I:unhenmm~, V~I RFlP .bind.log activity i~ required for the specific association uf Vgl RNA to microtuhales m yarn. These data sug~esl a gtncral model for how specific RNAs can be localized Io particular slt¢~ via common ¢ytoskcletal clean'hiS. Eli,;ha. Z.. Ilavin, L.. Ringel. I.. and Y[~oeli, J. K. 144 The EMBO Journal 14(20):$109-$114. 199.5. Prom the Department of Anatom~ :=;:d Embryology and Department Pharmacology. llehrcw University Medic:,~ School. Jerusalem. Israel. CEU. DEATI h PROGRAMMED, APOPTOSI$, NECROSIS, OR O13 IER? There are at least two major types of =clive or physiololi,~l ¢¢11 death. The most well.known form. apolXosis or T~pe l, involv= e=dy nude=' collapse. ¢onden. sullen of chums.tin, generation of n~cleosomal ladders. ~nd cell fra~mcnl~iou with li111¢ or no narly altcrllion of lysosomes, h is most commonly seen in ~lla deduinB from highly mitotic lines, and Ihc cells are phagocytoscd b~ nelghhecln= cells m" infiltrating mncmphages, In met-',morphosiug o¢ secr¢lo~ cell=, =a~ uedcr ¢o~'~|l|ons where Ihe majorily of cells die. the bulk ¢£ the cytoplasm is ¢omumcd by expensinn of Ih¢ lysn.~nmal system well before nuclc-'ar collapse is Inmtif~l. This fom~ Of cell death has been Icrmcd Type II c¢11 death, and ~ rcvcrt to this temdnology. The requiremenl for protein synlhesis is me~: eheracteristic or Type II cell dealh in devel~pment-'d slmations than it is for T.v,.',e ! cell dcalh. "I'he vm'ial|ons seen I'o~'e • rca.s.'~¢s.'¢mcnt of Ihos¢ aspects of phys|.=~',&ic=l cell death that are Italy unive~,al. Iherchy focusing altcntion on the hlnlng~ 0=" the Im~es~, A hetler undersllu'lding of Ihe hiolngy and morphology of dying cc!i; will help elm'iry the s|gnificanee of the nmlccul.'w :rod hinchem~al findings. Z=kerl. Z.. Bursch. W.. Tennlswood. M.. =u~d Leekshln. R. A. Cell Dealh and Differenti'ation 2:87-96..'~,.wll 1995. Olhcr support: National lnstitule on A~,.:.'tg. P.S.C. CUNY. Medical Reseerch Council of Canada. and Notional Cance." !'|stilUlc m~d Human Froutier~ of Science Program. From the Depanmen! or Biology. Queens College ~d Graduate Center of CUNY. Flushing. NY. Institut fur Tumorbiologi.- und Krebsforschung, Universtl=l Wten. Austria. and W. Alton Jones Cell $clcnee Cent~. L~© Placid. NY. IV'. Developmental B~ology EMBRYOGENESIS IN VITRO: ~'I'UDY OF DIFFERENTIATION OF EMBRYONIC STEM CELL~ Embryogeocsis is Ihe fundamenl|! lU, OCCSS or diffcrenli~tion or all tissues Eem z fertilized egg. This process involves establishment of dislin¢l slem cells Ih~ will 145
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later dlfl'ercnllate Io all cell types. RecentJy, liocs oir embryonic stem cells have been established in culture rrom hlastocysts, The cells arc pluripotent and can dil'fercut;ate in t'irn Io all lineages, InlercsfinRly, differcntiation ol Ihese cells can also be induced in t'izro. Morphological and molecular events that are characteristic of Ihe develop- ment of the emb~o can be mimicked in t~zro by I~rowing Ihe cells under ca•rolled condillons, Furthermore, the process o1" early development can now Ix: sludied ;nd mce~rK~e.laled in ,',,ro and specific ,in,ges or stem cells can he o,),aioed and grnwn in Dushnik-Lcvinson. M. and flenvenisty. N. Bioloj~y o~'tbe Neonate 67:77.83, 1995, Giber support: United Slates-lsruel Binati~nal Sci'.nce Fnundation. From the Deparlmcnl oir Genetics, Institule of LiI'¢ Sciences, The llehrcw University, Jenasalem, ls~-.~l. MEK 2. A CA£NORIIABDITI$ F.LEGztNS MAP KINASE KINASF.. FUNCTIONS IN RA$-MEDIATED VULVAL INDUCTION AND OT! DEVELOPMENTAL EVEN'iS Activat.ed Ras in_illales a cascade or sequenli-I phosphon/lalinn cvem,~ including the p~olcin ','inases Ka/', MEK and MAP klnase. The Let-6() Ras mediated signal Iransduclion pathway controls vulval induction in Coe~nrha~iti$ ele&an;. Both Lin- 45 R~" ~d Sur-I MAP klnase have been dclennlned Io be essential factors during w~lval induction', however Ihe C. e/eRan.t melt gent has ~ol I~en identified. In this paper we have cloned a C. ete&ons mel~ gene, reek-2, and d¢monstrated Ihal MEK-2 protein possesses biochemical properties o1" MAP kinase kinases: The C. ele&ans MEK-2 protein can phosphoP/late and activate • human MAP klnase (ERKI) ~ MEK-2 ilselt can be phospho~lated and activaled by immunoprccipl. rated mammalian Rat. The reek.2 gene plays a key role in the 1¢l.60 ra,~-mediated vulvaJ induction palhway as Ioss-ol'-I'uncdon tool•lions in Ihe gene (~'.i14 and significantly reduce the signal I~ansmilted through Ras. mek.2(k.fl4) completely suppressed the Muhivulva (Muv) pheno~ype ot a hyperactive le#-60 r~x mutation and animals homozygon$ I'or mek-2(k.ll4J also displayed a partial larval lethal pheno. type. Animals homozygous lot" r~ek-2(It29¢) exhibited a highly penelranl sit.to and Vulvales$ pbeno~ypc,. Microin.~ction o1" • gain-ol'-fuoction mr~.2 mutation resulted in Muv and other rout.ant phenotypes, whereas micro|n~ection or a dominanl-ne/:~. live mutation no~ only suppressed the Muv phenotyp¢ or an nelly•ted let.60 • maliGn b~l also caused an egg-laylng del'cetive phenotyp¢ in olhen,v;se wild type animals. Our results demonstrate that me~:-2 ~cl~ bet~n lin.#3 roland sur-llmpk.I in a signal Iransduclio~ pathway used in the conlrol of" vulval dil'l'ercntiarion and olher developmental evenls. Wu. Y., ila,, M.~ and Ouan, K.-L Genes x- Development 9:742-755, 1995. 146 Other support: U.S. Public Health Service n:;,J the American Cancer Society. From the Departmenl or Molecular. Cellular, lad Dev¢lopmenlal Biology, University o1" Colorado ,'t Boulder. Boulder. CO. and Department or Biolo¢ical Chemistry and the Institute of Geronolology, University or Michigan Medical School, Ann Arbor. ML ,¢UR-2, A NOVEL GENE, FUNCTIONS L~TE 7 3 IN THE LE'r.60 MEDIATED SIGNALING PATIlWAY D~ ;~'ING CAENORIMBDITi$ ELEGANS " VULVAL INDUCTION We dc~:rih¢ hcrc a new ~cne actin~ d: .a tst~com o~frl.~O ra.¢ in the vulval nalin~ pathway olr Cornnrhalxlili~ ele&an~. "':e ~;~r.2 (l.gLqxcssor or [as) ¢¢n¢ is defined hy eight mutations identified in ~ :enetio screen For xuppRssor~ of the Multivulva phenolype orter-60(nlO#6), an..';rated lel-6Oras tool•lion..~1~r-2 mala- lions result in pleiotropic, incomplelel. "~netrant phenntypes Ihal include a Vulvaless phenotype in hennaphrodiles. ~.,:.".'cls in development ot the male lull, ¢on,'~lal .hnonnalitios, and larval lethalily, i.:.;lio~ilng • role I'or the .~ur-2 eene i~od. ucl in multiple dcvclopmenlal evcms. Gc~,, : epislasi$ analyses su¢gesl thai ,s.r.2 required lair in Ihe vulval si~n-'qin¢ palhw~,), downslrcam oriel.G0 R~. and is Io ~Cl" downslream or" Ihe P~f/MAP Kinase .:•soMe. We ©loncd Ihe ~r.2 ¢¢n¢ by DNA-mediated transform•lime, a~d have sh ', ;'fl thai il ¢l¢odes a Gavel protein. We alsn show that a .~ur.2::tm'Z Imns~cne is • :~ssed In the vulval pRc~rsor cells Ih¢ time or vulval determination. Slash, N. and II-n, M. Genes &: Develop•cut 9:2251-2265. 1995. Olher suppotl: U.S. Public Health Service at~ :k.e Locille P, Mm'l:ey Foondalion, From Ihe Departmenl or Molecular. C.- l.,lar, und D©v¢lopmenlal Biolol~y. Universily oF Colorado al Boulder. Boulder. CO. TIlE DROSOPHILA EXTRA $~X COMBS !'.~.01~IN CONTAINS V/D MOTIF~ ~SEN~AL ~R ~S ~N~ON AS A P.F~R~SOR OF HOME~C GEN~ ~¢Zrn ~e.~ r~n~.¢ is I •emir of the Pa.~ ~nth G~ ~en~ w~ ~cls ~ Bilh~ax and An~na~dla c~xes. ~ ~-O ~ei~ ~ ~iRd Io m~inl~in the spalL111y reslr~ted d~alm o¢ homenllc ,..,.c ex~i~ ~tabli~d ~ t~ ~. sicntly expressed RpRSs~. ¢.¢ h~ach~t~. ,~.~ a¢¢ ~ ~ui~ rot the 147
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oF these e~tly repressot.s. This implies two distinct modes nf repression and raises the question how does the transition fcom early transient repression to s able Pc-O-medi- ated Fop•salon occur? While other Pc-C prolens arc required conlinuoosly through- out development, the esc RNA is only present Iranslently in early embnjos, suggest- ing that e;c may play a role in mediating this transition to stable long term Pc-G- mediated rep~esslon. The predated moral', Fou•d in G-I~'oteln ~ subunits as well as non-G proleins involved in diverse eellul~ Functions including transcriptional repression. The ~qucncc alteration¢ oF a number o1' ¢.~c mutations muse amino acid substitutions within tbc WO zcFats, identifying them as essential I'o¢ the [unction or the esc protein ~.,~ a rcprc~,m.." of" homcollc gone expression. Other WD proteins ~r~ comlmnents el" reversible mac~'o- ranlecular assemblies and the WD motif has re.oily Ix:on direclly implicated in mediating inler~clious with other ix'oleins in such complexes. We prnlm~e that the e;c" protein is similarly involved in the initial recruitment el" Pc-G rcpres.~rs t~ the hnmeotic genes to establish their stable long term repressi(m. Salhe. S. $. and Ilarle, P. J. Mechanisms o1" Development ~2:77-E7. I From the Deparlmenl or Genetics. School or Medicine, Cuse Western Reserve University, Cleveland, OII. TIlE EXTRA SEX COMBS PROTEIN IS FIIGllLY CONSERVED BETWEEN DRO$OPlflL4 VIRlU$ AND DRO..COPIIlLA MEf.J~VOGAa"TER Ezzra se.r comics (esc) is one or the Polycomh Group Bones. whose products ~m required For long term mdmenanca of Ih~ spatially restrlc~ed domains of he•colic gcoe expression initially established by the products or the segmentation genes. We recemly show~d that the esc IXotein contains five collies of the WD motif, which in olher proteins hu been direcdy implicated in prolein-pmtein interactions. Mutalions affecling the WD repe•Ls or the esc protein indicate that they are essential For its I'unetlco ~s • repress•" of the homeo~ic ge~.s. We proposed that they may mediate interactions between es~ md Olhe~Pol.~om~ Group proteins, recruiting them to Ihelr tU~el genes, perhaps by eddidou'd intersclicos wilh tr~nsienlly expressed repressor~ such as hu~cM~uc~. To Further Investigate the Funcllon-',l importance or the WD motirs ~nd identify o~hce re•clio•ally important regions o1" the esc protein, wc have begun to delerminc ils evolutionary coec~rvatlon by characleri~.ing the e~¢" gone from Drosophito t~irili$. • disl~ntly related Dare.philo species. We show that Ihe esc pro- lean is highly conserved between Ihese sixties, parliculady its WD molars. Their high degre~ or consen~alion, particularly al Posilions which arc not conserved in the WD consensus derived F~om alignment of" all known WD retail's, suggests that each 14g o1" the WD repeats in the e$c protein is Fuoctior~lly speelzlized ~ that this sps~:ixl- izztion has been highly conserved during e~.olutio.. IL~ highly chzxged N.termlnus exhibits the ~reatest divergence, but ~¢n d,er.e differences ~re conservative or its predicted physical properties. These obscrvat|ons suggest that the t.~t ~olcin is Functionally eompxct, nearly every residue m~ing an impe~lant contdbutlon to its Fuoction. Sathe. S. S. and llmrte~ P. 3. Mechanisms or Development $2:225-232. 1995. From the Department or Genelies. Scbobl of Medicine. Case Western Rcse~c University. Cleveland. Olh IDENTIFICA'rlON OF FAT-CELL ENHAI~CER ACI"IVITY IN DRO$OPIIILA MEL~NOGASTER USING P-ELEMENT EI.~HANCER TRAPS To identify genes impotlam in fiat-eelt .,,etabolism and development, we h~e screened Drosophila stocks can'ying an ~.~r:emd transposable ekmenl that can reveal the presence or nearby enhancer r foments. We have identi~'icd those "cnhancu.trap lines" that contain transpo:. ,~,~: P clcmenlS integrated near I'sl..ccll specific enhancer clemenls, W¢ snticip~' "~'at the genes Is•oct•led wilh th©sc enhencers will provide information co•cct.:.;~g Fst-¢cll Function ~en~s For studyin~ rat-cell specific ~cnc ex, -,;ssioa. Furlhermofe. Ihe idcndl]catio, of cohenccr.lr-',p lines active in the dcvelop!nJ~./'at cell should pmvlde an entry point into the molecular and i~cn¢llc analysis of e~:ly Fat-ceil developmen¢. Analysis two liocs has revealed that the transcrip(|o¢ %clors ~. • slemid-hemlonc reeel~or. sod Kr, • zinc-ringer Ixolein. arc pro•col in ~;:¢. lit body; ~ Faclors m likely Io Ix: involved in rat-¢~ll ~ne expression. In twu. "her lines. ~3-plsclosldsse was cklecled in • subset or adq)ithelid cells thai may b L¢ Wtcutsocs Io Ihe sdull I'st cell. ,t, nd finally, in a single line t~ans~ene •clarity ;. ,,~scnt in the Ixo~enitor cells o1' embr~onic Fat body. ~ g,~es sssoeiate-. ,,,tb these cnhanccr.lrap lines may Ix: involved in Fat-cell development. iloshlukl~ D, K.~ I..unz, R., Ghosh, M., and Jot~son, W. Geoom¢ 3~(3):497-506, 1995. Other support: America• Cancer Society, Illinois Divlslo•, American lleart Assu~iatlon, ~d the Campus Rese~,reh From the University or Ilia•dis College of ~ledicine at Chic,,lo, Depunment of Biochemistry, Chic•l~o, ]L, and Univer.~;ty College oF ~cdic|ne at Chicago, Department or" Immunology and Miczoh?olua.v, Chicalo, IL. 149
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O PROTEIN KINASE C ISOZYMES IN PROGRESSIVELY TRANSFORMED RAT EMBRYO RBROBLASTS "TEe role of individual pmle|n Einase C (PKC) tan[arms in progressive transfor- m•clan or z ~t cmt~o fil~oblast cell line (REFS2 cells) has been eva~uated. Nom~l (REF A) co|Is wcre transfected with SV401o produce (he paniall), transformed REF B cell line, REP B cells m'e mo~hntogically transformed but have only limited capacity ror growth in soft ag~r. Message and protein levels [or PKC-= ~nd PKC-& were simil~ in REF A and B cells, indicting that expression or SV40 large T did not dirtily influence the ~mounts of PKCs. However. PKC-a localization was influ- enced. PKC-a was associated with (acal contacts or REF A but not REF B indicating teal changes in location rather than content are an cady event in REF$2 cell If•as[actuation. Clones at REF B cells were selected for ;rowth in soft IREF C cells), Levels of PKC-~, but not PKC-o or e, were increa.~d in s~veral of these clones, suggesting thai increased PKC-5 content may f'acililale anchorage-lade- pcndcm graunh. In olher studies, we have determined that PKCs inter•el wile their binding pratc|ns/subslratcs through lhelr regulator,/damn|as (RD; L. l.fan el EiochemislO', 33: 1229.1233, 1994). Thes~ r~,ulc; pe=Jict lh~ RDs could pnlenlially inhibit PKC signaling by competing with endoBenau-s wild.tyi~ PKCs for hindlnl~ protci~subStr~tc interactions. Ovemspresslun of the RO al" PKC-~ inhibited ~towth in s~l alzr ol'one ~epresentativc REF C clone, whereas ovcrcxprc~inn of the RD of PKC-c~ promoted growth in soft ugh'. Th~se ~sutts surge.,• that RD c~pression may be a useful approach for dominant ncptivc PKC inhibltors with po~ntlal isoxyme Spccihciiy. Lisa, L., Ramsay, K., ~nd JuEen~ $, Cell Growth & Di[l'erenilation S:l Ig$-1194, Nov~znbcr 1994. O~h~r sul~rl: National Inslitutes or lie•liE. From ~e W, Allan ~on~s Cell Science Center, Lake Placid, NY. HMGI? IS A CHROMA11N-SPECZFIC TRANSCRIPTIONAL COACTrlVATOR THAT IN~pe THE EFFICIENCY OFTRANSCRIPTION INItiATION We have ~nined ~ dTcel or HMGI7 on trmseHixion by RNA polymerase il b,/the assembly =~1 ~lysis or HMGI7,~x~taining ¢hromalin lemplales consist- in~ or ~ularly sp~'ed nocl-osomll ~n~ys. Slmctur~l ~t~lysis or the chren~t;n indi- ¢~! (hat ltMG 17 is i~corpom~l iron chmmein in • physiological mama" with full comptemem of core Eistoaes, The ttlascrlpdonzl studies revealed that HMGI7 stimulates tmn .s.~lXion in conjunction with tee SCClU~nce-specific activator GALA. VPI 6. THIs effect was observed with chromstin, but not with non-nucteosom•l tem- plates, and required the presene¢ at IIMOI? during chromalin assembly. The incor. par•lion oFHMGI? into chxomzlin resulted in a ?- In 40-fold stimulatloo o~ GAL4. VPI6-~-'~iv~l~d lranscH~llon Io levels that were comparable to those observed wile histonc-frec DNA templates, in coou'ast, transcription ream IIMGl?-containine chxamatin was not detectable in the ahsence of GALA-VPI6 or with a GAL4 deriva. WIRING DIAGRAMS: REGULATORY CIP,~ UITS AND 1118 CONTROl. OF SKELETAL MYOGENESIS Durinl~ the past ye,'u', largeted mutsl;enesis in mice h~s be~zn to claN[7 the roles" or individual members of Ihe MyoD family of myogenic regulators in vertebrate 151
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O developmenl. In 1his review, we discuss these studies both in the context of tissue interactions neccssan/to induce skelel;I muscle precursor cells during embryogene- sis and the molecular circuicry 1hat regulates the terminal dil'f'erenfiatinn of Ihese cells. l.assar, A. and Munsterberg, A. Cur~enl Opinion in Cell Biology 6:432-442. 1094. Oil,or suppor[: Nalional Sclcnce Foundation. l.ucille P. Markey Chnrilahlc Trusl. Muscular Dyslropby Associalio~. and Ihe March of. Dimes Birth I)cl'ecl.s Fn,ndallnn. From Ihe Dcpartmen! of Biological Chemislry and Molccular Pharmacology. lion, oral Medical School. Boston. MA. COMBINATORIAL SIGNALS FROM TIIE NEURAL TUBE. FI.OOR PLATE AND NOTOCiIORD INDUCE MYOGENIC hllLll (iENE EXPRF.SSION IN TIlE $OMITE The neural tube. floor plain and nolochord are axial tlssucs in Ihe ver[ehrale embryo which have been demonslraled Io play a role in som;Ic morphogene.sis. Using in vicrn co-culture or llssu¢ explanls, we have monilorcd inductive inleraclions of these axial tissues with the adjacent semitic mesodenn in chick emhnlos. We have found lhal signals from lhc neural lube and floor plale/nolnchord are neccsxar~ l'or expression or the myogenic bllLll regulators Myol), MyI'.S and myogenin in the semite. Eventually semitic expression orthe myogenic bllLll genes is maintained in lho absence of the axial lissues. In or~an cu!lurc, al carl), developmental slages (llll I I-), induclioo oi" myogenesls in lhe lhrce most recently rormnd somiles can be mediated by lhe neural Iube tOgelher wilh lhe floor [~lalc/nolochord, while in more rostral somitcs (slages IV.IX) the neural lube wi[houl lhe floor platclnotochord is sufficient. By recomblnlng somites and neural tu[0es from dilTcrent axial levels oi" the embryo, we have found lhal a second signal is necessary Io promote coml~Icnce of. lhe somile lo respond to inducing signals from the neural tub:. Thus, we propose lhal at leasl lwo signals from axial tissues work in combination to induce myogenic b}ILH gone expression; one signal derives from lhe floor plale/noloclmrd and other signal derives from regions orlhe neural luhe other lhan the floor plale. Munste~herg, A. E. and Lassar, A. B. Developmehl 121:651-66~, 1995. O~hcr support: National Science Foundation, Lucilla P. Murkcy Charilablc Trust, Muscular Dystrophy Associatioo. and lhe March of Olmcs llirlh l)cl'ccls Foundalion. From lhc Departmenl of Biological Cl,emislry and Molccuh~r Pharmacology, llarvard Medical School. Boslon, MA. 152 BCL.X~ IS TIIE MAJOR BCL-X mRNA FOR~ ~ ~PRESSED DURING MURINE DEVELOPMENT AND ITS PRODUCT LC L.,UZE5 TO MITOCHONDRIA Most examples or celt death in animals ~',c conlmllcd by n gentile IXogmm thai is acllvalcd within Ihe dying cell. ~ alx~lo|,.'," process is fudhcr ~gululed of genes thai act us rcprcssors or cell death. (?~ Ibese, bd-2 is expressed in a v~l~ely or embryonic and postnatal IL~sues which su~.l~ests a ¢~tical role for bd.2 in genesis and tissue homeoslasls. Suq~risingty, m~ll~nt mice with I~rgettd dlsmptlo~ o1' bd-2 oppear normal at birth and complete matu~tloo or lymFho|d tissues bcl'o~ soccumhlng In f, lminant lymphopenla and p~lycyslic. I~nll discas~ by 2-5 weeks o1' age. This su~csts thai there may I~ genes Gig'.or than brl.2 Ihl! elm r~gulate sis during development. To beKin to investi~a:e, this possibility, we have cloned ~nd characterized the routine b¢/-x gone. whose human counterpart displays strikin| homology to bci.2. The predicted murinc bc~ xr.gcne [~duet cxhlblts u I/iBh level o1' amino acid iden|ily (97%) to ils human c(~..~ltcrparl. Jusl llke Bcl2. the murine f~cl-xL gent product can acl ~s a dominanl inlr;:~itor of cell de~th upon growth f~ctor wilhdrawal. In addilion, tim bulk or Ih¢ bd..~, ixoduct loclliz~s Io Ihe IX~Hphery of mitochondria as assessed by a bd-x~-Iae expression system, suggesting thai bolh Bcl-2 and hd-xr proteins prevent cell devil h'~, a similar mechanism./~'l-.t~ is the most abundant bd.x mRNA species expres.~ .~ [n emlx~x~ic ~nd ~dult tissues. levels of hcl.~r mRNA appear hil~her than t~'~ ~ o1"6d-2 during ~nbcymlal de,lop- meat and in several adult organs includlhg t.r~e manow, bllin, kidney mid thymus. In addilion to bcFxo we have idenlificd an:.:,:r form of/~'l-x mRNA. bd:t~, thai results from an unspliced/~d-.~ Inmscriph ' ,'!-.r~ mRNA is expressed in ~lrlous emh~j, onic and poslnatal tis.~uca. Surprlsin~.l,.: the eapmsslon of bd-.r~ (a ne|atlve regulator or programmed cell dealh) w~s t,~_,.:ectuble by n scnsilive $1-nuclcasc z~say and polymetase chain n'Jctlon analysi, or" mou~ llssues. 13ascd on ils tissue and devclopmcnlal patterns or expression, il a~ear~ Ih~l bc/-.~ may play ~n impor- long role in Ihe regulation of cell death durln~ ,;-.velopmenl ~d tissue homeosllsis. Gonz~lez.Garcia. M.. Perez-Ballestero, R.. Diag. L.. Duan. L., Boise. L.H.. Thompson. C. B~ and Nunez~ ~. Oevelop'm-ml 120:30.t3-.'1042. 19~4. Other support: U.S. Public Health $ervlcr, American Conccr Society. and the 5andoz Foun~latlon for Gcronlological Rcsca: :h. From the Department or Patholosy, Un|ve~i.y of' Mlchi~n Medical School, Ann Arbor. MI. and Howard Hughes Medico! ~nslitut=, Dcparlments o/" Medicine. Molecular Genetics and Cell Biolog),. Universe.,/of Chlcago, Chica~o. PLATEI,tTT GLYCOPROTEIN lib OENE i..,¢ ~,,IESSION AS A MODE[. OF MEGAKARYOCYTE-SPECIFIC EXPRES~,;C,~'I GlycopnSein (GP)llb/illa is ~n [nlegHn c,,,npl~ nonn~ly Its~ricled [n its expm~.
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MATURATION-DEPENDENT CHANGES ~': THE REGULATIO~ OF LIVER- SPECIFIC2 GENE EXPRESSION IN EMBR~,'."/AL VERSUS ADULT PRIMARY LIVER CULTURES During rol liver development, which Slan,~ on day 10 of ¢rnt~ol~encsls (1~10), and until EIb, all pureuchymal cells are thou, ~. m be I homogeneous PopuLation o/. bipolenlial progenitors, able IO give rise In I:, "', hepalocyles and bile duel epithelial cells. We established prima~ liver colluRs :, :;, emlx~onic Ilver~ at v:u{ons opmcntal sieges. I'rom El4 to neonates, as mrll ',s adult rats. Gene expression regulation hy three known dif/.erenlinting agents. Kaput|n, dimelh)'lsul/.oxhle (DMSO), and sodium hut),rale, were examinc~f in these primer/cultures. Alpha. leloprotein (a-FP). albumin, samma-glulat+.),llra•speptidase (GOT), and lathione-$-Irans/.era.m.P (Yp) wen: expresse~: +~, cultured liver ceils Ihmulh felal development, whereas insulin-like growth fa:. ,~-!! (IQF II) Rceixor. expressed in I'elal pnrenehymal cells, was nm present i• cui.+ --d ncunalal cells, IIq~'in inere~"d e~.FP levels in fetal liver cells, but not i• cells +"~alned ali~r bi~lh. Tic exixeasion o/. GGT and Yp was coordtnatel), r~gul~(ed. The Iv-:, ~enes were up.m~lal~,d by sodium butyrate and down-regulaled b), DMSO in c'a[+nred Iiv~ c~lis/.ram all emlx)'noal ales tested, Ilowever, Ihe regulation o1" these two genes b), sodium belyrale and DMSO was hal apparent in neonatal and ~d~:It liver cultures. Sodium bul),rat¢ increased o-FP and albumin mRNA expressio~,, i• El4 and hIS cells, but not in El6. neonatal or adult cultures, and its addition ca: ,-~, heleroCeonus exlxessio~ ol .~bu.. mln. We conclude that the ~egulalio• of gene ,",: :=sslno in Fdm~, liver cullures 5), the Ihroe agents tested is allured a/.ler b;rlh. |~, ."l.d cells, sodium b~lyrate ser~¢$ ,,~ • strong general dilrcrenli~ion aleut, iucseasing .,.;prcssinn of s~Lt or lanes charac1~. isllc/'or beth hepatocyleS and bile duel cells. L y co.real, DMSO suppresses sets o!' genes expressed in bile-duct cells, e.g. GOT, Yp, and cylokeralin 19 (CKI9). Drill, S., Zvibel, !. and Reld~ ~-- M. Differentiation 59:95-102, 1995. Other supporl: America• Cancer Soclel),. ~.. ,-hal Institutes o/' Health, and The' Richard Molto Foundation. From the ~ancer and Liver C'~mle~S, Alhe~l E+r,.;teln College o/' ~edicine, Bronx, NV, Departments o1" Molecular Pharmacology, ~::¢rt Einstei• Colk:ge or bledicine, and Microbiology and lmmuno!og),, Albert F.~ ,':cln College o!' Medicine. Bronx. NY. PRIMARY CULTURE OF DRO$OPliiI.A EM,'I~.YO CELLS Primary cultures of Drosophila embryonic ¢~.lis offer I unique system Io sled), the lransilions ot" undiffercnliated cells Into • vmt;.-ty or cell lypes. Coupled with the power of Drosophila classical and molecular grr-.llcs, Ibe analyses I~lal a~ possible in v/Ira using differenlialing embl~O celts wi~"- eontln~¢ In oonlrlbme to • dcqx:r understanding of developmenl. Prepamtinn .,:, rimanj' embryo cell cullurea was - developed independently in the laberatorias of ~cecof and his colleagues (Succor
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and Unanue. 1968) and Shields and Sang (1970). The procedures for p~eparing cul- tures urn technically u~demandlng and can he adapted to solve a wide range or b~o. logical problems. Important variations includ~ tl~ culture of single embryos (Cross and Sang. 1978). as well ~s the dcvclopmcm of techniques filr isolallng IlK precur- surs of different cell types such as neurons and muscle c~lls (Furst and Mahow;,Id. Sal~alervao P. M. and llayashl, I. Cell Biology: G Labov~loP~ Handbook. ~cadcmic Press. pp. 393-3gg. 1994. Other suppo~: National Institu,¢s of Health. From Ihe Deparlmenl of Molecular GencllcS. hackman Research lnslilule of ilia City of Ilope. Duane, CA. MYOGENIC REGULATORY FACTORS: DISSECTING TI IEIR ROLE AND REGULATION DURING VERTEBRATE EMRRYOGENIL~;IS surge of research aclivily Ihal promises Io brid~e molecular ond embryological approaches Io music rcsca~h. In Ihls ~v~w. we con~idcr Ihe arguments IhOl would place MRFs in a key dcle~at~ role for I~ skclelal muscle ]inca~ brala embeD. Amphibion. lvian, and murinc syslcms are compared anti rcccnl dcvelo~¢nls in Ihe ml~ular em~lo~y of myogcncsis are ~vicwcd. Wonted Io occoun~ for I~ a~Rm di~Rp=~s in concl~[ons drawn from stud- ies usin~ cultural calls I1~1 indicale a role for the MRFs in dcle~inallon, nod its of MRF and muscle gcnc ¢x~ss~ i~ riro Ihal indicale = ode in diffcrcnlialion ~l nol in lineage Dcvclo~cmal Bioloey 156:1 I-2~. From Ihe Depanmcnl o( Bi~hemislw. Bo~l(m Universily School of Medicine, Boston. MA. DIGIT TIP REGENERATION CORRELAI~S WITI I REGIONS OF M.TXI tlIOX 7~ EXPRESSION IN FETAL AND NEWBORN MICE We ~ Ihal d~ng ~ reid dcvclo~nl I~n~c~ipt~ of Af~.~l ~nd M¢~2 ~come ~ogressively ¢~riclcd to cells that will [o~ m~c dlslal digil slruclurcs; the kfsw2 execs:ion domain is mlwo~s m~ dLstal than Af~.¢I. At bldh ~lh M.¢d 156 and Msx2 arc c~pr¢ssed in cells ol'the nail ~ -'~ and hair follicle. We have found that Ihe regeneralive ability of mouse dlgil dps i *;~ Io larch in which ~he ~. ladon plane is wkhin Ihe Rglon o[ M¢~I...~ n~ M¢¢2. ex~ssion digils ~nd Io levels where ~h M~i ~nd ~1- ,. am ex~ ~ late reid tel dicils. Feint digit tip regenemli~ is ~pk. ~.:~ ~mpIctc4 by 51n11. w~ ~nl. tel digit lip rcgenemti~ ~quiRs 4 weeks ~] is ~llm~ SmoKer. In ~th f¢lal and neonatal digits, we find that ~h M~xl ~.. : J~ am cxp~ssed dudn~ lion. ~t nol during wound healing ~i~', .~ Wilh p~l ~llli~s where mgcncrmive ms~n~ is obse~cd. ~c~ de;: .u~ I~ hy~hesis Ihll sion of M¢~ gun= a~e im~a~t f~ digit cet~. ~,~ initiate a~ pnki~te b a ~c~r- alive rcs~nse. Reglnell;, A. D., Wnng. Y,-Q., Stem~ D~" .. Men,ks, ~ Developmc~ 121:1~.1076, ~rom the ~pa~nt of Cell e~ Molecula~ .~t~dogy, and t~ Mo~eltr Binlogy Pmgnm, Tulane Univenily, ~ew Cdean~, Bosloa Upivenily Schml of Medicine. Boston. MA. RESTRICrED EXPRESSION OFTYPE.I! TGF~ RECEPTOR IN MURINE EMBRYONIC DEVELOPMENT SUGGES'.,"" A CENTRAL ROLE IN TISSUE MODELING AND CN$ PATTERNING ~ lyp~-ll TGF/~ ~¢cep~or mcdillas In~.l.~ ~.,f the biolocical esponse$ io'TGF/~, An examinalion of Ihe expression of the ,)','e-II TGF~ rcceplor during mouse embryogenesls therefore provides specific L~¢.~'mallOn shoal the role or TGF/~ dur- ing emb~ogenesls than has t~een available to dale. We have isolated the genomi¢ merlne homologoe of the human typeoll TG~:ff receplor corresponding lo exon 2. The routine and human sequences show a t::gh degree of homolngy. Using the murlne probe we found that lype-II 'TGF~ ~ece.-,:of expressio~ is ~egulated in bolh a spatial and a temporal fa.~hion by using in~it~. ~.~ brldj~al|on and Hlxmuclease lion a¢~ys. Ty[,e.II TGF/) receplo¢ ¢apre~si~," ~: localized to the mesenchym¢ def. lag critical ialcracllons wilh adjaccnl epiihei~,m suCh aS developing hair follicles, whisker follicles and t~olh snlage. In the ccmlral nervous syslcm, lype-II TGFp receptor expressio~ is highly rcslrlcled to the 1]~o¢ plale. Sl~'oflg exFes$ion i.~ aim dcleclcd in mi~-allng neural er~l ¢¢11s. meni.'~£-.s and chomid plexus. Specif¢ mcs- enchymal Iocal;x-',tion of type-II TGF~8 reccp,'~' Is also observ,.~l in lung, kidney, lnlesline, ston~ch, and bladder. "i~e reslrkle,J exl~cssiou of lyp¢-II TGF/~ in mesenchymal cells at sites of q~hhellal-mescnchymal inleractlofls sufgeSlS Ihal lype.ll TGFp receptor plays n ma~or role in mediating Ihe es~ablishmcnl of eml~y. onic ~'~.'m systems. The highly reslricled expr~.,d~n of lype-II TGF/~ developing CNS su~gexls an imponnnl role ftw u ~rlnchhreonlne klnnse in p~lient- ing of the nan, out system. Wang, Y..Q., Sizelond, A,o Wang, X.-Fo, end ,q~ssoon, D,. Mechanisms of Development '~2:275-289.1995. 157
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From the'Departmcn! of Biochemistry. Boslon University School of Medicine. Boston. MA. Whitehead Institute for Biomedi~l Research. Cambridge. MA. and Dcparlment of" Pharmacology. Duke Unlversily Medical Center. Durham. NC. ECTODERM-MESENCIIYME AND MESENCIIYMP--MFJ;ENCIIYME INTERACTIONS REGULATE MSX-I EXPRESSION AND CI':LLUI.AR DIFFERENTIATION IN TIlE MURINE LIMB BUD "]'he apical eclodennal rldce (AER) is a spcclali~.ed thickening oflhc distal limb me.~Jlehym¢ Ihal has bccn dcmonslratcd Io sup~orl limb oulBrowth and pr.pcr llmb development. The homcobox gone. Msx-I, is associated will, the distal limb enchyme (progress, zone) and ils expression depends upon Ihc presence of the AER in chick limbs. We demonstrate here thai Ibe expression of' Msx-I is dependcn! ulx~n the limb ectoderm in Ih¢ mouse, but thai Ihe inducl|Ve capacity of' murin~ limb derm is not reslricted In Ik¢ AER. Max. I can also be mainlained in limb mes- eneh)'mc by Ih~ substitution or FGF 4 for the eClOderm; however, we see Ihat local cell<ell interaclions are required for high levels of" expression, Dismplion of" cell interaclions in the llmb mesenehym© rcsulls in a dr.~mati¢ de.ease in Msx-I levels and a precocious expression of MyoDI. suggcsllng that Ilze limh environment represses differentiation and promo,ez cell proliferation during early development. BMP 4 and FGF 2 can also maintain Msz-I expression in limb mesenchyrnc --,,; well ~ retinolc acid which is usually associated with polarizing activity in Ihc curly limb. Msx-2 expression does nnt appear Io be dependent nlxm cell.cell interactions measured in these expcrimcnl~;. T~en Iog©tber, our dale suggest Ih-',t Iho expression of" Msx.I. but hal Ms~-2. hal only requires raclors from Ihc limb ecloderm, but also relies upon cues from local cell inleractlons and 1hat the spati-I distribution of indtK:- lay© capacities in limb eclodcm~ diff"c~s hclwccn the avian and murinc syslcms. Wang, Y. and Sa,ss~n. P. Developmental Biology 168:374-~82. 199~. Other support: National lnslitules of" Heallh. From Ihe Deparlment of Biochemislry. Bostnn University School of Medicine. Boston. SECRETED SPITZTRIGGERS TIlE DER SIGNALING PATIIWAY AND IS 6. LIMITING COMPONENT IN EMI~RYONIC VF.NTRAL EC'TOI)FJtM DETERMINATION The ~pitz Cent encodln¢ n TGF-cz homolog, ha..~ been shown Io affccl a suh~t of dcvelopmenl~J processes that am similar Io Iho~ rc~ulalcd by DER. Ihe I')roxnphil~ EGF receptor homolog. This work clcmonstmt"s thz4 Spilz triggers the DER signal- ing cascade. Addilion of • ~lcd. ~1 not I~ mem~nc-~ialed ¢~ ~ Spitz In $2 Drosopldt~ cells expmssin~ DER ¢iv~ ri~ to ~ ~ zymsi~ lotion or D~R. Following ~,lophosphowloli~. DER ~iales wilh pr~cin. Con~q,cnlly. aclivoli~ or MAP kin~ is ob~wed. ~ profile or MAP klnx~e ocllvolinn provides ~ q.nnlilalivc o~y for DER ~ctivat~n. ~lWC~ Ihe levels of Spit~ and MAP ki~ activity w~ ob~ed. Spilx prNetn w~s expressed in ¢m~os In ns~ hs binl~i~l mlvily. An in cell rates w~s ohsuvcd in th~ vcnlml ~1~. z~h Ih~l I~lcml c¢11: vcnlnd-mn~! fates. ~ rcs,II indical~ I~ ~1 ~ivalioo o£ d~ DER may nnmzolly glv¢ ri~ In ~ m~rloire of di~c~le cell f~les in I~ vcnlml ¢~1~ml. S~li~lly reslricled pr~cssin~ of Split may ~ R~nslble tar this ¢~ed ~ R~m~id (Rhn) and Slur p~cins wcm su~¢=1c4. ~ t~ ~is ~enel~ ~tions. to act ~ m~tdat~ of OER stealing. No ~tt~d~ in ~R w~n the Rho a~ Star pint,ins w¢~ c~xpm~d with DEE in ~ cel~ mutual ~or rim ~ Slur Ihc venl~lizin¢ effect or ~led Spilt is epis~li¢. 1h=1 Rho a~ Star may no~dly facilil~e ~sin~ ~ the S~lz ~, Schwcilzcr. R., Shoh~beny. M., ~er. R.. ~d ShiM, B.-~ Gen~ & ~vdopmcnt 9:151 R- 1529. I Olhcr sup~rt: N~li~nal Inslilulcs or Heallh. Go.an Ca~er Rese~h Cen~er, WoIE~o. Fatal. oral Ihe Min~ F~fld~ti~. Fr~ zhc Dcp~nm¢n~s or Molecular G¢~li~ ~ Virology and Mem~ Resca¢h and ~io~yslcs, W¢izm~nn Insdlulc or Scarce. R~vm. REGULATION OFTIIEXENOPU.~ LAgML HOMEODOMAIN GENES. IIoxA I AND l{oxDl: ACTIVATION BY R...................~NOIDS AND PEPTIDE GROWTll FACTORS Verlebrale homolo~ues of Drosophila kd~'nl are likely Io play key anlcropnsterinr axis formation. However. lilllc ;s known alx~ut the re|ulalion of Ihcsc genes during verlebratc developme~. Here we examine the ¢xlxcssloo regulalion or the Xtnopux luht~! hom~doma|, genes, HoxAI ~nd Ho~DI. IloxAI was expressed ~round the dorsovenlral circumference or Ih¢ Irunk in neu~ula emb~os, with later expression in spinal cord, midbraln, hindb~in, and endolym. phallc duct. By mid gaslmla, HoxDI was pcdomlnanlly expressed in do~zolateral and ventral .cct~xfc~m. with a g~p in expression al Ih¢ dorJzal mldline. By neurola, venlrnl cxpr~,xioe had dccllned with mO~l expm~sM~ reslrieted to do~Jolalerel mesa. dcm~ and ¢ctndcn~. Retinoic acid strongly indoced IloxAI and lloxDI Ihro~gho~l th~ ectoderm ~nd mesendnd~rm o1" patrol,, stages, while in old~" emb~os relinoids induced cctopic expression of these cones in mar limiled ~l~ions. Induction by , ~clinolds w~s indep©ndc.t of pmzcln symhezls. Suq~Isln~ly, Iloxkl was expiated 159
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C) at high levels in isolatad animal caps in the absence of rcdnole acid. "l~e ~ptidc Browlh factors bFGF and activin A slrongly imtuccd exprcsx;nn nf lloxDl, hut nnl IIoxAI, in animal caps: however, RNA accumulalcd only many hours alice the application of th~s¢ faclors, Ovcrexprc~sMn of thyroid hmmonc rcccplnr (c.erhA) prevenl£~ inductimt of IIoxDl by retino;c acid in animal caps, c.~rbA also ahlalcd expression of l{oxDl in whole embr/os, suggcsling a r(:le for ~dn~cnous rctinohls in tha regUlalion of HoxDl expn:ssion. Oomlnm.lt inlcrrering activin and I"GF recep- tors pravenlcd c~pression of lloaOl in t4¢~, implicallng thc,;e faclors in the normal induction of }IoxDI. Our dala indicate Ihat induclion of labial-like homeodo~ain genes is Complex and may ~equ|m many f~ctor~, Kolm. P. L and .~ive, !1.1~ Developmental Biology 167:34-49, 1995. Other suplmrl: National Science Foundulinn. From Ihe Whitehead |nslilute f~ Biomedical Research and Dcp~rlment of l'llnlogy, Massachusetls Inslilule for Technology, C-',mhrhlgc, EFFICIENT i IORMONE-INDUCIBLE PROTEIN I~JNCTION IN XENOP~$ La£t'l$ A major problem in analyzing ganc funcliOn during Xen,pltt development has been Ihe inahilily Io induce gone expnesslon in a teals)rally controlled manner. We have attcmplcd Io solve Ihls problem with a system of hormnnc-a¢livatcd prolcin funclion, using the myogenic gone MyoD as a paradigm. We show that microM]ec- lion of RNA for MxoD fused to the llgand-binding domain of either the csuogcn ~ glucoconicoid reeeplor resulls in hormone-dependent -,:dvatinn of MyoO function, as assayed by ectopic induclion of muscle-spacJflc acdn mRNA. Ind~lion is tightly regulaled in both isol~ed animal c~ps and inta¢l embeds, whh ectol~c mas¢lc-spe- cilia actin expression inducible alter 2 hr nf hormon© trealmenl. Illgher levels of MyoD.receplor fusion proteins thai native MyoD prntein arc present in cmhz~/os, apparenlly u rcsull of incrcasnd ruslon pr~cin slability. This is tim first demonstra- tion Ihat hormone.lnducibic fusion prol©ins can work effectively in a complex embryo. Kolm, P, and Siva, II, L. Devctopmenta! Biology I -/! :26"/-272, 19%. Other support: National Science Foondadon. From the Whitehead Institute for Biomedi~l Research and Depanmem of Biology, Massachusetts Insthule ol'Tachnotogy, Cambridge, MA. 160 A RI IESUS MONKEY MODEL TO CHARACTERIZE THE ROLE OF OASTRIN-RELF.ASING PEPT1DE (GRP) IN LUNG DEVELOPMENT-- ~.Wn~N~ FOe STt~U~tON O~ ^tnwAv ~ow'nt Gastrin-rclcasing peptlde (GRP) is developmentally eXl~eSSed in human feint lung and is a growth [actor for normal and neoplastic lung ~! its role in no~n~ lung devclo~menl has yet to I~ clearly deiince. In this study we have char~cte~zcd Ihe expression or GRP and ils receptor in I'clel dr:sun monkey lung and determined the effects of bomhesin on fclal lung developmc~ff in e/taD. By RNA blol analysis, GRP mRNA was first delectable in fetal monkey inltg at 63 days gestation, itach~ high- esl levels at P;0 days gestatinn; and then declined to nc~' Mull levels by 120 gestation; n pallcm closely paralleling G~.P expression in human f¢~al lung, At in human lung, in .fftu hybridizalion localizt'J ORP mRNA IO I~¢mocndocrina celll though du~'mg the canalicular phase of d~elopmcnt (l~tw~n 63-gO d=ya GRI' mRNk was present nol only in classic pakn~m~y ,¢moendocrinc cells, also in ceils nf budding airw=~nL Immtmohistuchemist~ showed that bombcsi~like intmunore-octivily w¢~ I~esent in ncumendocdnc rails, but nol in budd~ag suggesting that in budding ai~ways either tM OAt mRNA |s m~ trm~sltled, Is tepidly secreted, or a relnled0 Iml different RNA is pn:sen~. RNas= pmtectkm analysis a prtxt',e to the monkey GRP receptor demtmsttated thll IM tlm~ co~n~ of RNA expression closely paralleled the lima cou~ oT GRP RNA ©xi:~ion. f~ ~m hyhrid|zatioo showed thee GRP ~-ceptors we~ p~imsrily eIpres~d in apilhelisl cells of the developing airways. Thus. ORP wo~ld appcm" to I~ secreted from dncrine cells In ice on larval cells in developing ai~vays. This hypmhesis was con- firmed by organ oullure of felal monkey lung in Ihc presence of bumbesin and bombesln anta~onlSlS. Boml~sln Irealment at I and 10 nM slgnil'~-anll)~ increased DNA synthesis in airway epithelial cells Imcl signilicanlly iuc~ascd Ihe number and size of airways in cullured felal lung. in f~-'~, a~lluring 60 d f~lal lung for 5 d wilh 10 nM bombcsin increased ai~.ay size and numt~' nearly Io lira! observed in aullured 80 d trial lung. The cffccls of bombcsln could hu Mocked by sp~Ific GRP ~ntagonists. "~us Ihls Sludy demonslra(es (hal GRP rec~o~ In: eqxessed o~ air- way epithelial cells in developing fetal lung and thai the inle~-Ih~ orORP whh the ORP receptor stimulates ainvay dev¢lopmcn(. Jonmal of Cllnlcal Investigation ~4:160~-1615. Oclob~ 1994. OIh©r ~upporl: National in.,~thutes of Heallh and Nalional InslllUle of Child Ilaallh and Iluman.D~velopmcnt Population ~ese~'ch ~entcy From tha Division of Neurosclence, Oregon Reg[onll P/areal© Re~h Center, Beavc~lon. OR. DEVELOPING AND REGENERATINO SYSTEMS AS MODEI~ FOR TROPIIIC EFFECTS OF ACTII NEUROPEPTIDES Neurons undergoing drastic melabolic changes, such ~ IX:l'~pheral nerve I~'CEV- c~ng From nerve trauma, central neurons in tissue culture, nigmstrlat~t n~urons 161
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lowing chemical lesions, or neuronal circuitry in Ihe dynamic process oF develop- meat. are plasl|c enough to serve •s models f.or the stu(ly ol" II~e neurotmphic effects of the mclannconins. It musl he kep~ in mind, however, th-',t one is capturing nnly • small ~vindc)w in lime and place in which tissue susceplihilhy is manirc,~led. This provides us wilh • strong Jm~x:lus Io dctcnltinc Ihe underlying nmk'cular encchani~s which decide such r~epdvlty, for i! is indeed Ihls ~ttem hi" resrxmslvcness Ihat rcg- ulalcS the preci.~ chronologlcal order hi" dcvch)p,ncnt and regencralhm in you,; system, nor- Strand. F. l_, Alves, S. F... Antonawich. F. J., Lee. S. ]., I.ce. T. S.. ond Zucc,'~clli. L.A. . In: Palomo. T.. Archer. T~ and Beninger. J. (Eds.): Sir•logics for Studying Brain Disorders. Vol. 2. Schizophrenia, Movemenl Disorde~ and Age Related Cognitive Disorders. Fan'and Press. London. pp. ~89-4U7. 1094. Olhcr supporl: Biomeasur¢ Inc. From the Biology Dep•rtmenl •nd COoler For Neural Sciences. New York Universily, Washinglon Square, NY. DEVELOPMENTAL REGULATION OF (~-FETOPROTEIN EXPRESSION IN INTESTINAL EPITHELIAl. CELL3 OF TRANSGENIC MICE The a-rcloprotcin (AFP) gene is transcribed in mosl epilhelial cells lining Ihc fetal mou~ small talc•line, but Ir~nseriplion pc~isls in cmly a suhsel of enlerncno docrlne cells RpRSenting less Ihan I'~ of. Ihe Iolal intcsllnal epilhclial cells in Ihe • dull. The decrease in AFP expression •leer hh'lh is mediated in p',n by a rcpre,c,;or elemenl lying belwcen -838 and -250 bp of Ihe AFP gone. Delcli~m of this element from AFP mlnlgene constructs resulls in high.level minigene expression in the inteslines of adult trans~enic mice. Although high levels of AFP minicene RNA expressed, Ihc felal patl~'n of expression is nol maintained upon deletion ol" repro•sot element. Instead. Ihe number of" cells in which Ihe minlgcne is expressed increases from less Ihan 1% to •pproximately 10~ of. the ephheli:~l cells in the adult small tale•line, and includes the majority of the goblel cells in addition Io enterncnducrine cells. In contrasl, the pattern of" AI=P mlni~eon expression in the enterocytes is unaffected by deletion of Ihe repros•or clement and cominues In decrease in the neonale. Thcs~ studies indlca~c Ihal the identified AFP rcprcssor is • clive specifically in gohlc! cells. The decrease in AFP expression in Ihe enterocytcs may Ix: merit•led by • ~parale cis-~ctin¢ element Ih~ is contained in Ihe AFP mini. gone conslmcl. Allemalivcly, it is possible thai malure enterocytes lack some of" posilive factors required for inillalion a.d maintenance of. mini~ene transcription in the absence'of" Ih¢ identified negalive element. Cirilln,. L A.. Emerson, ]. A.. Vacher. ].. and l"yner, A. !.. Developmental BiolnBy 161(:395-405, 1995. OIh,'r suplmct: National In•tholes of" lie•lib. 162 From Ihe Dep,,rlment of. Biology. Carlel~'n Colle+e, Norlhrield. MN, I.R.C.M., Monlre,al. Quebec, C-had•, ~nd Departmc.-.l of. Genelics, Univer;hy of Illino|s College o1" Medicine, Chicago. IL. KB ENI lANCER-BINDING COMPLEXES TllAT DO NOT CONTAIN NF-KB ARE DEVELOPMENTALLY REGULATED IN MAMMALIAN BRAIN DNA.bindin~ complexes which are lempozally regulaled in developing ral brain he, re been identified by Iheir ability to inter•el with the KB enhancer sequence in eleclrophm¢lic mobillty-shift assays. T~s~ ,:emplexes. rcl'en~d Io as devdoplnp hrnin facto~ (DBFs) I and 2. arc •bond•n! ~n nuclear extracts from developing rat brain through posln,l,,I day 2 and decline to ,,~;ldy undclectahle levels by day ?. DBFs were nol detected in extra, Is f.rom euhured (:ell lines o¢ in tissues than the developing br•in. The highest level or DBF DNA-hindln¢ Ktivily observed in developing corlex, ond the Iowesl in cerchellum. In UV cmsslinking experimenls, ,, lnlx:led KB oligonuclcolid¢ pmhe crosslinl~ed I IO- and I IS.kD- ~ reins trrom DBF complexes. DBFs am likely to ~ involved in Ihe re~ulalion of. Iran. scriptional events which t-',k© place during br:,tn developmenl. Cmllcy. K. nnd ~/ermn, |. M. Proceedings or the N~lional Academy ot Sciences 91"..190-.'194. Jnnu~ry 1994. Olhcr supporl: National lnstilules or Hcallh. American Cnncer Soci¢ly. and H. N. aml Frances C. Berger Found•lion. Frnm Ihc Molecular Biology and Vimln~.: L~hor•to,/. The S,,Ik lnslilure. Diego, CA. V. Epidemiology POLYMORPIIISM OF GLUTATHIONE $-TRANSFERASE M I AND LUNG CANCER RISK AMONG AFRICAN-AMERICANS AND CAUCASIANS IN LOS ANGELES COUNTY, CALIFORNIA ilackgrc~und: Glulalhio~c S.Ir~nsrerase MI (GSTMI) is aclive in Ihe dcloxica. lion of• number of carcinogens. Including polymomsti¢ hydrocm'bons, snch ~ IhOSe present in cigarette smoke. In about 3(Y~, -5~.% of indlvldonls, dependln$ on the elh. nic gr~mp, there is a virlual absence of. O$ :'MI enzyme activity duc IO delelion of bolh copies of Ihe GSTMI gent (GSTMI m,ll gcnol),pc}. ~'his genclie polymor- phism of the GSTMI gone locus has been prul,osgd ~ I risk factor for lung e=ncer. Ilowcver. results across studies -,re i~consix~en~ Purist: We condoc~d • ca~..¢on. Irol sludy of p.atlcnts w|lh incldcnl lung car~er and population comrol suhj~ls'to 163
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C) examine the as,~ocialion between homozygous deletit)n o1" Ihe C,s'rM I gent and I~g ~c~ risk amon~ A~can.Am~c~s ~od Ca~asians. Af~zhz~l.~: At 35 hospitals in ~s Angeles County, Califomla. we identified patients with a first diagnosis of lung c~ccr ~lwacn Sc~lem~ I. I~0. and Jan~a~ 6. 1994. Of li~e 8~9 ~lenli~lly ell- gable case patients. 207 had died by the dine their physici=.s h:~ ~eceived ~ur request ~nr ~issiun ~o contact them. Wc enmll~ 3~6 clipihlc case patients (167 A[rican-Amer~ans and 189 ~ucasians) and 731 eligihlc contrnl snbjects A[rican-Amcricans and 473 Caucasians. all rcsidcms n[ Los Angeles County). SampTes or white bl~ cell DNA su~cient [or dclc~in~i~ of Ihe G~ I gcno- ty~ by ~ pnlymcra;c chain rcacli~-hased a~ay were .htnlncd ~r~ 342 case ~en¢. in lolal and a[ler Slralificalian by a humor o[ ~levanl chataclcfislics, were ¢slimal~ by IogislJc rcgr¢~i~ analysis. Rtsull,¢: F~e ~alJCIIl~ wilh all hmg canc¢~ tom.ned, Ihe G~l null gc~ly~ w=~ ~s=¢ial~l wilh ;.= OR of 1.2~) (9~% CI = 0.94-1.77~. ~le OR w~ similar =m~& African-Americans (OR 1.20; 95 0.72-2.~) and Cau=si~ns (OR = I.~7; 9~% Ci = O.P1-2,06). 11~c a¢~¢~iali~ strongest foe squamous ¢efi ~¢inoma (OR = 1.57; 95% CI = (U)~-2.6]). obse~ed an OR or 1.77 (95~ CI = I.I 1-2.82) for I~ GSTM I null gcm~ty~ lion amon~ heavier smokc~ (OR = O.~; 9~ CI ~ 0.56-1A4). C, mchl;i,.ex: d~a do not sup~ a substanlJal ass~ial;~ MlWeen h~ozygous dclcti~ GSTMI gone and Ihe risk of lung ~n~r overall in Ibis ~Jpulolion, Ilnwcvcr, our data do su;g~l an clcvalcd risk r~ lighlcr smo~c~ with Ihi~ g~noly~, lmpllc,~i.ns: ~e~usc the ~wcr e[ our analy~s within SIrdlD of li[cllmc sin.king history was lim- ited. las&er studies will ~ n~ed to confi~ the~ ~d~. S. J.. ~aly. A. K.. C~. ]~ Nav/dl. W. C. ~ntcr, C. I~ and Idle, J. Journal o~the National C~ccr Institute B7(16):1246-1~3. August 16. 1995. Ot~r sup~: Stale of Clti[omil Tobacc~Rclatcd Disease Research Program. C~lifmnia Public lleahh F~nd~d~, H21ional Cance~ In~tltote. Institu~eso[ Heolch. From the Division of Occupational and Environmental Medicine. Division Biostalistics, ~anmenl or Preventive Medicine, Unlvcrs;ly of ~omhcm Calirnrnia School of Medicine, Los Angeles. CA. and Department ~f Pharm=col.gic~l Sciences, Unive~ily of Ncw~sde M~,I Sch~l. UK. Vl. Gene~ies SITE-DIRECTED MUTANTS OF THE ~ SUIIUNIT OF PROTEIN KINASE CK2 DEMONSTRATE TIIE IMPORTANT ROLE OF PROohl] The following amino acids of the X.ennpn~ tueL'i.~ [J subunit of pcotcin kanaka CK2 (casein kinase 2) were chanced to alaninc: Prt)-SK (p..,}; Asp-.~9 and flu-N) IM and flu 61 (/~,,e.,,~'); His IS1-153 (.~_..,~.}. The llsl 37 mm~o ~ids of I~ ~ar- "~xyl ~d were deleted (~,,~.). Slimulall~ o[ CK2e ~lal~ sub.it ~lJvily was measured wilh casein as subsirale and the (ollowing selalivc activilics were simil~ Io ~ in ~ducin¢ ~2¢ binding Io r~ulls indicate Ibal ~lh P~-58 and I~ ~r,~nding acidec clusler p~ay ~,¢s m dare,in& lhe a~ivad~ o~ CK2a and that inlc~Cllop with IIinrichs. M. V.. Galiea. M.. All~de. C. C.. Olher sup~: lnlem~li~a} Centre ~or Genetic From lhe ~amcnlo ~ Bi~imica. F~ule~d de M~ici~. and ~iologia. Facullad ~ Cicnci~. Univc~idad dc A$CARI$ AND CAENORHABDITI$: COMP'.~MENTARY ORGANISMS FOR STUDYING GERMLINE DETERMINA'ilOb~ As an approach IO unde~Slandiflg how tile ;¢nn|il~ is clelenn;ned in nematodes, we have cloned and have begun Io characluiz¢ two families or genes in Ihc lode, lhe Rile and cyclin rents. These genes are polenllal eomponcnla germltne-specil~c P.granMcs. A role for g~h pm~cins in ~ermline speeific~dun has become more likely with ore' I~ccnl Ending, .sktg ~mibodin.to lib. ¢h.~t leln(s) sc~gsle with the gem'dine pReursor ¢¢11s in the C. erel~ans emr~jo, we alSO cxpccl thai lhe Y UTRs of each gent in these {'amil[es, which appear Io have mold- ple reRulalory molil's, may posl.transcripl;onally ¢oordinal© lheir expl'cssion in development. The avallabilily of unlimilcd st-,¢e- -nd llssue-specifi¢ RNAs from A~cnrls could allow us Io isolale olhcr RNAs yel {o have bc~n identified lhat may be bound by glh zlnc fingers In lhe P-granule'tcasm¢ chest." Roussell. D. L.. Gruldl. M. E., Kreutz~, M. A., Richards,.[. P., and Benlle|l, K. [.. In: 9ooth~'oyd. J. C. and Komunieekl. R. teals.): Molecular Approaches to P~rasitology. Wiley-L{sso Inc., pp. 321J40, 1995. O~her support: NallonaI Sci~c¢ Foundallon. F~om the D~parlm,'nl or Molecular Microbiology and Immunolor~', School dr Medlcinc. Onivcrsily or Missouri. Columbia, Me. 165
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C-) EPITOPE-Tag VECI'ORS FOR EUKARYOTIc PROTfilN PRODUCTION We report on the C~l~cli~ ~d u~ or two cnkarynl~c cxp~sinn VCCl~rs which add wcll-ch~clc~z~ epilo~ la~s to the N I=~ini of pr~eins. ~c ulilily these ~cto~ is demonsl~tcd r~ del~ling I~ cxp~ssi~ or a variety of ~o~cins. the ~Jlion or lh,e epko~ la~ ~ in some cases ohviale the need to cJ~ anl;scra to ~ch individual ~lein. thc~ v~lo~ provide a f~Jlc means ~th m~it~ prolcin cxp~ssion ~d Io pufi~y such ex~csscd proteins. Sells. M. A. ~d Chern~, 3. Gent 152:187-189. I~. O~er supra: Hali~al lnslhut~ of Fr~ the Fox ~a~ Caner Center. ~iladel~hia. PA. MURINE CI IROMOSOMAL LOC.~TION OF F.I(;I IT MEMBERS OF TI IF. NEPATOCYTE NUCLEAR FACTOR 3/FORK llEAD WINGED IIELIX FAMILY OFTRANSCRIPTION FACTORS A 100-amino-acld DNA-binding multi', known as the winged helix, w~ first IdenliF~,d in the mammalian hepa~oc~te nuclear factor-3 (HNF-3) and DrosnpIdlo I'ork head family o1" Iranscrlplion I'aclors. Subsequendy. more than 40 differenl thai contain the winged hcllx multI" have been identified. In Ihe sludies described here, we have dclecmlned the muting chromosomal location ot'eigh! members of Ihis gene family, flF}l-I, HFH-3. HFH-4. I|FH-$. HFil.6. IIFll-8. DF-I. and BF-2. by thin,specific backctos$ analysis. These genes, dcslgnat©d I INF-3 fork head homolog I (llJl~l). IlJh3. IIJ&l. HjTd, llj'kE, lug&, 11~9. and IIj'hlO. respCCllvel.y, mapped Io different mouse autosomes and are thus well dispersed throughoul the mouse genuine. Based on this mapping information, we predict the chremosomal location of these genes in humans and discuss the potentlal of these genes as canal;dales unc|oned mouse mulations. Avra~am, K. B.. Fletcher. C. Overdier, D. G.. Cicvidcnce. D. E.. La;. E.. Costa, R. H., Jenkim,. N. A.. and Colxdand. N. G. Genomlcs 25:385-393. 1995. Other snpporl: Nulio~al Cancer Inslilulc, American Cancer Soclc~y. National Instilules of Hcahh and Nallonal |nstitule ol'Geneml Medical Sclcnc~. From the Mammalian Genetics Lahorslo~/. ABL-Basic Research Program, NCI- Frederick Cancer Reseaxch and Devcloixn~nt Center. Frederick, MD, Dcpazlmenl of Biochemlst~, College of Medicine. The Univcrsily of Illinois. Chicago. IL. and Division of Endocrinology and Cell Biology and Gcncllcs Program. Memorial $1oa~-Kcltedng Cancer Center, New York, GENETIC ANALYSIS OF GROWTH INHIBITION BY GAL4.1xB.~ IN .T/~CCIIAROMYCES CEREVI.TIA£ IKB proteins bind to and regulale Rel/~IF.KE Inmseriplio~ faClOre. W¢ showed previously thai a f~L~ion protein (GALA.F40) comainlnI the DNA-bindinl domHn of GAL4 and sequences of chicken IKB-,,(p40) inhibits |rowlh in the Sac¢llarnm.yres rtrtt,i.tin~. We now show thai pdO must bc Ix:)und IO DNA to inhlhll ye-',~t grnwth, p40 protclos, bound to DNA ehher as GALA or LEXA luslo~ pmlelns. inhihil yeasl growth. In conlrasl, p40 prolcins Ihal cannot bind to DNA. such as full- Ionglh p40. a GAL4-1KB Fusion ~olein ¢ootaining• mulaot GALA DNA-bieding domain. -~nd a fusion proleln (GAD-p40) ¢Ofllaining the Imnscrlpllonsl aclivalion domain or GALA fused Io p40. each failed Io Inhibit cell gnswlh. As wilh GALA- VPI6. GALA-p40 needs a funclional cellular ADA2 gene IO each il~ |rowlh. inhihitory'effccl in S. ttlt~J~ot. Using • high-copy supl~esslon Slllletjt. we h~ve isolalcd three .T. ¢¢rtt'istn¢ genes thai reslo¢~ normal growth In yeasl expressing GALd-p40 or LEXA-p40. We have lenncd these rescuing genes collectively ~s $1K genes, for'Suppressors of l~B." Exp~ssio~ of Ihe $1K genes specifically supi~'sscs the growth-lnhlhitory activity o!" GAL4-pdO and LEXA-p40 beemlse $1K genc cxpressinn canna! Mock (3ALA-VPI6.mu:haled ~rowth inhihltlnn in $. ,TIKI enemies a novel I~'oleln Ihat coelalr, s • COOII.termlnal IcpeM thai has been found in many microlubulc-hinding prot,:t~:s. $1K2 encodes NIl~.tcnnlnal aCelyl. transfc~se, and $1K~ encodes the year4 ribosomal $4 protein. Hone Ol" the $1K Icing hinds direc¢ly to pdO sequences in v~lm, suggesting that Ihe $1K pmleins are I~kcly to ncl ,h~wnstrcnm ol" the di~cl lmlnt or gn~Wlh Inhthillm~ hy GALd-1~4|I. O~r resulls may be u.,.cful for devising slra~eg|cs for idenlifyiog vcrtebrale inhlbhors I~B proleins ~nd of other proteins tha¢ inhibit growth in $. ¢~r~'isiu~. Mnrln. P..f.. Downs../. A.. Snedgrass, A. M., and Gilmore, T, D, Cell Growth & Differentiation 6:789-'/98,.I~1y 1995. Other support: Massachusefls DJvls[on of the Amerie~m Cr, neer Snciety, and the Children's Lcukemla Research Association, Inc. From the I~anmenl of Biology, Bnslon University. Boston. MA. ISOLATION OF A cDNA ENCODING A NO'gEL CHICKEN CHEMOKINE IIOMOLOGOUS TO MAMMALIAN MACROPIIAGE INFLAMMATORY PROTEIN- I [3 A eDNA encoding a novel chicken chemokine homologous [o mammalian chemokine macmphage inllammalory protein I1~ (MIP-1~3) was isolaled and chcuae. lerlsed. The cDNA encndcs u pint©in whh:h is ?.~-R0t~ homolo~m~s Io human and mouse MIP-II3. All conserved u.mino ncids charaelcrislic of Ihe mammalian chemoklne family have been evolulioearily preserved in chicken MIP-Ip. suggesl- ing similar pmrein foldln8 patlems and functionsl propellics. Pelrenko, O., lschenko. I.. and Enrlcllo, P. J, • 167
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C) Gent 16~.'30~-306. 1995. O,her supporl: National Institut~ or ~eahh. From Ihe Depa~mcnl or Microbiology, Stale Universily or New York if Slony Or~k. Slony Br~k, NY. . ISOLAT/ON OF MICRODISSECTION CLONES FROM RAT CIIROblOSOME 10 We construcled an RNOI0 specllic microlibr~T and isolated DNA markers of ral C~r I0. We ~ cun~nt|y continuing to/solale as many additional m~kers from Ihls mierolibrary as possible, since it could be useful in genetic mapping, not only I'or the Eker rat genc. bul also olher disease genes located on rat Chr 10. Kobayashi, T., K'awaguchi. T., Kishino, T., Matsumo¢o, N., N i . • • Levan, G., Klinga-Lcvan, K.. and Hino, ~). i kawa. N.. Mar,, M., Mammalian Ganome 6.'216-218. 1995. Olher supporh Minlsm/of Educilion. Science, and Culture or.rapan. FTroOm Ihe Depanmenls of Expe~menta/Pathology and Pathology, Cancer Institute, J~yo, Japan. Depar~men! of Human Genetics, Nagasaki University School of Medicine. Nagasaki. Japan, Inslilule or Laborstor7 Animals Fac ~_y.o_!o unp©...i,y, gyoto. S.p,n. and Other,men, U~reDorg, UOl¢.Oorg. Sweden ..... v,~,,ctlcs, university el VII. Immunology and A daptive Mechanisms A~TIVATION OFSRC-RELATED TYRO$1NE KINASES BY IL-3 The activalion o1" .~rc-rel~led lylmine kinases following 11.3 stimulation was examined in 32Dcl3 ceils Three src.relatcd tyro~inc ki~sscs wcrc a ing IL-3 stimulal" • ~,,~ ,t,.i. ~a, ......... ctivatcd follow- ien.~ ......... ,, ,,,,,. ~ucu ce~s Were lransfecled with retroviral vcclors exl:~.sslng each or Ihese Ihrcc klnases and independent elene v !ng c.lch klnase were isolated In cells over .... : ....... s o crc~pres$. • " • " --~-~-~,,& eimerlyn or twk, IL-3 slimu- ~ateo a rap~d ,ncrease m ca~alyllc acllvily, which remained elevated longer compared with the kinelics observed in pa~nlal 32Dcl3 Cells, An increase in the number of lYsoslne-phosphorylated proteins ;n Ihe presence and absence of IL- i " was obsen, ed in cells overex ressine~v k 1. "¢' ..... _ ] sl mule.hen P _lynor..cx. ',"-~,fectmn of 32Dcl3 cells wdh a retroviral vector cncndlng I.Vn also resulted in an elevated level or klnasc activity, " although Ihe increase was not as dramalle as Ihal observed wilh j'yn or Consistenl with observations in parenlal 32Dc1~ cell~, a high basal level or kinase ~ctlvlty was observed in unslimulsted [yn.l~sfec~ed cells and IL-3 slimuls- lion resulted in an approximate thceefold incre~e in klnase activity. Ovet~xp~ssion of c-~rc in 32Del3 did n~t result in IL-3- stimulated activation of c'-src, Indic,~tln| spccif'¢ity for fyn. hck, and I.yn. While the overexpression ol'[yn, hal:, or I.vn In 32Dcl3 cells resulted in increased k|nase activity taxi IL,.3 stimulated ~msine p/ms. phmylalion, it did onl render the cells mo¢c sensitive to IL,-3. These re,ha the! in addldon to d~e ]AK2 tyrosine kinasc..~rc.relnted kin:w, es muy pl,,y n signifi- cant role in signal transduetion by cytokine receplors. Anderson, S. M, and ]orgensen, B. The/oumal el'Immunology 155:1660-1670, 1995, Giber suppo~l: Hew York Community Trust and the Hat;anal Cancer Institute.. From the Department of Pathology. University of Colorado Health Science Center, Denver, CO. COORDINATE AND COOl?ERA'lIVE ROLES FOR NF-AT AND AP-I IN REGULATION OFTHE MURINB IL,-4 The transcriplion I'aclor NF-AT plays ~ eiscutld role in Iha Inducible Iran. scription of several cytoklne genes during T cell ~Clivatioc'rhe distld NP-AT site the murinc !1-2 womotcr binds both NF-AT end AP-I prote~s, and,thus relm:sents a composite regulnto~ site that integrates Ca"'- and PKC-dependen¢ signaling path. ways in T cell activation. However. the Ind;v;dual coatn'bolious el" the NF-AT AP.I compooents to promoter activity via thls composltc site have not been resolved, owing to the absence of a clearly defined AP.I binding site. which, when mutated abolishes AP-I binding. We dcscrlbe hem an i~pIrenlly analogous NF. AT/AP- I composite site in the mudne IL-4 promoter, which ~ ~ mutated to lively block the reenshm~mt of each compo~¢nL We sbow that Ihe ¢oo~r~ive and ¢ooedinate Involvement oflx~h NF-AT and ?.P-I is neeessa~ for full acfivily of the NF-AWAP-i eomposhe she, and, ultimately, the e~llre !!.,.4 p~mo~er. Rooncy, J. W., Hoe~, T., and C, Ilmcher~ I.,. Immunity 2:473-4R3. May 1995. elect support: Tularik. Incoq~o~ted. From Ihe Deparlments of Genetics a~d Medicine, Haeva~d Medieal School, Tul,~dk, Incorpo~ed, Sooth Sun Fmnebco, CA, and Department or Cancer Biology, ! I.~rv~rd School o~ Public lignitE, Bos~o~, MA, 169
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C) TIlE PROXIMAL PROMOTER OFTtlE |L-4 GENE IS COMPOSED OF MULTIPLE ESSENTIAL REGUlaTORY SITF~ 3'IIAT BIND AT LEAST TWO DISTINCT FACTORS Immune responses to pathogens often lead to the generation of polarized T helper subsels designated Thl and Th2. Thl ceils, characterized by the production of IL-2 and IFN--f, stimulate cellular immune responses important for protection against intracellulzr pathogens. In contrast, Th2 ¢¢11s. which produce IL-4. am polcnt stimulators of B cells and stimulale proteclinn against ¢xtracellular pathogens. IL-4 has also emerged as • key cytokine in T cell differentiation since il has bee• show• to dirccl Ih¢ dev¢lopmenl of naive T oclls toward a Th2 phenolype. Recent sludics have provided insights into Ihe transcriptional regulation of IL-4, including the ;den- tification of multiple binding sites for a ~bunh of the lL-2 transcription factor NF- AT. In IhJs study we dn~ribe Ihe characlerizalion of an csscnl;al rcginn of Ih¢ IL-4 promoter located immediately upstream of Ih¢ TATA clement. Iligh-rcxnlulion mutagenesis of this 33-bp region revealed multiple sites indispensable fro' inducible IL-4 transcription. Included in this region are nverlappi.g binding sites for the ¢yclo~porin A-sensitlve factor NF-ATp and a novel constitutively expressed factor designated PCC. An additional sequence adjacent to the TATA element i.~ shown to be critical for IL-4 transcription i• Th2 cells. I Io~ge. M. R~ Ronney. J. W.. and Glimeher. I, IL The Journal of Immunology 1:~4:6397-6405. 1995. From the Department of Cancer Biology. Ilarvard School of Public llealth and Depzrlments of Genctlcs and Medicine. Ilarvard Medical School. Boston. MA. RECOMBINANT SOLUBLE CDI4 PREVENTS MORTALITY IN MICE TREATED WITH ENDOTOXIN (LIPOPOLYSACCI IARI DE) Endotoxic shock is a life-threatening condition mediated by cymkincs rclca~d after exposure m bacterial LPS/end0toai•. Activallon of mrmncytes and neutmphils by the binding of LP5 to the membrane receptor. CDI4. plays a key role in this response. Furthermore, a soluble form of the CDI4 receptor enhanc¢~ the endothelial cell response ¢o LiU3. We show hem Ihaz de,pile the a~onist effects of soluble CDI4 on the endothelial cell response to LPS. ~ecomhinant soluble CDI4 is able to prelect mice from loS-induced lethality. This pmteclion apfx:arz to be associated with the inhibition of TNF-= release, These results suggest that the soluble CDI4 receptor may represent • new form of therapy for endotoxi¢ shock in humans. llzziot. A.. Rongo G. W.. Lin. X.-Y.. Silver. J.. and Goyerl. S. M. The Journal of Immunology 154:6529-6532. 1995, Other supporl: National Institute~ of Health and the American Ileal1 Association. New York Slate Affiliate. From Ihe Division of Mnlceular Medicine. No~lh Shore University I Iospital/Cnmell Universily Medical College. Manha~et. NY. 170 CDI4-1NDEPENDENT RESPONSES TO LPS REQUIRE A SERUM FACTOR . THAT IS ABSENT FROM NEONA'llES Monocytes/maerophages and neutrophih can =~,=pond to endo~oain via a high- affinity receptor (CD 14), requiring low h,/els ef LPS (<1 ng/ml) It well tz tlwough another pathway(s) thai requires high levels of LPS (>10 ng/ml). Both pathways result in the sec~lion of high levels of cylokines, such =s TNF-a. and Ihe up-regain- lion of various other cffcctor molecules. To further define Ihe a~tivatlon of cells by LPS via • palhway Iha! does no~ involve CDI4. we ha~© used an exp~mental model that dlstinguishe.~ CDI4-dependenl from r...~)|4.|ndependent ~'sponsea ming ~lumt- ing •mounts ol an =ntJ-CD 14 Ab to block Ihe COI4-dependent response. Analysis of the ability of varic, es individuals Io resp~..;J to LPS via l~h the CDI4<k'pend~t and Cl)14-1ndel~ndent pathways shows tha~ adults can respond via both pathways; furlhennorc, in Ihe presence of 100 ng of LPS/ml. the primary ~pt~se i| CDI4 imle~emlent. In cnnlmsl Io the rcspralse by •dulls. •co•ales can (rely I~pt~d via CDI4 dependent pathway. Fmtbe~ analysis has shown that the CDI4-|ndepemk.~ pathway requires a nem-CDI4 plasma pmlein lax'sent in aduh plauna Ih-I is eilher missing or nonfunctional in neonate (cord) plasma. Cohen. U., llaziol. A.. She•, D. R.. Lin. X.. Sia. C.. Hmper. R.. Silver. J.. and ~oyert, S. M. The ]ournal of Immunology 155:5331'-5342. 1995. Other support: National I•stitules of Health and Amerieaa Heart A~ocia~ioo. New York State Aff~llale. From the Divisious of Nconatology. Dep.'ulment of Pediatrics. Molecular Medicine. Department of Medicine, and DepartmonI of OB/GYN. North Shore Un|ver=hy llospltal/'Cnmell University Medical Colic~e. Manha~,¢t. NY. FUNCTIONAL VAUDATION OF LIOAND MIMICRY BY ANTI-RECUR ANTIBODIES: STRUCTURAL AN D THERAPEUTIC IMPUCATIONS Several anti-receptor monoclonal antibodies have be,:n described Ihz~ compete with the ligand for receptor binding. The po~ibilily thai slrUClUmS within Ib~ hyper. variable or complemenl.qrity-detenn|ning reg|ons (CDR) of these anlibodlea might display similarity with Ihe receptor llgand hr~ been documented. Ihereby validating this imt~nnologi¢ approach In explore figand.receptor interactio~. Taub. P,.. and Geeene, M. Io Biochemistry 31:7431-7435, 1992. 171
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C) Other supporl: National Institutes o~" lleateh, Nation~t Eye lnstltute. Am~rlean Cancer Society. and Mackey Olaritohle Trust. From Ihe Dcpartmcnt o1" Genetics and Howard Ilughcs Medical Inslilulc and Deparlmenl ot" Palhology. University of Pennsylvania School of Medicine. Philadelphia. PA. INVF.TrlGATION OF NEUTROPI III. SIGNAl. TRANSI)UC. nON USING A SPI~CII:IC INI IlIIITI')R OF PI II')SIq IATII)YI.INOSlTOI. 3-KINASI.~ Neulrophils cofllain a muhlcompone~l NADPII oxid~sc system Ihat is involved in Ihe produclion of microhlcldal osldanL~. Slimulalin~ of human nculmphils wilh Ihe Pepdde FMLP ~livat~s this re~pir~lory borsl enzyme Io produce superoxide and also has b~cn shown to result in •city•lion of phnsphalldyllmJsilnl (Pldlns) 3-kinase. Trcatmenl of human neulmphils with 2-(4-morphollnylJ-g.phenyl-411. I-lcnznpyran- 4.one (LY294002), • potent and slrccific inhibitor of Pldlns 3-ki.'mse, rcsulled in complete inhibition of Pldlns J-kin•st activily as well as in inhibition ol'supcmxide production in FMLP<realed neulrophils in suspension; FMLP-slimulaled axial•hi production in adherent cells was also ebolished. Treatment of human neulmphils with PMA rcsullcd in prodUCliOn or snperoxide without acdvalion of l~dlns 3- kin•st; LY294002 did not block SUl:~Joxid¢ prodocliou in ncutrophils exposed Io PMA. In •dditlon. LY294002 did not inhibit ¢¢llfrce NADPII oaldase activation. CDI Ib-dependcnt adhesion, actln Ix)lymeriz~lion in response In FMLP. or FMLP- induced calcium flux. These results suggest Ihal lilt signal Iransducllon palhway or the FMLP-receptor involves aclivzden of Pldlns 3-ki~a.,¢. w.~;ch is requlrcd I'nr sub. scquem SUl~-roxide production induced by the chemotacdc pcplidc. Furthermore. Pldlns 3-kina~e may be Iocaled directly upslream of protein kln-',se C or olher l~oteln 14oases, which in lure activate the NADPII oxidase system. Vl~hns, C. J.o Mailer, W. F., Brown, R. P., Traynor-Kaplan, A. F, lleyworlh, p. (;,, Prossnilz, E. R., Yen R. D., Marder, P~ Schclm, J. A., Rolhru~,;, K. J., Serlin, I]. and Simpson, P. J. The Journal of Immunology 154:2413.2422. 1095. Oth~r supporl: Nallonal Inslitoles o[ Health. From Ihe Cardiovascular Research, Lilly Research Laboratories. Ell Lilly and Company, Indianapolis, IN, Dcparlmenl of Medicine, Universily of CaliFornia al San Diego Medical Center. San Diego. CA, OepmmenlS of Molecular and Esperlmcntal Medicine and Immunology, The Scripps Research Insdtule, La Julia, CA, and Dcpanmenl of Biological Science.% OcPauw University, Grcenca~tle, IN. B LYMPHOCYTE RECOGNITION OF CY'i'OC'HROME C: HIGIIER FREQUENCY OF CELLS SPECIFIC FOR SELF VERSUS FOREIGN ANTIGEN EARLY IN THE IMMUNE RESPONSE AND V GENE USAGE IN TIlE RESPONSETO SELF ANTIGEN To study immunoglol~lin geno usage in Ihe anlibody response of mice to self anligen (Ag) mouse cytochmme c (cyt). B cell hybridomas w~rc i~epan~! from splcnic B cells of immunized BALB~c m|c¢ p~or to II~c onset or sO~allC mulal~OO. Lc. ~ days alter injecting ov•lbumin (OV.~-p~imcd mice with mon~e cyl co~pled OVA. Monoclonal antibodies (mAb} fro~: ,'.;I or Ihe ~¢veo I~mary hylxldomas we obtained were sensitive ¢o a single ~mino acid s~bslilulion from asimllc acid Io lamlc acid al Posilion 62 in mouse Cyl. Th!s is the specificily or Ihe vial m•jo~ly of B cells rr.~ponding Io mouse eyl as detennlned from assays of il cells Klivaled in splenic frogmenl co|lures. Six of the m;',b ~edv© I*rom the 19.1.2 J$5~ V. which L~ nl.~o slued in Ih¢ rcspo~s~ In o~ (I-°$) d~almo a~l 1h¢¢ or I1~'~ mAb dcl~vc from Ihc R9 V, gone, • member of Ihe Y.O4-1 fandly. The olber mAb derive distinct, altlK~gh s|milar,V, genes. Attcm;::s Io obl,,in hybddom~ ~'~linl~ primary (unmutated) mAh specific I'or cyt fo~|gr~ .o m;ce have been hampered by Ihe much lower frequency of II cells sespondinl,' em|y In I'omit~n cyt in con~p.'~.~o Io Ibe sell" rlALBIc mice B lymphocyles specific fo~' • :ingle epltol~ on I~lf Cyl •~ pl¢'~nl io higher'frequency Ihan B lymphocyles sperific for similar cpilope~ Go fo~il~ eyh Possible exphmallons for Ibis rcsull include biased exlxcsslon in Ihe B till Rix-noirc of the particular combination of V I~e~ea ¢ocodin¢ mo~xe ¢~jI-ap¢¢ific mAb posilive selection of d~veloping E lymphocylel by endogenous Minnerath. J. M.. Mueller. C. M.. BurGh. $.. and Jemmers~o, R. European ./unreal of Immunology 25:784-791. Other support: National Science Foundation. U.S. Public He•lib Sen, ice. and National Instilules of He,,llh. From the Deparlmenl of Microbiology, Un|ver~lty of Minnesota Medical School. Minneapolis. MN. A Till C'ELL'LINE (3E9.1) FROM RESISTANT A~ MICE INHIBriZ: INDUCTION OF MACROPHAOE PROCOAGULAbrr ACTIVITY IN VITRO AND PROTECT~ AGAINh"r MHV-3 MORTALITY IN VIVO Induclion of immul~e coagull~tl has bee~l Implic|ted in Ih© pllhogenesia of mnrlne hepatilis vires str•in 3 (MHV-3).|nduced l'ulminanl hepatic necrosis. Pmvinus work from our labotllOf~ h~s shown Ihal the induclion o1' activity (PCA) cormlzles wilh Ihc rcsistance/smceFl~illty to dise~ Rcombin-nl inlxed (RI) air, ins of mice. M~croFhagea from susceFllble, but am reslsiam, strains of mice expt'essed incp~scd le~©ls of i~A in response 1o MI[V-J stimulation. 1" lymphocyles, however, had z marked re~ulalmy role in Ihe final cxpr~ion o1' macrophagn'l~A. CD3" CD4" CDg" I)~mphecyles from RI 11-2 com-
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C) p-',dble suscelxlb~c mice were able to instruct macmphages from s,sceptihl¢ mice to ~.P2ress significantly augmented levels of ~A. w~re~s CD]" lymphm~,~s ~r,m RI comp;fib~e MtlV-]-immunized r~slan~ mice were =hie Io suppress induction of PCA. In Ihis prescnl study. T-cefl lines were derived (mm ~raini,~ lym~ n~cs f~ resisl~l ~] mice. which had ~cn tram,nixed wile MIIV.]. Aft T~cll li~s showed markc~ proli~eral~n m MIIV-~ and MIIV.JIIM which was major hist~mpalibi~i~y ~plc~ (MHC) r~lricled. All cell lin~s were CO3". or ~Eese were C~" and ~e was ~-. All orlhc C~" cell lines produced IL-2 ~(I ~wo pr~uced imcffcmn.y (l~.y). c~sisl=nl whh ~ ~ll ~lokin~ pmfi~=. c~ll lln~ (3E9.1) was able Io i~ibi~ tee inducl~n oF m~mph=ge ~A Ih~h C~" T-cell lln~ c~rc~d ~i~ Io intoned and su~cp~ih~e AX~ mice. ~sulls ~m~Ira~c ~hat ~c existent era ~I xu~pul=li~ of cells wi~ a Io~ c~¢1 on macro~ag~ ~A ind~li~ in MllV-3-inrccind mice con,throes lee rcsJsl~ce o~tEe A/] st~Jn o[ mice to MilV-3 inrcclion. Chung. S.. Go~czynski. R.. Cruz. B.. ~n~erote. R.. Skamenc. E.. Perlman. S.. ~ibowilz. ]~ Fun~. L.. Ro~,. M.. ~d ~vy. G. ~mmunology ~3:353-361. 1994. OIEer su~: Medical Re~h Cou~il of Canada. Amcrican Ilcan As;~iati~. ~d National lnstlmles o~ Fr~ the University or Tmomo. Toranla. Canada. Mnntrcal General Ilnxpital. Montreal. Que~c. Canna. Unive~il~ ~ Iowa. Iowa Cily. IA. and Uni~crsily Texas lithe Sc~nc~ Cent~. Ifoust~. TX. ALTERING THE,4uN11BODY REPERTOIRE VIA TRANSGENE IlOMOLOGOU$ RECOMBINATION: EVIDENCE FOR GLOBAL AND CLONE-AUTONOMOUS REGULATION OFANTIGEN-DRIVEN B CELL DIFFERF.NT~AT~ON Antibody Vw transgen~ conlaining small amo~nls of nalural 5" and 3" Nanking DNA un~'go nonr~:iprncal homologous racemE;nation with the endogenous ~ ml| ~ IOlly InnC11mlil. tlndergoing sOmlllc hypernmlallon and iS~ype clIss swilching. We hive used Ibis Rcombinallon pilhwly Io inlroduce • somili~olly mulcted vlrilbl¢ (Y} region wilh 111 ~JnUll/llly high affinily for lh¢ haplen p- IzopEen¥11~-~o~ma (Ars) inlo l~c pmimmune lalibody repertoire. ~ afi~nily of lhls V rcgim for A,s is lO0-fold higher Ibm any unmnlaled anl~-Aes ~lib~dy Enviously ch~rlcledzed. Expl~ssion of ihe Iransgene-~ncoded V region did n~l affect m~my Ispacts of in|ieen.drlven B cell differcnlilliOn, including som-lle hyp~nnulolio~, in eilhcr Ars-spe~i/~c Irlnsgcnc- ce endogenous V gent-expressing chines. Thus, lhc Rgulslion of lhes¢ processes Ippelra IO ~l'~le in a "global" fiL~hlon, in machanlsms Involved are im~rccp~ivo of lhe ¢alalive afflniliaS flit ~Inllgen of ~ilibodlas ~prassed by B cell clones pmlcipaling in lha immuo~ l~$ponse. In con- 174 Irasl. the selection or V mgimz mutants L--adlng to al]]nily ma'ur~lion mzd mc~ cell fo~zti~ was ~nd to ~ slmnily i~flu~ by t~ I~sicnk V ml~. ~t only in clones cx~ssing this V R~ion. ~ly~idom~ dcdvcd k~ I~ns~enc- I~ cndogc~us V r~n.axpressing ~mo~ cells we~ bobl~ at s~il~ fRq~s horn indlvldunl transgenic mice. ~ V A.Si~s expRsscd ~ by~d~tn~ in ~h or Ihcse groups ~d 2- In 30-¢old greater z~finily ~or Am I~n t~lr unmut~ted ~cur. sors, dcspltc the fact that the Iransgcn~cnc~ed ~ccu~ts had I~-f~ld hi~hcr affinily than their cndogc~s c~nlc~s. ~sc ¢sul~ s~w t~t t~ cdtct~ f~ entry into the ~mn~ c~m~l is ~taMi~cd ~ by I~ aWmity o[ a B ccll'~ V region relative to all other V tcgions e~prcssed durin~ the ~sc. but by the affinity of this V rcglm ~clativc Io il~ ~.~utaled ~u~. ~us. I~ dcvcl~cnt of B ~11 mcmn~ is Rgulaled in a "cl~.: ~l~m~" Vote. K. A. a~ Manser. T. Journal of Ex~rlmcntal Mcdlcine IRE2, .. ~gl. Jan9a~ Olhcr sup~: National Inslitut~ of H~ilh. From t~ ~parlmcnt or Micmhioloff and I~uado¢y ~ ~ ]¢ffc~ ~r Institute. ~oma~ Jcffc~on Medical College, ~dladclphia. PA. EFFECTS OF ANTI-CD4 ANTIBODY: ENI IANCEMENT OF LYMPII NODE PPD-MEMORY T CELL RESPONSE In vitro incubalions of CD4" T =lls with Imti-CD4 mennclonal amibody (Mab) dcmonstraled ~atumlitm binding and inhihi!cd helh PPD.slimulaled pmliremtk~ , delayed-type hypersensitivity (DTll) after passive transfer of these cells into ndve rals, In coml~rison, in vls'o ndmlnlstrdtinn of enti-CD, I M-.h inlo mls. 2 weeks al~r CFA immunizalion (PPD priming), salurated CD4 molecules e~prasscd on T lym- phocyte.,~ and depleted sienificanl Ix:rcen:lg,~ or CD4. T cells from blood, spleen. and lymph nodes, yet failed Io cause sigoificanl inhibllioa of PPD-slimulalcd DTil ear swelling. Only repeated in vis~ ad|ninislralion of anli-CD4 Mabs during Ih¢ CFA/PPD antigen priming caused signifieam decreases (36 - $'~) in PPD-~imu. lated DTH ear swelling. T cells isolated from the blood and spleen showed trot|nail- cant relative d~'g~ses in PPD-slimulited ptolif©nuion ah~r aad-CD4 trcalmcltls. contrasl. PPD-stlmulated proliferaden of T cells tsolmed from lee draining nodes a/let anti-CD4 Ireatmenls showed dramallc inc~.as~s in PPD ~activily. PPD stimulation of Tcells isolated from lee draining lymph node~ of and-CD4 Mab,4real. ed rats Ixoduced relaliv¢ phcno~yplc ch,,nl~es in CD4 ¢oexpmssin~s whh CD4$RC (inverse memoP~), CD49" (inflammatory Idhesion inteerin VLA-4). Ii~d OX40 (CD4" hl,'t.~4). Rpresenl~ive of m~mm~ CCq" T cell blasts wills Rdu¢¢d cap,Icily for endmhellal infiltrallon. These data demoasltata that depiction or CD.I' T cells by anti.CD4 Mab iojcellom, administered ¢ilher during m" -,f~" anligen priming. lively cnh,'mced raemory r~ponses in T lymldmC~eS to lee draining lymph nodes. Morrison, W. J., Kennedy. N. J., Offncr, H. nnd V~nb~k. A. A, • 175
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Cellular Immunology 163:106.112. 1995. OIhcr support: Depanmenl of Vctcrans Affairs. From Ihe Neuroimmunology Research ~raw~ Y¢lc~an • ". • Cenlcr. Porllznd OR ..a ,k. ~ ..... ~._ s Admlmslr~hon Medical ' ..... ,,,~ ucpm[mcnts Ol Pharmacology, Neurology, and Micmbiolo~y~mmunoio:y. Oreson Ilcallh Sciences Unlvcrshy, P~land, ECL-2 AND DCL.X: REGULATORY SWITCI IF.~; FOr( IoYMPIIOID DF.A'Ill AND .~I/RvIVAI. Th~ survival and dealh or lymphoid cells is under the cnntrol ore gcnetic pro- cram. Cell dCalh is aClivaled at dlFFerenl ~,laiics (d'dcvelnpmcnl aml xcrvcs to removo unnecessary and autoreac¢ive lymphocyle% as well as Io lim|l Ihe |ram,he respnnse. The survival oFcclls is regulalcd hy a sol oF gcncs lime oct as rcprc~snrs oF the cell dcs[b mcchan|sm, OF these, bcl-2 and Ix:l.x exhibit a striking pattern o[r regulation durin[~ lymphoid maturation and can inhibit several Forms of apo~ntotic cell death. Here, Gabriel Nuflcz and colleagues rcvlcw rcccnt devclopmems in Ihe field, panic- ula~ly focusing on the role oF the Dcl-2 and Bcl.x prmc;ns in regulating lymphoid de~,lh and survival. Nonez, ~. Merino. R.. Grillm, D.. and Gon~.alez-Gareia. M. [mmunoloZy Today I ~( 12):$82.J~7, 1994. OIher supporl: National lnslilu[os oF Ilcalth, American Cancer Socicly and The Sandoz SoCiely I'or Gcronlological Resca~b. " From the Ocpmlmmll of Pathology. "r~ OnivcrsJly o1" Michignn. Ann Arbor. MI. INDUCTION OF THE TRANSCRIPTION FACTORS NF-,cB. APo I AND NF-AT DURING B CELL STIMULATION THROUGH TIlE CD40 RECEFrOR To address ¢lemenls thai might uniquely characlenze CD40 mediate " 11o • ° Ihe clear ex ssm~ oF t ,. ,~.o..:~: ..... d s,gnal|ng. i~e hre ..... -.,,lama ~a~¢ors was evacuated following B ¢~11 slimulalio~ by CD40L and b~ anli.lg antibody. Cross-llnknd CD40~ was Found to induce nuclear ©alxesslon or NF.~B, AP-I and NF.AT with a dmc course ~d iotcn. sily similar Io Ihat prodoced by anli-lg. Exminalion or NF-~[! in more delail demonslraled Ihal the CEM0 medialcd expression o1" I)NA binding ~omplcx~s cone- lated whh induclion or rre~-aclivallng actlvlly which a in allained followine eross-linkin- r.rx~n _ . ~ miler levels lion r.,.,;.. :..,._.~__. ? .o~ ~ .,~_.,. and.alg?_ _D_e._.sl~te the mar~cd s;milarily in Iranscrl • "; .....u,~-,,on mgger~ mrough CIMU and slg, dilTcrencc.s in the inlroccllul~ 176 sigrmling pxlhv, mys utilized were apparem in did no~ zffeci C~O mediated indu~ e~ ~i~KB ~cn as in~ct~ by ~i-I~ was a~llshcd. ~se ~sul~s SUggest Ihat a 'final c~ ~lhway" ~ c~vc~e~ or (tanscriplion fa~ induction may exist fo~ two di~i~ indlvidually capable or lfig~e~g cell cyc:¢ ~og~i~. despile inl~cellul~r si~aIin; ~lhways lha[ di~ aloe fac~ ind~li~ w~ slmil~ r~ C~CL ~d ~i-lg e~ly ~. susie di~e~s in ex~sed NF-~6 and AP- I nuclco~icln ~plexes differences m~y play n ~e in d~c~inin~ the varianl effe~Is ~ B ~ells o[ sdmula. lion dwo.ph ll~se lwo r~. F~ncis. D. A.. Ka~s. J. G.. he. X.. ~n. R.. Inlcmai~nal Immunology ~{2):I~ 1-16l. Oll~r ~np~: An~rlcnn C~cer S~[cly nnd From the Depa~nl~ or Pal~gy. Med~ne ~d M~mb;ology. Evem Mm~al ~p~mcnl or Clinic~l Re~ch, B~l~ Unive~ity Medical Cc~r. ~. MA. ~d Dcpa~mcnl or ~inlogy. Ro~nsllcl Conl¢~, REGULATION OF CDI4 EXPRESSION DURINO MONOCYTIC DIFFERENTIATION INDUCED wml In,2S-DIHYDROXYVrrAMIN D3 COl4. a monocsjte/mocrop~lge ~-ptor for th~ Cmr~plex el' Li~ ~! Li~ ing protein, is I differenlialion marker Fo" lhe mo~qlr~m~q~h~|e Ilneale. We have analyzed Ihe regulation oF C'DI4 ex~,.cssion durln~ 1a,2~.dihydmxyvll~ln D3 (VitD~)-induced monocylic diffcrenlialk'.~. Usinl FAC~, Honhem blollin~, and nuclear run-on analyses, we demonsu~le lh.'-: ~he up-~gulalion of CDI4 during monoc3~ic cell malumtlon is re|elated mlnly at Ihe level or lone Ir~s,~p. lion, end Ihal new pcoic~n s).mhesls is i~-q~i~cd ForCDI4 induction. We have rcc~lly cloned Ihe CDI4 $' upstream sequence and ~::mo~slraled IIs tissue.specific pmmoler ac~ivily. Using stable Iramreclion of the mon~ U937 ceil I~ne whh a delelion mutants oF~e CDI4 $' upstr~m sequence coupled Io u ~'Fo~r i~e con- struct, we show thai hp -128 Io-?0 is Ihe crilicul eegion rot ~he andre:lion or con expression This ~ion contains two blaine sites for she Spl ~-~plion f~clo¢. A 3-bp mutation at the dislal Spi-b[ndin~ she ~ only ellm|nales Spl iMem¢lio~ also abolishes most of the V|tO~ induclion of CDI4 exlxession. Eleclro~ic mobility shill analysis does not detccl a dlrccl Iot~tlon oF flu: CDI4 db~d Spl- binding silt with the vilamin D3 reccplor and lls parlncr, the relionkJ These dale demonslrale llm VitO~ induces C'DI4 indirectly throvlh sonde inlem~edi, ary Foclor. and sup~geSl a cdtlcal role lo~ Spl in this Zh.nn~. D..E., Hclherin~lon. C. L, Gonz~lez. D. A., Chen. II.-M.. and Tenth, D, The ]oum.l or Immun~logy I$3:3276.-3~R4. 1994. 177
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Other suppo~l: U.~. Public Ifcahh $c~vlce. From the Division of }lcmamlogy/Oncology. Oeparlmenl or Medicine. Beth Israel Hospital and Ilarvard Medical School. Boston. MA. MONOCY'T~ CIIEMOA'ITRACTANT PROTEIN-I GENE IS EXPRESSED IN ACTIVATED NEUTROPIlILS AND Ri~TINOIC ACID-INI)UCED IIUMAN M YELOID CELL LINES We have u~;ed di{`fcmmial display Polymera~e chain rc.'ction In ,de,lily genes Ihat are upregulated af, er rellnoic acid (RA} trca,nen, of human myelohlaslic" ILL.60 cells. Three Of lee cON/~s cloned hybridized In RA.inducible IranseripIs on No.hem b|ols, one oF which was shown Io encode sequences {'or m.nocyte chcmoallr~Cla/}!/xnlc|n.I (MCP- I ). a rectally described cyt.k/nc thai is chemm~.lic /or monocytes hul nn! For neutrophiL,~. Nuclear run-on analysis indic•led thai dee upregulalion of" the MCP-I gent occurs al the IranscripIinnal level in 111.-6(I MCP-I Iranserlpt levels al.~ increased after RA treatment o/" Ihe NB4 acute promy- ¢lncyllc cell fine. MCP-I lean.ripe, wcru undoteclable in fr,',~hty isolated ncu- trophils by Nm'lhem analysis or reverse transcriptiou-polymera,~e chain rcacllon were readily detectable in neutmph|ls aft~" incubalion in media a137"C {'or 20 hm,rs. suggesling Iha! an actJvalion event can le~d Io MCP-I expression in neulrophils. Immunocylochemistry confirmed Ibe presence of MCP-I protein in aclivatcd neu- Irophils. This is Ihc first ~'epor~ Ihat Ihe MCP-I Bent is R^-responsivc in myeloid cell lines and is expressed in neutrophil.¢ MCP-I expRssioe by act!valed may play an imporlant role in atlr, lcllng monocyles Io lhe Silt o{` el.see damage or inf'¢ction. Bum. T. C.o Pelrovick, M. S.. Hoheus, S~ Rollins, B. J., and Tenth, D. G. Blood 84(8):2776-2783. October I$, 1994. Other ~upport: National InslilUles or Health, Nalional Cancer Ioslilute. and the Leukemia $ociely or America. From Ihc I|emalology/Oncology Division. Beth Israel I;c~nital. Department of Medicine. and Program in Cell and Developmeol Biology. Hot'yard Medical School, and the Depallmen! of Medicine, Dana-Father Concer Inslilute. Bns~ou. M/~. STRUCTURE. ASSEMBLY AND INTRACEI.LULAR TRANSPORT OF TIlE T CELL RECEPTOR FOR ANTIGEN The T cell rcccpIor/'or anligen (TCR) is respon,,~ihle/.or ehc rcco~ni|ion otr anti- ~¢n as,oct•led with the major histocompalibilily complex (MIle). The TCR expressed on th~ ~d'aee of T ceils is a~sceialed Wilh an invarJant struclure, CD3. CD.3 is assumed to be responsible for inlmedlullr signaling following occapency of the TCR by I~g,,nd. The TCR/CD3 complex cons,sis of six dilTerenl polypeOlldc~. and represents a uniquely complex mulllsutmnit assembly problem fro" Ibe cell. The cell copes with this prob}em by regulatin.~ abe inlracellular assembly of the complex. Within the endn plnsmic reliculum, the ncwly.synthesised chains assembtc into c(~npIcte sllln:lUre prior to Imnsport to Ih~ cell sud'ace. There are a relies of L~oforms o/. the reccplor involving difl'crenllal use o~ the TCR heRmdlmer (~-p o~ • y-E). ~-I'amily member, and CD~3 7 or ~ ¢haihs. These are presumably linked Io dlf. ferent TCR/.unctions. Assembly of Ihe "IC'R/~D.3 complex compelcs wilh speciF¢ de~mdalion of unassemh~cd potypo~lld¢.~. ,',"he f~le of Ibe ~¢ceptof dee, cads oe presence of sublle signals on individual c., .ins which delermlne pairing and ~ss~m. bly or degr;~d01ion. The T cell is thus a' '.. to select a completely asRmMed r,,lly funclional series of dL~tincl TCPJCD3 ¢o :. lexes for expression ot the cell sud'ace. Exley, M.. Terhorsl, C., and Wileman, T. scmln.~ in IMMUNOLOGY ~:2~3-297. From ti~c L~ho~tnry of Molecular Immunolo~.y, Dana Father Cancer Inslitule. Dcpanmcnl nf PalholoGy. Ilnn~.nl Mcd|cn! School. BeniGn, MA. MURINE B-CELL SUBSETS DEFINED fly CD23 The B-cell companmenl is composed o.r a complex mixture o¢ dislincl sub.Is, Three anatomic silts that are p~iculady .'~'.~ in B-cell sublx~poladoes are Ibe man'ow, spleen, and periloneal cavity. Ou~ ~a.tx~alo~ he,/.erred CD23, Ihe Ily IgE Fc receptor (FceRII), to be or yah.." In discrimin.ling a numMr or mur~ne B- cell subsets, especially when used in c~,'.d~:,lion with olher B-cell mad~c~. In Ihe bone re•now, the site o{` matumlion, the .~:esencc of CD23 ,patti',rally delineate-. mature recimulating B celL% where~s its ~',~.":¢e denotes the ma,urlng B-¢¢11 Popula- lions. In Ih¢ spleen. CD23 is cOnSlilUllVe:: ,=.qxessed on Ihe folllcul~ or m=nlk B cells and is absenl on the marginal zooe e:~ imm|tur~ B cells, These lacier Iwo pelm- lotions can be dislinguished by differing l~.~.'is ol" Ihe bes! slable antigen (II~A). In Ihe perkone-I cavily, B2 (cnavenlinnal) B cells display the CD23 antlpen whereas BI (COh.,.Isister) subsel does not. kdd[tlorml studies e~.:~mlnln8 the expression CD4.'I (Icukoslalin) have found Ihls anligen Io sa've as a reciprocal real'her Io CD2.'I in m~ny cases. In the boot manow, • ~ anti.CD4~ anlilx~ d¢lecls IhOS¢ B cells Ihat are CD2~ nee•live, and in the spleen -nd per|leGal c-Juily. Ihe majesty of CD.~.I ntis- alive BI cells m~ CD43 positive. TOeelh--'. Ihexe sludles demon~rale the utilily ~,f CD2.'1. CD43. HSA, nnd o~her Ir~r~ers IO .';scrim|n~te between B-cell sub.Is and allow {`or more precise purification and anal:~;is or ibese Popubdoos. ricer, c. G,. Kemp,]. D.. Wsld.,~hmldl. T. J. METIlODS: A CampaiGn to Mclhnds in En~,ymology 8:3-10, 1993. Other supper1: National In,climes of Heahh. From lhc [~par~ment or P-',lholo~y and Immunology Gmdu~e l~ogr=m, Univc~ily or Iowa College of Medicine. Iowa Cily. IA.
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c) TIlE EFFECT OFIN Vtl~O ILo7 DEPRIVATION ON T CELl. MATURATION A number of l~avinus sludies have sucgeSl~d a key folc for inlcdcukln 7 (lk7] in the maluralion of T lymphocytes. To belier a,;~ess Ihe funclion of IL-7 in lym- phop~iesis, we have deprived mice or IL-? in vA,o hy hmg-lcrm adminl.',lrnllon or a neulrnlixlng ami-lL.? antibody. In a previous rep~rt ((|r.'lhslein, K. II .... Waldschmidl, F, D. Finkclman. IL W. Iless, A. R. 'Alpcrl, N. E. Boianl, AT. Namen. and P..L Morri~sey. 1993. J. E~p. ll'/e#. 17R:2~7-2f~4). we used this system Io demonslrale Ihe critical role or IL-7 in FI cell macurafi.n. Al'lcr a brief` perkxl nr anti-11..-7 Irealmem, n~sl nf`lhe pro-B cells and -II ~d'the pen-i| "-,nd immalurc 13 cells ~ere depleted from Ibe bone marrow. In Ibe pre.:cm ml.m, we hove inj~cled anli-lL. 7 anlibody for periods of up Io 12 wk to delcrmine Ihe ef`f`ecl of`in I,in,~ IL-7 depdva- liOn on the Ihymus. The resulls demonslrale a >~.~% redu¢lion in Ihymi~ cellularily M'ler cxlcnded periods or nnlibody admlnlstralinn, r:xamin~inn of Ihymic CD4- and CD~- defined subsels revealed Ihal, on a pml~mlonal I~si.% Ihe CD4÷, COlt+ subsel w-',s mosl deplelcd. Ihe CD4 and CDlt single positive cells remained csscnllal- ly unchanged, and Ihe CD4-, CDg- comparlmenl aclunlly increased In --.~ll~ of the Ihymus. Further examlnaliun of lhe doable negalivc Ihymocyles demonstrated thai IL-7 deprivation did, indeed, dcplcle Ihe CDa-, CD4-, CD~- precursors, wilh e~l~ansiOn or ihis subscl being inlempled al Ibe CD44÷, CD25~- singe. The pmpr, r. llonal inerea.~e in Ihc CD4-, CDg. compmlmem was found to he due Io an accumu- lalion o1" CD3% T cell rcceplor a,/~ + double new,live T cells. Aeklilion.~l noalysis revealed Ihal anli-lL-7 Ireatmenl suppressed lbo oudilion/~lcclinn precis of` T cells, ns shown by a siBnificanl reduclion hi" single imsi~ive cells caprcssin~ CDI~9 ~nd heal slihle anl|gcn. Finally, Ibe effcels or IL-7 deprivation on lie Ihymu~'wcre I'ouod In be reversible, wilh a normal i~llem o1" Ihymic suh,~s relumin~ 4 wk al'ler ccss;i. lion of IrealmenL The pre~nl ~'esulls Ihus indic~lc n cenlr-,I ale f`~ 11.-7 in lhe ration of` thymie.d~rived T cells. Dhatia. S, K., Tygrell, L. T., Grabstein, K. I!., and Wald~hmidl, T. J. Journal of` ExpeHmenlal Medicine I B I: 1399.1409, April 199~. Olher supporl: Nalioeal lnslilules of` H~hh. From Ibe Dcpnrlmenl of` PalhO~0gy, Uoive.ity or Iowa ColleEn of` Medicine Cily, IA, and Immone~ Cotpo~alion, Seallle, WA. ' IMMOBILIZED ANTT-CD3 ANTIBODY ACTIVATES T CELL CLONES TO INDUCE THE PRODUCTION OF INTERSTITIAL COLLAGENASE. BUT NOT TISSUE INIIIBITOR OF METALLOPROTEINASES. IN MONOCYTIC TI IP- I CELLS AND DERMAL FIFIROBLAST~ In Ihi.; qudy we have |nvcstlgaled Whelh~r direcl cell In cell c~ml--.cl helween acllvmed paraformMdehyde-~cd T cell clones obtained from synovial llssue of pallCnlS wilh oslaoanhHliS {OA) or dleumsloid ailhrilis and Im'/~¢l monnoylic cells or dermal I'il~'oSl&~;Is influenced Ihe balance belwccn inlcrslhlal collagen:Jan and ils ape. cific inhibilor lissu¢ inhibhnr of` melalloproteina~c~ (TIMPI produced hy Ihc latlcr 180 cell lypes. PIIA/PMA-ucliVUled fixed T cell clones o¢ Ihe|r membranes slmn~ly induced Ihe prodoclion ol'collagcnnse belh in monceylic THP-I cells and in dermal fllxoblasls. In conlra~l, only low levels of ..~MP were induced in THP-I cells and no chnnge of` TIMP expression was obre~'cd lu fibroblz~ls .e a ~sull or slimulali~n wllh PIIA/PMA.~elivalcd T cells or T ce~! membranes. AnlI.CD.l.zclivatcd T cell clones stimulalcd Ihe product;on of colloids.nasa bolh in TIIP-I cells and fib~oblailS. whereas TIMP levels were nol influenced. ~ollagcnasc p~0ducllon ;11 TIIP-I cells induced by nnli-CD] ncllvaled T cell cl~,,:s was I) dcpendcm on the dose 04" anti. CD3 u~d Io sfimulnlc Ihc T cells. 2) inili-';tcd o~ly when CD~ was cross.linked, and 3} inhibiled when cy¢losporin A was inch.~.cd during T cell aCliv~ic, fl. O~r dais c~l- lecflvcly Indicalc that acfivaled T cells in :onlaC! wilh monceylic cells or rd~oblms may slier Ihe balance between imersti;i-'-! collagenase and its spceific inhibilor "lIMP. This selccllve |nduc~;on o1' n modifier prelate represen~live of maIHlt breol~- down as a resull of Inrgel cell interaclinn wilh m:llvaled T cells may be an iml~xlanl Fnclor in Ihe local process of lissuc dcst~.;~ion thai chnracledzes oslceanhrhls and rheumatoid anhrilis, I~il(enburg. A. M. M., Lnc~z, S.. Well:us, II. O., nod Dayer, Journal of" Immunology l Y4:265:Y-2667. 1795. O:hcr support: Swiss Nalionnl Science F,~undallon. nod Ihe Nallonal Inslilu~es of" llcnhh. From Ihe Division of Immunology and Allergy. Hans Wilsdorf Luborulory. Deparlmcnl of Medicine. Univorsily II~spllnl. Geneva. Swllzerlnnd. and Division of` Dermatology, Wnshlnglon Unlversily School of Medicine u! Jewish llospl~=l. SL Louis. VIII. A'I"FE~UATION OF AGONIST-INDUC~D DF.~ENSITIZATION OF TIlE RAT SUBSTANCE P RECEPTOR BY PROGI~?,S$1VE TRUNCATION OFTHE C-TERMINUS Wc have investigated Ihc C-terrains! I~,il of Ih¢ rat subslanoc P re~ptor (SPR) as a domain csscmial for ngonbt.ioduced ,_," .;cnsitiutlon. Four pmg~'ssivcly shmler mutants, using prem~lure termination in I;,e C-terminus, were constmcled and pared wllh the unnllered SPR using eCl(;pic expression el' wild-lyl~ :rod mulanl rcceplors in X~m~x ooc~tes. Thes~ mulp.'lla were desi~nt~ted DI6, D4"/. DT0 and D96 wilh 16. 47. "~0 and 96 amino ac;ds r~:;dues deleted fm Ihe In;I. respeCllvely. Wild lype SPR. DI6 and !)47 cxhibked normal cun~-m resixmses when challent.ed wilh sul~tance P. but DT0 ood !)96 had ~-'doc~ maximal cun~l responses and 5% of wild type SPR. rcspa¢lively). DT0. howev©r, exhihiled stihslanllul reals. 181
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C) Lance to substance P-induced dcsensilizat;on in that $5%, versus 8% I'¢~r wild lylx: SPR, of` the peak cu~cn| o1" lee first r~sponse was preserved on second challcni;c with substance P. Therefore, • domain rmm residues 3~R to 36() of the rat SPR, Ihnugh no4 necessary roe the functional activily of' Ihe receplnr, plays an essential role in agnni',t-induced desensilization. Sasakau~a, N.. Sharif, M., and llanle}', ~,|. R. FEBS Lellers 347:!81-184. 1994. niece supporl: Nalional InqilUles of lie•lib. From Ihe Depa~Imenl of Biological Chemislry, School nf Medicine, University of' California al Davis, Davis. CA. TRANSIEN'~ ISCI|EMIA STIMULATES GLIAL FIBRILLARY ACID PROTEIN AND VIMENTIN GENE EXPRESSION IN TIlE GERBIL NEOCORTEX. STRIATUM AND I{IPPOCAMPUS Astrocydc aclivalion plays • major role in homeostali¢ malnlenance o1" Ihe ccn- I!11 heinous system in response Io neuronal damage. To assess the reactivily el" •sire- ¢ytes in Irons•col cerebral ischemia of Ih¢ gerbil, we .~udlcd the levels of glial fihril- la~y acidic prolein (GFAPI and •Is mRNA. GFAP mRNA increased hy 4 h M'ler carolld ane~ occlusion, re~ched peak levels by 72 h with a 12-f'old increase over control lind Ihen slarlcd declining as early as 96 h postischcmia. An examination the specific regions of the brain revealed an increase in GFAP mRNA associated with Ih¢ rorebraln, midbraln, hippocampus and stdatum. GFAP mRNA in the nnn- :schcmic cerebellum however, remained expressed at ¢onslilutively low levels. Immunoblol analysis with nmi.GFAP •re•bodies demonstrated a 2- In 3-fold increase in the protein after 24 arid 48 h of" Rperl'uslon. Pretreatment wizh pentoharbital and I-($'-oxohexyl)..3-mclhyl.7.pmpyl xanthlne (HWA 285), the ~/~s ihal have been shown to pro~¢c! alains[ ischcmlc damage, prevented the increase in GFAP mRNA in Ihe cortex following ischemic injury. Fo~ebrain ischemia also induced virnenlin mRNA and protein quantilics by ! 2 h of reped'uslon in the cortex. The levels of • nd preproenkeph•lin mRNA increased rapidly WJlhir, I h a/'~er ischemic inju~, demoosu-alin~ a lemporal dilTerenc¢ in mRNA changes I'ollowing ischemia. These results indicate that ~n increase in GFAP and vimentio, Ih¢ two glial'inle~nedlate ~il- ament prmeins in the are• o/" lee ischemi¢ lesion may be assneialed with a/;lial respome to in.iury. Kind~, M, S., /]hal, A. N. and Bha~, N. R. Molneula" Brain Research 13:199-206, 1992. From the Depallment of Biochemistry. Laboratory of Cellular and Molecular NcurobJoloey. Center of" Excellence on Stroke or Ihe Sanders.llrnwn Center on A¢in¢, Chandler Medical Center, UnJversily o1" Kentucky, Lexington, KY. 182 TYROSINE PilOSPIIORYL~TION OF MICROTUBUI.~-ASSOCIATED PROTEIN KINASE A~R TRANSIE~ ISCH~IA IN ~E GERBIL BRAIN ~e lymsi~ pho~pho~lalion ~ micm~c~le-~s~i~ ~lein (MAP) kindle w~s examined in the ~¢rbil br~in Ifler transient ischemia and repcrrusion. I~m~phn~l~d~n hr MAP kiuase wn~ maxim.t .~i~h~ I mln or ~rru~i~ rnllowin~ 5 rain Of isch~nia nnd rcmmcd Io conlml I~" ,. ~ ~rly ~ 5 gr~tcxt in~a~c ~ MAP kln~se ~msplm~'lal~t,n wos ~t~tcd wilh minor incre~s in olhcr L~ch~ ~¢;,~4 of t~ ~. Seve~l I~ine.~ pImrylnlcd proteins ~r¢ clelccled in Ihe ~c.~.' hidings: ho~ver, .nd rc~ffusinu injury ~ly i~=~tl lyms~',; e4~l.li~ incase in lyres•no phospho~l~lion was ,,, .vcnled by Ihe (NMDA) ~ceplm blmku (+)-MK-801, wl,~-~s a .on-NMDA ~¢~ Mocker. 6- cyann-?.nilr~lulnox~llne-2.~-dinnc, was i~, ':~cliv¢. P~I~aI~M o( eer~ls wilh calcium channel ~l~kcrs also prcvenl~ : - I~sine ph~p~rylalio, of MAP kinnse in I~ isc~mic brain, AIIog~r, Ihe~ msulls imply an male Rcepio~ and calcium during ~he ly~,,.,;n¢ pbosp~lad~ ~ MAP k~a~. Tyr~ine phosphnWl~li~ w~ also ~vem~ : "#hen i~mia conduclcd undu hy~lhe~ic c~diti~s. ~. ~-h ~ecl aeai~ ~ur~eee~mlive damage. ~cse findings implicot¢ • role rm >~,'~P kin~¢ in ~mn~! ing [r~ i~hemia ~d ~ff~si~. Cam~s-Go~le~ R. end Kind7, M. S. ]oumnl o[ Ncu~hemisl~ 59:1955- I 9~. l~" ~. From lee ~mcnls or Su~e~ an~ Bi~,'~..'.~;slW. ~¢ Lucille R M~kcy ~¢r Cooler, a~ ~nlc~ on A¢ing and Slrokc. U,~vc~ily of K~l~ky MeScal I~xin~lon. KY. INHIBITION OF TYROSINE PHOSPHOR¥i.ATION PREVENTS DELAYED NEURONAL DEATII FOLLOWING CEREI~RAL ISCHEMIA Prolcin lyroslne phosphorylzlion pla).s :"~ important role in Ihc n~zulalio~ of` neuronal runellon. We examined the ¢~l'ecls ~: ;nhib~lio~l o( lyres•no on ischemie neuronal damage in lee CA, ,.:.~..'.n or the hippocampus. In Ih¢ lerbil hippocampus./;an•stein and lav'endustin A, tymsina kin•so inhib~tor.~, were ndmlnls. toted 30 mln bernie initiation of 5.mln isch.':,,ia and reperfusinn. Bod~ gcnlsleln and lavendustln A blocked lyres•no phosphm':.",:tion end prevenled delayed neuronal death {DND). However, genLslin, an inner; ..: m~aloguc of genistein, did not block DND. Genis~elo was dose.dependem in d,~" : ,bib•lion of DND •ft~ i~ch~mia and rcperl'usinn. Administralion of ~euislcin 5 ~ i,) mln •her ischemi• •nd reperrusinn was ineffective in hlockin~ DND in Ih¢ C/~. r:¢ion of the hippocampuao The lyre- sine kinas¢ inhibitors sele¢lively blneked II, e ;)hosphmylalinn of mlerotubole-zs~o¢i. ~led protein (MAP)-2 kinase following ~. '=mia and ~eperfuslon injury. These result.,; ~;u[,'l;cst Ihal'tymsinc phosphr~jlat|. • " the i.,,chemic brain is
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neuronal injury and that MAP-2 klnase may pl',y a role in ,he on~t or d~layed neu- ronal death. Kindy. M. S. ~ouma| o~'Cet~brat Blood ~ow and Mct~li~m 1~:372-37~. I~J93. From the ~pa~menl or Bi~hcmlsl~. Stroke Pr.gram .f II~ ~:nders-Brnwn C~ler ~ A~in¢. University of Kemucky, ~xln~ton. KY. NMDA RECEFI'OR INIIIBITION USING Ai'qTISENSFo OUGONUCt.F-..OTIDES PREVENTS DELAYED NEURONAL DEATII IN GERBIl. IIIPPOCAMPUS FOLLOWING CEREBRAL I$CIIEMIA Glutam~le plays a key role in central ilervous systent I~y modutallng synap/i¢ plasdClty of Ihe neurons as well as neuronal cell deolb. These proce¢,~cs are medialed IhrouBh Ihe activalion of N-melhyI-D.asparlate (NMDA} receptors e,nd Ihe rcsuhlng influx of Ca". We examined the effects or inhibition of Ihe NMDA rectplorx at Ihe molecular level on ischemlc neuronal injury in the CA, regkm of the hip/~campus. In the gerbil hippocampas, antisen~ olignnuelantidcs (CJDNs) dif~t~d ag;~i~.,it the NMDA ~¢ceptot (NMDARI) ~duecd the I~el or NMDA reCCl~Or mRNA and tlgand binding in the CA, Rgio¢ Sen.~ or nom, efl~e oUgnnuclcntides had no cffccl upon the NMDA receptor. Adminislrallon of antJsense NMDARI ODNs prevented delayed neuronal dealh (DND) following 5 mln of cerebral ischemia, whereas, sense ODNs did not ~her Ihe effects ol' ischcmia. These results suggest Ihat the NMDA recepltx is involved in the pm(:ess of DND during ccre~al ischem;m. Klndy, ~L S. .Ncuroscience Research Communiea!ions 14(3): I ?~- I R3, 1994. Giber suppon: Nulional In,qitutes o1" Itcahh. " From Ibe Dcpaflment or Biochemist~,, Stroke Program ol'thc Sanders-Brown Center on Agin¢. University or Kenlucky, L~xington. KY. BLOCKADE OF ORNITHINE OECARBOXYLA.~E F.NZYME PROTI~.CTS AGAINST ISCllEMIC BRAIN DAMAGE Polyamines are derived from omithin¢ by the zcsioos of omithine dec~rboKylase (ODC). which is the rate-limiting step in this palhway. Polyamines play a role in ecll growth, neoplasia, differenlialion, and response to injury. Wc have down Ihal trnn- sienl cerebral ischemia gives ri~ Io i~cRa.5-'d ODC mRN~ and cm,.ymc activily in 184 the gerbil brain. ODC and polyamines a.'x- thought to be important in the generslio~, of edema n~d the neuronal cell loss ass~..iated with ceRbral ischemia. To lest this lheor)', we examined the ODC ~clivity., ": CA. region of the hippocampus foils',. ~. ischcm|| and rcpcd'asio~ injury in tl~e absence and presence of an iuhibltor ~,: ~DC activity, ,.-difluoromethylomithlne {DFMO}. Pmtmelmcm of anlmnls with ;' ~."MO I~sulted in |llenuMio~ or ih¢ ODC aclivily following 5 sin of Jschcmia and . vented the increase in polyam|r~ levels..~- ~c~crmlned by in Ihe isehemi¢ brain. These a|lcrations ~'--~ not d~ to chan~el im OOC mRNA level. Further analysis reve~led that DFI-'~O cell death in th~ CA, re,ion o~ the hi~'x'am~s that a~mp~s isch~ia rc~dusi~n inju?. Admlnlst~i~ of D~" ~'~ ~sulted in effect u~ fi~mnal cell su~ival. ~e.~ ,~u~ suggest ~t O~ ms~ ~livity and the ~fion or~l~m~ play c .:~niC~ m ischemic in~. Kind7. M. Sq Hu. Y., a~ ~Fey, R. ' Joummt o¢ Cereal BI~ R~ and M~t,,t~llsm 14:IN~IN~, I~, ~r supS: Native1 Institutes of lles~.h. From Ihe Slfoke Program. Sanders-~.,:~n Cenler on Aging, Depaflmenl of Bi~hemislry and Division of Neuro¢: v,,e~. Unive~lly ot Kenl~ky Medial Cenler. ~xlng~on, REGULATION OF ALPHA,.ADRENERr~IC RECEPTOR SIGNALLING Atpha~.adrenergle f~ceptom (¢,FAR~, mediate a number of m¢~l~c e~s of cat~l~es ~ ~m ~ of a lqe f~lty which t~sd~e their ~11 signals thigh I,~d~d¢ gum~ ~tei~. Utili~ng the %~ ~e~o~.~tm u a ~ive s~b~l~ of G.~tein coupl~ recc~o~, we ~ a.u,~mg to ~fim oete~ m to~s in dlffeRnt celt ~enu~s a~ ~t¢~in~ how diKcmn~ in ~se m~ ~vironm~ ~d ~ll amhltc~um ~t~i~te to ~11 ~ ~i~ ~ceptor. ~e strategy entails the u~ of %-AR su~s stagy ex~d i~ ~1~-~ fi~o5luts a~ the p~h~t~a c. "; ~ ~-12. two ~ll inf~structure and in ~tch the ~epto~, ~ple tO diff~l Although R. G ~d E arc t~ ms~ enti'~ invotv~ ~ si~ ~pt,~. ~r ass~iated ~tcins may ~ c~lkal for ~ :ficlen~ ~ I~i~¢ ~¢s¢ ~eins ~y ~ ~toskel~sl elen:¢nls ~l~ling ~ c~nlsl~li~ ~d "l~al~ed signal Iran~ucli~" or mole~l~ t~ ~te 185
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C) G-E interface, A more complelc undcrslanding of cellular plas¢icity whh respect ¢o signal eransduclion may lead to a new level of then~peu¢ic specificity and the devel- opment of thcrapeutlc •ppmaches lhat perhaps targcl ¢he R-G or G.E ineerface as opposed to the reccplor's hormone bindin= sit©. L~,nler, S, M. Pharmacology Communications 6( 1-3): 133-137. 1995. Giber support: N•tional lnslilules of Ilcallh. From the Deparlmem of Pharmacology, Medical Univershy of South Carolina, Charleston. SC I IETEROGENEITY AND REGULATION OF NICOTINIC ACETYLCIIOI.INE RECEPTORS Since 1986 there has been a rem~kable avolulion in our conceptual understand. ing of the nicotinic acnt).Ichollne receptor Syslem. It is now clear thal there is helero- geneity among mcmlx:~s of an extended nAChR family at gone, protein, and fuoc- tional levels. This heterogeneity poscs challenges m • clear underslanding of the function of these importanl molecules. It is also becoming clear thai divcrsily in mechanisms involved in the regulation of nAChR expression and function build on the dlvcrslty or ih¢ nAC'hR family. Coetioued. exciting advances in these fields will undoubtedly occur under • deluge of appliealions of skills of • number of invesliga- tars working in a vatlely of disciplines who will bcncfi! from reflection on the basic principles of ncurotransmiitcr-receptor biology revealed by posl studies of the nAChR system and hem • heighlencd antlcipa¢ion tha¢ man)' conven¢ional and provincial views of" nAChR blolo~y and regulation will require aleeration or aban- donmenl by 1996. Lukms. R. J. and I~cncherif. M. In: Imemsflonal Review of Neumblology Vol. 34. Academic Press. Inc, pp. 2~-13 I. 1992. Other support: Nalional Insliluees of Health. From the Division of Ncurobioiogy. Barrow Hcumlogical lnsliluee. Phoenix. AZ. AGING-INDUCED Cl IANGF.S IN TIlE AUTOREOUI.AT1ON OF ACETYLCIIOLINE RELEASE IN TIlE RAT BRAIN In scnilc Fischer mate r~ls (33-mouehs-old). the spontaneous and evoked releas- es of ACh from cerebrum were dcpmssnd. The defecl in the vesicular sloral~e and release of ACh is Ihe most am/amain paramct©r which ms)' contribute Io the cholin- crgic dcfieit in the cerebrum of 33.month-old rats. The feedback systems for antorcgulation of the release of ACh AOs ~l~sc triggers the ~silive-f~d~k system which in~e~s t~ ~tc of ~1~ of SP thsring aging. ~crc is no ~cd for :;;c negative f~d~k s~cm. ~f~. MEK release w~s dcpr~d, ~e in.talc in the ~le of SP ~le~se dufinl sgin~ o~ns a new avenue For d~g dc~lo~em. ~¢~-~plldc SP-sm~l~ists m~y ~ use- Ful in the t~tmcnt o~nile in: Haman. I. ct ~1., (cds.): AIz~clmcr's ~.d ~klnsm's Dises~s. Plenum ~ss, New York, ~. 275-282. Othcr supra: U~. Public Health Settee ~d ~e Study Center For A~sthesis Toxicology of Vanderbilt Prom the ~pm~nts oF Anest~siolozy ~d ~s~y, V~,E~I Unlvcaily Medical Center, Nashville, ENDOGENOUS GANGUOSIDE OM I ,'/.." -~)UL.ATE.S L-TYPE CALCIUM C! IANNEL ACTIVITY IN N I 8 NEUROI: '..ASTOMA CELI.~ Digieal imaging fluoresconc= microscopy was u~d Io inVeSligale the elTocl of the fl subunit of cholera toxin on calcium homeostas,s in neumblas~oma Nil cells, The 13 subuni¢, which binds spocifically to gangliosidc GMI in the ouetr Icaflel o1" the ccll membrane, was found to induce • sustained in~-.asc of intracellular calcium concentration (|Ca"|.). The increase in |Ca;'|, was not obsen, ed in the absence of cxtraccllular calcium, or in ehe I~eSCnce ¢: the calcium chelator EGTA. lad was blocked by nickel. Th~ B subunit was also found to induce sn influx of m~nganeso ions, as indicated b)' • quench or the anita ,.llular furs.2 fluorescence. These dais suggest that the B subunil induces •n .:,"" -ase in calcium influx in NI~ cells. Potassium.lnduced depolm'izalion also slb' , ,led manganese influx~ however, the onset or depolarlzntion-inducrd influx, ~:.c B subunlt ~ no funbcr clT6"cl. This occlusion suglcsts involvcmenl of voltaze .l~,tndent calcium channels. with BayK~644, • dih)'dropyridin~ agonis! selecti,,,c fro" L-type c•lc|um channels, induced msn~•nese inllu~c that w~ not a;:ered by the B suhunil and sppamntly blocked the effect of the B subunit itself, F'u,-lhennore, Ihe dlhyd~dln= L-I)'pe channel antagonists niguldlplne or nlc~tCIpInc completely inhibited B Subunil- induced mangxn~s© influx. Thus, the B s:,"mil-lnd~ccd manganosc influx Is likely due In activation of an L-type vollagc-,~e.,.ndcnt calcium channel, Sponl~cous influx of m~ngaocse ions was -Iso inhib; d by nicardipine or niguldlplne ~nd by exogenmss gnngl|osldcs. G',nglioside GMI : as more potent Ihaa O~3, bul I~lobo- side had no slgnlflcant effect. TI~e mo~l,.:..iioo of L.type c,,Iclum ch•nnelz by endogenous ganglioside GMI has Importar, t i.npllcatlons for its role in neural devel. opmem, diffcrontlatlo~, and reZoneratiou ~,J also for its potential function in the elcclrlcal excilability of neumnX. Carlso~o R. 0,, Masco, D.. Brookcr, G.o nnd Spiegel, S.
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O The Ioumal of Heumscienee 14(4):22"/2.22gl, April 1994. Other sup/x~n: National |nstitmes of Heahh. From the Department of Biochemistry and Molecular Biology, Georgelown University Medica| (2enter, Washington. DC. DIFFERENTIAL SPATIOTEMPORAL EXPRESSION OF K" CIIANNEL POLYPEI~I'IDE5 IN RAT HIPPOCAMPAL NEURONS DEVELOPING lIV SITU AND IN VITRO |llppoeampal neurons ar~ highly plaSlin in their excitable I~OlX:nies. lx~h d~- ing development ~nd in the adult brain. As vo|tage-sensitive K' channels are mawr dctenninams of membrane excitability, one mechanism for gener~ling pl~llcity is through ~gulation of K" channel ~ctivily. To gain insights into the regulation ol" K" channels in the hlppocampus, we have analy~d the spatlotemlxnal expm,~siou pat- terns of five K" channel pelypeptides in rat hipponampal neurons developing ~'n and in vilro. Delayed rectifier-type channels (Kvl.5. Kv2.1, and Kv2.2) are expressed on all neuronal samara and proximal dendrites, while A-type channels (Kvl.4 ~nd Kv4.2) are presem distally on dislincl subpopu}ations of neurons. The d~relopm~nt of U~.se patterns in ~itu is monotonic; that is, while the time and spatial development v~ies among the ch~cls, e~ch K" channel sublype inilially spears in its ~dul| pattern, suggesting Ihat the mcchanlsms underlying spatial patleming o~r- ate through development. Immonobiols confirm the differc'mlal temporal expression o1" K' chan~.ls in the developing hippocampus, and dcmnnst~te developmentally regulated changes in the microheterogeoeity of some K' channel polypeptide species. Temporal expression patterns of all five K" channels observed in shu are retained in vii~o, while cer~in ~specrs o[ cellul~" and subecllular localiz~tlon are alrew.d for some ol" Ihe K" charmel polypeF/kles studied. Similarilies in K' polypel~ide exp~ssion in xim s~d in s¢~ro indicate that the same ~gulato~/mecha- nisms are controlling spatlotempe~l patterning in Ix~h situations. However, dil'l'c~- enccs betwcen lcve|s of exp~sslm for al! subtypes sludled eacepl Kv2.1 indicate sdditional m~ehanlsms operating in slr~ bet absent in ~,i¢~o that ~re important in detenninlng polyFep6de abundance. Malet|c-Savat|¢, M.o Lenn. N. J., and Trimmer, J. S. "l~e ~onma| of Neurmcience |$($):3840-3851, May 1995. Other support: N~ion~l Inslltutes of Heahh. From the Departments of Neurology, and Biochemistry and Cell Biology, Slate University o1' New Yodc, Stnny Br~k, NY, ASSOCIATION AND COLOCALIZATION OF K' CHANNEL a- AHD ~-SUBUNIT POLYPEI~IDES IN RAT BR Recent cloning or auxiliary subunits ns~ .elated w~th volta~e-gate~ km ehannds and their subsequent conxpmssion with the ~.;;,'u~nel forming ,,hsubuftltt he5 ~eveat~ that the expression level, g~in; ~d condu~t:~'e prope~ies or the expressed el~n- nels can be profoundly affected by the p~c:.-..,'.e of an auxilia~ subunit polyr, cPti~k. In the present study, we raised antlhedies ~:.',imt the ~-su1~nlt asscei,~ed whh the bovine dendro~oxin s~nsitlve K',,ch~n~| c~mplex and u~d there ~ntibudi~ to displayed a s~ciflc m~Cllon on immonobTot': o1 rat l~'aln memmnes 'nn a 38 kDa polypeptlde, and a minor 41 kDa I,u~ptide, which cor~six~d closely to the predicted sizes of the Kvp2 and Kv~,; ~-subuni¢ Folypep/ides, ~sFc~ively, ~ecently cloned from ,'at brain. RceipwrA coimmonopr~lpitatinn exFetimcnt~' ~eve.',led that the ~3.~bunlt pol)'peptld~s ~-~.. ~i|ted wi~h Kvl~ ~d Kvh4, but no~ Kv2.1, a.s,bunlts. Immunobistocbem'. " staining re~ealed that the polypeptides were widely dist;ibuted in ad:, .'at braln. Moo:over, the cellul" dial,d" button of ~-subonit immuonreactivity em:~" .,.~ded closely wilh immunm~l|V~y for Kvl.2, and to • lessu extent Kvl.4, bus no~ with Kv?.l. The~ ~,su|ls that neuronal mech~isms may exist to direct the selecllvc inleractlon of K" a- and p-subunit polypeptides, ~d that the ~opedies of K" cha~nels in specific suh- cellular domains may be I~gulated by the l',~ .q~im of heleromultimerk K" charnel complexes co~ainin~ s~cJr~c combimlli~l'; t,~' el. I1~ Rhodes, K. I,. Keilbaugh. 3. A.. Barrezuel& N. X.. Lopez, K. L.. and THmmer. TI~ loumat of Neuro~ci~nce ! ~(?):~360-~37 I. luly 199~. Other ~uppo~: Lederle Laboratories. the Cente~ fo~ Bio~eclmotogy at Stony Bank. and ~ew York State Science and Technology Foondatim. From the Department of CNS Biological ~,esesmh, Medical Rescan:h Division, Lederle Laboratories. American Cyan:.~,id Company. Pearl River. NY. and Deparlment of Biochemislry and Cell Biology. Sta~e University or New Yak. Stony Brook, NY. IMPAIRED RETENTION BY ANGIOTENEIN 11 MEDIATED BY TIlE AT, RECEPTOR We demons~cd il~evlou'dy that hipi~: :~:mFal d.e.nlate..gyms ..neu..m~. s we. I~. ~.n. sltive to angio~ensin II (All) and ~ccnlly c~:',overed that All appnea at~'~'lly in m~ dentate gyrus inhibited gm~ulc cell long ,:,m i~enlialion induelion and that Ibe inhibition is mediated by the AT, I~-eptor ,.,; ¢m be blocked by Ioslala, • spceift~ t~...o....,..;., 'rh,. n~rn~e of Ihe m~.sen: s:edy was to examine the eff'e~ts of All of an inhibitory shock avoidance reslxmse ~m to aetermme tr the msmtam imps r- meat atr retentim can be blocked by tos~'-,~,, A total of 12 groups el' rats i~ Ihme experiments were studied. Three indepem~¢nt relX:titio~ .of 5 ng _All ed.m. iniste .,~d bilaterally to the dentate gy~us demonsmt:o • clear impatm~ent oK fcten.m unacr 189
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O "-4 0 these experimental conditions and Ih~l the impairment can t~ eirl'ecdvcly prevcnled by prelreatmenl with 20 m~g or Iosarlan IP. Lee. E, IL Y., Me. Y. I_, Wzyner. M. 3.. and Armstrong, D. L. Peplldes 16(6):1069-1071. 1995. Other support: lnsdlute o1" Biomedical Sciences, Academia Stoics,. Taiwan, Repnbiic of China. From the Institute or Biomedical Sciences. Academia Sinica, Taipei. Taiwan. Republic o1" China, and Division or Life Sciences, Universily of Texas at San Antonio, San Antonio, TX. DOSE AND TIME DEPENDENCY OF ANGIOTENSIN I! INI IllZITION OF lllPPOCAMPAL LONO-'I~..RM POTENTIATION We previously reported Ihzz injeclion of 1.0 Izl of" 4.78/,dlt' angiolensin II (All) above the hippocsmpus in rats inhibils long Icrm polcntiation (LTP) induction in medial pefforanl path-stimulated dcnlate granule cells. The present experiments were conducled in urelhane-aneslhelized Sprague-Dawley rals. LTP was measured in lerms of" Ihe relalive change in slol~ o1" the populalion EPSP compared In hasclinc. Ell'eels o1" 0.48, 0.956. 1.195, 2.39, and 4.78/J.Af All end lime delays at" 30. (~t. 90 •nd I~0 rain wilh the 4.78 ~ dose were title,mined. Resulls w~rc slgnific=nt and demonslrale ~ All inhibilion or LTP in denlale granule cells b bolh dose and lime depcndenl. The I~eshold is ap~oxim~cly 1.0 Final of peplide. Inhibilion duc to Ihe 4.78/aM All begins slowly aflcr I h and is complete over the nex130 rain. cominuos [or anolhcr 30 rain. and then fully recovers by Ihe cod o/'the nex130 min. This time dependency conM be due to the intemalizatlon ol'lhe All. inlcraetion wilh -', cyloso- lic. receptor, ~d cvcnlual degradation. Wayner, M.J,~ Polan-Cu~in. J.. and Armstrong, D. L. Peplides 1~(6):1079- 1082, O~her support: Na~lonal Ins~ilules o/'Health. From Ihc Division o1" Lifo Scienee~, University o1" Texas al San Anlonio. San Anlonio. TX. IX. Pharmacology" ACUTE ETilANOL EXPOSURE SUPPRESSES TI IE REPAIR OF O'- METI|YLGUANINE DNA LESIONS IN CASIRATED ADULT MALE RATS Alcohol has cleazly been Isso~a/ed whh an incre~e ol~canccrs in numerous Its. sues, including the respiralot~j tract, colon, reClum, liver, but especially Ihe ¢sopha. 190 Bus, I-~jnx. ph.~,nx, and mouth. A~cohel alone has ~ been ~own !o ~ a mul~en until It is converled to no.aldehyde and, c~a~inogen. ~vl~s data h~s s~wn tha: a~ol ~entmti~s inhibils DNA ~ir. and in lighl of I~ wid:~ ~mpl~. or al~ol~ ~vcr- ag~ with alcohol conlcnls ~ngin ~ino~n~is hz~ ~ighlc~d. All~gh ~vJ~S sl~, in ~t ~s, hl~ inveili- gnled the off.is o~ c~ic alc~ol cx~ h~ve ~un to sdd~ Ihe effects of ~u:: ~" ~in~c" ~k~! ex~s~ on m~- malian DNA ~pnir. Tow~ thb end. we..~.o:z the inbi~t~ ~ ~.~thylgu~l~- DNA mcthyltrans~crssc (MGM~ by ctha~l in ~ull mslc c~tratcd ~lS. ~is i: "iti~ lasted r~ st Ic~124 hr. We ~1~o ok~cd • d~e.res~se e~! I~l~ ~ls. ~c finding o~ cl~nol's inlacl ~aLq implies a ho~n! c~i, ~GMT DNA ~sir ~, ~hkh h~ only ~en alluded to in p~t reset. Wilson II!, D. M., Tootler, J. J., ~mey. ?.. Wils~. T. M.. a~ Kelley. M. R. Alcoholism: Clinical and Experim..sl Research 18(5):1267-1271, S~em~r~lo~ 1994. Other sup~: Nati~nl lnslilules ~ ! lcahh. From the Department of Pcdialrics. Scclion or Pediatric End~i~lo~y. Wells ~nlcr [m Pcdi~ric Rescamh. Ril~ l~?~l Molccolar Biology. indiana Unlvcrsil Molc~lar Biology Pm~m s~ Dcpa~u:-'4 of Bimhcmist~. ~o1~ Univcaily Medical ~hool. Mny~. I~ TIlE TOXICITY OF AZIDOTllYMIDINF, . qZ'T) ON HUMAN AND ANIMAL CELLS IN CULTURE AT CONCENTRAT, .NS USED FOR ANTIVIRAL TIIERAPY AZT, a chain lerminalot of DNA syn:" ;is originally developed for chemother- apy, is now prcscrihed Is an antihuman i,.~,;lcnedeficicncy vires (HIV) drag al to 1500 mf,/peaon/day, which con~.spo~lr ,~ 20 to 60 DM AZT. The human is ba.~d on • study by the m~ornc~.ru, .~ drag md Iheir collalx~lors, which reported in 1986 that the inhibitor/dose .'.'" IIlV repl;catlon wIs 0.05 to 0.$ AZ'I" and thai l'or human T-cells was 20:'-~ ," 20,000 times hi~ber, i.e. 1000 AZi'. This su~cslcd Ihal HIV COMd be s::'..;j inhibited in h.mans at 20 to 60 p,M AZT. llowcver, al'ler Ihe licensing orAZT ::,' ,m anli.lllV d~.~. several independent studies reported 20 to 1000-fold lower IN'. " "..~'y doses of AZT for humm ~d ani- mal cells Ihan did Ihe msnufscturer's stc;: . mngi~g from I to 50 p.M. In KccoN wilh Ibis. life threatening toxic effects wet.. -.~.potlcd in humans lRatcd with AZT 20 to 60 p.M. Therefore. wc have re-cxe|,.:., :d the growlh inhiblto~y doses of AZ'r for the humen CEM T-cell line and sever-,, ntber human and animal cells. It was 191
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o found Ihal al I0 pM and 25 pM AZT, all cells arc inhibited at Icast 50~ alter 6 In 12 days, and between 20 and 100% after 38 to 48 days. Unexpcclndly, variants of all cell lypes emerged over time thai were partially resislant Io AZ'F. !1 is conclndrd the/ A'TT, ,,! Ih¢ dosage prescribed as an anli-IIIV drug, is highly Ioxic Io human cells. Chgs. D. T. and Duesher|, P. !1. Genetics 95:103-11Y), 1795. Other support: Priv,,te Donations. From the Department of Molecular and Cell Biology, Universily of" California, Berkeley, CA. HIV AS A SURROGATE MARKER FOR DRUG USE: A RE-ANALYSIS OF TIlE SAN FRANCISCO MEN'S HEALTH STUDY Our analysis of drag use and morbidity dala from a coho~1 or 1o34 men yields the following results: I) HIV infection is a strong indicalor of drag use---lllV-positive respondents reix~ed an average lifelime dose of rccrcaxional drags (excluding mad- juana) 2.3 limes higher lhan lllV-neg-live respondents. 2) llmnnscxualJly is a slrong indicalor of drag use--hommexual respondenl.~ repnrtcd In average lifelimc dine 2.0 limes higher lhan helCrOScxual respondents, 3) "llle incidence of AIDS-defining d;s- eases w&$ nol limilcd Io fcspondenls infccled wilh IIlV, bul w~,t almosl complelely limiled (98~) Io respondcols who reposed using drags. We alxa nddres.¢ a p~cvious rc[x~tl (Ascher el oL. 1993) lhal was based o~ lhe ~ dalaba.~ and [mrporled IO show lhal I[IV alone coaelal~ w|lh lhe dc*velolwncnl of AIDS. Spccil]c~ll¥. we shaw lh:~ lhe rclalio~|p hclwccn HIV infection and CD44- T Cell loss is weaker lhan rqx~ed by Aschcr/*I of,, and provides tilde evlc}euce for a causalive relalionship. These rcsulls support the hypOlhesis thal Ion~*lemL habitual drug use can cau~ lhe conditions known ~s AIDS (indepcndcnl of lee wescnce of IIIV). and ferule the hypothesis that HIV alone causes Ibese conditions indcpcn~nl of drag use. Ellison, B. L, Downey. A. B., and Duesbu~, P. II. OenellCa 9~:165-171, O,her support: l~vme Do~adons. From the Deparlmcnt of. Molecular and Cell Biology, University of California. Berkeley. CA and EECS Computer Science Division. University of Callfornia. Berkeley. CA. ATTENUATION OF AOONIST-INDUCED DESENSITIZATION OF TIIE RAT SUBSTANCE P RECEPTOR BY MICROIN|ECTION OF INOSITOL PENTAKIS- AND HEXAKISPHOSPHATES IN XENOPUS LAEVI$ OOCYTF.S Rccen|ly. inosilol hcxaki~phosphat¢ (ph)qic acid) was shown to bind to photore- 192 captor aweslin and Mock its inte~ct|o, with .hodopsin. Such an inter,,ction mlgh~ predict Ihal inosilol polyphosphaleS could ~]lcr O pmleln-¢oupkd ~cceptor dcsens|t|- zation. To investigate the possible ales of Itigh~ inosilol polyphosphates on mcep Inr dcsc~itiz~lion, we have expressed the r~t subslsnce P rCcel~or in Xenopus Inevls oocylcs. The functional expression of' s.~nce P RcepIor was mac|laced by volt- age-clamp rccordln~ of substance P-indz,c'.d Ca*+-dependent CI- caveats. COnlrol oocytes were stimulated with sub~;~, ace P (~0 nM)o alter 10 mln or washing Ihe second responses Io subslnnco P wc, e ~ : .,woximately 15"~, or the firSl Cytosolic injection or inositol pcnlakisph.,.**~hat¢ (100 pM) or inosilol hcx~klsphos- phale (100 pM) inhibited the reduction oi [he second subslance P-induced corral responses, mainlaining the second rcspop~es to 57-55~, of the initial responses. The prolective cff'ccts of jnositol pcntakis; ;:,~phlte and inn|teal bexakisphosphale a~,aiost a~odistoinducnd dcseositlzation w.".~e O~'lC¢lttraliOII lad tlmc dq~qldenl structurally s~cific, in thai inosilol hex,'.-,',.~;fale and inosltol ~s- and tctraE|sphos. phate i~omers were inactive. Microinjceti,;'~ of inositol hcx~kls~osi~te did not change the Potency of" suhslznce P or ll~ .=nlilivity of Ibc exixcsscd substance receptor to substance P. (b) |nh|bk 12.O.t.-.trsdceanoylphorbol-13-lcetatc-indoced loss of subslance P.inducnd can-ca responses, or (¢) Mler Ih¢ ©urrcnt$ elicited by microinjeclion of inosilol-l.4.S-trisphosphate. These Rsults SuSges! that pentakisphosphate and inositol hcxakisphosphat¢ have spccW, c inhlblto~ cl]'~cls on the agonist-inducnd loss of responsivcoq~ cr lhc nl substance P ~cepto~. Moszover. these protective effects o£ inosilol hex-'@ ~pho~phale agaimt desensilizalioo also o~rvnd with the endogenous lysopL;..~)hMidi¢ i¢id/phosphatldic Icid indicalln~ thai Ihis mechanism is hal slx:c!:~e Io ¢ctopio Rceplof~. These IeSulls gest that inositol pentakisphosphate and i,lositol h=xakispho~phsle may be novel pharmacological tools for tim sludy of. a[~enisl.|nduced desensitizal|on. Sasakawa. N., Fer~uson, ]. E., gh~r, M., 'nd lhlnley~ M, R. Molecular Plmrm~ology 4~:3R0-355, 199;, Giber supporl: Nalional Instilules of. From the Department of Biolo~icM Chemistry. School of' Medicine0 Univcnily of Call foals at Davis. Davis. CA. EVALUATION OF CALCIUM INFLUX FACTORS FROM STIMULATED JURKAT T-LYMPHOCYTES BY MICRO|NJECTION INTO XENOPU$ OOCTYTES Acid exlracts of thapsignrgin-actlvr,;~.. Jurkai ceils have been shown to Mve intraccliular activity in inducing • do~e-¢', pendent tepid chlor|dc current upon micminjection in Xennpux tae~,ls oneytes. ? he extmeLt Ict by ¢levll|on of c~lcium through cslclum entry. The lictor(s) rcsp~n:|ble fo~ th~ activity hav~ ~o tcm~cd calcium influx factor (CIF) and have bccn [ound to b~ small. ,=L~tively polar re.ale- cures (< I(XX) daltons) ~vhosc activity is ~bolL~Eed by alkal|nc phosphatase tsestmcnt and potentiated by co-lnjectloa of okadaio acid (a Ixote|o phosphstase inhibilm). CIF 193 i
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is produced in a time-dependem manner f.ollow|ng thapsilprgin Irealmcnt o~ tells. ~in= tim ~l~val~d a~ve b~al levels ~y 2 mln. Inlracellular CIF acdvily is complelely Ibscnl ~mm NGI I~01L ~uro~! ~lls, which lack, ca~cilafive On Ihis basis, it ap~ thM Jmkzl cells, a~ivstcd by slimulJ thai deplete internal c~lcium stores, produce on~ or mort CIF aclivilics acling imraccllularly, and Xrnopns ~ytcs may ~ a ~weffu] 1~ to puffy and charactcrlzc CIFs. ~om~. D. and llanley, M. R. ~ Journal of Biological ~cmistw 27~ 12):~29-M32. March 24. Other sup~: National [n~ilules of l[cahh. From the ~pa~nt o~ Biological ~emi~lW. Univc~ity of California Sch~)l of Medicine. Davis, CA. C|IROMATOGRAPIIlC RESOLUTION OFAN INTRACELLULAR CALCIUM INFLUX FACTOR FROM TIIAPSIGARGIN.ACTIVATED JURKAT CELLS: EVI[~.NC~ FOR MULTII~.~ ACTIVITIES INR.UENCiNG C&I.~IUM ELEVATION In Acid exlracts of thapsiga~g;n-stimulated Jmkit cells rcvc~Icd both intracellular and extracellolar ~'livilies slimulallng Ca~'.depcndent CI - cun'enls on Xcnopus lacvls occy[es. Chromato~=phic fractionation or these extracls on Rot fil|r-~lion separated two aCliVC rracl;ons of M, approximately 600 and 40{l. Moreover, lho M, 600 fraction eahibhcd both intracellular and exit',cellular ac[ivldcs. [[owcver, the in[raccllular aclivily was nbscnl l'rom extracts or unstlmula[ed Jurkat cells, suggesting ils produco lion was stimulated by thapsigarg;n. The further p0riF~calinn of" this l'racl;on hy high p~rronn~nce thin layer chromatography resolved a single fraclion which war active only on microln~clioo and which rcqulr~d calcium enlry rnr activation o{" corrcn[ responses. These resuhs suggest lhat a single authcnlic c'alcium inllmc factor can bc resolved by purification l'rom co, rounding aclivities dclecled in crude acid extracts. Kim, H. Y., Thomas, D., and l[anley, M. R. The Journal or Biological Chemisi~ 270(17):9706.9708. April 28. O~her suppo~l: National Institutes of H~ahh. From lhC D~parlmem or Bio[og|cal C'hemiso'y, School or M~dlcine, Universlty o{" California. Davis. CA. PIIENCYCLIDINE INIIIBITS EPINEPHRINE-STIMULATED PLATELET AGGREGATION INDEPENDENTLY OF H|G|| AFFINITY N-METllYL-o- ASPARTATE INMDA)-TYPE GLUTAMATi~RECEPTOR$ The psycholomlmclic analr, esic phcncyclidlne (PCP), which hinds to a flash 194 afl~nily site on the neuronal N-mclbyi-t. :spaaale (NMDA).sensitive ~lulam~,te receplor, has previously been [round Io bh;d Io plitc~els wilh h;~h al~nily and Io s~ifically delay ~ ~SCl or epin~hrinc-~mulzlcd platclcl m~#rcgali~ (Jamiesnn cl el. (1992) Bi~hem. ~. 2g~, ~-39). We ~zve now sh~ lhol binding ar~nifi~ or 14 symhelic ~ aM~ogs z[ lhe high ~nily binding sale on plalclcls ~s nm parallel lhc rank order er their z~niliel in blndin~ membranes, i~aling lhat lhc high argot:)" P~ bindlng site in pblelels is dlsli~l from lhe neuronal NMDA rcccplor. ~c c~ .¢ of ~le~ of six or l~e ~Io~ In delaying lhc case[ or cpincphri~-sllmul~t~d plafelcl 18$~Mi~ zl~ did nol ~l- Icl zhe ~nk order or their binding affinities r~ plzIclel ~ dale indicate lhat lhe ahilily or ~P unzlo~ In inhibit cpincp~limulMcd gallon is not sol,led Io lh~i~ ability Io ~n~: ~,. I~ high ~finlsy pl=telel ~P bi~in~ site. Funh~orc. (+)MK-g01, which blnd~ Io ~ ~ high afflnily bindin~ ~ile in • [rccls on plalclcl sccr¢linn a~ nm involv~ in ~P-nl~i~d inhi~l~ of phrino-induccd ~lalclc[ From Ihe Dcp~nm~nl or C¢11 Biology. A~cI~ R~ Cross. R~kv~l~. Ki~n~y Disea~s. Oclhes~a. 3'-AZIDOTI IYMIDINE (ZIDOVUDINE) LNiilBIT~ GLYCOSYLATION AND DRAMATICALLY ALTERS GLYCOSPI{~,NGOLIPID SYNTII "ESIS IN WHOLE CEIJ.S AT CLINICALLY RELEVANT CONCENTRATIONS Recent in rl;ro wc~k with Golgi-~nd.chc~ ~n~mlx-~s showed ~at 3'-Izldmhym[. dlne-5'-monophosphalc (AZTMP), Ihe I.ir" 'y imracellular metabolile of. 3'-azi- dothymldine (AZ'T), is a po(em inhibitor o," ' -.osyladon macdonz (Hall ¢~ eL (Igg4l J. Biol. Chtm. 269, 14355.1435~) and Ixe, ,~:,.-d Ihal AZT IrCalmen! of whole cells should cause similar inhlbillon. In Ibis re~,, ",e verify Ibis prediclion by'#J~ow]n¢ ~ha! trcalment ol K562 cells wi!h AZ'T inhibits ::~.:d and protein ¢lycosylation. AZ'F heat. mcnl dram~lically alters Iho pal!era of glp.. ~p.Singolipid biosynlhcsis, nearly abolish- ing ganglioslde synthesis al clinically ~elcvz.,; concemralons (I-$ I~), ~ the incoq~om!ion o~" bo~h sialic acid and gal.'lose into proteins. Con~l experlmenls dcmonsl~ate thai Ihese changes do nol resuh Iron) nonspeclfic eIT~ts on either sccrelo~ apparatus or protein synthesis, C,': :he other hind, aludics using isolaled nuclei as a model syslcm rot ch~:)mosoma~ ::P!A Rplicatio~ show IbM AZTTP is u very weak inhibitor of" DNA synlhezis, ~v.:~,. obr.-q'valions suonBly zuggcsl that Ihe myelosuppressive effecls or AZT in rhY~ a~ ~~.'e to inhlbldon or protein a~d or lipid glyco.~ladon and not to el'f.ecls on chromos~,.~ ..I DNA rrplicallon. Yon. J,-P., Ilsley. D. D., Frohlick, C., Slccz, R. llall, E, T., Kurhta. R. O.,, apd Melancon, P, 19.S
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C-) The ,/oumal of' Dinlogical Chemistry 270( 39).22936.22/~4 I. • . . September 29. 1995. Other support: National Institutes of" Heahh and American C-~ncer S~ciety. From the Deparlmenl of" Chemistry and F~iachemlstry. University of Colorado. Boulder. CO. NICOTINE EFFEC~ ON PRESYNAI~IC RECEPTOR INTERAC13ONS Presynaptic regulation of ncumtransmilter release in the brain inlluenccs the gmount of I~¢Urolreusmlner released. This may or may ncl del~nd on nerve activity, •nd can be modulated by oth~" racine• tha~ influence the membrane Ix-~enti:d of Illc nerve ending. The neurolransmitter ilself can inhibh ils own release by acting on presynapliC aUloreceplors. Other Irgnsmillcrs can aim aCl pre.'~ynaplically to mndu- lale IransmJller OUlflOW. "/l~e emplexily and divcr.,~ily .f .~uch m(~lulalory mecha- nisms of release have received much allcntion. Wc were particularly interested in reeel~or-reCeplor inlet•eden• influenced by participilinn of nicotine in tim eenlral sctinns of nicotine involving presynaplie effects. The mechanisms ~f" Ih¢.~c reccpt-r-rccel~lur internclinn.~ have I.:cn tli.~cu.~cd recemly. In receptor heleroregulation, neur~a~nsmilters or neuron|(alulatm's, hy binding Io receptors on Ihe neuronal membrane, may he able to regulate the charac- terisl~s and function of Ihe recognilion sile~ of another Ir~nsmlncr or modulator receptor. Whereas aotoreceptor mechanisms regulate the sensitivity oFa receiver and ~¢ modulated by Ihe levels of ils ligand flu'curb pOSilive or negative I'ccdback loops. heleroreceptor mechanisms include direct of indirect inleractions between dil'ferenl receptors and Iransmillers. involving the plasma membrane, inlracylnpla:cmalie loops, or inleractlons at the nuclear levd. ~ershen. !!., Toth, ~, LaJth% ,A., and visa, E. 5. Annals of the New York Academy ofSclences 757:23~-244. May I0. 199~. From the N.S. IQine |nstilule for Psychialrlc Research. Center f"or Neurochemistry. Omngebur8, NY. NEUROCHEMICAL EVIDENCE OF HETI~ROGENEITY OF PR ""~YNAI~C AND SOMATODENDRITIC NICOTINIC ACRTYI,CI IOIJNE R ECEIq'ORS Wc dcscr~he studies of" the eff"ecls of" differen! nAChR agent•t• on the release of" differenl Wansmiltcrs and the modulateW role of presynaptic nicotinic receptors in chemical signal Iransmlssion. In addition, using diffctenl nAChR agent•Is and nnlag- 196 onists we atlempted to characterize the presy,.:,£:ic crgic axon tc~inals in the hi~ampes I~ '~s ~e~ns ~ the ~il~enddt~ nA~Rs I~lted on the myenlcHc plexus. ~. idyllic ~ Rlea~ s~ies Is thai one can ~ certain whoa Iha transistor cot'-': ~r~, ~d thus c~ I~ate the site or action of different nA~R agent•is ~ an,t: ~isl~ m~ing the c~cledzati~ of the ~e~s ~lher Vizi, E. S.. Scrshen. If., Ball•. A.. Mi~e. ;~.. 'Windisch, K.. Ju~nyi. ~jlha. A. Annals or Ihe New York Ac~cmy of Scion, ~hcr sup~: llung~ Re~ch Fund From the Department of Pharmacology. '.islltule of Experimental Medicine. Ilungarian Academy of Sciences. Buda~l, ~lunga~. and ~e Nathan $. Kline lnslitute for Psychialdc Resorb. Center for ~¢~ SUBTYPE-SPECIFICITY OF THE PRESY~ ~.£'TIC %-ADRENOCEPTORS MODULATING I IIPPOCAMPAt, NOREP;:,i~i'I IRINE RELF, A,qE IN RAT !~ rive brain microdialysls and hi~h-pcr'wmance liquid chmmato|raphy with eleclrochemic;d detection were used Io slur. "" the effect of diffecenl seleclive a~- anllgonisls on hippocampal norepinephrine (.~.") release in freely moving •wake Val. Systemic ndminiSlmlion (0.5 mg/kg i.p.) of el, Ihe et~o-•ntagonisl ARC 239 did not signJfie,,-:;': change the basal releas~ of HE. At • higher dose (S mg/kg i.p.) ARC 239 we, ° ~:| Ineffective. whereas BRL, 4440~t caused a slgnifleanl increase of the extrace:¢~ .~ level of NE. SImilw results were oblained from in vitro perfusion experlmen~ . • at hlppacampal slices were Iolded with I'll]HE and the electrlenl stimulilion..'.oked release of I'll]HE was deter- mined. The acantagonisls were applied in -'" • .x:colretion range of I0" Io 10"" M. ARC 239 was ineffeclive, whereas BRL 444 • ~ignific-',nlly increas~ t he elecl~eal. ly induced release of |'HINE. In •~recment :,~.:t '.he dill of mlcmdialysis ~ perfu- sion experiments. BRL 4440~ displaced [Sll|)'.,,imblne from hippocampal and corti- cal membranes of rat brain with high affini::, • hereas ARC 239 w~s less effective. The pK~ values of elght different e~.admnergi,'..omlx~nds shewed a very good cor- relation (r = 0.98, slope = I.I I P < O.000l) it,' :f,pecampus and frontal cortex where the %--ndrenoceplm~ have bean characterize0 as am-subtype. Our data indicate Ihil hippocampal NE release in rat is regulated by ,'%-ad~nncepCoP~. a species variation of" the human ctt,,-subtype. Kiss, J. P.. Zsitla. Q.. Mike, A.. Zelles. T.,'l'ot~ .~, LaJIh•, Brain Research 674:23g-244, 1995. Other supped: llungarlan Resa~ch Fund ants " n From the Department of Pharmacology..~:,:ttlut¢ of Experimenlal Medicine. tlungsrlan Academy o.f Sciences. Bu:',,;:est. Hungary. and Center for Neurochemistry. H.S. ~llne Institute rof Psych~;:lric Research. Orangcberg. HY. 197
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C) DETERMINANTS OF%-ADRENERGIC RECEPTOR ACTIVATION OF O PROTEINS-" EVIDENCE FOR A PRECOUPLED RECEPTOR/G PROTEIN STATE The ability of agonlst-occupicd a~-ndmnergic receplms to aclivate G pmlcins was measured in membranes from PC-12, cells s~bly expressing the cloned receptor, using guanosine-S°.O-{3l,,Slthio)triphosphalc (["SIGTPyS) binding as an endpoint. Epinephrine (EP]) stimulated I"SlGTP~,S binding in a Mg"-dependcnt manner, showin~ both micromolar and mill!molar cation arlinili~. Prior treatment of cells with per~ussis toxin completely eliminated the EP[ stimulation. The presence o1" GDP dccroased basal ["S]GTP-yS binding and incroarcd Ihe pmporlinn of EPI-stimulated binding. Increasing concenlrations or No" also reduced basal I"SIGTi~S hinding hut had less effect on EPl-stimulatcd binding, such thai the agonisl response was propor- tionalely greate¢ at higher No' levels. In sutural|on binding slnd|es with I"SlGTP~S only low affinity binding was observed in the pre~nc¢ or I(X) mM Na'o whereas in Ihe absence of No" u high aft'rally component was also proscnt, indicating a No'regu- lated rcceptor/G proteio interaction. EPI induced high all, oily I"S|GTP',/S binding in Ihe presence or No" and increased the affinity of the high ar/~nlty comimncn! undc¢ Na'-fro¢ conditions. 31~¢ selective %,-zdrenerglc antagnnisl rauwolscine produced rightward shills of EPI dose-response curves and decreased Ihe basal level of ["S]G'rP-tS binding across Ihe some range of concenlrations. The cxrenI ot' decrease was dependent upo¢l the ~-~drenerglc receptor expression Icvcl, indicating that c~- ndrcncr¢ic receptors contrlbul¢ to basal G protein aclivalion in the ab~nce of ago- hist. The ability of rauwolscJnc to decrease basal I"SIGT~S hlndin~ was dimin- ished as the level or No" was increased, sug~esling Ihal bolh agents act to reduce rcceptor/G protein interaction, by distincllv¢ mechanisms. ,'.,-Adr©nergic receptor amagonlsls Rcluend basal G pmlein acllvalion wilh • rank o~dcr for maximal elTce- tlvcness th,,e was dif£crcm rrum their receptor binding olfinlties. These resulls sop- port the cxlslcocc or precoupling between a~,-adrenergic recepIors and G proleins; coupling can bo diminished by both No" and anlagonisls, whereas agonisL¢ increase the el~ci~cy of receptor/G IXOleln coupling. :'tan. W.-N~ Duxlc. F. M., l.anler~ S. M, and Dclh. R. C. Molecular Pharmacology 45:$24-5~1.1994. O~her support: U.S. Public lleallh Service. From the Department of Phan~aceutical Sciences. Northeastern Unive~ily. Boslon, MA, and Dcpanmen| of Cell Molecular Pharmacology. Medical Univusity of South Carolina. Charleston. $C. USE OF HIGH AFFINITY. RADIOIODINATED PROBES FOR IDENTIFICATION OF IMIDAZOLINE/GUANIDINIUM RECEPTIVE SITES Various pharmacologically active compounds with an imidar.oline or guanldin. ium moiety arc recocniz~d by membrane Ix~nd pmlcins thai appear structurally and functionally distinct from known hormone receplors. Such cot!tics arc termed imida. zolinc binding sites, I receptors, or imldazoline/guanidinium rcceplivc sites (IGRS). 198 To f~cilitate the identification and stroctural ~.';lysis or IGRS. we develo~d rune. tionalized molecular prob~ exhibiting high ; " "~ity and selectivity for IORS. Tic molecular Wol:~s are stn~clurall), related to c: ~.oline. an imidazolioc thai exhibits high affinity for IGRS in both central and pcr;vl.:,'al liSSU~S. The parenl molecule 2- [3-aminophenoxylmetbyl imidazol|ne was ~ ~.:'t:,iodinaled to yield 2-13.amino-t- I"11 iodophenoxy]melh)'l imidazolinc (,hi. ,! !!PI), which w',s used SO idcotify IGRS in brain and peripheral tissues. "I-AMh ~ .~v=s converled to the pho~osenshlve arylazide dcrivalive ("I-AMIPI) and u~ed to L'- ~.tiry the M, of the liBand binding suhuni! of IGRS in various lissues incledln~ ::~ai,s. pancreas, kldncy, and liver. The results of these sludics indlcore that there arc ~;mldpk: binding proleins I'or these molecules thai dilTcr io Ihcir npparent mnlecu;,~ welgM0 liSSne dislrlbullon, introits. suc location and ligand recognition properties. l.onler, $. M., L~nier, B., Rakthav~halam. V.. ',,;cO~ath, C. R., Ind Hcumeyer, J, L, Annals or the New York Aendcmy of Science." ;'~.'1:10~11 I, July 12, 199.~. Oilier support: National ]nslilures or Ilealth :l~...! the Medical Onivc~[ly of Sonlh Carolina. From the Dep,,rlment of Pharmacology, M~. Ileal Unive~sil¥ of South Carolina, C~arleston, sen and Research Binchemicals h, ";nat[guM, NMICk, MA. NICOTINE EFPECTS AND WORKING ME;." ")RY PERFORMANCE Nicotine and nther nicotinic drugs m~y :.ave a vat|ely of Iherap~otlc uses. Cognilivc cnhanccmenl [s one clTccl or nlco(i~c which shows I~mlis~ for lh¢ treat- mcnl or disease slalcs. Similes rrum oor luhnr,~ln.~' and ~lhers h~ve shown that line improves cognitive performance. Improvemeots in ~esls of attentiveness and mcmo~ in humans, monkeys ond rats have I..c.,t found in many bot no~ all slndlcs. We have conducled a series or csperiments e~. "ntng nlco[[ne and nk:ol~i¢ antago- nist efrecls on rats in working tnemo~ lask ;~ the radial-~nn m~e ~nd T.mate. Chronic subcutaneous nlco~ine infusion con.,:::-nll}' impro~J cholc~ accmacy o4" normal adult rals in Ihe win-shift working m=~.~v~ ~xlon of the radial.ram maze task. This chronic nicotine exposure also attc,-...ates the menmry del'~cils in radial- • rm maze choice accuracy caused by leskms Io ~lo.~cfions oflh¢ hlppouampos the bas~l rorebrain, two areas affected in AIz~m~'s discu¢. The nk:oti~e-indu~d improvement was robos! in the race or boll-....~is! ~d &nta¢onlst challcn~j~ wilh dopamincrglc drugs. In contrast the same chr~. ~ ," -ficodne adminlslradon did no[ I|~- nircandy allot choice accuracy in • T-maze -~'.;ical alternation work|rig meroor~ task. This lack of efl~cacy or nlcotio¢ in the ", ~ :a~o lack may have be~n due Io ~roater importance or pru..~'ll~e interference i'.;-,:Rnt in II~s task. Nicotine m' olher nicotinic .~gonists may he u~rul tma!n~nts fo' ~ld¢|mcr's disem¢ and other syn- ¢lrom~.¢ characlcrized by c~gnltlve impalnnenl , I,evln, E. 199
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In: Recent Advances in Tobacco Science, Volum~ 20. Proceedings of a Symlx~sium Presented al the 48th Meeting or the Tobacco Chcmisls" Re~carch Conference, Greensboro, NC, September 25-2g. 1994. OIh~r sup~: R. J. Reynolds, Alzbeimer's Foundatinn. and lhe Hn~ional Science Foundation. From Ihe Neuro~havio~l Re~ch ~ra~o~, ~panmcnl o~ i'sychial~y. Duke Univeni~y M~ical Cenler, Durham. NC. WORKING MEMORY PERFORMANCE AND CIIOLINERGIC EI.'FEcrs IN TIlE VENTRAL TEGMI~NTAI. AREA AND SI]FIS'FANTIA NIGRA The n;col;nic ;mtagonist mccamylamino has been found In imp;dr working memory performance in the radial-arm m~o (RAM) al'lcr s.c. t)r i.c.v, admitdstnt- lion. Mecamylsminc has important interactions with dopaminergic (DA) systems. Mecamylamine-induced memory deficits in the RAM are pntcntlalcd by the antagonist raclopride and reversed by the D~ agonist quinpirnle. The nicolinic ago- nist nicotine has been found to improve w~klng mcmnry pcrfom~ance in I1¢ RAM • fief s.c. or i.c.v, administralia¢~. Nicotine-induced memory improvemeul in the RAM is potentiated by the D~ agonist quinpirole- The midMain DA nuclei, the sub- st-',nlia nigra (SN) and the venlral legmental •re• (VTA) have relatively dense con- centrations of nicotinic receptors which may be critical sites of action for mecamy- lamine and nlcoline. In the current stud)', the of feels of mecamylamlne (I, 3.3 and I 0 pg/s|de) infusions into the SN (n =: 12) and VTA (n -', 13) on working memory in the radial-arm maze ',,,'ere examined in adult female SpfaP.ue.Dawlcy rats. The ttg/sidc dose of mecamylamine significantly impaired radial-arm maze wo~king memory ped'onnancc when infu~d into.either the SN or VTA. No significant effects of m¢camylamine on response latency were seen. The nlcot~Pic agonlsls eylislne (0.1, 0.33 and 1.0 p.g/side) and nicollnc (0.3, 1.0 and 3.3 ;tgJside) were administered in a ¢oentcrbalanced order. The high dose of cytisine ( I pg/side) nearly caused a signlfican! deficit in choice accur'ocy. Nicotine slightly depressed choice accnracy but not slgolficantly in this study. The inlefaClion of niculinc and enccamyl,'mdnc was then studied. A dose of i.0 itg/slde of ok:aline cau~d a significant decrease in choice accuracy. Interestingly, this was significantly rever~ed by • 3,3-p.g/side dose of meea.mylamine. Studies of the mu~arlnlc antagonist .~opatamine (I, 3.3 and 10 pg/slde) and the muscadolc agonisl pilocarpinc (3, 10 and 30 iJ.g/sldc) did hal dclecl significant effects on RAM choice accuracy. These dais support the involvement of ni~tlnic innervation of the mldlwaln DA nuclei in memory function. Levin, E. D.~ Bsi~g.% S, L, U'~rislopbero N. C.. and Auman. J. T. Brain Research G57:165-170o 1994. Other support: N•tional Science Foundation. From Ihe Neumbehavlnral Re,arch I..abaralo~, Deparlment at" Psyehialry, Duke University Medical Center, Durham, NC. 2130 NICOTINE EFFECTS ON MEMORY PERFORMANCE Nicotine has been found by many but nul all studies to improve and ,mccamy. lamine to impair pcKormance on cognilive tas~ in hununs and cxpcrimcnl,tl animtls. In our laboratory, a very repruducible finding I~ been that chronic infusion of nk'o- line significantly improves memo~ performance |n • win-shill vcnlon or the radial- arm maz.c. Nicotlnc attenuates the memory d..r .':'-s caused by lesions of the fimbria- fomix or the medial basalocorfical pm~cclion. ~,~xoelc co-adminlslrallo~ of the nk'o- line antagonist mecamylamine eliminates the r icntinc effect. Some ~pe~ of Ih¢ cog. nltive dcfich in Ab.hcimer's disease are anenu~t..,'l by ~kollne. cu~gesting Ihe therapeutic use of nicotine o¢ other nicolini: tl.-.ands for cognitive Levin. F, D. andTony. D. In: Clarke. P.. Quik. M.. Adlkofcr. F.. Thum:.;cds, K. (~ds.): Effects of Nicotine on Biological Syslems II. Advances in Phemnacuh~g|cal Sciences. lIirkhauter Verlag Ua~l. pp. 329-335. 1995. Olher support: R. J. Rcynnlds. AIzhelmer's ' ~st~ia~inn. and the N•tlonal ,~knce Foundation. From Ihc Dcpa~ments of Psychiatry and Ph:.,'~a~cology. Duke University Medkal Ccntcr. Durham. NC. and Depaamcnl of llldchemlstry. Unlvershy of Bath. UK. ACUTE AND CHRONIC NICOTINIC INTE~.'~ --"finNS WITH DOPAMINE SYSTEMS AND WORKING MEMORY PEPFURMANCE Nicotine has been found Io improve men:~*.'y performance in • variely or lesls In rats. monkeys, and humans. Intcraclions of u!.. ~tinlc sDten'~ with dopamlae (DA) systems may be importanl for this effect. W¢ ¢~,nducled • ~¢rlcs or studies or nico- linic agonist and ant~gonisl internclions with ~A Syslems using ms in a wln-shill worLiog menmry task in the radlal.ann maze. "il~e wo~king memo~/del'tch caused by the nicotinic antagonist mecamylamlne w~-s potenllaled by the DiDe DA antago- ni.q haloperidol and the specific Da anlagoni "',clopridc. In cona-tsl. Ihe mecamyo lamine-induced deficit was reversed by co-au:n;~.islmloa of the DJD~ ogonisl qulw pirole. Nicoline also has significant iflleractio..s ~.'ilh dopaminc drags with regmd to working memory ped'ormaoce in the radial-arm maze. The DA xgonist pergollde did hal by itself improve radial-arm maze mem,'~ry performance, bul when given together with nicotine it produced an elevaled dose-dependeo! incRas¢ in choice accuracy. The Dt agontst SKF 38393 signific~nlly impaired radial-am, i maze choice accuracy. Nicotine was effective in ~eversing |:,|.~ del'~:it. W~cn g|ven to,ether with nicotine, the D~D, agonlst quinpirole impro,P'~ RAM choice accuracy relative In either drug •lone. Acute local infusion of me,',~.:£qamin¢ Io the midMaln DA nncle| effeclively impairs working memo~ funclion :. ~e radial-am maze. I~ t'ontrasl to acme nicotinic mantpulallons, considerably !~o evidence exists thai the effncls of chronic nicotine administration are Influen~-: by DA systems. This may be an example of the differcnl neural subslrates Ih,~l 'Jndedle the memo~' impmvemenl, cau~d by acute and chronic nlenline. Levln, I~. D. and Rose. J. E. 201
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C) Annals o/. 1he New Yock Academy o/' Sciences 757:24S-252. May 10. 1995. From the Departmem of Psychiatry, Duke Un|versily Medical Cooler Research Serv;ce. ~nd VA Medical Center. Durham, EFFECTs OF RETINAL LESIONS UPON TIlE DISTRIBUTION OF NICOTINIC ACETYLCIIOLINE RECEFirOR SUBUNITS IN TIlE CIIICK VISUAL SYSTEM Immun0hlslochemislry was used in Ihis sludy Io evaluate Ihe effecls o/. relinal lesions upon the distrlbulion of neuronal nicotinic acetylcholine recepIor subunJls in the chick visual syslem. I'mllnwing unilalcral relinal lesions, the ncumpil .staining wilh an anlibody against Ihe ~2 receplor sub,Jail, a major ¢omponenl of Ioxin-inscnsilivc nicolinlc receptors, was dramatically reduced or complelcly elimi. nal.Cd, in el! .o/. Ihe. conlralaleral re~inor~cipien! struclures. On ehe other hand, ncumpil slammg w~m anl~bOdles agaln.~l Iwn a-bungafnlo~in-~nsitivc n.'cepIor suh~,nils, and u~, was only slighlly alTeclcd arlcr rclin;d leslnns. I)~c.'n:-',~c(I ne,mpil slaining /'or faY-like immunoreacdvily was only observed in ~h¢ nucleus ol" the basal optic ~oo! end layers 2-4 and 7 of the optic leClum, For ,,g.like immunnreaclivily, sligh! rcduclion of neuropil slaining could I~ observed in Ihc vculral geniculale complc~. criscum lecli, nucleus laleralis an|erinr, nnclcus lentifnnnis m¢~,encephali, layers 4 and 7 o/' Ihe leclum, and nucleus supr, chiasm;,|icus. Taken Iogcll,er will, Ca~a on Ihe Iocalizalion o/' nicotinic rcceplors in the reline, Ihe preach1 msuhs indi- cate thai the [32 subunil is Iransported from rclinal ganglion cells Io their cenlral tar- gels, whereas the o7 and ,',8 subunil immunoreaClivily ai~..a~ to have a cemral ori- gin. The source o1" these immunoreacdvilies could be, at least in part, II~ stained Perikar~a Ihal were obsc~ved to conlain a? and ag subunits in all relinorccipiem areas. In ngrecmenl with Ibis hypothesis, the [32 subuni! of the nicotinic acclyl- choline receptors was rot fRqurmly/.onnd in Fcrikar),a of Ihe same areas. Brillo, L R. (3., Tartan, A. $., Hamassaki-Brhlo, D. E., Mpodnxis. ]., Keyser, K. T., Lindstrom, J. M., and Ka~len, II. J, "~e Joum-~! of Comparative Neurology 350:473-484, 1994. 0lEer supporl: Nalional Inslilutes o1" Heallh, Council/'or Smokeless Tobacco Research, Muscular Dyslrophy Associalion, California Tobacco-Related Disease Reseatth Program, FAPESP and CNPq (BrazE), and Fondccy! (Chile). From lee Deparlmcnls o/' Physiology and Biophysics, Depa~lmcm o/' Illslology Embryology, Inslimle o1" Biomedical Science,. Univusky at San Paulo, San Paulo Brazil. Dcpanmenl o/' Biology, College of So;cocos, Unlvrrsity o/. Chile, Sanlia¢o, Chile, Del~lmcnt ol'lqcurosclences, Universily o/' Cali/.omia Sen Diego, La .~olla, CA, and Depanmenl of Neuroscicnce. Univen~hy ol'Pcnnsylvania. Philadelphia, PA. NEURONAL NICOTINIC RECEPTOR STRUCTURE AND FUNCTION The besl characlerlzed ~blypc from Ihe Ixanch oflhe neuron.I nicotinic reccp- 202 tar (nAChR) gone family which does no~ b:.,,~ u-bung~otoxtn b~s Ihe subuni¢ slot. chiomee~ (a4)~(132), and ~counts for >~" mammalian b~ins. ~mni¢ tRatmonl o[c~'" :~s~eclcd whh a4~2 A~ an increase in ~ ~moun~ of ACER wilh p~,-.. cou~c I~ parallel the nlcotine-iMuc~ In~ ~., .so in brain ~4~2 A~. ~is is the resull of a decrease in lumovcr o¢ s~ A: . ~nenlly [uncllonally ina~iv~ ~e p~e:'- neutral A~R [ene family which binds ~ uniB. =7 A~Rs p~edomlnale in ~in, wh;, : ~7~8 A~Rs are a minor com~nenl in ~.~. liss~s. ~7 similar channel pm~i~, but ~8 h~on -~ and nalive ~ ACIIRs have lower affinity for ~-DTX and higher affinity for .mall hom~c~ and ~7 A~Rs. ~¢ high Ca" p:...~abilily ~d mp;d ~7 and ~8 A~Rs may enable them Io pa~i P~I¢ in unusual sy~ ~h~isms. Lind~lrom. J. Anaml. R., P~. X.. end Ge~',:;:¢h. V. In: Effects of Nicotine on ~io]ogic~l Sysle,,~s II. Advances in ~ha~colo¢~al ~icnccs. ~irkhanscr Verier. Rasel. Swilz~rln:~:l, pp. 4~.52. S~keless Toh~cu R~ea~ch Cou~il, Inc. Iqdh,~lphi,, CYTOCllALASIN MODULATION OF NICOTINIC CHOLINERGIC RECEPTOR EXPRESSION AND MUSCARINIC RECEPTOR FUNCTION IN HUMAN TE671/RD CELLS: A POSSIBLE FUNCTIO;oAL ROLE OFTHE CYTOSKELETON Previous studies have shown Ihat ceils of the TE671/RD human clonal llne cxp~ss musclt-t)~pe nlcolln|c ~cetylcholiue r~epto~ (flAChR) ~d m3.ty~ mus- carlni¢ acelylcholinc rcccpIors (mAChR) who~ numbers and runclion ~,~ legulale~ by agonist Irealmen! and second messenger modulation. Here we show thai lasin lrealmenl, which causes disruplion of aclin nClWO~k$, induces marked in the nurnbc~ and distribution o/. nAChRo bul ,1ol mAChR, Moreover, wherea~ cytochalasln Irealmen!/.ntis to aher nAChR/'c., ::ion slenIficamly, il ~'ulely polenli- • lea mAchR-medlaled phospholnos|llde hydr~..--,is. T~almem of TE6? I/RD cells wilh dilTerenl cyluchalasln analogues (rank o: ,¢," clTca¢~ al 5 p,g/ml is H > J C: D > A : E) l~duee~ = two- In loud'old in~;~se i~ numl~-rs of mcmbr~n¢-tm~md nAChR (F~_ in unils of R~ceific "l-labeled ~'...~.nngaroloxin t~nding per milligPam or membrane prolcin), nAChR upregulalion is o',P'f.'.nl afl~ I-2 da~ of cylochalasin B ex~.spre, is maximal after 3-6 days of drei ~realrl~nl. and is dominated by an npproximalely 10-fold increase (per col~. ;o an iolracellular nAChR pool. Cytochalasln-lodu¢cd nACkR upte~ulalio~ is ~;milar in mash|lode IOo bul hal live with, up.regulalion ofnAchR following r: "~,11c expmum Io nicoline or osier. Nonhero blot analysis shows • four- Io -'-tom ~oordlnalc incense in levels o1" mRNA Ihat encode nAChR a, [3. "y. or ~ ..~mnhS |n cylochdasln-Ilcaled calla, 203
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suggcstinB Ihal nAChR up-reBulalion has a possible Ir-"nscri I'd " ' d°ne using | ~'Rb" el'flux assay indicate thai vl olaf; ...... p t. n. al h~sls.. S.~dlcs " ¢._OCh ......... ~almcot has no $1~nlllCan| efrecl on nAChR funclion. By conlrasI, cylnchalasin Ireatmenl has no cf/'ccI on the numbers of mAC~R as asse~ed hy binding r.iudies wilh die radioanlu~nnisl 'll- labeled qulnuclidinyl benzilale, hut it induces marked enh:meemeo! of carbachol. slimulated, bul no! basal, pJmsphoino,~illde hydrolysis. "l'hesc ~lmlie,; .~uggesl ehal presumed modulation by cylochalasin trealn~enl of cyl(P;kcletal microrilamen! inlcEril), can differentially in[lueuce expression and runclinn nFmAChR (a, ptomlylx: of Ihe mclahnlrnpic rcCeplnr sulx:rfamily) add nAChR la prolol)./x: of Ihe ligand. Baled !.n-channel sued'emily). The resells ul.,m sui;gesl ~s,~ihl¢ new roles fro" the Cyloskelelon in regulalinn of membrane receplor cxpr~si.o. Functinn. and cross lalk. Bencher!f. M. and Lakes, R. J. Journal of Neurochemis|ry ~l(3):gJ2-R64, 1993, Other supix'.n: Men's and Women's Boards oflhe B-"now Neurological I'-ound~don and Epi-Ilab Phoenix. Inc., Arizona Disease Conlrol Research Commission. Alcohol. Drug Abuse and Menial Ileallh Adminislralinn. and II,e Smokeless Tobacco Rose.oh Council. From the Divison of Neurohiology. Barrow Neurological Inslilule. Phoenix. AT. DIVERSITY AND PATTERNS OF REGULATION OF NICOTINIC REC~.PTOR SUBTYPES In our slodlcs we explored Ihe f`unclianal relevance of nAChR diversily, in pan f`rom Ih¢ ~rspcclivc nf nAChR a.~ ideal target,; f`or regulatory influence.,;, including Ihose medlaled via actions of ligands a! Olher *'inlcmclin;" rccept(~s. We expln~cd possibic n~chanisms for nAChR regulation ~ roles played hy nAChR subtype and suhunll dive~ily in Ihose proces~s. We showed ~hal regulalory I'aclors can influence nAChR numbers al Iranscriplional and postwan~ril~ioual levels aml can affcel nAChR funclion and suhecllulm" dislribulion. We also demonstrated Ihai nAChR expression can be influenced (I) by nlcolinlc liB~nds. (2) by second messengers. (3) by gruwlh factors. (4) by agenls ta~BelinB Ihe nucleus, and (5) by agoras largefing !he cyloskele- Ion. We found common el'f`eClS of` some regulalory influences an mare Ihan one nAChR sublype, and we fonnd insl~ces where regulalory influences differ for differ- enl cell and nAChR types. Eveu from !he very lira!led number of these ini¢ial studies, it is evidenl Ihal nAChR subunil and sublype dive, ally. which a/one can provide diversl- ly in nAChR f`unclions, localization, and ligaod semldvily, dovelails with divcrsi!y in ccllulm" signaling mechanisms Ihat can affecl nAChR ex~x'e.~ion In amplify Ihe lial f`unedonal plus!icily of' cholioneeptive cells. As examples, we disused poleniial roles for nAChR diversily and regulatory plaslicily in synapse remmicling .~nd in changes in neuronal circuit condilioff'¢. 'Ibe~ exa~npIcs ill,xlrai~, how nAChl~ divee~hy could play import~n! roles in Ihe regular!tin nf ncn~ous syslem funcli~n. Lukas. R. J. Annals of the New York Academy hi" Silences 7~7:1~3-16~. M;~y Ill. 2(~4 Olher sup)on: Hal!anal lnslilule on Drug ~.;,,use. Nalio~! InstilUle or Neumloll¢~l Disorders and S!roke, Smokeless Tobac~, Research Council. Arizona Disease Con!rol Rcseureh Commission. and !he Amrri,:an P-,kinsou Disease Associallon, From !he Division of Neurnbinlogy. Ban'ow ,~eurological lost!lute. Phoenix, AZ. I IIGll AFFINITY IIINDING OF TIlE E/V'I::.,fOEBA IIISTOL)'TICA LECTIN TO POLYVALENT N.ACETYI.GALACTOS/,'..~;NIDES Entamneha hi3tolyti¢o Irophozoi!es in~.:aic palhogenlc colonizalion by adhcr. Once Io hosl glycneonjugnlcs via an amebic -office leclin which b~nds IO l:alaclo~ (Gel) and N-acelyl~al~ctosamine (OalNAc) ;¢sidues. Monovalenl and mullivalenl carbohydralc llgands were screened for inl;?,ilio~ of` £. hijtolytico iccdn.medlaied human red cell hcmagglulJnalion, revealing thai: (1) Ihe synthelic muh|valenl ncoBIP coproteln GalNAc,.BSA (having an overall,. ~ 39 GalNAc residues linked Io bovine serum albumin) was 140.IX)O-fold more pal, ~,' an inhibiloe Ihan monovalcnl C, alNAc and $00,000-f`old more po!en! Ihan mouov-',:.-. : G;d; and (it) small s]ml~elk: mukivs. lent ligands which hind wilh high affinily ," "he mammalian hepatic Gal/GalNAc lectin do no! bind vi!h high all'rally Io Ihc :. his~olydco I~lin. R~dloli|a~d binding studies revealed salunthic binding at "'I-G. " I^c,,BSA Io E'. ~l{s't/~).t/'fo (K~ -- l0 _ 3 aM. I~ ~, 0.9 ~' |).0B pmol/:'l~ rncothlcmc ~elrl). Maximal binding required the presence or calcium chic!de (JCJ p,M) O¢ sodium ehloride (50 raM). and had a broad pII maximum (pH 6-9). GalN^c,,BSA was 200.O00-fold mine Ix~enl Ihan monovalcnl GalNAc ;n blocking radio!i,:',~d binding. Among synlhetie ride-derivalized line.at polymers. Ihe GalNP.: z-; nnd GalNAc=JOall3 derivalives the mosl poeenl, wilh GalNA¢,- and Gal. ~ ' c(~3(Fuc(~2)Gall] tier!relives much weaker. The dale suppa~! a model in wh',, • unique pallem or spaced mull!pie GalNAc residues are Ihe hi.ghesl af~nky earle:: for ~he E. Idstnl.wt¢o Adler, P.. Wood, S. ~.. Lee, Y. C.. Lee. R. T.. ~elri, W. A.. Jr. ar~ Schn~r. R. L. The Journal of' Biological Chcmislty 270(IC;.:.'~ IM-$171. M~ch !0. 1995. Olhcr suppo~:: U.S. Army C~emical and L.;,.ogical Defense Command, EdCcwood Research Dcv¢lopmenl and Engineering "'.~.-.Icr. and Ihc Nal|onal Inaiilulcl of I Icahh. From the Depanmem or Pharmacology ~nC l,¢oloeular Science. The Johrd Hopki~ School of` Medicine, Ball!more, MD, U.S. ,'.'my Chemical Rcseaxch Command. Aberdeen MD. Deparlmenl pf Biology. Johns Hopkins Unlversily. Baltimore. MD. and Ihe Dcpa~lmcnls or Mid!nine. Palhology and Microbiolol:y, U,,iversily of Vir¢inia School or Medicine. Ch-,'lonesville, VA. STABLE EPISOMAL TRANSF~.~i'ION OF ENTAMOEBA 1115"TOLYTICA The enteric prolozoan Etltanmet~ h/st'~l~lira Is Ihe causative agent or nmebPa- sis. The foclors required for virulence of the or~nism arc ill.d~l'med ~ stodics or 205
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ca) the parasite at the molecular level have Ix:on limilcd by the lack of a DNA transrec- lion system. Recently. methods for transient transf'ecfion of E. hisrnlytirn were dcvcloix:d. We rein here th~ dcvcl~mcnt of a s~ab~e t~ns[ccfion sys~ rot £. kiJlol)~ica t~ugh I~ use of drag seleclion. To dclc~inc i~ lhe neomycin ~a~c g~c (nOB) could ~ used is # scl~- table m~kcr, G418 a[ co~emrali~s o[0-50 pg ml" was added in ~al dilutions m amebae growing in TYI-S~3 medium. Arler 72 h o~ ¢roweh a~ ~7"C, G418 ~cen- ~a~ions over 3 pg ml" killed I~ o~lhe ameba. We concluded d,a~ E. ki.unly~ic~ is qui~e sensitive Io G418 a~ Ib#l non could ~ u~d as ~ ~Icclahlc marken Vines, R. R., ~rdy, J. E,, Ra¢land. B. D., Samuel~n, ~., Mann, R. ].. and Pelrl, W. A., Jr. Molecular an~Bi~mical Parasilolngy ? 1:265-267, I~)~. From Ihc Oepa~mcnls of M~dlcine, Microhiol~y, a~ Palhology. University o~ VirBtnl#, C~Holl~ville. VA. an~ ~pan~nl o~ Tropical Puhlic I lcallh. I la~ard Sch~l o~Publlc Ile#lch. Boslon, MA. PIIYSIOLO~ICAL DIVERSITY OF NICOTINIC AC~'TYLCIIOLINE R,~CEPTORS EXPRESSED BY VERTEBRATE NEURONS "The I~ d~ade has rev~lcd ~ asl~;ng de~ of physiological and rural div~ity in ~orotnn~ittef-~ct~vated ~cc~ms. in ~icu]ar, molecular stud- its o~ lhe aceiylcholine.galed ~e~ors in vc~¢~¢ muscle cells :~ Torpedo oleo- ~ylcs pvc ~nb w a new e~ o~ ~ch on s rclalcd su~amily o£ nicotinic rece~ ~o~ (hAiRs} cxprcs~d ~ cenlml and ~fi~eral nouns. ~is review f~s on the nA~R su~y~s cx~c~d by ~u~s. view~ ~inly ~r~ a ~ysinln&ic~ s~li~. It iS ime~ed Io c~plcm~ ~v~l ~cellem ~eccnl reviews ~ musclc- ly~ A~Rs ~ ~ lhe ~ammical disld~lion, blmhemisl~, and molecul~ biol~y o[ neum~l nA~. ~c ~ summ~ in this ~w ~ ~l~d ~ a l~g-st~ing flow c~ t~ ~ll-~own ~havi~l. c~nidv~ ~ ~ivc cff~ ~ n~ine ~il~ ~ ~ ~ly o~ cvi~e ~ CNS s~ w~c ~he l~sm~ion is mcdil~cd by nicol~ic rcc~? ~e Id~lificalion o~ s large [amily o[ neutral ~R ~[t g~ ~ ~ ~y or ~u~i~}y dislin~ hAiRs h~ giv~ a b~is ~or ~c divc~ c~ o[ nicmi~. Pe~he~ ~ im~n~l is emerg~l evidc~e [or ~io~ m~ Of ~, ~ welt ~ ~, ~S hAiRs that c~[e lo extensi~, m~late ~milt~ rel~. ~ t~in eff~ ~e focally. We finl summ~ze Ihe ~lccul~ Sol~y ~a~ h~s calalyzed I~ Iransillon the cuncnt "slalus nicodnicus," ~xt discuss I~ divcfslty in suhunil com~shlan, i~ic ~eability. I~li~llon. ~d m~ulalion of neural nAChRs, and finally ~nslder physi~ogi~l ~ for I~ exlem of nA~R c~nne} dive~sily ~d ~ fulure te~arch M~ehee. D. S. and Role, !. W. 206 Annual Review of Physiology 57".521-546. ; Other support: National lnstilules of Ih~'th and the McKn|ght and llirsehl Foundations. From the Depanmcnl of Cell Biology and ,' qatomy in Ihe Center foe Neuroblology anti Behavior, Columbia University. Collcg,~ ..f l~hysicians and Sergeons. New Yo~k, NY. NICOTINE ENIIANCEMENT OF FAST EXCITATORY SYNAIq'IC TRANSMISSION IN CNS BY PRF.SYNAPT;C RECEPTORS Tbc hehavinr.~l and cognilive niT, Is of ..;cotinc sugfcsl thai n|colinic ac~tyl- cho|ine receptors (oACbRs) panicipnte in £,":..el nervous syslem |CNS} funclio~. AIIhnugh nAChR suhunil messenger RNA (s,,RNA) and nicotine blndln~ sites are coramun in .he brain, ill-'n: is little evidence f. r synapses medlal~l b)r nAChRs in Ihe CNS. To tesl whether CNS nAChRs mif: ' ,.~lify ~lhcr Ihan mcdmtc Iransmls. sion. the rcgttlation of excitatory synapdc trot....ission by I~ese reccplocs was exam- incd. Nannmolar concentrations of nicoline eat' raced both glulamaterglc and cholin- ergic synaptic transmission by activation n. pmsynaplic nAC'hRs that increased presyn-,ptie [Ca*'|.. Phan'nacotogieal and s, ~>.'~it dclctlon exl)c~iments I~veal that thes~ presynaptlc nAChRs include the a? s, ;:unit. These findings fl:vcal Ih~l CNS nAChRs enhance fast excltalory Iransmlssim,. providing | likely mechanism for the complex behavioral cffecls of nicotine. McGehee, D. S., Ileath, M. J. S., Gell~r, S,. [~:vay, P~ and Role, u W. Science 269:1692-1696. September 22. 1995. O~hcr support: National Institutes of llealth. ;,tcKnight Fotmdat[on. Foundallon for Ancsthesla ~,ducation and Re,auth. mad fro~.' From the Delx'mment of Cell Biology and ,*... ,t,,my in the Ccnt~ for Neurobiology and Behavior. Columbia University. College -" • :,ysicians and Surgeons. New Yolk. NY. and Deparlmcnt of Anesthesiology, Cole ..,.:a Univ=~,zity. N~w York. NY. DISTRIBUTION AND RETENTION OF N!t, , INE AND ITS MEYABOLn'E. COTININE. IN THE RAT AS A FUNCY[O: ". -'rIME Nicotine is oxidized to its major melabO;"e c~lln[ne, which has • long biologi- cal half-life (19-24 h). The plasma conccnl~. • ..n of cmininc has I~en used as an index of Iobacco smoke exposure. Colininc :ibly Incmmses the mmo~cr r~W or platel©t.actlvadng factor (PAF) because it ~ ~ potem activator of PAF hydmlase. and it may play a signiBcant rote in tot~cco-:.; ~.nced an~'iat thrombosis. Therefor. we sludicd Ihc distribution and rclcntion of nicollno as it ~ metabolized to co, thine° in Ihe rah Nicotine (I mfJkg. ~ ttCi/kg)'wa'~ o~:minlstcn:d Into Ihe femoral vein of 207
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male Sprague-Dawley rats under nembutal anesthesia..4,t cliff.teen! times (:5-60 mln) alter nicotine administration, nicotine and its melabolile, ¢otinine. were dr.lcrmlned by IIPLC in plasma, liver, kidney, heart and hraln. Within 5-10 rain alter administra- tion, nicotine concenlfatlons reached peak values in pla'~ma (2.16Opmol/ml) and the organs analyzed. The plasma level or nicotine decreased hy 5()"/,,, wilhin 211 rain (half-lime) alter ils intravenous adminlstratlnn. The half-time of. nlcolin¢ in the hr~in wa,; about St) rain. The hall-times ol" nicnlion for Ihe miter ~'~:lns were aboul 2(I-25 rain. The major melabolite, cotlnine, accumulaled in plasma, and by al~tul 30 rain Ihe conccnlrafions or n;cotlne and cotlnine in plasma were about equal (It9(I. pmol/ml). While cotlnine accumulated in plasma, nicotine was elimlnaled hy the kidney. While the nicotine concentralions decrca~d wilh time in all organs, cntlnine concentrations remained constant. 1'hose ohservalions indic•It tea! nicoline is tonally eliminated or metabolized to cotinine while cotlnlne exhihits a long releotion lime and accumulates in plasma. This plasma accuntulalinn may comrih,,te to •city•lion o1" PAF turnover rate and to Ihrembosis. Saslr.v,/I. 'V. R., Chance. M. B., Singh, G., lforn, J. L., Jansnn, V. E. Pharmacology 50:128-136. 199;5. O~her support: U,S. Department of. lleahh and lluman Se~ices-Natlnnal ln~tht~l¢ on Drag Abose, and the Sludy Center f.or .Aneslhesla Toxicology. From Ihe Deponmenls al" Pharmacology and Anesthesinh|gy, Yandcrbih Univer~;ity School ol" Medicine. Na'~hville, "T'N. SMOKING, PLACENTAL FUNCTION. AND FETAL GROWTI ! Solon/liCe literature published durin¢ Ihe past century indicates Ihal ciga~clt¢ smoking dur|n~ Ixcgnancy has si~nificanl advers~ elTecls on Ihe dcv¢lopmcnl or Ihe I'elus and Ihe heahh ~d development of Ihe newborn baby. The h-',z~rdous ¢ffccL~ of smolcing du~n~ pregnancy include possible abortion, feral death, relal growth retar- dation and short- or long-lotto developmenlal dcl~cits in lhe |of.ants. Concern aboul Ihe exposure or preCn~ll wom~n In Iobacco dust and smoke has bc~n eapressed since 1865. AIx~lion was I'rcqucn! among women workers in tobacco lractories in Europe. There was also a high rule o1" mortality among Ibe intanls o1" females in tobacco-relaled occupalions. In 19350 Soma~ and Wallace observed Ieal cigasellc smoking by prcgnanl women incre~.~--d I'etal heart rate. and Ihey speculaled Ihat Ibis was probably due to transplaccm~l Iraosf.er ol" nicotine iron felal circulation. Campboll observed Ihat a woman who smoked heavily durin~ pregnancy w.~,,~ llkcly to have more dil'l'icuhy during the course olr pregnancy, panurilion, and laclalion Ihan a nonsmoker. In 1940. E,~nbcrg ~nd collaborators investigated the influence or nicotine and Iol~.cco smoke on pregnant alhlnn r-',ts. "phey feared that the young or olcollne.lrea~ed as well as Inhucco.smnk¢.cxix~sed rals were unde~wcii:ht compared Io Ihose o1" control unlreated rats. These sludi~s raised important questions about clT¢c|s or Iobacco smoke and nicotine on intraulerine I'eral growlh retardalion (IUGR). In 1957, Simpson repotled teal babics or women smokers had significantly lower birth weich| than those of nonsmokers, $impson's sludies were confirmed hy several o~hers involving more |ean halir a million h|rlhs. These studies have been dis. 208 cussed in detail in reports ot the U.S. Sue. Jn General. In |ener~, I~h~es or nanl w~ smokers are 2~ & liBhtet ,~'.n ~bies ~ to women whn a~c nnnsmoke~. ~ m~h~: .~ms o~ ~okin~-induccd IUGR and rnlc o[ placenl~! ~u~d~ in IUGR have yet to ~ established, 1995. Olhcr snp~rt: ~tudy ~¢nlcr for ~ncslh, '¢~ Toxi¢oJo~y, ~ashville, T~. U.~. ~paflmcnl o~ Ile~ll~ ~,~ Hum~ Sc~s, ~ali~l Insdlules of I leal~h, and the ~alional Insfilule on D~ .... bose. From Ihe Dcpaflm~nls of AncslhcsJolo~y.,., ~acolo~y, ~bill U~Jve~J~ Medical C~nler, Nashville. TN. OPIOID ADDICTION. PLACENTAL FI.t~'..qON, AND FETAL GROWTll Illlcit u,~e olr up|old drugs is a major ~" .,: long.slandlng problem in the United Slalr.s. The most widely used illicit up|old is heroin. The opiold-dm|-usln$ popola. lion includes a significant porccntage of ,e ..-rrcn. The pmponio~ o~ women i~ulicl. paling in drug IrcalmCnl programs supp,,.':d by Ih~ Nalional lnslilule on Drug Abase reached a level of 30~ in Ihe 1980.;. ~ ma, iodly ol'lheso women ~u~ o~ child- bearing age. In the United Sl~eS, an eslim".l:4 10,0O0. children annually have been born to women usln~ oplold dn~gs, mainly r:.=;,~hlne and hem~n. Opiold use durime pregnancy has Lc.'~; ;nvestiga~cd darin~ the p~| 35 with regard to ils ¢lTcclS.On Ihe woman, el%. irelus, and the child. |Is us~ presenls medical, obstelrical, and managemen! probl¢,ns, including Ib¢ dill~cully in deloxlry- ing the pregnant heroin addicl~ I'e|al dtsl~'., due Io maternal abstinence I'mm dm~. which is even more harml'ul to the fi.l|.~ Ihan passive dependence; and withdrawal symploms in the I'elus and inf',.::..4, comFeehe~slv¢ mel~done nancc Ire•(meal (MMT) wilh approprla~ ".|cthadone dosage levels can possibly rech~ce the incidence of obstelrical -',nd fel-,I c~.mplicalions. gasify, I]. V. R. In: S.~try, 9. V. R. ted.): Placental Toxic ~ ~,y. Chq)l~ 4. CRC Press. pp. 1995. Olher supporl: .~t~dy Center I'or Anesll,, • Toxicology. Vamlerhih Unlverxily. Nashville. "IN. U.S. Deparlment or Heel;;....~ lluman Services, and Ihe National loslilutc on Drug Abuse. From the Depanmenls of" Anestheslolo&y z.zul Phannacobgy, 'Vandc;hilt Unlv~ip~ Medical Center, Nashville. TN. 209
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COCAINE ADDICTION. PLACENTAL FUNCTION. AND FETAL GROWTll One o~" Ihe serious and 8rowinl problems which will hlve an impacl od Ihe heallh and wcll.lx:ing or pregnant women iS coca~ne.~cr~ck abuse. |I is eslimaled lhal 11% of the Uo S. p~pul.don regularly use cocaine; :2 to 3% are heliev¢(l In use .c~caine during pregnancy. A survey in 16 states and Ihe DislHct of Columbia involv- ing 50,0¢X) people indicales that women of childbearing a~-" (18 to :34 years) cnnsli- rule 15% of all regular users of oncalne. The human fetus is dcpenden! upnn malernal cnvlmnmenl for its sa/'cty, health, growth, and development. O~caine and other abused d~s in~c~led ~ ~c~n~l w{~n may dan~gc Ihe dcvcb)pmenl nr Ihc fclus by many mcchankm~. C~aine and ils mcla~li~cs may inlcKere wilh no~al physiological p~scs of p~e~nancy, resuhin~ in fclal Ins~, prcmamril~, and Hcal complicalion~ Ih~ca¢cnlng m ~lh malcm~l and infanl hcahh. C~aine and/~r ~cla~li~es may cn~cr Ih~ pla~nla and ime~cre willl ph~colal Ira~ ~ nu[r~ms In {he fc{us. ~us. Ihcy cul Ihc maternal su~l~ li~ ~o Ihe developing feint. ondl~ ils mela~lilcs ~ss I~ placenta a~ inlc~cre with gmwlh and dcvdopmenl. ~lh ~ys~csl ~nd mental, causinB ~uccd birth wci~h~, h~nh c~fccls, Icam~n~ and ~havioral district, and ~w~m distress. ]~¢ immature cn~y~ sy~lc~ns in devel~ing fclus canno~ demxify drags. ~crcforc, c~a/,c use may cau~ cmnplica- lions due In ils effects ou I1~ ~lher. pl~cnla, fetus and nowhere Saslry, ~. V, R. 199~. Olhcr sup~: Sludy Cenlcr ~or Ancslh~ia Tax;colony, YandcrbHI Univc~ity, Nashville. TN. U.S. Dcparlmenl of Hcallh and Human Services. and National Inslilu~c on Drag Abuse. From Ihe ~panmcnls o~ An~t~siol,gy ~d ~a~ac~logy, Ysndcrhil( University M~dical Center, Na~hvifl~, TN. STUDIES ON THE DIVERSITY OF MUSCARINIC RECEPTORS IN TIIE AUTOREOULATION OF ACETYLCIIOLINE RELEASE IN TIlE RODENT CEREBRUM USING FURAN ANALOGS OF MUSCARINE Previous studies have indicated Ihal Iwo feedback mechanisms, one positive and the olher neRalive, regulale the rule of acelylchnl|ne tACk) refuse. The posilive feedback syslem hls al leaSl lhr¢e componcnls: I muscarlnlc receptor (Ms). release of substance P (SP). and influx of exlr~cellular Ca". Ir lhc amnunt of ACh from lhe p.resynapl|C nerve lermlnal is low. lhe released ACh aclivales Ms. which. slimulates the ~elease of SP. Subs|ance P Increases Ca" influx, resulling in lhe release of furlher quantilics ~d'ACh fmr cffecliv¢ chollnergic Iransmissinn. ~imilarly. lhrec compunenls Ire prescnl in lhe negative feedback sys:em: a presynaptic carlnic receplor (Mi). release of mclhionine ¢~kephalln (MEK). and inhibition Ca~" influx. If ~he ACh in lhe syn~pli¢ gap is hi~h. il acllvalcs Mi. rcsulling in Ibe rele~.'~ ~f MEK. Mclhi,nL'~ enkephalin decreases Ca*" influx which dccre.-~cs rate of release of ACh from lhe chulinergic nerve terminal. "11~e crhical first slcp in 210 bath of th~se I'e~dback systems is acllvatio.~ of pt~'~aptlc musc;+~n(c ~.'ceptors. Ms a~ Mi. No seleclive ~¢~ist~ h~ve ~en ~.:d~d f~ ~sy~ mu~n~ ~ep. tors. Thercr~e, we studied Ihe effecls of ~-melhylfurfu~hrimelhylammonlum (5-M~ n~ 5-hydmxyh~rfurllfime~bylan,nlonlum (5-11M~) on Ihe simuh~cous release of ACh, SP. and MEK from lip eu~rrus~ mon~ cereal slices in our search got ~lccdvc ~onisls for Ms and ~Z ~ecc~. 5-M~ ond 5-HM~ were sclccl¢d ~or ll~is p.~ ~cau~ I~y ha~-'. ,flvc~e eff~ls in I~ ~dpheml ne~'ous ~aslr~. II. V. R.. T~ycb, O. ~.. end Jnl~wal., ~. Annal~ o~1~ New York Ace~my or Sc~c~-¢s ~57:1~-196. May I0. 1~5. Olhcr snp~: U~. Dcpnnmenl at lle~hh :r'J Iluman ~e~. Halbnal lnslilUle m ~g Ahu~c. ~nd ~ Slody C~tcr ~or A~,e:t~ Toxicolopy. From Ihe Dcpa~menls o[ An~slhesJolney • ;'l ~a~acoloBy, Vandcfb[ll Unive~lly Mcdlcal C~er. N:~hville. NEUROPllARMACOLOGY OF NICOT1HE: EFFECTS ON THE AUTOREGULATION OF ACETYLCIIC,.'.dE RELEASE BY SUBSTANCE P AND METIIIONINE ENKEPIIALIN IN P. 3DENT CEREBRAL SLICF..S AND TOXICOLOGIC^L IMPLICATIONS I. The neuronal release of aCelyl¢ho:b ~ IAC~) and |15 autor~gulaliOn by neum- modulator, snhstnnee P (SP) and melhio~,' , ~kephdin (MEK). hive b~n SlUdled using supcrfused rodcnl cerebral ~lic+;, " :oti~e e~e~ts signifieanl effects on uumrcgul~tion of ACh release, which ma.~ ;,. ~. Ioxicolol~iCal impliealions. 2. Posidv¢ and ne~talive fecdlmck sy. :: bare been postulaled for autoreeula. lion of ACh release. Th~ components of Ih+..':)sitiv~ rccdback syslcm include a c,~rlnic (Ms) rccepIm'. SP, aml ~¢livnli~ c~ .:a:' influx. Low levels of AC'h in blopha~ o1" the choliner~.ic synapllc gap P..ay Iriltr~'~" Ihe posilive fecd~ck system, and high levels of ACh may Iriglter the ne~;~'~t e fecdl~:k syslem. 3. There are also neuron+';I pnlhway: ~.., '!iRcl mcil~'ocal re~Ulalions of SP and MEK, 4. Low concenlr-',tious of nicolin¢ IriS.,. "l.+ Ihe ~lcase of AC'h followed by MEK and SP. R¢le~e of SP causes ncumsenle i,', -'.-~mallm. 5. Nicotine and ils metatx~lite, colinin¢, ~.livale plalekl aclivalia¢ I'~cm~ hydrolasc w~d theRby enhance lhe lumove- :;J¢ of PAF. This ell'eel rely In tobacco.induced arlcrial thromhosls in I~' .~'al and cemral oeP~ou$ systems, $~slry. B. V. R. Clinical aml Exrerimcnt~l PharmacoloBy snd Physloln~y 22:2RR-290. 1995. Olher sup~n: U.S. Dcl~flmeni of Heallh ...4 Human Services. Nalional |nSlilule Dru~ Abut. and The Study Center rot. Anc..-.hesia Toxicology. • Frmn th~ r)cpa~Imcnt~ or Phnm1.~:olo~.y -'m.1 An~lheslnln~y. Vand~bill Univershy Medical Cenl~r. Na~hville. TN. 211
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EVALUATION OF TI IE NATURE OF RAT RETINAL ACETYLCIIOLINESTERASE USING A SPECIFIC SUBSTRATE AND A SPECIFIC INIIIBITOR The occun'cncc or cholJnestcrases (ChE) has bccn denlonstrntcd in retinas of several mammalian specics. Using BW21t4C$1 and Iso-OMPA as sclectivc inhlbimrs of acctylcholineslcrase (ACHE) and hutyrylcholine.,;Icrasc (fiChE}, rcspectlvcly, il has been demonstrated thai the rat ~linal ChE is predominantly ACHE. Thercfo~ the kinetic nature of inhihilion or the rat rcdnal AChE by RW2g4C5| was studied using acclyl.~-m~'thylthiocholin¢ (AMTCh) as a scl/:cllvc suhslrate at' AChI~ AChE -ctivily of Ihe ra; relinal sonicates was as.,myed using AMTCh a~ the substrale in the presence of $.5-dithiobis-2-nltrobenzoate and yellow 5-thio-2-nilrohen~.oic anion was measuRd by Ihe absorplion ai 41:2 rap. using I speclrophotometer. The subsl~le (AMTCh) was varied between 0.1 and (1.5 raM. The inhibitor concentratinns used were 2.1 and 4.2 nM. Double-reciprocal plots between suhslrale ¢onc~nlralio~s and the velocities I'nr the enzymatic hydrolysis of AMTCh in the pre.senco and absence or inhibilor were conMrucled. This sludy gave Ihe following resuhs: IIW2XdC51 was a potenl inhibitor of the hydrolysis of AMTCh by rat retired AChE (ICSfl. 5.2 nM). The nature of the inhibition was found Io Ix~ compelhive us Ihe double reciprocal plots wilh and withoul the inhibilo¢ crossed on the ordinate. Saslry, Bo Y. It., Singh. G.. Log. P.. and Janson, V. E. Journal of Ocu|ar Pharmacology and Therapeutics I I ()):401-409, 1995. Other support: The Study Center I'm" Ancslhesia Tnxicoh)gy and Ihe S~nokclcss Tobacco Research Council. From ~he Depanmems or Aneslhesioloey and Pharmacology. Vandcrbil! Universily Mcdical Center. Nashvill,'. TN. X, Pulmonary and Respiratory Systems DECENTRALIZATION OF TIlE SUPERIOR CERVICAL GANGLIA IN IIIBITS MAST CELL MEDIATED TNFa.DEPENDENT CYTOTOXICITY. I. POTENTIAL ROLE OF SALIVARY GLANDS Decentrallzallon or ganglioneclomy at' the superior ccrvical ganglia ($CG) reduces pulmoaary inflammation, as well as ¢hemolaxis and aclivulion or circulating ncutrophils, llowever, the pcolecl|ve effccl or decent~lizatiem was abolished when combined with removal of Ihe submandibular g:ands (sialadencctomy) in the same animals. Thus, it has been postulated Ihal Ihc submandlhular glands (SMG) release an anli.|nFlammaloty f=clor(s) thai is conlrolled by cervical sympalhctic nerves. l:)ccenlrallzadon of SCG did no~ modify in vilrohlslamine relea.,~e or in vi~'n levels ol' rat ma$1 cell pro~eas© II. bul il reduced m~! ucll (MC).mediated minor necrosis I'ac- lot (~ (TNFe,).dependen! cyloloxlcily. Combined decemralizallnn/sialadeneclomy abrogated the inhibition of MC cy¢otoK~c activity, as we have shown previously for pulmon~y inflammalion and oeulrophil fun¢lion$. Ilowever. sialadeneclomy alo~e 212 inhibited MC-medl-',lcd TNF~-depende~.: • .:olo~icily0 an observation which sug- gesls Ihat SMG prndt~ce a I'aclof(s) Ihal cme "o:enl|Mc MC cyloloxic Klivily. at' Ihe el'feels of SMG-derived I'aclors, " . :~ as epidermal growlh rlclor, nerve growth factor, ~nd ~nsfornltn~ |~owlh f,~ .~ r p O'GF,), showed d~ll only mcnl of MCs with TGF, 10' gJml inhibileo :,lC-medialed TNFm-depe~denl icily. Thus. IhC mo~ulallon of MC-mcdlai-.,.: -. NFm-depondenl c)'toloxicity I~ cal sympalhctic inncrvalion and SMG is r, ,.~plcx and disllncl fn)m the mndulslion of pulmonary inflamm,qion and neulroph;; :~ ncdons idenlifled prevloudy. Bissonnelte. F_ Y.. Mathison, R., Caner, I avison, J, S., and Berus, A. D. Brain, Behavior, and Immunily ?:293-30(~.. 993. Giber suppon: Alberta Ileritage Foundaiio;:. r Medical Research. From the Deporlmenl of Medicine, ~'-:.ersity of Albe~a, Edmontolt, and Deparlmenl of Medicnl Physiology. Uni ,:ratty of Calgary. Calgary. Alberla. Can,~da. TEMPORAL ANALYSIS OF THE ANTI- ..~'I.~MMATORY EFFECR~ OF DECENTRALIZATION OF TIlE RAT St. "';RIOR CERVICAL GANGLIA Bilateral deccntralizalion of the sup.,.;r cecvical ganglia (SCG) reduced the pulmonary inflammation that develops ~ h slier inducllon of an|phyl|xls in I~ippo~trongyhtx brasillen~i~.sensillzed r, .'ilslamine levels in perko~eal lavage fluid and ra! m~! col| prolcasc type ii in .~'." .~ wc~c inc~ased Io comparable levels in sham-oper~led and decenlralzed r~ls. In ~'~, ~ sllmulallon of ulveolar macro~hages (ALM) with lipopolysacchadde (LPS) pro: *ked lumm" necrosis I'a¢lor.a (TNF.~) release thai wax Iwo 1o Ihree limes greater w;~h unchallenged d¢cenlr~lJ~,¢d mls than wilh sham.opcraled rals. Ilowever. arler all:rsen challenge Ll~-sdmlllaied TNF.~ release I'rom ALM of,,ham-ol~r~led mls In¢;:ased th~efold and l|sled II lell124 h0 whereas with decentralized rats release o1"~! :. -)1ok|he Kltmlly dcceta_sed ai 4 md| h. The increase in the phagocylosis Ind Iespi~..,'~, burst of eiteulaiing nemmph|ls seen at 4 end R h after allergen ¢hldlenge in ~ham Ol~mled rals was reduced by dccenlralil:ulion. These resuIIs suggesl thai Ihe ullenualion oi' induced p~lmonary inflammatio~ that occ~:,~z ~, ~h ducentralivadon of Ihe SCG is Pri. marily associalcd with downrtgulation of n'..~cphil and macrophage I'u~.'lions. Mathlson, R.. Caner. L. Mowat. C.. Bisso~.,'t~e. E.. Davlso~. J. S, and Befus. American ]o~mal of Physiology 26605):RI ..'/-RI543. 1994. From the Deparlmenls of Medical Physic . :;. Faeully of Medk:ine. UniverSily or Calgary, Calgary, Alberla0 Cari|da. and ;-. hnonary and Cell Biology Res¢~c:h Group. Ilerilage Medicbl Rese|tch Centre, Universily of Albell|, Edmonlon. Alberla. Canada. 213
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PRETREATMENT WITI I AN ANTIBODY TO INI"ERI.EUKIN-$ PREVENTS LOSS OF PULMONARY M~ MUSCARINIC RECEPTOR FUNCTION IN ANTIGEN.CilALLENGED GUINEA PIGS • !nhalalional chellcnge with antigen decreases Ihe funclino or inhihltooj M, mus- ea~m~c autoreceplors on parasympathetic n~es in Ihe lung. inc~'caslng line release or acctylcholinc from the vagus Itcr~cs arid potenllaling vagally induced hronehocon- striction. It is possible that eosioophils cause M, eccepIn¢ dysrnnclinn, perhaps by r,'leasing positively charged proteins Ihat are M~ receptor amagoni~ls. Because or the probable role of inlcrlcukin-$ in gallanting and maintaining tie coslnophil infiltration, wc tested the funclion or neuronal M= recelSors in antigen-challenged guinea pigs ahcr pretrcatmen! with a monoclonal antibody IO interlcukin.$ (TRFK-5). Ovalbumin w~ g|ven intrapedto~eally 1o sc~s|l;za the animals. Three weeks later, the animals were in,~-cled intrapcriloneally with ¢ilher TRFK-5 (240 p.gJkg i.p.) or saline. Beginning three days lalero Ihey were ehellcngcd with an ovalhumin aerosol for $ mln on each of four consecutive days. M, rcccplor function was tested 24 h arler the last snl|gen challenge. Electrical stimulation of bo~h vagi caused bron. choconstrlcdon arid bradyeardiL In conlrol animals, pilocarpin~ altcnualCdo and lamioc polPJIllated, vagaily induced bronehoconslrlction by sdmolaling and Mncklng neuronal Me musca~Jnlc receptors, respeclivcly. Jn challenged anim01s Ihal did not receive TRFK-5 these cffeds were markedly z'cduced0 cunfinnlng M~ receptor dys- function. In TRi=KoSotreatcd guinea pigs. Ihe cffccls of hath pilocarpine and lamin¢ ~rc Ih¢ ~am¢ as Iho~ in control animals, demonstrating normal M~ reccwor function. Pr©traalment wilh TRFK-5 selectively inhlhiled the migration or aosinophils into the lungs as measured by lung lavagc. Thus die funcdon of M~ mus- czrinic receptors in antlgen-challenged guinea pigs can be prnlcctcd by inhibiting cosinophil inl~ux into Ihe Inn¢s, Elbon, C. L., Jacoby. D. B.. and Fryer. A. D. Amebean Journal of Respbatoz7 Cell and Molecular Biology 12:320-328. 1995. Other support: National Heart, Lung. and Blood Insritule. American Lung Associalion, mad the Amczlcan Heart Assoc[,~ioo. From Ihe Depzrlment of Environmental Health Sciences. School or Ilygicne and Public llealth0 and Division of Pulmonary and Critical Care Medicine, Johns Ilopkins Asl~ma and Allergy C©n~er. ;alms Hopkins University. Dal6rnmc. MD. C[GARL~'rE SMOKE-INDUCED BRONCHOCONSTRICTION AND RELEASE OF TACHYKININS IN GUINEA PIG LUNGS Two ~¢ries or experiments wcz¢ carried o~t to detcrmlnc whether the relca.~ of ~chykin;ns is involved in the bro~checonslriclion induced by inhalalion of ¢igareue smoke in guinea pigs. In Ihe first ~'i~. cigarc[le smoke consistently induced bron- chocoostriction {~.RL = +203% and ACdyn =. --46%) in anesthetized guinea pigs. and the response was only p,ulially blocked by bilateral ten, anal vagotomy. IIowevcr. the smoke-induced bronchial conslriction was completely abolished in animals 214 e©ccivlng u systemic c,~psaicin prelrealmet.: to de-my tic lacbykln[n-ccmla]nlng C- rier aKcrcnls. In I1¢ s~ond series, Ih¢ brnn~ 3~onsld~ive creel,or ci~uc smoke was increased by approx. Ihrec limes in ;~laled ~rfused guava pig lungs when ph~ph~mid~ (3 X of lachykin;~ after Iheir ~lc~. Mo~ovcr. "~c cn~ctl ~c~mt~ ms~ to snmkc was accompanied by nn ove~ of,.omkinin A-like immnno~livily (LI) anti ~lcit~in gear-related ~pdde showed Ih~ cigamlte smoke leiden the r,:;c~e ~ ~achykinins in the lungs, whkh plays an tin.Rant role in the smoke.iodu,:~d ~h~slrkli~ e~ccI in lulnea pigs. ~e. L-Y.. L~. Y.-P.. Ilong. ].-L, ~d ~::,4herI. & M. Respirali~ ~ytiolo~ 99( I ): 173-181. ]ant ~ I ~5. Olher sup~: Swedish Medical Rcsea~;, Council. S~dish Taboo C~y. Swedish Natio~l Environmental P~olecllon Boa~. Nalional Instilm~ of and I~ Unive.ily of Kentucky Tob~co an4 Ileallh Rc~mh Insthute. From the ~panmcnt and Dcpa~nl of ~tysiol~y. Unlve~ily ,',~ Kenl~ky. ~xlnglon. KY. X l. TIlE PRODUCTION OF NITRIC OXIDE IN ::.NDOTHEUAL CELLS BY AMPIIIPHILES Lysophosphatidylcholinc0 on cndo~'.~,.; detergcm is an ¢mdolheliom-depen. dent smooth muscle relaxant, which acls :'.~,ngh Ibe release of nilrlc oxide. [I is ship between detergent action, relaxation <'r ~Oxbelium-|nlac! rabbll Iollic slI~pS and the release of nitric oxide, we consid-.. -- the possibility that ¢Hher an~hiphiles al~o produce nilriC oxide from cndolbellal .,. ~. We therefore invest|gated the of digiton|n on rellxallon of precontracl, • *,~bbil aortic strips and Ihe release nil~c oxide from freshly ha~estcd bovine r ;'belial cells as detcnn|ned by clu:ml. luminescence. We found that both digltot, h..,td LPC release nild¢ oxide and Ihal this proocss is inhibited by Ihe NO synlh='.; :nhibltor Nc~-Nitro-L-At, ginlne Methyl F~er (L-NNAMU). Conforlo. A.. Dudek. R., lloffmann, M.P... ~ Bin|, R. J. Life Sciences 54(IG}:! 143-1153, 1994. Olher support: The Margarel W. and }[c~be~l Hoover, Jr. Fotmdallofl, Pu~Cfll. CA, 2l$
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C) From lh¢ California Institute of Technology. Dcpartmcn! of Environmenlal Engineering. Pasadena. CA. and Departmenl of Experimental Cardiology. lluntington Medical Research Institutes, Pasadena. CA. INVOLVEMENT OF INDUCI'LE NITR IC OXIDE S YNT! IASE IN TIlE INFI,AME,'IATORY PROCESS OF MYOCARDIAL INFARCTION Inducible nilric oxide symhase finns), which calalyze~; the reaclion or I.-argi- nine to L-cilrulline and nilric oxide (NO). plays an imporlanl role in immune-mediated cardiac disorders. The prc.~nl repot1 summari;rcs and discusses findings on Ihe induclion of NOS in myocardial ;nfm'ctlon of rabbits, iNnS was sigoiflcanlly increased in infarcted myocardium 4R h after coronary "Jrlery ligalinn. Tllc el'feel persisted for 14 days and declinnd Ihereafler. Immunohi~;Iochemicnl Incnlizalinn revealed maccopha~es as a major source of iNnS cxpresslon; iNnS expression was also Fresco! in infarcled human myocardium. Increased iNOS activily appear~J !o be related to lhe induction of.apOplosis in Inl~ItrallnR mucrophagcs and cardiomyocytes. Moreover. prefemnlial inhibition of iNnS by $-mclhylisothiourea sulfate (SMT) rcsulled in sil:nifican! improvemen! of let! ventrlcular p~rfonnance and incr¢=sed rc~ional myoc','dial blood flow. These findlnss suggest thai seine|ire inhibition of iNnS activity may provide a Ihcrapeulic strateBy in cardiac disorders such as myocardial infarction. Wildhin. S. M., Dudek. R. R~ Suzuki. |L, and flinR. R. J. International .toumal of" Cardiolocy 50:253-26 I. 1995. Olher supporl: Gustavus and Loui.~ Pro/f for Research Found:.i~n. Redhmds. CA. Margaral W. and }lcrhcrl Ilocwcr. Jr. Foundation. and the I.I.clh~ M.rcy Murphey Foundation. Pasadena. CA. From Ihe Depanmenl of" Experimental Cxrdiolo£y. llunllug~on Medical Reseamh lost/rules. Pasadena. CA. T}iE REACTION OF NO" WI'T}I O;" AND HO;: A PULSE RADIOLYSI$ STUDY The rcaclians of NO" wilh O,~'" and with HO; were studied using the poise radi- olysis technique under pseudo I~rsl order coati/lions whera (IO,"1 + [110;I ) > INO'I at pl-! 3.3-10.0. The role conslanl or Ihe reaclion or NO" with O~'" wa~; determined both by monilorins !he decay of O~'" a1250 nm and the I'om~alioo of ONOO" nm to be (4.3 _ 0.5) x 10' M'~s'', independen! of ionic slrcnglh and pll in Ihe range 216 O~r 6.1-10.0. The rap constant or!he reoct;~ of NO" with HO;" was dc|ennlned by I'ollowin& the decay of IIO; a! 250 nm to Ix: (:q ~' _ 0.3) X 10' M'~s-' al pH 3.3. Golds!eta, 5. and Czapski, Free Radical Biology & Medicine 19(4):50'; .:.~0. 1995. Other supporl: [srnel Academy of Sciences. Frnm the Dcparlment or Physical Ct~emismd. The llcbmw Unlvmlty or ,I,'msalcm. Jen,~s~lem. Israel, NITRIC OXIDE REGULATION OF SUPE,~. ]XIDB AND PEROXYNITRITE- DEPENDENT LIPID PEROXID,~'rlON: S'c'~,., --~'O~MA'noN O~ NOVEL NIT~OGEN-CONTA|NINO OXIDIZED UlqO DERIVI ~.'~'~.~ S,peroxlde (O'~). n/eric oxide ('NO). ;' "J :heir re~Clion Ixoduct peroxynilrile (OON00") have all been shown Io indepcr.cfemly exerl !oxic tax,tel molecule reuc. lions. I~ecause Ihcse reactive species are r'.~,.) gcncr~cd in excess durln~ dive~ iol']ammalon/and olher patholocic circum~r~,.-:.:s, wc Isscsscd Ih¢ inl~nece or'NO on membrane lipid peroxidation induced b: ~,*~, H~O~. and "ON derived from xan- Ihine (XO) and ONOO'. Experimenlal coc. it..as in lipid oxidation Wsl~ms were adjusled to yield diffcren! rules or deliveP; :-" "~lO relative to rules of O~ and II~O~ generation, by infusion of cilhor "NO et ",'ix "NO released from $-nltroso.N. aeelylpenicill.',minc $-nilrosoglulathione. Pemxldalion of phosphalidylcholin¢ lipo- stones was ns~e~ed by foment/on of thioharhilurle ucid-reactlve pmduels lind by llq.id chrnmalngraphy.mnss speClrometry. L;posomos e~Fot.ed to XO.dedved re~¢. tire species in Ihe presence of"'NO exhibited .'-.~h stimulation and inhibilkm or lipid pemxidadon,.depending on the ralio or !he r:tees of reactive oxygen species Froduc- llon and "NO mtrmluclion inlo re-¢lion sy~',rn.s. Nit~c oxide alone did nol induce lipid pemxidation. I.Inolenic ecid emulsio~.~ t~.'mxldized by XO.dedved reaellVl species showed similar dose-dependent re, u.:~tlon of lipid peroxldalion by "NO. Mass spectral analysis or oxid|lion produ,.,, showed I'mm~ion of nill~lO-, nilm., nltrosoperoxo-, and/or nitrate~ lipid o~i,"...i'~n adducls, demonslratin~ Ihal 'NO serves as a potent ~erminator of radical c.~-',, ~ropagation reacllon|. Eleclron |pin resonance (ESR) analysis of" incubation mix,_...-, provided no e~idencn rm- formation or I, aramagnelic iron-lipid-nitric oxide coml, k,: ..s in reaclIon systems. Peroxynlt~le. dependent lipid peroxidalion, which pred:~,: molly occurs by melal-lndependcn! mechanisms, wasalsoiohlbltedby'NO, Perr.: .'.*ilrile.medialed henzoale hydmxyla. lion was pan/ally/oh/h/led by "NO, inferrin ; .;,¢llon belwcen "NO ~d ONOOH. !1 is concluded Ihat "NO can bOlh slimulal© C' ":'i,O~/'OH-induced lipid oaid~lion and mediate oxidanl-proleclive reaclions in me~'...'anes at higher rules of'NO produc. lion, with Ihe prooxidant t'ersus ~nlioxidant ¢~,tcome ¢dllcally dependenl on relalive concenlralions ol" individual re:clive species. [~moxidanl Ruction| oI"NO will Occur hi'let O~ reaction wi~h "NO Io yield Fotem second,~, oxld~ls such as ONOO" ~d 217
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C) the antioxidanl ell"ccts of'NO a consequence or dlrcct reactiun with alkoxyl ~d per. oxyl radical intermedialcs during lipid peroxidatinn, thus tcrminaling lipid radical chain propalzatio~ reaclions. Rubbo. H.. Rzdi. R.. Tmjillo. M.. Tellcri. R.. Kalyanaraman. M.. and Freeman. R. A. The ,~ournM of. Biological Chcmistry 269(42):2~.0~6-26075. Octolx:r 21. 1994. Other supporl: Fullbright Foundation. Halional Instltules of. lleallh, and the Universidad de la Republica and Consejo de Investig.-,cioocs Cicotil~cax F, om the Dapanmeots of ,Anesthesiology. Biochemistry and Molecular Genetics, Pcdlatrics, and Pharmacology and lhe Mass Spectromelry Shared Facillty. Compreh©nslve Cancer Cenler. University of'Alabama at Bin, ingham. Rirmingham. AL. DcpaHmento de Biociulmlca, Faculli Rcpoblica. Montevideo. Uruguay, and Ihe Biophysics Researdt Institule, Medical College of. Wi~o~sin. Milwaukee, STABLE Mn(lll) PORPIIYRINS MIMIC SUPEROXIDE DISMUTASE IN VITRO AND SUBSTITUTE FOR IT IN WVO Several manganic porphydns, with subslitucots on the mclhin¢ bridge carbons. wen: prepared and examined for slability, rcdox behavior, catalysis or the dismuta- lion ol" supesoxide radical (O;). and the abilily to protect a su~roxid,, dismutase (SOD)-null strain ol" E. co//against dissolved oxygen a~d a SOD-competent strain against paraquat. All or the compounds tried exhibited reversible rcdox Ix:havlor and were slablo In EDTA in both Ihe oaidlzcd and reduced slalcs, and .~veral wcra able Io calalyze the dismulallon of O; With the rate conslanls or -10' M' $'. The mxr~:ed prutecllve erJ'ecL~ or some of. thes© compounds exceeded that which coold Ix: anticipated on the basis of such rate conslanL~. The Ictrzkis (l-melhyl.4-pyridyl) compound was Rduced enzymatically at the expenr,¢ or NADPI I and no~enzymali- tally by GSH and was kcpl in the reduced stale wilhin E, colt. Since the rate ¢OllSlanl for re.oxidalio~ of Ihe reduced form by O; is 4 x I(r M' s', it apl~ars that this com- p~u.n.o acts in viva as an NADPHIGStl:O; oxidoreducta.~ ralher Ihen a.,~ an SOD rmm,c. Its ability to I'acililate a~obic growth or ih¢ SOD-null strain can he explained on this basis. Faulkner, K. M.. Liochev. $. I,. and Fridovich. I. The Journal of. Biological Chemistry 269(3B):23471-23476, September 2"1. 1994. Olher suplxxl: 3ohnson and ,fohnson Focused Giving Program -',nd Eul(m';o. Inc. From the Departmcnl of. Biochemistry, Duke University Medical Center. Durham, 215 ESCIIERICIrlA COU EXPRESSES A COPPE; .. AND ZINC.CONTAINING SUPEROXIDE DISMUTASE A mutanl of E.tcherirl#ia cnli, unable to prc.l :co m~nganes¢- or imn-co~tslning supcroxide dismutase (SOD). was found to ¢~..z, in modest levels of. an SOD that was judged to he a copper- and zinc-comalnh'~" -'end on the b,~is or inhibition by cyanide and inactivation by either H~O~ or d!:t,'.yldithioc:~bamate. Moreover. the diethyldlthiocarbamat¢-ina¢livatcd enzyme cm,~d ,"¢ rcectlvatcd with Cu(ll). and Ibis reconslitutcd enzyme, like the native enzyme. ~,,:s unaffected by EDTA and was inhibited by cyanide. This enzyme was. furlhe,. ,-;.',~, seleclively Rleascd by osmoli¢ shock, in keeping wilh a periplasmic Iocalizati, . .,t~d It was slrongly induced aerobic growth. This enzyme was also presto! |" .he SOD.competent parallel IIZ'ain. Failure to detect it previously ,":,n Ix: attribute ' o its pedplasmlc Iocallzadon. thor. real lability, scnsitivily Io pll. and to its rel~ti~,..~::city. !1 will now he interesting Io explore the phCnolypic consequences impo~d t.:" :he absence or this SOD. Benny, L. T. and Frldovich, |. The ,~ournal or Binln~icnl Chemistry 269(4 I):2.',." ~0-25] 14. October 14. 1994. Other suppo~l: ,Inhns~ anti Johnson Focu,;ed G~':~J Prod.ram and Eukarlon, From the Dcpartm~t of. Biochemistry. Duke ",.,cnlty Medical Center. Dmham. NC. SUPEROXIDE AND PEROXYNITRIT~ INA~." "'/ATE ACONITASES, BUT NITRIC OXIDE DOES NOT The Eschtrichia call ~ Rcomhtnmll hum:" ¢ .'.'tosolk: acooltascs are ina~ivaled by O~. wilh • rate constanl o£ -3 x I0' M" :.. he ¢ofTcsponding value for Ihe Pornlnc mitochondrlal aconita.~ is -,O.R x 10' .,,r' ~ '. Nil~ic oxide, which is !o in*citrate nconilasc, did not do so at a perle.';"...; rate. while incuballon wilh For. oxynilrile led Io a rapid loss of. ,,cOnil~e activi;. ,:,) I~OpOSe that the repotted in~c- livation of =conil~sc hy nilrlc oxide in viva h .. ,ally mediated through peruayni- trile. Ih," product of" Ihe rcaclion b¢lw~n O~ an~"~ • "): Ilausladen. A. and Fridovich. L 1'he ,lournal ol" Biological Chemistry 269(47):27a ~$.2940R. November 23. 1094. Olher support: Johnson and Johnson Focused Gi, ".-. Program and Eu~ulon, |he. From the Dcparlmcnt of" ~iochcmlstry. Duke U.;venlty Medical Center. Durham, 219
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Exposure or a superoxlde dismulase.nul! (.Tr~JA sndtg) slrain nr same heal slrcss applied ~crobically h~ a negligible ~[r~cl. la~e-com~lcnl palatal xlrain wx~ resislanl In I~ Iclhal eHccl o[ Ihc acmhic healing. h follows Ihat aerobic heating imps an oxi~alivc bu~]cn major compel ~is C~l is nm ~en al ~] C. prcsumahly ~ca.~c, a; Ihi~ higher tom.lure, di~ Ihe~labilily o~ vilal c¢11 cnm~ncnls su~oxlde radicals. Benny. ~ and Fri~n~icb, I. Journal or Bacl~riolo~y 177( I I ):~3~-~. June 199~. From ~he Dep:~mcnl o~ Di~h~isl~, Duke Univcrsily M~di~l Cenl~. Durh~. A SUPEROXIDE DISMUTASE MIMIC PROTECTS Soda $OdB E$CIIERICIIM COL! AGAINST AEROF)IC !I-FATING AND ST^TION^R Y-I'IIASE DF.^TII Superoxlde appears to be a major cause of stationaPj-pha~e death and heat kill. In suppo~l of this conclusion are the I'ollowing ob..,crvatitmx: (a} Stationary-ph.,~sc death was apparent in t~ soda sodB. but nm in Ihe supcro~idc distnuh'~,;c compclenl parenlal slfala; (b) Statinnary-pha~ dealh in the .~NJA s~xlll sir;tin was dioxyg¢o.dcpeodem: (c} A m~nganic porphy~in, which calalyzes Ihe dismutalion ol" superoxide, protected Ihe soda $odl'J slrain against statio.ary-pbase death; (d) |lealJng.lhe soda aodD slmln Io 42"C caused a lu,~s (d" viahilily nat seen wilh the $OD-compelent parental strain and prevenlable by Ihe munganic porphyrin. Exposure Io aerobic conditions induced anlibiolic resistance in the .,mdA hal in the parental slrain and Ihe manganic poq~hyrin prcvcnled Iha! induction. This again indicates its ability In substkute I'o~ SOD in E.rcherlrhln Benny. L and Fr|dovieh, L Archives ol" ~]iochcmisiP/and 8iophysies 322(!):291-294. Scpteod'cr IO. From Ihe Dep'llmen! of Biochemisl~. Duke University Medical Cenler. Durham. NAPIITllALEN'ESULPilONAMIDES BLOCK NEUTROPIIIL SUPEROXIDE PRODUCTION BY INTACT CELLS AND IN A CELL-FREE SYSTEM: IS MYOSIN LIGHT CIIAIN KINASE RESPONSIBLE FOR TI IFJ;E EFFECTS? Selective anlsgonists o£ myn.sin light chain klna~ (MI.CK) ]e.g. ML-7; I-(.';. todonaphlhalcne-l-sulphonylpIII.hexahydrO.lo4.dlaxcpinc hydrnchl.rldcl were 220 found to inhibll superoxlde (O,') ~l~=s~ from -...,.,ulalcd nc~ln:~bils. TIc ~- lions or ML-7 that wc~c inh~it~ were ~bs~:~ ~lly I~r I~ th~ ~d for selcclivc anlagonisl or protein kinl~ C [i.e. "~-7; I-(~-i~uinoli~sul~yl)-2- ~lhylpi~inc dihyd~hloride], M~7 dso ~'~ccd I~ phosp~lali~ of Ihe 4T k Da subunil o[ Ihe NADPi I.oxi~o syslem (p Z I .'h~ and bl~ked Imnd~al;~ of Ibis pmlein In I~ T~lon X.l~.i~olublc rm~ "- in slimul~ c¢lb. M~7 also i~ibilcd O," W~uclion in I call-[ :..~em ~d~ f~ ,¢ul~il~ ¢oncenl~lions similar Io IIm~¢ Ihal wc~ ¢[~¢c ~ ~'i~. ~is ~ll-r~ sy~¢m ,m ~uire ~ ~d i~ inscnsilivc IO all oIL • ..:aibilm or ~cin k~aRi includin~ some highly cK¢clive against ML~ Tl.¢ sl~ms~d~). ~us. I~ i~l~d may inlcmct dircclly wilh a su~nil o( '~, uxida~. ~¢ ~¢ site f~ ML- 7 may provide ~ valuable larg~l [~ inhibilin¢ ~ , mfl~ ~s or cyl~ Icuc~yl~ by na~lhalcnc~l~=mide: J .~i;~d Io l~k ocliVily a~ain~l loin kisses. 9i~hemical Joumol ~11:~1-87. O~r sup~n: N,ti~al lnslitulcs of ll~hh. From Ihe Dcparlm~nl o~ Mol¢col~r and E/.;.rimonlal Medicine. The Pharmacology. Ilarvard Medical Sch~l. R ~lcn, MA. and Boeton Bi~dical REMODELING OFTIIE PLASMA MEMBP.J.. ~ AFTER STIMULATION OF NEUTROPHILS WITII F-MET.LEU-PHE AI':~: DIHYDROCYTOCHALASIN B: IDENTIFICATION OF MEMBRAHE SUBDGI<..',INS CONTAINING NADPH OXIDASE ACTIVITY Superoxide tOt') production by neutmpl-".;~ st;mublcd with chemolaclic pep. ddu [e.g., fonnylmethlonylleucyl-phenylalani~.~ (I~ILP)] is IrM~slcnl ~ in rate and duration mylar pr~lmatmcnt of th. cells with dihydrocylochalasin B (dhCB). suggesting z possible role I'or Ihe plasr.~, membrane 8rid membma~ skeleton in the regulation or the O~" gcn~'ating syslem. A~l:l~ia or pl.a.$ma mere .brae from these cells by isopyrn[¢ sucrose donate:, ~,3:d;cnt sedimentation showed Iher¢ were on significant vBialloo in the disz;, z ,ion o1" plasma mcmbm," belween coetml and dhCB.treatcd cells, where: : signil'JCZ~l rediswibulion ol'pla.srna membrane markers was ot~srrved in dhCB + ..-'4LP-Irealed celia. Inslead of s~d[. menlin; to 31-35% sucrose, as in IhC fom~er t~.." :roups. plasma membrane were broadly dislrihuled over 25.50'7, aucrme ";, ,he dhCB + IMLP4reatcd cells. In addition° -80% drgranulation was achieved '.r "cse cells, whereas little granule release (<.5~,) was (~,erved in cm~trol and dh;.'F .'.'~atcd cells. Analysis of'the g~di. rot fracl;ons for membrane akclelal tacit, a.~d fndrin) and NADPH oxidase 221
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(cylochromc b and p47.phox) componenls in dhCB + tMl.P-trcalcd cells deraon. sir•led that the disl~buli~ of r~n, ~,in. cyl~hr~e b. and ~7.phox followed ~he broad disl~bmion of plasma mcm~n= markers, with an overall ei~hlrold i~reasc in m~bran~ass~ial~d •clio. ~p~le the broad ~ktri~dnn o~ plasma m~m~= m~ers, the dlstri~ or O~- ge~ling activily rcmai~d confin~ ~o a n~ower ~ a~ -~0~ s~m~. ~ ~ulls demonslratc th01 a race m~mb~ or hi~her densit~ whh a dirr~dal di;~ribut~n gon~rallng acdvil~ arc creeled utter dhC~ + ~LP wealmcnl; however, ~maln structure is c~ in ~he new mem~ane, whh only a suh~raction or Ihc rcor;a. nlz~ plume m~mbra~ conlainlng all or t~ com~n~m~ g~neratlon. Our rcsuhs sup~ a role for plasma membrane Ime~al nrganlz~ion and p~icipalion o~ the mcm~e skH~mn in the re~uladon .r lhc O~ ~cncradng s~. lcm. ~ukh~rjcc. G., ~u;nn. M. T.. IJnner. ~. G.. and Jet.ilk, ]numal o~ ~uk~= Biolo~ ~S:68S-694. June Other supine National In~dmles or Ileallh. From th~ De~nmcnl of Micmbinlo;y. M~tana Slale Unive~;ty, ~.z=man. MT. IMMUNOCYTOCIIEMICAL DETECTION OF LIPID PEROXIDATION IN PtlAGOSOMES OF llUMAN NEUTROPIlILS: CORRELATION wrrll PRESSION OF FLAVOCYTOCHROME B Oxldanls generated by the NADPII oxic~se or ac|ivaled neulrophils can re•el with a number of tissue largels Io form toxic met•baltics such as 4-hydroxynonenal (4-11NE). 4-1INE is • lipid l~roxidalion producl generated by free radical altack on ~u-6 polyunsuluratcd laity acids and is • marker for membrane lipid pcroxldali~. In this study, we examined the accumulation oi" 4-11NE.~'olein adducls in phagosomcs or n~utrophils Oblained from u male patienl wilh homoeygous X-linked. flavocy- tochromc/~defcienl chnmlc ~anulomatous disc&~ (COD). his l~tcrozygous molh~r, and his normal l'ather. Specific FOlyclonal •nlibodics rccognlzln~ 4.11NE-Im-)lein .addoc~s and gpgl-phox (flavocytoch~ome h large sub•nit) were prepared and used to Immunocytochcmieully detect these anligons in c~yofixed, molecular distillation. dried neulrophils. No 4-HNE-proleln •dducts were delected in llavocytochrom¢ defcient cells from lhe homozygous palicnt or from the helerozy~ous COD cartier, However, in gp9 l-p~s'~.t-posilive cells from both the normal and heterozygous ca~rler, .s|gniflcant 4-HNE-protein addu¢! labeling was observed, primarily in the phagosom-'s. When dale from single- and double.labeled cells were combined, th~ .frequency distribution or the labels in phaeosomes supported this observation, show- mi that neutrophils from ;..% 5;;erazy~ous COD carrier were 71% 4-11NE-ixotcin add•el-positive and $6~ ~p91-phoz-posltive, while cells from the normal i'•thcr were >97~ positive for both 4-HNE-Ixoteln •dducts ~,nd ~pgl-pAo~. These resulls confirmed ~be nitroblu= telrazolium tests of 100%, 60 + 2%, and ~% po.~ilive for the ralher's, mother's, and son's cells, respectively, and dcmonstrated that 222 rein sdducl antibodies are usd'ul and accuru'te p~obes or oxldali~ la ~,ivo. We conclude that 4-HNE ~neml~d as a resuh of NADPll oxidase ~:vily in I~ pha~o~es or h~ neu- I~phils ~d ~hal these lipid ~roxidali~ ,,,~u~s may comri~le to microbial kiliin~ ~dl~ damage of ~utrophil pha[ol3 ~ :~mal ~ei~ ~uinn, M. T,. Unnu. J. G.. Simms=n, ~ J~mlz. E. A., Bucscher. E. 5.. and Olhcr sup~: National I.~flmles or Ilealth ~:,.~ the U~, ~flm~l of A~cullu~. From Ih~ ~pa~menls of Microbinl~y an," Slale Univc~sily, ~ozamnn, MT, and Dcp ¢lmenl of Ptdillrics. Easlcm VirBinia Medical Sch~l. Nnrrnlk, VA. STRUCTURE OF I IUMAN PI IAGOCYTE .", TOCHROME b AND IT~ R ELATIONS! IIP TO MICROBICIDAL St:e'..'. OXIDE PRODUCTION The lmman immune syslcm dcpemt~ o " • phaguc~les for on-demand lion of" toxic oxyKcn spcci~ at sites or infe¢- -. invasion, or injur]. The~ sp.'c;es are I'om~d because of Ihe production of SU. ;.'-~xidc anion (O~'-) by the phagocyte nicollnamide-adenin¢ dinucleollde phospha:-, h'^DPH) oxidase. This mellicompo. nenl. acl~vatahleo membrane-enzyme camp! ~ ~'~nsre~ hi|h el~ef.t]t ¢lcelro~s cyloph'Lsmic NADPll to molecular oxygen. ~ - railling praduclion ot" topologically extraccllular Oa'-. At the funclional cenlcr o|. ;s syslgm is a un|que Integnd brahe b-type cytochrom¢ Ihat functions as t~ , redox "middleman" ia Ihis produ¢. llon. In the la.,ct 3 years, new s:lmclural and (.'r.ctioeal inl'ormalion alx~u¢ the o~ida~ has emerged Ihal s.pports the functioning ol. h~l the molecule as an eleclron tran$- fcrasc rot Ihc rc:lclion: NADPil + 20~-.N^I .~ 4. H" + 20,'.. Jesuilis, A. J. The Journal of" Imraunology: 32116-32R8, 1995. Other support: U.S. Public Ileahh Service. From the Departmenl of Microbiology. Mon,',.na Slate Unlversily. Bozcm~. MT. SUPPRESSORS OF OXYGEN METABOLI~ ::~ FAIL TO REDUCE VEIN GRAFT INTIMAL IIYPERPLA$1A ObJecliYe: To cvalualc the role of reactive oxien met•baltics in the Initiation of intimal hypeq',lasia o1" vein grafts inserled inlo the mlerlal cigculation. 223
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Settlng: University padmlogic ~eseareh labomt,r~. Animals: Spraguc-Dawiey rats. |nlervention: Animals were randomized Io rcccivc either the xanlhinc oxidlsc inhibitor alloptirfnol, Ihe iron chelating agent starch-con.jui~tnd del'eroxamioe, or the I'ree-ridical scavenger 21.aminostemld U74311BG. Control animois were included for each l:roup. The cpigislrl¢ vein was inserled iron the righl common I'emoral ahead. Vein gratis were harvested ;30 days posto~ralively. The degree of intlmal hyl~pla- sis at the two anastomoses as well is al the midgral't was calculated. Main Outcome Measures: The vein grafts were divlded into three sections design•red proximal anastomosis, midBral't, and distal anastomosis, latimal and medial areas were determined in in observer.blind/.ashion and cxpr~s.~ as intimal area-medial area ratios. RiSu|ls: Preireatmenl or the animals with any o1" these agenL, I resulted in no slg. nil'leant reduction in the del~ree of intimll hyperplisia in ~ny treated groups com. pared wilh the coulrol animals 30 days postolx:ratively. Conclusions: Arlerlal [cconslrucliml of'ten involves intel'posilion olr vein meats inio the Irlerial circulaiion. These veins ai¢ suhjccl to ischemia and reped'u- slon, wilh the polential/'or generation o/.lel:tive oxygen reel•hollies and suhscquenl vein gra/.! injun/, resulling in intimal hypeqdasi-', We hyp,~sthcsl;,cd Ihai i~-io ra lay " e pharmacologic intervention either to .%"avenge or to red.co the: pr(xlucti~n re.cllve oxygen metabolires would aRcnuate Ihe initial vein grab injury and thus limit the subsequem development of inlimal hypcq)las;a. These data create donht to Ihe influence of" reactive oxygen metalmlites in Ihc ioitiaticm .r intimal hyper~la- ~ia in Ihe vein graft. Ga~es..~. D.. |l;rsch. G. M.. and Karnow.:r.~y, M. J. A~'chives ol'Surgery 130:976-980. September 199.5. Olher suppo~l: The Up]ohn Co.. Kalamazoo. MI. From the Departments or Sur&e~ and Palholngy. Ilarvard Medical School. Brigham and Women's Hospital, and Massachus~ll$ General I Io.~pital. Boston. MA. THE KINETICS OF THE OXIDATION OF L-A$CORBIC ACID BY PEROXYNI'i'R ITE fonnPeroxynitdte IO-NOO-. osop,*rosonitratc..I.)] is a strong oxidant Ihal may be ed i~ vit'n by the re.orlon of O;" and NO'. Oxope~'oxonilrete(I-) react,, Wile moleenlcs in aqueous ~idio solulions via pathways Ihat involve Ihe highly reactive hydrogen o~loperoxonltrato eilher is in tnlcnncdiare in • Irlr.il-n~er reaction or as a relclivc agent in z simple iecond-orclar reaclion. F..SR expuim~nts sh.w thai hydro. Ben oxopcroxonilrale o=id!-'.ci monohydrol~en L-ascorhate hy one ¢lcctron: when mixed iI pH ca..5 and passed Ihrou~h z Ilow cell wilhio O.I s, the two.lio~ ESR sag. hal or the Iscorbyl radical anion (i,, ,- 018 "/', g ,= 2,005) is obc, a~ed. The ovarlll aioichiometry o/. the Raclion was I tool o/. Iscmbaze oxidized per tool o1' oxop~ox. onitrate(I-) added. The i'inelics ol" the rcactlon were studied over the pH I'an~e 224 4.0-7.5 hy slopped-flow spectrometry. Hy4mEe~ oxopemxoni~'ai¢ observed helwecm 300 and 350 nm, and the oxoperoxoni~te(I.) anion at 302 rim, dlsappeaz taster t~an .l~.edict.ed (or the first-order isomu;zaiion to NO;. The rate incRas~ rmm pH 4 to =.s, ,,n~ then decreases wilh increasing pH. The rate v~z~llon su~esls • blmolecot-. reaction either between the oxoperoxonitrmtc(|.) anlcm and ascorbic mold or between hydrogen oxopemxonilrate and the monohydrogen ascorhalo anion. AlthoUgh d~c Iwo pathways are kinetically indistinguishable, the pK. v~ues or ascod)i¢ ~¢id and hydrogen oxoperoxoni|rat¢ slrongly sug~.sl Ihai Ihe re.ling species IR hydrogen oxopcroxonitrala and monobydmgen ascod~te. The second-order talc ¢o~slar4 |o~' Ibis Raclion is 235 ¢ 4 M'~s" at 25~C. ~ enthaIpy and enlropy o~'aclivaiion axe .~!1' = 9.3 • 0.5 kcal/mol and '~S' " -16 "" 2 ¢'al/(mol K). respectlvcly. Barlle.. D. C~urch. D. F.. Boands. P. L. and KopFeool, W. !!. Free Radical Biology & Medicine 18(I):~5-92. 1995. Other support: National Institulcs of Health. From the "Depmtmcnls of Chcmislry and Bicchemisw/md Biodynamk~ lnslil~, Loulsi,na Sl=~le Uoiversily, B-~on Rouge, LA. and I)¢Fmment oir Cl~mistP/~ Physics. Southe~em Louisiana SIIle Univm'ity. Hlmmond. ~ OXIDATIVE STRESS BY MFJqADIONE AFFEC13 CELLULAR COPPER AND IRON HOMEOSTASIS Menadlone IXnduces DNA s~nmd Im:a~cs eDNA st)) in cultured Chin¢,~ hamster ~fiypo(broblas.ts which •re. to • great ¢x1~111, mediated by OH radical. A rcnso~ble heSlS iS Ihal XiOf • pilxluc| Of nlai~ion~ inelilx)llsm, relcls wllh nnclelr lion and produces OH radied in ~im. Conslsicnl wilh liill, 1.10-ph~mllhlolllta pmvenls menidione-indueed DNA sb al low (< 200 lIM) chelator. However, It higher PHEN coneenUllions, Ihe ell'eel is r~ersed llld in enhlnc~meol or DNA sb is observed. The PHEN.irdneed l~comes move avidenl al hlih (> 60 p.M) mella'ione conc~nlnlllons and ii smxl|l# provenled by neocoproin¢ (NEO), •n ¢lTiclcol copper chelllor. Xowavcl', offers only • dight pml~Cllon liainst DNA Ib Cllsed lit resulls are con/islent wilh Ihe following evenls: (i) the prodllcls of menldloile melabolism cnus~s copier iotl relmlso from som~ cellillr compiml~n¢; (it) ~ce or PHEN, i Cu(PHEN), conlplex is fom~ (ill) Ihe Cn(PHEN) comldlx is known Io be very cllslogenlc, iad.c;ng DNA dlmale ill • m#ueinI envlnmrnenL Evidence is also pres~led Ihat m~lldlol~ nl~atlolism clu~ an incmisa in inimcellullr chelalable irotl: in Ihe ~ of • tonsil 2,2'-dlpyl'Myl eonctnlll- li0a. Ihe DNA sb prndu~ed by lncvelsing ¢o~lConlillon$ of m~lldicon bx:ome Pro- lressively less s-~eplible Io klhlbilion I)7 Ihe chelilor. Themfme the DNA damage or;gloated from menadicme m¢labolism seems to caused by two conjug:lcd and synergistic events, via., I~ IXed,*ctlon oxygen species and 1he releas~ of coS~cr a~d ima f~mn • cellular SlOrS~ site into • "tree' roan Ix)ol, c, pable or cmlyz~ns DNA dam=gang reactions. 225
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Calderam. M.. M~ins. E. A. L, and Meneghint, R. Molecular and Cellular Biochemistry 126:1"/-23. 1993. Giber support: FAPESP (Brazil). From the lnslitule of Chcmislry, Oepar~menl of Biochemistry. Un;vcrsity of Pauto. SaG Paulo, Brazil. IIYDROGEN PEROXIDE AND DNA DAMAGE Vcl7 clearly F¢ ions play an impotlant role in DNA damage produced by reac- tive oxygen spocics. This is Ix:cause DNA has Fe(ll) ions as one or its ¢o~nlcrions. H~O~ reaches the: nucleus it tin relCl with Fe(II) ion~ and produce an OH" redical. which in turn attacks i~ ;ilu Ihe sugar or the base. i~oducing strand breaks and base modifications. These DHA lesions may initiate m~tatlon and caminosenle events. In this context it is noteworlhy Ihal epldemlological studies have indicated a correlation belween body iron stores and rise of cancer (Steverm et oL. 198R). II remains to hc established why and how F¢(II} ions reach the DNA. Ilec~use int~cetlular iron s~¢ms ro be.handled very carefully one would nol expect its presence so close such • vital ~'~el. Menechlnl, R. and Mar~ins. E. L. In: Amoma. O. T. and Halliwell, B.. (F-~. s.): DNA and Free Radicals. Ellis Horwood Lira.. London. pp. 83.93. 1992. Giber supporl: FAFESP (graxJl). From the lnslitule of Chcmislry. Deparlmcnt ot" Biochemistry. University of Paulo. San Paulo, Brazil. CELLULAR DNA DAMAGE BY HYDROGEN PEROXIDE !$ ATTENUATED BY HYPOTONICITY Chinese transfer fib~o~lasts (llne V'~9) withst:nd well exposure for 30 mht to hypotonic medium, ¢otTcslxmding to 25% physiological phozphate.bulTered saline {PB$}. Under Ihese condilions, the cells become resistam to two erfccls of II~O,: DNA d~mage and inhibilio~ or cell ekm¢ I'onnalion. The normal scnsltivily to the DNA-damaging action o1" H,O, is n'..stored if, after exposure to hypolonic cells are incubated in ismonic cell.cuhure medium. However, resloration of sensitiv- ily is no( observed on incubalion in isolonic PB$. The normal sensilivily also Rstored if one of the following Rduclng agent.'; i~ raided to hypotonie PBS: 226 ascorb~te, NADH and NADPH, in this order o1" decmxsing elT~ey. ef senskivky ~o H~O~ by ~e is ~mplelely inhi~ ~ I,l~h~i~, ~sitivily !o I~ DNA-damaBin~ acli~ of H~O~ is nm a ~li~l~ o¢ h~l~iC PBS, sincc it ap~a~ to ~ caus~ by hy~l~ty in Gemini: k is sl~ obs¢~ in culture medium oF 25% the isotonic ~tmli~. explanation r~ lhis phe~menon is that hy~ I~s ~ads Io I depl~i~ of ~uclng s~ies, in paflicular a~co~te. U~er I~ oxMiz~d In Fdlll) and Ihe F~lon c~mi~ is dcrin$ it lass Kccssi51c to H:O~, of that it ~uatcs the C~:'-a~iv~l~ close, indued by oxidative st¢css. Matins. E. A. ~ and MeneRh~l, R. Bi~hemlst~ Journal 2~:117-140. I~. ~ supS: FA~P (B~I). From ~he ~p~ment ~ Ri~hcmlst~, lnslilme Paulo, 5~ ~ulo, B~zil. XII. Virology PAPILLOMAVIRUS DNA REPLICATION The DNA replication o1" papiltomlvimsex has been studied in clinical speci. men•. in Ir~nsf'ormed cells, following transient Iransfections. and in cell-tree s~. terns. Togclher, these rasulLs suggest that papillomaviroses have thre~ modas of DNA replication: an establishment Phase of modest amplificalion upon cnt~ into basal stem cells ot a wouodcd squamou$ epithelium: II s|cady-st~e, low.level main- finance mode. enough to keep up with division oflhe basal nod pmubesll cells, • vegetative amplification mode for th~ productlon of progeny vlms in diffc'Rntialnd cells. Bolh the vital El and £2 I~O~ein$ ~e requ[Rd to ~ the o¢/~ad i~ltlice viral DNA replic•tioe, while the host cells provide Idl Ihe other repllcaliOn enzymes. The eel, Ice•led in Ihe upslrcam ~gulatmy Rglon. enmisls or biml'mg xkes for Ihe El and ~z ~eir~ R. ePv l, El is ,he p~m~ o~ ,~..am.on ..p~. ~ w.he .,.~,~. for HPV ! I, und p~rhaps for HPVs in general, the E2 pm~enn ~s t~ re.cut on pn~etn. The papillomaviruscs provide | superb ~yslcm Io sludy euka~oli¢ DNA replica- llon. D¢in~ impmlant human pathogens. ~ uce Ihe subjcel el' intense scrutiny in the search for antlvird pham~ceuticel i~duc~ ned ftmher sere u excelicm the disregolalion of the viral E6 und E7 once/series, the p~'KlU¢lS m wmcn mice•el wilh and InnctlvMe th~ heat IUmOr SU~'~"SSOr 227
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Chow, L.. T. and Broker, T. R. |nterv|mlogy 37:150-158, 1994. Othu suppotl: U.S. Public Health Service. From ~he Departmenl otr Biochemisl~y and Molecular Genetics. Sehnols of Medicine and Demistry. University of Alabama at Birmingham. Birmingham. REGULATION OF II "EPATITIS B VIRUS ENI ENllANCF.R ACTIVITY I IEPATOCYTIZ-ENRICllED TRANSCRIIqlON I:AcroR I INI-.'I llepatitis B vires (IIBV) -V..NI ~hancer can autivate lee expre.,,sinn of IIBV and non-I IDV genes in a Iiver-specil-~ manner. I1¥ Frfunning 1Ira electrophorelic mobil- ily-sbifl assays, we demonslmred Ih,,t Ihe Ihme related, liver-,'nrlched. Iransc~ilxiOn faClOrS, HNF3cz, IlNF3~, and HNF3"~ could all bind Io lee 2e sil¢ of the ItBV ENI enhancer. MUlItions inlroduced in lee 2e site to abolish lee binding by HNF3 reduced Ibo enhenc~, uc~ivily Ippmximatdy 15-trold. Mmeover, expression of HNF3 anlisense sequences to suppress the expression of IINF3 in Ilu~.7 hepaloma cells led to reduclion oftbo ENI en~--er ac~ivily, These resulls indicate Ihal llNF3 posilively regulates the ENI enhancer aClivity Ind Ibis regulation is most likely medialed throulh the 2c site. The ~-quimment of HNF3 trot the ENI enhancer aclivily could explain lee liver specil'mity of this enhancer element. Chen. M. llleng, S., Qian, X., Coslu, R., and Ou, J.-ll. Virology 20~:127-132, 1994. O~her support: National Inslitutes of Ilealth. From the Deparlmenl of Microbiology. Unlvet;sity of Southern California. Los Angeles, CA, Ind DepIrtment of Biochemistry. Universily of Illinois College or Medicine. Chica~o, It.. AIDS: VIRUS OR DRUG.INDUCED7 : We have seen above that the Dru~-AIDS hypolhesis cmrectly predicts all aspects of American/European AIDS. On the other hand ~he IIlV- hypolhesls has not predicted AIDS conectly; more imperlant, il has failed Io produce an), decrease in monalily, cure, ot I vaccine. AIIhough probable, the Drug-AIDS hypolhesis, until experimentally proven, remains an intcllec~oal CmSlmCt, Because of Ihe number of patients suffering from AIDS. lee implicalions of this hypnlhesls to Public Ileallh, 228 and lee polenlial for prevention of new AIDS cases, tesllnl lee drag hylX~hesis should hsve i high Ixiotily. Because Ibis hypolhasis makes verifiable pr~dk'*io~s, cxpe~memal IeSli~g Is legible ~d p~lical, Dm~ wxlcity c~ ~ I~ cx~m~ly In ~im~s, hum~ ~lls in tissue cuhur~ in ~iti~, d~ tox~[I7 c~ ~ t~cd e~mi~o~i- ~lly in hum~ who ~ ~icZed to ~1 ~s. ~ ~ ~ t~s c~ ~ ~led at a smull f~li~ of ~ ~1 in~t~ in ~ HIV h~s~ ]C Ihc dmc h~is proves to ~ c~ A~S ~ ~ =nli~ly venlabl~: - A~, curly the m~l toxic d~, ~ ~ ~C~d to its ~li~ indication, t~t is t~ ~mpy of met~lui~ ~n. • ~Ucallon uculnst illicit ~llonal ~ls w~ld ~du~ ~ ~v~t AIDS ~ by I~ ~mm~lated Ioxlcily of I~ dm~ ~st ~ ~ti~ Ipi~t inc has pr~d ~ app~iabla ~1~ in I~ ca~. - AIDS I~mpy ~ld ~ ~hi~ed ~ I~ ~li~l ~ ~al~ d~l ~ Ilwough Ir~n~l of AIDS dtse~ f~ I~ir ~iflc ciu~s, e.B. wile C~ ~l~llon, ~lhcr lean t~at ~h of I~ un~lated di~s wile cell-killer ~ ~luli~ to the AIDS epidam~ ~y ~ ~ cl~ as u ~ u~le ~c~e illicit dines, ~d to c~fine ~ to i~ a~ate ~lcat~. Ou~be~, P. If. A~ Joumul of Conlinuin¢ ~ucat~ in Nu~i 7:31 ~, I ~. ~r sups: ~vatc ~atlo~ From the Dcpa~mant or ~ol~ular and Cell Biolo~, Unive~ily of C~ifomi~ B~elcy, CA. FOREIGN-PROTEIN-MEDIATED IMMUNODEFICIENCY IN HEMOPHILIACS WITH AND WITIlOUT HIV Hemophilia-AIDS has been inteq~eted in lento of IWO hypotheses: lee lotion. protein-AIDS hypothesis and the Human lmmunedcfickney Virus (XIV)-AIDS hypolhesis. The fo~;gn-ixo~e;n.AIDS hyix)~esh bulds ~ Im)lek~ cot~minatir~ cor~merclal clotting facto~ Vlll cause immenosuppresskm. The foteiga.pc01ein hypolhesis, but no~ the HIV hypothesis, cmmtlly Ixtdicts seven char~eri,tit~ or hemophilia-AIDS: I) TEe increased life span of Ame~an hemophiliacs in 1~ two de~tdes before 19~7. altbo~gh 75% I~.came infecled by HIV~ Ixeau~ ftctof VIII treatment, begun in ~he 1960s, extended ~eir I|ves md simeltmenudy dlt~ninated hatmless HIV. After 19g? the life span or henx)pbillacs ap~m to buv~ decreased aguln: probably Ixcuuse of widespread tmalmem with the c,~o(~¢~;~ mi-HIV Az'r. 2) The d;stlnetly low. 1.3-2%, anntml AIDS risk of hemo~, se~. comical the hlghef 5-6% annual risk of intravenous drag men and male Eomo~xual aF~. dislac drub users--because Im~fuslo~ of foreign pmte~s Is less |mmu~osup~res- 229
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C) slve than recreational drag us~. 3) "r~ age bias at" hemophilla-AiDS, i.e. that annual AIDS risk increased 2-fold for each J0-year incre~e in agc~anse immunosupp~si~ ~ a ru~i~ or the JffetJ~ d~c of fnrel n f~ l~sfus~s 4~ ~ .... ~-.' ....... "g p~cins ~ceiv~ • ~ ,,,; ,-=,-~-~ m n~opflzuz-~]DS to immun~ficic~ dis- eases~u~ foreign p~cins ~nnot cz~ n~-immu~deficic~ ~IDS dis. ~s. llkc Kz~i's ~o~ ~) ~ z~c~e of AIDS di~es a~v¢ ~eJr background Jn ~xual pzn~rs or hemophilJacs~eaus¢ lransrusi~.m • immu~loxicily is n~ ¢~ts~s 6) ~c ~c of im ........ ~d~d Zcm. ~) Slzblli=Zi~, ~cn ~c~l~, ~ immunily of iliV.~sitiv= by Ion~.zc~ treatment with pu~ f~l~ VIIL ~is shows thai ~h~r HIV n~ ~act~ Ylll plus HIY ~ immunusu~ivc ~ l~m~lv.. ~c~r~. AIDS prevented by climinalion of HIV rr~ the bZ~ supply and cannot ~ ~lionally t~at~ with len~oxic ~liviral dm~ like ~, instead, h~philia-AIDS can prcv~zed ~d ~ wen ~n reve~cd ~ I~at~t with po~ factor G~clicz P~:51.70, Olher supra: ~vaz~ ~zli~s. From Ihc Dcp~mcnt or Molcculw ~d Cad Biology, Univc~ity of Cali(omia Berkeley. Beskc~cy, C~. DNA RECOMBINA'IION IS SUFFICIENT FOR RETROVIRAL TRANSDUC'I"ION On'eugenic retrovlmscs eany coding sequcn~s that are t~msdnced from cellula~ pmtooncogenes. Natural transdncdon involves two nonhomologous recombinations and is thtJs exlrenlcly rate. Since transductinn has never been reproduced experimen- tally, its mechanism has been studied in terms of two hypotheses: (1) the DNA model, which pint, dates two DNA reonmbinations, and (ii) the RNA modal, which jx~tulates • $' DNA n~'ombination ~cl a 3' RNA recombination occugring during revere transc~pti.on ofv~. and pmtooncogene RNA, Here we use two viral DNA cons•nuts to test the prediction of the DNA model that the 3" DNA recombinstion is achlued by conventional Integration of t relrovirzi DNA 3' of the chromosomal Wotoeegogc~ coding region. For the DNA mockl to he viabie, such recombinant vbuses must Ix: ioJ'ectinu~ without the poqxxtedly essential polypurine uact (ppQ that Jxccedcs th~ 3' long tcrminzi repeat (LTR) of 811 relmvimses. Our constructs consist oft rox coding r~gion from Ila~ey sarcoma virus which is naturaly linked at the :5' end to • reuovird LTR and mlificjzily linked at the 3' end either directly (con- start NdN) or by a cellular seqtw.n¢o (construct SU) to the 5' LTR or• retrovims. Both constructs Jack the pp(. and the LTR of NdN even lacks 30 nucicot;d~ at Ihe $' end. Both cons•nuts ixoved to he infectious, producing viruses at tile83 or io, fecus. fonning units per mL Sequence analysis proved that both viruses were coliecar with input DNAs and that NdN vires lacked n pp( and the .5' ~0 nucleotides of the LTR. 230 The results Indicate that DNA rccomhinstio~ is sufflcicnt for ~n)vi~t tmuduction and that neither the pp¢ nor the complete LTR is mtial for relxovims ~ication. DNA recombination explains the following ob~.'t~ations by othe~ thai cwmot Ix: reconciled with t.he RNA model: (i) expevlmentsl tmnsdnctlen is independent of the packaging efficiency of viral RNA. and (ii) experimental tmnsduction my invert sequences with respect to other~, as expected for DNA rccomh;nation during trans- feet|on. $chwmlz, J. R.. Duesherg. S.. ~d Due,~erg, P. Proceedings of the National Academy of Sciences USA 92:2460-2464, M~tch 1995. Other support: Private Donalions. From the Deparlment of Molecular and Cell Biology. University of California. Berkeley, CA. CHIMERIC PROTEINS COMPOSED OFJUN AND CREB DEFINE DOMAINS REQUIRED FOR INTERA~rlON WITH TIlE HUMAN T-CELL, LEUKEMIA VIRUS TYPE I TAX PROTEIN The regulation of human T-cell Isukemia vires type I (lfFLV-I) long tennlnal repeal gent expression is dependent on three- nix.acting elements known -, 21-bp repeats and the tran~ctivator protein Tax. Mutagenesla has demonstllled that .s~'q_t_t_t_t_t_t_t_t_t_~. in each of the. 21-bp repeats can be d~vMed into ~ dom~ns de, ignored ^, u, and C, Tax ~limulates the binding oI'CREB to Ihe B domain, which is for Tax activation ol" HTI.V-I gent expression. In th~ study, wc demon.mate Tax will stimulate the hlodlng of CREB to the HTI.V-I 21-bp ~ts but dncs aM ~ffnulate CREB t~edlng to the consensus cyclic AMP Ire~xx=e element (CRE) ele- mcnt found in the somatostmln promoter. Howc~-r.'iax stimulates CREB Mnding to z consensus CRE in the contest of the 21-bp repe,ats, iodlr~ing the impmlancc of these sequences in stimulating CREB binding. To dctcrmlne the mcchaxism by which Tax stlmuistes CREB binding ~d determine potentiM intemctiom belween Tax and CREB, we used the memmali|n two-hybrid system in co~junelio~',wilh b~ v~tro binding maJ gel retardation assay=. TWo-hylxid ~mdyzls indicated that tions in either the basle or leuclne zipper region of CREB prcvenled |otevactions wilh Tug. Since several studies have demonstmled that Tax will alto stimulate the binding or s variety of different he,in region-leucine zipper p~leins Io their cogn~le .binding sites, we assayed whether chimeric Ixote~ ~ of portin~s of CREB and another basic reglon-leucine zipper imp(tin,/tin, could he used to map required for intemclions with Tax. The~ ~nd;es we~ possible beeau.~ we d[d not detect in vivo or i~ vitro interactions ~ TI~ lind ]en. ~ re|no KM or the CREB basic reglae and a pertio~ of its legcine zipper were ~'qul~l for ie ~vo and ia vhrn interactions with Tax and inct~,~l binding or CREB to the 21. bp n:peats in response to Tax. These st.die5 defin~ the dual;as in CREB p:quired for both in ~'vo and in vitro interactions by lhe HTLV-! Tax Wotcin. Yin. M. J. Psulssen. F_. S~eler. L, and GaTnor, P_ B. 231
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Journal o!" Virology 69(I 0):6209-6218. O¢lober 1995. O|her suppo~l: National Insdtotcs of Heallh. From Ihe Deparlmenls of Internal Medicine and Microbiology, Divisions of Molecular Virology and Hematology.Ontology. Universily of Tcx,~s Soulhw~stem Medical Cenler. Dallas, TX. PROTEIN DOMAINS INVOLVED IN anTI! IN VIVO AND fN VrrRO INTERACTIONS BETWEEN HUMAN T-CELL LEUKEMIA VIRUS TYPE I TAX AND CREB Gc~¢ cxpresslou from Ihe human T-cell leukemia virus type I (liTLV.I) long terminal rep:at (LTR) is mcdialed by Ihcee cis-actin8 regulalo~ clcmems known as 21-bp repeals and Ihe lransaclivato¢ protein Tax. The 21-bp repeals can bc sulxJi. vided into mree motirs known as A, B. and C, each of which is impor~an! I'or maxi- real gene expression in response Io Tax. The B molif coulains nuclcoddc sequences know• as a cyclic AMP response elemenl (CRE) or to.r-re.~lmnse element which I~nds memhers of the ATFJCREB family o1" transcription raclnrs. Though mulalions or lhis clement in Ihe HTLV-I LTR climinule tax nclivstion. Tax will nm acdvale runs! olh~" promote~ conlaining these CRE sites. In Ihls sludy, we invesdgaled ~he mcchanlsm by which Tax acdvales gene expression in conjunction wilh members of Ihe ATFJCREB family. We found thal Tax enhanced Ihe binding at'one memicr o~" the ATF/CREB I'amily. CREB I, Io each oflhe Ihrec HTLV.I LTR 21.bp repeals bul not another member designated CRE-BP! or CREB2. Tax enhanced the binding or CREBI Io nonpalindromie CRE binding sites such as those found in the HTLV.I LTR. but Tax did not enhance Ihe binding o1" CREBI In palindromic CRE bindinG sites such as fated in Ihe $omaiostaii• Ixomotcr, This finding may hell) explain failure of Tu to activate promoters comaining consensus CRL silts. Tbese ~,udlcs were extended by use of the mammafia~ two-hybrid syslem. Tax was dcmm~aralcd to interact diRcdy wilh CREBI b~ no! wilh other bZIP proteins, including CREB2 and Ju•. Sire.directed mulagenesis o/' both Tax and CREBI dcmonslratcd thai amino lemdnus o/" Tax and both Ihe basic ~ the leuci~¢ zipFcr regions or CREEl w~re required for direc~ inlmcliom hetwcer~ these proteins I~h i• vlvo and [• vitro. This inloraclion occurRd in vit'o and Ihus did hal Rqulre Ihc ixcsence oCthe FrTLv.I 21obp repeats, as Ixevious|y su~cslcd. These reselts dcfin© the domains required for inleracliOn bclw,o.en Tax and CREB that ar~ likely critical for the eotlvallon o~r HTLV-! gone exlxcssio~. YIn, M,-J., Paulssen, E. J., Sealer, J. S~ and C,•yno% R, Journal or Vi~osy 6g~6}:342G-3432, ~une 199.5. From the Division of Molecular Vhology. Dcpa~lmcnls or Inlcrual Medicine and Microbloloiy, Unive~slt¥ oCTcxas Soulhwe~tem Medical Cenler, Dallas, TX. 232 HUMAN IMMUNODEFICIENCY VIRUSES REGULAI-rJ) BY AL,TEP-~ATWE TRAN$-ACTIVATORS: GENET/C EVIDENCE FOR A NOVEL NON- TRANSCRIPTIONAL FUNCTION OFTAT IN VIPJON INFEC11VIl~ Thirteen genelically allered HIV-I pmviruses were creeled. These v~dovs gcnomcs can he segregated into three gmup¢ (i) m set o!'~1(-) vilusel thai have n [uuclional H'TLV-I Tax inserted in nt~,, (el) a set at laK-) viruses wllh Gel4 binding sites inse~lnd in U3 and e Gal4--VPI6 eDNA insc~lcd in nt~ sad (ill) a HIV genoese thld ar~ $' and 3' TAR(-) and urn Gul4-blnding-silc(+) in U3 and Oul4-.-VPI6(+) in st/. We found Ih~l viruses in Croups (i) and (it}, ~llhouSk wee Cully copplcmented fo~ viral gene exlx~ssion hesed ~ quanlhadve measu~- meals of viral prolein synlhesis a~. on Ih~ visuallzaiion by elcclron mic-mscopy Ihe pro~r as.~,:mbly o1" mocpholo¢lcully corRct vidoes, lnlm:slln¢ly, ~j~up (i) and (it) vlrio~ worn defective in u spreading cytopIthi¢ infc¢lion whm Iss~yed in lymphocytcs. G~oup (ill) viruses, although cqmble o( Ix~ducin¢ intact T~ Ix~cin, al~ could ,~ot use Tat fo~ transcrIplio~/|ene exlxesskm because ol'lhe TAR(-) ICglo- lye. Ilowcvcr. Ibis class o1" vi~al ~nom~s produced viroses Ihai v~ highly inl'~'- tious end cytopathic in pdmuw and in conlinuously propagated T-lymphocytcs. Th~se d~re~ groups of viroses are all Ir~nscdplio~lly Tel--TAR imkpendenl. Their distinct diITe~nces in inl'ecdvity/cy~oFx~hicby lXovide genetk: evidence Ihai T~ rides • tran.~.'~iptio~ally independent funclkm in dclen~ining infeclivily ~ ¢~lo- pathici~ in lie: setting ol'a slxcadin¢ viral inCccdo~ Give Ihat all HIV vlrlons nor. rosily comuin four iotucl copies of TAR RNA, our findings suggest I to.examination of whether Tat could I~ u virion-TAR-associaled protein and Ihe Ixmible impliea- lions of this ~'or virus inrccllvily/~ylopathicity. Huun¢. L. Joshi, A., Willcy, R., Oremleln, J., and 3e~ngo ~ EMBO Journal 13(12):2886-2896,1994. Other support: Nalio~ul Insfitulcs ol'Health. From the Molecular Virology Section, Labor~lor~ of Molecule" Microblolosy, National Inslitute or Allergy xnd lnfectio~s Diseases, Belhesda, MD. and Dcpurlmcnt or Palhology, George Washington Unlvcrsi~o W~hin~o,, DC. GENE TFIERAPY FOR HUMAN IMMUNODIg:ICll~CY VIRUS GENETIC ANTIVIRAL STP~TFJ31ES AND TARGETS FOR INTERYFJeTION Gent lhe~pguli¢ sl~ile$1es for Iha lxeaimgn~ ol'human imm~no~r~n~ vim type I (HIV-I) inl'cctlo, have Rceived ira:reused nitration due In luck ol'¢hemod~c~- six:uric drugs or vaccines thai show lemg-term efl'~cucy in vtvo. An el~q~|n| referred Io here as "genetic anllv~rals." ~s revi~vcd. Gentian antivireh a~e defined as DNA or RNA elcmenls the! are transl'cn~'d h~o cells and all"ucl Iheic targels either direclly, or s~er expression as RNA or ~ "they include anlisense ollgonuclenlides, rlbozymes. RNA decoys. Iraasdomlnanl mulanls, toxins, and immunogens. They niter Ihe FO.~ibillty to ta~gel s~mullanco~ly m•ldple s~es in IlIV gcnome. II~ereby minimizing Ihe produclion of resislanl ¥1mses. We re¥1©w 233
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C) moleculat, mechanisms of'genetic anlivirals, lheir HIV too|ocular targets, and discuss ,ssues concerning their application as ahti.HIV agents. Dmpul;c. B. and./ezng. K.-T. Human Gone "l~crapy 5:927.939, August 1994. Other support: National Institutes of Hcahh. From the Molcculat Virology Section, L~bor~tory of Molecular Microbiology, Nalional lnslilules or AIl~.gy and Infectious Diseases, National Institutes of Ileahh. Bethesda, MD. EXPRESSION CLONING OF GENES ENCODING RNA-IIlNDING PROTEINS RNA.binding proleins arc important rcgulalory factors in gone cxprcsslon. Studlcs on the stmclure and r, nctio, of these i-o~clns ~ complicated by limitations in the technology for their isolation. One approach is to purify the prolein, to Per- fn'm microsequoncing or amino acids, In construct degenerate ol|gonucleolidcs, and then to sen:on lilxaH~. An dtemalivc to this is Io isolate expression cONAs direcdy. Here, we dcsc6he a method to isolate cDNAs em:oding RNA-hinding prolclns, using a direct screening assay for RNA-prolein.bi~ding inleractions. Fo¢ this puqx)s~, we modified the ixocedure commonly used in isolating cDNAs encoding sequcncc.spe. cilic DNA-blnding proteins. Using this I~otocol, we have isolated • I.~-kb cDNA thai codes for • human pmlein Ih~l binds to Ihe human immunndeficiency tyl~ I (.HIV-I) Tal.reslx~se elcment {TAR) RNA. Modifica|ions and delails or our tech- tuque ate presen[nd below. Gatignol, A. ~nd Jeang, K.-T. In: Methods in Molecular Gcnelles, Volume 4, Academic Press, Inc., pp. 1994. Other sul)lXxl: National Instltutcs of Health. From Ihc Molecular Virology Seclion, L~borstoq~ of Molecular Microbiology, N|lional lnstltuzes of Allergy and Infect|ous Diseases, N-'ional lnslltutes or Ilcahh, Bethesd~, MD. COMPARISON OF REGULATORY FEATURES AMONG PRIMATE LENT|VIRUSES Thls review highlights the gene~l similatities ~nd soma specific diffe~nc¢s in the re~ulalory mcchani.m~ u.,~'d by the l~imale lenljvjmse.~, From the perspective or Ihe rcgulalory prolcins Tal and Rcv and Ihcir sespcclivu RNA tatgels, TAR md RRE, one c~n con¢lud~ Ih~l m,~h is cons~cv~d b:lw~n HIV-I and HIV.2~IV. TEe fact Ihxt non-re~ilxocal effects h~ve been desulbed for bulb l'ran~-|ctiv,lion axes reinforces the ide~ thai there rum:in unique i~e~iel individu|liznd to the diffcrem species. The clear abstract of the accessory t~ gone in Ihe H]V.2~IV subgroup is consislcnt wilh this. Having slated the above., it is cleat Ih~ SIV, with its |bility to infec! CD4" cells and Io cuuse AIDS in monlc~.'Ts, ~'l~S~nts for in vivo HIV-I imlhogoncsis. The recent clucidallon of Iome o1' Ihe I)~tbogcnic dctcrminanls for SlY (DEwlIUII$'r el al. 1990;, K~n.E~t el al. 1991; DANm.I. el al. 1992: L~NO cl ,,I. 199:!) should go a long way toward onr understanding of HIV-I. Jenng, K..T. ~nd Gatigonl, A. In: Curt, on! Topics in Microblnlngy and lmm~olo~.. VoL IIl[I. Spc~ngef-Vedag, Bedin-Ileidelherg, IT~. 123-144, 1994. Olher support: National Institut=s ol'llcshh. From the Lahomlor? of Mnlccul~r Micmbinlogy, Nali~mal lr~titute of Allergy and Inl'cclious Diseases, N~ional Instilules of II¢~lth, B~hesda, hiD, DEFINITION OF A MINIMAL ACTIVATION DOMAIN IN HUMAN T.O_~LL LEUKEMIA VIRUS TYPE i TAX Fourteen muumla ~ein of human T~ell le~ia vlms I~ I. In ~ ~y m~g s ~-~ (O~) fus;~ ~cin ~ • ~sive ~ ~t~ning Gall ~ ~in~ ~1~, wc f~M chat ~iva~ in trans. ~n Tax mu~ wc~ t.~ ~ ~khing. m~y ~ eff~ly againsl G~x. Howcvu. Ih~ to inhibit activity. In p~icul~. Ih~ mutinls I~1 w~ ex~ sllbly, w~h ly. On ~ ~ ~M. mu~s wi~ ~ivM~l ~ at m~ ~M 3. 9. ~. 41. 273. ~d 3~7 ¢ff~mly s~aMy exp~d. In ~nle exxon, w~n f~ ~!~ Io ~ DN~b~ing doma~ of Gal4. mino ~ids 289 Io 322 of Tax ~r~ t~ ~tlval~ ability. ~is fusion ~cin • mino ~ids 2R9 ~o 322 dcfi~ a ~gi~ ~ ~ m ~nlial ~ fm~ ~ Ihll this ~gi~
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Other support: National Institutes of lleal.h. From ~e Molecular Virology Scclion. Laboratory of Molecular Microbiology. National lnslilute ol'Allerl~y and lnrcclious Di.~eas~s. Bathe•dan MD, GENETIC MAPPING IN HUMAN AND MOUSE OF THE LOCUS ENCODING TRBP. A PROTEIN THAT BINDS THE TAR REGION OF THE IIUMAN IMMUNODEFICIENCY VIRUS (HIV- I) Productive infection with HIV-I, Ihe virus responsible for AIDS, requires involvement orhost cell factors for complcliou of Ihe replicaliv¢ cycle, hu! Ihe aden. tification o,~ these rac~ors and elucidalion o1" their specific funclinns has bcon dil'l~- cult. A human eDNA, TRBp, was rceonlly cloned and characteriT.cd as a positive reeulalor of" ¢ene expression Ihal binds to the TAR region of' the IIIV.I go•nine. Iler¢ we demonstrale that this factor is encoded by • gene. TARBP2. that maps to human chromosome 12 and mous~ chromosome 15, and we al~ identify and map one human pseudoeenc (TARBP2P) and two mouse TRBP-rcla|cd scqucnccs (Tarbp2.rsl, Torhp2-rs2). The map location or Ihe exp'es~ed ;ene identi/ics it as a candidate for Ih¢ previously ida•lifted factor encodc~ on human chrommome 12 thai has bcco shown In he impolll~t for expression of HIV-I genes. Western hlnltlng incficales thai dcsp|le high sequence co•sen, alarm in human and mouse, the TARBP2 protein dikers in apparent sizc ;n prlmale and rodenl cells. Kozak. C. A.. Gatisnol. A.. Graham. K.. Jeans, K.-T., and McBride. O. W. Generates ~'q:66-72, 1995. Other suppml: Nalional lnslilutes or Hcallh. From the Labor•too, of Molecular Microbloloey, Nalional Institutes or Allergy and |nfeclious Diseases. and Laboratory of" Biochemislry. National Cancer Instilulc. National Institutes of He•lib. Bclhesda. MD. WHAT REASSORTS WilEN REOVIRUS GF.NOME SEGMENTS R "EASSORT? Rcov~us genome segment Rzssorzmont is clearly • complex phenomenon. On Ihe o~e hand. vastly different gc~ome se.~mcnl$ are Radily steepled, like the ST2 S I genorr~ seemenl into the ST~ gconmc; |! and Ihe RT.'t R I gcnnme ~el;ment have diverged 59% toward randomness in their sccoud hase cndon posillons (K). Thcm is, bowevu. • requirement for the exchange In Ix: ¢ffccl~d: and Ih-"t is two mutations in the ST3 $4 ¢enome Sel~meot. These same Iwo mulallons ,,re required for the lance o1"•11 ST2 genome segmenls introducllous inlo Ihe ST'3 ~enome. includin¢ thal of. the ST2 M2 gcnomc segment. the s~cond base code• position or which has diver~¢4 In random•ca• from that of the S'T3 M2 ge~ome segment by only 2% ($). ; This suggests Ilut wh,,t is n¢¢dcd he~ is not mulalio~u 81 t~ RNA I~cl in ~r to ~it RNA s~c~s Io rc~¢nJze e~h elba. ~1 ~ ~ ors ~l ~cin ~3 (which Js z c~em or ~ ssRCCs ~d dsR~ (~ n~)~ Hoover. ~is d~ not apply Io the 9 ~1-1 ~ MR s~lem, whe~ hetem~g~s gc~ seg- mon~ arc ~ce~ed with ~ to four muttli~ lest ~ I~m ~ simi~ to I~ gcnomc s~m~t ~nl t~y ~pl~e. ~t wJ~ut I~ G~ ~ G~ ~ltti~s ~ t~ ~1 54 ¢cno~ ~g~nl. N~ d~s it z~ In t~ ~7 s~tem, ~ ~ muhti~ in Ihe S I gcoo~ ~¢mcnt am surreal In ~t I~ ~l~ or ~ = ~ p~ segment by the Is' $2 gc~ ~gmaot, which diWm f~ iZ ~ ~ly ~e ~i. Jokllk, W. ~ ~d Roner, M. R. Olher su~R: U.S. ~hlic Health Fr~ Iha Department of M~mbiol~y. ~ka Univmity ~diczl Center. ~, NC AN RNA-PROTEIN CONTACT DETERMINED BY 3-BROMOURIDINE SUBSTITUTION. PHOTOCROSSLINKING AND SEQUF.NCING An analogue of the Rplicsse lransladonal operator of" bacteriophage RI?, thai contains a 5-bromouddine at positio~ -$ (Rb/A I), c~mplexes with • dimer of Ihe coat protein and photocrosslinks In the coat protein in hlr, h yleM upon excitation at 308 nm with 8 x~ou chloride cxclme~' laser. "rzTp(ic dieestlon of Ihe cmsslinked nuclcoprotcln complex i'ollowcd by Edman de/z'ld~tiou ord~c ~'yplic rmgn~nt hear- ing the RNA indicates eros•linking Io lyre•inn ItS el" the coat protdn. A control exJ~dment wilh n Tyr RS Io Sor 85 ,~rlm~t c~t Im~cin showed bi~d~ but no photo. can•slinking •t saturating protein ce~mlx~ion. This is consislen( wi~h Ihe oh•cry•. tio~ from model compound stndi~s of Werem~izl p~otocro~slinking or BrU to the electron rich •remade amino acids ll~tpIophan, lydia•c, Imd hislidine with 30g nm cxcltatlon. Willis. M. C.. LcCuycr, K. A., Mciscnbcimcr, K. M.. Uhlcnbcek, O. C.. Ind Koch, T.II. Nocieic Acids Re~aeh 22(2.1):4947,4952, 1994. Other suppo~l: N*'ional Institute ~ Gcncrd Medical Sciences. From the. Deparlmont of Chamist~ sad BiochemislPJ, University of Colorado," Bouidcr, CO. 237
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ASSOCIATION OF MOUSB HBRINOGEN.LIKE PROTEIN WITH MURINE HEPATITIS VIRUS-INDUCED PROTHROMBINASE ACTIVITY Previously. we demonstrated induction of • unique macrophage prothrombinasc during infection of BALB/cJ m|ce by mouse hepatitis ~ims strain 3 (MHV-~). By immunologic scfconing, a clone m~'csendng Ibis prolhromhina~ was isolated from • eDNA library and sequenced. The sequence ~denlified Ibis clone as r"prescming part or • gene, musjT/~lp, Ihat encodes • fibrinogcn-like pmlein. Six additional clones were isolated, and one clone, pl I-3- I, encompassod Ihe entire coding region nf mu.r- J~blp. Murine macrophages did not constilutively express mn.rJ'/blp but, when infected with MIIV-3, synthc~z.ed mu~J/hlp-specillc mRNA. mlt.~hlp mRNA indue- lion was earlier and significanlly grealer in BAf..B/cJ than A/J macrophages. ~.hro_m.b.[n. _as~..~llv!!y was de.monslratcd when mus~blp was exp'e.~sed from p ll. -~ m K^w zb4.7 ce,s. These oat• suggest Ihlt mu~hlp encodes Ihe MHV-induccd prothrombinase. Parr, R./_, Fung,/.., Rencku, J, M)'c~-Mason. Journal of Virology 69/8):~0:~3.S038, August Other support: Medical Research Council of Canada and National Instilut,-s of Health. From Ihe Dcpartmen! of Palhology and L~heralocy Mcdlclne and [}epanmem of Microbiology and Molecular Genetics. University of Texas Medical School at Houston, Houston, TX, and Oepartmcnf of Medicine, T~onlO Ilospital, Univc~ity of Toronto, Toronto, Canada. PATI~.RH OF DISEASE AFTER MURINE HEPATITIS VIRUS STRAIN INFEClION CORRELATES WITH MACROPHAGE ACTIVATION AND NOT VIRAL REPLICATION di Mu~.~:..h~.pali.tis vlms s.train 3 (MHV-3) produces • strain-dclx:ndcnt pattem sense wmcn n~s I~-en useo as • model for fulminam viral hep~il~ This sludy was undcn,~en to examine whether there was • co•elation between macrolphagc act|v•. tin• and susceptibilily or resistance to MHV-3 infection, Pcriloneal mgcfophases were isolatM from resistam AJJ a~l eusecpl~lc BALBIcJ mice and. following stim- ulation wilh MHV-3 or lipolmlysaccharide (LPS). analyzed for transcription of mRNA and production or int~leukin.I (IL,-I). tumor necfosis faclor •Iphe (ThIF.~). ttansfom~ing growth factor ~ (TGF-~). mouse fibr|nogen-like protein (musfihlp). tissue factor (TF). leuheViene B.. and prose•gin•din E~ (PO~). Macrophagea from BALBIc~ mice produced grealer amounls of" IL-I. THF--o. TGF.J3. leukotrlene and musfiblp following MHV-3 infection Ihan mlcrophagcs from resistant All mice. whereas in ~csponse 1o LP~. equivalenl •mou~ts of" IL-I. THF,~. TOF-~3. and TF were produced by mncrophages from bolh strains of. Rice. Levels of. mRNA of THF-ao and mu, sfiblp worn greater and more pcrslstenl in RALBIcJ than in AIJ macrophages, whereas the levels and kin•lies of IL-i. THF.e~. and TF mRNA lowing LP~ sllmulatlon were idemical in mac~oi~ages from bulb s~raius of mice. 238 I Levels of poxtucdon of PG~ by MHV-3-stimulated macrophages from ~slmm and suscel~ible mice wcfe equivalent; however, the lime courJe f.or induclk~ of PGF~ dilTered, but the total q•antit7 of I~E~ pmduecd was insufl'~ent In i~ibk induction of. musfib~p, a pmcoagulant known to conelale with development of fuhe|. • ant hepatic necrosis in susceptible mice. "l~ese ~sulls demonstr~c mmked diffcf- trices in produClio~ of inflnmmato~ mcdiatms to MHV- 3 infcclion In m~cn~ges from resistant ,~JJ and susecplible BALB/cl mice. which Ray explsln tbu n~lkcd hepatic necrosis and fibrin depo.~ilion and uccounl for the lelh•llly of MIIV-3 in sus- ceptible mice. Pope. M.. Rntstein. O.. Cole. E.. Sinclair. $.. Parr. R.. Cruz. B. F'm~emte. R.. Chung. S.. Gorczynski. R.. Func. L.. Leibuwllz, J., Ran. Y. S.. and Levy. G. .Toumal of" Virology 69(9):$2S2-~260. Septcmlx~' 1995. Other support: Medical Research Council of Canada and National Instil•tea of Health. From the Departments of Surgen/and Medicinc. Tommo Hospital. Unlver~¥ of Toronto. Toronto. Ontario. Canada. and Department or Pathology. University of Texas l|callh Sciences Center. Houston.'lX. KEY ROLE OF A CCAAT ELEMENT IN REGULATING HEPATITIS B VIRUS SURFACE PROTEIN EXPRESSION Two sap•role promoters, the upstream ~,SI and the downstw~m 5 promoten. give rise to Imnser~p{s encoding three forms.of Ihe hepelitis B vires surface p~lein. Ovet~roductio~ ef large surface proleln I~:ause of inceeased to a block in secretion of all forms of. the surface pr~ein and of vi~m pmlicl~s. We show h~e Ihat • CCAAT element in the S prom~cr nee only bcro~ses the arno•nl of" S transcdl~S, but also ducreases the ~unount of tx~51 Comcqucntly, mutations in this elcm~t c~use intraecllular lecun~lllio~ of surface Ix'Me|as because of the secset.of7 block. Thefefo~r., this CCAAT etcmenl qq~'ars to he C~ilical for mainCalning the high ratio of. S versus Ixr.SI transcripts ahec is neces- sary for Ihe viral life cycle. Lu. C.-C., Chen. M.. Ou, J.-H., and Yen, T. S. B. Virology 206:11~$-I I~$, 199~. Other support: Halional Institutes of. Hcallh. From the Dcp~nmen! of Pathology, University of California, San Francisco. CA, Depanmcm of Micmbiolngy. University of Soulh~ CalWocn]a, Los Angeles, CA, and Anatomic Pathology Service. Veterans AITa~z Medical Center. San FrancisCo. CA. 239
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PHOSPHORYL~TION AND NUCLEAR LOCALIZATION OFTllE 14EPATITIS 13 VIRUS CORE PROTEIN: SIGNIFICANCE OF SERINE IN TI IE TItREE REPEATED SPRRR MOTIFS Hepatitis B ,~irus core prole~n (antigen) is an imporl-',nt serologic marker of hcpatitls B virus infection. This protein is round in the cyloplasm or the nuclei, or bol~. o1" |nfected hepatocytes. A nuclear luc•llzalion signal has previously Ix:co aden- tiffed in the core prmein sequence. This signal overlaps three rape•led SI~RR molifs. In this repo~1, we demonstrate that subslilulion of all o1' the seslne residues in Ihese Ihree SPRRR motifs with el•nine can prevent almost cnllrely Ihe phospho~la- don of the co~ pmteln in Xuh-? hap•iDa• cells, enhance nuclear lucali~tlon of core prmein in 5o4h Huh.? and nonhcpatlc ceils, and at~llish cell cycle regulatio~ of" nuclei" Iocalizaiinn of the core pro(tin. Since the throe core protein mutants which retained only one sedne residue of each of the th~-'~ SPRRR motifs could be phos- phon/laied to similar degree. Ihese three •edna residu,'s likely could serve as acceplor sites for phosphon/laiion with equal el'ficicncy, These results. Iogether with the observation that Ihc three SPRRR mural's overlap the nuclear locallzatirm slgnol of the core protein, raise Ihe possibility thai nuclear Iocalix,~tton o1" the core protein is ncgailvely regulated by phospho~laiion of the •ulna r~sidues in the SPRRR molil's. LieD. W. and Go. J.-II. Journal of Virology 59(2):1025-1029. February 1995. Other support: Natlon•l ]nstltutes or Health. From the Department of Molecular Microbiology and Immunology and D~parlmcnt oF Pharmaceutical Science, Univelsity or So•them C•llfomla. Los Angeles. CA. MULTIPLE SUPERINFECTIONS FAIL TO ACTIVATE DEFECTIVE I IUMAN IMMUNODEFICIENCY VIRUS-I (HIV-I) INFEC'~ION OF RABBITS Supcflnfcction or human immunodeficiency virus (HIV)-I-infecled rabbits with Treponema pallldum. Mycobacterium ovlum, herpes simple• Candid~l albieans. My~oplam.~ incoinilus, and malignant eam,~h~ fe~e~ vires as well as irndiaiion or cortisone treatment. I'•ils to activate production of infectious virus. For up to 6 momhs •her Infeclion of rehbils wilh HIV-infcetcd cells or free vim•. them ~re nei- ther dinlc,,t symptoms nor poshive labcx~X(~J le.~ts for deP:ction of IlW. However. uher supcr~ufcction with o~hes agents, lily sequences may he transiently found in poripher~! blood mononuclcar cells by polymer•st chain reaction (PCR), and mulli- pie antibodies to HIV antigens may be detceted by Western blo~ting. Bulb the PCR posilivity and Western blot reactivily become negatlvc with time al~er superlnfec. lion. Other than delayed healing of the skin lesiaos produced by T. I~llidtme and vac. cinia in HIV-infc,:ted rabbhs, them is no evidence of any immune •bnonnalily. Aflcr death, gag sequences •re dr.l~ctable in the splenocytes of essentially even/HIV. infected rabbil, and the splenouyles of ei~h! of ?,~ infected rabbils responded by pro- liferadon to HIV pcpiides. In ~ddition,/Cog sequences a~ detent•bin in rat~its that ~re ln.iccted with lymphoid cells [mm HIV-infected rahbils, llowevcr, after muhlple 240 ~ testing of l~th peripl~erat blood of living mhbll~ and oigans of mbbhs thai died or were killed (spleen. t~ain, lymph nodes, liver, and ~ot.. nlesttnal..~1.)., no via.b.lu virus has ever been convincingly detected by in rilro CUIliYadM will1 tuolcB|or C~llS. In contrast to some other published repom, the~ a, tl indic~n that HIV-I lore•lion of rabbits duel not provide • model for AIDS I~thogenesis therapy or ix~v,~tioo, hut it may 5o useful as • model to study the relative res~anc~ oi'• small h'~:tkm of the human population to development of AIDS af~f HIV infection. S~|l, ~. and Tseng. C, K. Journal oi" Acquired Immune D~ficiency Syndromes and Human R~rovimingy 9:2t 1-226, From the Department of Pathology and Laborato~ Medi¢in~, Unlver~y of Texu Houston Hea)lh ~ciel',¢e Center, H(mslon, TX. Xlll. Miscellaneous 241
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C> KINETICS OF PEP'TIDE SYNTHESIS STUDIED BY FLUORESCENCE OF FLUOROPHEN',;L The kinetics of Ihe reaction of Boc-allnine-lril]uomphenyl, Roc-alanin¢.tctra. fluorophenyl. Boc-alanioe-penlalluorophonyl, and PSoc-alanlne.p.chlomtctrafluoro. ph~nyi esters (BocAlaOTff, BocAlaOTfp, BOCAlaOPI'p. and BocAlaTfc, respective- ly) with Icucine snide and wilh valine methyl ester have been m~sured using changes in fluornphcnyl chrom~hore emis.~ion at 375 nm. The kinetic data be well fit wilh a simple s.~cond.ord,.r reaction ~h,:m~.. Mea~remenls of the roan- lion k|nclics al different concenlrations of I1~ rcagcms showed Ihat the cx esslon for Ihe reaction rate is V:/~C~.~L"~? in which ~, :- ,~- ...... _pr. ". concentratlonofe|lherL~u n ~, al ""-n;w.~a~lonratecon.stant, C, isthe NH, or V OCH:, and C,~ rs the concentrallon of the f]uo- rophcnyl esrer. This reaction equalion indi~t¢: a complex, probably chain-like, rcaclion mechanism. The order of reaclivlty for these active esters with ValOCH, is BOCAlaOT[c>BocAlaOP~p>EneAIIOTrp>BocAlaTIT. The apparent rate constant, for the retction with LeuNHw is higher Ihan thai for the reaction with VaIOCH:. Permyakov, E. A., Mcdvedkln. V. N., Klimenko. L. V.. Mitln, Y. V., and Pennyakov, S. E., Jr., (Krels|nger, R.) Intemalional ]onmal of Pep~ide & Pro~cio Rcseamh 44:472-476, 1994. From Ihe Theoretical and ExpeAmenlal Biophysics, Russi,m Academy or Sciences. Pushehino, Moscow, Russia, and Instltu~e of Protein Research. Russian Ac~emy of Sciences. Pushchino, Moseow. Russia. P-CtlLOROTET~AIR.UOROPNENYL ESTERS OFN-PROTECTED AMINO ACIDS p-Chlorolclrafluorophenyl (T~'¢) esters of pcotecled amino acids and peptides are m~.rc rea.c, tive tha_n Ire t.he w.¢ll known pentslluorophenyl {Pip) esters. Two ~a~nes, p'cnto~letrallUOrOf)llenyltrifluoroaczlate (TI'c-O'ITa)and di-{pchloml©lra- fluorophcnyl)c'adoonat¢ (di-Tfc-carbon~e). can be used for their synlbeses, thereby avoiding use or Ihe allergic dicycloEeaylcarlx~ilmide. This is especi~dly impm1~nt for I~lk Ixcpar'atlons. Mam~ Fmoc- and Boc-c"nino acld-OTfc esters have been syn- U~esiznd I~1 characterized. The hexadecamed¢ landem repeal H-(AlaAlaLysPro),- OH was synlhesizcd using di-Tfc-cartxmate for the ix~aralion of Tic.esters. Medv~lkin, V. N., Klimenko, L N., Mitin. Y. Y~ KreLdnger, R, I!., Shabanowitz, J., Zabolotskikh, V. F., and Podgornova, N. N. lntemalional Journal ot" Peptide & Pmtcio Researeh 44:477-41~4. 1994. ~om the l~,titut¢ of P[otein Research, Russian Academy of Science, Pushchino. oseow Region, Russia, D:p~rtmcnl or Biology, Unlvcrsity of Virginia, Charloll©sville, VA, Department or Chemistry, University of Virginia, _Ch~u'lo¢tesville. YAi and Penn Branch of tim Ir~ilule of Appllcd C~¢m;slry. Pcm~. Kussia. and Biochemical Firm "Biolar', Olaine, Latvia. 242 DEVELOPMENT OF A HIGH-AF~NITY RADIOIODINATED UOAND FOR IDE~ITIFICATION OF IMIDAZOLINFJOUANIDINIUM RECEFTIVESITES (IOR$): INTRATISSUE DISTRIBUTION OF IORS IN LIVER, FOREBRAIN. AND KIDNEY Imid"zoline/guanidinium receptive sites (IGRS) ate membrane ixoteins thai exhbit high alTinity for various compounds wilh an imidazoline or gu~idinbm moiety. TI~ ~lruclure of these hindin~ sties lu~ tl~r signil'~--,u~e in the broad phar. macolngical nction or such li~.,.md~ sre uncles. To address this issne, we devcloFed selective high sfTinity compounds Ihat could be rudiolodineled and used us molecu. lar probes for struclurO| characterization of" these pmleins. This ~ describes the synthesis and charact¢rizalio~ of" such u molecule. 2-(3-amlno.4-["lliodophen. oxy)melhTlimidazoline (["IIAMIPi). ["IIAMIPI is s~mcturally rclaled to clr'¢zollne, an imldazolioe axhibiling high affinity for IGRS lad the r~nily of rehtcd imidazo- line binding sites. The ph-'nyl-substituted analogue of ¢irazoline, 2-(3.1minoFhe. nosy)metbylimidazoline, was gene~tcd by |lk),latlo~ ol" Icetlmidophenol wilh 2- ch_lo._mm.elhylimidazoline. 2-(3-Aminophenoxy)methylimidlzoline exhibiltd hilh a|im,ty for IGR$ in rabbit kidney mc~brai~s, as determined i~ comi~tltlo~ blndh~ sludles wilh [~H]ldasoxan (K, - 12..5 + 7_5 aM), and was radioiedinalcd by ramioc.T oaidation IO yield ['-"IIAMIPi. The Idnding pmperlics of ['~IJAMIFI determined in membranes prepared from two ~talive tissues, ra6bit kidney cortex and rat liver. Six.cil'lc blndln¢ of ['~'lJAMIPI was saturable and orhi/k a/hat. ty. us de(ermined by Scatchard analysis of" umraliou binding isod~eams (tablet kid. he},, K. = 2.0 * 0.9 aM, B,.. ,,, 5~ 4. 201 fmo~/mI. five CXlXllm~ rat li~er. K. • 2.6 4. 1.3 nw,. B=,. ,,, 73 4. I~ fmolfmg, thRe experiments). ["'iIAMIPi binding in rub. bil kidney memtwu~cs was inhJbiled by vaxlous imid~oliv~s and 8uanidinium pou~s, wilh the following rank order of Fo~cncT: cirazolin¢. (K, ,= 3.6 ~ 0.96 aM) > idazoxan (high-all'mary site K. = 0.2 + 0.1 nM md Iow4ffinity she K. u, "/6 4. 30 n~t) > guan.~enz (high-s~nity sale ~ • 1.7 4. 0.t~,4 nM and Iow--,mnlty site K. ,= 2OI + ?5..7 aM) • -,milodde (K, ,. 625 4. 130 aM) • clo¢lidioc (K, ,, 2.'q30 4. ~ ~M) > p-Imln- oclonldlne (K, • 3422 4. I"/2 aM), ['uiIAMIPI hindini was net inhibiled by the admner~ic receptor anlagonisl rauwolseine ~ endolenous agoalsts for ncum~ram,. milter reccp(ors (cpinephrlne. histamine, serolonln, and dof~lmine). The rank on~r of competiog ligands is consist~t wilh the d~l'mili~ oi' Ibe ['t'I]AMIPi I~ndiog site as an IGRS. Rccc~or autor~diography wes u~d Io detemdne the inlratissu¢ dlstribu- lion of IGRS in ral liver, rat foRbraln, am| celiac kidney. Autotudiolrams indicltcd homog~cous specific binding of ["IIAMIPi in livc~. Rend I"i|AMIPl bindin¢ was ohseryed us dls~ci¢ corlJcsl rays. Automdlo~sms cd" r~ fo~cl~ain tissue soclions indicated high dcnsilioS of six:earle blndinI in abe h3qx~lha|amlc lucuale nucJcus and the subl'omJcal orgy. ['"i]AMIPI bandana was inldbilcd by bolh knidlzoline a~r~d guanldinium ligamls bul was no( comp¢led fro' by lhe selecllve arsenic recep- tor antagonist rauwolscine. These dais indi¢lte tlut [mI]AMIPi is t high afi~nily probe thai is specifically ~-co~ized by the |GRS ~d Ihus should racilkale cological tad molecular characterization of this ca(it7. Ivkovlc, B., Baklhavaehalam. V., Zhang, W., Psrini, A.. Diz, 0,, Bosch. $., Neumcycr, J. L.. and L, anler, S. M. Molecular ~am~acology 46:13-23, 1994. Olher supposl: National |nslitut,'-- of Health, Medical Universi~ or Sonlh and Cootrut de R~¢'berchc Externe |nstitut National de In Santa et de la R~beRbe Medicale. 243
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From the Dope•men! of Pharmacology, Medical University or Soulh Carolina. Ch~leslon. SC. Research Biochem;cals Inlematlonal. Natick. MA. Pharmacologic • -- .- ' : . u,v,=,on m ",urgcry. uowman-Gray School of Medicine winston-Salem. NC. • COMBINED IMMUNOCYTOCHEMISTRY AND ENZYME CYTOCIIElVlISTR y ON ULTRA-TIIlN CRYOSECTIONS: A NE~' METIIOD We combined immunocylocbemismj and enzyme cytochemisln/to ]ocalize two diffgrenl proteins on the same ullra-~in cpjo~-cllon. In this m~thod the immunocy- Iochcmical Iocalizalion is visoalizcd with colloidal gold probes and Ihe enzym~ cyzo- chemical dezcclion iz achieved with cerium ns Ihe caplure agcnl. Tize immunocylo- chemistry is ¢o~cled J'~ so Ihat any pet~ttial ndver~ e|£ccls nr the enzyme eylo- chemical procedgrc will ~ ,,lzer the antibody binding properties of Ta~iz~wa. T. and Robinson, J. M. ~ Journal of" Hislochcmisl~ and Cyloch~misl~ 41 (I I ): 16iS- I Other support: Nalto Fonndaden. From the Dcpsrtm~t of Cell Biology, Neurohiolo~. and A~alomy. Ohio Stale Universily. Columbus. OH. COMPOSmON OF T/IE TRANSFER MEDIUM IS CRUCIAL r<)R HIGH.RESOLUTION IMMUNOCYTOCHEMISTRY OF CRYOSECTIONED HUMAN NBUTROPHILS O~a of" the more routine steps associated with cWm,~tioning or ullrz-lhin and semi.the1 ~'¢lio~s fO'/immu~ocytocbemi~lp/is the transfer or ScCllons to EM grids or cove~slips. In a sludy or ihe ~ranulcs o1" human neutrophils, we have found Iha! d¢ comFmidon or the Iransf'er medium wls c~ucial Io the success or these experi- ments. T~izawa, T. and Robinson, J. M. The Journal of.HJstochcmistry and Cylocbemistn~ 42(8): ! 157- 1159. 1994. Other suppo~l: Nalto Foundation. From the Dcparlment of Cell Biology, Ncurobiology, and Anatomy, Ohio Slate Unlversily, Columbus, OH. 244 USE OF 1.4.nm IMMUNOGOLD PARTICLES FOR IMMUNOCYTOCHEMISTRY ON ULTRA-THIN CRYOSECI~ONS W¢ p~cscnl a new appllcatloo ~'or the use or snail immunogold panldcz (',,,I.4- nm diameter) for ultraslruclural immunocylOCbemlslpj,. Theso small gold have been used ~n ultr~-Ihin ¢ryo~clio~s in conjgnclkm with • silver procedure th~ do~s nol dc~md¢ ul.zmnclural doing. We h~e used Ihe hunch trophll is a model syslem, in which known pcateln markenz of' Iwo dilTe~m plasmi¢ granules were Iocaliz.ed, in the development or this prmcduz¢. ~ 1.4.nm immuno~old peel•des coupled wilh silver enh~nccmcnl yield intense labeling for localization of I~¢lofenin, • ma..ker for the specific emnules, sod myelol~mxidzs~, • marker for the •zurophil ~ranul~s. Double l~¢lin| in which ~ze antilCn was visual. ized with 1.4.nm gold m~d silver eolzancemcn! and • second ~u~i~cn was wilh colloidal lzold-lpG on Ihe same ullra-lhln cryoseclinn w~s a-cceasl'ully • chieved. We o1~ show thnt 1.4-nm di,,me~er immuno~zold i'~lides penclml© cryoseedoned neulrophila Io *', Bee•tee extent Ihan S-rim or 10-nm'immuao|old probes. These resulls show that small immunolold panicle•, aloog Wilh silve~ enhancemenl, •re z useful addhinn to Ibe Immmml,,lx:lin~ methods cyril•hie for use with ullr'4.thin c~joseclions. Takizzwa, 1". and Robinson, 3. M. Tbe ~oumal of Hi,~ochcmist~ and C~,lochemlslry 42(12):1615-1623, 1994. Other support: American tleatl Assoe|•tlon, Ohio Al'flli•t¢ and the Nailo Foundation. From the Department olr Cell Biology, Neumbiolo~t, and Anatomy, Ohio SIIII Universily, Columbus, OH. AQUEOUS TWO-PHASE PRO'I:EIN AFFINITY PARTITIONING A LABORATORY DEMONSTRATION OFA BIOTECHNOLOGICAL PROCESS Tbe ¢cchniq~c described be~ is one solution to the complex Ix~blem of fractio~tion. Its I'acilily is due in pen Io Ibe fact that.lhe cofllixNleftla OJ" Ih~ Iwo- phase system are also household psoducts. PolyvinylpTm~lkkme (PVP) can M I'ou~d in many tyl~S of'lations and hair cam imx~-Is whlk~ mallodeatrlns •m used in many lypes o1" rood. This exl~r~menl dcmonslr~s • #iabl© indusld•l Im~¢as. and hoped that it begins Io clemysllfy at lint one Ispect of. modem biol~:hnoloey. Some f"onn of this experiment will mosl lihely fit inlo the Ix~ei~ ebemist~y Redo• or d~, bi~¢bemislr)' lahor~tor~ cun'lculum rot unde~mduatcs or po~iMy ,,dv~w~d scc- ond~xy,.Ic~el studcnls. ' 245
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Giullano. K. A. (Taylor. D. L.) ]oumul of Ch:rolcal Educalion 71(7):590-591. July 19P4. Olhcr support: Nulional Science Foundation Science and Technolo£y Center. From he University or Piltsburgh and C~megie Mellon University. Piilsbureh. PA. INDIRE@T USE OF IMMOBILT~ED METAL AFFINITY CHROMA'~;RAPHY FOR I$OLA31ON AND CHARACI--~.RIZATION OF PROTEIN PARTNERS Immobilized m~tal MT'mity chromatograph), in co~junction with modem ~ccom- binant DNA technology, allows efficient immobilizztion of • I•~cd prOl¢in In u .matrix with • consisten¢ trod defined spatial oti~tadon. The unique nalum of this ;-p¢l,l~ lag. typically six c~ig~us h;sddine ~sidu,. ~ilc cicnl to allow s~g and s~i~ ~te~ti~ wi~ a chcla~d racial, offe~ minimum inle~e[en~ wilh most ~otcin ~li~s. ~h cnzymfic ~d ~yslcal. TOgelher. • ~e ch&~sli~ dcF~ a s~s~em Ihal illows the ~mMy or ~ggcd ~lcin plex~ ~ir [~ile ~Aficat~ ;n a native ~d (~li~zlly ~li~ ~atc. and a ~o d/ss~l I~ tractions t~m~. In I~ r~. ~hem~al sludi~ involving ~lcr- ~l;~s ~lween ~l~id, ~ ot~r ~molecul~ will m~t likely scc IMAC p]ay;ng ~ In~ingly im~n~t m~. Szwa~go. M. ~d Van DTke. ~. W. go: Sellow. ]. K. (~.): Gcn~ic ~gin~ng. Yol. 17. P]~um Pr~. New Ymk. pp. 5~5. Olher su~: Nzzion~ Instit~, ~ Hcallh and Ihc Roan A. Welch ~un~li~. Froro the Department or Molecular Genetics. ~parzmcnz or Tumor Biology. Univmily or Text. M.D. An~ ~ Comer. Houst~. ~. 246 i Active Projects Following k a list of the principal investl~lors, or institutim~s, wlmsc projects ~ under way ~ were ~ivatcd in tim ~ s~c t~ ~vi~s Re~, to~¢t~r wkh I~ r~ctive project lilies. Compkled ~cts u~ Ils~ in a I~er s~. PRINCIPAl. I NVF.qTI~ ATOR OR IN.~'Tn'UTION CORY T. ARATF~ Assistant Professor of Neu~oscienc¢, Comet fo~ Advanced Rit~cchnolo~y and Medicine. Univc~{ly of M~llcine and tistry. Roberl Woo~ |ohn~cnn Medical Schm4. Pi~ataway. NJ. . SYF.I) S. At |MAD. M.D.. ~LD. R~-~.~ch As~oclalc Pn~rc~.w4. Ten,k: Un|- ~si~y. I~il~lph|a. 5TEVFJ~ A. AKMAN. M.D. Pmrc~m ~" Medicine ~ml C~nccr 9owing. Gray School of Medicine. Wake F~x~ U.i~mlty. Wir~mm.Salcm. NC. PAUL ~ A~. JORGE F. A~ ~.0. Pmfc~ of Bim~mt~l~ Unlvmily of Ass~i:~c Pmre~r. Unive,ily ~f Cot- ~. ~er. ~. IIARRY N. A~ADF~. ~:.D. KIMT. ARN~. ~I.D. I b~r, NY. SUSAN ~ ~IN. S~eior Membu. Instilule for NARAYAN G. AVADHANI. ~l.D. ~fes~ of Bim~mist~. Univ~hy of JONA~AN M. BA~ER. M.O. A.~anl ~fcs~r. AI~ Eiaslein C~- Ic;e of Mcd~inc. ~. NY. VIII Risnlutlon Of lxleislcen-llikld ONA m I~ h.mm Immune elTeelor meehanisms in mhelo. Si|nal tmductioe via pml~in ki~s and Silnul t~ansductiofl by Ihe interlcukin-3 recq~or. Gm~lh r:mor~ and nxe~on ie hum:m pmllr. cralive di~mkr~ The role or I~ei. ICms~me 2A in me ~II Mechanisms or nuclear.mh~dal lhmtlhllklyllnolllol 3'-kinase snd Inlulln Ktmn 24?
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O PRINCIPAl. OR IN.~T|TffrI(IN SRILATA BAGCIfl. Rt.D. Assistanl ~r,~'c~,,~'. Ccn~er fm M~lL'cular Bio|ngy of 01at ~V~ BALK. M.D.. h~O. V~AS A. BANKA~. ~t.D. Un;versi~ or Alabama al DiminEhem. DAVID W. BARNF.% ~.D. Associale Pto~essor of Bi~chemislry/- Biophysics. Ore~nn Slale Un;vcr~i~y. ~PII~N B. BAYLW. M.D. icing. The ~ohns llo~klns ~h~ or M~i~n~ A~i~l Pmr~ ~ Pa~h~y ~ I~ ~e. ~1. WILLIAM F. D~EDI~. M.D. Profesmr ~ B;m~lo~y. Blyl~ Col. KAR~ ~ B~N~. ~I.D. Assi~ ~f~. U~v~Jly ~ Mi~. Cdum~a. MO. NISSIM B~I~. M.D. ~[D. DAR~N K. B~G. ~.D. Pmfes~ a~ Chilean. ~anmem of Biology. Univefsily of California. San ~LE J. B~G. Assistant ~fcs~. Ha~a~ Unive~sily. C~ge. MA. Sll~ L B~GER, R;.D. Ass~nl ~es~. ~l~a. )UD~I BERMAN. ~[D. As~iale Pmfe~r. Unlve~ify of Min- ima. 5L Paul. MN. BRUCE BE~ER. M.D. As~ialc ~css~ of In~l M~i~. Unlv~ly of Texas ~h~s~ M~ical Cent. Da~l~, TX. I'ROJECr "ITI'I.E Bk~chemic~ funclkxlx of I~ human pa~ll~ Regulalkm ,d CDI gc~ exp~es~l~ dur n hunch ~ ~m~u~yle devck~nem Mulanl ~sphnlipid transfer ~m~eins and I~mmh~ ~nc~gcnic ~ran~fn~malinn in serum-~ree cuhum Rcgulalion of the I)NA melhyla~ gone in I~lldmxal hemnlngues cell migr~hm The role .r Ih¢ rcllm~hi-smma £cn¢' in lun¢ P-~.tanule componenls of Ih~ germline cell flneag,' Isol~lhm ~J characlerbatiml of larc¢~ ethos hwmyc ~.tivily Nic~in]c ~ceplor~ Rculatlng seco~l Function and imeracdons of I~e T cell ¢i1"¢ ly~mine kim~.T~;K Tramcriplinnal acfivalinn by lira tum~ sup "~l~ ale of hnRNPs in leh~mere xln~u~e =ml Molecular ckming of the f4~s eDNA 2411 PRINCIPAL INVF.CrlGATOR OR IN.VrlTUTION RICilARD !. fllNO. M.D. Pmfe~w of Medicine (F.medtus). Uni~- • ily ~ ~ C~if~ia ~d d ~1. ci~, ~ An~ele~ Vi~tlng A~b~. Ex~rimenlal C~rdinl~gy and 5cienlifie D¢velopmenl. Ilunlin¢lon MeBical BARBARA K. fllRSI ~IN. ~.D. ~f~. Alan ~m~e~ Co~ege of M~ ~AIU A. BISIIOP, Ax~ixlanl ~fc~ of MJcmbi~o~ Inlemal Medicine. Unive,sily of Iowe ~ly. DAVID A. BO~AK. M.D. As~ial~ Pmfe~. Univ~sily ~r Vat. ginla. ~rlollesville. VA. BRUCE M. flOMAN. M.D. ~.D. • I~ Unlve~ily. ~,,he. NIL MRR~D~I ~ND, ~*.D. Axx~iale Stuff. ~se Clevela~ ~in[¢ F~al~. Oevcb~. OIL H~RY R. BOS~ R*.D. Prof, s. Univc~ily of Tcxa~ ~ Austin. ~S DAVID BOYD. Assblanl Pmf~, Unive~ily of Tex~ R~ ~ BRE~. RLD. Assk~l ~of~. ~ild~n's Hospi~l. O~ MA. ~N~ ~ BRIN~W, RI.D. ¢hemislry. Datlmoulh Medical I I~mwcr. NIl. ~IOMA5 R. BROKE~ Pmfexsm of B~hcmixt~, Univcnily Alabama. $ch~l of Medlci~ a~ PROJECT .m-l.~ Analysis of ~¢ R|;o~ 3' of ~¢ hum~ IIII |e~e cluster B ctll sub~¢l Icllvslioa El nostril lad aulo- immune mice $1n~-tut~ a~l fuacllm d dm |lmily o! GTP. binding pmleim A~chidonic acid and myoflbdlla. ~ein I~os~ylatk~ h~ ischemla Mutations in the C-lerminws of v-tel aed Iheir sic in emc~cr~--sis Rde of umkl~se and hs ¢an~'t invasim~ ~ic a~lTsls of Rpllc~km pmlcln A;m Rec~m~ fo~ SAA in cell~u~ bduc~k~ H~man i~llomlvims RTlicatio~ and le~e eXl~'.lon ;~ IXlm~ human kem|nec~q
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PRINCIPAL INVESTIGATOR OR INSTITUTION |EANNErTEc EULINSKI. M.D. Aside ~es~. C~ll~e or • Surgeons. Columbia New Y~. NY YURI BUSI IKIN, Assistant Member. Poblie Ileellh Re- ,arch In,dime. Hew York. NY. KA~ IRYN CAI~MF, Professor. Columbia Unlve~;ly. Ileallh Sc~¢s. Hew Ymk. NY. ANNE L CAI~F, AssisI~ ~. Unlve~hy of CIIW~- EDWARD J. CAMPBEL~ As~i~e ~o~es~ e~ Medici.. Unive~i. ~y of U~ Sail ~e ~y. ~. ~ic~. Rc~vm. BI~N~ IE CA~ M~I Cemer. ~h~. MARIA ~ CARD,AS. ~cD. M~I Cemer. ~m. PA~ICK L ~Y, ~.0. Ass~iale P~ofcs~r. Duke Unive~lly ANDREW J. CAT~. ~LD. As~iatc ~re~. ~ wistx Institule Anally ~ Biol~y. ~1~¢)~. PA. Ve~ ~. h~. ~Y. AssJstlnt ~cs~r. Unlvmlly o~ ~i- • ~way. ~J. ~o~cs~ and Head. ~nmem o~ TA-YUAN CIIANG. P~o~ess~. Oa~lme~th ~edi~al ~ch~t. PROJECT TITI.R |nleracllnn of mierotuhulcs wile c¢|l cycle efl'ectm's Expression and I'unct|,m of p2m-f~c MIIC cla~ I A~snCilllnn el' ~opr~ein c.myc with I~n- ~ri~Em ~ul;~ Idemi~yin~ ~s a~ial~l wile ~- sis in ~C mammalian nff~ epi~hum lluman lung el•alia Properties of the ALL-I protein with human acul¢ leukemia Cell ~dgnaling intcraclhms in te~li~ e(ml tm- Immum~philln.c•lclne•fln co•pleura with a,d widmul imutun~m,pp'c~.sanl.~ Struclute and ~unclinn of p;otcln I'•rnesyl- Iran~r,~a~¢ Tnlerar~ce and |utnimmunily Io inllu~nz= IIA as • Imnsgcnic Role of the czhB.'l pmtei~ in growlh factor- coupled St|haling Exit•cellular, matrix degra~atlon in the lung NeuroJmmune inKeracllons: mechanism c~helamine effects on lympho~lc rune- Mapping ¢h¢ stcml binding sites mr ACAT - • PRINCIPAl. INVF_qTI~ATOR OR IN.RT|TfJTION lEAN-PIERRE CllANQEUX. ~ltJ3. Profm. IrL~titut Pasteur, Paris. Fc~nce. IONATI tAN CIIRRNOFF. M.D. I~t.D. Inslilut¢ for Ca~K'¢¢ Research. Phil•del- phi•. KATIII.F~.N R. CLIO. In.~lmClt~'. ~p~l~i|¢l|| of Palhok~y. The lohnx I Inl~ins I Iosplt.~l. llaldmme, MD. RANDOLPII D. CIIRISTF~I. M.D. A.~ixlanl Adjuncl Pm~c-~m~. Unlver~ily Callfomiu. San L~e ¢.o. Le. I~41•. CA. CIlRNG-MING CHUONG. M.D.. P~.D. A.¢,;;slanl Profea.,,ot of' Pal~y. U~Ivm~- AAROH J. C1ECllANO~J~ Professor of Biochem|slty. Technlon- Ixr~l In.~ltute of Technology. Haif~, Israel. PAUL B. CLARKF. I~.I). . . Asr~:iul¢ p.olcxs~r. McGIII Umvef~ity. Monlre~l. w, L. CLEVELAND. R~.D. Research Sclenlld..~1. Luk¢'s-R~evelt Ih~pil•l Cemer. N~.w Y~. MELAHII~ !1. ~BB. RLD. Sm~hw~ Medal Cenl~. Dal~. Axs~cill= Profc~or. Toffs Untv¢r~ily JF~EY P. ~NN. hI.D, Ass~ille ~nf~. Emmy Unlve~lly. Allam~ ~A. IV ~ Tex~ M.D. A~ ~es~ ~ Ri~mistw. Univc~ily of M[~. SL ~ul. MN. ROBERT !1. ~A, ~.D. ~sist.nt Protest_of fli~icd ~m- GIDEON ~KI. Professor, The Ilehr~ P RO$ ECT 'T'TT LE Mnd~latlo~ oi' Ihe cellula~ ~ge mpm~ Re~mvk'al mnduL~;em o~ OCC (~k~4 k~ eel. mce~l c.a~ionms) im CNS uc~s ~f n|coti~: Identificatlmt or I~l im~ in the I~tme Roles or the MAP kl~s~e I~thway in cell A rowel cell cycle pmleln Ihsl k~-r~t~ wile physlologkal mica of mhiple mu~c~d~le
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PRINCIPAL INYF.STIGATOR OR INSTITUTION bETA DEAN. ~. Anlsl.I Ptd¢s~r. SlUe Universily New Y~. Stay ~. NY. ~ERI~ C. DE BE~ M.D. ~fe~ ~ M~i~. Oniv~y ~ Ken. I~y C~e ~ M~. ~in~t~. K Y. WALTER A. OE~CII. verily. Ratnn R~. ~. RO~T C, DIC[SON. I~i.D. ~mf~ n~ ~hemktey. Unevenly Ktm~ky. f~.i~gt,. KY. I~WRF~ A. ~)NEI lOWER. Assisllfll P~oressor, Uaylor College of M~ici~. filmily. TX. A~i~¢ P¢~¢~. U¢i~l~ ~ M~i- W~ J~nlo~ Mdicd Sch~. Pi~al. ~ofes~ ~ Mic~iology ~ ~cs~ af Mole~lar ~O Cdl Biolocy. Un{v~ky ~ Cdif~i~ B~¢t~. WI~AM R, ~RERG, ~o~c~ ~ ~o~y. Ibw~ Unlve~lly. W~p~. D.C, E~INE A. E~ON, Assislanl Pmfesso¢. IlarvsN Medi.I ~RRY S. ELRN. cot Re~hl~d Educallon. I~.. P~Io Al~o. C~ PAU~ J. F~RI~O. ~.D. Univ~ily ~Ncw Y~.S~y g~. NY. A~IaI¢ R~arc~r. Unive~lty d Calf f~a. I~ An~ale~ I'RCURCT 11'rLK Pmlein ~ing to Ihe ¢olgi complex Funcli~n of.~er~m nmyloid A Im~lein (SAAI Oxidative r,l,e. ~ Ih~ cukaryotic ~cpni~ of A~.~nt ¢~f Ioz~; ;rod nlul:~pnic elf~:cls in p.S]-¢lcfidcfll Mechanism and role of retrovi~l-mediated Ir~vluctlo~ of ~:llular .,;:que~ccs Malecular mcchan|.~ms nI alum;num, a~znic md nickel t,xit:tly Rc~roviral n~c ~e~es and cellula¢ proln unc cc~'cvlsi~¢ Role of cdothelin in Ihe plhngenesis oir hypoxic pulmnnan/hypcnens~ |n~racllons mdiatnd by glutamate oplake Ir'a~lers heween ~cum~s a~d i:lia Mn,,lily Ircods amnng smokers and ram- tRINC|PAL, INVF..SI"IGATOR OR INb'TITUTION AgRAHAM FAIN$OO. I~.D, L~clurer. Hebrew Univefsily-Hadas--h Mecli¢~l Schod. J¢~.lem. Is~d. DOUGLAS V. FAI I ER. M.O~ rmfc.or oF Medtcirl¢, glo~hemi~j'. ~fries and pxlholo¢y. Bn~lon Univ¢.ily. Bm~. MA. ~MAS G. FANNING, ~.D. DANI~ ~. lu ~pi~oW. Ila~ard Medical ScUd. g~. MA. ~ ~ ~RD. M.D, A~lal¢ ~f~, Un~vmi~y DAVID A. ~ ~.D. A~ble ~f~, Ilunlu ~ollc~ of ~Unive~ly of Hew Y~. New Y~. AL~ J. ~SO~. R~D. Aui~al ~. Unlv~ly ~ a~ A~ioln~. ~i~hcm- i~ a~ ~dialr~s. Unie.q ~ Alab~ m. Bi~ham. IRWIN ~l~ViOl. Pmf~ of Bim~ist~. ~ke Unlvc~. Professor, Technion-ls~d Institute of A~ON D. ~Y~ ~.D. li~.MD. " XIN-YUAN ~. ~I.D. ~ M~ici~. New ~NNA GA~OUS~ ~I.D. Univc~iliu C~lq¢ of Mcdlci~. R~s. Iown. OI L FROJECTTITU~ Molecular mccbnismx conlrdllng blood Function of LINE-I relrolransposo~s In fm chin in DNA RFak Melt|pie inlrxdlelx, sl|~l|q ~-cha~sm ~ic ~nd I~xh~mk:al ~lysis of c~lkd~' Vuc~b" o~d~l;d b;nd~l d' x all~ kv.' od- Telrihelicxl DNA binding p~olelns f~om the lung Mokc~hl' m~t~nlu~ of :,t|v:lk~ ¢' STAT 2,q.1
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PR(IJ~..CT TITI.E Regulatle~ of neelonal nieminl¢ AC~ ~c~W tnr Cl~r~cted~tinn or tlm cellular facmcTIdF The molecular basis for the effccl of c~ll ~hesi~n o~ rnalicna~ Iran~r~mal~ ~t~riUli~ or rff~ gen~ f~ I~ ~1 ~eins C'l~'actedzlli~ of the mdm2 t~ncogene and its imeracllnn with p$) U3 ~mRNA md riho~nm|l RNA pmcc~siug Recel~or m~ch=nism~ in carcinn~cnesiq~ Factors uss~ialed with an EIA.llke ~'~cin rn~n Novd activities associated wilh ~el "inhibi- pl3OJRb2 • novel target for Ihe AdS EIA The llpnd bi~ing site.el'. FGF r~el~n~s as • target for growth mOOUlate~ Regulslimt of the intufeukin-4 gent Rcgulalion of mRNA Im~|ati~ PRINCIPAL INVF.qTIGATOR OR |NSTITUTtON SANNA ~,NY. SERGEI A. GRAN~. M.D., ~LD. ~ate ~f~, Unlv~ily ~ Min~- ~& Min~lls, MN. JF~EY A. GRAY, ~I.D. Pmfe.or ~ Iletd, ~afl~m of Pay* c~lo~, I~;(ule of Psychiat~. ANN M. GRAYDI~ Pmf~ I~t~e of T~, Carnage, Asskl,~ P~C~ or Pml~n~, N~h- western University Medlclt School, MARK I. ORF~F~ ~a,D. ~Mok~Gy. Comer for ReC~I~ 11~In~y, Unlve~ity of Pcnn~ylvlmu 5¢~! of Mctlk{~. I~i~kl~*~ PA. ~R~ W. GR~JDER. MARK & GRI~. ~LD. ~I~EY ~ge ~ WL~si~ Milwau~. WI. yOS~GRU~BA~. ~.D. JUN-U~ GUAN. ~LD. physics ~ ~clics, Uni~ily el Col- SUBRATA llA~AR. ~cD. fc~ Univcnily. Jcffe~m ~r
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PROJECT TITI.IE. Design;n; an RNA.Mmtim.. proq¢in Roles of sur- I and olhcr ,.~:ncs in ~a~-medial. ed cell :dgnalling d.l'in/~ animl devclup- Re;alaSofy ¢~Ics of tl~ C-~e~ini ~f muldpl= lach~i~n T~cnl ~ e~p~km define high.densely ~mwlh ~e,l in n~m~l c¢11~ Ca~lyl~ ~lalhm~hlp ,f t~P ~nlll~ ~d dCMP hydm~clhyl~ The molecular b~sis or stable lone Rc~a(i~ of ~palili~ ~ ~imx ~ M~l~r ~¢1~ h)r tis~ue~cific allcmallye RNA ~icing Regulal~ or h.~. ncul~il nxy~ ~i- Cil ~ucl~n hy c~c~nllc Ma~in¢ m~l~ ¢en¢~ cat,~ing ~ ~1 or eady rsl~ll ~vcl~l Role of NAD(PII h quinoflc o.idclccluclase 1 (NQOll Jn carcinn;en ~ncI dre¢ melahO- $1ep-xrr©sl variants of F.Ip.recomblnase: 236 PRINCIPAI~ INVF.qTIGATOK OR INSTITUTION KUAN-TEH ]EAf~G, M.D., Llhl~lo~' of M~lccul~ Biology. Nadnn:d Imthu1~ nf | I~|lh. Bed~e~da. MD. WILFRED JEFFERIES. Asslstnnt i~'ofc~r. Uoivcrs|ly of British Columbia, Vancr, uver. C:m~hl. ALGIRDAS J. JF, SA ~S. As~isl~nl Prof--, U,Ivc~sily of Texas ~lhw~cm M~l~al Cenlcr. Dallas. As~iale Pm¢~. Unive~ily ~thw~cm M~al Conics, Oall~. TX. WOL~ANG K. JOKUK. D.~tI~ Chai~an. ~ps~menl o[ Mic~bioloo Jerusalem. Iladlssah-Medical AsSillanl P¢ores~or of Medicine and Pal~o¢y. ~c Unlvc~ily atria. Johns Hopkins Un versily, ualll- JOilN F. K~NEY. M.D. A~si~ant ~f~ ~f B~ltt~y PRoJECT 'ITTL£ Role o1" Tat and cellular facm~ in lily es. p,~slm : In Iowefln| cell svfflce ex .l.~__lllO~ ol Ilia rno~cvl¢~ and in vi~l ~ Mo~ tar;cllng ~ ~ants ~. ~i- ~ st~m ~ [u~d~ of ~ims ~ Abheime~'s dkea¢ Mcchanb~s ©f ;cr,¢ mivado~ in DiffeR. ~tisl ;e~¢ expression in ¢pilhelial somil~ d~milns Motec~llr mechenisms of ¢on|*.'t~llal and ~-quircd f~c~o¢ V dcr, cicmy m ~d cx~gcncs Fmm~cfli In6A.T The l~tioxldanl properlies of ella|ca IM EORF ~*tkm ~ mclal~ism Archaebacte~|al prole|n phosphatise i~d
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PRINCIPAL INVF3;TI~ATOR OR INSTITUTION AMY KENT~R. Pli.D. Asslslznt Professor of ~icrobiology z~d immunology. Univcrsily ~ago. I~ Plof~. Uni~ni:y of Wi~si~ Mid|. MICIIAELP. New Y~. IIANNAII I. KleIN. As~ial¢ Pn~. N~ Y~ tJ~ve~ily M~i~ Cent. Ntw Y~k. NY. ERIC B. KMIEC, ~.D. TAD H. K~II. ~0[¢~ O[ ~m}51~. UnlvmJly of C~- ~IOMAS ~. KODAO~. ~t.D. A~lin. ~. HEI~ KOIILB. K~k~ Med~ C~. ~x~k:. KY. vc~ity, Bal~ R~:e. ANDREW S. KRA~. M.D. ~rofcssor o¢ Medicine. U~ivetslty of Alab~l, Bi~in¢~. AL ROBERT II. ~SlNGER. Pto~¢s~r of Bi~y. University o~ Vir. SKAIOR~ KRISES. ~e.D. v~i~y. Sm D~o, CA. AssJseant Pr~es~r. Universily or Col- HSIHGJI~ KUHG. ~.D. atty. Oevd~ OIL RYOKO KURIY~M~. RLD. Neu;~zlomy. ~]z:iveruly of Mlme~. Mi~li~, MN. PROJR(.*I" TITI.I.: Mo|ccular assays fo¢ Ig swltclt recombinatloo Molecular analyses of growth and diffcrcntia- liOfl in the nematode Cacnnrhahdilis ele- Dclctiems of mit~n~rlal DNA and Re~ulstlnn of ~e~fiC tea.eaSement ~7 ri~. ~ling "ln~luW" F~i~ t~ trois Cm t~ study of Anli~y m~iafcd dcliv~ a~ ex~sslnn o~ ~e in~erac(inn of ~toxynil~a~e whh mclal cmnplexes Regulation of mRNA t~anscriWinn by Ta~ teazle in ~ein~ N~lear DNA replicalion: m~hanism and M~ular anal~ls of an ¢~.~ciF~ m~ MAP PRINCIPAL INVF.qTIGATOR OR INSTITUTION ABEL LAJTII& Dinar'for, Center Ira' Neurochem|stl~. The Nathan $, Kliae Insl|lul¢ for Psychiatric Re.arch. Oran~bmg, NY STEPIII~I LANIER. A.~'dst,.m Pr~'~.m of Ph~rrn~colow. Med- ic,,| University or $o.th Caroti~a. Chadc.~nm, SC. ANDREW ~SSAR. Assisl~nl Prnrcsso¢. I1~=~ ~cl~d. ~. MA As~t~l=m Prefer. Colmmln Sl~c U~i- ~y~cchn~ ln~imlc hl~kx~ VA. ~key, Unive~ily ~Texas Ifcallh Ccm¢~. Sm hn~. TX. Plofcssor and Dir~lor, Insfilul¢ of ¢b~i%t~. Univ~ly of Tex~ I le:llh ~e Center. ~an Am~io. or=tory Medicine. U~l~c~hy ~[ Texas In~lruc~ i~ Pcdi~lr}cs. F~nz p~f~r, Universily ~lif~nlm. San F~=~. Sm F~. . As~b(e Pmres~, Unlve~ily I~ ~ ~ ~tic;~. ~t~, MO. PROJECTTITLE
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C~ PRINCIPAL INVESTI(;ATOR OR INSTITt,ri'IOH sCOTt w. R~ERS. ~h ~e Cky. UT. ~RNA W. ROL~ ~.D. Ass~t ~of~ of An~y mad Cell GE~LD M. ROSEN. PII.D. Pm~e~s~ o~ ~acu~o~y a~ gy, Univ~nil~ ~ h~ryland Sch~l PEssary, Ballimm~. NADIA ROSE~ IA~ Assisllnl P~o~essor nf Bioehemlsl~y. TIIO~AS ~ RO~I~N. Associat~ Professor of Medicine M~i~o~. Bml~ Uni~ky Medial Cenler. Bos~. IIARRY RUBIN. ~.O~ D.V.~. Pmf~ ~ Molecula~ B~y. U~v~i- ly of Cali~ia, Berkeley. CA. K~T W. RUNG~ ~.D. I~ Relish ]nslim~ Cl~la~. OIL Asstslanl ~of~r of McO~i~. 5lanfmd FABIO P. RUPP, Pll.~. A~i~lanl ~es~. Joh~ Ih~inq Unl. versily S~I of Medici~ ~allimme. MD. ANIL K. BUll, M.D. Assistant Pf~ts~r ~ M~Ic[~. KEVIN RYDER. R¢D. A~iate Mem~r, I~sdtu~e fo~ R~mc~ ~il~el~b, PAUL M. ~ALVA~, PH.D. Research Scientist. Brc~man Re,earth In~ilu~e. Cily Of H~, Duafl¢, CA. Assislanl P~o~essor, Case Rc~e Unlv~nily, ~cvcbM. OIL P~ SARNOW. U.ive~ily or ColD.do licallh 5cie~ rcm~r. ~nvu. CO. 2M Fr~e radical mediated cytotoxicily Molecular mechanisms of muscle flhe¢ diversification in Iranq~enlc mice TR~AP.I DNA binding ~',~ein'~ in II lyre- (~nlilitlve ~udies of spom~nacms trln~/'or- matkm in c~ll c~llu~e Telomem f'unct~on in chromosome slabilily sad n~lear stmctme Pmlicipatlcn of mast cell t~l~z~e in ainv=y.~ infl~mati~ Agrin, nicotinic neuyon=l acelylcholine Gone ~t~ulali~m and p,'mcln function ld~nlific~,tio~ cf the t:cne Ill|ell of OntO- |chic jvn lWOl~ir,~ Molecular and biolngic|l aspecls of a neu- folrophie f~lnr in Dr~snpSila Regulalinn or zbern~dve pre-mRNA splicle~ by set, a Drosophila snRNP ~o~ein Initiation o~ Iranslstkm by intcn~l ,ibomme PRINCIPAL INVFJ;TI~ATOR OR INSTrI"UTION DAVID A. SASSOON, P~t.D. Assislaat P~fcs.~m'. Moun¢ Sinai Medical Center. N~ Y~. NY, 3. DENRY SA~. Research Scicntisl. W. Alton J~s Cell ROBERT II. Assignat P~ofe~r o~ Toxicology, lbr- v~rd Sch~l of Public Health. Boston, ~slsta~t Pror~ or ~d~i~. Colum- 51a Onive~ily. New Y~. NY. BRUCE L S~ tNA~, ~LO. Associate Prorcs~r. llarvl~d Mcdlcnl ~ IRI~PIIER P. Rcsca~h l~cl~. Onlvushy of N~h GIN~ SERRERO. ~l.D. Assislanl Professor. Sc~ of M~c~. B~. MA. ANN-BIN SIIYU, DAYID ~ SIRB~KU. ~hD, AU[SlIfll Pmrc~. Colom~ Univ~lly, New Ymk, NY.

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