Wednesday. Degradation of Extracellular Matrix (2518-2519) 433a

2516 2519 SIMILARITY BETWEEN GROUP-I PHOSPHOLIPASE A2(PLA2) AND MANNOSE INFLUENCE HYPOXIC HUMAN RECEPTORS AND PLA2-INDUCED INVASION OF ECM BY NIH 3T3 CELLS ((A.Krtolica1 J.W.Ludlow''2)) ((G. C. Kundu and A. B. Mukherjee)), Section on Developmental Genetics, Dept. Biochemistry;

HGB/NICHD/NIH, Bethesda, MD 20892. Center, Rochester, Rochester, (Spon. Hilf.) Phospholipases A2 (PLA,s) are a class of key esterases involved in the production of proinflanmatory lipid mediators. Group I induces several cellular responses, Ischemic PLA, microenvironments. Hypoxic including cell proliferation, chemokinesis and smooth muscle contraction, via receptor- microenvironments a mediated pathway. Here, we report that InI-PLA27I (porcine pancreatic) binds with responsiveness receptor on NIH 3T3 cell surface with high affinity and specificity and this binding is subpopulations displaced by cold porcine pancreatic and Nqja noja PLA2s but not by bee venom and tensions Cotalw adamas PLA2s. Scatchard analysis of competition binding of mzI-PLA27I chemotherapy, we with cold porcine pancreatic or N4ja-naja PLA2s using NIH 3T3 cells yielded stress properties We characterized nM dissociation constants (K,) of 0.5 and 2.25 nM respectively. Affinity crosslinking cells by gelatin zymography, vitro of I25-PLA2-I on NIH 3T3 cells using disuccinimidyl suberate (DSS) followed by SDS- assays. PAGE and autoradiography, identified a receptor with a molecular mass of 180 kDa. activity vitro This radiolabeled receptor band was virtually abolished when competed with cold in some but pancreatic and Naja n4ia PLA2s. Interestingly, competition affinity-crosslinkling that at populations experiments with mannose-BSA, galactose-BSA and N-acetylglucose-BSA suggested metastasis even oxygen to repopulation patient can from the receptor but there were that these ligands specifically displace m2BI-PLA27I even surgical no displacement when mannan, mannose, glucose, fucose and N-acetyl glucosamine tissue. were used. Deglycosylation (by PNGase-F) of the receptor after cross-linking drastically reduced the molecular mass (M, -150). However, deglycosylation before binding showed no radiolabeled receptor band indicating that carbohydrate moieties are required for PLA2 binding. Moreover, there was a dramatic increase in cellular invasion of artificial ECM (Matrigel") when NIH 3T3 cells were treated with PLA2-I. Our results suggest that:(i) PLA2-I receptor is present on NIH 3T3 cells and this receptor may bind ligands other than porcine pancreatic PLA5;(ii) N-glycosylation is essential for PLA2 binding and (iii) since PLA5-I is found in circulation, receptor- mediated invasion by NIH 3T3 cells may have physiological significance. Structure and Function of Membrane Proteins II (2520-2523)

2520 2521 CONTROL OF GLUT1 AND GLUT3 GLUCOSE TRANSPORTERS IN A BICARBONATE DEPENDENT H+-EXCHANGER IS UPREGULATED IN CHO CHICKEN EMBRYO FIBROBLASTS. ((P. Wagstaff, H.Y. CELLS ADAPTED TO GROWTH AT pHe 6.7. ((RA Coss, DB Leeper, ML Wahl and Kang, P.J. Robbins and M.K. White)) Department of CS Owen)) Thomas Jefferson University, Philadelphia, PA 19107. Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC Heat sensitization of mammalian cells in culture by low extracellular pH (pHe) is attributed to a reduced intracellular pH (pHi). However, it has been reported that 27858. explanted tumor cells are not sensitized to hyperthermia at reduced pHe. Also, mammalian cells adapted to growth in reduced pH medium become less sensitive to When chicken embryo fibroblasts (CEFs) are heat killing at the reduced pH. This decreased heat sensitization may result transformed by the v-src oncogene or stimulated because one or more of the celkular proton extrusion mechanisms are upregulated with mitogens, glucose transport was increased to maintain pHi at acceptable levels. Inhibition of the upregulated proton transport with an associated elevation in GLUT3 uRNA but no mechanism(s) should decrease the pHi in the phenotypically adapted cells and change in GLUT1 iRNA. This was the converse of resensitize them to hyperthermia at reduced pHe. We tested this hypothesis using the pattern of regulation in rodent cells where amiloride, an inhibitor of the Na+/H+ antiporter (the major proton exchanger in GLUT1 but not GLUT3 mRNA was modulated. We have mammalian cells), and CHO 10B cells adapted to growth in pH 6.7 medium. isolated and sequenced a chicken GLUT1 cDNA which, Adaptation was tested for by comparing the survival of adapted and nonadapted unlike its mammalian counterpart, was not induced cells heated at pH 6.7 and 7.3. Maximal adaptation of the heat response was this observed after 180 days of growth at pH 6.7 (in contrast to that observed for OvCa by v-src, serum or TPA. Nevertheless cDNA cells, which occurred within 10 days). Furthermore, adaptation was readily and mammalian GLUT1 have 95% amino acid sequence observed for 42°C but not for 45°C. Amiioride (0.5 mM) sensitized control and similarity and there is also a conserved 120 adapted cells comparably to 42°C at pH 7.3. However, the heat sensitization of the nucleotide subdomain in the 3'-UTR containing an adapted cells by amiboride at pH 6.7 was only a fraction of that observed for the ATTTA motif. Thus there is strong evolutionary control, nonadapted cells, inplying that the sodkirrproton arTtiporter was minimally conservation of GLUT1 structure but not of its upregulated in the adapted cells. Upreguiation of proton extrusion, measured by role in transport induction. In contrast, the analysing intracellular BCECF excitation spectra, was documented in cells grown at chicken GLUT3 isoform was induced by v-src and pH 6.7 for 180 days. The upreguiation was predominantly bicarbonate dependent mitogens in CEFs. Nuclear run-on studies showed (+0.025 pH unis in the absence of NaHCO3; +0.17 pH units in the presence of NaHCO3). These results suggest that pHi homeostasis in CHO10B cells adapted that induction of GLUT3 occurs at the level of exchangers Thus avian GLUT3 is like rodent to pHe 6.7 mainly occurs by upregulation of bicarbonate dependent transcription. (e.g., the bicarbonate-choride exchanger) other than or in addition to the amiloride GLUT1 in that its transcription is inducible. sensitive sodium/proton antiporter. (Supported by PO1CA56690 and T32CA09137 from NCI, NIH, DHHS).

2522 2523 FUNCTIONAL ROLE OF CYSTEINE RESIDUES IN HUMAN AEI, THE ERYTHROCYTE BAND 3 LIGAND IMMOBILIZES RECEPTOR AND ERYTHROCYTE MEMBRANE ANION EXCHANGE PROTEIN RIGIDEFIES MEMBRANE. ((S. McGee, D. Knowles, D. Anstee, N. ((J.R. Casey and R.R. Kopito)) Department of Biological Sciences, Stanford Mohandas, and J. A. Chasis)) Division of Cell and Molecular Biology, University, Stanford, California 94305-5020, U.S.A. Lawrence Berkeley Laboratory, Berkeley, CA 94720.

Sulfhydryl-specific protein chemistry is a powerful tool to analyze protein structre Erythrocyte band 3, well-characterized as an anion channel protein, and function. After removing all cysteines from a protein, cysteines may be appears to also have the potential to regulate membrane mechanical reintroduced and used to probe specifically the structure at particular sites. We have properties. Utilizing ektacytometry and fluorescence recovery after constructed a cysteineless version of human AEI (AEIC-) in which all five photobleaching (FRAP), we measured the effect of band 3 ligand on cysteines of AEI (band 3) were mutated to serine. In this investigation we have membrane deformability and receptor lateral mobility. MoAb BRAC 18, characterized the role of native cysteines in the function of AEL. Wildtype and which binds to the 3rd extracellular loop of band 3, induced a dose- AEIC- were expressed by transient transfection of human embryonic kidney cells. dependent rigidification of the membrane. This rigidification was not AEIC- retains its ability to bind to the immobilized anion exchange inhibitor, 4- induced by binding of BRAC 18 Fab but was present after binding of the acetamido-4'-isothiocyano-stilbene-2,2' disulfonate (SITS), suggesting that its Fab fragment plus goat anti-rat IgG implying that a bivalent ligand was strscture is not grossly altered. The cytoplasmic domain of AEI anchors the cxucial for ligand-induced rigidification. To aacterize the effect of BRAC erythrocyte cytoskeleton to the plasma membrane, via ankyrin. Two of the mutated 18 binding on the lateral mobility of band 3, band 3 molecules were labeled cysteines in AElC- are in a region previously shown to be involved in ankyrin with eosin-5-maleimide (EMA) and the cells were incubated in varying binding and ankyrin binding is sensitive to the oxidation state of these cysteines. concentradons of unlabeled BRAC 18. EMA-labeled band 3 had a mobile The Kd for ankyrin binding was 14 nM for AEI and 22 nM for AEIC-, suggesting fraction of 47.5%. As the concentration of BRAC 18 incrased, the mobile that AEI cysteines are not essential components of the ankyrin binding site. The fraction of band 3 decreased in a dose-dependent fashion. At 20 ltg/ml formation of covalent dimers of AEI and AEIC- following treatment with the BRAC 18 the moblle fraction was < 1%. BRAC 18 was then labeled with homobifunctional crosslinker bis(sulfosuccunimidyl)suberate (BS3)indicates that FITC and its effect on band 3 lateral mobility analyzed. At saturating cysteine residues are not essential for the native oligomeric stucture. Fmally, anion antibody concenaions, the moble faction was < 10%. We conclude that transport by AEIC- is indistinguishable from AEI, as shown in a reconstituted band 3 ligand-induced membrane rigidification is associated with microsome sulfate transport assay. This transport was inhibited by 4,4'- immobilization of the receptor molecule and speculate that ligand binding diisothiocyanodihydrostilbene-2,2'-disulfonate (H2D1DS). We conclude that the induces oligomerization and clustering of band 3 which impedes the sulfhydryls of AEI have no identifiable role which cannot be replaced by serine. deformation of the spectrin-based membran skeleon Supported by a Medical Research Council of Canada postdoctoral fellowship (JRC), by NIH grant GM38543 (RRK) and Americam Cancer Society grant BE-164 (RRK). 434a Structure and Function of Membrane Proteins I1(2524-2529). Wednesday

2524 2525 ALTRED HUMAN ERYTHROCYTE BAND 3 GLYCOSYLATION IN DETECTION OFALTERED CELL SURFACE CAROCHYDRATES IN MAMMALIAN CARBOHYDRATE-DEFICIENT GLYCOPROTEIN SYNDROME TYPE H. CELLS CULTURED IN INHBITORS OF GLYCOPROTEIN PROCESSING. (J. ((J.H.M. Charukl', J. JaekenO, H. Schachter3 and R. Reithmeierl)). RIPKA) ALDEN BIOMEDICAL, NEW YORK, NY 10017. 'MRC Group in Membrane Biology, Dept. Medicine, University of Toronto, Toronto, M5S lA8, Canada; 'Dept. Pediatrics, University Hospital Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium; Chinese hamster ovary (CHO) cells were grown in the presence of 'Dept. Biochemistry Resarch, The Hospital for Sick Children, 555 inhibitors of cellular glycosylation. The three inhibitors swainsonine, University Avenue, Toronto, M5G 1X8, Canada. castanospermine and 2-deoxy-D-glucose were shown to alter the resistance of CHO cells to the toxicity of several plant lectins. The Carbohydrate-deficient glycoprotein syndromes (CDGS) are multi- pattems of resistance and hypersensitivity to these lectins are unique for systemic diseases characterized by deficient glycosylation of each inhibitor. Growth of CHO cells in the presence of all three inhibitors secretory glycoproteins and lysosomal enzymes. Patients with CDGS results in an additional unique lectin resistance pattem (see below). type II have severe psychomotor retardation and greatly reduced Consequently, phenotype testing with lectins may be used to detect altered N-acetylglucosaminyltransferase II (OnTII) activity. We compared cell surface carbohydrates by inhibitors of glycoprotein processing on the lectin binding properties of erythrocyte ghost membrane proliferating hamster cells. proteins obtained from a CDGS type II patient, his family members, a congenital dyserythropoietic anemia type II (HEMPAS) patient Lectin Fold-Resistance and normal controls. Tomato lectin binding to CDWS Band 3 was Inhibitor OcnA W Riin L-PHA significantly reduced relative to control or family member samples Swainsonine (R) S3 S3 R25 R>20 but the amount of N-linked polylactosamine on CDGS Band 3 was CastaneenrTnine R2 S2 Wr (R) R10 still greater than that of HEMPAS Band 3. Both CDGS and HEMPAS 2-Deoxy-D-Glucose R3 S2 S2 R2 R>10 Band 3 had elevated Concanavalin A reactivity indicative of a high Combination R,12 S7 S2 Rio R>5 mannose structure. A 50% reduction in GnTII activity of mononuclear cells derived from the brother, mother and father CHO cells were grown in the presence or absence of inhibitors, and tested revealed they are heterozygous for this disorder. We conclude that for resistance to five plant lectins. Fold-resistance, R; fold-sensitivity, a 50% reduction in the cellular level of OnTII is not rate limiting wild type, WT; less than twofold the of for of S; resistant, (R); magnitude biosynthesis complez polylactosaminyl carbohydrate resistance or to the lectins are shown relative to structures on Band 3 but an almost complete absence of this enzyme sensitivity cylotoxic results in a severe reduction of polylactosamine glycan on Band 3. CHO cells grown in the absence of inhibitors. Supported by the Medical Research Council of Canada.

2526 2527 A NOVEL FORM OF SIALIC ACID, DEAMINATED (KDN) POLYSIALIC CHARACTERISTICS OF COMPLEMENTARITY-DETERMINING ACID, IS WIDELY DISTRIBUTED IN ANIMAL TISSUES BUT FOUND REGIONS OF T CELL RECEPTORS FOR ANTIGEN. ((G. ON SELECTED PROTEINS ONLY. Roth, M. B. Z. C. Johnson and T. T. Wu)) Department of Bio- ((J. Ziak, Qu, Xulei, chemistry, Molecular Biology and Cell Biology, Zuber, A. Kanamori *, and Y. Inuoe *)) Division of Cell and Molecular Northwestcern University, Evanston, IL 60208. Pathology, Department of Pathology, University of Zurich, CH-8091 Zurich, Switzerland, and * Department of Biophysics and Biochemistry, Faculty of While the CDRH3s of antibodies have extensive of length and sequence variations, the corres- Science, University Tokyo, Tokyo 113, Japan. ponding CDR3s of TCR alpha and beta chains have much restricted lengths. Furthermore, shorter The natural occunrence of a deaminated analog of sialic acid, KDN (2-keto-3- CDR3s of alpha chains are usually associated deoxy-D-glycero-D-galacto-nononic acid), was first reported in rainbow trout with longer CDR3s of beta chains and vice and later in the vitelline KDN- versa. Thus, the sums of their iengths also egg polysialoglycoprotein envelope have very limited variations, in agreement glycoprotein of this fish. In the latter a large number of (-+8KDNa2-+)n units with the suggestion that the CDR3s of alpha are attached through O-glycosidic linkages to the core protein. We have now and beta chains are primarily in contact with produced a mAb against the a2-+8-linked oligo/poly KDN epitope. This IgM the processed antigen peptides in the grooves class monoclonal termed reacts with the of MHC class I or II molecules. The other antibody, kdn8kdn, a2-8-linked CDRs of TCRs are difficult to identify by their oligo/poly KDN structure, (-*8KDNa2-+)n (n > 2). The antibody was variablility plots due to the presence of in to detect the numerous peaks and valleys. Unlike the other I successfully applied immunogold labeling techniques presence of this novel form of sialic acid in various and adult rat tissues. In CDRs of antibodies, these TCR CDRs are in developing contact adult rat KDN with the processed antigen peptides as tissues, oligo/poly was found in epithelia and mesenchymal well as the relatively conserved helices of MHC elements of all studied tissues but the brain and liver. Embryonic tissues, molecules. Thus, for each of these CDRs. the including brain and liver however, expressed the epitope recognized by mab variability plot should not display a single kdn8kdn often in a fashion. Westem blot peak, but rather a split peak, in the sense developmentally regulated analyses that the middle of each CDR loop will be in of tissue homogenates revealed the presence of a major reactive protein contact with the MHC molecule and thus have species of apparent molecular mass of - 150 kD in the various organs and of a low variability. As an example, the human TCR single reactive of 300 kD in the These data demonstrate that beta chain CDR1 is bracketed by Cys 23 and Trp protein 2 kidney. 34. There is a invariant His 29 in a2-48-linked KDN exists on selected of a of nearly the oligo/poly glycoproteins variety middle. The variabililty plot is shown here, animal tissues which is in contrast to the limited distribution in adult tissues of with a valley two peaks. a2-+8-linked poly N-acetylneuraminic acid of NCAM and brain sodium CDR2 anddistinctthe CDR formedbetweenby the "fourth" loop channel. show similar characteristics. L c: H w

2528 2529 CLONING OF HUMAN AND BOVINE ENTEROKINASE, A SERINE CATHmEPN B, D AND L LocAuzATN AND POSSIBLE INVOLVEMENT IN EXTRACELLULAR PROTEASE THAT INITIATES INTESTINAL DIGESTION. ((Y. PROTEOLY5B ATTHE PLASM MEMRANE OF MACRCPHAGES AND THYROID EPTEALCELLS. Kitamoto,* X. Yuan, Q. Wu, and J.E. Sadler)) HHMI,Washington ((K. Brix and V. Herzog)) Ins_tue for Cell Biology, Universi of Bonn, Ulrich- University School of Medicine, St. Louis, MO 63110, and *3rd Dept. of Hab.rland-Str. 61a, D-53121 Bonn, Germany. Internal Medicine, Kumamoto University, Japan. (Spon. by L.J. Pike.) The 60 kDa dimeric gFyopratein thyroglobulin (TG) is the precuorfor the thyroid Enterokinase is a serine of the small hormones T3 and T4. In the folikcuar lumen of the thyroid gland TG is stored in a protease intestinal brush border that covslerilycroeanked form Cell Biol. For the converts to (J. 1992,118:1071-1083). maintenance trypsinogen trypsin. Assuggested by Pavlov in 1899, this of thyroid fundion, .e. thyroid hormone relase, solubWzaton must preceed reaction initiates an cascade to activate other enzymatic pancreatic dqgraddon odTGfiD rgh e of T3 and T4. Recenfy, we have show ttdegradation cDNAs for zymogens. Full-length human and bovine enterokinase were TGOted NNdacea priorto endocytoals of TG (J. CNin. Invest 194, 93:1388- cloned and Northern sequenced. By blotting, a -4.4 kb mRNA for 1396). From in o dudles kil noown thatTG dograded by aminoe -lae N and enterokinase is expressed specifically in small intestine. The deduced the cdh epinsB, D L The sequence of the enzymes iwolved In TG degradation amino acid sequences indicate that active two-chain enterokinase is derived is unknown, but the cellular localizlon mt be a tool to analyze the possIle from a single chain zymogen. The junction between the heavy and light proteolyllc cawcade. Here, we hav analyzed the localkatlon of TG degradig chains is 1TPK-IVGG in human enterokinase and VSPK-IVGG in bovine enzymeIntnhethyroldand, thyrid pIheal cell tines and In a macrophage cell kIe f enterokinase, indicating that another protease activates proenterokinase by whIch isable relem thyroid hormones from TG. Interesingly, the maorportion of cleaving this Lys-Ile bond. Membrane association of enterokinase may be the pro4ornsoftoe clhepains D L were found secretd and twfth the menbranes of or ma Mature mediated by a conserved signal anchor sequence near the N-terminus or by plasma trocyla cathepsins were a conserved N-myristoylation motif (MGSKR...). The N-terminal heavy mostly rWide biracellular poo buta minor par of the enzyme waspreentat chain contains (noncatalytic) domains that are homologous to segments of the plsma membrn. Sine the pro-form of cahpsn hav redual protmolWyc adelythese rinudli -mlNa hfndion of secrebd and cathepsins In the the LDL receptor, complement components Clr and Cls, the macrophage partal degradton of TG prior to endocyloeis. This n is in line wIth our scavenger receptor, and meprin (a membrane-associated metalloprotease). obs ion that thyroid hormone reaem from TG at the plasma membrane was The C-terminal light chain is homologous to trypsin-like serine proteases. dlin by inhibiton of cysteiyl- (CB, CL), aspertyl- (CD) and metallprotae The substrate specificity of enterokinase for the DDDDK-I sequence of (APN). Analyi of the protin comnposrion of membranes be hmmunolsolated phsma trypsinogen may explained by complementary basic amino acid residues from thyrocyis or mcrophag showed imtiNati betwen the two clustered in S2-S5 subsites. potential The isolation of cDNA clones for cetiuAr ss T sinbhr,ourresult indbcebeVt enzymes classicellthought enterokinase provides the means to address the regulation and structure- to function in lysosoma proteolyal might be involved In extaceluar protein function relationships of this ancient,essential protease. dqgdadonnbn bdlh,lmcrp --sand epl cells, such asthyrocyle. (Supported bytheDeubche Fohg. chafi SFB 284, andtheFondsder Chemischen Industrle.) Wednesday. Structure and Function of Membrane Proteins II (2530-2535) 435a

2530 2531 PARTIAL PURIFCATION AND BIOCHEMICAL CHARACTERIZATION OF MEMBRANE INTEGRATION AND TRANSMEMBRANE TOPOLOGY OF GAP AN UNGLYCOSYLATED MEMBRANE PROTEIN, THE GAP JUNCTION JUNCTION CONNEXINS: POLYTOPIC, CHANNEL FORMING MEMBRANE PROTEIN CONNEXN43: ARE THE REAL AND PREDICTED pIS AT PROTEINS. ((M. M. Falk, N. M. Kumar, and N. B. Gilula)) Department of Cell ODDS? ((E.L. Hetberg, T.C. Chan, and R-A. Corpina)) Dqment of Nuro- Biology, The Scripps Research Institute, La Jolla, CA 92037. scenoc, Albert Eistein College ofMedicine, Bronx, New York. Connexins (Cx), the proteins that form gap junction (GJ) channels in the plasma membranes (PM) of adjacent cells are structurally conserved non-glycosylated It has been rported that isoforms of connexin43 (Cx43), especially its unphos- members of a multigene family. Hydrophobicity plots and the analysis of Cx phorylated form, are soluble in non-ionc detergents (Musil and Goodenough organized in GJ channels suggest that Cx are class Ill membrane proteins that (1991) J. Cell Biol. 115:1357-1374). We have used this discovery as part of our traverse the membrane bilayer four times oriented with their NH2- and COOH- effort to purify Cx43 from rat heart and brain for biochmical, structural and termini located cytoplasmically. Various experiments indicate that following biophysical in Using a combination of subcellular fractionation and synthesis, Cx follow the classical secretory pathway of the cell to reach their final chromatogrsphic procedures, we have succeeded in substantally enriching Cx43 destination in the PM. In this study, the translocation of five different Cx into the with effrts to achieve frther purification underway. During the course of chroma- membrane of the endoplasmic reticulum (ER) was analyzed, first in vitro, by toraphic it became apparent that the soluble forn(s) of Cx43 do not synthesizing Cx in translation competent cell lysates supplemented with pancreatic behave as a protein with a pl of 10.2 as predicted by sequence analysis of its microsomes, and second in vivo, by expressing Cx in several eucaryotic cell types. Cx are oriented with their cDNA clone (Beyer et al. (1987) J. Cell Biol. 105:2621-2629). Because binding of Although NH2-termini in the cytoplasm, the translocation was an efficient Cx43 to accompanied by signal peptidase processing at a detergent-solubilized charged chomagaphic matrices might be site, when Cx were expressed in vitro. In addition to full-length to .cryptic' Cx, resticted due inaccessibility of many charged groups for interacbon with dte similar processed Cx cleavage products were also identified in the ER membranes of marx, we exended our analysis to include gel electphoresis and isoelectric Cx overexpressing cells. The results demonstrate that the Cx contain a 'cryptic' focusing in the presence of non-ionic det nts. Although we have not, as yet, signal peptidase cleavage site that can be processed by this enzyme in vitro and in been able to assign an exact experimntal pL available data are consistent with a pI vivo in association with their insertion into membranes. Consequently, a specific of about 7 for dte unphosphorylated form of Cx43. Among several possibiliis, factor or condition must prevent this aberrant processing under normal conditions in these data may suggest the presence of p nsla charge modifications dher the cell. Possible mechanisms for regulating this processing will be analyzed. dtan glycosylation or phosphoylation that may be necessary for trafficking, Finally, this presentation will include also a detailed analysis of the transmembrane topology of Cx in the ER membrane and in the PM. [Supported by assembly and/or regulaon of gap juncos and their intercellular channels. Supported by NIH gant GM30667 to E.L.H. grants GM 37904 (to NBG), GM 37907 (to NBG and NMK), a grant from the Lucille P. Markey Charitable Trust, and a Deutsche Forschungsgemeinschaft grant Fa 264/1-1 (to MMIv).]

2532 2533 CHARACTERIZATION OF MONOCLONAL ANTIBODIES SPECIFIC FORTHE THE TRANSPORT OF ORGANIC ANIONS SUCH AS TAUROCHOLATE MANNOSE 6-PHOSPHATE/INSUUN-LIKEGROWTH FACTORII RECEPTOR. INTO THE CYTOSOL AS WELL AS THE ENDOPLASMIC RETICULUM ((M. Brzycki-Wessel, D. HIN, J. Alvarez, D. Messner, and N.M. Dahms)) LUMEN OF HEPATOCYTES IS MEDIATED BY TWO DISTINCT Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI TOPOLOGICAL FORMS OF MICROSOMAL EPOXIDE HYDROLASE. 53226. ((D. Levy, C. Alves, M. Amoui, R. Stellwagen and P. von Dippe)) University of Southern California, School of Medicine, Dept. of The extracytoplasmic region of the mannose 6-phosphatefinsuin-like Biochemistry and Molecular Biology, Los Angeles, CA 90033. growth factor 11 receptor (M6P/IGF4I receptor) consists of 15 homologous bile acids such as domains. The two mannose 6-phosphate binding sites have been localized Hepatocytes transport taurocholate (TC) across the basolateral membrane as well as into the lumen of to domains 1-3 and domains 7-9 while domains 5-11 contain the plasma the smooth single reticulum This has been shown to be IGF4I site. To furher extend our structure/function studies on endoplasmic (SER). process initially binding the mediated, in part, by microsomal epoxide hydrolase (mEH), based on receptor, we have characterized two monoclonal antibodies (86f7 and proteoliposome reconstitution studies and the modulation of taurocholate 2G1 1) spocific for the M6P/IGF-II roceptor. The epitopes of these uptake in intact cells and in right-side-out sealed SER vesicles by monoclonal monoclonal antibodies wero localized to speciflc regions of the receptor by antibodies. The transport properties of mEH were further established by expressing varkou truncated forms of the receptor in transfected COS-1 demonstrating that sodium-dependent TC uptake was expressed in the plasma cells. The reactivity of 86f7 and 2G1 1 to these truncated receptors was membrane of COS and MDCK cells upon transfection with the cDNA for assessod by Western blot analysis and immunoprecipitation of mEH, with a Km value similar to that observed in hepatocytes. The use of [35SImethloninelabeled Cos-1 cell extracts. The results demonstrated that monoclonal antibody reagents in conjunction with transport and tryptic the opitope for 86f7 is located within domain 5 while the opitope for 2G1 1 digestion assays has also estabLished that a common epitope of mEH is found is located within domain 6. Incubation of the construct encoding domains on the surface of hepatocytes as well as on the cytoplasmic (80%) and luminal 5-11 with IGF41 inhibited its immunopreclpitation by 2G11, whereas surfaces (20%) of right-side-out SER vesicles, suggesting the expression of two distinct forms of mEH in the where the luminal of a construct containing domains 5-10, which does topological SER, epitope immunoprecipitation form serves as the precursor of the plasma membrane form. These results not bind IGF4I, was unaffected. These resuits indicate that either the demonstrate that mEH is functionally expressed at multiple sites in Opitope for 2G11 is a component of the IGF4I binding site or that the hepatocytes and exists in two distinct topological orientations in the SER as binding of IGF4I aters the conformation of the receptor, including the recently described for P-glycoprotein. Topological properties may thus serve opitope for 2G 1 1. Studies are cuntiy underway to distnguish between to regulate mEH cell targeting and transport characteristics. (Supported by these two possibiltes. (supported by NIH grant DK42667) NIH grant DK 25836)

2534 2535 IDENTIFICATION AND CHARACTERIZATION OF PROTEINS RESIDUES REQUIRED FOR THE EXPRESSION OF A FUNCTIONAL G ASSOCIATED WITH HMG-CoA REDUCTASE IN YEAST. ((S. A. PROTEIN- COUPLED RECEPTOR AT THE CELL SURFACE. ((A. Shah Castillo and R. Wright)) Depatment of Zoolgy, University of Washington, and Seattle, WA 98195 L. Marsh)) Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. The cellular mechanisms that signal synthesis of specific membranes have not been characterized in any organism to date. One approach to uncover The last two amino acids of a highly conserved region among Gprotein- these mechanisms is based on manipulating the cellular levels of. the coupled receptors of three species of yeast were mutated in the alpha- transmembrane protein, 3-hydroxy-3-methylglutaryl coenzyme A reductase factor receptor of S. cerevisise to determine the region's significance. (HMGR). This enzyme catalyzes the rate limiting step in sterol Four substituted receptors (W295L, W295S, A296D and A296V) were biosynthesis, and increased levels of this protein in yeast induce created. W295 and A296 lie at the end of the seventh putative characteristic nuclear-associated stacks, termed karmellae. Karmellae membrane spanning domain bordering the cytoplasmic C- terminus of formation is independent of the catalytic activity of the enzyme. However the receptor. The mutations decreased expression of functional receptors karmellac induction may depend on the catalytic domain presence to form at the cell surface as measured by ligand binding assays. Immunoblots its characteristic structure. Consequently, unique proteins that are found in using anti-receptor antibodies indicated that substituted are membranes be receptors karmellae may involved in membrane synthesis and stability. abberantly a defect in or I am and the glycosylated suggesting receptor topology isolating identifying major proteins that are associated with conformation. Immunoblots also indicated that the mutant HMGR within karmellae. My approach is to fractionate membmne vesicles receptors are generated from strains over-expressing this proteins, cross-link and as stable as wild-type and are therefore not routed to the vacuole for Indirect immunoflouresence immunoPrecipitate HMGR, and analyze the protein complex. The ability to degradation. usingP-galactosidase tagged did not with the isolate HMGR complexes and to analyze its protein composition may receptors colocalize endoplasmic reticulum (ER) provide insight into the components needed for formation of nuclear chaperone, BIP indicating that the substituted receptors are not retained associated membrane stacks. In addition, identification and manipulation of in the ER. Surprisingly, substituted receptors and an underexpressed wild these proteins will also be important for understanding the regulation of type receptor were able to support signal transduction suggesting that sterol biosynthesis. S. cerevisise express 'excess' receptors and that residues W295 and A296 are not necessary for signal transduction. The C-terminal region of the seventh hydrophobic segment of the alpha-factor receptor is needed for topogenesis or folding but not for signal transduction once the receptor achievesits proper conformation. 436a Structure and Function of Membrane Proteins II (2536-2541). Wednesday

2536 2537 CLONING AND CHARACTERIZATION OF THE INTRINSIC TRYPTOPHAN FLUORESCENCE OF RAB5 IS SENSITIVE TO SCHIZOSACCHAROMYCES POMBE CHOI+GENE INVOLVED GUANINE-NUCLEOTIDE AND Mg2+ BINDING ((Y. Pan, J. C. Sanford, and IN PHOSPHOLIPID BIOSYNTHESIS. ((M.I.Kanipes and M. Wessling-Resnick)), Harvard School of Public Health, 665 Huntington S.A.Henry)) Department of Biological Sciences, Carnegie Mellon avenue, Boston, MA 02115 University, Pittsburgh, PA 15213. Rab5 is a small GTP-binding protein that functions in membrane traffic. Other The phospholipid methyltransferase encoded by the Schizosaccharomyces Ras-related GTP-binding proteins have been identified to contain tbyptophan pombe chol+ gene catalyzes the two sequential methylations of phosphatidyl- residues with fluorescence properties that are sensitive to guanine nucleotide N-monomethylethanolamine (PMME) to phosphatidyl-N-N- binding conformations of the molecule. When excited at 290 nm, GDP-bound or dimethylethanolamine (PDME) and finally to the end product, empty Rab5 exhibits intrinsic tryptophan emission spectrum with intensity phosphatidylcholine (PC). S. pombe mutants which are choline auxotrophs peak wavelength at 340 nm. GTPzS binding by Rab5 causes an increase in the were isolated and placed into two complementation groups. The first group fluorescence intensity. Mg2+ alone can also cause this effect in a dose- consisted of cholk mutants that require either dimethylethanolamine (DME) or dependent manner, reaching a maximum at concentrations of 5-10 mM. We choline for growth. This phenotype indicates that chol- mutants are blocked in studied the fluorescence properties of two truncation mutants of Rab5, Rab51- the second methyltransferase, since they do not grow on MME. The second 198 and Rab523 198, the latter of which does not bind [32PIGTP on blots. While group consisted of mutants that will use monomethylethanolamine (MME), fluorescence properties of Rab5l-198 are similar to Rab5WT, Rab523-198 does DME or choline for growth. These mutants are defective in the first methylation not display any fluorescence changes upon incubation with either GTPyS or reaction that converts PE -+ PMME. These mutants are believed to be Mg2+. Even though low concentrationsof Mg2+ (05mrM) are required forguanine analogous to the S. cerevisiae opi3 and cho2 mutants, respectively. We report nucleotide binding, our studies show that high Mg2+ concentrations (5mM) the of the of the inhibit both [3HIGDP and [35SIGTPyS binding to RabS and that this effect is cloning chol+ gene by complementation cholP mutation. due to inhibition Sequence revealed no to lnown of GDP release from the protein. Inhibition may be exerted analysis significant sequence homology any a In vivo through interactions of Mg2+ with second binding site within the first 23 methyltransferase. labelling assays revealed that the chol+ gene amino of the was able to acids protein, correlated with conformational changes detected product catalyze the two sequential methylations which convert by the fluorescence studies. We are currently studying fluorescence and PMME -o PDME -e PC. We are in the process of further characterizing the nucleotide-binding properties of a mutant Rab5, Rab5S34N, whose Mg2+ chol+ gene and its gene product. (Supported by NRSA GM14827-01). binding site in the GTP-binding pocket is deleted. The results of these studies will help us to confirm the presence of the speculated second Mg2+ binding site, and provide mnore insight into it's role in regulating guanine nucleotide binding.

2538 2539 a AND B SPECTRIN DEFECTS IN A KINDRED WITH HEREDITARY EXPRESSION OF THE Rh GLYCOPROTEIN WHICH MAY EXIST AS A ELLIPTOCYTOSIS. ((J. Burke, S. Zail & T.L. Coetzer)), Department of COMPLEX WVTH Rh PROTEIN ((K Suyama, R. Lunn, B.L. Smith, and J. Haematology, School of Pathology of the University of the Witwatersrand Goldstein)) The New York Blood Center, New York. NY 10021 and Johns and the S. A. Institute for Medical Research, Johannesburg, South Africa. Hopkins University School of Medicine, Baltimore, MD 21205. We have investigated the underlying spectrin (Sp) abnormalities and their The elucidation of Rh antigenicity is of upmost importance because of its role in inheritance in a kindred with hereditary elliptocytosis. The father has a mild transfusion reactions and the hemolytic disease of the newbom. Since the Rh glycoprotein (Rh5OA) is postubltd to be involved in Rh we were Sp ankyrin binding defect and the mother is normal. Both children have a antigenicity, interesd in the expression, both endogenous and in vitro, of this protein. We severe binding defect, decreased Sp content and defective Sp selfassociation detected endogenous expression of both a 32 kDa Rh glycoprotein and the 32 implying inheritance of a silent factor from the mother. Sp consists of an Of kDa Rh(D) protein in K562 cell lysates. Since inbta K562 cells express Rh(D) and B subunit, which participate in Sp dimer selfassociation, whereas binding protein on their cell surface, this finding supports the theory that Rh(D) to ankyrin is via BSp. To investigate the silent maternal contribution we antigenicity may involve Rh(D)-Rh glycoprotein complexing. We prepared ascertained the effect of each subunit on the functional abnormalities. ca and RhXlIl(Rh(D)) cDNA and Rh5OA cDNA from human bone marrow and human erythroleukenic K562 cell libraries using PCR, and then subdoned B were either anion or SDS PAGE these Sp purified by exchange chromatography cDNAs into pSVL vector (pSVLRh(D),pSVLRh5OA) and pcDNA3 vector and electroelution. The renatured monomers were reassociated to form (pcDNA3Rh(D), pcDNA3Rh5OA). In vitro expression using pcDNA3Rh(D) and hybrids between the control and patient (P) subunits. Sp structure was pcDNA3Rh50A was perfomied in a rabbit reticucyte lysate system. The analysed by limited tryptic digestion, isoelectric focusing and SDS PAGE. Rh5OA protein produced migrated to the 30 kDa region on SDS-PAGE as a PcaSp lacked the 35 and 30 kD aIIfragments and exhibited an acidic pH shift non-glycosylated form, and the Rh(D) protein migrated to the expected 32 kDa of 0.13 pH unit in the 46 kD aII peptide. These structural changes were not region. Transient expression of Rh5OA protein and Rh(D) protein in COS-1 influenced The interaction was assessed cells was also accomplished using the above constructs. COS-1 cells by BSp. Sp-ankyrin by binding I25I transfected with pSVLRh50A produced a 32 kDa protein band (glycosylated labelled to inside out vesicles and showed that the reduced Sp Sp depleted form) that was immunoprecipitatd with rabbit anti-Rh glyprotein, while COS- was due to Exon ankyrin binding partly PBSp. 26 of the BSp gene encodes I cells transfected with pSVLRh(D) yielded, as expected, a 32 kDa protein that part of the ankyrin binding domain and was analysed by PCR, subcloning reacted with rabbit anti-Rh. These systems (K562 and COS-1 cells) should and DNA sequencing. The patient was heterozygous for a silent prove useful for studying the transport of Rh poteins within the cell and the polymorphism, T-C, at residue 356 of exon 26, but both alleles were expression of Rh antigenicity at the cell surface. Supported by O.N.R. Cont N- otherwise normal. 00014-93-1-0364 and N.I.H. grant RO1 HL 33991.

2540 2541 A DISSECTION OF THE RAT LIVER ECTOATPase/C-CAM/ppl2O/BILE ACID THE LIMBIC SYSTEM ASSOCIATED MEMBRANE PROTEIN IS ONE TRANSPORTER GENE. ((A.F. Knowles)) Department of Biochemistry OF THE SUBSTRATES FOR ECTOPROTEIN KINASE ACTIVITY ON and Molecular Biology, SUNY Health Science Center, Syracuse THE SURFACE OF NEURONAL CELLS. ((V.A.Zhukareva, Y.H.Ehrlich, NY 13210 P.Levitt)). Dept of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, Piscataway NJ 08854; College of Staten Island, The rat liver ectoATPase has reportedly been cloned. Sequence CUNY, Staten Island, N.Y. 10301. analysis indicates that it is identical to a rat hepatocyte cell adhesion molecule (C-CAM) and the endogenous substrate of The utilization of exacellular ATP by different ectoprotein kinaseslocated the rat liver insulin receptor/kinase, ppl2O. Functional on the cell surface has been implicated in developmental process, and studies by different groups showed that transfected cells (1) provides a novel role for phosphorylation of specific proteins on the cell acquired the tendency to aggregate, (2) manifested ATP- surface. Intracellular communication and interactions can be regulated by dependent taurocholate transport. EctoATPase activity of the phosphorylation of receptors, integrins and NCAM, through ectoprotein transfected cells was either not evaluated or inordinately low. kinase activity (Erlkch et aL Ai. N.Y. Acad. of Scitce, 603,401-16, 1990). The The cDNA has been called the rat liver ectoATPase/C-CAM/ppl2O/ limbic system associated membrane protin (LAMP) is a new memberof Ig bile acid transporter gene in recent literature. We previously superfamily, and is a GPI-anchored protein, with patterns of expression established that the mercurial-insensitive (MI) ectoATPase of restricted to limbic system regions of the developing brain. Sequence human hepatoma Li-7A cells is enzymatically similar to the rat analysis of LAMP reveals eleven potential phosphorylation sites in the liver ectoATPase. In cloning the human enzyme by utilizing the molecule. The phosphorylation of surface proteins by acelular ATP was "rat liver ectoATPase cDNA" as a probe, a clone was obtained studied in the SN56 immoralizd brain cell line, which is known to express which contained a partial cDNA of the carcinoembryonic antigen LAMPat the surface. We also studiedculted primary neurons from limbic (CEA) which has no demonstrable ATPase activity but is related (perirhinal cortex, hippocampus) and nonlimbic (olfactory bulb) areas of the to rat C-CAM. The "rat liver ectoATPase cDNA" hybridized weakly fetal rat brain at different ages. Several membrane proteins are with a 2.3 kb mRNA of Li-7A cells. Its amount was not increased phosphorylated under conditions that promote ectoldnase activity. More in cells treated with EGF, cholera toxin and hydrocortisone specifically, immunoprecipitation of LAMP from cultured cells, following a which markedly induce the MI-ectoATPase activity. It is brief incubation with extracellular ATP resulted in specific phosphorylation concluded that the "'rat liver ectoATPase cDNA" codes for C-CAM/ of the protein. The results indicate that post-translational modifications ppl2O, but probably does not code for an ectoATPase. It was through phosphorylation and dephosphorylation may be an important discovered that the excpression of the CEA gene in Li-7A cells mechanism to regulate the functon of LAMP and other neuronal surface was increased EGF and further cholera by stimulated by toxin, proteins during growth and circuit formaton. Sqpeu by NMHHraN MH 45507. Wednesday. ER to Golgi Transport (2542-2547) 437a

2542 2543 RAB 1B REGULATES EARLY STEPS IN EXOCYTIC TRANSPORT OF AN INTRACELLULAR DEGRADATION PATHWAY INDEPENDENT OF ALZHEIMER'S 8-AMYLOID PRECURSOR PROTEIN. ((J.M. Dugan*, C. De ENDOSOMES PREVENTS THE ACCUMULATION OF TROPOELASTIN Witt, L. McConloguet, and W.A. Maltese*)) Weis Center for Research, Geisinger IN ELASTOGENIC CELLS FOLLOWING BREFELDIN A TREATMENT. Clinic, Danville, PA 17822*, and Athena Neurosciences, South San Francisco, ((Elaine C. Davis and Robert P. Mecham)) Department of Cell Biology and CA 94080t. Physiology, Washington University School of Medicine, St. Louis, MO 63110.

Alzheimer's disease is characterized by neural amyloid deposits containing B- Elastin is secreted as soluble tropoelastin monomers which are then crosslinked in amyloid peptide (AB), a 4 kD fragment derived from proteolytic processing of the presence of extracellular microfibrils to form insoluble elastic fibers. Although an integral membrane glycoprotein, B-amyloid precursor protein (BAPP). BAPP the secretion of tropoelastin is thought to be mediated and targeted by an can undergo altemative secretory processing resulting in the release of a soluble intracellular chaperon complex, the intracellular route taken by this protein and the N-terminal fragment (APPS) and a 3 kDa peptide (p3). Secretory processing of role of such a chaperon complex remain undefined. In the present study, the BAPP precludes formation of AB. To better understand the intracellular specific pathway of tropoelastin secretion was investigated in fetal calf ligamentum trafficking of BAPP, we have begun to investigate the possible involvement of nuchae (FCL) cells using brefeldin A (BFA) to disrupt the secretory pathway. small GTP-binding proteins of the Rab family, which have been implicated as Consistent with the reported effects of BFA on intracellular organelles, electron mediators of specific steps in vesicular transport pathways in mammalian cells. microscopic studies of BFA-treated FCL cells showed extensive dilation of the Initially, we focused on RablB, which has been shown by others to control ER to endoplasmic reticulum and a perinuclear accumulation of tubulo-vesicular Golgi transport of VSV G-glycoprotein. BAPP751 was co-expressed in cultured elements. FCL cells labeled for 4 hours with 3H-leucine followed by immuno- 293 cells with RablB(wt), or RablB mutants that have a reduced capacity to precipitation with a tropoelastin antibody, SDS-PAGE and autoradiography bind guanine nucleotides (RablBNi21I or RablBS22N). Processing of BAPP was revealed a band of metabolically labeled tropoelastin in both the cell lysate and assessed after a 10 min pulse with [35S]methionine followed by a 1-h chase. medium. When labeled in the presence of BFA, however, radiolabeled tropoelastin Immunoprecipitation and SDS-PAGE of labeled BAPP75, revealed a marked was not detected in the cell medium, nor was there an increase in the amount of decrease in conversion of Endo-H sensitive BAPP (108 kDa) to the mature 0- radiolabeled tropoelastin in the cell lysate. Northem analysis of mRNA levels in glycosylated form of BAPP751 (130 kD) in cells expressing RablBNi2i1 or FCL cells showed that the message for tropoelastin was unaffected by the BFA RablBS22N, compared to cells expressing RablB(wt) or BAPP alone. The block treatment thus suggesting that the newly synthesized tropoelastin was being in BAPP processing resembled that observed in cells exposed to Brefeldin A. degraded. In control cells, inhibition of acidic proteases in the endosome/lysosome Immunoprecipitation of radiolabeled APPS from conditioned medium showed a pathway resulted in an increase in the amount of radiolabeled intracellular dramatic decline in the secretion of APPS in cells co-expressing BAPP751 with tropoelastin. In the presence of BFA, however, the inhibitors had little effect on the RabIBN121I. The results indicate (1) that RablB is required for ER to Golgi amount of radiolabeled tropoelastin in the cell lysate. These results suggest that the transport of BAPP, (2) that secretory proteolytic processing of BAPP occurs in a accumulation tropoelastin in the endoplasmic reticulum as a result of BFA late compartment of the exocytic pathway, and (3) that similar mutations in treaument leads to rapid degradation of the protein by a non-lysosomal pathway. other Rab proteins may help to define specific routes of BAPP transport involved in the generation of AB. Supported by NIH grant CA34569 (WAM).

2544 2545 IDENTIFICATION OF THE PRINCIPAL ATPASE OF RAT LIVER TRANSITIONAL INVOLVEMENT OF PROTEINS IN SORTING OF IgM TO DEGRADATION IN B ENDOPLASMIC RETICULUM. ((L. Zhang and D. J. Morre)) Department CELLS. (I. Shachar, D. Winitz, A. Kerem, and S. Bar-Nun) Department of of Medicinal Chemistry, Purdue University, West Lafayette, IN Biochemistry, Tel Aviv University, Tel Aviv, Israel. 47907 B lymphocytes produce two forms of IgM, a ,rm-containing membrane form Transfer of membranes from endoplasmic reticulum to Golgi and a s-containing secretory form. These two types of heavy chain differ only apparatus occurs via 50-70 nm transition vesicles which derive in their carboxy termini. The 41 residues in sm which include an hydrophobic from part-rough, part-smooth transitional elements of the transmembrane domain are substituted in lss by 20 hydrophilic residues endoplasmic reticulum (TER). Vesicle budding from the TER is designated I.L% However, during the course of differentiation, these two forms of ATP-dependent both in vivo and in vitro. An ATPase with a IgM exhibit distinct intracellular fate. On the surface of B cells Wm2L2 is displayed, monomer molecular weight of 100 kD by SDS-PAGE was isolated from while plasma cells cease to produce the rm. On the other hand, plasma cells TER and designated as TER ATPase. A 19 amino acid sequence of efficiently secrete (iSsLs)s assembled in the trans-Golgi, whereas B cells fail to the TER ATPase having 84% identity with valosin-containing assemble these polymers due to preceding degradation of 1sS2L2. This indicates protein (VCP) was obtained. Antisera to a 15 amino acid portion the operation of developmentally regulated sorting mechanisms that distinguish of the sequence of TER ATPase recognized a 100 kD protein from between i's and ism. We have shown by transport blockers and in permeabilized TER. cDNA clones encoding TER ATPase were isolated by B cells that degradation occurs in a post-ER compartment. In our attempt to immunoscreening a rat liver cDNA library with the affinity- identify cellular proteins involved in the retention/degradation of sIgM in B cells, a purified antibody. The deduced amino acid sequence revealed TER 3OkDa polypeptide, designated p30, was isolated by co-immunoprecipitation with ATPase to be the rat equivalent of porcine valosin-containing IgM. The ability of p30 to bind slgM specifically was further demonstrated by protein, a member of a novel family of ATP binding, homo- ligand blot analysis. Purification of p30 was accomplished by affinity oligomeric proteins including the N-ethylmaleimide sensitive chromatography, utilizing its specific association with eAs. Interestingly, this fusion protein. The native TER ATPase was characterized as a association appears to be hampered in the presence of P-mercaptoethanol or homo-hexamer of six 100 kD subunits by gel filtration and calcium perturbants, conditions that we have recently shown to interfere with electron microscopy. TER ATPase antisera inhibited the ATP- sigM sorting. Analyses by anti-p30 antibodies raised by us, as well as by affinity dependent cell-free formation of transition vesicles and chromatography, suggest that p30 is maintained during differentiation, at least in inhibited transfer of radiolabled material from endoplasmic hybridoma cells. The efficient secretion of slgM implies that the interaction of with is obstructed in these cells. reticulum to Golgi apparatus in a reconstituted membrane p30 slgM normally transfer system. The results suggest that the TER ATPase is obligatorily involved in the ATP requirements for budding of transition vesicles from the TER.

2546 2547 DEGRADATION OF IgM IN PERMEABILIZED B CELLS. (S. Bar-Nun, D. Winitz, LACK OF THE INTEGRAL MEMBRANE PROTEIN P24B RESULTS IN 1. Shachar, and M. Samuelov) Department of Biochemistry, Tel Aviv University, DELAYED SECRETION OF INVERTASE AND GPI ANCHORED GASI P. Tel Aviv, Israel. ((F. SchimmOller, B. Singer-KrOgerl, S. Schr6der and H. Riezman)) Biozentrum, Uriversity of Basel, Switzerland; I Present address: EMBL, B lymphocytes produce two forms of 1gM, the migM membrane form and the Heidelberg, Germany. slgM secretory form. Although they differ only in the carboxy termini of their respective R& heavy chains, lm and Fs, these two forms of IgM exhibit distinct A novel yeast protein (p24B) was identified in a partially purified intracellular fate. Monomers of mIgM are displayed on the surface of B cells, endosomal membrane fraction of Saccharomyces cerevsisae (Singer- while im is no longer produced by plasma cells. Polymers of sIgM, assembled in Kroger et al., JBC268,14376-14386, 1993). p24B, encoded by the or beyond the fan-Golgi, are efficiently secreted from plasma cells, whereas EMP24 gene (GENEMBL databank: X67317), is a type I transmembrane they fail to assemble in B cell. Instead, monomers of slgM are degraded prior to protein. An immunoisolation approach demonstrates that the protein is the n>s-Golgi. This indicates the operation of developmentally regulated not associated with yeast endosomes. p24B shows overall 24% identity mechanisms of that between and to gp25L. a mammallan protein that was copurfied with calnexin from retention/degradation distinguish mlgM sIgM. A mutant To an to these mechanisms, we have studied degradation in rough microsomes (Wada et al., JBC266, 19599-19610, 1991). gain insight sIgM lacking p24B (e&p24) grows normally but, as analyzed by pulse-chase vro. in B occurred when of This degradation, permeabilized cells, only transport experiments, Is defective In secretion of periplasmic invertase and sigM from the ER was reconstituted. This is in agreement with the in viv glycophosphatidyl Inositol (GPI) anchored Gaslp. In cells devoid of degradation which, based on its sensitivity to brefeldin A, occurs in a post-ER p24B, invertase and Gasip acquire modifications charactedstic of the compartment. In addition, the involvement of a short-lived protein and a cysteine Golgi apparatus with reduced kinetics suggesting that the protein the inhibition exerted protease in the in vivo degradation was indicated by strong functions early in the secretory pathway. In addition, part of the invertase by cycloheximide and calpain inhibitors, respectively. Indeed, upon incubation secreted by errp24A cells Is aberrantly glycosyated. Lack of p24B does with dithiothreitol, but not with f-mercaptoethanol, both ;s and ;an were not intertere with the secretion of a-factor nor whh transit of vacuolar degraded. However, this was not due to activation of the cysteine protease, but proteins through the biosynthetic pathway to the vacuole. We conclude rather the consequence of dissociation of the heavy chain from the light chain. that p24B shows a novel activity influencing the secretion of a subset of This resulted in transport-independent degradation of the unassembled and secretory proteins. unfolded chain within the ER, as corroborated by the insignificant effect of brefeldin A. In addition to a process that did not distinguish between gs and zrn, this ER degradation occurred in both B and plasma cells. Moreover, this is analogous to the recently described degradation in a light chain deficient variant and it probably reflects the situation in the pre-B cells. 438a ER to Golgi Transport (2548-2553). Wednesday

2548 2549 BREFELDIN A INHIBITION OF INTRASOMAL EVENTS PRECEDING INFLUENCE OF CYTOSOLIC FACTORS ON GOLGI-ER RETROGRADE FAST AXONAL TRANSPORT IS NOT READILY REVERSIBLE TRANSPORT. ((J. Hidalgo, M. Muftiz, and A. Velasco)) Department of tR Hammerschlag', J Bobinski', F-F Chu2, H Chan3 and RS Smith3)) 'Div Neuroscience and 2Dept Med Oncology Res, City of Hope Med Ctr, Duarte, Cell Biology, Faculty of Biology, University of Seville, 41012- CA 91010; 3Dept Anat Cell Biol, Univ Alberta, Edmonton, Canada T6G 2H7. Seville, Spain. Incubation of bullfrog spinal ganglia in brefeldin A (BFA) inhibited the transfer of newly synthesized protein to the rapid phase of intra-axonal Misfolded and ER resident proteins can eventually reach the Golgi transport within the sciatic nerve (Smith et al, J Ncurochem 62:1698,1994). The greater sensitivity and shallower dose-response to BFA found in these neurons complex where they can be recognized and retumed back to the relative to the responses of mammalian secretory cells led us to extend our ER by retrograde transport. Here we describe a semiintact cell of BFA effects on fast axonal transport. Drug reversibility was observations system that can help to identify the requirements for this initially tested by preincubating spinal ganglia in BFA for 1 h, transferring to [3H]leu-supplemented BFA-free medium for I h, and incubating for 16 h at pathway. Streptolysin 0-permeabilized cells incubated with a high 18°C to allow accumulation of transported l3H]protein at a nerve ligature. concentration (5-10 mg/ml) of cytosolic proteins and ATP- Reversibility was not significant at 1 ,ug/ml BFA, .30% at 0.1 gg/ml, and generating system exhibit Golgi disruption and redistribution into =85% at 0.01 gg/ml. At I gg/ml BFA, recognizable Golgi stacks were no longer present by 30 min, remained undetectable after a 12 h drug-free the ER. This process requires ATP and is non affected by previous incubation, and retumed to normal levels by 24 h. Further, in contrast to microtubule depolymerization. Ultrastructural observations findings in mammalian cells (Lippincout-Schwartz Ct at, J Ccll Biol 112:567,1991), indicate that Golgi disassembly occurs by budding of coated incubation of ganglia in 100 .M forskolin (FSK) for 16 h did not reverse the transport-inhibiting effects of I h treatment with either 0.1 or 0.01 .g/ml BFA. vesicles. This stage of the process is regulated by the activity of Finally, we found that the inhibitory effects of 5 gg/ml BFA on secretion of trimeric G proteins. On a later stage, vesicles lose their coats and [3Hlprotein was rapidly reversible in primary hepatocytes prepared from either fuse with the ER. This part of the process does not occur in cells rat or frog liver. Our findings suggest that the mechanism of BFA inhibition incubated at either 150C or 200C, or exposed to N-ethylmaleimide. of intracellular transport differs for pathways preceding secretion and axonal transport. Supported by NIHINS27173 (RH) and MRC of Canada (RSS).

2550 2551 NEW GENE PRODUCTS THAT INTERACT WITH THE COATOMER COMPLEX IN LOCALIZATION OF RABlp TO THE EARLY COMPARTMENTS OF THE YEAST ((Rainer Duden, Brian Whitcomnb and Randy Scheknian)) HHMI and Molecular SECRETORY PATHWAY ((J. Saraste, U. Lahtinen* and B. Goud**)) Depart- ment of Biochemistry and Molecular Biology, University of Bergen, N-5009 Cell Biology, U.C. Berkeley, CA 94720. Bergen, Norway, *Ludwig Institute for Cancer Research, Stockholm Branch, Non-clathrin coat proteins (COP I) have essential functions in biosynthetic membrane Box 240, S-171 77, Stockholm, Sweden and **Unite de Genetique Somatique, traffic. Yeast cells harboring conditional alleles of genes encoding coatomer subunits, Pasteur Institute, 75724 Paris Cedex, France. SEC21 (yeast y-COP), SEC26 (yeast n-COP) and SEC27 (yeast 0'-COP), are defective in We have studied the localization of the small GTPase rablp using polyclonal ER-to-Golgi transport under non-permissive growth conditions. Overexpression of the antibodies prepared against the rablA isoform of the protein. IF of NRK and genes encoding Boslp, Betlp and Sec22p partially suppresses the temperature-sensitive mouse myeloma cells showed the association of the protein with the Golgi defects of the mutants sec27-1 and sec21-1 (Duden et al., 1994: JBC, in press). In a search complex and peripheral sites, where it colocalized with p58, a marker protein for genes whose products may interact with coatomer, we screened a yeast genomic library of the intermediate compartment (IC) and cis-Golgi. Rablp and p58 also had similar distributions in membrane fractions derived from rat pancreas micro- for high copy suppressors of sec21-1. We have isolated several novel genes that suppress somes. Both were concentrated in two intermediate density subfractions bet- both sec21-1 and sec27-1. As expected, the screen also re-isolated the genes encoding ween the rough ER and trans-Golgi, whereas rab6p, previously localized to Sec2lp, Sec22p and Sec26p. DNA sequencing of the strongest suppressor revealed a 8OkDa middle and trans-Golgi, was concentrated in light density trans-Golgi fraction. protein. Another suppressor encodes a 109kDa hydrophilic protein which in addition IEM revealed the association of rablp with 1-2 cistermae and vacuolar and tubulovesicular membranes in cis-Golgi, where labeling was seen between suppresses a temperature-sensitive sec22 allele. The phenotypic consequences of depleting closely opposed membranes. The peripheral, rablp-positive IC elements had yeast cells of these proteins are being investigated. We are constructing epitope-tagged a predominantly tubulovesicular appearance in NRK cells, whereas in myeloma versions of these gene products to study their subcellular localization and membrane cells they consisted of vacuoles surrounded by tubules and vesicles of hetero- association. Several other suppressors are being sequenced. These novel proteins may be genous size. Interestingly, rablp was mainly associated with the tubulovesi- cular domain of these IC elements. The rough ER and the nuclear envelope in involved in the regulation of coatomer function in ER-to-Golgi transport or may be required these cells contained negligible labeling. These results do not support a role for retrieval of membrane proteins from the Golgi to the ER, i.e. retrograde transport. for rablp in vesicle budding from the rough ER but rather suggest that the protein may function in membrane fusion events within the IC and cis-Golgi.

2552 2553 A RAB PROTEIN IS REQUIRED FOR THE ASSEMBLY OF SNARE FUNCTIONAL ANALYSIS OF RAB2 IN ER TO GOLGI COMPLEXES IN THE DOCKING OF TRANSPORT VESICLES TRANSPORT ((M.Sogaard, K.Tani, R.R.Ye, S.Geromanos', P.Tempst, T.Kirchhausen', (( E.J. Tisdale and W.E. Balch)) Dept. of Cell Biology, The Scripps T.Sollner and J.E. Rothman)) Cellular Biochemistry and Biophysics Program Research Institute, 10666 N. Torrey Pines Rd., La Jolla, CA 92037 and Molecalar Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.+ Dept. of Cell Biology and Center for Blood Members of the Rab family of Ras-related small GTPases are MA Research, Harvard Medical School, Boston, 02115. associated with cellular compartments of the exo- and endocytic vesicle but the pathway. In previous work we reported that the Rab2 protein, a Rab proteins are generally required for transport docking, resident of intermediates is required for protein traffic precise role they play remains unknown. We now report in vivo studies pre-Golgi between the ER and complex. Mutations in its GTP or GDP exploiting yeast secretion mutants demonstrating that a rab protein is required Golgi for v-SNAREs and t-SNAREs to assemble, eventhough the rab protein is not binding domains resulted in proteins which inhibit transport. To itself a part of the docking complex thereby formed. This suggests that, in a further study the function of Rab2 in ER to Golgi traffic, we have broad sense, rab proteins participate in a reaction that catalyzes SNARE generated double mutant forms of the protein containing either assembly to dock vpsicles, and in so doing rab proteins could help impart an GTP or GDP binding domain point mutations and deletions of the additional layer of specificity to vesicle docking. This mechanism likely N terminus (A11414,A26) based on the observation that the N- involves a member of the SEC] gene family, Slylp, which we identified in terminus of H-Ras and Rab5 are necessary for function. The effect isolated docking complexes. We also report the identification of a novel v- of N-terminal deletions in wild type and mutant rab2 forms SNARE (Ykt6p) component of the yeast ER-Golgi docking complex that has suggest that the N terminus is required for Rab2 function. To a CAAX box at its C-terminus and is predicted to be lipid-anchored rather further explore this observation, a peptide was synthesized to the than an integral membrane protein. The surprising finding that a docking first 13 amino acids of Rab2. This peptide was a potent inhibitor complex can contain many distinct species of SNAREs (Sed5p, Boslp, of ER to Golgi traffic, in vitro. The inhibition was specific; Sec22p, Ykt6p and likely Betlp) implies that it is multimeric, a feature analogous N terminal peptides to rabl, rab3, and rab5 had no expected of the fusion machinery, and may also be a means to improve the effect. Combined biochemical and morphological studies fidelity of vesicle targeting according to a combinatorial code. indicated that the peptide blocks at a late, pre-fusion step to the Golgi complex. Wednesday. ER to Golgi Transport (2554-2559) 439a

2554 2555 THE KDEL-RECEPTOR ACCUMULATES AFTER BREFELDIN A DOMINANT NEGATIVE MUTANTS OF RABIB ARE DEFECTIVE IN TREATMENT IN VESICULAR STRUCTURES (BIVS) WHICH ARE PRENYLATION. ((A.L. Wilson, K.M. Sheridan, F. Castellano, and W.A. DISTINCT FROM THE ER-GOLGI HYBRID COMPARTMENT. Maltese)) Weis Center for Research, Geisinger Clinic, Danville, PA 17822. Dement of of (a. FOlkcrug and G. Mieskes)) Clinical Biochemistry, University Rab proteins comprise a major subgroup in the superfamily of Ras-related Gwtingen, 37075 G Cttingen, Gemany. (Sponsored by Prof. Dr. E. Neher) GTP-binding proteins. Studies performed with mammalian cells expressing mutant Rab proteins with altered guanine nucleotide binding properties have The subcellular localization of endogenous KDEL-receptor/ERD2 gene product established roles for specific members of the Rab family as mediators of protein was invsated in Brcfeldin A (BFA) trated Vero and NRK cels using imuno- trafficking along the exocytic and endocytic pathways. Although such mutants have been used extensively, the exact mechanism underlying the dominant fluorescence and suboellular fiatin. ERD2 and an ie iate ERGIC-53, negative phenotype remains unclear. Since interaction of Rab proteins with cmartment marker protein, showed overlapping stuctu with ERD2 having a Rab:Geranylgeranyl transferase (GGTase II) involves structural domains distinct more p d Golgi staining. Howev, after BFA treatment both proteim accu- from the carboxyl-terminal prenylation site, we asked whether mutations that mubated in doty, presumably vesicular struct throuout the cell and were no alter guanine nucleotide binding (S22N and N1211) and GTP-hydrolysis lor disi le. These BFA iduced vesicular structures (BIVS) are not (Q67L) might affect the prenylation of RabIB. Recombinant wild-type and identical to the reently desribed BFA bodies as they were not associaxed with mutant proteins were assayed in a cell-free system containing [3H]GGPP and GGTase RablB constructs were also B-COP. They are also disinct from the ER-Golgi hybrid a n marked by analyzed by transcription and translation in vitro in reticulocyte lysate containing [3H]GGPP. In both types of assay, the ER for a former calreticulin for and a-mannosidase II resident Golgi protein. RablBS22N and RablBN12lI were extremely poor substrates for prenylation. Overexpression of ERD2 tagged with a C-terninal nyc epitope and subsequent RablBQ67L was a relatively good substrate when assayed in the presence of GDP, BFA treatnt resulted in an ER-like patter indicatig that the additional amino but was a poor substrate when assayed in the presence of GTP, suggesting that acids interfere with the normally occurring accunulatsoring of ERD2 in the GGTase H prefers the GDP bound form of RablB. Consistent with this finding, BIV structures. Slowg down th response to BFA by decrasing the tempwAtur prenylation of the wild-type protein was reduced when it was assayed in the indicated an early sorting event between ERD2 and resident Golgi proteins. Subcel- presence of GTPyS. In vivo analysis of mutant RablB proteins by expression and metabolic labeling with [3H]mevalonate in 293 cells demonstrated that lular fractionatiw of BFA treated and control cels centn- by equilibrium dessity RablBS22N and RablBNI2I! were poorly prenylated in intact cells. Prenylation of fugation de the presenc of ERD2 in fracbos with a similar density as RablBQ67L was similar to that observed for wild-type, possibly because the ER. Velocity crolld ntriatio resuted in two subpultios of the interaction with a Rab GAP in vivo may overcome the intrinsic defect in GTP KDELIrtor of which one was separated from the ER. Although the position of hydrolysis. The results suggest that failure to undergo prenylation may the trans-Golgi marker UDP p l in both gradients ckarly shifted contribute to the dominant negative phenotype exhibited by Rab proteins with in upon BFA treatment, the distribution ofERD2 remained almost unchaged. defects guanine nucleotide binding. Supported in part by NIH grant ROI CA 34569.

2556 255T GOLGI-INDEPENDENT, BREFELDIN A-SENSITIVE PROTEIN A CONSERVED PROTEIN DOMAIN REQUIRED FOR SEC7P TRANSPORT IN GIARDL4 LAMBLLA (Hugo D. Lujin#, Alex Marotta, FUNCTION. ((S. DItz, I Qvdiy, and A. PsumM) 1sqn m Michael R. Mowatt, Noah Sciaky', Jennifer Llppincott-Schwartz & Ms& l *ido*yand D=k cf (tk and Sias B*igy, Theodore E. Nash) Laboratory d Parasitic Di_sses National Indilule UaMnk43fSsw&HArsm=CbnI,D,CO8)S of Allergy and Infectious Dieases, National Institutes of Heakh, Bethesda, Maryland, and *Cal Bioogy and Metabolsam Branch, Natiorl Aozmvedpr1sthnds(gm Scc7doman) has been found in a number Institute d Ctild Health and Human Development. Natioal Institutes d of p from a wide range of species We have been swtying this Health, BEhesda, Marylan. domain in the context ofyeast Sec7p functo Sec7p is a 230 kD, cytolic p that titly associates with ellulr Fnzuoffet d, A fundamental characteristic of eukaryotic cells is the presence of 1991, J. Cell BioL 112: 27-37). The evidence fom in viw and in vibt and membrane memane-bound comartmenet tansport pathways in experients suggests that Sec7p is a constituent of the protein co of which the a central role in the seective Golgi complAex plys processing, sctyvesftransport feta4 1992, Nature 355: 173-175). and secretion d The Giara sorting proteins. paraitic protozoan bembia AnaleIeofSEC7has been found to contain a mnuan in the Sec7domain bekongs to the earliest identified linege among eUiaryotes and therelore in a lapaxsiedlOl vb c We repor that offear unique insight in the pr n frm pritive to more complex reufng addion ofsyn c peptidesand affinit ant eukaryotic coas. Here, we describe the properties of the secretory s ific for the pathway in both noenysting and encysting dages of Girdia, uing Sec7 d to an in virm ER to transport assay blocks vescle markers for Golgi membranes, costoner, and ADPI-ribosyation fcor budding and proe raffic. These re sugges that the Sec7 domnain is (ARF). lnhbition of protein socretin by brefldin A (BFA) and BFA- involved in the membrane of Sec7p during the fmaton of senskive membrane asocition of ARF and D-COP were observed in btrport vesices Addinl evidence is ped that ges simila both develmta stages of Gidia indicating its secretory machinery mechanisms may be at work in the fornatio of mammlin exocytic is similar to higher eukaryotes. Despite this, no biochemically or vesicles. morphologically evident Golgi apparatus existed in nonencysting cells. Upon encystation, however, during which car aerih cyst wafl componerts are synthesized, Golg sert enzyre biosynthesis was induced and a mophologicly Wdnifiable GoC i co appeared within these calls. These findings have irplications both for defining the minimal machinery for protein secretion and for examining Golgi biogenesis and function within eukaryotic cals.

2558 2559 ESSENTIAL DOMAIN OF THE S. cerevisiae PROTEIN TIP) DIFFERENT FUNCTIONAL DOMAINS OF ADP-REBOSYLATION ((G. Frigerio, D. J. Sweet, and H. R. B. Pelham)) Division of Cell FACTORS (ARFS) INVOLVED IN CHOLERA TOXIN AND Biology, MRC Laboratory of Molecular Biology, Hills Road, PHOSPHOLIPASE D (PLD) ACTIVATION IDENTIFIED WITH ARFI AND ARF-LIKE 1 (ARLl) CHIMERIC PROTEINS. ((G.-F. Zhang, W. A. Cambridge, CB2 2QH, UK Patton, F.-J.S. Lee, M. Liyanage, J.S. Han, S.G. Rhee, J. Moss, and M. Vaughan)) Laboratory ofCellular Metabolism & Laboratory of Biochemistry, The products of the S. cerevisiae genes TIP) and SEC20 are NHLBI, National Institutes of Health, Bethesda, Maryland 20892. required for exit of proteins from the ER. In contrast to genes necessary for vesicle from the ER or fusion with the ADP-ribosyladon factor 1 (ARFI) and ARF-like 1 (ARLI) are strucurally budding related -20-kDa GTP binding proteins with 57% identity in amino acid Golgi apparatus, absence of functional TIP) or SEC20 lead to sequences. ARF1 stimulates cholera toxin A subunit (CTA) ADP- accumulation of ER membranes as well as clusters of vesicles. ribosyltransferase activity whereas ARL1 does not. ARF proteins have Although the TIPI protein has been shown to interact directly recently been reported to be activators of phospholipase D (PLD). To with the integral membrane protein Sec2Op, the function of the determine whether the same funcdonal domains are responsible for CTA and PLD activation, two chimeric proteins were constructed by switching the complex is still unknown. In order to identify regions of TIP) amino uminal 73 amino acids of ARFI and ARL1. The recombinant rL73/F essential for viability, a number of amino and carboxy tenninal protein, in which the amino terminal 73 amino acids of ARLI replaced those deletions were generated. Some of the truncated proteins had no of ARFI, retained CTA activation, whereas the rF73/L protein, in which the detectable others were defective at N-terminal sequence of ARF1 replaced that of ARL1, did not activate CTA. phenotype, high temperature, Although the activity of rL731F with CTA required GTP and a third failed to at all. In this a and group support growth way phospholipidldetergent, thus resembling that of wild type rARFl, the GTP miniprotein of roughly half the size of wildtype Tiplp could be concentrtion rquired for half maximal activation (EC5O) ofcholeratoxin was defined which in addition to binding to Sec20p must provide any nearly 100 times that required with rARFl. Interestingly, the two chimeric further activity required for the overall function of the complex. proteins behaved in quite the opposite way for PLD activation. The rL73IF sensitive mutations in this essential protein failed to activate PLD, whereas rF73/L activated PLDin the presence Identification of temperature of GTP. These results are consistent with the conclusion that the amino domain with normal protein stability and wildtype affinity for terminal region of ARF1 is not critical for its action as a GTP-dependent Sec2Op should provide a tool to further analyse the process activator of choleratoxin, whereasit may be important for the GTP-dependent dependent on membrane bound functional SEC2O/TIPl complex. activation of PLD. 440a ER to Golgi Transport (2560). Wednesday

2560 ISOLATIONOF ANAMINO-TERMINAL-DELETED RECOMBINANT ADP-RIBOSYLATION FACTOR(ARF)1 [rAl3ARFl] IN AN ACTIVATED NUCLEOTIDE-FREE STATE. (J-X.Hong, J.Moss and M.Vaughan.)) Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda MD 20892. ADP-ribosylation factors (ARFs) are 20-kDa guanine nucleotide-binding proteins, initially discovered as activators of cholera toxin (CI), and later shown to participate in vesicular trafficking. Mutant rAl3ARFl (ARFI lacking the first 13 amino acids) synthesized in E. coli produced equally high stimulation of CTA-catalyzed ADP-ribosylation with added GDP, GTP or no added nucleotide, in part due to the presence of bound GTP. Nucleotide-free ral3ARFl (rAl3ARFl-F), prepared by dialysis against 7M urea, was active without added nucleotide in the absence of detergent or phospholipid but inactive without added GTP, or with GDP, in their presence. Renaturation of rAl3ARFl-F in the presence of GTP or GDP produced rAI3ARFl-GTP or rAl3ARFI-GDP that was active or inactive, respectively. The effects of phospholipids and detergents on kinetic properties of the rARFI and r&l3ARFl differed. These studies are consistent with the conclusions that: (1) r&l3ARFl is active in the absence of bound GTP. (2) The protein can be isolated in a GTP-bound state. (3) rAl3ARFl differs from rARFl in nucleotide-binding properties and effects of phospholipids and detergents. It appears the ARF amino terminus plays an important role in nucleotide binding and the molecular conformation associated with it. Golgi to Cell Surface Transport (2561-2564).

2561 2562 PRIMARY STRUCTURE OF MG-160, A MEMBRANE SIALOGLYCOPROTEIN OF 5 CEREVISIAE sacl MUTANTS EXHIBIT ELEVAIED LEVELS OF HIGHLY THE GOLGI APPARATUS.(JO Gonatas,Z Mourelatos and NK Gonatas) MODIFIED INOSlTOL SPHINGOtP AND AN INOSIML AUXOTROPHY THAT Department of Pathology and Laboratory Medicine, University of IS CORREClED BY DISRUPTION OF ThE CDP-CHOLINE PATHWAY FOR Pennsylvania School of Medicine,Philadelphia,PA 19104 ,(WS Lane) PHOSPHOLIPID BIOSYNTIESIS. ((B.G. Keaens, and V. A. Bankitis)) Deparmeent of The Biological Laboratories, Harvard University,Cambridge,MA 02138, Cell Biology, University of Alabama at Bismingham, Bimingham, AL 35294. (J Brosius) Mount Sinai Hospital and Medical CenterNew York 10029. The S. cereiua SAC) gene was originally identiled on the basis of two disiguishig 60 is a phenotypes associd with loss of funcion mutios: (i) the ability of sucd mutatios to MG-1 membrane sialoglycoprotein residing in the medial cisternae suppess yeastacti defect in an alespecfic manner, and (ii) the ability ofsacl of the Golgi apparatus of rat neurons, pheochromocytoma (PC12) and mutations to effect bypss ofthe normally essential requir t forphospaidylinositol other cells (Gonatas,JO et al. J.Biol.Chem. 264:646-653,1 989).The cDNA (PI) kospatidykbDeh (PC) nmsfer poin (SEC14p) function in Golgi sectry encodes a polypeptide of 1,171 amino acids with an Mr of 1 33,403 processes and oell viability. Fiaully, we noled that sacl strains exhibit an inositol Daltons. An intralumenal cleavable signal peptide is followed by a Pro-Gin auxotopy, and suggsed that this inositol auxotophy mightreflctsorm acamted rich segment and sixteen contiguous, ca.60-residue-long, regtlarly spaced al turover ofphspoipidptiuay modtio As aresuLt, wehave cysteine-rich segments showing sequence identities ranging from 15 to characerized theinositol s oipidprofile in sac) stains of yeasL Our data idiate 35 percent. The lumenal domain is followed by a single membrane that sac) yeawt stais exhibit a Signiant enricblanet in the most highly modified spanning domain and a 13 amino acid carboxy-terrninal cytoplasmic tail. inosit sphingolipid, M(IP)2C, that represents mannosylinositolphospho mide modified with an additional inoidtol phosphate. It is currently believed tat this ultimate The protein contains 5 potential NXT glycosylation sites. inositol phospate is donated by phosphatidylinositol. As yeast ephingolipids receive The sequence of MG-160 is 90% identical to a basic and acid fibroblast tbher final modiaon i the Golgi compex, these data are consstent with the notion growth hctor receptor(CFR)isolated from membranes of chicken embryos that sac) stains might expaiencoelevated turnover of P in Golgi menbaes. However, (Burms et al.,Mol. and Cell Biol. 1 2:5600-5609,1992). we have been able to uncouple the sphigolipid penotype frm the inostol auxotrophy. MG-160, purified from rat brain, binds basic human fibroblast Wheeas aU the sacl alleles we have tested maifest the elevaed M(IP)2C ph e,y not growth factor (bFGF). In PC1 2 cells bFGF is internalized in an endosomal- all such alkkls rest in a lo phbenotype. Iteresngly, we have found that the Ioo Iysosomal compartment but not in the Golgi apparatus.MG-1 60 shows no pheotpe ofAracl strais is completely and efically alleviad by inactivabon of the sequence similarities with fibroblast growth factor receptors involved in CDP-cdolioe pathway, but not the PE methylaton pathway, for PC biosynthesis. These signal transduction. These findings are consistent with the hypothesis findings suggest a reladonship between SAClp funcio and thatof the CDP-choline that MG-160 is involved in the traffic and processing of certain pathway. Elucidation of tis reladonship will provide new informaio into the endogenous FGFs. mechanism by which inactivation of esther SAClp or the CDP-choine pathway results in Supported by Grants NS05572(NK Gonatas) and MH3881 9(J Brosius). a 'bypass SEC14p' phenotype.

2563 25" A STRUCTURE/F)NCTION ANALYSIS OFTHIE RAT PHOSPHATIDYL- OVERPRODUCTION OF THE YEAST IN02 GENE PRODUCT CORRECTS THE INOSITOL TRANSFER PROTIN USING A YEAST SURROGATE SYSTEM. INOSITOL AUXOTROPHY ASSOCIATED WnTH bsd2-1, A BYPASS SEC14p ((J. G. Alb, Jr., and V. A. Banitis)) Department of Cel Biology, University of MUTATION IN S. CEREVISIAE. ((S. Kagiwada, T.P. MoGee, and V. A. Bankaitis)) Alabama at Birnmgham, Binnmingbam, AL 35294-0005. Deparmnt of CeUl Biology, University of Alabama at Birmingham, Birmingham, AL 35294. (Spon. by DiM. BodwelL) The metoa phospbatidyHnositol (Plphosphadtylhline (PC) iranserprotein (PI-lP) is ahighy coservedpolypepde originally idendfied on its ability to The bsd2-1 allele confes yon Saccharoycescerevuia thecomplete nce from catalyze the exchange of Pi and PCbeween mebrane bilayers in vitro. However, a the normally essential requirement of SEC14p, the yeast detaied approciaton of the ii wio fwmcion for this protein in higher eukaryotes is phosphatidylinositol/p loline tsfer prein, for Golgi secrtory functio not yet availble. Moreover, how ths protein recogizes andbindsiransfers its and cell viability. This 'bypas SEC14p penotyp is a dominant traiL An additioal popolipidligands is not entiod. Toatdress the lalerissue, we contructed phenotype assocated with bsd2-1 is an inodsol auxotephy that exhibits codomant secl4 yeastsais tatexhbitedadependence on tat PI-TPexpression forcell c aistca in most strs. la atempting to rcover clones of the RSD2 gene on the gowth and viabilty at 37CC; theey prviding a yeast saoga system by which basis of compl on of the bsd2-1 inositol auxoboy, we isolated a yeast funcional studies of tat PI-TPcouldbe executed. To this end, werandomly rentroioeric plaamid th a geanmic hsert that did not cwry BSD2 (as determid by mutagenized the rat PI-TPgen and utied the yeast surrogae sys tem to sceen integrative genetic m_ping) butnoetheless Ief ntly supressedthlnopheotypeof mutagenized cones forbiocal function. Of6000 mutagenized genes screened, 33 bsd2-1 strais. The identity of the suppreso gene was determined by a nember of faied to complement the grwth and secrtory defects of secl)4 yeast srins at criteria to be IN02, a posidve regulator of pospolipid biosynthetic 370C. Eight of these defective rat PI-TPgenes were deemed of interest ofthebasis genes. This finding suggested that the inositol auxotophy of bsd2-1 strains might be of their ability todrive exprsi of reWvdy stable and full lengt rat PI-TP related to an inability of these stins to derepress the expression of pospholipid antigen as deteninedby Wesem analysis. Wehavedfcarized thesemutust biosynthetc gSens when inotol (dte primay conttoro reprson) is removed frm proteins on the basis of PI and PC transferproiles rdative to the WT PI-TP, and the medium. Condsie with this noio, quantiave pospolipid analyses indicate have identified twomutant lasses. The fourcbss II mutations awe typified by an mased defects in the iuer sutges of the PE methyimion patway for PC biosynthesis in oximale 65% eduction in steady-staterat PI-IP lvels. The most intresting bsd2-1 strains These ults, when coupled with other data that will be presented, feature of tbese mutants, however, is hdr _ tally complete and specific defects in suggest that the bsd2-1 gene prduct sifs at the divergence of two disti celular PI-trasfer capability, but little n_dnbledefcts i PC-tsfer capability. pathways; one that is wlvt to SECl4p and Golgi secretry function, mid the second Furdtermo, two ofthe four class sidues define, or immediately adjacent to, that lilely involves a senoy palhway that lads bacl to the i reguion of cosens pi kinase C (PKC) ph ylaio sites. Class I mtatius ame also pIo-_olipid biosynteiic genes. typified by an approxise 65% reductio in seady-state rat PI-TP levels with ommensrate reducto in both PI and PC tsferacdvity. Two Class I resdues also involve consenu PKC phosporylaio sequences. The finding that4/6 mutatedresidues define, or am immediatly adjacent to, PKC pbospbohyaion sietes suggests the possibility that PL-transfer/binding is regulated by PKC in vivo. Wednesday. Golgi to Cell Surface Transport (2565-2570) 441a

2565 2566 CHARACIERZATION OF SFH1, AN S. CEREVISUE GENE ENCODING A RAB 6 AND ARF ASSOCIATE WITH EXOCYTIC MEMBRANE CARRIERS IN RAT PROTEIN WITH HIGH PRIMARY SEQUENCE IDEN1TIY TO SEC14p. ((M. HEPATOCYTES. ((L. Saucan and G E. Palade)) Cellular and Molecular Medicine, Keams, M. Fang, B. Keamns, and V. A. Bankaitis)) Department of Cell Biology, University of Califomia, San Diego - School of Medicine, La Jolla, CA. 92093 University of Alabama at Birmingham, BinniDgham, AL 35294. We have shown that in rat hepatocytes membrane and secretory proteins are SEC14p is the S. cerevisiae phosphatidylinositol (Pl)/phosphatidylcboline (PC) segregated, presumably at a post Golgi site, in two different classes of vesicular tansfer protein, and it is required for Golgi secretory function and cell viabiity. Current carriers (Saucan and Palade, J. Cell Biol. 125:733-741, 1994). Starting with a Golgi data indicate that the in vivo role of this proin in S. cerevisiae is to effect a light fraction (SM) which bands at the 0.25M-0.86M interface in a discontinuous downiregulation of PC synthesis va the CDP-choline pathway in yeast Golgi sucrose gradient and processing it through immunoisolation on magnetic beads menibranes by specific inhibitiko of the rate-limiting enzyme of this pathway. SEC14p coated with an antibody against the polymeric IgA receptor (plgA-R) cytoplasmic tail, exhibits high conservaum at the pimay sequence level throughout the unicellular we end up with a nonbound (NB) and a bound (B) subfraction. In our pursuit for fumgi. This sequenc conservation manifests itself in functonal tenms as expression in specific proteins belonging to each class of vesicular carriers we found that rab 6, a S. cerevisiae of SEC14ps frm Kluyveromyces lactis, Yarrowia lipolyrica, and small GTP binding protein, previously localized to the Golgi and trans-Golgi network Schizosaccharomycespombe which results in an efficient complementation of the (Goud et. al., Nature 345 553-556, 1990), is associated with the smaU vesicular carriers lethality associated with sec)4 null mutations. We now report the identificaton of an (present in the B subfraction) that transport membrane proteins in a constitutive-like S. cerevasiae gene that has the potential to encode a protein with high pimary sequence fashion to the sinusoidal plasma membrane. Rab 6 however is not detected in the identity to SEC14p, and we designate this gene SFH1 (SFC Eourteen tiomolog). The large secretory vacuoles (present in the NB subfraction). Moreover, when an antibody putative SFHlp is predited to represent a 310 amiDo acid polypeptide with a 63% against rab 6 is used for immunoisolation, the same type of vesicles, enriched in pigA- identity and 78% similarity to the 304 rsidue SEC14p; a level of identity that eitber R are isolated from our starting preparation. Our preliminary experiments show that equals or exceeds the identities to SEC14p exhibited by the SEC14pa from Y. lipolyNica over 90% of the rab 6 protein in SM separates in the B subfraction, can not be and S. pombe. Moreover, SFHlp exhibits conservation of the amino acid motifs that removed by either high salt or high pH treatments and partitions in the detergent we find to represent invariant signattues of fungal PI/PC-tansfer proteins. SFHlp, phase during Tx-114 extraction. These findings are in concert with the current however, is not essential for yeast vegetative growth, Auihl mutations do not show thinking that rab proteins are preferentially distributed to specific relays of the synthetic lethality or any other measurable effect when coupled with secl4ts mutations, secretory and/or endocytic pathways. In vitro incubations of our SM, NB and B fractions with liver and an ATP system, and Asihi does not compromise the ability of cki, cct, cpt, bsr3, sacl, bsdl, and bsd2 cytosol, ATP, GTPyS regenerating show that ARF is recruited onto in mutations to effect a bypass suppression of secl4 null mutations. This lst point is of (ADP ribosylation factor) vesicles present the SM and B but not in the NB subfraction. ARF was identified critical impotance as it demonsrates that the 'bypass SEC14p" phenotypes associated by a-13NP]-GTP overlays and by immunobiotting. However, we do not know which ARF molecule is recruited onto the with the mutations in these seven genes, phenotypes whose bypass chaacter has been small vesicular carriers because the antibody we have used does not distinguish insumental in the formulation of a nusber of conclusions cocerning SEC14p between different isoforms of the ARF Work is in to function vinw,s are not the trivial cseencs of amplified activity of a redundant protein. underway trying identify other molecules for one or the other class of vesiles as well as SEC14p-l&le ftmction. These findings suggest the presnce of multiple members of a specific secretory SEC14p protein family, each devoted to regulating distinct cellular processes. trying to identHy the functional roles of rab 6 and ARF presence in the small exocytic carriers immunoisolated in our protocol. Supported by NCI grant CA58689.

2567 2568 HOW MHC CLASS MOLECULES REACH THE ENDOCYnIC ISOLATION OF MAMMALIAN CELLS W1T- DEFECTS IN PATHWAY. EXOCYTOSIS. ((R.-H. Wang, C.-Y, Kao, N.T. Ktistakis and M.G. Roth)) Department of Biochemistry, University of Texas Southwestern ((M.Yilla*, P.Benaroch*A, G.Raposo!, H.J.Geuze! and H.LPloegh*)). Medical Center at Dallas, TX 75235-9038. Center for Cancer Research, of Department Biology, Massachussetts Institute We have developed an efficient method for isolating animal cells with of Technology, Cambridge, Massachussetts 02139 Prsent address: defects in secretion. Using as a reporter of secretory activity the ICGM.UPR-0415, 22 Rue Mechain, 75014, Paris. Department of Cell fluorescent lipid analog C5-DMB-Ceramide followed by fluorescence- Biology, University of Utrecht Medical School. 3584 CX Utrecht, The activated cell sorting, we have identified mutant Chinese hamster ovary Netherlands (Spon. by M.Yilla) cells that are resistant to the drug Brefeldin A (BFA) and others that are defective in the transport of glycoproteins to the cell surface and in the We have examined trafficking of MHC class II molecules in human B cells morphology of the Golgi complex. Ten lines of cells that show dominant exposed to concanamycin B, a highly specific inhibitor of the vacuolar H+- resistance to BFA have been isolated that differ in growth characteristics ATPases required for acidification of the vacuolar system and for early to late or Golgi morphology in the presence of the drug. Seven lines of CHO endosomal transport. Neutrlization of vacuolar compartments prevents cells with temperature-sensitive (ts) defects in secretion have been breakdown of the invariant chain (Ii) and blocks conversion of MHC class H isolated. Some of these cell lines have ts defects in transport between the molecules to peptide-loaded, SDS stable op dimers. Ii remains associated with endoplasmic reticulum and Golgi apparatus, some lack an identifiable a and this complex accumulates internally, as ascenained biochemically and by Golgi apparatus at the non-permissive temperature, and some retain the immuno-electronmicroscopy. In concanamycin B-treated cells, a slow increase Golgi complex but are inhibited in transport between the Golgi and (>20-fold) in surface expression of li, mostly complexed with CEO, is detected. plasma membrane. Only one of these cell lines has defects in fluid-phase This surface-disposed fraction of cipli is a minor population that eaches the cell endocytosis, suggesting that the other six do not belong to previously surface directly, or is routed via early endosomes as internediary stations. In identified complementation groups of CHO mutants. Assignment of these inhibitor-treated cells, the bulk of acpi is no longer accessible to fluid phase mutants to genetic complementation groups is in progress. Using endocytic markers It is concluded that the majority of 4Ii is targeted directly fluorescent lipid as a reporter of membrane traffic is an efficient, general from the TGN to the compartment for peptide loading, bypassing the ceU method for isolating mammalian cells with defects in the secretory surface and early endosomes en route to the endocytic pathway. pathway that will provide useful reagents for investigations of the mechanisms which control the function, stability and biogenesis of secretory organelles.

2569 2570 VAPP14 IS INVOLVED IN THE FORMATION OF HEPARAN SULFATE EXPRESSION AND LOCALIZATION OF GLUT4 IN SKELETAL MUSCLE CELL PROTEOGLYCAN-CARRYING SECRETORY VESICLES FROM THE LINES ((E. Ralston, B. E. Flucher, M. J. Quon and *T. Ploug)) Laboratory of TRANS-GOLGI NETWORK ((W.Nickel, A. Barthel, C. Tonko, J. Ftillekrug, NINDS and *Diabetes National Institutes of D. FaBhauer, and H.-D. Soling)) Institut fur Kinische Biochemie, Georg- Neurobiology, Branch, NIDDK, August-UniversitAt Gottingen, D-37070 Gottingen, Germany. (Spon. by Health, Bethesda, MD. 20892 Felix T. Wieland) One limitation to the study of insulin-stimulated glucose transport in muscle VAPP14 has been described as a peripheral membrane protein associated is that most muscle cell lines have been reported to express little or no with heparan sulfate proteoglycan (HSPG)carrying TGN-derived secretory GLUT4, the major insulin-responsive glucose transporter. We have vesicles isolated from rat hepatocytes (Nickel, W., LA. Huber, RJA Kahn, reexamined expression of GLUT4 in the mouse muscle cell line C2 at N. Kipper, A. Barthel, D. FaBhauer, and H.-D. Soling. 1994. J. Cell Biol. different stages of differentiation. Immunoblotting experiments showed that 125:721-732). Investigations using an antibody raised against VAPP14 GLUT4 is expressed in C2, beginning in confluent myoblasts and increasing revealed that the protein is also present in the GolgitGN and in the steadily during myotube maturation. Immunofluorescence localized the cytosol. Following treatment of NRK cells with Brefeldin A VAPP14 was to the area the with found to dissociate from strongest staining trans-Golgi surrounding nuclei, Golgi membranes. Unlike known coat proteins additional and, in mature a finer close to VAPP14 is a punctate staining myotubes, staining cytosolic not part of high molecular weight complex like the the cell surface. The is in and to that coatomer T. and J.E. Rothman. staining comparable intensity pattern (Waters, M.G., Serafini, 1991. Nature in [Lond.] 349:248-251) as it behaves as a monomer during gel filtration. observed rat muscle fibers. In mature, spontaneously contracting cultures, VAPP14 displays ubiquitous tissue distribution and, therefore, appears to some myotubes showed a membrane staining in areas that also show be a general component of the late constitutive secetory pathway. On the enhanced staining for the transferrin receptor (TfR), although GLUT4 and TfR basis of an in vitro-assay described previously for PC12 cells (Tooze, S.A are not colocalized. Transient transfection with a plasmid encoding a GLUT4 and W.B. Huttner. 1990. Cell 60:837-847) we have measured the budding cDNA with an exofacial tag confirmed that, in some myotubes, GLUT4 is on of HSPG-carrying secretory vesicles from the TGN using a cell-free system the cell surface. No changes in GLUT4 localization were observed after from a rat hepatoma cell line. The budding of HSPG-carrying secretory insulin stimulation of young myotubes. Only in cultures that had been vesicles from the TGN was inhibited by GTPyS and stimulated by contracting for several days and in which myotubes appeared striated did palmitoyl-CoA. A rabbit preimmunserum and a control antibody (a-rab5) insulin cause an increase in membrane staining and a decrease in trans-Golgi had no effect on the budding process. In contrast, both a purified IgG staining. GLUT4 was also observed in the Sol8 and BC3H1 muscle cell lines. fraction directed against VAPP14 as well as corresponding Fab fragments We conclude that 1) GLUT4 is at detectable in inhibited the release of expressed easily levels all strongly secretory vesicles from the TGN. We muscle cell lines tested 2) expression of GLUT4 is not sufficient conclude from these data that VAPP14 function is essential for the export endogenous to confer insulin since the former the latter of HSPG from the TGN en route to the cell surface. responsiveness precedes by several days in C2 myotubes. 442a Golgi to Cell Surface Transport (2571-2576). Wednesday

2571 2572 REVERSIBLEpH DEPENDENT INTERACTION BETWEEN REGULATEDSECRETORY PROTEIN KINASE C REGULATES CONSTITULIVE MEMBRANE TRANSPORT FROM PROTEINS AND LIPIDS OF THE SECRETORY GRANULE MEMBRANE. ((J. THE TRANS-GOLGI NETWORK TO THE CELL SURFACE. ((Buccione R., Santone I., Lai", Di Tulio G.. Baldassarre M., Luini A. and *De MaEeis MA)) Laboratory of Molecular M. Beat, and D. LBelO) Departm of Biology, Universit de Sheibrooke, Neuobiology and Unit of Physiopathology of Secretion, Consorzio Mario Negri Sud, 66030 Sherbrooke, 0C, CANADA, J1K 2R1. S. Maria Imbaro (Chieti) Italy. (Spon. by Anna Teti)

We have previously shown (Nature, 364:818) that the binding of ADP-ribosylation factor We have pHdependent n ofa e ed srtory rwnsdttedth into reguted (ARF) and the coat protein -COP to the Golgi complex in vivo is stimulated by activation of protesis wi memne takes ple i vNo in thw t s-Gog network (TGN) by the IgE receptor in rat lbphiic leukemia (RBL) cells thrugh the stimulation of the signal usiNg pa tic ogens as rglated protes, and purild granule me transduction enzyme protein kinase C (PKC). This was also demonstated in vitro on isolated fractions activation of PKC with The as the target. By addhig puified sectty ganule mer to zymogns in a rato Golgi by phorbol esters. fungal metabolite brefeldin A (BFA), which causes detachment of ARF and the coat proteins, contasted the effect of PMA. one in the native in simibr the ymogen granule ontions of the Ai vito assay of Here we show how the activation of PKC leads to the acceleradon of constitutive transport aggregin d l" d in the rat (BWw. J. 291. 289, 1993), we wr abble to from the trans-Golgi network (TGN) to the plasma membrane (PM). Tbis was carried out by spalyand quantilatively pel toh membns at pH 5.6, cditions cwparable to studying the transport and exocytosis of both a fluid phase bulk-flow marker such as glycosaminoglycan (GAG) chains, and an integral membrane protein such as the those beiaved to exist in the c acnar cell TGN. Addo of vesicular panc pre-aggregaed stomatitis virus G-prtein (VSV-G). RBL cells, pulse-labellnd for 5' with [35S]sulfate, released to mne braes was 30% less ficient. Coss-inked cnstitutive proins granule 2 to 3-fold more GAG as compared to controls when stimulated with phorbol myriste acetate secretory proteis (Igs) were ieffective inthe systsm. Aggrgation and interacion wkih (PMA). BFA inhibited both basal and stimulated release. This was due to activation of PKC mwbmanes we i p of cadium ions. Even i he patnm ofaggregationofpig since a) the stimulation by PMA was completely inhibitable by the highly specific PKC nogen wasdiferentfromth one of rat in tens of and inhibitor Ro82-20 and b) inactive phorbols failed to exert any stimulatory effect. RBL cells zymogens ficincy optimum also feaure regulated exocytosis from secrtory granules. A fraction of GAG accumulates in pH of agWegation, pig zymoge polted rat mebtbmes. Reversely, rat zynogens these granules and can be released by stimulating degranulation with Ca ionofore. In RBL pileted pig mem ns. To know whether lipids or proteins of the granule membrane cells, however, stimulation of PKC alone does not induce the release of granule contents. PKC-dependent stimulation of constitutive tanwsport of the integral membrane protein VSV-G were aocoable forthe iterction, liids wer extracted and reconsttued in vesicles. was also studied The rate of appearance of [35S]methionine-labeled VSV-G on the PM of As whole int ed in a and reverse did membranes, pid vesicles pH-dependent VSV-infected RBL cels, as assessed by cell-surface biotinylation, was increased by PMA. The manner wih aggrgted regulatedscretory proteis. These obseriatons sWgestthat acceleation ofTGN to PM transport is not a fea unique to RBL cells. We have studied and the membran lipidsaresuffident tosa as receptorsforbiding ageated rogdted paially characterized this aspect in different cell lines, most of which lack regulated secretion. In all cases PMA effectively increased the rate of release albeit with different klietcs proteins under the acidic dions the TGN, and thus to facilite eul of and effectiveness dependingon the cel type. in vesices to these aggrgates depting build up secrtoy granules. [Supported by Acknowledgmnts. This work wa partially supporud by the Aenszia per 1 Promozione e lo NSERC, Canadi CF Foudaton and Fonds FCAR (Oufbec)I Svihlppo del Mezogino (PR-2) and by tie CNR (Convenzione CNR-Mario Negi Sud)

2573 2574 PROTEIN KINASE C IS A TONIC POSMIVE REGULATOR OF COAT PROTEIN HETEROGENEITY IN VESICULAR COAT PROTEINS. (a. A. Wbitney, D. BINDING TO GOLGI MEMBRANES AND OF CONSTITUTIVE SECRETION Sheff, R. Kahn*, T. Kreis§ and L Mellman)) National Cancer Institoe, NIH, ((G.Santini*, R.Buccioneo, M.Baldassarre*, G.Di Tullio°, A.Fusella*, A.Luini°, and Bethesda, MD. §University of Geneva, Geneva, Switzerland. and Yale University M.A.De Matteis*))*Unit of Physiopathology of Secretion and Laboratory of Moleallar School of Medicine, New Haven, CT 06520-8002. Neurobiology, Consorzio Mario Negri Sud, 66030 Santa Mana Imbaro (Chieti), Italy (Spon. by A.Pavan.) We are interested In ldentifying and characterizing proteins Involved with the soring of membranes and proteins in the endocytic and exocytic pathways. We We have that kinase reported protein C (PKC) stimulation through plasma membrane have tacen advantage of recent advances in our underanding of the bo ry activation or can receptor by phorbol esters induce the binding of the coat proteins ARF of sorig and vesicular coat binding to Identfy compartment specific roles for coat and fCOP to Golgi membranes (Nature 364: 818-820). Here we examine the possibility and coat-associated proteIs. Heterogeneity in coatproteins was emined thrgh that a tonic PKC acdvity may have a role in regulating coat protein assembly on Golgi binding of cytosollc proteins to free-flow electrophoresis (FFE)- enriched membranes and constitutive membrane traffic. To this end we have used the most specific endosomal, Golgi, andER membran. Bound protens were compared using 1-D PKC inhibitors presently available: calphostin C (acting on the diacyl-glycerol binding and 2-D gels. Similar, but distinct, pattern of proteins bound to the each site of PKC) and Ro 82-20 (acting on the ATP-binding site of PKC). The binding of membrane fraction. In particular, two imunologically related but disa MCOP ARF and uCOP to Golgi membranes was evaluated in rat basophilic leukemia (RBL) proteins were deteted in endosomal and ER/GolgibInding fractions, while -COP cells both in vivo (by immunofluorescence utilizing specific antibodies and Helix Is replaced by an unrelated protein on endosomal membrane Specificity was also Pomatia lectin as a marker of cis- medial-Golgi) and in vitro, on isolated Golgi found in ARF interaction with FFE-enrched membranes In paticular, ARFI and membranes incubated with cytosol and GTP or Al/F or GTPyS. Constitutive secretion ARF5 bind bothGolgi and endosomal membranes, while ARF6 isfound asdated was followed by the release of 35S-sulfated glycosoaminoglycan (GAG) chains with only the plasma membrane. Based on the her detcted in prteins synthesized on exogenous xyloside in RBL cells exposed to -"S-SO4. Both PKC binding to various organdIes, the cytosollc pool of coatomer was also examined for inhibitors caused the detachment of ARF and nCOP from Golgi membranes within 5-15 microhetogeneity of coatomer subunitsthat may in trn be dier ay boundto min with an EC50 of I ILM for calphostin C and 5 pM for Ro 82-20. These inhibitors, various vesicular populations. Coatmer complex was purified to homogeneity. however in contrast with brefeldin an known to induce the A, agent cytosolic Heterogeneity with regard to I but not MKW. was detected for p.., and £- COPs. redistribution of ARF and did PCOP, not cause the tubulation of Golgi complex or its Furher, phosphorylation of F- and e-COPs was demo tedratei Preliminary data with The intermixing endoplasmic reticulum. effect of PKC insibitors was not due to also suggest phosphorylation of a- and 6- COPs. lhe hetogeneit detected In non-specific cytotoxic activity since it was partially counteracted by phorbol esters or cytosolic coatomer may in part explain the antigenic heterogeneity detected in fl. phosphatase inhibitors, such as okadaic acid. Both calphostin C and Ro 82-20 inhibited COP binding to endosomal and golgi vesicles. Fsher, heterogenei detected in the binding of ARF to isolated Golgi membrane in response to GTP and Al/F. Fsally, p-, 6- and a- COPsmay allow interactions with diffent AF proteins to produce both PKC inhibitors inhibited by 50% the release of 35S-GAG from RBL cells, compartment-specific vesicular coats. Interaction between these coats and suggesting that a tonic activity of PKC may have a role in setting the rate of constituive targeting signals may In turnplay a role In the sorting mechanism. transport at least from TGN (the site of GAG-chain sulfation) to the plasma membrane. Acknowledgments. This work was partially supported by the Agenzia per la Promozione e lo Sviluppo del Mezzogiomo (PR-2) and by CNR (Convenzione CNR-Mario Negri Sud).

2575 2576 MOLECULES INVOLVED IN EXOCYTIC VESICLE FORMATION CYTOPLASMIC DYNEIN BINDS TO BUDDING MEMBRANES INAN IN FROM THE TGN: INTERACTIONS AND REGULATION. ((S.M. Jones, ViTRO GOLGI STACK BUDDING ASSAY. ((G.M. Tsimbu, KR. Fath and J.R. Crosby, J.L. Stow* and K.E. Howell)) University of Colorado School D.R. Burgess)) Departnent of Biological Sciences, University of Pitsburgh, of Medicine, Box B lll Denver, CO 80262 and *MGH East, Charlestown Pittsburgh, PA 15260. MA 02129. Micro= bules (MT) are required for dte efficient tunsport of branes from We have identified a cytosolic complex (62kD kinase and a small GTPase) the tras-Goli and for tranacysoss of vesicles from the basolaterl membane which associates with the cytoplasmic domain of TGN38/41 and is to the apical cytoplm in p epithelia. MTs in these cells are primly essential for the of vesicles from the TGN. et budding exocytic [Jones al., oriented with theirplus near the Golgp derm nus ends the These studies used washed membranes. non- 1993]. high pH Golgi When apical cyoopla Golgi membran isolad from inestnal epithelal cells washed CHAPS solubilized Golgi membranes are used in similar possess the actin-sed moor myosin-1, the MT minus-end directsd motor experiments, antibodies against TGN38/41 co-precipitate p200. In abstract, cyopls icdyneinandkisinvWromotilityacdvatwdynacin(pl5t1SGed)(J.Cei Crosby et al., [ASCB, 1994] we present evidence that p200 (originaly Biol. 126,inpress).Thesem _embrancannlocaaeonMTsiniutvbromotility described by Namla et al. 1992) is also required for exocytic vesicle asmays. Golgi sacks, which lack dynein but contain myosin-l can bind soluble budding and is a candidate for the lace-like coat found on vesicles budding dynein sWlied frm added cytosol. In cell-fre Golgi stack budding assys, from the TGN [Ladinsky et al., 1994]. In the membrane (the CHAPS sacks can be induced to bud orreleaesmallrrmmbanes when incubued with solublized complex of TGN38/41, p62 complex and p200 and perhaps cytosol and ATP.Dynein appea to bind to Golgi membranes destined to bud other proteins) is 1.3 MD by sizing column chromatography. In cytosol the becauw dyneinispr eint budded baes,but absentfrom the sucsafer p62 complex is 85kD while p200 is found in two pools, one of 44OkD and budding Myoin- is also associasedwith budded embrnan The pesence of the other of 220 kD. Pulse-chase analysis indicates that the components of dynein on budded membranes, after inubatc with GTPyS, which ha been the cytosolic p62 complex turn over very rapidly, with t/2 of -120 min. shown to cause accumultin ofcoated vesicles, was ma to levels found The kinase and GTPase form a complex by 30 min and both are degraded in comlesebudding eactions. I contst,about10foldmo dyneinwas found by 120 min. These short half-lives are similar to those of regulatory on budded incubated with NBMK whichhas been shown tocauw an proteins. On the other hand, p200 has a half life on the order of 80 h which accumuato of smooth, non-cotd vesicle We prose that cytoplasmic is similar to the half-lives of chain and other structural clathrin heavy dynein bxing t Golgi manesccurdunrngtheClaet1ages0of memrne- proteins. These data indicate that the p62 complex and p200 form a large protein ta thrugh theGolgi. We asoproposethatdynn transports MW complex with TGN38/41 and suggest that these interactions are a part budded GolgiM salongMlstothe ellcorexwh myon provides of the lace-like coat assembly essential for exocytic vesicle budding from local deltrich cysokleo to the apical membran the TGN. Supored by NIHDK 31643. Wednesday. Golgi to Cell Surface Transport (2577-2582) 443a 2577 2578 DISTRIBUnON OF HETEROTRIMERIC G PROTEIN ALPHA SUBUNITS IN THE RAT INTERACTION OF NADH OXIDATION, PROSTAGLANDINS AND BREFELDIN A PANCREAS. ((S. DenkerT, J. M. McCaffery, P. Insert, and M.G. Farquharq) IN THE TRANSFER OF HICHOLESTEROL FROM GOLGI APPARATUS TO DIvison of Celluar and Molecular Medicine' and Department of Pharmacoblg, PLASMA MEMBRANE IN A CELL-FREE SYSTEM FROM RAT Unvrsly of CaWomb, San Diego, La Jolb, CA 92093. LIVER. ((B. Hoffman, D. J. Morrd, J. Lawrence and D. M. Morrd)) Department The heerotrmnerc G protens Gczu and Gas have recn been irnpiated h the of Foods and Nutrition and Department of Medicinal Chemistry, control of renbrane traffiddng events aong the exocytic pathway. In the present Purdue University, West Lafayette, IN 47907 study, subcelluar fractionation and nmnocalzation were used to investigate the distribution of these alpha subunits In pancreatic acinar cells. By Cell-free transfer from Golgi apparatus to plasma membranes immunofhuorescence on semithin cryosections prepared from pancreas lobules, of rat liver was shown previously to be stimulated by 0.1 mM GaIS and Gas were loalzed to both the Golgi and the plasna membrane (PM). NADH (Rodriguez et al., Biochim. Biophys. Acta 1107, 131-138). GCi3 stann was concentrated h the Golgi area, whereasGaw s s8t to appeared To measure cell-free transfer, Golgi apparatus donors were extend into the apical reglon of the cel. However, secretory granules were not labeled with [3H]cholesterol by exchange. Rat liver labeled. Double label showed a co-localization of GaC wIth a- plasma experiments partial membrane sheets as acceptor were immobilized on mannoskdase 11, a marr of cis though trans Golgi cstenae In the panras, as wel qitrocellulose as with Rabla and P-COP, markers of ER to Golgi and intra-Golgi transport vesicles strips. In the presence of NADH, transfer of [ H]cholesterol and the irtemedate compartment. Irterestingly, Gas showed strn co-obcalzation was stimulated by 12.5 AM prostaglandin A2 and inhibited by with caveolin. By subcelkular fractionation combined with enzymatic and inmunoblot brefeldin A (2 to 20 u4M). In the absence of NADH, both analysis of gradlent fracftions, Gu6 was found to fractionate exclusively with smooth prostaglandin A2 and brefeldin A inhibited cell-free transfer. membranes (d-1.12-1.16 gIml) containing Golgi and PM as monitored by a- When added together in the absence of NADH, the net result of an&mnxidase 11 and akalne phosp dleserase activiles. Using a fttation gradient the combination of prostaglandin A2 and brefeldin A was still desIogned to separate PM from total microsomes, Gas was resolved iro two pools - inhibition. However, in the presence of NADH, the combination a broad peak associated with PMs and a second peak of higher density (1.16 g/mnl). of prostaglandin A2 and brefeldin A resulted in either a more This heavier fraction was enriched in TGN38 and rab6, and contaied ARF, P-COP marked inhibition or a more marked stimulation of transfer and the P subunit of the heterotrimeric G proteins, but was not enrIched in early than either drug alone depending on concentration and ratio of endosomes, marked by the transterrin receptor. These data show that Gas: 1) is prostaglandin A2 to brefeldin A. The results indicate an a of membranes present in population intermediate in density between Golgi and interaction between prqstaglandin A, and brefeldin A in the PM, and 2) co-loalizes with caveoin, TGN38 and rab6, suggesting that Gas is cell-free transfer of [ H]cholesterol from Golgi apparatus to associated with vesicles involved in transport from TGN to PM. The precise plasma membranes dependent upon the presence of NADH. An functional role of Gas in transport along the exocytic pathway(s) remains to be inhibition of NADH oxidation activity of rat liver Golgi defined. Supported by NIH grants CA58689 (to MGF) and GM40781 (to PI). apparatus by brefeldin A that was accelerated by GDP was reported earlier (Morrd et al., FEBS Lett. 346, 199-202).

2579 2580 SEC9 IS A SNAP-25-LIKE COMPONENT OF A YEAST SNARE IDENTMICATION OF TWO DISTINCT POPULATIONS OF SECRETORY COMPLEX THAT MAY BE THE EFFECTO OF SEC4 FUNCTION IN VESICLES THAT ACCUMULATE IN LATE SEC MUTANTS. ((E. Harsay and EXOCYTOSIS. ((P. Brennwaldl2, B. Kcans3, K Champion3, S. A. Bretscher)) Biochemnistry, Molecular and Cell Biology, Cornell University, Kerlnen4, V. Bankaitis3, and P. Novick2)) IDeprtment of Cell Biology and Ithaca, NY 14853. Anatomy, Cornell Univasity Medical College, New York, NY 10021. 2Departmentof Cell Biology, Yale University School ofMedicine. Saccharomyces cererrsiae mutants that have a block late (post Golgi) in the secretory pathway accumulate 100nm vesides carrying plasnu membrane and cell 3Depsrment of Cdl of Alabama-Birmingham. 4VTF Biology, University wall componentsl. As these vesicles are homogeneous in size, they are Biotechnology and Food Reavh. easily purified by gel filtration chromatography2. While the various vesicle enzyme markers copurify by this method, they can be separated into two groups To idenify Sec4effects, we isolated s of a by potential high-copy equilibrium isodensity centrifugation. The major population of vesides contains Sec4 effector domain mutanL The most potent oftese was found to be SEC9. Bgl2p, an endoglucanase that is a cell wall component3, as well as a large number a gene required for post-Golgi unspor The sole essentidaldomain of Sec9 of mannoproteins. These vesides also contain an exoglucanase and an ATPase has ssgmifcant sequence similarity to the nronal prein SNAP-25, which is activity. In addition, an 18KDa protein that has been suggested to be a a of the SNARE complx, licatedin vesicle wteng and synaptobrevin4 copurifies with this group. Another fusion Analogous to SNAP-25, Sec9 is bond to the yeast plasma membane vesicle population contains the periplasmic enzymes invertase and acid phosphatase. Both vesicle and is absent fom post40ogi vesicles. Furthermre, Sec9 is physicay populations accumulate in the late mutants secl, and sec6, assoied with the Sso and Sncproteins, the yeast homologs ofsyntain and seretory src4, while synapiorein which are mnts of the nmunal SNARE complex. Like neither is detected in wild type cells or early ser mutants. The secl3/src6 double mutant does not accumulate either veside populations, indicating that are the nuronal SNARE complex, the Sec9/Sso/Snc complcx appears to they both derived from the normal Thin disassemble in the presence of Mg/ATP but is stable in the presence of secretory pathway. section EM shows that both vesicle groups have an average diameter of 100nm. The presence of two Mg/ATPyS. Our rest idendfy Sec9 as the yeast cognate ofSNAP-25 and populations of late secretory vesicles suggests a divergence in the post-Golgi part suggest that SNARE complexes acting at specific sages ofvesiculr tansport of the secretory pathway. (We thank F. IGebl for providing antibody to Bgl2p.) serve as the ultimate targets of regulaton by members of the Sec4Yptl/Rab of GTPases. family lNovick, P.,C. Field and R. Sdhekman. 1980. Cell 21:205-215. 2Walworth, N. and P. Novick. 1987. J. Cell Biol. 105:163-174. 3Mr1a, V., F. Klebl and W. Tanner. 1993. J. Bacteriol. 175:2102-2106. 4Protpopov, V., B. Govindan, P. Novick and J. Gerst 1993. Cell 74:855-861.

2581 2582 SECRETION OF PROTEOGLYCAN AND SECRETOGRANIN FROM IN VITRO FORMATION OF RHODOPSIN-BEARING POST-GOLGI THE TRANS GOLGI IN PC12 CELLS. ((M.A. Lynch and R.B. Kelly)) VESICLES. ((D. Deretic, B. Puleo-Scheppke and C. Velez)) Department Department of Biochemistry and Biophysics, University of California, San of Patholgy, UTHSCSA, San Antonio, TX 78284-7750. Francisco, CA 94143-0534. We have characterized rhodopsin-beanrng post-Golgi vesides formed in in isolated retinas We are of secretion from the trans in vivo frog (Deretic and Papermaster, 1991, J. Cell Biol. studying pathways Golgi apparatus 113:1281-1293). In order to themolecular mechanisms PC12 cells, a rat adrenal cellline, which has both study underlying pheochromocytoma polarized sorting of rhodopsin we have developed an in vitroassay that constitutive and regulated pathways of secretion. In this cell line, both reconsiues dopein-bearing post-Golgi vesile formation. Isolated frog heparan sulfate which is secreted the constitutive proteoglycan (hsPG), by retinas are radiolabeled for 1 hour and after removal of the rod outer pathway, and secretoganin [I (SGII), which is secreted by the regulated segments, retinas are homogenized in 0.25 M sucrose. After low speed pathway via dense core secretory granules, aresulfated in late Golgi centrifugation, supematants are incubated in the presence of an ATP- compartments. 35SO4-hsPGand 35S04-SGII thus can be used as markers regenerating or ATP-depleting system for 2 hoursat 200C or on ice. In to asses the of for the constitutive and regulated secretory pathways, from the order role cytosolic proteins in this process, retinal respectively, are a Golgi apparatus to the cell surface. To monitor the of homogenates centrifuged through cushion of 0.5 M sucrose and kinetically transport membranes that enter the cushion are from soluble hsPG and SGII from the trans cells are labeled with separated proteins. Golgi, PC12 pulse Post-Golgi vesicles formed in vivocontain -30% of the newly synthesized radioactive sulfate and chased for varying times in the absence or presence rhodopsin after the 2 hour chase. A similar amount of rhodopsin is found in of compounds which may affect vesicular budding, utansport, and/or fusion. the post-Golgi vesicles formed in vitro in the presence of cytosoland ATP, Using this strategy, we characterized the effects of treating cells with NaN3 while ATP depletion arrests rhodopsin in the Golgi. Cytosolremoval as and As were NaF. expected, intracellular ATPlevels reduced and well as 1 ILM GTPyS reduces the amount of vesicle formation in vitroby unstimulated secretion of sulfate-labeled hsPGand sulfate-labeled SGIIinto -50%, consistent with an existing pool of membrane-bound cytosolic the culture medium was inhibited. Consistent with this inhibition of proteins and GTP-activated G-proteins at the onset of incubation. When secretion, smallvesicles containing sulfate-labeled hsPG accumulated retinal homogenates are incubated with ATP on ice, >40% of newly intracellularly and maturation of sulfate-labeled SGII-containing secretory synthesized rhodopsin is found in newly formed post-Golgi vesicles, while granules was inhibited. Supported by ACS grant #PF-3698 and NIH grant ATP-depletion completely blocks this process. Thiscold enhancement is DK33937. mimicked by the addiffon of 50 tiM nocodazole in the retinal media suggesting that microtubule removal favors post-Golgi vesicle formation. Supportedin part by EY 6891. 444a Golgi to Cell Surface Transport (2583-2587). Wednesday

2583 2584 DOSE-DEPENDENT EFFECT OF CHLOROQUINE ON SECRE- SYNERGISTIC INHIBITION OF THE TRANSPORT OF HERPES TORY AND ENDOCYTOTIC TRAFFIC IN THE NELANOTROPH SIMPLEX VIRUS-1 (HSV -1) GLYCOPROTEIN gD BY ((N. Back, S. Soinila)) Department of Anatomy, INTERFERON AND Institute of Biomedicine, FIN-00014 University CHLOROQUINE. ((A.K.Singh, G.S.Sidbu & of Helsinki, Finland. (Spon. by I. Virtanen.) R.K.Maheshwari.)) Department of Pathology, Uniformed Services Unriersity ofthe Health Sciences,Bethesda, MD 20814. (Spons. by G.S. Sidbu) Chloroquine (chl)(200 AM) causes a shift from regulated to constitutive secretion in the AtT- We have previously shown that mouse interferon a/0 (100 LU/ml) 20 cell. Lower doses (40 AM) which still effectively abolish intracellular pH-gradients inhibit the transport of gD protein from TGN to the plasma membrane in do not affect processing of POMC in AtT-20 mouse LMtk- cells which contain the gene for the gD protein and cells or melanotrophs. We studied the morpho- constitutively express gD protein. In this study, we investigated the effect of logical effect of chl on the melanotroph. 40 AM low doses of chloroquine or interferon on the transport of gD.LMtk cells chl for 2 h caused occasional dilatation of were treated with chloroquine (lOtM) or IFN (10 I.U/ml) alone or with both secretory condensations in the TGN. Ferritin IFN and chloroquine, and the transport of gD was examined by indirect was internalized into rounded endosomes and delivered to the TGN. 200 AM chl for 2 h caused immunofluorescence using monoclonal antibodies against gD and FITC extensive dilatation of the TGN with inhibition labeled immunoglobulins.Data shows that gD was accumulated intracellularly of secretory granule formation. Endocytosis was in the cells treated with IFN and chloroquine together whereas chloroquine arrested in multivesicular bodies containing or EFN alone does not block the transport ofthe gD. DAMP distribution was 30-50 nm tubules. The cytoplasm vas vacuolized deternined by fluorescence microscopy using an anti DNP antibody. Low and myeloid bodies characteristic of chl tox- doses of chloroquine or IFN could not inhibit the distribution of DAM, icity frequent. The study shows only minor effects of perturbation of pH gradients by chl however, DAMP distribution in the cytoplasmic vacuoles was inhibited in cells while higher doses with a TGN effect are treated with both interferon and chloroquine, suggesting that pH nay be associated with toxic changes and arrest of responsible for the block in the transport ofgD protein. endocytotic membrane traffic.

2585 2586 MECHANISM OF RETINOIC ACID AUGMENTED APICAL SIALOSYL BASOLATERAL SORTING OF EPIDERMAL GROWrH FACTOR RECEPTORS IN LEWIS' SECREION IN COLORECTAL CARCINOMA CULTURES. ((T. MDCK CELLS REQUIRES SEQUENCES LOCATED IN THE REGULATORY Beringerl, C. Icard-Liepkalns2, D. Eboue3, A. Kulksis4, and VA Liepkalns3)) 5Cell CARBOXY TERMINUS. ((C.S.Lazar, R. Wu. and G.N. Gil)). Department of Biology and Biophysics, University of Missouri-Kansas City, 21.ab Genetique Medidne, University of Califomia San Diego, La Jolla, CA 92093. Molecular Neurotransmission, Gif-sur-Yvette, France, 3Department of Biochenmistry and CNRS, University of Paris, Orsay, France, 4Banting and Best Department of Epidermal growth factor receptor (EGFR) expression Is resrictod to the basolateral Medical Research, University of Toronto. (BL) surface of polrized epithelial cells. To Investigate strctural features of EGFR reoquied for specific sorling to the BL surface we expressed mutnt human EGFR in Intracellular oriins of 3jiM retinoic acid (RA) augmented secretion of sialosyl polarzed MDCK cells and determined surface disbutIon using human receptor- Lewis (19-9) molecular species (SiaLeams) bycuturedSW1116 colorectal carcinoma specific monoclonal antibodies. Tyrosine kinase activw was not required for BL sorting of EGFR but sequences located in the regulatory C temhinus were essential. cells were studied. RA treated cells showed large increases in sialosyl Lewise lvels Removal of the C terminal regultory domain of sa in a light membrane subfraction plus stimulated incorporation of (3H)palmitate into 228 abolIshed preferential to the EL surface but fusion of a of the dbsl C aa membrane subfractions and SiaLeams. Intracellular lipid radioactivity bound to solid targWng fragment terminus, 1024.1186, to the tyrosine kdnase core at aa 958 was sufficient to restore dominant phase 19-9 antibody behaved chromatographically like ganglioside or NH2OH-labile BL sorting. This fragment also restored EL taeting to a mutant EGFR (c888) acyl groups; (3H) bound to SiaLeams from incubation media behaved like base-labile iacing the tyrosine kinase domain. This C terminal fragment of EGFR transferred fatty acyl groups or free fatty acid. Secretion of lipid radioactivity after 3 hrs was efficient endocytic function to EGFR truncated after (c958) or before (c68) the almost exclusively apicaL Electron microscopic immunocytochemistry revealed high kinase domain. Neither EGFR endocytic code I nor exon 16 of the insulin receptor localization ofmonoclonal antibodyagaist sialosylLewis(19-9) to apical membrane. that contains 2 endoytlc codes wer effective in restoration of EL targWting of Oligosaccharides from SiaLeams of membrane subfractions and apical media truncated EGFR. We conciude that sequencoes required for efficient EL targeting presented much higher (3H)fucose incorporation in controls than RA samples. are located in the C terminus of EGFR where endocytic codes reside but not all Northern blots showed dramatic decline in mRNA encoding fucosyl transferase endocytic motifs are effecive In movement from tho trans Golgi network to the (FTIII) but unaltered sialyl transferase (ST4) mRNA. We conclude sialosyl Lewis basolateral surface. Exposure of sequences is important but, In contrast to the induction involves lower expression of Lewis fucosyl transferase mRNA and less process of endocytosls, neither ligand binding nor kinase activity are required for BL subterminal fucosylation of G1cNAc permitting prior sialylation of terminal Gal targeting. moieties of oligosaccharide prior to Golgi export. Fatty acylation of SiaLeams may be associated with altered glycosylation and direction to apical surface. Lipid analysis is consistent with ganglioside chaperonage of SiaLeams to apical surface where N- fatty acylated gangliosides remain integrated in the bilayer but oxyester or thioester bonds may be cleaved to release Sialeams to apical medium.

2587 HIERARCHY OF SIGNALS FOR APICAL AND BASOLATERAL SORTING IN MDCK CELLS. ((R. Cescato, K Fiedler, J. L. Brown, and M. Spiess)) Department of Biochemistry, Biozentrum of the University of Basel, CH-4056 Basel, Switzerland. (Spon. by M. Horst)

We studied the intraceuklar sorting of two chimeric proteins, ANI and TRN, In which the cytoplasmic domain of aminopeptidase N (ApN) was replaced by that of ether subunit HI of the asialoglycoprotein (ASGP) receptor or the transferrin receptor (TtR), respectively. ApN Is a resident apical plasma membrane protein with the apical sorting information blcaized to its exoplasmic domain. TtR and HI are basolateral membrane proteins that are endocytosed and recycled constitutively. The signals for endocytosis and basolateral transport are contained within their cytoplasmic domains. While TtR and ApN function as dimers, subunit HI forms at least trimers. All three proteins, are shine-spanning type 11 membrane proteins. MDCK cel lines expressing AN1 or TRN were created and the polarized delivery to the surface was determined. While TRN was delivered to the basolateral surface, ANI was found predominantly on the apical surface. Moreover, biochemical and immunofltorescence studies using Fab fragments qualitatively showed that both chimneric proteins are endocytosed. The cytoplasmic domains of HI and TfR are thus sufficient to mediate endocos. However, only the cytoplasmic domain of TIR but not that of HI is sfflicient for polarized sorting of the chimeric protein to the basolateral surface. This suggests that basolateral sorting signals may function differently. Aitematively the basolateral sorting information of HI is functional only in higher oiigomers andnotin dimers. Wednesday. Exocytosis: Regulated Secretion (2588-2593) 445a

2588 2589 VISUALIZATION OF SECRETION-RELATED STRUCTURES AT THE PROTEIN PROCESSING AND MORPHOGENESIS OF CELL SURFACE USING SCANNING FORCE MICROSCOPY (A. Spudich SECRETORY GRANULES IN PARAMECIUM ((L. Madeddu, M.-C. and D.P. Braunstein) Dept of Biochemistry, Stanford Univ. School of Gautier and L. Sperling)) Centre de Gedntique Mol6culaire, Centre Medicine, Stanford, CA 94305 National de la Recherche Scientifique, 91198 Gif-sur-Yvene Cedex, FRANCE Secretion is one of the critical events of the signal transduction cascade in cells. the surface SUMMARY The ciliated protozoan Paramecium provides a model many eukaryotic Cross-linking IgE receptors initiates system for the study of regulated secretion, featuring architecturally secretion in RBL cells with concomitant spreading of the cell body (1). complex secretory storage granules - trichocysts - docked at the plasma Scanning Force Microscopy (SFM) has proved to be a unique tool for membrane ready to respond to an exocytotic stimulus. The trichocysts are studying cell surface structures, subcellular organelles and surface characterized by crystalline contents that confer upon the organelle a dynamics of living cells at high resolution (1,3,4). Using SFM to image cell defined shape which can be altered by single gene mutation. The surfaces under different stimulation conditions that either permit or inhibit crystalline trichocyst contents is built up from a heterogeneous set of small acidic polypeptides generated by proteolytic maturation of a family secretion, we demonstrate the presence at the cell surface of structures of precursor molecules, suggesting an important role for protein -1.5 am in diameter that relate to secretion spatially and temporally. The processing in this system. We have recently shown [Il that the primary size and position of these surface pits suggest that they are related to the defect in several secretory mutants lacking functional trichocysts is in dense core granules positioned along the cytoskeletal filaments in intracellular trafficking rather than protein processing. However, analysis detergent extracted, unactivated RBL cell processes. The position of these of how these defects lead to altered trichocyst shape supports the notion structures SFM is also coincident with the location of that the protein processing is essential for morphogenesis. Preliminary imaged by secretory results of a cloning project reveal that an extensive multigene family granule membranes at the surface of stimulated RBL cells detected by an (-100 genes) codes for the trichocyst matrix proteins. Deduced amino acid antibody previously shown to stain granule membranes (5). Topographic sequences of putative processing sites indicate that (at least) two distinct SFM images of the cell surface at 2, 5, and 35 min after activation show processing reactions are probably involved in the maturation of these that the structures persist and show changes in the cross-sectional profiles proteins, and allow us to speculate that each reaction may control a key with time. The possibility that these structures may relate to the event of trichocyst biogenesis. membrane retrieval mechanism of cells after intense stimulation, as proposed by others (6), is being explored. We thank JA. Spudich and C.F. Quate for their support. 1) D.P. Braunstein and A. Spudich, 1994 Biophy. 1.,":1717. 2) J.R. Pfeiffer, et at., 1985 J. Cell Biol 101 2145. 3) L Chang, et at., 1993 Biophys. J. 64 1282. 4) E.Henderson, et at., 1992 Science 257 1944. 5) J. Bonifascino, et at., 1986 J. Cell Biol. 102:516. 6) P. Thomas, et at., 1994 J. Cell. Biol. 124:667.

2590 2591 POSSIBLE ASSOCIATION OF PKC WITH COATED VESICLES INADH TARGETING OF PROTEIN KINASE C-c TO PRESYNAPTIC CYTOSKELETAL STIMULATED TOAD URINARY BLADDERS. (('A.J.Mia,2L.X.Oakford PROTEINS DURING THE PHORBOL ESTER-DEPENDENT ENHANCEMENT and 2T.Yorio)) Jarvis Christian College and 2The University ofNorth Texas OF GLUTAMATE EXOCYTOSIS. ((R. Prekeris, M.W. Mayhew, and D.M. Health Science Center at Fort Worth, Fort Worth, Texas 76107. Terrian)) Dept. Cell Biology, East Carolina Univ. School of Medicine Greenville, NC 27858. There is increasing evidence to indicate that protein kinase C (PKC) may participate in vasopressin (ADH)-induced water transport across toad uinary The protein kinase C (PKC) activator, 46-phorbol dibutyrate (PDBu), bladder epithelia. Mezerein (MZ), an activator of protein kinase C (PKC), increases the availability of releasable vesicles and potentiates the Ca2+- may induce exo- and endocytosis very similar to that seen following ADH dependent release of glutamate. This presynaptic action of PDBu appears challenge. Immunogold localization ofPKC subtypes alpha, beta and gamma to involve a novel mechanism that is not sensitive to inhibitors of PKC- were reported in toad urinary bladders in concert with ADH-induced osmotic dependent catalytic acitivity, but is completely blocked by cytochalasin D. water flow. PKC subtypes were found to be distributed singularly and in In the present study, we have identified the isoforms of PKC which may be discrete isolated clusters in the cytosol as well as on the membrane domain, responsible for mediating the PDBu-dependent enhancement of glutamate Western blot revealed that suggesting a possible link with the outward transport of water channels exocytosis. analyses PKC-a, e, 6, and C, but (aggrephores). Recently, using the techniques of ultratin section and freeze not PKC-8, y, or rj are present in the isolated nerve endings we have fractures replicas, we observed the presence of coated vesicles in the employed in these studies; the hippocampal mossy fiber boutons. cytoplasm of ADH-stimulated toad urinary bladder tissues. These coated Synaptophysin and neuromodulin were also found to be localized in these terminals. The results of our translocation vesicles occurred in a circular array surrounding a central vacuole. presynaptic assays demonstrated that PDBu redistributed in the Immunogold localization of PKC, subtype gamma, and protein A gold, selectively PKC-e, presence of 10 mM EGTA, into a cytochalasin D-sensitive component of the Triton- revealed that a similar distribution patten ofgold particles in circular arrays insoluble fraction of the mossy fiber terminals. This binding of PKC-e to was seen associated with the coated vesicles. These observations suggest that cytoskeletal elements was abolished when particulate proteins were treated PKC either a role in the translocation of coated vesicles or may play may with trypsin prior to Triton extraction, but was not phosphatidylserine- itself be translocted to the membrane on coated vesicles apical during dependent and, once established, was not reversed by the removal of US DAMDI7-19-C-1096 and DK46550. exocytosis. Supported by Army Rl5 PDBu. Thus, the availability of synaptic vesicles may be influenced by the formation of stable, kinase-independent, PKC-e interactions with cytoskeletal proteins.

2592 2593 DYNAMICS OF CORTICAL GRANULE FUSION AND THEIR RELATIONSHIP TWO DISTINCT PATHWAYS FOR STIMULUS-SECRETION TO THE CALCIUM WAVE FOLLOWING FERTILIZATION OF SEA URCHIN EGGS. ((J.C. Matese and D.R. McClay)) Developmental, Cell and Molecular COUPLING COEXIST IN BOVINE CHROMAFFIN CELLS. ((E.P. Biology, Duke University, Durham, NC 27708. Seward, N.L Chernevskaya, KL.Engisch, and M.C. Nowycky)) Dept Anatomny and Neurobiology, Med. Coll. Pa., Philadelphia, PA 19129. Fertilization is followed by an increase of free intracellular calcium, exocytosis of the cortical granules, and elevation of the fertilization envelope. Both cortical Ca2+-secretion coupling in bovine chromaffin cells was studied with patch granule exocytosis and the increase in free calcium move across the egg in a clamp asurentsof in cellmemrane as an wave initiated at the point of sperm entry. It is known that an increase in free changes capacitance (Cm) indicator of calcium can directly promote cortical granule fusion in vitro and in vivo. We exocytosis. Exocytosis was stimulated with trainsof depolarizing decided to closely investigate these two events to learn more about their pulses. In standard solutions with extracellularCa2+, most Cm responses are relationship in vioo. Using video enhanced DIC microscopy, populations of coupled to periods of active Ca2+influx and proceed at peak rates of -1000 cortical granules undergoing fertilization-induced fusion were observed in living fF/s. Ba2+substitutes weakly for Ca2+in this stimulus-coupled pathway. urchin eggs. Movies were digitized and employing a background subtraction Howevcr, the bulk of total Ba2+-induced Cm increases occurs via a kinetically method, we were able to visually isolate fusing granules. Exocytosis events were distnct pathway.These Cm changes are delayed, and proceed as slow (-35 analyzed and it was found that granule fusions within any one region are rises that the distributed over a window of time. Individual cortical granules were observed fF/s), prolonged, outlast initiating influx by 10-50 s. In perforated to fuse to the plasma membrane in a stochastic manner, which as a population, patch recordings, Ba2+induced Cm incases are kinetically similar, but last could be described as a broad wave. The pattern of release along the wave front approxiimately twice as long,reflecting the slower removal of Ba2+when it is appears to be random, and dearly the release is not a simultaneous explosive not free todiffuse into the pipette. Similarslow inceases of Cm (5-35 fF/s) are of all vesicles as the wave This fusion wave front is exocytosis passes by. dosely observed when cells are continuously pefused intracellulariy with 1mM Ba2+ followed by a peak fusion rate 2.4 ± 15 sec later and then by a long tapering rate in whole-cell configuatio Thus, the stimulus-decoupled pathway appears to lasting over 30 sec. The fusion peak is propagated along the egg at a velocity of respond to of rather than to 3.7 ± 1.5 pm/sec. Simultaneous visualization of cortical granule fusion and free triggering levels Ba2+, the elevated sub-membrane calcium levels was also done in an attempt to resolve the relationship between Caz+levels required for stimulus-coupled secretion. Finally, the response is not cortical granule fusion dynamics and calcium dynamics. We found that the affected by the removal of MgATPfrom the pipette solution. It is therefore cortical granule fusion wave front does not coincide with the wave front of unlikely that the SNARE/NSF/SNAP complex that requies MAgTP for increase, delayed until the fiee calcium level plateaus maximally function is involved in the slow, stimulus-decoupled pathway. within region The calcium and corticalgranule dynamic wave fronts are staggered by sec, have comparable velocities of propagation. 446a Exocytosis: Regulated Secretion (2594-2599). Wednesday

2594 2595 CALCIUM ENTRY THROUGH THE NICOTINIC RECEPTOR OF REGULATED MUCIN SECRETION FROM SPOC-1 CELLS, A CELL-LINE FROM BOVINE CHROMAFFIN CELLS TRIGGERS EXOCYTOSIS. ((P. THE AIRWAYS. ((L.H. Abdullah, E.R. Lazarowski, and C.W. Davis)) Moilard, E.P. Seward, and MeC Nowycky)) Dept. Anat & Neurobio., Med. Departments of Physiology and Medicine, University of North Carolina, Coll. Pa., Philadelphia, PA 19129. Chapel Hill, NC 27599. The SPOC-1 cell [a spontaneous cell line derived from rat tracheal Neuronal nicotinic receptors are wealdy pemnant to Ca2+. Despite the small epithella cells that differentiates Into a goblet cell phenotype when seeded flux, we find that nicotinic stimulation in the absence of activation ofvoltage- in denuded tracheas and implanted into immune-defcient animals) in culture. Perfused of cells gated Ca2+ channels elicits exocytosis in chronmffin cells. Single cells were secretes mucin when grown cultures SPOC-1 on Transwell-Col supports, exhibit a baseline mucin secretion rate of 4.0 patch clamped and held at -60 or -90 mV. Nicotine caused an inward cunent 0.4 ng-min-'; this rate is stimulated by extracellular ATP (1 04 M) to a peak that was and in a in (Inic) blocked by d-tubocurarine resulted jump plasma of 21.5 ± 2.6 ng-min' (n-9) over a period of 30 to 40 min. Dose-effect membane capacitance (C,), presumably reflecting exocytosis. Average Cm studies showed ATP, UTP, and ATP-y-S to have nearly identical efficacies, jump amplitude was propoonal to the integral of Inic in a saturable relation- with apparent affinities in the range of 6 pM, whereas 2-methylthio-ATP ship. Cmjumps were blocked by 10mM BAPTA in the pipette, but unaffected had no effect. These results suggest a P2U purinergic receptor. RT-PCR of by doubling intracellular Na+ indicating that Ca2+, not Na+, elevation was SPOC-1 cell RNA, using probes against conserved regions of the P2U gene, yielded a strong band of the predicted size and nucleotide sequence. The critical. We combined Cm measurements with recordings of fluorescent Ca2+ SPOC-1 P2U receptor appears to activate phospholipase C, since purinergic or entry a dyes (fura-red fluo-3). Both Inic and voltage-gated Ca2+ evoked challenge stimulated 'H-inositol phosphate production by 720 to 920 %, rapid increase in cytosolic Ca2+ that peaked in 1-2 sec and decayed over 10-20 and increased Ca,' (fura-2 fluorescence) from SO to a peak of 350 nM. sec. Pretreatment with the Ca2+-ATPase inhibitor, thapsigargin, (1 gKM, 30 Mucin secretion was stimulated by both ionomycin (apparent K, S5 pM and 7.5 elicited a peak response 80.7-fold over baseline) and phorbol min; TG) strongly diminished only the -evoked A Cm, implicating the par- ILM Inic myristic acid (PMA, apparent K,,- 70 nM, and 300 nM yielded a peak ticipation of intracellular Ca2+ stores. Surprisingly, the Ca+ signal in re- response 52.9-fold over baseline). Finally, these two agents used in sponse to Inic was unaffected by TG. Thus, Ca2+ increases contributed by TG- combination had approximately twice their Individual effects (1 58-fold sensitive stores must be local and not detected by spatially averaged Ca2+. In increase over baseline). Thus, P75-purinergic activation of SPOC-1 cells conclusion, the secretory response to activation of mcotinic receptors is appears to stimulate mucin secretion through parallel regulatory pathways kinases and kinase C. determined both by the Ca2+ermeabiliy of the receptors and the degree to involving Ca--dependent protein Supported by grants from the NIH, Cystic Fibrosis Foundation, and Glaxo. which they are coupled to Ca2+ release from internal stores.

2596 259' CLONING AND SEQUENCING OF PARAFUSIN, A CALCIUM- CROSS-UWNKING OF EXTRACELLULAR THYROGLOBUUN FROM HUMAN DEPENDENT PHOSPHOGLYCOPROTEIN IOLVED IN THYROIDS ALLOWS STORAGE AT HIGH CONCENTRATIONS AND IN EXOCYTOSIS ((S.V. Subramanian, E Wyroba, AP. Andersen and OSMOTICALLY INERT FORM. ((U. Bmrdorfer and V. Herzog)) Institute for Cell Satir)) Department of Anatomy and Strucural Biology, Albert Bio, UnIersity of Bonn, D-53121 Bonn, Germany. Einstein College of Medicine, Bronz, NY 10461. The secretory pathway of thyroglobulin (TG) involves thre major steps: 1. synthes of TG and export, 2. storaoe In the follle lumen and 3. A cDNA for parahfsin, an evolutionarily conserved phoshoy otein ondocytosis and proteoltc rele of thyroid hormones. Storage of TG Is an Important step involved in exocytosis, has been cloned and sequenced from a unicelllar because It serves the maintenance of constant evbls of thyroid hormones in tho tetnuwvia. A Panrecum CDNA was eukaryote, Pa_mecum hray cIrcultin. We hae recently obseved that storaog of TG In a highly condensed screened uing an oligonucleotide probe sthesized to an internal amino forn alow isolton and lago scale preparation of intact luminal contents (J. Cell isolated e was 3kb in with an acid sequence of parafusin imsert length 8iol. 118:1071-1083, 1992). Human thyroid tssue was colleted from surgical open reading frame of 1.75 kb. Although the deduced amino acid specImen and mined wlth razor blades Into small fragmnts. Low sped sequence had 50.7% identity to rabbit muscle pho ase, ce_rlfuo and repead washing rested In pelets of large spherical translucent Southern bybridization analysis suggests that pr sin and gobuies (dameer 100400 pxm) which consisted of TG and reprsented In form and phoshoglucoiutase P ec are products of different genes. In surfac charapierlatcs a precIse replica of the corresponding follile lunen. Isolated addition, isolated parafin has no detectable hosqhoglucomutase thyroid glbueswer insoluble in PBS but comptdely dissociable by treatment with activity. Separation of these two proteins was taned usglingid dith eloIt (DTT) and sodium dodecylsufate (SDS) Indcatng Vt TG in human chromatography, enzymatic activity assay and globules Is covntly cros-linked by dlulde iges. DTT appled wthout SDS with the skec4icparafin peptide, tibody. P u fracons rutedI nsw nofglobusto a 3-5-fod volume whereas dsocation into singl incorporated V5'-StJUDP-Glc but not [3JS]Glc-F-P whereas Pawnecim moecules began only after the addIion of SDS indicating th the fomiation of lcomutase fractions incorporated [3JSGc-1-P but not disule bridges reduces the osmoaly of extraellular TG and that additinal fores, presumably hydrphobic Interacons, play an impotant role during TG The deduced van amino acid sequence had four raoss-linkdng. We conudet Intemocuar cross-lInking of extracelular TG is an inserts and two deletions whic t confer on the protein unique important means to allow storag of TG at high concentrations (400-00 mglml or functions in signal transduction events related to ezoctois Furthermore, searches for potential phosphorytion sites showed the more) without osmotic selling of the luminal contet. In addition, storg of TG In a covalently croslinked form requires mechanisms which enable th cells to, e. g. of a protein Csite specific to PCR presence kdnase (KDFSFR) pra proteicall, dissocIate stora-TG befo endocytosis. (Supported by analysis revealed that there are no introns m the ene. We Gradulertenkolleg Funktolle Proteindomnnen' and by Deutsche propose that pamfusin and phosphoglucomutase, though in Forschunggemneinschaft SFB 284) cuntion, are members of a superfamily that conserve homologies important for the terdary structure of the molecules.

2598 25" IDENTIFICATION OF A COMMON AMPHIPATHIC LOOP MO- DETERMANTS OF THE SUBCELLULAR TARGETING OF THE SYNAPTIC TIF IN TEE N-TERMINAL REGION OF POMC AND PRO- VESICLE PROTEIN SYNAPTOBREVIN STUDED IN CHINSE HAMSTER ENKEPHALIN AS A SORTING SIGNAL FOR THE REGULATED OVARY CE IS AND CULTURED HIPPOCAMPAL NEURONS. ((A. E. West C. C. Overly, and K M. Buckley)) Dq nen_t of N , Hvd Medical SECRETORY PATHWAY. ((A.M. Bamberger, D.R. Cool, C.R. Snell SchooL Boson, MA 02115. and Y.P. Loh)) Section on Cellular,Neurobiology, Lab. Dev. Neurobiol, NIH/NICHD, Bethesda, MD 20892 and Sandoz Institute for Medical Research, The prin s evn sored by nroes andndocre cUs io synaptc London, England. vesicles, whe it is pcposed to be a key plyer in the exocytoic fuso machiny. We bhe boe studying the tgetig patway to diis spcalize d rge in two Pro-neuropeptikes are targeted to the regulated secretory pathway in neuroen- _sysas Ez_dssion in ie non-n n cel l e CHO has preiuy boee shown to rveal dienc in dhe soig patwas of snptc potins In docrine cells. We have previously provided evidence that sorting of the pro- ve*le tbae cells synate is twrted to an intracellular orgnelle which may be an neuropeptide pro-opiomelanocortin to the regulated secretory pathway requires endosome,a ase ed by the similarity between the poa of apt evin a signal encoded by a 13 amino acid amphipathic loop which is formed by a in Oreacee ad tha of endeoma makers sc an the rnsferin receptor disulfide bridge (CysSCys20) at the N-terminal of the molecule Another pro- or expresed hysin. other protei tgetd to this pahway, aomatc amino bee to neuropeptide, pro-enkephalin, also contains disulfide bridges in the N-terminal acids bave show be key demet of taget sequence. Howee, mutaon of te tyrosie ad pbenylaline residu in the cytpsmic region and is sorted to the regulated pathway. Immmunocytochemical and stim- domain of evin failed to resl in a redisWbon of te proftin to tde ulated studies were cells swretion performed using Neuro-2a transfected with surface an ayed by immusofluorescece. We plan to assess moe subtle eDNA encoding a chimeric protein consisting of the N-terminal 31 amino acids differeces in in i lkics betw te mua uig bmhelly epiope of pro-ensephalin fused in frame with CAT reporter protein The results sug- tged preinm to e_Ame tmene of endocytosiL Our scond experimental sysem emply cultues of disocatd rat E19 nuron Owce plad, gest that this region is suffict to cause sorting of the chimeric protein to the hibocana neeo ude a dharctristc aeepe of polaity coiniden with the secretory case regulated pathway. In the of pro-enkephalin, sorting information soring of a as Iaber of prti iow either ndI t-c or axona domains. may also be contained in the C-terminal part of the molecule Molecular mod- Like other synaptic vesicle protei, sy"aptrevia becomes progrsively eling of the N-terminal regions of both POMC and pro-enphalin suggest that cncen ed iDthe axon n poaty develop. To evalse prtein sequences a common conformational motif, the amphipathic loop stabilized by disulfide for this even co amansfectedae int the neurons wih cationic Upid compounds, and the expresed protein is localized by bridge, may act as the sorting signal for targeting both these molecules to the imusocmyche y. n ofcellubrvin, the b ge of regulated secretory pathway. ynaptrevin, has reaed tht tis protein is not axonaly sregaed, impicadg regswhich betwen two conseved moicules in more specifictbe sorting pathway of synaptobrevin. Wednesday. Exocytosis: Regulated Secretion (2600-2605) 447a

2600 2601 INHIBITION OF SECRETION FROM ISOLATED ALVEOLAR IIDENTIFCATION OF RAB3D IN RAT BASOPHILIC LEUKEMIA CELLS ((N. Kapp, EPITHELIAL TYPE II CELLS BY CALPAIN SPECIFIC INHIBITOR. S. Bar. and B.S. Wilsm)) DepL of Padtoqgy and C Reseach and Teatment Cee ((U-J.P. Zimmerman and M. Wang)) Institute For Environmental Medicine, Univerity of New Mexico Bealth Sciens Cener. Albuqurque, NM 87131 University of Pennsylvania School of Medicine, Philadelphia, PA 19104. We have used PCR-based method to scr for rab3 RNA expsson in rat bophilic leikemia (RBL.2E3) ceols. Our PCR prmers wer based upon highly ccsaee regios of Stimulation of isolated type II pneumocyte with various secretogogues results homoly betwemn rab3 protins. (Sense 5'- CAG AA(CO TI(Cr) GAC TA(TC) ATG TMC in a rise in intracellular calcium level and enhanced secretion. We examined AA - 3'; Antisn: 5'(GA)TC CAC (CGA)AG (AG)CG (TC)TC (AG)AA-3'] Tbhse prs whether or not the endogenous calcium-dependent neutral protease (calpain) tart highly conserved stresic acids that are both common to all kmown members of the rab3 group of proeins & D) and muique in compison to cloely is involved in the secretory process of type II cells using a cell permeable (rab3A.B.C otds mised rabs. Paenfth indicte the lvel of degeracy to accmodate codon use for the inhibitor specific for calpain II. Secretion was monitored by the release of identcl nino acid sequence amwog the known rab3 genes mRNA was prepaed from RBL- [3H]phosphatidylcholine from type II cells cultured in medium containing 2H3 cells, revased tansibed to cDNA md prmer extenions were prfamed by polymerse [3H]choline. After 18 h of culture, cells were placed in fresh medium and chainreactio (PCR). cDNA from rat bain mRNA or a plasmid ontaining complet cDNA preincubated with calpain inhibitor for 45-60 min. After a 3 h incubation, the squence for rab3A (provided by Dr. Yoshimi Taki ofKobe Univeity) were used as a poitivXe secretory activity of the inhibited cells was compared with those of the cotrol order to detmine cyln ctditions. The rsultig PCR product rpenti 70% untreated cells and the cells stimulated with agonists for f-adrenoreceptor (10 of the full length rab3, was squenced using de di-deoy drain trminatio mehod. analysed using fte GCG pdcage, and found to be 100% identical to rat rabl6 (Eferink. et aL 1992) and pM terbutaline), purinoceptor (1 mM ATP) or calcium ionophore (1 pM 95% identical to mo rab3D. Upon close inspecti, the sequnce epoted for rabl6 A23187). The basal level of secretion was defined as the secretory activity of appred to have an unusually stort ai tminu sequen upstm of the first cosved the untreated cells after 3 h incubation. Terbutaline, ATP and A23187 Anine.encleotide bing motif. We hypoteszed that rabl6 was indeed the rat equivalent of augumented the basal secretion by 1.5, 2.5 and 4 fold, respectively. In mose rab3D md that the cDNA reportd by Elfink et aL (1992) lacked contrast, cells treated with calpain inhibitor had activities depressed 40-60% approimatly 63 nucotids inciding the rue iniiang methionine codo We ested this below the basal level (n=3). Stimulation with A23187 of the calpain inhibited by nmer speofic to mose rab3D d carbocty termi. which en the hyperariale rgions of the rab3 proteis. m the PCR amplification. We cells did not lead to augumented secretion. Rather, the activity remained at obtained full-length rab3D PCR pnrodut in this raction. The PCR oduct was inserted into or below basal level on the depending inhibitor concentrations. These results a Bhescript sequeingvector ad full length equce dotained. We ae currently investgating indicate that calpain has a specific role in the process of constitutive secretion the potnti role for rab3D in mast coil seceay responses using RBL-2H3 cells as a model. of alveolar type II cells. (ROI HL 49982)

2602 2603 RECOMBINANT RAB3B COLOCALIZES W1TH NATIVE RAB3A AND AN EP1THELIAL PROTEIN THAT BINDS SPECIFICALLY TO THE PROMOTES Ca++-DEPENDENT CATECHOLAMINE RELEASE IN HYPERVARIABLE C-TERMINAL DOMAIN OF RAB3B. ((S.C. Francis DIFFERENTIATED PC12 CELLS (( E. Weber, T. Jilling, KIL Kirk )) and K.L. Kirk)) Department of Physiology and Biophysics, University of Department of Physiology and Biophysics, University of Alabama at Alabama at Birmingham, Birmingham, AL 35294. Binningham, Binningham, AL 35294. (Spon. by T. Jilling) Rab3B associates with epithelial tight junctions where it likely regulates Rabs 3A and 3B are highly homologous monomeric GTP-ases that are apical and/or junctional protein traffic. We identified a rab3B-specific expressed in neuroendocrine cells and in epithelial tissues, respectively. Each binding protein that may interact with this monomeric GTPase in the of these GTPases targets to specialized intercellular juntons in its respectave regulation of polarized membrane traffic in epithelial tissues. Metabolically tissues; i.e. rab3A to presynaptic nerve terminals, where it likely regulates labeled HT29 epithelial cell lysates were incubated with recombinant synaptic vesicle recruitment and/or dockdng, and rab3B to epithelial tight glutathione-S-transferase (GST)-rab fusion proteins in the absence of added junctions, where it may regulate apical and/orjunctional protein traffic. We nucleotides. A 30KD protein (p30) was obseed to bind to GST-rab3B but have tested the hypothesis that these tissue-specific isoforms possess similar not to GST alone. Maximal binding occurred within 60 minutes at 40C and targeting and functional properties by stably expressing recombinant rab3B at submicromolar concentrations of rab3B. Binding assays performed on in PC12 neuroendocrine cells; cells which normally express rab3A but not high speed membrane pellets and supenatnts (ie. cytosol) revealed that p30 rab3B. Each isoform targeted to regions of cell contact rich in is predominantly cytosolic. The binding of p30 appears to be specific for norepinephrine-containing, large dense core vesicles (LDCV); namely, to the rab3 isoforms; namely, p30 also binds with high affinity to rab3A but not to lateral cell margins between undifferentiated PC12 cells and to the neurite rab5. p30 also binds to fusion prtein containing only the hypervariable C- tips in PC12 cells that were induced to polarize by treatment with nerve terminal domain of rab3B, i.e., that region of rab3B that confers targeting growth factor (NGF). Immunoblot analysis of PC12 membranes fractionated specificity. The rab3-specific binding of p30 and its interaction with the by Ficoll gradient centrifugation revealed that recombinant rab3B and native hypervariable C-terninal domain of rab3B implicate this protein as a rab3A were each predominandy associated with a dense membrane fraction candidate regulator and/or downsream effectorofrab3B function in epithelial that was enriched in secretogranin II, a marker of LDCVs. In addition, tissues, perhaps facilitating the specific membrane targeting of this following NGF treatment rab3B- overexpressing PC12 cells exhibited a 3-4 monomeric GTPase. fold greater capacity for ionomnycin-induced norepinephrine release than mock- or untransfected cells. Our results support two conclusions: (i) rabs 3A and 3B are functionally related monomeric GTPases with similar targeting properties; (ii) rab3B is capable of regulating a spadally segregated secretory pathway in polaized neuroendocine cells.

2604 2605 IDENTIFICATION OF A RAB3D-LIKE ANTIGEN IN RAT PANCREATIC RAB1 IS AN APICALLY LOCATED SMALL GTP-BINDING PROTEIN ACINAR CELLS. ((E.M. Konieczko, D. Sengupta, L.H. Tang, F.D. Gumskowski, IN DISCRETE UPPER GASTROINTESTINAL EPITHELIAL CELLS. ((P. M.M. Carrasquillo, A.C. Lee, R. Fan and J.D. Jamieson)) Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510. Cameron, J. Smith, H.D. Vaughan and J.RGoldenring)) Institute for Molecular Medicine and Genetics, Medical College of Georgia and the We previously reported the presence of at least seven low Mr GTP-binding Augusta VA Medical Center, Augusta, GA 30912. proteins associated with pancreatic acinar cell zymogen granule membranes (ZGM) (Padfield and Jamieson, 1991, BBRC, 174:600-605). Recendy, we demonstrated that a rab3-like protein is associated with ZGM and recycles back to Small GTP-binding proteins have been implicated in the regulation of many the Golgi following stimulation of regulated secretion (Jena et al., 1994, JBC, aspects ofvesicular trfficking. Rabl 1 is a component of secretory granules in We now the presence of a rab3D-like in rat 124:43-53). report antigen pancreatic PC12 cells as well as cell tubulovesicles. We have a acinar cells. Rab3D was first isolated from a rat adipocyte cDNA library (Baldini gastric parietal generated et al., 1992, PNAS, USA, 89:5049-5052). We have carried out confocal murine monoclonal antibody against recombinant rabbit rabl 1 which is microscopy, electron microscopy, and 1D and 2D Westem blot analysis using a specific for rabI 1, and does not detect its closest relative, rab25. The rabbit polyclonal antibody directed against the N-terminus of rab3D (Baldini et distribution ofrabI 1 in rabbit tissues was evaluated indirect al., 1992). One-dimensional Westem blots revealed a band of -27kD in rat using pancreatic ZGM, similar to that found with the mouse monoclonal rab3A/B immunofluorescence. In the esophagus, rabl1 was present in the highest levels antibody. However, 2D Westem blot analysis using the rab3D antibody revealed in cells located in the middle layer of the squamous epithelium. In the gastric -4 with ZGM in the exocrine at least two proteins of pI associated pancreas, while was observed in cells in a the rab3A/B antibody recognized proteins of slightly different molecular weight mucosa, prominent labelling parietal punctate pattem and pI. Confocal microscopy of unstimulated pancreatic lobules incubated with consistent with a localization on tubulovesicles. In both surface mucous cells the rab3D antibody revealed an intense apical granular stain. Stimulation of and chief cells, a punctate subapical staining was observed. In mucus cells of pancreatic lobules with IIM carbachol led to the loss of apical staining and a the antrum, punctate subapical staining was also noted. Pancreatic acinar cells relocation of the rab3D-like ismmunoreactivity to a perinuclear region, as revealed by confocal microscopy. The apical localization of the rab3D-like antigen in demonstrated a subapical staining patten similar to that seen in chief cells. resting pancreas was confirmed by electron micrographs of homogenized Apical staining ofpancreatic ductal epithelium was also observed. pancratic lobules incubated with the rab3D antibody. Membranes of zymogen Hepatocytes showed punctate staining deep to their bile canalicular surfaces. granules were heavily inuninodecorated with the rab3D antibody. We suggest that the rab3D-like protein may be involved in regulated exocytosis or No staining was observed along sinusoidal membranes. The results suggest comnpensatory membrane retrieval in the pancreatic acinar cell. (Supported by that rabl 1 is present as an apical vesicular component in discrete populations USPHS Grants DK17389 & CA46128) ofepithelial cells. 448a Exocytosis: Regulated Secretion (2606-2608). Wednesday

2606 2607 CARBOXYL METHYLATION OF LOW MOLECULAR WEIGHT (LMW) GTP-BINDING ASSOCIATION OF MSS4 WITH A SUBSET OF RAB GTPASES IN MAMMALIAN PROTEINS IS NOT REQUIRED FOR GTPyS-INDUCED PEPSINOGEN SECRETION CELLS ((J.L. Burton and P. De Camilli)) Dept of Cell Biology and Howard FROM GASTRIC CHIEF CELLS. ((R.D. Raffaniello and J-P. Raufman)) Hughes Medical Institute, Yale University School of Medicine, New Gastrointestinal Cell Biology Laboratory, Department of Medicine, SUNY- Haven, CT 06510. Health Science Center at Brooklyn, NY 11203-2098. Mss4 is a mammalian protein that was originally identified as a suppressor of a yeast secretory mutant harboring a point mutation in A role for LMW GTP-binding proteins in regulated secretion has been the GTPase Sec4. Mss4 was shown to promote GDP-release from Sec4, suggested by studies in yeast and mammalian secretory cells. In dispersed Rab3a, and Yptl but not yeast Ras2, suggesting that Mss4 is an chief cells from guinea pig stomach, GTPyS-induced pepsinogen secretion is accessory protein for the Rab GTPases (Burton et al., 1993. Nature 36.L: independent of changes in cellular calcium or cAMP, or activation of protein 464). In this study, we have performed a biochemical characterization of the Mss4 and kinase C. Several LMW are protein have demonstrated its association with the GTP-binding proteins (20-30 kD) localized Rab GTPases in mammalian cells. Antibodies against the Mss4 protein predominantly to chief cell membranes. Because studies in other cells have reveal that it has a widespread tissue distribution and migrates at indicated that carboxyl methylation of LMW GTP-binding proteins may l7kDa in SDS-PAGE. Mss4 is primarily soluble but is also detected in a regulate association of these proteins with cell membranes, the objective of particulate fraction. In tissue extracts, Mss4-linked beads bind to the present study was to investigate whether this post-translational GTPases and by a gel overlay assay we show that this interaction is modification is required for the actions of GTPyS in chief cells. Pre- highly specific for Rab proteins. Using recombinant GTPases, we further define this interaction to a subset of the Rabs, which permeabilization (20 min) of chief cells with streptolysin 0, to allow the include Rabl, Rab3a, Rab8 and RablO, Sec4 and Yptl. Strikingly, all of these egress of cytosolic constituents, did not alter GTPyS-induced pepsinogen Rabs belong to the same subfamily branch. Furthermore, Mss4 secretion. Preincubation (20 min) of cells with up to 100 pM of two potent stimulates GDP-release from, and the association of GTPyS with, only inhibitors of carboxyl methylation [N-acetyl-S-famesyl-L-cysteine (AFC) this subset of Rabs. Mss4 is more efficient at stimulating GDP- than and N-acetyl-S-geranylgeranyl-L-cysteine] did not alter GTPyS-induced GTP-release from Rab3a and was able to bind both prenylated and pepsinogen secretion. Moreover, AFC (100 F&M) did not alter [3H-methyl] non-prenylated forms of this protein. Immunoprecipitation from a methionine labeling of chief cell proteins. These results indicate that, in Triton X-100 soluble brain extract demonstrates that Mss4 co- precipitates with Rab3a and not Rab5, in agreement with the dispersed chief cells from guinea pig stomach: (1) GTPyS stimulates specificity of the Mss4-GTPase interaction revealed in both the gel pepsinogen secretion by interacting with a membrane-bound component(s); overlay and GDP-release assays. These results suggest that Mss4 and (2) carboxyl methylation of LMW GTP-binding proteins is not required behaves as an accessory protein for a subset of Rabs to influence for GTPyS-induced pepsinogen secretion. distinct vesicular transport steps along the secretory pathway.

2608 RAB3D, A SMALL GTP BINDING PROTEIN IMPLICATED IN REGULATED SECRETION, IS EXPRESSED IN RAT HEPATOCYTES. ((.M. Larkin, BJ. Oswald, VJ. Balan, N.F. LaRusso, and M.A. McNiven)) GI Basic Research Center, Mayo Clinic, Rochester, MN 55905. Rab proteins, members of the ras superfamily of GTP binding proteins, are small GTPases which regulate vesicle-mediated transport in several cell types. Various rab3 isotypes have been implicated in both regulated secretion and polarized membrane targeting. Since hepatocytes traffic both membrane and proteins in a polarized fashion, we hypothesized that a rab3 isotype is expressed in hepatocytes and is involved in the regulation of polarized membrane targeting and secretion. Messenger RNA repared from isolated rat hepatocytes was screened by RT-PCR ussng Specific primers for rab3D. A 151 base pair fragment was generated that corresponds to amino acids 24-74 of rab3D protein. By nucleotide sequence analysis, the 151 base pair fragment was 100% identical to the published sequence of rat rab3D (Oberhauser et al. FEBS Lett. 339:171-174, 1994). GTP overlay and immunoblot analyses were performed using a polyclonal antibody to a synthetic peptide repesenting the N-terminal hypervariable region of murine rab3D. This antibody recognized a -25kD GTP binding protein in poladzed hepatocytes (in short-term culture and in intact liver) but not in clone 9 cels, a hepatocyte-derived cell line. A survey of hepatic subcellular fractions showed that rab3D was predominantly localized to the cytosol and was enriched in a l.15M crude vesicle carrier membrane fraction reported to contain transcytotic vesicular carriers involved in apical membrane trafficking. These molecular and biochemical data demonstrate that polarized rat hepatocytes express rab3D which may play a role in the regulation of apical membrane targeting and secetion in the hepatocyte. Cell-Cell AdhesionJunctions (2609-2610).

2609 2610 IDENTIICATION OF A NEW CATENIN: THE TYROSINE KINASE SUBSTRATE CAT1NIN BINDING TO THE E-CADHERIN CYTOPLASMIC DOMAIN IN P120 (CAS) ASSOCIATES WITH E-CADHERIN COMLEES. ((A.B. Renolds, VITRO. (a. A. Marrs, D. B. Stnwrt sad W. J. Nelos)). Department of J. Daniel, P. McCrea,+ MJ. Wheelock,# J. Wu,* Z Thang*)) Dept of Molecular Cellar Phydlogy, Staford Umlversky, St_mfrd, CA 9435. Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN 5426. (Spoi. by A. Bari) 38105. of and Molecular of +Dept Biocheistry Biology, University Texas, Ca_dheies are dependentce adhesion molcule which geamt ceil-uo.c Houston, TX 77030-4095. #Dept. of Biology, University of Toledo, Toledo adhesion. Cadhin f is ao depeaden on cytopsq mic domain, in OH, 43606. pscul, poriaor ofdie cyqbunic domain which binds cysplmc p_ti ndy-cae ha-eas sequo ece ident w b vinc Beta- dt pl20Cas (Cas; Cadherin-assoclated Src-substate) is a tyosinekinase catnin s_ae. sequence identity sd fwmonal similaries with armado gt substat previously ilicated in ligand induced receptor signaling through the jsnthnCt from DraGophI&(ST fua I n -eein-eXp_ eggiM EqdhaftCyuoplSMkq domain sequec been enginoW_ddexpemd in E. We do da,t-. EGF, PDGF and CSF-1 receptors, and in cell transformation by Src. Analysis 0- ad y-caxim in deegn exua sval bind tothed of the cDNA sequence of Cas revealed sequece simlity to the cadhin qeicsllAy cadberin cyapkami domain within fuso poteins. In ad4di we find dti binding protins, and suggesting that Cas have a P-catenin plakoglobin, might anoth arwslo family membs, da arc kinse I p12, is bound to the role in cell adhesion. Using specific antibodies to Cas, E-cadherin, oa-catenin, E-cadhmin cytoplasmic domain vitro. We we in-rtd in stdying cadhi- I-camin, and y-tni (plakoglobin), we have found that Cas is tighdy cas bi ces in oelr exacts In con_at to MDCK ad HCTI16 cells, in associated with protein complexes containng E-cadherin. FrthecI, E- which canins ae net availab for binding exogeno_ cadherin, we foud tat SW480 cell have a pool of caim amwno amocited with ad cadherin and its associated cateins precisely cololized with Cas in vivo in cadbin, both nomrml and Src-tansfornied cells. Cas binds direty to E-cadhein and hactioam wit quperent molecuw weight expeced fbran (ap)- or (aqcamin complx by gd fiuaton. Wealso fosd in SW480 cels hdt dwe is*arg anoun the binding inotaton ismediaed by therepated amm lo modfs. These of a-. P- ad y-camnin avaiabl for binding da E-caiu cytplasmic domain OST results indicate that Cas is a new catenin and strongly suggests a catenin-le fusion prein. Simnlr amounts of p120 fom SW480 ad HCT116 exa we The role for Cas in cell adhesion. tyrosine phosphorylation of Cas and avaiabl for binin do E-cabuein cyoplmic domain Ibis ind_ic thatpl20 is catenin in Src-transfonmed cells has been reported priously. We show here n additio compoem of da cadhri complx ad h s a disint disrbu that plakoglobin is also yrsine phosphorylated in Src-transfomied MDCK betwee cadhein-bound and unboud pools from dat of a-. p- and .ctmi cells. In addition, we have identifed fourmajor isofomns of Cas which were Analys of diffaetcell provide sa expensecal sysm for ivestgatig differentially expressed in different cell types. All of the isoforms interacted thegubae aembly in Wiro. with cadberin complkees suggesting that cels might regulate cadhin function by controlling the isofomsu of Cas that are expressed in cells. Wednesday. Cell-Cell AdhesionJunctions (2611-2616) 449a

2611 2612 E-CADHERIN AND THE TUMOR SUPRESSOR APC COMPETE FOR DELINEATING THE BINDING SEQUENCE OF E-CADHERIN, THE INTERACTION WITH -CATENIN AND THE CYTOSKELETON. a-CATENIN, AND 3-CATENIN USING THE YEAST 2 HYBRID ((J. Huelsken, J.Behrens, and W. Birchmeier)) Max-Delbrueck-Center for SYSTEM. (( T-S Jou, J.A. Marrs, and W.J. Nelson)) Department of Molecular Medicine, Robert-Roessle-Strasse 10, 13122 Berlin, Germany. Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305. -Catenin is a key regulator of junction formation in epithelia, it interacts Cadherin-mediated cell-cell adhesion plays critical roles in cell-cell with the cell adhesion molecule E-cadherin and the tumor suppressor gene recognition, morphogenctic development, and the generation of polarized product APC, and its Drosophila homolog armadillo transduces distribution of proteins. The function of this physiologically important morphogenetic signals. We have reconstituted both the cell adhesion and the junctional complex depends upon the interaction of an integral membrane APC complexes by ectopic expression of recombinant proteins in Neuro2A protein-E-cadherin and its cytosolic countrparts: a-catenin and p-catenin. cells and could demonstrate that E-cadherin and APC directly compete for Perturbation of any of these protein interactions abolishes the normal binding to 0-catenin. By use of deletion mutants of -cateninwe identified function of the cadherin complex. These three proteins are known to be similar structures for the two E-cadherin or APC associated with one another because they are coimmuno-precipitated in a strikingly complexes: (i) complex using antibodies for any one of them. However, the sequential bind to a loop structure built up by the internal, armadillo-like repeats of order of protein-protein interaction that generates the cadherin-catenin 0-catenin, and(ii) the N-terminal domain of i-catenin links the two complex has not been clearly delineated. We have used the yeast 2-hybrid alternative complexes to the cytoskeleton associated a-catenin. Plakoglobin assay, and the activation of two reporter genes ( HIS3 and LacZ), to (y-catenin), which is structurally related to 3-catenin, mediates identical investigate the order of protein-protein interactions in this complex. We interactions and can replace -catenin in both complexes. This new model demonstrated that a-catenin only associates with E-cadherin through the of the molecular interactions within the adherence junctions and the APC bridging of p-catenin, and P-catenin directly interacts with E-cadherin in complex suggests that 3-catenin/plakoglobin is a central integrator of cell the absence of a-catenin. We also mapped a binding domain at the N- adhesion, cytoskeleton and tumor suppression. terminal domain of r-catenin for P-catenin. This study shows that the yeast 2-hybrid system can be used to rapidly screen interactions between known proteins. We demonstrated that assembly of the cadherin/catenin complex involves a sequential order of protein binding between E- cadherin and 0-catenin, and then a-catenin binding to 0-catenin.

2613 2614 ARMADILLO REPEAT DOMAIN OF -CATENIN INDUCE SECONDARY AXIS IN EVIDENCE OF DISTINCT REQUIREMENb FOR CADHERIN- XENOPUS EMBRYO. ((N. Funayama, P. McCrea', B. M. Gumbiner)) Cellular MEDIATED CEI AGGREGATION VERSUS JUNCTION ASSEMBLY, Biochemistry and Biophysics program Memorial Sloan-Kettering Cancer AND OF CADHERIN-INDEPENDENT EPrrHEIAL MORPHOLOGY. Center, New York, NY10021. 'Department of Biochemistry and Molecular (( H. Chopra and S.W. Craig)) Dept. of Biological Chemistry, The Johns biology, UTMD Anderson Cancer Center, Houston, Texas 77030-4095. Hopkins University School of Medicine, Baldmore, MD. 21205. 0-catenin associates with the cytoplasmic domain of cadherin cell adhiesion Interaction of cadherins on neighboring cells is involved in assembly of molecules and with the tumor suppressor protein APC. It is the homologue intrellular junctions and in establishment and maintenance of epithelial-like of the Drosophila segment polarity gene, armadillo, which acts downstream morphology. To test for a role of E-cadherin in rcruitment of vinculin, we of wingless, a member of the wnt growth factor family. In previous study, transfected the cadherin-negative L929 fibroblast cell line with cDNA coding injection of the affinity purified FAB fragments of anti-p-catenin for murine E-cadherin. Clones expressing levels of E-cadherin similar to antibodies cause axis duplication in Xenopus embryo. These data implicated MDCK cells exhibit epithelial (clone SA22) or non-epithelial (clone T10.6) 0-catenin has a role in dorsoventral pattem formation in Xenopus, which is morphology. Clones that do not express E-cadherin also exhibit epithelial also known to be affected by wnt expression. To study the function of 0- (SA44-) or non-epithelial (clone 2B8) morphology. The expressed E- catenin in induction of secondary axis, we injected mRNAs encoding full cadherin mediates Ca2+-dependent cell aggregation and upregulates and length f-catenin or deletion mutant --catenin. Overexpression of full length associates with a- and p-catenins and plakoglobin. TEM shows that E- P-catenin in the ventral side of cleaving embryos induces a secondary cadherin does not mediate assembly of gap junctions, adherens junctions, dorsoventral axis. Furthermore, the intemal repeat domain of 0-catenin, desmosomes, or tight junctions and does not affect formation of the non- (armadillo repeat domain) alone can induce axis duplication in Xenopus. The classical intercellular contact that does exist between the cells. Both SA22 repeat region is also responsible for the interaction of P-catenin with and SA44- lines display poor cell communication in dye transfer assays and cadherins and with APC, but not with a-catenin, which interacts instead express similar levels of connexin 43. The presence of E-cadherin does not with the N-terminus of --catenin. Armadillo repeats are found in a number affect localization of F-actin and vinculin in epithelioid clones. These results of other proteins. Therefore, we propose that the armadillo repeat is a new separate the requirements for E-cadherin-mediated cell aggregation from protein motif for protein-protein interactions involved in intracellular those for junction formation and organization of actin and vinculin. In signalling. addition, they provide evidence for a cadherin-independent pathway for generation of epithelial-like cell shape, provide an initial characterization of the non-classical intercellular junction of L929 cells, and document a regulatory effect of E-cadherin expression on levels of a- and pcatenins and plakoglobin in L929 cells.

2615 2616 TYROSINE PHOSPHORYLATION REGULATES CELL-CELL MAST CELL GRANULES CAUSE CONFLUENT ENDOTHELIAL AND CELL-MATRIX ADHESIONS IN RAS-TRANSFORMED CELLS TO LOSE CADHERIN 5(C5) FROM THEIR JUNCTIONS HUMAN BREAST EPITHELIAL CELLS ((M.S. Kinch and K. AND RECOVER MOTILITY ((D.Lagunoff, A. Rickard, Z.M. Goeckeler and Burridge)) Deparanent of Cell Biology and Anatomy, University of North R.B. Wysolmerski))Dept of Pathology, St. Louis University, St. Louis, MO Carolina at Chapel Hill, Chapel Hill, NC 27705. (Spon. by L Romer.) 63104. Transfomed epithelial cells exhibit altered cell-cell and cell-extracellular matrix interactions. Our laboratory has identified defects in the We have previously described the formation of holes In endothelial cell mono- layers when confluent cultures are exposed to mast cell granules. Heparin pro- cytoskeletal organization of human breast epithelial cells tansformed with tooglycan is the responsible granul constituent. Extended time-lapse video Onogenic vanants of ras. Ras trniormation is chauacterized by a loss of phase mIcoscopy has been used to examine the formation of the holes. When adherens-type junctions between cells in favor of the formation of focal bovine pulmonary artery endothelbal cells are seeded at subconfluent densities, adhesions at sites of cell-subsate binding. The loss of cell-cell junctions the cells are motile. Over a period of 3-8 days as the cells approach and achieve reflects decreased interactions between the cadherin/catenin complex and confluence, call motility largely but not entirely cases. The loss of motility of the actin cytoskeleton. The transformed cells display an altered cells within a confluent monolayer is associated with the formation of complete organization of actin filaments, with stress fibers and focal adhesions circumferential adherent junctions as defined by immunostaining with antibody to becoming prominent. Elevated tyrosine phosphorylation regulates this cadherin 5(C5). Between 2 and 8 hrs after the addition of granules, the cells in transition of the actin cytoskeleton as treatment with protein tyrosine confluent monolayers begin to pull apart focally and revert to a motile state. Con- kinase inhibitors restores adhrens-type junctions in the ras-tansformed trary to the impression derived from fixed, stained preparations, the holes that MCF1O epithelial cells. Conversely, phosphatase inhibitors induce focal appear In a monolayer are not stable but are evanescent, forming and then fill- adhesion formation in the normal epithelial cells. Tyrosine ing, while new holes form as a result of the combination of cell separation and phosphorylation of p-catenin correlates with the decreased cell-cell cell motility. The separaton of calls end the reappearance of motility following junctions of ras-transformed cells. We have also identified tyrosine exposure to granules correlates temporally with the progressive loss of lmmu- phosphorylated focal adhesion proteins which appear to promote focal nostaining of cell margins with antibody to cadherin S(CS). By 18 hrs after gran- adhesion assembly. These data suggest extensive interplay between ule addition, there is virtually no stainable cadherin remaining at the call margins. signal uansduction pathways involving protein tyrosine kinases and ras. Based on our previous results and those reported here, we propose that heparin This work supported by grants from NIH and an ACS post-dctoral proteoglycan binds to sites on endothellal cells and induces disassembly of researchfellowship (MSK). cadherin-dependent adherent junctions resulting In the loss of cell cohesion and the return of call motiliy thereby producing fenestratos in a previously conflu- ent monolayer. 450a Cell-Cell AdhesionJunctions (2617). Wednesday

2617 CADHERINS AND CATENINS IN BRAIN CAPILLARY ENDOTHELIAL CELLS. ((T. Hashimoto, K. Herrenknecht and L. L. Rubin)) Eisai London Research Laboratories Ltd, University College London, London WC1 E 6BT, UK. Tight junctions between endothelial cells in brain capillaries are distinguished by having extremely high electrical resistance, when compared to those present between endothelial cells in peripheral capillaries. To understand the molecular basis for this difference, we have begun to characterize junctional proteins in the two types of endothelial cells. Adherens junction proteins are known to be important for tight junction formation, therefore we have begun by studying endothelial cell cadherins and catenins. We have used specific peptide-directed anti-catenin antibodies to immunoprecipitate endothelial catenins and their associated cadherins. Both types of endothelial cells have 3 catenins, presumably corresponding to a, p and y. In brain endothelial cells, the major associated cadherin has an apparent molecular weight similar to that of epithelial cell E-cadherin. The presence of an E- cadherin in brain endothelial cells was unequivocally established by PCR using cadherin subtype-specific primers and by the cloning of full length E-cadherin from a cDNA library derived from these cells. However, peripheral endothelial cells also express an E-cadherin (in addition to 2 or 3 other cadherins). Further studies will be required to determine if differences in the cadherin/catenin complexes account for the differences in tight junction permeability in the two cell types. Membranes: Other (2618-2621). 2618 2619 FILIPIN VERSUS ENZYMATIC LOCALIZATION OF CHOLESTEROL MEMBRANE COMPOSITION OF THE COLD ADAPTABLE FOOD BORNE IN TESTICULAR CELLS ((R.-M. Pelletier and M. L. Vitale)) Dep. PATHOGEN USTERIA MONOCYTOGENES ((.K Mastronicolisl2, J. B. German2 and Anatomy and Neurobiology, Univ.Ottawa Ontario, Canada KIH 8M5 G. M. Smith2)). 'Dept. of Chenustry, School of Nat Sci, Univ. of Athens. GREECE 2Dept of Food Sd. and Tech, UC, Davis, CA 95616 G. M. (RMP) and Instituto de Neurobiologia, Serrano 665, 1414 Buenos Aires, (Sponsored by Smith). Listeriosis is a public health concern because of sporadic outbreaks associated with Argentina (MLV) ingestion of contaminated food such as salted foods, soft cheeses, vegetables and meat. L. monocytogenes (LM) is a Gram-positive pathogen that causes septicemia, meningitis and death in susceptible people such as pregnant women, the very old or very young To test the validity of filipin cytochemistry for localization of cholesterol, and those with AIDS. LM grows well at refrigerator temperatures and survives in we compared the results obtained using this technique with results obtained osmotically stressful environments. It does not compete well in mixed cultures and is using a two-step enzymatic method involving cholesterol esterase and susceptible to bacteriolysins. Therefore, food handling and storage practices (high osmolarity and low temperature) that suppress competitors promote its growth. A cholesterol oxidase. In all animal models tested, i.e., guinea pig, mink and mechanism of osmotic adaptation found in bacteris involves intracellular accumulation mallard duck, the disappearance of subsurface filaments along Sertoli cell of "compatible solutes' (e.g., glycine betaine, GB) to counterbalance osmotic pressure. junctional membranes was accompanied by a significant increase in the Bacterial adaptation to cold is poorly understood, but may involve modification of number of filipin-cholesterol complexes per Asm2 of membrane. With the membrane lipid composition (homeoviscous adaptation). In bacteria in which enzyme enzyme histochemistry, free cholesterol was stability sets the lower limit of growth, cryotolerance could be conferred by the intra- localized in the membranes cellular accumulation of protein stabiling compatible solutes, some of which are also surrounding multivesicular bodies, in membranes included in lysosomes, in known to stabilize membranes, suggesting a relationship between psychrotrophy and mitochondrial membranes, and injunctional membranes accompanied or not osmotic tolerance. This is supported by our observation that GB transport is stimulated by subsurface filaments. The enzymatic technique also allowed selective by either high salt or by cold. We have analyzed the polar lipid fatty acids (FA) of LM, visualization of cholesterol esters in lipid grown at 300 C. Total lipids were extracted by the Fokch method which yielded 7±1 mg droplets. It is concluded that 10 total lipids/ml wet cesl. The polar lipids were separated from neutral lipids by solid Filipin mapping of cholesterol induces false-negative results. 20 The phase extraction and fractionated by 2D TLC into 12 components. FA methyl esters enzymatic method is superior to filipin cytochemistry in that it allows (ME) were prepared from the bulk polar lipids and from each of the 12 components. localization of free cholesterol in junctional membranes and permits The FA ME were analyzed by GC on a polar column and by GCMS on both polar and non-polar columns. FA of the polar lipids were primarily branched, saturated chains. visualization of cholesterol esters in lipid droplets. Funded by MRC Grant There was a striking lack of cis unsaturated FA, especially in certain polar lipid dasses. MT-11160 to RMP. The preponderance of branched chain FA in lipids of LM may indicate a functional advantage to branched chain structures in cold tolerance.

2620 2621 HYPERGLYCEMIA INDUCED LOSS OF MEMBRANE PHOS- CHARACTERIZATION AND CLONING OF TIHE RAT LIVR CYTOSOIUC O-GleNAc PHOLIPID ASYMMETRY IS MEDIATED BY INCREASED PAS- TRANSFERASE ((M. A. Blomberg, L K. Kreppel and G.W. Hart.)) Department of SIVE LIPID FLIP-FLOP. ((M.J. Wilson, T. Jose, V. Patel and Biochemistry and Molecular Genetics, University of Alabama at Bimingham, AL D.L. 35294-005 Ph 205-934-7120 Daleke)) Department of Chemistry, Indiana University, Bloomington, IN 47405. Fac 205-934-0758 Glycosylation of nudear and cytosolic proteins by a single N-acetylglucosamine Hyperglycemia induces a concentration- and time-dependent loss of transmem- 0-glycosidically linked to Ser/Thr residues (O-GlcNAc) occurs in all eukaryotes brane phospholipid asymmetry in normal erythrocyte membranes. Nondiabetic examined to date. This post-translational modification is highly dynamic and often human erythrocytes exposed to hyperglycemic buffers for extended occurs at sites of proline-directed protein kinases. On some proteins O-GlcNAc periods appears to have a reciprocal relationship with protein phosphorylation. While on of time have increased phosphatidylserine (PS) and phosphatidylethanolamine other proteins the levels of O-GlcNAc and phosphorylation appear to be regulated (PE) and decreased phosphatidykcholine (PC) and sphingomyelin (SM) in the in parallel. O-GlcNAc may therefore serve as mediator of protein regulation in outer leaflet of the cell membrane as assayed by exposure to exogenously-added conjunction with phosphorylation. We have purified the O-GlcNAc transferase phospholipases. Furthermore, these erythrocytes activate purified factors of (UDP-GlcNAc:polypeptide B-N-acetyl-glucosaminyl transferase) to apparent homogeneity from rat liver. It appears to be a hetero-trimer composed of two the prothrombinase complex, consistent with exposure of PS in the membrane catylitic 110 kDa subumits and one 78kDa (regulatory?) subunit We have generated outer monolayer. Co-treatment of the erythrocytes with a combination of hy- polyclonal antisera against synthetic peptides based on internal peptide sequence, drophilic and lipophilic antiodidants reduces membrane scrambling as assayed as well as polydonal antisera raised against gel purified pl 10. Interestingly, these by both the phospholipase and prothrombinase assays implying that glucose- antisera show high specificity toward both the pllO and the p78 subunits, induced oxidation is involved in the loss suggesting they are related polypeptides. This relationship is confirmed by peptide of asymmetry. Calculations based on mapping Many other regulatory enzymes, such as kinases are regulated, at the a two-state model for endogenous PC distribution predict a 1.6 fold increase in transcriptional and post-translational levels. Possible post-translational the rate of endogenous PC flip-flop to account for the observed loss of asymme- modifications of the transferase include glycosylation as well as Ser/Thr or Tyr try. Flip rates of exogenously added short chain PCs in erythrocytes treated phosphorylation. We have shown that anti-phosphotyrosine mAb will react with with hyperglycemic buffers were measured using a cell assay. The the enzyme by Western analysis and that enzyme activity can be retained on a anti- morphology phosphotyrosine mAb agarose column. We are investigating whether this rate of didecanoylphosphatidylcholine flip increased 6-fold and lyso lauroyl-PC modification is involved with either the activation of enzyme activity or the flip increased 3-fold in hyperglycemic cells. These results confirm that passive association of the subunits. Using the anti plO antibodies and the anti- transbilayer lipid movements are increased in hyperglycemic erythrocytes and phosphotyrosine antibody we are characterizing the biosynthesis of the O-GlcNAc support the hypothesis that increased flip rates account for the observed loss transferase in various tissues as well as the subcelular localization of the enzyme. We are utilizing synthetic oligonucleotides derived from intemal peptide sequence of transmembrane phospholipid asymmetry. as well as the antibodies described above to done the cDNA for the enzyme. Supported by NIH HD13563 Wednesday. Membranes: Other (2622-2625) 451a

2622 2623 ULTRASTRUCTURAL EFFECTS OF A NOVEL ANTI-HIV DRUG N- ISOLATION AND CHARACTERIZATION OF INERMEDIATE STAGES BUTYL-DEOXYNOJIRIMYCIN. ON HL-60 CELLS. ((G. R. Neises, F. IN THE INFECTION AND ASSEMBLY PROCESSES OF VACCINIA M. Platt*, and R. L. Ornberg)) Monsanto Company, St. Louis, MO 63167 VIRUS. ((V. Ley, M. Ericsson, S. Cudmore, J. Krijnse Locker and G. Griffiths)) Cell Biology Programme, European Molecular Biology Laboratory, and *University of Oxford, Oxford, UK. Meyerhofstrasse 1, 69117 Heidelberg, Germany

The imino-sugar, N-butyl-deoxynojirimycin (NB-DNJ) is an inhibitor of Vaccinia virus, the best charactrzed member of the Poxvirus family, is a complex glycoprotein and glycolipid synthesis. NB-DNJ has potent anti- large, complex, DNA-containing virus that is made up of about 100 proteins HIV activity, in vitro. The effect of NB-DNJ on the morphology of and which has two distinct infectious forms that assemble sequentially during cultured HL-60 cells has been examined to assess the structural effects of the infection process. We have recently shown that the first of these forms to assemble, the intracellular matwe viIUs (IMV), is enclosed by at least two NB-DNJ on cells. At in vitro, anti-viral doses, three distinct effects were membranes. Moreover, our data argue that these (outer) two membranes in the thickness and stain observed after 72 hours; an apparent increase originate in the infected cell firom the intermediate compartment between the density of the plasmalemma, a rearrangement and compression of the Golgi -rough ER and the Golgi complex (Sodeik et al, J. Cell Biol. 121: 521-541, apparatus, and a loss of density core material in secretory granules. The 1993). In order to facilitate a further understanding of the assembly of the IMV, time course of these effects at the antiviral concentration was different. The we have used sucrose gradients to isolate intermediates in both the assembly plasma membrane effect appeared after 30 minutes, while the full effect on and dissassembly of the virus. For the assembly intermediates, we took advantage of the ts-16 temperatu-sensitive mutant of vv (Condit et al., the Golgi and secretory granules required at least 8-24 hours. These effects Virology 128:429-435, 1983) (kindly provided by Drs. R. Condit and S. were reversible but with different time courses. After 72 hours of treatment Shuman). At the non-permissive tempeame this mutant is blocked at an followed by NB-DNJ removal, the Golgi apparatus was reverting to a intermediate stage in the assembly, a stage that appears to coincide with the .normal" morphology after 4 8 hours, while secretory granules with dense entry of the newly syntheszed DNA into the assembling virus. For the cores required 8 hours of culture. The plasma membrane effect disappeared isolation of the intermediate form of the dissassembly process we used purified after 24 hours of drug-free culture. A study of the dose response after 72 virus, double labelled with 35S-methionine and 3H-thymidine and allowed the hours essentially paralleled the kinetics and reversibility data. Low doses infection to proceed in the presence of cycloheximide which blocks the synthesis of viral uncoating factors. Consequently, after the neutral pH fusion or and altered the plasma membrane but not the Golgi secretory granules event that occurs duing entry, an intermediate particle accumulates that is high doses accelerated all three structural changes. The relationship of the devoid of the outer membrane of the IMV. We will present our morphological biochemical activity of NB-DNJ with these changes is under investigation. and biochemical results on these two isolated intermediate stages in the context of our model for the assembly of IMV.

2624 2625 EXPRESSION AND PURIFICATION OF ACTIVE RECOMBINANT FORMS OF NADH-COENZYME Q REDUCTASE FROM LIVER PLASMA LACTOFERRIN (Lf) FROM E.COLI. (M.P. Sitaram, B.Maloney, and D.D. McAbee) MEMBRANE: PURIFICATION AND ROLE ON Department of Biological Sciences, Univ. of Notre Dame, Notre Dame, IN 46556. TRANSMEMBRANE ELECTRON TRANSPORT. ((J.M. Villalba, F. Navarro, F. C6rdoba, A. Serrano, A. Arroyo, and P. Navas)) The ability to express and purify recombinant froms of Lf from E.coli presents a Departamento de Biologia Celular, Universidad de C6rdoba, potentially powerful tool for the study ofstnrcture-fimction relationships through site- C6rdoba, Spain. directed mutagenesis. We report here, for the first time, the expression of bovine and mouse Lf in prokaryotes. The bovine Lf clone, pN16b (lacks the first 70 amino acids A requirement for the CoQ in the plasma membrane electron transport is a clue for in from its amino termini) and the rat Lf clone pT267 were a gift from Drs. F. understanding this system and its function cell metabolism. Extraction of Co from plasma membranes Schanbacher and C. Teng, respectively. They were cloned into 17 expression vectors inhibited the transmembrane NADH-ascorbate free radical (AFR) and as gene 10 fusion proteins in Ecoli, BL21 cells. Initial expressed (DE3) reductase. The addition of CoQ to both extracted and non-extracted characterization of the expressed bovine and mouse lactoferrin proteins consisted of membranes stimulated the activity. A 35 kDa protein (p35) immunoblots using rabbit anti-bovine Lfantibody. A prominent immunoreactive band displaying NADH-CoQ reductase was purified from pig liver of lOOK Da was detected, consistentwith expected molecular weight of the gene lI-Lf plasma membrane and catalyzed the reduction of different electron fusion protein (rLf 100). Soluble exracts of E.coli cells with and without acceptors but not AFR. However, the addition of extra amounts of pN16b, following IPTG induction were prepared and assayed for their ability p35 to CoQ-supplemented and -nonsupplemented plasma membranes to compete with native 125I-Lf for binding to high affinity(Kd_20nm) Ca+2- stimulated the NADH-AFR reductase. These activities were inhibited by both WGA and dicumarol. c dependent binding sites on isolated rat hepatocytes. Extracts containing rLf NADH-cytochrome reductase as marker for cis-electron transport on the cytoplasmic l00(vector with pN16b cDNA), but not extracts from cell without rLf side of plasma membrane was insensitive to both extraction or l00(vector alone), showed a 10 fold reduction in specific 125-ILf binding to high addition of CoQ, and was also not affected in p35-supplemented affinity sites on rathepatocytes. These data suggest that the first seventy amino membranes. These results support the involvement of p35 as first acids, are not required for Lf binding to high affinity sites on hepatcytes. step for the transplasma membrane electron transport chain These studies demonstrate that recombinant Lf variants can be produced in E. oxidizing NADH and CoQ as an intermediate electron carrier betweenNADH and AFR. Supported the coli and suggest that it will be possible to use a bacterial system to produce by Spanish DGICYT grant PB92-0714. other structual variants of lactoferrin to map it's various functional domains including it's cell binding domain. Gene Structure (2626-2627)

2626 2627 MOLECULAR CLONING OF THE HUMAN THE NUMBER OF KRINGLE IV AND PROMOTER ACTIVITY ARE CREATINE TRANSPORTER RELATED TO PLASMA LIPOPROTEIN (a) CONCENTRATION. ((J.H. of and ((R. Proujansky, G. Iyer, V. Funanage)) Wu, S.C. Kao, and M.S. Wen)) Department Microbiology of Medicine and and Department of Clinical research Immunology, Chang Gung College Technology and Medical Cardiology Section, Depatment of Internal Medicine, Chang Gung Memorial Cell Biology, A.I.duPont Institute,Wilmington, Hospital, Kwei San, Tao Yuan, 333 Taiwan. DE 19803. Upoprotein (a) [Lp(a)J concentration was higher in patients with coronary heart disease (mean*SD, 233*188 mg/L; n=1l1) and stroke (236*219mg/L; We have isolated a cDNA for a creatine n=29) than in the control group (125a129 mg/L; -ZT277), hypertnsion group transporter from a human intestinal library. The (144*133 mgtL; n=59) and diabetes melitus patients (153*186 mg/L; nucleotide, as well as the amino acid sequence n=146). Western blotting revealed an inverse relationship between Lp(a) concentration and the of show a high degree of homology with both the size apolipoprotein (a) [apo(a)]. Eight samples [with an Lp(a) concentration ranging from 15 to 536 mg/L] with apparently rabbit and a rat creatine transporter choline homozygous alleles as shown by Westen blotting were selected for kringle transporter which has also been shown to IV enumeraton by PCR-cloning-hybridization. The number of kringle IV was transport creatine. Northern analysis detects the inversely reated to Lp(a) concentration. Seven of these samples were screened for promoter of PCR and to of expression of the creatine transporter mRNA in activitiy apo(a) by cloning plasmid reportr luciferase and the promoters were tested in HepG2 cells. Strong human brain, muscle as well as intestine, contrary promotractivi wasobserved more often in samples with high Lp(a) than in to findings in the rabbit. Creatine uptake studies low Lp(a) concenrtions. The results indicated that the higher the Lp(a) in the human colon carcinoma cell line (Caco2), concentration, the lower the number of kringle IV repeats and more often and transfection studies should help identify the associated with high apo(a) prooter activity. Both tanscriptional and post- transcriptional processes are probably involved in Lp(a) concentration role of this metabolite transporter in the human regulatiol intestine. 452a Gene Structure (2628-2633). Wednesday

2628 2629 PARTIAL CHARACTERIZATION OF A PROMOTER REGION FOR CD36. A SEQUENCE ANALYSIS OF THE PROMOTER OF THE HUMAN ((K.T. Taylor, Y. Tang, E. Medved, D.M. Eckert, and R.H. POLYUBIQUITIN GENE UbC. ((M.Nenoil, KMita2, S.lchimura2, Lipsky.)) Cell Biology Department, J.H. Holland Laboratory, M.Yamauchi3, H.Tsuji3, and I.L.Cartwright4)) ITraining School, 2Division of American Red Cross, 15601 Crabbs Branch Way, Rockville, MD Biology, 3Division of Genetics, National Institute of Radiological Sciences, 20854. Chiba-shi 263, Japan. 4Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH We have studied the human CD36 gene organization to understand structure/function relationships. We and others 45267, USA. have determined the genomic structure of the CD36 gene (Taylor genes shows an interesting pattern of regula- et al., 1992, Gene, 133:205-212; Tang et al., 1994, JBC, Polyubiquitin of eukaryotic cells 269:6011-6015; Armesilla and Vega, 1994, JBC, 269:18985- tion. We previously showed that the human polyubiquitin gene UbC is in- 18991). The coding exons and a 3'-untranslated alternative ducible by UV light and a phorbol ester TPA, which has been speculated to be a on the observa- exon of CD36 occur on the long arm of chromosome 7, near the mediated by transcription factor API the basis of following centromere, spanning approximately 30 kb. The 5'-untranslated tions; 1) the induction of the UbC gene is rapid and transient with the maximal region contains at least four exons; two (exons 2a and 2b) induction being achieved in 2.5-3h after UV irradiation or TPA treatment just undergo mutually exclusive alternative splicing. However, as observed in the inductions of APl-regulated genes such as collagenase and transcripts containing either exon 2a or 2b can be found in metallothionein genes, 2) inhibition of a de novo protein synthesis by cyclo- the same cell lines or tissue samples. Exons 2a and 2b are heximide does not repress the induction of the UbC gene. In order to investi- approximately 100 bp apart and 250 bp upstream of the first gate the regulation passway, we determined the nucleotide sequence of the pro- moter of the UbC gene, and found two sites (TGACT- coding exon. 5'-RACE has been used too identify two terminal region APl-recognition CA) at -1884 and -2611 from the transcription initiation site, together with nu- exons. The 5'-terminal exons are at least 6 kb upstream of exon 2a. Recently, the nucleotide sequence of one promoter merous Spl sites and one HSE. On the other hand, when we sequenced the region has been published. We are gathering information on promoter region of the Chinese hamster polyubiquitin gene CHUB2 which is the second promoter region to determine which factors are the evolutional equivalent to the human UbC gene, no AP1 site was found. important in the transcriptional regulation of CD36. As, contrary to the UbC gene, the induction of the CHUB2 gene by UV light or TPA is not detectable, our DNA sequencing analysis supports the specula- tion that the UbC gene is regulated by AP1. The speculation could be still more strengthened by an in vivo footprinting experiment with the LMPCR method, although under way now, which showed a footprint at the AP1 site at -2611.

2630 2631 CENTRIN EXPRESSION IN MAMMALIAN RETINAE. ((U. WolfruMI,2 NUCLETIIDE SEQUENCE OF 16S rDNA OF BACTERIAL ENDO- and J.L. Salisbuy1)) IDept. of Biochistry and Molecular Biology, Mayo Clinic SYMBIONTS IN AMOEBA PROTEUS ((K.J. Kim and K.W. Jeon)) 37996. Fondafmo, Rochester, MN 55905; 2Zoogsches Institut I, Universitit Karisruhe Department of Zoology, University of Tennessee, Knoxville, TN (T.H.), Komblumenstr. 13, D-76128 Karlsnihe, Germany. (Spon. by J.W. Koontz.)

to the phylogenetic position of unidentified X-bacte- Centrin, a 20 kDa cytoskeletal, Ca2+-binding protein is a conserved component of In order determine living inside the strain of A. proteus as obligatory basal apparatuse of ekaryotic celis and vertebrate centome, the major ria xD endosymbionts, we sequenced nucleotides of their 16S rDNA by directly amplifying microtubule anition centers of thes cells. In mamnmaian retinae, cenrin is rDNA of isolated endosymbionts and sequencing. amplified a 1.5-kb the VW loalized at the centaoome of neuronal cells, the basal bodies of photoreceptor DNA segment by asymmetrical PCR by using synthetic primers specific cells (PRC), but also in their ciliary part, the so-called conneting ciliurn. Centrin for eubacteria and sequenced it by the cycle-sequencing method. The was also idenfiod in isolated human retia and purifiod rat photeceptor clLs by nucleotide sequence was the same for 16S-rDNA amplified from purified insnunoprecipitatio or Westem-Blot with eihr poly- or monoclonal anti-centrin chromosomal DNA of X-bacteria and that obtained from a genomic antibodies. Previous reports on centrin squences have disgished two different expression library, that were sequenced by the same method. A compari- human centrin forms, one, also called human caracin in HeLa cells, and a second son with known 16S-rDNA sequences showed that the X-bacteria be- form found in a hunan testis cDNA library (Lee and Huang (1993) Proc Natl Acta longed to the gamma subclass of proteobacteria. The most closely related were aeruginosa, mendocina, 1216: 126-128; Errabolu et al. (1994) J Cell Sci 107: 9-16). We sought to organisms Pseudomonasflavescens, P P detmine whether one of these cenrin isoforms is specific for nro-retinal tissue. and Flavobacterinum lutescens in the pathogenic Pseudomonas genus. The 16S rDNA of X-bacteria had a 94.9 - 97.1% nucleotide-sequence identity Using RACE-PCR techniques we characrized in the rat retina, one centrin cDNA with that of the above bacterial species. It was interesting to note that X- which is ientia to a cDNA we obtained from the rat brain. However, in human bacteria were more closely related to Legionella species or Coxiella we charaiz two diffeet centrin showing about 100%/o retina specific cDNA, burnetti on the bases of their lipopolysaccharide composition and the identity to the sequences dcribed above from different origins. Therefore, two nucleotide sequence of heat-shock gene. The possible relationship bet- diferent centria are expressed in the sanm tissue, indicating that both centrin forms ween X-bacteria and other bacterial species will be discussed. r[Te work may have dfferent cellular fuwctions. Supported by the DFG (Wo 548/1-1) and the was supported by a grant from the National Science Foundation.] NIH (HD 29366).

2632 2633 ALANINE tRNA GENES OF NEPHILA CLAVIPES ((E. Luciano, A. NONSENSE ALLELIC MUTATIONS IN HUMAN TRANSCOBALAMIN Plazaola, E. Vazquez, M. Gonzalez and G.C. Candelas)) Department of II (TCII) DEFICIENCY. ((N.Li1.,D.S.Rosenblatt2 and B. Biology, University of Puerto Rico at Rio Piedras, San Juan, Puerto Rico Seetharamt) .Depts.of Medicine,Medical College of 00931-3360 Wisconsin1 Center,Milwaukee,WI,and McGill University2,Montreal-,Quebec, Canada.) Dissection of the molecular events involved in elicited fibroin synthesis in the large ampullate glands of Neoh*la clavige.e reveals the involvement of Plasma TCII promotes tissue uptake of cobalamin alanine tRNAs in the process. As a response to the stimulus for fibroin (Cbl;Vitamin B1). Human TCII deficiency lead to Cbl synthesis, disproportionate levels of alanine tRNAs prelude fibroin deficiency manifested as megaloblastic anemia and production in the glands. Two alanine tRNAs are generated by the glands neurological deficit.In the most comon form of one of them, of tissue-specific nature whose synthesis correlates with that TCII deficiency, lack of plasma TCII is due to TCII molecules within the of fibroin. In order to be able to study the role of these mRNA deficiency (Li et al.Biochem.J.In press). The of their production, alanine tRNA genes have process and the regulation molecular basis of TCII mRNA deficiency is not been isolated. Using the method of Sanger a sequence of 2.3 Kb of Nephila's genomic DNA has been determined. Its analysis has revealed the known.RT-PCR has been used to amplify, clone, and presence of an alanine tRNA gene cluster composed of four irregularly sequence TCIIcDNA from fibroblasts of two unrelated spaced genes. All the genes display the same orientation. Although they TCII deficient patients. A child of consanguineous display a high degree of similarity in their sequence, differences are marriage contained single nucleotide(nt) deletion observed in both their coding regions and their flanks. The genes' at position 258 in both alleles, while the child computer predicted tRNA structures suggest that the products encoded by from an unrelated parents revealed a nonsense the four genes may be functionally equivalent. Additional searches for mutation at position 1206 in one allele and a alanine tRNA genes are being conducted since it seems unlikely that single nt deletion at position 483 in the other genes under different transcriptional controls be Interspersed within the allele. Both the single nt deletions introduced a gene same cluster. We are currently involved in the characterization of a premature termination codon(nonsense mutation). exhibiting a considerable of divergence from the clustered genes in is 5' hybridization flank. A where differences between the tissue-specific and Allele specific oligonucleotide region confirmed all the mutations. These data suggest constitutive gene are to be expected in Polymerase Ill genes. Computer predicted tRNA structures of all genes isolated to date suggest they that heterogeneous nonsense mutations cause human encode functionally equivalent alanine tRNAs. TCII mRNA deficiency. (Supported by grants NIE,DK- 26638 and VA Merit 7816-OlP) Wednesday. Developmental Control of Gene Expression (2634-2639) 453a

2634 2635 NQ-I, A MODEL SYSTEM FOR TE STUDY OF FARLY SEIECIVE EXPRESSION OF C-JUN, JUN B AND JUN D PROTEINS REGUTlAORS IN MYOGFNESE ((Y.-P. Hwung, F. Rastinejad RELATED TO OSTEOBLAST DEVELOPMENT. ((L.R. McCabe, H. and H.M. Blau)) Department of Molecular Pharmacology, Hoffmann, C. Banerjee, J.L Stein, J.B. Lian and G.S. Stein)) Dept. of Cell Stanford University, Stanford, CA 94305. (Spon. by M.-J. Tsai.) Biol., U. Mass. Med. Ctr., Worcester, MA 01655. Developmental and transgenic animal studies implicate the fos-jun family of The helix-loop-helix (HLH) myogenic proteins are critical for transcription factors in the regulation of bone specific gene expression and myogenesis, however, little is known about how cells become bone tissue formation. To investigate if the representation of JUN protein is modulated during bone development, nuclear extracts were isolated from muscle cell precursors and begin to express these genes. To calvaria-derived rat osteoblasts at different stages of differentiation: address this question by a genetic approach, we have generated a proliferation, extracellular matrix (ECM) maturation, and ECM muscle cell mutant, NQO-1, which was isolated as a non- mineralization. All JUN proteins were at highest levels during the osteoblast differentiating mutant from C2F3 myoblasts mutagenized with proliferation stage. c-JUN levels rapidly declined after proliferation to undetectable levels, while JUN D levels were low but detectable throughout nitroquinoline oxide. The NQO-l cells do not express any of the the time course. JUN B levels declined to less than 40% during osteoblast four myogenic HLH regulators, myoD, myogenin, myf-5 or MRF- ECM ma ao but inreased during the latter part of matrix minealization. 4, as analyzed by Northern. Furthermore, when myoD was Surprisingly, unlike c-JUN and JUN D, JUN B exhibited a dramatic change was in protein migration rate at the onset of matrix mineralization. To access introduced into these cells, myogenin induced, bona fide whether protein levels correspond to DNA binding activity, band shift myotubes formed, and expression of biochemical markers such as analyses were performed with the AP-1 consensus sequence and supershift myosin heavy chain was detected as in wild type muscle cells. antibodies specific for each family member. A parallel relationship was Cell hybrids between NQO-l and C2F3 differentiated like wild observed between the level of each JUN family member and the extent of type cells, indicating that the defect in NQO-l can be DNA binding activity assayed by antibody supershift. This was true for JUN B, despite apparent protein modifications. In summary, JUN family member complemented by factors from C2F3 cells. Taken together, these expression and DNA binding activity are selectively modulated over the findings suggest that the mutation in NQO-l cells is likely to be course of osteoblast development. These temporal differences may be in an early step of myogenesis which controls expression of the involved in regulating osteoblast gene expression and differentiation. HLH regulators, and that genetic complementation of NQO could lead to the characterization of such regulators.

2636 2637 EXPRESSION OF THE TRANSCRIPTIONAL NEGATIVE REGULATOR HXT ENCODES A BASIC HELD(-LOOP-HELIX TRANSCRIPTION FACTOR ID-2 IS MODULATED DURING HUMAN CYTOTROPHOBLAST THAT REGULATES DEVELOPMENT OF PLACENTAL TROPHOBLAST CELLS. DIFFERENTIATION. ((M.J. Janatpour, M.T. McMaster, M.A. Israel and SJ. (g.C. Crossl, M.L. Flanneryl, M.A. Blanar2, and Z. Werbl)) 1 Laboratory of Fisher)) Departnent of Stornatology, University of California, San Francisco, Radiobiology and Environmental Health, and 2 Hormone Research Institute, CA 94143 University of California, San Francisco CA 94143. (Spon. by C. Alexander) Formation of the placenta depends on differentiation of specialized epithelial cells termed trophoblasts. One differentiation pathway, analogous to tumor Trophoblast cells which are the first lineage to form in the mammalian formation, gives rise to a subpopulation of cytotrophoblasts (CM) that invade conceptus, play an essential role in the placenta; defects in their function are the uterus and its blood vessels. We study stage-specific antigen regulation associated with common dlinical problems including early embryonic death and during CTB differentiation along this pathway en viVo by immuno- intra-uterine growth retardation. Despite this, factors that regulate trophoblast histochemistry and in situ hybridization on tissue sections of human placenta. cell commitment and differentiation are unknown. We have found evidence for a We perform fction-pertrbation experiments using a cell culture model in role of basic helix-loop-helix (bHLH) transcription factors in this process, and which highly purified CTB stem cells differentiate along the invasive have cloned a new bHLH transcription factor. This factor was called Hxt because pathway. In both systems CTB invasion is characterized by dramatic changes it is preferentially expressed in e2itra-embryonic cells that make the placenta, m the cells' expression of integin cell-ECM receptors, HLA-G(a trophoblast- induding trophoblast and transiently in mesoderm. A dose relative of Hxt that specific MHC class I molecule) and the 92-kDa type IV coilagenase, was called Hed, shares 85% amino acid identity with Hxt in the bHLH domain molecules that important roles in invasion and tolerance. play immunologic but is the product of a separate gene and is not expressed in the placenta; it is we show that (TB also modulate their of a transcriptional Here, expression expressed at high levels in the deciduum, maternal tissue that surrounds the regulator, Id-2. Members of the Id family are helix-loop-helix (HLH proteins that lack a DNA-binding region. T'hey inhibit positive transcriptional implantation site. Hxt encodes a nuclear antigen that is expressed at higher regulators of the basic-HLH family by dimerizing with them, thus blocking levels in more differentiated trophoblast cells as they begin to produce placental the interaction with a second basic-HLH protein required for subsequent lactogen, MMP-9 and al integrin. Over-expression of Hxt in Rcho-1 trophoblast binding of DNA and transcriptional activation. In setu hybridization to Id-2 cells induced their differentiation by promoting changes in cell adhesion, mRNA in placental tissue sections revealed that expression of this gene is whereas the HLH negative regulator Id-I was inhibitory. Hxt also induced downregated as CTB differentiate along the invasive pathwa. Northern transcription of a co-transfected trophoblast interferon promoter. Over- blot analysis showed that as C(B differentiate in vitro, Id-2 mRNA levels expression of Hxt in cells of deavage stage mouse embryos restricted their decrease. Immunocytochemistry confirmed this downregulation at the protein development into trophoblast and exduded them from the inner cell mass. These level. We are now testing the hypothesis that Id-2 gene expression plays a data suggest that bHLH factors regulate trophoblast differentiation and that critical role in transforming CIB from a stem cell population to a invasive Hxt has trophoblast cell-inducing activity. Supported by NIH (HD 26732) and differentiated, phenotype. DOE (DE-AC03-76-SF01012).

2638 2639 GENOMIC IMPRINTING INFLUENCES PARTHENOGENESIS IN CBA, THE LCR-MEL CELL IXFRION sYmK ((D.-A.Hdle-y , RIClodkeyt and C57BL/6 MICE. ((E.S.PLATONOV, L.I.PENKOV, and AJ.Dldko)) 5Biocbelsstu Divia, 12, Scho of Biobog Sdeaes, Usivenrty O.V.Mironova)) Vavilov Institute of General Genetics, of Maacheater, Oxford Road, Moachester, 1M13 9PT, UK tZeaea Pb.araatlcal Russian Academy of Sciences, Moscow. (Spon.by AdelyPark. Malsfld, Cheshire, SKI 4TG, UK 0r- P.M.- BAYLEY) Murme aythrolekeam (MEL) cdls are aytroadprogstors blockd om their diffaentces pathway due to infecti by the Friend Vms caplex Diffaeatice down the aythocyte The effect of genomic imprinting on postimplantation li age can be riggedb a uhmber ofchemical r physical agets. This pence is development of diploidized parthenogenetic embryos of isd by a dratc imcreae in hasnoglobn produclxa md the cesstia of cel CBA and C57BL/6 mice as well as of their reciprocal divisio Tle ontrol of 0-gobe in_duon at the klvel oftraipboohas bees attributed to hybrids has been studied. C57BL/6 embryos did not the locu cotrol rego (LCR) found 6 to 22Kb 5 the A-globui locb. Trunsfectie of MEL with expeo ces cetaminigthe the globn promoter and the develop to the somite stages, while CBA, (CBA x LCR, hunm grwth hee (hW)cDNA gives positien-adepesdnt, copy numsber-depesdtnt C57BL/6)Fl, (C57BW6 x CBA)F1 embryos reached dif- expreso ofhGH atte induct with dimethyt sulfoxide (DMSO). This offers a high-level ferent stages of somite formation in 45%, 30% and 14% inducible mu_olian exrssion sy3tom withhOtl production of 7OpgIfl being obained of cases, respectively. The latest stages of develop- after idueti ofthe CBS hOH ME cell line with 2% DM80. However, use ofthe ment observed in CBA and hybrid embryos were of 33-35 lsemlobin-dpecifi stain 3,3',54cr h (TB)eBveadthat only 20% ofthe somites. These data show that genomic imprinting is ells w produiong h_eeoglobin nder the s canoi s. Also, cell divisi wa not realized differently in these inbred strains of mice. inhibited bythe presene ofDM5O. Thus the populatin wa htrognu with respect to Probably it is important from what strain the inac- its ability to rspesd to DM80, and also the possibility of ceepetitn bedwe the 2 indeprendnt gnes the coatrol of the LCR ws rand To mveatgate thin relass tivated X-chromosome takes its origin. The action of the cells cloned. Analyms ofthe clene geneated highlighted eval ckca that display a DNA methylation inhibitor 5-azacytidine was studied a positive ocerelatin between dif ti obmnd hGH mRNA inductio and GH in vitro in parthenogenetic one-cell embryos, at the ecetien One asch eke rcesot a poorpoulaion in tems of TMB aining (12%) and blastocyst stage and in vivo after implantation in the hOHproducto (Sg/unl) In mofrast othe rdens both sbin well with TIB (up to 95%) uterus of pseudopregnant mouse females. The obtained ad produe greaSer leves ofKh;II( l3pto /nl This is eflected m the mRNA evels, data indicate that demethylation of the genome of with the 'highp oducing clasmhevig up to 5-fold greae kl of P-globn ad bhH parthenogenetic embryos results in the activation of mRNA. These clnes will provide usful tools to help eucidate the in_tanimochaniss cenolig MEL cell diffetat ind eprss fies the LCR. genes repressed by imprinting but is not sufficient for their expression without an additional transac- tivating factors. 454a Developmental Control of Gene Expression (2640-2645). Wednesday

2"0 2"1 MOUSE EMBRYONIC STEM CELLS: USE AS A NOVEL SCREEN FOR CHARACTERIZATION OF A DEVELOPMENTALLY REGULATED DIFERENT ION INDuciNG FACTORS. ((1. Jacoby, C. Lindberg, M. FORKHEAD TRANSCRIPTION FACTIOR IN PULMONARY AND Mills and J. Dinsmore)) Diacrin, Inc., Building 96, Thirteenth Street, Charlestown, MA 02129. REPRODUCTIVE EPITHELIUM. ((B.P. Hackett, S. L Brody, and J.D. Gitlin)) Department of Pediatrics, Washington University School of Medicine, We have developed methods to induce differenion of mouse embryonic St. Louis, MO 63110. stem (ES) cells at high de ency intoeidernronsrskeletal muscle. We wanted to use mouse ES cells as a genetic screen for novel factos capable Determinants of cell fate during mamma development remain lagely of inducing diffrentdation. Thus, we have optmized parameters for such a unknown. Forkhead transcription factors appear to play important roles in cell screen. The screen involves the intoduction of cDNA expression libraries fate determination and cell-specific gene expression. To investigate the role into ES cells, seecton of cells indcedto diffeeniate and isolaionof of these factors in pulmonary epithelial cell differentiation, a member of the clones ble for inducingdifeentiaon In this way, we can screen family, HFH-4, was isolated from a rat bronchiolar epitielial cell cDNA large numbers of genes in celluldiffertiation. cDNA forinvolvment library using PCR. Screening of a rat lung cDNA library with the isolated exression libraries were made from severl tses and used to trnsiendy in a a tansfctES cells. Electroporstion was used for ction, and we have partial clone resulted cDNA with 606 amino acid open reading frame. optimed condidons for transient ransftion ofES cells which differ from RNA blot analysis of adult rat tissues revealed a 2.4 kb HFH-4 tanscript in that reported for stable tection ofES cells. Under opdmal conditions, lung, testis, and oviduct. By in situ hybridization, transcript was localized to we found that 30 % of cclls express a E-gal reportr gene, with peak the bronchiolar epithelium within the lung. During lung development, HFH-4 expression at 24 hrsm post-transfection. Additionally, several promoters transcript was first detected at E14.5 and increased in abundance to E17.5 were testedin ES cdls, with the cytomegalovirus promoter giving highest followed by a decrease in abundance. Embryonic expression in the lung was expression. Afteroptimizing conditions forthe expression ofa p-gal repotergene, we demost thatES cells were induced to differentiate restricted to the developing pulmonary epithelium. In the testis, transcript was by the uwnsient expression ofa known inducer of differendation, MyoD. specific to germ cells with no transcript in Leydig or Sertoli cells. RNA blot ES cells responded to the transient overexpression of MyoD and analysis of HFH-4 expression in postnatal testis and testis from qk and hpg differentiated, with aroximately 6% ofthe cells having differentiated after mice revealed that expression was associated with haploid germ cells. transfection. Finally, we were able to recover plasmids from tnnsiendy Detailed examination of the testis revealed stage-specific HFH-4 expression transfectedES cells up to 5 days after transfection. Sevenl cDNA libraries These of HFH-4 have been transfectedinto ES cells, and we are currendy analyzing the during spermatogenesis. unique patterns expression suggest results of our initial screens. a role for this factor in cell fate determination during lung development and regulation of post-mneiotic gene expression in the testis.

2642 2"3 DIFFERENTIAL DNA BINDING AND TRANSCRIPTIONAL PROPERTIES OF GENE EXPRESSION IN CULTURED EMBRYONIC RAT LUNG ((A. HOX PROTEINS MAY CONTRIBUlE TO TIR SELECTIVE FUNCTIONS Meneghetti, W. V. Cardoso, J. S. Brody, and M. C. Williams)) The DURING DEVELOPMENT. ((C. Schnabel, L Pellerin and C. Abate)) Centr for Pulmonary Center, Department of Medicine, Boston University Advanced Biotechnology and Medicine, Piscataway, NJ. School of Medicine, Boston, MA 02118. which share a conserved DNA The murie hox gene comprise a multigene family Cultured embryonic lungs form airways, alveolar sacs, and med a DNA seque t the hoieobox.The homeobox encodes biding connective tissues with patterns of differentiation similar to those in domain (the homeodomain)present amonUggnuerouslmatory vivo. We used RT-PCR and in situ hybridization (ISH) to compare proteis that fucion during development. In conras;t to sties that have established an essental role for hox gems in regutng murine embryogenecis, the gene expression of in vivo lungs with their cultured lung properes of their protin products as transcriptional regulators have not been counterparts. Fetal day 13 rat lungs were explanted and cultured for examined extensively.Towards this end, we have tize the DNA biding 5 days in serum-free media and compared to fetal lungs excised on and transcriptional prperties of several Hox protein. We show that these various day 18. We assessed the following developmental marker genes: Hox protdns intract preferentially with a consenus DNA site conaining the surfactant proteins A, B, and C (SP-A, -B, -C), Clara cell secretory sequence (C/G)TAATTG, however, they exhibit subtle, but distnct, prefeences for protein (CCSP), and Tla, a type I cell-specific gene in adult rat lung. DNA sites containig vadations of the consensus modtf. Moreover, we show that RT-PCR reveals SP-C and Tla mRNAs in uncultured day 13 lung but for the consenus DNA site. Hox proteins differ in their relative affinity Intriguingly, SP-A, SP-B, and CCSP mRNAs are not detected. In vivo fetal day 18 the differences corelate with the of their respet on the in affinity position genes lungs contain all five marker mRNAs as do day 13 lungs cultured for hox duster, sgestn an altrnadve avease of the "Hox code." We have 5 ISH of mRNA in excised examined lie transcriptional properties of several Hox proteins. In particular we days. By patterns expression day 18 lungs and 13 cultured for 5 are similar. SP-C is confined to show that Hox A7 fucions as a trancrptiona repressor in vivo. bterestngly, the day lungs days homeodomain contributes direy to repression, suggesting a novel function for this the tips of the epithelial tubules whereas SP-A and SP-B mRNAs are domain. In adtion, we have mapped a potent on al activator domain to the expressed in tips and their immediately proximate tubules. CCSP is amino termnus of the Hox A7 protebnTherefore, various fuional domains expressed in large and small airway epithelium of both cultured and imparting distinct transciptional activities may contribute to teir ability to uncultured lungs. Little Tla is expressed in either the most proximal differentially regulate gene expession. In summary, these biochemical studies will or distal tubules; most Tla expression is immediately proximal to the allow insight into te molecular mechaniss by which hox gene products function SP-C expressing cells. Therefore cultured lungs largely mirror the selectively as transcriptional regulators during embryogenesis. pattern of gene expression seen in utero and can serve as useful models for lung development studies. That explanted lungs express cell-specific genes at appropriate times and locations without serum or added factors indicates that information for pattern formation and cell differentiation is contained within the lung itself.

2644 2645 IDENTIFICATION OF DEVELOPMENT-SPECIFIC GENE EXPRESSION TB ANF PROMOTER IS TRANSCRIPTIONALLY ACTIVE IN THE ATRIA AND IN CHICK EMBRYONIC CHORIOALLANTOIC MEMBRANE BY mRNA VENTRICLES OF STAGE 14-23 EMBRYONIC CHICIEt HARTS. ((S.A. DIFFERENTIAL DISPLAY. ((E. Suyama and R.S. Tuan)) Dep ent of Fisher* and C.J. Forehand*)) *Dept. of Medicine, Case Western Reserve School of of Thomas Jefferson PA 19107. Medicine, Cleveland,OH 44106;*Dept. Orthopaedlc Surgery, University, PhiladelphIa, Anatomy and Neurobiology, Univ. of Vermont, Burlington,VT 05405 The chorioalbntoic membrane (CAM) Is an extraembryonkc membrane which Is responsible for calcium mobilkatdn from shl to embryo during We have previously reported that stage 10-17 embryonic chick chick development. After incubation day 1u. the CAM has grown to hearts are transfected in vivo with plasmid DNA-liposome completely surround the chick embryo and bacomes attached to the shel complexes and that this method can be used to map adlacent to the egg shel, and calium bansport initiated. The transcriptional regulatory elements (J Cell Bioch 18D:496). epitheliai ceHu-iar morphology of the CAM dramatlcally changes at this point, When these embryos are incubated for 48 hrs. after with the peance of transporting cell typesu est spefic celluar transfection, the hearts develop from a tube into a four differtiab ,events. In this study, we used n DIffrena Dspla chambered organ with morphologically distinct atria (A) and We have initiated these studies to to to identfty genes which are ex ed in a develont- ventricles (V). attempt (DD) technique elements which the atrial and manner In the CAM. Ths technique is on identify cis-regulatory specify specific ibaed olgo-dT-prmed ventricular compartments. Two plasmid constructs, one reverse transcription and PCR amplIfication of mRNAs uing aary 5'- containing a -610 bp 5' fragment of the rat ANF gene (pANF), ollgo-amplime; the dstribution of 3'-termlna message is then d bspyedby and the other the RSV LTR (pRSV), each linked to a luciferase denaturing poIyacrylamlde gel electrophoresls. We omad non-alcium reporter, were used in transfection experiments and ranspo CAM (incbation day 8) and calcu CAM luciferase activity measured independently in atrial and (day 14). After the isolation and re-amnpifcatIon ventricular homogenates. In 8 of 10 embryos transfected with from the denatured gel, Northem biot analysis confim the differel pANF, the luciferase activity was greater in the atria than expression on day 14 of one of the amplified mRNAs. The correspondg the ventricle (A:V 11.8+3.5 mean+SBM) while in 5 of 7 embryos cDNA was suboloned and Its sequence was determined by the transfected with pRSV the luciferase activity was greater in dideoxynucieotide method. Analysis of be cDNA sequence ompaion to the ventricles than the atria (A:V 0.43+.15). These data GenBank database showed no sIgnIficant homology to anY known indicate that the -610 bp ANF fragment is sufficient to of the in the atria and ventricles sequences. Current effort Is devoted to Isolaing full-ength oDNA for this promote transcription gene in the of these gene by screenIng cDNA of CAM very early stages morphogenesis. Moreover, library day1W4 localking gene data differences in ANT expresson bY h slfu Ion various stes of to gain insght suggest quantitative promoter hybirI on CAM, activity between A and V at these stages. The validity of into the fnction and regution of this gene. (Supported by NIH HD 1582, this suggestion is being explored utilizing a co-transfected HD 29937 and ES 07005) control plasmid to normalize for interanimal differences in DNA uptake between the two compartments. Wednesday. Developmental Control of Gene Expression (2646-2651) 455a

2646 2647 EXPRESSION OF HSP90ax AND 90p GENES DURING ZEBRAFISH CLONING, SEQUENCING AND EXPRESSION OF A HOX 1.3 EMBRYOGENESIS. ((JB Sass, and PH Krone)) Dept. of Anatomy, HOMOLOGUE IN THE MEXCAN AXONAI AMBYSTOMA University of Saskatchewan, Saskatoon, Canada S7N OWO MEXICANJM. ((A. Gaur, D.K. Dube, ME. Fransen, LF. Lemanski)) Department ofAnatomy and Cell Biology, SUNY Health Science Center, Heat shock proteins are a highly conserved family of proteins expressed at Syracuse, New York 13210. high levels during exposure of an arpnism to heat stess, as well as during exposure to numerous chemicals and toxins. The 90 KDa heat shock protein family (hsp90) are the focus of this study. Under non-stress An ecewllent model for sudying heart development is the cardiac conditions members of the hsp90 family have been shown to play a role in ledulmutman(gmnc) mian xk , sm mxcnum In order to steriod receptor function, in cellular protein translocation, and in the cormect facilitate our analyses ofthe mutant system we have undertaken a search for assembly of polypeptides. We have recently isolated cDNA's encoding moicula markers of specific stages in the development of the axoloti hsp90a and 90, from a zebrafish embryonic cDNA library. Northern embryo. We have ted on homeobox genes as suitable candidates analysis of total RNA revealed that the mRNA is present at for wacing molculrchanges during development A 280 bp probe hsp90 high encoding a portion of the axolod homeobox gene Ahox-I was levels constitutively from late cleavage stages to 4-day old generated by swimming PCR from a stage 18 axolod embryonic cDNA library. Tlhe probe was then larvae. Levels increased 2-3 fold upon heat shock in early stage embryos. employed to sacreen a X-gt 11 stage 18 cDNA library using modeately Alternately, the hsp90a gene is expressed at very low levels under normal stmngent condibons and six diffcrent honmobox cDNA cones wer isolated. temperatures, and is strongly heat-inducible at all stages of development One of the clones has been further analyzed and it shows very high except the cleavage stage. Analysis by whole mount in situ hybridization homology to human, mouse and rat Hox 1.3 (83% nuclic acid and 99% with antisense RNA probes revealed that during heat stress of embryos amino acid identity in the homeobox coding region). higm hybridization undergoing somitogenesis hsp90a was expressed in the head region and the and Northm blot analyses am bing used to follow the expression of this dorsal aspect of the body and tail region of the developing embryo. gene during axolod development. Prlimina results suggest dtat this gene is exprssed in the chordamesoderm of stage 13 embryos as well as in other Interestingly, heat-inducible accumulation of hsp9Oa mRNA was not embryoni tissues at later stages. Future experiments include expression of detectable in the dorsal midline, in the region of the notochord and neuml the whole cDNA as wel as demonstratio of its DNA sequence binding tube. In contrast, at this stage of development hsp900 was expressed specificity by gel retadation assays. (Supported by NIH grants HL-32184, throughout the embryo at both control and heat stress temperat . This HL-3770 and an Amerian Heart Associaion grant to LFL) work was supported by NSERC and HSURC of Saskatchewan.

2648 2649 THE XENOPUS GASTRULA IS RESPONSIVE TO GROWTH FACTOR CONTROL OF FERRrTIN GENE EXPRESSION IN SEA URCHIN EMBRYOS: SIGNALS. ((C. Domingo, R. Keller, and E. Amaya)) Department of Molecular EFFECTS OF IRON ON APPEARANCE OF FERRrTIN AND FERRITIN and Cell Biology, University of California, Berkeley, CA 94720. MN SENGER RNA. ((T. Hamilton, J. Milanovich, and D. Fromson)) Depsrtmeit of Biological Science, California State University, Pullerton, CA 92634-9480. At the onset of gastrulation several signaling systems are working to organize the vertebrate embryo. These signals are responsible for. 1) defining the organi- This study focuses on elucidating the mechanisms corntrolling the appearance of zer region that consists of the notochord and anterior somites; 2) dorsalization ferritin in control and iron-treated embryos. Sea urchin embryos (Strongylocentrotus that recruits lateral mesoderm to differentiate into posterior sormites; and 3) purpuratus) were grown in the presence and absence of added iron (ferric ammonium neural induction that induces the dorsal ectoderm region to form the neural citrate, 25 mg/mn sea water) throughout development, and the amount of ferritin plate. How does the embryo orchestrate these events? Cell transplantation were quantitated colonimetrically by measuring the amount of imn released from experiments have shown that cells from the early gastrula are pluripotent and TCA-precipated protein fractions. The amount of fersitin in iron-treated competent to respond to several signals depending on the context of the graft. In pregastrular embryos was substantially higher than in controls. The ratios of the particular, the prospective posterior neural ectoderm can respond to notochord amounts of ferritin in iron-treated to controls was 65 in cleavage stage embryos, 7.9 inducing signals when grafted to the prospective notochord region at the onset in monsla stage embryos, and 48 in early blastulae. This ratio dropped below 2.0 in of gastrulation. In order to exanmine the nature of the notochord inducing signal early gasrulae. The amowunts of ferritin increased following gastrulation; the iron- at the gastrula stage, we added purified growth factors to dorsal marginal zone treated-to-control ratio rose to 4.0 in early prism stage embryos. To determine if the explants composed of axial mesodemi and neural ectoderm from gastrulating appearace of ferritin was dependent on utilion of stored mRNA or new mRNA, Xenopus embryos. We found that purified Xenopus basic fibroblast growth the amounts of ferritin mRNA were nmssred using digoxygenin-labeled rat ferritin factor (bFGF) altered the morphology of the explants. In particular, the cDNA as a probe. The resulting slot blot analysis (quartitated by densitometry) mesodermal region composed of notochord and somite tissue hyper-extended, revealed that in pregaatrular embryos, treatment of iron resulted in recruitment of possibly at the expense of the neural ectoderm. Furthermore, we constructed mRNA from cytoplasmic, nonpolyribosomal fractions. However, in postgastrular dorsal marginal zone sandwiches in which one half originated from embryos embryos, iron treatment appeared to cause an inaease in the total amount of ferritin injected with FGF RNA at the one to four cell stage and the other half from mRNA and in the amount of polyribosome-associated ferritin nRNA. The uninjected embryos. These sandwiches developed much like the dorsal marginal cytological localization of ferritin was determined by indirect immunofluoresence zone explants exposed to FGF protein. They had a hyper-extended notochord microscopy. In both control and iron-treated embryos, the ferritin appeared to be and somite region that again appeared to be at the expense of the posterior prmarily locaized in the mesenchyme plate and then the archenteron cells with neural region that was morphologically absent from these sandwiches. These some fenritin localized in the ectoderm cells. In addition, ferritin was loalized in results indicate that dorsal axial tissues from the gastrula are still responsive to the arcenteron cells in control and iron-treated exogastrulae. The intensity of FGF and that FGF may be involved in a signaling process that patterns the fluC in iron-treated embryos was noticeably higher. (Supported by CSUF mesoderm and neuro-ectoderm during gastrulation. Supported by HD25594. DAC Grants to TH and CSUPFFaclty Research Grants to DF.)

2650 2651

METAMORPHOSIS ASSOCIATED CHANGES IN FAT BODY CELL THE DROSOPHILA NK-4 HOMEODOMAIN TRANSCRIPTION FACTOR TRANSDUCES NUCLEAR PROTEINS IN A LEPIDOPTERAN (C. Wang and A. K. SIGNALS FROM TWISTTO THE NK-3 HOMEOBOX GENE. ((Y. Lee, K.Chung, T. Park, and Y. Kim)) of Moleculr Beshesda, MD Kumaran) Department of Biology, Marquette University, Milwaukee, WI Laboraty Cardiology, NHLBLNIH, 20692. 53233 Previous genetic sdie and the ep p of NK-4 () and NK-3 (a )homeobox genes, together with those of in duing embryogeesis suggested that these genes may Ecdysteroids in insects activate or repress gene expression in a tissue and regulate each other and teeby may cotrol meaodermal cell In oarder to stady stage specific manner. Depending on the presence or absence of juvenile transcriptional control of these genes, trnsient expression assays were used with ch nh as a goe. hormone different sets are affected The col acetyltaferse (CAT) reporter By measuring CAT activities in (JH) gene by ecdysteroids. stage exs from C2C12 myoblast cells cosfected with GSBCAT and various GAL4:NK4 specific response to ecdysteroids that this hormone which initiates implies fusionconsructs we have de ined that the amino-termina region (aa 1-110) of the NK-4 in the absence of metamorphosis JH, activates a set of genes whose products promin isa tranipia activaordoma. Becameour nucleotd sequence analyus of the S cause a modified response to ecdysteroids after metamorphosis. These upr region of the NK-4 promotr revealed two chuters (El,3 copies of ACATATO from - ecdysteroid responsive genes should, in theory, code for DNA binding 933 -903; E2,3 coi of CACTTGA from -632 to -602) of B-box seqces (CANN ), and becsewefound that nuclearxru fromkilam embryos showed bindigacvitis proteins including transcription factors which are different from the acx early to thewe E-box chuser, we have tested whethr twist is a direct activator of the NK-4 gene. the Ashburner model et response genes predicted by (Ashburner al, 1974, When we measured CATactivities in extat fronCVI cells coansfected with CAT repoter Cold Spring Harbor Symposia Quant. Biol. 38, 655-662) and isolated in plasnids containing vriots 5 uprsean re gions of the NK-4 promotr and the twist expression studies on Drosophila (see Karim et al, 1993, Devt. 118;977-988).The vector, CMV-Twist, we observed a 5-fold msease in CAT activities frm exacts tnsfected with Fl (150 from -950 to consruc. furher mutation arylphorin gene in Galkria mellonella larvae is bp, 401) Indeed, analysis within the transiently repressed by El duster and gel-shift asays with a punfied GST:Twist fusion proteinexpressed in E&li ecdyseroids, but is ireversibly repressed after initiation of adult development stongly suggsted that theEl cluster is ible for the direct uvat of the NK-4 eneby (Kumaran et al, 1993, Ins. Biochem. Mol. BioL 23,145-151). The arylphorin twist We have also observed that the F3 rion (150 bp, from-651 to -502) contained s- gene promoter contains a putative ecdysteroid responsive element (ERE) ang el reiuble for by NK-4. Mraeover, E-box sequenc within the which in gel retadation analyses binds to nuclearextractsof day-O last instar E2 cluster were found to be bindig for the NK-4 protein by both gel-shift assays and DNael assa with a GST:NK-4 fusion protein. larval and day-I pupal fat body cells when a 191 was used. But footprinting punified Fmally, from the bp fragment cotnfection assy with the NK-4 expresion vector and reporters driven by the NK-3 nuclear proteins only from a fat cells, but not from 0 day-i pupal body day promoter we have deermined that NK-4 is an activtor of the NK-3gene. Further deletion instar larval fat last body cells, bind to a 78 bp long subfragment containing analysis of the rm re of the NK-3promoter revealed that the proximalregion to the the putative ERE. This DNA binding protein may belong to the class of NK-3 promeer(- 271 to +2S) inwhich two copies of theNK-4 binding site are located,was impornt for the aivain of the NK-3 gene NK-4. nuclear proteins that are synthesized at metamorphosis. Ecdysteroid induced by Taken together, these results is a dirct tget ofxig which is a mesoderm deterninant in lkjila genes during larval-pupal metamorphosis are being isolated from a cDNA embryos. and that the NK-4 protein, in sin.auoacivate the NK-4 gene itself and finally library from last instar larval prepared ecdysteroid injected ligated day-5 NK-3 that a cascade of gene regulators may play an mRNA. (Supported by NSF grant DCB 89-10757 and Wehr Foundation) roamt leX mdwAn ing Stb> . 456a Developmental Control of Gene Expression (2652-2654). Wednesday

2652 2653 TRANSCRIPTIONAL UP-REGULATION OF A METHYL EXPRESSION PAITERNS OF SPECIFIC SOYBEAN VEGETATIVE TRANSFERASE BY SIGNAL TRANSDUCTION DURING LIPOXYGENASE GENES IN RESPONSE TO DEPODDING AND WOUNDING. CHL4MYDOMON4S FERTILIZATION. ((V.Kurvari, F.Qian, and ((T.W. Bunke, D.S. Koetjel, LS. Stevenson, and H.D. Grimes.)) Department of W.Snell)) Dept. Cell Biol. & UT TX Botany, Washington State University, Pullman, WA 99164-4238. IDeptrtment of Neurosi., Southwestern, Dallas, 75235. Biology, SUNY, Fredonia, NY 14063. Chlamydomonasgametes ofopposite matingtypes interact through flagellar The enzymatic ability of soybean seed lipoxygenases to add molecula 02 to cis, cis adhesion molecules called agglutinins, leading to a signal transduction pentadiene regism of unsaurae faty acid such as aracbidmic acid (animals) or linoleic cascade that induces cell wall loss and activation of mating structures along and linolenic acids (plants) has been well charcterzed. In animals, lipoxygenases are with other cellular responses that ultimately result in zygote formation. To important in intig leuktniene biosyntheais Related biochemical pathways may be identify molecules involved in these complex cellular events, we have initited by Lipoxyg se in plants. However, a physiogial fumcton for plant to their biochemical also employed subtractive and differential hybridization methods lipoxygenase unrelaed activity has reeiy be discovered. using cDNA Lipoxygenases in plants appear to fuwton as protein atom for nitrogen since from agglutinating mt gametes and non-signallingvegetative cells. Multiple lipoxygenase accumulate in the leaves of mature soybean plants (along with vegetative screenings of approx. 50,000 cDNA clones identified 55 clones, which were stepge protins VSPa and VSPP) in response to contnuous dpeding. Because the characterized further using Northern and Southern hybridizadons and lipoxygense gene family has seveml members, however, it is uncetin as to whether nucleotide sequencing. The transcript for one clone, F43, was up-regulated single or multipl lipoxygenase genes are responsive to source/sl manltions. 18-fold in gametes undergoing prolonged sexual signalling induced by the Likewise, whether specific members ofthe vegetadve lipoxyygenase gene family are more response to woundig or other strese, is unknwn. addition ofmt' whereas the levels ofthis 3.6kb flagella, expression transcript To circumvent this problem, our laboatory consuucted a cDNA library from the leaves remained unaffected in vegetative cells undergoing cell wall regeneration. ofdepodded soybeans to ficilitate dte isolaion ofnitrge-responsive lipoxygease Sequence analysis ofthe 1.2 kb cDNA insert revealed an open reading frame cDNAs. Sceeng of this library yielded three disict lipoxygeme cDNAs which were of 360 amino acids. Database analysis and sequence alignment indicated aized and sequenced. Two of thede were idenical to the previously identified vegetative lipoxygenase cDNAs. The third lipoxygenase we term about 40% identity and 50%-60% similarity at the amino acid level to a class (which v

2654

SINGLE-CELL ANALYSIS OF DNA NLIX OPENINS WRIN IN-VIVO CELL DIVISION AND DIFFENTIATION. ((J.. Freaster)) Physicia' Educatioaal Series, Atherton, CA 94027-5446.

DNA molecules uadergo helix opeaings during active geae trans- cription, and these helix openiags can be detected within sin- gle cells by high-resolutioa probe electron microscopy (Ann. N.Y. Aead. Sci. 567, 334 (1989). Human bon* marrow cells were aspirated and rapidly fixed in cold glutaraldehyde, followed by acridine orange probe insertion and DNase-I digestion. Probe sites were visulized at high resolution as electron-dense renction products, and ranged in length from 25-700 m, corres- ponding to 70-2000 base pairs in length of DM. Probe sites were fouad exclusively within the zxtended suchromatin micro- fibrils of the cell nucleus, where they correlate most highly in location with sites of active gene transcription. Probe counts and sizes were detemined for marrow cells in each of the distinct *t&5** of cell mitosis, ad in each of the dis- tinct stages of granulocytic sad erythrocytic dlfferentiation. Probe sites were found to decrease in size and number l divid- ing from prophase through metaphase to asaphase, licreasing again in telophase. Probe sites were also found to decrease in size and number fro the earliest cells In the gruaulocytic and erthroeytic sries to the most mature nucleated cells within each of the series. It Is wocluded that high-resolution probe electron microscopy rveals a progressive reduction in the size and the number of DNA helix openings during the course of in- vivo human cell division and cell differentiation. Nuclear Envelope Import, Export, and Structure (2655-2656).

2655 2656 Analysis of a Nsplp containing nuclear pore subcomplex ((V. Doye, P. Grandi, A BRANCHING HOLLOW CABLE SYSTEM INTERCONNECTS THE N. Schlaich, H. Tekotte, C. Wimmer& E. Hurt)) EMBL Heidelberg. INTRANUCLEAR COMPONENTS OF NUCLEAR PORE COMPLEXES IN XENOPUS OOCYrES. Exchange of macromolecules between nucleus and cytoplasm occurs nuclear through ((H.Ris)), Dept. of Zoology and Integrated Microscopy Resource, pore complexes (NPC). Since the NPC is a conserved we use University of structure, S. cemvsiae Wisconsin, Madison, WI 53706. as a model system to identify and study NPC components by combined genetic and biochemical approaches. The nuclear pore protein Nsplp is involved in nuclear import The nuclear pore complex(NPC) is a large macromolecular of reporter proteins, but seems not to play a role in nuclear of It assembly inserted into export poly(A)+RNA. the nudear pores, and is unlikely, that NPC proteins exert their function independently, but rather in concert controlling import export of proteins and nucleic acids. Recent studies using field emission electron with their neighbors or components with overlapping function not physically attached. 'in-lens'scnning microscopes have greatly advanced our nowledge of NPC structure, particularly the components the We set up a biochemical procedure to purify NPC proteins under non-denaturing con- facing cytoplasm and the nuclear space. The cytoplasmic face consists of a 120 nm ring ditions. This way, Nsplp was shown to be complexed with 3 other proteins, Nic9fp, carrying 8 cylinders, 2Ox4Onm in size. On the nudear side is another 120 nm ring, Nup57p and Nup49p. Strikingly, these components could also be found in a genetic with 8 filaments, 5nm thick and 50 nm long, each ending in a small sphere. The wcreen designed to isolate synthetic lethal mutants of Nsplp. Sequence analysis allowed 8 spheres are to form a 50nm top of a to group Nup57p and Nup49p into the family of GLFG repeat containing nucleoporins. joined ring.the fishtrap'-like structure. In published images this ring is obscured by associated fibrous material Both contain amino-terminally GLFG repeats, which are dispensible for growth, and usually that forms irregular connections between adjcent NPCs. To prevent nuclear like Nsplp, an unique carboxy-terminal domain organised into heptad repeats. The swelling, which causes stretching and disruption of pore connecting structural organisation of Nic96p is more complex: amino-terminally, it contains hep- fibers, have used tad repeats, possibly required for stable interaction with the other nucleoporina of the McGregors buffer, isotonic to cytoplasm. Adding 5% sucrose to the buffer prevents the contraction of the nuclear gel and makes it soluble. NPC subcomplex Then follows a middle domain with stretches of hydrophobic amino Treatment of the isolated nucleus with 0.1% Triton X-100 and 1% In buffer acids, in which single amino acid substitutions yielded thermo-sensitive nic96 mu- glutaraidehyde this removes the tant aleles. The carboxy-terminal domain is not required for physical interaction with membrane. The orhogonal net of lamin fibers is now visible. The fishtrps and attached 50 nm fibers are preserved. Nsplp, but mutations in this part cause synthetic lethality with a thermo-sensitive weR Other nudear envelopes were plunge- frozen in liquid ethane, freeze-substituted and embedded in epon. epon was nspl alleL Since conditional lethal mutants of the four proteins display a defect in The extracted from 200 nm sections (Ris and Malecld,J. Struct Biol. and import of an NLS-containing reporter protein, a predominant function of the Nsplp- 111,148) Imaged FESEM. cross we containing subcomplex could be in nucleocytoplasmic transport. by In sections see side views of flshtaps with 50 nm fbers attached at their small ing. Unextracted sections imaged by HVEM reveal that this fiber is a hollow tube with a wall consisting of eight Snm fibrils arranged to give a periodic structure.In summary, on the intranudear side, groups of NPCs are interconnected by branching holow cables. A colaboraftve study is now in progress to determine the role of these hollow cables in the export and import of nuclear RNAs. Wednesday. Nuclear Envelope Import, Export, and Structure (2657-2662) 457a

2657 2658 IDENTIFICATION OF NTF2, A CYTOSOUC NUCLEAR PROTEIN IMPORT FACTOR WHICH INTERACTS WITH NUCLEAR PORE COMPLEX PROTEIN p62. ((B. STRUCTURE OF THE NUCLEAR ENVELOPE IN PROLIFERATING AND Paschal and L Gerace)) Depts. of Cell and Molcular Bblogy, The Scripps Research QUIESCENTCELLS ((Ermon6J.H. Adam and Stephen A. Adam)) Department Instiute, La Jolla CA 92037 of Cell and Molecular Biolgy, Northwestern University Medical School, Chicago, Illinois 60611 The active transport of macomoculs between the cytoplaomlc and nucear corparmnts nvolves irteractions between the rnclear pore corrplex (NPC) and Using digitonin permneabized MDBK cells we have investigated the muliple cytosollc factors. We are analyzig the functns of these cyosolic strucure tarapot of the nuclear factors usig digionin permeabized cels and a new flow cytmetry-based envelope in proliferating and quiescent cells. After digitonin detecton system Here we report the pfixcation and inal chwateIaion f a perrreablizatfon or nitrocellulose perforation of the plasma membrane, the factor (NTF2) which interacts functonaly with NPC protein p62. p62 quiescent cell nuclear membranes (NM) vesiculate with gross rearrangement was ed on Sepharse beads and used to deplete HeLa cel cytosol of an of nucear pore compls. The NM of proliferating cells display a normal essential tranport Usin we the aciviy. bbchemial corplementation, purIfied morphology. Pore complexes appear to be excluded from vesiculated regions activity 1200-fold from a high speed cytosolic extrad. NTF2 stimulates rncbar of the NM when detected with wheat germ agglutinin or MAb414 and the the protein knport of p62 pretreated cytosol in a saturable manr, with masximal inyoe occurrhg at 200 nM NTF2. NTF2 is an abundant ptein (-106 copies percel) and lamina appears deformed but intact Vesicleformation is due to hyperosmolarity purifies as an apparert dimer of -14 kDa subunits. We obtained a cDNA encoding of the peninuclear space relative to the permeabilization buffer as indusion of human NTF2, prepared recombinant protein, and showed that it stimulates transport 1M sorbitd during permeabilization is required to prevent vesiculation. to the same maxinal level as the native protein. To determine whethr NTF2 is a rate Vesiculabtion of the NM leads to dramatic and reversible compacfion of the lnkitng component of an early (pretransbcation) step of nuclear protein impon we nuclear interior. Membrane "ysis with Triton X-100 causes collapse of the depleted ATP from the assay and examined the accumulation of transpot gand at the nuclear envelope. Under this 'docking condition, p62 pretreated cytosoi drhi vesicles and a return to normal nuclear morphology. We have investigated the the accumulaton of Ugand skmilar to mock treated cytosol. This indicates NTF2 is not nature of the pernnuclear osmolyte. Intracellular sorbitol is sequestered in a ikety to be required for NLS recognition orfor the Initial bding of an NLS receptor- membrane bound compartment but levels ofthis osmolyte do not differ between igand complex to the cy surface of the nuclear pore. These data are proliferating and qulescent cells, suggesffng that another osmolyte is involved. consistert with a for NTF2 afterthe nitial docking to requirement step but prior NM vesiculaton is not unique to MDBK cells although it is most pronounced in translocation through the central channel. Since p62 is localized to the cytoplasmic these cells. These resuits suggest that mechanical properties of the nuclear and nucloplamic skies of the NPC, NTF2 may function in both nclear potein inport and RNA export. Supported by the Damon Runyon-Wakter Wunchell Cancer envelope may be involved in the regulation of gene expression through forces Research Fund and the NIH. transmitted to the karyoskeleton.

2659 26"0

EXPRESSION AND ASSEMBLY OF NUCLEAR PORE PROTEINS TPR IS A 265 KDA PROTEIN WIH A LARGE COIL C:OIL DOMAIN ASSOCIATED WITH PERIPHERAL SUBSTRUCTURES OF THE WHICHLOCAUZESTOTHEPERl!RIPHICYrOPIASMICREGIONOFTHE PORE COMPLEX (R. Bastos, A. Un and B. Burke) Dept. of Cell NUCLEAR PORE COMPLEXC ((D.Byrdt, D. Sweet, N. Patet, U. Aebif, K Biology, Harvard Medical School, Boston MA 02115. Konstantino, T. Guant, P. MicheII, C Cooper+, and L Geracet)) tTheScipps Research nsitute, La Jolla, CA 92037; Univrsity of Basel, Swizrlnd; and + Inste We previously reported the location and interaccions of a number of of Ca Research, Sutton, Surey, UK protein subunits of the nuclear pore complex (NPC) of higher cells From a p-wl of monoclonal antibodies raised agintactios of rat by using the QE5 monoclonal antibody (Pante et al., J. Cell Biol. 126 nuclear enveopes (NEs), we idendfied an antibody, RL30, whih reacts with novl (1994) 603-617). In this study we report the identification and nudea pore complex (NPC) antigen tat ae no 0-glycosylated. in characterization a pore mmu of novel complex protein, p75. This protein op-of acured cls, RL30reacts emrusivelywith the NE, in a puncte was pattr identified by virtue of its co-immunoprecipitation using QE5 and that coinddes wih tht of defined NPC protein In muool ec was purified from high salt /detergent extracts of rat liver nuclear RL30 label only the cgopasic sface of the NPC, ly in envelopes and subsequently cloned. It does not belong to the 0- regions RL30 recogizes a 265 kDa band in of isolated rat linked glycoprotein family and it contains neither GLFG nor XFXG NEt culured rat cell In addition, the antibody reacts wih a seies of 17s- repeats. p75 is physically associated with Nup 214/Can which is a 265 kDa bands rat iv NEs that are appart proteolytic producs of p265. of constituent of the cytoplasmic filaments of the NPC. These Seqencing peptides from the 175 and 265 kDa RL30 antens of rat ivrreva filamentous structures of the NPC have been proposed to represent that they are both losly related to previously descried hu6ia Tpr, a protein whose amio terminal L50-250 amio acds fusons docking sites for proteins destined for import into the nucleus. This appear onenic with the kiase proposition is currently under investigation. We have also carried domains of the Met,Raf ad Trk protoono Moreo, in vivo transation of a mRNA for human Tpr yields a major 265 kDa band. Considered out a deletion analysis of human Nupl 53, a component of the tgethr, data indicate that the 265 kDa RL3O am in de NPC is rat Tpr. Interestingly Tpr nuclear baskets of the NPC, in order to study its targeting and contins an e-eptiony long predicted i-coi domain (1600amino ais). in view assembly in vivo. Our results ndicate that only the N-terminal region of its loalizatio and sequence, Tpr is likely to be a componet of the cytoplasmi of this protein is required for corred its localization since this domain fis of the NPC implicated in import Elnd docking. Imm is able to target a heterologous molecule to the NPC and appears to microscopy shows tat during NPC re_asbly at the end of mitosis, Tpr becomes be involved in the self-association of Nupl53. Deletion of the central concentrated at the NE significant later 0-lnk including p62. zinc finger domain and/or the C-terminal XFXFG repeat domain This idcatesthat reassembly of the NPC following mitosis is a stepwise process, and have little or no effect on localization. that the Tpr-containg peripheral are asmbled later than p62.

2"1 2U2

ALDOSTERONE-REGULATED EXPRESSION OF NUCLEAR PORES IN cdk-SrrE REGULATED SIGNA-DEPENDENT NUCLEAR KIDNEY CELLS. ((H. Obether and G. LOCALIZATON OFTHE YEAST TRANSCRIPION Depament Folprecht)) Physology, University of W0rzburg, 97070 FACIOR SWISIN MAMMALIAN CEI S W0rzburg,Gemany. (Spon. by R. Benavente.) ((D. A. Jans ,T. Mol2, I. Nasmyth3, R. Peters4 and P. JansI)) for Biochemistry & Mokcular John Curtin School of aldosterone 1Divrision Biology, nulei taret enhances ansiponal acty Medical the Research,AusinNaionalUniversity, Canbera,Australia;2Vienna follwed by transot specific RNA molecules across nuclear International Researh Cooperation Center, Vienna, Austria;3Institut fur envelope. Tffing b n cel nuceus and cytopasm octurs via nucear the Molculare Pathologic, Vienna, Ausria; flIr Medizinische pores (NPs) lcated double-layered nucear envelope. We 4Institut Physik, invesigated the nuceocytplsic tansport route by sure-functon vWyerm , Mnster, Gemany t subcellular level in analysis qulscent and on ad We are interested in the regulation of the nuclear of cells. Wth atomic force (AFM) we iwaged indvidua pores of nsport transcription factors (TFs), which represents a level of gene regulation integral to many nucear cultred Madin-Darby canine kidney cels and cellular processes such as related differentiation, transformation and signal tenumber pores/pm2 to nucear envelope conc (G,. per transduction. lTe yeastTFSWISis excluded from the nucleus in a cell cycle- Pm2) eecialy by current into eisolated nuleus. jcbon dpndebnt fashion,m iad by cyclin-dependentkinase (cdk) phosphorylaon, octagonal subs e and te 30 channel in center nm tepore most likely by C&-kinase. fusion resolved AFM. hours of SWI5 could by Six aldsteron exposue(0.1 PM Ssi proteins canying amino 633-682 the - ieased acids (including nuclearlolization sequence NLS amno te NPs per pm2of nuclearsurfaoefrom 7.4 ± 0.4 to acids 636-655) wer expressed in b ia, ified, labelled, and nu cimort ( ± At same ± (n=12; 0.01). the timeG, rose from 6900 520 to kinetics measurd inHICrat hepaon and VeroAfican Green monkey kldney 9600 ± 610 pS/Im2 (n=18; P ( 0.01). Assuming that NPs represent the cells using microinjection and quantitative confocal laser microscopy. The we to the sole in proteins a nucleusin fashion, conductive pathway tenuclarenvelope we caclated a mean aanNLS-dependent w eby the 980 to Ala the serines single conductance pS, beforeand after aldeone mutation of cdkftt -site (§er646 and 5er664) in the of the inc the exposure, respectively. vicinity SWI5-NLS sed maxima level of nuclear acc laton from about Ito over 8-foldTheseresults conclude hat do ofacilitates nucleocytoplasmic support ourpreviousobsevion for transport by the inhibition of number of NPs SV40large nuclea transport ieasig fe but does not change th blph c tumr-antigen byW-kinase,the mod mmamlian CDC28 correlate, in that cdk-site phosphoryladonreduces the p gstle pore cond . maximal level of nuclear lation The reslts show for the first timethat a yeastNLS and inputularits regulation, can fiucion in higher eukaryotes, implying the un verslnatureof NLSs and mrgulaonof their funcdon. 458a Nuclear Envelope Import, Export, and Structure (2663-2668). Wednesday

2663 2668 NUCLEAR ACCUMULATION OF NLS-P-GALACTOSIDASE RJSION PROTEINS IDENTIFICATION OF THE NUCLEOLAR TARGETING SIGNAL OF BY FACILITATED TRANSPORT AND INTRANUCLEAR BINDING. ((I. Vancwovs, HUMAN ANGIOGENIN. . Moroianu', and J. F. Riordan)) 'Howard J. Rupes, and P. Paine)) Department of BIologIcal Scnces, St. Johns Univerit (a Jamaica, NY 11439. Hughes Medical Institute, Rockefeller University, Laboratory of Cell Biology, New York, NY 10021, Center for Biochemical and Biophysical We previously employed an oil-isolated nuceus (OIN) to sudy nucearaccumuatIon Sciences and Medicine, Harvard Medical School, Boston, MA 02115. of NLS-contaning protins and showedthat aprototplc NLS-prdn,n, nucleoplesln (Np), (I Is translocated through the nuclea pore complex by energy-dependent Angiogenin is endocytosed by subconfluent endothelial cells, located faciitatedttrnsport(f.t.) - but not 'aclve transpor, and (ii) is accumulated by to the nucleus and accumulates in the nucleolus. It also localizes into the intranuear We report here as binding((b.). similar studies using transportant a nucleolus of digitonin-permeabilizadendothelial cells. Colchicine and -ad of NLS-PI-gal fusion protein (gawn sly prvoded by R. Petes), incuiding nocodazole, which thecellular microtubulenetwork, prevent variants containng different portion the SV40 lgT NLS region. We expressed depolymerize fusion proteins in E.ooil and radolabe-ed them in vio. We find: (1) Nucler the nuclear import of angiogenin, suggesting that intactmicrotubules are accumulation the variant (p4K) wIth the wildtype NLS region T resIues111- required for this process. The peptide RRRGLcorrespo to residues 135) is several-fold greater than reported for fluoresoently-Iabed p4K intssue 31-35 of human angiogenin specifically targets non-nuclearcarrierproteins cuiture cNs, posbly beause in vivo lbNlng better preseves native molecular such as albumin, an anti-human nucleolus monoclonal antibody and R33A characteristis of p4K. (2) As in cuitured calls, the varit (p41) with the single angiogenin to the nucleolus of permeabilized endothelial cells. Proteins K128-T128 substitutionis not (3) .b. detectably trnpoted. Thef.tfl modeldeveloped with a 'mutant' RRAGL, are not imported. for Np also holds forthe fusion proteins; I.e.,Np's behavioris not unique. conjugated peptide, P-gal Sinc Fluorescein RRRGLisalso importedinto the fusion proteins,ike Np, bind within thenuclus, intranuclar bindig of NLS- isothiocyanate-conjugated rapidly proteins can be attributed to their NLS region rathe than some other donisi(s). (4) the nucleus and localized to the nucleolus, whereas the 'mutant' peptide A variant(p1 K) containing only theminiml lgT NLS (P126-K-K-K-R-K-V132) is not is not. R33 is essential for nuclear translocation and R31 and R32 appear detctablytransported.Apap nty,this minimal NLS engages the fadliad tnsport to modulate this process. Thus, ,RRRGL,, is a nuclear localization signal mechandsm infficienly,if a"at (5) In contrast, varlants (p8K, p7K) containing the responsible for the nucleolar targeting of human angiogenin. minimal NLS plus either one ofthe two p h sites onits amino flank (S1 1,S112 [CKIIJ andT124 [cdc2J, respectively) do engage faclitatedtransport and do subsequently bind within the nuclus. Thus, these two phosphoryktion sites, previously shown toinfluence transport in cuitured cells, have a profoundeffect on NLS-proteintransportinto the OIN.

2665 2666 Rapid nucleo-ytoplaanic shuttlHg of the p53 tumor uppreor observed THE ASSOCIATION OF THE NUCLEOPORIN, p62, WITH MEMBRANE by Scamp, anewphotoble g technique. ((A. Th S. Jenovai, M. Tschdrich- VESICLES: A MECHANISM FOR TARGETING NUCLEAR PORE Rotter, U. Kubitacec, J.Bischoff#, andR. Peters)) Institut fr Mediziwche Physik uDd COMPLEX COMPONENTS TO THE CHROMOSOME SURFACE. ((D. A. Biophysik, 48149 Mnster, Germany. # Cold Spring HaborLaboratory, Cold Spring Warren andM.J. Lohka)) Department of Biological Sciences, University of Habor, NY 11724. Calgary, Calgary, Alberta, CANADAT2N1N4. The shuttling of proteins between nucleus and cytoplasm may play an important role in the coordination of nuclearand cytoplasmic functions. However, in all casus de- Xenopus egg extracts capable of nuclear envelope assembly can be separated into scribed so far (e. nucleolin, HSP70, progesteron reeptor) shuttlingis slow (hours to two distinct particulate fractions, Nuclear Envelope Precursors-A and -B (NEP-A Here we demonstrate for the first time that shuttling of an important days). regulatory and Vigers & Lohka, J Cell Biol. 112: 545. 1991), which are both required bothin vivo and in vitro. Human fluorescetly was -B; protein can be rapid, labeled p53 in vitro. Vesicles in NEP-B bind chromatin, whereas those in NEP- into the cytoplasm of HTC polykaryons containing 2-6 nucleiand found to be for assembly injected A do not. We have that the nuclear pore protein, p62, associates nuclei a few minutes. Shuttling was previously found accumulated in the within studied byscanning with chromatin whenever NEP-B vesicles bind. Confocalmicroscopy of bound microphotolysis (Scamp), a recently developed technique (Wedekind et aL, J. Microwc., showed that the distribution indirect in press) permitting to photobleach areas of arbitrery geometry and to assess molecular vesicles of p62, visualized by immuno- transport at the full resolution of confocal imaging (Kubitschecket aL, Biophys. J., in fluorescence with mAB 414 (Davis & Blobel, Cell 45: 699. 1986), was almost press). When one nucleus of a p53-injected polykaryon was beached its fluorecene identical to that of vesicles, detected by the lipophiic dye DiOC6. Immuno- and incraed within about 10 min while the fluoresence of non-bleached nuclei cy- magnetic beads coated with mAb 414 depleted NEP-B vesicles from egg extracts, toplasm decreasedslightly. Conversely, when a cell was bleached except for one of its whereas those coated with a control mouse IgG did not. When the mAb 414- nuclei the fluorescence of the noss-bleched nucleus strongly decrease within about 10 coated beads were examined by electron microscopy, vesicles, similar in min while the fluorescence of other nuclei and cytoplasmslight increased. The in ivo morphology to those that bind to chromatin, were found on the surface. observations were complemented by in vitro studies with digitonin-permeabilised cells. However, no vesicles were seen on beads that had been coated with the control The that shuttling of a protein assuming a prominent role in the observation regu- These results indicate that p62 is a surface component of membrane vesicles lation of arapid process for its physiological IgG. cell proliferation is argues importance. in the NEP-B If the NEP-B vesicles were by treatment with Supported by Deutsche Forscsungsgemeinschaft (Pe 138/14-1). fraction. solubilized Triton X-100 before they were mixed with chromatin, p62 failed to bind. However, if NEP-B vesicles were solubilized after they had bound chromatin, p62 remained associated with the chromatin. Since solubilized p62 did not bind to chromatin, a protein other than p62 is likely to target vesicles to the chromosome surface. Supported by N.S.E.R.C. Canada.

26|67 266 POTENTIALKINASE INVOLVEMENT IN NUCLEAR THEDROSOPHILA YOUNG ARREST PROEINIS AN ESSENTIAL ENVELOPE GROWTH. ((DIK Shumakr andK.L. Wilson)) DEVELOPMENTALLY REGUIATED COM NENTOFTHE Department ofCell Biology and Anatomy, The Johns Hopkins NUCLEARLAMINA. ((J.M. Lopez and M.P. Wolfner)) Section of University School ofMedicine, Baldmore, MD 21206 Genetics and Devekoment, Conell University, Ithaca, NY 148532703. nucw encoded the wwlg Using an in vitro nudear assmbly asay derived from Xenopus lalamina proten by Drosophilafs(l)Ya, arreu(), laevis eggs we have tested for potential roles of various kinase geneisessentialfortheinitdatiof eimbryonicnucearcycles This depedentproin, which is only found in during nuclear formation. In general, nulear envelope (i.2). ellcye envelope developing egg cambers ad emlry, is theonlyknownexampleofa assembly involves the following events: vesicle bining to nu lanapotel forwhich ade_Ie function has been to form an nulear chromatin, vesidle fusion endosed envelope, genetically de ed(ls Thelauniniso obetheachitetral pore complex lamina assembly, followed bychromatin backbone of the nuclear envelope, maybean anchg t forinterphse decondensation and furthe nuclear envelope gowt Inhiitors DNA mdmaybe esental forinidatnOfDNA ei n(^ specific for PKC, PKA, tyrosine kinese and Ca/CaM kinase Emlw Ya ip nvelopes wer tested. The data suggsts that tyrosine kinases and (5) Taken crth dhesedata suggest that the roleof Yamaybein nuclear or sruuu. Ca/CaM kinase II are involved in nuclear envelope growth in envelopc formation Toimvestigae his, a)We are vitro. So far we have deterined that vesicles are targeted to implentinq gXIn vitro nuclearofmo asys to test the abliy of mutant todeconden DNA and form normal nuclei (6,7). Our oatn, fuse to form a nuclear envelope, and nudlear pores are emhxyonic exuac show with wild is cruitedto assembled and func when the inhibit are presnt. Our cunentreults that typexexrcs, Yaprotein decondensed chomain and to vitro formed nucei. And b), we are reults su t that kinas acvity maybe required for sein Ya is involvedin the association ofDNA with the nuclear ofnudear envlope gowth. The specific investigatig if aspect sep(s) inhibited ec c Ya protein localizes to nuclar invesigtd are being and associates cb_ Oon latterresult Woam Ya pmmin may as a pu of itarole in nuslenvelope. i. mL wdw(1991)cel 64,49-622. Laps eL aL (1994) Dey. lio. 163: 202-211. 3. Nswpsrt ed Feoba. (1987)A.L Rev. Blioc 535-6. 4. Jkba at aL (1993) J.Cel Se 106,275-255.5. Lia. at aL in psp. 6. Cevel sad Coil (1991) DMO 10. 4361-4369. 7. Buls rd Av;i (1990) lxp. Cel las 191, 64-70. Wednesday. Nuclear Envelope Import, Export, and Structure (2669-2674) 459a

2"6 2670 IP3 RECEPTOR ANTIBODIES INHIBIT NUCLEAR VESICLE FUSION. THE MOLECULAR CHARACTERIZAnON OF NUCLEAR ENVELOPE ((K.M.C. Sullivan and KL. Wilson)) Department of Cell Biology and COMPONENTS IN YEAST. ((C. Strambio de Castillia, G. Blobel, and M. Anatomy, Johns Hopkins University School of Medicine, Baltimore, MD Rout)) Laboratory of Cell Biology, Howard Hughes Medical Institute, The 21205. Rockefeller University, New York, NY10021. (Spon. by T. A. Chen.) We investigated the role of the inositol 1,4,5-trisphosphate (IP3) receptor in nuclear vesicle fusion using polyclonal antibodies raised against different Thenuclear envelope (NE) is a double membraned organelle that defines the domains of the IP3 receptor. At the end of mitosis in higher eukaryotes boundary between the nucleusand the cytoplasm. We are studying the struc- nuclear vesicles reform the nuclear membrane by binding to chromatin and ture, composition and function of the NE in the yeast Saccharomyces. The fusing with each other. The IP3 receptor is an 1P3-gated channel that NE controls the exchange of material between the nucleus and cytoplasm and stores of the reticulum and releases Ca2- from the lumenal endoplasmic is to be involved in the organizaton of chromatin and the regulation nuclear membrnne. Using Xenopus egg extracts, we previously showed that thought of its metabolism. In addition in the NE contains the organizer heparin, a competitve inhibitor of the IPs receptor, inhibited nuclear vesicle yeast, spindle fusion and that this inhibition was specifically reversed by competition with and forms a significant part of the endoplasmic reticulum (ER). We have excess IP3 (Sullivan et al., 1993). We obtained antibodies which were made developed a method which produces large quantities of a NE fraction from against glutathione S-transferase fusion proteins containing sequence from the yeast S. uvarun. This material is proving useful inultrastructural studies either the cytoplasmic C-terminus (3'0) or the coupling domain (M) of the and the localization of previously identified NE components. The NE frac- 1 The 3' and M mouse cerebellar type IP3 receptor (Lin etal., submitted). tion can be stipped of non-lumenal peripheral proteins to yield a nuclear antisera were found to cross-react with the type 1 IP3 receptor Xenopus membrane fraction enriched in NE membrane found in eggs. Pretreatment of the extract membranes with either antiserum (NM) highly integral proteins. inhibited both nuclear vesicle fusion and IP3-stimulated calcium flux Both the NE and NM fractions appear to be morphologically intact and 80- through the IP3 receptor, compared to controls pretreated with the 90% pure on the basis of microscopical examination, the coenrichment of corresponding preimmune sera. In addition, the M antiserum decreased IP3 known NE components and the partial coenrichment of Inown ER proteins. binding to membranes, suggesting that the M antibodies blocked IRs As part of the ER, it was found that both fractions are competent for the post- receptor activation by inhibiting IP3 binding. These results demonstrate a translational translocation of vitro. Various derivatives from these strong correlation between IP3 receptor activation and fusion and support proteins fractions were used to monoclonal antibodies, which will be used to our proposal that Ca2+ flux through the IP3 receptor is required for nuclear produce vesicle fusion. define and localize novel components of the NE.

2671 2672 PROTEIN-PROTEIN INTERACTIONS BETWEEN HUMAN NUCLEAR LAMINS CALCULATED DIFFUSIVE HISTONE FLOWTHROlGH NUCLEAR PORES INTOTHE of NUCLEUS Departrent of School EXPRESSED IN YEAST. ((Q. Ye, and HJ. Worman)) Department of Mediclne and S-PHASE ((MJ. Alen)) Biological Chom

2673 2674 ANNULAR PROTEIN OF THE NUCLEAR ENVELOPE INTERACT IDENTIFICATION OF NOVEL CYTOSOLIC FACTORS INVOLVED IN NUCLEAR ((D.J. Sweet and L Gerace)) (Spon. by J. Westendot) WITH THE PERINUCLEAR CHROMATIN. ISOLATION OF cDNA NKPORT The Scrpps Research Irnite, La Jolla, CA 92037 THAT ENCODES A 70 kD PROTEIN OF THE NUCLEAR ENVELOPE. ((R. Herrera-Esparza, R. Herera-Diosdado and E. Avalos)) lIport of proteins into the nucleus via nuclear pore complexes is a highly Centro de Boogla ExpemrIental. UniverSida Aut6noma de Zacatecas. selhete process specified by nuclear loalization signals (NLSs) on the of Guadalupe, Zacatecas. 98600. Mexico. (Spon. by E. Avaloe) trnported proteins. A number cytosofic transpo factors have been identfed, inludiog the NLS receport and the GTPase RantTC4". but t is icely that many etoe ae also iolved. We are studying cytosolic nucear inport factom using an To address the quesin wheter annular DNA recognized by ant-nDON auto hi vft assay system consisg of digitonln permeablllaed cells supplemented with ibodies, is conplxed wth te protei of the nucear envelope. A Lupus serum exognous cytosol. Addition of bacterially expessed Ran to assays containing a with annular antinDNA auto antbodie resitant to DNAase digstion, was select ha saurating concenron of cytosol resukts In stimultion of transport to the from a group of Lupus patient, and used for the isolation of sevea cDNA lones maxdu bvel, inicratig that in our system Ran is the maJor ratellmItlng cytosoic from a gtl I HeLa ceirs lbrary. componeit Ran is also able to stimulae trnvspor wiout exogenous cytosoL so il that, under sandard preparation conditons, the cells retain The coned fuion protein n the annular anb-nDNA auto antbodis appem peod a significant fraction of the tansport factors. To screen for and the inmunofinify auto antboies elutd from the 0rcobinant Biters, necessary cytosollc novel factors, we have modified the import assay In a way desined to reveal other prduced around the nuclus in fluorescent assa on HEp-2 celis; annularpatem rat-nsng cytosooic componerts besies Ran. In these assays, the also mcognize a 70 kD from cell exract, by Westen blot they proin HEp-2 cels are subJected to a prolonged pre-incubation on ice to enhance loss of E and the induced poin was of Anrular-gt logens made in cob Y1089, hsion cyloplasmic proteis, thereby increasing the dependence nuclear import on the ecognized in Wetem bot by the sera from Lupus patent with annular anti-nDNA exogenous cybosol, and Ran is added to a saturating level so it is no onger rate- autoantibodies. The abily of the annular reconina protin to bind DNA litig. Using this modified assay, we have identiied two apparently novel amssed hin vw, using a minidce of DNA, resuled in a trdation of the DNA cyoic tansport facos that are presnt In both HeLa cel and rat iver cylosol, and -lrsi mobiliy. are currently purifying them to enable fulther cerisation. Our result suggest: 1) The annular DNA in some ukriotic celis is complxed 1. Adam SA & Geacs L Cell 66 (1991) 837-647 2. Meichior F et al JCB 123 (1993) 1649- wih potenfrom the elope. 2) The anuaninuNa autontiboles 1659 3 Mor MS & obs G Nau 365 (1993) 66143 from patient cross react wih per-nucear proten. 3) Intracton between DNA and te 70 kD protin is inducbl i vitro. The ifiance of this n in cell physiology, sat unknown 460a Nuclear Envelope Import, Export, and Structure (2675-2678). Wednesday

2675 2676 THE GTP-BOUND FORM OF THE YEAST RANITC4 CHARACTERIZATION OF A CYTOSOUC FACTOR NECESSARY FOR HOMOLOGUE INHIBTS BI-DIRECTIONAL TRANSPORT NUCLEARLOCATION SEQUENCE-MEDIATEDBINDINGTOTHE NUCLEAR ACROSS THE NUCLEAR ENVELOPE. ((G.Schlenstedt, C. ENVELOPE ((Neil Chi, Ermon6J.H. Adam, and Stephen A. Adam)) Department Saavedra, C. Cole and P.Silver)) Department of Biological Chemistry of Cell and Molecular Biology, Northwestem University Medical School, and MolecularPhmacology, Harvard Medical School and Dana Chicago, Illinois 60611 Farber Cancer Institute, 44 Binney Street, Boston, MA 02115 and Deparment ofBiochemistry, Dartouth University Medical School, Hanover, NH 03755 Nuclear protein import can be separated into at least 2 disfinct steps: binding to the cytoplasmic side of the pore complex followed bytranslocation across the nuclear envelope. The binding step can be reconstiu in vitro with two Ran/TC4, a ras-like GTP-binding protein, and its nucleotide exchanger, RCC1, have been implicated in control ofvarious nuclear- cytosolic factors: a nuclear locafton sequence (NLS) recqptor and a recently related events including tansport ofproteins and RNA. The yeast described 97kD protein (Adam and Adam, J. Cell Bid. 125:547-555). Both Saccharomyces cviae conins twhomoues of the mammalian factors are required simultaneously for NLS-mediated binding to the nucear RaWTC4 encoded by the GSPI and GSP2 genes. Ispl makes up envelope in a permeabilized cell assay. A monoclonal antibody, 3E9, specific about 0.5% of total yeast protein. Yeast strains have been constnuted for p97 localizes the protein to the nuclear envelope and the cytoplasm by that overproduce 2- to 4-fold either wild-type Gspl or a form ofGspl indirect immunofluorescence and cell fractionation. Furthermore, 3E9 with glycine 21 converted to valine (Gspl-G21V), which should specifically inhibits nuclear transport in vitro yet does not inhibit the binding stabilize the GTP-bound form. Cells producing Gspl-G21V have step. A human cDNA clone has been isdated and sequenced and encodes a defects in localization ofnuclear proteins; seveal different nuclear in the cytoplasm following induction ofGspl- 97,233 dalton protein. The protein contains a unique sequence and none of the proteins accumulate repeat sequences identified in nucleoporins. We are attempting to identify G21V. Similarly, cells Gspl-G21V have a defect in export producing interacting with the NLS receptor/p97 complex. Potential RNA the to the cytoplam In an in vitro nucleoporins ofpoly (A)+ from nucleus nuclear system using semi-intact yeast cells, cytosol prepared from cells nuceoporin candidates have been identffied from partially purifed producing the mutant Gspl does not support nuclear protein import as envelopes. well as cytsol from cells overproducing wild-type Gspi. These findings suggest that hydrolysis of GTP by Ran1rC4 is important for propr -directonal traffic across the nuclear envelope.

2677 2678 PURIFICATION OF A RAN INTERACTING PROTEIN THAT IS DIFFERENTIAL ROLES OF HEAT SHOCK PROTEIN 70 IN THE IN VITRO ((M.S. NUCLEAR IMPORT OF GLUCOCORTICOID RECEPTOR AND SIMIAN REQUIRED FOR PROTEIN IMPORT INTO THE NUCLEUS. VIRUS 40 LARGE TUMOR ANTIGEN. ((D.B. DeFranco, N. Xiao and J. Moore and G. Blobel)) Laboratory of Cell Biology, Howard Hughes Yang)) Department of Biological Sciences, University of Pittsburgh, Medical Institute, The Rockefeller University, New York, NY 10021. Pittsburgh, PA 15260.

Previously we reported the isolation of two cytosolic fractions (A Nucler import of glucocorticoid receptors (GRs) was ualyzed in vitro using and B) from Xenopus ovary that are required sequentially to support digitonin permeabilized cells (S.A. Adam, R. Stere Mar, and L. Gerace, J. Cell Biol. 111:807-816, 1990). Indirect immunofluoreAcce metbods were used protein import into the nucle of digitonin-permeabilized cells (Cell to monitor the transport of GRs from rat hepatm and fibroblast cell cytosol 69, 939-950, 1992). Fraction A is required for nuclear localization into HeLa nuclei. In vitro nuclar import of GRs was shown to be hormone- sequence recognition and targeting to the nuclear envelope while dependent, NEM-sensitive, and require ATP, GTP, and incubation at ambient fraction B Is required for the subsequent translocation of the bound temperatures (i.e. 30C). Hormone-dependent dissociation of GR-bound substrate into the nucleus. The first protein required for fraction B proteins such as the 90 kDa beat shock protein, hsp90, is part of an was ran nuclear activation process that is obliptory for the expresion of the rceptor's DNA- actMty to be purified the small GTPase (Ms related binding acdvity. Inhibition of in vitro GR activation by Na2MoO4 blocked protein) (Nature 365, 661-663, 1993). We have now purified the hormone-dependent nuclear import demonstrating that receptor activation is second (and final) protein required for fraction B activity. By SDS- required for nuclear imporn The addition to GR-containing cytosol of PAGE, the purified protein (p10) appeared as a single band with an antiserum directed against the cytosoUic 70 kDa heat shock protein, hsp70, apparent Mr of 10 kD but the native protein fractionated upon gel while effective in blocking the nuclear import of simian virus 40 lrge tumor filtration chromatography with an apparent Mr of 30 kD. In addition, antigen (SV40 TAg), did not affect hormone-dependent nuclear import of endogenous, futl length GRs or an exogenonly added truncated OR protein (ie. ran an regard to nuclear p10 and appeared to form active (with XGR556) that tacks a hormone binding domain but posses a constitudvely import) complex of 55 kD. Substrate prebound to the nuclear active nuclear localization signal sequence (NLS). Likewise, depletion of envelope with fraction A could be chased into the nuclear interior by hsp7O from HeLa cell cytosol did not affect the nuckar import of exogenously the subsequent addition of p10 and ran indicating that these are the added XGR556 but led to inhibition of SV40 TAg nucka import. Since both only two soluble proteins necessary to catalyze this stage of nuclear GR and SV40 TAg were shown to be associated with bsp7O under nuclear in import conditions, the formation of an hsp7O-karyophile complex is not import digitonin-permeabilized cells. necessarily predictive of a requirement for hsp7O in the nuclear import process. We are currently investigating whether distinct structural or contextual differences between the GR and SV40 TAg NLSs are responsible for differential hsp70 requirements in nuclear import. Fertilization III: Egg Activation (2679-2680).

2679 2"6 THE RISE IN INTRACELLULAR pH AT FERTILIZATION IS NOT AN ABSOLUTE ABSENCE OFA SIGNIFICANT CANGE IN INTRACELLULAR pH AT FERTLIZATION IN SEA REQUIREMENT FOR THE ACCELERATION OF PROTEIN SYNTHESIS OF THE MOUSE EGG. ((D. Kline and J. ZWray)) Depament of Biogical Sciencs, URCHIN EGGS. ((B.B. Rees, C. Patton, J.L. Grainger, and D. Epel)) Deparment d Kent State Unie , Kent, OH 44242. Biological Sdcnces, Stnford Unheralty, Pacfc Grove, CA, 93950 and Bolgy Depament, Sant Clara Uniesty, Santa Ciara, CA, 95053. The intracellular pH of ft mouse eg was meestrd durng fert1ization to dtrmire We hav reevaluated the premed requirement for an elevated Intraelar p1H if an increase in pH accompanies activation of the mous eg. The intracliar pH of (pH,) in the aceraIon of protein synth which occum at the tme of ferllzaton hI some invortebate and amphbian egs increases at fetilizafton and, in some cases, eggs the sea urchin Ly.d m pkbu. Early emryo and ly- vd suggets that alkainizatn of the oplsm may be an inpoa cmpornnt acfvfad egp were himbated hI sa water at a low pH (6.85) containing a pefmant of activation (Gould and Steph , 1993, DovBioIl5 608). The dye, weak acid to prevent or reverse the rbe in pH, that noally enstue upon fertlzatlon. was introuced into tie Usn thefluorheent pH probe 7,r-b(carboxy y)-5(6)-cayesce, we BCECF [2,7'.bis(2caaboxytyl)5(and-6)c yflureaci mouse egg in BCECF-AM or midrolaction showed that these teatmnts hold the pfiA, near the urfertdized value. Rates of by incubation by iioporaton of adoled ieucine nto protein, hower, rose to at least 50% d BCECF. Thecellswweralso dedwiththe DNA-peci rchome, Hoect3334Z contro alues in more han hal the eerime, and exceeded 90% d control to confirm fetiatn by obrvaon of Hoectsained, decondensing perm heads in value inhabot equter d the eerit. We also assesedleucine the cytoplasm. The ratio of emi ins for th dye (495/440 nm exctaton incoporation icbaton of and embryos hi sodkum-free sea water or sea during egp wavenhs) was moadno continuousy wh a oto-oun phoomulpr tue. water containlng arnilorlde, two addlormnal tratnt that block the p-, rise. In the Ther was no increase in the ratio Xth re Contol e9g presec dofrillorkde, Iucdne incorporation inreesd upon ferdtIaton, whereas In ftle or no hcrese was obswevd hI sodIufee sea water. We provide evidence displed the ced incrase in pH when expsed to NHCl. other exeriments, that the low ratesdoucle icporaton hI sodium-fre sea water result from the intracluar pH and intaceluarCa+ wer motoesimunouduringfel* 1t tendncy forth ep im condIon t iowerthe pto valu sgnfcany lower The ewsr injoecd wih BCECF dextran and Fura dexan. Fluor embion aseml of rlbowm into than the pH, In urderfllzed egs. The poysomes, was recorded at excitaion let 4o nm (BCECF, pH enIt waveength) anodtr index of proten syntheI, incrsed to asbot 50% of the control vale in and 385 nm (Fura, Ca" sensitive w ). A dcm emIsn intensiy at 385 embryos whose piH, was lowered to thte unertIzd oevel withIn minutes ferldzatlon. These flndngs cal hto doubt the bel that a rise in pH, is esenti for rnm excitaton clarly marked th repe CaWWs at eg activat. The was emittdat 495 angebse of the on of huk pioesin sythei. We suggest that the increase in p1, Is no chadw in theflu sc rnm excitation, any only one d severa sIgnals involved, and that in ome case stImulatIon by other increase in intracelular pH. These results inicae hat itacear ahlinization of th sgnals may be sufficen for the accelratIon d protein synhesis at the time d cytoplsm does not accompary activation of this ve fertizaton In sea urchis. Wednesday. Fertilization III: Egg Activation (2681-2686) 461a

2681 2682 TRYPSIN ACTIVATION OF STARFISH EGGS.((D.J. Carroll and CLONING OF g69,64 AND OVOCHYMASE FROMXEVOPUS L.A. Jaffe)) Department of Physiology, University of Connecticut LAEVIS EGG& ((.C. Yang andJ. L. Hedrick)) Sectko of Molecular and Health Center, Fa ming CC 06030. of California, 95616. (Spon. by U.A Urch)Cdlular Biology, Univesity Davis, Appication of the endoproteinases trsin or chymotrypsin to eggs of the starfish Asterinaa mniafa caused responses like those seen at Upon ferilization, thevitelline envelope of Xenopuslaevis egg undergoes fertilization. Within 1 min after exposure to 100 tg/inl trypsin (bovine limitedbydrolys a the result of cortical granle exocytoai. Exocytosis pancreas or recombinant) or chymotrypsin (bovine pancreas), cortical actvates a chymotypainlike protease (ovodcymase) which removes a 3 kDa granule exocytosis andfertilizaton envelope elevation occred. These C-terminal firgment fromgp69,64. We are cloning gp69,64 and ovochymase proteases also simulated an increase in intracellular free calcium, as to study their finon retions. An oocyte cDNAlibrary of and strur detected by use of the calcium indicator calcium green dextran, Xenopus waa constructed in XZAP and saeened with anti-deglycosyated stimulated DNA synthesis, as detected by 2-bromodeoxyuridine pp69,64 antibodies. Poitive clones were plaque purified and the cDNA incorporation. Egg activation was not seen if the enzymes were inserts were sequenced by the dideoxychain Mination method. Degenerate pretreated with the protease inhibitorac2-macroglobulin, or if a pimscoesponding to the N-terminal peptidesequence ofovochymae and recombinantbypsin with a point mution thateliminated its proteolytic to theflankixg ine active site of seine proteases were prepared to PCR activity was used. Several other proteases (plaswin, thrombin, LysC, or amplif thecoding sequence for ovochymase. A 550 bp PCR firgment was Arg C) had no effect on the eggs. Trypsin orchymotrypsin tatment of subcloned using the TAcloninglkt and sequenced. This sequence was PCR munmture oocytes did not cause a calciuminrease or exocytoss, and did extended to the ends ofthe protein using the cDNAhlbrary. The cDNA clone an a and asequnce naling not stmula germnl vesicle breakdown. However,ifimnua ure ooytes ofgp69,64 had open readingframe, stop codon, molecular weretreated with trypsn and theninduced to mature byapplying 1- polyadenylation. The ORF contained 376 amino acidswith a methyladenne,their abilitytobefeilimzed was derased.If thetrypsin- weight of 42.2 kDa, smaller than the predicted size of 54 kDa and indicaing treated oocytes were cultured for 16 18 hours beforebeing matd, missing of the N-terminal end. When compared to published sequences, the the ability tobe ferdlized was reacquired. These reslts suggest that partial sequence ofgp69,64 was509/e similar and 30/ identical to a region of trypsin and chymotrypsin act on an egg surface protein that is required mammalian ZP2. Other featues common to gp69,64 and ZP2 included for egg actvation atfertlizaon. multiple Nlinkced glycoyation sites, conserve cyseine residues, and a predic-td C-trminl brane following the Arg-Xaa- Lys/Arg-Arg motif for finn cleage. The amino acid sequence of ovochymase cDNA was 40/ idenical to chymotiypsinogens from different forming the were homologous toenzymes that are phenylalanine-specific. (Supported in part byHD4906.)

2683 2684 A PROTEIN OF THE SEA URCHINCORIICAL GRANULES CONTAINS AN SEPARATE CALCIUM DEPENDENT PATHWAYS CONTROL FERTILIZATION IN LDL-RECEPTOR-LKE MOTIF. ((G.M. Weel)) Department of Mocular and TRIGGERED GLYCOSIDASE RELEASE AND CORTICAL CONTRACTIONS and C. Sardet1)) CeDular Biology & Bioehemistiy, Brown Univernity, Providence, RI 02912 ASCIDIAN EGGS ((C. Lambert1l2, A. McDougal1, 1-UA 671 CNRS/Paris VI, StationZoologique, 06230 France; 2- Calif. State Univ., Corfical granules ofeggs contain a population of heterogeneous proteins that are Villefranche-sur-mer, Biology, Fullerton, CA 92634 sequestered selectively within the cortical granule dunng oogenesis and that participate in the block to polyspermy during the ferfilization reaction. To beginto understand the Fertilization or rapid release of cell targeting mechanism of proteins to this organelle,we have identified cDNA clones that ionomycin induces surface responsible for the block to encode proteins ofthe corfical granule. Herewe characterize the protein encoded by N-acetylglucosaminidase polyspermy andcortical contractions leading to ooplasmic the cDNA, SFE 9, which is packaged specifically into the spirallamellae ofthe cortical segregation in Pballusia mamillata eggs. Glycosidase release granules in the sea urchin puepuratu. The SFE 9 protein showed a Strongylocentrotus without cortical contractions occurs in response to 100 iM molecular mobilit in PAGE of 150 kDa, close to the estimated 128 kDa predicted by isoproteronal, 2 mM carbachol and 100 pM acetylcholine. and contained three regions of distinct repeating sequence. Eachtype cDNA sequence, Ionosycin (2.7 piX) in Ca free SW causes cortical contractions was of different length, frequency, and amino acid composition,but of repeat sequence without glycosidase release. Both processes are blocked in a ofconservation within a repeat type. A fourth repon of eachtype showed high degree BAPTA-AM loaded eggs. Thus the two events are Ca dependent but N- sequence showedsimilasity to the low densityhpoprotein (LDL)-receptor motif The can be independently activated. The glycosidase is released a terminal2/3 ofthe protein contained an abundance of cysteine residues with regular and few seconds after fertilization but the first cortical conserved spacings. Upon fertilization, SFE 9 was secreted and was rapidly incorporated contractions occur 5 min. later. Activation by ionomycin into the fertilzation envelope. The integration of SFE 9 into the fertlization envelope results in the first contractile peak occurring at around 1 was complete within 5 minutes post-fertilization and was mediated by ovoperoxidase min. In calcium green injected eggs the cortical contractions catalyzed crosslinking. Incorporation of SFE 9 into the fertilization envelope was not occur 40 sec after the Cai has increased by either sperm or uniform. Instead it concentrted to the microvillar casts both in normal and in ionomycin and further support the role of raised Cai ovoperoxidase inhibited fertilization envelopes. Thus SFE 9 shows molecular triggering the contractions. Taken together, these experiments by Ca heterogeneity within the fertilzation envelope and the targetedintegration is suggest that the cortical contractions are induced independent ofovoperomidase activity. The strong repeat regions and the LDL-receptor release from intracellular stores and that actual Ca influx may be more important for the glycosidase release. Thus motifofSFE 9 are poulated tobe used in specific protein-protein interctions that events that occur either duringtrafficking to the cortical granules during biogenesis or to the ascidian eggs are activated by two Ca requiring fertilization envelope during the fertilization reaction. are functionally independent. Supported in part by NSF grant DCB-9004735

2685 2686 CA2' WAVE PROPAGAnON FOLLOWING FERTlIZATlON IN FROG EGGS EFFECT OF SULFATED POLYANIONS AND CAFFEINE ON SPERM INVOLVES PIP2 HYDROLYSIS AND DOES NOr REQUIRE CA--INDUCED CA2 INDUCED ACTIVATION CURRENTS AND ELEVATION OF RELEASE ((Paula Snow, Sunita K. Saini Eric D. Zee, Jeffrey D. Leibow and Richard IN SEA URCHIN EGGS. University of California, Davis, CA 95616. CYTOSOLIC [Cae]i VOLTAGE CLAMPED Nuccitelli)), ((T. Mohri, P. I. Ivonnet, and E. L. Chambers)) Department of Physiology Sperm triggr the hydrolysis of PIP2 is the frog (Xenopus laevis) egg plasma menmbrane to and Biophysics, University of Miami School of Medicine, Miami, FL 33101. podc Ins(1,4,5)P, whicb rdeases Cae from the eidoplasnic reticulum. We have measred the endogenous production of both Ins(1,4,5)P, and PIP2 during the sperm-induced Ca(2, wave examine the two mechanisms of intracellular Ca2' release (IP3 2 To further eg and find that Ins(t,4,5)P3 increases 3- to 5-fold, peaking as the Ca wave is the frog induced Ca2' release or 11CR, and Ca2' induced Cae release, or CICR), reacas the antipode. While one might expect PIP2kvels to fall as a resit of hydrolysis, we were out voltage clamped at -20 fnnd PIP2 increases following fertilization (about 5-fold) as originally reported in sea urchin simultaneous measurements carried (eggs the increase of eggs. The unfertilized egg membrane fraction exhibits 0.2 pmoles PIP2 per egg and this mV) on the Ca2' dependent activation current, Ip, and increases to 1 pmole per egg by the time the Ca2 wave is half-way across the egg. We are cytosolic Ca2' (using Calcium Green-I) induced by sperm. Eggs were also exploring the mechanism of Ca2 wave propaation and find that Ca2t-induced Ca2. inseminated eggs only) after (a) microinjection with 0.5-1.0 Thapsigargin (monospermic release (CICR) is not required for sperm-induced Ca2 wave propagation. (5-6,000 MW), (b) exposure to 10 mM caffeine starting 5 min specifically inhibits SERCA-type Ca2 pumps in frog eggs without triggering Ca2 release from mg/ml heparin to both with heparin and exposure to 100 blocks CICR in response to the microinjection of well defined, prior insemination, (c) microinjection the ER. pM apsigargin delayed the time buffered Ca soohtions in 60% of the injcted eggs, but sperm-induced Ca2 waves still occur caffeine as in (a) and (b). Heparin at all concentrations (1) of in thapsigargin-injected eggs, exhibiting a 50% increase in propagation velocity compared of occurrence and amplitude of Ip, and (2) delayed the onset of increase with controls. This suggests that CICR requres the pumping of Ca2' into the ER for wave [Ca2ji, slowed the rate of increase of [Ca2']i and diminished the peak Pura-2 ratio-imaging of thapsigargin-loaded eggs during the microinjection of propagation. amplitude of the [Ca2ls increase. The only major effects of exposure to Ca2e buffers rveals the expected local [Ca2+]i increase at the injecion site, but we observe peak amplitude 50% caffeine were to decrease the amplitude ofIp and diminish the no regenerative release. Inslead the [Ca2+1, increase spreads passively, falling by about increase. The combined microinjection of heparin and exposure in magnitude 250 pm away, and by 91% 500 jam away from the point of injection. We ofthe [Ca2ei of Our conclude from these studis that CICR is not absoheldy requd for sperm-induced Ca2' wave to caffeine obliterated Ip and nearly eliminated the increase [Ca2li. initiation or propagation. Howev, under normal conditions (without thapsigargin) CICR data suggest that a caffeine sensitive Can- release mechanism (CICR) is probably does contribute to wave propagation. present in Lyechinus variegatus eggs, and that when 1CR is inhibited by (Supported by NIH gn ND-19966) heparin, other Ca release mechanisms (e.g., CICR) may not be able to fully replace the deficit in intracellular Ca?' release. Supported by NIH HD19126. 462a Fertilization III: Egg Activation (2687-2692). Wednesday

2687 2688 INHIBITORS OF CYCLIC-GMP-MEDIATED CALCIUM RELEASE IN PI TURNOVER AFTER FERTILIZATION. ((D. Roberts, A. SEA URCHIN EGGS DO NOT BLOCK THE RISE IN CALCIUM DURING Ferdensi, T. Smart, R. Espinoza, B.J. Stith))Department of Biology, University of Color- FERTILIZATION. ((S.J. Lee and S.S. Shen)) Department of Zoology & ado at Denver, Denver, CO 80217. Genetics, Iowa State University, Ames IA 50011. After fertilization of Xenopus eggs, the mass of A number of different agentsmay trigger the release of calcium from intracel- inositol 1,4,5-trisphosphate (IP3) and sn 1,2- lular stores in the sea urchin egg. These calcium release agonists include both diacylglycerol (DAG) increase. DAG increased independently of elevated intracellular calcium cyclic ADPnbose (cADPR) and inositol 1,4,5-trisphosphate (InsP3), and dif- and peaked at -45 min after insemination. This ferent pathways for their production during fertilization have been proposed. increase was -1000-fold larger than that of the One proposed pathway for regulating calcium release during fertilization is the IP3 peak which occured at 5 min. As opposed to conversion ofP-NAD to cADPR by cGMP-dependent protein kinase activa- IP3, DAG mass declined during cleavage. Following tion of,B-NAD glycohydrolase. We have tested this possibility of cGMP-me- a similar time course as DAG mass, there was an increase and decrease in translocation of diated production of cADPR during fertilization using specific inhibitors of protein to a particulate fraction (as detected by protein protein kinase and glycohydrolase activities. Rp-pCT-cGMPS and H8 are two kinase C antibody). To determine the source of specific inhibitors of cGMP-dependent protein kinase, which blocked cGMP- DAG, phospholipids from Xenopus eggs were separat- mediated calcium release in both egg homogenates and unfertilized eggs. ed by TLC, metabolized to the diglyceride and However, in eggs preloaded with heparin to block the action of InsP3, the ki- derivatized. After GC, the molecular species of each phospholipid was compared with DAG obtained nase inhibitor did not block the transient rise in calcium during fertilization. A after fertilization. We suggest that phosphati- potent inhibitor ofP-NAD+ glycohydrolase is 3-aminopyridine NAD, which dylcholine breakdown contributes to the production also blocked calcium release induced by cGMP but had no effect on heparin- of DAG. We have also shown that IP3 increases insensitive calcium release during fertilization. These observations suggest during cleavage and now report that colchicine, which prevents cleavage the cGMP-mediated calcium release in the intactegg may occur through cGMP- in XenoRus zygote, did not prevent the IP3 increase. dependent protein kinase activation ofP-NAD glycohydrolase, but this path- way is not required for calcium release during fertilization.

2689 2690 ACTVATION OF SICYONIA INGENTIS OOCYTES BY EXTRACELLLAR ROLES OFHETEROTRIMERC AND MONOMERICG PROTEINS IN MAGNESIUM: ROLES OF IP3, G4PROT NS, AND TYROSINE KINASES. ((LL SPERM-INDUCED AClVATION OFMOUSEEGGS. ((G.D. Moore, T. Undesy and W.H. Chrk Jr.)) UnIversiy d CallfornrI Dav Bodep Maie Ayabe, P.E Visconti,R.M. Schultz RM.*, G.S. Kopf)) Dept of Ob/Gyn., aboratory. Bodepg Bay, CA 943 Div. of Rerd. Biol, and Dept of Biology*, University of Pennsylvania, Philadelph, PA 19104. (Spon. by G.S. Kopf) Oocytes of the maern shrimp Sicyos hgefl are naturly acvated upon Spe-induced egg activation has several featues in common withG conta with _aear We inetgated the mechan d Mge-4nduced mchanisms in somatic Mgf'. preceptori sipal many iracedular Csd rel cortca cortraction through trmt of PFIxo3- Cells. We rent hat of GDPpS into metaphase i-arrested oaded oocytes wth varius acvators and Inhlbitors d sIgn aeon egs sperm-n ced eg activation. Since GDPFSinacdvates patways and =amnation usig conioca no . Injedon of oocyte wih both e and mes of£G proins, the involvement of the seond en _gr inodol 1,4,lephosphste reet*ed In an kmed deb In members ofeach ofdtese familie in sperm-indced e activation was ktracedkiarCP' and nornal orntIcal conrton Bycor eon hdtheGTP evaluated. Neith perassis toxin etment ofeggs nor micrinjection of anaog guao 5-04(3%thmirhoephlnt) to actvate Gipotelns did not a1ec eggs with inhibitory andbodies towad Gaq blocked sperm-induced egg ltracwlular CW+ but did Idue cordcal contraction. Tyroelne kna atvation. Nevertheless, mcrnjon ofphoducin, aprotein that binds spefially hhlbrs (r tand podne) uwPp d the Mg+nduced Cs+ ris tightly tofree G ptein pysubunits, inhibited polar body and contraction, and the Inhibton could be overcom by with Inok ol isson, the faelizaon-evoked decse of HI inase activity, and 1,4,54trlbhepho lmunopreclpion -of orin-containing proen us formain (PN). Phosducin did not, however, inhibit the from oocyte Iys showed dt a 110 kDa protein was ph whIn on induced ons of the zonapelucida (ZP). Inacdvation of seconds of oocyte poeur to Mg+. Phopr on this protein was Inhlbited the noc Rho famuily ofG proiS with C3 transferae from by typhostr. Pretreatent of oocyte wth the prot trypsin abolhed eIr Closridwm bondinws inhibitedpolarbodyemission and cleavage to the two ablity to refem Cs+ In reepones to .xtacear Mg!+, Indiang a rob for a cil cell stage, but did not affect the mdi ns of the ZP, orPN formation. sufce protein duringnormnl oocyte actvaton; reated oocyte could be reeued Micrinjecdon of Rasv'112, which is aconstitutively active form of Ras, did by ietd 1,4,5trphophe IedorL Thene sgge S. s not result in egg activadon in the absence of spem. Moreover, microinjection oocytes ae acivated though a Mg!+ Ifeceptor which acivates a tyroeine khn_ ofeitheran anti-Ras neutralizing antibody (Y13-259) oradominant negative and In the Inoed to produton 1441rphoephate reemb Itroular form ofRas (RasT) did not affect events of spem-induced egg activation. In Ca+ stores and Indue corcal contction. A G-proten/GTPas may abo be contst, m njdon of RasT inhibited cleavage to the two-cell stage. iwolved In the pathway eadIng to Coril conrat This work wu These results suggest that both hermeric and n c G proteins are by USDA Gmnt 87 CRCR-1-2514, and CalionIa Sea Grant NAtfM-D-SG140. involved in varous aspects of periniced egg ativation. Supported by the NIH (HD 22732, 074,22681), Rockefeller Fdn. and LalorFdn.

2691 269 PROTEIN KINASE C ACTIVATION INDUCES CYTOSKELETAL CHANGES IN CDC2/CYCLIN B KINASE (MPF) AND MITOGEN ACTIVATED REORGANIZATION IN HAMS, BUT NOT IN MOUSE EGGS. ((G.D. PROTEIN KINASE (MAP KINASE) FOLLOWING FERTILIZATION OF MOUSE Moore, G.S. KCopf, ad R.M. Sdcultz*)) Dept. of Ob3yn, Div. Reprd. Bhlo, and EGGS AND DURING THE FIRST CELL CYCLE ((J. Moos.1t2 p. E.Vownt5l, G. D. Dept of Blolbgy, Univeity of Penmylvai PA 19104. (SpOnIby Moors,1 G.S. Kopfland R.M. Schultz2)) Div. of Reprod. Biol., Det. 10betet. and RM Scbltz) Gynecol and 2Biol., Univ. Pennsylvania, Philadaiphia, PA 19140 (Spon. by S. Heyner) Pro lkae C(PKC) acivaon by diacyllycerol (diCS) or orbol 12- mytistte-3-actut (PMA) indtce events of egg For Chnges In the activl o the p34cdc2lcydh B complex (MPF) and MAP Wnca have example. fensmpdon of the cell cycle, soond polr body ssio d been analyzed following hi vkro fertlization or after calium onophore-Induced wem, followIng AbrdI nae arrestd activation in the mouse. While the histone Hi kinase activt of MPF decreased to bsl ( het aL 1993). Tratoment s with P ordiCS. leveb within I h following insemination, the kinas activIy towad myslin basic protein ationofth wDC ID that obscied (MBP) ubltrae pept,de which is atrbuted to MAP kina, deceasd more slowiy and levei -7 h insemination. When in vhm or in vIo ftizatlo otl w notobrved t A folowing fetiiized but cycle (Endo 1987). th were cuitured to 2-cell stage and analyze for the kinke actvis, a sharp Sice observatIon ih r mous gg wee p d by two difet rie In HI kinas and, to a ber extent. MBP kinase aciviy was found 2-3 prior to labs, we evauatd Ift c n se t_ were due dtfo cs in the cleavage. Le, the inceas in kinas acthvis correld wth etry t M phae. Both used forPKC a nordue spcie dffnc M ase H- xelku activite declined again prior to cWavage. The chang in the acdiviy of HI kinase were _aster ormonse were stImled wh PMA forS min. or iarrestd egc (100 nM) consisent with the destruction and reynthesis of Cyclin 01. as assesse by d wih diCB (250 pM) for lh. PKC actvato inhansewegp resulted In 1) the immunobotn. and with chae in th state of p34cdc2 onof"seoodp rbody-Mm" stuctr (PBS) 5 im after k*Shitbtrphorecin mobily. On the othr hand the state of P n ia 2) a Incse filiuneos acwdn in die region of these PBS; kine did not change ethr within 2 h folow or 2-3 h prior to 3) the sssembly of spindl drosbules. Cell cycle reumpIon, assesed clvage ahouh MBP uinas activty caed dramatialy the same time. by DAPI stI and a dmese In Hi kInae acvity, w not decetd. PKC Imm experiments demontrated that caes In MAP kinase-fke acAtIty were lIkely due to the abily of to the MAP kinase actvaon in moue eg did rensut in the bmaton of a PBS, did not cause an MPF phosphorilste phoephoryistion ite on MOP. MAP kinase was -aed at least 7 h after incrae In udn, ad did not result in cel cycle sumpton; t did, Insmination and once dphwphoryated it was not throughout the however, luidcdsmsembly of snle We conclude that fte rephobphorytd duration of the first ce1 cycle and during the entry into the scond cell cycle. "spperent acdvatlonofhatereg by PKC activaor is due to the effect of the Dephoephof_yist of MAP kWnes (erk 1 end of2) cfordW tempy with the onet rnts on re ttII Of 1ronucleus (PN) rman Moreove tr nt dfeIzed gs with okakdac acid (OA) lmilar ptaorbodies mid that the changs occur in the abence prevented both th depprylation of MAP kWne and P foration. In adddIion, ofcell cye ti. lbe dIfferene soen in mouse nhamster egg ae likely once a PN formed. OA induod precocius nucear nvelop breakdown tha was n due to dl I senvity of cy l rean Induced by correlatd with MAP kinas r-phoehoryistion and with an min M OP kinase actvty. PKC acivaor Supponed by tie NI (HI 22732,06274,22681), Rockefeller and Thes changes occurred en in cycloheximidetreated enbryos, where no Inrse in Hi lalorFdn. kinass actvIy was found. Taken together, these finings suggst active MAP kinas mayo6daton.he Ivolved in aspects of PN dynamics Supported by AID, NIH and the Rockefeler Wednesday. Fertilization III: Egg Activation (2693-2698) 463a

2693 269 SPECTRIN AND ACTIN CYTOSKELrON OF THE ZEBRAFISH BGG REGULATORY ENZYME INDING IN SEA URCHIN EGGS: TO CORTEX. ((K.A. Becker and N.H. Hart)) Department of Biological Sciences, Rutgers University, Piscataway, NJ WHICH PROTEIN(S) DOES GLUCOSE4-PHOSPHATE 08855. DEHYDROGENASE BND? ((H. KIBAK and D. EPEL)) Department of Bbbgy, Stanford University, The actin cytoskeleton of the zebrafish egg undergoes Hopidne Mrine Station, Padci Grove, CA 93950. dramatic reorganization following fertilization. We have previously shown that filamentous (F-) actin, non-filamentous The flow d carbon through the hexose Mn_phosphate shunrt hcreaes actin, and syosin-II are constituents of the unfertilized egg markedy by two minutes after fertilization. This pathway is the cytoskeleton. The cytoplasmic faces of cortical granules principal (CGs) appear enriched for actin monomer and myosin-II. Upon souce of reducng power in most cos, crul for biosyrhetic reactions and egg activation, actin gradually assembles at individual sites redox-reguisted prcees. The rae-*ing enzyme is _e of CG release. We are extending these studies to include the dehydrgenase (G6PDH), which catayz the oxidation of gkxcose6- identification and spatial localization of a-spectrin in phosphate and th reducon d NADP+. In unfertlized Strangybcenfrtus unfertilized and activated eggs. SDS-PAGE and immunoblot pcapwatw egg hotmges G6PDH is associed wih a low-speed of unactivated fractions have identified two analysis egg particulate frction and is hilM y inhibited. Upon fertlilzation G6PDH actvity closely apposed bands of approximately 215 and 220 kDa which inceses and the enzyme is bund in the d isanoreact with sea urchin egg a-spectrin antibody. Whole aupematart homogenates. This egg mounts, cryosections, and fragments of egg cortices were uranWdn and release fror inhbifton of G6PDH may be a general component prepared for fluorescence localization of a-spectrin, actin, of cel acdvation as the ph en has also been seen in mammalian cell and myosin-II. Several distinct intracellular domains of after growth facor stimulation.t1) In this study we attempt to identity spectrin were observed. In the unfertilized egg, a-spectrin the celber constit involved in the revwibie, and probably colocalized with F-actin at the plasma membrane and was reguiory, binding of G6PDH. The capacity to bind G6PDH remains with associated with microplicae and the subplasmalemmal the partulate fraction even after extaction with 5% triton X-100, 2 M KOA and cytoplasm. The largest domain of a-spectrin was present as a dense meshwork throughout the cortical cytoplasm. 8 M urea, but is ba upon trea_tmet wih protease. By incubation overnht in Cryosections specifically detected a domain of spectrin 8 M urea, 100 mM DTT at pH 9, we were able to solubilize some of the binding around the surfaces of intact CGs. These differences in activily as determined by a Far Western' approach of probing blotted proteins spatial distribution suggest that spectrin has multiple with G6PDH. The blots were then assayed for G6PDH activity using in the zebrafish functions egg cortex. These include a mehosute niro blue tetazoium dye precipitatin. Several of structural barrier to release in the F-actin-spectrin CG theMhighly Insoluble proteins In the 30 to 100 kDa range bind G6PDH. Their unfertilized egg, facilitation of CG movement during pper n renbl termedi flament secretion, and transformation of evacuated CG sites following proteins. exocytosis. mSon, R.C. a L (1W1) J. Bi. Cm. 2S12442-12448

2695 2696 MICROTUBULE CONFIGURATIONS IN OOCYTES, ZYGOTES, AND MICROTUBULES, BUT NOT MICROFILAMENTS, ARE REQUIRED FOR PRONUCLEAR MIGRATION DURING BOVINE FERTILIZATION: EARLY EMBRYOS OF A MARSUPIAL, POWELPFIS IMPLICATIONS FOR CENTROSOME RECONSTITUTION DOMESTICA. (( W.G. Breed,- C. Simerly, # C.S. Naaa, # J.L ((C.S.Navaral.2.S, N.LFirstl.2 and G.Schattenl.3)) 'Cell and Molecular Bilogy and of 2Meat and and VandeBergO, & G. Schatten#)). * Dept. Anatomy and Histology, U. Adaide, Program Depts. Animnal Scence 3Zoology. UniversItof Wisconsin-Madison, Madison WI. South Australa 5005, # Dept. of Zoology, UW-Madison, WI 53706, and- Dept of Genetics, SW Foundation for Bbmed. Rea., San Antonio, TX. 78228. Mcrotbule namics during bovine fertiizaon imic those of most oher anhnals, xVaslanudingC. , D iVi n_a d sea urchins To explore the oruhg Nd mode of tromie hi a non- This investfgatfon ex ores the requirements of microtubules and rnirofliamenIa1. oocyte; 2. ronudlw euthran mammal, mrubules and DNA were Imaged I nmarin oocyts, hntheunfertilizedbovine during migraton, and 3. for the fomiaton of the first =itotic spindle. Pronudear monospermic and polyapennic zygotes, N gtaactvated oocyts mgon was blocked In bovine zygobs plced culture with noodazole mid eary embryos of the short grey-taled opoaum. The unfertlzd oocyte or cocmid after extusion of the second body. Recovery at the diapayed mroubules only In the short, anar, brrel-shaped meotic pronudewr stage resuted In a mico spermn aster fored only spinde which was oriened radialy to the cel surasce. After sperm with the male pronudeus. Rean at first mitosis resuted in two bipolar crporation, mirotbules bfrmed a sperm aster eocluively around the spindles, one with each set of DNA. In contast to results from mouse oocytes, cytochalasn had no effect on pronudear migration, but caused male pronucleus. The cytoplasm deveped a disnct heterogeneity after egg extive bebbing the plama m . Taxol was used to determine activation between a and one in which microtubules microtubule-ich region if the unfertilied bovine oocyte has centosomal materbl disibutd in were largely absent. In the early embryo, microtubues were deced the troughoutthe cy . ocytes treated wih taxol display much ar outer cortical regbns of daughter blastmeres and In the extruded yolk mass. melotic spindles and numerous cytoplasmic asters, suggestive of te In polyspermic eggs, sperm aster fomed adacent to each male pronue presnc of non-spindle mkrotubule nudeaing material. zygotes treated with taxol form one lre aster with male while In parthenogenetially activated oocytes only disarayed microtubules sperm ssodated fe pronudeus. These resut Indcate there is a centbeomal dlshtuted In the were detected. Nocodazol or cold dsruption of the melotic spidle and taxol corrponent c*bpksm of the unfertlzed oocyte that may be recruitd to the sperm aster stabilzation of cybplasmic mirotubules sugested that mirotubules may via t mirotubuies of that aster. Perhaps in the presence of rubue not be dynamic as in the oocytes of emhran mammalL concuded druugs this dspwsed in the coplasm at the centroso is paternaly nherited In th species, and that If the micro IhIbiting drugs are removed durin prophe the sperm microtubues organized by the spem are crical for pronuear appositon aster is sl able to recnmt these mat however ithe dnrs perst until mitos thistcentosoma matrial be used for bipolar spndles with and the cytoplasnic polarity which results in yolk ma extusion. may each set of DNA. Supported by the USDA and the NIH

2667 2696 CENTROSOME INHERITANCE DURING FERTILZATION IN MORPHOLOGICAL CHANGES IN ThE ENDOPLASMIC RETICULUIM ASSOCIATED HUMANS. (( C. Smeriy-, G-J. Wu, S. Zoran, T. Ord+, R. Rawlns, J. WITH MATURATION OF THE MOUSE EGG. Jonest, C. Navara, M. Genrity, J. RhneharP, Z. Blnoe, R. Asch & G. (( LN. Mehimann', M. Twasald', LA. Jafe', D. Kline' )) 'Denp of Biological Schatten')). *Dept of Zoology and WI PRegona Prmate Research Ctr, UW- Moan, WI 53706, *Ctr. for Reprod. Healh, UC-Irvine Med Center, Sciences, Kent Stat U r, Kent OH 44242. Labcratory of Nerobgy, NINDS, Orne, CA 9268, fSection of Reprod. Endocrinology & Infert, Rush NIlH, Betesda MD 20692. Deparent of Physiology, Urvs of Conc Heal U., Chcago, IL 60612, 'Ass Reprod. Tedbgies Prgran, Glenbrook C4enr, Farminglon, CT 06032. Hospital, Glnview, IL 60025, ItD . of Ob)Gyn, UW-Madison, Wi 53706. The endoplasmic tculum (ER) oft maure, metase Il-arrested mou oe was Centrome inheritance is exloed In human oocyte and embryos by cmpared to the ER d the immae, p hse Iarrsted mouse oot, using laser imaghg microtubudes and DNA thoughout fil in exoes in- Seminated material ob minformed, consenting donors who were scanning confoca mioscp aftersbrta with a fluresce ipophidicarbocyn u rgoi hMv l n or GIFT p . d human dye (Dil). A satrabd solution of Dil, dssoved in soybean oil, was mcoincted into oyes display mictubus inhFe second motc spinde but lact cyo- the oilsand fedye spad t t cypwsm. The matueegg contained a fire pamic mroue. The spie is anas l, melotic orietd radily to rece neworkof ER thru t cel and dense accumulats of membrane in th the egl surfaoe, Nd with a focuss pole abutting the cortex cortax. Thes accumulatons of membrae, 1-24Lm in dianet, we bund within 5 aid a broader pole fIdn the cyoplasm. The inWpo human spem Im of ft ce mmbr and wer generally not deper or a rdial rr of mioubuls, S sperm aster, witn 3 hour found in ft cytplsm. A similar of tb found In the mkdbody stnin wasobsenvdwhen fthegwerefixed wiin one minut of Injection, prviding structure but not In oher regions of the cyt m. Duin q evidence th corca accumuitions of nmmbane are part of a contnus ER the aster enlg develpmet, sperm becomes symmetric the mewbra ssbm. Clusters of membrane wer not osved in ft cortx adjacent to prnce near te cortex, and split ognize a bipOar the meioic (the corical free ar); thus ft dib of ER mirotu aray that ewmans from the tghtly apoed prudel. Al accumuaons matchd the pobrizd dstirbuon of cortical granules and sperm prometaphese, the dchromome begin to merge on the aatral barre- eported rthe matr In ER accumulatons shaped spindle on concludes. Micotbule inhibidion by recepty preous conrast, wwe noodaz blocks srm bmallon and adap-slor The rar found in ft cortex of th hnmaue, poase Ifrested oot, but similar, lss obevtOf supemulry sprm aW py ooc defied membrane cusers ware fbond thr fot toplasm d in t oxyte. supports hypotme the the at The eppeaanes dER dusr i the ortx folblowg oocyte ma on correlates brtlzaton In huma Apavd by tos Lkdis bAM &M StAMs Rsd wih an ncrased abity dte matu egg to relem calcium at felization. 464a Growth Factors in Development (2699-2704). Wednesday

2699 2700 TGFaAS A REGULATOR OF TERMINAL DIFFERENTIATION IN Late Onset Morphological Changes In The Brain Of A TGF-a ZYMOGENIC CELLS. ((D.E. Bockman, R. Sharp, and G. Merlino)) Deficient Mouse, Waved-1. ((R.C. Burrows, P. Levitt)). Department of Department of Cellular Biology and Anatomy, Medical College of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Georgia, Augusta, GA 30912-2000, and Laboratory of Molecular Jersey, Robert Wood Johnson Medical School. Piscataway, NJ 08854. 20892. Biology, National Cancer Institute, NIH, Bethesda, MD The Waved-I mouse is a naturally occurring mutant characterized by a wavy coat and kinked vibrissae. It has recently been shown that this phenotype is The localization of transforming growth factor alpha (TGFa) in correlated with a reduced level of transforming growth factor-alpha (TGF-a), normal gastric mucosa raises the possibility that it regulates a Inown mitogen that acts through the epidermal growth factor receptor. This differentiation of gland cells. To test this possibility, we used signaling pathway is known to play critical roles in the development of many organ systems, but little is known about its role in the brain. High levels of transgenic mice that overexpress TGFa to detect its effect on TOF-ac found in various regions of the CNS, including the striatum, suggest zymogenic (chief) cells in the stomach. In these mice mRNA for that it may play an important physiological role. Initial screening of adult pepsinogen C is present at normal levels during the neonatal period, brains revealed a dramatic enlargement of the lateral ventricles with possible then decreases markedly. Zymogenic cells are present in the loss of specific telencephalic regions. To examine this in more detail, stomach during the neonatal period, but are missing in transgenic animals were sacrificed between postnatal day 23 and 100. Paraffin and adults. The bases of gastric glands, normally rich in zymogenic cells, frozen sections were collected and stained with either Nissl or acetylcholinesterase to delineate brain regions, including the neostnatum and are occupied by undifferentiated cells and mucous neck cells, the amygdala The area of the ventricle as well as the area of the neostriatum was precursors of zymogenic cells. To test for a general regulatory role of measured at three levels along the rostro-caudal axis of the brain. The results TGFcx in differentiation of zymogen-producing cells, salivary glands show a late onset age-dependent increase in the area of the ventricle and a from transgenic mice were studied. Zymogen granules in concomitant decrease in the area of acetylcholinesterase-rich staining. These submandibular glands of transgenic female mice are reduced in changes are likely to stem from a progressive partial loss of the striatum cells in the and/or the amygdaloid complex and suggest that TGF-a may be critical for number. Zymogen granule-containing parotid gland maintaining certain neuronal populations in the mature brain. This work was undergo redifferentiation to form tubular complexes, collections of supported by NIMH grant 45507. ductular-like structures like those formned in the transgenic pancreas. TGFa therefore is a major participant in the regulation of terminal differentiation of zymogenic cells.

2701 2702 CELL MATRIX INTERACTIONS MODULATE INDUCTION OF ce-SMOTH MUSCLE GLUCOCORTICOID-GLUCOCORTICOID RECEPTOR SIGNAL ACTIN (aLSM EXPRESSION BYTGF p1 IN CULTURED FIBROBLASTS. ((P.A. Bany, TRANSDUCMIONPATHWAYANDTGF-,BEXPRESSIONDURING RD. Cavanagh, W.M. Petroll, and J.V. Jester)) Dqeatment of Ophtasnl, Unverty of MOUSE PALATE DEVELOPMENT A. T. Jaskoll, M. 75235. ((H. Choy, Texas Southwesern, Dalas, Txats, Melnick)) Laboratory for Developmental Genetics, University of Southern Los CA 90089-0641. The expression of ce-SM actin by fibroblasts in cultue was studied as a marker for in vio California, Angeles, myofibroblast tanformation. effects of TGFpt, known to induce ceSM actin expreson, wer evaluat pe y cona katoyte (fibocyte) ineaeun- Glucocorticoid (CORT)-induced cleft palate in mice has been an fre coonditis. Exprossion of oe-SM actin, fibroncin, ad asPI integrin wa evaluated by important animal model for half a century. We investigated the CORT- precspitsti ad Western blotting using xmo monoconal glucocorticoid receptor (GR) signal transduction pathway during antibodies to hun_nca-SM actin (Sigma), human anti-fibronectin and rat menolooal anti- embryonic palate development in B1OA mice. The 96-kDa GR is simila to In situ Under srm-free conditon keaocyte appeared snosphologically present in embryonic (E13 to E15) palates and can be activated to bind cofneal keratocyte having a broad, stellate morpholog, no ce-SM actin expression and an f- a DNA CORT element The actin or as determined by FlTC-phalloidin staining, limited to the cell cortex response (GRE). electrophoretic mobility but Addition of TGFpl (1-10 ngm/ml) drmatically induced morphologic t o ao to of the GR-GRE complex does not vary between days of gestation, fibroblatc-like clls ad the exprsion of ce-SM actin localized to prominent intucellular increasing levels of DNA-binding with progressive development are stess fibers in 100% of cells. Tbis effoct wa blocked by anti-TGFi1 nnstrizing observed. These data suggest a role for GR in palate formation. To antibodies (15 ig/ml). TGFpI also incresed cell growth, ha extraceular fibronectin identify the molecular mechanism in GR-mediated cleft palate, we synthesis, and induced expression of ce5Bl integrin. In order to test the hypotdeis tat studied the effect of CORT on growth factor gene expression, intractions between extraeulr fibronectin and surface membrn 5l integr modulate specifically TGF-0. Pregnant mice were injected on day 12 of gestation the TGF effect, cells wee grown in the prooese of GRGDNP (RGD, 0.01-0.0S mM) or I with a dose of CORT which induces 90% cleft and the GRADS (RAD, 0.1 mM). RGD abolished induced expression of ce-SM actin palate steady- TGFpjI state of TGF-32, and mRNA in were without influencing cell aachment while RAD had no effect A simi respone to TGFp1 levels TGF-,1, TGF-,B3 palates and RGD was obsesved in rabbit sclrl fibroblasts, human foresicn fibroblasts, and human evaluated by Northern analysis. CORT treatment lowers TGF-U1 and corneal fibreblasts. Overall, the data ggests that induction of ce-SM acin by elevates TGF-02 and TGF-,B3 transcript levels. Our results indicate that TGFpI is modulated by cel-matrix i actions between extracellular fibrOnectin in its high the CORT-GR signal transduction pathway could participate in the affinitY rcPtor c1 integrin Supportedby NIH 7348 and SSRA from RPB, Inc., NY. pathogenesis of deft palate by modulating TGF-,B gene expression. Supported by NIDR DE10376.

2703 2794 FGF AND TGF-B INTERACTION IN THE REGULATION OF NEWT IRIS IMMUNOCYTOCHEMGCAL LOCALIATION OF ACIDIC AND BASIC FIBROBLAST EPMITELIAL CELLS (IECs) TRANSD TION IN VITRO. ((*LD. GROWTH FACTORS (aFGF, bFGF) AND TRANSFORMING GROWTH FACTOR Taores, CL. Camacho, N.M. Zombrn, G.I Perez, I. Garcia, *M. Rosario, *P. BETA CTF4) IN NEWT EYE DURING LENS REGENERATION. ((M. LoAm, J. Rios Luperon, *V. B ero-Aldhndo and JR. Ortiz)) Dept of Biob, Univ. of Puerto G.I Prz, 5L Gacia, S.Y. Ra_re, P.O. Santigo, LD. Torrs, and JR. Ortiz)) Dept of Rico, Rio Piedr Campus, San Juan, PR. 00931-3360 and *Natural Sciences Biology. Univ. of Puato Rico, Rio Piedas Campus, San Juan, P.R 00931-3360 and Dqwrtent. Univ. of Puero Rico at Caoi, Carolina, P. 009844800. Natual Sciene Dqepan, Univ. of P. R. at Caroina Caolia PR. 00984-4800.

Les regeneration in the adult newt N. virsdesce involves the disaiffcrentidon and FGF is a modulator during eye onsogenua and regeneration pocesses in vertbnra We redifrIation of ECs fim the pupilary mag of tde drsl iris into a new les. It have peviously demonstaed tht aF0F, bPGF and TG-B e involved in the regulaton of has ben postuated that growth fact sereted by the nural rtina regulate this iriepith cels (IECs) _ i in o and immulic d de gowth regenerative process. We have previuy shown that FGFs (aFGF and bFGF) fac in nwteye. In dte naml tissu aF, bFGF and TOF-B differenally sdmula5e ECs growth and d i_ in culture codion, whras TGF-8 localized in the cornea, dorsal d vental ises, in the les fiber, lens epithelhum, and inhibits the respoes and abolishes the bFGF effect Recet experimenU bave neu retina TeaPGF dbl7FsIgnls thenvenal ad doral is are amocied with deonstated that bFGF simulates ECs migrtio f-maxia smulati dose - is sroma and IECs nucli whra no FORs si wu observed the [ECs plasma EDsf- 1.0 ngml) wbile TGF-B inhibited th response (lDstj 0.5 ng/mI). TGF-B aso membrane. TGF-B dudon showed a simnirpaten, but the sigl ws moer lens vena iris in par y inhibited IECs outgrowh frm explants; moreover IECs cultured in the fibers and epithum, and in dorsal ad epihian, especially IECs nuclei and regeneation an Prescene of an antibody against TGF-B sbowed outgowth fom explants. The plama membran Lens suts I-M aFGF and bFGF signals increased progressed. IV -VI regenerm a signal an speficity of the bFGF resoe was examned by exposig ECs to polyclonal noclr expnsion Suges cels showed frm nucle to membrane, transition cells sti antiodies aginst bFGF in antbodies tested inhibited cytplsm plasma ato wa zome showed the nuclear signaL of sages rvealed a in the les ECs ugowti folm dorsal iris expl, migration cel division (half maximal Analysis VI-X stong signal differiati sin oeutralizaion dose, ND50- 0.25 sgAnl; 0.4 pgmI). When dorsa iri explts vesicle zone censer. There a a marked plama membrae clls to the dorsal iris ad prospective epihelium. Fimal stage rener (XI), in exposre to anti-bFGF was discoatined norma IECs outgrowth, migrato and cel which detchment of new lens occurs, showed a ster sigal in lens eptheli cell divisin resumed. FuIrmore, st_set ofexpbs widt RIFreepor antibody (Ab- membne in lens cente fiber However, the is no i the dorsl is resulted in the inhibi of the outgrowth response (D)50- 1:100). Our signal ppiluy IFPR-1) margin whnlkdetchieocenrs. Aagaagdietwsobservedding ther_egrt the idea datRP and TGP-B are involved in the mechanm rIts furh espport dtat ln to nucii - process, being so i res gener, we_erin trantonl zoe, andresriclnd b "11 iOik lmom fiben Ion regulaes IBCs dwiD _gmo Swppcned in the distal dorsal iris epubeim. TOF-8 ws detcted, the sae stgs, moody the by NIH MARC GRANT OMO 7821-14, NIH GRANT 08102-22, NIH BRIDGES regenert prospcve lens epiubehism. Our results sbow dtt FGFs ad TOF-B are GRANT 1 R25GM48987-01, FIPN Office, UPR, and UPR-RCA CARP. differentdnlly pre in the lns regenerate. Thes resul fth pporttn these fact in of in newt. IH MARC GRANT OMO 7821-14, NIH GRANT 08102-22, NIH BRIDGES GRANT I R25GM48987-01. FIP! Office, UPR. ad UPR-RCA CARP. Wednesday. Growth Factors in Development (2705-2710) 465a

2705 2706 TISSUE SPECIFIC DISTRIBUTION AND EXPRESSION OF TGF-B1,2,3 BY RAT GROWTH FACTOR INDUCTION OF EPITHELIAL DIFFERENTIATION IN HUMAN CALVARIA: POSSIBLE ROLE IN SKULL MORPHOGENESIS. ((LA Oppernan, A RENAL PROGENITOR CELLS GROWN IN VITRO. ((C.R. Burrw, and P.D. Nolen, S Casirghino1, M Centrella, TL McCarthy1, RC Ogle)) Neurosurgery, Plastic Wilson)) Division of Nephrology, Johns Hopkins School of Medicine, Baltimore, Surgery and Cell Biology, University of Virginia, Charlotnesvile, VA 22908; 1 Plastic MD 21205. Surgery, Yale University School of Medicine, New Haven CT 06610 Renal pgenir cells ( oblsts) can be isolated from human Idneys (12-17 Cranial sutures are the major grwth regions of cavarial bones, requiring tissue weeks gestation) and grown indefinitely in culture in the presence of nephroblast interactions with underlying dura mater, which involve heparin-binding conmponento growth factor (NB-GF; PNAS 90: 6066). Marker arnaysis suggess that these cells to resist osseous obiteration. As TGF-81,2 and 3 are heparinbndn factors represera early, uninduced mesanephric mesenchyral cells: vimentin, syndecan, known to be podced by calvaria, and to regulate plferation and differentation N-CAM and c-met positive, Pax-2, cytoketin E-cadherin and alkaline of osteogenic cells in vitro, their localization in fetal day 19 (F19) through neonatal phophataseh (ALP) negative. If these cells were proved to be capable of epithelial 21 rat was to to day ( N21) calvaria estabished, examine possible links caiial in vitr, this would provide a model system for the analysis of the morphogenesis. Rat coronal and lambdoid sutures, which never fuse, were molecular and cellklar events involved during devebpmfent to generate the compared to intrafrontal sutures, which begin fusing by neonatal day 15 (N15), diverse ephelil cell types in the mature nephron. To test this, primary and serially possibly due to a change in growth factor gene expression or concentration. pessaged, suspension cuitures were subjected to removal of NB-OGF and addition Imrtnobocalization of TGF-B1,1.2,2 (pan-specific antibody) and TGF-B3 (specific of seumn and other growth factors. Morphological analysis demonstrated antibody), demonstrated the presence of either TGF-B1,1.2 or 2 in periosteum adherent, epilhelial foci aftertreatmert wih 10% serum or hepatocyte growth overlying the calvaria, and in matrlx of all sutures and underlying dura mater at F19, factor, while EGF, TGFa, FGF, PDGF, IGFl and 11 had no effect. Marker analysis of F20 and NI, afterwhich levels and declined by N5 disappeared by N15. TGF-83 semru stimlated cells showed a characteristc protein expression pattern for renal was distributed throughout periosteum and dura mater, and was loalized within epithela: vimeritin and syndecan negative; cyto-keratin, E-cadherin and ALP sutures to osteoprogenitor celis comprising the blastemas on either side of the positive. Sice many post-induction everts in vivo nephrogenesis involve the suture, in F19 to N21 coronal and lambdoid sutures and in F19 and Ni intrafrontal transient expression of proteins, tepwoal marker analysis was cafried out 3, 7 and sutures. Proliferation assays demonstrated that Ni dural cells in primary cullure 10 days after onset of exposure to differen-tiation conditions. The transcrption responded biphasically to TGFB1, while cavarial osteoblasts were inhbited. factor, Pax-2, was transiently expwsed in early epithellal foci (4-8 cell), N-CAM However, the latter to both and non- responded TGFB1 by enhancing collagen was rapidly swilhed off while ALP was expressed in foci only. These results collagen protein synthesis. Results indicated that TGFB1,1.2 or 2 were present in suggest that in vitro induction of epithell diWferentiation in this model system dura mater and suture matrix of immature sutures, at the time sutures required the recapitaes the evers in rogen in vivo and that soklue growth factors presence of dura mater to resist ossifiation, and disappeared from these tissues play signfta role. once the suture was estabished and resistant to obliteration. Supported by NIH grants DE05622-03 & RO1-DE10369-41A2.

2707 2708 NEUROTROPHIC ACTION OF STEM CELL FACTOR (SCF) AND MELNOOPmC EFFECT OF BFGF ON THE EXPRESSION OF OTX2, DLX2, AND DLX5 ACTION OF THE SCFqNERVE GROWTH FACTOR (NGF) COMBINATION. HOMEOBOX GENES IN PRIMARY CULTURES OF CELLS FROM ((J. L Saskowsidl, C. J. langtimm-Sedlk1 J. Canarh2, ad M. Siebe-Blum*nI Meal EMBRYONIC DAY 13.5 RAT TELENCEPHALON. ((L.Robel, M.Ding. Col. of Wisconsin, Milwauke, WL 53226 and 2Amgen Ctr., housand Oks CA, 91320. A.J.James, A.Simeone, J.F. Leckman and F.M.Vaccarino)) Child Study Center, Yale University School of Medicine, New Haven, CT, The embryonic neural crest gives rise to a variety of different phenotypes in the adult 06520 USA.(Spon. by P. De Camilli) orgi, includig all astnomic and prmary smnomy netro, meanocyte, neve of supporting cclls the PNS, and cranial structres Duing migrain and at stes uf rminal The characterization of the domains of expression of homeobox differeati, many neural aest cdls ae pluripotent, indiatig that signuls from the embryonic microenvironmenpatciae in cell type sificatin Thusthe influence of two genes specifically expressed in the mammalian forebrain(Dlxl, growth fact that we pejmnt in nwal cra migary pathways, SCF and NGF, on neural DIx2, D1x5, DIx6, Emxl, Emx2, Otxl, Otx2) led to the hypothesis crest cel proliferato and diffentien was determined by in Wiro coloy asy. In the of its eatly regionalization in neuromeres. So far, the role of presence of SCF more colnies coninWd cells uimunoreactive for the stage specific homeobox genes in cell specification has not been clearly embryonic antigen-l (SSEA-1) than in control cultres SSEA-1 is a marker for quail demonstrated. To addres this question, we studied the effect of basic senxsry neuroa Morover, the size of colonies fonned by pluripoen neural crest cd, fibroblast growth factor (bFGF) on the expression of these but not by commited meanogen cdls, w aso sgnifficty inreased in the prsence of homeobox genes in cultures of cells obtained from embryonic day SCF. In the presence of the SCF/NGF combination the size of coloies derived from 13.5 rat telencephalon. In this in vitro preparation, stem cells commited melanogenic cclls was inesed, whereas in the presence of SCF alone or NGF multiply and then differentiate into aspartate-,glutamate- and alone no noiceable effect was observed, Moreover, the erogenic and trophk effects GABA-containing neurons, as well as in glial cells. We have obseved with SCF alone wereaboishd m the presence of SCF plus NGF. TheNGF effects could shown that bFGF i treatment be mimicked by other ssuch as eur 3 (NT-3) ad brin-drived previously leads to a 3-fold increase in facto (BDNF). Cell cycle analysis with bomodtoxyuridine ndicated tat the the number of glutamate-containing neurons. Using a RNase observed increases in colny sizes were due to an icse mitotic activity of the cells protection assay, we observed an increase in the expression of Ihe r t suggest that SCF has neurouophic acivity and that ae involved in Otx2, Dix2, and Dlx5 at 3 and 5 days in vitro, corresponding with molanogenesis: 1) SCF seems to increase thepbability that pi t neural ces clls active neuronal differentiation in cultures. We are currently will gerate senory neu prctrsor nd 2) to ic e the survival of the offspring of studying the effect of bFGF in cultures of basal and dorsal these precursor 3) the combination of SCFand any rin tested (NGF, BDNF,or telencephalon separately, since we demonstrated that the NT-3) increases the survival of progeny derived frm committed melanogenic cclls, expression of Emx, Otx, and Dlx genes in cultures of basal and indicating that the known trophic ction of SCF in molsogeesis most likely also requires dorsal telencephahon reflects the regionalization in vivo. the presence of a nurorophin. Supported by USPHS grantHD21423 ad a Resech Grant from theFamilial Dysautonomia Foundation

2709 2710 HEPATOCYTE GROWTH FACTOR (HGF) PROMOTES MOTOR DOMINANT NEGATIVE EGF RECEPTOR EXPRESSION REDIRECTS ES NEURON SURVIVAL AND ENHANCES CNTF-STIMULATED CELL DIFFERENTIATION. ((J-X Wu and E.D. Adamson)), La Jolla Cancer CHOLINERGIC DIFFERENTIATION. ((V. Wong, R. Arriaga, D. Research Foundation, CA 92037. Glass, G.D. Yancopoulos, R.M. Lindsay, and G. Conn)) Regeneron EGF receptors are expressed on most fetal and adult cells but their precise Pharmaceuticals, Tarrytown, NY 10591. (Spon. by C. Hyman) roles are not clear. We previously reported that in P19 embryonal carcinoma cells the expression of kinase-negative EGFR inhibits retinoic acid (RA)- induced differentiation Hepatocyte growth factor was originally identified as a potent to nervous tissue (Wu and Adamson, Dev. Biol. 159, for cultured 208-222, 1993). Embryo Stem (ES) cells differentiate into a wide range of mitogen hepatocytes. Ciliary neurotrophic factor tissue types after the removal of the cytokine LIF from the culture (CNTF) has been shown to promote survival of neuronal medium, many but the direction cannot be controlled. We demonstrate that some early types during development, including motor neurons. In this study, markers of differentiation , tPA and Endo A, are inhibited in clones containing we have found that c-met (HGF receptor) mRNA is present in the kinase-negative mutant EGFR. After 34 days of differentiation, the cell types rat spinal cord and is developmentally regulated. The peak present in mutant and control cultures differed. Mutant clones produced expression of c-met (at E14 and E16) coincides with the period of frequent cardiac muscle and endoderm as the predominant cell types; other naturally-occurring cell death in motor neurons, suggesting a cel types were sparse or absent. In control cultures, many fibroblasts and possible role in the regulation of this process. HGF and CNTF epithelial cells were observed as well as endoderm and cardiac muscle. Since treatment both increased the survival of cultured motor neurons by mutant differentiated cultures had fewer cells and more cell death, we 2-fold. HGF correspondingly elevated CAT activity 2-fold, whereas concluded: 1) mutant EGFR does not allow some cell types to persist such that tissues that do not CNTF produced a 5-fold increase. When saturating doses of HGF require EGFR are favored; 2) EGFR activity is not and CNTF were added to motor necessary for cardiac muscle and endoderm formation; 3) apoptosis occurs in simultaneously neuron cultures, tissues that EGFR for their CAT activity was elevated 1 4-fold, almost doubled the sum of 2 require survival, development or function. the Analysis of the expression of several transcription factors during factors alone. Co-treatment with the 2 factors resulted in an the differentiation of control and mutant EGFR ES cell clones indicated no additive 4-fold increase in motor neuron number. Our findings detectable differences in Fos, Jun, Egr-I and RARiprotein expression indicate that HGF is a survival factor for motor neurons and it although stage-dependent distinctive espression occurred RARD expression enhances CNTF-stimulated cholinergic differentiation in these rose and fell twice during spontaneous ES differentiation, once at 2h to 24 h neurons. By extrapolation, HGF may enhance the efficacy of CNTF and again from 3 to 7 d, this change in expression appears to be stimulated by in vivo. aggregation in the absence of any added RA other than that in serum. The implications of these findings are discussed. Supported by grants from the PHS: CA 28427 and HD 28025. 466a Growth Factors in Development (2711-2716). Wednesday

2711 2712 LOSS OF INTESTINAL ELECTRICAL RHYT1MCITY WHEN EXPRESSION OF TNF-a IS REGULATED BY GLUCOCORTICOIDS TYROSINEKINASE FUNCTION OF c-kit PROTEIN ISIMPAIRED. DURING MURINE LUNG DEVELOPMENT IN VIVO. ((P.D. Boyer, T. ((S.M. Ward, S. Torihashi, A. Burns and K.M. Sanders)) Department of Jaskoll, M. Goicoechea, and M. Melnick)) Laboratory for Developmental Physiology, University ofNevada School of Medicine, Reno, NV 89557. Genetics, University of Southern California, Los Angeles, CA 90089. Mammalian lung development requires classical epithelial-mesenchymal a antibody (ACK2) to the receptor (c- Injection of monoclonal tyrosmine kinase interactions. Previous work in our laboratory has shown that kit) into neonatal mice disrupts the development of normal small intestinal glucocorticoid injection of B1 O.A dams accelerates embryonic lung motility. c-kitprotein-like immunoreactivity (c-kt-LI) was exprsed selectively maturation and histodifferentiation, and that gene(s) which map to the by interstitial cells of Cajal (ICs) in the muscularis externa of the small bowel H-2 region of chromosome 17 mediate this effect. Since growth factors and colon. Cells with similar morphology and distribution as the cells with c-kit- and their receptors are likely to act as mediators of LI were stained with methylene blue, an histochemicalmarker for ICs in the epithelial-mesenchymal interactions, we have selected TNF-a as one mouse. Immunocytochemistry verified that ICs were labeled with ACK2. candidate growth factor for embryonic lungs. The TNF-a gene both maps within the H-2 region and has been shown to respond to Multipleinjections ofACK2 immediately after birth caused a dramatic reduction glucocorticoids in other systems. In the work presented here, we of ICs at day 10 compared to control animals. Intestinal muscles inthe number demonstrate by immunolocalization that TNF-a and its receptor are from animals treated with ACK2 had no electrical slow waves. The distribution expressed in embryonic lungs in vivo with progressive development. By of ICs and electrical activity of small intestinal muscles of White-spotting (W) Western blotting we identify TNF-immunoreactive species of 17,26, and mutant mice in which thetyrosine kinase fimction of c-kit is impaired were also 68 kDa, as well as a ladder of apparent multimers of the 17 kDa species. studied. W/WV mutants had few ICs in the myenteric plexus region compared to In addition, we show that glucocorticoids reduce the expression of normal (+/+) siblings. Intestinal muscle strips from +/+ siblings had electrical TNF-a gene products by embryonic lungs in vivo. We conclude that slow waves, but electrical rhythmicity was absentin W/W"mice. These findings glucocorticoids exert their effects upon embryonic lung development, at least in part, through negative regulation of the TNF-a gene. are consistent with the hypothesis that ICs are necessary for the genertion of electrical rhythmicity in intestinal muscles. ACK2 treated animals and W locus mutants provide a powerful new model for evaluating the function of electrical rhytmicity in gastrointestinal reflexes and transit (Supported by NIDDK 41315)

2713 2714 OVEREXPRESSION OF FGF-2 (BASIC FIBROBLAST GROWTH FACTOR) LOCALIZED STEEL-FACTOR ACTlVllY DIRECTS MELANOCYTE IN TRANSGENIC MICE: ALTERATION OF SKELETAL DEVELOPMENT. PRECURSOR DISPERSAL ON THE LATERAL CREST MIGRATION ((I: Sasse, D.Coffin*, P. Govindraj, D. Pearson, and T. Ganey)) Shriners PATHWAY. ((B. Wehrile-Haller and J. A. Weston)) Institute of Neuroscience, Hosr.tal for Crippled Children, Immunology Section, Tampa, FL 33612 and University of Oregon, Eugene, OR 97403 *McLaughlin Research Institute, Great Falls, MT 59405. Trunk neural crest cells see from the neuroepidhium and enter a "migration staging area' (MSA) lateral to the embryonic neural tube. After some crest cells have We have studied skektal development in mice expressing the human FGF-2 begm to migrate on the medial pathway, a subpopulation of crest-derived cells remaining the MSA can be detected. These cells express mRNAs for the receptor transgene. Using RT-PCR we demonstrated largely elevated levels of FGF-2 tyrosine inase, c-idt, and the tyrosinaerelated protein (TRP-2), charrisc of gene expression in all tissues examined in the tranAgenic mice. Expression of meacyte. These putative manocyte precursors (Ms) then migate on a lateral the human transgene was detected by Western blotting in a wide variety of pathway toward the dermatome, and subsequently disperse into nascent dennal tissues including brain, heart, lung, liver, kidney, and spleen. Radiographs of mesenchymeMPs transiently require the c-kitligand, Steel factor (SlY) for survival. transgenic mice revealed profound differences in the proportion of their In addition, SLF mRNA is known to betransiently loclized in the dorsal dermatome before the omset oftrunk neural crest cell dipersal on the lateal pathway. To assess extremities. Knees of the transgenic animals showed an enlarged diameter of the role of SLF in MP disper and fate on the lateral pathway, we anayzed two the metaphyseal bone and an elongated patella. Mineralization within the different SLF mutants, SI and St. SI is a null mutation that lacks SLF, whereas St' meniscus was increased in the transgenic mice. In addition, growth plate lacks cel surface-associated SLF but produces a soluble form of SLF. No MPs were thickness was noticeably increased in the transgenic animals while the number detectd in the dermatome of embryos homozygous for the SI allele, whereas in of chondrocyte columns and number of proliferating chondrocytes within each embryos honozygous for the Sid allele, MPs disperse normally on the lateral pathway, but subsequently were not detected in the dermis. We conclude, threfore, that soluble column was decreased. In addition, the area of calcified cartilage at the SLF is required for the onset ofmelanocyte precursor migration, whereas membrane diminished chondro-osseous junction of the metaphysis was notably in the bound SLF plays a role in their survival in the dermis. To analyze other transgenic animals as was the spongiosa of the metaphysis. Radiographic and environmental conditions involved in MP migration and survival, we examined histological examinations show growth disturbances in a multitude of skeletal emnbryos homozygous for the recessive-lehal coat color mutation, Patch, a deletion of the a-subunit of the PDGF receptor, in which severe in demal tissue are tissues and cell types as well as profound changes in the pattern of defects observed. In such embryos MPs leave the MSA and form cell clusters on the lateral extracellular matrix depositon and calcification in the transgenic animals. pathway. Later, no MPs can be detected in the dermis of Patch homozygotes. It that an important of These data support the notion FGF-2 represents mediator remains to be determined if the Patch mutation acts cell-autonomously on MPs, or if skeletal development and plays a role during cell differentiation, cartilage and it affects localiztion of SLF in the embryonic dermis. Supported by EMBO ALTF bone growth and extracellular matrix and calcium deposition. 169-93 and NIHGrant DE-04316. (Supported by a grant from the Shriners Hospitals for Crippled Children)

2715 2716 RECEPTOR TYROSINE KINASE (RTMN, ELK, IS EXPRESSED IN VASCULAR CELLS TARGETING OF INSULIN-UKE GROWTH FACTOR-la (IGF-1) AND DES-(1-3)- AND DEVELOPMENTALLY REGULATED. ((E. StehI, H.O. Sdchecdmann and T.O. IGF-la (DES) TO THE MOUSE MAMMARY GLAND THROUGH EXON DanIel)) Dpad d Phmacology, Medicine and Cl BbIoloy, Vandebit REPLACEMENT IN A RAT WAP TRANSGENE. ((D.L. Hadenl, N.M. Grenberg, Unveraty School d Medicine, Nashvle, TN 37232. and J.M. Rosen)) Departmet of Cel Bbbgy, Baylor Colge of MedicIne, Houston TX 77030. A Wge bfmy d p uy 'orpha RTKa, Indudhg eck,eph and elk, densa cil-type rsftr emgeIn and are hniplcated hI d eat-on. Reewt work ha Efficlert ransge exprslon has been shown to require the preence of hItrons. defned the lgand for th eck RTK as B-M, a product d TNFa actvated endrAea In agreement wkh tths obseeraton, mnnigene constr generaly show greater n cel To expore whather endcthell coea tp RTKs of this fwnly, we ueed penetrance and greater expre han promoter-cDNA fusIon consucts. SInce gene degenerat cilg_nucle eIhi a PCR-ba_ed donhg strategy to klely RTa the mere size of the 80 kb hunm IGF-I makes mlnigen consucton by hi human renal mir_ovectlar wnkh_I cel (HRMEC). convertbnal methods kIpossble, a novel PCR-mlaed appach was used to oed cDNA shgutenom ancodhi t k K obtaInd a ftJbeh prcIsely reploe the exons wItn the 3 kb rat whey acidic poteI gne wth DNA hurmn elk and pedll rne eJk cDNAa fom lambda llrals& Norten -n fragmenrts derived from the human IGFIa cDNA. The resuIng ranegee, or a demonsrat ht lWvel mRNA _bep lon hI HRMEC an hun umbllcal vehn modication thereof, was used to target IGF-I (WI) or DES (WD) expressIon to the endothelll dle, lower lWel expreeIon I dchorlcanma (JAR) and mamnwary glands of lactating mice. Northem analysis of total RNA from the oseecora (HOS) cdl lInes Ek nRNA _ereon we IdentrIed hI adult ratteauea mammary tsusu of trakgec mice at 10 days of lactation shwed the presence of fom brah, teo and hea a 0.7 kb band in 100% of the Wl mouse Wnes and 86% of the WD Ines. Steady- Nortm and RT-PCR aniyea of mulrne embryorIc mRNAs howed dk mRNA state concertratIons of transene mRNA ranged from 0.1 to 10% of the at (5.0kb) k deop from EM5 to E15.5, with p expreseion at E125; erpresslon Is endenous mouse WAP b,aer but was observed up to 43-lId over that of Ibe a_tr E15.5. In batIon (whole mourt) murhne embryoe the endogenous IGF-I transcrIt found hi Iver RNA from a lactating mouse. ahowed abudt elk opeeon hI developIng mdbmah/hhidbraI, onstr_ with Analysis of total RNA by RT-PCR supported the conclusion that transcripts prvIous repor ths elk Ia tpreed hI nural lesue. Low lWel qren hI produced from these novel transgenes were spliced correctly and prduced the aevphg may repreet an In vt correle to cuiured endothell cell correct open reading franws for pre-pro4GF-I and pre-pro-DES. This conclusion exp $ a potertW role for elk hi vawar was confrmed by sequence analysds of the RT-PCR prodcts Westem blot analysis of milk whey fractons. A bossa bad on hicrporan of [HHlclne kito protein of rat L6 myoblast showed the translatlon poducts of these trangenes were biologically active and secreted into mnlk at concentrations up to -80 agrn. These mice represent a valuabie model for dudyIg the role of IGF-I and IGF-bInding proteins hI mammary development, ctatIon and tumorgenesis. Supported by NIH grant #HD07618-03 and USDA grant #92-372063. Wednesday. Growth Factors in Development (2717-2722) 467a

2717 2718 DETECTION OF ERYTHROPOIEIIN RECEPTOR TRANSCRIPTION IN bFGF/IGF INDUCE EXPRESSION OF ID mRNA DURING THE THE DEVELOPING MOUSE BRAIN. ((C. Liu, Z.Y. Liu and C.T. SYNCHRONOUS ACTIVATION OF QUIESCENT C2C12 Noguchi)) LCB, NIDDK, National Institutes of Health, Bethesda, MD MYOBLASTS INTO S PHASE ((Y.-M. Vinagre and S.R. Farmer)) 20892 Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118. Binding of erythropoietin to its specific cell surface receptor is believed to be the primary stimulation of erythroipoiesis. We have produced transgenic Idl is a member of the Id family of helix-loop-helixtranscription factors mice containing a human erythropoietin receptor (EpoR) gene which exhibit which lack a basic DNA-binding domain. Idl has been shown to negatively specific expression in hematopoietically active tissue and low but persistent regulate muscle cell differentiation and is also postulated to be involved in expression of the transgene in the brain, even after birth. Examination of cellular growth. Due to the difficulty of synchronizing muscle cells without mouse endogenous EpoR expression revealed significant levels of EpoR inducing differentiation, however, the extracellular factors and the mRNA in the embryonic brain at day 10, but this decreased with development intracellular mechanisms which may be involved in the regulation of and was absent at birth. We now report on the localization of the endogenous growth by Id have not yet been elucidated. We are now able to reversibly EpoR mRNA by whole body in situ hybridization to nine day mouse arrest proliferating C2C12 myoblasts in Go by suspending them in embryos. Specific high level staining with murine EpoR antisense RNA was methylcellulose-containing medium for 72 hr, without inducing muscle- detected in the region of the brain and neurocoel. Lesser staining was specific gene expression. Using Northern blot analysis, we have shown that observed in the regions of the major posterior blood vessels. Perfusing the IdlImRNA expression is down-regulated five fold during suspension embryos to remove residual blood did not alter the pattern of staining. culture. Adhesion of these quiescent cells to a collagen coated dish in 20% Probing with murine EpoR sense RNA was negative under identical serum (GM), 1 nM bFGF, 10 nm IGF, or 1 nM bFGF/10 nM IGF activates conditions. An antisense RNA probe made from brachyury cDNA as a their entry into GI followed by a synchronous progression to S phase. The control stained tail bud as expected. Expression of EpoR mRNA in the defined growth factors also induce Id mRNA within the initial 2-5 hr developing brain may be related to our recent observation of EpoR mRNA postplating along with other immediate-early genes. The extent of this and functional EpoR in human umbilical vein endothelial cells (P.N.A.S. induction correlates closely with the ability of each growth factor to induce 91:3974, 1994). Since the need for blood as a means of oxygen delivery proliferation. Immediately following the attachment of the suspended cells depends on both the generation of blood and the vessels that transport it, to the extracellular matrix, prior to the induction of the immediate early these two processes may use similiar regulatory mechanisms and share gene program, there is a transient drop in Id mRNA below those levels common factors. Conversely, it is possible that brain EpoR is more directly expressed in the quiescent cells. This response appears to be due to a related to stages in neural or glial development. destabilization of the mRNA. When the suspended cells are replated in the absence of growth factors they do not proliferate and they express almost negligible amounts of Id mRNA.

2719 2720 VASCULAR ENDOTHELIAL GROWTH FACTOR REGULATES IDENTIFICATION OF FUNCTIONAL DOMAINS IN Wnts VASCULOGENESIS, AN IN VIVO ANALYSIS IN THE QUAIL THROUGH EXPRESSION OF CHIMERIC PROTEINS IN XENOPUS EMBRYO. ((CJ. Drake and C.D. Little)) Cardiovascular Developmental EMBRYOS ((S.J. Du*, J.L. Christian+, R.T. Moon*#)) #Howard Hughes Biology Center, Medical University of South Carolina, Charleston, SC 29425 Medical Institute, Department of Pharmacology, University of Washington, Vascular endothelial growth factor (VEGF) is a potentmitogen specific to School of Medicine, Seattle, WA 98195.+Department of Cell Biology and endothelial cells. Considerable work has been reported on the role of this Anatomy, Oregon Health Sciences University, Portland, OR 97201. cytokine during angiogenesis. Microinjection of femtomolar quantities of recombinant VEGF (rhVEGF,65) into early quail embryos, caused profound Wnts are secreted signalling factors which may act through receptor- alterations in vasculogenesis (de novo blood vessel formation). Analysis of mediated signalling pathways to influence cell fate and cell behavior in embryos cultured for seven hours aftermicroinjection showed: 1) alterations developing embryos. Overexpression in Xenopus embryos of a Xenopus to the overall pattern of the intraembryonic vascular network, 2) a response by Wnt, Xwnt-8, leads to a duplication of the embryonic axis. In embryos endothelial cells that appeared distinct from the angiogenic response generated ventralized by UV-irradiation, Xwnt-8 restores expression of the putative by the addition of rhVEGF165 to the chick chorioallantoic membrane (Wilting transcription factor goosecoid, and restores normal axis formation. In et al. 1992, Anat. Embryol.) and 3) a localized elaboration of vessels that contrast, overexpression of Xwnt-5A generates defects in anterior structures, resulted in the formation of abnormal vascular plexuses connecting the dorsal without inducing goosecoid, or an axis in normal or UV-irradiated embryos. aortae to the pimordia of the developing atria. The vasculature of rhVEGFi65- Wnt receptors have not yet been identified nor is it established whether Wnts injected (n=27), control-injected (n= 50) and control-cultured (n=44) embryos are composed of discrete functional domains. To investigate whether distinct was analyzed using the antibody QH-l, which recognizes quail endothelial regions of Xwnt-8 and Xwnt-5A were sufficient for eliciting the observed cells. Additionally, endogenous VEGF was localized in whole-mounted effects of overexpression, we generated a series of chimeric Xwnts, and embryos using anti-rhVEGF165. VEGF had a wide distribution that was not injected RNAs encoding the chimeras into normal and UV-irradiated exclusively localized to vessels; it was observed as granular foci arranged Xenopus embryos. Analysis of the phenotypes of the embryos, and of along extracellular matrix filaments. Microinjected, endogenous VEGF goosecoid levels, reveals that chimeras composed of carboxy terminal labeled, and QH- I labeled embryos were analyzed using laser scanning regions of Xwnt-8, and amino terminal regions of Xwnt-5A, are confocal microscopy, including control embryos. This in vivo study indistinguishable from the activities of native Xwnt-8, and that the reciprocal establishes that vascular endothelial growth factor regulates the behavior of chimeras elicit effects indistinguishable from overexpression of native Xwnt- endothelial cells engaged in vasculogenesis. 5A. We conclude that the carboxy terminal halves of these Xwnts are candidate domains for activating Wnt receptors.

2721 2722 AN ANTI-DORSALIZING MORPHOGENETIC PROTEIN DEVELOPMENTAL CONTROL OF CELL MIGRATION IN EXPRESSED IN SPEMANN'S ORGANIZER. ((S. Wang, M. DROSOPHILA: C/EBP, FGF RECEPTOR, THE RAS PATHWAY Krinks, and M. Moos Jr.)) Center for Biologics Evaluation and AND MORE ((D.J. Montell, C. Andrews, T. Lee, A.M. Murphy, B.Z. Research, FDA, Bethesda, MD 20892. Shilo, R. Tinker)) Department of Biological Chemistry, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205. Using degenerate PCR, we have identified a novel member of the (Spon by) TGF-8 superfamily of peptide growth factors in Xenopus laevis. Expression levels are highest at the onset of gastrulation, most To investigate the mechanisms controlling developmentally regulated prominently in the Spemann organizer. Injection of mRNA encod- cell migrations we have been studying a small group of migratory ing the gene into dorsal blastomeres results in dose-dependent follicle cells in the Drosophila ovary, the border cells. Initiation of ventralization, even in the presence of lithium chloride. We there- border cell migration requires the slow border ceUs (slbo) locus which encodes the Drosophila homologue of the transcription factor C/EBP. In fore designate this gene product Anti-Dorsalizing Morphogenetic a screen to identify downstream target genes we found that the I (ADMP-1). Overexpression of the gene downregulates Protein breathless locus, which encodes a Drosophila FGF receptor, was, in the the dorsalizing factors noggin, goosecoid and follistatin, as well as ovary, specifically expressed in border cells. This expression could be NCAM, muscle actin, and MyoD; expression of XWnt-8, XHox-3, altered by reducing expression of C/EBP or by expressing C/EBP XBra and cardiac actin is unaffected. The gene is induced by ectopically. Furthermore, a C/EBP-independent bd transgene was able lithium chloride treatment or activin and repressed by UV irradia- to rescue slbo migration defects specifically. These results suggest that tion, a behavior heretofore associated with dorsalizing messages. the FGF receptor is a key downstream target for C/EBP in controlling Overexpression of ADMP-1 resulted In abnormal development only border cell migration. The rescue of slbo mutants by btl was not If injected in cells giving rise to the regions of normal expression. complete, indicating additional genes involved in the migration. We We therefore propose that this factor serves as a homeostatic have identified two additional genes whose border cel expression regulator that moderates dorsalizing influences expressed in the depends on C/EBP. One of these is also expressed in a gradient along Spemann organizer. This interpretation supports the recently ad- the migration pathway, maling it a candidate for guiding the migration. that activin-like signalling inhibits neuralization. Progress in the molecular characterization of these genes will be vanced contention presented. 468a Growth Factors in Development (2723). Wednesday

2723 CLONING OF AN INSULIN-LIKE RECEPTOR IN HYDRA AND ANALYSIS OF THE BIOLOGICAL ACTION OF INSULIN AND OTHER GROWTH FACTORS IN THIS SIMPLE METAZOAN. ((Michael P. Sarras Jr. Ninh Mai, Sue Tatarewicz, Katherine Rauen, Robert Steele)) Dept. of Anatomy & Cell Biology, Univ. of Kansas Med. Center, Kansas City, KS and Dept. of Biol. Chem., Univ. of California., Irvine, CA. Previous studies have indicated that pattern formation in hydra is in part regulated by activator and inhibitor factors that exist as gradients along the longitudinal axis of the organism. Recent findings suggest that hydra might utilize proteins with some homology to vertebrate growth factors as potential regulators of cell division and cell differentiation during establishment of pattern formation. We therefore tested the biological activity of purified human insulin, mouse EGF, bovine bFGF, and activin in hydra polyps and in hydra cell aggregates. Various concentrations ofgrowth factors (or BSA) were either DMSO-loaded into the interepithelial space of polyps or incubated from time zero on (-DMSO) with hydra cell aggregates. All growth factors tested (at m lOng/ml) stimulated 'H-thymidine incorporation at about 30 hr after the initial exposure. By BrdU incorporation, growth factor-treated groups indicated that all growth factors (insulin, EGF, bFGF, and activin) caused a stimulation in the division of epithelial cells and Interstitial cells (0-cells). Using PCR to clone fragments ofprotein-tyrosine kinase genes from hydra, we have identified a gene (HTK7) which encodes a receptor protein-tyrosine kinase closely related to the vertebrate insulin and IGF-I receptors. Amino acid sequences conserved between the hydra and vertebrate receptors include those in the cysteine-rich ligand binding domain, suggesting that the hydra receptor can bind an insulin-like ligand. Hydra treated with hydroxyurea to remove multipotent stem cells and committed precursors of cells in the I- cell lineage still contain HTK7 mRNA. (Supported by NIH grants RR06500 and RR09755) Aging (2724-2727).

2724 2725 ALTERATION OF THE ADRENAL ANTI-OXIDANT DEFENSE SYSTEM INDUCTION OF AGING & EXCITOTOXICITY PROCESSES IN HIPPO- DURING AGING. ((S. Azhar, L. Cao, and E. Reaven)) Dept. of Veterans CAIPAL SLICE CULTURES. ((BA. Bahr, G.Y. Park, M. Sl-gh, and G. Affairs Medical Center, Palo Alto, CA 94304. Lyncb)) Ctr. Nesroblol. Learning & Memory, UaIv of CalIf., Irvine, CA 92717. The apd brIn, especIally In patlents wIth Albhelmeres disease, Is notable for Early studies from this laboratory have shown that homogenates from the 1) the bulid-up of amyloldogenlc peptides in aeuronal cell bodies, 2) abnormal adrenals of aging rats (24-27 mo) challenged with prooxidants are far more lntranearonal filaments, 3) cytoskeletal degradation, 4) the loe of syaaptic susceptible to oxidative stress then are similar samples from young mature (5- structure, and 5) Increased vulnerabillty to excitotoxlc Insult. Long-tem 6 mo) rats. The present study was designed to determine if these findings are hippocampal slice cultures were used to develop a model system that can associated with the recognized decrease in adrenal steroidogenesis with aging, reproduce cellnlar events prominent in hnman brain aging and dementia In and if such changes could be accounted for by age-related alterations in the days Instead of decades. The cultured dlices maltain the high synaptic adrenal anti-oxidant defense system. As such, endogenous levels of various denaity characteristic of the brain and, more importantly, express a stable non-e= aticantioxidant-t (i.e.,vitamins E and C, and reduced glutathione synaptic compositio for months. Ly sosotropic agents induced abnormal [GSH]), and enzymatic antioxidan (i.e. superoxide dismutases [SODs], protein proceasiag in the slices which was accompanled by features of aging catalase [CAT], and selenium-dependent glutathione peroxidase [Se-GPX] lcluding the build-up of I) P -amylold coataining peptides, 11) neurofibrillary were measured in adrenal tissues of male rats. The results show that the tangle-like epitopes, and 111) lysosomal cathepslas, and reductions In pre- and adrenal content of vitamins E and C decreased 60-70% while the content of postsynaptic proteins and evoked potentials. The age-related effects were GSH decreased 20-30% in the old rats compared to the younger controls. influenced by another potential contributor to bral aging, In particular Likewise, the activity of both cytosolic and mitochondrial SOD enzymes and excitotoxIcity. A brief pretreatment with the excitotoxis kalaic acid altered Se-GPX declined by 50-60% in old animals. Indeed, only the expression of the events following from a subsequent lysosomotropic infudon. The onset of both of aging features was more rapid and, more Importantly, did not reverse upon CAT remained unchanged with age. A decline in the levels protein wasbout of the agent. Kalaate treatment alone had no effect oa amylold or and mRNA content for SODs and Se-GPX in aged adrenals (measured by synaptic markers, but caused the appearance and accmulatioa of amino- Western blotting and RNase Protection Assay, respectively) paralleled the ± ± in (BDPN; 147 4 kDa, mean±sad, null) and carboxy-termlaal (BDPC; 152 4 decrease in the expressed activities of these enzymes. As such, the changes kDa, not) spectr fmragments mediated by calpain and measred with two activity appear to arise from changes in the mRNAs coding for the enzymes. antibodies developed to peptide sequences on either side of the calpain Insitu hybridization experiments on the adrenals show that the decline in cleavage site. Antibodies to the intact cleavage site and to spectran labeled mRNAs of the enzymes is not due to an age redistribution of the mRNAs both proteolytic products as well as a third BDP of 144 ± 4 kDa (aT7). The within the various adrenal zones. These observations are consistent with the BDPs are signs of excitotoxicity and were also Induced by NMDA (10-300MM, idea that aging in rats is associated with a deterioration of protective oxidative 5-60 sms) and AMPA In dose- and time-dependeat manners, and blocked by mechanisms, and in the adrenal these may lead to oxidative damage in calpain ilhibitor 1. These findings Indicate that certain aspects of brain membranes which are crucial for cholesterol processing and steroidogenesis. aging reflect a synergism between lysosomal disturbances and excitotoxicity.

2726 2727 EXTENDED LIFESPAN OF HUMAN FIBROBLASTS AND LOSS OF A MODULATION OF MUSCLE SPECIFIC GENES EXPRESSION DURING AGING CHROMOSOME 6. ((T. Tsutsuil and J. C. Barrett2)) 'Department ((*A. Musaro, #C. Ciocarsli., *A. Germanl. -M. Mdolo and #B.M. ZanI)) 'Irnite of Pharmacology, The Nippon Dental University, Tokyo 102, ot Histdogy and gen. Embryology Univrsiy of Rome 'La Sapbrzn ; #Dep of Japan and 2Laboratory of Molecular Carcinogenesis, NIEHS/NIH, Experr Medlcino Unh ersity of L'Aqula. Italy. NC 27709. Research Triangle Park, detemnt factor myoD myoge0nin. My5, MRF4 and MEF-2C Myogenb of Hybrids between immortal cells and normal fibroblasts have been ahown to play a regulatve role In the ersso musci specifc genoa cells must lose one or (myodn, AChR, MCK) in vivo as wel as hI vm. The bcto are dffely senesce, which indicates that immortal nvlrmta h vivo and hI more senescence genes for immortalization. To examine a expresseddependIng on: 0 muscle coil type., 1) hfor, UI) possible involvement of gene alterations in extended lifespan vlro d nferertatlon stage. human fibroblasts We dUdId the epresion of the myogenlc btor durIng rnwh muscle agig The of normal human fibroblasts, normal (WHE-7) on detcted derived from an embryo of 7 weeks were periodically irradiated restt dae hidklate that MyoD and myogenin mRNA expr ae by 2 days afterbrth,dgcra up to 5 moths.In the 2 y oldmie amoWt Of with X-ray. Treatment of WHE-7 cells 9 times with X-ray (2 Gy the oar hand the an extended but MyoD and myogernn rncripl m rkedy icrea. On qpr_ln at 2 Gy/min) resulted in cells with lifespan of MRF4 varatons dug posnatal le Alhugh the not immortalization. These cells had as a sole karyotypic Is detcted wlhoit sgdcatlve alteration, monosomy of chromosome 6. Loss of heterozygosity tancrlpt of myf-5 Is knwn to be detected prefentially durig emnbryog _ne we locus at fin ta li exprsseed In the esaly potnal ie Ik decrease hI young mou and of the repeat sequence on chromosome 6 (D6S87 6q22) accum es In adult and sene muscle. The tanci of MEF2 omponet of the was confirmed by polymerase chain reaction. Single stranded of muscle HLH gn eprsio during DNA conformational analysis revealed that X-ray irradiation regulaory loop specwfic the cells. The poMn Ile wlth a consit incease sinoe 5 month up to 2 ywr The epeno did not induce any mutations of p53 gene in of MyoD iniIory factor Id. durig aging. rem ns con_ The tn l results suggest that loss of a gene on chromosome 6 without of human of tansacvad genes foreAChR and MCK perlel lhose fd MyoD and myoge p53 alterations results in extension of lifespan dwig agig Intedr y myosin ranin Is c y u . fibroblasts without immortalization. Immunolluoreeence eperiment on sectoned muncie from young. adtlt and senie mouse dened that myogenin Is detab in young and eJnle muncle nuce and s completely abeent In adult muscle, confrmn the remita on mRNA excprselaon Thene data ndiate iat, during aging, skele musne undergoes a epr of myogenesis medlated by a set of muscle speifc regulatorY tors an c ry mechaniam for the dedifferentte process (Supported by CNR PF4NVECC0IIAMENTO and MURST B.M.Z) Wednesday. Aging (2728-2729) 469a

2728 2729 EXOGENOUS AMYLOID ABl-42 STIMULATES ACCUMULATION OF INFLUENCE OF THE HYDROPHOBIC DOMAIN OF THE ALZHEIME'S AP AMYLOIDOGENIC CARBOXYL-TERMINAL FRAGMENTS OF APP. PEPTIDE ON iTS SURFACTANT AND SELF ASSEMBLY PROPERTIES. ((B. Soreghan, J. Kosmoski, D. Burdick and C. Glabe)) Dept of Molec. Biol. and Biochen., ((A. Yang, D. Burdickc, J. Kosmoski and C. Glabe)) Dept of Molec. Biol. Univ. of Calif., Irvine. CA 92717 and Biochem., Univ. of Calif., Irvine. CA 92717 (Spon. by D. Fosket) The amyloid f-peide (Af) is the major proteinaceous component found in extracellular amyloid deposits charactristic of Alzheuness diseas. Te sequence of is Exogenous amyloid peptide, AB1-42, is internalized from the culture medium prnmary A,t amphipathic: residues 29-42 consist of sequences within the hydrophobic tr an and accumulates intracellulrly in late endosomes and lysosomes in human domain of the amyloid pcursor proein (APP) and the aminoterminal segment of AP is cells where it is resistant to furither degradation. We have analyzed the effect primarily hydrephilic as it is comprised of sequences within the extracedlular domain of of internalized ABI-42 on the metabolism of the amyloid precursor protein APP just prior to the membrane domain We have found that AP displays sevral (APP) in stably transfected 293 cells by 35S-methionine metabolic labeling, properties of detergents or srfa5ctants. Increasing co aons of AP analogs of 1-36 followed by immuno-precipitation. We found that the amounts of C-terminal, residues or longer lower the surfa tension of water up to a critical concentration above which litle further decrease in strfyce tension is observed. At concentrations above this amyloidogenic fragments, are dramatically enhanced by the treatment of the critical concention, increasing amounts of non-covaknt aggregates of Aft are rmadily cells with ABI-42, but not ABI-28, which does not accumulate in cells. In observed by SDS ge elephoresis and gel filtration chromatography. Furthermore, the particular, a 16 kDa C-terminal fragment is highly enriched in the insoluble formation of Af aggregates is correlated with the appeance of a hydrophobic environment fraction of the cell lysate and is colocalized with internalized 125I-labeled which excludes water as meansued by the partitioning of diphenylhexatriene. These rmslts peptide. The stimulatory effect is not due to an increase in the rate of APP siggest that the strucre of AP in solution is aially amphipathic and that the pnnciples synthesis or an inhibition of the non-amyloidogenic processing pathway for which govern micelle formation may also apply to the mechanisms of amyloid aggregation assembly. To test whether nature or amino APP. Pulse-chase analysis of the 35S-met labeled 16 kDa fragment indicates the hydrophobic the specific acid sequence of the hydrophobic carboxyl terminus of Af is critical for its nrfatant-like properties, we that it is much more stable in AB1-42 trated cells, while it degraded in rapidly substitued residues 29-42 with sequences derived from the rnaebane domains of the untreated control cells. Our results suggest that the 8-peptide may stimulate P-adrenergic rcepto and the LDL rceptor. Although the sequence of these chimeric its own production by aggregating with newly synthesized amyloidogenic anaologs is amphipathic, they do not lwer the suLrfa tension of water. Furthermore, fragments, thereby protecting the AB domain from degradation. These results although the chimeric Af anologs display some similar properties as the Af pedeks, only suggest that amyloid accumulation could be a "run away" process once AP forms typical 6 mm amyloid fibrils. These results sugges that the specific sequence of the hydrophobic domain of and not nature, is important for its amyloid aggregates have nucleated inside the cell. Supported by the AP, soldyits hydrophobic surface activity (its linear amphipathic stmcture in solution) and its ability to self assemble American Health Assistance Foundation and NIH NS3120. into amyloid fibrils. Neurotransmitters, Peptides, and Receptors (2730-2733)

2730 2731 EXPRESSION OF THE VASOPRESSIN GENE BY HUMAN BREAST CANNABINOID RECEPTOR EXPRESSION IN MACROPHAGE LIKE CANCER. ((MJ. Fay, X Yu, V. Menoli*, and W.G. North)) Departments CELLS IN RAT BRAIN. ((D. Sinha, K.L. Mitro, and L.A. of Physiolgy and Pathology*, Dartnouth Medical School, Lebanon, NH Matsuda)) Department of Psychiatry and Behavioral 03756. Sciences, Medical University Of South Carolina, Charleston, SC 29425. The purpose of this study was to determine if the vasopresn gene is normally expessed by breast cancer, and if this ex on leads to the Major histocompatibility complex classII (Ia) fomation oa cell surface mara for thindisease. We have prviously antigens on antigen-presenting cells are involved in esablished in small-cell lung cancer that exprssion of the vascpressin gene the recognition of antigen by T cells. In the brain, leads to the formation of 20 and 42 KDa cell face markrs (NRSA, Ia expression is confined to some macrophage-like nearophysin-related cell surface antigen). Vasopressin gene epession by cells in the meninges, especially cells around blood 19 breast tumors, prpared as acetone fixed specimens, was examined by vessels. Here we report the expression of the avidin-biotn (ABC) immnoiso y using antibodies dircted against cannabinoid receptor on these macrophage like cells vasopressin (VP), the brdging pepide region of the vasopressin precursor by in situ and Northern hybridization techniques (pro-VP), vasop_esin-assocated human neurophysin (VP-HNP) and during rat brain development. These findings are vasopressin-associated glycopeptide (VAG). All tums testd stained interesting in light of the fact that it is presently positively with anti-VP, and anti-VAG. Of 14 tumrs examined with anti- hypothesized that the differentiation of monocytes (pro-VP), 11 de positive staining, while only 1 of 19 tumors was into microglia takes place exclusively during posiive for anti-(VP-HNP). Surrounding normal breast tissue gaec negative embryonic development but not in the adult. We have staining with all of these antibodies. Wester-blot analysis from SDS-PAGE also developed monospecific antibodies to the brain using 4 biopsied human brast tumors and a monoclonal antibody directed cannabinoid receptor which may be used to show against VP-HNP rvealed protein products with apparent molecular weights whether this receptor plays a role in the secretion of 20 and 40 KDa. direct immunofluoescece with anti-VAG and flow of cytokines by cultured microglial cells. These cytoessetry analysis using live MCF 7 breast cacinom cells vealed cell studies address the possibility that the cannabinoid surface i unoreactivity which was 7 fold grater than negative controls receptor is involved in degenerative neurological These studies suggest that vasopressn-gene related products are commonly disease, or that drugs which target this receptor may expressed by breast carcina cells, and that this expssion in cultued cells prove useful in treating central nervous system leads to the production of a cell-surface antigen. disease. (Supported by DA08104).

2732 2733 LOCALIZATION OF mRNA FOR THE MUSCARINIC Ml RECEPTOR CHARACTERIZATION OF THE HUMAN VASOACTIVE INTESTINAL IN RAT STOMACH, COLON AND BRAIN. ((H.F.Helander, K.Bamberg, PEPTIDEi RECEPTOR-MEDIATED INTRACELLULAR CALCIUM INFLUX K.G.Helander, D.Melle, G.Sachs)) Wadsworth VAMC/UCLA, Los IN STABLE TRANSFECTANTS. ((S.P. Sreedharan, M. Xia, Y. Kong, and Astra Sweden. and E.J. Goetzl)) Departments of Medicine, and Microbiology/ Angeles, CA, Hassle AB, M6lndal, Immunology, Univ. of California, San Francisco, CA 94143.

The muscaninic Ml receptor is one of several types of receptors for Vasoactive intestinal peptide (VIP) has potent effects acetylcholine. Aims: To localize cells in the oxyntic mucosa which on nervous system, lung, gastrointestinal tract, and immune transcribe the Ml receptor. Methods: We used 3H or 35S labelled functions. Human embryonic kidney 293 cells were stably- riboprobes, 375 nucleotldes long, to localize the mRNA for the Ml transfected with human VIP1 receptor (HVR) cDNA, receptor by in situ hybridization on paraffin sections of rat stomachs (a generating 293V5 cells which expressed lO HVRs/cell that specifically bound VIP (Kd - 0.2 nM). VIP evoked normal one, and another one including a 3-day old experimental ulcer, dose-dependent increases in levels of intracellular cAMP and respectively); sections from colon and brain were included for calcium ([Ca2+l,) concurrently in 293V5 cells, and in comparison. Hybridization was detected by autoradiography, and after cells bearing native HVRs. The increase in [Ca2+i] was staining with hematoxylin-eosin the sections were studied in the observed in the absence of extracellular calcium and was microscope. Resultso In sections of oxyntic mucosa hybridized with the abolished by pretreatment with 10 nM thapsigargin, antisense probe there was a fairly strong accumulation of silver grains implicating release from intracellular stores as the source of the Pertussis toxin treatment over the cells and a weaker accumulation over the mucous calcium. (PT) partially zymogen decreased the VIP-induced increase in in 293V5 It was not to detect in ICa2+N] cells. possible any hybridization the parietal cells cells, suggesting a role for Go and/or Gq proteins. or the lamina propria. In the ulcer margin and in the colon, there was a VIP additionally stimulated phosphatidylinositol hydrolysis moderately strong hybridization in the epithelial cells of the mucosa. In in 293V5 cells in a dose-dependent manner (ECso - 12 the brain, hybridization occurred in the hippocampus, the choroid nM), indicating that the VIP-induced release of intra- plexus, the pyramid cells, the medial habenular nucleus and the cellular calcium depended upon the activation of arcuate nucleus. The sense probe showed no phospholipase Co, that was mediated by HVR via specific binding. PT-sensitive G proteins. Conclusion: mRNA for the muscarinic Ml receptor is transcribed by the (Supported by NIH grants DK 44876, AI 29912, and AI 34570) gastric zymogen and mucous cells, the colonic epithelium, and several regions of the brain. 470a Neurotransmitters, Peptides, and Receptors (2734-2739). Wednesday

2734 2735 FUNCTIONAL PROMOTER CHARACTERIZATION OF THE A 60 kD PROTEIN, EXPRESSED PREDOMINANTLY IN RAT RAT SEROTONIN la RECEPTOR (,ENE BRAIN, BINDS TO 5-FITIc AND 5-HT2 mRNA. ((H.T. Kao, S.R. ((A.C. Chin and R.D. Ciaranello)) Department of Psychiatry, Stanford Ghaf(x)ri, and R.D. Ciaranello)) Nancy Pritzker Laboratory of ULiiversitv Stanford. CA 94305-5485. (Sponl. by A.C. Chin) Molecuilar and Developmental Neuirobiologv, Stanford University Medical Center, Stanford, CA 943(05. The neutrotransmitter, serotoniin (5-hydroxytryptamnine, 5-HT), plays a ma,jor role in nulimierouis behavioral and physiological functions including The 5-HT-T and 5-HT2 receptors are members of the serotonin 5-HT2 sleep, paini perception, sexuial behavior, memory and control of mood. The family of receptors. sharing similar phamiacology, signal transduction evidence inidicates that adirenial steroids inflLuence binding at 5-HTla receptors pathways. and sequenice honmology. The nmRNAs for both receptors and that the restilting chaniges in serotoniergic activity may play a role in the contain utntranslated reg ions ii-ore than twice the length of the coding development of depression and anxiety disorders by affecting the ability of region, suggesting that post-transcriptional regulation may be an the animals to adapt to stress. Adrenalectomy (ADX) results in an increase in important mechanism for regulating these receptors. Since many post- binding at 5-HTj;j receptors in the hippocampus. Our lab has recently transcriptional processes, sLich as the control of RNA stability or demonstrated that ADX also resuilts in a rapid increase in the transcription translation, are exerted by the action of RNA binding proteins, we rate of the 5-HTlti gene and that this tipregulation is abolished in the presence began ottr investigations by searching for brain proteins that bind to of gluicocorticoids (Zhong and Ciarainello). To investigate the mechanism by RNAs encoding 5-HTIC aiad 5-HT2 receptors. We observed several which glticocorticoids regtilate 5-HTta gene expression we isolated the proteins capable of biniding to both 5-HTIC and 5-HT2 receptor RNAs, corresponding rat genomic clones. Primer extension, reverse transcriptase- wheni binidinig was detected by crosslinkinig radioactive RNA to rat brain and nuclease pcr protection assays have been employed to map the cortical extracts. Most of the bindinig is confined to the 3' UTR and transcriptioni initiation site. SeqLtence analysis of approximately 2kb of 5'- coding region of the RNA. We initially focsised on one protein, a 60 kd revealed the absence of a GRE. In order to flanking regioni conlsensus define protein, that binds in a sequence-dependent nmanner and recognizes AU the cis-anid trait.s- elenienlts that Linderly this regulation, we have established rich donmains. 5-HTlc RNA seqttenices can compete against binding of both transietit and stazble traitsfectioni systems in cell lines that display this prteiti from 5-HT2 RNA, suggesting that the same protein is expression of the endogenous 5-HTta gwene. ResuIlts of our studies capable of binding to both RNAs. Saturation kinetics indicate that the with 5-HT1 condtIcted promiloter-reporter deletion plasmids will be affinity of the interactioni is high (Kd = 1.2 nM). The 60 kd protein is presetited. expressed predomiaiilntlsv in the central nervoLsS system, with highest levels in the cortex and hippoxampus. We hypothesize that this protein is an important post-transcriptional reulIator of a class of brain RNAs, which incltides somiie tmiRNAs encoding neurotransmitter receptors.

2736 2737 ENHANCEMENT OF CALCIUM SIGNALS EVOKED BY THE RAT CHANGES IN THE ASSOCIATION OF G PROTEIN SUBUNITS WITH SUBSTANCE P RECEPTOR (SPR) LACKING THE 83 C-TERMINAL THE CLONED MOUSE 8 OPIOID RECEPTOR UPON AGONIST AMINO ACIDS. ((W.S. Saltsmanl, IL Lil, 0. Hauaer2, N.D. Boydl, J.E. STIMULATION. ((S.F. Law and T. Reisine)) Department of Pharmacology, Krause3, S.E. Leemanl adJ.RBlusztajnl)) iBono Univ. Sch. Med., University of Pennsylvania, Philadelphia, PA 19104. Boston MA 02118; 2McLean Hosp., Belmont, MA 02178; 3Washington Univ. Sch. Med., St. Louis, MO 63110. The primary action of opioids is to inhibit the modulation of synaptic Two isoforms of SPR, pes generated by proteolytic processing, have transmission in both the central and peripheral nervous systems. Opioids been detcted in rat salivary glands. They differ by the presence or absence induce their biological actions through association with three classes of of an 80-100 C-temina tail. Binding of SPand SP-evoked amino-acid-long receptors, 8, Y., and a. At the cellular level, 8, opioid receptors inhibit adenylyl changes in intracelularcalcium concentrations (LCa2ii) (using Fura-2) wae cyciase and Ca++ conductance, while stimulating K+ conductance, and deteamined in Chinese haster ovay cells stably unsfcted with the trnm- Na+/H+ exchange. Opioid receptors are coupled to these effector systems cated (SPR324) and full length (SPR407) Both reepto bound by G proteins. With the recent cioning of the 8 opioid receptor, the ability to well M. The numbereceptors cell was SPequally (KO.1 ave per study its association with various G protein subunits in isolation from other 0.3x 105 (SPR324)and1.0x105 (5PR407). SP rapidly elevated [Ca2+iin the tawfectants with similar potency (ECso values: 12 nM and 0.78 nM for opiold receptors may allow insight into the signalling process between 8 SPR324 and SPR407, respectively), however, the magnitud and time corse opioid receptors and effector systems. To answer the question of which G of the sponses mediated by the two receptors differd greatl . Activation protein subunits associate with the cloned 8 opioid receptor, G protein of SPR324 by 1 nM SP causedarapid initial elevation of [Ca2+]ifrom 30 to directed antisera were used to immunoprecipitate 8 opioid receptor/G protein 780nM followedbyafallto a prolonged plate of 400 nM. Activation of complexes. Our resuits show that 8 opioid receptors stably associate with SPR407 led only to a slowerrise in [Ca2+ji to a similar plateau Enhance- Gial, Gio3, Goa, Gpl, and GO2. Upon binding of agonist to the receptor, the ment of calcium signals evoked ativation of SPR324 was also observed by G protein association changes such that Gial is no longer coupled to the in calcium-frce medium; 1 nM SP devated [Ca2+]J by an avrage of 283 nM and 106 nMin cells transected with SPR324 and SPR407,irespedveIy. By receptor, whereas the receptor now associates with Gia2, and remains coupled to analogy to p adrenergic receptors (PAR), which are desitid by PAR Ii- Gia3, Goa, Gpl, and GP2. Our findings reveal aiterations in 8 receptor/G attenuation of in nase, ftese signals SPR407-tansfected cels may be due to protein coupling that occur as a resuit of agonist binding . These dynamic of phosphorylafion multiple se/t sites in the tail of this SPR by a similar changes in 8 receptor/G protein associations may be the underlying basis for The data that the two SPR isoforms distinct ldnase. suggest have physiologi- the activation of the 8 opioid receptor signal transduction pathway. cal functions.

2738 2T39 IN VITRO MORPHINE-INDUCED DESENSITIZATION OF MOUSE PERITONEAL CLONING OF A SECOND ALPHA ACETYLCHOLINE RECEPTOR SUBUNIT FROM MACROPHAGES. ((E. Zapata-Tomei and F.L Renaud)) Bblogy Department, _.M"qIR-Ixams. ((P. Gamez', T. Valdez', T. Miranda', C.-M. Jen', S. University of Puerto RIco, Rio Pledras Campus, San Juan PR 00931-3360 Alcala', S. An', J. Lye', T. Barnes, and J. Lewis)) 'Division ofLife Sciences, Univ. of Texas at San Antonio, San Antonio, TX 78249, 2Dept. ofGenetics, Washington Univ. Drugs of abuse have been postulated to serve as a co-factor In Infections Medical School, St Louis, MO 631 10, and 'Dept ofBiology, McGill Univ., Montres], PQ that afflict drug addicts such as AIDS. Reports from our laboratory and H3A lBI others suggest that this may be due to a depression of the immune system by drugs such as morphine. We htad shown that an acute morphine exposure. In a shotgun genomic sequencing effort to define the nature oflet-354, one of us (J. Lye) Inhibits phagocytosis of opsonized sheep RBC (SRBC) by mouse peritoneal found sequence fiagments with high homology to nicotinic alpha acetylcholine receptor macrophages In vitro, whereas a chronic exposure caused desensitization to (AChR) submits. The seques were contained within cosmids capable ofrescuing un-74 the effect of the drug. We now report a detailed study of the kinetics of mutants. wsc-74 mutation is associaed with loss ofnicotinic acetykholine receptor function desensitization and explore how other phagocytic functions are affected. A in C elegau. wc-74 mutants are pharmacologically unresponsive to all nicotinic dose-response study showed a biphasic effect with 50-100 nM morphine acetylcholine agonists effective on the wlld type and re also severely deficient in biing of Inducing desensitization optimally, whereas higher concentrations had no ['Hmeta-aminolevamisole, a nicotinic agonist ofnematode AChRs. A screen of aC effect. A time course study showed that in desensitized cells morphine may elugancDNA lbwry produced two positive clones at a frequency of I in 100,000. actually stimulate phagocytosis of SRBC. However, under certain Sequencig ofan almost full-klgth cDNA confms that the cDNA encodes a nicotinic alpha condtions, morphine withdrawal will inhibit phagocytosis in desensitized subunit as shown by its high homology to the wuc-38 alpha subumit sequence ofC egants cells. Since addicts are usually exposed to opiates intermittentiy, we tested and to the sequenesofother nown nicotinic AChR alpha subunits. Like unw-38, the new the effect of aitemating periods of exposure and withdrawal of different alpha subunit conis about 10 addiional amino acids that are not found in alpha submits of lengths on the development of desensitization. Regardless of the total insects between the Cys loop region and the vicinal cysteines equivalentto Cys-192 -193. exposure period, withdrawal for 4 hrs was enough to prevent RFLP mapping studies are underway utlling rasoson andy-ray-induced mutants of desensitIzatIon. Flow cytometric analyses demonstrated that the production wsw-74 to detemine if the new alpha subunit is indeod encoded by ww-74. The possibility of reactive oxygen intermediates was stimulated by acute exposures to thatthe new alpha subunit is ww-74 posesa mild pardox. Mutant studies show that both morphine but Inhibited by a chronic exposure; a simgar inhibition was anw-74 and ww-38 ae essential to produce a phamacoogically fumctional levamisole detected on the production of nitric oxide and secretion of Il-la. We nicotnic AChR Ifboth are alpha subunits, it appan that the nematode levamisole AChR is conclude that the muftiple effects of opbids on macrophages may have an obligate heterodimer ofalpha subunits. escalating effects on other cells of the imune system, thus contributing to lowerng the defenses of the drug addict. Supported by NIH Grant S06 GM- 08102 Wednesday. Neurotransmitters, Peptides, and Receptors (2740-2745) 471a

2740 2741

INFLUENCE OF GLYCOSYLATION AND AMINO ACID SUBSTITUTIONS IN TWO C. ELEGANS AVERMECTIN-SENSITIVE GLUTAMATE- CONFERRING a-TOXIN RESISTANCE TO RECOMBINANT DNA DERIVED GATED CHLORIDE CHANNEL SUBUNITS: STRUCTURE AND NICOTINIC RECEPTORS. ((S.H. Keler, H. Kreienkamp, R. Maeda, and P. EXPRESSION OF THE GENES. ((M. Hamelin, M. Chou, D. Cully, Taylor)) Department of Pharmacology, University of California, San and D.K. Vassilatis)) Merck Research Laboratories, Diego, La Jolla, CA 92093. Rahway, NJ 07065-0900. (Spon. by J. Barker).

Sequences of c-bungarotoxin resistant and susceptible -subunits of the cDNA clones encoding for two subunits of a C. elegans nicotinic acetylcholine receptor have been elucidated, and include avermectin-sensitive glutamate gated chloride channel substitutions encoding glycosylation at amino acids 189 (cobra) and 187 have recently been isolated by expression cloning (Cully (mongoose), a substitution from proline to leucine at amino acid 194 et. al., submitted). These subunits, GluCla and GluCl$, when (P1 94L), and an additional glycosylation signal at amino acid 111 coinjected in Xenopus oocytes, form a heteromeric (cobra). Our previous study using site-directed mutagenesis and channel that reproduces the electro-physiological mammalian cell expression of the intact receptor on the cell surface properties seen with total mRNA. The GluCla and GluClp demonstrated that N-linked glycosylation signals at positions 189 and 187 peptides share 45% identity in their amino acid sequence, conferred up to a 600 fold reduction in a-bungarotoxin affinity, whereas and represent a novel class of invertebrate glutamate- P1 94L alone had little or no effect (Kreienkamp et. al., 1994. J. Biol. gated chloride channels. The genes encoding GluClc and Chem. 269, 8108-8114). The mutation P1 94L was introduced into GluClI are on distinct loci. Furthermore, their intron cDNA templates that code for the 187 and 189 glycosylation sites, and the positions are not conserved, suggesting that the two genes 111 glycosylation site found in the snake was added to the template diverged early in evolution. We will present data containing the 189 glycosylation site and the P194L substitution. concerning the nature of the RNA transcripts from both Preliminary experiments using glycosidase treatments and mobilty shifts loci and the tissue localization of their expression. on SDS-PAGE indicate that oligosaccharides are expressed at the 11 1, 187 and 189 glycosylation sites. The insertion of P194L with the 189 glycosylation site confers up to an additinal 80 fold reduction in affinity, and the 111 glycosylation signal further decreases affinity two fold. Analysis of the rates of a-toxin association and dissociation suggest that glycosylation places a constraint on the receptor in producing a high affinity configuration. Supported by USPHS grand GM 24437.

2742 2743 DIADENOSINE TETRAPHOSPHATE (Ap,A) MODULATES CLONING, EXPRESSION, AND FUNCTIONAL CHARACTERIZATION OF A BIOGENIC AMINE METABOLISM IN PC12 CELLS AND BRAIN HUMAN LUNG SECRETIN RECEPTOR. ((D.R. Patel, and S.P. SYNAPTOSOMES. ((E.B. Pivorun)) Department of Biological Sciences, Sreedharan.)) Department of Medicine, University of Clemson University,Cemson, SC 29634. (Spon. by J.P. Wourms.) California, San Francisco, CA 94143

Membrane preparations from PC12 cells and synaptosomes exhibit The 27-amino acid neuroendocrine peptide secretin specific receptors for Ap.A. Chromaffin cells and neurons of a diverse stimulates tyrosine hydroxylase activity in the CNS and array of vertebrates co-release ApA suggesting a modulatory role in the fluid and electrolyte secretion in the GI tract, inhibits PNS and CNS. The role of extracellular Ap,Ain the nervous system is gastrin and gastric acid release, and affects cardiac and unknown. HPLC with electrochemical detection had been used to renal function. Specific secretin receptors (SRs) have been extraelular on dopamine release from monitor the effects of Ap,A and detected on pancreatic acini, gastric glands, neuroblastoma metbolism in PC12 cells. Ap,A resulted in a selective dose dependent cells, and liver cholangiocytes. We have isolated a 1613 bp Increase in extra- and intracellular leveis of dihydroxyphenylacetic acid human SR (HSR) cDNA from lung tissue, which encodes a 440 with no effect on the other The (DOPAC) metabolites. increasein amino acid G protein-coupled HSR protein (MW - 50 kDa), with DOPAC levels suggested that Ap,A stimulates monoamine oxidase an homology of 82% with rat SR and 37% with human vasoactive (MAO) activity. PCI2 celis and brain synaptosomes were preincubated intestinal peptide, receptor. Stable transfectants of with exogenous radioactive dopamine and serotonin, respectively. Both HSR in 293 cells (293S12) expressed 10l binding sites preparations responded to with dose and time dependent (K Ap4A inaeases for secretin/cell d 3 nM). PACAP-38, VIP, and in DOPAC and 5-hydroxyindoleacetic acid (5-HIAA) production. The glucagon were less potent than secretin in displacing bound synaptosomes responded to 10 and 20 min with Ap,A exposures a 215I-secretin by 4 orders of magnitude. Secretin maxmal increae In5-HIAA leves of 60-80% over control levels. A more concurrently evoked dose-dependent increases in intra- profound increase was noted in PC12 cells A 30 sec exposure resultedin cellular cAMP and calcium levels, and stimulated a 5-fold a 75-100% Increasein DOPAC levels; while 5 -20 min exposures resulted increase in phosphotidylinositol hydrolysis in 293S12 cells. in approximately 3 to 4-foldincreass in DOPAC levels. Preincubation Northern blot analysis of human tissue aRNA revealed the with MAOinhibitors prevented the observedincreases in DOPAC relative intensity of a 2.1 kb HSR transcript being pancreas levels. A hyperbolic dose response curve was observed with maximal > kidney > small intestine > lung > liver, with trace 1500-1800 elevations occurring at doses of IM Ap,A This study suggests expression in brain, heart and ovary. that extracellular Ap,A is a potent and rapid activator of MAO activity. This purinergic agent may have a neuromodulatory role in the vertebrate (Supported by NIH grant DK 44876) nervous system by stimulating biogenicamine metabolism.

2744 2745 MULTPLE INOSITOL 1,4,5-TRISPHOSPHATE RECEPTORS IN NlE- ENKEPHALIN AND PROHORMONE CONVERTASE 2 (PC 2) 115 CELLS ((I. Kovacs, J.S. Coggan, A. Maranto* and S.H. Thompson)) EXPRESSION BY THE NEURO-2A NEUROBLASTOMA CELL Hopkins Marine Station and Department of Biological Scienwcs of Stanford LINE. ((A.M. Bamberger, L.P. Pu, and Y.P.Loh)) Section on Cellular Neuro- University, Pacific Grove, CA,*Tufts School of Medicine, Boston, MA. biology, Lab. Dev. NeurobioL, NIH/NICHD, Bethesda, MD 20892. Mouse neuroblastoma Neuro-2a cells were examined for the expression of pro- In NlE-l 15 neuroblastoma celis several G-protein coupled receptors activate enkephalin mRNA and met-enkephalin ([met]Enk). Cells were cultured in Dul- phospholipase C andIP3 production. Three types of InsP3 receptors have been beoco's modified Eagle's medium with 10% FBS and harvested or fixed when cloned and identified in differenttissues and species We examined NIE-l 15 they were about 80% confluent.In situ hybridization demonstrated the presece po cells for InsP3 receptortypes. Reversetranscriptase ymeras reaction chain of pro-enkephalin mRNA in these cells. Pro-enkephalin expression was shown by (RT-PCR) was used to level of mRNA for the analysis estimt the thrce type immuonocytochemistry using an antibody which recognizes the precursor form. InsP3 MessengerRNA forall of receptors of receptors. three types was By RIA using a [metlEnkspecific antibody, tmetiEnk was detected, indicating detectable with relative abundance: typeI<1<11. Cell ines usually express hig that these cells can process the precursor to yield the bioactive pentapeptide. receptor may in cell level of type InsP3 which have afumction growth. We Because PC2 has been imPlicated as an important enzyme in pro-enkephalin used stable antisense transfection to create clones lacking the expression of the Processing, we have also investigated PC2 mRNA expression by the Neuro-2a type receptor. Antibody specific for the type III receptor showed cels. Using in situ hybridization, we demonstrated thatPC2 mRNA is in- immunoreactivity at 250 kD with the protein isolated from the wild type cells deed being expressed by thesecells. Moretver, double labeling experiments, but not from the dones. There is no significant difference in the mRNA ratio combining in situ hybridizationfor PC2 with immunocytochemistry for pro- for the different types of insP3 receptors between the dones lacking the enkephalin, showed coocalization of PC2 mRNA and pro-enkephalin in the same expression of the M receptor protein and the wild type.Cells lacking the cells. Thus, the Neuro-2a cell line can be an excellent model to study type receptor have lower growth rate and have altered morphology not only pro-enkephalin gene expression, but also pro-enkephalin processing by manifested by a greater number of neurites. PC2. 472a Neurotransmitters, Peptides, and Receptors (2746-2750). Wednesday

2746 2747 ANTHO-RW AMIDE IMMUNOREACTIVITY IN GRANULAR VESICLES CYltYTOXICITY INDUCED BY INTRACEI LULAR ZINC CHELATION IN RAT OF ORAL SPHINCTER NEURONS IN SEA ANEMONES. ((J.A. CORTICAL NEURONS. ((R. E. Sheridan and S. S. Deahpande)) Neurotoxicology Westfall, K.L. Sayyar, C.F. Elliott, and C.J.P. Branch, U.S. Army Medical Research Institute of Chemical Defense, APG, MD 21010 Grimmelikhuijzen*)) Department of Anatomy and Physiology, Kansas State University, Manhattan, KS 66506, *Department of Cell Biology and Anatomy, Cytotoxicity produced by chelation of intracellular Zn+5 was studied in isolated rat University of Copenhagen, DK-2100, Copenhagen, Denmark cortical neurons uDg the membrane permeant compound N, N, N', N'-etreakis (2- pyridylmethyl) ethylenediamine (TPEN). CeLs were removed from fetal rat cortex and Immunofluorescence microscopy using antisera to the cultured under standard conditions on polylysme-coated dihe for 2 weekcs. Cultures novel sea anemone peptide Antho-RWamide reveals were then exposed to TPEN in a DMSO vehide for 2 to 24 hours at 37eC. Morphological immunoreactive bipolar neurons crossing the mesoglea damage was seen with phase-contrast microscopy in cells exposed to 210pM TPEN for and extending into the circular sphincter of 24 hours. This consisted of granular opacities in neural processes and cytoplasmic blebs Calliactis parasitica. Using immunogold electron of the soama. In 50 100pM, cellular detachment from the substrate was common. These microscopy we found evidence of Antho-RWamide changes were correlated with release of cellular LDH into the supernatant. Both some immunoreactivity in granular vesicles of slender naxphoogical damage and LDH release were not significant at 2-4 hours of exposire but neuronal processes traversing the mesoglea and developed over time. However, brief 2-hour exposures to 100pM TPEN followed by bundles of entering the V-shaped myonemes making up washing produced damage, equivalent to that in continous 10pM TPEN, by 24 hours. the oral sphincter. In addition, the immunogold Ihis suggest initiation of cytotoxic events during the first few hours of exposire. The marker was found in synaptic vesicles aligned at paired electron dense membranes of neuromuscular cytxic effects of TPEN were apparendy linked to chelation of intracellular Zn" and junctions. These vesicles were smaller (54 nm in could nt be replicated with a 1:1 complex of Zn-TPEN, with the membrane impermeant diameter) than the granular vesicles (124-143 nm in chelator dipicolinic acid or with equivalent concentrations of the DMSO vehicle alone. diameter) of the neurites and were devoid of dense Incationd of the cortical cells with the competitive NMDA receptor antagonist APV or granular material. Similar granular vesicles (100-120 the noncompetitive antagonist MK-801 did not prevent TPEN-induced damage but did nm in diameter) were immunogold-labelled in some block gueimiced toxicity. Patch-clamp recordings of spikes in the cortical cultures neurites of the adjacent gastrodermal nerve plexus. Ashwed no significant increases in sonwnmw activity during the initial 2 hours of TPEN These results provide further ultrastructural evidence applications. Thus, the mechanism of TPEN cytoxicity does not seem to be mediated of peptidergic neurotransmission in sea anemones, by enhanced presynaptic release of endogenous glutamate acting through NMDA which belong to the first group of animals to evolve receptors. a nervous system. Supported by NSF grant IBN-9120161.

2748 2749 IMMUNOCHEMICAL ASSESSMENT OF EXPRESSION OF NEUROPEPTIDE NEUROTROPHIN-3 INDUCED SIGNAL TRANSDUCTION EVENTS IN PRIMARY RECEPTORS IN MOUSE LUNGS DURING PULMONARY IMMUNE HIPPOCAMPAL PYRAMIDAL CELL CULTURE. ((M.N. Marsh* and H.C. Palfrey.)) RESPONSES. ((S. Ichikawa, M.L. Warnock, S.P. Sreedharan, J.M. Beck, P.K. Dept. Pharmacol. and Physiol. Sci., Univ. of Chicago. Chicago, IL 60637. Byrd, E.J. Goetzl, H.B. Kaltreider)) Medical Service, V.A. Medical Center and The trkC gene encodes a tyrosine kinase receptor, gp145trkC, for Neurotrophin-3 (NT-3). Departments of Medicine Microbiology, University of California, San Francisco, CA To understand the role of gpl45trkC in the nervous system, we have investigated its 94121. expression in rat hippocampal pyramidal neurons and examined the effects of NT-3 on signal transduction in these cells. Neuronal cultures were prepared from embryonic day 17 Intratracheal (IT) administration of sheep erytocytes to systemically - primed mice rat hippocampus and grown for 10-14 d above a confluent astroglial layer. Previous PCR induces pulmonary immune responses characterized by: a) maximal mononuclear analysis showed that pyramidal neurons but not astrocytes express trkC-specific transcripts (Marsh et al., 1993. J. Neurosci. 27: 19448-19454). To determine if NT-3 leukocyte (ML) and neutophil (N) infiltration of the lungs by days 4-6, b) 3- to 10- initiates signal transduction in pyramidal neurons several parameters were measured. fold increases in the concenttifons of substance P (SP) and vasoactive intestinal Neurons were glia-deprived for 2 h prior to experimentation. Immunoprecipitation of peptide (VIP) in bronchoalveolar lavage (BAL) fluid by days 1-4, c) inceaaed levels p2lras from 32P-prelabelled cells showed that NT-3 (20ng/ml) increased GTP bound form of mRNA encoding receptors (Rs) for SP and VIP in MLs of BAL fluid by days 4-6. of the protein 3-fold over controls within 5 min. MAP kinase activity also increased and To elucidate the types and distribution of cells expressing SPRs and VIPRs during was maximal by 2 min (320% of control) relurming slowly to baseline over the next 60 pulmonary immune responses, fixed cryostat sections of lungs obtained on days I to min. NT-3 rapidly increased [Ca2,ji from -125nM to ItM with a slow recovery to 12 after IT antigen challenge were stained with rabbit antibodies to the Rs and basal levels over 10 min as measured using fura-2 based fluorescence microscopy. In the developed with peroxidase - labeled second antibody. Prior to challenge, VIPRs were presence of NT-3. the neurons showed a sustained elevation of ICa2+1i (eight-fold to reach detected in smooth muscle of some bronchi and blood vessels, and in airway titM) with a slow recovery to basal levels over 10 minutes. To assess a possible epithelium from bronchi to terminal bronchioles. By day 2 after antigen challenge, mechanism of Ca2+ mobilization in these cells, the ability of NT-3 to activate PLC-y was examined. Tyrosine phosphorylation of PLC-y increased after NT-3, as determined VIPRs disappeared from epithelial cells. receptors were present on incoming Both by antiphosphotyrosine staining of immunoprecipitated PLC-y, suggesting an increase in MLs (VIPR>SPR) and Ns (SPR>VIPR) in perivascular and airway infiltrates, but PLC-y activity. These results indicate that gpl45trkC constitutes a functional receptor not on lymphocytes. R-bearing leukocytes peaked at day 2 and nearly completely for NT-3 in hippocampal pyramidal neurons, and that the neurotrophin rapidly activates disappeared by day 12. The close temporal relationship between increased several signal transduction events. Supported by NS32150. concentratons of VIP and SP in BAL fluid and influx of leukocytes expressing VIPRs and SPRs suggests that these neuropeptides may regulate the expression of pulmonary immune and inflammatory responses to IT antigen. Supported by NIH Grants Al 29912 and Al 34570.

2750 DEPOLARIZATION INDUCES THE PHOSPHORYLATION OF MacMARCKS/MRP IN PC12 CELLS AND IN RAT BRAIN SYNAPTOSOMES ((Chang, S., *H.C. Hemmings, Jr., and A. Aderem)) Laboratory of Signal Transduction, The Rockefeller University, and *Dept. of Anesthesiology, Cornell University Medical College, New York, NY 10021

MacMARCKS/MRP is a member of the MARCKS family of PKC substrates. MARCKS binds both actin and calmodulin, and is believed to regulate actin membrane interactions during neurosecretion, membrane trafficking, and phagocytic activation. By contrast, the function of MacMARCKS/MRP is completely unknown. We report here that MacMARCKS/MRP is present in both PC12 cells and in rat brain synaptosomes. It is phosphorylated upon PKC activation, and phosphopeptide mapping demonstrates that the same residue are phosphorylated in vivo as in vitro. When PC12 cells are depolarized with 60mM K+, MacMARCKS/MRP phosphorylation is increased 5 fold over basal levels, and this increase is Ca 2 dependent. Immunofluorescence confocal microscopy localizes the protein to varicosities along neurite processes, and in the cell body. In purified rat brain synaptosomes, both phorbol esters and depolarization markedly increased the phosphorylation of MacMARCKS/MRP. Temporal analysis indicates that MacMARCKS/MRP is more rapidly phosphorylated after depolarization than MARCKS. These results suggest that MacMARCKS/MRP may have a role in neurosecretion. Wednesday. Neurobiology: Other (2751-2756) 473a

2751 2752 EFFECT OF BRAIN ISCHEMIA ON CDC2-LIKE PROTEIN IMPORTANCE OF PHOSPHOGLUCOMUTASE'S STIMULUS-SENSMTIVE KINASES. ((S.L. GREEN, P.R. VULLIET)) Department of Molecular MEMBRANE ASSOCIATION FOR Ca'' HOMEOSTASIS IN THE NEURON. ((N.A. Biosciences, School of Veterinary Medicine, UC Davis, Davis ,CA Veyna and RB. Marchase)) Department of Cell Biology, University of Alabama at 95616. Birmingham, Birmingham, AL 35294. Our laboratory has previously documented the stimulus-sensitive glycosylation of the Neuronal cdc2-like protein kinases phosphorylate cytoskeletal proteins glycolytic enzyme phosphoglucomutase (PGM) in rat cortical synaptosomes (RCSs). We now which are involved in axonal transport, cell differentiation and process present data indicating that a population of PGM is associated with a membrane structure of outgrowth. The objective of this study was to examine the effect of brain RCSs. Both western blot analyses and enzymatic data in synaptosomes that had been lysed and separated into membrane and cytosolic fractions showed membrane-associated PGM. ischemia on the specific activity (pmoles/min/mg) and subcelluar Interestingly, depolarization leads not only to increased glycosylation of PGM but also to an distribution of cdc2-like protein kinase in neocortex and hippocampus increase in the amount of PGM associated with the washed membrane fion. Enzymatic using a postdecapitative rat model of complete forebrain ischemia. At 5, assays for the cytosolic marker protein lactate dehydrogenase (LDH) were carried out in 15, and 25 min ischemia, there was a significant (p<0.01) decrease in the parallel to demonstrate the purity of the membrane fraction. Membrane association of PGM neocortical cytosolic fraction of cdc2-like activity to 74%, 30%, and 29% was first observed by Lee et al. in rabbit muscle sarcoplasmic reticulum. It was also in this system that it was first that PGM be in the of of control values, respectively. Comparatively, cdc2-like protein kinase suggested may important regulation cellular We believe that the role of PGM in the of cellular Ca is an indirect one, in the Ca:. regulation activity hippocampus declined to 83%, 34%, and 30% of controls carried out through the regulation of the levels of one of its primary substrates, glucose-6- after 5, 15, and 25 minutes of ischemia. In addition, western blot analysis phosphate (G-6-P). G-6-P has been reported to increase the amount of r5Calsequestered by using anti-cdc2 antibody showed evidence of proteolytic degradation of liver microsomes. We have observed a sinilar result using the Ca' sensitive dye Fura 2 in cdc2-like protein kinase after 25 minutes of complete ischemia. This data microsomes isolated from rat cortex and from cortical synaptosomes. In these systems G-6-P suggests that postischemic decline of cdc2-like protein kinase activity may significantly increased the rate at which Ca" was sequestered in the presence of ATP. This as not be mediated in effect is specific to G-6-P, several other sugar phosphates tested did increase the part by proteolysis. kinetics of Ca" sequestration. Glucose-l-phosphate, however, did increase the rate of Ca" Supported by the Alzheimer Disease Research Program, Department of uptake provided the microsomes were preincubated in its presence for 60 seconds prior to the Health Services, State of California addition of ATP. This would be sufficient time for G-1-P to be converted to G-6-P by the action of PGM. Therefore the membrane association of PGM may be critical to either modulating or localizing PGM activity, thus potentially altering G-6-P levels in a specific region.

2753 2754 DEFINING THE MOLECULAR CONSEQUENCES OF MUTATIONS IN SUPEROXIDE PROCESSING OF HUMAN f5-AMYLOID PEPTIDE PRECURSORS IN YEAST DISMUTASE 1 THAT CAUSE FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS. SUGGESTS A NOVEL PROTEASE WITH SPECIFICITY IDENTICAL TO I(D.R. Borchelt, M. Guarnieri, P. Wong, C. Pardo, M. Lee, H. Slunt, Z-S. Xu, S. MAMMALIAN a-SECRETASE. V.Hines2, M.Innis2, and Sisodia, N. Jenkins, N. Copeland, D. Price, and D. Cleveland)) Departments of ((W.Zhangl, Pathology, Neuroscience, Neurology, and Biological Chemistry, Johns Hopkins D.L.Millerl)) 1Department of Molecular Biology, NYS Institute for Basic Research, University School of Medicine, Baltimore, MD 21205, and Mammalian Genetics Staten Island, NY10314; 2Department of Microbial Expression, Chiron Corp., Laboratory, National Cancer Institute, Frederick, MD 21703. Emeryville, CA94608. (SpOn. by D.L.Miller) Mammalian cells can metabolize transmembrane f-amyloid peptide precursor Mutations in superoxide dismutase 1 (SOD11) have been linked to familial (APP), which is thought to be isnplicated in Alzheimer's disease, in several pathways amyotrophic lateral sclerosis (FALS), a dominantly inherited motor neuron which result in the secretion of the ecto-domain and f-peptide. Yeast contains disorder. Because SOD1 is a homodimeric dimerization of mutant enzyme, and precursor-converting enzymes homologous to their mammalian counterparts, and is wild-type SOD1 subunits could dominantly alter the or activity, stability, susceptible to genetic manipulation. As a first toward the identification of APP localization of SOD1 holoenzyme. To test this hypothesis we used transient and step secretases, we investigated the processing of human APP (695 and 751 isoforms) in stable gene expression paradigms to overexpress mutant human SOD1 in the Scha presence of wild-type mouse and/or human SOD1. In both systems, the activity yeast transformed with plasmids expressing APP cDNA of wild-type SOD1 subunits was unimpaired, and the kinetics of wild-type subunit controlled by the glucose-repressible ADH2/GAPDH hybrid promotor. In yeast degradation was distinct from that of mutant subunits. Our data indicate that strains transformed with the plasmids, high levels of APP (1% of total protein) were SOD1 subunits can readily dissociate, which in effect precludes the possibility detected on immunoblots. Like mammalian cells, yeast can cleave APP in the that mutant HuSOD1 could dominantly alter the function of wild-type SOD1 in middle of the fl-peptide region to generate a soluble C-terminal truncated fragment, heterozygous FALS individuals. Instead, mutant SOD1 appear to acquire while a complementary C-terminal fragment (7kD) accumulated in the membrane. injurious properties. In transgenic mice, expression of a mutant human SOD1, Amino acid sequencing demonstrated that the secretory cleavage site of APP was 12 a to encoding Gly Arg substitution at codon 37, causes motor neuron residues away from the membrane-spanning sequence and was identical to "a- degeneration. Because human SOD1 (G37R) subunits retain full nearly specific secretase" site in mammalian cells. A revealed the turnover of activity, loss of SOD1 activity cannot be the cause of motor neuron death in pulse-chase study FALS. The injurious property acquired by mutant SOD1 remains unclear; six out APP with tl/2 30-40 min and the parallel appearance of soluble APP and the C- of six mutant subunits retain full solubility in nonionic detergents, and appear terminal fragment, which further confirmed that they were the products of samne structurally similar to wild-type SOD1 subunits as they retain resistance to mild protease(s). Processing of APP occurred efficiently in a vacuolar protease-deficient protease digestion. Transgenic mice provide a means to investigate the strain and in an aspartyl protease (YAP3) null mutant. We conclude that yeast mechanisms by which mutant SOD1 cause motor neuron death. possesses a distinct protease that resembles mammalian a-secretase, and therefore can serve as a model for studying mammalan APP secretases.

2756 TRANSGENIC MICE SEQUENCE ANALYSIS OF A 23 kDa LEAD-INDUCED PROTEIN IN CHIMERIC HUMAN-MOUSE PRION PROTEIN (PrP) ASTROGLIAL CELLS. ((L.A. Opanashuk and J.N. Finkelstein)) Department of SPECIES-SPECIFIC FACTOR IN PRION Environmental Medicine, University of Rochester School of ((G.C. Telling, Scott, Schatzl, D. Foster, S-L.Yang, Medicine, Cohen, DeArmond Rochester, NY 14642. Torchia, F.E. S.B. Prusiner.)) University of California, Francisco, 94143-0518 (Spon. by S.B. Prusiner) Astroglial cells preferentially accumulate lead (Pb) and are less sensitive to its Transgenetic scrapie Syrian (SHa) and mice (Mo) overt deleterious effects than are neurons. However, there is some evidence barrier species transmission prions resides within the suggesting that Pb may induce precocious differentiation of astrocytes. To sequence. Transmission (Hu) prions from patients dying of begin to clarify the relationship of altered protein expression and Pb exposure, (CJD) >1.5years occurs with this study evaluated the effect of with on frequently apes monkeys, infrequently mice. We surmised exogenous Pb proteins synthesized in that prions to apes and mice reflected primary astroglial cultures. Cellular proteins were labeled with 13HI-leucine homology: apes compared to Hu and 89% and subsequently analyzed by SDS-PAGE and fluorography. The most notable hypothesis, transgenic (Tg) were constrcted that 10% Fchange was the elevated synthesis and accumulation of a 23 kDa protein (p23) express Tg(HuPrP) and non-Tg mice developed which persisted throughout a 14 day treatment period. Treatment of cells with neurologic days inoculation CJD human prions. To actinomycin D or cycloheximide to the addition conundrum, of Pb explore differs prior this Hu/MoPrP from MoPrP by 9 precluded and induction of Two-dimensional was constructed and designated p23. electrophoresis of cellular proteins Tg(MHu2M) mice developed neurologic disease -200 days revealed that p23 was resolved into at least nine charged species with pi brain homogenates from three patients dying of CJD. values ranging from 4.6-7.8. Cleveland mapping of the individual isoelectric Tg(MHu2M) mice with CJD prions produced MHu2MPrPSc; species indicated that 8 of the nine isoforms were related with the most acidic Mo prions produced MoPrPsc. The patterns of MHu2MPrPSc (p23, 4.6) isoform being a different Partial N-terminal MoPrPsc accumulation protein. sequencing of the brains of Tg(MHu2M) mice were distinct this acidic protein that it was a controlled The susceptibilities Tg(HuPrP) and Tg(MHu2M) mice to Hu suggested translationally growth prions additional associated species specific factor(s) is involved in prion protein (TUMP). The other proteins were N-terminally blocked. replication. species factor is a chaperone protein that catalyzes a Intemal sequence analysis indicated that these proteins were glutathione-s- change PrPCthat results in PrPScformation or it is an transferase X(GST-x). The identity of this protein was confirmed by Western enzyme PrPcprior its conversion into to be PrPScremains blot .Moreover, cytosol frornthe brains of Pb-exposed rats contained elevated Diagnosis, prevention and treatment of human prion diseases levels of GST-x as assessed Westernblot. Alterations in facilitated by protein expression by Tg(MHu2M) mice. may begin to provide an explanation for the molecular events leading to the protective and developmental effects observed with Pb exposure. 474a Neurobiology: Other (2757-2760). Wednesday

2757 2758 B-ADRENERGIC RECEPTOR BLOCKADE REDUCES ASTROCYTE BRAIN VAXUIAR RENIN EXPRFSSION IN DES AND NON DES TREATED RESPONSE TO INJURY IN VITRO AND IN VIVO. ((J. F. Reilly, L HAMSTERS. (( A.H.DODGE, I.A. REID, T. INAGAMI )) Bair, and V. K. Menon)) Dept. of Cell Biology & Human Anatomy, Departs., B.S., Calif.Coll.Pod.Med., S.F., CA; Physiol., School of Medicine, University of California, Davis, CA 95616. UCSF, S.F. ,CA; Biochemt., Vanderbilt, Nashville, Ten In response to injury, astrocytes undergo morphological changes leading DES treatment has been reported to induce hamster renal to the formation of a glial scar. The present study used a previously rat et tumors with renin positive secretory granules (Dodge et al, described model of mechanically wounded cultured astrocytes (Yu, 1988). DES has also been reported to induce renin to test the effectiveness of a AJA, al., J. Neurosci. Res., 34: 295-303, 1993) positive nonjuxtaglcnerular renal vasculature (Dodge et al, 8-adrenergic antagonist in reducing the astrocyte response to injury. A9CB meeting, 1990). Renin positive fetal vascular smooth were at the time Cultures treated with DL-propranolol (10-4 M) beginning muscle has been observed prior to the of the of wounding and continuing for three days. Immunohistochemistry for development apparatus AR, 1993). The present glial fibrillary acidic protein (GFAP) revealed that the injury response juxtaglcnerular (Dodge, study was undertaken to determine the action of DES treatment was attenuated by propranolol treatment. This effect was reversed by the addition of basic fibroblast growth factor (bFGF, 20 nghml). Addition of on renin expression in nonrenal vasculature. 15 male hamsters were treated with DES (59-257 days). Renin imnunohistochemical anti-bFGF (100 and 10-4 M propranolol to the culture medium gg/ml) staining procedures that were used have been described was no more effective than propranolol alone. This suggests that propranolol reduces GFAP expression by inhibiting the endogenous previously (Dodge, 1993). Brain renin positive vascular smooth production of bFGF by wounded astrocytes. The effectiveness of muscle was observed in 12 of the 15 DES treated hamsters. Plasma renin activity was higher in 7 of the DES treated was in an in vivo model of traumatic brain injury. propranolol also tested for nonDES For five days following a stereotaxic lesion to the rat brain, DL- hamsters than the activity reported previously et fran propranolol was injected (5 mg, 3 times per day) or infused (350 l±g/hr) treated control hamsters (Dodge al, 1988). Brains neonates and subcutaneously. Astrocyte hypertrophy was reduced, as demonstrated adult nonDES treated males, 15 day fetuses, These results that old adults (737-758) were stained for renin. Two of the old by GFAP immunohistochemistry. suggest propranolol brains had renin positive vasculature, but all other treatment effectively reduces the astrocyte response to injury, and provide specimens were negative. Therefore, DES treatment induced a useful model of decreased astrogliosis for future studies on CNS NIH grant AG 06159.) the expression of renin in sane brain vasculature; however, regeneration. (Supported by the correlation between this expression and plasma renin activity varied.

2759 2760 CHARACTERIZATION OF THE HUNTINGTON'S DISEASE GENE A PROTEIN RELATED TO HUMAN HUNTINGTIN IS ASSOCIATED PROTEIN PRODUCT, HUNTINGTIN, IN HUMAN TISSUES. ((N. Aronin, WITH TRANSPORT VESICLES IN RAT BRAIN NEURONS. ((M. DiFiglia, K. Chase, E. Sapp, C. Schw , A. Meloni, E. Martin, S. Reeves, F. MK E. Sapp, K. Chase, C. Schwartz, A. Meloni, E. Martin, S. Reeves, F. M. Boyce, J.P. Vonsattel, R. Carraway, M. DiFiglia)) Departnents of Medicine, Boyce, J.P. Vonsattel, R. Carraway, N. Aronin)). Departnqts ofNeurology, Cell Biology, and Physiology, University ofMassachusetts Medical Center, Pathology, and Neurosurgery, Massachusetts General Hospital and Harvard Worcester, MA and Departments ofNewology, Pathology, and Neurosurgery, Medical School, Fruit Street, Boston, MA and Departuents of Physiology, Massachusetts General Hospital and Harvard Medical School, Fruit Street, Medicine and Cell Biology, University ofMassachusets Medical Center, Boston MA. Woreester, MA

The gene defective in Hu n's disease, IT15, encodes a protein, huntingtin, The Huntington's disease gene, IT15, was recently isolated and found to encode which has no known function. As a first step in identifying the loction and a protein "untnn" with a predicted molecular mass of 348 kD. IT15 mRNA functional properties ofhunngti in normal tissues, we generated anti-peptide is widely exprssed in neuronal and non-neuronal tissues but the localization and antisera to three regions ofthe predicted human protein. The antigenic sites function ofthe encoded protein are unknown. Anti-peptide antisera directed included the N-terminal residues 1-17 and internal residues 647-660 and 2929- against the N-terminal 1-17 amino acids ofhuman huntigtin identified a protein 2942. Antisera were affnity purified and tested for their selective epitope which co-migrated with thyroglobulin at about 300 kD on Western blots of specificity using E coli-expressed recombinant proteins to corresponding human neuroblastoma and rat brain extracts. The detection of the 300 kD regions ofhuntingtin. Western blot analysis was performed with protein huntingtinreated protein in rat was significantly increased synaptosomal extracts derived from human cortex subcellular fractions and the human fractions ofrat cortex and caudate tissue. Immunohistochenical analysis in rat neuroblastoma celi line SYMY. Antisera directed to internal sites in the protein brain showed labeling ofneuronal cell bodies, dendrites, and axons throughout identified a single high molecular weight species at about 350 kD, the predicted the brain at the light microscopic level. Electron microscopic study revealed molecular mass ofhuntingtin. The antisera directed to the N-terminal immunoreactivity on the membranes of synaptic vesicles and in vesicles in recognized a single band at about 300 kD. Immunohistochemical localization in transport within dendrites and axon fibers. Staminig was also associated with the neuroblastoma celis showed a pennuclear cytoplasmic localization with antsera membranes and vesicles ofthe Golgi complex. These results suggest that a to all three sites. Results suggest that two related species ofthe huntingfin protein related to human huntingtin may be involved in vesicle transport in the protein may exist in human tiues. rat brain. Chloroplasts and Mitochondria (2761-2762).

2761 2T62 THE PRECURSOR TO A EUGLENA SOLUBLE CHLOROPLAST CONSTRUCTION OF A RZDOz-SENSITIV5 MALATE DESYDROGENASE PROTEIN IS TRANSPORTED FROM THE ER TO THE ((5.H. Muslin, D. Li, M. Schiffer, F.J. Stevens, N.I. CHLOROPLAST AS AN INTEGRAL MEMBRANE PROTEIN ((S. Donnelly, and L.B. Anderson)) Department of Biological Sciences, The University of Illinois at Chicago, Chicago, IL Chidananda, N. Rivenburg and S. D. Schwartzbach)) School of 60607-4833, and Center for Mechanistic Biology and Biological Sciences, University of Nebraska, Lincoln, NE 68588. Biotechnology, Argonne National Laboratory, Argonne, IL 60439-4833 The precursor to a Euglena cytoplasmically synthesized chloroplast protein, the small subunit of ribulose bisphosphate carboxylase (pSSU) is Light-dependent reduction of a target disulfide results in a change in the activity of the chloroplast NADP-linked malate a polyprotein composed of 8 mature SSUs. To determine the transport dehydrogenase. Tertiary structure modeling [Li et al., route from cytoplasm to chloroplast,and the site ofpolyprotein processing, Biophys. J. 67, 29 (1994)] suggests that oxidation of two Euglena was pulse labeled with 5S5-sulfate, organelles separated on crucial cysteines to cystine will restrict motion around the sucrose gradients, pSSU and SSU immunoprecipitated from gradient hinge which connects the two domains of the enzyme. we fractions and separated on SDS gels. After a pulse, unprocessed pSSU reasoned that if light activation involves these cysteine was found in the ER and Golgi apparatus but not in chloroplasts. After a residues and this mechanism, we should be able to construct chase, the amount of pSSU in the ER decreased and the amount in the redox-modulatable enzymes from the enzymes of non- photosynthetic species. We chose the NAD-linked malate was Golgi increased. Mature SSU found only in chloroplasts and never dehydrogenase from Sscherichia coli. Our model predicts that in the Golgi identifying the chloroplast as the polyprotein processing site. the cysteine residue in the chloroplast malic dehydrogenase ER and Golgi localized pSSU was not removed from membranes by corresponding to S,. coli residue 305 is close enough to form Na2CO3 but it was degraded by exogenous protease indicating pSSU is a disulfide with the residues on either end of helix 5 inserted into but not translocated through the membrane. In vitro (corresponding to E. coli residues 122 and 131.) We therefore constructed two double mutants, N122C-L305C and V131C-L305C. synthesized pSSU was co-translationally inserted into canine microsomal Consistent with our prediction, one of these mutants, N122C- membranes without signal peptide removal. The in YiyQ and in Ybw L305C, is reductively activated by DTT and inactivated by the studies demonstrate that Euglena cytoplasmically synthesized proteins are oxidant diamide. Potentially many two domain enzymes can be inserted into the ER and transported as membrane bound precursors to the converted into a redox-modulatable form by mutation of the Golgi apparatus prior to chloroplast localization. appropriate residues to cysteines. Wednesday. Chloroplasts and Mitochondria (2763-2768) 475a

2753 2764 FATTY ACID OXIDATION IN ISOLATED CHLOROPLASTS FROM MOLECULAR CHARACTERIZATION OF AN INTEGRAL ENVELOPE THE GREEN ALGA ANADYOMENE STELLATA. ((M.V. Mikhailoval, HSP 70 HOMOLOGUE INVOLVED IN PROTEIN IMORT INTO D.L. Bemisl, J.N. Norris2, and R.S. Jacobsl)) tDepartment of Biological CHLOROPLASTS. ((F. Tian, and D.J. Schnell)) Department of Biological Sciences and Marine Science Institute, University of California, Santa Sciences, Rutgers, The State University of New Jersey, Newark, NJ 07102 Barbara, CA. 93106, and 2Department of Botany, National Museum of Because of its dual genetic origin, a majority of chloroplast proteins Natural History, Smithsonian Institution, Washington, D.C. 20560. are synthesized in the cytoplasm and posttranslationally imported into the organelle across the double membrane of the chloroplast envelope. We Fatty acids containing conjugated tetraene bonds were detected in have reported the isolation of a set of six chloroplast envelope proteins that extractions of isolated chloroplasts from the marine algae Anadyomene associate vith bound and partially imported precursor proteins during the stellata (Chlorophyta). Additionally, the enzymatic conversion of protein import reaction. We refer to these proteins as import intermediate polyunsaturated C16 to C22 fatty acid substrates by A. stellata was shown associated proteins (IAPs). Four of the LAPs are components of the outer to result in accelerated production of sirnilar conjugated tetraene structures. membrane import machinery. One of these outer membrane LAPs is a 75 To characterize the enzyme system involved, three hour incubations were kD integral membrane hsp 70 homolog that is oriented with the bulk of its conducted on a chloroplast fraction and a 105,000 x g fraction with each of mass in the intermembrane space( IMS) between the outer and inner six fatty acid substrates (palmitoleic acid, 6,9,12,15-octadecatetraefoic acid, envelope membranes. We propose that this hsp 70 LAP acts as a molecular arachidonic acid, eicosapentaenoic acid, cis-7,10,13,16-docosatetraenoic chaperone that binds precursors as they emerge from the outer membrane acid, and cis-4,7,10,13,16,19-docosahexaenoic acid). The enzymatic import machinery into the IMS. The binding of the hsp 70 lAP would products of the incubations yielded a UV signal typical of conjugated prevent folding of the precursor in the IMS, allowing the precursor to tetraene structures. Preliminary evidence indicates 6,9,12,15- engage the inner membrane import machinery and thereby complete octadecatetraenoic acid has a higher affinity for the chloroplast fraction and envelope translocation. We will present our recent results on the molecular cis-4,7,10,13,16,19-docosahexaenoic acid has a higher affinity for the characterization and functional analysis of the hsp 70 LAP. 105,000 x g fraction. NMR analysis of methyl ester derivatives revealed the presence of five distinct fatty acid products, one of which is a previously unreported 22:7 fatty acid. The presence of these five unique compounds suggests the existence of an enzyme cascade that plays a significant role in fatty acid oxidation in A. stellata. Funded in part by the California Sea Grant College Program, grant NA36RGO537 (R.S.J.), and in part by the Smithsonian Marine Station at Link Port, Florida (J.N.N. and R.S.J.).

2765 2766 RUPTURE OF THE MITOCHONDRIAL OUTIER MEMBRANE MMM1 ENCODES A MITOCHONDRIAL OUTER MEMBRANE IMPAIRS PORIN ASSEMBLY. (M. Smith*, K. Baker+ and R. PROTEIN ESSENTIAL FOR ESTABLISHING AND MAINTAIN- McCauley*)) *Department of Pharmacology, Wayne State University, ING THE STRUCTURE OF YEAST MITOCHONDRIA. ((S. Detroit, Michigan 48201 and +Department of Molecular Biology, Genentech Burgess, M. Delannoy, and R Jensen)) Dept. of Cell Biology and Anatomy, INC., South San Francisco, California 94080. Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Outer membranes isoated from yeast mitochondria were capable In the yeast Saccharomyces cerevisiae, mitochondria are elongated organelles mediating the in vto insertio of As with the outer membrane of porin. which form a reticulum around the cell periphery. To determine the mechanism intact mitochoedria, the inertio was ATP and the inserted dependent, by which mitochondrial shape is established and maintained, we screened yeast porin was res_tt to trpsin treatment after deteret solubilization. mutants for those defective in mitochondrial morphology. One of these mutants, However, the extent of porn insertion into isobted outer membranes was mmml, is temperature-sensitive for the external shape of its mitochondria. At much less per mg of outer membrane protein that with intact the restrictive temperature, elongated mitochondria into mitochondria. of intact mitochondria was not due quickly collapse large, The greater efiiency spherical organelles. Upon return to the permissive temperature, wild-type to contact site mediated a isolated contact sites were kss mitochondrial structure is restored. The morphology of other cellular organelles able to insert than ioated outer and blockade of the porin membranes, is not affected in mmml mutants, and mmml does not disrupt normal actin contact site channel in intact mitochondria did not affect pori insertion. or tubulin organization. Cells disrupted in the MMMI gene are inviable when However, mitochondrn that had been subjected to osmotic shock grown on nonfermentable carbon sources and show abnormal mitochondrial sufricet to rupture the outer membrane and deplete the contents of the morphology at all temperatures. The lethality of mmml mutants appears to intermembrane space (i.e. mitoplasts) lost most of their abilit to insert result from the inability to segregate the aberrant-shaped mitochondria into porin. Since outer membranes are isoated from mitoplsts, the low daughter cells. Mitochondrial structure is therefore important for normal cell nertio activity of mitoplsts exphins the low effciency of insertion into function. Mmmlp is located in the mitochondrial outer membrane, with a isobted outer membranes. These results also indicate that, unlike protes large carboxyl-terminal domain facing the cytosoL We propose that Mmmlp tht are imported to the inner membrane and matix of the maintains mitochondria in an elongated shape by attaching the mitochondrion mitochondria, porin's assembly i severly reduced by breaching the outer to an external framework, such as the cytoskeleton. membrane and depletio of the intermembrane space contents.

2767 276T RAT UVER MITOCHONDRIAL PROCESSING PEPTIDASE: SITE- PYROPHOSPHATE EFFECT ON GLUCAGON-INDUCED CHANGES IN DIRECTED MUTAGENESIS OF ZINC BINDING MOTIF. ((F. Kalousek, P. MITOCHONDRIAL ADENINE NUCLEOTIDE CONTENT AND SUCCINATE OXIDATION RATES. Department of and H.-M. Striebel)) of ((P.V. Blair)) Biochemistry Rysavy, Department Genetics, Yale Uuniversity and Molecular Biology; Indiana University School of Medicine; School of Medicine, New Haven, CT 06510. Indianapolis, Indiana 46202-5122 Mitochondrial processing peptidase (MPP) plays a crucial role in the Adenine nucleotides (AdN) accumulated in rat liver cleavage of mitochondrial precursor in the mitochondria (RLM) during glucagon treatment is retained proteins synthesized throughout isolation. The increased AdN content is cytoplasm. The native enzyme in rat is a 105 kDa protein consisting of accompanied by increased energy-linked oxidation activities. two non-identical subunits, a- (55kDa) and 1- (5OkDa); nucleotide The AdN, accumulated through the MgATP/phosphate exchange sequence encoding these two subunits has been recently cloned in our mechanism, can be removed from the mitochondrial matrix by laboratory. The B-subunit contains an unusual inverted zinc binding site, extramitochondrial pyrophosphate exchanging for intramitochondrial ATP via the AdN translocase required for HXXEH, which also found in E.coli Ill and is prototns instuin-degrading oxidative phosphorylation. Mitochondria isolated from sham- enzyrnes. To investigate the involvement of the particular residue of treated rat livers have an AdN content of 11.7±0.6 nmoles/mg this motif in the catalysis of MPP, we generated a series of mutants by RIM protein and an ADP-stimulated succinate oxidation rate of sitedirected mutagenesis. Substution of glutamate t04 to aspartste 181±16 ngatoms oxygen/min x mg RIM protein and an uncoupler- has very little effect on MPP activity whi to stimulated rate of 211±9. Glucagon perfusion of rat livers or subsftiution glutamine glucagon injection of intact rats can increase the AdN yielded a fuUly inactve enzyme. When we substituted histidine 101 or content of subsequently isolated mitochondria to 20.7±1.4 105 by argirire or inverted the zinc binding motif to HEXXH, the acbvity nioles AdN/mg RIM protein with accompanying succinate decreased to les than 1%. We detected slighty higher (about 3%) oxidation rates of 229±17 and 266±19 for ADP and uncoupler, MPP activity after substitution of histidine 101 The respectively. AdN loaded RIM incubated 5 minutes at 30°C with by glutamine. 0.2 mel pyrophosphate lowers the AdN content to 8.5±0.5 dimeric form of ths was mutant retained even after partial purification, nmoles/mg RIM protein and the ADP-stimulated succinate that the a- indicating mutation did not affect the interaction of and oxidation rate to 133±18 ngatoms oxygen/min x mg RLM protein subunits. Addition of the wild type B-subunit restored MPP activity in and uncoupler-stimulated rate to 167±20. Subsequent all mutants. Supported by NIH grant DK 09527. incubation of the RIM having reduced AdN content with 5nM ATP restores the AdN content and succinate oxidation rates. It is concluded that glucagon increases RIM oxidative activities by stimulating reactions that lead to net accumulation of AdN in the mitochondrial matrix. 476a Chioroplasts and Mitochondria (2769-2770). Wednesday

2769 2770 Import Mechanisms of Plastid Protein in Marine Algae. ((K.E. MITOCHONDRIAL ABNORMALITES IN REFRACTORY ANEMIA WITH RING Apt, D. Bhaya, A. Sukenik,A.R. Grossman)) Carnegie Inst. of Washing- SIDEROBLASTS. ((B. Bader-Meunier, J. Breton-Gorius, MiieooF. P.Rustin,.M. ton, Dept. of Plant Biology, Stanford, CA 94305. Lavergne, A. Rotig. A. Munoich, 0. Tchernia.)) Hopital Bicetre, U. INSERM, Hopital Necker, France (Sponsored by N. Mohandas) There are at least two general pathways of plastid protein import among the different phyla of plastid-containing organisms. Red algae and higher Iron reduction, a mandatory preliminarystep for bane synthesisisccomppisd o on the inner mitochondrial of plants have douhle membrane-bound plastids without additional periph- membrane by the respiratory chain as a source reducing equivalents. As 13 out of the 70 respiratory chain proteins are encoded by the eral membranes. Both of these groups of organisms apparently use a mitochondrial genome, mitochondrial DNAabnoaecan lead to ionacccmuulaton similar mechanism of protein import that involves a tar- posttranslational sierobl wanemia. Inthetwo children initially presenting with isolated maccytic geting of proteins to the plastid. Diatoms,, rown algae and other chrys- amnmisan ring idoeblasis ononne mrow smears i l disorder wasdiagnosed ophytes have plastids that are completely surrounded hy a second set of by a decrease of the activity of the respiratory chain complexes I and IV in muscle ad in one case and the of 2 miochondra DNA, one membranes referred to as the plastid ER. Nuclear encoded plastid proteins fibroblasts by presence populions of being partialy deleted, in lymphocytes, bone marrow and muscle cells in the second case. must first transverse the plastid ER prior to import into the organelle and Large cytoplasmic vacuoles were present in erythroid and granulocytic precursors. are prohbaly synthesized on rihosomes hound to the plastid ER, which Cytogenetic examination of bone marrow cels was normal and X-lilned methylation requires the presence of an ER signal sequence.Following import into the analysis of peripheral neutrophils and lymphocytes displayed a polyclnal pattern. At the unicellular mirochondrial mosiacism was is poyrls by plastid ER, a second signal also present on the presequence facilitates levd, demonstrated some ultrasiuctural examination showing 2 popution of mitochondriac one beig of normal movement across the plastid envelope. size (0.3iL) and appearance and the other giant (2.5-3j±) with various feaues of iron overloadin more mature erythroblasts. Sideroblastic anemia with vacuoles has already been reported in micondrial disorderssuch as Pearon and Kcams-Sayre sndromes or after high dose chloramphenicol which inhibits the respirao chain complex 1. A mitochondrial deletion may play a role in the pathogeny of some constitutional or acquired sideroblastic anemia.Theheteroplasmic segregation of mitochndrial during cellular division accounts for the finding of a double population on ultrastuctural examination. The morphological heterogeoiety of erythropoietic cells as well as the polyclonal pattern of peripheral cells suggest that in sideroblastic anemia involving mitochondrial abnormalities is not a trly clonal disease. Peroxisomes (2771-2774). 2771 2772 EFFET POIInIICTEra CATLM TIUIW ((O. Hu sa)), (R. gm jm)), _ IMMUNO-MAGNETIC ISOLATION OF PEROXISOMES. ((G. LOerst, R. (Ul U)a) )and ((J. S ilve)). It_sa ds Fssewlaim y Hartig2, M. Hausmann2, H.D. Fahimi1, C. Cramer2 and A. V8lkl')), Institute Toesdlogia Histologia, Facultad de redicim, U.A.N.L. Apdo. Posta 146, Col. of Anatomy and Cell Biology1 and Institute of Applied Physics2, University delUalle, Gwza Gweia, N.L. Nix. of Heidelberg, Heidelberg, Germany. The genus Karvinskia includes diverse species of plants froi which have been Immuno-magnetic separation has been used to isolate distinct vesicular isolated several toxic compounds characterized to be diaeric anthracenones. The fractions of the endocyticand secretory pathways but has not yet been T-514 or peroxisomicine is one these compounds.Ultrastructural studies made to applied for the isolation of peroxisomes (PO). With recent evidence on the elucidate the mechanism of action of this toxin have demonstrated that heterogeneity of peroxisomes, the isolation of highly purified peroxisomal peroxisomicine produces ass irreversible and selective damage on the peroxisomal We have embrane of yeast cells in vivo. We now report the inhibitory effect of subpopulations becomes mandatory for their detailed analysis. from rat liver peroxisosicine in vitro on liver catalase activity from three several animal established a method for immunoisolation of peroxisomes sources: bovine, dog and mouse. Catalase activity determination Was performd using magnetic beads. A polyclonal peptide-antibody against the by a spectrophotometric method. Inhibition degree on catalase activity caused cytoplasmic C-terminal 11 aa of the rat 70 kD peroxisomal membrane by peroxisomicine determined using a fixed enzyme concentration and several protein (PMP 70) was covalently bound to magnetic beads (Dynabeads@9 coricentratios of peroxisomicirie. Kinetic coiisLants (Km and max) were also M-450), which were incubated with a crude peroxisomal fraction from rat determined for each liver catalase. Peroxisomicine produces a noncompetitive liver. The beads were separated by a ContinuousImmuno Magnetic Sorter inhibitiong ons Lhe bovise, dog and mouse catalase and the inhibition degree is (CIMS), which permits continuous fractionation of magnetically labeled proportional to peroxisomicine concentration. In order to test if particles by magnetic deviation. Scanning electron microscopy revealed peroxisomicine causes also this effect on liver tismuecaLalase activity, we decoration of with particles measuring 150 - 400 nm in diameter. applied a histochemical technique, incubating fresh mouse liver with beads fragments These were as detection differesaL cosicentratioras of peroxisomicifse prior to fixation with partides identified peroxisomes by cytochemical of catalase activity by the DAB-reaction and by immunoblot analysis of glutaraldheyde, and further processing with DAB indicator. Controls incubated of with a known catalase isihibitor and without any reatment were ruimed at the proteins representing the matrix, core and membrane compartments same time. Results failed to demonstrate any inhibition in the smples peroxisomes. Recovery of catalase, the marker enzyme of peroxisomes, incubated with peroxisomicine, where ds the known inhibitor caused a complete was higher in immunoisolated fractions than in fractions isolated inhibition of the histochemical reaction. Based on these findings, we conclude conventionally by density gradient centrifugation. These results indicate that the enzyme "in situ" is protected against the inhibitory effect of that immuno-magnetic separation provides an alternative approach for a- peroxisomicine. either at the level of the peroxisomal arrangemnt of the reliable and specific isolation of highly purified peroxiomes. This study was enzyme or at the tissue level. supported by the Deutsche Forschungsgemeinschaft (DFG Vo 317/4-1).

2773 2774 INTERACTION OF PEROXJSOMES WITH MICROTUBULES IN VIVO AND SACCEMCfrEEk'WUE POXI AND CHCIGENES SHARE THEIR PROMOTER IN VITRO. ((M. Schrader, J.K. Burkhardt1, E. Baumgart, G. LOers, A. Volki, REGlONS BUT ARE RECRJLATED DFFERNTLY. ((I.V. Kphm, Y. Luo aid and H.D. Fahimi)) Institue for Anatomy and Cell Biology11, Urnversity of Small)) Dept of Cell Blol1gylAnatomy, Mount Sinai Mmdeal Contmr, NwYook, Heidelberg and European Molecular Biology Laboratory1, Heidelberg, NY 10029. (Spon. by T. Barks) Germany. Our previous tudle have Identfd and a numbwr of regulatory this study we have the of the In investigated role cytoskeleton in elmen in the proXotmr of POXI geoe encodIg acylCcA oxddmae. Therm ar determining the shape and intracellular distribution of peroxisomes. We used at eM thee regulo elmmentm (two negative URSI and URS2, and an olae- the human hepatoblastoma cell line HepG2, which is quite well responsive acivating mquence - UASI) that are invohld In the complex differendated, exhibiting a large number of morphologically heterogenous regution of POXI expresmlon. We exmnmdd our mudsma in ore to find pomeibe peroxisomes (Schrader et al., Eur. J. Cell Biol., in press). To study the regulatory emmntm further upstream from the POXi promoter. Using the effects of cytoskeleton-active drugs on peroxisomes, HepG2 cells were Inver PCR techIque a DNA fragment contaning the dimtal pmrt of the POXI cultured in alpha-MEM, drug-treated and prepared for immunofluorescence promoter and an adjacont region h been clond and partally s"qunced. Comput anal of t sequence data led us to conclude that POXI pmoter using an antibody to catalase, a peroxisomal marker enzyme. A number of meence is a_mn to clathn havy chain gwn (CHC1), whch is encodod on microtubule-depolymertring drugs (cokcmid, nocodazole, virblastine) the oposit stand. Thus, the two gene share this romoter region and the induced significant increases in frequency of tubular and in peroxisomes ditance betwen the InII ATG codons Is only 650 bp. Northm blot analyIs of some cases led to the formation of peroxisomal Taxol, a aggregates. total RNA extacted from yat coel grown on olt, glycrol and glcrol + microtubule stabiltzing drug, affected neither the shape nor the intracellular oleats mdIa showed that synthe of the CHCI mRNA Is 10-20 times lowe oQ distribution of peroxisomes. For furher in vitro studies highly purified rat liver glycerd and glyweol oleate than on glucom. auggtng th UASI does not peroxisomes were isolated by Metrizamide density gradient ceilfugaton. function an actIv emnt for CHC1. In order to tSt for the protin Video-enhanced microscopy revealed strong binding of isolated bindin mes In the 'CC1H po tho swared promote, a correspondIn DNA peroxisomes to taxol-stabilzed microtubules from bovine brain. The binding fragment was used In DNA moblt assays. The fragnt gave rIs to a cell extract from was sensitive to protes tratnnlt of peroxisomes and to high sait specifc protein-DNA complbx with gkucoe-gown yeat, that the contakn a g reponse olmmnts). n conditions and occured equally well in the presence or absence of cytosol. indicating ICHC1 portion- appears to be an activation sequence Wponiv to glucose. In These data show for the first time specific binding of peroxisomes to contr to geneal feature of yest repression and vaticang eleme1t which that microtubules play a role in determining microtubules, suggesting the are usualy able to exet the effects In a dItance and orlsntation-4ndpendent shape and intracellular distribution of peroxisomes. Supported by SFB 352 manner, our preliminary data suggsts that th elments In the POXI/CHCI and LFSP of the State of Baden-WOrttemberg, Germany. promoter may not follow these rule. Wednesday. Peroxisomes (2775-2780) 477a

2775 2776 TRANSCRPTIONAL REGULATION OF THE POXI GENE ENCODING PEROXISOMAL PMP24 FROM SACCHAROMYCES CEREVISIAE IS HOMOLOGOUS TO ACYL-OOA OXDA N SACai4REVCES GEEVLE ((Y. LUO and G kt SMALL)) PMPS 31-32 OF CANDIDA BOIDINII. ((P. A. Marshall, Y. I. Krmkevich, R. Departmwnt of Coll Bology and Anatomy, Mount Sinai School of Medicine, New Lark, and J. M. Goodman)) Department of Phanmacology, The University of York, NY 10029 Texas Southwestem Medical Center, Dallas, TX 75235.

Biogenesis of peroxisomes and expression of peroxisomal enzymes are highly Our laboratory has been identifying and characterizing the proteins of the mentxane, the goal of their function and reguitaed In mammals and yeasL In S. cerevisiae transcription of peroxisomal yeast peroxisomal with understanding acid can be assembly. The most abundant memnbrane protein of the peroxisome of fatty B-oxidation enzymes Induced by fatty acid (oleate) and Saccharomyces cerevisiae has an apparent molecular mass of 24 kD rPrssed by In order to understand fatty acid induction mechanism, (McCammon et al. 1990. JBC 172: 5816-5827) and is termed PMP24. We we studied th nscr regulation of POXI, encoding peroxisomal acyl- have coned the gene that encodes this protein. The sequence revealed that it CoA oxidase. We have aracrized the 5' upsem region of POX1 by delebtion is a homologue of PMP31 and PMP32 from the methylotrophic yeast Candide analpis using lacZ as a reporter gene. Three regulatory elements have been boidinii. PMP24 is exclusively peroxisomal, as assayed by cell fractionation identified: two URSs (Upstream Repression Sequence) and one UAS (Upstream and immunobotting with an affinity-purified anti-peptide antibody. Additionally, Band shift Activating Sequence). assays demonstrated that the these elements PMP24 is predicted to span the membrane at east once by a Kyte-Doolittle srve as protein(s) binding sites. Specific protein(s) were found to bind to the analysis, and this protein partitions completely to the detergent phase in a UAS only when extracts from oleate-grown cells were used In a DNA band shift Triton X-114 extracfion. The protein is induced about 100-fold by oleic acid, assay. Thus this UAS is an olsata aplflc response element that mediates the which causes peroxisomal prolferation in yeast. In order to determine the transcriptional activation of POXI. The UAS was able to activate transcription function of this protein, a genomic disrWtion was performed. Surprisingly, no through a heterologous CYCl promoter In an oleate Induction-dependent manner. phenotype of the mutarnt has been detected thus far. Thus, there is no growth We have a UAS difference seen in the disruptant in a variety of carbon sources including oleic identifed POXI related sequence in the upstream region of the acid. Additionally, other than the absence of PMP24, the biochemical gene encoding human peroxisomal acyl-CoA oxidase. This homologous human composition of peroxisomes of the disruptant appear identical to those of wild sequence was able to compete with the POXI UAS for protein binding. This resuit type. We are continuing to study the disruptant, and a syrnthetic lethal screen suggests that the fatty acid regulatory mechanism may be conserved through is underway in order to identity a possible functional homologue of PMP24. evolution. In order to understand the fatty acid induction pathway, we are cioning the gene(s) coding for the trns-acting factors that mediate the transcriptional activation of POXI. In order to identify the protein that binds to the UAS we have characterized, we are purifying this protein by affinity chromatography. To identify and cione other trans-acting factors that activate the expression of POXI we have devised a screening strategy to Isolate mutants in this induction pathway.

2777 2778 PMP27 ENCODES A PEROXISOMAL MEMBRANE PROTEIN FROM IMPORT AND FOLDING OF PEROXISOMAL PROTEINS ((C.C.M Ruigrok, H.F. SACCHAROMYCES CEREVISIAE INVOLVED IN PEROXISOME Tabak and 1. Braakmran)) Department of Biochemistry, University of Amsterdam, PROLIFERATION. (R. Erdmann and G. Blobel) Laboratory of Cell Meibergdreef 15,1105 AZ Amsterdam, The Netherlands. (Spon. by G. van Meer) Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. Peroxisomes are defined as organelles bound by a single membrane containing catalase and a hydrogen peroxide producing oxidase. Peroxisomal proteins Peroxisomal membranes from oleic acid induced Saccharomyces generally are synthesized on free ribosomes and posttranslationally imported into cerevisiae cells were isolated and proteins of 27kDa, 46kDa, and the peroxisome. Targeting of the proteins is regulated by at least two peroxisomal 96kDa identified as the major peroxisomal membrane proteins. targeting signals: PTSI is a C-terminal SKL-motif, while PTS-2 is N-terminal. It The 26kDa peroxisomal membrane protein (Pmp27p) was purified is still unknown where peroxisomal proteins fold in relation to the translocation and the corresponding PMP2 7 gene was cloned and sequenced. process. To study the folding process of peroxisomal proteins, we fused residues 39- The isolated gene has the potential to encode a protein of 26,879 280 of the top domain of the Influenza virus hemagglutinin (HA), thereby Daltons. Peroxisomal localization of Pmp27p was confirmed by deleting the signal peptide for translocation across the ER membrane, to either an subcellular fractionation studies, immunofluorescence SKL-peptide (HA-SKL) or to the SKL-containing firefly luciferase (HA-luc). microscopy, and immunogold labelling. Biochemical data as well Using a recombinant vaccinia virus system, we expressed the fusion proteins in as immunogold labelling confirmed the protein to be tightly HeLa cells. The transfected cells were pulse-labeled with radioactive amino associated with the peroxisomal membrane. Expression of Pmp27p acids and the labeled proteins were immunoprecipitated with several was highly inducible by growth on oleic acid medium. A p mp 27 conformation-specific monoclonal antibodies directed against HA. By combining null mutant cells were unable to grow on oleic acid medium and this approach with subcellular fractionations we intend to kinetically relate the growth defect could be restored by complementation with the possible changes in conformation of the fusion proteins to the different import corresponding PMP27 gene. Oleic acid-induced mutant cells stages. So far, we established that both fusion proteins are correctly expressed and showed the presence of typically 1-2 giant peroxisomes instead of that it is possible to immunoprecipitate them with a polyclonal antibody directed the 10-20 small ones usually found in wild type cells under the against HA. Immunofluorescence of HeLa cells expressing HA-luc and HA-SKL same conditions. Because of this mutant phenotype, we suggest gave rise to a punctate fluorescence pattem, indicating that both fusion proteins Pmp26p to be involved in peroxisome proliferation. were targeted to the peroxisomes. In the ER, due to its oxidative environment, disulfide bridges are formed in HA, which are detectable using non-reducing SDS- PAGE. In our fusion proteins, however, which contain 6 cysteines in the HA- domain, we couldn't detect a difference between the reduced and non-reduced forms, suggesting that disulfide bridges cannot be formed in peroxisomes.

2779 2780 FURMER CHARACERISATION OF -LKM PEROXISOMES IN CHARACTERIZATION OF PAS8p, A PEROXISOMAL MEMBRANE FIBROBLASTS FROMPATIE1IS W1IHZLLWEGE SYNDOME PTSI-RECEPTOR IN THE YEAST, PICHIA PASTORIS. ((MForste, D. Riegelnegg LSalmhofer, and G. Hoefler)) Universty of ((S.R. Terlecky, W.M. Nuttley, and S. Subramani)) 0raz, Instituteof Pathology,Graz Austia and Sws Fedeal Institute of Department of Biology, University of California, TechnologyZurich, Insdtute for CellBiology, Zurich, Switzland. San Diego, La Jolla, CA 92093. (Spon. by S.J. Singer.) Zellweger syndrome (ZS), the most sevempo disorder, is cdacteriedby i complete absence of ma logically detxble The peroxisomal targeting signal I (PTSI), peroxsoVes (POs).Upon density centrifugatinof fibroblast consisting of the carboxy-terminal tripeptide Ser- frmm a patent with ZS we found catlase notin the cytoic fionsbut at a Lys-Leu, directs polypeptides to the peroxisome density betwe that of lysoses and m i Thse pidesam also matrix in a variety of organisms. Previous studies found in n frm colfibroblaststoge r with peoxisomes of in the methylotrophic yeast Pichia have nomal esen was perfo antibodies Rastoris using against identified a 68 kDa protein, PAS8p, as an important calas,acyl-CoAtthidase,and integral poxisomalmemane proes component of the PTSI-import machinery. We have FlDblasts wee eibr Sedby _utmen with n e- eat-200C or with further characterized PAS8p and defined several new X-100 atrooMcamalaseture Punctuate properties of this molecule. For example, PAS8p is immunofluorecesac was observd whe ie firobshad been fixated in localized to, and tightly associated with, the nethanoVaceuebut nt when ftated with fum1kdehyd drionwheeas peroxisome membrane. Furthermore, purified thiolaseand theper Inu nrae showed a punctae %S8p u ouefini binds an iodinated-PTSI containing peptide ([ I]- inzmunofluorefxetuern independent Of die fixi medtod used, We also PTSI) with high affinity (nM dissociation anysisof celula fracti constant) pedba sub detecting catlae in an in vitro binding assay. Using a IpMUJUgml nnMtIm oteinem and die 44 kD thiolase novel Pam =oin tde membrane assay, we have demonstrated samedensity range. The per soal if al enzym, h ,could filtgation not binding of IJ-PTSI to the peroxisomes of wild- be detected, could VInVOmpesdfiefly lucidee We furtder ated tueaeI b bype type, but not Rasg (null) mutant Pichia Rastoris detecin asocied wiparch nphogclly cells. In the wild-type cells, I]-PTSI can be tlase cross-linked We assume to a 68 kDa peroxisomal protein using learly flefrom thesepardcksae the amine-reactive usor in PObiogenesis canntt beprocessed crrdy in patients with primary cross-linker, bis- unable to (sulfosuccinimidyl suberate). Our preliminary t beom fuctional POs, ie. they i- proteinsthat evidence on the suggests this 68 kDa protein is dependthe C-tmminal 5Kmtif for isation to POs. PASSp. 478a Peroxisomes (2781-2786). Wednesday

2781 2782 THE PICHIA PASTORIS PASIO GENE ENCODES A ZINC FINGER A ZINC-BINDING PROTEIN IN THE PEROXISOME MEMBRANE IS AND IS REQUIRED FOR PEROXISOME ASSEMBLY. ((J.E. Kalish, J. REQUIRED FOR AN EARLY STEP IN PEROXISOME ASSEMBLY. Morrell, and S.J. Gould)) Kennedy Krieger Institute and Departmnt of Cell ((Stephen J. Gould', Christiane Thedal, James C. Morrelli, Jeremy M. Biology and Anatomy, Johns Hopkins University School of Medicine, 707 Berg2, and Jennifer E. Kalish )) IThe Kennedy Krieger Institute and North Broadway, Baltimore, MD 21205. Department of Cell Biology and Anatomy, 2 Department of Biophysics and Biophysical Chemistry. The Johns Hopkins University School of The PASIO gene is essential for peroxisome assembly in the yeast Pichia Medicine, 707 North Broadway, Baltimore, MD 21205. (Spons. by S.J. pastoris and encodes a 48 kDa protein, PaslOp. PaslOp was found to be an Gould) integral component of the peroxisomal embrane and hydropathy analysis revealed a single hydrophobic region long enough (25 amino acids) to span a We have cloned and sequenced PAS7, a gene required for peroxisome lipid bilayer. Computer analysis also showed that PaslOp contained a region assembly in the yeast Pichiapastoris. The product of this gene, Pas7p, is near its carboxy-terminus with similarity to the consensus sequence for the an integral component of the peroxisome membrane. Analysis of the PAS7 C3HC4 zinc-binding domain. However, PaslOp only contains half of a sequence revealed that Pas7p is a member of the C3HC4 family of putative classical C3HC4 domain, suggesting that PaslOp may bind only one zinc per zinc-binding proteins. We demonstrate that the C3HC4 domain of Pas7p is protein molecule rather than the two zinc ions bound by a full C3HC4 indeed a zinc-binding domain, coordinating one or two zinc ions per protein domain. The putative zinc-binding domain of PaslOp is able to bind zinc in molecule. Point mutations that alter conserved residues of the C3HC4 motif vitro and is necessary forPASO function in vivo. Protease protection abolish PAS7 activity, suggesting that Pas7p binds zinc in vivo and that assays showed that the carboxy-terminus (which contains the zinc-binding zinc-binding is essential for PAS7 function. The observations that Pas7p is domain) is oriented toward the cytoplasm. Absence of PaslOp not only an integral membrane protein of peroxisomes and that the zinc-binding blocks import of proteins into peroxisomes but results in fonnation of domain of Pas7p is located within the peroxisome makes DNA-binding an peroxisomes with abnormal morphology. Specifically, these peroxisome unlikely function for this particular C3HC4-containing protein. Deletion of "ghosts" are markedly smaller than normal peroxisomes and are somewhat PAS7 not only results in an inability to assemble functional peroxisomes, sausage-shaped. In several cells we detected defective peroxisomal but causes an accumulation of doubie-membrane sheets in close proximity structures surrounded by additional membranes, suggesting that they nay be to the vacuole. Our results suggest that an early step in peroxisome degraded by autophagy. assembly may involve the vacuole, possibly as a source of phospholipid for the peroxisome membrane.

2783 2T84 THE CYTOSOUC HUMAN PEROXISOMAL TARGETING SIGNAL 1 THE PICHIA PASTORISPAS4 GENE ENCODES A UBIQUlTIN- RECEPTR (PTS1R) RESCUES THE PEROXISOMAL ASSEMBLY CONJUGAT`IG ENZYME REQUIRED FOR PEROXISOME DEFECI IN A PATIENT W1TH NEONATAL ADRENOLEUKO- ASSEMBLY. ((J.E. Kalish, D.L Crane, and SJ. Gould)) Kennedy Krieger DYSTROPHY ((G. Dodtl, N. Braverman2, C Wongl, D. Valle2, Institute and Departmentof Cel Biology and Anatomy, Johns Hopkins SJ. Gouldl )) Kennedy Krieger Insdtute and Dept. of Cell Biology and University School of MediMc, Baldmore, MD 21205. Anatomyl and Ctr. for Medical Genetics and Howard Hughes Medical Institute2, Johns Hoplins University Sch. Med., Baltinore, MD Our lboratory has cloned and sequenced the Picia pastirosPAS4 gene and l_ d~PIi its geneproduct Pas4p. A varety of In the yeast Pichia pastons, the PAS8 gene encodes the receptor for per- veao that P#4p is a 24Da pai l n inhanepic atocites with the plmic surface peioe. oxisomal tagting signal 1 (PTS1)-containing proteins. Mutations in cy of Compueranalysis of the PAS8 result in the inability to imporproteiDn with the PTSl into peroxi- PAS4 sequence showed sigf t i to ubiquitin-conjugating somes but do not affect import of PTS2-containg proteins. We report enzymes, p la in theregon song the puatv actie-site here the identification and charcterization of PTSlr, a human homolog of cyseine e. Subsdtuion ofa ine or seine for this cyssen resultedin the Pichia pastoras PAS8 gene. The PTSlr protein shaes 26% amino acid loss of PAS4 funcm F a highar molecular mass fornof Pas4p sequence identity with Pas8p, binds PISl-containing peptides, and is able (32 kDa), which isthe predicted sizeof a Pas4p-ubiquitin conjugate, was to partally compledentthe protin import defect of the yeastPAS8 mu- detected both in Wvo and in vitro. Additional experinents that mass tant. In humans, peroxisome assembly defects can result i neonatal conclusively dtis higher molcula form of Pas4p does ideed adrenoleukodystrophy (NALD), a genetc disordercharaterized by the contain ubiquitin. Electron miroscopy of nutant strains laddng Pas4p lossof0multipkeperol enzymaic actiitis, severe neurological de- showed an acumulation of sall, cuboidal, single ntbran-bound structures fects, andpremature death. Expression ofPSlr in fir from an within cells. eeuniquevesicles are clustered lie poxisomes NALD patient, with a specific defect in the import of PTSl-cntaining of wild-type cells, but are n cy smaller. Subcellular frctionaion of deleonells that some cataa at a proteins, rescued the proxisom inport defect of this cell line. This pa- pas4 acvity exits density that is of p s, tient carries a mutation in the PTSlr gene that affects a reidue conserved tpical suggesing that the small proxisone-ie between PISrandyeastPAS8, changing the upagine at position 489 to stuctues oboavedby EM may contain matrix ptein A mu nal ofthe a Iyuine (N89K). Inrsteingly, we find that the human analyis PAS4 gene is cuetiy uderay to demine mI'Sl receptor is present in the1c while a smal percentage is found PAS4's role in peroxisome assembly. in association with peroxisome. This PISl receptor may function as a recognition factor that dircts newly syntheized Pl-containing proteins from the cytosol to peroximes.

2T85 2T86 IDENTFICATION OF THREE DISTINCT TYPES OF PEROXISOMAL THE HUMAN GENE FOR L-PIPECOLIC ACID OXIDASE SHOWS PROTEN IMPORT DEFECTS IN PATENTS WITH DISORDERS OF SEQUENCE SIIMARITY TO SARCOSINE OXIDASE AND ITS PEROXISOME BIOGENESIS ((M.Slawecki, G.Dodt, A.Moser, PRODUCT IS TARGETED TO PEROXISOMES. H.Moser, and S.J.Gould)) The Kennedy Krieger Institute and Departmnt ((S J.Mihalik, D.G. of Cell Biology and Anatomy, The Johns Hopkins University School of Kim, S.J. Gould, G. Dodt)) Kennedy Krieger Inst. and Depts of Medicine, 707 North Broadway, Baltimore, MD, 21205. Pediatrics and Cell Biology and Anatomy, Johns Hopkdns Univ. Sch. Med. Baltimore, MD 21205. Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum's and classical rhizomelic hun disease, chondrodysplasia punctaa are Loss of L-pipecolic acid oxidase activity and accumulation of the genetic disorderscharac by defiecies in multiple peroxisonal metabolic pathways. Skin fibrobastcell lines from 59 patients with these substrat are found patients with generalized disorders. diseases were tested for ther ability to import the two known clsses of We invesiated this proten in order to understnd its tiip to human peroxisomal matrix proteins: those which contain the carboxy- peroxisomal disorders. The pxtin, purified from Macoaca mulatta tenninal tripeptide peroxisomal tgeting signal PTS1, and those which liver, was cut with endoproteinase Glu-C and cyanogen bromide, and contain the anmno-terninal PTS2. Three different types of peroxisomal pepddcs wee HPLC purified and sequenced. Based on prtin protein import defects were detected in cell lines from these patients. The sequence, degenerate oligonucleotides were designed and used to first of these, the Type-I peroxisomal protein import defect, was generate a 180 bp PCR product from human liver RNA. When a characterized an to inport into by inability PTSl-containingproeins human liver lambda was screned with this PCR peroxisomes but nonnal impert of a PTS2-containing protein. In the Type- gtll library product a 2 defect, import of PTS2-containing proteins was blocked but import of 1600 bp clone was detected. The 5' end of the gene was derived by 5' PI'Sl-containing proteins appeared normaL The Type-3 peroxisomal RACE. The open reading frame contains 1173 bp and encodes a protein import defect was ch iz by a loss of, or reduction in, the protin of 391 amino acids. The terminal 3 amino acids are AHL, an iport of both PISSl-containing and PrS2-containing proteins. These three identified peroxisomal targeting signal 1 (PTS 1) sequence. When types of protein import defects have also been observed among yeast expresed in CV-1 cells the gene product was targeted to peroxisomes. peroxisome assembly (pas) mutants. The strong similarities between the peroxisomal protein import defects of the patients' cell lines and yeastpas Analysis of the amino acid sequence revealed significant homology mutants suggest that defects in human homologs of yeast PAS genes may with the sarcosine oxidase from Bacillus sp. NS-129 but no homology be rsponsible for the human perxisome biogenesis disorders. with peroxisal D-amino acid oxidases. (Supoted by NIH grant HD10981) Wednesday. Peroxisomes (2787-2791) 479a

2787 2788 LONG- AND VERY LONG-CHAIN ACYL-CoA SYNTHETASE ACTrIVTY CARBON CATABOLXTE REGULATION OF PEROXISOME PROTEIN IN PICHA PASTORIS PEROXISOMES AND MITOCHONDRIA. EXPRESSION IN THE YEAST SACCHAROMYCES CEREVISIAE. ((J.J. ((P.A. Watkins, J.E. Kalish, C.I. Chen, and S.J. Gould)) Taub, C. Williams, and J.A. Rothblatt)) Department of Biological Sciences, Kennedy Krieger Institute and Depts. of Neurology and Cell Biology and Dartmouth College, Hanover, NH 03755. Anatomy, Johns Hopkins Univ. Sch. Med., Baltimore, MD 21205. In the yeast Saccharomyces cerevisiae, availability of the preferred carbon In yeast, U-oxidation of fatty acids is exclusively a peroxisomal process. source, glucose, represses the expression of multiple genes involved in Activation of fatty acids to their CoA derivatives precedes degradation by the utilization of alternate carbon sources (e.g., galactose). Yeast peroxisomes, U-oxidation pathway. We investigated the subcellular distribution of acyl-CoA which are the principal site of fatty acid p-oxidation, have been shown to be synthetase activities in P. pastoris. An organelle fraction containing subject to glucose repression. When yeast cells are shifted to growth medium supplemented with oleic acid as the sole carbon source, a dramatic increase in mitochondria and peroxisomes from a wild-type strain grown on oleic acid the number of morphologically detectable peroxisomes and in peroxisomal activated as well as acids. When this very long-chain long-chain fatty organelle oxidation enzyme activities has been detected. We have examined the behavior preparation was fractionated by sucrose density gradient centrifugation, all of the of 0-galactosidase and the yeast imidazoleglycerolphosphate dehydratase very long-chain acyl-CoA synthetase (VLCS) activity was associated with (HIS3 gene product) when the genes coding for these enzymes are fused to the peroxisomes. In contrast, most of the long-chain acyl-CoA synthetase (LCS) upstream regulatory domain of POX), which encodes peroxisomal acyl-CoA activity was associated with mitochondria; less than 10% of LCS activity was oxidase. As predicted, J-galactosidase activity is not detectable in glucose- found in peroxisomes. Yeast strains deficient in genes required for peroxisome grown cells. Furthermore, the regulated copy of HIS3 is unable to confer assembly (pas mutants) were also examined. Organelle fractions from three growth on histidine selection plates containing glucose. In contrast, growth on different pas mutants had normal LCS activity but significantly reduced VLCS galactose-supplemented medium derepressed 0-galactosidase expression. activity. Furthermore, no VLCS activity was found in pas mutant gradient Galactose-grown cells exhibit histidine prototrophy and growth is detectable in fractions having the same density as wild type peroxisomes. We conclude that the presence of the HJS3 enzyme inhibitor, 3-aminotriazol, at concentrations the VLCS activity in P. pastoris organelle fractions is mainly peroxisomal; up to 8 mM. Galactose supplemented with oleic acid (18:1) induces even therefore, this organism may be useful as a model system for investigating the higher levels of 0-galactosidase activity. Quantification of endogenous human disorder X-linked adrenoleukodystrophy in which peroxisomal VLCS peroxisomal thiolase and AOx mRNA and protein levels in time-course studies activity is impaired. (Supported by NIH grant HD10981.) of their expression in gal/OA medium following preculture in glucose- or galactose-containing media showed that the t1n2 for AOx and thiolase mRNA appearance is quite rapid, ranging from -5 min following a shift from galactose to gal/OA to -7-8 min in a glucose to galVOA shift.

2789 2790 S. CEREVISlAE SSH2 GENE PRODUCT, HOMOLOGOUS TO HUMAN THE HANSFJULA POLYMORPHA PERI GENE IS ESSENTIAL FOR ALDP AND PMP70, IS REQUIRED FOR PEROXISOME FUNCTION IN PEROXISOME BIOGENESIS AND ENCODES APEROXISOMAL PROTEIN VIVO. ((E. Swartman, M. N. Viswanathan, A. E. Emerick and J. Thorner)) W1TH TWO TARGETING SIGNALS.((IL R. Walesam, J. bt Cregg and M of Molecular and Cell of Department Biology, University California, Veenhui)) Laboaty for Electron Miceocopy, Universty of Groningen The Berkeley, CA 94720-3202 Nedtlands andDepartmenofCham Bitry, and Molecular Biology, Oregon Graduate Insttute of Science and Technology, Portland, Oregon 97291- The polymerase chain reaction was used to amplify sequences from the 1000. genome of the yeast S. cerevisiae that are highly related to the ATP- binding-cassette (ABC) domain of the STE6 gene. Ste6 is a yeast plasma membrane-associated ABC-transporter responsible for the export of the In the cours of our sdes on the biogenesis of peroxisomes in yeast mating pheromone, a-factor. Multiple distinct products were obtained and we have cloned ihe PERI gene of the yop- yeast H. pomoipha. Tbe used as probes to isolate the corresponding genes from a yeast genomic gene was cloned by funcional of a perl mutant of H. DNA library. One of these genes, SSH2 (STEd. kiomologue), was found to pobwzorpha, wich was Impaied in the impontof peoiasomal matrix proteins encode a polypeptide with striking similarity to two peroxisomal proteins (Pimo phenotype). The DNA sequence of PERI predi that PERlp is a from human cells, ALDP and PMP70. Patients with X-linked adreno- polypeptde of 650 amio acd with nO dgnificat sequence milarity to other leukodystrophy have defects in the gene encoded by ALD and are unable to protns pres in cment datases. Promoter-stdes revealed that PER) efficiently metabolize very-long-chain fatty acids in the peroxisome. expression was low but sgnficant in wildype H. polmorpha growing on However the roles of both ALDP and PMP70 in peroxisomal function glucose and inereasd duing growth on subsrae whch Induce proliferation of and/or biogenesis remain uncertain. S. cerevisiae requires functional peroxsones. lbe PER) gene prdu, PERlp contains both a catboxy- (PTSl) to utilize unsaturated such as as peroxisomes fatty acids, oleate, sole carbon and an amino-trminal (PTS2) p o l targeig signal which both were source for growth. Yeast cells from two different strain backgrounds that demonstrated to be capable of ig bacte -lactamase to the organcees. carry an ssh2 null mutation were unable to grow in liquid medium In wild-type H. pobymopha PERlp is a protein of relatvely low abundance containing oleate as the carbon source, but were able to grow at the same which was demonstrated to be localized in the peroxisomal matix. Our results rate as wild type on medium containing glucose or glycerol as the carbon that the of into is a for the source. Furthermore, cells grown in oleate medium show a 10-fold increase sugges import PERlp peroisomes prerequite of matrx and we a function of in the steady state level of SSH2 mRNA, as compared to cells grown in import attiosla proen propose regulatory glucose medium. Thus, further analysis of the function of the yeast SSH2 PERlp on pexisomal protein import gene product may provide insights about peroxisomal biogenesis and function and may help elucidate the molecular basis for human ALD.

2791

THE PER3 GEN PRODUCT OF PICHIA PASTORIS IS A PEROXISOMAL INTE.RALMEMBAN PROEINREQU PORPEROXISOME BIOGENESIS. ((JA.M Cregg,. Liu, Mi Veenhuis,* and X Tan)) Department of Chemistry, Biochemistry, sad Molecular Biology, Oregon Grduate Institute of Science & Technology, Portad, OR 97291, *Laboratory for Electron Microscopy, University of Groaligen, Haen, The Netherlands. (Spon. by C. Enna)

We have cloned a DNA fragment containing the P. pastoris PER3 gene. DNA sequencing of the complementing region revealed a single open reading frame capable of encoding a proten of 713 ano acids (-81 kDa). PER3 produces a constitutive but methaol-inducible tanscript of approximately 2.0 kb. The predicted amino acid sequence showed no significant similarity to other reported proteins nor to consensus sequence motifs that were examined. The three carboxy-terminal amino acids of Per3p are Ala-Lys-Leu, a common targeting signal for peroxisomal matrix eazymes. However, Per3p was shown to be an integral membrane protein of the peroxisome. A chemically induced per3 mutant (per3-1) ws defective in import of severl PTS1- containing enzymes but not thiolase, a PTS2 enzyme. However, a strin in which PER3 was deleted (per3A) was defective in import of both PTS1 and PFS2 enzymes. Furtheore, per3-1 cells contained possible peroxisomal ghost structures, whereas per34 did Models to explain these results will be presented. 480a LeukocytesII (2792-2797). Wednesday

2792 2793 CYOTACTIN TENASCIN) BLOCKS THEMIGRATION OF HUMAN CD50 IS PHOSPHORYLATED ON TYROSINE IN HUMAN MONOCYIE-DERIVED MACROPHAGES ACROSS MATRIGEL ((J.D. NEUTROPHILS. ((K.M. Skubitzl, K.D. Campbelll, K. Ahmed2l4, and Loike, L. Cao, S.C. Silverstein, and S. Hoffma)) Depatmet of A.P.N. )) 1Department of Medicine, of Laboratory Physiology and Cellular Biophysics, Columbia University New York, NY Skubitz2Z3 2Department 10032; Department of Medicine, Medical University of South Carolina, Medicine and Pathology, 3Biomedical Engineering Center, University of Charleston, SC 29425. Minnesota Medical School, and 4VA Medical Center, Minneapolis, MN 55455. Leukocytes must traverse extracellular matrices as they migrate from the vascular system to sites of infection or tumor formation. Since cytotactin is a CD50 (ICAM-3) is expressed at a high level on resting blood matrix protein found primarily in the adult inareas of wound repair and tumor granulocytes, monocytes, and lymphocytes. The constitutive high stroma formation, we examined its effect on monocyteerivedmacrophage expression of CD50 on resting leukocytes suggests that it may be an (MO migration. Polyethylne terephthalae filters (8-pm pore size) in Becton- important LFA-1 ligand in the initiation of the immunefinflammatory Dickinson cell culture inserts were coated with 20 pg of reconstiuted response. By radiolabeling with [y-32PJATP, we found that CD50 basementmembrane (Matrigel). 40-50 x 104 M+, purified by adhernce and monoclonal antibodies immunoprecipitated a -125-170-WDa phosphoprotein mainained in culture for 24 hr, were added to the upper compartment of the from human neutrophils. Phosphorylation was increased following inserts and a chemoatra added to the lower compartment. The filters stimulation with the chemotactic agent N-formyl-met-leu-phe, platelet were incubated at 37' C. for 24 h at which time -6 x 10' MO migrated activating factor, 12-O-tetradecanoyl-phorbol-13-acetate, and the calcium across Matrigel in response to TNF (5 x1I7M), LTB4 (10-7 M) or fMLP ionophore, A23187. This increase in phosphorylation was transient, with (ICfM). In the absence of a cheoatactant <10' cells migrated into the the maximal phosphorylation being observed by 1min. Phosphoamino acid lower compartment. The absorbtion of 0.2 pg/filter of cytotactin to the analysis revealed that CD50 contained predominantly phosphotyrosine. Matrigel reduced MO migration in response to TNF,fMLP or LTB4 by at While this radiolabeling technique was initially designed to detect ecto- least 60%. However, cytactin did not inhibit MO migration when MO were protein kinase activity, subsequent studies have shown that membrane pre-incubated with 10 pg/ml of an anti-a6, integrin antibody or when an anti- proteins can be phosphorylated on the cytoplasmic domain under these cytotactin antibody was present in the upper compartment of theinserts. In conditions. When CD50 was immunoprecipitated from solubilized contrast, cytotactin inhibited MO migration when cells were pre-incubated with neutrophils, protein tyrosine kinase activity associated with CD50 was 10 pg/ml of anti-B2 or anti-a5 integrins. These results show that cytotain is detected in the immunoprecipitate. The data suggest that phosphorylation a stop signal for MO migration across reconstituted basementmembrane and of CD50 on tyrosine by an associated tyrosine kinase may play a role in the that this signal can be overcome by inhibiting the interactions of,B integrins function of CD50. with cytotactin.

2794 2795 HUMAN EOSINOPHIL ROLLING IN INFLAMED VENULES IS MUTATIONAL ANALYSIS OF THE MEMBRANE PROXIMAL MEDIATED BY L-SELECrIN AND VLA4 INTEGRIN. ((P. Srirmsao, CLEAVAGE SllE OF THE L-SELECIIN ADHESION MOLECULE. U. H von Andrian-, E. C Butcher, M. A. Bourdon, and D. H. BroideG)) ((G.I. Migakd, J. Kahn, and T. K. Kishimoto)) Departnent of Immunology, Boehringer Ingelheim Phannaceuticals, Inc., Ridgefield, Connecticut La Jolla institute for Experimental Medicine; 'Stanford University, Stanford; 06877. *University of Califomia, San Diego, CA, USA L-selectin expression is actively regulated through a rapid and inducible The interaction between circulating eosinophils and vascular endothelial cells cleavage event from the surface of leukocytes and is constitively down regulated from the surface of COS cells tranafected with L-selectin cDNA. are important early events in the recruitment of eosinophils to sites of chronic The down regulation of L-selectin from the surface of neutrophils has been inflamnation in vivo. Using intravital microscopy, we have determined the implicated as asignificant event in the adhesion cascade kading to relative contribution of various adhesion receptors in mediating human neutophil reitment to sites ofinflammation. We have previously eosinophil adhesion to inflamed endothelial cells under conditions of blood identified the cleavage site of L-selectin between Lys 321 and Ser 322, in a consensus repeat and the flow ina modified rabbit mesentery preparation. At physiological shear rates, region located between the second short (SCR) Uansamembrane domain We demonstate that replacing the cleavage to 'roll" in venules but not arterioles or human eosinophils were found domain of L-selectin with the corresponding region of E-selectin capillaries. Eosinophil rolling could be stimulated by activation of significanly inhibits L-selectin shedding from thesurface of transiently endothelium with IL-1. This step of eosinophil adhesion was mediated by transfected COS cells as judged by the generation of the 68 kD soluble and L-selectin, and in addition, by VLA4 integrin. In vivo rolling ofeosinphiils 6 kD transmembrane cleavage products of L-selectin. In addition, short truncations of the wild type cleavage domain appear to could be significantly inhibited with monoclonal antibodies to L-selectin and L-selectin inhibit that to anti-CD18 antibodies. In comparison, neutrophil significantly L-selectin down regulation. Mutations appeared VLA-4a, but not with inhibit L-selectin shedding resulted in higher levels of L-selectin rolling could be inhibited only with anti-L-selectin but not with anti-VLA4 expression on the cell surface, consistent with a lack of proteolysis from monoclonal antibodies. The inhibition of eosinophil rolling in mesentery the cell membane. However, point mutations of the cleavage site as well venules by anti-VLA4 antibodies was not due to modulation of eosinophil as mions of multiple conserved amino acids within the cleavage domain do not significandy affect L-seecdn shedding. Thus proteolytic processing L-selectin or CD18 expression. These results provide evidence for a L- of L-selectin appears to have a relaxed sequence specificity at the cleavage selectin and VLA-4 dependent mechanism of eosinophil rolling and suggest site and may be dependent upon the physical length of the cleavage that in contrast to neutrophil rolling, multiple adhesion pathways may mediate domain. early events eosinophil adhesion in vivo.

2796 2797 Glycyrrhitmnic Acid Glycoudes Are Sialyl Lewis X PHOSPHATASE INHIBITORS PROMOTE HOMOTYPIC AGGREGATION OF LEUKOCYTRS VIA ICAM AND LFA-1 Mimics, And Function As Selectin Inhibitors B. N. N. Rao, M. B. ACTIVATION. Joseph D. Imoro, Subbash Dhnwawf, and J.H. Musser, C. Foxal, Y. K. Bradbury, D. Asa and Anderson, O0a, Benjamin S. Weeks. *Hamilton College, Bidogy Department, BK Inc. 860 Atantic Ave., Alameda, CA Brandley. Glycome4 195 College Hill Road, Clinton, NY 13323, * Division of Transfuslon Transmitted Diseases, Center for Biologlcs The seleet are a family of adhedon receptors Implicatd In the Evaluation and Research, Food and Drug Administration, MD by iniffal Interaction between leukocytes and the vasculr endothdlum. Rockiwlle, 2052. (spon. J. Inorso) During the iiial stages ofinflmmai, leukocytes agggate with A salyl Lewis x [sLed, CD1Ss, or NenAc three sdeeets recognze one anothw (hoypc agggaon). Hr we invetgatd the role oa-3 Gal p1-4 (Fuc al-3) GlcNAc], sldyl Lewisa [sleA, or NeuAc a2- of cellular phosphatses in homotypic aggregaton of l ys by 3 Gal Pl-3 (Fuc al-4) GIcNAcJ and related We adding phosphatase inhibitors okadaic acid (OA) and calyculin A oNlgosarchsaides (CA) to cultures of the human pr U937 cell line and the used oo onal energy comput and igh feld NMR In human T-lymphocyte Jurskat cell line. We found dtt OA produces a copiunction with structure-functon studies of svral dAe anlog, 120 fold increase of homotypic aggregation with Jurkat cells and a to dine the crtcal funcdonal groups of sLex as well their 200 fold increase with U937 cells. Calyculin A increased dIbt parissLex wph used to search a aggregation of Jurkat cells 10 fold and U937 cells 88 fold The gg are three-Idmenslond databmeof chenal Compounds leukocyte cell surfac recepors invovled in cell-cell tion of groups the lymphocyte function-associated molecule-1 (LA-1) and the whih had a simlar spatial relaionship funtonal were intercellulr molecules Monoconal Usig adhesion (ICAMs). antibodies tested as Inhbitors of selectn binding. ths approach, toLFA-1 completly inhibited the OA and CA ind aggregation glycyrrhin, with an -I y p a natural product in both cell lines, whereas the monoclonal antibody to only and carmhic add were dicovered to block selecin blind to aLex inhibited U937 cell aggregation. This data demonstrates that and to sulfated ligands Four 0-lycodde derivatves were generated cellular phosphatase inhibition can acdvate the inflammatory in by replacin the glroic acid reside with other sugpr Of these response leukocytes. compounds, the fucose derivative ws found to be the most broadly actves. A C-fcoldde of glycyrrhitinc acid, desiped to optimIze spacng between- the fuene and late group, wa more actve than the parent nn Its ablty to block tn acwdvity. Wednesday. Leukocytes II (2798-2803) 481a

2798 2796 HUMAN POLIOVIRUS RECEPTOR IN MONOCYTES AND RETROVIRAL STIMULATORS ARE AN ESSENTIAL FACTOR FOR THE BRAIN PRODUCTION OF REVERSE TRANSCRIOPTASE RELEASED BY MONOCYTES FROM PATIENT W1TH[ RHEUMATOID ARTHRITIS ((M. S. Freistadt, D. A. Stoltz, and K. E. Eberle)) Department of ((A. M. Al-Sunidaie, J.C. Woodrow)) Department of Medicine; University of Microbiology, LSU Medical Center, 1901 Perdido Street, New Orleans, LA Liverpool, England. 701 12 Thbe evidence for an association between retroviruses and human rheumatoid Although the cellular receptor for poliovirus (PVR) was identified and arthritis has been accumulating. Our previous studies showed that monocytes, from cloned in 1989, its normal function remnains unknown. The objective of thi patients with rheumatoid arthritis, release reverse transcriptase (RI), suggesting the work is to determine the normal function of PVR. Lack of knowledge of presence of a retrovirus. This study was designed to investigate the effect of viral the precise tissue distribution of active cell surface PVR protein makes it stimuators, nsch an, dexamethasne (DM), 5-Azacytidinme (5-Aza), and phorbol dIifficult to assess its normial andi pathogenic roles. Earlier work revealed msyristate acetate (TPA) on viral releas by these cells. that primary human CD14-positive cells (monocytes) express PVR. Dual Peripheral blood monocytes from 12 patients with rheumatoid arthritis were staining, frationation and infectious centers assays suggest that the miajosnty incubated in Eagle's medium supplemented with 10% fetal calf sesmm, Paralleled of monocytes take up poliovirus, but a minority produce infectious virus, cultures were set up with 10O4mDM, 7sg/nsl 5-Aza, or 330 ag/mI TPA. After 6 days' Immunocytochemical experiments, using the anti-PVR monoclonal incubation, the aqiernatant was centrifuiged at 600 g for 30 msinutes, fdtered through antibody, D171, and primary human brain tissues showed that PVR protein 220 -m nilropore filter. The filtrate was centrifuged at 100 000 g for 1 hour. The is present in a susceptible (motor cortex) and at least one nonassceptible part pellet was suspended in TNE buffer, and RT activity was assayed using a standard of the human brain, the olfactory bulb. The extracllular portion of PVR method, No activity was detected without stimulatoms In the presence of DM the consists of 3 immunoglobulin domnains, while the short cytoplasmic tail of activity was 10 ± 0.4; omea, SEM pmol dCTP incorporated/10'6 monocytes, and PVR has a `TAM' (tyrosine-based activation motif) that functions in signal signifscantly increased in the presence of 5-Aza (42 ±2; mean, SEM). However, transduction in other cell susface molecules. Metaboic labeling experiments higher and significat (p40.0001, Student's "t" test) RT activity was detected in the showed that PVR is phosphorylated in Hela cells. Cell surface PVR in presence of TPA (425 ± 15; mean, SEMW This study clearly indicates that, viral monocytes is increased with gnmma interferon btratment. Taken together, tidmulators are essential for the release of the retrovinis present in monocytes from these observations suggest that PVR may have a role in adherence anainswtdhnaodarhIi,adTAgv ihe n infcn ciiy activationomonocyteso neurons to arget cells.These observations strongly mupport our previous findings, which soggest the presence of a retrovimos in monocytes from patlents with rheumatoid arthritis. The role of this retrovimos in the pathophysiology of the disease remains to be established.

2800 2801 COLUMN EMMUNOADSORFI'ON AND CELL SORTING FOR BULK DIFFMENIAL EXPRESION OF NAD OLYCOHYDROLASE AND CD38 SEARATION OF LYMPHOCYTES FROM PERIPHERAL BLOOD: B IN DiFFERENf1ATED HL60 CELILS BY PH-ORBOL ESTER AND RETINOIC CELL ACTIVATION LEVELS. ((MA. Reline)) Departmnest of Genetics ACID ((U.-H. Kim and MAK Han)) Department of Biochemistry, Chonbuk CelIPro, Inc., Bothell, WA 96021 National University Miedical School, Chonju, 560-182 Korea Selection ofcells onthe basis ofmemsbrane aision expression frequenitly sisusulates Human leukemic HL60 cells are terminally differentiated into granulocytes, the selected populaio to upregulate sur6c activation mattes. mU pmnt study monocytes or macropliges by a variety of inducers. Retinoic acid (RA) and comprestheeffetstofcll spartio mehodson uma peiplsralblodu B phorbol ester (12-0-tet-adecanoylphorbol-13-acetate (TPA)) make the cells to celativit PMvels usin CD sexparessionletelsds animndicatrbofatvtodtn granulocytes and mar lig-ke cells respectively, with expressions of several studyissientratesasignficantreducton ~ ~ ~ CD19 enzyme activities, including NAD glycohydrolase (NADase). NADase is an via We pa B cells after 24 hsours in adar Mom o vdn kDaectoessymneNADaseanchoredfrom rabbit esythrocytesglycosylphoaplatidylinositolthrough solubdlizationlinkage.with thepurifiedutreatent6.5 coated heads in a column (CEPRATEO0 LC19) compared to controls of non- of bacterial phosphiatidylinositol-specific phospholipase C. Recently, it was poesdcells and the non-adsorbed flow-through cell fraction There was no reported that a novel NADawe was identified as human leukocyte antigen CD38 imdae(

2802 2803 DIFFEENTITION OF HL.60 CELLS IN ROTATING-WALL VESSELS: EVIDENCE THAT AURANOFIN INFLUENCES CELL CYCLE INDUCTION By SIMULATED MICROCRAVrFY. ((Tj. Goodwin', T.L Prewett', PROGRESSION IN PHA AND EL-2 ACTIVATED PEIHRLBLOOD D. Rislin', D.L Pleron', and M.L Jones-)) *NASA/Johnson Space Center, Houston, MONONUCLEAR CELLS.((B. A. Lafitte, K. Q. Tran and R. A. Ace)) TX 77058,' ICRUG Lffe Sciences, Houston, TX 77058, 'Department of Anesthesiology Dept of Chemistry and Biohmsr, Cal. State Univ., Loeng Beah,CA and Surgical Oncology, M.D. Andersn Cance Institute, Houston, Tx 773o;t 90840, and(( K~R. Jadus ))DepL ofPtogy Veterns Amnsrto "Department of Pathology, Univessity of MichIgan, Ann Arbor, MI 48109. Medica Center, Long Beah, CA 980 HL.60 promyelocytic leukemia cells cultured in T-flaka seeded (mpasg 2D) wete Aa oi[2346et0aey--& * IUO,rw--Xrehl at 2 x 10' cells/mI into rotatin-wall vessels (RWVs) with Cytodex-3 mircrir. phosphsine) gold(I) is cufrrently used in the treatment of rheumatoid arthritis. T-flasls with and without mcoareawere seeded identically to serve as controls. While the compound has hbes reporte to have anti-proifeatiectvty, its Mficrocarrimr were used to provide a substrate for adherent differentIated exact mode of actio is unknown. We report here on fth effects of this drug populations that were generted. Cultures weregrown in RpMI-164osupplemented on the poieaonof PHA and IL-2 activate husman peripheral blood with 2D% fetal bovine serum at 37'1 C under 95% alr/5% CO2 conditions without the monula cells. Cells were isolated from whole blood on Ficoll addition of induding agents. RWV cultures were sampled daily to meaure pit Hsoau est rdet n ctvtdwt H 21 sIl rI- dinslved CO2 and Oa, and glucose utIlization. RWV cultusts achieved between 4.25- 1AM edniygdebm Wae ihPA(-0jgw rE- 5.85 x 10' cells/ml while T-flak controls achleved between 3.75-4.25 x 10' cell/mL (10041000 Uhml). Cell proifeainwas monitored by 3H-thymidine(H-dT) Cell counts and viability assessments were performed every 2 days. Cultures in inoporaion Cells were cultured with or without auranofin and both thseRWV and T-flskcontros maintaIned greter than90% viability throughout pusdwt Htydiefr6hsrorocletonWenels(0.l1tM) ee the 13.day culture period. PrelImhinrImmunofluorescet date asn meaured by epodsed wthda-tlymdoses fofa 6rnf eh er by dircoectaditioofwhen drugoere flow cytomsetry fur the panleukocyte maturation marker cD4s, the macrophage pwtodfyossfauafi,ewbyirtadi fthdugr maturation marker CD14, and the adhesion nmarker CD11b (monsocytes) wer by relacmetof the ctilture medium, the drug sgicatyinhibited 3H-dT evaluated at day 4 of the culture. In multiple experiments CD45 increased from ttcr lebo.Te effec was evident readesof the activator used to 0.6% In unit gravity (r-flask controls) to 9.9% in simulated mkicogravity (RWV). simli the ceIlls in oonast, in PItA activated cells cultisre continousy for SWbimry, CD14 in%creased from 1.0% in T-flsks to 14.8% in RWV and CD11b 72 hours witho.ta any manipuatio of fth medium, 3H-dT incorporation increased from 40% to 54% under RWV conditions. Wright-Gleinsa saimied cell remained elevated in (k5geIaed cell relative to the unfteated cells Analysis smears were performed every 2 days to assess the presence of diffentiation. At by flow ctmery shows thatte deevatelevde of 3H-dT i is day 8, the RWV showed Iitil sgns of cels In the process of differentiation and by comelatd with a sificat nmber of cells remang e S-pse of the day 12. a lre population of differentiated cells was prt Differentiated cel cyle le data sugge that the drug idelf s D syntesis in populatiens of cels generated in the RWV represented neutrophil, basophl, activaed PBMCs and a putative meaboile may imfluence smsequent monocyW, and ropaes chancts ed by the Gienas stains. p n thrgh the cdl cycic Suprted by NIH AR420I0-1. *Author(s) and/or presenter(s) have noted that there is a potential conflict of interest. 482a Leukocytes II (2804-2806). Wednesday

2804 2805 LYMPHOCYTES AND PROMONOCYTES ATTACH TO A THE APPLICATION OF TWO FLUORESCENT-BASED VIABLE CELL STAINS, SYNTHETIC ITYR5*1 2, LYS7 1 - POLYPHEMUSIN If PEPTIDE PKH26 AND CALCEIN AM TO THE DETECTION OF CARCINOGEN-INDUCED (T22). Benjamin Weeksl, Motoyoshi Nomlu2, Akirs Otska3, MUTATIONS IN HUMAN LYMPHOBLASTOID CELLS. ((L.D. Taylor, N.D. Goosby, Christi Westonl, Akiko Okuusul, Hirokszu Tamamura3, Aklyoshi and D.A. Caseisno)) Division of Genetic Toxicology, National Canter for Matsumoto4, Nsoki YamamotoS, Nobutaks FuJli3. IDepartment of Toxicogicsl Rearch, Joffeson, AR. 72079. Biology, Hamilton College, Clinton, New York,13323, USA, 2National Institute of Dental Researcb, National Institutes of Human risk due to exposure to potential mutagns or carcinogens, can be Health, Bethesds, Maryland 20892, USA, 3Kyoto University, Sakyo- assessd by determining the frequency of mutaons at the hprt gone locus in km, Kyoto 60X, Japan, 4SEIKAGAKU Corporation, Cbuo-kiu, Tokyo human cell lines. Mutagenesis assys can be time consuming and inclds identifyin mutant clones and expansion of these clones for moecular 103, Japan, 5Tokyo Medical snd Dental University, School of aysis. The purpose of this study was to develp a roliable method to identify Medicine, Bunkyo-ku, Tokyo 113, Japan (Spon. by Weeks) 8 hprt m us using vital dyes and flow The wer SUMMARY: The [Tyr5't2, Lys71-polyphemusin 1I peptide (T27) has cytometry. dyes teted a) PKH26, a red Upophilic dye which membras Calcein acetoxy been shown to inhibit HIV-l replication in lymphocytes. The sbeis coll and b) mety sar (Ciceln AM), a fluorescein-based substa for mechanism ofT22 inhibition of HIV-I replication is not known but may intracelulr esterase which only sains viable Two human lymphoblastoid cell lines, involve T22 competition with HIV-I for attachment sites on the plasma cells. AHH-1 (hprt*, wild) and TK8 mutant) wero cultured in 96 well plates at a membrane oftargeted cells. Here we find that three human immunocyte (hprr, cell lines (H9, Jurkat, and U-937) attach to T22. The phorbol ester, 12- ratio of 99.9:0.1 in RPMI 1640 medium supplemented with 2.5 ug/mi 6- O-tetradecanoylphorbol 13-acetate (TPA) has been shown to activate thiouanine (8-TG) which elct for th growth of hprr cl. Prior to culturing, intracellular protein kinase C and to stimulate lymphocyte attachment to celi w lbeed with 2X104M PKH26 for 2 min at room tomp and the dDution of the red siga associated with the growth of hprr cells was various substrates through specific cell surface receptors. Here we find monitored by cytometry on day 2,4,6. In a second set of experiments, cells that TPA treatment enhances attachment of the immunocytes to T22 by flow were cultured in medium + with 1 ug/mi AM on day three- to four-fold. These data demonstrate that T22 binds to 6-TG, stained Calcein 2,4,6 immunocyte cell surfaces and supports the hypothesis that T22 may flow cytmety was done. Data indicate both fluorescent reagnts labed cia insely and wer of 6-TG resista hprt mutant inhibit HIV- I replication by competing with the virus for a common cell capable idenying surface coNs over the 6 day period. Finally, we concluded that both asys could be receptor(s). esily adapted into automated methods for determining carcinogen-induced mutant freuency.

2806 USE OF PCR DETECTION METHOD TO REVEAL U-937 CONTAMINATION BYK-562. ((YA Reid, K. O'Neill, C. Dorotinsky, L McGuire, M. Macy, R. Hay)) Cell Culture Department, American Type Culture Collection, Rockvhlle, MD 20852

Recent PCR analysis ofU-937, a human histiocytic lymphoma cell line, revealed that some of the token, seed and distribution stocks were contaminated with K-562, a chronic myelogenous leukemic cell line. This result was also confirmed by karyological analysis. The PCR assay was performed using D1S80 primers to human minisatellite regions. The K-562 contaminant was detected by PCR analyses in some of the token, seed and distribution stocks. Based on karyological data, the contaminant was estimated at about 0.6% in the initial token freeze and at 3% in the seed stock. The contaminant increased to a detectable level when the cells were grown in culture for multiple passages. The negative results in some token, seed and distribution freeze lots indicate that selective pressures exist that prevent overgrowth by the contaminating cell line.

Cells and Tissues: Other (2807-2808).

2807 2808

LONG-TERM MAINTENANCE OF LIVER SPECIFIC FUNCTION OF CRCERTZEATIOtI OF A NE CELL LINZ DERIVED FROM EMBRYOS OF TEE DIScOOLossID FROG, BONrINA oR.tENTArIS. ((R.L. Lindquiat and HEPATOCYTE SPHEROIDS. ((F.J. Wu', A. Lazar2, and W.S. Hu')) N.A. Delgado)) Department of Biology, Southeast Missouri state 'Department of Chemical E ing and Materials Science, University of University, Cape Girardeau, ND 63701. (Spon. by W.V. Lilly.) Minnesota, Minneapolis, MN 55455, and Department of Biotechnology, Israel A new continuous cell line has been eatablished by culturing Institute for Biological Research, Ness-Ziona, Israel. (Spon. by D. L. Clapper.) cells froe embryos of the Korea fire-bellied toad, BombIna orlentalls. In order to utilize this cell line as an in vitro Freshly harvested primary pig hepatocytes cultivated as spheroids, or ystm for studying differentiation and carcinogenesis, the multicellular aggregates, have been observed to exhibit prolonged viability, cells must first be characterizd in term of morphology, growth, viability, and cr content. Morphology was enhanced liver-specific function and differentiated state compared to hepatocytes charcterized and documented by phase-contrast mLcroscopy and plated in a monolayer. Extensive cell-cell contacts and tight junctions are photography. growth characteristica, includLng serm dependence and population doubling tim, were determined by plotting growth observed between hepatocytes in spheroids, as seen by tansmission electron curves generated by a NTT proliferation assay. Cell viability microscopy. Microvilli-lined bile-canalicular-like networks have also been was measured by Trypan blue exclusion staining and counting with a hsmacytcmter. Cells grown for chroosom counts were observed within spheroids. Cells in the interior of the spheroid exhibit a arrested at metaphase with demecolcine, and chraosom cuboidal morphology, in contrast to the outermost layer of cells which are preparations were stained with gimeaa for counting. Bor 3 cells flatened. Results indicate that the that exhLbit a fibroblast-like morphology, anchor nd nt cytoarchitecture of spheroids resembles growth, and contact inhibition of proliferation. During log of an in vivo liver lobule. Collagen entapment of hepatocyte spheroids appears growth, cultures typically exhibit >90% viability. Frozn to contribute to the longterm maintenance ofliver-specific functions. Shortm stocks, mintained at -80'C in mdium containing 10% DM8O, exhibit approximately 37% viability when thawed and replated. entrapment not to the or collagen does appear alter gross organization initial proliferation assays indicate a population doubling tim morphology of cells within spheroids, as seen by scanning electron micopy. of approximately 48 hours in mdium containing 10% fetal bovine serum The times of cells in 1% entrapment of hepatocyte into a hollow fiber bioreactor is (78). population doubling grown Collagen spheroids and 5% 788 were significantly lower. Preliainary results currently being evaluated as a bioartificial liver (BAL) device. Assessmnt of suggest that the cell Lin consLsts primrily of oells with a liver-specific fimctions withmi a spheroid-entrapment BAL indicates significant norml diploid chromosome content. Experimnts to determine karyotype, plating efficiency and cloning efficiency are in improvement in device performance compared to entrapment of a suspension of progress. Based on our data, the Bor 3 cell line is a single-cell hepatocytes. A spheroid-entrapment bioartificial liver wafrants relatLvely untransformed, chromosomally unaltered, embryonic fibroblast cell line, which represents an ldeal systm for in further sudies for potential human therapy. vitro studies of developmntal processes. Wednesday. Cells and Tissues: Other (2809-2814) 483a

28W 2810 EXPRESSION OF CARCINOEMBRYONIC ANTIGEN IN MIP101 COLON A 2D GEL PROTEIN DATABASE FOR SACCHAROMYCES CEREVISIAE CARCANOMA CELLS IN ROTATINGWALLVESSELCULTURE. ((W.S. Fikzgerald, ((J. I. Garrelsl, B. Futcherl, R. Kobayashil, G. Latterl, B. *J.N. Craig, *G.F. Spaulding, 'J.M. Jessup, *T.J. Goodwin)) *KRUG Life Sciences, Schwenderl, T. Volpel, C. McLaughlin2, and J. Warner3)) QUEST Houston, TX 77058; 'NASA/Johnson Space Center, Houston, TX 77058;'Deaconess Protein Database Center, Cold Spring Harbor Lab, Cold Spring Hospital, Boston, MA 02215. Harbor, NY 11724; 2) Univ. of Calif., Irvine; 3) Albert Einstein Coll. of Med., Bronx, NY. MIP101, a metastatic colon cardnoma eal kee, was cullured in T-fasks, petri dishes, and rotating wal vessels (RWVs). Cultures were initiated at a eel density of 3 x 10' Two-dimensional (2D) gel analysis gives a global view of cellular ceaN/mi with 5 mg/ml Cytodex 3 microcarriers in the RWVs and petri dishes. protein synthesis, protein degradation, and protein modification. Cukures were grown in GTSF-2 medium and maintained for 14 days wlth refeedings In a genetic system such as budding yeast, 2D gel analysis can at 24 to 8 hour intervale with concomkant to measure decreasing samping pH, pCO2, reveal the responses to genetic and environmental In pO0, and glucose. Cel counts were performed every 2 days on al cuktures. changes. Samples order to correlate our findings with the existing knowledge of were taken at 4, 8, and 14 days for immunohistochemical analysis. Glucose yeast gene we have identified of the utizkaion rates in RWVs exceeded 35 mg/dfhr during the experiments. expression, many protein Oxygen spots using direct protein sequencing, overexpression of genes tension was typlcasy 30-90 mm Hg, and ranged between 40-110 mm Hg in the pCO, on multicopy plasmids, and amino acid composition analysis. Our RWV cuktures. Average cel densities were 6.7, 5.8, and 5.9 x 10' cob/ml in RWVs, database contains data on more than 100 identified proteins and petns, and T-flasks, Cea in the RWVs formed three dimensional respectively. many unidentified proteins. The metabolic changes in these aggregates 2-4 mm in diameter; three dimensional aggregates formed in the petri have been studied in to several dishes to a limited degree and T-fasks were restricted to monolayers. proteins response types of heat Normaly shock, in to shifts in carbon source, and in MIP101 doss not express carcinoembryonic antigen (CEA) in standard tissue cukture. response pulse-chase Immunohistocheniical with anti-CEA monoclonal antibody showed that experiments. Protein modifications have been studied by analysis CEA with radiolabeled and accumulated wihin the MIP101 cals grown in the RWVs, and this expression labeling phosphate through studies of the increased over time. Ceab grown in petri dishes showed weak temperature-sensitive N-acetyltransferase mutant natl. Protein expression of CEA localization has been studied cell and monolayer cuktures remained negative. CEA expression appeared to be by fractionation. For each of escited these have as a consequence of the three dimensional growth alowed by the sinulated experiments, unsuspected findings been obtained for the identified as well as for of the microgravity evironment of the RWVs. Medium sampls analyzed using clnical proteins many yet unidentified Because our chemistries and EUSA's showed that CEA was not secreted into medium proteins. protein identification effort is closely the in any with the of the cultures. Differential display performed with mRNA extracted from MIP1 01 coupled yeast genome sequencing effort, we are a number of for which ceab showed an ateration in gene expression between monolayer and RWV cuktures studying proteins nothing is yet known except the protein sequence and the 2D gel expression data. of 6 bands in the molecular weight range of 3.4 - 0.6 Kb.

2811 2812 A TRUNCATED, SECRETED FORM OF THE FURIN/IfLCE ENDOPROTEASE PHORBL 12-MYRISTATE 13-ACETATE (PMA) INHIBITS ANEFFECT EFFECTS PROPEPTE CLEAVAGE IN CHO CELL OF PIOGLITAZONE ON GENE EXPRESSION DURING DIF- LINES EXPRESSING RECOMBINANT HUMAN FACTOR IX ((M.A. Hamilton, T S. Charlebols)) Mammalian and Microbial Cell Sciences, Genetics FERENTIATION OF 3T3-L1 PREADIPOCYTES. ((C.D. Drace and J.W. Institu. Inc., Andover, MA 01810 (Spon. by J.A. Winkles) Aiken)) Endocine Pharmacology, The Upjohn Co., Kalamazoo, MI 49001. Human Factor IX contasins an 18-amino acid N-terminal propeptide domain Pioglitazone (Pio) enhances insulin stimulated differentiation of3T3-L1 that directs the vitamin K-dependent gamma-carboxylation of twelve adjacent oells treated with dexamethasone (dex) and increases expression of glutamic acid residues. Biological activity is dependent on proteolytic removal of the propeptide region from the carboxylated Factor IX precursor. In order adipocyte fatty acid binding protein (aP2) by an interaction with a Pio to express recombinant human Factor IX (rtFIX) at suitable production levels respone element (PioRE) on the aP2 gene promoter (Harris, P.K.W. and for clinical use, the rbFEX gene was intnoduced and amplified in Chise Kletzien, R.F.: Molecular Pharmacol. 45:439-445, 1994). This effect of Hamster Ovary (CHO) cels, resulting in oe on of rhFDI At high Pio was studied in 3T3-L1 cells transfected with one of two chimeric levels of rhFIX expression, propeptide cleavage was inefficient 20-30% of the reporter genes: one contained 7kb of the aP2 promoter coupled to rhFIX secreted from a number of independent cell lies retained the chloramphenicol acetyltransferase (CAT) (aP2/CAT) and the other propeptide domain To augment the CHO cells' limited ability to cleave contained 4 proFIX. cell lines expressing suitable levels of rhFlX were tansfected with a repeats ofthe PioRE coupled to the SV-40 promoter and CAT gene encoding a truncated sereted form of furin/PACE lacking the 79 amino (PioRWSV-40/CAT). We confirmed that enhanced CAT expression was acid C-terminal transmembrane domain and cytoplasmic tall (PACE SOL; induced by Pio in both cell lines over a 6 day period following addition of Rehemtulla and Kaufnan. Blood 12 2349-2355, 1992). No steps were taken dex and insulin, and this occurred concurrently with differentiation ofthe to amplifly the PACE SOL gene. In all cases, beterologous PACE SOL cells. PMA at 1 nM to 1 FM, added within the first 24 hours, had no expression dramatically reduced the proportion of proFIX relative to mature FIX effect on basal CAT expression after 6 days of hormonal treatment, but secreted from these cells. The level of PACE SOL mRNA was found to it suppressed be simila to that of the bomologous CHO endoprotease in these cell lines. induction of CAT by Pio in a concentration-dependent The relative importance of the levels of both the endogenous ast manner. This effect ofPMA occurred without inhibition ofdifferentiation beterologous aota and the significance of extracellular PACE SOL or cell viability. The effect ofPMA occurred at similar concentrations in activity in propeptide cleavage were evaluated. Our results indicate that co- both the aP2/CAT and PioRE/SV-40/CAT transfected cell lines, thus the expression of PACE SOL and rhFIX in CHO cells is an efficient system for site of action of PMA is likely prior to a direct interaction of a response ensuring the production of rhFIX with appropriate propeptide pocessing, factor with the PioRE. Some other pharmacological agents, including thereby improving the ability to express high-quality bFIX at levels compatible with the development of a viable manufacturing process. modulators ofio channels, had no effect on Pio-induced CAT expression. These results suggest that Pio activates gene expression via a pathway that is modulated by a protein kinase C activity.

2813 2814 ATRIAL NATRIURETIC PEPTIDE IN ADULT CARDIAC VENTRICULAR HYPERBARIC OXYGEN INCREASES GLUCOSE UPTAKE IN SKELETAL MUSCLE. MUSCLE CELLS IN CULTURE. (D.E. Schultz, N. Lee and A.C. Nag) ((VE Matndak ad PA Dixon)) US Air Fore. 59th Medical Win& Wilferd Hall Medical Department of Biological Sciences, Oakland University, Center. San Anoio. TX 78236. Rochester, MI 48309-4401. Hyp ic cecygan (100% oxygen at 2.36 atmospheres abslute) increases glucose uptake by Atrial natriuretic peptide (ANP), consisting of 28 amino freshl diced esc This nrease is greaer th tha inuced by inSUlin (100 millihunts acids that function in natriuresis, diuresis, vasodilation (mu)>Mmemaibiaflnhynicctygadgtiondinsidiennsreb mghucoeuptake and inhibition of aldosterone is known to be synthesized, not di (P>0.05) fon that induced byhypabic aoye alc This is conan with the stored and released predominantly by cardiac atrial cells. hypods otime d ghxlose uptke in the pr oecihypabuic oyemakesuse cfthe Previous studies (Lee & Lee, Jpn. Heart J., 31:661, 1990; .pn-gmacbleguoe rter, peaps by activatigedwexercise c glucs utake Morri et al., J. Cardiol. Pharm., 13:55, 1989) indicated that p_dwy. Dmqisapzuwediseefrom mniliBJb C mid piat under light tension in gluoos-free certain pathogenic conditions cause increased level of HEPES buffer, pH 7.4, for a 30 minutcui folowed by a 10 miute synthesis of ANP in adult ventricular myocytes. Normal myicose-freeHEPES bu&rwthrediolabeled gluwose analogs Two glucose nlogs were usle, H-3-0-alguceose. which equilbr inside and ventricular myocytes have very little or no ANP at all. The rapdy otside cells, rving as s cell vume indihcir. edt which is taken present studies focused mainly on the ANP content in cultured -C-2-deoyglucose, up and phoshosylated, thereby trpped withn to a adult ventricular cardiac myocytes with immunolocalization Tlmratioco'C H within given tme is therfo a direc meae of the glucs uptakeovertinewith respec to cell volumn. A control technique. Cultured adult ventricular myocytes contained cognt g 10 pM cyhlasin B alows sbtation of in extreliular or ranocrd abundant ismsunopositive ANP, which were distributed in labelbtrpped apac, by nospecifc transport into cels The remuke, normalized tontrol, were as follows, granular, vesicular or amorphous form. The perinuclear distribution of ANP found in adult atrial cells was also Preina,30 m_ Amy, 10mim Results observed in many adult ventricular myocytes. Our studies on cultured embryonic ventricular cardiac myocytes revealed that CO, incubao, 37eC CO, incaor, 37°C 100% the ANP content present in these cells was comparable to that found in CO, inubor, 37-C CO2 incubor, 37°C 140%, P<0.05 cultured embryonic atrial cells. It is evident from 100 mu/ml insulin 100 mu/mI insulin these studies that young and adult ventricular cardiac muscle cells in vitro are capable of producing considerable ANP in hyrkbaricchnbem , 37 C hypabaic chamber. 37 C 212%, P<0.05 contrast to their counterparts in vivo. (Supported by Research Excellence Fund of O.U.l ccenber, 37-C hypabaric chmab, 37-C 228%,P<0.05 0 100 muAmliniin 100 mum insulin (163% preradhn control + insulin, P

2815 2816 MODULATION OF IGF-1 GENE EXaRESSION IN THE RAT HEART EFFECTS OF ASCORBIC ACID ON THE GROWTFH OF CULTURED AFTER COMPLETE AORTIC COARCTATION. ((D. L. Buchana K. MOUSE LEUKEMIA CELLS IN VARIOUS MEDIA. ((C. S. Tsao, M. Whte, and D. E. Buetow)) Department of Physiolgy and Biophysics, Young and K. Miyashita)) Linus Pauling Institute of Science and University of Illins, Urbana, IL 61801. Medicine, 440 Page Mill Road, Palo Alto, CA 94306. The growth of a mouse leukemia cell line was studied in three Cardiac cell hypertphy suting from chronic presre oveload was different culture media with and without added ascorbic acid. The examined with a high-renin model system: complete coarvtation of he rat rate of cell growth was virtually the same in all three media. In the abdomial aorta betw the ogins of the sunal astery. In const to presence of ascorbic acid, cell growth was significantly reduced. previous stuies, the focus here is on the early (1 to 7 days) posaurgery Ascorbic acid was found to be cytotoxic to this cell line. The penod. Mean carotd arterial blood pressure mained at about 144 mm Hg cytotoxicity of ascorbate was greatest in Fsscher medium with 10% thrugh the 7 days in the shams while it nceased rapidly in coarted rats horse serum, followed by DMEM with 10% horse serum and RPMI (CR) to 210 mm Hg (<.001) by day 3 and then rmained costant Heart 1640 with 20% fetal bovine serum. Gas chromatography studies weight (HW) of the CR incrased over the shams 10% by to 2 days indicated that ascorbate decomposed rapidly in these culture media (P<.025) and 19% (P<025) by 7 days. Tbe weight of the pess-loaded and that many oxidation and degradation products were formed. The rate of decomposition of ascorbic acid was greatest in Fischer right lkdney in CR was sgnificantly ireaed thoughout the 7 days while the medium in comparison with DMEM and RPMI medium. The presur-prtected left kidney was significantly decread frm day 2 onward. results suggested that certain reactive compounds produced upon In contat, when fed the Agio n II converting enzyme inbiNtor, the oxidation of ascorbic acid in the media were cytotoxic agents. coawted rats were like the shams for a 30% Captopril (CCR) except lower The sulfhydryl groups in the components of the serum provided heart weight on day 7. The ealy snrese in HW in the CR is paralleled by protection against ascorbic acid oxidation. The cytotoxic effect of a positive change in insulin-like growth factor-i (IGF-1) gen expremsn in ascorbic acid was reduced by the addition of catalase, a hydrogen this tissue. By day 3, the steady state level of IGF-1 mRNA in the hearts of peroxide scavenger, suggesting that hydrogen peroxide was one of the CR was sigdflcsty higher by 44% (P<.05) than in the same day shams. the cytotoxic agents. The results indicate that the cytotoxic effect of On day 2, the IGF-1 level in the CR averaged 25% higher than the shams ascorbic acid is medium-dependent. Medium components are while the level in the hearts of the CCR was 22% lower. Results suggest a important considerations when comparing results between link between an inceased IGF-1 mRNA level and the renin-Angiotensin laboratories. In vitro cell culture conditions of experiments must be identified and controlled when ascorbic acid is present. system in producing hyperuphy of the pessure-loaded heart

2817 RIBONUCLEASE H (RNASE H) CHARACTERIZATION IN CULTURED RAT MESANGIAL CELLS. ((M. Ailenberg and M. Silverman)) Department of Medicine, University of Toronto, Toronto, Ont., M5S 1A8. Antisense synthtic deoxyoligonucleotides (oligos) are being evaluated as potential therapeutic agents in human diseaaes. RNase H, an endogenous ribonuclease that hydrolyzes hybrid DNA-RNA complexes, plays a pivotal role in degradation of the RNA part of the oligo-mRNA hybrid. Since mesangial cells (MC) play a key role in the pathophysiology of glomerular function, we undertook to measure and characterize their RNase H activity. Primary cultures of rat MCs were prepared (JASN 4:1760, 1994) and used after 3-11 passages. Cells were lyzed with lysis buffer, spun, and the cytosolic fraction used for further analysis. Poly(dT) and 3H-poly(A) were hybridized at a 2:1 ratio and used an substrate for RNase H. One unit of RNase H activity is derined as 1 nmol RNA hydrolyzed /30 min at 37 0C. After 30 min incubation of enzyme with substrate, 10% TCA was added. Acid insoluble material was removed by centrifugation and supernatant radioactivity was determined. Our findings show that MCs express 0.623 and 0.259 units RNase H /105 cells using Mg2+ and Mn2+ respectively, as cations. The enzyme is optimally active in the presence of 10-15 mM Mg2+ or 0.4-1 mM Mn2+, and is completely inhibited in the presence of EDTA. Other divalent cations like Ca2+ or Ba2+ are unable to restore activity, whicb is also completely abolished after 5 min boiling. Rat mesangial RNase H is sensitive to a variety of 18-mer phosphorothioate oligos with 50% inhibition obtained in the presence of luM of these oligos. We conclude that rat MC are potential candidates for antisense targeting. Methods: Other (2818-2819).

2818 2819 POST-EMBEDDING IMMUNOCYTOCHEMICAL LOCAUZA- MICROCULTURE TEIRAZOIUM BIOASSAYS: XIT v MTS. ((CJ. Goodwin, SJ. TION OF PHOSPHOUPD ANALOGS ((L Ghitescu)) Department Ho, S.Dow ad NJ. Marshall)) Depll_entof Molua Pathy, Univerity Colegp of Anatomy, University of Montreal, MontreaL Quebec, Canada. London and *DepL snaPaithbology, Institue Of UscSresU Post-embedding immuocytochemistry was assesed as a method to Miro tetrazolium mas (MTAs) we bein widy applid to probe relashps detect and locaize phospholipids at the ultrastuctural leveL bewee cell survival gowth and d abo So inestig associas. betwe conproisd cel molim ad prgramned ell deah as ocu aspopsis. MTAs rdy Unilamellar liposomes prepared from a lipid iture containing non- upon the bioredction of a eteraolio ak much as MIT, so ia inensly coltared fo*suan. exchangeable, bead grmodiSed analogues of phosehatidylethan- These onorimesric asays, which use cells in mrpae wel yprc and lamine (Nditropheyed derivives) have been u ed with technically amesable. We have compared The use of 2 ew euaz m alt, XTr ad Speiens fd in an macrophages. aldehydes and po-fied by MTS which, unlke the commonly used MFT, fom soluble formazss. This has she were m osmnium-tannicacidiumprotocol embedded epoxy or acrylic advantage t it renders obsolete tbe error-pon step reqied for M1T- resins Labeling of the thin sectons with anti DNP antibodies and fo_maz Significant biordion of XlT and MBIS requirs the addism of protein A-gold provides a strong and specific signal on the liposomal intermdiate electron acceptor, phebnaine methosulpe (PMS). Using optimised membranes The presence of a spacer between the DNP goup and the concnoaio,nsth a molar ratio of 1:30 for PMS:XTr, we found this mixte to be polar head of the phospholipid does not influence the intensity of inhmerny unstable. Nucleato ad crystal fommatio in the reet miture, prepared in PBS, labeling. The lipidic components of the carrier liposomes and an could occur witin 2-3 minues There w a coanquent mared mud prgresve decline D entrapped polypptidic material can be sim uy revealed when formazn prducion in sbwsqu ITAL For inl 1ffA which used Nb2 st both moieties are differentally haptenated. Equally detectable by this lympboma cdls, formazan prducto decind by 50% if the PMS/XTr mue was added 3 techniqle are the phosp ds carrying the hapten (7-nitro-2-1,3- mis after ia preparation. Formazan procto was reatred by addg fresh PMS, but sot bennozadiazol) on the fatty acid chain, phospholipids which can be XTT. Ths suggst dhsPMS depletion wa de tothe formato of fco exchanged between the liposomal and cellular membranes. These betwreen PMS and XI. PMS carries a potive charg, whes two SOB groups bhve bee results open the possibility to study at the ultrastructural level the introduced intoaromatic rings in XT inre the of its fonnas Loweig dte from to gain mechanismbywhichliposomes delier their macromolecclarcontent to pH 7.4 6.5 produed only a mgina in XITPMS sbiliy. In conusss, no smch i_bhty wa found whe XIT wurpoced witb MS. whh has ly ane SOfgrop. cells Similarly, the EM detection of exchangeable phospholipid analogs We demons with 4A- sa forgwth hormoe,which usai ofMrAs represents a mean to inv e with high resolution the intracellular which ae widely used for potency estmates of grwth fa tht SjMPS prvides traffic of lip (Supported MRC Cada). bisys wbich ae free from the arasfcos aecosard with XITYPMS, nd perior within-assay precin odu we advocatete ue of fM in prefereuce to XTr for the new genat of micoculsue _trzolim smaya Wednesday. Methods: Other (2820-2825) 485a

2820 2821 FLUORESCENCE AND DYNAMIC STUDIES ON THE BINDING OF FISH IN FLOW: CELL ANALYSIS BY PCR, SIMULTANEOUS BIOLOGICALLY IMPORTANT LIGANDS W1TH HUMAN APOLIPOPROTEIN D FLUORESCENCE IN SITU HYBRIDIZATION (FISH) AND ((RC. PatelT, K. Asoaer, T. Jovinx, W. McConat}y, S. Sureah, Y.C. Patel' and S.C. IMMUNO-PHENOTYPING IN FLOW CYTOMETRY. ((K.E. Patelt)) TClarkson Univ., Potsdani, N; 5VAMC, Newington & UCONN Health Center, Yokobata, N. Ostrerova, M. Janszen, V.C. Maino, and K.L. Fannington, CT; 'Max-Planck Institute, Gttngen, Gmany, fUNTHSC, Forth Worth, Lohman)) Becton Dickinson Immunocytometry Systems, San TX; 'McGill Univ., Montreal Canada Jose, CA 95131. Apolipoprotein D (apo D), a member of the lipocalin-calycin suefunily of small hydrophobic ligand carrier proteins, has been implicated in the transport of cholesterol, The ability to rapidly enumerate and characterize cells containing hemin, steroid hormones, and a variety of similar ligands which may have important specific DNA sequences in a large population of cells would roles in diseases including neurodegenve disorders. Molecular details of the provide a powerful tool for clinical researchers of infectious binding process, however, have so far been lackng. We have therefore undertakn the diseases, genetic diseases and cancer. We are developing a follownmg studies: (1) Nanosecond tine resolved sectoscopy of the four intrinsic muitiparameter flow cytometry assay to detect and classify human tsyptophans in apo D, to understand the conformational states of the native protein. (2) peripheral blood mononuclear cells (PBMC) containing specific Tbermodynamic, stopped-flow, and temperature-jump studies on an ideally suited DNA sequences. In our model system, T lymphocyte-enriched indicator (BIS-ANS) which shows tight binding with apo D, and serves as a competitive PBMC are classified by labelling with CD4 antibody, followed by ligand with cholesteroL pregenolone, progesterone, etc. The rather high 'on' rate DNA sequence detection using in situ polymerase chain reaction constant with BIS-ANS (about 10 M' sec-' ) when compared with the diffusion (PCR) of and in situ controlled limit, indicates that binding specificity will be largely governod by the 'off' amplification target sequences hybridization rate constant, and that protein conformational states are of critical imponance. (3) with labelled oligonucleotide probes. Mufticolor fluorescence Several fluorescent labelled apo D samples (at CYS-116 or the amino terminus) have signals from the cell surface staining and FISH probes are been prepared (e.g. with pyrene, fluoreein, eosin) so that they can be utilized for a analysed by flow cytometry. Currently, we are optimizing the assay variety of studies (energy ansfer to map distance relationship to the binding pocket, by evaluating male and female PBMC with PCR primers and triplet anisoeropy for size/aggregation state of apo D, in situ observation of apo D in probes directed to Y chromosome sequences, and developing a living cells by state of the art fluorescence lifetime imaging microscopy (FI;M) and set of assay controls using HLA-DQ alpha sequences. By using dynamics of cholesterol/steroid interaction from lime resolved tnplet decay). (4) primers and probes directed to the gag and pol genes of the Opfical studies with phosphorescent hemin, metallo-porphyrins, and their respective human immunodeficiency virus (HIV-1), we are applying this analogs, including cholesterol conjugates, to develop a better u anding of the method to detect and quantitate HIV-infected T cells from patients. requirements of apo D as a carrier, in particular the nature of the binding pocket

2822 2823 AN ELECTRICAL METOD TO CONTINUOUSLY MONIOTOR VASOPRESSIN - INDUCED WATER FLOW IS MAINTAINED IN MORPHOLOGY AND MOTION OF CELLS IN CULTURE. ((C.R Keese and L DIGITONIN-PERMEABILIZED EPITHELIAL CELLS. ((N. Giaever)) SchOol of Science, Rensselaer Polytechnic Institute and Applied Franki, F. Macaluso, and R.N. Hays)) Dept. of BioPhysics, Inc., Troy, NY 12180. Ned., Albert Einstein Coll. Med., Bronx, NY 10461. Introduction of antibodies into the living permea- Great advances have been made in quantifying biochemical and physiological bilized cell provides a sethod for determining the activities in cultured cells. It has, however, been difficult to quantify changes of importance of a specific step in a metabolic se- cell morphology. A method has now been developed that can continuously and quence. A requirement is that the cell retain its non-invasively track morphological changes of adherent cells and provide physiological properties. The chromaffin cell, for quantitative data from both sparse and confluent cultures. In Electric Cell- example, has been perseabilized by digitonin or substrate cells are streptolysin 0, permitting entry of antibodies to Impedance Sensing (ECIS), cultured on small (0.001 cm2) gold spectrin or a-actinin, while maintaining catechola- film electrodes whose impedance is measured with a I micro amp current, mine secretion in response to calcium. We have generally at 4000 Hz; normal tissue culture medium serves as the electrolyte. used digitonin, a membrane cholesterol complexing When cells attach and spread on these electrodes, their insulating membranes agent, to permeabilize the apical membrane of toad constrain the current and force it to flow beneath and between the cells. This urinary bladder epithelial cells. Bladders treated results in large impedance changes. Furthermore, alterations in cell morphology with 10 gg/ml digitonin for 15 min. show extensive give rise to variations in impedance that can be numerically analyzed to report cylindrical ridges on the apical surface, extrac- levels of cell motility and, indirectly, cell metabolisnL The approach is tion of cytoplasm, and entry of immunogold-labeled exceedingly sensitive and is capable of detecting changes in cell morphology on IgG. The bladders retain 68% of their normal water the order of nanometers, well below the resolution of an optical microscope. In flow response to vasopressin (AVP). Incubation of addition to use as a general research tool to study cell spreading and motility, the perseabilized cells with an anti-cyclic AMP several potential applications of ECIS are being investigated including in vitro antibody reduced AVP-stimulated water flow by 23 i toxicology, drug efficacy and discovery, mammalian cell reactor monitoring, and 3% compared to permeabilized controls not receiving of metastatic antibody (p < 0.05). Digitonin permeabilization is determination potential. a promising technique for the study of AVP action in target cells. (Giaever, I. and Keese, C.R., Nature 366, 591-592 (1993).

2825

MOTILITY ASSAYS FOR TOMCITY TESTING THE CHARACTERIZATION OF CYTOTOXICITY OF SURFACTANTS ((S.A. J.R. McGuire)) Department of Biological Sciences, UTILIZING AN IN VITRO TEST APPROACH.(( J.F. IL BAr1ERY Hamberger, University, Chicago C.J. Peters, and C.B.Jessee)) Bausch & Lomb, Inc. Rochester, NY 14692

Diatoms, form aquatic golden algae, are sensitive to many lhe mostcommon model used to determine the irritancy of a compound to the types aquatic pollutants. Because of this sensitivity, and because ocular environment has been the Draize rabbitocular irritation test. Thismethod ecologically important contributors to almost all aquatic can be prohibitive in screening large numbers of compounds based upon ethics, for environments, species many years to test for the as well as cost. Our laboratory has utilized alternatives to the Draize testin order toxicity many potentially harmful substances found in and lake, stream, to more fully investigate compounds in a more ethical and cost efficient manner. However, of these tests majority have used cell Anin vitro test batery approach was used to determine and compare the cellular growth analysis, requiring the diatoms to be incubated in test substances for toxicity of srfactants commonly used inpharmaceutic and OTC ophthalmic days weeks, prior determining the effect of the potential toxins formulations, wettingand contactlens cleaning agents. Doses of two such growth. part studies on environmental effectors of surfarfanspresent in'multi-purpose' contact lens disinfecting and cleaning movement, of approach using diatom motility as a products, Poloxamer and Tyloxapol, were used to expose monolayers of L929 physiological cell vitality, and have measured diatom movement mouse fibroblass. Responses were measured over timeusing three assays: a concentration timeincubated in the toxin. Neutral Red Dye Release assay masuring lysosomal and membrane integrity, an used the supematant from aqueous alamarBlue Recovery assay toxic meuring metabolic acativity of the cellsfollowing an resuspensions previously characterized sediments and from sludges exposure to test agents,and a Real-Time Viability assay determing cellular solutions. Our studies show a time and dose viability. Results indicated that Tyloxapol exhibited greater cellulartoxicity than the of dependent motility presence the toxins, with some 50-80% Poloxamer tested at equivelant ions. The Neutral Red Dye Release showing motility over 90 minutes. Such assay was the most sensitive assay likely due to the damage of the cellular suggest analysis of diatom motility may provide an accurate, caused by the urfactn prior to lysis. Tbe alamarBlue much more Recovery quantitative, rapid assay for the of determining toxicity assay gave indications that a release of nntral red dye did not always indicate suspected agents communities. This aquatic impending lysisand that it was posible fora rcovery of the cellsover time.The supported part by DePaul Research Council University Grant, Viability assay gave confirmatimthat high amounts of neutral red dye release Faculty Enhancement Grant from the of Arts College Liberal was indicative of celldeath. The test battery approach, while not a complete Sciences, # IBN-9407279. substtute for invivo testing, can serve a useful purpose inchacterizig the toxicity of arge number of compoumnd quickly and efficnly, as wellas reducing the amount of in vivo tests performed. 486a Methods:------Other- ---- \---- Wednesdav, v (2826-2829). . 2826* 2827 VALIDATION OF PREDICTIVE MODEL FOR HYBRIDOMA CELL MONOCLONAL ANTIBODIES TO HUMAN IMP DEHYDROGENASE CULTURE IN HOLLOW FIBER BIOREACTORS. ((H. Stump+, J. ISOFORMS: DEVELOPMENT OF ISOFORM-SPECIFIC ELISAs FOR Godoy+, J. Newcomb+, K. Sterne+, and R. Goffe*.)) +Procept, Inc., HUMAN IMP DEHYDROGENASE ((M. Sendal, M.R. Welchl, S.F. Cambridge, MA 02139 and *Unisyn Technologies, Inc., Tustin, CA 92680. Carrl, Y. Natsumedal*, D.R. Webb2 and J.S. Kenneyl)) 1The Institute of Biochemistry and Cell Biology, 2The Institute of The hollow fiber bioreactor cell culture method offers several benefits in Immunology and Biological Sciences, Syntex Discovery Research, the production of biomolecules. These include: small footprint low capital Palo Alto, CA 94303 *Present address: Schering Plough K. K. 2-3-7 cost systems; high density long term perfusion culture; and relatively Hiranomachi, Chuo-ku, Osaka, Japan concentrated clean supernatants. Such supematants are particularly amenable to rapid downstream processing techniques (such as affinity Monoclonal antibodies (McAbs) for I and/or II purification). A common problem scientists and specific type type facing engineers is how to human IMP dehydrogenase (IMPDH) were developed. McAbs predict the productivity of a particular cell line in large scale hollow fiber were generated using splenocytes from mice immunized with cell culture systems. We have conducted a comprehensive study of five purified recombinant type I or II IMPDH. hybridoma cell lines in a large scale hollow fiber cell type Hybridoma culture system (Cell cultures and clones were characterized and selected by Pharm 2000) in an attempt to validate a predictive model. The hybridomas radioimmuno based selected produced commercially significant antibodies of varying isotypes assay (RIA) upon the binding of 1251-labeled recombinant I and/or II IMPDH. Three distinct (such as anti-CD4, IgG2a) that were employed in type type types of efficacy studies. McAbs were isolated: those specific for type I IMPDH, those Extensive static culture studies were done in 6-well plates, T-75 flasks, specific for type II IMPDH, and those which bind both I and and T-150 flasks for each cell line. Using these data and the type predictive type II IMPDH. The McAbs were for their to model, we analyzed capacity have determined the number of days in culture required to bind IMPDH by Western blot and to inhibit the produce unit quantity of active antibody with the Cell Pharm 2000 system. analysis biological activity of type I or type II IMPDH. We have established ELISAs Results are presented and discussed relation in to the validity and utility of using McAbs which can measure the amount of IMPDH protein, the Unisyn Predictive Model. quantitatively and isoform-selectively. The McAbs and ELISAs make possible the analysis of protein levels in cells and tissues of the IMPDH isoforms and their relative contribution to guanylate metabolism. This study was supported in part by Grant 1R55 CA-60344-01 from the U. S. National Cancer Institute.

2828 2829 DETECTION OF IMMATURE MAST CELLS USING A MONOCLONAL A SENSIlVE, SPEOFIC IMMUNOBIOASSAY FOR QUANTITATION ANTIBODY (MAB AA4) TO MAST CELL SPECIFIC GANGLIOSIDES. OF HUMAN INTERLEUKIN 6. ((M.C. Jamur', A.N. Moreno', A.C.G. Grodzki', L.O. Lunardi2, and C. ((T. Krakauer)) Applied Research Division, U.S. Army Medical Oliver3)) 'U. Federal do Parana, Curitiba, PR, Brazil, 2Fac. Med. Ribeirao Research Institute of Infectious Diseases, Frederick, MD 21 702-5011. Preto-USP, Sao Paulo, Brazil, 3Office of Naval Research, Arlington, VA, USA. Picogram quantities of human interleukin 6 (h-IL-6) were detected by a two-step method. A microtiter plate coated with anti-h IL-6 The lack of immunological or morphological markers makes identification of monoclonal antibody was used to capture the IL-6 present in immature mast cells difficult. We have previously shown that mAb AA4 binds biological samples. An IL-6 dependent B cell line (7TD1) that to rat mast cell specific a-galactosyl derivatives of the ganglioside GD,b on the proliferates in response to IL-6 was added to the captured IL-6. The surface of mature mast cells (Oliver, et al., J. Cell Biol. 116:635, 1992). The lower limit of detection for this immunobioassay with serum or cell present investigation extends these observations to immature mast cells. culture supernatants was 5 pg/ml. The specificity of the assay was Immature mast cells were obtained either by peritoneal lavage 2 days after achieved by the antibody used in the first step. The sensitivity was injection of distilled water (dH20) or from bone marrow from Wistar rats. provided by the IL-6-dependent cell line. The method also allows for Cells were fixed by microwave irradiation in dilute aldehydes, immunostained the removal of inhibitors, metabolites, antagonists or activating with mabAA4, and examined by electron microscopy. In bone marrow, mAb agents used to induce IL-6. This immunobioassay has theadvantage AA4 staining was restricted to morphologically identifiable mast cells, in all over other current methods in that it measures immunoactive as well stages of maturation, and to a population of small poorly differentiated cells. as biologically active IL-6. When this method was used, inhibitory These undifferentiated cells had large lobated nuclei, a well developed Golgi activities of IL-6 were detected in serum samples from patients with apparatus and virtually no granules. In peritoneal lavages, 2 days after Korean hemorrhagic fever. injection of dH20, mAb AA4 staining was restricted to immature cells that were morphologically identical to those seen in the bone marrow. No mature mast cells were present. Therefore, mAb AA4 should be a useful probe for identifying immature mast cells and elucidating their early stages of differentiation. College Science Education (2830-2831).

2830 2831 CONTEMPORARY BIOLOGY IN AN INTEGRATED LIBERAL ART1 "FIRST YEAR INTFRODUCING STUDENTS TO UBRARY RESEARCH TECHNIq IN CELL CLUSIER" AT WELLESLEY COLLEGE. ((D.M. Smith)) Department of Biological BIOLOGY. ((R.H. Colby)) Biology Program, Stockton College. Pomona, NJ Sciences, Wellesley College, Wellesley MA, 02181. 08240.

Wellesley CoUege is a liberal arts a College for women with traditionaUly strong A handout wig be posted and distributed, adapted from one used in a one-credt, Science program. For ten years, the College has offered a "Cluster Program for First- course for Year required college juniors, 'Preparation for Research." Students Students' Up to seventy five entering students may elect to take the program as prepare bibliographies on topics of research Interest, under faculty a mnajor portion of their first year studies. The program is sponsors. designed to approach the The following steps/tools are called for and explained: develop keywords; use liberal arts using a set of six related specialty courses which focus on a commnon science encyclopedias, Ulrich's International Periodicals Directory, Ubrary of theme. Each student takes two of the six specialty courses. In addition, each faculty Congress Subject Headings, the Online Public Access Catalog (OPAC) in subject member presents a lecture to the entire group of cluster students. These group mode, OPAC in keyword mode, OPAC in call-number (shelf list) mode, Books in lectures are particularly important in stressing the inter-relationships between the Print; browse serial books (e.g. Annual Review of Cell Biology, International courses. Examples of the fields involved in clusters include Biology, History, Art, Review of Cytology, Methods in Cell Biology), review (Seminars in Literature, Philosophy, Classical and joumals Studies, others. The last two clusters have Cell Biology, Current Opinion in Cell Biology); use Index to Scientific Reviews, incorporated cellular and molecular biology into a course within the framework of Index Medicus-Bibliography of Medical browse the Reviews; semi-popular review general theme. This has worked very well, in that it has allowed the students see journals (Bioessays, Trends in Cell Biology); use hard-copy Indicies to contemporary biology as an of their important part liberal arts education. They leam periodicals (Index Medicus, Current Advances in Cell and Developmental the science in the context of the other courses, which allows them to appreciate the Biology, Current Opinion in Cell Biology), Medline, undergraduate-level value of scientific literary in general, and depth of knowledge in a focused sub-field. indices on CD (Intotrac, Wilsondisc); browse research This works particularly wel for the joumals (JCB etc.), biology because importance of the field is use Dissertation Abstracts on Science reinforced by many articles in the popular press CD, Citation Index, Biological Abstracts, dealing with the particular subjects Current Contents and and to discussed in the course. As an example, the 1994 cluster topic is 'Human Identity and (hard-copy diskette versions), indices govemment The Body: Biological, and documents. Students present preliminary bibliographies to their faculty Cultural, Historical Perspectives" The Biology course - in a within the cduster is -The Biological Boundaries of sponsors tutorial session, and are asked to actually read reviews and Humanity". This course indudes research for at a sections on organization of the biosphere, cell papers presentation second tutorial session The search topics structure/function/division, protein often to senior Students who on to synthesis and DNA replication, Mendelian and molecular genetics, and reproductive iead projects. go graduate school generally report favorably on the experience. It might also be appropriate as a technologies. All of these topics, plus some others, are presented as they relate to the component of a first-year graduate seminar. would be happy to share my overall theme of "the body". In Summary, the duster is an effective means of creating with these and maintaining interest in science within a liberal arts education. experience library tools, and in other library matters.

*Author(s) and/or presenter(s) have noted that there is a potential conflict of interest. Wednesday. College Science Education (2832-2836) 487a

2832 2833 CELL BIOLOGY AS PART OF AN INTERDISCIPLINARY CURRICULUM THE EFFECTIVENESS OF SUPPLEMENTAL INSTRUCTION IN ((S.K Sommers Smith, P.E. Busher, and S. Hammer)) Division of Science and TEACHING CELL BIOLOGY AT ILI-XNOIS BENEDICTINE Mathematics, College of General Studies, Boston University, Boston, MA COLLEGE ((D.B. Taylor, C.R. Moore, R. Dixon-Kolar and M. Retzer)) 02215 Departmt of Biology and Academic Support Center, Illinois Benedictine College, Lisle, 60532. The Biology course at the College of General Studies at Boston University is a year-long laboratory-based course which is requird of all second-year The Instncional Assistance Program (IAP) at llinois Benedictine College students. The College, which admits the second largest first-year class in the (IBC) is designed to assist students in difficult college courses by integrating University, is a two-year core curriculum program, which views laboratory learning and study strategies into course content. The program follows the science as an essential component of a liberal education. Although most of Supplemental Instution (SI) model which targets courses with a high rate of our students do not continue in the University as science maors, they do withdrawals, below average grades, or are essential foundational courses for take scientific experience and perspectives into their concentrations. Cell furher study in the major and prepration for graduate studies. In 1993, Cell biology is an integral part of the biology curriculum. Integrated klcture- Biology (CB), a critical core course for science majors at IBC, was adopted laboratory-discussion modules are designed to show structure-function by IAP. A SI leader led review sessions twice weely for a penod of two relationships in cells, tissues, and organs. The course focuses on a single hours The SI leader, trained in interactive lemaning and study strategies, was a topic each week Definitions and broad conceptual background are former CB student who had demonstrated content mastery and possessed introduced in a general lecture. More detailed and spedalized lectures are stong interpersonal and orgnizational skills. Her role was to direct then given to smaller teams of students, who proceed to a related laboratory collabative leang, not to re-lecte or serve as a tutor. Review session and exercise. Last, a student-centered discussion section considers problems postexam surveys indicate that IAP participants have leaned "what questions raised by the week's work. The progressive narrowing of focus throughout to ask and how to analyze and predict the results." These acquired cooprative the week is complemented by reducing the size of student groups at each skills result in student ownersip over their education and direct improvement step. Course topics are centered on the evolution of the cell, and include in the cours. For academic year 93-94, students who participated in SI membrane systems, replicator molecules, metabolic systems, evolution of frequently sueeded at a higher rate than infrquent participants or non- the eukaryotic cell, and cellular cooperation and specialization. Tlhe course is attendees. In Fall 93, the combined 'D, F and W" rate for SI participants who team-taught and stresses student writing as well as critical thinking. atended nine or more sessions was 11.1%. The combined "D, F and W" rate for those who attended less than nine sessions was 42.7%. In addition, the combined "A, B and C" rate for the SI group was 88.9%; for the less frequent attedees the rate was 57.3%. These preliminary results demonstrate the effectiveness of our program and provide the basis for a model that can be implmented across scitific disciplines.

2834 2835 A QUANTITATIVE APPROACH FOR TEACHING DEVELOPMENTAL NEW PERSPECTIVES ON SALAD DRESSING: DEMONSTRATION OF ANATOMY. ((R. Blystone and R. Cooper*)) Department of Biology and CONCEPTS IN BIOLOGICAL CHEMISTRY WITH COMMONPLACE *Depqarent of Mathematics, Trinity University, San Antonio, Texas 78212. MATERIALS. ((Christopher Cullander)) Departments of Pharmacy and Pharmaceutical Chemistry, School of Pharmacy, University of Califomia, San Undergraduate students of developmental anatomy often overlook the Francisco, 94143 of the of We have quantitative aspects microscopic study embryos. We have developed a series of quick lecture demonstrations of biological developed a series of exercises utilizing starfish cleavage to reveal elements chemistry concepts which rely upon commonplace materials and everyday of sampling theory, statistics, and the geometry associated with this dynamic experiences, and are suitable for use in a large class. For example, the concept developmental process. Students begin with digital video image capture of of partitioning between hydrophobic and hydrophilic environments is whole mount starfish embryos resenting five each of the first five demonstrated by using an oil and vinegar salad dressing to show how a colored cleavage divisions. Using NIH-Image software (ftp: Zippy.nimnlih.gov) on substance soluble in both (e.g., the red vegetable colorant in paprika) can move Apple Maintosh microcomputers, 2-dimensional measurements are tamen of from one phase to the other. SimDariy, dissolutlon and diffusion are illustrated by the embryo images and compiled in a database. Variations in individual comparing the results of dropping highly-colored crystals of instant drink mix into a student and pooled class data are noted and explanatons sought Available column of water and a column of oil. Demonstrations of this sort, which are on the class server are optical sections of whole embryos and rotatable 3- recognized as an extremely effective teaching tool, are often employed in dimensional reconstructions. With their data and the reference images, chemistry and physics courses, but there are very few such available for biological students the dimensinal of whole mount chemistry, and those that do exist (e.g., dialysis through a membrane) are often explore pspecibve embryos. either ditficuit to demonstrate to large groups, or take too long. The intent here is Using computer graphics students can rewonstruct the effect of angles of to not only to enliven rather dry subject material using rapidly-paced illustrations onentation on the collcted measurements. These observations lead to with a strong visual impact, but to point out the connection between the discussions ranging from sampling techniques, sampling error, indices of phenomenon discussed in the classroom and everyday experience, thereby roundness, to surface area / volume ratios. With these experiences, the making it more accessible than something that is perceived by the student as course lecture can explore the anatomical correlates associated with the occurring only in the laboratory. Additional benefits of such 'kitchen chemistry' activation of the embryonic genome. By using this quantitative approach to demonstrations are that (a) they are particularly helpful to visually-oriented developmental anatomy, the students gain a more interactive appreiation for students in learning the concept, (b) the demonstrations are easy to remember, in microscopic anatomy and the dynamic processes associed with that and (c) students find them useful explaining the concepts to others, and thus anatomy. (Supported by NSF USE-9152675 and Trinity University.) leam by teaching. All of the demonstrations are designed for overhead projection.

2836 GLUCOSE TRANSPORT IN CULTURED ANIMAL CELLS: A LABO- RATORY EXERCISE ((M.L.S. Ledbetter)) Dept. of Biology, College of the Holy Cross, Worcester MA 01610.

Membrane transport is a fundamental concept that students understand better with laboratory experience. Other formal teaching exercises are unbiological (dialysis tub- ing), qualitative (Elodea plsmolysis, erythrocyte hemolysis) or require intricate and time-consuming preparations (everted intestinal sacs). In contrast, attachment- dependent animal cells can be grown on miultiwell culture trays and readily manipulated in sitU to yield reproducible, biologicallb relevant, quantitative data regarding key as- pects of membrane transport. The exercise can also be readily adapted for student projects. Each 24-well tray of cultures allows 8 conditions to be tested in triplicate. If different groups test different conditions or different cell types, data can be shared for an even broader experience. I usually use uptake of the non-metabolizable glucose analogue, [3H --2 - deozy - D - glucose, but amino acid or nucleotide analogues are equall suitable The radioactivity is readily contained in safely disposable form, and the amount needed can be purchased without a liense Sterility need not be main- tained over the 1-hr uptake period. If cel culture facilities are lacidng, cells can be ordered from the ATCC and grown in HEPES-buffered media or in incubated desic- cators purged with 5% CO2 in air. If a scintillation counter is not available on site, samples can be prepared, saved for a time, and transported elswhere for countin& Students have successfully tested the effects of phloridzin, insulin, serum, Na+ - free medium, ouabain, and various other sugars on gucose transport in both epithelial and fibroblstic ceBs. One student even measured the Km and V.. of the glucose transporter for her project. Students find the nutritional and medical implications of glucose transport and its regulation intriguing; they also learn to handle radioisotopes and cultured cells.