摘要:

AbstractA biotinylated derivative of murine epidermal growth factor (EGF) was prepared by covalent attachment of the terminal amino group of EGF to N‐biotinyl‐ε‐aminocaproyl‐N‐hydroxysuccinimide. The stoichiometry of biotin incorporation was in the range of one biotin moiety per EGF molecule. The biotinylated EGF (biotinyl‐ε‐caproyl‐EGF, BioEGF) binds to EGF receptors on intact Ehrlich ascites carcinoma (EAC) cells with an affinity similar to that of native EGF and displays the same mitogenic activity as EGF in a soft agar test system with normal rat kidney (NRK) cells. BioEGF was visualized on cultured cells and tissue sections of a head and neck tumour by commercial streptavidin/avidin detection systems. Cytochemical analyses of certain tumour forms can be easily performed using

ISSN:0730-2312    

DOI:10.1002/jcb.240410202

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractWe have examined the mechanism of membrane protein insertion in the ameboid spermatozoa ofCaenorhabditis elegansusing two monoclonal antibodies which recognize the same set of eight sperm‐specific polypeptides. Previous electron microscopic studies demonstrated that these antibodies label surface and cytoplasmic populations of antigen. Cells whose surface antigen had been removed by proteolysis were able to localize new membrane protein insertion at the tips of pseudopodial projections.C. eleganssperm do not contain the protein synthesizing machinery needed for delivery of new membrane to the cell surface. It has, therefore, been of interest to determine how localized membrane assembly occurs. Here we have determined the subcellular location of each of these eight polypeptides. A closely positioned doublet of bands around 97 kD (comprising 40% of the total antigen in sperm) represents surface (larger member of doublet) and cytoplasmic (lower member) forms of protein. Proteolysis of live cells eliminated this surface form from immunoblots but did not affect the cytoplasmic protein. When cells were allowed to reinsert new protein following removal of the enzyme, this surface form was regenerated. Since sperm are unable to synthesize new protein, this higher molecular weight species may arise from a posttranslational modification of proteins in the cytoplasmic pool. We present evidence suggesting that the surface protein is generated from this cytoplasmic pool by addition of fatty acid. Fatty acid acylation would account for both the observed decrease in electrophoretic mobility of the surface form and provide increased hydrophobicity to the protein which may allow for its insertion into the lipid bilaye

ISSN:0730-2312    

DOI:10.1002/jcb.240410203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractTenascin is a large, disulfide‐bonded glycoprotein of the extracellular matrix. The predominant form of tenascin observed by electron microscopy is a six‐armed oligomer, termed a hexabrachion. We have determined the molecular mass of the native human hexabrachion to be 1.9 × 106Da by sedimentation equilibrium analysis and by electrophoresis on non‐reducing agarose gels. On reducing polyacrylamide gel electrophoresis (SDS‐PAGE), human tenascin showed a single prominent band at 320 kDa and minor bands of 220 and 230 kDa. The molecular weight of the native human hexabrachion is thus consistent with a disulfide‐bonded hexamer of the 320 kDa subunits.Upon treatment with neuraminidase, the apparent molecular weights of all human and chicken tenascin subunits on reducing SDS‐PAGE were decreased by about 10 kDa. Prolonged incubation with α‐mannosidase, however, caused no apparent change in the apparent molecular weight of tenascin subunits. Sedimentation in a cesium chloride gradient gave a higher buoyant density for human tenascin than for fibronectin, suggesting that it has a higher degree of glycosylation. The far‐UV circular dichroism spectrum indicates a predominance of β‐structure and a lack of collagen‐like or α‐helical structure.When human hexabrachions were reduced and acetylated, the resulting fragments were single arms which sedimented at 6 S in glycerol gradients and migrated at 320 kDa on non‐reducing gels. Treatment of tenascin with trypsin and α‐chymotrypsin also produced large fragments which were fractionated by gradient sedimentation and analyzed by non‐reducing SDS‐PAGE and electron microscopy. We present a structural model for the assembly of the observed fragments into

ISSN:0730-2312    

DOI:10.1002/jcb.240410204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractMedium conditioned by the established lung tumor cell line A549 was used as a supplement to culture cells from primary solid lung tumors. Of 36 cases placed into culture, primary cells were obtained in 33 (91.7%). Of 29 cases in which subcultures were attempted, 18 (62.1%) were successful. Nine cell lines have been established by this technique to date. In growth assays, conditioned medium (CM) was found to stimulate both monolayer colony formation and growth in semi‐solid medium of cells cultured from primary solid tumors. CM has been found to contain factors with the properties of both transforming growth factorα (TGFα) and insulin‐like growth factor‐I (IGF‐I). The addition of a combination of these factors as purified peptides to basal medium at levels found in CM (0.1–0.5 ng/ml) stimulated colony formation of lung tumor cells by up to fourfold. These results indicate that secretion of growth factors may be important in tumor growth in vivo, and that use of CM may be a valuable tool for obtaining cultures from primary s

ISSN:0730-2312    

DOI:10.1002/jcb.240410205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractA rat liver gap junction (GJ) cDNA probe that detects mRNA encoding the 32 Kd GJ‐protein (connexin 32) was employed to study GJ‐protein gene expression in rat liver tumors induced by a single exposure to diethylnitrosamine (DEN) followed by exposure to 2‐acetylaminofluorene (AAF)/CCL4/AAF or induced by systemic administration of N‐ethyl‐N‐hydroxyethylnitrosamine (EHEN). All carcinomas generated by these carcinogens showed markedly reduced levels of GJ‐protein mRNA. This may indicate that GJ‐protein levels and gap‐junctional intercellular communication (GJIC) capacity are also severely compromised. Moreover, all hyperplastic nodules also showed a reduced level of GJ‐protein mRNA. Taken together with our earlier finding that the liver tumor promoter phenobarbital inhibits GJ‐protein gene expression, these results suggest that deranged GJIC is a relatively early event in liver multistage carcinogenesis. A range of other cDNA probes was also used to characterize gene expression in the DEN‐induced tumors. Induction of expression was seen for glutathione S‐transferase (placental form) (GST‐P), γ‐glutamyltranspeptidase (GGT), and c‐r

ISSN:0730-2312    

DOI:10.1002/jcb.240410206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractIn this report, we have examined whether (6R)‐tetrahydrobiopterin (H4biopterin) modulates the binding of interleukin 2 to high‐affinity sites of the cloned mouse cytotoxic T‐lymphocyte clone CTLL‐2. Scatchard plot analysis of the equilibrium binding data reveals increased affinity when the cells are exposed simultaneously to interleukin 2 and to the pterin. The Kdvalues are statistically significantly reduced from 1.4 × 10−11M to 0.78 × 10−11M interleukin 2. The dissociation kinetics of the ligand were followed at 4°C after equilibrium binding under high‐affinity conditions (1.2 × 10−10M interleukin 2). In the presence of H4biopterin, the dissociation rate constant (k−1) decreases from 6.2 × 10−3min−1to 3.0 × 10−3min−1and the half‐time of dissociation increases from 106.8 min to 218.0 min. As a third approach interleukin 2 was bound to the surface of cells under high‐affinity conditions by incubation in the cold and the internalization kinetics upon warming were determined. Sigmodial‐shaped kinetics of endocytosis in control cells indicate that the internalization rates increase only gradually. The presence of H4biopterin causes an apparent immediate transition from higher‐order kinetics of a linear response so that maximum internalization rates are reached immediately upon warming. The data show that lymphocyte‐derived H4biopterin in vitro at concentrations ranging from 2–8 × 10−7M modulates interleukin 2 high‐affinity binding and that H4biopterin potentially participates i

ISSN:0730-2312    

DOI:10.1002/jcb.240410207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

ISSN:0730-2312    

DOI:10.1002/jcb.240410208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

ISSN:0730-2312    

DOI:10.1002/jcb.240410209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

ISSN:0730-2312    

DOI:10.1002/jcb.240410201

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY