摘要:

AbstractSDS‐polyacrylamide gel electrophoresis was used to study the effects of the thiol inhibitor monobromobimane (MB), EDTA, and prostaglandin E1(PGE1) on the formation and composition of the platelet cytoskeletal core (Triton‐insoluble residue) and its association with glycoprotein (GP) IIIa. Stimulation or aggregation of platelets in response to ADP or thrombin increased the amount of Triton‐insoluble myosin. Aggregation resulted in incorporation of [125I]GP IIIa and a new band at about 210 kDa into the cytoskeletal core. EDTA and PGE1caused little disaggregation of platelets that were aggregated in PRP with ADP and that had secreted the contents of their granules. In contrast to EDTA, PGE1decreased the amount of Triton‐insoluble residue and its association with GP IIIa. MB added after ADP‐induced aggregation caused an increase in the amount of cytoskeletal core despite marked disaggregation and a substantial decrease in core‐associated GP IIIa. With aspirin‐treated platelets that had not secreted, EDTA, PGE1, and MB all caused disaggregation and loss of cytoskeletal GP IIIa. MB diminished, but did not reverse, thrombin‐induced aggregation of washed platelets and arrested GP IIIa incorporation into the cytoskeletal core. Concanavalin A (Con A) cross‐links glycoproteins on a single platelet and induces incorporation of GP IIIa into the Triton‐insoluble residue in the absence of platelet aggregation. This induction was not inhibited by MB, although this reagent, as well as aspirin, inhibited Con A‐induced secretion. Since GP IIIa incorporation caused by ADP‐induced aggregation differs from that caused by Con A in its susceptibility to MB, it seems unlikely that thiol groups are directly involved in the association of GP IIIa wit

ISSN:0730-2312    

DOI:10.1002/jcb.240390402

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

年代:1989

数据来源: WILEY

摘要:

AbstractPrevious studies have shown that human colon carcinomas contain elevated amounts of chondroitin sulfate proteoglycan (CS‐PG) and hyaluronic acid, and that the major site of synthesis of these products is the host mesenchyme surrounding the tumor. These findings have led to the proposal that the abnormal formation of the tumor stroma is modulated by the neoplastic cells. The experiments of this paper were designed to explore further this complex phenomenon in an in vitro system using co‐cultures of phenotypically stable human colon smooth muscle (SMC) and carcinoma cells (WiDr). The results showed a 3–5‐fold stimulation of CS‐PG and hyaluronic acid biosynthesis in the co‐cultures as compared to the values predicted from the individual cell type cultured separately. The increase in CS‐PG was not due to changes in specific activity of the precursor pool, but was rather due to a net increase in synthesis, inasmuch as it was associated with neither a stimulation of cell proliferation nor with an inhibition of intracellular breakdown. These biochemical changes were corroborated by ultrastructural studies which showed a marked deposition of proteoglycan granules in the co‐cultures. Several lines of evidence indicated that the SMC were responsible for the overproduction of CS‐PG: (i) SMC synthesized primarily CS‐PG when cultured alone, in contrast to the WiDr, which synthesized exclusively heparan sulfate proteoglycan; (ii) only the SMC in co‐culture stained with an antibody raised against the amino terminal peptide of a CS‐PG (PG‐40), structurally and immunologically related to that synthesized by the SMC; (iii) the stimulation of CS‐PG in SMC could be reproduced, though to a lesser extent, using medium conditioned by WiDr, whereas medium conditioned by SMC had no effects on WiDr. In conclusion this study has reproduced in vitro a tumor‐associated matrix with a proteoglycan composition similar to that observed in vivo and provides further support to the concept that production of a proteoglycan‐rich extracellular environment is regulated by specif

ISSN:0730-2312    

DOI:10.1002/jcb.240390403

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe role of aspartic acid‐49 (Asp‐49) in the active site of porcine pancreatic phospholipase A2 was studied by recombinant DNA techniques: two mutant proteins were constructed containing either glutamic acid (Glu) or lysine (Lys) at position 49. Enzymatic characterization indicated that the presence of Asp‐49 is essential for effective hydrolysis of phospholipids. Conversion of Asp‐49 to either Glu or Lys strongly reduces the binding of Ca2+ions, in particular for the lysine mutant, but the affinity for substrate analogues is hardly affected. Extensive purification of naturally occurring Lys‐49 phospholipase A2 from the venom ofAgkistrodon piscivorus piscivorusyielded a protein that was nearly inactive. Inhibition studies showed that this residual activity was due to a small amount of contaminating enzyme and that the Lys‐49 homologue itself has no enzymatic activity. Our results indicate that Asp‐49 is essential for the catalytic action of phospholipase A2. The importance of Asp‐49 was further evaluated by comparison of the primary sequences of 53 phospholipases A2 and phospholipase homologues showing that substitutions at position 49 are accompanied by structural variations of otherwise conserved residues. The occurrence of several nonconserved substitutions appeared to be a general characteristic of nonactive phospholipas

ISSN:0730-2312    

DOI:10.1002/jcb.240390404

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

年代:1989

数据来源: WILEY

摘要:

AbstractWe investigated the action of cholera toxin on the intracellular ionized calcium [Ca2+]i increase induced by anti‐CD2 and anti‐CD3 monoclonal antibodies in the leukemic human T‐cell line Jurkat. Cholera toxin inhibits in a dose‐dependent manner these two pathways of human T‐lymphocyte activation but with different half maximal inhibition doses (75 ng/ml for CD3, 30 ng/ml for CD2). This effect cannot be accounted for only by the increase in cAMP induced by cholera toxin because forskolin, which raises cellular cyclic adenosine monophosphate (cAMP) to the same levels, induced only a small inhibition of the [Ca2+]i increase in similar conditions. Cholera toxin induced a decrease in the surface expression of the CD3 molecule, suggesting a down‐regulation of the CD3 molecules. On the other hand, the expression of CD2 remained unchanged. Cell surface disappearance of the CD3 molecule cannot account for all the inhibitory effects of cholera toxin because CD2 molecule expression was not affected (no modifications in the half maximal binding of anti‐CD2 monoclonal antibodies). All together, these results suggest that cholera toxin acts on substrates, possibly G proteins, that could regulate the [Ca2+]i increase induced by anti‐CD2 and anti‐CD3 mAbs in Jurkat cells. In addition, the present study demonstrated that the rise in cellular cAMP partially inhibits the [Ca2+]i increase induced by anti‐CD

ISSN:0730-2312    

DOI:10.1002/jcb.240390405

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

年代:1989

数据来源: WILEY

摘要:

AbstractPreviously using PKC isozyme‐specific antibodies for immunoblot analysis, we demonstrated the heterogeneous distribution of PKC isozymes in various regions of monkey and rat brains and that type I PKC was most abundant in cerebellum, hippocampus, amygdala, and cerebral cortex (Huang et al.:J Biol Chem262:15714–15720, 1987). Using these antibodies, we have also demonstrated that type I, II, and III PKC are products of PKC genes γ, β, and α, respectively (Huang et al.:Biochem Biophys Res Commun149:946–952, 1987). By immunocytochemical analysis, type I PKC‐specific antibody showed strong reactivity in various types of neuron in hippocampal formation, amygdala, cerebellum, and neocortex. In hippocampal formation, granule cells of dentate gyrus and pyramidal cells of hippocampus were heavily stained. By immunoblot analysis, relative levels of PKC isozymes in several areas of monkey cerebral cortex involved in the visual information processing and storage were determined. Both type II and III PKCs appeared to be evenly distributed and at moderate levels, type I PKC formed a gradient of increasing concentration rostral along the cerebral cortex of occipital to temporal and then to the limbic areas. Neurobehavioral studies have demonstrated that the neocortical and limbic areas of the anterior and medial temporal regions participate more directly than the striate, prestriate, and posterior temporal regions in the storage of visual representations and that both hippocampus and amygdala are important in the memory formation. As type I PKC is present at high levels in hippocampus, amygdala, and anterior temporal lobe, we predict that the type I protein kinase C may participate in the plastic changes important for mnemoni

ISSN:0730-2312    

DOI:10.1002/jcb.240390406

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

年代:1989

数据来源: WILEY

摘要:

AbstractPolycationic molecules were studied either for their ability to displace the binding of basic fibroblast growth factor (bFGF) to high‐ and low‐affinity membrane interaction sites and/or to modulate bFGF‐induced proliferation of fibroblasts. Heparin‐binding polypeptides, such as polylysine, protamine, histones, and thrombin‐displaced [125I]bFGF bound to bovine brain membrane receptors. The most displacing polypeptides were those with the strongest affinity to heparin. Two of these polypeptides, protamine and polylysine, inhibited (at 5 μM) by more than 90% the mitogenic effect induced by bFGF on Chinese hamster lung fibroblast cells (CCL39). At the same dose, no effect was observed with basic proteins that do not bind to heparin, such as cytochrome C and lysozyme. An interesting observation was that protamine at 1 μM potentiated by 1.5‐fold the mitogenic activity of bFGF, while it acted as an inhibitor at higher

ISSN:0730-2312    

DOI:10.1002/jcb.240390407

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

年代:1989

数据来源: WILEY

摘要:

AbstractThis report demonstrates that the expression of melanoma growth stimulatory activity (MGSA) mRNA can be modulated in a positive fashion in the Hs294T human melanoma cell line by PDGF and MGSA. There is close correlation between MGSA expression and the pattern of cell growth in Hs294T cells.

ISSN:0730-2312    

DOI:10.1002/jcb.240390408

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

年代:1989

数据来源: WILEY

摘要:

AbstractInsulin stimulated autophosphorylation of the β‐subunit of the insulin receptor purified from Fao hepatoma cells or purified from Chinese hamster ovary (CHO/HIRC) or Swiss 3T3 (3T3/HIRC) cells transfected with the wild‐type human insulin receptor cDNA. Autophosphorylation of the purified receptor occurred in at least two regions of the β‐subunit: the regulatory region containing Tyr‐1146, Tyr‐1150, and Tyr‐1151, and the C‐terminus containing Tyr‐1316 and Tyr‐1322. In the presence of antiphosphotyrosine antibody (α‐PY), autophosphorylation of the purified receptor was inhibited nearly 80% during insulin stimulation. Tryptic peptide mapping showed that α‐PY inhibited autophosphorylation of both tyrosyl residues in the C‐terminus and one tyrosyl residue in the regulatory region, either Tyr‐1150 or Tyr‐1151. Thus, a bis‐phosphorylated form of the regulatory region accumulated in the presence of α‐PY, which contained Tyr(P)‐1146 and either Tyr(P)‐1150 or 1151. In intact Fao, CHO/HIRC, and 3T3/HIRC cells, insulin stimulated tyrosyl phosphorylation of the β‐subunit of the insulin receptor. Tryptic peptide mapping indicated that the regulatory region of the β‐subunit was mainly (>80%) bis‐phosphorylated; however, all three tyrosyl residues of the regulatory region were phosphorylated in about 20% of the receptors. As the phosphotransferase was activated by tris‐phosphorylation but not bis‐phosphorylation of the regulatory region of the β‐subunit (White et al.:Journal of Biological Chemistry263:2969–2980, 1988), the extent of autophosphorylation in the regulatory region may play an important regu

ISSN:0730-2312    

DOI:10.1002/jcb.240390409

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

年代:1989

数据来源: WILEY

摘要:

AbstractA receptor for fibroblast growth factor (aFGF, bFGF) was partially characterized in intact cell cultures, cell plasma membranes, and tissue plasma membrane preparations. Analysis of 24 different cell types from four species identified a 165‐kDa FGF receptor present on the cell surface of most mesodermal and neuroectodermal cells. Chemical crosslinking of125I‐aFGF to its cell surface receptor was specifically blocked by a 100‐fold molar excess of either aFGF or bFGF. In contrast to the similar molecular weight of FGF receptors, different cell types exhibited significant variations in binding of125I‐aFGF to intact cultures with low values of 8 pM and 700, to high values of 60 pM and 30,000, for the Kdand receptor number per cell, respectively. A binding assay was developed for quantitation of125I‐aFGF binding to cell‐ and tissue‐derived membrane preparations. Membranes prepared from baby hamster kidney cells exhibited a Kdof 55 pM, while a similar Kdof 67 pM was determined for intact baby hamster kidney cells. Although ten different adult bovine tissue membrane preparations and human term placental membranes exhibited no specific binding of125I‐aFGF, FGF receptor was detected in embryonic murine tissues (17 days gestation). These results support the existence, in a variety of cells, of either a common FGF receptor that binds both aFGF and bFGF or closely related FGF receptors that cannot be distinguished by molecular weight. The differential binding of FGF to its receptor in embryonic vs. adult tissues suggests a potentially broad role for FGF in embryonic development and a more restrictive ro

ISSN:0730-2312    

DOI:10.1002/jcb.240390410

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

年代:1989

数据来源: WILEY

摘要:

AbstractRecently, we have obtained evidence in favor of a structural interaction between the epidermal growth factor (EGF) receptor and the Triton X‐100‐insoluble cytoskeleton of epidermoid carcinoma A431 cells. Here we present a further analysis of the properties of EGF receptors attached to the cytoskeleton. Steady‐state EGF binding studies, analyzed according to the Scatchard method, showed that A431 cells contain two classes of EGF‐binding sites: a high‐affinity site with an apparent dissociation constant (KD) of 0.7 nM (7.5 × 104sites per cell) and a low‐affinity site with a KDof 8.5 nM (1.9 × 106sites per cell). Non‐equilibrium binding studies revealed the existence of two kinetically distinguishable sites: a fast‐dissociating site, with a dissociation rate constant (k−1) of 1.1. × 10−3s−1(1.0–1.3 × 106sites per cell) and a slow‐dissociating site, with a k−1of 3.5 × 10−5s−1(0.6–0.7 × 106sites per cell).The cytoskeleton of A431 cells was isolated by Triton X‐100 extraction. Scatchard analysis revealed that ∼5% of the original number of receptors were associated with the cytoskeleton predominantly via high‐affinity sites (KD= 1.5 nM). This class of receptors is further characterized by the presence of a fast‐dissociating component (k−1= 2.0 × 10−3s−1) and a slow‐dissociating component (k−1= 9.1 × 10−5s−1). The distribution between fast and slow sites of the cytoskeleton was similar to that of intact cells (65% fast and 35% slow sites). Incubation of A431 cells for 2 h at 4°C in the presence of EGF resulted in a dramatic increase in the number of EGF receptors associated to the cytoskeleton. These newly cytoskeleton‐associated receptors appeared to represent low‐affinity binding sites (KD= 7 nM). Dissociation kinetics also revealed an increase of fast‐dissociating sites. These results indicate that at 4°C EGF induces the binding of

ISSN:0730-2312    

DOI:10.1002/jcb.240390411

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

年代:1989

数据来源: WILEY