摘要:

AbstractChick transferrin (Tf) is essential not only for growth and differentiation but also for the maintenance of chick myotubes in culture. Its removal from the culture medium gives rise to degeneration of the myotubes. The analysis of this process revealed that the removal resulted in decrease in total and messenger RNA content in the myotubes; this was mainly due to a decrease in RNA synthesis. Activity of in vitro RNA synthesis in isolated nuclei from myotubes cultured without Tf was lower than the activity in nuclei from myotubes cultured with Tf and increased with the addition of FeCl3. Although RNA degradation in myotubes was also enhanced following Tf removal, the degree was small. The synthesis of most proteins was reduced. In contrast to this, a few new proteins of unknown nature were synthesised in myotubes cultured in Tf‐free medium.The role of Fe ion carried into the cells by Tf in promoting myogenic cell growth and differentiation and in preventing the myotubes from degeneration can be explained, at least in part, on the basis of its effect on RNA synthesis.Since we have found that Fe is required for activation of RNA polymerase purified from embryonic muscles (Shoji and Ozawa, 1985b), these effects may be ascribed to this activating effec

ISSN:0021-9541    

DOI:10.1002/jcp.1041270302

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

年代:1986

数据来源: WILEY

摘要:

AbstractWe have constructed a hybrid gene in which the SV40 T‐antigen coding gene is driven by a mouse rDNA promoter and we have compared its expression to that of an SV40 T‐antigen coding gene under the control of its own promoter. The comparison has been carried out in microinjected cells, in transfected cells, and in stable cell lines carrying the respective T‐antigen coding genes in an integrated form. These cell lines were derived from ts AF8 cells, a mutant which is temperature sensitive for RNA polymerase II activity. The hybrid gene clearly expresses T‐antigen, albeit less efficiently than when the T antigen coding gene is under the control of the SV40‐promoter. We also show that the expression of T‐antigen by the hybrid gene is 50% inhibited by an antibody against RNA polymerase I. In tsAF8 cells carrying the hybrid gene, T‐antigen is still expressed at the restrictive temperature (where RNA polymerase II is inactive) at a level again about 50% of controls. However, our findings also confirm those of Smale and Tjian (Mol. Cell. Biol. 5:352, 1985) that such hybrid genes are in part transcribed by RNA polymerase II and generate abnorma

ISSN:0021-9541    

DOI:10.1002/jcp.1041270303

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

年代:1986

数据来源: WILEY

摘要:

AbstractIn vitro, high density monolayer cultures of vascular smooth muscle cells can be induced to form multicellular nodules. The nodular cells appear to be morphologically differentiated smooth muscle cells. Sodium dodecyl sulfate (NaDodSO4)‐polyacrylamide gel electrophoresis was used to compare the proteins synthesized and secreted by monolayer and nodular cultures of smooth muscle cells. Although most proteins appeared to be similar, the nodular cultures contained a unique heparin binding protein of Mr = 38,000 (38kD protein) (Millis, A.J.T., Hoyle, M., Reich, E., and Mann, D.M., 1985, J. Biol. Chem., 260: 3754–3761). The 38kD protein was glycosylated and its apparent molecular weight was shifted to Mr = 32,500 after synthesis in the presence of tunicamycin or digestion with endoglycosidase F. The production of 38kD protein by nodular cell cultures did not appear to result from the degradation of a high molecular weight precursor in nodular conditioned medium. Further, it was not detected in monolayer cell conditioned medium that had been incubated with nodular cells. Finally, its synthesis was not induced in monolayer cell cultures that had been labeled in nodular cell conditioned medium. The 38kD protein appears to be uniquely associated with nodular cultures of smooth muscle ce

ISSN:0021-9541    

DOI:10.1002/jcp.1041270304

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

年代:1986

数据来源: WILEY

摘要:

AbstractA novel, serum albumin‐binding glycoprotein of molecular weight (mw) 43,000 (43K protein) was initially purified from the culture medium of bovine aortic endothelial (BAE) cells (Sage, H., Johnson, C., and Bornstein, P., J. Biol. Chem.259:3993–4007, 1984). Its secretion by normal mesenchymal cells and by transformed cells of both ectodermal and endodermal origin suggested a general role in cellular function. To examine the effect of sublethal injury in vitro on the biosynthesis of 43K protein, BAE cells were exposed to endotoxin. At concentrations which produced minimal cell detachment and lysis, the cells secreted 70–100% more protein compared to control cultures, and the relative increase in 43K protein over total protein was approximately three‐fold. A second type of cellular injury, manifested by rapid cellular proliferation and migration in response to sparse plating density (a condition that we have termed ‘culture shock’), was also accompanied by a significant increase in the secretion of 43K protein.Pulse‐chase studies revealed that the initial product secreted within 1.5 h was of Mr38,000, and that between 6 and 21 h this molecule was converted to the final form of Mr43,000. The 43K protein was not associated with RNA or glycosaminoglycan, but appeared to be linked to complex oligosaccharides containing peripheral sialosyl residues. Treatment with tunicamycin produced lower mw forms that displayed reduced affinity for albumin. By immunologic criteria, peptide mapping, and amino acid analysis, the 43K protein was shown to be structurally distinct from several proteins of Mr40,000–50,000 associated with endothelium or with serum, including tissue factor, a plasminogen anti‐activator, and several apolipoproteins. In addition, the 43K protein was not present in the extracellular matrices of endothelial, fibroblastic, or smooth muscle cells, nor was it found in plasma, serum, platelet releasate, or alveolar lavage fluids.These studies identify a unique Mr43,000 glycoprotein that is associated with cellular stress or injury in vitro. As a secreted but nonmatrix macromolecule, this protein may be part of a ‘survival kit’ used by the endothelium to cope

ISSN:0021-9541    

DOI:10.1002/jcp.1041270305

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe major [35S]methionine‐radiolabeled nuclear matrix proteins of mouse 3T3 cells were isolated, and most of these were found to be similar in molecular weight, charge, and protease fingerprint to the nuclear matrix proteins of African green monkey kidney cells, which are found tightly bound to simian virus 40 chromosomes. These nuclear matrix proteins were found to be synthesized in quiescent and serum‐stimulated cells and to be turned over slowly during pulse‐chase experiments. In contrast, a 70‐Kd (kilodalton) neutral protein identified as lamin a was found to be turned over rapidly, producing a 68‐Kd protein with a similar isoelectric point. In addition, we observed a decrease in the amounts of two chromatin‐bound matrix proteins and a relative increase in lamin a following labeling in the presence of 2 μg/ml actinomycin D. However, these effects do not appear to be a result of inhibition of transcription, since they were not observed with other inhibitors (α‐amanitin and 5,6‐dichloro‐1‐β‐D‐r

ISSN:0021-9541    

DOI:10.1002/jcp.1041270306

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

年代:1986

数据来源: WILEY

摘要:

AbstractCollagen types synthesized by murine bone marrow cells were studied and the effect of lithium chloride on collagen biosynthesis in vitro was investigated. In the liquid culture system used, an adherent, mixed cell population supports hemopoiesis. Radioactive labeling of cell cultures and subsequent fractionation with ammonium sulfate, enzyme digestion, immune precipitation, and gel electrophoresis indicated that the bone marrow cells synthesized precursors to collagen types I, III, and IV, and fibronectin. A previously undescribed molecule or fragment with an apparent molecular weight of 17,000 daltons that was susceptible to bacterial collagenase and containing no interchain disulfide bonds was also identified in the culture media of both control and lithium‐treated cells. Lithium treatment did not affect the types of collagen synthesized, although the relative proportions of collagen types may differ from controls. However, lithium does have an effect on the appearance of some, as yet unidentified, non‐collagenous components in the cell culture me

ISSN:0021-9541    

DOI:10.1002/jcp.1041270307

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

年代:1986

数据来源: WILEY

摘要:

AbstractInterleukin 3 (IL‐3) is a regulatory glycoprotein required for the proliferation and differentiation of cells from many if not all hemopoietic lineages. With the emergence of the competence‐progression model of cell proliferation, which predicts that growth factors function at specific stages of the cell cycle, we examined the possibility that IL‐3 functions at a specific stage of the cell cycle. C‐63 cells were developed as a cell line from normal murine bone marrow. They have a mast cell phenotype and require pokeweed‐stimulated spleen cell‐conditioned medium (CM), a rich source of IL‐3, for their continued growth. Exponentially growing cells were transferred from growth medium, which contains CM, to medium lacking CM or IL‐3. After 24 hours, cell viability had decreased 40–50%. The remaining viable cells did not incorporate3H‐thymidine, and displayed a single peak at G1 in a DNA histogram. Restimulation of these cells with CM or IL‐3 resulted in a dramatic rise in3H‐thymidine uptake 20–24 hours after restimulation. DNA histograms of restimulated cultures indicated that the cells were progressing in a wave‐like fashion throughout the remainder of the cell cycle. The length of time necessary for cells to be in contact with CM or IL‐3 before they could progress into the remainder of the cell cycle was also examined. Cells incubated with CM or IL‐3 for less than 16 hours could not progress into S phase, whereas cells incubated for 16 hours or longer could progress into S phase and through the remainder of the cell cycle. These data suggest that IL‐3 exerts its function at a

ISSN:0021-9541    

DOI:10.1002/jcp.1041270308

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

年代:1986

数据来源: WILEY

摘要:

AbstractInsulin‐like growth factor I (IGF‐I) regulation of the sequence of transcriptional, translational, and postranslational events during G1that are necessary for DNA synthesis, and the induction of thymidine kinase and in vivo thymidylate synthase activities was studied in synchronized confluent BALB/c 3T3 mouse fibroblasts. IGF‐I was the only growth factor necessary from 6 to 2½ hours before S phase for onset of DNA synthesis. 5,6 dichlororibofuranosylbenzimidazole (DRB), an inhibitor of transcription, was ineffective in blocking DNA synthesis and increase of both enzyme activities during an interval of about 2½ hours before S phase. Cycloheximide, an inhibitor of translation, was ineffective in blocking DNA synthesis and the increase of in vivo thymidylate synthase activity during an interval of about 1½ hours before S phase but was effective in blocking the increase of thymidine kinase activity up to the G1/S boundary. The results demonstrate that IGF‐I is dispensable beyond the R‐point (2–3 hours before S phase), the time when serum factors are no longer necessary for the initiation of DNA synthesis, and that IGF‐I regulates transcriptional events necessary for both DNA synthesis and the induction of thymidine kinase and in vivo thymidylate synthase. The results also demonstrate a posttranslational interval for in vivo thymidylate synthase, suggesting posttranslational modificatio

ISSN:0021-9541    

DOI:10.1002/jcp.1041270309

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

年代:1986

数据来源: WILEY

摘要:

AbstractInterferons (IFNs) are proteins which have antiviral and antiproliferative properties and are known to affect various immunological processes. Some of these activities have been shown to be potentiated by increased temperatures. When cells are subjected to a rise in temperature, the synthesis of the heat‐shock proteins (HSPs) is ‘switched on.’ In this report we demonstrate a synergistic effect of IFN and stress (arsenite treatment or elevated temperature) on the heat‐shock response. On the one hand, IFN pretreatment enhances the accumulation of HSP mRNAs and the corresponding protein synthesis after a mild stress and, on the other hand, it amplifies the decrease of the total protein synthesis after a severe stress. Thus in IFN pretreated cells the range of temperatures leading to the heat‐shock response is shifted towards common physiologic

ISSN:0021-9541    

DOI:10.1002/jcp.1041270310

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe bovine aortic endothelial cell (BAEC) cytoskeleton is a complex structure modulated by many stimuli including release from contact inhibition and various components of the extracellular matrix (ECM). Transduction of information from the ECM to the cell nucleus proceeds via several complex pathways including the cytoskeleton. We have demonstrated the presence of an immunoreactive isoform of the human erythrocyte cytoskeletal protein band 4.1 (4.1) in BAEC. BAEC 4.1 is similar in molecular weight to the erythroid protein by immunoblot analyses and produces a similar pattern of cysteine specific cleavage products consistent with a cluster of cysteine residues previously described in the erythroid molecule. We have also examined the effects of defined ECM proteins on the distributions of cultured BAEC 4.1 and actin filaments (AF) at confluency and following release from contact inhibition. The distribution of 4.1 in BAEC on a plasma fibronectin substrate is complex, having partial codistribution with cytoplasmic AF and a unique perinuclear staining. In contrast, on a collagen type I/III substrate, 4.1 is localized, in part, to peripheral areas of cell‐cell contact distinct from the dense peripheral band staining of AF. During migration on this substrate, 4.1 had a filamentous distribution having partial codistribution with AF. Indirect immunofluorescence staining of cross‐sections of bovine calf aortae revealed a cortical staining pattern in the aortic endothelial cells with staining noted on the luminal and basolateral aspects of the cells. These data suggest that, in endothelial cells, protein 4.1 is a cortical membrane protein which may function to link actin filaments to other skeletal proteins such as spectrin. These findings also suggest an active role for protein 4.1 in cytoskeletal reorganization events which can occur in response to external stimuli, such as the extracellular matrix or contact with other ce

ISSN:0021-9541    

DOI:10.1002/jcp.1041270311

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

年代:1986

数据来源: WILEY