摘要:

AbstractThe patterns of radioactively labeled proteins from cultured chicken embryo cells stressed in the presence of either D2O or glycerol were analyzed by using one‐dimensional polyacrylamide gel electrophoresis. These hyperthermic protectors blocked the induction of stress proteins during a 1‐hour heat shock at 44°C. The inhibitory effect of glycerol but not D2O on the induction of heat shock proteins could be overcome by increased temperature. By using transcriptional run‐on assays of isolated nuclei and cDNA probes to detect hsp70‐ and hsp88‐specific RNA transcripts, it was shown that the D2O and glycerol blocks occurred at or before transcriptional activation of the hsp70 and hsp88 genes. After heat‐stressed cells were returned to 37°C and the protectors were removed, heat shock proteins were inducible by a second heating. This result and the fact that the chemical stressor sodium arsenite induced stress proteins in glycerol medium indicated that the treatments did not irreversibly inhibit the induction pathways and that the stress response could be triggered even in the presence of glycerol by a stressor other than heat. In principle then, cells incurring thermal damage during a 1‐hour heat shock at 44°C in D2O or glycerol medium should be competent to respond by inducing heat shock proteins during a subsequent recovery period at 37°C in normal medium. We found that heat shock proteins were not induced in recovering cells, suggesting that glycerol and D2O protected heat‐sensitive targets from thermal damage. Evidence that the heat‐sensitive target(s) is likely to be a protein(s) is summarized. During heat shocks of up to 3 hours duration, neither D2O nor glycerol significantly altered hsp23 gene activity, a constitutively expressed chicken heat shock gene whose RNA transcripts and protein products are induced by stabilization (increased half‐life). During a 2‐hour heat shock, glycerol treatment blocked the heat‐induced stabilization of hsp23 RNA and proteins; however, D2O treatment only blocked RNA transcript stabilization, effectively uncoupling the hsp23 protein stabilization pathway from hsp23 RNA stabilization and transcriptional activati

ISSN:0021-9541    

DOI:10.1002/jcp.1041390202

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

年代:1989

数据来源: WILEY

摘要:

AbstractA monensin‐resistant mutant Monr‐31, derived from Chinese hamster ovary (CHO) cell line, has been shown to have a reduced number of insulin receptors and a reduction in glucose uptake in response to insulin. We have further investigated the possibility that altered glucose uptake in Monr‐31 cells is related to an alteration in the activity of the insulin receptor. Uptake of glucosamine, 2‐deoxy‐D‐glucose, and 3‐O‐methyl‐D‐glucose in Monr‐31 cells was one‐half to one‐third that of CHO cells. The cellular content of the glucose transporter in Monr‐31 was reduced to about one‐third that of CHO as assayed by use of an antiglucose transporter antibody. After transfection with the human insulin receptor cDNA, we obtained clones CIR‐0 from CHO, and MIR‐2 and MIR‐15 from Monr‐31; CIR‐0 expressed a tenfold higher level of the insulin‐binding activity than did CHO, and MIR‐2 and MIR‐15 expressed a 20‐fold higher level than did Monr‐31. Glucose uptake in both CHO and CIR‐0 was significantly enhanced by exogenous insulin, but not in Monr‐31, MIR‐2, and MIR‐15. The β‐subunits of insulin receptor in CHO, CIR‐0, Monr‐31, and MIR‐2 were similarly phosphorylated. The decreased glucose transport activity in Monr‐31 cells is discussed i

ISSN:0021-9541    

DOI:10.1002/jcp.1041390203

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

年代:1989

数据来源: WILEY

摘要:

AbstractExpression of c‐fosis induced by a number of signals in several cell systems. Although the exact function of the c‐fosproduct is unknown, it has been implicated to be of importance for both cell growth and differentiation (Verma and Sassone‐Corsi, 1987). To analyze how c‐fosexpression relates to in vitro myogenic differentiation, the kinetics of c‐fosmRNA expression during spontaneous in vitro differentiation of L6J1 myoblasts was examined; c‐fostranscripts were most abundant at day 4 of the differentiation process. Multinucleated myotubes and expression of α‐actin and myosin heavy chain (MHC) mRNA appeared later, at day 6 or 7, and increased to maximal levels after 10 days in culture. To analyze further the relation between c‐fosexpression and L6J1 myogenic differentiation, L6J1 myoblasts were transfected with expression vectors containing the murine c‐fosgene driven by a metallothionein promoter. The growth rate of c‐fos‐transfected L6J1 cells did not differ from that of control cells. However, formation of myotubes was significantly reduced in c‐fos‐transfected L6J1 cultures compared withneo‐transfected controls. Myotube formation and expression of the myogenic markers α‐actin and MHC were reduced in subclones expressing high levels of c‐fos, but not in subclones with lower levels of c‐fosexpression. These results indicate that a marked elevation of c‐fosexpression at least partially i

ISSN:0021-9541    

DOI:10.1002/jcp.1041390204

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

年代:1989

数据来源: WILEY

摘要:

AbstractIn situ hybridization has been used to study the accumulation of colonystimulating factor (CSF) mRNA in single cells of a T lymphocyte clone (E9.D4) following antibody‐mediated (F23.1) activation via the Ti‐T3complex in fillerindependent bulk cultures. The specificity of hybridization for cellular RNA was demonstrated by pretreating the cells with the Ca2+‐dependent enzyme micrococcal nuclease by using a novel protocol developed for use with riboprobes. Maximal levels of granulocyte‐macrophage (GM) and multipotential‐CSF (interleukin 3) mRNA were detected after 8–10 h, with GM‐CSF mRNA being detected earlier and at a lower concentration of stimulus. The rise in intracellular mRNA was accompanied by an increase in the corresponding CSF bioactivity in the supernatant. In situ hybridization was of comparable sensitivity to Northern blot analysis and revealed significant heterogeneity in the accumulation of CSF mRNA within individual cells of the clone following stimulation with F23.1. This could account for the corresponding heterogeneity in CSF production by single cells. Under optimal conditions at least 25% of cells contained both transcripts. The method has been used to examine CSF production by normal spleen cells and should be useful in the further analysis of lymphokine gene regulation in s

ISSN:0021-9541    

DOI:10.1002/jcp.1041390205

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

年代:1989

数据来源: WILEY

摘要:

AbstractPlatelet‐activating factor (1‐O‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine [PAF]) is a vasoactive ether lipid produced by activated blood cells. To examine the molecular traffic and sites of metabolism of PAF released in the vascular wall, we used a coculture system in which endothelial cells are grown on micropore filters suspended over confluent cultures of vascular smooth muscle cells. The endothelial cells took up PAF 5–7 times more readily from the apical than from the basolateral surface, converting it to 1‐O‐alkyl‐2‐acyl‐sn‐glycero‐3‐phosphocholine (2‐acyl‐PAF) and other minor metabolites. Intact endothelial monolayers effectively shielded the underlying smooth muscle cells from PAF present in the apical fluid; after a 30‐min incubation with [3H]‐PAF, only 1% of the radioactivity was transferred to the interstitial fluid. By contrast, PAF readily entered the interstitial fluid when the endothelial monolayers were injured by exposure to xanthine and xanthine oxidase. PAF did not significantly increase the permeability of endothelial monolayers to albumin. Smooth muscle cells took up and metabolized interstitial PAF more quickly and more completely than did endothelial cells; 65% was converted to 2‐acyl‐PAF in 15 min by the smooth muscle cells. PAF enhanced the proliferative effect of PDGF on smooth muscle cells, as assessed by [3H]‐thymidine incorporation. These findings suggest that endothelial cells form a barrier to PAF released at the luminal surface, but PAF released in the vascular intima interacts primarily with smooth muscle cells,

ISSN:0021-9541    

DOI:10.1002/jcp.1041390206

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

年代:1989

数据来源: WILEY

摘要:

AbstractIntracellular pathways that rapidly stimulate the expression of a mitogeninducible, zinc‐finger encoding gene, EGR1 (Sukhatme et al., Cell 53:37–43), have been characterized in two human fibroblasts strains (WI‐38 and HSWP). Serum and epidermal growth factor (EGF) were each found to strongly stimulate EGR1 expression in both cell types. Comparably high levels of expression could also be induced by treatment with the phorbol ester TPA. In cells rendered deficient in PK‐C, serum and EGF were each still capable of inducing high levels of EGR1 mRNA. demonstrating that additional non‐protein kinase C pathways are capable of stimulating EGR1 expression. In both fibroblasts strains, stimulation of EGR1 expression by all these agents exhibited rapid, transient kinetics and could be superinduced if protein synthesis was inhibited through the addition of cycloheximide. Finally, various agents, known to stimulate/inhibit the activation of another early mitogenic response, the activation of Na/H exchange, were analyzed for their effect on EGR1 expression. Interestingly bradykinin, vasopressin, and Ca ionophores, which dramatically stimulate Na/H exchange, were only weak stimulants of EGR1 expression. Conversely, EGF, which stimulates Na/H exchange poorly, strongly activated EGR1 expression. Hence while EGR1 expression could be triggered by multiple intracellular pathways, its expression does not appear to require the prior activation of Na/H

ISSN:0021-9541    

DOI:10.1002/jcp.1041390207

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

年代:1989

数据来源: WILEY

摘要:

AbstractWe have isolated and purified a cell surface sialoglycopeptide (SGP) from bovine cerebral cortex cells that previously was shown to be a potent inhibitor of cellular protein synthesis. The following studies were carried out to characterize the potential ability of the SGP to inhibit DNA synthesis and to arrest cell division. Treatment of exponentially proliferating Swiss 3T3 cells with the SGP inhibitor resulted in a marked inhibition of thymidine incorporation within 24 h. When the SGP was removed from inhibited cultures, a sharp rise in3H‐thymidine incorporation followed within 3–4 h that peaked well above that measured in exponentially growing cultures, suggesting that the inhibitory action of the SGP was reversible and that a significant proportion of the arrested cells was synchronized in the mitotic cycle. In addition to DNA synthesis, the inhibitory action of the SGP was monitored by direct measurement of cell number. Consistent with the thymidine incorporation data, the SGP completely inhibited 3T3 cell division 20 h after its addition to exponentially growing cultures. Upon reversal there was a delay of 15 h before cell division resumed, when the arrested cells quickly doubled. Most, if not all, of the growth‐arrested cells appeared to have been synchronized by the SGP. The SGP inhibited DNA synthesis in a surprisingly wide variety of target cells, and the relative degree of their sensitivity to the inhibitor was remarkably similar. Cells sensitive to the SGP ranged from vertebrate to invertebrate cells, fibroblast and epitheliallike cells, primary cells and established cell cultures, as well as a wide range of transformed cell

ISSN:0021-9541    

DOI:10.1002/jcp.1041390208

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

年代:1989

数据来源: WILEY

摘要:

AbstractMicrovascular endothelial cells (MEC) must use a set of surface receptors to adhere not only to the vascular basement membrane but, during angiogenic stimulation, to the interstitium. We examined how cultured MEC Isolated from human foreskin interact with their subendothelial matrix. MEC were able to attach to diverse extracellular matrix proteins, including fibronectin (Fn), vitronectin (Vn), laminin (Ln), type I and IV collagen, as well as to fibrinogen and gelatin. Adhesion to Fn, but not to laminin or collagens, was specifically blocked in the presence of Arg‐Gly‐Asp (RGD)‐containing peptides. When surface radioiodinated MEC were solubilized and subjected to affinity chromatography on Fn‐Sepharose columns, two polypeptides of 150 and 125 kD, corresponding to the integrin heterodimer α5β1, were identified. MEC also express a complex of 150 (α) and 95 kD (β3) that is related to the Vn receptor. Immunofluorescent staining of MEC cultures with antibodies to the integrin β1subunit demonstrated receptors on the basolateral surface at focal adhesion plaques that co‐localized with vinculin and with Fn‐positive matrix fibers. Occasionally, antibodies to the Vn receptor stained the vinculin‐positive focaladhesion plaques that frequently co‐localized with the β1complex. However, in cultures of MEC that were attached to substrates coated with alternating strips of Fn and Vn, the β1complex was preferentially localized to the Fn substrate, while the Vn receptor was concentrated on the Vn substrate. The results indicate that MEC express at least two different heterodimer adhesion receptors that belong to the integrin superfamily and appear to have distinct ligand specificities: the Fn receptor and the Vn receptor. These receptors mediate cell adhesion to the extracellular matrix and presumably have an important role in hemostasis

ISSN:0021-9541    

DOI:10.1002/jcp.1041390209

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe effects of cell surface heparan sulfate proteoglycan (HSPG) prepared from log and confluent monolayers of a rat hepatoma cell line on hepatoma cell growth were studied. When HSPG isolated from confluent cells was added exogenously to log phase cells, it was internalized and free heparan sulfate (HS) chains appeared transiently in the nucleus. Concurrently, the growth of the treated cells was inhibited, but the cells resumed logarithmic growth as the level of nuclear HS fell, and the cells grew to confluence and became contact inhibited. When HSPG prepared from log‐phase hepatoma cells was added exogenously to log phase cells, it was internalized but very little of the internalized HS appeared in the nucleus, and there was no change in the rate of cell growth. However, when the rate of cell growth was reduced by culture of the cells in serum‐ and insulin‐deficient medium, HSPG prepared from log‐phase cells stimulated the growth rate of these slow‐growing cells. The cell cycle dependency of HSPG uptake and growth inhibition was studied in cultures synchronized by a thymidine/aphidicolin double block. When [35SO4]HSPG from confluent cells was added to synchronized cells just as they were released from the second block, a portion of the [35SO4]HSPG was internalized and [35SO4]HS appeared in the nucleus. However, at mitosis the [35SO4]HS disappeared almost completely from all of the cellular pools, and after mitosis, more of the [35SO4]HSPG was taken up and [35SO4]HS reappeared in the nucleus and remained in the nucleus until the cells divided again. When cultures were released from the aphidicolin block, both control and HSPG‐treated cells progressed through the S, the G2, and the M phases of the cell cycle. However, the length of the G1phase of the cycle was increased in the HSPG‐treated cells. The treated cultures then progressed through the second S, G2,and M phases. Thus, the inhibition of cell division occurred in the G1,phase of the cell cycle, prior to the G1,/S boundary. Addition of the HSPG to the synchronized cultures just after the first mitosis resulted in an immediate arrest of the cell cycle in G1,These results support the earlier suggestion (M. Ishihara, N.S. Fedarko, and H.E. Conrad 1987 J. Biol. Chem. 262:4708–4716; M. Ishihara and H.E. Conrad (1989) J. Cell Physiol., in press) that the HSPG formed by confluent hepatocytes plays a role in the prevention of cell division, whereas the HSPG formed by exponentially growing cells plays a role in the stimulation of cell division. The inhibition of cell growth results from a block in the G1,phase of the cell cycle prior to the

ISSN:0021-9541    

DOI:10.1002/jcp.1041390210

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

年代:1989

数据来源: WILEY

摘要:

AbstractA significant interindividual variation in the growth rates is found in normal cultured human mesothelial (NHM) cells derived from different donors. This variation is observed when the mesothelial cells are incubated in medium containing serum and when the potencies of several separate growth factors are measured by using defined media. Depending on the donor, gamma‐interferon and interleukin‐2 can be toxic, have no effect, or stimulate the growth rate of NHM cells. Cultured NHM cells can be induced to multiply by growth factors that are released by activated macrophages. Thus, interindividual variation in NHM cell growth control could play a role in the pathogenesis of mesothelioma for a person exposed to asbes

ISSN:0021-9541    

DOI:10.1002/jcp.1041390211

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

年代:1989

数据来源: WILEY