摘要:

AbstractDuring the development of atherosclerotic and fibromuscular proliferates/lesions, smooth muscle cells (SMC in the media, particularly near the lumen, are activated to migrate into the intima, where they continue to proliferate to form an intimal thickening. It is to date unclear whether SMCs situated adjacent to the adventitia possess a lower capacity to proliferate because they are a special subpopulation of medial SMCs or because the adventitia excerts an inhibitory effect. We have, therefore, developed an in vitro system whereby we have attempted to clear up this uncertainty. The following observations were made from the in vitro experiments:Media‐explants from rabbit aorta were laid on a polycarbonate filter with pores 5 μm in diameter. The SMCs migrated through the pores and formed a fibromuscular proliferate on the other side of the filter.Endothelial cells were seeded on one side of the filter before media‐explants were laid on the other side of the filter. The confluent endothelium inhibited migration of SMCs through the filter pores.Media‐explants were placed between two polycarbonate filters (pores 5 μm diameter). In this “sandwich” arrangement SMCs migrated through both filters, i.e., in both directions. The quantity of migrating and proliferating cells through both filters was almost identical. This suggests that there is no difference in the migratory and proliferate capacity of SMCs in the inner and outer layers in the media of arteries.To investigate the influence of the adventitia on medial SMCs, media‐explants were placed between a lower (5 μm) and an upper (0.2 μm) filter adventitia‐explants were laid above the media‐explants. The 0.2 μm filter prevented migration of SMCs from the media‐explant into the adventitia and migration of fibroblasts from the adventitia into the media. Interestingly, the adventitial tissue inhibited proliferation of SMCs at the abluminal and migration and proliferation at the luminal side of the media‐explant; the number of cells migrating through the 5 μm pores at the luminal side was diminished, suggesting that the adventitial tissue has an antiproliferative influence on SMCs. Moreover, it was found that in media‐explants near the filter with adventitia, the medial SMCs were in a better preserved condition than at the de‐endothelialised luminal side.As a control, cultures consisting of media‐explants were incubated without filters (i.e., explant organ cultures). The proliferates in the concavity (luminal side) exhibited a pattern of proliferating SMCs different from that of the cells at the abluminal convexity. This finding indicates that the different texture of vessel walls near the adventitia and near the lumen may also influence c

ISSN:0021-9541    

DOI:10.1002/jcp.1041470302

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

年代:1991

数据来源: WILEY

摘要:

AbstractWe have studied the ability of bFGF to traverse and be trapped within basement membranes. An extract of EHS tumor (matrigel) coated on culture chamber filters was used as an in vitro model of basement membranes. Our results showed a slow diffusion of bPGF dependent on the amount of low affinity binding sites present within the matrigel. High amounts 01 bFGF and heparin increased the initial rate of diffusion by displacement of bFGF bound to matrigel. An in vitro corneal endothelium model (endothelial cells overlying matrigel) was also developed. This monolayer, with a weak permeability, decreased the kinetic rate of bFGF diffusion compared with matrigel alone. These results indicate that modulation of bFGF distribution in a tissue by a basement membrane is dependent on bFGF concentration, basement membrane composition, permeability of associated cells, and local presence of heparin. This selective control may be a regulating step in bFGF action.

ISSN:0021-9541    

DOI:10.1002/jcp.1041470303

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

年代:1991

数据来源: WILEY

摘要:

AbstractIn the present study, we have examined the effects of a putative antagonist of prostaglandin E2(PGE2), AH6809, on chondrogenesis in serum‐free cultures of mesenchyme from distal tips of stage 25 chick limb buds in order to test the hypothesis that endogenous PGE2, through receptor‐linked adenylate cyclase (AC), initiates differentiation of cartilage in limb mesenchyme. Daily addition of 10−4M concentrations of AH6809 produced marked inhibition of chondrogen‐esis over a 5‐day period of cell culture as evaluated by Alcian green binding to cartilage matrix components. Inhibition of chondrogenesis by this compound was further shown to be reversible and treatment of cells with the antagonist limited to periods when chondrocytes had differentiated and were actively secreting cartilage‐specific matrix components had little effect. Preincubation of control cells in 1094 M concentrations of AH6809 inhibited PGE induced activation of AC by greater than 80% without significant (P>.05) inhibition of basal activity by the antagonist. Responses to parathyroid hormone, which increased AC activity by 7‐fold, and forskolin which increased AC activity by 23‐fold in control cells, were also uninhibited by preincubation in AH6809. The results demonstrate that blockade of PGE2‐AC linked receptors in prechondrogenic limb mesenchyme inhibits chondrogenesis supporting the hypothesis that endogenous PGE2concentrations in undifferertiated limb mesenchyme play aninitiating role in the differentia

ISSN:0021-9541    

DOI:10.1002/jcp.1041470304

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

年代:1991

数据来源: WILEY

摘要:

AbstractOncoprotein c‐myc is expressed in proliferating but not quiescent mammalian cells, and its overexpression or inappropriate expression is associated with malignant transformation. However, in spite of an intense interest, the normal function of this protein has remained elusive. As a step towards the elucidation of the function of c‐myc protein, we studied its distribution within several types of cells, including HL 60, K 562, COLO 320, and CHEF/18 cells. In all of the cells studied, c‐myc protein was detected in high molecular weight protein fractions, in 350–600 Kd range, in gel‐exclusion chromatography and sucrose gradient centrifugation. This distribution of c‐myc protein coincided with the distribution of DNA polymerase α and several other enzymes necessary for DNA replication. The data suggest that c‐myc product may be a component of the replitase complex of enzymes involved in nuclear D

ISSN:0021-9541    

DOI:10.1002/jcp.1041470305

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

年代:1991

数据来源: WILEY

摘要:

AbstractPlatelet‐derived growth factor (PDGF) exists as a homodimer or a heterodimer comprising either PDGF‐A or PDGF‐B subunits, and each isoform occurs in various tissues, including bone. Although the stimulatory effects of PDGF‐BB have been studied in cultures of bone cells and intact bone fragments, the influence of other isoforms that may arise locally or systemically in vivo, has not been reported. Therefore recombinant human PDGF‐BB, PDGF‐AB, and PDGF‐AA were evaluated in osteoblast‐enriched cultures from fetal rat bone. Within 24 hours these factors produced a graded response in bone cell DNA and protein synthesis, with half‐maximal effects at approximately 0.6, 2.1, and 4.8 nM PDGF‐BB, PDGF‐AB, and PDGF‐AA, respectively. Increases in collagen and noncollagen protein synthesis were abrogated when DNA synthesis was blocked with hydroxyurea. Furthermore, each factor reduced alkaline phosphatase activity, PDGF‐BB being the most inhibitory. Binding studies with125I‐PDGF‐BB or125I‐PDGF‐AA and each unlabeled PDGF isoform produced discrete ligand binding and displacement patterns:125I‐PDGF‐BB binding was preferentially displaced by PDGF‐BB (Ki ≈0.7 nM), less by PDGF‐AB (Ki ≈2.3 nM) and poorly by PDGF‐AA. In contrast,125I‐PDGF‐AA binding was measurably reduced by PDGF‐AA (Ki ≈4.0 nM), but was more effectively displaced by PDGF‐BB or PDGF‐AB (each with Ki ≈0.7 nM). These studies indicate that each PDGF isoform produces biochemical effects proportional to binding site occupancy and suggest that receptors that favor PDGF‐B subunit binding preferentially

ISSN:0021-9541    

DOI:10.1002/jcp.1041470306

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

年代:1991

数据来源: WILEY

摘要:

AbstractReplacement of media in cell cultures during exposure to hyperoxia was found to alter oxygen toxicity. Following 100 hr of exposure to 95% or 80% O2, the surviving fraction (SF) of Chinese hamster fibroblasts, as assayed by clonogenicity, was less than 1 × 10−3when the culture media was replaced only at the onset of the O2exposure. Media replacement every 24 hr throughout the hyperoxic exposure resulted in SFs of 1.7 × 10−1(95% O2) and 1.9 × 10−1(80% O2) at 95 hr. Cellular resistance to and metabolism of 4‐hydroxy‐2‐nonenal (4HNE), a cytotoxic byproduct of lipid peroxidation, was examined in cells 24 hr following exposure to 80% O2for 144 hr with media replacement. These O2‐exposed cells were resistant to 4HNE, requiring 2.6 times as long in 80 μM 4HNE to reach 30% survival as compared to density‐matched normoxia control. Furthermore, during 40 and 60 min of exposure to 4HNE, the O2‐preexposed cells metabolized greater quantities of 4HNE (fmole/cell) relative to control. The activity of glutathione S‐transferase (GST), an enzyme believed to be involved with the detoxification of 4HNE, was significantly increased in the O2‐preexposed cells compared with controls. Catalase activity was significantly increased, but no change was found in total glutathione content, glutathione peroxidase, manganese superoxide dismutase, and copper‐zinc superoxide dismutase activities at the time of 4HNE treatment in the O2‐preexposed cells relative to density‐matched control. The results demonstrate that in vitro tolerance to the cytotoxic effects of hyperoxia can be achieved through media replacement during O2exposure. Tolerance to oxygen toxicity conferred resistance to the cytotoxic effects of 4HNE, possibly through GST‐catalyzed detoxification. These results provide further support for the hypothesis that toxic aldehydic byproducts of lipid peroxidati

ISSN:0021-9541    

DOI:10.1002/jcp.1041470307

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

年代:1991

数据来源: WILEY

摘要:

AbstractThe molecular basis for increased metallothionein concentrations in copperresistant hepatoma cells was examined. The copper‐resistant cell line HAC600, which is maintained in 600 μm copper, had increased steady‐state mRNA levels for both the metallothionein‐1 (MT‐1) and the metallothionein‐2 (MT‐2) genes. Levels of mRNA were increased 11‐fold for MT‐1 and 15‐fold for MT‐2, with no significant change in α‐tubulin mRNA content. HAC600NM cells, which are copper‐resistant cells kept in a normal copper concentration for over 1 year, also had eight‐ and tenfold increases in MT‐1 and MT‐2 mRNA levels. Nuclear run‐on assays showed that MT‐1 and MT‐2 gene transcription was increased nine‐ and eightfold in HAC600cells and seven‐ and tenfold in HAC600NM cells, respectively. Southern blot analysis showed amplification of both metallothionein genes in HAC600and HAC600NM cells. Thus the molecular basis of increased metallothionein in these hepatoma cells involved a stable gene amplification of both MT genes. The greater increase in metallothionein mRNA levels in HAC600cells relative to the changes in transcription suggests that posttranscriptional mechanisms of gene reg

ISSN:0021-9541    

DOI:10.1002/jcp.1041470308

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

年代:1991

数据来源: WILEY

摘要:

AbstractIn intact reticulocytes, but not in fragmented membranes, the loss of adenylate cyclase activity during cell maturation followed a biphasic time course. A rapid phase (t½≈ 2 h) during which the initial activity was reduced by 40–50% was followed by a slow phase with t½close to 3 days. The fast decay seemed to occur on the adenylate cyclase level since (‐)isoprenaline‐ or forskolin‐stimulated activities behaved similarly and bacterial toxin‐monitored Gsand Giproteins remained stable. The mechanism of the initial decrease in hormonal responsive ness was further analysed in hybrid cells prepared by fusing reticulocytes with Friend erythroleukemia (MEL) cells. The hybrids contained reticulocyte‐derived β‐adrenoceptors and MEL cell‐derived adenylate cyclase and G proteins. Fusion of reticulocytes to native MEL cells caused adenylate cyclase activity to drop by 30% at 2 h and 45% at 18 h after fusion. By contrast, hybrids prepared after dimethylsulfoxide‐induced differentiation of MEL cells showed stable or increasing rates of receptor‐coupled cAMP formation between 2 and 18 h after fusion, concomitant with the enhanced activity of the Gsprotein in these cells. A cyclase‐stimulating factor present in the cytosol of MEL cells and of reticulocytes appeared not to be involved in short‐term regulation of hormonal responsiveness. We conclude that the strength of β‐adrenergic responses in erythroid progenitor cells is primarily regulated by modulating G protein‐mediated receptor cyclase coupling while reticulocytes, during early maturation, seem to rely on direct inactivation of adenylate cyclase, probably vi

ISSN:0021-9541    

DOI:10.1002/jcp.1041470309

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

年代:1991

数据来源: WILEY

摘要:

AbstractTaurine (2‐aminoethanesulfonic acid) is a unique sulfur amino acid derivative that has putative nutritional, osmoregulatory, and neuroregulatory roles and is highly concentrated within a variety of cells. The permeability of Percoll density gradient purified rat liver lysosomes to taurine was examined. Intralysosomal amino acid analysis showed trace levels of taurine compared to most other amino acids. Taurine uptake was Na+‐independent, with an overshoot between 5–10 minutes. Trichloroacetic acid extraction studies and detergent lysis confirmed that free taurine accumulated in the lysosomal space. Kinetic studies revealed heterogeneous uptake with values for Km1= 31 ± 1.82 and Km2>198 ± 10.2 mM. The uptake had a pH optimal of 6.5 and was stimulated by the potassium specific ionophore valinomycin. The exodus rate was fairly rapid, with a t1/2 of 5 minutes at 37°C. Analog inhibition studies indicated substrate specificity similar to the plasma membrane β‐alanine carrier system, with inhibition by β‐alanine, hypotaurine, and taurine. α‐Alanine, 2‐methylaminoisobutyric acid (MeAIB), and threonine were poor inhibitors. No effects were observed with sucrose and the photoaffinity derivative of taurine NAP‐taurine [N‐(4‐azido‐2‐nitrophenyl)‐2‐aminoethanesulfonate]. In summary, rat liver lysosomes possess a high Km system for taurine transport that is sensitive to changes in K+gradient and perhaps valinomycin induced diffusional membrane potential. These features may enable lysosomes to adapt to changing intracellular concentrations of

ISSN:0021-9541    

DOI:10.1002/jcp.1041470310

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

年代:1991

数据来源: WILEY

摘要:

AbstractThe synthesis of metabolically labeled proteoglycans and glycosaminoglycans from medium, cell layer and substrate attached material by rat glomerular mesangial cells in culture was characterized. The cellular localization of the labeled proteoglycans and glycosaminoglycans was determined by treating the cells with Flavobacterial heparinase. Of the total sulfated glycosaminoglycans, 33% were heparan sulfate; 55% of the cell layer material was heparan sulfate; 80% of sulfated proteins in the medium were chondroitin sulfate/dermatan sulfate. Putative glycosaminoglycan free chains of heparan sulfate and chondroitin sulfate were found in both the medium and cell layer; 95% of total proteoglycans and most (90%) of the putative heparan sulfate free chains were removed from the cell layer by the heparinase, whereas only 50% of the chondroitin sulfate and 25% of dermatan sulfate were removed. Large amounts of hyaluronic acid labeled with3H glucosamine were found in the cell layer. In summary, approximately 60% of total sulfated glycoproteins was in the form of putative glycosaminoglycan free chains. Thus rat mesangial cells may synthesize large amounts of putative glycosaminoglycan free chains, which may have biological functions in the glomerulus independent of proteoglycans.

ISSN:0021-9541    

DOI:10.1002/jcp.1041470311

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

年代:1991

数据来源: WILEY