摘要:

AbstractMarrow stroma has been shown to have osteogenic potential. Here we report the characterization of a unique stromal cell line derived from mouse bone marrow (MBA‐15), which expresses osteoblastic phenotype in vitro and forms bone in vivo. More than 70% of cells in culture were histochemically positive for alkaline phosphatase. The enzyme levels were enhanced threefold when cultures were treated with dexamethasone. Gel electrophoresis of [3H]‐proline‐labeled cultures showed that MBA‐15 cells produced only type I collagen. These cells were responsive to PTH, as indicated by a 50‐fold increase in intracellular cAMP. Prostaglandin E2, but not calcitonin, stimulated cAMP up to 70‐fold. When cultures were grown to confluence and fed daily with ascorbic acid and β‐glycerophosphate, the cells formed a Von Kossa positive, thick extracellular matrix, shown to contain hydroxyapatite crystals. MBA‐15 cells produced mineralized bone when implanted in diffusion chambers. These results indicate that the MBA‐15 cell line possesses osteoblastic features in vitro and osteogeni

ISSN:0021-9541    

DOI:10.1002/jcp.1041400102

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

年代:1989

数据来源: WILEY

摘要:

AbstractWe examined the effects of dexamethasone on creatine kinase (CK) activity and insulin‐like growth factor I (IGF‐I) binding in two skeletal muscle‐derived cell lines (mouse, C2C12; rat, L6) and in one cardiac muscle‐derived cell line (rat, H9c2). Dexamethasone treatment during differentiation of cultured cells caused a dose‐dependent increase in CK activity as well as an increase in the degree of myotube formation in C2C12 and L6, whereas H9c2 cells did not exhibit significant CK activities during culture or dexamethasone treatment. Dexamethasone treatment of C2C12 did not stimulate proliferation in differentiating cultures, but a dose‐dependent increase in the number of nuclei was observed for L6 concomitant with increased CK activity. In L6 the increased CK activity may therefore reflect a dose‐dependent increase in proliferation. Short‐term (48 hr) treatment of C2C12 with dexamethasone (20 nM) did not appear to alter myoblast fusion but reversibly increased CK activity. In C2C12 the observed increase in CK, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities with dexamethasone treatment suggest modulation of protein expression and/or turnover. Although the data for dexamethasone effects on CK activities varied in each of the cell lines, consistent behavior was observed in all three cell lines when IGF‐I binding was examined. IGF‐I binding to dexamethasone‐treated cells (50 nM for 24 hr the day prior to confluence) resulted in an increased number of available binding sites, with no effect on the binding affinities. Affinity cross linking and autoradiography indicated that the increase in IGF‐I binding was the result of dexamethasone up‐regulation of type I IGF receptors. Our data for all three muscle cell lines suggest that similar heterologous hormone receptor modulation of type I IGF receptor sites occurs wit

ISSN:0021-9541    

DOI:10.1002/jcp.1041400103

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

年代:1989

数据来源: WILEY

摘要:

AbstractA novel effect of carnitine and O‐acylcarnitine derivatives has been described. The presence of these compounds has been shown to inhibit the aggregation of erythrocytes otherwise elicited by the addition of clusterin or fetuin. The specificity of carnitine action has been investigated by comparing influences of chemically related compounds. The concentrations required for inhibition by approximately 50% of aggregation of erythrocytes by clusterin under in vitro conditions defined were determined to be 1.5 mM for L(‐) or D(+) enantiomers of carnitine; 0.5 mM for decanoyl(−)‐ or (+)‐carnitine; 0.13 mM for lauroyl(−) ‐or (+)‐carnitine, and 0.05 mM for myristoyl(−)‐ or (+)‐carnitine. In contrast, concentrations up to 12.5 mM of dimethylcarnitine, deoxycarnitine, acetylcho‐line, acetyl‐β‐methylcholine, or inositol had no detectable inhibitory effect on aggregation elicited by clusterin. Clusterin addition also resulted in the aggregation of three other cell types examined (guinea pig spermatozoa, a cell line derived from testes of neonatal mice called TM4 cells, and Sertoli cells from testes of 20 day‐old rats). As in the case with erythrocytes, the presence of carnitine inhibited aggregation of spermatozoa, TM4 cells, and Sertoli cells in suspension. We consider possible mechanisms by which carnitine inhibits aggregation of erythrocytes and other populations of dispersed cells incubate

ISSN:0021-9541    

DOI:10.1002/jcp.1041400104

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe cellular basis of the membrane‐limited state of glucose utilization and the mechanism of the endogenous regulation of hexose uptake in dense monolayers of C6 glioma cells were investigated. In an earlier study, it was shown that at high rates of glucose transport and phosphorylation combined with the inhibition of glycolytic adenosine triphosphate (ATP) production by iodoacetate, an endogenous regulatory response occurred that resulted in rapid, periodic variations of the glucose uptake rates (Lange et al., 1982). Similar time‐dependent periodic changes of uptake rates also occurred during incubation of C6 glioma cells with 2 mM 2‐deoxyglucose (2‐DG) without pretreatment of the cells with iodoacetate. These changes were accompanied by variations of the intracellular ATP content, by distinct alterations of the shape and arrangement of microvilli and lamellae (lamellipodia) on the cell surface, and by changes of the cytoskeletal F‐actin content. Because the changes of 2‐DG uptake rates occurred independent of the intracellular 2‐DG concentration, the bulk of this 2‐DG pool was assumed to be localized apart from the membranal transport sites. Downregulation of 2‐DG uptake appeared to be triggered by a rapid decrease of a small pool of the cellular ATP involved in the phosphorylation of transported hexose. Scanning and transmission electron microscopic observations of cells fixed in different states of the endogenous uptake regulation supported the assumption that the interior of lamellae and microvilli may represent a small entrance compartment for transported hexoses in which occurred the observed close coupling between hexose transport and phosphorylation as well as the rapid variations of ATP content. Hexose uptake is supposed to be regulated by cytoskeleton‐mediated changes of volume and diffusional accessibility of this compartment, modulating the degree of its metabolic coupling with the cytoplasm

ISSN:0021-9541    

DOI:10.1002/jcp.1041400105

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe KC gene is a cell cycle‐dependent competence gene originally identified in platelet‐derived growth factor‐stimulated BALB/c‐3T3 cells. This gene is also induced in murine peritoneal macrophages in response to activation stimuli. We have examined the expression of the KC gene in cultured porcine aortic endothelial cells following treatment with bacterial lipopolysaccharide (LPS) as a first step in defining the early molecular events involved in endothelial cell stimulation by physiologically relevant modulators. LPS markedly elevated the steady‐state level of KC mRNA in confluent endothelial cells; maximum induction of KC occurred in the cells following exposure to 10 ng/ml LPS for 2 h. LPS did not increase the growth fraction of the cells, nor was the KC mRNA level changed in dense endothelial cells stimulated to enter the cell cycle with epidermal growth factor. However, KC mRNA expression was elevated by addition of serum to starved, subconfluent endothelial cell cultures. Treatment of endothelial cells with phorbol myristate acetate (PMA) and 1‐oleoyl‐2‐acetyl‐glycerol (OAG) also induced KC gene expression. A maximum response was obtained with 10 nM PMA, the effect decreasing with higher levels of the phorbol ester. The calcium ionophore A23187 exhibited little stimulatory activity alone; however, the ionophore did cause a doubling in the PMA‐stimulated KC expression. The increased expression of KC induced by LPS and PMA was inhibited by the presence of 1‐(5‐isoquinoline‐sulfonyl)‐2‐methylpiperazine (H7), a protein kinase C inhibitor, but not by HA1004 (an H7 analogue with little protein kinase C inhibitory activity). No cytotoxicity was observed in inhibitor or LPS‐treated endothelial cell cultures. These results demonstrate that KC gene expression is stimulated by LPS in vascular endothelial cells in a proliferation‐independent process. Second, unlike LPS‐induced KC expression in macrophages and platelet‐derived growth factor‐induced KC expression in 3T3 cells, LPS induction of KC in endothelial cells appears to

ISSN:0021-9541    

DOI:10.1002/jcp.1041400106

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

年代:1989

数据来源: WILEY

摘要:

AbstractTo begin defining the factors regulating neurotransmitter receptor expression, we examined beta‐adrenergic receptors in rat liver in vivo and in primary liver cultures under defined hormonal conditions. β‐receptors described a remarkable developmental profile in vivo, increasing fivefold between embryonic days 16 and 20, and decreasing tenfold by early adulthood. The developmental decrease reflected reduced receptor number without a change in receptor properties. The ontogenetic decrease appeared to be specific for β‐receptors α‐receptors developed in a hyperbolic fashion, reaching high plateau values by the third postnatal week.Adult rat liver cells plated into culture re‐expressed high β‐receptor levels, exhibiting a 4‐8‐fold increase. A similar pattern of expression of the β‐receptors, having similar pharmacological properties, was observed in primary liver cultures maintained in serum‐free medium, in a serum‐supplemented medium or in several variations of a serum‐free, hormonally defined medium designed for primary liver cultures. Thus, the degree of expression of the β‐receptors was not found affected by various hormones, by serum, or by any medium condition. By contrast, the degree of expression of the β‐receptors was markedly sensitive to cell density. High expression of the β‐receptors was observed at low cell densities (1‐3 × 106cells/150 mm dish), and low expression or no expression was observed in confluent cultures (10‐20 × 106cells/150 mm dish). Our experiments suggest that β‐receptor expression does not follow an immutable program, but may be regulat

ISSN:0021-9541    

DOI:10.1002/jcp.1041400107

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe growth regulation of human diploid fibroblasts by platelet‐derived growth factor (PDGF), epidermal growth factor (EGF), insulin‐like growth factor I (IGF‐I) (somatomedin C), dexamethasone, and transferrin was investigated in a serumfree, chemically defined culture system. Cell‐cycle kinetic parameters were determined using 5′‐bromodeoxyuridine (BrdU) incorporation and flow cytometric analysis with the DNA‐specific dye Hoechst 33258. We found that PDGF and EGF regulate the proportion of cells capable of entering the cell cycle from the quiescent state, with smaller effects upon the rate of cell transition from G1into S phase. IGF‐I, on the other hand, regulates the rate of cell exit from G1without affecting the cycling fraction. Transferrin and dexamethasone showed less effect upon the cell‐cycle kinetics under these culture conditions. The data provide functional evidence that PDGF and EGF regulate similar cell‐kinetic parameters in human fibroblast cultures. IGF‐I is functionally distinct from both PDGF and EGF in its role of regulating G1exit rate without affecting the cycling fraction. These observations made by BrdU‐Hoechst flow cytometric techniques provide a novel perspective on the regulatory effects exerted by different classes of growth factors, and suggest a mode of interdependence of these mitogens in regulating the net growth rate which could be a feature of growth regulation in vivo. These data also provide a different perspective on the regulation of the growth of fibroblast‐like cells than that of the “competence/pr

ISSN:0021-9541    

DOI:10.1002/jcp.1041400108

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

年代:1989

数据来源: WILEY

摘要:

AbstractBasic fibroblast growth factor (bFGF) binds to heparin‐like molecules present in the extracellular matrix (ECM) of transformed fetal bovine aortic endothelial GM 7373 cells. Binding of bFGF to ECM can be competed by heparin or heparan sulfate, and ECM‐bound bFGF can be released by treating the cells with heparinase or heparatinase. After binding to ECM, bFGF is slowly released into the medium in a biologically active form, as shown by its capacity to induce an increase of cell‐associated plasminogen activator activity and cell proliferation. The increase is prevented upon removal of ECM‐bound bFGF by a neutral 2 M NaCI wash. Soluble heparin and heparan sulfate reduce the amount of ECM‐bound bFGF released into the medium, possibly competing with ECM polysaccharides for heparinase‐like enzymes produced by endothelial cells, suggesting that these enzymes are involved in the mobilization of ECM

ISSN:0021-9541    

DOI:10.1002/jcp.1041400109

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

年代:1989

数据来源: WILEY

摘要:

AbstractWhen bovine capillary endothelial (BCE) cells were treated with 10 ng/ml of basic fibroblast growth factor (bFGF) for 10 or 30 minutes at 37°C, washed extensively with phosphate‐buffered saline (PBS) and incubated in bFGF‐free medium, plasminogen activator (PA) production was stimulated to the same extent as in cells exposed continuously to bFGF. Three methods of removing bFGF from heparin‐like binding sites in the extracellular matrix, but not from bFGF receptors, abolished this long‐term effect of a brief exposure to bFGF. First, BCE cells exposed to bFGF for 30 minutes were washed with 2M NaCI and incubated in bFGF‐free medium. Second, BCE cells were incubated with bFGF for 10 minutes in the presence of heparin, and cells were washed with PBS and incubated in bFGF‐free medium. Third, BCE cell cultures were treated with heparinase and exposed to bFGF. Each of these treatments abolished the long‐term (24‐48 hours) stimulation of PA production normally observed after brief exposure to bFGF. In each of these experiments, incubation of cells in bFGF‐containing medium after the treatments resulted in normal stimulation of PA production, demonstrating that the treatments did not harm the cells. Stimulation of DNA synthesis was observed when cells were exposed to bFGF for 2 hours at 4°C, incubated in bFGF‐free medium for 24 hours at 37°C, and assayed for3H‐thymidine incorporation. However, no stimulation was observed if the 2 hours incubation at 4°C was carried out in the presence of heparin. Thus, long‐term stimulation of PA activity and DNA synthesis after a brief exposure to bFGF seems to be a consequence of bFGF binding to the extracellular matrix. The extracellular matrixmay act as a physiologic buffer, binding bFGF when concentrations are high and releasing it later for interaction with its receptor. This interaction with matrix may be required for

ISSN:0021-9541    

DOI:10.1002/jcp.1041400110

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe ability of mouse thymocytes and peripheral blood lymphocytes from rats to synthesize and secrete proteoglycans in the presence of a variety of mitogens and lymphokines was studied in vitro, and it was confirmed that such lymphocytes synthesize and secrete significant quantities of proteoglycans. Mitogenic stimulation of the cells with phytohaemagglutanin (PHA) induced a fourfold increase in proteoglycan synthesis; stimulation with interleukin‐1 stimulated proteoglycan synthesis up to fivefold. Proteoglycan synthesis could also be stimulated by culturing the cells in the presence of interleukin‐2. To determine if this response was related to cell proliferation, the cells were cultured in the presence of PHA and either cyclosporine or prostaglandin E2two agents that inhibit lymphocyte proliferation. Under these conditions, proteoglycan synthesis remained elevated, indicating that this effect may be independent of cell proliferation. Chemical analysis of the proteoglycans indicated them to be composed of chondroitin sulfate and heparan sulfate. Their molecular size was small compared with cartilage proteoglycans but similar to the small dermatan sulfate proteoglycans synthesized by fibroblasts. On the basis of molecular size, three proteoglycan populations were identified, and their relative proportions were altered by mitogenic stimulation of the cells. Taken together, these findings imply that proteoglycan synthesis is intimately associated with lymphocyte activation and may be related to cellular function in immune respon

ISSN:0021-9541    

DOI:10.1002/jcp.1041400111

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

年代:1989

数据来源: WILEY