摘要:

AbstractThe addition of anti‐laminin IgG to the basal surfaces of rat Sertoli cells in culture in a two‐chambered assembly results in a perturbation of F‐actin arrangements, including disruption of the pericellular circumferal rings, impairments of the Sertoli cell permeability barrier, and subsequently focal defoliation, followed by cell reaggregation. The pentapeptide YIGSR, which competes with the laminin receptor for laminin (Kleinman and Weeks: Curr. Oph. Cell Biol., 1:964–967, 1989; Graf et al.: Biochemistry, 26:6896–6900, 1987) also elicited focal defoliation of Sertoli cells from the extracellular matrix‐coated filter in the two‐chambered assembly. Addition of YIGSR to Sertoli cell cultures resulted in cell detachment within 2 to 3 h. In contrast, the irrelevant peptide YIGSE had no detectable effects. The anti‐laminin IgG was effective only when added to the chamber in which access was readily available to the basal surfaces of Sertoli cells, but YIGSR was effective when added either to the outer chamber or to the inner chamber. These data were interpreted to indicate that the Sertoli cell barrier generated in the two‐chambered assembly allowed a relatively rapid diffusion of YIGSR between chambers, but prevented the rapid equilibration of anti‐laminin IgG between compartments. Addition of anti‐laminin IgG to the basal, but not to the apical surfaces of Sertoli cells, resulted in more rapid rates of equilibration of [3H]‐methoxyinulin and [86Rb]Cl across the Sertoli cell monolayer. This evidence of impairment to the integrity of the barrier was detected prior to the disruption of stress fibers and focal defoliation, but after evidence of dissolution of the circumferal F‐actin ring, which occurred within 1 h after addition of anti‐laminin IgG. We consider the possibility that a transmembrane link exists between extracellular laminin and cytoskeletal elements which modulates the circumferal F‐actin ring. We further postulate that this linkage can influence the nature of tight junctional complexes, and thereby the integrity of the Sertoli cell ba

ISSN:0021-9541    

DOI:10.1002/jcp.1041560102

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe development of tetrahydrobiopterin synthesis during lectin stimulation of resting human T lymphocytes (Kerler et al. [1989] FEBS Lett.,250:622–624), the interferon‐γ induced neopterin production by human monocytes/macrophages (Huber et al. [1984] J. Exp. Med.,160:310–16), and the control of tetrahydrobiopterin synthesis in activated T cells by the synergistic action of interferon‐γ and interleukin 2 (Ziegler et al. [1990] J. Biol. Chem.,265:17026–17030) were previously explained by modulation of the apparent GTP‐cyclohydrolase I activation. In this study we demonstrate that increases in GTP‐cyclohydrolase I activity which occur after lectin induction and after cytokine treatment correlate with increased steady state mRNA levels specific for this enzyme. The enhancement of interferon‐γ induced enzyme activity in primed T cells by interleukin 2 also corresponds to further increases in mRNA expression. The steady state GTP‐cyclohydrolase I mRNA levels in primed T cells, however, do not correlate with the steep decline which follows the culmination of enzyme activity 44 hours after treatment. This indicates that the down‐regulation of apparent GTP‐cyclohydrolase I activity is caused by posttranslational modification of the protein

ISSN:0021-9541    

DOI:10.1002/jcp.1041560103

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe effects of soluble chondroitin sulfate proteoglycans (CSPGs) purified from the rat brain on proliferation of and neurite outgrowth from PC12D cells (Katoh‐Semba et al., J Neurosci Res 17:36, 1987) were investigated. When PC12D cells are cultured under standard conditions, they proliferate with a doubling time of about 2 days, irrespective of the presence or absence of NGF. However, the addition of a mixture of several types of purified soluble brain CSPG (50 nmol uronic acid/ml) to the culture medium prevented the increase in the number of PC12D cells as well as the nerve growth factor (NGF)‐induced neurite extension. The dose for 50% inhibition (ID50) was 1.6 nmol/ml for cell proliferation and 2.7 nmol/ml for neurite elongation. The increase in cell number seemed to stop around 6 h after exposure to culture medium supplemented with brain‐derived CSPGs, and even substratum‐attached CSPGs were able to exert such inhibitory effects. Only brain‐type CSPGs, not a cartilage‐derived CSPG (PGH) or a hyaluronate‐binding PGH, had such inhibitory effects. Furthermore, these inhibitory activities were associated only with the core proteins of brain‐derived CSPGs, and not with polysaccharide chains from brain‐derived CSPGs. Incorporation of [3H]thymidine into DNA did not decrease for at least the first 12 h. Consequently, the amount of DNA per cell after 48 h of culture was about twofold higher in cells treated with brain CSPGs than in nontreated cells after exposure to the medium with CSPGs. Microspectrophotometry revealed that the population of cells with a high DNA content was greater in the culture treated with brain‐derived CSPGs than in the control culture. These findings indicate that purified soluble brain CSPGs block the cell cycle of PC12D cells at the G2 phase with resultant cessation of cell proliferation and the inhibition of neurite outgrowth. ©

ISSN:0021-9541    

DOI:10.1002/jcp.1041560104

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

年代:1993

数据来源: WILEY

摘要:

AbstractWe have previously shown that supplementing cultured porcine pulmonary artery endothelial cells (PAEC) with exogenous oleic acid (18:1ω9) alters the fatty acid composition of the cells and reduces oxidant‐mediated cytotoxicity. Because the mechanisms by which lipid alterations modulate oxidant susceptibility have not been defined, the ability of 18:1 to reduce hydrogen peroxide (H2O2)‐mediated PAEC dysfunction was evaluated. PAEC monolayers on polycarbonate filters were incubated for 3 h in maintenance medium supplemented with either 0.1 mM 18.1 in ethanol vehicle (ETOH) or with an equivalent volume of vehicle alone. Twenty‐four hours later monolayers were treated for 30 min with 50 or 100 μM H2O2in Hanks' balanced salt solution (HBSS) or with HBSS alone (nonoxidant control). As a functional index of PAEC monolayer integrity, the permeability of monolayers to albumin was then measured for 3 h. Treatment with 100 μM H2O2caused cytotoxicity and progressive increases in PAEC monolayer permeability that were attenuated by 18:1 supplementation, whereas 50 μM H2O2caused only a transient increase in permeability without cytotoxicity. Supplementation with 18:1 also attenuated H2O2‐induced reductions in PAEC adenosine triphosphate (ATP) content and disruption of PAEC microfilament architecture. The ATP content of PAEC monolayers was reversibly reduced in the absence of oxidant stress by incubation with glucose‐depleted medium containing deoxyglucose and antimycin A. Metabolic inhibitor‐induced ATP depletion increased monolayer permeability and altered cytoskeletal architecture, alterations that resolved during recovery of PAEC ATP content. These results demonstrate that ATP depletion plays a critical role in barrier dysfunction and suggests that the ability of 18:1 to reduce oxidant‐mediated PAEC dysfunction and injury may relate directly to its ability to preserve PAEC ATP content. © 19

ISSN:0021-9541    

DOI:10.1002/jcp.1041560105

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe biosynthesis and assembly of the peripheral sector (V1) of the vacuolar protontranslocating adenosine triphosphatase (V‐ATPase) was studied in a bovine kidney epithelial cell line. Monolayer cultures of cells were metabolically radiolabeled with Tran35S‐label and the V‐ATPase subsequently immunoprecipitated using a monoclonal antibody raised against the bovine brain‐coated vesicle proton pump. The V‐ATPase immunoprecipitated from the bovine kidney cell line has a subunit composition very similar to that of the bovine brain‐coated vesicle proton pump and the V‐ATPase prepared from other kidney tissues. Radiolabeling the cells for increasing times showed that the V1or peripheral portion of the V‐ATPase is assembled within 10–15 min; the intact V1V0complex is also detectable within 10–15 min. Fractionation of the cells into cytosolic and membrane components prior to immunoprecipitation revealed that there is a significant pool of V1in the cytosol; a similar complex is also found in bovine brain cytosol. Pulse‐chase studies suggest that this cytosolic pool is not an obligate precursor for membranebound V1V0and does not exchange with the membrane V1population at later times. No qualitative differences in assembly were observed when pulse‐chase studies were performed at 15°C or in the presence of brefeldin A. This suggests that assembly of V1V0is probably completed in the endoplasmic reticulum prior to distribution of the enzyme throughout the cell, with a cytosolic pool of V1of unknown function existing in parallel with the fully assembled complex

ISSN:0021-9541    

DOI:10.1002/jcp.1041560106

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

年代:1993

数据来源: WILEY

摘要:

AbstractCobalamin (Cbl, vitamin B12) metabolism was analyzed in cultures of human chorionic villus (CV) cells obtained at 9–10 weeks of gestation. CV cells were shown to synthesize transcobalamin II (TCII) and to possess a high affinity receptor for that molecule. The cells bound and internalized radioactive cyanocobalamin (CN[57Co]Cbl) complexed to TCII. This internalized CN[57Co]Cbl was found to be converted to both methylCbl and adenosylCbl, the two intracellular coenzyme forms of Cbl, and bound to the two known intracellular Cbl requiring enzymes, methionine synthase (MS) and methylmalonyl‐CoA mutase. Both enzyme systems were found to be functional in the intact cell by demonstrating the incorporation of the radioactive label from both [14C]CH3‐tetrahydrofolate and [14C]propionate into acid insoluble products. MS activity was also detected in lysed cell material. CV cells were shown not to be auxotrophic for methionine since they were able to utilize homocysteine in place of methionine for cell division. Since CV cells are capable of performing many of the complex events associated with Cbl metabolism, it may be possible to use these cells to diagnose genetic defects of Cbl metabolism. © 1993 Wiley‐L

ISSN:0021-9541    

DOI:10.1002/jcp.1041560107

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe effect of transforming growth factor β1 (TGFβ1) on the proliferative response of aortic smooth muscle cells (SMC) in vitro was investigated. TGFβ1 substantially inhibited the growth of human and bovine SMC. Rapidly growing SMC and quiescent serum‐stimulated SMC were inhibited by TGFβ1 with an ID50of approximately 0.5 ng/ml and maximal inhibition was observed at 10 ng/ml TGFβ1. In the presence of TGFβ1, quiescent serum‐stimulated SMC progress into the G1 phase of the cell cycle, but become reversibly arrested at a point temporally located 1–2 hours from S phase. Release from this late G1 TGFβ1 arrest point results in S phase entry within 2 hours. Associated with this inhibitory effect is a decrease in the histone H1 kinase activity of p34cdc2protein kinase while TGFβ1 has no effect on the transcription or translation of p34cdc2. Under these growth inhibitory conditions, TGFβ1 is still capable of upregulating the expression of fibronectin mRNA. These results suggest that TGFβ1 growth inhibition in SMC is associated with the regulation of p34cdc2activity in late G1. © 1993

ISSN:0021-9541    

DOI:10.1002/jcp.1041560108

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe regulation of RNA degradation by specific amino acids and insulin was investigated in cultured rat hepatocytes from fed rats previously injected in vivo with [6‐14C]orotic acid. The effects of three groups of amino acids were compared to those of a complete amino acid mixture. The first one consisted of the eight amino acids (leucine, proline, glutamine, histidine, phenylalanine, tyrosine, methionine, tryptophan) previously found to be particularly effective in the control of proteolysis. The two other groups were defined from our study with single additions of amino acids, one consisting of proline, asparagine, glutamine, alanine, phenylalanine, and leucine and the other including the latter group with serine, histidine, and tyrosine. The results showed that these three groups were able to strongly inhibit deprivation‐induced RNA breakdown at one and ten times normal plasma concentrations but to a lower extent than the complete amino acid mixture. Six amino acids (proline, asparagine, glutamine, alanine, phenylalanine, leucine) inhibited individually RNA degradation by more than 20%. However, the deletions of proline, asparagine, glutamine, or alanine from the group of these six amino acids were not followed by a loss of inhibitory effect. On the contrary, an important loss of inhibition was observed when leucine and phenylalanine were deleted. Furthermore, only these two amino acids exhibited an additive inhibitory effect. Thus leucine and phenylalanine could be considered as important inhibitors of RNA breakdown in cultured rat hepatocytes. Finally, insulin which had no significant effect on RNA degradation in the absence of amino acids, was able to potentiate the inhibitory effect of different amino acid groups. © 1993 Wiley‐Lis

ISSN:0021-9541    

DOI:10.1002/jcp.1041560109

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

年代:1993

数据来源: WILEY

摘要:

AbstractActivator protein‐1 (AP‐1) complex plays a central role in the regulation of both growth and differentiation in many cell types. Monocytic differentiation of HL‐60 cells by TPA (12‐0‐tetradecanoyl phorbol‐13‐acetate) has been reported to be paralleled by increased AP‐1 binding to DNA and by elevated c‐jun expression, suggesting transcriptional level of control. We show that two forms of AP‐1 complex, designated AP‐1/1 and AP‐1/2, can be demonstrated in logarithmically growing HL‐60 cells, that the exposure of these cells to 10−8M 1,25‐dihydroxyvitamin D3(1,25(OH)2D3) results in increased binding of these complexes to the AP‐1 DNA element, and that the AP‐1 complex can be resolved into at least three forms in differentiated cells. Binding to, or competition with, a mutated form of the AP‐1 binding site shows that the most slowly migrating complex (AP‐1/3) binds to DNA with greater specificity than do complexes AP‐1/1 and AP‐1/2, while antibody inhibition and binding studies performed at 37°C indicate that jun proteins predominate in AP‐1/2 complexes. Exposure of extracts from differentiated, but not untreated, HL‐60 cells to 2 mM ATP increases the prominence of AP‐1/3 complexes, and reduces the DNA binding of AP‐1/1 complexes. Treatment of the extracts with phosphatases abolishes the binding of AP‐1/2 and AP‐1/3 to DNA, and increases the binding intensity of AP‐1/1. When extracts from differentiated cells are mixed with extracts from undifferentiated cells the AP‐1/3 complexes become less prominent, suggesting than an inhibitory activity in undifferentiated cells prevents the formation of AP‐1/3 complexes. These studies show the association of multiple forms of AP‐1 complex with the mature monocytic phenotype, and suggest several levels o

ISSN:0021-9541    

DOI:10.1002/jcp.1041560110

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

年代:1993

数据来源: WILEY

摘要:

AbstractStudies designed to better understand the involvement of cellular resistance to oxidative stress in mechanisms of cellular resistance to cisplatin were undertaken using H2O2‐resistant variants of the HA 1 Chinese hamster fibroblast cell line. H2O2‐resistant cell lines were resistant to clonogenic inactivation mediated by cisplatin with dose modifying factors at 10% survival of 1.5–3.0, relative to HA 1 cells. The most cisplatin resistant of these cell lines (OC5) also demonstrated fewer DNA–DNA crosslinks induced by cisplatin, relative to HA 1. Since H2O2‐resistant cells contained increased catalase activity as well as total glutathione (GSH) content, the involvement of these cellular antioxidants in the resistance to cisplatin toxicity was evaluated. Treatment of HA 1 and H2O2‐resistant cell lines (OC5, OC14) with 9 mM aminotriazole reduced catalase activity by 60–65% but had no effect on the cytotoxicity of cisplatin. In contrast, treatment with 5 mM buthionine sulfoximine reduced total GSH by 90% and sensitized the cells to cisplatin cytotoxicity. Furthermore, extracellular reaction of GSH with cisplatin prior to treating HA 1 cells reduced the toxicity of the compound, indicating that this reaction is capable of participating in the detoxification of cisplatin. These results indicate that cellular adaptation to oxidative stress renders cells resistant to DNA damage as well as to cytotoxicity associated with cisplatin treatment. Furthermore, increases in total GSH content (but not catalase activity) appear to partially account for cisplatin resistance demonstrated by H2O2‐resistant cells. © 1993

ISSN:0021-9541    

DOI:10.1002/jcp.1041560111

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

年代:1993

数据来源: WILEY