摘要:

AbstractProgressive stenosis or occlusion of bi ateral internal carotid arteries by fibrocellular intimal thickening results in cerebral ischemia in moyamoya disease. The etiology is unknown. We examined cultured arterial smooth muscle cells (SMC) from scalp arteries of five patients with moyamoya disease. In this study we investigated the responsiveness of the cells in culture to serum mitogens including platelet‐derived growth factor (PDGF), a major mitogen of SMC, and compared the response to that of cells derived from age‐matched control patients. SMC from patients with moyamoya disease proliferated less rapidly in a medium with 15% serum than did control SMC and responded poorly to the addition of PDGF to 5% serum. PDGF alone did not stimulate SMC in a quiescent state to initiate DNA synthesis in moyamoya disease, without serum factors other than bovine serum albumin, though it significantly stimulated the controls. Simultaneous additions of epidermal growth factor, insulin‐like growth factor‐I, and PDGF stimulated initiation of DNA synthesis in cells from moyamoya disease, but not as much as PDGF alone did in the controls. Although direct correlations with the pathogenesis of the disease remain to be clarified, the results indicate altered interrelations between serum factors and the cellular responses in vessels of moyamoya

ISSN:0021-9541    

DOI:10.1002/jcp.1041470202

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

年代:1991

数据来源: WILEY

摘要:

AbstractTreatment with 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) and incubation with lipopolysaccharide (LPS) induces Interleukin 1β (IL‐1β) production in the histiocytic lymphoma cell line U937. Here we investigated the effect of treatment with both TPA and 1α,25‐dihydroxyvitamin D3(1,25(OH)2D3) on LPS‐induced IL‐1β production in U937 cells. To clarify the mechanism of IL‐1β production, the possible role of polyamines in this process was examined. Combined treatment with TPA and 1,25(OH)2D3for 72 h followed by incubation with LPS for 24 h caused synergistic induction of both IL‐1β release and mRNA expression. On the other hand, TPA increased the numbers of vitamin D3receptors, which may be one mechanism of this synergistic induction. Ornithine decarboxylase (ODC), a rate‐limiting enzyme for polyamine biosynthesis, was also induced by these compounds biphasically: the first peak of ODC activity was observed at 4 h of the incubation with the two compounds and the second peak was at 4 h after the addition of LPS. To find whether these peaks were related to IL‐1β production, DL‐α‐difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC, was added together with TPA and 1,25(OH)2D3. DFMO decreased the cellular levels of putrescine and spermidine and suppressed IL‐1β release and IL‐1β mRNA expression by 65%. Exogenous putrescine, but not spermidine, abrogated these kinds of inhibition. Similar results were obtained with DFMO and the polyamines during the differentiation of the cells up to the monocyte or macrophage stage. These results thus suggest that changes in either of these intracellular polyamines, especially putrescine, help to regulate the differentiation of U937 cells, resulting in partial co

ISSN:0021-9541    

DOI:10.1002/jcp.1041470203

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

年代:1991

数据来源: WILEY

摘要:

AbstractIncreased numbers of mast cells are commonly seen at sites of increased bone resorption and in osteoporosis. Long‐term administration of heparin, a major component of mast cell granules, causes osteoporosis. We therefore tested the effect of heparin on bone resorption by osteoclasts disaggregated from neonatal rat long bones. We found that, in the absence of serum, heparin was without effect on osteoclast function. However, in the presence of newborn calf serum, rat serum, or bovine platelet‐poor plasma‐derived serum, heparin, in the range 25–100 μg/ml, induced an increase in osteoclastic bone resorption. Heparin appeared to act through binding and enhancement of an osteoclast resorption‐stimulating activity (ORSA) present in serum. A number of known factors that show an affinity for heparin, including transforming growth factor‐p, platelet‐derived growth factor, insulin‐like growth factors I or II, acidic or basic fibroblast growth factors, fibfonectin, or laminin, could not substitute for ORSA, suggesting that the activity may represent a novel heparin‐binding factor. The ability of glycosaminoglycans (GACs) and related molecules to enhance resorption was dependent on the degree of sulfation and on their size: The high molecular weight GAG heparan sulfate and polysaccharides fucoidan or dextran sulfate showed a similar effect, while low molecular weight heparin, chondroitin‐2‐sulfate, chon‐droitin‐4‐sulfate, and chondroitin‐6‐sulfate were without effect. We propose that mast cells or heparin therapy increases bone resorption through augmentation of the activity of a factor invoked in the locd and systemic regulation

ISSN:0021-9541    

DOI:10.1002/jcp.1041470204

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

年代:1991

数据来源: WILEY

摘要:

AbstractUsing methods designed for isolation of mutants defective in receptor‐mediated endocytosis, a novel L‐cell mutant was obtained that exhibits resistance to three different protein toxins as well as alterations in secretion. This rrutant, LEFIC, is resistant to modeccin,Pseudomonasexotoxin, and ricin. Thesi toxins, which enter the cytoplasm via receptor‐mediated endocytosis, are thought to penetrate into cells at the level of late endosomes or the trans Golgi network. Early endosomal acidification appears to be normal in the mutant based on its accumulation of iron from transferrin and its sensitivity to diphtheria toxin A chain‐transferrin conjugate. Within the secretory pathway twodelays in transport ofvesicular stomatitis virus (VSV) G protein wereobserved in LEFIC: a 20–30 min delay in acquisition of Endo H resistance and a 1–2 hr delay in appearance of newly synthesized G protein on the cell surface. Movement of endogenous proteins along the secretory pathway was also affected in LEFIC. Fibronectin secretion was delayed by 15 min, and membrane proteins were delayed in arrival at the cell surface. The phenotype of LEFIC is consistent with a defect in a component or compartment shared by both the late endocytic and constitutive secret

ISSN:0021-9541    

DOI:10.1002/jcp.1041470205

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

年代:1991

数据来源: WILEY

摘要:

AbstractMouse bone marrow‐derived cultured mast cells proliferate on +/+ mouse embryo‐derived 3T3 fibroblasts, but not onSI/SIdmouse embryo‐derived 3T3 fibroblasts, in the absence of IL‐3 and IL‐4 (Fujita et al:Proc. Natl. Acad. Sci. U.S.A.86:2888–2891, 1989). To further characterize the mast cell‐fibrpblast interactions and the effects cfSImutation, we tried to analyze the adhesion of cultured mast cells to 3T3 fibroblasts in vitro. Mast cells plated onto NIH/3T3 fibroblasts showed marked adhesion within 30 min, which reached a plateau after 3 h. The numbers of adhered mast cells were linear over the range of 103to 5 × 105cells inoculated into each (2 cm2) of 24 multiwells. Adhesion required active energy production and the presence of divalent cations. It was not inhibited byan RGD‐containingpeptide, an anti‐LFA‐1 antibody, or asialofetuin. Mast cells adhered efficiently to the eight 3T3 cell lines derived from +/+ mouse embryos, but not to the eight 3T3 cell lines derived fromSI/SIdmouse embryos. Adhesion to mouse spleen‐derived fibroblasts lacking mast cell‐supporting activitywas comparable to that toSI/SId/3T3 cells. The failure of mast cells to adhere to fibroblasts with theSImutations was not due to a production of a diffusible inhibitor by the latter. These results indicate that production of wild typeSIgene product by fibroblasts is mandatory for adhesion/migration, as well as for proliferation of mast cells on them, and that the coculture system should be useful for the biochemical and molecular analys

ISSN:0021-9541    

DOI:10.1002/jcp.1041470206

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

年代:1991

数据来源: WILEY

摘要:

AbstractAnalysis of gene expression following stimulation of growth‐arrested cells has beei the main approach for identification of growth‐associated genes. Since the activation of these gene sequences is dependent on both the stimulatory agent and theitate of quiescence of the cell, the activation and role of the same genes may be entirely different in non‐growth arrested, actively proliferating cells. We have addressed the question of growth‐associated gene expression during active growth by analyzing gene expression during G‐1 of cells which have jusl exited mitosis without first leaving the cell cycle. We were able to isolate, by a non‐inductive, drug free system, a population of highly synchronized Swiss 3T3 cells within mitos is (>90%) in numbers sufficient to determine the pattern of expression pf a large number of representative growth‐associated genes. Our results show that after replating the mitotic ceils into conditioned medium: (1) growth‐associated gene expression is not constant during G‐1 of actively proliferating cells, and (2) while a number of genes (e.g., JE, c‐myc, ODC, p53, and histone) exhibited patterns of expression similar to that reported in the quiescent systems, others (e.g., nur‐77, vimentin, calcyclin) exhibited patterns which were completely different. From these results, we can begin to construct a temporal map of G‐1 progressi

ISSN:0021-9541    

DOI:10.1002/jcp.1041470207

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

年代:1991

数据来源: WILEY

摘要:

AbstractMouse L929 cells were exposed to the nonionic detergent Brij 58. As has been shown in some other cell types, protein leaked from Brij 58 exposed cells only after a lag phase. In the current study we have extended the observations of the kinetics of protein efflux using cultured L cells subjected to treatment with buffers containing Brij 58. The results show that while the cells become permeable essentially at first exposure to the detergent, proteins do not scape immediately. This lag in efflux is at least partly dependent on the concentration of detergent such that a greater lag is seen in cells exposed to the lowest concentrations of Brij. Data are presented that are most readily interpreted as protein leakage having occured fairly rapidly from individual cells and that show that the time course of protein efflux results, to a large extent, from different sensitivities of individual cells to the detergent. The perrneabilized suspension cells consist of only two types, whereas the conversion of cells from one type to the other occurs through the loss of protein to the permeabilization medium. Only two bands are seen in continuous density gradients and there is a conversion of the more dense type to the less dense with longer exposure to detergent. Moreover, the less dense cells contained about half of the protein per cell as the bottom banding cells, and the proteins of the more dense cells appear to be the sum of those released into the permeabilization medium plus those found in the less dense cells.

ISSN:0021-9541    

DOI:10.1002/jcp.1041470208

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

年代:1991

数据来源: WILEY

摘要:

AbstractWe report the investigation of the growth properties of tumorigenic and reverted nontumorigenic Wilms' nephroblastoma cells when cultured in serum‐free medium. Wilms' tumor, a pediatric nephroblastoma, has been associated with deletions encompassing the p13 band of chromosome 11 and an independent loss of heterozygosity at 11p15. Weissman et al. (Science236:175–180, 1987) transferred a human der(11) chromosome into the G401.6TG.6 Wilms' tumor cell line via the microcell‐mediated chromosome transfer technique. The resulting microcell hybrids were nontumorigenic when assayed in nude mice; however these cells retained all of the in vitro growth and morphological characteristics of the tumorigenic parental cells in 10% fetal calf serum (FCS). Segregation of the der(11) Chromosome from the nontumorigenic microcell hybrid cells resulted In the reappearance of the tumorigenic phenotype in vivo. In vitro culture of these cell lines in serum‐free medium supplemented with 0.1% bovine serum albumin (BSA) and 10 ng/ml Na2O3Se resulted in sustained growth of both the tumorigenic parent and the tumorigenic segregant while the nontumorigenic microcell hybrids were unable to divide. The separate addition of either 10 ng/ml of epidermal growth factor (ECF) or 5 μg/ml of insulin did not alter this effect. However, the addition of 5 μg/ml of transferrin stimulated the nontumorigenic microcell hybrid cells to grow at a rate comparable to the tumorigenic cells. In addition, conditioned serum‐free medium from the tumorigenic parental or tumorigenic segregant cell lines was able to stimulate the growth of the nontumorigenic microcell hybrid cells, whereas the reciprocal experiment had no effect on the growth of the tumorigenic cells. These data suggest that the inability of the microcell hybrid cells to grow in serum‐free conditions is correlated with their genetic nontumorigenic phenotype and that a specific growth factor, transferrin, can bypass or alter this negative growth regulatory pathwa

ISSN:0021-9541    

DOI:10.1002/jcp.1041470209

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

年代:1991

数据来源: WILEY

摘要:

AbstractTumor‐promoting phorbol esters and insulin produce similar effects in Reuber H35 rat hepatoma cell proliferation, including increased ornithine decarboxylase (ODC) enzyme activity, DNA synthesis, and mitogenesis. We investigated ODC mRNA accumulation in cells treated with either insulin or 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). Both agents caused rapid accumulation of ODC mRNA: for TPA, it was maximal 3 hr after treatment (4–6‐fold greater than control cells) and returned quickly to control levels; for insulin, it was significantly longer, continuing to increase for at least 6 hr. Simultaneous treatment with TPA and insulin led to additive effects on ODC mRNA. Induction of ODC by TPA was blocked by down‐regulation or inhibition of protein kinase C (PKC), consistent with a PKC‐mediated mechanism. In contrast, PKC down‐regulation had little effect on ODC induction by insulin. Furthermore, although both agents stimulated ribosomal S6 protein phosphorylation in cells containing normal amounts of PKC, the response to TPA was abolished in PKC‐depleted cells; the effect of insulin was only slightly inhibited. TPA caused a rapid redistribution of essentially all of the PKC activity from the cytosolic to the membrane fraction of the cells, whereas insulin had no effect on PKC distribution. These results suggest that although insulin and TPA share some common cytoplasmic signalling pathways, their effects on phosphorylation of nuclear proteins and transcription of ODC may be media

ISSN:0021-9541    

DOI:10.1002/jcp.1041470210

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

年代:1991

数据来源: WILEY

摘要:

AbstractThioglycollate elicited peritoneal (TG‐Mϕs) but not resident peritoneal Mϕs (R‐Mϕs) were found to bind the lectinGriffonia simplicifoliaisotype I‐B4(GSI‐B4). This was demonstrated by ultrastructural studies and FACS analyses. Membranes from TG‐Mϕs were isolated, separated on SDS‐PAGE, electrotransferred onto nitrocellulose, and exposed to peroxidase‐labeled GSI‐B4. These procedures revealed two major membrane glycoproteins of molecular weights 180,000 and 94,000 daltons that bound the lectin GSI‐B4which has a specificity for recognizing terminal α‐galactosyl residues. The presence of these epitopes on the two membrane glycoproteins was further substantiated by the fact that treatment of the membranes with α‐galactosidase destroyed their capacity to bind GSI‐B4and that α‐D‐galactopyranoside but not N‐acetyl‐D‐glucosamine competitively inhibited GSI‐B4from binding to the glycoproteins. Treatment of TG‐Mϕs with GSI‐B4reduced the capacity of interferon (IFN) and lipopolysaccharide (LPS), or IFN alone, to induce Mϕ mediated cytotoxicity towards tumor cells by as much as 40%. GSI‐B4also caused alterations in the pattern of biosynthetically35S‐methionine labeled secreted proteins as early as 2 hours after contact with TG‐Mϕs. Out of 35 discernible proteins on fluorograms of SDS‐PAGE separated proteins, 5 were down‐regulated and 9 were enhanced. It is suggested that the two novel Mϕ membrane proteins may play a role in regulating the response of

ISSN:0021-9541    

DOI:10.1002/jcp.1041470211

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

年代:1991

数据来源: WILEY