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排序方式: 共有100条查询结果,搜索用时 15 毫秒
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Migratory behavior of cells on embryonic retina basal lamina 总被引:1,自引:0,他引:1
In order to study cell translocation in vitro on a physiological substrate a novel cell migration assay was developed using the inner limiting membrane of the avian embryonic retina. The matrix sheet consists of a laminin-rich basal lamina covered by a dense layer of neuroepithelial endfeet. The retina basal lamina does not contain fibronectin. Cells translocating on this substrate displace the neuroepithelial endfeet, leaving behind tracks in the endfeet monolayer. Motility of cells and the relative forward to lateral migration can be quantitated by measuring lengths, widths, and areas of the tracks. Using this assay system, the conditions and patterns of cell migration for a variety of cells have been examined. In the absence of serum all cell types show only minor migratory activity and addition of serum to the culture medium always enhances the rate of cell migration in a saturable, dose-response manner. The serum cannot be replaced by fibronectin or vitronectin (serum spreading factor). For maximum cell migration, serum has to be constantly present in the medium; however, 58% cell migration is obtained in serum-free medium when the matrix is preincubated with serum. According to the area and linearity of the tracks, the migratory behavior of the different cells can be classified into three groups: (i) fibroblasts and the nonpigmented Bowes melanoma cells form straight and long tracks; (ii) glioma, sarcoma, and carcinoma cells from straight but short tracks, and (iii) neuronal tumor cells, epithelial cells, and pigmented B16 melanoma cells form wide and short tracks. Comparative studies with low and high metastatic clones of tumorgenic cell lines show that migratory activity and metastatic potential of cells do not necessarily correlate. Finally, we show that fibroblasts deposit fibronectin fibrils on their paths as they migrate on the basal lamina. Fibronectin trails are also seen when fibroblasts are cultured on plain basal laminae that are pretreated with detergent to remove the endfeet monolayer. Likewise, when fibroblasts are cultured in the presence of antifibronectin antibodies, the fibronectin secreted by cells is detectable. Due to antibody treatment the cellular fibronectin is precipitated and its normal fibril formation is inhibited; however, the translocation of fibroblasts is not impaired. 相似文献
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Brain cells and glioma cells in culture release a protein which induces neurite outgrowth in neuroblastoma cells. This neurite-promoting factor (NPF), which has been purified from serum-free glioma conditioned medium, has an apparent mol. wt. of 43 000. NPF inhibits urokinase as well as plasminogen activator-dependent caseinolysis or fibrinolysis. NPF and urokinase form an SDS-resistant complex. The fact that this glia-derived NPF is a potent protease inhibitor indicates that glial cells modulate the proteolytic activity associated with neuronal cells and suggests that this phenomenon is one of the biochemical events involved in the regulation of neurite growth. 相似文献
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Protease-nexin I as an androgen-dependent secretory product of the murine seminal vesicle. 总被引:3,自引:0,他引:3 下载免费PDF全文
J D Vassalli J Huarte D Bosco A P Sappino N Sappino A Velardi A Wohlwend H Ern D Monard D Belin 《The EMBO journal》1993,12(5):1871-1878
A search for inhibitors of urokinase-type plasminogen activator (uPA) in the male and female murine genital tracts revealed high levels of a uPA ligand in the seminal vesicle. This ligand is functionally, biochemically and immunologically indistinguishable from protease-nexin I (PN-I), a serpin ligand of thrombin and uPA previously detected only in mesenchymal cells and astrocytes. A survey of murine tissues indicates that PN-I mRNA is most abundant in seminal vesicles, where it represents 0.2-0.4% of the mRNAs. PN-I is synthesized in the epithelium of the seminal vesicle, as determined by in situ hybridization, and is secreted in the lumen of the gland. PN-I levels are much lower in immature animals, and strongly decreased upon castration. Testosterone treatment of castrated males rapidly restores PN-I mRNA levels, indicating that PN-I gene expression is under androgen control. 相似文献
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Interaction of vitronectin with glia-derived nexin (GDN), thrombin, and the complex GDN-thrombin was demonstrated in direct binding assays that indicated the formation of binary and ternary complexes. The concentration of vitronectin necessary to obtain 50% saturation of the immobilized GDN-thrombin complex binding sites (EC50) was about 1 nM. Under similar experimental conditions, the EC50 of vitronectin for the immobilized antithrombin-III-thrombin complex was about fivefold higher. A tight complex was also formed between vitronectin and immobilized GDN (EC50 approximately 1.5 nM) but when vitronectin was immobilized, GDN displayed a reduced affinity for vitronectin (EC50 approximately 10 nM). These results suggest differences between the immobilized and free conformations of GDN and/or vitronectin. In contrast, vitronectin displayed negligible affinity for antithrombin III. Biotinylated GDN was used to characterize further the binding of GDN or the GDN-thrombin complex to vitronectin. The interaction of the biotinylated GDN-thrombin complex with immobilized vitronectin (EC50 approximately 2 nM) was completely blocked by nonbiotinylated complexes of thrombin with either GDN or antithrombin III, whereas free GDN, free thrombin and the GDN-trypsin complex were only weak competitors. Active-site-blocked urokinase and the complex GDN-urokinase also strongly competed for binding of the biotinylated GDN-thrombin complex to vitronectin. Binding of biotinylated GDN to immobilized vitronectin was specific, saturable and was competed with decreasing efficiency by the GDN-thrombin complex, free GDN and free antithrombin III. These interactions between the adhesive component vitronectin and the serine protease inhibitor GDN may relate to localized control of thrombin and/or urokinase action at certain extravascular sites. These results are discussed in terms of binding sites for vitronectin on GDN, thrombin, and the GDN-thrombin complex. 相似文献
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The phosphatidylethanolamine-binding protein is the prototype of a novel family of serine protease inhibitors 总被引:11,自引:0,他引:11
Serine proteases are involved in many processes in the nervous system and specific inhibitors tightly control their proteolytic activity. Thrombin is thought to play a role in tissue development and homeostasis. To date, protease nexin-1 is the only known endogenous protease inhibitor that specifically interferes with thrombotic activity and is expressed in the brain. In this study, we report the detection of a novel thrombin inhibitory activity in the brain of protease nexin-1(-/-) mice. Purification and subsequent analysis by tandem mass spectrometry identified this protein as the phosphatidylethanolamine-binding protein (PEBP). We demonstrate that PEBP exerts inhibitory activity against several serine proteases including thrombin, neuropsin, and chymotrypsin, whereas trypsin, tissue type plasminogen activator, and elastase are not affected. Since PEBP does not share significant homology with other serine protease inhibitors, our results define it as the prototype of a novel class of serine protease inhibitors. PEBP immunoreactivity is found on the surface of Rat-1 fibroblast cells and although its sequence contains no secretion signal, PEBP-H(6) can be purified from the conditioned medium upon recombinant expression. 相似文献
10.
Differential inhibition of human immunodeficiency virus type 1 fusion, gp120 binding, and CC-chemokine activity by monoclonal antibodies to CCR5 总被引:7,自引:0,他引:7 下载免费PDF全文
Olson WC Rabut GE Nagashima KA Tran DN Anselma DJ Monard SP Segal JP Thompson DA Kajumo F Guo Y Moore JP Maddon PJ Dragic T 《Journal of virology》1999,73(5):4145-4155
The CC-chemokine receptor CCR5 mediates fusion and entry of the most commonly transmitted human immunodeficiency virus type 1 (HIV-1) strains. We have isolated six new anti-CCR5 murine monoclonal antibodies (MAbs), designated PA8, PA9, PA10, PA11, PA12, and PA14. A panel of CCR5 alanine point mutants was used to map the epitopes of these MAbs and the previously described MAb 2D7 to specific amino acid residues in the N terminus and/or second extracellular loop regions of CCR5. This structural information was correlated with the MAbs' abilities to inhibit (i) HIV-1 entry, (ii) HIV-1 envelope glycoprotein-mediated membrane fusion, (iii) gp120 binding to CCR5, and (iv) CC-chemokine activity. Surprisingly, there was no correlation between the ability of a MAb to inhibit HIV-1 fusion-entry and its ability to inhibit either the binding of a gp120-soluble CD4 complex to CCR5 or CC-chemokine activity. MAbs PA9 to PA12, whose epitopes include residues in the CCR5 N terminus, strongly inhibited gp120 binding but only moderately inhibited HIV-1 fusion and entry and had no effect on RANTES-induced calcium mobilization. MAbs PA14 and 2D7, the most potent inhibitors of HIV-1 entry and fusion, were less effective at inhibiting gp120 binding and were variably potent at inhibiting RANTES-induced signaling. With respect to inhibiting HIV-1 entry and fusion, PA12 but not PA14 was potently synergistic when used in combination with 2D7, RANTES, and CD4-immunoglobulin G2, which inhibits HIV-1 attachment. The data support a model wherein HIV-1 entry occurs in three stages: receptor (CD4) binding, coreceptor (CCR5) binding, and coreceptor-mediated membrane fusion. The antibodies described will be useful for further dissecting these events. 相似文献