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Studies of proteins that inhibit tissue factor activity have generally been conducted using either an extracted tissue homogenate ("thromboplastin") or tissue factor protein reconstituted into phospholipid vesicles rather than with tissue factor expressed in cell membranes (its physiological environment). In the present study, a human fibroblast cell strain was used to evaluate the effects of lipoprotein associated coagulation inhibitor (LACI), placental anticoagulant protein (PAP), and apolipoprotein A-II (apo A-II) on human tissue factor in cell membranes. LACI was tested from 7.8 to 500 pmol/L on fibroblasts cultured at cell densities ranging from 3,500 to 9,925 cells/well, and caused a progressive inhibition of tissue factor activity. PAP was tested from 3.9 nmol/L to 1 mumol/L at cell densities ranging from 4,500 to 15,400 cells/well and caused up to 83% inhibition of tissue factor activity. Inhibition by these proteins appeared to be influenced by cell density as well as whether the cells were intact or disrupted. Apo A-II, up to 1 mumol/L, did not inhibit the tissue factor activity of intact or disrupted fibroblasts at any cell density examined even though it did inhibit the activity of tissue factor in phospholipid vesicles. Of these inhibitors of tissue factor-dependent activation of factor X, LACI was the most effective in suppressing the generation of factor Xa activity. The effects obtained with apo A-II are clearly dependent on the nature of the tissue factor preparation with which it is tested. The disparity between the inhibitory effect of apo A-II on the activity of tissue factor reconstituted into lipid vesicles and the absence of effect on the activity of tissue factor remaining in cell membranes serves to reemphasize the necessity of reexamining results obtained with model systems using as nearly physiological reagents as possible. 相似文献
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Regulation of intestinal guanylate cyclase by the heat-stable enterotoxin of Escherichia coli (STa) and protein kinase C.
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J K Crane M S Wehner E J Bolen J J Sando J Linden R L Guerrant C L Sears 《Infection and immunity》1992,60(12):5004-5012
The heat-stable enterotoxin of Escherichia coli (STa) stimulates membrane-bound guanylate cyclase in intestinal epithelium and induces fluid and ion secretion. Using the T84 human colon carcinoma cell line as a model, we observed that phorbol esters markedly enhanced STa-stimulated cyclic GMP accumulation in T84 cells (C. S. Weikel, C. L. Spann, C. P. Chambers, J. K. Crane, J. Linden, and E. L. Hewlett, Infect. Immun. 58:1402-1407, 1990). In this study we document that the phorbol ester treatment increases 125I-STa-binding sites as well as membrane-bound guanylate cyclase activity in T84 cells and provide evidence that both effects are mediated by phosphorylation. Guanylate cyclase activity was increased approximately 50% in membranes prepared from intact T84 cells treated with phorbol-12,13-dibutyrate (beta-PDB) and after treatment of homogenates with beta-PDB in a manner dependent on ATP, MgCl2, and cytosol. Similarly, treatment of membranes with purified bovine brain protein kinase C in the presence of appropriate cofactors and beta-PDB resulted in an increase in STa-stimulated guanylate cyclase activity of about 70%. Likewise, the number of 125I-STa-binding sites was increased by about 25 to 40% in membranes prepared from intact cells or homogenates treated with beta-PDB; no effect on binding affinity (Kd = 0.15 nM) was noted. These experiments suggest that protein kinase C may phosphorylate the STa receptor-guanylate cyclase or a closely related protein and increase guanylate cyclase activity. The stimulatory effects of protein kinase C on STa-sensitive guanylate cyclase are opposite in direction to the profound inhibitory effects of the kinase on atrial natriuretic peptide-stimulated guanylate cyclase, demonstrating differential regulation by protein kinases within the guanylate cyclase-receptor family. 相似文献
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Myosin VIIA gene: heterogeneity of the mutations responsible for Usher syndrome type IB 总被引:8,自引:1,他引:8
Levy G; Levi-Acobas F; Blanchard S; Gerber S; Larget-Piet D; Chenal V; Liu XZ; Newton V; Steel KP; Brown SD; Munnich A; Kaplan J; Petit C; Weil D 《Human molecular genetics》1997,6(1):111-116
Usher syndrome is recognized as the most frequent cause of hereditary
deaf-blindness. Usher syndrome type I (USH1), the most severe form of the
disease, is characterized by profound congenital sensorineural deafness,
constant vestibular dysfunction, and retinitis pigmentosa of prepubertal
onset. This form is genetically heterogeneous and five loci (USH1A-E) have
been mapped thusfar. However, only the gene responsible for USH1 B (which
accounts for approximately 75% of USH1 cases) has been characterized. It
encodes a long-tailed unconventional myosin, myosin VIIA, with a predicted
2215 amino acid sequence. Primers covering the complete myosin VIIA coding
sequence as well as the 3' non coding sequence were designed, allowing
direct sequence analysis of each of the 48 coding exons and flanking splice
sites in seven patients affected by USH1. Four novel mutations were thereby
identified. The possibility should now be considered of a sequence-based
prenatal diagnosis in some of the families affected by this very severe
form of Usher syndrome.
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