Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Serpins are responsible for regulating a variety of proteolytic processes through a unique irreversible suicide substrate mechanism. To discover novel genes regulated by transforming growth factor-beta1 (TGF-beta 1), we performed differential display reverse transcriptase-PCR analysis of NRP-152 rat prostatic epithelial cells and cloned a novel rat serpin that is transcriptionally down-regulated by TGF-beta and hence named trespin (TGF-beta-repressible serine proteinase inhibitor (trespin). Trespin is a 397-amino acid member of the ov-serpin clade with a calculated molecular mass of 45.2 kDa and 72% amino acid sequence homology to human bomapin; however, trespin exhibits different tissue expression, cellular localization, and proteinase specificity compared with bomapin. Trespin mRNA is expressed in many tissues, including brain, heart, kidney, liver, lung, prostate, skin, spleen, and stomach. FLAG-trespin expressed in HEK293 cells is localized predominantly in the cytoplasm and is not constitutively secreted. The presence of an arginine at the P1 position of trespin's reactive site loop suggests that trespin inhibits trypsin-like proteinases. Accordingly, in vitro transcribed and translated trespin forms detergent-stable and thermostable complexes with plasmin and elastase but not subtilisin A, trypsin, chymotrypsin, thrombin, or papain. Trespin interacts with plasmin at a near 1:1 stoichiometry, and immunopurified mammal-expressed trespin inhibits plasmin in a dose-dependent manner. These data suggest that trespin is a novel and functional member of the rat ov-serpin family.
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PMID:Identification and characterization of a novel rat ov-serpin family member, trespin. 1198 14

Thrombin appears to underlie myometrial contractions in response to intrauterine bleeding. In a similar fashion, thrombin generated within the uterus in the absence of active bleeding could also produce contractions. These studies sought to determine whether functionally active prothrombin is expressed in the pregnant and nonpregnant rat uterus. Uteri were obtained from proestrus/estrus and timed-pregnant Sprague-Dawley rats. Western blots were performed using antithrombin antibodies. Immunohistochemical studies were performed using the same antibodies along with the Vector Elite ABC kit. Qualitative reverse transcriptase-polymerase chain reaction studies were performed using rat prothrombin-specific oligonucleotide primers. In vitro uterine contraction studies were performed using Taipan snake venom (an exogenous prothrombinase) and components of the plasma prothrombinase complex (Factors Xa and V) with and without pretreatment with thrombin inhibitors (heparin or hirudin). The Western blots demonstrated prothrombin peptides in myometrial tissue from estrus and pregnant rats. The immunohistochemical studies confirmed prothrombin peptides in both the circular and longitudinal myometrium, along with the endometrium. The reverse transcriptase-polymerase chain reaction studies demonstrated prothrombin mRNA in the endometrium and placenta, but not in the myometrial smooth muscle. The Taipan snake venom stimulated a significant increase in contractions, which were suppressed by pretreatment with heparin and hirudin. The Factor Xa and V complex also significantly stimulated uterine contractions, which were likewise inhibited by hirudin. These studies provide evidence supporting the expression of functionally active prothrombin in the pregnant and nonpregnant rat uterus. Based on the presence of its mRNA, prothrombin appears to be synthesized in the endometrium and placenta; in contrast, the myometrial smooth-muscle cells appear to sequester preformed prothrombin. These results support the hypothesis that intrauterine thrombin could play an autocrine/paracrine role in the regulation of contractile activity.
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PMID:Intrauterine expression of prothrombin in the sprague-dawley rat. 1238 11

We previously demonstrated that cyclic ADP-ribose (cADPR) elicits Ca2+ release in airway smooth muscle (ASM) cells through ryanodine receptor channels. CD38 is a cell surface protein that catalyzes the synthesis and degradation of cADPR. In inflammatory diseases such as asthma, augmented Ca2+ responses and Ca2+ sensitivity contribute to increased ASM contractility in response to agonists. In this study, we investigated the regulation of CD38 expression and the role of cADPR-mediated Ca2+ release in airway inflammation. Human ASM cells in culture between the second and fifth passages were exposed to tumor necrosis factor alpha (TNF-alpha), interleukin 1beta, or interferon gamma, or bovine serum albumin (controls). CD38 expression was measured by reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, and Western blot analysis, and ADP-ribosyl cyclase activity was assayed with nicotinamide guanine dinucleotide as the substrate. Ca2+ responses to acetylcholine, bradykinin, and thrombin were measured in fura-2AM-loaded cells by fluorescence microscopy. Cytokines caused significant augmentation of CD38 expression, ADP-ribosyl cyclase activity, and Ca2+ responses to the agonists, compared with the control. TNF-alpha effects were greater than those of the other two cytokines. The cADPR antagonist 8-bromo-cADPR attenuated the Ca2+ responses to the agonists in control and cytokine-treated cells, with the magnitude of inhibition correlating with the level of CD38. This study provides the first demonstration of a role for CD38-cADPR signaling in a model of inflammatory airway disease.
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PMID:CD38/cyclic ADP-ribose-mediated Ca2+ signaling contributes to airway smooth muscle hyper-responsiveness. 1251 17

Activation of protein C by thrombin bound to thrombomodulin is enhanced by endothelial protein C receptor. This pathway may inhibit inflammation. We investigated effects of protein C and activated protein C on neutrophils as well as whether an endothelial protein C receptor is involved in mediating protein C effects. Neutrophils were from venous blood of healthy donors. Cell migration, respiratory burst, phagocytic activity, and apoptosis were studied by micropore filter assays and fluorometry. Receptor expression was investigated by reverse transcriptase-polymerase chain reaction (PCR) for mRNA, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of immunoprecipitated receptor protein, and fluorescence-activated cell-sorter scanner (FACS) analysis using the anti-endothelial protein C receptor antibody RCR-252. Neither protein C nor activated protein C induced migration, yet both of them inhibited neutrophil chemotaxis triggered by interleukin-8, formyl-Met-Leu-Phe, antithrombin, or C5a. A protein C activation-blocking antibody against endothelial protein C receptor diminished inhibitory effects of protein C or activated protein C on migration. No effect of either protein C preparation was seen in neutrophil's respiratory burst, bacterial phagocytosis, or apoptosis assays. Endothelial protein C receptor immunoreactivity was confirmed on neutrophils by FACS. De novo synthesis is suggested by endothelial protein C receptor mRNA expression as demonstrated by reverse transcriptase PCR and immunoprecipitation SDS-PAGE analyses. Data suggest that an endothelial protein C receptor is expressed by human neutrophils whose active site ligation with either protein C or activated protein C arrests directed cell migration. Inhibitory effects of these components of the protein C pathway on neutrophil function may play a role in the protein C-based treatment of severe sepsis.
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PMID:Expression and function of the endothelial protein C receptor in human neutrophils. 1271 92

Eosinophil recruitment to airway tissue is a key feature of asthma, and release of a wide variety of toxic mediators from eosinophils leads to the tissue damage that is a hallmark of asthma pathology. Factors that control the release of these toxic mediators are targets for potential therapeutic intervention. Protease-activated receptors (PARs) are a novel class of receptors that are activated by cleavage of the N terminus of the receptor by proteases such as thrombin or trypsin-like enzymes. To date, PAR1-4 have been identified, and there are several studies that have demonstrated the expression of PARs in airway tissue, particularly the respiratory epithelium. We have investigated whether eosinophils express PARs and if activation of these receptors will then trigger a functional response. Using a combination of reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometry analysis, we have demonstrated that eosinophils express PAR1 and PAR2. FACS analysis showed that PAR1 could be clearly detected on the surface of the cells, whereas PAR2 appeared to be primarily intracellular. Trypsin and the PAR2 agonist peptide were seen in trigger shape change, release of cysteinyl leukotrienes, and most obviously, generation of reactive oxygen species. In contrast, thrombin had no effect on eosinophil function. The PAR1 agonist peptide did have a minor effect on eosinophil function, but this was most likely down to its ability to activate PAR1 and PAR2. These results demonstrate that PAR2 is the major PAR receptor that is capable of modulating eosinophil function.
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PMID:Expression of and functional responses to protease-activated receptors on human eosinophils. 1283 43

The serine protease thrombin present at the site of vascular injury triggers fibrin formation, platelet activation and different cellular responses including angiogenesis. We report a role for thrombin in the human monolayer cultured endothelial cell growth and angiogenesis in 3D collagen gel angiogenesis assay. The angiogenic activity of thrombin is, in part, related to the expression of the vascular endothelial growth factor (VEGF)165 mRNA, assessed by reverse transcriptase-polymerase chain reaction, either in monolayer cultured endothelial cells or in endothelial cells forming capillary-like structures in the 3D collagen gel assay. This expression of VEGF mRNA is associated with a VEGF secretion in the supernatant of thrombin-treated human umbilical vein endothelial cells. The thrombin-induced VEGF165 mRNA expression is associated with the regulation of hypoxia-inducible factor 1alpha, analyzed by Western Blot, in endothelial cells.
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PMID:Thrombin induces angiogenesis and vascular endothelial growth factor expression in human endothelial cells: possible relevance to HIF-1alpha. 1287 82

Fibrin deposition in the peritubular capillaries and along the tubular basement membrane is commonly observed in several renal diseases and suggests the involvement of blood coagulation in tubulointerstitial damage. It has been demonstrated that tissue factor (TF) is present in tubular epithelial cells of animal models of nephritis. Tissue factor pathway inhibitor (TFPI) regulates the extrinsic pathway of blood coagulation through its ability to inhibit TF activity and it is now thought to be produced mainly by the vascular endothelial cells. We examined whether human proximal tubular epithelial cells (PTEC) could produce TFPI and attempted to clarify the regulatory factors affecting TFPI production. Cultured human PTEC were used. The procoagulant activity (PCA) in PTEC lysate was quantified by measurement of the one-stage recalcification time. TFPI in the cell supernatants was measured by ELISA. The mRNA of TF and TFPI in PTEC was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). PCA which is compatible with TF activity was present in the PTEC lysate. TF mRNA and TFPI mRNA were detected in PTEC. The amount of TFPI increased over time in the cell supernatants. Immnoblot analysis revealed 40 kD protein of TFPI, and TFPI antigen was demonstrated in PTEC by immunofluorescence. The concentration of TFPI was significantly increased following incubation with thrombin and heparin in a dose- and time-dependent manner, although the amount of TFPI mRNA was not changed. Our study showed that TFPI is produced in cultured PTEC and added one more cell type that produced TFPI other than endothelial cells. Thrombin and heparin stimulated TFPI secretion from PTEC. TFPI of PTEC may act against generation of thrombin and tubular fibrin formation induced by tissue factor activation. The augmentation of TFPI secretion by heparin may play an important role in the modulation of anticoagulant properties of PTEC.
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PMID:Tissue factor pathway inhibitor production by human proximal tubular epithelial cells in culture. 1289 29

Thrombin, a serine protease, plays an important role in the progression of atherosclerosis. How atorvastatin could limit the pro-inflammatory response to thrombin was studied in cultured rat aortic smooth muscle cells. The variations in expression of interleukin-6, heme oxygenase-1, p(22phox) and Mox-1 mRNAs were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Interleukin-6 release was determined using the B9 cell assay. Nuclear factor-kappa B (NF-kappaB) translocation was analysed by electrophoretic mobility shift assay (EMSA) and RhoA protein translocation by Western blot. Thrombin activated interleukin-6 secretion and mRNA expression in smooth muscle cells in a dose-dependent manner. The greatest effect on mRNA expression was obtained after 1 h of stimulation. Preincubation (72 h) of the cells with various concentrations of atorvastatin prevented this effect. Simultaneous addition of mevalonate overcame this statin effect. Thrombin was without effect on p(22phox) and heme oxygenase-1 mRNA expression but, after 3 h of stimulation, induced a two-fold increase in that of Mox-1. Preincubation with atorvastatin dose-dependently downregulated this Mox-1 mRNA expression. In addition, thrombin induced NF-kappaB translocation and membrane translocation of RhoA in smooth muscle cells which were both prevented by pre-treatment of the cells by atorvastatin. These data demonstrate the ability of atorvastatin to prevent the induction by thrombin of a pro-inflammatory phenotype in smooth muscle cells.
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PMID:Atorvastatin limits the pro-inflammatory response of rat aortic smooth muscle cells to thrombin. 1292 59

Thrombin, a serine protease generated by the activation of the blood coagulation cascade following vessel injury, induces vascular endothelial growth factor-(VEGF) release. However, the molecular mechanism of thrombin-induced VEGF release is largely unknown. Anagonist of protease-activated receptor-i (PARI), SFLL-RNPNDKYEPF, mimicked thrombin-induced VEGF release in human vascular smooth muscle (HVSM) cells, as determined by enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction, and Northern blotting. In contrast, the agonist of PAR3, TFR- GAP, did not affect VEGF release or expression. SFLL-RNPNDKYEPF, but not TFRGAP, up-regulated [Ca2-]i.Moreover, the calcium ionophone A23187 was found to trigger VEGF release in HVSM cells. Thrombin-inducedVEGF release was blocked by anti-thrombin, heparin, a synthetic thrombin receptor inhibitor E5510, the calcium chelator BAPTA, the protein kinase C inhibitor calphostin C, and the MEK1/2 inhibitor U0126. Thus, our data show that thrombin caused VEGF release via PARI activation in a manner dependent on [Ca2+]i and p44/42 downstream from the receptor activation.
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PMID:The agonist of the protease-activated receptor-1 (PAR) but not PAR3 mimics thrombin-induced vascular endothelial growth factor release in human smooth muscle cells. 1451 37

Most tumors have constitutively active tissue factor on their surface, capable of generating thrombin in the surrounding environment, and thrombosis is associated with cancer. Thrombin is known to induce a malignant phenotype by enhancing tissue adhesion and cell growth in vitro and in vivo in mice. Because tumors require angiogenesis for growth, we examined whether thrombin induces neoangiogenesis in a physiologically intact in vivo model. Thrombin (0.1 U mL-1) induced neoangiogenesis in the chick chorioallantoic membrane over a 24-72-h period by approximately 2-3-fold. This was inhibited by the potent thrombin inhibitor, hirudin and shown to have its mode of action by ligation of the thrombin protease-activated receptor, PAR-1. The thrombin receptor activation peptide, SFLLRNPNDKYEPF (200 microm) also enhanced neoangiogenesis c. 2-3-fold. Thrombin-induced neoangiogenesis was accompanied by the induction of vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) mRNA at 24-48 h (approximately 2-fold) as determined by semi-quantitative reverse transcriptase-polymerase chain reaction. Thrombin-induced neoangiogenesis was inhibited to baseline level by the specific angiogenesis receptor inhibitors KDR-Fc (vs. VEGF) and Tie-2-Fc (vs. Ang-1 and Ang-2), as well as the non-specific angiogenesis inhibitor thrombospondin-1. Thrombin-induced neoangiogenesis was also inhibited to baseline level by agents known to inhibit thrombin receptor signaling in other cells: G-coupled protein receptor inhibitor, pertussis toxin (40 pg per egg), protein kinase C inhibitor, bisindolylmaleimide (1 microm per egg), MAP kinase inhibitor, PD980598 (10 microm per egg) and PI3 kinase inhibitor, LY294002 (0.25 microm per egg). Thus angiogenesis is stimulated by thrombosis, which could help explain the enhancement of experimental tumorigenesis by thrombin.
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PMID:Thrombin induces neoangiogenesis in the chick chorioallantoic membrane. 1452 87


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