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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), which allowed easy purification of the fusion protein by affinity chromatography on immobilized glutathione. Inclusion of a thrombin cleavage site between the GST carrier and the viral enzyme facilitated the release of purified mature EMC virus RNA polymerase from the GST carrier by proteolysis with thrombin. The purified recombinant enzyme has a molecular mass of about 52 kDa and is recognized by polyclonal immune serum raised against a peptide sequence corresponding to the C-terminal region of the protein. The recombinant enzyme comigrates with immunoprecipitated EMC virus RNA polymerase from infected mouse L929 cell extracts when run in parallel lanes on a sodium dodecyl sulfate-polyacrylamide gel. The enzyme exhibits rifampin-resistant, poly(A)-dependent poly(U) polymerase activity and RNA polymerase activity, which are both oligo(U) dependent. Template-size products are synthesized in in vitro reactions with EMC virus genomic RNA or globin mRNA. The availability of recombinant EMC virus RNA polymerase in a purified form will allow biochemical analysis of its role in the replication of the virus as well as structure-function studies of this unique class of enzyme.
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PMID:Expression, purification, and properties of recombinant encephalomyocarditis virus RNA-dependent RNA polymerase. 185 68

The complete nonstructural NS5 gene of dengue type 1 virus, Singapore strain S275/90 (D1-S275/90) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein (126 kDa). The GST-NS5 fusion protein was purified and the recombinant NS5 protein released from the fusion protein by thrombin cleavage. The recombinant NS5 had a predicted molecular weight of 100 kDa and reacted with antiserum against D1-S275/90 virus in Western blot analysis. The purified recombinant NS5 protein possessed RNA-dependent RNA polymerase activity which was inhibited (>99%) by antibodies against the recombinant NS5 protein. The polymerase product was shown to be a negative-stranded RNA molecule, of template size, which forms a double-stranded complex with the template RNA.
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PMID:Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity. 860 61

1. The biological activities of the proteinase-activated receptor number 2 (PAR-2)-derived peptides, SLIGRL (PP6) SLIGRL-NH2 (PP6-NH2) and SLIGR-NH2 (PP5-NH2) were measured in mouse and rat gastric longitudinal muscle (LM) tissue and in a rat aortic ring preparation and the actions of the PAR-2-derived peptides were compared with trypsin and with the actions of the thrombin receptor activating peptide, SFLLR-NH2 (TP5-NH2). 2. From a neonatal rat intestinal cDNA library, and from intestinal and kidney-derived cDNA, the coding region of the rat PAR-2 receptor was cloned and sequenced, thereby establishing its close sequence identity with the previously described mouse PAR-2 receptor; and this information, along with a reverse-transcriptase (RT) polymerase chain reaction (PCR) analysis of cDNA derived from gastric and aortic tissue was used to establish the concurrent presence of PAR-2 and thrombin receptor mRNA in both tissues. 3. In the mouse and rat gastric preparations, the PAR-2-derived polypeptides, PP6, PP6-HN2 and PP5-NH2 caused contractile responses that mimicked the contractile actions of low concentrations of trypsin (5 u/ml-1; 10 nM) and that were equivalent to contractions caused by TP5-NH2. 4. The cumulative exposure of the rat LM tissue to PP6-NH2 led to a desensitization of the contractile response to this polypeptide, but not to TP5-NH2 and vice versa, so as to indicate a lack of cross-desensitization between the receptors responsive to the PAR-2 and thrombin receptor-derived peptides. 5. In the rat gastric preparation, the potencies of the PAR-2-activating peptides were lower than the potency of TP5-NH2 (potency order: TP5-NH2 > > PP6-NH2 > or = PP6 > PP5-NH2); PP6 was a partial agonist in this preparation. 6. The contractile actions of PP6 and PP6-NH2 in the rat gastric preparation required the presence of extracellular calcium, were inhibited by nifedipine and were blocked by the cyclo-oxygenase inhibitor, indomethacin and by the tyrosine kinase inhibitor, genistein, but not by the kinase C inhibitor, GF109203X. The contractile responses were not blocked by atropine, chlorpheniramine, phenoxybenzamine, propranolol, ritanserin or tetrodotoxin. 7. In a precontracted rat aortic ring preparation, with an intact endothelium, all of the PAR-2-derived peptides caused a prompt relaxation response that was blocked by the nitric oxide synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME) but not by D-NAME; in an endothelium-free preparation, which possessed mRNA for both the PAR-2 and thrombin receptors, the PAR-2-activating peptides caused neither a relaxation nor a contraction, in contrast with the contractile action of TP5-NH2. The relaxation response to PP6-NH2 was not blocked by atropine, chlorpheniramine, genistein, indomethacin, propranolol or ritanserin. 8. In the rat aortic preparation, the potencies of PP6, PP6-NH2 and PP5-NH2 were greater than those of the thrombin receptor activating peptide, TP5-NH2 (potency order: PP6-NH2 > or = PP6 > PP5-NH2 > TP5-NH2). 9. In the rat aortic preparation, the relaxant actions of the PAR-2-derived peptides were mimicked by trypsin, at concentrations (0.5-1 u ml-1; 1-2 nM) lower than those that can activate the thrombin receptor. 10. The bioassay data obtained with the PAR-2 peptides and with trypsin, along with the molecular cloning/RT-PCR analysis, point to the presence of functional PAR-2 receptors that can activate distinct responses in the gastric and vascular smooth muscle preparations. These responses were comparable to those resulting from thrombin receptor activation in the same tissues, so as to suggest that the receptor for the PAR-2-activating peptides may play a physiological role as far reaching as the one proposed for the thrombin receptor.
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PMID:Rat proteinase-activated receptor-2 (PAR-2): cDNA sequence and activity of receptor-derived peptides in gastric and vascular tissue. 876 73

We have examined protease-mediated activation of the mitogen-activated protein (MAP) kinase cascade in rat aortic smooth-muscle cells and bovine pulmonary arterial fibroblasts. Exposure of smooth-muscle cells to trypsin evoked rapid and transient activation of c-Raf-1, MAP kinase kinase 1 and 2 and MAP kinase that was sensitive to inhibition by soybean trypsin inhibitor. The actions of trypsin were closely mimicked by the proteinase-activated receptor 2 (PAR-2)-activating peptide sequence SLIGRL but not LSIGRL. Peak MAP kinase activation in response to both trypsin and SLIGRL was also dependent on concentration, with EC50 values of 12.1 +/- 3.4 nM and 62.5 +/- 4.5 microM respectively. Under conditions where MAP kinase activation by SLIGRL was completely desensitized by prior exposure of smooth-muscle cells to the peptide, trypsin-stimulated MAP kinase activity was markedly attenuated (78.9 +/- 15.1% desensitization), whereas the response to thrombin was only marginally affected (16.6 +/- 12.1% desensitization). Trypsin and SLIGRL also weakly stimulated the activation of the MAP kinase homologue p38 in smooth-muscle cells without any detectable activation of c-Jun N-terminal kinase. Strong activation of the MAP kinase cascade and modest activation of p38 by trypsin were also observed in fibroblasts, although in this cell type these effects were not mimicked by SLIGRL nor by the thrombin receptor-activating peptide SFLLRNPNDKYEPF. Reverse transcriptase-PCR analysis confirmed the presence of PAR-2 mRNA in smooth-muscle cells but not fibroblasts. Our results suggest that in vascular smooth-muscle cells, trypsin stimulates the activation of the MAP kinase cascade relatively selectively, in a manner consistent with an interaction with the recently described PAR-2. Activation of MAP kinase by trypsin in vascular fibroblasts, however, seems to be independent of PAR-2 and occurs by an undefined mechanism possibly involving novel receptor species.
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PMID:Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases. 900 84

Chinese hamster ovary cells (CHO cells) do not exhibit any Ca2+/calmodulin-stimulated cAMP phosphodiesterase (PDE1) activity. Challenge of CHO cells with agonists for endogenous P2-purinoceptors, lysophosphatidic acid receptors and thrombin receptors caused a similar rapid transient induction of PDE1 activity in each instance. This was also evident on noradrenaline challenge of a cloned CHO cell line transfected so as to overexpress alpha 1B-adrenoceptors. This novel PDE1 activity appeared within about 15 min of exposure to ligands, rose to a maximum value within 30 min to 1 h and then rapidly decreased. In each case, the expression of novel PDE1 activity was blocked by the transcriptional inhibitor actinomycin D. Challenge with insulin of either native CHO cells or a CHO cell line transfected so as to overexpress the human insulin receptor failed to induce PDE1 activity. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1C isoform, did not amplify any fragment from RNA preparations of CHO cells expressing PDE1 activity, although they did so from the human thyroid carcinoma FTC133 cell line. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1A and PDE1B isoforms, successfully amplified a fragment of the predicted size from RNA preparations of both CHO cells expressing PDE1 activity and human Jurkat T-cells. Sequencing of the PCR products, generated using the PDE1A/B primers, yielded a novel sequence which, by analogy with sequences reported for bovine and murine PDE1B forms, suggests that the PDE1 species induced in CHO cells through protein kinase C activation and that expressed in Jurkat T-cells are PDE1B forms.
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PMID:Receptor-mediated stimulation of lipid signalling pathways in CHO cells elicits the rapid transient induction of the PDE1B isoform of Ca2+/calmodulin-stimulated cAMP phosphodiesterase. 900 15

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRP), which is the central catalytic enzyme of HCV replicase. We established a new method to purify soluble HCV NS5B in the glutathione S-transferase-fused form NS5Bt from Escherichia coli which lacks the C-terminal 21 amino acid residues encompassing a putative anchoring domain (anino acids 2990-3010). The recombinant soluble protein exhibited RdRP activity in vitro which was dependent upon the template and primer, but it did not exhibit the terminal transferase activity that has been reported to be associated with the recombinant NS5B protein from insect cells. The RdRP activity of purified glutathione S-transferase-NS5Bt and thrombin-cleavaged non-fused NS5Bt shares most of the properties. Substitution mutations of NS5Bt at the GDD motif, which is highly conserved among viral RdRPs, and at the clustered basic residues (amino acids 2919-2924 and 2693-2699) abolished the RdRP activity. The C-terminal region of NS5B, which is dispensable for the RdRP activity, dramatically affected the subcellular localization of NS5B retaining it in perinuclear sites in transiently overexpressed mammalian cells. These results may provide some clues to dissecting the molecular mechanism of the HCV replication and also act as a basis for developing new anti-viral drugs.
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PMID:RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus NS5B protein truncated at the C-terminal region. 962 34

1. Dysregulated vascular endothelial growth factor (VEGF) expression has been reported in several pathological states based upon evidence of elevated serum VEGF levels. Using two immunoassays for VEGF, this study determines normal plasma and serum VEGF ranges, determines which are more likely to reflect circulating VEGF levels and investigates a potential contribution of VEGF from platelets to VEGF levels detected in serum. 2. The presence of soluble VEGF receptor, sflt-1, at a molar excess of 7:1 significantly reduced measured VEGF levels in both assays. Serum VEGF levels were higher than plasma levels in children [(mean +/- S.E.M.) 306.1 +/- 39.4 versus 107.4 +/- 24.9 pg/ml, P < 0.0001] and adults (249.4 +/- 46.4 versus 76.1 +/- 10.7 pg/ml, P < 0.0001). Serum VEGF increased with clotting time (P = 0.0005 t0 compared with 2 h samples); plasma VEGF levels were not affected by time between sampling and centrifugation. 3. Calcium-induced clotting of platelet-rich but not platelet-poor plasma induced VEGF release with a proportional response between platelet count and VEGF level and isolated platelets released significant quantities of VEGF upon incubation with thrombin. Reverse transcriptase-PCR studies confirmed that platelets express VEGF121 and VEGF165 mRNA. 4. These data suggest that plasma is the preferred medium to measure VEGF levels; a significant and highly variable platelet-mediated secretion of VEGF during the clotting process invalidates the use of serum as an indicator of circulating VEGF levels in disease states.
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PMID:Vascular endothelial growth factor (VEGF) is released from platelets during blood clotting: implications for measurement of circulating VEGF levels in clinical disease. 964 Mar 45

Osteoblasts express protease-activated receptor-1 (PAR-1), which is activated by thrombin or by synthetic peptides corresponding to the new "tethered ligand" N-terminus of PAR-1 created by receptor cleavage. Both thrombin and human PAR-1-activating peptide stimulate an elevation of [Ca2+]i in the human SaOS-2 osteoblast-like cell line, but the peptide stimulates receptor-mediated Ca+ entry, whereas thrombin does not. Stimulation of proliferation in rat primary osteoblast-like cells is greater in response to rat PAR-1-activating peptide than to thrombin. Because the PAR-1-activating peptides are now known to activate PAR-2, the current study was undertaken to investigate whether osteoblasts express this receptor and, if so, whether this could account for the observed discrepancies between responses of osteoblasts to thrombin and to PAR-1-activating peptides. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemical studies demonstrated expression of PAR-2 by primary cultures of rat calvarial osteoblast-like cells. In immunohistochemical studies of embryonic mouse bones, osteoblasts showed positive staining for the presence of PAR-2. Activators of PAR-2 include trypsin, mast cell tryptase, gingipain-R, and synthetic peptides corresponding to the PAR-2 tethered ligand sequence. Treatment of primary rat osteoblast-like cells with rat PAR-2-activating peptide (SLIGRL), or SaOS-2 cells with human PAR-2-activating peptide (SLIGKV), caused a dose-dependent increase in [Ca2+]i. Trypsin or gingipain-R also induced an increase in intracellular calcium concentration, and caused reciprocal cross desensitization. Activators of PAR-2 caused a sharp peak in [Ca2+]i followed by a sustained plateau; [Ca2+]i returned to baseline levels upon treatment with ethylene-glycol tetraacetic acid (EGTA). Treatment of rat osteoblast-like cells in vitro with SLIGRL did not affect thymidine incorporation or endogenous alkaline phosphatase activity. The results presented here demonstrate that osteoblasts express PAR-2, and that such expression is able to account for the observed discrepancies between thrombin and PAR-1-activating peptides in their ability to evoke calcium entry, but not proliferative responses.
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PMID:Expression of protease-activated receptor-2 by osteoblasts. 1061 51

Protease-activated receptor-2 (PAR-2) is distributed throughout the gastrointestinal systems. The present study investigated the role for PAR-2 in the rat salivary glands. PAR-2 mRNA was detected in the sublingual, submaxillary, and parotid glands by a reverse-transcriptase polymerase chain reaction. In the isolated sublingual gland that exhibited the strongest signal for PAR-2, Ser-Leu-Ile-Gly-Arg-Leu-NH(2), a PAR-2-activating peptide, and trypsin, a PAR-2-activating enzyme, but not thrombin that can activate PARs 1, 3, and 4, triggered secretion of N-acetylneuraminic acid, an indicator of mucin, that was a unique major sialic acid detectable after hydrolysis of the sublingual mucin with 0.1 N HCl. The PAR-2-mediated secretion of mucin was attenuated by genistein, a tyrosine kinase inhibitor, but not by inhibitors of protein kinase C and phosphatidyl inositol 3'-kinase. Thus, PAR-2 is expressed by the three distinct salivary glands in the rat, and sublingual PAR-2 appears to play a role in triggering mucin secretion, at least in part, via activation of tyrosine kinase.
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PMID:Activation of protease-activated receptor-2 (PAR-2) triggers mucin secretion in the rat sublingual gland. 1073 43

Development of the primary palate involves a series of processes including cell growth, differentiation, and morphogenesis. To study the molecular and cellular processes during mouse primary palatogenesis, mesenchymal cells were isolated from the primary palate of BALB/cBy embryos (day-11, hour 20). Most of the primary palatal mesenchymal (PPM) cells were morphologically similar to fibroblasts. The population doubling time was about 36 h. At concentrations of 5 and 10 unit/ml, alpha-thrombin significantly stimulated the proliferation of these palatal cells by 2- to 2. 4-fold compared to untreated controls over a 72 hour incubation period. Reverse transcriptase-polymerase chain reaction using primers based on the mouse type 1 protease-activated thrombin receptor (PAR1) detected PAR1 mRNA in the PPM cells, the authenticity of which was confirmed by partial DNA sequencing. Blocking of the alpha-thrombin proteolytic site with the highly specific inhibitor D-phenylalanyl-prolyl-arginyl chloromethyl ketone significantly suppressed the mitogenic effect of thrombin on the PPM cells by 71%. These results suggest that PAR1 is present on PPM cells in the mouse embryo and that serine protease activity is important for the receptor activation.
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PMID:Expression of functional type 1 protease-activated thrombin receptors by mouse primary palatal mesenchymal cells in vitro. 1097 55


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