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)

HMG-CoA reductase inhibitors (statins) are cholesterol-lowering drugs and reduce the risk of myocardial infarction and stroke. In this study we investigated whether rosuvastatin, a new, potent HMG-CoA reductase inhibitor, upregulates endothelial nitric oxide (NO) expression and activity and protects from cerebral ischaemia in mice. Endothelial cells in culture and 129/SV mice were chronically treated with rosuvastatin. The expression and activity of endothelial NO synthase (eNOS) was determined by reverse-transcriptase polymerase chain reaction (RT-PCR), Western blotting and arginine-citrulline assays. Cerebral ischaemia was induced by occlusion of the middle cerebral artery (MCAo) for 2 h and infarct size was determined after 22 h of reperfusion. Treatment of endothelial cells with rosuvastatin concentration- and time-dependently upregulated eNOS mRNA and protein expression. In aortas of 129/SV wild-type mice, treatment with 0.2, 2, and 20 mg kg(-1) rosuvastatin subcutaneously (s.c.) for 10 days significantly upregulated eNOS mRNA by 50, 142, and 205%, respectively. NOS activity was significantly increased by 75, 145, and 320%, respectively. Stroke volume after 2-h MCAo was reduced by 27, 56, and 50% (for 0.2, 2 and 20 mg kg(-1), respectively). Serum cholesterol and triglygeride levels were not significantly lowered by the treatment. The novel HMG-CoA reductase inhibitor rosuvastatin dose-dependently upregulates eNOS expression and activity and protects from cerebral ischaemia in mice. The effects are independent of changes in cholesterol levels and are equivalent or even superior to the protective effects by simvastatin and atorvastatin in this animal model.
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PMID:Rosuvastatin, a new HMG-CoA reductase inhibitor, upregulates endothelial nitric oxide synthase and protects from ischemic stroke in mice. 1203 49

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) essential for virus replication. Several consensus sequence motifs have been identified in NS5B, some of which have been shown to be critical for its enzymatic activity. A unique beta-hairpin structure located between amino acids 443 and 454 in the thumb subdomain has also been shown to play an important role in ensuring terminal initiation of RNA synthesis in vitro. However, the importance of these sequence and structural elements in viral RNA replication in infected cells has not been established, mainly due to the lack of a reliable cell culture system for HCV. In this study, we investigated the effect of several single amino acid substitutions and beta-hairpin truncations in NS5B on viral RNA replication by using the subgenomic replicon cell culture system. A strong correlation between in vitro polymerase activity and viral RNA replication was observed with most of the substitutions. Interestingly, truncations of the beta-hairpin (by four and eight amino acid residues, respectively), which did not reduce the in vitro enzymatic activity, completely abolished the ability of the replicon RNA to replicate in Huh-7 cells, demonstrating its essential role in viral RNA replication. Furthermore, a conservative substitution in motif D, from an arginine residue (AMTR(345)), which is conserved among all HCV isolates, to a lysine residue, resulted in significant improvements in both transient RNA replication and colony formation efficiencies. This result also correlates with a previous observation that the enzymatic activity of NS5B increased by about 50% when the same NS5B substitution was introduced (V. Lohmann, F. Korner, U. Herian, and R. Bartenschlager, J. Virol. 1997, 71, 8416-8428).
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PMID:Mutations in NS5B polymerase of hepatitis C virus: impacts on in vitro enzymatic activity and viral RNA replication in the subgenomic replicon cell culture. 1208 28

Results regarding the nitric oxide (NO) system in uraemia are contradictory. L-arginine, the precursor of NO, is also metabolized by arginase to form ornithine and urea. In the present study, endothelial NO production and arginine metabolism in uraemia were assessed. In addition an in vivo model was used to examine excess consumption of NO in uraemia. NO and amino acid measurements were made from basal and stimulated (by bradykinin) uraemic and control endothelial cells. Reverse-transcriptase PCR was used to assess endothelial NO synthase (eNOS) and inducible NOS (iNOS) expression. Finally, aortae of uraemic rats were stained for nitrotyrosine (a marker of peroxynitrite). Basal uraemic cells produced more NO than the control cells. L-arginine levels were greater in uraemic (supernatants/cells), but ornithine levels were higher in control (supernatants/cells). Following stimulation, NO levels in supernatants were similar, but the rise in NO production was greater in control compared with uraemic cells; l-arginine levels still remained higher in uraemic supernatants/cells. Differences in ornithine concentration (supernatants/cells) disappeared following bradykinin stimulation, due to a rise in ornithine levels in the uraemic group. There was no difference in eNOS expression, nor was iNOS detected in either group. Only aortae from uraemic rats showed evidence for nitrotyrosine staining. These studies demonstrated increased basal NO release in uraemic endothelial cells, perhaps by inhibition of arginase and hence diversion of arginine to the NO pathway. The increased NO produced under basal conditions may be inactive due to excessive consumption, resulting in peroxynitrite formation. Interestingly, bradykinin appears to restore arginase activity in uraemia, resulting in normalization of NO production.
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PMID:Altered L-arginine metabolism results in increased nitric oxide release from uraemic endothelial cells. 1209 1

The hepatitis C virus (HCV) encodes an RNA-dependent RNA polymerase (NS5B), which is indispensable for the viral genome replication. Although structural comparison among HCV NS5B, poliovirus 3D-pol, and human immunodeficiency virus-reverse transcriptase RNA-dependent polymerase reveals the canonical palm, fingers, and thumb domains, the crystal structure of HCV NS5B highlights the presence of a unique A1-loop, which extends from the fingers to the thumb domain (amino acids 12-46), providing many contact points for the proposed "closed" conformation of the enzyme. The polymerase also possesses a tunnel, which starts at the active site and terminates on the back surface of the enzyme. This tunnel of 19 A contains five basic amino acids, which may be engaged in NTP trafficking. In the present study, we exploited the crystal structure of the enzyme to elucidate the involvement of these two structural motifs in enzyme activity by site-directed mutagenesis. As predicted, the replacement of leucine 30 located in the Lambda 1-loop is detrimental to the NS5B activity. Heparin-Sepharose column chromatography and analytical ultracentrifugation experiments strongly suggest a local alteration in the structure of the Leu-30 mutant. An analysis of amino acid substitutions in Arg-222 and Lys-151 within the putative NTP tunnel indicates that Arg-222 was critical in delivering NTPs to the active site, whereas Lys-151 was dispensable. Interestingly, the substitution of lysine 151 for a glutamic acid resulted in an enzyme that was consistently more active in de novo synthesis as well as by "copy-back" mechanism of a self-primed substrate when compared with the wild type NS5B enzyme. Burst kinetic analyses indicate that the gain in function of K151E enzyme was primarily the result of the formation of more productive pre-initiation complexes that were used for the elongation reaction. In contrast to the recent observations, both the wild type and mutant enzymes were monomeric in solution, whereas molecules of higher order were apparent in the presence of RNA template.
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PMID:Modulation of hepatitis C virus RNA-dependent RNA polymerase activity by structure-based site-directed mutagenesis. 1214 89

The aim of the present study was to investigate the effects of melatonin on non-adrenergic, non-cholinergic (NANC) relaxant neurotransmission in the gastrointestinal tract, which is mainly mediated by nitrergic and peptidergic mechanisms. Melatonin (10(-7)-10(-3) M) had no effect on the basal tonus of the rat gastric fundus smooth muscle. Relaxant responses following electrical stimulation(40 V; 0.5 ms pulse duration; 10 s stimulation duration) under NANC conditions on a 5-hydroxytryptamine (5-HT, 10(-7) M) contraction plateau were elicited at frequencies in the range of 0.5-16 Hz. Melatonin significantly reduced these inhibitory NANC responses (16 Hz without melatonin: -103 +/- 6.3%; melatonin 10(-5) M: -80.4 +/- 7.5%; melatonin 10(-4) M: -39.1 +/- 17.1%). Intracellular recording was carried out in a mouse colonic preparation. Electrical neural stimulation of the mouse colonic neurons caused biphasic intracellular hyperpolarization in smooth-muscle cells. The initial fast component is apamin-sensitive, and the following slow component is dependent on nitrergic mechanisms, as it is abolished in the presence of NG-nitro-L-arginine (L-NNA). Melatonin significantly reduced the nitric oxide-dependent slow component of neurally transmitted hyperpolarization, whereas the initial fast component was left unchanged. In a synaptosomal preparation of the enteric nervous system of rat intestine, enzymatic nitric oxide synthase (NOS) activity was significantly reduced by melatonin at concentrations ranging from 10(-7) to 10(-4) M (basal preparation including cofactors: 61.2 +/- 9.4 fmol/mg; melatonin 10(-4) M: 39.2 +/- 6.9 fmol/mg). Reverse transcriptase-polymerase chain reaction (RT-PCR) studies were conducted to investigate the melatonin receptors (mt(1), MT(2) and MT(3)) present in the esophagus, stomach and ileum of the rat. The presence of mt1 mRNA expression alone, but not of mRNA expression for MT(2) or MT(3), was demonstrated in the tissues. In conclusion, this study demonstrates that melatonin reduces the functional inhibitory NANC response. It shows that this effect may be the result of a reduction of the nitrergic component of the smooth-muscle inhibitory junction potential (IJP) and related to direct inhibition of NOS activity in enteric synaptosomes. The presence of mt1 receptor transcripts adds supportive evidence for a possible physiological role of melatonin within the enteric nervous system.
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PMID:Melatonin reduces non-adrenergic, non-cholinergic relaxant neurotransmission by inhibition of nitric oxide synthase activity in the gastrointestinal tract of rodents in vitro. 1215 44

The arginine deiminase system (ADS) is of critical importance in oral biofilm pH homeostasis and microbial ecology. The ADS consists of three enzymes. Arginine is hydrolyzed by AD (ArcA) to generate citrulline and ammonia. Citrulline is then converted to ornithine and carbamoylphosphate via ornithine carbamoyltransferase (ArcB). Finally, carbamate kinase (ArcC) transfers a phosphate from carbamoylphosphate to ADP, yielding ATP. Ammonia production from this pathway protects bacteria from lethal acidification, and ATP production provides a source of energy for the cells. The purpose of this study was to initiate a characterization of the arc operon of Streptococcus rattus, the least cariogenic and sole ADS-positive member of the mutans streptococci. Using an arcB gene fragment obtained by degenerate PCRs, the FA-1 arc operon was identified in subgenomic DNA libraries and sequence analysis was performed. Results showed that the genes encoding the AD pathway in S. rattus FA-1 are organized as an arcABCDT-adiR operon gene cluster, including the enzymes of the pathway, an arginine-ornithine antiporter (ArcD) and a putative regulatory protein (AdiR). The arcA transcriptional start site was identified by primer extension, and a sigma(70)-like promoter was mapped 5' to arcA. Reverse transcriptase PCR was used to establish that arcABCDT could be cotranscribed. Reporter gene fusions and AD assays demonstrated that the operon is regulated by substrate induction and catabolite repression, the latter apparently through a CcpA-dependent pathway.
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PMID:Characterization of the arginine deiminase operon of Streptococcus rattus FA-1. 1500 49

A gel mobility-shift assay was used to demonstrate the binding of the Sindbis virus transcriptase to the promoter for the synthesis of subgenomic (SG) RNA. The assay made use of a P15 fraction (the cell fraction that is pelleted at 15,000 x g) from cells infected with recombinant vaccinia virions expressing various Sindbis virus nonstructural proteins (nsPs) and a (32)P-labeled 24-mer oligoribonucleotide representing the minimal sequence with SG promoter activity. By itself, nsP4, the viral RNA-dependent RNA polymerase, did not bind to the SG promoter; rather, all four nsPs were required for the binding of the transcriptase to the promoter. UV crosslinking of the transcriptase to a thiouridine-containing SG promoter, followed by V8 protease digestion of the complex, generated a peptide fragment that was bound to the SG promoter. This peptide fragment contained a sequence that corresponded to residues 329-334 of nsP4. This peptide may be in the fingers domain of nsP4. The peptide that was identified contained Arg residues at positions 331 and 332. Another Arg is present at position 327. By changing each of the Arg residues to Ala, we demonstrated that only the Arg residues at positions 331 and 332 were required for binding nsP4 to the SG promoter.
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PMID:Identification of the amino acid sequence in Sindbis virus nsP4 that binds to the promoter for the synthesis of the subgenomic RNA. 1519 79

The human kallikrein 12 (KLK12) gene is a new member of the KLK gene family, some members of which are implicated in the initiation and progression of cancer. In this study, we examined 50 non-cancerous tissues from Japanese patients with primary gastric cancer to determine the presence of genetic polymorphisms in the KLK12 gene using polymerase chain reaction (PCR)-single-strand conformation polymorphism and sequencing. Four different types of genetic polymorphisms were identified: one at a splice-donor site of intron 4 (c.457+2T>C), two in exon 6 (c.618_619delTG:p.Cys206fsX72 and c.735G>A:p.Met245Ile), and one in intron 3. The c.457+2T>C polymorphism was observed at a high frequency (allele frequency:0.63), compared to the frequencies of the two polymorphisms in exon 6 (allele frequency:0.01). Reverse transcriptase (RT)-PCR and Western blot analyses revealed that the c.457+2T>C polymorphism was associated with a splicing abnormality and that the expression of the human KLK12 protein (hK12), corresponding to the putative serine protease, was absent in individuals with a c.457+2C/C genotype but not in individuals with the T/T or T/C genotypes. We also found that recombinant His6-tagged hK12 has activity that cleaves chromogenic substrate (H-D-Pro-L-Phe-L-Arg-p-nitroaniline dihydrochloride), that is, serine protease activity. These results indicate that individuals with the c.457+2C/C genotype have no substantial expression of hK12 serine protease.
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PMID:Splice-site genetic polymorphism of the human kallikrein 12 (KLK12) gene correlates with no substantial expression of KLK12 protein having serine protease activity. 1530 Aug 58

Reverse transcriptase is an essential retroviral enzyme that replicates the single-stranded RNA genome of the retrovirus producing a double-stranded DNA copy, which is subsequently integrated into the host's genome. We have previously reported that processive DNA synthesis of Moloney murine leukemia virus reverse transcriptase (MMLV RT) is severely compromised by substitution of an Ala for the fingers domain residue Arg 116. In order to further investigate the role of Arg 116 in interactions of MMLV RT with nucleic acids, we have determined the crystal structure of the R116A N-terminal fragment and characterized the binding of two self-complementary DNA duplexes [d(CATGCATG)2 and d(CGCGCGCG)2] to both the wild-type and R116A fragments by isothermal titration calorimetry. The resultant thermodynamic profiles extrapolated to 25 degrees C reveal that binding of the wild-type N-terminal fragment to both DNA duplexes is enthalpy-driven and characterized by an unfavorable entropy. Although the temperature dependence of the respective protein-DNA binding enthalpies is markedly different reflecting distinct heat capacity changes, the binding free energies are nearly identical and relatively invariant to temperature (DeltaG approximately -6.0 kcal x mol(-1)). In contrast to the wild-type fragment, the R116A fragment exhibits no measurable affinity for either DNA duplex, yet its crystal structure reveals no significant changes when compared to the wild-type structures. We suggest that hydrogen-bonding interactions involving the fingers domain residue Arg 116 are critical for DNA binding as well as processive DNA synthesis by MMLV RT.
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PMID:Structural and energetic characterization of nucleic acid-binding to the fingers domain of Moloney murine leukemia virus reverse transcriptase. 1532 91

The characteristics and function of human lymphocytes in tuberculous morbid site were studied. Exudative-sensitized lymphocytes in tuberculous pleural fluid reacted to the specific antigen more effectively and produced higher titers of cytokines including interferon gamma (IFN-gamma) than circulating lymphocytes. CD4+/CD8- T-cell subset is responsible for the antigen-specific IFN-gamma production in pleural T lymphocytes of patients with tuberculous pleurisy. Thus, activated T lymphocytes concern the production of cytokines at the morbid site and they effectively exert local cellular immunity through the action of such cytokines. Immunofluorescence study showed increased production of inducible nitric oxide synthase (iNOS) and peroxynitrite in BCG-inoculated human alveolar macrophages (AM). Reverse transcriptase-polymerase chain reaction methods also revealed the higher expression of iNOS-coding mRNA. Colony assay demonstrated that human AM effectively killed BCG in their cytoplasm. However, treatment of AM with NG-monomethyl-L-arginine monoacetate resulted in markedly reduced killing activity. These results clearly show that BCG-induced NO and its reactive product with the oxygen radical, peroxynitrite, could play an important role in BCG killing in human AM. We measured the pleural concentrations of IFN-gamma, interferon-gamma-inducing cytokines; interleukin (IL)-12 and IL-18 and interferon-gamma-inducible chemokines; IFN-gamma-inducible protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T cell alpha chemoattractant (I-TAC). These cytokines and chemokines in tuberculous pleural effusions were much higher than those in malignant pleural effusions. These findings indicate that IFN-gamma plays an important role in the cell mediated immunity in tuberculosis.
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PMID:[Tuberculous infection and biological response in man]. 1555 42


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