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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Type I plasminogen activator inhibitor (PAI-1) is a 50 kDa glycoprotein belonging to the serine protease superfamily. PAI-1 is consistently and dramatically upregulated in a variety of fibrotic diseases. The aim of this study was to compare PAI-1 expression in normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanism that may lead to induced PAI-1 expression. Twenty-five OSF specimens and six normal buccal mucosa were examined by immunohistochemistry. The activity of PAI-1 from cells cultured from OSF and normal buccal mucosa were assayed using reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blots. PAI-1 expression was significantly higher in OSF specimens and expressed mainly by fibroblasts, endothelial cells, and inflammatory cells. In addition, OSF exhibited higher PAI-1 expression than normal buccal mucosa fibroblast (BMF) both in mRNA and protein levels. To verify whether arecoline, a major areca nut alkaloid, could affect PAI-1 expression by human BMFs, RT-PCR and Western blots were used. The results demonstrated highly elevated PAI-1 mRNA and protein expression in normal human BMFs stimulated by arecoline. Taken together, these results suggest that PAI-1 expression is significantly upregulated in OSF tissues from areca quid chewers, and arecoline may be responsible for the enhanced PAI-1 expression in vivo.
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PMID:The upregulation of type I plasminogen activator inhibitor in oral submucous fibrosis. 1267 56

Laser capture microdissection (LCM) enables the dissection of heterogeneous components of tissues, helping to investigate the molecular properties of these tissues. We have reported gene expression profiles in human benign prostatic hyperplasia (BPH) using LCM and quantitative real-time PCR. In the current work, we extended the previous observations. Firstly, we studied the relationship between the number of dissected acini and amplification by PCR, and found that androgen receptor (AR) and 18S rRNA transcripts were successfully amplified from total RNA obtained from one prostate acinus. Furthermore, LCM-dissected samples were applicable to methylation-specific PCR of E-cadherin promoter gene after bisulfite modification of genomic DNA. Next, we performed cDNA microarray analysis to screen gene expression profiles in the epithelium and stroma. RNA was amplified by T7-RNA polymerase and labeled with Cy3 and Cy5. Epithelium-related or stroma-related genes were identified through cDNA microarray. We confirmed true gene expression levels by quantitative real-time PCR. In epithelium, E-cadherin and serine protease 2, Kunitz-type gene expression levels were significantly elevated, while the connective tissue growth factor gene expression level was significantly elevated in stroma. Thus, LCM-dissected samples were applicable to various molecular examinations including methylation-specific PCR and cDNA microarray, and this will contribute to a precise understanding of the molecular profiles of prostate glands.
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PMID:Gene expression profiles of human BPH (II): Optimization of laser-capture microdissection and utilization of cDNA microarray. 1268 Feb 12

Thrombin is a serine protease involved in many physiological functions and its receptor. the protease-activated receptor-1 (PAR-1), has a wide tissue distribution. We hypothesized that PAR-1 is expressed in gastric epithelial cells and that thrombin can modulate defence mechanisms through PAR-1. The rat gastric epithelial cell line (RMG1) and gastric biopsy specimens from gastritis patients were used in the study. Reverse transcriptase polymerase chain reaction analysis showed that the thrombin receptors PAR-1, PAR3 and PAR-4 are expressed by RGM1 gastric epithelial cell line. Immunohistochemical and electron microspcopic studies also showed PAR-1 expression in human gastric epithelial cells. Thrombin stimulated the secretion of mucin and prostaglandin E2 (PGE2) formation in RGM1 cells in a dose-dependent manner. PAR-1 agonist also stimulated PGE2 formation. In addition, thrombin significantly increases the expression of the PGE2 receptors EP2-R and EP4-R in RGM1 cells. In conclusion, the results of the present study showed for the first time that gastric epithelial cells express thrombin receptors and that these receptors may play a protective role in the gastric mucosa.
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PMID:Expression and cytoprotective effect of protease-activated receptor-1 in gastric epithelial cells. 1273 39

Degenerate oligonucleotide primers derived from conserved serine protease inhibitors were used to amplify a 90-base pair (bp) amplicon from an Ancylostoma caninum adult-stage complementary deoxyribonucleic acid (cDNA) library by polymerase chain reaction (PCR). The amplicon was labeled and used as a probe to screen the library, and a 2,300-bp cDNA clone was identified. The 5' end of the molecule was obtained from adult cDNA by 5'-RACE. The complete sequence named A. caninum Kunitz-type protease inhibitor (Ac-kpi-1) was 2,371 bp and encoded a 759-amino acid open reading frame. The deduced amino acid sequence had a calculated molecular weight of 84,886 Da and contained an amino terminal signal peptide, suggesting that the protein is secreted. Analysis of the predicted protein sequence indicates 12 highly conserved Kunitz-type serine protease inhibitor domains connected by short, conserved spacers. On the basis of sequence analysis, the first 11 domains are predicted to be active serine protease inhibitors based on the P1 amino acid. Domains 5-8 have identical amino acid sequences, and the remaining domains are 38-88% identical. Domain 12 lacks several of the conserved cysteine residues and has an atypical amino acid in the P1 position, suggesting that it is nonfunctional. Reverse transcriptase-PCR indicated that the Ac-kpi-1 messenger ribonucleic acid is present in egg, L1, L3, and adult stages but is most abundant in the adult stage. Ac-KPI-1 is most similar in domain architecture to several extracellular matrix proteins involved in cellular remodeling during insect development. In addition, there are 44 nematode proteins containing one or more Kunitz domains in GenBank, including several with multiple domains.
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PMID:Molecular cloning of a novel multidomain Kunitz-type proteinase inhibitor from the hookworm Ancylostoma caninum. 1276 Jun 67

Serine at position 624 of PA subunit of the Influenza A virus RNA polymerase is the active site of a serine protease domain. To examine the role of this protease activity in the viral infection cycle, we compared the growth and the pathogenesis of influenza A/WSN/33 (WSN) and the virus encoding a PA with a S624A mutation (S624A virus), which were generated by the plasmid-based rescue system. The growth of S624A virus was less extensive than that of WSN in cells. The LD50 of S624A virus and WSN for intranasal infection in Balb/C mice was 4.0 x 10(4) and 9.3 x 10(3) PFU, respectively. That for intracranial infection was 460 and 200 PFU, respectively. These data indicated that Ser624, the active site of the serine protease activity of PA, is not essential for viral growth and pathogenesis, but is required for the maximal viral growth.
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PMID:Ser624 of the PA subunit of influenza A virus is not essential for viral growth in cells and mice, but required for the maximal viral growth. 1450 82

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

TMPRSS2 is a type II transmembrane-bound serine protease that has gained interest owing to its highly localized expression in the prostate and its overexpression in neoplastic prostate epithelium. Once activated, the serine protease domain of TMPRSS2 is released from the cell surface into the extracellular space. PAR (protease-activated receptor)-2 belongs to a family of G-protein-coupled receptors (PAR-1-4) that are activated by specific serine proteases, which are expressed in many normal and malignant cell types. Previous in vitro studies on prostate cancer cells suggest a role for PAR-2 in prostate cancer metastasis. A polyclonal anti-human TMPRSS2 antibody was generated against the TMPRSS2 serine protease domain. The antibody showed specific reactivity with recombinant expressed TMPRSS2, and so was used to extract and purify the cleaved active TMPRSS2 protease from prostate cancer cells. Reverse transcriptase PCR and Western blot analysis were used to show the expression of both TMPRSS2 and PAR-2 in the androgen-dependent LNCaP prostate cancer cell line. Treatment of LNCaP cells with the cellular immunopurified TMPRSS2 protease induced a transient increase in intracellular calcium, which is indicative of G-protein-coupled-receptor activation. This calcium mobilization was inhibited by cellular pre-treatment with a specific PAR-2 antagonist, but not with a PAR-1 antagonist; inhibition of the protease activity also failed to mobilize calcium, suggesting that TMPRSS2 is capable of cleaving and thereby activating the PAR-2 receptor. The calcium mobilization was also inhibited by cellular pre-treatment with suramin or 2-APB (2-aminoethoxydiphenyl borate), indicating that a G-protein pathway is involved and that subsequent calcium release is mainly from intracellular stores. The present study describes how TMPRSS2 may contribute to prostate tumour metastasis via the activation of PAR-2.
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PMID:The membrane-anchored serine protease, TMPRSS2, activates PAR-2 in prostate cancer cells. 1553 83

Worldwide over 170 million people are chronically infected with the hepatitis C virus and hence at high risk to develop fatal liver disease. There is no vaccine available and the standard therapy [(pegylated) interferon alfa plus ribavirin] is only effective in 50-60% of patients and is associated with important side-effects. The discovery of novel antiviral strategies to selectively inhibit HCV replication has long been hindered by the lack of convenient cell culture models for the propagation of HCV. This hurdle has been overcome first with the establishment of the HCV replicon system in 1999 and, in 2005, with the development of robust HCV cell culture models. In recent years also mouse models have been elaborated that will be instrumental in assessing the in vivo efficacy of novel drugs. The viral serine protease and the viral RNA dependent RNA polymerase have shown to be excellent targets for selective anti-HCV therapy. Clinical studies with a limited number of HCV protease and polymerase inhibitors resulted in encouraging results. However, and not unexpected, preclinical evidence suggest that the virus may become rapidly resistant to such inhibitors. Combination therapy of drugs with different mode of action and resistance profiles may thus be required. Alternative strategies, such as the use of non-immunosuppressive cyclosporin A analogues with potent anti-HCV activity, may prove important, in particular since such compounds may have a resistance profile that is very different from that of protease or polymerase inhibitors.
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PMID:Selective inhibitors of hepatitis C virus replication. 1684 38

Polyamine analogs are known to inhibit tumorigenesis at least in part by mimicking some of the regulatory roles of natural polyamines. To begin the identification of those signaling pathways that are involved in differential cellular responses to the synthetic conformationally restricted polyamine analog CGC-11093, we conducted gene expression profiling, proteomic, and genome-wide DNA methylation and histone acetylation analyses of the HCT116 colon adenocarcinoma cell line after treatment with this analog. Gene expression analysis was performed using Affymetrix GeneChip human genome U133 Plus 2.0 arrays. Changes in protein expression were evaluated using 2D polyacrylamide gels followed by LCMS/MS. DNA methylation was measured using 6,800 element CpG island microarrays. Treatment of cells with CGC-11093 at concentrations ranging from 0.1 to 10 microM caused inhibition of cell growth and metabolic activity, but only minimally affected cell viability. Gene expression analysis showed concentration-dependent effects of CGC-11093 on the DNA/RNA binding transcription factor, cell cycle, signaling, transport, cytoskeletal/structural, and serine protease genes. Functional gene analysis revealed distinct expression patterns related to inhibition of cell cycle control, TGF beta signaling, proteasome and RNA polymerase pathways, upregulation of the aminoacyl-tRNA synthesis pathway, and perturbations in the MAPK and Wnt signaling pathways. Microarray results were validated for selected genes with real time RT PCR. Proteomics analysis showed correlative changes in the expression of proteins involved in the regulation of proteasome function (proteasome subunit Y) and tRNA synthesis. CGC-11093 treatment did not produce any detectable changes in DNA methylation or histone acetylation in cells. This study validates specific target pathways for a specific conformationally restricted polyamine analog and suggests the utility of combined gene and DNA methylation microarrays along with proteomic analyses as a useful approach to the evaluation of the mechanisms of action of anticancer drugs.
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PMID:Pharmacogenomics of the polyamine analog 3,8,13,18-tetraaza-10,11-[(E)-1,2-cyclopropyl]eicosane tetrahydrochloride, CGC-11093, in the colon adenocarcinoma cell line HCT1161. 1712 31

The next generation of anti-HCV therapeutics agents will fall into new interferons and interferon inducers, alternatives to ribavirin, specific HCV inhibitors and immune therapies. According to HCV variability, the clinical success of HCV-targeted drugs will depend on their ability to suppress all viral variants as well as prevent the emergence of resistant viruses. Examination of these properties are limited in the absence of convenient animal model of HCV infection and limited possibilities studies on HCV replicons. The NS3-4A serine protease and the NS5B RNA polymerase have emerged as popular target for antiviral intervention. Blockage ofNS3 activity is expected to inhibit HCV replication by direct suppression of viral protein production and restoration of host responsiveness to IFN. The experience with synthetic agonists of Toll-like receptors 7 and 9 have begun to show their potential in controlling HCV infection. The new anti-HCV agents have progressed through early-phase clinical trials. Based on HCV infection history it seems that IFN will be the gold standard of the therapy and new agents probably will administer in combination with IFN.
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PMID:[Treatment of chronic hepatitis C--new drugs, new hope]. 1768 54


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