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)

1. We have used the patch-clamp technique to study modulation of the inwardly rectifying K+ current (IK(IR)) in cultured bovine pulmonary artery endothelial cells (CPAE cells). In whole-cell mode, IK(IR) was defined as the Ba(2+)-sensitive current. In single channel recordings, we observed a strongly inwardly rectifying and K(+)-selective channel with a conductance of 31 +/- 3 pS. 2. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and functional data suggest that the endothelial IRK is most probably Kir2.1. 3. Intracellular ATP is required to prevent run-down of IRK in whole-cell mode. Single channel activity disappeared in inside-out patches exposed to ATP-free solution and in cell-attached patches on cells exposed to metabolic inhibition (KCN, 2-deoxyglucose). 4. The non-hydrolysable ATP analogues, ATP gamma S and adenylyl imidodiphosphate (AMP-PNP), did not prevent run-down. Run-down did not occur in the presence of okadaic acid, a phosphatase inhibitor, but was enhanced in the presence of protamine, an activator of phosphatase 2A (PP2A). 5. GTP gamma S and AlF4- inhibited IRK, also in the presence of ATP. GTP beta S antagonized the GTP gamma S effect. Pretreatment of the cells with PTX did not affect the GTP gamma S-induced inhibition. Okadaic acid, however, slowed this inhibition. 6. Neither activation of protein kinase A (PKA) nor activation of protein kinase C (PKC) affected IRK. Additionally, neither cytochalasin B nor a high concentration of intracellular Ca2+ affected the time course of IRK run-down. 7. We conclude that run-down of IRK is probably due to dephosphorylation by PP2A. Activation of a PTX-insensitive G protein inhibits this current by a mechanism that is neither mediated via the PKA and PKC pathways nor by intracellular Ca2+, but supposedly by a G protein-dependent activation of a phosphatase.
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PMID:Modulation of inwardly rectifying potassium channels in cultured bovine pulmonary artery endothelial cells. 940 63

We have recently detected de-novo transcripts of the predominantly muscle-specific myotonin protein kinase gene in human preimplantation embryos from the 1-cell to the 4-cell stages. Others have shown de-novo transcripts of the Y-linked genes, ZFY and SRY, in the 1-cell zygote. In order to assess the significance of early transcription of these predominantly tissue-specific genes in preimplantation development, we have analysed individual human oocytes and preimplantation embryos for the presence of transcripts of two further tissue-specific genes, alpha-globin and beta-globin, and two house-keeping genes, HPRT and APRT. Reverse transcriptase polymerase chain reaction assays were developed to the required single cell sensitivity, using human red blood cells and fibroblasts, prior to their application to human oocytes and embryos. As expected, transcripts of the house-keeping genes, HPRT and APRT, were detected at all stages of preimplantation development. Transcripts of 'tissue-specific' alpha-globin were readily detected in preimplantation embryos from the 1-cell stage. However, transcripts of beta-globin were detected only rarely (in only one of the 11 embryos analysed). This difference may be due to the fact that alpha-globin contains a CpG island. A survey of the data on gene expression in early human development suggests that CpG-island-containing genes may be expressed in preimplantation embryos. Expression of these genes in gametes and early embryos may be involved in the survival of CpG islands in evolution.
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PMID:Transcription of tissue-specific genes in human preimplantation embryos. 940 90

In response to dibutyryl cyclic AMP (dbcAMP) and all-trans retinoic acid (ATRA), HL60 cells differentiate into granulocyte-like cells. Membrane-associated phospholipase D (PLD) activity in response to guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or phorbol myristate acetate (PMA) was upregulated by these treatments. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that both hPLD1a and hPLD1b mRNAs were expressed in HL60 cells and that their expression levels increased during differentiation. hPLD2 mRNA levels rose dramatically during differentiation. These results suggest that the PLD genes undergo changes in transcriptional regulation during granulocytic differentiation of HL60 cells.
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PMID:Increased mRNA expression of phospholipase D (PLD) isozymes during granulocytic differentiation of HL60 cells. 951 45

1. The effects of 5-hydroxytryptamine (5-HT) in rat cardiac preparations were studied. 5-HT up to 10 microM failed to affect contractility in papillary muscles. However, in electrically driven (1 Hz) left atria 5-HT exerted a positive inotropic effect that started at 1 microM and attained its maximum at 10 microM (312+/-50% of predrug value, n=8). 2. 5-HT 10 microM stimulated the content of inositol-1,4,5-trisphosphate but not of cyclic AMP in rat left atria. 3. Plasma and serum levels of 5-HT amounted to about 0.3 microM and 15 microM, respectively. 4. The selective 5-HT4 receptor antagonists GR 125487 (10 nM and 1 microM) and SB 203186 (1 microM) did not attenuate the positive inotropic effect of 5-HT in rat left atria. In contrast, the 5-HT2 receptor antagonist ketanserin (5 nM, 50 nM, 1 microM) resulted in a concentration-dependent diminution of the positive inotropic effect of 5-HT in rat left atria. 5. Reverse transcriptase polymerase chain reaction with specific primers detected mRNA of the 5-HT2A receptor in rat atria and ventricles, while expression of the 5-HT4 receptor was confined to atria. 6. It is suggested that the positive inotropic effect of 5-HT in electrically driven rat left atria is mediated by ketanserin-sensitive 5-HT2A receptors and not through 5-HT4 receptors.
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PMID:Receptor mechanisms involved in the 5-HT-induced inotropic action in the rat isolated atrium. 955 3

The ATP requirement of influenza A virus RNA-dependent RNA polymerase was studied during in vitro transcription reactions. In complete transcription reactions, the Km for ATP was 10-fold higher than the Km values for the other NTPs. However, during transcription elongation the Km for ATP was as low as the Km values for the other NTPs, suggesting a special requirement for ATP during transcription initiation. Gel analysis of RNA products of transcription initiation reactions showed that the incorporation of AMP into nascent RNA was more efficient at positions 4, 6 and 7 relative to the template RNA than at position 5. The polymerase produced short, abortive transcripts with lengths corresponding to positions 3 and 4 relative to the template but never to position 5 or longer. These results suggest that incorporation of AMP at position 5 induces the influenza A virus polymerase to go through a transition from a transcription initiation to an elongation complex. This functional change of the polymerase complex rather than a requirement for ATP beta-gamma bond hydrolysis is the most likely reason for the particularly high Km for ATP during the early phase of transcription. This conclusion is supported by the fact that the ATP analogue ATPgammaS [adenosine 5'-O-(3-thiotriphosphate)] can efficiently replace ATP in in vitro transcription reactions and shows a comparable drop of Km between transcription initiation and elongation.
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PMID:Variation in ATP requirement during influenza virus transcription. 960 18

The cytokine, interleukin-6 (IL-6), is produced by osteoblasts and may in part mediate parathyroid hormone (PTH)-stimulated bone resorption. The goals of the present study were: (1) to examine PTH induction of IL-6 expression in 7-day-old mouse calvarial organ cultures; (2) to assess the role of intracellular signaling pathways in this model; and (3) to determine whether PTH regulates IL-6 expression by a transcriptional mechanism. Northern blot analysis of calvarial RNA showed that PTH(1-34) at 0.1-100 nmol/L induced IL-6 mRNA at 0.5 h with a peak at 2 h. Forskolin at 10 micromol/L and 8-bromocyclic-AMP at 3 mmol/L also induced IL-6 mRNA with a peak at 2 h. Phorbol myristate acetate induced IL-6 expression, whereas ionomycin and PTH(3-34) amide, an N-terminal-truncated PTH analog that has reduced ability to activate the cAMP-PKA pathway, were much less effective. PMA pretreatment of calvariae greatly blocked IL-6 mRNA induction by a subsequent dose of PMA and decreased induction by PTH and forskolin to a much lesser extent. A reverse-transcriptase polymerase chain reaction (RT-PCR) assay was used to measure IL-6 heterogeneous nuclear RNA (hnRNA) and mRNA. A 5' primer spanning exons 1 and 2 and a 3' primer complementary to exon 5 of the murine IL-6 gene were used to detect IL-6 mRNA as a 638 bp product. A 5' primer corresponding to intron 4 of the murine IL-6 gene and the 3' primer were used to detect IL-6 hnRNA as a 370 bp product. RT-PCR of total calvarial RNA showed that the induction of IL-6 hnRNA by PTH and other agonists was similar to their induction of IL-6 mRNA. These data support the conclusion that PTH transcriptionally induces IL-6 gene expression in murine calvarial organ cultures mainly through the cAMP-PKA signaling pathway.
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PMID:Parathyroid hormone induces interleukin-6 heterogeneous nuclear and messenger RNA expression in murine calvarial organ cultures. 976 44

The gene (dctA) encoding the aerobic C(4)-dicarboxylate transporter (DctA) of Escherichia coli was previously mapped to the 79-min region of the linkage map. The nucleotide sequence of this region reveals two candidates for the dctA gene: f428 at 79.3 min and the o157a-o424-o328 (or orfQMP) operon at 79.9 min. The f428 gene encodes a homologue of the Sinorhizobium meliloti and Rhizobium leguminosarum H(+)/C(4)-dicarboxylate symporter, DctA, whereas the orfQMP operon encodes homologues of the aerobic periplasmic-binding protein- dependent C(4)-dicarboxylate transport system (DctQ, DctM, and DctP) of Rhodobacter capsulatus. To determine which, if either, of these loci specify the E. coli DctA system, the chromosomal f428 and orfM genes were inactivated by inserting Sp(r) or Ap(r) cassettes, respectively. The resulting f428 mutant was unable to grow aerobically with fumarate or malate as the sole carbon source and grew poorly with succinate. Furthermore, fumarate uptake was abolished in the f428 mutant and succinate transport was approximately 10-fold lower than that of the wild type. The growth and fumarate transport deficiencies of the f428 mutant were complemented by transformation with an f428-containing plasmid. No growth defect was found for the orfM mutant. In combination, the above findings confirm that f428 corresponds to the dctA gene and indicate that the orfQMP products play no role in C(4)-dicarboxylate transport. Regulation studies with a dctA-lacZ (f428-lacZ) transcriptional fusion showed that dctA is subject to cyclic AMP receptor protein (CRP)-dependent catabolite repression and ArcA-mediated anaerobic repression and is weakly induced by the DcuS-DcuR system in response to C(4)-dicarboxylates and citrate. Interestingly, in a dctA mutant, expression of dctA is constitutive with respect to C(4)-dicarboxylate induction, suggesting that DctA regulates its own synthesis. Northern blot analysis revealed a single, monocistronic dctA transcript and confirmed that dctA is subject to regulation by catabolite repression and CRP. Reverse transcriptase-mediated primer extension indicated a single transcriptional start site centered 81 bp downstream of a strongly predicted CRP-binding site.
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PMID:Inactivation and regulation of the aerobic C(4)-dicarboxylate transport (dctA) gene of Escherichia coli. 1048 2

1 In primary unpassaged rat brain capillary endothelial cell cultures (RBECs), using reverse-transcriptase PCR with primers specific for P2Y receptor subtypes, we detected mRNA for P2Y2, P2Y4 and P2Y6, but not P2Y1 receptors. 2 None of the various nucleotides tested reduced forskolin elevated cyclic AMP levels in RBECs. ATP and ATPgammaS, as well as adenosine, enhanced cyclic AMP accumulation in the presence of forskolin. 3 Comparison of the concentration response curves to ATPgammaS with those for ATP and adenosine, at different incubation times, indicated that the response to purine nucleotides was not wholly dependent on conversion to adenosine. Adenosine deaminase abolished the response to adenosine but only reduced the response to ATP by about 50%. These results suggest the participation of a receptor responsive to nucleotides. 4 Isobutylmethylxanthine and 8-sulphophenyltheophylline prevented the cyclic AMP response, while neither 8-cyclopentyl-1, 3-dipropylxanthine nor SCH58261 were effective antagonists. 2-chloradenosine gave a robust response, but neither 2-chloro-N6-cyclopentyladenosine nor CGS 21680 were agonists. 5 These results show that adenosine and ATP can elevate the cyclic AMP levels of brain endothelial cells by acting on receptors which have a pharmacology apparently distinct from known P2Y and adenosine receptors.
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PMID:Regulation of cyclic AMP by extracellular ATP in cultured brain capillary endothelial cells. 1051 Apr 59

The 1282 bp cDNA of an isoenzyme of fructose-1,6-bisphosphatase was cloned from rat muscle. It shows 70% positional identity to the cDNA of rat liver fructose-1,6-bisphosphatase and is clearly the product of a gene different from that coding for the liver enzyme. After cloning of the coding region of the rat muscle fructose-1,6-bisphosphatase cDNA in an expression vector, the recombinant enzyme could be detected in E. coli cell-free extracts by activity determination and Western blotting. Overexpressed fructose-1,6-bisphosphatase was found to be allosterically inhibited by AMP comparably to the enzyme isolated from rat muscle. Analysis of steady-state mRNA levels of various rat tissues with reverse-transcriptase polymerase chain reaction (RT-PCR) and Northern blotting revealed one or the two fructose-1,6-bisphosphatase isoenzyme mRNAs in most tissues tested with significant quantitative differences. Quantitative PCR using a homologous competitor showed that 1 microg of total RNA of rat muscle contains 1.7 x 10(6) molecules of rat muscle fructose-1,6-bisphosphatase mRNA. 3 x 10(4) copies of this message were found per microg total RNA of heart and kidney, respectively.
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PMID:Rat muscle fructose-1,6-bisphosphatase: cloning of the cDNA, expression of the recombinant enzyme, and expression analysis in different tissues. 1054 45

Several organic anions are actively extruded from intestinal epithelial cells into the lumen and vascular sides. To examine the role of the multidrug resistance-associated protein (MRP) family in the intestinal efflux of organic anions, the function and expression of these proteins were investigated with Caco-2, a human adenocarcinoma cell line that retains many of the characteristics of normal enterocytes. [(3)H]2,4-Dinitrophenyl-S-glutathione (DNP-SG) and [(3)H]17beta-estradiol 17-beta-D-glucuronide (E(2)17betaG), typical substrates for MRP1 and cMOAT (canalicular multispecific organic anion transporter)/MRP2, were taken up into brush-border membrane vesicles (BBMVs) from Caco-2 in an ATP-dependent manner, with K(m) values of 16.9 +/- 7.2 and 9.4 +/- 1.2 microM, respectively. The uptake of [(3)H]DNP-SG into BBMVs was osmotically sensitive and stimulated to some extent by other nucleotide triphosphates (GTP, CTP, and UTP) but not by ADP or AMP. An ATPase inhibitor, vanadate, inhibited the ATP-dependent uptake of [(3)H]DNP-SG to some extent. Reverse-transcriptase polymerase chain reaction resulted in the amplification of MRP1, MRP3, and MRP5. Northern blot analysis indicated extensive expression of cMOAT/MRP2 and MRP3 and only minimal expression of MRP1 and MRP5. Although cMOAT/MRP2 was continuously expressed throughout the culture period, MRP3 was not expressed immediately after the confluent state was reached. Collectively, the presence of ATP-dependent transport systems for DNP-SG and E(2)17betaG was demonstrated in Caco-2 cells. Because cMOAT/MRP2 and MRP3 may be expressed on brush-border and basolateral membranes in epithelial cells, respectively, the transport activity associated with BBMVs may result from the function of cMOAT/MRP2.
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PMID:Function and expression of multidrug resistance-associated protein family in human colon adenocarcinoma cells (Caco-2). 1060 57


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