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)

The initiation of Escherichia coli DNA replication is a highly regulated event with many parameters exerting positive and negative effects. The activity of the dnaA protein (the initiator protein) is profoundly influenced by the tight binding of the adenine nucleotides ATP and ADP. Further regulation of dnaA protein activity may occur through dnaA protein-cell membrane associations. A replicatively inactive form of dnaA protein is found aggregated with phospholipids; enzymatic treatment of the aggregates with phospholipase A2 or dnaK protein liberates dnaA protein with restored replication activity. Proper DNA structure is essential for replication. The energy stored in the DNA's supercoiling is crucial for dnaA protein's ability to initiate replication. Under conditions where strand-opening by dnaA protein is inhibited, such as low free superhelicity, an R-loop formed by RNA polymerase activates the origin at a distance by aiding strand-opening. A novel protein has been identified as a specific inhibitor of the initiation of DNA replication. This 33-kDa protein binds to the AT rich region of oriC and inhibits strand-opening by dnaA protein.
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PMID:E. coli minichromosome replication: regulation of initiation at oriC. 192 9

Drugs with affinity for phospholipids, such as chlorpromazine, verapamil, tetracaine and imipramine, were found to inhibit accurate transcription from adenovirus 2 major late promoter in a nuclear extract of Ehrlich ascites tumor cells. The transcription activity of the nuclear extract inhibited by chlorpromazine was restored by addition of acidic phospholipids. The nuclear extract was also shown to lose transcription activity when treated with phospholipase A2. Chlorpromazine was found to inhibit transcription at the step of initiation, not elongation. Moreover, it did not affect the activity of purified RNA polymerase II, suggesting the interaction of phospholipids with transcription factors in the nuclear extract. Some transcription factors in the nuclear extract were found to have affinity for cardiolipin, and were precipitated with excess cardiolipin. The transcription factors precipitated with cardiolipin could be solubilized with guanidine hydrochloride, and restored the transcription activity of the cardiolipin-treated nuclear extract.
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PMID:Inhibitory action of phospholipid-interacting drugs on transcription initiation in a nuclear extract of Ehrlich ascites tumor cells. 200 95

This study examines how interleukin-6 (IL-6) expression by human luteinizing granulosa cells is regulated. IL-6 was assayed in culture supernatants, mRNA in cells by in situ hybridization and by a competitive reverse-transcriptase polymerase chain reaction (RT-PCR). TNF alpha (100 pg-1 ng/ml) induced IL-6 mRNA and protein. Phorbol 12-myristate 13-acetate (PMA) (50 nM) mimicked this effect. DibutyrylcAMP (1 mM) and 10 microM forskolin. C2-, C6- and C8-ceramide (15 microM), all had no effect. The inhibitor of protein tyrosine kinase (PTK), genistein (100 micrograms/ml) reduced tumor necrosis factor (TNF) effects. The inhibitors of protein kinase C (PKC) (staurosporine, 10 nM), of phospholipase C (U73122, 2 microM), of phospholipase A2 (PLA2), (indomethacin 30 microM, mepacrin 50 microM, nordihydroguaiaretic acid 10 microM, ONO-RS-082 3,5 microM), none prevented it. Hence, IL-6 is induced by TNF alpha via activation of PTK. Protein kinase A, phosphoinositide and conventional PKC, sphingomyelin and PLA2 pathways are not implicated.
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PMID:Tumor necrosis factor-alpha induces interleukin-6 mRNA and protein in human granulosa luteinizing cells via protein tyrosine kinase without involving ceramide. 908 55

Indirect evidence suggests that the renal and vascular production of prostaglandins is increased in cirrhosis with ascites. However, the activity of the enzymes regulating the prostaglandin pathway has not been investigated in cirrhosis. The aim of the current study was to determine the activity of phospholipase A2 (PLA2), the key enzyme in the regulation of prostaglandin synthesis, in kidney and vascular tissue obtained from rats with carbon tetrachloride-induced cirrhosis and ascites (n = 9) and control rats (n = 6). PLA2 activity was assayed in vitro using [14C]arachidonyl-phosphatidylcholine (PC) and [14C]arachidonyl-phosphatidylethanolamine (PE) as substrates in the presence of Ca2+. Kidneys from cirrhotic rats had significantly higher PLA2 activity compared with control rats, with both PC and PE (35 +/- 5 and 40 +/- 6 vs. 21 +/- 2 and 26 +/- 3 pmol/mg/min, respectively; P < .05 for both). PLA2 activity was increased in the renal cortex as well as in the renal medulla. Fractionation of the kidney extracts by Mono-Q anion-exchange chromatography showed that the elution position of PLA2 activity corresponded to the cytosolic PLA2 isoform (cPLA2). Increased amounts of cPLA2 protein were found in kidney extracts immunoblotted with an anti-cPLA2 antibody However, reverse-transcriptase polymerase chain reaction (RT-PCR) analysis did not detect any difference in cPLA2 mRNA. PLA2 activity was also higher in aortic tissue from cirrhotic rats than in controls (PC 38 +/- 5 vs. 26 +/- 1 and PE 66 +/- 8 vs. 41 +/- 3 pmol/mg/min; P < .05 for both). Incubation of renal and aortic extracts from cirrhotic rats with anti-cPLA2 antibody reduced PLA2 activity by 64% and 88%, respectively. In conclusion, PLA2 activity is increased in kidneys and vascular tissue from cirrhotic rats with ascites. This can be accounted for by an induction of cPLA2, which would mediate, at least in part, the increased renal and vascular production of prostaglandins in cirrhosis.
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PMID:Increased renal and vascular cytosolic phospholipase A2 activity in rats with cirrhosis and ascites. 942 15

The immunosuppressive effect of portal venous blood transfusions in organ transplantation has been well established and may be mediated by increased Kupffer cell production of the immunosuppressive arachidonic acid metabolite prostaglandin E2 (PGE2). In this study, butyrate, a short-chain fatty acid known to enhance gene transcription, is hypothesized to enhance Kupffer cell PGE2 production by altering cyclooxygenase or phospholipase A2 (PLA2) activity, thus augmenting the immunosuppressive effect of portal venous transfusion. Lewis rats were given a portal venous transfusion of Wistar-Firth blood or saline 1 h prior to Kupffer cell harvest. The in vitro effects of butyrate on Kupffer cell PGE2 production, cyclooxygenase, and PLA2 activity were assessed. Kupffer cell tumor necrosis factor-alpha (TNFalpha) production was also assessed due to its sensitivity to PGE2 and its proinflamatory effects. Kupffer cells from portally transfused animals produced significantly more PGE2 than saline-transfused controls. Addition of butyrate to the culture medium further increased PGE2 production by as much as sevenfold in Kupffer cells of portally transfused animals. Other short-chain fatty acids, propionate and hexanoate, did not increase PGE2 production. Butyrate added to Kupffer cells from transfused animals slightly upregulated inducible cyclooxygenase (COX-2) mRNA levels as measured by both Northern blot and reverse-transcriptase polymerase chain reaction and increased PLA2 activity fivefold as measured by Western blot. Kupffer cell immune function was also affected by in vitro butyrate treatment with a significant decrease in the production of TNFalpha. Thus, butyrate may be a useful immunoregulatory agent in organ transplantation protocols which seek to enhance transcription of immunosuppressive molecules.
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PMID:Sodium butyrate upregulates Kupffer cell PGE2 production and modulates immune function. 973 8

The current study examined mechanisms that account for the selective release of arachidonic acid (AA) from cells by secretory phospholipase A2 (sPLA2). Initial studies demonstrated that low concentrations of group I and group III PLA2 isotypes and an sPLA2-enriched extract from bone marrow-derived mast cells (BMMC) selectively released AA from mast cells. Much higher concentrations of group II PLA2 were required to release comparable quantities of AA. Group I PLA2 also selectively released AA from another mast cell line (CFTL-15) and a monocytic cell line (THP-1). In contrast, high concentrations of group I PLA2 were required to release fatty acids from a promyelocytic cell line (HL-60) and this release was not selective for AA. Binding studies revealed that cell types (BMMC, CFTL-15 and THP-1) which selectively released AA also had the capacity to specifically bind group I PLA2. However, group II PLA2, which did not selectively release AA from cells, also did not specifically bind to these same cell types. Additional studies revealed that sPLA2 binding to the mast cell receptor was attenuated after stimulation with antigen or ionophore A23187. Reverse transcriptase-polymerase chain reaction analyses indicated the presence of mRNA for the sPLA2 receptor in BMMC, CFTL-15 and THP-1 and the absence of this mRNA in HL-60. Final studies demonstrated that p-aminophenyl-alpha-D-mannopyranoside BSA, a known ligand of the sPLA2 receptor, also selectively released AA from mast cells but not from HL-60 cells. These experiments indicated that receptor occupancy alone (without PLA2 activity) is sufficient to induce the release of AA from mast cells. Together, these data reveal that specific isotypes of sPLA2 have the capacity to selectively release AA from certain cells by their capacity to bind to sPLA2 receptors on the cell surface.
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PMID:Mechanisms that account for the selective release of arachidonic acid from intact cells by secretory phospholipase A2. 974 13

We studied the expression of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), an enzyme capable of hydrolyzing platelet-activating factor (PAF), PAF-like phospholipids, and polar-modified phosphatidylcholines, in human and rabbit atherosclerotic lesions. Oxidative modification of low-density lipoprotein, which plays an important role in atherogenesis, generates biologically active PAF-like modified phospholipid derivatives with polar fatty acid chains. PAF is known to have a potent proinflammatory activity and is inactivated by its hydrolysis. On the other hand, lysophosphatidylcholine and oxidized fatty acids released from oxidized low-density lipoprotein as a result of Lp-PLA(2) activity are thought to be involved in the progression of atherosclerosis. Using combined in situ hybridization and immunocytochemistry, we detected Lp-PLA(2) mRNA and protein in macrophages in both human and rabbit atherosclerotic lesions. Reverse transcriptase-polymerase chain reaction analysis indicated an increased expression of Lp-PLA(2) mRNA in human atherosclerotic lesions. In addition, approximately 6-fold higher Lp-PLA(2) activity was detected in atherosclerotic aortas of Watanabe heritable hyperlipidemic rabbits compared with normal aortas from control rabbits. It is concluded that (1) macrophages in both human and rabbit atherosclerotic lesions express Lp-PLA(2), which could cleave any oxidatively modified phosphatidylcholine present in the lesion area, and (2) modulation of Lp-PLA(2) activity could lead to antiatherogenic effects in the vessel wall.
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PMID:Lipoprotein-associated phospholipase A(2), platelet-activating factor acetylhydrolase, is expressed by macrophages in human and rabbit atherosclerotic lesions. 1059 68

We present a modification of mRNA differential display in which increased throughput results from the use of an automated fluorescent sequencer. The sequence analysis is performed directly on purified fragments without further cloning. The amplified fragments carry a T7 RNA polymerase promoter sequence tag for in vitro transcription of riboprobes for nonradioactive in situ hybridization. We compared changes in gene expression in the liver and colon of group II phospholipase A2 transgenic and group II phospholipase A2 deficient mice during the course of experimental Escherichia coli infection. Fluorescent mRNA differential display comprising a 7 x 24 set of primers was used to study a total of 31,257 amplified cDNA fragments. Sequence analysis of the displayed fragments associated with infection identified classical acute-phase proteins in the liver and host defense proteins in the colon. The displayed mRNAs associated to transgenicity were the transgene itself, i.e., human group II phospholipase A2, and glutathione-S-transferase in the liver. In the colon, the displayed mRNAs associated with transgenicity were the pancreatitis-associated protein and mucin. The results show that fluorescent mRNA differential display is a reliable method to identify differences in the expression of the genes of acute-phase proteins.
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PMID:mRNA differential display of acute-phase proteins in experimental Escherichia coli infection. 1100 10

Arachidonic acid (AA), a metabolite of membrane phospholipids, and its metabolites are increased in Mg2+ deficiency. We examined whether the extracellular Mg2+ concentration affects AA production and whether AA regulates a putative Na+-dependent Mg2+ efflux pathway in renal epithelial NRK-52E cells. We used the cells cultured in 5 mM Mg2+-containing medium for 2 days because they enable us to detect Na+-stimulated Mg2+ efflux that was not observed in normal culture medium. Removal of extracellular Mg2+ increased AA release both in the absence and presence of extracellular Na+. This was inhibited by methyl arachidonyl fluorophosphonate (MAFP, 10 microM), an inhibitor of cytosolic phospholipase A) (cPLA2) and Ca2+-independent phospholipase A2 (iPLA2), and bromoenol lactone (BEL, 10 microM), an inhibitor of iPLA2. However, LY-311727 (10 microM), a secretory phospholipase A2 (sPLA2) inhibitor, had no inhibitory effect. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that NRK-52E cells express cPLA2 and iPLA2 mRNAs, but not sPLA2. In the mag-fura 2 fluorescence measurements, extracellular Mg2+ removal caused slight decrease in the intracellular free Mg2+ concentration ([Mg2+]i) in the Na+-free condition. The addition of Na+ caused a rapid decrease in [Mg2+]i, indicating the presence of a Na+-dependent Mg2+ efflux pathway. The Na+-dependent [Mg2+]i decrease was suppressed by MAFP and BEL. On the other hand, AA metabolite inhibitors, nordihydroguaiaretic acid (NDGA) (50 microM), indomethacin (10 microM) and 17-octadecynoic acid (ODYA) (10 microM), enhanced the Na+-dependent [Mg2+]i decrease. Furthermore, the addition of exogenous AA (30 microM) enhanced the Na+-dependent [Mg2+]i decrease, which was significantly inhibited by imipramine (0.1 mM), a putative Na+/Mg2+-exchanger inhibitor. These results suggest that extracellular Mg2+ removal elevates AA release mediated mainly by iPLA2 and that AA upregulates the Na+-dependent Mg2+ efflux in NRK-52E cells.
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PMID:Arachidonic acid-activated Na+-dependent Mg2+ efflux in rat renal epithelial cells. 1464 27

Homeostasis of phosphatidylcholine (PC) is regulated by the opposing actions between CTP:phosphocholine cytidylyltransferase (CT) and the group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)). We investigated this process during the cell cycle. PC mass doubles during late G(1) and early S phase when its rate of catabolism is lowest. We show that iPLA(2) activity is cell cycle-dependent with peak activity during G(2)/M and late S phase. iPLA(2) activity declines during G(1) and is lowest at the G(1)/S transition and early S phase. The accumulation of PC correlates with decreased iPLA(2) activity, suggesting that regulation of this enzyme contributes to phospholipid accumulation. The levels of 80 kDa iPLA(2) protein do not change and thus cannot account for changes in enzyme activity. Reverse transcriptase and real-time PCR experiments show that splice variant iPLA(2) mRNAs are preferentially expressed during G(2)/M. Immunoblot analyses with an antibody directed against the N terminus of iPLA(2) revealed a approximately 50 kDa protein that is of appropriate size to be the truncated protein encoded by the ankyrin-iPLA(2)-1 splice variant mRNA. The levels of truncated iPLA(2) protein were high in cells in late G(1) and S phase cells that had low iPLA(2) activity and low in G(2)/M cells that had high iPLA(2) activity. The truncated protein co-immunoprecipitated with full-length iPLA(2), indicating a physical interaction between the two proteins. Together, these data suggest that truncated iPLA(2) proteins associate with active iPLA(2) and down-regulate its activity during G(1). This down-regulation may contribute to phospholipid accumulation during the cell cycle.
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PMID:Cell cycle dependence of group VIA calcium-independent phospholipase A2 activity. 1538 40


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