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)

This study was undertaken to determine whether receptor and non-receptor components of the adenylate cyclase (AC) cascade were altered in brown adipose tissue (BAT) of 14-day-old pre-obese (fa/fa) rats, before endocrine status is strongly modified by fa gene expression. Activity of the AC catalytic subunit did not differ between the two genotypes. In fa/fa rats compared with control Fa/fa rats, there was a 50% decrease in the activity of alpha Gs (stimulated by NaF or guanosine 5'-[gamma-thio]triphosphate) but no change in protein content (Western blotting). alpha Gi function, assessed by the inhibitory action of low concentrations of guanosine 5'-[beta gamma-imido]triphosphate upon 10(-4) M forskolin-stimulated AC activity, was equally low in both genotypes. Analysis of dose-response curves for different beta-agonists revealed that (i) both the basal and the maximally stimulated activity of AC were 2-fold lower in fa/fa rats than in Fa/fa rats; (ii) BRL37344 and CGP12177 (beta 3 agonists) were less potent in fa/fa than in Fa/fa rats (Kact. multiplied by 2); (iii) noradrenaline and isoprenaline (Iso), at the low-affinity site (beta 3-AR), were less potent in fa/fa than in Fa/fa pups (Kact. increased by 30 and 20% respectively). At the high-affinity site (mainly beta 1) these two agonists were more potent in fa/fa than in Fa/fa rats (Kact. decreased by 40 and 80% respectively). In good agreement with the latter result, the beta 1-adrenergic receptor (beta 1-AR)-selective antagonist CGP20712A had more effect on the Iso-stimulated AC activity in pre-obese than in lean pups (2-fold decreased in IC50). Binding experiments with [3H]CGP12177 show that in BAT of suckling rats, beta 3-ARs represent 80% of the total beta-ARs. Bmax values for the two sites were not affected by the genotype, although the beta 3-AR mRNA concentration in BAT (quantitative reverse-transcriptase PCR) was 3-fold lower in fa/fa rats than in Fa/fa pups. In conclusion, these results provide evidence for alterations in beta 1- and beta 3-AR signalling in BAT of 14-day-old suckling pre-obese Zucker rats with a decreased activity of alpha Gs. The impaired AC responsiveness to catecholamines might be a primary contributor to the development of this genetic obesity.
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PMID:Early alterations in the brown adipose tissue adenylate cyclase system of pre-obese Zucker rat fa/fa pups: decreased G-proteins and beta 3-adrenoceptor activities. 855 20

In Bordetella pertussis the expression of virulence factors is coordinately regulated by the BvgS and BvgA proteins, members of the bacterial two-component signal transduction family, BvgS being the transmembrane sensor and BvgA the regulator. Activation of virulence gene expression requires phosphorylation of BvgA. On the basis of observed differences in the regulation of individual genes, the existence of accessory regulators has been postulated. They were supposed to be necessary for expression of genes encoding adenylate cyclase toxin (cya) and pertussis toxin (ptx), but not required for the expression of fha, encoding filamentous hemagglutinin. To clarify this issue we investigated the mechanism of activation of the BvgAS-controlled genes by performing in vitro run-off transcription experiments. We show, using purified RNA polymerase of B.pertussis, that phosphorylated BvgA is sufficient for transcriptional activation of the major virulence genes, thus providing good evidence that BvgA regulation operates directly with the transcription initiation machinery at the promoters of the virulence genes without a requirement for accessory activators. In addition, our results indicate that activation of the different promoters may involve distinct mechanisms. We suggest that the previously observed differences in regulation of individual virulence-associated genes reflect differences in the phosphorylation state of BvgA.
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PMID:Phosphorylated BvgA is sufficient for transcriptional activation of virulence-regulated genes in Bordetella pertussis. 859 92

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin (CyaA) which is related to the RTX family of pore-forming toxins. Like all RTX toxins, CyaA is synthesised as a protoxin (proCyaA), encoded by the cyaA gene. Activation to the mature cell-invasive toxin involves palmitoylation of lysine 983 and is dependent on co-expression of cyaC. The role of the cyaC gene product in the acylation reaction has not been determined. We have developed an efficient T7 RNA polymerase system for over-expression of cyaA and cyaC separately in Escherichia coli. Each protein accumulated intracellularly in an insoluble form and could be collected by centrifugation of lysed cells. A single-step purification was achieved by extraction of the aggregated material with 8 M urea. Active cell-invasive CyaA was produced in vitro when the proCyaA and CyaC proteins were mixed with a cytosolic extract of either E. coli or B. pertussis. Activation was assumed to occur by an acylation reaction requiring acyl carrier protein (ACP) as cofactor, as the cytosolic factor required for toxin activation was lost if the S100 extract was dialysed before use and the cytosolic factor could be replaced in the in vitro reaction by ACP charged separately in vitro with palmitic acid, as reported previously for activation of the homologous E. coli haemolysin (HlyA). The in vitro activation system may be used to investigate the mechanism of the CyaC-dependent acylation of proCyaA and the effect of variation of the modifying fatty acyl group on target cell specificity and toxic activity of CyaA.
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PMID:Bordetella pertussis adenylate cyclase toxin: proCyaA and CyaC proteins synthesised separately in Escherichia coli produce active toxin in vitro. 897 51

The pco determinant of Escherichia coli plasmid pRI1004 encodes inducible resistance to the trace element copper. The identification of two copper-dependent transcriptional initiation regions within pco that each contain a similar upstream hyphenated dyad motif is described. Deletion constructs showed that this 'copper box' motif was essential for copper-inducible activity at both pco promoters, PpcoA and PpcoE. The placement of the motif differs in the two promoters, and PpcoA contains an extended -10 nonamer typical of promoters for which RNA polymerase does not bind specifically to -35 sequences. PpcoE does not contain this motif and is the more strongly expressed promoter. The transcript from PpcoA contains the pcoABCDRS genes, while PpcoE expresses only pcoE. The induction profiles for PpcoA- and PpcoE-IacZ fusions were flattened sigmoidal curves with a gradual response to increasing copper concentration. On high-copy-number plasmids, zinc was found also to induce transcription from both promoters in vivo. Both promoters showed inducible activity in the absence of pcoRS, the plasmid-borne two-component regulatory system, indicating that a second trans-acting regulatory system is present on the chromosome. The pcoR product showed repressor action in the absence of pcoS, while still allowing induction, suggesting the chromosome encoded a similar two-component system to pco. TnphoA insertion mutagenesis identified chromosomal genes which affected promoter expression, including ptsH, ptsI (sugar phosphotransferase system) and cya (adenylate cyclase). The results support that idea that pco-encoded copper resistance is an auxiliary mechanism for handling copper, the regulation of which is integrated with the chromosomal regulation of cellular copper metabolism.
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PMID:Copper-inducible transcriptional regulation at two promoters in the Escherichia coli copper resistance determinant pco. 914 82

Cyclophilin A (CyP-A), a member of a highly conserved family of proteins, immunophilins, is the major intracellular receptor for the immunosuppressive drug, cyclosporin A (CsA). CyP-A is widely expressed in many tissues, but is found in the highest concentration in brain tissues and may perform critical neuronal functions. CsA is a known neurotoxin. Therefore, understanding the regulation of CyP-A levels in nerve cells, particularly by CsA, is important. We have utilized murine neuroblastoma (NB) cells as an experimental model to investigate this issue. Our results show that CsA alone was sufficient to induce morphological differentiation in undifferentiated NB cells and to increase CyP-A levels as determined by immunostaining. However, inducing terminal differentiation by elevating adenosine 3',5'-cyclic monophosphate (cAMP) levels using either 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO20-1724), an inhibitor of cyclic nucleotide phosphodiesterase, or prostaglandin E1 (PGE1), a stimulator of adenylate cyclase, was not sufficient to increase CyP-A levels. CsA was required to increase CyP-A levels in both RO20-1724- and PGE1-induced differentiated NB cells. Increases in CyP-A levels, however, occurred without any change in the expression of the CyP-A gene as determined by reverse-transcriptase polymerase-chain reaction analysis using (CyP-A)-specific primers. These results suggest that CsA regulates the level of its own binding protein, CyP-A, in both undifferentiated and cAMP-induced differentiated NB cells in culture.
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PMID:Cyclosporin A regulates the levels of cyclophilin A in neuroblastoma cells in culture. 1045 54

Chinese hamster ovary (CHO) cells stably expressing alpha(2) adrenergic receptor (alpha(2)AR) were pretreated with cholera toxin (CTX) and then treated with or without PMA. The alpha(2A)AR-mediated inhibition of forskolin-stimulated cAMP accumulation was completely ablated by CTX pretreatment only after additional treatment with PMA. Although the addition of cycloheximide (protein synthesis inhibitor) and H-89 (cAMP dependent protein kinase inhibitor) did not completely counteract the negative regulation, the elevation of cAMP was a primary factor for negative regulation by treatment with CTX and PMA. In contrast with the cAMP response, the inhibition of membrane adenylate cyclase activity and the agonist competition curve were not influenced by treatment with CTX or PMA, suggesting that a cytosolic factor was involved in this negative regulation. The m2-muscarinic-acetylcholine-receptor-mediated inhibition of the forskolin-stimulated accumulation of cAMP was also attenuated by treatment with CTX and PMA. The ablation of alpha(2A)AR-mediated inhibition was not observed when alpha(2A)AR was expressed in Rat2 fibroblast cells, suggesting that this negative regulation is not dependent on the receptor type but is instead a phenomenon common to G(i)-coupled receptors in CHO cells. Reverse-transcriptase-mediated PCR and Northern blot analysis showed that the expression of GOS8/RGS2 mRNA, which is a member of the regulator of G-protein signalling (RGS) group of proteins, was considerably increased by pretreatment with CTX. These results indicate a novel regulatory pathway, whereby a cytosolic factor induced by the elevation of cellular cAMP levels negatively regulates G(i) signalling in a protein-kinase-C-dependent manner.
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PMID:Negative regulation of alpha2-adrenergic receptor-mediated Gi signalling by a novel pathway. 1049 14

Pharmacologic tools were used to identify receptors in functional studies by measuring either transepithelial current (I(sc)) in strial marginal cells (SMC) or cAMP production in stria vascularis (SV). Further, receptors were identified in SV as transcripts by cloning and sequencing of reverse-transcriptase polymerase chain reaction (RT-PCR) products. Experiments were performed using tissues isolated from gerbils unless specified otherwise. I(sc) under control conditions was 1090 +/- 21 microA/cm(2) (n = 213) in gerbil SMC and 2001 +/- 95 microA/cm(2) (n = 6) in murine SMC. Direct stimulation of adenylate cyclase with 10(-5) m forskolin but not with 10(-5) m 1,9-dideoxy-forskolin resulted in an increase in the I(sc) by a factor of 1.14 +/- 0.01 (n = 6). The vasopressin-receptor agonist 10(-8) m Arg(8)-vasopressin had no significant effect on I(sc) in gerbil and murine SMC. The beta-adrenergic agonists isoproterenol, norepinephrine and epinephrine stimulated I(sc) with an EC(50) of (6 +/- 2) x 10(-7) m (n = 28), (3 +/- 1) x 10(-6) m (n = 40) and (7 +/- 2) x 10(-6) m (n = 38), respectively. Isoproterenol stimulated cAMP production in SV with an EC(50) of (5 +/- 2) x 10(-7) m (n = 8). The beta-antagonist 10(-4) m propanolol completely inhibited 2 x 10(-5) m isoproterenol-induced stimulation of I(sc). The beta-antagonists atenolol, ICI118551 and CGP20712A inhibited isoproterenol-induced stimulation of I(sc) with a K(DB) of 1 x 10(-7) m (pK(DB) = 6.96 +/- 0.15, n = 14), 1 x 10(-7) m (pK(DB) = 7. 01 +/- 0.14, n = 15), 2 x 10(-9) m (pK(DB) = 8.73 +/- 0.13, n = 19), respectively. CGP20712A inhibited isoproterenol-induced cAMP production with a K(DB) of 1 x 10(-10) m (pK(DB) = 9.94 +/- 0.55, n = 9). RT-PCR of total RNA isolated from SV using primers specific for the beta(1)-, beta(2)- and beta(3)-adrenergic receptors revealed products of the predicted sizes for the beta(1)- and beta(2)- but not the beta(3)-adrenergic receptor. Sequence analysis confirmed that amplified cDNA fragments encoded gene-specific nucleotide sequences. These results demonstrate that K(+) secretion in SMC is under the control of beta(1)-adrenergic receptors but not beta(2)-adrenergic or vasopressin-receptors and that the beta(1)-subtype is the primary beta-adrenergic receptor in SV although SV contains transcripts for both beta(1)- and beta(2)-adrenergic receptors.
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PMID:K+ secretion in strial marginal cells is stimulated via beta 1-adrenergic receptors but not via beta 2-adrenergic or vasopressin receptors. 1083 29

The presence and functional significance of the extracellular calcium-sensing receptor (CaR) on human pancreatic beta-cells were investigated. Reverse transcriptase-polymerase chain reaction with primers for the extracellular domain of the CaR expressed in human parathyroid-secreting cells identified a product of the expected size in human pancreatic mRNA. Immunocytochemistry using an antibody against the extracellular region of CaR showed extensive immunoreactivity in insulin- and glucagon-containing cells but not in somatostatin-containing cells. In perifusion experiments, elevations in extracellular Ca2+ produced initial transient increases in insulin secretion, followed by a concentration-dependent and prolonged, but reversible, inhibition of secretion. Microfluorometric measurements of intracellular Ca2+ ([Ca2+]i) in isolated human beta-cells demonstrated that elevations in extracellular Ca2+ (0.5-10 mmol/l) caused rapid elevations in [Ca2+]i. Increases in extracellular Ca2+ caused small increases in the cyclic AMP content of whole human islets. These studies demonstrated that human beta-cells express an extracellular CaR and that activation of the receptor inhibits basal and nutrient-stimulated insulin secretion. The transduction mechanism that mediates this inhibitory effect is unknown, but our results suggest that it is unlikely to be through the adenylate cyclase-cyclic AMP pathway or through the phospholipase C-IP3 pathway. This CaR-mediated inhibitory mechanism may be an important autoregulatory mechanism in the control of insulin secretion.
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PMID:The extracellular calcium-sensing receptor on human beta-cells negatively modulates insulin secretion. 1086 62

The effects of polyamines on the synthesis of various final sigma subunits of RNA polymerase were studied using Western blot analysis. Synthesis of final sigma(28) was stimulated 4.0-fold and that of final sigma(38) was stimulated 2.3-fold by polyamines, whereas synthesis of other final sigma subunits was not influenced by polyamines. Stimulation of final sigma(28) synthesis was due to an increase in the level of cAMP, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. Polyamines were found to increase the translation of adenylate cyclase mRNA by facilitating the UUG codon-dependent initiation. Analysis of RNA secondary structure suggests that exposure of the Shine-Dalgarno sequence of mRNA is a prerequisite for polyamine stimulation of the UUG codon-dependent initiation.
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PMID:Polyamine enhancement of the synthesis of adenylate cyclase at the translational level and the consequential stimulation of the synthesis of the RNA polymerase sigma 28 subunit. 1127 25

Based on the analysis of molecular interactions of proteins with DNA binding sites, a new approach to developing mathematical models describing gene expression is introduced. Detection of hierarchical structures in metabolic networks can be used to decompose complex reaction schemes. This will be achieved by assigning each regulator protein to one level in the hierarchy. Signals are then transduced from the top level to the lower level, but not vice versa. The method is shown by a simple example with two interacting proteins. A comparison of simulation results shows good agreement between a model taking all interactions into account and a model developed with the new approach. Finally, the method is applied to the crpA modulon in Escherichia coli, which controls uptake and metabolism for a number of carbohydrates. Here, RNA polymerase represents the top level, CrpA the second level, and the lactose-specific repressor LacI the lowest level, respectively. Besides the lactose operon, the method is applied to the adenylate cyclase gene and the gene for the regulator CrpA.
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PMID:The organization of metabolic reaction networks. II. Signal processing in hierarchical structured functional units. 1128 90


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