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)

Tyrosine aminotransferase mRNA was quantitated by translation in a cell-free system derived from wheat germ followed by specific immunoprecipitation of the newly synthesized enzyme subunit. Hepatic poly(A)-containg RNA prepared from rats treated for 4 h with N6, O2'-dibutyryl cyclic AMP and theophylline was approximately 5.6 times more active in directing the synthesis of the tyrosine aminotransferase subunit relative to untreated controls. The overall template activity of the RNA prepared from control and cyclic AMP-treated animals was virtually identical, demonstrating that the cyclic nucleotide effect was specific for the tyrosine aminotransferase mRNA. At all times, after a single injection of dibutyryl cyclic AMP and theophylline, the increase in hepatic enzyme activity was accompanied by corresponding induction in the level of functional tyrosine aminotransferase mRNA. Other inducers of tyrosine aminotransferase, such as glucagon and hydrocortisone, also increased the level of tyrosine aminotransferase mRNA in proportion to their effect on enzyme activity. The RNA polymerase II inhibitor, alpha-amanitin, completely blocked the dibutyryl cyclic AMP-mediated increase in tyrosine aminotransferase mRNA activity. These studies demonstrate that, in intact animals, the induction of tyrosine aminotransferase activity by dibutyryl cyclic AMP can be completely accounted for by a corresponding increase in the level of functional mRNA coding for the enzyme.
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PMID:Increase in hepatic tyrosine aminotransferase mRNA during enzyme induction by N6,O2'-dibutyryl cyclic AMP. 2 49

Synthesis of malic enzyme was rapidly and markedly stimulated by the addition of triiodothyronine to chick embryo liver cells in culture. Alpha-Amanitin, an inhibitor of DNA-dependent RNA polymerase II, blocked induction. The kinetics of induction and de-induction of malic enzyme synthesis suggested that the most stable event in triiodothyronine induction had a half-life of 18 to 20 h. However, malic enzyme synthesis decayed with a half-life of 2,4 h when transcription was inhibited with alpha-amanitin. Thus a long-lived event in thyroid hormone stimulation of malic enzyme synthesis occurred prior to transcription of a specific messenger RNA (mRNA), presumably malic enzyme mRNA. Malic enzyme synthesis decayed with a half-life of about 2 h when glucagon was added to pre-induced liver cells. The similarity of decay rates after inhibition of transcription with alpha-amanitin or inhibition of malic enzyme synthesis by glucagon suggests that glucagon may inhibit the transcription or processing of a specific mRNA required for malic enzyme synthesis.
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PMID:Regulation of malic enzyme synthesis by thyroid hormone and glucagon: inhibitor and kinetic experiments. 56 41

Colloidal gold is an electron-dense, lyophobic colloid that readily forms a stable electrostatic interaction with a variety of macromolecules. Monodispersed colloids ranging from 3-150 nm in diameter can be produced to provide the researcher with flexibility in selecting the optimally sized probe. Gold labeling of antibodies and lectins has been extensively used to study surface antigens and cell components. Recently, the use of gold labeling has been extended to study receptor-ligand binding, enzyme-substrate reactions, and transcellular pathways. Published applications include gold labeling of metabolites (low-density lipoproteins), enzymes (DNAase and RNAase, RNA polymerase, thrombin, collagenase, elastase), hormones (insulin, epidermal growth factor, glucagon), circulating plasma proteins (asialoglycoprotein, alpha 2-macroglobulin, factor VIII-von Willebrand factor), and endotoxins (tetanus toxin, cholera toxin). This broad spectrum of applications emphasizes the versatility and usefulness of colloidal gold as a probe in areas of cell biology related to receptors, endocytosis, transport, and functions of proteins.
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PMID:Colloidal gold: a pluripotent receptor probe. 635 33

A transgene consisting of an upstream glucokinase (GK) promoter fragment linked to coding sequences of the human growth hormone gene was expressed in certain neuroendocrine cells of the pancreas, pituitary, brain, gut, thyroid, and lungs of mice. In pancreas, the transgene was expressed in a nonuniform manner among beta cells and in a variable but substantial fraction of the other islet cell types. In pituitary, it was expressed in corticotropes, and in brain, it was expressed in cells of the medial hypothalamus. Within the gut transgene expression was detected in a subset of enteroendocrine cells of the stomach and duodenal epithelium, some of which also exhibited glucagon-like polypeptide-1 immunoreactivity. In thyroid, transgene expression was observed in C cells of neonatal animals, whereas in the lung, it was expressed among rare endocrine cells of the bronchopulmonary mucosa. RNA polymerase chain reaction analysis of human growth hormone mRNA corroborated the tissue-specific transgene expression pattern. Prompted by the finding of transgene expression in specific neuroendocrine cells, we sought to determine whether GK mRNA and GK itself was also expressed in the brain and gut, tissues not previously associated with the expression of this enzyme. Using rat tissues, GK mRNA was detected by RNA polymerase chain reaction in both the brain and intestine and was localized to specific cells in the hypothalamus and enteric mucosa by in situ hybridization. A high Km glucose phosphorylating activity was detected from isolated rat jejunal enterocytes that displayed a chromatographic elution profile identical to hepatic GK. GK immunoreactivity was detected in cells of the medial hypothalamus with many of the same cells also displaying GLUT2 immunoreactivity. Together, these studies provide evidence for upstream GK promoter activity, GK mRNA, and GK itself in certain neuroendocrine cells outside the pancreatic islet and lead us to suggest that GK may play a broader role in glucose sensing by neuroendocrine cells than was thought previously.
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PMID:Analysis of upstream glucokinase promoter activity in transgenic mice and identification of glucokinase in rare neuroendocrine cells in the brain and gut. 810 9

Transcription initiation of the gene encoding phosphoenolpyruvate carboxykinase (PEPCK) is stimulated by glucocorticoids and glucagon, via cAMP, and dominantly inhibited by insulin in rat liver and H4IIE cells. Lysolecithin-permeabilized H4IIE cells recover completely and continue to multiply, yet are transiently penetrable by macromolecules. These cells, after various hormonal treatments, were utilized for in situ DNase I protection studies of the PEPCK promoter. Nearly all of the sites of protein interaction observed in vitro are protected in vivo as well as several additional sites. The DNase I protection pattern is the same in cells without or with any of the hormone treatments, suggesting that hormonal modulation of transcription does not involve addition or removal of factors from the hormone response elements of the promoter. We focused on the organization and stability of the transcription initiation complex as well as the dynamic nature of distal promoter factors in their interaction with DNA. The transcription initiation complex was detected, and it appears to be co-existent with a short region of naked single-stranded DNA over the TATA box on the template strand, as determined by potassium permanganate reactivity. This complex is quite stable, even under conditions of much reduced RNA synthesis, which suggests that the complex is not broken down and reformed with each round of initiation by RNA polymerase II. Other factors bind to the PEPCK promoter with half-lives ranging from a few minutes to more than 40 min. The cAMP response element apparently involves transcriptional modulation achieved through modification of a bound factor (presumably cAMP response element-binding protein), whereas the glucocorticoid/insulin-responsive region of the promoter functions through factors which are involved in a rapid exchange, suggesting quite different modes of transcriptional regulation.
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PMID:Dynamic aspects of DNA/protein interactions in the transcriptional initiation complex and the hormone-responsive domains of the phosphoenolpyruvate carboxykinase promoter in vivo. 822 59

Shifting rats to a protein-free, carbohydrate-rich diet, although not starvation, resulted in the appearance of mRNA for, and activity of, 3-phosphoglycerate dehydrogenase (3-PGDH) in liver as well as in a marked decrease in plasma cystine concentration. Refeeding with protein caused a 50% decrease in the mRNA in 8 h and its complete disappearance within 24 h, followed by a slower disappearance of the enzymic activity. Intraperitoneal administration of cysteine or methionine to protein-starved rats decreased the mRNA by 50-60% after 8 h. However, the repeated administration of cysteine failed to cause the complete disappearance of this mRNA in 24 h. In hepatocytes in primary culture, cysteine plus methionine and glucagon had, independently, an approx. 4-fold inhibitory effect on the abundance of the 3-PGDH mRNA and caused its almost complete disappearance when tested together. Insulin had an approx. 2-fold stimulatory effect, which was antagonized by cysteine plus methionine but was still apparent in the presence of glucagon. Nuclear run-on experiments and analysis of the stability of the mRNA with 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA polymerase II, suggested that the effect of cysteine plus methionine was due to destabilization of the mRNA, whereas the effect of glucagon was exerted on transcription. Cysteine, but not methionine, inhibited the accumulation of 3-PGDH mRNA in FTO2B hepatoma cells. In conclusion, the dietary control of the expression of the 3-PGDH gene in liver seems to involve the negative effects of cysteine and glucagon and the positive effect of insulin.
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PMID:Role of cysteine in the dietary control of the expression of 3-phosphoglycerate dehydrogenase in rat liver. 1054 28

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

Phospholipase C (PLC) activity was investigated by stimulation of membrane preparations obtained from insulin (beta-TC3)-, somatostatin (Rin 1027-B2)-, and glucagon (INR1-G9)-producing pancreatic cell lines using the non-hydrolyzable GTP analogue GTPgammaS alone, the C-terminal octapeptide cholecystokinin (CCK-8), or gastrin. All compounds caused a significant 2- to 4.4-fold stimulation of PLC activity in the different cell lines, which was diminished by the non-hydrolyzable GDP analogue GDPbetaS. CCK receptor subtypes were characterized by radioligand binding experiments. High-affinity binding sites for tritiated CCK(A) receptor antagonist L-364,718 (K(d) = 0.24 nM) and tritiated CCK(B) receptor antagonist L-365,260 (K(d) = 0.13 nM) were only present in Rin 1027-B2 cells. High-affinity binding sites for both ligands were not found in beta-TC3 or INR1-G9 cells. Competition binding experiments with non-labeled CCK receptor antagonists CR 1505 (CCK(A) receptor-selective) and CR 2945 (CCK(B) receptor-selective), as well as microphysiometry experiments, resulted in the same receptor distribution. Reverse transcriptase-polymerase chain reaction confirmed the CCK receptor distribution pattern for Rin 1027-B2 cells, but in addition showed the existence of CCK(B) receptors in beta-TC3 cells. Immunoblocking experiments with C-terminal antibodies against different G-protein alpha-subunits demonstrated inhibition of CCK-stimulated PLC activity in beta-TC3 cells by G(q/11)alpha antiserum (70%), in Rin 1027-B2 cells by G(q/11)alpha antiserum (70%) and G(i)-3alpha antiserum (23%), and in INR1-G9 cells by G(q/11)alpha antiserum (60%) and G(o)alpha antiserum (45%). We conclude that CCK receptor subtypes with different G-protein-coupling specificities to PLC are present in the different hormone-secreting cells of the endocrine pancreas.
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PMID:Activation of phospholipase C by cholecystokinin receptor subtypes with different G-protein-coupling specificities in hormone-secreting pancreatic cell lines. 1093 May 42

Members of the TGF-beta superfamily of cytokines have been implicated in pancreatic cancer, pancreatitis and in regulation and differentiation of pancreatic endocrine and exocrine cells. Different TGF-beta members signal through phosphorylation of different signal transduction proteins, which eventually form oligomers with SMAD 4 and translocate to the nucleus. Reverse transcriptase-polymerase chain reaction showed that SMADs 1, 2 and 4 are expressed in pancreatic islets. Immunostaining revealed that SMAD 1 and 4 predominantly were expressed by islet insulin and glucagon cells. Since SMAD 1 is known to transduce signals from receptors binding bone morphogenetic protein (BMP) these results indicate a previously unknown role of BMP-like ligands in islet function.
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PMID:Expression of SMAD signal transduction molecules in the pancreas. 1168 56

Hepatocyte function is regulated by several P2Y receptor subtypes. Here we report that 2-methylthioadenosine 5'-diphosphate (2-MeSADP), an agonist at P2Y(1), P2Y(12), and P2Y(13) receptors, potently (threshold 30 nM) stimulates glycogen phosphorylase in freshly isolated rat hepatocytes. Antagonism by N(6)-methyl 2'-deoxyadenosine 3',5'-bisphosphate (MRS 2179) confirms that this response is mediated by P2Y(1) receptors. In addition, in these cells, both 2-MeSADP and UTP inhibited glucagon-stimulated cyclic AMP accumulation. This inhibitory effect of 2-MeSADP was not reversed by the P2Y(1) antagonists, adenosine-3'-phosphate-5'-phosphate (A3P5P) or MRS 2179, both in the range 1 to 300 microM, indicating that it was not mediated by P2Y(1) receptors. This contrasts with the increase in cytosolic free Ca(2+) concentration ([Ca(2+)](c)) induced by 2-MeSADP, which has shown to be inhibited by A3P5P. Pertussis toxin abolished the inhibitory effect of both UTP and 2-MeSADP. After culture of cells for 48 h, the ability of 2-MeSADP to inhibit cyclic AMP accumulation was greatly diminished. Reverse transcriptase-polymerase chain reaction analysis revealed that during this culture period, there was a decline in the ability to detect transcripts for P2Y(12) and P2Y(13) receptors, both of which are activated by 2-MeSADP and negatively coupled to adenylyl cyclase. However, in freshly isolated cells, the P2Y(12) and P2Y(13) receptor antagonist, 2-propylthio-beta,gamma-dichloromethylene-d-ATP (AR-C67085) (10 nM to 300 microM) did not alter the ability of 2-MeSADP to inhibit glucagon-stimulated cyclic AMP accumulation. We conclude that 2-MeSADP regulates rat hepatocyte glycogen phosphorylase by acting on P2Y(1) receptors coupled to raised [Ca(2+)](c), and by inhibiting cyclic AMP levels by an unknown G(i)-coupled receptor subtype, distinct from P2Y(1), P2Y(12), or P2Y(13) receptors.
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PMID:Regulation of rat hepatocyte function by P2Y receptors: focus on control of glycogen phosphorylase and cyclic AMP by 2-methylthioadenosine 5'-diphosphate. 1515 27


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