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)

A human thioredoxin cDNA was modified to optimize Escherichia coli expression and subcloned into the plasmid pACA, a vector for T7 RNA polymerase-directed expression. The substitution of structural (noncatalytic) half-cystines in human thioredoxin (hTrx) was made by site-directed mutagenesis. The recombinant wild-type (wt) hTrx and its mutant C61S, C72S, and C61S/C72S were expressed and purified to homogeneity. Characterization of the wt and mutant hTrx was done with respect to redox activity with thioredoxin reductase (TR), tryptophan fluorescence, and effects of incubation with GS-Se-SG, which is believed to be the major metabolite of inorganic selenium compounds in mammalian tissues. The Km and kcat of wild-type hTrx for human placenta thioredoxin reductase (HP-TR) at pH 7.0 were 2.0 microM and 2800 min-1, respectively. The mutant proteins C61S, C72S, and C61S/C72S had Km and kcat values similar to those of the wt thioredoxin. Tryptophan fluorescence measurements showed that the wt and mutant proteins had similar stability to a denaturing agent. Incubation of fully reduced thioredoxin with 0.1 molar equivalent of GS-Se-SG resulted in continued oxidation of SH groups. After 3.5 h only 0.5 of initially 4.6 SH groups/thioredoxin remained. With the oxidized protein, a pronounced lag phase in thioredoxin reductase-dependent insulin disulfide reduction was present. Disulfide-linked dimers of the protein were present. The results clearly showed that noncatalytic cysteine residues in hTrx were oxidized accompanied by dimerization and inactivation. The activities of the mutant proteins C72S and C61S/C72S were unchanged after 3 h of incubation with GS-Se-SG. No dimer appeared of the C72S thioredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mutagenesis of structural half-cystine residues in human thioredoxin and effects on the regulation of activity by selenodiglutathione. 837 74

Inositol 1,4,5-trisphosphate (IP3) functions as a second messenger for many neurotransmitters, hormones and growth factors. It causes the release of Ca2+ from intracellular stores by binding to specific receptors that are coupled to Ca2+ channels. Recent studies have shown that there is a family of IP3 receptors, and the complete sequences of two members of this family and partial sequences of two others have been reported. We have determined the complete sequence of a third IP3 receptor, designated IP3R-3, and characterized its pharmacological properties and sites of expression. Rat IP3R-3 is 2670 amino acids in size, has 62 and 64% identity with IP3R-1 and IP3R-2, and is predicted to have a similar structure including a region of eight potential membrane-spanning segments at its COOH terminus, which presumably functions as a Ca2+ channel. Expression of recombinant rat IP3R-3 in COS-7 cells showed that it bound IP3 as well as inositol 1,3,4,5-tetrakisphosphate and inositol hexakisphosphate. Immunohistocytochemical studies of cells expressing recombinant IP3R-3 indicated that it has a preferential cellular distribution in the endoplasmic reticulum. RNA and protein blotting studies indicate that IP3R-3 is expressed in a number of different cultured cell lines including insulin-secreting RINm5F cells. The IP3R-3 is also expressed in adult pancreatic islets, kidney, gastrointestinal tract, and brain. Reverse transcriptase-polymerase chain reaction amplification of IP3R-1, -2, and -3 mRNAs in adult rat pancreatic islets indicated that IP3R-3 was the predominant subtype expressed in this tissue and thus may be responsible for mediating the effects of IP3 on insulin secretion.
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PMID:Sequence and functional characterization of a third inositol trisphosphate receptor subtype, IP3R-3, expressed in pancreatic islets, kidney, gastrointestinal tract, and other tissues. 838 91

Inflammatory cytokines, particularly those produced by Th1 type lymphocytes, are hypothesized to play a major role in the pathogenesis of autoimmune diseases. The present studies investigated this hypothesis in the BB rat. Diabetes-prone (DP) BB rats develop spontaneous hyperglycemia and thyroiditis. Coisogenic diabetes-resistant (DR) BB rats do not develop either disorder spontaneously, but both diseases are induced by depletion of RT6+ T cells. Reverse transcriptase-PCR was used to measure mRNA encoding type 1 and type 2 cytokines. In both DP and RT6-depleted DR rats, IFN-gamma mRNA was present in islets before and during disease onset. IL-2 and IL-4 mRNAs were minimal or undetectable in infiltrated islets but present in activated peripheral T cells. IL-10 mRNA was present at low abundance in infiltrating T cells. These observations suggested a Th1 type inflammatory response, and consistent with this interpretation, we observed that mRNA encoding the p40 chain of IL-12 was also present before and during disease onset. Similar cytokine mRNA profiles were observed in the thyroids of RT6-depleted DR rats and in the islets of DP rats treated with prophylactic parenteral insulin to prevent diabetes. We conclude that IFN-gamma and IL-12 may play a major role in the expression of insulitis and thyroiditis in the BB rat, that Th1 lymphocytes may predominate over Th2 lymphocytes in these inflammatory lesions, and that prevention of diabetes by insulin is not associated with an alteration in the cytokine gene profile of islet infiltrating cells.
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PMID:Cytokine gene expression in islets and thyroids of BB rats. IFN-gamma and IL-12p40 mRNA increase with age in both diabetic and insulin-treated nondiabetic BB rats. 855 12

We previously established transplantable rat thyroid carcinoma cell lines in vivo from primary thyroid tumors induced by N-bis-(2-hydroxypropyl)nitrosamine (DHPN). In the present study, an insulin-like growth factor I (IGF-I)-responsive cell line (TRTC-G1-C-A4) in culture was derived from one (well differentiated papillary type) of these carcinoma cell lines G1. TRTC-G1-C-A4 cells were found to exhibit specific saturable binding of IGF-I with a Kd of 1.16 nM at approximately 43.6 fmol/10(5) cells. Inclusion of IGF-I (10 and 50 ng/ml) in the culture medium resulted in a significant increase of [3H]thymidine incorporation and marked cell proliferation. IGF-II (10 ng/ml) and insulin (1 microgram) produced no such effects. The molecular weight of IGF-I receptors on the cell membrane was determined by Western blotting analysis, a single band of binding proteins with a molecular weight of 125 kDa being evident under non-reducing conditions. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that the TRTC-G1-C-A4 cells contained IGF-I receptor mRNA with a sequence corresponding to that determined from rat uterus. These results demonstrate that the IGF-I receptor can be expressed in a thyroid carcinoma with an important contribution to cell growth.
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PMID:Establishment of a rat thyroid carcinoma cell line in vitro demonstrating high DNA synthesis in response to insulin-like growth factor I. 862 Apr 77

A macromolecular fraction was isolated from ovine bone marrow and designated the active fraction (AF). Two distinct enzymatic activities were detected in the AF: (1) a RNA-dependent RNA polymerase, and (2) a reverse transcriptase. RNA polymerase uses an endogenous RNA molecule of the AF as the template while the product of this RNA polymerase reaction forms the template for the reverse transcriptase. Synthetic reactions are initiated exclusively upon the exposure of the in vitro system to one of the external proteins, selected at random, ovalbumin or insulin. Ovalbumin specific (OS) and insulin specific (IS) AF fractions were prepared. OS-AF binds 14C-ovalbumin and the IS-AF binds 14C-insulin, but not vice versa. Reverse transcription activity of OS-AF is stimulated only upon its exposure to ovalbumin and not to insulin while the reverse is true for the activity associated with IS-AF. Indirect evidence indicates that the enzymes which synthesize nucleic acids are closely associated with the antigen receptor on the B-lymphocyte plasma membrane, the mlgM, and that the antigen binding to its receptor helps in the activation of these enzymes.
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PMID:Reverse transcription in a macromolecular complex of ovine bone marrow: possible involvement in the antigen receptor mediated signal transduction process. 868 97

The vaccinia virus/T7 bacteriophage expression system was used to express human decorin in HT-1080 cells by co-infection with vTF7-3, encoding T7 RNA polymerase, and vDCN1, encoding the decorin core protein fused to a polyhistidine-insulin signal sequence fusion-protein cassette. Overexpression using the vaccinia virus/T7 phage system resulted in secretion of approximately 30 mg of decorin/10(9) cells per 24 h which enabled purification and separation of multiple glycoforms under native conditions. Cells were cultured in the presence of [35S]methionine or a mixture of [3H]glucosamine and [35S]sulfate, and recombinant glycoprotein purified by metal affinity chromatography which resolved the secreted decorin into two classes, a proteoglycan form and a core protein form. About 25% of the recombinant protein was secreted into the culture medium as core protein devoid of glycosaminoglycan chains. The decorin core protein was resolved into two forms (approximately 49 and approximately 53 kDa) that differed in the extent of N-linked oligosaccharide substitution (2 and 3 N-linked oligosaccharides, respectively). Deglycosylation of the recombinant proteoglycans and core proteins resulted in a single band migrating with an apparent molecular mass approximately 43 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. Far-UV circular dichroism spectra of native decorin proteoglycan showed a minima at 218 nm, consistent with a secondary structure that is predominantly beta-sheet. Circular dichroism spectra of bovine decorin extracted from articular cartilage and recombinant decorin similarly treated revealed a minima of 205 nm indicating a loss of secondary structure. The affinity of decorin proteoglycan and core protein for collagen-like molecules was demonstrated, with the complement component C1q exhibiting the most striking affinity for decorin, although adherence to collagen types I and V was also observed. The extensive secondary structure maintained in the purified recombinant protein is likely to be important for the biological function of decorin.
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PMID:Recombinant decorin glycoforms. Purification and structure. 870 52

The effects of retinoic acids (RAs) on development of seminal vesicles (SVs) of neonatal mice were investigated in vitro. SVs from 0-day-old male mice were cultured for 2-6 days in serum-free, chemically defined medium containing transferrin and BSA supplemented with 5alpha-dihydrotestosterone (DHT; 10(-8) M) and insulin (10 microg/ml), alone and in combination. Before culture, SVs from 0-day-old mice consisted of an unbranched epithelium surrounded by mesenchyme. SVs cultured in medium with DHT plus insulin or DHT alone formed numerous epithelial branches after day 2 of culture, whereas epithelial branching did not occur in SVs cultured with insulin alone. All-trans-RA or 13-cis-RA (10(-9)-10(-6) M) added to medium containing DHT plus insulin or DHT alone inhibited epithelial branching in a dose-dependent manner. This inhibitory effect was reversible after removal of the retinoids from the medium on day 4 of culture. These RAs also decreased [3H]thymidine labeling indexes of both epithelium and mesenchyme of SVs cultured in medium with DHT plus insulin or DHT alone and inhibited the increase in their protein contents. 9-Cis-RA was less inhibitory than all-trans-RA or 13-cis-RA on epithelial branching, [3H]thymidine labeling indexes of epithelium and mesenchyme, and protein content of SVs cultured in medium with DHT and insulin. In the absence of DHT (insulin alone), all-trans-RA did not affect either the [3H]thymidine labeling indexes of epithelium and mesenchyme or the protein content of cultured SVs. Reverse transcriptase-PCR demonstrated strong expression of transcripts for mouse RA receptors (RARalpha, RARgamma, and RXRalpha), with lower levels of expression of RARbeta, RXRbeta, and RXRgamma in neonatal SVs. The present results indicate that RAs reversibly inhibit androgen-dependent development of neonatal mouse SVs, most likely through RARs.
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PMID:Inhibitory effects of retinoic acids on androgen-dependent development of neonatal mouse seminal vesicles in vitro. 877 Sep 10

1. There are two functionally different isoforms of the insulin receptor in humans and rats. We hypothesized that a change in their relative proportion could be of relevance to insulin resistance in hypertension. 2. A reverse-transcriptase polymerase chain reaction technique was established for the detection of mRNA for the exon 11+ and exon 11- isoforms and the proportion of each was determined in 3, 6, 9 and 12 week old spontaneously hypertensive rats and Wistar-Kyoto rats, as well as adrenocorticotrophin (ACTH)-induced hypertensive rats and controls. 3. The proportion of the exon 11+ form (approximately 95%) and exon 11- form (approximately 5%) was similar in the liver of all rats studied. 4. We conclude that there is no change in insulin receptor isoform expression in the liver in the models of hypertension studied.
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PMID:No difference in the proportion of insulin receptor exon 11 +/- isoform mRNA in the liver of rats after development of hypertension. 880 May 98

Cholecystokinin (CCK) has been suggested to modulate insulin output. We have shown that Otsuka Long-Evans Tokushima Fatty (OLETF) rats show little or no expression of the CCK-A receptor gene in the pancreas. We examined whether the CCK-A and CCK-B receptor genes are expressed in the islets and the role of CCK-A receptor in insulin secretion. Gene expressions of CCK receptors were determined by the reverse-transcriptase polymerase chain reaction (RT-PCR) followed by Southern blot hybridization and Northern transfer analysis using LETO rats as controls. Pancreatic endocrine function was examined in perfusion (exogenous CCK stimulation) and meal ingestion (endogenous CCK stimulation) studies. CCK-A receptor mRNA was detected in the islets of LETO rats but not OLETF rats. Expression of the CCK-B receptor gene was detected in both strains by RT-PCR. Insulin secretion was impaired in OLETF rats, but the insulin contents of OLETF and LETO rats were not different. No abnormalities were detected histologically in either strain. These results suggest that the occurrence of pancreatic endocrine dysfunction in OLETF rats may be due to a defect in expression of the CCK-A receptor gene, not to insulin deficiency.
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PMID:Pancreatic endocrine dysfunction in rats not expressing the cholecystokinin-A receptor. 883 Mar 28

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel neuropeptide that produces its biological effects by interacting with G protein-coupled receptors. Molecular cloning of the PACAP receptor revealed the existence of five splice variant receptor forms differing in the third intracellular loop region, with four variants activating both adenylyl cyclase and phosphoinositide phospholipase C and one variant activating only adenylyl cyclase (Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P. H., and Journot, L. (1993) Nature 365, 170-175). Here, we report cloning of a novel PACAP receptor variant, designated PACAPR TM4 (transmembrane domain IV), that differs from the previously cloned short form of the PACAP receptor (PACAPR) primarily by discrete sequences located in transmembrane domains II and IV. Reverse transcriptase-polymerase chain reaction and primer extension analyses demonstrated tissue-specific differential expression of mRNAs encoding PACAPR TM4 and splice variant forms of the PACAP receptor. PACAPR TM4 and PACAPR possess identical intracellular domains, implicated as primary determinants of G protein recognition by rhodopsin-like receptors. However, unlike the PACAPR, PACAPR TM4 does not activate either adenylyl cyclase or phosphoinositide phospholipase C in response to PACAP in either transient or stable expression systems. However, PACAP stimulates increases in [Ca2+]i in cells expressing PACAPR TM4 by activating L-type Ca2+ channels, a response not elicited by stimulation with vasoactive intestinal polypeptide. The signaling phenotype of PACAPR TM4 is characteristic of the PACAP receptor involved in regulation of insulin secretion from pancreatic beta islets, a tissue expressing transcripts for PACAPR TM4 but not for PACAPR or its longer splice variant forms. These findings are consistent with a role of PACAPR TM4 in the physiological control of insulin release by PACAP in beta-islet cells. The finding that PACAPR TM4 has a unique signaling phenotype, although it possesses intracellular domains identical to those of the PACAPR, suggests that receptor-G protein recognition by rhodopsin-like receptors can be determined by sequences other than those located in intracellular receptor domains.
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PMID:Molecular cloning of a novel variant of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor that stimulates calcium influx by activation of L-type calcium channels. 894 80


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