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)

We investigated the influence of age and a 20% or 52% reduction in dietary calories (caloric restriction) on expression of mRNA for a number of transcription factors and signal-transducing proteins using 4, 16, and 30-month-old female mice of the long-lived C3B10RF1 strain. In all age groups, 52% caloric restriction, which extends maximum life span by approximately 33%, increased insulin receptor mRNA by 15% to 25% over the levels in animals fed ad libitum. Aging increased insulin receptor mRNA and glucocorticoid-receptor mRNA in all dietary groups. A similar increase in glucocorticoid receptor mRNA was not observed for male mice of three other strains, suggesting the change is sex- or strain-specific and not a general feature of aging. These changes appear to be specific. Neither caloric restriction nor age had an effect on the level of mRNA for insulin-like growth factor-I, RNA polymerase II elongation-factor S-II, or transcription factors Sp1, CCAAT and enhancer binding protein, or proto-oncogene c-jun.
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PMID:Aging and restriction of dietary calories increases insulin receptor mRNA, and aging increases glucocorticoid receptor mRNA in the liver of female C3B10RF1 mice. 194 74

To investigate myeloid cell maturation, we established a panel of monoclonal antibodies that recognize myeloid cell nuclear antigens. One of these monoclonal antibodies was used to purify a specific protein complex (PC) from a human spleen. This PC, which is present at high levels in peripheral blood monocytes and granulocytes, contains a protein that is the cystic fibrosis (CF) antigen. The purified PC was shown to inhibit the activity of casein kinase I and II but not cAMP-dependent protein kinase, protein kinase C, v-abl tyrosine kinase, or insulin receptor tyrosine kinase. The observed Ki values for casein kinases I and II purified from several sources were 1 microM or less. Furthermore, the addition of the purified PC to a nuclear extract from human cells was able to prevent protein kinase-mediated stimulation of RNA polymerase activity. The unique inhibitory character of the PC and its elevated levels in monocytes and granulocytes and of the CF antigen in CF patients implies that this complex may be associated with myeloid cell functions and perhaps with the cause or consequence of the clinical manifestations of CF.
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PMID:A protein containing the cystic fibrosis antigen is an inhibitor of protein kinases. 265 77

The murine adipocyte lipid binding protein (ALBP) has been cloned into Escherichia coli, purified from expressing cultures, and its ligand binding and phosphorylation properties studied. In the cloning strategy, the recombinant, pT7-5 rALBP, was transformed into E. coli strain K38 harboring plasmid pGP1-2 which directs the synthesis of T7 RNA polymerase. Upon shifting the temperature from 30 to 42 degrees C to induce T7 RNA polymerase expression, the 14.6-kDa recombinant ALBP (rALBP) was expressed for approximately 2 h and accumulated to about 1% of total E. coli protein. The recombinant ALBP was soluble in E. coli extracts and resistant to bacterial proteolysis. A procedure for purifying rALBP was developed utilizing immuno-chemical detection based upon reactivity with anti-murine ALBP antiserum. A combination of acidic ammonium sulfate fractionation, gel permeation chromatography, and carboxymethyl ion-exchange high performance liquid chromatography separation was used to prepare homogeneous rALBP. Sequence analysis of rALBP indicated that the initiating methionine residue had been removed and the amino-terminal cysteine residue was not blocked. Purified rALBP exhibited stoichiometric, saturable binding of oleic acid (n = 1.0, K0.5 approximately 100 microM) and retinoic acid (n = 1.0, K0.5 approximately 170 microM). Incubation of rALBP with wheat germ agglutinin-purified insulin receptor, ATP, and 100 nM insulin resulted in a 5-fold stimulation of rALBP phosphorylation above the basal state. Kinetic analysis of rALBP phosphorylation by the 3T3-L1 insulin receptor kinase yielded a Michaelis constant (Km) of 50 microM and a maximal velocity of 1 mol of rALBP phosphorylated/min/mol insulin binding sites. Phosphoamino acid analysis indicated that phosphorylation occurred upon tyrosine. These results indicate that murine ALBP has been cloned and expressed in E. coli, purified to homogeneity, and is a substrate for the insulin receptor tyrosyl kinase in vitro.
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PMID:Cloning of murine adipocyte lipid binding protein in Escherichia coli. Its purification, ligand binding properties, and phosphorylation by the adipocyte insulin receptor. 268 57

We used a ribonuclease cleavage assay to screen for insulin receptor mRNA sequence alterations in 12 patients with syndromes of severe insulin resistance. Uniformly labeled [32P]antisense RNA probes complementary to insulin receptor mRNA were prepared by an SP6 or T7 RNA polymerase transcription reaction. Four probes ranging in size from 670-1470 bases were used to examine the entire 4.2-kilobase receptor protein-coding region. Patient RNA samples were hybridized to individual probes in solution, and mismatched sequences were detected by susceptibility to cleavage by a mixture of RNAses A and T1. The method was validated with insulin receptor mRNAs from cells transfected with cDNA constructs bearing known point and deletion mutations. Alterations in the insulin receptor mRNA sequence of two patients were detected. A patient with the type A syndrome of severe insulin resistance (A2-Boston) had a mutation in the insulin receptor beta-subunit mRNA sequence that localized to the region coding for amino acid residues 1174-1211 near the tyrosine kinase domain. The second alteration was a sequence polymorphism in the insulin receptor alpha-subunit mRNA in a patient with lipoatropic diabetes (LA-2) that localized to a region within amino acids 268-272. Direct sequence analysis revealed that the ribonuclease cleavage sites in patients A2-Boston and LA-2 were due to distinct single base changes in the insulin receptor gene and mRNA. Additional insulin receptor mRNA sequence polymorphisms were also identified as mismatches between the labeled RNA probes used and mRNA from several cultured human cell types. This study demonstrates that ribonuclease cleavage can rapidly detect and localize insulin receptor mRNA sequence mutations and polymorphic variations as small as single base changes. Further analysis of insulin receptor mRNA sequence alterations identified in this way may elucidate a possible genetic basis for functional insulin receptor defects in patients with severe insulin resistance and can also reveal some insulin receptor sequence polymorphisms that occur in the population at large.
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PMID:Insulin receptor messenger ribonucleic acid sequence alterations detected by ribonuclease cleavage in patients with syndromes of insulin resistance. 273 94

Modulation of the three beta-adrenergic receptor subtypes (beta-ARs) by insulin was investigated in mouse 3T3-F442A adipocytes. Saturation and competition experiments measuring binding of 125I-labeled (-)-cyanopindolol to adipocyte membranes demonstrated that cell exposure to insulin for 4 days caused a 3.5-fold decrease in the density of the major beta-AR component of the adipocyte, the beta 3-AR, while beta 1-AR sites remained unchanged and beta 2-ARs were undetectable. This correlated with a lower potency of the beta 3-AR-selective agonists CGP12177, ICI201651, and BRL37344 in stimulating adenylate cyclase. Northern blotting analysis indicated that insulin induced a rapid and sharp decrease in beta 3-AR mRNA levels. This effect was detectable at low insulin concentrations (EC50 = 3 nM) and was not observed in the presence of insulin-like growth factor I, suggesting an insulin receptor-mediated phenomenon. Reverse transcriptase-PCR analysis showed that, in contrast to its dramatic down-regulatory effect on beta 3-AR mRNA, insulin did not modify the levels of beta 1- and beta 2-AR transcripts. As assessed by nuclear run-on assays, insulin inhibited the beta 3-AR gene transcription rate by 90% within 30 min. mRNA turnover experiments showed that the half-life of beta 3-AR mRNA was short (90 min) and remained unaffected by insulin. These findings demonstrate the genetic control of a beta-AR subtype expression by insulin and reveal a mechanism for the regulation by this hormone of cAMP-dependent biological processes in adipocytes.
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PMID:Transcriptional down-regulation by insulin of the beta 3-adrenergic receptor expression in 3T3-F442A adipocytes: a mechanism for repressing the cAMP signaling pathway. 820 47

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

Chinese hamster ovary cells (CHO cells) do not exhibit any Ca2+/calmodulin-stimulated cAMP phosphodiesterase (PDE1) activity. Challenge of CHO cells with agonists for endogenous P2-purinoceptors, lysophosphatidic acid receptors and thrombin receptors caused a similar rapid transient induction of PDE1 activity in each instance. This was also evident on noradrenaline challenge of a cloned CHO cell line transfected so as to overexpress alpha 1B-adrenoceptors. This novel PDE1 activity appeared within about 15 min of exposure to ligands, rose to a maximum value within 30 min to 1 h and then rapidly decreased. In each case, the expression of novel PDE1 activity was blocked by the transcriptional inhibitor actinomycin D. Challenge with insulin of either native CHO cells or a CHO cell line transfected so as to overexpress the human insulin receptor failed to induce PDE1 activity. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1C isoform, did not amplify any fragment from RNA preparations of CHO cells expressing PDE1 activity, although they did so from the human thyroid carcinoma FTC133 cell line. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1A and PDE1B isoforms, successfully amplified a fragment of the predicted size from RNA preparations of both CHO cells expressing PDE1 activity and human Jurkat T-cells. Sequencing of the PCR products, generated using the PDE1A/B primers, yielded a novel sequence which, by analogy with sequences reported for bovine and murine PDE1B forms, suggests that the PDE1 species induced in CHO cells through protein kinase C activation and that expressed in Jurkat T-cells are PDE1B forms.
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PMID:Receptor-mediated stimulation of lipid signalling pathways in CHO cells elicits the rapid transient induction of the PDE1B isoform of Ca2+/calmodulin-stimulated cAMP phosphodiesterase. 900 15

cDNA clones encoding human (h) Grb7 and a previously unknown protein with high homology to hGrb-IR and mGrb10 (where m indicates mouse) were found by screening expressed sequence tag data bases. hGrb7 mRNA expression is greatest in pancreas and restricted to a few other tissues. The second protein termed hGrb-IRbeta/Grb10 contains an intact PH domain and lacks the 80-residue mGrb10 insertion. Expression is greatest in pancreas and muscle but occurs in nearly all tissues. hGrb-IRbeta/Grb10 and hGrb-IR likely arise as alternative mRNA splicing products of a common gene. Reverse transcriptase-coupled polymerase chain reaction shows both mRNAs in muscle. In cells, Grb-IRbeta/Grb10 protein translocates from cytosol to membrane upon insulin stimulation, most likely due to direct interactions with the insulin receptor. These interactions are mediated by the SH2 domain and additional regions of the protein. Studies with mutated receptors and synthetic phosphopeptides show that the hGrb-IRbeta/Grb10 SH2 domain binds at least two sites in the insulin receptor: the kinase activation loop > the juxtamembrane site. hGrb-IRbeta/Grb10 also binds a 135-kDa phosphoprotein in unstimulated 3T3-L1 adipocytes; binding is reduced upon insulin stimulation. In addition, the c-Abl SH3 domain binds Grb-IR/Grb10, whereas Fyn, phosphatidylinositol 3-kinase p85, and Grb2 SH3 domains do not. The site of c-Abl SH3 domain interaction is highly conserved within the Grb-IR/Grb10/Grb7/Grb14 family. hGrb-IRbeta/Grb10 also binds platelet-derived growth factor and epidermal growth factor receptors, suggesting a broader role in the signaling pathways of numerous receptors. We conclude that hGrb-IRbeta/Grb10 is a widely expressed, PH and SH2 domain-containing, SH3 domain-binding protein that functions downstream from activated insulin and growth factor receptors.
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PMID:Human GRB-IRbeta/GRB10. Splice variants of an insulin and growth factor receptor-binding protein with PH and SH2 domains. 900 1

Cell-free translation/translocation systems are broadly applied to examine gene expression and characterize the structure-function relationship of gene products. We present the characterization of Xenopus egg extract (XEE) translocation and processing of proteins synthesized in rabbit reticulocyte lysate. The XEE was prepared from eggs laid by adult female frogs that received serial injections of gonadotropins. The eggs were then dejellied in 2% L-cysteine-HCl and the cytoplasm extracted by centrifugation at 10,000 rpm for 15 min. The in vitro translocation and processing of XEE was examined with a cell-free translation system containing reticulocyte lysate, and appropriate messenger ribonucleic acid (RNA) or complementary deoxyribonucleic acid plasmids with RNA polymerase. Cell-free production of the following proteins were used to assess posttranslational modifications: Escherichia coli beta-lactamase for signal sequence cleavage, Saccharomyces cerevisiae alpha-mating factor for translocation and N-linked glycosylation, the soluble protein luciferase for functional activity, and the membrane-bound human insulin receptor for translation efficiency. All translation products were identified by [35S]-methionine labeling, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The results demonstrate that (1) XEE produces near-complete signal sequence and N-glycosylation processing of proteins synthesized in reticulocyte lysate, (2) XEE contains endoplasmic reticulum-equivalent microsomes, which allows for protein translocation and protease protection, (3) the addition of XEE in the translation reaction does not affect synthesis and chemiluminescence activity of luciferase, (4) XEE is efficient in processing the nascent 160-kDa human insulin receptor precursor, a transmembrane protein, and (5) as compared to canine pancreatic microsomes, XEE translocation efficiency is minimally decreased with the addition of dimethylsulfoxide. These results are the first description of the combined use of XEE with reticulocyte lysate and clearly demonstrate a higher efficiency of translocation and processing compared to canine pancreatic microsomes. This method of cell-free translation and processing allows for more extensive in vitro examination of posttranslational modifications of secretory and membrane-bound proteins.
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PMID:Efficient translocation and processing with Xenopus egg extracts of proteins synthesized in rabbit reticulocyte lysate. 1093 32

In this study the expression of uncoupling protein 3 (UCP3) and its regulation by insulin-like growth factor 1 (IGF-I) and insulin in human neuroblastoma SH-SY5Y cells were characterized. Reverse transcriptase-PCR, Western blot, and immunofluorescence analysis showed that SH-SY5Y cells express UCP3 natively. IGF-I induced a time- and concentration-dependent induction of UCP3 protein reaching a twofold expression after 72 h with 10 nM IGF-I. Extremely high insulin concentrations (860 nM) and 10 nM trIGF-I, a truncated form of IGF-I with the same affinity for the IGF-I receptor as the full-length IGF-I, but with lower activity on the insulin receptor, also upregulated UCP3. We conclude that SH-SY5Y cells express UCP3 natively and that the expression is regulated by IGF-I via the IGF-I receptor.
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PMID:Insulin-like growth factor type 1 upregulates uncoupling protein 3. 1158 36


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