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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous investigations have shown that CCAAT/enhancer binding protein (C/EBP) can function as a trans-activator of the promoters of several adipocyte-specific genes--i.e., the 422 adipose P2 (422/aP2), stearoyl-CoA desaturase 1 (SCD1), and glucose transporter 4 (GLUT4) genes, in 3T3-L1 mouse preadipocytes. We now describe a cell-free system prepared from nuclei of 3T3-L1 cells that carries out transcription directed by these promoters. To measure transcript formation, we employed a polymerase chain reaction-assisted analysis. Nuclear extract from 3T3-L1 adipocytes that express C/EBP supports a higher rate of transcription of chimeric 422(aP2) promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs than nuclear extract from preadipocytes that lack C/EBP. A competitor oligonucleotide containing the C/EBP binding site sequence and antibodies raised against C/EBP inhibit transcription directed by the 422(aP2) promoter. The factor limiting transcription by nuclear extract from preadipocytes appears to be C/EBP, since recombinant C/EBP (rC/EBP) markedly activates transcription of the 422(aP2) promoter-CAT gene with preadipocyte extract but not with adipocyte extract. rC/EBP also activates cell-free transcription of SCD1 promoter-CAT and GLUT4 promoter-CAT chimeric genes. Point mutations within the C/EBP binding site in the 422(aP2) promoter markedly decrease transcription activated by rC/EBP. Consistent with activation by cAMP of the 422(aP2) promoter in intact preadipocytes, cAMP-dependent protein kinase activates transcription through this promoter with the cell-free system, this effect being independent of C/EBP. Thus, regulation of transcription directed by the 422(aP2) promoter in the cell-free system resembles that which occurs in intact 3T3-L1 cells.
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PMID:Cell-free transcription directed by the 422 adipose P2 gene promoter: activation by the CCAAT/enhancer binding protein. 168 37

Long-term regulation of mammalian steroid hormone synthesis occurs principally by transcriptional regulation of the gene for the rate-limiting cholesterol side-chain cleavage enzyme P450scc. Adrenal steroidogenesis is regulated primarily by two hormones: adrenocorticotropin, which works via cyclic AMP (cAMP) and protein kinase A, and angiotensin II, which works via Ca2+ and protein kinase C. Forskolin and 8-bromo-cAMP stimulated, while prolonged treatment with a phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA]) and a calcium ionophore (A23187) additively suppressed accumulation of endogenous P450scc mRNA in transformed murine adrenal Y1 cells. In Y1 cells transfected with 2,327 base pairs of the human P450scc promoter fused to the bacterial gene for chloramphenicol acetyltransferase (CAT), forskolin increased CAT activity 900% while combined TPA plus A23187 reduced CAT activity to 15% of the control level. Forskolin induced the P450scc promoter as rapidly as a promoter containing two cAMP-responsive elements fused to a simian virus 40 promoter, a system known to respond directly to cAMP. Basal expression was increased by sequences between -89 and -152 and was increased further by sequences between -605 and -2327. This upstream region also conferred inducibility by cAMP. TPA plus A23187 transiently increased CAT activity before repressing it, reflecting the complex actions of angiotensin II in vivo. Repression by prolonged treatment with TPA plus A23187 was mediated by multiple elements between -89 and -343. Induction of CAT activity by forskolin was not diminished by treatment with TPA plus A23187, nor were the regions of the promoter responsible for regulation by the two pathways coisolated. Thus, the human gene for P450scc is repressed by TPA plus A23187 by mechanisms and sequences independent of those that mediate induction by cAMP.
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PMID:Human P450scc gene transcription is induced by cyclic AMP and repressed by 12-O-tetradecanoylphorbol-13-acetate and A23187 through independent cis elements. 170 Feb 77

The dihydropyridine derivative B859-35 inhibits phospholipid- and calcium-dependent protein kinase C (PKC) in cell-free extracts from NIH3T3 cells. Inhibition is competitive with regard to phosphatidylserine. At 1 microM phosphatidylserine, half-maximal inhibition (IC50) is obtained at approximately 2.5 microM B859-35. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-dependent activation of the Na+/H+ antiporter was used to determine whether the enzyme is also affected in intact cells. The activity of the antiporter was monitored by following the dimethylamiloride-sensitive cytosolic alkalinization. It is demonstrated that B859-35 depresses the TPA-induced alkalinization with an IC50 of 5 microM, indicating that PKC in intact cells and the enzyme in cell-free extracts are equally sensitive to the drug. TPA-induced expression of the c-fos gene was used as an additional marker for intracellular PKC activity. Activation of c-fos expression was determined by measuring chloramphenicol acetyltransferase (CAT) activity in cells transfected with a c-fosCAT construct in which the CAT gene is expressed under the control of the endogenous human c-fos promoter. The studies revealed that 2.5 microM B859-35, a concentration equivalent to the IC50 in cell-free extracts, significantly depresses TPA-induced c-fosCAT expression. B859-35 inhibited cellular proliferation of NIH3T3 cells with an IC50 of approximately 5 microM. This is close to the IC50 for the anti-PKC activity of B859-35. It is suggested that the inhibition of PKC contributes to the growth inhibition following exposure to B859-35.
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PMID:Inhibition of cell proliferation, protein kinase C, and phorbol ester-induced fos expression by the dihydropyridine derivative B859-35. 171 84

Since cAMP has a number of important effects regulating activity of adrenergic receptor pathways, we wondered if expression of the alpha 2A adrenergic receptor gene is regulated by this second messenger. We have examined the effects of a cAMP analog (Bt2cAMP) on the expression of alpha 2A adrenergic receptors in HT-29 cells. Bt2cAMP induced a 5.3 +/- 0.8-fold increase in alpha 2A receptor mRNA abundance as did forskolin and vasoactive intestinal polypeptide which both increase cAMP accumulation in these cells. Bt2cAMP increased alpha 2A receptor number up to 2.4 +/- 0.3-fold. The rate of alpha 2A receptor gene transcription increased 7.8 +/- 3.2-fold in cells treated with Bt2cAMP for 2 h; after 24 h, the transcription rate was 3.7 +/- 1.7-fold higher than in controls. The increased rate of transcription occurred in the presence of the protein synthesis inhibitor cycloheximide. The half life of the alpha 2A receptor mRNA in cells incubated with Bt2cAMP for 2 h increased by 1.5-fold but returned to the original value after exposure to Bt2cAMP for 24 h. The increased expression of alpha 2A receptors was associated with an increased efficacy of inhibition of cAMP accumulation mediated by the alpha 2 adrenergic agonist UK14304. Using a chloramphenicol acetyltransferase (CAT) reporter plasmid containing 5'-flanking sequences of the alpha 2A receptor gene, we found that co-transfection of JEG-3 cells with expression vectors containing cAMP-dependent protein kinase regulatory subunit cDNA with mutations at both cAMP binding sites inhibited basal and Bt2cAMP-stimulated expression of CAT activity. These results demonstrate that an alpha 2A adrenergic receptor gene is regulated by the second messenger cAMP via cAMP-dependent protein kinase, mainly by controlling the rate of transcription, which leads to an increased expression of these receptors.
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PMID:cAMP regulates transcription of the alpha 2A adrenergic receptor gene in HT-29 cells. 184 58

Gonadotropins (follicle-stimulating hormone (FSH), luteinizing hormone, and human chorionic gonadotropin) and beta-adrenergic agonists have been shown to stimulate expression of the proopiomelanocortin (POMC) gene in ovarian granulosa cells. The current studies investigate the intracellular mechanisms by which gonadotropins regulate gene expression. Primary cultures of rat granulosa cells were transfected with the plasmid POMC-CAT-150, which expresses the chloramphenicol acetyltransferase (CAT) reporter gene under the regulation of the rat POMC 5'-flanking region. CAT activity was stimulated by treatment of the cells with either 20 ng/ml FSH or 1 microM isoproterenol. To assess the role of protein kinase A (ATP:protein phosphotransferase; EC 2.7.1.37) in the gonadotropin and adrenergic response, an expression vector, MtR-AB, encoding a mutant RI regulatory subunit was cotransfected with POMC-CAT-150. The mutant protein kinase A regulatory subunit encoded by MtR-AB lacks functional cAMP-binding sites but effectively binds and specifically inhibits the catalytic activity of protein kinase A. The results of this analysis demonstrated that gonadotropin and adrenergic agonist stimulation of the POMC-CAT reporter construct in primary cultures of rat granulosa cells were abolished by cotransfection with MtR-AB; whereas a control SV40-promoter construct was unaffected by either gonadotropin treatment or cotransfection with MtR-AB. Basal expression directed by the POMC promoter was also decreased by cotransfection with the MtR-AB, implying that basal expression from the POMC promoter may also depend on protein kinase A. Deletion analysis of the POMC sequence indicated regions (-40 to -33 and +4 to +63) important for basal and FSH-stimulated expression. These studies suggest that both gonadotropin and adrenergic stimulation of the POMC promoter are mediated by protein kinase A and that regions proximal to the promoter are essential for gonadotropin-regulated expression from the promoter.
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PMID:Specific inhibition of protein kinase A in granulosa cells abolishes gonadotropin regulation of the proopiomelanocortin promoter. 190 60

H2O2, like other oxidants, is known to act as a mitogen at low concentrations in resting Balb/3T3 or mouse epidermal JB6 cells. We described previously that H2O2 induces some early response genes in Balb/3T3 cells. We extended these observations using another cell line, MC3T3 (mouse osteoblastic) cells by examination of transcriptional activity of these genes and by using inhibitors of protein kinases. H2O2 increased the expressions of c-fos, c-jun, egr-1 and JE genes which are known to be early response genes and are induced by mitogenic stimuli in many types of cells. Exogenous addition of H2O2 increased the mRNA levels of these genes, the kinetics of increase being similar to those of their inductions by a phorbol ester or serum. Nuclear run-on transcription showed that this induction occurred at the transcriptional level. H2O2 at 0.1-0.2 mM induced maximal expressions of c-fos and c-jun, whereas 0.3 mM H2O2 was required for induction of stress-induced heme oxygenase mRNA. The inductions of c-fos and c-jun were inhibited by 50 microM H7, a protein kinase inhibitor that is relatively specific for protein kinase C, but were not affected by H9, relatively specific for cAMP-dependent protein kinase. In cells pretreated with 12-O-tetradecanoylphorbol 13-acetate, however, in which protein kinase was supposed to be downregulated, H2O2 induced c-fos and heme oxygenase as efficiently as in untreated cells. H2O2 did not increase the phosphorylation of p80 protein, which is known to be a substrate for protein kinase C. Thus, H2O2 seemed to induce c-fos and c-jun by activating protein kinases distinct from protein kinase C. Activity of the chloramphenicol acetyltransferase gene under control of the serum-response element of human c-fos genes was increased by H2O2 treatment, whereas that under control of cAMP-response element was not affected. These results indicate that the inductions by H2O2 of c-fos and possibly other early response genes are mediated through activation of the serum-response element in their enhancer.
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PMID:Transcriptional activation of early-response genes by hydrogen peroxide in a mouse osteoblastic cell line. 191 80

cAMP-dependent protein kinase appears to play a role in cAMP-induced gene expression in mammalian cells. There exist two major types of cAMP-dependent protein kinase, type I and type II, which are distinguished by their regulatory subunits, RI and RII, respectively. We investigated the role of type I and type II protein kinase in the cAMP-induced gene expression by either stable or co-transfection of RI alpha, RII alpha, or RII beta gene in an expression vector together with somatostatin-chloramphenicol acetyltransferase (SS-CAT) fusion gene using a cAMP-unresponsive mutant pheochromocytoma cell line (A126-1B2). Introduction of the RII beta gene restored the capability of these cells to induce the SS-CAT gene expression in response to forskolin stimulus and induced a changed morphology which resembled that of wild type. The RII alpha gene also induced SS-CAT gene expression but to a lesser degree than that achieved by the RII beta gene, whereas the RI alpha gene had no effect. The induction of SS-CAT gene expression by the RII beta gene was specifically blocked by the 21-mer RII beta antisense oligodeoxynucleotide. These results show for the first time that type II but not type I regulatory subunit of cAMP-dependent protein kinase is essential for a cAMP-induced gene transcription.
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PMID:Type II regulatory subunit of protein kinase restores cAMP-dependent transcription in a cAMP-unresponsive cell line. 197 35

A cAMP response element (CRE) has been identified in the proximal 5'-flanking region of the rat glucagon gene, and activation of the cAMP-dependent pathway in fetal rat intestinal cells leads to an increase in the levels of glucagon mRNA transcripts. In contrast, the human glucagon gene does not contain a similar CRE, and the results of studies using immortalized rat and hamster islet cell lines have suggested that glucagon gene expression may not be regulated by cAMP. To reconcile these observations, we have studied the control of glucagon gene expression. Incubation of primary rat islet cell cultures with forskolin in the presence of low (0.5 g/liter) or high (2.0 g/L) glucose resulted in a 2- to 3-fold increase in the levels of glucagon mRNA transcripts. Forskolin also stimulated the secretion and synthesis of immunoreactive glucagon. The importance of the protein kinase-A-dependent pathway in the regulation of glucagon gene expression was also examined in hamster islet InR1-G9 cells. Cotransfection of a glucagon-chloramphenicol acetyltransferase (CAT) fusion gene containing the glucagon CRE and a cDNA encoding the catalytic subunit of protein kinase-A resulted in stimulation of glucagon-CAT activity in hamster islet cells. Catalytic subunit cotransfection also activated somatostatin-CAT, but no activation of RSVCAT was detected. The results of these experiments suggest that the rat glucagon gene is regulated by a protein kinase-A-dependent pathway in the endocrine pancreas.
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PMID:The rat glucagon gene is regulated by a protein kinase A-dependent pathway in pancreatic islet cells. 198 32

cAMP regulates the expression of several genes by activation of a promoter consensus sequence which functions as a cAMP-response element. Evidence indicated that this is accomplished via cAMP dissociation of cAMP-dependent protein kinase into its regulatory (R) and catalytic (C) subunits. Our investigations of the role of these two subunits in gene expression provide direct and quantitative evidence that the C subunit is required for cAMP stimulation of the cAMP-response element in the vasoactive-intestinal-peptide gene in rat pheochromocytoma cells. After cotransfection of a metallothionein-regulated C-subunit expression vector (pCEV) and a vasoactive-intestinal-peptide--chloramphenicol acetyltransferase construct containing a cAMP-response element, we could demonstrate expression of transfected C-alpha-subunit mRNA (truncated size 1.7 kb) by Northern blot and a concentration-dependent C subunit stimulation of chloramphenicol acetyltransferase activity. Basal activity was stimulated 12- and 50-fold by pCEV (30 micrograms), in the absence and presence, respectively, of Zn2+. Metallothionein-regulated expression of C was demonstrated by results that showed a 2-4-fold increase in chloramphenicol acetyltransferase activity in the presence versus the absence of 90 microM Zn2+. In contrast, overexpression of the R-II beta regulatory subunit did not stimulate chloramphenicol acetyltransferase activity, and R-II beta transfected together with C (ratio 2:1 and 4:1) inhibited the stimulation by the C subunit 70% and 90% respectively. Our results indicate that transfection of cAMP-dependent protein kinase subunits results in functional expression of both C-alpha and R-II beta subunits. Expression of the C subunit mediated cAMP-regulated gene expression but this expression could be inhibited by cotransfected R-II beta subunit, indicating intracellular reconstitution of the inactive holoenzyme of cAMP-dependent protein kinase.
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PMID:Regulation of gene expression by transfected subunits of cAMP-dependent protein kinase. 215 96

The rate of transcription of the beta 2-adrenergic receptor gene is increased in response to beta-adrenergic agonist stimulation of the receptor at the cell surface. This effect is mediated by stimulation of adenylyl cyclase and elevation of intracellular cAMP levels. We have previously shown that this responsiveness to cAMP resides in the 5'-flanking region of the human beta 2-adrenergic receptor gene (Collins, S., Bouvier, M., Bolanowski, M. A., Caron, M. G., and Lefkowitz, R. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4853-4857). A 34-base pair sequence derived from the beta 2-adrenergic receptor promoter region (-70 to -37 base pairs), containing the sequence GTACGTCA, confers responsiveness to cAMP when present in either orientation 5' to the thymidine kinase promoter on the chloramphenicol acetyltransferase reporter gene. Overexpression of the catalytic subunit of protein kinase A fully substituted for forskolin in inducing expression through this sequence, indicating that the cAMP induction is mediated through this kinase. Mutations within the GTACGTCA sequence completely abolished the stimulation. A 43-kDa transcription factor (cAMP response element-binding protein) confers cAMP responsiveness through binding to specific sequences. In gel mobility shift assays, purified cAMP response element-binding protein bound to the 34-base pair oligonucleotide from the beta 2-adrenergic receptor gene with an affinity similar to that for the well-characterized cAMP response element from the human glycoprotein hormone alpha-subunit gene, and failed to bind to mutated elements. Thus, positive autoregulation of the beta 2-adrenergic receptor gene appears to occur through receptor-mediated stimulation of adenylyl cyclase, with consequent activation of cAMP response element-binding protein and stimulation of beta 2-adrenergic receptor gene transcription. These results demonstrate a novel mechanism by which a receptor (beta 2-adrenergic receptor) stimulatory for adenylyl cyclase can exert positive feedback regulation on its own expression.
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PMID:A cAMP response element in the beta 2-adrenergic receptor gene confers transcriptional autoregulation by cAMP. 217 52


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