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)

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family of related proteins, is expressed by stromal fibroblasts and acts on epithelial cells in a paracrine fashion. To understand the mechanisms responsible for regulating normal KGF expression and how these might be altered in disease, the 5'-flanking region of this gene was cloned. The presence of two KGF transcription initiation sites was suggested by ribonuclease protection assay and confirmed by primer extension analysis. Examination of the genomic DNA sequence revealed the presence of the putative promoter sequences TATTTA and CCAAT, located 31 and 50 base pairs upstream, respectively, from the first of the two mRNA start points, and putative initiator sequences surrounding each transcription start site. Transient transfection into murine NIH/3T3 fibroblasts demonstrated that the region required for basal level KGF promoter activity was located between bases -225 and +190. Inclusion of sequences between -1503 and -775 markedly reduced promoter activation, indicating the presence of negative regulatory element(s) in this region. A similar pattern of promoter activation was detected in human fibroblasts and in murine C2C12 myoblasts. In contrast, no chloramphenicol acetyltransferase activity was observed in macrophages and epithelial and lymphoid cells transfected with the same constructs. Northern blot analysis revealed a strong correlation between KGF RNA expression and promoter activation in all cells tested. Activation of the KGF promoter could be induced by the proinflammatory cytokines interleukin 1 and interleukin 6 and by the adenylate cyclase activator forskolin. Taken together, these results indicate the existence of cis-acting element(s) responsible for selective activation of the KGF promoter only in cells that express KGF mRNA and may provide a mechanistic basis for KGF gene expression during inflammation.
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PMID:Cloning and characterization of the promoter region of the human keratinocyte growth factor gene. 774 56

Vasoactive intestinal peptide (VIP) is widely recognized as a regulator of tyrosine hydroxylase via a mechanism of trans-synaptic activation. Subsets of adrenal medullary cells and postganglionic sympathetic nerves coexpress the peptide neurotransmitter neuropeptide Y (NPY) with catecholamines. Using PC12 cells transiently expressing a fusion gene in which the bacterial enzyme chloramphenicol acetyltransferase (CAT) is under the control of 700 base pairs of the 5' flanking region of the NPY gene, we have studied the role of VIP and the related peptide pituitary adenylate cyclase activating peptide (PACAP) in regulating NPY gene transcription. Both VIP and PACAP stimulated expression of the NPY gene through activation of cAMP-dependent protein kinase. PACAP was 1000-fold more potent in eliciting this response compared to VIP and activity resided in its N-terminal 27 amino acids. Both VIP and PACAP caused a subpopulation (approximately 50%) of PC12 cells to undergo profound morphological changes in that the cells extended long, slender neurites with prominent growth cones. This change in morphology was unaffected by preincubating cells with inhibitors of either cAMP-dependent protein kinase or calcium/phospholipid-dependent protein kinase. A trophic role for either VIP or PACAP in regulating sympathetic nerve function is proposed.
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PMID:Vasoactive intestinal peptide stimulates neuropeptide Y gene expression and causes neurite extension in PC12 cells through independent mechanisms. 796 4

This report establishes that increasing the activity of cyclic AMP-dependent protein kinase (protein kinase A; PKA) potentiates glucocorticoid-mediated signaling in embryonic day 5.5 (E5.5) chicken retina. Expression of a glutamine synthetase-chloramphenicol acetyltransferase (CAT) fusion gene is not induced by treatment with glucocorticoid hormone in transfected E5.5 retina. However, treatment of the retina with forskolin, an activator of adenyl cyclase, or cotransfection with an expression vector encoding PKA is sufficient to render the fusion gene hormonally responsive. Similar results are obtained after forskolin treatment of E5.5 retina that have been transfected with a plasmid that contains the CAT reporter gene under transcriptional control by the thymidine kinase promoter and a 46-nucleotide enhancer with two glucocorticoid response elements (GREs). In contrast, forskolin augments but is not required to achieve glucocorticoid-inducible CAT gene expression in E5.5 retina transfected with a plasmid that contains the reporter driven by a minimal promoter with six juxtaposed GREs. Based on these results, we postulate that E5.5 retina contain glucocorticoid receptors whose signal transduction properties are enhanced by PKA. Unlike the transiently expressed glutamine synthetase fusion gene, however, activation of PKA does not render the endogenous glutamine synthetase gene glucocorticoid-inducible. Thus, its expression appears to be subject to an additional level of control in the developing retina.
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PMID:Protein kinase A activation of glucocorticoid-mediated signaling in the developing retina. 809 80

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to activate adenylate cyclase and stimulate PRL secretion in dispersed pituitary cells. We have employed the GH3 rat pituitary cell line to investigate whether PACAP can regulate expression of the PRL gene. PACAP increased cellular levels of cAMP in a concentration-dependent fashion (EC50, approximately 6 x 10(-9) M). PACAP also increased PRL mRNA levels in GH3 cells, implying that this peptide stimulates a step in expression of the PRL gene. In addition, PACAP strongly stimulated chloramphenicol acetyltransferase (CAT) activity in GH3 cells transiently transfected with a plasmid containing the first 187 basepairs of the rat PRL promoter cloned up-stream of the CAT gene, implying that PACAP stimulates transcription directed by the PRL promoter. The PACAP stimulation of CAT activity was observed at concentrations as low as 10(-11) M. We examined the action of PACAP on expression of a 5'-deletion series of PRL-CAT constructs. The PACAP response is completely lost when PRL promoter sequences between positions -187 and -113 are removed, implying that neither a previously described sequence resembling a cAMP response element nor the most proximal pit-1-binding site 1P plays a major role in the actions of PACAP on PRL gene transcription. This observation together with the ability of low concentrations of PACAP to stimulate PRL promoter activity without detectably increasing cellular cAMP levels suggest that the action of PACAP on PRL gene transcription might involve a cAMP-independent pathway.
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PMID:Pituitary adenylate cyclase-activating polypeptide stimulates prolactin gene expression in a rat pituitary cell line. 824 97

The 14-residue peptide (peptide 14) corresponding to Arg2410-Lys2423 of the insulin-like growth factor II receptor (IGF-IIR) can activate the adenylate cyclase-inhibitor guanine nucleotide-binding protein Gi, and the 15-residue beta III-2 peptide Arg259-Lys273 of the beta 2-adrenergic receptor (beta 2AR) can activate the stimulatory protein Gs. In phospholipid vesicles, IGF-IIR and beta 2AR activate Gi and Gs in response to IGF-II and isoproterenol, respectively. We constructed a chimeric IGF-II receptor (beta III-2/IGF-IIR) by converting its native peptide 14 sequence to the beta III-2 sequence. In cells expressing beta III-2/IGF-IIR, membrane adenylate cyclase activity markedly increased without IGF-II and was further promoted by IGF-II. This was verified by measuring chloramphenicol acetyltransferase (CAT) activity in beta III-2/IGF-IIR cells with cotransfection of a cAMP response element-CAT construct. This study shows not only the conversion of G-protein specificity of a receptor from Gi to Gs but also the simulation of G protein-coupled receptor signals by using a short receptor region and intact cells. These findings indicate that the G protein-activation signals are interchangeable, self-determined structural motifs that function in the setting of either a single-spanning or multiple-spanning receptor.
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PMID:Conversion of G-protein specificity of insulin-like growth factor II/mannose 6-phosphate receptor by exchanging of a short region with beta-adrenergic receptor. 826 25

In this study, the functional role of two cAMP-response elements (CRE) in the promoter of the chinook salmon GH gene and their interactions with the transcription factor Pit-1 in regulating GH gene expression were examined. A chimeric construct of the chloramphenicol acetyltransferase (CAT) reporter gene with the CRE-containing GH promoter (pGH.CAT) was transiently transfected into primary cultures of rainbow trout pituitary cells. The expression of CAT activity was stimulated by an adenylate cyclase activator forskolin as well as a membrane-permeant cAMP analog 8-bromo-cAMP. Furthermore, these stimulatory responses were inhibited by a protein kinase A inhibitor H89, suggesting that these CREs are functionally coupled to the adenylate cyclase-cAMP-protein kinase A cascade. This hypothesis is supported by parallel studies using GH4ZR7 cells, a rat pituitary cell line stably transfected with dopamine D2 receptors. In this cell line, D2 receptor activation is known to inhibit adenylate cyclase activity and cAMP synthesis. Stimulation with a nonselective dopamine agonist, apomorphine, or a D2-specific agonist, Ly171555, suppressed the expression of pGH.CAT in GH4ZR7 cells, and this inhibition was blocked by simultaneous treatment with forskolin. These results indicate that inhibition of the cAMP-dependent pathway reduces the basal promoter activity of the CRE-containing pGH.CAT. The functionality of these CREs was further confirmed by deletion analysis and site-specific mutagenesis. In trout pituitary cells, the cAMP inducibility of pGH.CAT was inhibited after deleting the CRE-containing sequence from the GH promoter. When the CRE-containing sequence was cloned into a CAT construct with a viral thymidine kinase promoter, a significant elevation of cAMP inducibility was observed. This stimulatory response, however, was abolished by mutating the core sequence, CGTCA, in these CREs, suggesting that these cis-acting elements confer cAMP inducibility to the salmon GH gene. The interactions between CREs and the transcription factor Pit-1 in mediating GH gene expression were also examined. In HeLa cells, a human cervical cancer cell line deficient in Pit-1, both basal and cAMP-induced expression of pGH.CAT were apparent only with the cotransfection of a Pit-1 expression vector. These results taken together indicate that the two CREs in the chinook salmon GH gene are functionally associated with the cAMP-dependent pathway and that their promoter activity is dependent on the presence of Pit-1
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PMID:Induction of chinook salmon growth hormone promoter activity by the adenosine 3',5'-monophosphate (cAMP)-dependent pathway involves two cAMP-response elements with the CGTCA motif and the pituitary-specific transcription factor Pit-1. 861 14

Basic fibroblast growth factor (FGF-2) is synthesized as different molecular mass isoforms all lacking the signal-peptide sequence. The high molecular-mass isoforms (21-24 kDa) possess a signal sequence directing their nuclear translocation. The role of each isoform is still poorly understood, however, modifications in intracellular signalling pathways could explain some effects of these peptides. In order to evaluate the role of FGF-2 isoforms on the adenylate cyclase (AC) signalling pathway, we retrovirally infected a rat pancreatic cell line (AR4-2J) with point-mutated FGF-2 cDNAs, allowing the expression of the 18 (A5 cells) or 22.5 kDa isoform (A3 cells) at a low level. In membrane preparations of A3 cells, unscheduled expression of the 22.5 kDa FGF-2 isoform induced a 2-fold decrease in both basal and forskolin-stimulated AC activity. Studies carried out on intact cells also showed decreased accumulation of cAMP in A3 cells in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Both FGF-2 peptides also induced functional modifications of G-proteins without affecting their levels. The 22.5 kDa peptide led to enhanced ADP-ribosylation of both alpha(s)-subunits in vitro, whereas the expression of the low molecular-mass 18 kDa peptide resulted in a 2-fold increase in alpha12 and alpha0 ADP-ribosylations. Furthermore, control CAT cells (AR4-2J cells transfected with the retrovirus containing the chloramphenicol acetyltransferase gene) and A5 cells were growth-inhibited by 8-Br-cAMP, in contrast to A3 cells. These data provide evidence that the expression of FGF-2 peptides could play a role in cell functions by modifying the AC signalling pathway. FGF-2 peptides are able to modulate both AC activity and the regulatory G-proteins. Finally FGF-2 expression may interfere with cAMP-regulated cell proliferation.
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PMID:Cells retrovirally transfected with fibroblast growth factor-2 isoforms exhibit altered adenylate cyclase activity and G-protein functionality. 861 38

Parathyroid hormone (PTH) activates both adenylate cyclase and phospholipase C in target cells, and cloned PTH/PTH-related protein (PTHrP) receptor can mediate both responses when expressed in host cells such as LLC-PK1 renal epithelial cells. Because calcitonin (CT) is known to augment 70-kDa heat shock protein (HSP70) mRNA by an adenosine 3',5'-cyclic monophosphate (cAMP)-independent mechanism in LLC-PK1 cells, we examined regulation of HSP70 transcription by PTH in these cells. Like CT, human PTH-(1-34) [hPTH-(1-34); 10(-10) to 10(-7) M)] increased porcine HSP70 mRNA and human HSP70 promoter-chloramphenicol acetyltransferase (CAT) expression within 4 h in LLC-PK1 cells that stably express > or = 100,000 PTH/PTHrP receptors per cell. The effect of PTH on HSP70 mRNA was not mimicked by cAMP analogues, forskolin, phorbol esters, Ca2+ ionophores, or alpha-thrombin; was insensitive to pertussis toxin; and was not due to increased mRNA stability. The upregulation of HSP70 gene transcription by hPTH (and CT) was clearly observed even after deletion of the functional heat shock consensus element in the promoter region of the human HSP70/CAT reporter. Upregulation of HSP70 transcription via endogenous PTH receptors also was observed in the osteoblastic cell lines SaOS-2 and ROS 17/2.8. Regulation of HSP70 gene transcription by PTH may be a common cellular response to the hormone, which, in some cells, may not be mediated by activation of adenylate cyclase or protein kinase C.
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PMID:Regulation of HSP70 by PTH: a model of gene regulation not mediated by changes in cAMP levels. 876 37

We investigated the inhibitory effects of intracellular cyclic adenosine monophosphate (cAMP) levels in regulating class 3 aldehyde dehydrogenase (aldh3) gene expression using cultures of primary rat hepatocytes and transient transfection experiments with HepG2 cells. In addition to regulation by an Ah receptor-dependent mechanism, expression of many members of the Ah gene battery have been shown to be negatively regulated. As was seen for the cytochrome P450 (cyp1A1) gene, aldh3 is transcriptionally inducible by polycyclic aromatic hydrocarbons (PAH), and this induction involving function of the arylhydrocarbon (Ah) receptor is inhibited by the protein kinase C (PKC) inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine di-HCl (H7) and staurosporine. However, PAH induction of ALDH-3 activity, protein, and mRNA was potentiated 2-4-fold by addition of the protein kinase A (PKA) inhibitors, N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide di-HCl (H8) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide HCl (HA1004). These PKA inhibitors had no effect on the PAH induction of the cyp1A1. Protein kinase A activity of cultured hepatocytes was specifically inhibited by H8 and HA1004 in a concentration-dependent manner, but not by H7, and there was an inverse correlation observed between potentiation of PAH-induced aldh3 gene expression and inhibition of specific PKA activity by the PKA inhibitors. The cAMP analog dibutyryl cAMP, the adenylate cyclase activator forskolin, and the protein phosphatase 1 and 2A inhibitor okadaic acid all dramatically inhibited both PAH induction and H8 potentiation of PAH induction of aldh3 expression but had no effect on induction of cyp1A1 expression in cultured hepatocytes. Both basal and PAH-dependent expression of a chloramphenicol acetyltransferase expression plasmid containing approximately 3.5 kilobase pairs of the 5'-flanking region of aldh3 (pALDH3.5CAT) were enhanced 3-4-fold by the PKA inhibitor H8 but not by the PKC inhibitor H7 (>20 microM). cAMP analogs, activators of PKA activity, or protein phosphatase inhibitors diminished expression of the reporter gene in a manner identical to the native gene in cultured rat hepatocytes. Using deletion analysis of the pALDH3.5CAT construct, we demonstrated the existence of a negative regulatory region in the 5'-flanking region between -1057 and -991 base pairs which appears to be responsible for the cAMP-dependent regulation of this gene under both basal and PAH-induced conditions. At least two apparently independent mechanisms which involve protein phosphorylation regulate aldh3 expression. One involves function of the Ah receptor which requires PKC protein phosphorylation to positively regulate both aldh3 and cyp1A1 gene expression and the other a cAMP-responsive process which allows PKA activity to negatively regulate expression of aldh3 under either basal or inducible conditions.
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PMID:cAMP-dependent negative regulation of rat aldehyde dehydrogenase class 3 gene expression. 901 60

Parathyroid hormone (PTH) functions in part by regulating osteoblast cytokine expression. We recently demonstrated that PTH induced a rapid and transient increase in interleukin-6 (IL-6) mRNA expression in rat bones in vivo. To determine the molecular basis of this effect, we analyzed the human IL-6 promoter fused (-1,179 to +9) with the chloramphenicol acetyltransferase (CAT) reporter gene in stable transfections into human osteoblast-like osteosarcoma SaOS-2 cells. We compared the effects of PTH on IL-6 expression with adenylate cyclase activator forskolin, PKC activator phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187, interleukin-1 alpha (IL-1 alpha), prostaglandin E-2 (PGE-2), RS-66271 (a parathyroid hormone-related peptide analog), and platelet-derived growth factor-BB (PDGF-BB). Analyses of cell clones showed that IL-6 promoter expression was extremely low in the unstimulated state. Exposure to PTH (0.001-100 nM) for 12 h stimulated CAT expression in a dose-dependent manner (200-500% of control). Treatment with IL-1 alpha was more potent than PTH in inducing transcription of the IL-6 promoter (900-1,000%). Activation of the cAMP-PKA pathway by treatment with forskolin induced a comparable level of induction with PTH. Together, the effects of PTH and forskolin were additive. RS-66271, previously shown to have PTH-like effects, induced a comparable level of IL-6 promoter expression. When examined together, PTH+RS-66271 effects were comparable to PTH effects alone. Exposure to PGE-2, PMA, PDGF-BB, or A23187 for 12 h did not significantly alter IL-6 promoter expression. These results demonstrate PTH, forskolin, the PTHrP analog RS-66271, and IL-1 alpha stimulate IL-6 expression by stimulating gene transcription. The response to forskolin suggests that the messenger system mediated by PKA is sufficient to induce IL-6 expression.
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PMID:Parathyroid hormone (1-34)-mediated interleukin-6 induction. 932 32


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