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)

The expression of the principal opioid peptide gene, preproenkephalin A, is exquisitely regulated by primary afferent inputs to the spinal and medullary dorsal horns. This regulated expression in response to neural synaptic activity has been referred to as trans-synaptic regulation. To define which DNA regions could mediate this trans-synaptic regulation, transgenic 'HEC' mice whose genomes include 193 bp of the human preproenkephalin A promoter fused to a chloramphenicol acetyltransferase (CAT) reporter gene were studied. Mice received unilateral electrical stimulation of the trigeminal ganglion or adjuvant injection into the hindpaw, stimuli known to regulate dorsal horn proenkephalin expression in vivo. CAT activity conferred by this promoter displayed trans-synaptic upregulation with both stimuli. Although the level of the upregulation was 2- to 3-fold higher than the change in the wild type gene, several features of this induction paralleled aspects of the behavior of the wild-type gene: the rapidity of responses to trigeminal ganglion stimulation, the stimulation intensity dependence of responses to trigeminal ganglion stimulation and the time course of upregulation noted following adjuvant injection. Regulatory proteins binding to this restricted promoter region are thus likely to mediate aspects of dorsal horn enkephalin regulation by pain and other somatic stimuli.
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PMID:Primary afferent stimulation acts through a 193 base pair promoter region to upregulate preproenkephalin expression in dorsal horn of transgenic mice. 131 94

Glial cells execute essential functions in central nervous system (CNS) development and are also believed to play important roles during gliosis in response to trauma or disease. These developmental and pathological states have also been associated with elevated expression of opioid genes. Because levels of the cytokine interleukin-1 beta (IL-1 beta) increase following CNS lesions, we examined the possible influence of IL-1 beta on the expression of opioid genes in astrocytes cultured from rat cortex. Proenkephalin mRNA expression was stimulated by IL-1 beta in a time- and concentration-dependent manner, being maximal with 5 U/ml IL-1 beta at 4 h. Although the beta-adrenergic agonist isoproterenol was also active, interferon, glutamate, and carbachol were not. Unlike isoproterenol, the actions of IL-1 beta were not associated with a cyclic adenosine monophosphate (AMP)-dependent pathway. Interleukin-1 beta also regulated a proenkephalin-chloramphenicol acetyltransferase fusion gene transiently transfected into astrocytes, with a dose-response similar to that active in proenkephalin mRNA. These effects of IL-1 beta were region-specific, not being observed with either cerebellar or hippocampal astrocytes; however, isoproterenol was active in the latter cell populations. Proenkephalin mRNA in cortical astrocytes was stimulated following a temperature stress. These results suggest that enhanced proenkephalin gene expression in astrocytes by IL-1 beta may be important in neuroimmune interactions and in trauma-induced CNS injury or stress.
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PMID:Interleukin-1 beta regulates proenkephalin gene expression in astrocytes cultured from rat cortex. 147 30

Preproenkephalin metabolism, in the rat, was studied in primary striatal neurons maintained in a chemically defined medium. Acute treatment (30 min) with forskolin (10(-5) M) or phorbol 12 myristate 13 acetate (10(-7) M) resulted, respectively, in a two- and seven-fold increase in methionine-enkephalin secretion. Chronic treatment with forskolin or phorbol 12 myristate 13 acetate (24 h) induced a 100% increase in methionine-enkephalin content (forskolin) and on the other hand a 50% decrease in methionine-enkephalin (phorbol 12 myristate 13 acetate). Both treatments increased preproenkephalin mRNA levels in a time-dependent manner, this augmentation being observable after 180 min by Northern blot analysis and in situ hybridization. These data indicate that under chronic stimulation, with either forskolin or phorbol 12 myristate 13 acetate, proenkephalin turnover is accelerated. However, after stimulation with phorbol 12 myristate 13 acetate, the more potent methionine-enkephalin secretagogue, increased peptide synthesis is not sufficient to replenish methionine-enkephalin intracellular stores. Preproenkephalin gene transcription was analysed by introducing the preproenkephalin gene promoter fused to the bacterial acetyl chloramphenicol transferase reporter gene into primary neurons. Chronic stimulation (48 h) by forskolin (10(-5) M) or phorbol 12 myristate 13 acetate (10(-7) M) of striatal neurons transfected with this fusion gene increased chloramphenicol acetyltransferase activity six-fold and the two effects were additive. These data suggest that the cyclic AMP and the protein kinase C pathways directly activate preproenkephalin gene transcription.
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PMID:Striatal proenkephalin turnover and gene transcription are regulated by cyclic AMP and protein kinase C-related pathways. 165 16

Glucocorticoids enhance proenkephalin gene expression in several cell types. To elucidate the mechanism(s) involved, we analyzed the potentiation by dexamethasone of the cAMP-dependent increase in proenkephalin mRNA levels elicited by forskolin in C6 rat glioma cells. This potentiation did not require ongoing protein synthesis. In nuclear run-on transcription assays, dexamethasone alone did not alter proenkephalin transcription, but strongly increased the magnitude and duration of transcriptional elevation by forskolin through a direct action not requiring ongoing protein synthesis. Dexamethasone did not alter basal or stimulated cAMP levels. To search for functionally cooperative glucocorticoid and cAMP regulatory elements, we transfected C6 cells with plasmids containing the chloramphenicol acetyltransferase (CAT) gene under the control of rat proenkephalin sequences from bases -5800 to +703. Maximum stimulation of transiently expressed CAT activity by forskolin required more than 145 and 190 or fewer base pairs of 5'-flanking sequence, implicating sequences up-stream from the previously described cAMP-inducible enhancer. Dexamethasone reduced forskolin-stimulated CAT expression from plasmids with 190 or more base-pairs of 5'-flanking sequence, an effect apparently involving multiple up-stream regions. Dexamethasone also reduced forskolin-stimulated CAT mRNA levels in C6 cells stably transfected with proenkephalin/CAT chimeric genes in the presence or absence of proteins synthesis. In summary, we demonstrate that glucocorticoids and cAMP synergize positively in regulating transcription of the endogenous gene, but interact negatively in regulating the chimeric constructs, which may lack the context or distal element(s) required for positive synergism.
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PMID:Proenkephalin gene expression in C6 rat glioma cells: potentiation of cyclic adenosine 3',5'-monophosphate-dependent transcription by glucocorticoids. 165 36

The rat and mouse proenkephalin genes each contains two distinct promoters, one of which is utilized exclusively by spermatogenic cells. The germ cell-specific promoter lacks TATA sequences, is G+C rich, and contains multiple initiation sites. To investigate the nature of the cis-acting elements that determine selective transcription of the proenkephalin gene in male germ cells, two rat proenkephalin-chloramphenicol acetyltransferase fusion genes containing the two different promoter regions as well as 1.6 or 0.3 kilobases, respectively, of 5'-flanking sequence were expressed in transgenic mice. Multiple transgenic lines were developed which expressed the fusion genes in testis, brain, and heart but not in tissues that do not normally express the proenkephalin gene. Fusion gene transcripts in transgenic mouse testes were localized to those spermatogenic cell types that utilize the spermatogenic cell promoter and were selectively and accurately initiated from the multiple rat germ cell start sites. Transgenic mice thus provide a useful model for the localization and characterization of cis-acting elements mediating transcription of the proenkephalin gene from its germ cell-specific promoter.
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PMID:Selective transcription of rat proenkephalin fusion genes from the spermatogenic cell-specific promoter in testis of transgenic mice. 174 59

A 203-base-pair sequence 5' of the latency-associated transcripts (LATs) of herpes simplex virus type 1 contains a 7-base consensus sequence TGCGTCA that is identical to the cAMP-response element of the proenkephalin gene. This consensus sequence is at -38 relative to the putative 5' end of the LATs with a TATA box at the -24 position. In transient chloramphenicol acetyltransferase assays in rat pheochromocytoma (PC12) cells, this enhancer region stimulated gene expression up to 3-fold in the presence of dibutyryl cAMP, forskolin, nerve growth factor, or phorbol 12-myristate 13-acetate. Mutation of the cAMP-response element to TGCG-CAA resulted in a 4-fold reduction of basal activity and a complete loss of inducible stimulation. In DNA gel retardation assays, purified cAMP-response element-binding protein and a nuclear protein from PC12 cells were shown to bind specifically to this element. Furthermore, it was demonstrated that the reactivation of wild-type herpes simplex virus type 1 from dissociated latently infected murine trigeminal ganglia was significantly accelerated (P less than 0.005) by the addition of cAMP analogs or adenylate cyclase activators. However, these reagents did not accelerate reactivation of a deletion mutant that lacks the putative cAMP-response element-containing promoter region, transcriptional start site, and 1015 base pairs of the LATs. These studies demonstrate that the promoter region of the LATs contains a functional cAMP-response element and that expression of the LATs is likely controlled by second messenger signal transduction and imply a role for cAMP in triggering viral reactivation.
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PMID:The promoter of the latency-associated transcripts of herpes simplex virus type 1 contains a functional cAMP-response element: role of the latency-associated transcripts and cAMP in reactivation of viral latency. 184 42

The rat gene encoding phenylethanolamine N-methyltransferase (PNMT) was cloned and a consensus sequence for a glucocorticoid response element (GRE) was found at -513 bp, 5' to the transcriptional start site. In order to define the function of this element, fusion genes containing the PNMT promoter and a chloramphenicol acetyltransferase (CAT) reporter gene were constructed. These constructs did not express after transfection into any of 7 continuous cell lines, none of which endogenously produce PNMT. A system for transfecting chromaffin cells in primary culture was therefore devised using constructs containing 200 bp of the proenkephalin (ENK) promoter, whose expression characteristics are well known. pENK beta GAL-1, containing the ENK promoter with a lac Z reporter, was introduced into these cells and beta-galactosidase activity was visualized in situ. Approximately 90% of cells transfected were chromaffin; transfection efficiency was 5%. High levels of CAT activity were measured in chromaffin cells transfected with pENKAT12, possessing a CAT reporter. In contrast to tumor cell lines, pENKAT12 induction in these cells by forskolin and phorbol esters did not require a phosphodiesterase inhibitor. In this chromaffin system, both basal and regulated expression of the PNMT fusion genes were detected. Dexamethasone (dex) induced expression of pPNMT3000 and pPNMT900, containing the putative GRE and 3000 bp or 863 bp of PNMT promoter sequence, 4- to 10-fold. Expression of pPNMT300 and pPNMT100, which lack the GRE and contain 273 bp or 99 bp of PNMT promoter sequence, was unaffected by dex. Addition of the PNMT region spanning -490 to -863 bp conferred full dex responsiveness to a thymidine kinase promoter. Deletion of the putative GRE sequence by site-directed mutagenesis abolished the dex response. These data identify the sequence at -513 bp in the rat PNMT gene as a functional, positively acting GRE. Primary cultures of bovine chromaffin cells provide a biologically relevant expression system for transcriptional studies of catecholamine genes and their related neuropeptides.
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PMID:Identification of a functional glucocorticoid response element in the phenylethanolamine N-methyltransferase promoter using fusion genes introduced into chromaffin cells in primary culture. 230 57

The expression of opioid genes was examined in isolated populations of glial cells in primary culture. Northern blot analysis of purified type I astrocytes, oligodendrocytes and mixed oligodendrocyte-type-2-astrocyte lineage cells derived from cerebral cortex demonstrated robust expression of proenkephalin mRNA exclusively in type I astrocytes. The expression of proenkephalin mRNA was stimulated by the beta-adrenergic agonist isoproterenol, and 8-(4-chlorophenyl thio)adenosine 3'-5'-cyclic monophosphate (cpt-cAMP). Both of these compounds regulated a proenkephalin-chloramphenicol acetyltransferase fusion gene transiently transfected into type I astrocytes. HPLC and immunoassay of the cell culture media revealed significant levels of unprocessed proenkephalin secreted by the cell and this secretion was stimulated by isoproterenol and cpt-cAMP. The relatively high levels of proenkephalin expressed suggest that enhanced expression in astrocytes may be important during neural development, in trauma-induced gliosis and in neuroimmune interactions.
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PMID:The regulation of proenkephalin expression in a distinct population of glial cells. 231 81

The regulation and possible function of the preproenkephalin gene in testis were studied in vivo in transgenic mice containing: (1) bases -193 to +210 of the human proenkephalin gene and an additional one kilobase of 3' proenkephalin flanking sequence driving expression of bacterial chloramphenicol acetyltransferase (CAT), and (2) the same promoter and flanking sequences driving expression of a rat proenkephalin cDNA. Five lines of mice, designated HEC1-5, expressed the first construct and 10, HER1-10, the second. Each HEC male and many HER males showed dramatic expression of the transgene in the testis, although much lower expression was observed in the brain and other enkephalin-producing tissues. High levels of expression in testis can thus be achieved with a very short promoter region and do not require intron A sequences previously considered necessary. Altered enkephalin expression may affect testicular function. One founder, HER8, displayed grossly abnormal testicular morphology and was completely infertile. A second founder, HER6, had low sperm motility. Two offspring from other lines also displayed subnormal fertility. These studies support a role for specific promoter sequences in testis expression and may further support a significant role for proenkephalin in testicular function.
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PMID:Proenkephalin transgenic mice: a short promoter confers high testis expression and reduced fertility. 791 79

Rat and mouse spermatogenic cells contain a family of 1700-nucleotide (nt) proenkephalin mRNAs that are generated from an alternate, germ cell-specific promoter. This promoter is located approximately 350 base pairs (bp) downstream of the promoter used in somatic cells, within the first intron for the somatic transcript. In a previous study, rat proenkephalin-chloramphenicol acetyltransferase fusion genes containing both promoters were shown to be transcribed selectively from the germ cell promoter and in the correct developmental pattern in spermatogenic cells of transgenic mice. In the present study it was found that spermatogenic cell-specific transgene expression was maintained after deletion of the upstream somatic promoter. This result establishes that the rat proenkephalin germ-line promoter is capable of functioning independently of transcriptional elements associated with the somatic promoter and localizes the requisite spermatogenic cell cis-elements to a 500-bp region encompassing the germ cell initiation sequences. A comprehensive analysis of binding sites for rat spermatogenic cell nuclear factors within this 500-bp region was performed using gel-shift and DNAse I footprinting techniques. Eight distinct binding regions were identified, each of which formed one or more cell-specific complexes with nuclear proteins from rat spermatogenic cells. These results suggest that multiple cis-acting elements may cooperate in the cell-specific and developmental regulation of rat proenkephalin gene transcription during spermatogenesis.
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PMID:The rat proenkephalin germ line promoter contains multiple binding sites for spermatogenic cell nuclear proteins. 823 18


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