The PalkBFGHJKL promoter is under carbon catabolite repression control in Pseudomonas oleovorans but not in Escherichia coli alk+ recombinants. (1/260)

The alk genes are located on the OCT plasmid of Pseudomonas oleovorans and encode an inducible pathway for the utilization of n-alkanes as carbon and energy sources. We have investigated the influence of alternative carbon sources on the induction of this pathway in P. oleovorans and Escherichia coli alk+ recombinants. In doing so, we confirmed earlier reports that induction of alkane hydroxylase activity in pseudomonads is subject to carbon catabolite repression. Specifically, synthesis of the monooxygenase component AlkB is repressed at the transcriptional level. The alk genes have been cloned into plasmid pGEc47, which has a copy number of about 5 to 10 per cell in both E. coli and pseudomonads. Pseudomonas putida GPo12 is a P. oleovorans derivative cured of the OCT plasmid. Upon introduction of pGEc47 in this strain, carbon catabolite repression of alkane hydroxylase activity was reduced significantly. In cultures of recombinant E. coli HB101 and W3110 carrying pGEc47, induction of AlkB and transcription of the alkB gene were no longer subject to carbon catabolite repression. This suggests that carbon catabolite repression of alkane degradation is regulated differently in Pseudomonas and in E. coli strains. These results also indicate that PalkBFGHJKL, the Palk promoter, might be useful in attaining high expression levels of heterologous genes in E. coli grown on inexpensive carbon sources which normally trigger carbon catabolite repression of native expression systems in this host.  (+info)

Characterization of cytochrome P450 expression in human oesophageal mucosa. (2/260)

The expression of cytochrome (CYP) P450 enzymes in human oesophageal mucosa was investigated in a total of 25 histologically non-neoplastic surgical tissue specimens by using specific antibodies in immunoblots and by RT-PCR mRNA analysis. The presence of CYP1A, 2E1, 3A and 4A enzymes was demonstrated by both techniques; CYP2A reactive protein was also detected by immunoblot. The presence of CYP4B1 mRNA was established but no specific antibody was available for detection of the corresponding protein by immunoblot. CYP2B6/7 mRNA was not detected in any sample. The mRNA transcripts for CYP1A1, 2E1, 4A11 and 4B1 were consistently detected in the majority of samples (>84%), whereas CYP1A2 mRNA was only detected in 11 of 19 specimens examined. An RT-PCR method to differentiate CYP3A4 and 3A5 mRNA was developed. This demonstrated CYP3A5 mRNA expression in all samples tested, whereas CYP3A4 mRNA was not detectable, suggesting that CYP3A5 is the major CYP3A protein in human oesophagus. There were significant interindividual variations in the amount of proteins, ranging from 8-fold for CYP4A to 43-fold for CYP2E1. For each patient, data on exposure to risk factors for oesophageal cancer were available, including tobacco smoke, alcohol, gastro-oesophageal reflux and hot beverage consumption. None of these risk factors or other patient characteristics (age, sex, tumour location and tumour stage) were correlated with the protein level of the individual CYP enzymes as determined by quantitation of immunoblot staining. However, the small series of samples precludes any strong conclusion concerning the lack of such correlations. There were no differences between squamous cell carcinomas and adenocarcinomas in either the qualitative or quantitative expression of the CYP enzymes. These data demonstrate that a range of CYP enzymes are expressed in human oesophageal mucosa and indicate that this tissue has the capacity to activate chemical carcinogens to reactive DNA binding metabolites.  (+info)

Role of the alternative sigma factor sigmaS in expression of the AlkS regulator of the Pseudomonas oleovorans alkane degradation pathway. (3/260)

The AlkS protein activates transcription from the PalkB promoter, allowing the expression of a number of genes required for the assimilation of alkanes in Pseudomonas oleovorans. We have identified the promoter from which the alkS gene is transcribed, PalkS, and analyzed its expression under different conditions and genetic backgrounds. Transcription from PalkS was very low during the exponential phase of growth and increased considerably when cells reached the stationary phase. The PalkS -10 region was similar to the consensus described for promoters recognized by Escherichia coli RNA polymerase bound to the alternative sigma factor sigmaS, which directs the expression of many stationary-phase genes. Reporter strains containing PalkS-lacZ transcriptional fusions showed that PalkS promoter is very weakly expressed in a Pseudomonas putida strain bearing an inactivated allele of the gene coding for sigmaS, rpoS. When PalkS was transferred to E. coli, transcription started at the same site and expression was higher in stationary phase only if sigmaS-RNA polymerase was present. The low levels of AlkS protein generated in the absence of sigmaS were enough to support a partial induction of the PalkB promoter. The -10 and -35 regions of PalkS promoter also show some similarity to the consensus recognized by sigmaD-RNA polymerase, the primary form of RNA polymerase. We propose that in exponential phase PalkS is probably recognized both by sigmaD-RNA polymerase (inefficiently) and by sigmaS-RNA polymerase (present at low levels), leading to low-level expression of the alkS gene. sigmaS-RNA polymerase would be responsible for the high level of activity of PalkS observed in stationary phase.  (+info)

Insulin differentially affects xenobiotic-enhanced, cytochrome P-450 (CYP)2E1, CYP2B, CYP3A, and CYP4A expression in primary cultured rat hepatocytes. (4/260)

Uncontrolled diabetes results in enhanced expression of cytochrome P-450 (CYP)2E1, CYP2B, CYP3A, and CYP4A. Because of the simultaneous and confounding metabolic and hormonal changes that occur in vivo as a consequence of diabetes, primary cultured rat hepatocytes provide an excellent model system for examination of the effects of insulin on P-450 expression and on xenobiotic-mediated P-450 expression. In the present study, we examined the effects of insulin on pyridine-, phenobarbital-, and ciprofibrate-mediated expression of CYP2E1, CYP2B, CYP3A, and CYP4A in primary cultured rat hepatocytes. Pyridine addition to primary rat hepatocytes cultured in the presence of 1 nM insulin or in the absence of insulin resulted in a 3.5-fold and 3-fold enhancement in CYP2E1 protein expression, respectively, in the absence of any pyridine-mediated increase in mRNA expression. In contrast, hepatocytes cultured in the standard concentration of 1 microM insulin resulted in only a 2-fold increase in protein expression. Thus, the fold-induction of CYP2E1 protein in response to pyridine was 1.5- to 1.8-fold greater in either the absence of insulin or in the presence of 1 nM insulin, respectively, than that monitored in the presence of 1 microM insulin. To examine whether insulin effects on xenobiotic-mediated CYP2E1 expression were selective, insulin effects on xenobiotic-mediated expression of transcriptionally regulated CYP2B, CYP3A, and CYP4A were examined. Pyridine- or phenobarbital-mediated induction of CYP2B mRNA and protein expression in hepatocytes was suppressed by as much as 80% at lower insulin levels (0 and 1 nM), relative to the level monitored in the presence of 1 microM insulin. Omitting insulin from the medium resulted in a 50% decrease in CYP3A mRNA levels in response to phenobarbital treatment and a 30% decrease in CYP4A mRNA levels in response to ciprofibrate treatment, relative to the level obtained in response to these treatments in the presence of 1 microM insulin. The results of this study demonstrate that decreasing the insulin level in the primary hepatocyte culture medium enhanced xenobiotic-mediated CYP2E1 expression, whereas lower insulin levels suppressed xenobiotic-mediated CYP2B, CYP3A, and CYP4A expression in this cell culture system.  (+info)

Hypoxia-induced production of 12-hydroxyeicosanoids in the corneal epithelium: involvement of a cytochrome P-4504B1 isoform. (5/260)

The corneal epithelium metabolizes arachidonic acid by a cytochrome P-450 (CYP)-mediated activity to 12-hydroxy-5,8,11, 14-eicosatetraenoic acid (12(R)-HETE) and 12-hydroxy-5,8, 14-eicosatrienoic acid (12(R)-HETrE ). Both metabolites possess potent inflammatory properties, with 12(R)-HETrE being a powerful angiogenic factor, and they assume the role of inflammatory mediators in hypoxia- and chemical-induced injury in the cornea in vivo and in vitro. We used a model of corneal organ culture that exhibits hypoxia-induced epithelial CYP-dependent 12(R)-HETE and 12(R)-HETrE synthesis for isolating, identifying, and characterizing the CYP protein responsible for these eicosanoid syntheses. Northern analysis revealed the presence of a CYP4A-hybridizable mRNA, the levels of which were increased after hypoxia. Reverse transcription-polymerase chain reaction analysis with primers specific for the CYP4A family led to the isolation of a 671-base pair fragment with a 98.8% sequence homology to the rabbit lung CYP4B1 isoform, of which the levels in the corneal epithelium were greatly increased under hypoxic conditions. Moreover, phenobarbital, an inducer of hepatic CYP4B1 in the rabbit, also induced 12-HETE and 12-HETrE synthesis. Antibodies against CYP4B1, but not against CYP4A1, inhibited hypoxia-, clofibrate-, and phenobarbital-induced 12-HETE and 12-HETrE synthesis. These results suggest the involvement of a CYP4B1 isoform in the corneal epithelial synthesis of these eicosanoids in response to hypoxia.  (+info)

Arachidonic acid and PGE2 regulation of hepatic lipogenic gene expression. (6/260)

N-6 polyunsaturated fatty acids (PUFA) suppress hepatic and adipocyte de novo lipogenesis by inhibiting the transcription of genes encoding key lipogenic proteins. In cultured 3T3-L1 adipocytes, arachidonic acid (20:4,n-6) suppression of lipogenic gene expression requires cyclooxygenase (COX) activity. In this study, we found no evidence to support a role for COX-1 or -2 in the 20:4,n-6 inhibition of hepatocyte lipogenic gene expression. In contrast to L1 preadipocytes, adipocytes and rat liver, RT-PCR and Western analyses did not detect COX-1 or COX-2 expression in cultured primary hepatocytes. Moreover, the COX inhibitor, flurbiprofen, did not affect the 20:4,n-6 regulation of lipogenic gene expression in primary hepatocytes. Despite the absence of COX-1 and -2 expression in primary hepatocytes, prostaglandins (PGE2 and PGF2alpha) suppressed fatty acid synthase, l-pyruvate kinase, and the S14 protein mRNA, while having no effect on acyl-CoA oxidase or CYP4A2 mRNA. Using PGE2 receptor agonist, the PGE2 effect on lipogenic gene expression was linked to EP3 receptors. PGE2 inhibited S14CAT activity in transfected primary hepatocytes and targeted the S14 PUFA-response region located -220 to -80 bp upstream from the transcription start site. Taken together, these studies show that COX-1 and COX-2 do not contribute to the n-6 PUFA suppression of hepatocyte lipogenic gene expression. However, cyclooxygenase products from non-parenchymal cells can act on parenchymal cells through a paracrine process and mimic the effects of n-6 PUFA on lipogenic gene expression.  (+info)

Kinetic profile of the rat CYP4A isoforms: arachidonic acid metabolism and isoform-specific inhibitors. (7/260)

20-Hydroxyeicosatetraenoic acid (HETE), the cytochrome P-450 (CYP) 4A omega-hydroxylation product of arachidonic acid, has potent biological effects on renal tubular and vascular functions and on the control of arterial pressure. We have expressed high levels of the rat CYP4A1, -4A2, -4A3, and -4A8 cDNAs, using baculovirus and Sf 9 insect cells. Arachidonic acid omega- and omega-1-hydroxylations were catalyzed by three of the CYP4A isoforms; the highest catalytic efficiency of 947 nM-1. min-1 for CYP4A1 was followed by 72 and 22 nM-1. min-1 for CYP4A2 and CYP4A3, respectively. CYP4A2 and CYP4A3 exhibited an additional arachidonate 11,12-epoxidation activity, whereas CYP4A1 operated solely as an omega-hydroxylase. CYP4A8 did not catalyze arachidonic or linoleic acid but did have a detectable lauric acid omega-hydroxylation activity. The inhibitory activity of various acetylenic and olefinic fatty acid analogs revealed differences and indicated isoform-specific inhibition. These studies suggest that CYP4A1, despite its low expression in extrahepatic tissues, may constitute the major source of 20-HETE synthesis. Moreover, the ability of CYP4A2 and -4A3 to catalyze the formation of two opposing biologically active metabolites, 20-HETE and 11, 12-epoxyeicosatrienoic acid, may be of great significance to the regulation of vascular tone.  (+info)

Regulation of P-450 4A activity in the glomerulus of the rat. (8/260)

We recently reported that an enzyme of the cytochrome P-450 4A family is expressed in the glomerulus, but there is no evidence that 20-hydroxyeicosatetraenoic acid (20-HETE) can be produced by this tissue. The purpose of present study was to determine whether glomeruli isolated from the kidney of rats can produce 20-HETE and whether the production of this metabolite is regulated by nitric oxide (NO) and dietary salt intake. Isolated glomeruli produced 20-HETE, dihydroxyeicosatrienoic acids, and 12-hydroxyeicosatetraenoic acid (4.13 +/- 0.38, 4.20 +/- 0.38, and 2. 10 +/- 0.20 pmol. min-1. mg protein-1, respectively) when incubated with arachidonic acid (10 microM). The formation of 20-HETE was dependent on the availability of NADPH and the PO2 of the incubation medium. The formation of 20-HETE was inhibited by NO donors in a concentration-dependent manner. The production of 20-HETE was greater in glomeruli isolated from the kidneys of rats fed a low-salt diet than in kidneys of rats fed a high-salt diet (5.67 +/- 0.32 vs. 2.83 +/- 0.32 pmol. min-1. mg protein-1). Immunoblot experiments indicated that the expression of P-450 4A protein in glomeruli from the kidneys of rats fed a low-salt diet was sixfold higher than in kidneys of rats fed a high-salt diet. These results indicate that arachidonic acid is primarily metabolized to 20-HETE and dihydroxyeicosatrienoic acids in glomeruli and that glomerular P-450 activity is modulated by NO and dietary salt intake.  (+info)

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