DNA methyltransferase is a downstream effector of cellular transformation triggered by simian virus 40 large T antigen.
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This paper tests the hypothesis that DNA methyltransferase plays a causal role in cellular transformation induced by SV40 T antigen. We show that T antigen expression results in elevation of DNA methyltransferase (MeTase) mRNA, DNA MeTase protein levels, and global genomic DNA methylation. A T antigen mutant that has lost the ability to bind pRb does not induce DNA MeTase. This up-regulation of DNA MeTase by T antigen occurs mainly at the posttranscriptional level by altering mRNA stability. Inhibition of DNA MeTase by antisense oligonucleotide inhibitors results in inhibition of induction of cellular transformation by T antigen as determined by a transient transfection and soft agar assay. These results suggest that elevation of DNA MeTase is an essential component of the oncogenic program induced by T antigen. (+info)
Pharmacological evidence that inducible nitric oxide synthase is a mediator of delayed preconditioning.
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Brief periods of myocardial ischaemia preceding a subsequent more prolonged ischaemic period 24-72 h later confer protection against myocardial infarction ('delayed preconditioning' or the 'second window' of preconditioning). In the present study, we examined the effects of pharmacological modifiers of inducible nitric oxide synthase (iNOS) induction and activity on delayed protection conferred by ischaemic preconditioning 48 h later in an anaesthetized rabbit model of myocardial infarction. Rabbits underwent a myocardial preconditioning protocol (four 5 min coronary artery occlusions) or were sham-operated. Forty-eight hours later they were subjected to a sustained 30 min coronary occlusion and 120 min reperfusion. Infarct size was determined with triphenyltetrazolium staining. In rabbits receiving no pharmacological intervention, the percentage of myocardium infarcted within the risk zone was 43.9+5.0% in sham-operated animals and this was significantly reduced 48 h after ischaemic preconditioning with four 5 min coronary occlusions to 18.5+5.6% (P<0.01). Administration of the iNOS expression inhibitor dexamethasone (4 mg kg(-1) i.v) 60 min before ischaemic preconditioning completely blocked the infarct-limiting effect of ischaemic preconditioning (infarct size 48.6+/-6.1%). Furthermore, administration of aminoguanidine (300 mg kg(-1), s.c.), a relatively selective inhibitor of iNOS activity, 60 min before sustained ischaemia also abolished the delayed protection afforded by ischaemic preconditioning (infarct size 40.0+/-6.0%). Neither aminoguanidine nor dexamethasone per se had significant effect on myocardial infarct size. Myocardial risk zone volume during coronary ligation, a primary determinant of infarct size in this non-collateralized species, was not significantly different between intervention groups. There were no differences in systolic blood pressure, heart rate, arterial blood pH or rectal temperature between groups throughout the experimental period. These data provide pharmacological evidence that the induction of iNOS, following brief periods of coronary occlusion, is associated with increased myocardial tolerance to infarction 48 h later. (+info)
Expression of the nlsLacz gene in dendritic cells derived from retrovirally transduced peripheral blood CD34+ cells.
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BACKGROUND AND OBJECTIVE: Gene transfer and expression of exogenous genetic information coding for an immunogenic protein in antigen presenting cells (APCs) can promote an immune response. This was investigated by retroviral transfer of a marker gene into CD34+ derived APCs. DESIGN AND METHODS: To achieve long term expression of a specific transgene in APCs, G-CSF mobilized peripheral blood CD34+ cell populations were retrovirally transduced with the bacterial nlsLacZ, a marker gene used here as a model, in the presence of IL-3, IL-6, GM-CSF and SCF prior to being induced to differentiate into dendritic and macrophage cells by GM-CSF and TNF-a. RESULTS: Addition of IL-4 was found to induce dendritic differentiation preferentially by inhibiting proliferation and differentiation of the macrophage lineage. As assessed by X-Gal staining, LacZ gene expression was observed in cells from both the dendritic lineage (CD1a+/CD14-) which still exhibits the highest immunostimulatory activity in mixed lymphocyte reaction and from the macrophage lineage (CD1a-/ CD14+). INTERPRETATION AND CONCLUSIONS: This study sets out the possibility of transducing dendritic and macrophage progenitors present in the CD34+ cell population and in using a marker gene such as nlsLacZ to study gene expression in antigen presenting cell compartments. (+info)
Curcumin inhibits cyclooxygenase-2 transcription in bile acid- and phorbol ester-treated human gastrointestinal epithelial cells.
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We investigated whether curcumin, a chemopreventive agent, inhibited chenodeoxycholate (CD)- or phorbol ester (PMA)-mediated induction of cyclooxygenase-2 (COX-2) in several gastrointestinal cell lines (SK-GT-4, SCC450, IEC-18 and HCA-7). Treatment with curcumin suppressed CD- and PMA-mediated induction of COX-2 protein and synthesis of prostaglandin E2. Curcumin also suppressed the induction of COX-2 mRNA by CD and PMA. Nuclear run-offs revealed increased rates of COX-2 transcription after treatment with CD or PMA and these effects were inhibited by curcumin. Treatment with CD or PMA increased binding of AP-1 to DNA. This effect was also blocked by curcumin. In addition to the above effects on gene expression, we found that curcumin directly inhibited the activity of COX-2. These data provide new insights into the anticancer properties of curcumin. (+info)
Comparison of the DNA adducts formed by tamoxifen and 4-hydroxytamoxifen in vivo.
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Tamoxifen is a liver carcinogen in rats and has been associated with an increased risk of endometrial cancer in women. Recent reports of DNA adducts in leukocyte and endometrial samples from women treated with tamoxifen suggest that it may be genotoxic to humans. One of the proposed pathways for the metabolic activation of tamoxifen involves oxidation to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. In the present study, we compared the extent of DNA adduct formation in female Sprague-Dawley rats treated by gavage with seven daily doses of 54 micromol/kg tamoxifen or 4-hydroxytamoxifen and killed 24 h after the last dose. Liver weights and microsomal rates of ethoxyresorufin O-deethylation, 4-dimethylaminopyrine N-demethylation and p-nitrophenol oxidation were not altered by tamoxifen or 4-hydroxytamoxifen treatment. Uterine weights were decreased significantly and uterine peroxidase activity was decreased marginally in treated as compared with control rats. DNA adducts were assayed by 32P-post-labeling in combination with HPLC. Two major DNA adducts were detected in liver DNA from rats administered tamoxifen. These adducts had retention times comparable with those obtained from in vitro reactions of alpha-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide with DNA. Hepatic DNA adduct levels in rats administered 4-hydroxytamoxifen did not differ from those observed in control rats. Likewise, adduct levels in uterus DNA from rats treated with tamoxifen or 4-hydroxytamoxifen were not different from those detected in control rats. These data suggest that a metabolic pathway involving 4-hydroxytamoxifen is not a major pathway in the activation of tamoxifen to a DNA-binding derivative in Sprague-Dawley rats. (+info)
Defective interleukin (IL)-18-mediated natural killer and T helper cell type 1 responses in IL-1 receptor-associated kinase (IRAK)-deficient mice.
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Interleukin (IL)-18 is functionally similar to IL-12 in mediating T helper cell type 1 (Th1) response and natural killer (NK) cell activity but is related to IL-1 in protein structure and signaling, including recruitment of IL-1 receptor-associated kinase (IRAK) to the receptor and activation of c-Jun NH2-terminal kinase (JNK) and nuclear factor (NF)-kappaB. The role of IRAK in IL-18-induced responses was studied in IRAK-deficient mice. Significant defects in JNK induction and partial impairment in NF-kappaB activation were found in IRAK-deficient Th1 cells, resulting in a dramatic decrease in interferon (IFN)-gamma mRNA expression. In vivo Th1 response to Propionibacterium acnes and lipopolysaccharide in IFN-gamma production and induction of NK cytotoxicity by IL-18 were severely impaired in IRAK-deficient mice. IFN-gamma production by activated NK cells in an acute murine cytomegalovirus infection was significantly reduced despite normal induction of NK cytotoxicity. These results demonstrate that IRAK plays an important role in IL-18-induced signaling and function. (+info)
NahW, a novel, inducible salicylate hydroxylase involved in mineralization of naphthalene by Pseudomonas stutzeri AN10.
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Two genes, nahG and nahW, encoding two independent salicylate 1-hydroxylases have been identified in the naphthalene-degrading strain Pseudomonas stutzeri AN10. While nahG resides in the same transcriptional unit as the meta-cleavage pathway genes, forming the naphthalene degradation lower pathway, nahW is situated outside but in close proximity to this transcriptional unit. The nahG and nahW genes of P. stutzeri AN10 are induced and expressed upon incubation with salicylate, and the enzymes that are encoded, NahG and NahW, are involved in naphthalene and salicylate metabolism. Both genes, nahG and nahW, have been cloned in Escherichia coli JM109. The overexpression of these genes yields peptides with apparent molecular masses of 46 kDa (NahG) and 43 kDa (NahW), respectively. Both enzymes exhibit broad substrate specificities and metabolize salicylate, methylsalicylates, and chlorosalicylates. However, the relative rates by which the substituted analogs are transformed differ considerably. (+info)
Inducing effect of diamines on transcription of the cephamycin C genes from the lat and pcbAB promoters in Nocardia lactamdurans.
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The diamines putrescine, cadaverine, and diaminopropane stimulate cephamycin biosynthesis in Nocardia lactamdurans, in shake flasks and fermentors, without altering cell growth. Intracellular levels of the P7 protein (a component of the methoxylation system involved in cephamycin biosynthesis) were increased by diaminopropane, as shown by immunoblotting studies. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase activities involved in biosynthesis of the alpha-aminoadipic acid precursor were also greatly stimulated. The diamine stimulatory effect is exerted at the transcriptional level, as shown by low-resolution S1 protection studies. The transcript corresponding to the pcbAB gene and to a lesser extent also the lat transcript were significantly increased in diaminopropane-supplemented cultures, whereas transcription from the cefD promoter was not affected. Coupling of the lat and pcbAB promoters to the reporter xylE gene showed that expression from the lat and pcbAB promoters was increased by addition of diaminopropane in Streptomyces lividans. Intracellular accumulation of diamines in Nocardia may be a signal to trigger antibiotic production. (+info)