Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00492 (hypoxanthine-guanine phosphoribosyltransferase)
 2,385 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rapamycin (RAP) disrupts signaling events implicated in cytokine-dependent proliferation of lymphocytes and other cells. This action is known to involve the formation of molecular complexes between the drug and intracellular binding proteins, termed FKBPs. However, the biochemical target(s) for the effector RAP-FKBP complexes remain uncharacterized. As an approach to explore the mechanism of action of RAP, we have isolated three independent sets of somatic mutants of the YAC-1 murine T cell line with markedly reduced sensitivity to the drug's inhibitory effects on proliferation and on IL-1-induced IFN-gamma production. These mutants were still fully sensitive to FK-506, an immunosuppressant structurally related to RAP whose mode of action also involves an interaction with FKBPs. Furthermore, the 12-kDa FKBP, FKBP12, was detectable in immunoblots from cytosolic extracts and eluates from RAP-affinity matrix in the mutants as in wild-type cells, suggesting that the resistance to RAP in the mutants is not due to a lack of FKBP12 expression. Cell fusion experiments were conducted to further define the nature of the alterations imparting RAP resistance in these mutants. Clones deficient in either thymidine kinase or hypoxanthine-guanine phosphoribosyltransferase, suitable as fusion partners for aminopterin-based selection of hybrids were generated from the wild-type or mutant lines. In most instances, the hybrids derived from the fusion between RAP-sensitive clones and RAP-resistant clones exhibited a RAP-resistant phenotype. Similar results were obtained with hybrids between RAP-resistant YAC-1 clones and the RAP-sensitive EL-4 cell line. Therefore, the mutations that confer resistance to RAP in the present system are dominant. Altogether, our observations are consistent with a model where pharmacologically relevant targets for the RAP-FKBP complex, rather than FKBP, might be altered in the mutants such that the inactivation of these targets by the effector complex is prevented.
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PMID:Dominant mutations confer resistance to the immunosuppressant, rapamycin, in variants of a T cell lymphoma. 753 11

A modified polymerase chain reaction (PCR) assay for analysis of cytokine gene expression from reverse-transcribed (R/T) RNA obtained from small numbers of cells is described in detail. This method employs a previously described dot-blot format and utilizes a target specific radioactive oligonucleotide probe which hybridizes to the PCR amplified product, thus increasing both specificity and sensitivity. This obviates the need for repeated electrophoresis gels and easily accommodates large experiments (e.g., numerous samples or kinetic studies), using small amounts of RNA from low cell numbers. Manipulation of many samples is further enhanced with the use of a PCR thermocycler, which like the dot-blot apparatus is designed in a 96-well format. We describe the use of the house-keeping enzyme hypoxanthine phosphoribosyltransferase (HPRT) as an internal standard, which is especially suitable since its range of detectability of expression is similar to that of the cytokines under test. This enables one to obtain an accurate measure of losses or degradation of RNA, as well as controlling for efficiency of the R/T and PCR reactions. These reactions are further controlled by inclusion of a standard curve consisting of a titration of a known amount of RNA from a cell line expressing the cytokine under test. As well as controlling for the R/T-PCR, this standard curve also enables one to obtain a semi-quantitative measure of cytokine expression by different cell populations during an immune response. We show that this method can be used successfully for studying differential expression of IL-10 in different microenvironments during infection of mice with Schistosoma mansoni.
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PMID:Detection of in vivo expression of interleukin-10 using a semi-quantitative polymerase chain reaction method in Schistosoma mansoni infected mice. 768 99

Chronic inhalation of carbon black can produce carcinomas in rat lungs. At present the mechanisms underlying the rat lung tumor response to carbon black are unknown, although a significant role for inflammation and cell proliferation has been postulated. To investigate the processes which may contribute to development of rat lung tumors after carbon black exposure, we characterized the effects of subchronic inhalation of carbon black by rats on mutagenesis in alveolar epithelial cells, pulmonary inflammation, inflammatory cytokine/growth factor expression, and lung histopathology. Briefly, rats were exposed for 6 hr/day, 5 days/week for up to 13 weeks to 1.1, 7.1, and 52.8 mg/m3 carbon black and the effects on the lung were characterized after 6.5 and 13 weeks of exposure and 3 and 8 months of recovery. Endpoints characterized after carbon black exposure included mutation in the hprt gene of alveolar epithelial cells, changes in bronchoalveolar lavage fluid markers of lung injury and inflammation, expression of mRNA for the chemokines, MIP-2 and MCP-1, and lung histopathology. Lung burdens of carbon black were also determined. After 13 weeks of exposure to 1.1, 7.1, and 52.8 mg/m3 carbon black, lung burdens were 354, 1826, and 7861 micrograms carbon black, respectively. The lung clearance of carbon black appeared impaired after exposure to 7.1 and 52.8 mg/m3 carbon black, with the effects being more pronounced at the higher exposure level. Subchronic inhalation of 1.1 mg/m3 carbon black did not elicit any detectable adverse lung effects. A significant increase in hprt mutation frequency in alveolar epithelial cells was detected immediately after 13 weeks of exposure to 7.1 and 52.8 mg/m3 carbon black as well as after 3- and 8-month recovery periods for the group exposed to 52.8 mg/m3. No increase in hprt mutation frequency was observed for epithelial cells obtained from rats exposed to 1.1 mg/m3 carbon black. The observation that genotoxic effects (i.e., mutations) on alveolar epithelial cells occurred only after carbon black exposures which resulted in significant inflammation and epithelial hyperplasia supports the hypothesis that inflammatory cell-derives oxidants and increased cell proliferation play a role in the pathogenesis of rat lung tumors in response to carbon black.
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PMID:Pulmonary inflammatory, chemokine, and mutagenic responses in rats after subchronic inhalation of carbon black. 861 46

A competitive PCR assay (cPCR) was used to quantify swine cytokine responses to parasite infection. Internal standards (deleted cDNA competitor molecules [DcDNA mimics]) were produced and tested for swine interleukin-12 (IL-12), interleukin-10 (IL-10) and hypoxanthine phosphoribosyltransferase (HPRT) from PCR generated cDNA cloned in plasmid vectors. Deletion clones for the cDNA competitor molecules (DcDNA mimics) were generated for IL-10, IL-12 and HPRT by PCR in a single step and verified by (1) amplification of the expected smaller PCR product with the original primers, (2) appropriate fragment size released by restriction digestion of the deleted clone, and (3) correct sequence of the new DcDNA insert. DcDNA mimics were used to quantitate cytokine gene mRNA production during experimental and natural infections of swine with the gastrointestinal nematode parasite Trichuris suis. Mesenteric lymph node cells were collected from control and infected pigs at the time of maximal pathogenicity (35 days after infection) and snap frozen. After RNA extraction, samples were reverse transcribed (RT) to cDNA. cPCR was performed using the housekeeping gene HPRT DcDNA mimic and HPRT specific primers to insure RNA integrity and concentration. Cytokine cDNA content in these samples was then quantitated using cytokine mimics and gene specific primers. IL-10 gene expression in MLN draining the colon of pigs experimentally infected with T. suis increased 10-20 fold at day 35 compared to control pigs. IL-12 gene expression was not detectable in MLN of these pigs, but was detectable in MLN of pigs exposed naturally to T. suis on a contaminated dirt lot that also exhibited signs of secondary bacterial invasion. Swine IL-10 and IL-12 gene expression can be quantitated in local mesenteric tissues. This cPCR assay will enable scientists to quantitate cytokine gene expression in swine and determine the nature of immune responses to important infectious diseases.
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PMID:Construction of internal cDNA competitors for measuring IL-10 and IL-12 cytokine gene expression in swine. 980 77

Hypoxanthine phosphoribosyltransferase (HPRT) is a purine salvage enzyme that catalyzes conversion of hypoxanthine and guanine to their respective mononucleotides. Because of its ubiquitous nature, HPRT is known as a "housekeeping" gene and has been frequently used as an internal control in reverse transcriptase-polymerase chain reaction (RT-PCR) quantification of cytokine mRNA. Cloning and sequencing of the gerbil HPRT cDNA sequence is an important step in the development of RT-PCR procedures in this model. Two forms of gerbil HPRT cDNA were isolated and molecularly characterized.
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PMID:Hypoxanthine phosphoribosyltransferase cDNA in gerbils (Meriones unguiculatus). 1009 10

Interleukin (IL)-1beta is a pleiotropic cytokine implicated in a variety of activities, including damage of insulin-producing cells, brain injury, or neuromodulatory responses. Many of these effects are mediated by nitric oxide (NO) produced by the induction of NO synthase (iNOS) expression. We report here that IL-1beta provokes a marked repression of genes, such as fragile X mental retardation 1 (FMR1) and hypoxanthine phosphoribosyltransferase (HPRT), having a CpG island in their promoter region. This effect can be fully prevented by iNOS inhibitors and is dependent on DNA methylation. NO donors also cause FMR1 and HPRT gene silencing. NO-induced methylation of FMR1 CpG island can be reverted by demethylating agents which, in turn, produce the recovery of gene expression. The effects of IL-1beta and NO appear to be exerted through activation of DNA methyltransferase (DNA MeTase). Although exposure of the cells to NO does not increase DNA MeTase gene expression, the activity of the enzyme selectively increases when NO is applied directly on a nuclear protein extract. These findings reveal a previously unknown effect of IL-1beta and NO on gene expression, and demonstrate a novel pathway for gene silencing based on activation of DNA MeTase by NO and acute modification of CpG island methylation.
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PMID:Methylation-dependent gene silencing induced by interleukin 1beta via nitric oxide production. 1058 50

Potential additive effects of ethanol consumption, a common life-style factor, and low-level benzene exposure, a ubiquitous environmental pollutant, were investigated. Ethanol is a potent inducer of the cytochrome P-450 2E1 (CYP2E1) enzyme, which bioactivates benzene to metabolites with known genotoxicity and immunotoxicity. A liquid diet containing 4.1% ethanol was used to induce hepatic CYP2E1 activity by 4-fold in female CD-1 mice. Groups of ethanol-treated or pair-fed control mice were exposed to benzene or filtered air in inhalation chambers for 7 h/d, 5 d/wk for 6 or 11 wk. The initial experiment focused on immunotoxicity endpoints based on literature reports that ethanol enhances high-dose benzene effects on spleen, thymus, and bone marrow cellularity and on peripheral red blood cell (RBC) and white blood cell (WBC) counts. No statistically significant alterations were found in spleen lymphocyte cellularity, subtype profile, or function (mitogen-induced proliferation, cytokine production, or natural killer cell lytic activity) after 6 wk of ethanol diet, 0.44 ppm benzene exposure, or both. This observed absence of immunomodulation by ethanol alone, a potential confounding factor, further validates our previously established murine model of sustained CYP2E1 induction by dietary ethanol. Subsequent experiments involved a 10-fold higher benzene level for a longer time of 11 wk and focused on genotoxic endpoints in known target tissues. Bone marrow and spleen cells were evaluated for DNA-protein cross-links, a sensitive transient index of genetic damage, and spleen lymphocytes were monitored for hprt-mutant frequency, a biomarker of cumulative genetic insult. No treatment-associated changes in either genotoxic endpoint were detected in animals exposed to 4.4 ppm benzene for 6 or 11 wk with or without coexposure to ethanol. Thus, our observations suggest an absence of genetic toxicity in CD-1 mice exposed to environmentally relevant levels of benzene with or without CYP2E1 induction.
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PMID:Effect of CYP2E1 induction by ethanol on the immunotoxicity and genotoxicity of extended low-level benzene exposure. 1066 33

The human population is exposed to both the ultraviolet A (UVA) and B (UVB) regions of the solar spectrum. UVB induces mainly dipyrimidine photoproducts in DNA by a direct photochemical mechanism, whereas UVA is absorbed by other cellular constituents and induces mainly oxidative damage indirectly. The proportions of the different dipyrimidine photoproducts, and the ratio of dipyrimidine to oxidative damage depend on the exact spectral output of a UV source. Irradiation of human epidermal keratinocytes induces release of cytokines, with cyclobutane pyrimidine dimers playing a significant role in the process. These cytokines may then modulate the activity of cells of the immune system. Freshly isolated human lymphocytes are exquisitely sensitive to UVB irradiation, because of their low deoxyribonucleotide pools. They also have a separate defect in removal of cyclobutane pyrimidine dimers from their DNA. We have observed that frequencies of mutations at the hprt locus in human T-lymphocytes and translocations involving the bcl2 locus in B-lymphocytes appear to be associated with sunlight levels over the period before the blood sample was taken. This may be an indirect cytokine-mediated effect, and may be relevant to the possible link between non-Hodgkin's lymphoma and sunlight. On the other hand, sunlight can have beneficial effects, and may protect against autoimmune diseases including type I diabetes and multiple sclerosis.
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PMID:Possible effects of sunlight on human lymphocytes. 1070 50

A variety of commercial DNA arrays specific for humans and rodents are widely available; however, microarrays containing well-characterized genes to study pathway-specific gene expression are not as accessible for domestic animals, such as cattle, sheep and pigs. Therefore, a small-scale application-targeted bovine immune-endocrine cDNA array was developed to evaluate genetic pathways involved in the immune-endocrine axis of cattle during periods of altered homeostasis provoked by physiological or environmental stressors, such as infection, vaccination or disease. For this purpose, 167 cDNA sequences corresponding to immune, endocrine and inflammatory response genes were collected and categorized. Positive controls included 5 housekeeping genes (glyceraldehydes-3-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, ribosomal protein L19, beta-actin, beta2-microglobulin) and bovine genomic DNA. Negative controls were a bacterial gene (Rhodococcus equi 17-kDa virulence-associated protein) and a partial sequence of the plasmid pACYC177. In addition, RNA extracted from un-stimulated, as well as superantigen (Staphylococcus aureus enterotoxin-A, S. aureus Cowan Pansorbin Cells) and mitogen-stimulated (LPS, ConA) bovine blood leukocytes was mixed, reverse transcribed and PCR amplified using gene-specific primers. The endocrine-associated genes were amplified from cDNA derived from un-stimulated bovine hypothalamus, pituitary, adrenal and thyroid gland tissues. The array was constructed in 4 repeating grids of 180 duplicated spots by coupling the PCR amplified 213-630 bp gene fragments onto poly-l-lysine coated glass slides. The bovine immune-endocrine arrays were standardized and preliminary gene expression profiles generated using Cy3 and Cy5 labelled cDNA from un-stimulated and ConA (5 microg/ml) stimulated PBMC of 4 healthy Holstein cows (2-4 replicate arrays/cow) in a time course study. Mononuclear cell-derived cytokine and chemokine (IL-2, IL-1alpha, TNFalpha, IFN-gamma, TGFbeta-1, MCP-1, MCP-2 and MIP-3alpha) mRNA exhibited a repeatable and consistently low expression in un-stimulated cells and at least a two-fold increased expression following 6 and 24 h ConA stimulation as compared to 0 h un-stimulated controls. In contrast, expression of antigen presenting molecules, MHC-DR, MHC-DQ and MHC-DY, were consistently at least two-fold lower following 6 and 24 h ConA stimulation. The only endocrine gene with differential expression following ConA stimulation was prolactin. Additionally, due to the high level of genetic homology between ovine, swine and bovine genes, RNA similarly acquired from sheep and pigs was evaluated and similar gene expression patterns were noted. These data demonstrate that this application-targeted array containing a set of well characterized genes can be used to determine the relative gene expression corresponding to immune-endocrine responses of cattle and related species, sheep and pigs.
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PMID:Construction and application of a bovine immune-endocrine cDNA microarray. 1526 89

In order to quantify in vivo the mRNAs of cytokines which play important roles in leptospirosis, we have developed quantitative real-time PCR assays for interleukin-2 (IL-2), IL-4, IL-10, IL-12p40, tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), transforming growth factor beta, and two housekeeping genes (encoding beta-actin and hypoxanthine phosphoribosyltransferase). We used a lethal hamster model reflecting severe leptospirosis in humans. The LightCycler system was used to quantify the gene expression levels with the SYBR green I detection format using external standard curves for each target. We compared the expression levels of cytokine mRNA in the peripheral blood mononuclear cells of both control (uninfected) hamsters and Leptospira interrogans-inoculated hamsters from 1 to 24 h and then 1 to 4 days postinfection. In this kinetic study, there was pronounced expression of Th1 cytokine mRNA (TNF-alpha, IFN-gamma, and IL-12), with transcripts being detected as early as 1 h postinfection. Expression of anti-inflammatory cytokines, such as IL-4 and IL-10, was prominent in delayed samples from 1 to 4 days postinfection in response to infection with Leptospira interrogans. Our data are the first to establish that pathogenic leptospires can stimulate in vivo the production of type 1 cytokines involved in cellular immunity by using this informative animal model. Measuring and assessing cytokine profiles may provide a useful method for accurate study of the mechanisms of anti-Leptospira immunity, indications of prognosis factors, and prospective evaluation of leptospirosis vaccine efficacy in humans.
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PMID:Proinflammatory and immunomodulatory cytokine mRNA time course profiles in hamsters infected with a virulent variant of Leptospira interrogans. 1679 Jul 92


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