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:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The fusion of thioglycollate-elicited peritoneal macrophages from lipopolysaccharide (LPS) non-responsive C3H/HeJ mice to an HPRT-negative variant of the murine macrophage cell line P388D1 has resulted in the derivation of a series of macrophage hybrids. Following exposure to LPS, these hybrids now produce the cytokine hepatocyte-stimulating factor (HSF) which induces the synthesis of the acute-phase reactant alpha 2-macroglobulin in primary rat hepatocyte cultures. The concentration of extracellular HSF was dependent upon both the duration and amount of LPS, with optimal HSF being detected after 72 hr incubation with 10 micrograms/ml of LPS. Parallel LPS-stimulated cultures treated with 10(-6)M dexamethasone did not secrete detectable amounts of HSF. Both the molecular weight (29,000 MW), and the fact that HSF activity was not inhibited by an antiserum directed against murine interleukin-1 alpha (IL-1 alpha), suggests that HSF and IL-1 are distinct cytokines. Therefore, macrophage hybrids have been derived which have acquired the LPS-responsive phenotype and which synthesize the cytokine HSF following LPS stimulation. This phenotype appears stable since similar results have been observed with these hybrids after in vitro culture for over 8 months.
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PMID:Restoration of the LPS responsive phenotype in C3H/HeJ macrophage hybrids: LPS regulation of hepatocyte-stimulating factor production. 332 5

A synthetic DNA construct has been developed as a standard molecule whereby murine cytokine mRNA molecules can be quantified by the reverse transcription-polymerase chain reaction (RT-PCR). The construct, designated Cytoquant 1, allows the quantification of murine IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IFN-gamma, TNF-alpha, TGF-beta, GM-CSF, CD4, CD8, HPRT and beta-actin mRNA levels. This technique is based on the amplification of a transcribed RNA molecule from Cytoquant 1 as an internal standard control in both the RT and PCR reactions. The quantification data from these analyses are expressed in absolute values, i.e. molecules/cell, which allows the data derived from separate experiments to be compared. In this study, mRNAs encoding beta-actin, IL-10, IFN-gamma and GM-CSF have been quantitated in both Th1 and Th2 cell clones with, and without, stimulation. The quantitative analysis data are highly reproducible and cytokine mRNA concentrations are reflective of restricted cytokine secretion patterns. Furthermore, constitutive cytokine mRNA levels are detectable in resting cells, eliminating the need for exogenous stimulation. The high degree of sensitivity and accuracy make this methodology uniquely suited for the study of T-cell subset cytokine expression in both in vivo and in vitro biological models.
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PMID:A synthetic standard DNA construct for use in quantification of murine cytokine mRNA molecules. 747 5

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

Competitive polymerase chain reaction (PCR) is a sensitive method for quantification of cytokine mRNA expression. Co-amplification of an internal standard serves as control for comparing the efficiency of PCR in different samples. We have developed a novel control fragment for multiple analyses of rat cytokine gene expression containing primers for IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, TNF-alpha, TGF-beta 1, IFN-gamma and MIP-2. Additional primers were incorporated to analyse the content of T cells (CD3), activated T cells (CD25) and housekeeping genes (beta-actin and HPRT). As an example we demonstrate analysis of IL-2 mRNA expression in small pieces of kidney tissue obtained from rats after kidney allotransplantation. The IL-2 expression decreased tenfold during treatment with an anti-rat CD4 monoclonal antibody as compared to untreated animals.
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PMID:A novel multispecific competitor fragment for quantitative PCR analysis of cytokine gene expression in rats. 782 31

It has been postulated that HIV-infected patients undergo an active production of virus and CD4+ T cell destruction from the early stages of the disease, and that an extensive postthymic expansion of CD4+ T cells prevents a precipitous decline in CD4+ T cell number. Based on the rebound of the CD4+ T cell number observed in patients undergoing antiretroviral therapy with protease inhibitors, it has been calculated that, on average, 5% of T cells are replaced every day in HIV-infected patients. To obtain an independent estimate of the recycling rate of T cells in the patients, we measured the frequency of cells carrying a loss-of-function mutation at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus. Assuming a recycling rate of 5%/d, an accumulation of 2.6 mutations/10(6)/yr over the physiological accumulation was predicted. Indeed, we observed an elevated frequency of HPRT mutants in the CD4+ T cells of most patients with < 300 CD4+ T cells/mm3 of blood and in the CD8+ T cells of most patients with < 200 CD4+ T cells/mm3, consistent with an elevated and protracted increased division rate in both subsets. However, in earlier stages of the disease the mutant frequency in both CD4+ and CD8+ T cells was lower than in healthy controls. The cytokine production profile of most HPRT mutant CD4+ T cell clones from both healthy and HIV-infected patients was typical of T helper cells type 2 (high IL-4 and IL-10, low IFN-gamma), whereas the cytokine production pattern of wild-type clones was heterogeneous. The cytokine profile of CD8+ clones was indistinguishable between HPRT mutants and wild type. Our data provide evidence of increased CD4+ and CD8+ T cell recycling in the HIV-infected patients.
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PMID:Frequency and cytokine profile of HPRT mutant T cells in HIV-infected and healthy donors: implications for T cell proliferation in HIV disease. 904 68

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

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

Reliable housekeeping gene controls are critical for measuring and comparing gene expression at the transcription level by Northern blot and RT-PCR. In order to develop such controls for studying cytokine mRNA expression in dogs, DNA sequence encoding a full-length canine HPRT protein has been obtained. Numerous primer pairs derived from the canine HPRT sequence have been tested on canine genomic DNA as well as cDNA. The data from the present study suggest that there may be processed HPRT pseudogenes in dogs. Three pairs of canine HPRT primers designed and tested in the present study were able to differentiate between cDNA and genomic DNA under specific PCR conditions. These primers would be useful controls for measurement of mRNA expression by RT-PCR in the dog.
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PMID:Selection of HPRT primers as controls for determination of mRNA expression in dogs by RT-PCR. 1511 53

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


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