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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this study we have designed and constructed an anti-Fas ribozyme and show that it can specifically cleave the Fas mRNA in vitro. Moreover, to test its efficacy ex vivo, we transfected the anti-Fas ribozyme into betaTC-3 insulinoma cells, using a RNA polymerase III promoter to drive its expression. Like pancreatic beta cells, betaTC-3 cells do not constitutively express Fas, but Fas expression can be induced with IL-1 and IFN-gamma. Transfected cells expressed an average of 5000 copies of anti-Fas ribozyme transcript per cell as assessed by reverse transcriptase-real-time PCR. After IL-1/IFN-gamma treatment, betaTC-3 cells transfected with the anti-Fas ribozyme expressed 80% less Fas compared with mock-transfected cells. In addition, the anti-Fas ribozyme also inhibited Fas expression in NIT-1 insulinoma cells and in primary cultures of dispersed pancreatic islet cells. Inhibition of de novo Fas expression in betaTC-3 cells expressing the anti-Fas ribozyme correlated with resistance to Fas-mediated apoptosis as determined by the number of cells exhibiting caspase 3 proteolytic activity. Hence, we have engineered a ribozyme capable of preventing Fas expression in the betaTC-3 pancreatic insulinoma cell line and conferring resistance to Fas-mediated apoptosis. We suggest that ribozymes may be potentially useful to engineer resistance to apoptosis in transplantable beta cells, a feature that may significantly improve the survival of islet cell grafts.
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PMID:Inhibition of Fas-mediated apoptosis in mouse insulinoma betaTC-3 cells via an anti-Fas ribozyme. 1081 Dec 32

Previous studies have suggested that large quantities of bacterial lipids may accumulate and persist within host cells during chronic stages of Mycobacterium avium infections. This study intended to assess the ability of purified M. avium lipids to affect TH-1-type responses in human peripheral blood mononuclear cells (PBMC) from healthy donors. PBMC were exposed to total lipids and serovar-specific glycopeptidolipids (GPL) extracted from M. avium serovars 4 and 8, which have been reported to predominate as opportunistic infection among AIDS patients. After 24 h exposure to lipids followed by PHA/PMA treatment, IL-2 and IFN-gamma were assayed in the supernatants. Reverse transcriptase polymerase chain reaction (RT-PCR) was used for a semiquantitative estimation of mRNA for IL-2 and IFN-gamma in cell pellets at various time points. Exposure of PBMC to M. avium total lipids significantly suppressed PHA/PMA-induced secretion of IL-2 and IFN-gamma as determined by ELISA. The GPL antigens from serovar 4 were more efficient at inhibiting TH-1 responses than GPL from serovar 8. CD4(+)T-lymphocyte enrichment of PBMC demonstrated that suppression by M. avium lipids was intact without the presence of other cell populations such as monocytes and B-cells. Preliminary RT-PCR experiments showed that the secretion of TH-1 cytokines was partially affected at the transcriptional level. The results obtained showed that M. avium lipids are indeed able to modify the induction of TH-1-type cytokines by human PBMC, and suggest that accumulation of M. avium lipids in the chronic stages of infection may play an important role in the pathogenesis of HIV infection.
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PMID:Exposure of human peripheral blood mononuclear cells to total lipids and serovar-specific glycopeptidolipids from Mycobacterium avium serovars 4 and 8 results in inhibition of TH1-type responses. 1087 86

Studies of mouse models of tuberculosis (TB) infection have indicated a central role for MHC class I-restricted CD8+ T cells in protective immunity. To define antigens and epitopes of Mycobacterium tuberculosis (MTB) proteins that are presented by infected cells to CD8+ T cells, we screened 40 MTB proteins for HLA class I A*0201-binding motifs. Peptides that bound with high affinity to purified HLA molecules were subsequently analyzed for recognition by CD8+ cytotoxic T lymphocytes. We identified three epitopes recognized by CD8+ T cells from patients recovering from TB infection. Those three epitopes were derived from three different antigens: thymidylate synthase (ThyA(30-38)), RNA polymerase beta-subunit (RpoB(127-135)), and a putative phosphate transport system permease protein A-1 (PstA1(75-83)). In addition, CD8+ T cell lines specific for three peptides (ThyA(30-38), PstA1(75-83), and 85B(15-23)) were generated from peripheral blood mononuclear cells of normal HLA-A*0201 donors. These CD8+ T cell lines specifically recognized MTB-infected macrophages, as demonstrated by production of IFN-gamma and lysis of the infected target cells. Finally, CD8+ cytotoxic T lymphocytes reduced the viability of the intracellular MTB, providing evidence that CD8+ T cell recognition of MHC class I-restricted epitopes of these MTB antigens can contribute to effective immunity against the pathogen.
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PMID:Antimicrobial activity of MHC class I-restricted CD8+ T cells in human tuberculosis. 1103 87

We investigated the kinetics of multiple cytokines and inducible nitric oxide synthase (iNOS) in experimental uveitis induced by bovine melanin protein (BMP) for the proper treatment of uveitis. Experimental uveitis was induced in male Lewis rats by injection of BMP. The levels of various inflammatory cytokines and iNOS mRNAs were semiquantified by the reverse-transcriptase reaction followed by PCR. The uveitis was started to develop at approximately day 14 and peaked around 21 days after immunization. The signs of uveitis disappeared by 4 weeks after immunization. When the inflammation was severest, TNF-alpha, IL-1alpha, IL-10, IFN-gamma and iNOS mRNA increased to their peak, which varied with the degree of induction and different time course. We concluded that both cytokines and iNOS might modulate the inflammation at different states of experimental melanin-protein-induced uveitis. Their combination will be necessary for an effective treatment of inflammation.
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PMID:The expression of multiple cytokines and inducible nitric oxide synthase in experimental melanin-protein-induced uveitis. 1172 Nov 85

In the murine model for respiratory syncytial virus (RSV) infection, cytokine patterns induced by vaccinations with either killed (i.e. formalin-inactivated, alum-precipitated) virus (KV) or live virus (LV) have been shown to influence disease expression. To determine the mRNA expression of the cytokines IL-4 and IFN-gamma in BALB/c mice challenged with RSV, a real-time quantitative reverse-transcriptase PCR assay was developed. This assay uses 5'-exonuclease fluorogenic probes and is performed on the ABI PRISM 7700 Sequence Detector System (TaqMan). The relative quantitative levels of mRNA for IL-4 and IFN-gamma were compared with those measured by an RNase protection assay (RPA) and an enzyme immunoassay (EIA), which are methods used to measure the levels of mRNA and protein, respectively. Results obtained by the TaqMan assay showed that mice primed with KV induces increased IL-4 mRNA production while LV induces increased IFN-gamma mRNA, which is in agreement with conventional methods. IL-4 and IFN-gamma relative quantities obtained from TaqMan were highly correlated to those determined by RPA (r=0.96 for IFN-gamma, P<0.01) and EIA (r=0.90 for IL-4 and r=0.75 for IFN-gamma, P<0.01). Assay reproducibility was examined by testing a same sample in triplicate at three experiments. Minimal deviation values were observed in both intra- and inter-assays. TaqMan, which is rapid, sensitive and reproducible, provides an alternative tool for the quantitative analysis of cytokine mRNA expression in the murine model of RSV immunopathogenesis.
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PMID:Cytokine expression in respiratory syncytial virus-infected mice as measured by quantitative reverse-transcriptase PCR. 1250 27

Both innate and adaptive immune systems are thought to participate in the pathogenesis of rheumatoid arthritis in adults and children. The experiments reported here were undertaken to examine how immune complexes, potent stimulators of inflammation, may regulate cells of the adaptive immune system. Human T cells were prepared from peripheral blood by negative selection and incubated with bovine serum albumin (BSA)-anti-BSA immune complexes that were formed in the presence or absence of human C1q. C1q-bearing immune complexes, but not unopsonized complexes, elicited both TNF-alpha and IFN-gamma secretion from human T cells. Secretion of both cytokines was time- and dose-dependent. Cross-linking C1q on the cell surface of T cells produced the same results. Cytokine secretion was not inhibited by blocking the C3b receptor (CR1, CD35) on T cells prior to incubation with immune complexes. Reverse transcriptase polymerase chain reaction (RT-PCR) of immune complex-stimulated cells revealed accumulation of both TNF-alpha and IFN-gamma mRNA within 2 h post-stimulation. IL-2 was not detected in cell culture supernatants, but IL-2 receptor alpha chain (CD25) was detected in low density on a small proportion of T cells activated by C1q-bearing immune complexes. Secretion of both cytokines was inhibited partially, but not completely, by IL-10. These experiments show that immune complexes, potent inflammatory mediators, may activate T cells through a novel mechanism. These findings have implications for chronic inflammatory diseases in humans.
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PMID:T cell activation by soluble C1q-bearing immune complexes: implications for the pathogenesis of rheumatoid arthritis. 1251 87

The role of T cells in eradicating leukemic cells has been well demonstrated for chronic myeloid leukemia (CML). Type 1 (T1) T-cell cytokines play a major role in this antileukemic immune effect. Studies in cancer patients have demonstrated a decreased T1 cytokine production, measured by enzyme-linked immunosorbent assay (ELISA), in cultures of peripheral blood mononuclear cells. This observation of malignancy-related suppressed T1 cytokines also occurs in untreated chronic-phase (CP) CML, raising the question of the influence of different CML treatment regimens on this immunosuppression. Intracellular flow cytometry (ICF) has facilitated the evaluation of cytokines on a single-cell level. This study analyzed T1 (interferon-gamma) cytokine production in purified peripheral blood T cells by ICF, comparing different therapy approaches for CML. Twenty-one newly diagnosed CP CML patients were compared with 24 patients treated with interferon-alpha (IFN-alpha) and to 30 allogeneic bone marrow transplant (BMT) recipients (BCR-ABL negative by reverse-transcriptase polymerase chain reaction, and free of, or having only limited graft-versus-host disease at the time of study). Thirty-seven healthy controls were included. Our results showed a significantly decreased T-cell IFN-gamma synthesis in CP CML patients in relation to healthy controls (P = 0.0007). Treatment with IFN-alpha resulted in a shift from immunosuppression--documented for the group of untreated patients--to immunopotentiation, with an increase of T-cell IFN-gamma production (P = 0.0266). Notably, BMT enhanced IFN-gamma production of T cells to a level not only exceeding untreated patients (P < 0.0001) but also healthy volunteers (P < 0.0001). The observation of T1 cytokine up-regulation with IFN-alpha therapy indicates that enhanced T-cell function may be achievable in patients with CML, even in the absence of an allo-response.
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PMID:Intracellular cytokine analysis of interferon-gamma in T cells of patients with chronic myeloid leukemia. 1260 98

Celiac disease (CD) is an enteropathy characterized by a Th1-type immune response to the dietary gluten. The transcriptional mechanisms or factors that control Th1 cell development in this condition remain to be elucidated. The aim of this study was to analyze in CD the expression of interferon (IFN) regulatory factor (IRF)-1, a transcription factor that regulates the differentiation and function of Th1 cells. Duodenal biopsies were taken from children with untreated CD and control children, and analyzed for IRF-1 by Southern blotting of reverse-transcriptase PCR products and Western blotting. IRF-1 DNA-binding activity was assessed by electrophoretic shift mobility assay. The effect of gliadin stimulation on IRF-1 induction was investigated in an ex vivo organ culture of treated CD biopsies. Enhanced IRF-1 was seen in untreated CD in comparison with controls. This was evident at both the RNA and protein level. Furthermore, untreated CD samples exhibited stronger nuclear accumulation and DNA-binding activity of IRF-1 than controls. In contrast, IRF-2, a transcriptional repressor that binds the same DNA element and competes with IRF-1, was expressed at the same level in nuclear proteins extracted from both untreated CD and control patients. In explant cultures of treated CD biopsies, gliadin enhanced both IRF-1 RNA and protein. This effect was prevented by a neutralizing IFN-gamma antibody. Furthermore, stimulation of normal duodenal biopsies with IFN-gamma enhanced IRF-1. These data indicate that IRF-1 is a hallmark of the gliadin-mediated inflammation in CD and suggest that IFN-gamma/IRF-1 signaling pathway can play a key role in maintaining and expanding the local Th1 inflammatory response in this disease.
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PMID:Enhanced expression of interferon regulatory factor-1 in the mucosa of children with celiac disease. 1278 88

Derangement of cellular immunity is central in the pathophysiology of adult autoimmune/idiopathic thrombocytopenic purpura (ITP). Herein we investigated cytokine gene expression in peripheral blood mononuclear cells (PBMCs) of adult chronic ITP patients and attempted to correlate cytokine polarization with the degree of thrombocytopenia. We used semiquantitative reverse-transcriptase-polymerase chain reaction (RT-PCR) to measure the expression of type-1 (interleukin-2 [IL-2], interferon gamma [IFN-gamma]) and type-2 (IL-4, IL-5, IL-10, IL-3, IL-13) cytokines by PBMCs from 21 patients and 11 controls. Plasma transforming growth factor beta1 (TGF-beta1) levels were measured by enzyme-linked immunoassay (ELISA). T helper 1 (Th1)/Th2 ([IL-2 + IFN-gamma]/[IL-4 + IL-5]) cytokine mRNA ratios, thought to reflect the Th deviation of the pathogenic disease-specific T cells, and type-1/type-2 mRNA ratios, thought to reflect the overall immune response polarization, were significantly increased in ITP patients. The Th1/Th2 ratio was inversely correlated with platelet counts. TGF-beta1 levels appeared suppressed in patients with active disease, though not significantly. Our findings show a clear type-1 cytokine polarization of the autoimmune response in adult ITP that persists irrespective of disease status.
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PMID:Adult chronic idiopathic thrombocytopenic purpura (ITP) is the manifestation of a type-1 polarized immune response. 1467 Sep 26

The high-affinity receptor for IgG (FcgammaRI) is a myeloid cell-specific and IFN-gamma-induced gene, and thereby serves as a paradigm for cytokine-induced cell type-specific gene responses. The expression of FcgammaRI is regulated by PU.1 and Stat1 transcription factors. We established an experimental model to analyze the individual functions of Stat1 and PU.1 in cytokine-induced transcription of the natural FcgammaRI promoter in U3A cells lacking both factors. PU.1 was required for both the basal activity and for the IFN-gamma-induced FcgammaRI promoter activation, while Stat1 alone could not initiate transcription. In contrast, in the context of a heterologous promoter, PU.1 inhibited the Stat1-mediated transcription. Systematic analysis of Stat1 and PU.1 mutants and FcgammaRI promoter elements revealed that activation of the promoter required the DNA binding, and the transactivation functions of both Stat1 and PU.1. PU.1 and Stat1 bound the promoter elements independently, and no physical interaction between the proteins was observed. The requirement of PU.1 for FcgammaRI promoter activity was supported by demonstration of in vitro interaction between PU.1 and components of the basal transcription machinery TBP and RNA polymerase II. Deletion of the acidic transactivation domain of PU.1 greatly diminished both the FcgammaRI promoter activity as well as the interaction with RNA polymerase II. In contrast, Stat1 did not interact with TBP or RNA polymerase II. These results define functional cooperativity between PU.1 and Stat1 in FcgammaRI promoter activation where PU.1 serves as an amplifier and bridging factor with the basal transcription machinery.
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PMID:Molecular basis of Stat1 and PU.1 cooperation in cytokine-induced Fcgamma receptor I promoter activation. 1473 12


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