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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytokines induce apoptosis in pancreatic beta cells, but the exact mechanisms and sequence of events are not clear. Here, we investigate a role for tumor necrosis factor alpha (TNF-alpha) in the apoptosis of beta cells. Using the ribonuclease (RNase) protection assay and the reverse transcriptase-polymerase chain reaction (RT-PCR) method, we confirmed that TNF receptor 1 (TNFR1), TNFR1-associated death domain protein (TRADD), Fas receptor-associated intracellular protein with death domain (FADD), and FADD-like interleukin-1beta-converting enzyme (FLICE) were expressed in the pancreatic beta cell line, MIN6 cells. Fluorescent microscopic examination using Hoechst 33342 dye (Sigma, St Louis, MO) demonstrated that TNF-alpha induced time- and dose-dependent apoptotic nuclear changes in these beta cells. In situ end-labeling (ISEL) DNA analysis revealed that 10 nmol/L TNF-alpha generated new 3'-OH DNA strand breaks. Moreover, qualitative assessment of the induced DNA damage on agarose gels showed that 10 nmol/L TNF-alpha produced characteristic apoptotic patterns of DNA fragments formed by internucleosomal hydrolysis of static chromatin. In addition, C2-ceramides and natural ceramides dispersed in a solvent mixture of ethanol and dodecane induced characteristic features of apoptosis in MIN6 cells, mimicking TNF-induced DNA damage. We also determined endosomal ceramide production after TNF-alpha (10 nmol/L) treatment in MIN6 cells using the diacylglycerol kinase assay. These results suggest that TNF-alpha can cause apoptosis in pancreatic beta cells through TNFR1-linked apoptotic factors, TRADD, FADD, and FLICE, and TNF-induced ceramide production may be involved in the pathways.
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PMID:Tumor necrosis factor alpha signaling pathway and apoptosis in pancreatic beta cells. 1059 77

The functional properties, regarding parasite growth inhibition in vitro, the cytotoxic potential and cytokine profiles of human gammadelta+ and alphabeta+ T cells, T-cell lines and clones stimulated with Plasmodium falciparum-antigen-or T-cell mitogen in vitro were investigated. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and specific primers, mRNA for the cytolytic molecules perforin, granzyme A and B, Fas and Fas ligand (FasL) were detected in both the gammadelta- and the alphabetaT cells. Despite this fact, only gammadeltaT cells inhibited, both Vdelta1+ and Vdelta2+, the in vitro growth of the asexual blood stages in a dose dependent manner. The inhibition required cell-to-cell contact and was not observed until the second parasite replication implied that the likely gammadeltaT-cell target was the extracellular merozoite or schizont. The failure of alphabetaT cells to inhibit the growth of the parasite suggests requirement of additional cytolytic molecules/signals or different receptor specificities exhibited by the gammadeltaT cells. Both the gammadelta- and alphabetaT cells expressed mRNA for a large number of cytokines. Interferon (IFN)-gamma, interleukin (IL) IL-5, IL-6, IL-8, tumour necrosis factor alpha (TNFalpha), tumour necrosis factor beta (TNF-beta)/lymphotoxin (LT) and T-cell growth factor beta-1 (TGF-beta1) were observed in all activated clones tested. No IL-3 was detected, while IL-1beta, IL-2, IL-4, IL-10 and GM-CSF were variably expressed. In conclusion, our data show that gammadeltaT cells in malaria nonimmune individuals inhibit the asexual blood stages of P. falciparum malaria, while similarly activated alphabetaT cells do not. Thus, it is likely that the gammadeltaT cells could play a mandatory role in the elimination of parasites and/or the regulation of the early immune response to malaria infection.
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PMID:Human gamma delta T cells that inhibit the in vitro growth of the asexual blood stages of the Plasmodium falciparum parasite express cytolytic and proinflammatory molecules. 1060 13

Dysregulation of apoptosis, particularly in the Fas/Fas ligand (FasL) pathway, is considered to be involved in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). Recently, a soluble decoy receptor, termed decoy receptor 3 (DcR3), that binds FasL and inhibits FasL-induced apoptosis, has been identified. Silicosis is clinically characterized not only by respiratory disorders but by immunological abnormalities. We have found that serum soluble Fas (sFas) levels are elevated in silicosis patients and that sFas message is dominantly expressed in PBMC derived from these patients. This study examined DcR3 gene expression in PBMC derived from patients with silicosis, SLE, or progressive systemic sclerosis (PSS), and compared it with that in healthy volunteers (HV). The relative expression level of the DcR3 gene was examined in PBMC derived from 37 patients with silicosis without clinical symptoms of autoimmune disease, nine patients with SLE, 12 patients with PSS, and 28 HV using the semiquantitative multiplex-reverse transcriptase-polymerase chain reaction (MP-RT-PCR). The correlation between the relative expression level of the DcR3 gene and multiple clinical parameters for respiratory disorders and immunological abnormalities in individuals with silicosis was analysed. The DcR3 gene was significantly over-expressed in cases of silicosis or SLE when compared with HV. In addition, the DcR3 relative expression level was positively correlated with the serum sFas level in silicosis patients. It is unclear, however, whether over-expression of the DcR3 gene in silicosis is caused by chronic silica exposure, merely accompanies the alteration in Fas-related molecules, or precedes the clinical onset of autoimmune abnormalities. It will be necessary to study these patients further, establish an in vitro model of human T cells exposed recurrently to silica compounds, and resolve whether the increase in DcR3 mRNA expression is a cause or consequence of disease.
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PMID:Over-expression of the decoy receptor 3 (DcR3) gene in peripheral blood mononuclear cells (PBMC) derived from silicosis patients. 1063 70

We investigated the cytotoxicity mechanisms of alloantigen-specific human CD4(+) and CD8(+) cytotoxic T lymphocytes (CTLs) using cells from family members with the Fas gene mutation. Alloantigen-specific CD4(+) and CD8(+) CTL bulk lines and clones were generated from 2 individuals by stimulation of their peripheral blood lymphocytes with allogeneic Fas(-/-) or Fas(+/-) cell lines that were established from B-lymphocytes of a patient with Fas deficiency and her mother, respectively. Both CD4(+) and CD8(+) CTL bulk lines and clones directed against allogeneic HLA antigens exerted cytotoxicity against Fas(-/-) and Fas(+/-) cells to almost the same degree. The cytotoxicity of CD4(+) and CD8(+) CTLs appeared to be Ca(2+)-dependent and was completely inhibited by concanamycin A, an inhibitor of perforin-mediated cytotoxicity. Messenger RNAs for the major mediators of CTL cytotoxicity, Fas ligand, perforin, and granzyme B were all detected in these CD4(+) CTLs with the use of the reverse transcriptase polymerase chain reaction. The majority of CD4(+) CTL clones that showed Fas-independent cytotoxicity were T(H)0, as determined by their cytokine production profile. These data, obtained with the use of a novel experimental system, clearly show that the main pathway of cytotoxicity mediated by alloantigen-specific human CD4(+) as well as by CD8(+) CTLs is granule exocytosis, and not the Fas/Fas ligand system.
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PMID:Granule exocytosis, and not the fas/fas ligand system, is the main pathway of cytotoxicity mediated by alloantigen-specific CD4(+) as well as CD8(+) cytotoxic T lymphocytes in humans. 1073 6

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

Fas receptor is a member of a superfamily of receptors characterized by cysteine-rich motifs in the extracellular domain of the molecule. Binding of Fas ligand to the receptor leads to receptor activation and the induction of intracellular signals that result in apoptotic cell death. In the present study, the expression of mRNA and proteins of Fas receptor and Fas ligand were examined in human submandibular gland ductal (HSG) cells treated with okadaic acid by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis. Six hundred and eighty-two bp of the PCR product of Fas receptor mRNA was detected in HSG cells and a protein with an estimated molecular weight of 58,000 was expressed in HSG cells. Treatment of HSG cells with an agonistic anti-Fas monoclonal antibody resulted in death of HSG cells, indicating that the functional Fas receptor protein is expressed in HSG cells. Fas receptor protein expression stimulated by okadaic acid was elevated in a dose- and time-dependent manner, with maximal expression at 20 nM and 48 h treatment. Fas ligand mRNA was also detected constitutively in HSG cells by RT-PCR. Okadaic acid stimulated the expression of Fas ligand protein in HSG cells in a time-dependent manner, while the expression of the ligand was low in untreated HSG cells. The molecular weight of Fas ligand was estimated as 68,000. An antagonistic anti-Fas ligand monoclonal antibody prevented okadaic acid-induced death in HSG cells in a dose-dependent fashion as determined by WST-1 assay. The results indicate that the expression of Fas receptor and ligand is regulated by protein phosphatase(s) sensitive to okadaic acid and is involved in okadaic acid-induced apoptosis in HSG cells. The results also suggest that the Fas receptor-ligand system might regulate apoptosis in HSG cells.
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PMID:Upregulation of the expression of Fas antigen and Fas ligand in a human submandibular gland ductal cell line by okadaic acid. 1086 77

Real-time PCR is a novel technology recently described to perform quantitative analysis of amplified products. Unlike classical quantitative PCR, this method is easy to standardize, does not required extensive manipulation, and is not reagent intensive, so that the risk of contamination is minimized. Therefore, we have chosen reverse transcriptase real-time PCR to quantitate CD95 (Fas) transcripts to test the cleavage efficiency of anti-Fas ribozymes in the mouse insulinoma cell line beta TC-3. Based on the melting-curve analysis of the amplified products, we determined the temperature at which to collect the fluorescent data used for quantification. After constructing a standard curve by plotting the log of the standards' copy number versus their fractional cycle number, the copy numbers of the unknown samples were automatically determined by interpolation of this curve. As we illustrate in this study, it is important, particularly while setting up the technique, to validate the melting-curve profile with standard gel electrophoresis analysis, achieved by matching melting temperature and size of the amplified product. The method is fast and reproducible: Excluding the isolation of RNA and synthesis of cDNA, the results can be obtained in less than 1 hr. The coefficient of variance is 15% in the range of 10(4)-10(6) gene copies. Accordingly, reverse transcriptase (RT) real-time PCR is a technique suitable for screening a large number of ribozymes.
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PMID:Assessment of ribozyme cleavage efficiency using reverse transcriptase real-time PCR. 1089 9

Fas antigen is a cell surface protein that mediates apoptosis via signal transduction from the plasma membrane. Using reverse transcriptase-polymerase chain reaction (RT-PCR), messenger RNA for Fas antigen was detected in the human oral squamous cell carcinoma cell line, SCC-25. In serum-free medium, a monoclonal anti-Fas antibody (CH-11) induced Fas antigen expression in SCC-25 cells, as determined by immunocytochemistry and Western blotting, using an anti-Fas polyclonal antibody (Fas D) as primary antibody. Fas antigen was localized to the cytoplasm and the cell membrane. The molecular weight of the protein recognized by Western blot analysis was 35,000, consistent with the value reported for the Fas antigen. The CH-11 antibody did not induce Fas antigen expression in serum-containing medium. To determine whether CH-11 could induce apoptosis in oral squamous cell carcinoma, we examined its effects on the survival of cultured SCC-25 cells. Anti-Fas monoclonal antibody in serum-free medium induced cytotoxicity in SCC-25 cells in a time-dependent manner up to 8 h, as determined by phase-contrast microscopy and WST-1 assay. Marked nuclear condensation and fragmentation of chromatin were observed in the CH-11-treated cells using Hoechst 33342 staining. This anti-Fas monoclonal antibody also induced DNA ladder formation in SCC-25 cells in a time-dependent manner. The present results indicate that the anti-Fas monoclonal antibody (CH-11) may mediate apoptosis by binding to the Fas antigen expressed in SCC-25 cells.
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PMID:Fas expression and Fas monoclonal antibody-induced apoptosis in a human squamous cell carcinoma cell line, SCC-25. 1089 May 58

Prolonged exposure of rodent beta-cells to combinations of cytokines induces the inducible form of nitric oxide synthase (iNOS) expression and Fas expression, nitric oxide (NO) production, and cell death. It also induces the expression of potential "defense" genes, such as manganese superoxide dismutase (MnSOD) and heat shock protein (hsp) 70. NO is a radical with multifaceted actions. Recent studies have shown that NO, in addition to having cytotoxic actions, may regulate gene transcription. It remains unclear whether NO mediates cytokine-induced gene expression and subsequent beta-cell death. Previous studies using NO synthase blockers yielded conflicting results, which may be due to nonspecific effects of these agents. In this study, we examined the effects of cytokines on gene expression, determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and viability, determined by nuclear dyes, of pancreatic islets or fluorescence-activated cell sorter (FACS)-purified beta-cells isolated from iNOS knockout mice (iNOS-/-, background C57BL/6x129SvEv) or their respective controls (C57BL/6x129SvEv). The combination of cytokines used was interleukin-1beta (50 U/ml) plus gamma-interferon (1,000 U/ml) plus tumor necrosis factor-alpha (1,000 U/ml). The lack of cytokine-induced iNOS activity in the iNOS-/- islet cells was confirmed by RT-PCR and nitrite determination. Cytokines induced a >3-fold increase in Fas and MnSOD mRNA expression in wild-type (WT) and iNOS-/- islets. On the other hand, hsp 70 was induced in WT but not in iNOS-/- islets. Prolonged (6-9 days) exposure of WT islets to cytokines leads to an 80-90% decrease in islet cell viability, whereas viability decreased by only 10-30% in iNOS-/- islet cells. To determine the mode of cytokine-induced cell death, FACS-purified beta-cells were exposed to the same cytokines. After 9 days, the apoptosis index was similarly increased in WT (39 +/- 3%) and iNOS4-/- (33 +/- 4%) beta-cells. On the other hand, cytokines increased necrosis in WT (20 +/- 4%) but not in iNOS-/- (7 +/- 3%) beta-cells. From these data, we concluded that 1) NO is required for cytokine-induced hsp 70 mRNA expression but not for Fas and MnSOD expression, 2) cytokines induce both apoptosis and necrosis in mouse beta-cells, and 3) cytokine-induced apoptosis is mostly NO-independent, whereas necrosis requires NO formation.
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PMID:Cytokines induce apoptosis in beta-cells isolated from mice lacking the inducible isoform of nitric oxide synthase (iNOS-/-). 1090 67

In the present study, we demonstrated that a close relationship exists between hepatitis C virus (HCV) infection of peripheral blood mononuclear cells (PBMCs) and cell-surface Fas expression in patients with hepatitis C, and showed the possibility of PBMCs apoptosis via a Fas-mediated system. The expression of Fas on PBMCs was found by flowcytometric analysis to be significantly increased in these patients. In addition, the treatment of patients' PBMCs with anti-Fas antibody induced cell death, with nuclear condensation and fragmentation and cellular DNA fragmentation. These data indicate that the patients' PBMCs expressed a large amount of functional Fas on the cell surface and were susceptible to stimulation against Fas, causing apoptotic cell death. We then quantified the serum-soluble Fas ligand (sFasL), which was known to bind to Fas and induce the apoptotic signals into the sensitized cells. The patients' serum sFasL levels were significantly higher than those of normal subjects and showed a good negative correlation with their PBMC number. To demonstrate the correlation between Fas expression and HCV infection, nested reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect HCV RNA. Interestingly, HCV RNA was preferentially detected from Fas-positive cells but not from Fas-negative cells, which had been isolated from PBMCs by magnetic beads. These results suggest that HCV infection of PBMCs might induce Fas expression and additional stimulation such as sFasL might induce apoptosis in these Fas-expressing cells. These mechanisms, in addition to hypersplenism, may explain the decrease in the number of PBMCs observed in patients with chronic hepatitis C.
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PMID:Fas-mediated apoptosis of peripheral blood mononuclear cells in patients with hepatitis C. 1093 Sep 83


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