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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Addition of pentoxifylline to lymphocytes caused a dose-dependent decrease in PHA-induced interleukin-2 receptor (IL-2R) expression. Expression of IL-2R protein and mRNA were inhibited by 60% at a concentration of 1 mM. Pentoxifylline also inhibited release of IL-2R into the medium by 85%. Treatment with recombinant IL-2 (50 U/ml) did not abrogate the effect of pentoxifylline. In addition to inhibition of IL-2R expression, pentoxifylline also decreased the expression of transferrin receptors and class I MHC antigens. Pentoxifylline also inhibited cell proliferation. However, aphidicolin, an inhibitor of DNA polymerase alpha inhibited cell proliferation to the same extent as pentoxifylline, but had no effect on IL-2R expression, indicating that inhibition of cell proliferation does not necessarily lead to inhibition of IL-2R expression. The inhibitory effect on IL-2R expression was also noted with other methylxanthines, theophylline and isobutylmethylxanthine, and with dbcAMP and forskolin. The inhibitory activity of pentoxifylline was prevented by W-13, a calmodulin antagonist, but not by HA-1004, a cyclic AMP-dependent protein kinase inhibitor. This suggests that pentoxifylline might act in part through a Ca2+/calmodulin-dependent mechanism. Pentoxifylline and other methylxanthines may prove useful in delineating the biochemical pathways involved in induction and expression of cell surface receptors.
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PMID:Pentoxifylline and other methyl xanthines inhibit interleukin-2 receptor expression in human lymphocytes. 170 25

Artificial DNA fragments encoding human interleukin-2 (133 a.a.) and its analogue (deletion of 14 C-terminal a.a.) were prepared by means of the DNA polymerase I mediated extension of synthetic polynucleotides having short overlapping sequences at their 3'-ends. The fragments were cloned in specially designed pFH-type plasmids and then excised by the FokI and other restriction endonucleases to yield the subfragments with the structurally predetermined 5'-unique cohesive ends. The complete synthetic gene was constructed by one or two-step ligation. The expressed IL-2 was tested by analysing the T-cell proliferation activity of E.coli crude lisates containing the pEXIL2 expression plasmid.
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PMID:[Effective synthesis and cloning of the human interleukin-2 gene and its analog: expression of the interleukin-2 gene in E. coli cells]. 177 62

Low-dose ionizing radiation caused definite stimulation of immune reactions both in humans and mice. The PFC reaction in response to SRBC immunization and the NK activity of the splenocytes were significantly enhanced after low-dose whole body irradiation. Activation of the T lymphocytes, especially the TH, with increased production of IL-2, might be a critical step in the whole process of immunoenhancement. A single dose of 75 mGy X-rays caused significant lowering of hypothalamic M-Enk content as well as serum corticosterone level. The increased serum testosterone level would exert an inhibitory influence on the CRF-ACTH-CS system to keep the blood corticosterone at a lower than normal level which might facilitate the immune reactions in the SRBC-immunized animals. The increased catecholamines in the spleen would probably reinforce this effect resulting in immunoenhancement. Low-dose ionizing radiation caused increased repair of the genetic material at both the molecular and subcellular levels. The UDS of human and murine lymphocytes was augmented by single or continuous low-dose irradiation. The stimulation of DNA polymerase activity might be responsible for such effects. Exposure to very small doses of low LET radiation could induce in different tissues an adaptive response which alleviated chromosome damage caused by subsequent larger dose radiation. Such an adaptive response could be induced both in vivo and in vitro in different animal species. The induced adaptive response faded away after 3 cell cycles could be re-induced by a second exposure to low-dose radiation. The mechanism of the inductive process needs further study.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Radiation hormesis. A new concept in radiological science. 251 54

The ubiquitous trace metal zinc has been discovered since a long time as an intrinsic element in all biological systems. However, its role other than structural or catalytic in enzymes is poorly defined. Zinc plays a determinative role both in primary and secondary T lymphocyte production. Experimental data support the notion that during intrathymic maturation, non-autoreactive, immunocompetent T cell clones are selected from the excess of immature thymocytes as a result of expansion of bone marrow derived prothymocytes in response to pleiotropically acting alarmon (s) and a subsequent escape via the thymic stroma cells from nucleotide-mediated "biochemical suicide". The activity of alarmon (Ap4A), nucleotide metabolizing enzymes (TdT, DNA polymerase, thymidine kinase, 5'-nucleotidase) and some of the soluble stromal cell products (FTS) require constitutive zinc. In the peripheral lymphoid organs the magnitude and duration of antigen induced, T cell mediated immunoreactions are regulated by T-cell growth factor (IL-2). Using receptor specific monoclonal antibody probes, it has been established recently that the intracellular role of IL-2 is probably to induce the phenotypic expression of high affinity transferrin receptors, known to be the main zinc transporter system in T-lymphocytes. The coordinative role of zinc in T lymphocyte development via the inducible metallothionein system is emphasized. Some clinical aspects of zinc metabolism are discussed.
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PMID:Zinc and immunity. 623 34

According to our data native Tth DNA polymerase displays higher reverse transcription activity than Taq DNA polymerase. This allows one to use Tth DNA polymerase in the complete reaction of reverse transcription and amplification (RT/PCR). We used this enzyme to synthesize the interleukine (IL-2 alpha) RNA template synthesized by the RT/PCR method in vitro. The conditions for RNA IL-2 alpha detection were optimized. The maximum yield of the specific product was obtained at pH 8.5-9.0. The influence of bivalent cations on the efficiency of RT reaction of coupled RT/PCR can be expressed as: Mn2+ > or = Cu2+ > Mg2+ > Cd2+ >> Co2+. The optimal ratio is 1.25-1.88 for Mn2+/dNTPs and 1.88-2.5 for Cu2+/dNTPs and Cd2+/dNTPs. The maximum yield of the RT/PCR product is found at Mg2+/dNTPs = 3.75. When Mn2+ is used instead of Mg2+ in the PCR reaction the efficiency of RT/PCR decreases. The RT/PCR method embracing thermostable Tth DNA-polymerase provides detection of 10(3) copies of RNA IL-2 alpha. An efficient method of the express-diagnostics of MDR-1 gene expression by coupled RT/PCR using Tth DNA polymerase is described.
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PMID:[Use of thermostable DNA polymerase from Thermus thermophilus KTP in a combined reverse transcription and amplification reaction of detecting interleukin 2alpha RNA and determining expression of the multidrug resistance gene (MDR-1)]. 747 58

The safety and efficacy of combined therapy with polyethylene glycolated (PEG) interleukin (IL)-2 and zidovudine was assessed in 19 human immunodeficiency virus type 1 (HIV-1)-seropositive subjects in a phase I/II open-label dose-ranging study. During courses of three weekly infusions of PEG IL-2, dose-limiting side effects were seen at 5 x 10(6) IU/m2 and reversible encephalopathy in 1 subject at 3 x 10(6) IU/m2. Significant increases were seen in CD4 cell counts (P < .01), NK cell activity (P < .05), and HIV-specific cytotoxicity (P < .01). Virologic monitoring (quantitative DNA polymerase chain reaction and p24 antigen assay) showed no evidence of increased HIV activation. Patients with CD4 cells < 200/mm3 were entered into a chronic dosing phase. PEG IL-2 was given at 14-day intervals at doses of 10(6) IU/m2 for 8 weeks and 3 x 10(6) IU/m2 for up to 16 weeks, resulting in mean CD4 cell count elevations of 16% and 33%, respectively. PEG IL-2 appears to warrant further investigation, especially in subjects with CD4 cell counts < 200/mm3, to determine whether increased lymphocyte numbers will translate into improved clinical outcome.
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PMID:Safety and efficacy of polyethylene glycol-modified interleukin-2 and zidovudine in human immunodeficiency virus type 1 infection: a phase I/II study. 809 58

The activity of DNA polymerase alpha in splenocytes of 24-month-old mice was about 35.6% lower than that of 3-month-old mice. Aged mice were intraperitoneally administered Ganoderma polysaccharides (GL-B) once a day for 4 days and then the activity of the enzyme was assessed. The results showed that GL-B at doses of 25 and 50 mg/kg-1 enhanced the activity of the enzyme in aged mouse splenocytes by 44.0 and 58.8% respectively. In addition, the mixed lymphocyte response to alloantigen, automatic proliferation and IL-2 production of splenocytes in aged mice declined as compared with that in young adult mice. GL-B (50, 100, 200 micrograms.ml-1) was found to restore those parameters to the levels of that of young mice in vitro.
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PMID:[Effects of Ganoderma polysaccharides on the activity of DNA polymerase alpha of splenocytes and immune function in aged mice]. 828 65

Antibody interaction with a specific epitope of the HLA class I alpha1 domain triggers apoptosis of activated but not resting T and B cells by a pathway which involves neither Fas ligand nor tumor necrosis factor-alpha. We have investigated at which stage of activation and proliferation T cells become sensitive to HLA class I-mediated apoptosis, using two monoclonal antibodies (mAb) which recognize the same monomorphic epitope of the HLA class I alpha1 domain (mAb9O, mouse IgG1, and YTH862, rat IgG2b) and can induce apoptosis of phytohemagglutinin (PHA)-activated peripheral blood lymphocytes. Sensitivity to apoptosis develops after the expression of G1 markers (CD69 expression) but it is accelerated by addition of recombinant interleukin-2 (rIL-2). Blocking the IL-2 pathway by cyclosporin A, FK506, rapamycin, anti-IL-2 or CD25 antibodies, prevented the development of sensitivity to apoptosis. Addition of IL-2 and, to a lesser extent, IL-4, reversed the inhibitory effect of cyclosporin A. Conversely, rIL-7 and recombinant interferon-gamma restored proliferation of peripheral blood lymphocytes stimulated by PHA in the presence of cyclosporin A but did not restore sensitivity to class I-mediated apoptosis. Finally cells stimulated in the presence of the DNA polymerase inhibitor aphidicolin did not enter into S phase of the cell cycle but secreted IL-2 and underwent apoptosis when exposed to mAb90 or YTH862. Together, the data indicate that sensitivity of peripheral T cells to HLA class I-mediated apoptosis depends on both activation signals and IL-2 or IL-4, but does not require cell proliferation. These data suggest that YTH862 and mAb90 might be used for achieving clonal deletion of antigen-activated peripheral T cells in vivo, provided that the IL-2 pathway is not blocked by other immunosuppressive agents.
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PMID:T cell sensitivity to HLA class I-mediated apoptosis is dependent on interleukin-2 and interleukin-4. 904 22

Extensive diagnostic and scientific investigations are often restricted by limited availability of material. Therefore, methods like multiplex PCR strategies are needed to conserve as much sample as possible. Unfortunately, the establishment of such procedures poses several difficulties. Here we describe the advantages of a new enzyme, AmpliTaq Gold DNA Polymerase, in multiplex and time-release PCR. The application of this thermostable recombinant Taq DNA polymerase allows the specific amplification of DNA/cDNA targets with very high sensitivity. With our protocol, the specific amplification of 13 different cDNAs of cytokines and cytokine receptors can be realized in three multiplex PCRs (IL-2R alpha, IL-2/15R beta, gamma c-chain, IL-4 and IL-4R alpha; IL-10, IL-15 and IL-15R alpha; and IL-2, IFN gamma, IL-7, IL-7R alpha and IL-9R alpha). The novel application of AmpliTaq Gold DNA Polymerase in a time-release PCR protocol allows specific amplification of target DNA/cDNA when only limited amounts of material are available or only low-copy-number DNA/cDNA is suspected. No IL-9 cDNA can be detected in peripheral blood mononuclear cells (PBMC) in the absence of any stimulation, thus it was difficult to amplify this target with routine PCR protocols. Here we demonstrate the reliable and reproducible amplification of IL-9 cDNA in the Hodgkin's lymphoma cell line KM-H2, in PBMC and in stimulated PBMC. Results with AmpliTaq Gold DNA Polymerase were more sensitive and specific compared with AmpliTaq DNA Polymerase, with and without manual hot-start procedure.
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PMID:Advantages of a new Taq DNA polymerase in multiplex PCR and time-release PCR. 945 68

Although the identity of T cells involved in the protection against Mycobacterium tuberculosis (Mtb) in humans remain unknown, patients with pulmonary tuberculosis (TB) have reduced numbers of Mtb-reactive, V gamma 9+/V delta 2+ T cells in their blood and lungs. Here we have determined whether this gamma deltaT loss is a consequence of Mtb Ag-mediated activation-induced cell death (AICD). Using a DNA polymerase-mediated dUTP nick translation labeling assay, 5% or less of freshly isolated CD4+ alpha beta or gamma delta T cells from normal healthy individuals and TB patients were apoptotic. However, during culture Mtb Ags induced apoptosis in a large proportion of V gamma 9+V delta 2+ peripheral blood T cells from healthy subjects (30-45%) and TB patients (55-68%); this was increased further in the presence of IL-2. By contrast, anti-CD3 did not induce any significant level of apoptosis in gamma delta T cells from healthy subjects or TB patients. Mtb Ag stimulation rapidly induced Fas and Fas ligand (FasL) expression by gamma delta T cells, and in the presence of metalloproteinase-inhibitors >70% of gamma delta T cells were FasL+. Blockade of Fas-FasL interactions reduced the level of Mtb-mediated gamma delta T cell apoptosis by 75 to 80%. Collectively, these findings demonstrate that Mtb-reactive gamma delta T cells are more susceptible to AICD and that the Fas-FasL pathways of apoptosis is involved. AICD of gamma delta T cells, therefore, provides an explanation for the loss of Mtb-reactive T cells during mycobacterial infection.
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PMID:Involvement of the Fas/Fas ligand pathway in activation-induced cell death of mycobacteria-reactive human gamma delta T cells: a mechanism for the loss of gamma delta T cells in patients with pulmonary tuberculosis. 968 24


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