Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

The levels or diadenosine 5', 5'''-p1, p4, tetraphosphate (Ap4A), a putative signal molecule associated with DNA synthesis, has been measured in murine T lymphocytes. The level or Ap4A detected correlated with the stimulation of DNA synthesis in murine T lymphocytes. In interleukin-2 (IL-2) dependent cells previously deprived of IL-2, new DNA synthesis can be induced by adding IL-2; the synthesis of DNA is preceded by an increase in Ap4A levels. A significant increase in DNA synthesis was observed after the Ap4A concentration exceeded the Kd of DNA polymerase alpha for Ap4A. Similarly, in cells blocked from synthesizing DNA by hydroxyurea, the levels or Ap4A are maintained only in the presence of IL-2. Once IL-2 is removed, the potential to synthesize DNA decreases and is preceded by decreases in the level or Ap4A. The DNA synthesis potential decreases rapidly after the Ap4A concentration fell below the Kd of DNA polymerase alpha for Ap4A. It is possible that Ap4A is a second messenger molecule required for the proliferation of lymphocytes and that the production of Ap4A in IL-2 dependent murine T lymphocytes is regulated by the homologous growth factor.
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PMID:Interleukin-2 regulation of diadenosine 5',5'''-p1 p4-tetraphosphate (Ap4A) levels and DNA synthesis in cloned murine T lymphocytes. 210 44

Twenty patients with HBe antigen positive, chronic active hepatitis receiving interferon-beta (HuIFN-beta) for 4 weeks were studied. Within the follow-up period (12.3 +/- 2.0 months; mean +/- SD), nine patients were seroconverted to anti-HBe positive and/or HBe antigen negative. In vitro synthesis of interleukin-1 (IL-1), interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) were determined from supernatants of peripheral blood mononuclear cells (PBMCs) cultured in the presence of lipopolysaccharide or concanavalin-A. PBMCs from patients before IFN-beta treatment secreted markedly reduced levels of IL-1 (p less than 0.01) and IFN-gamma (p less than 0.01) as compared with healthy controls. However, IFN-gamma synthesis in the patients was significantly increased (p less than 0.05) along with the IFN-beta treatment. IL-2 synthesis was similar in chronic active hepatitis B patients before and during IFN-beta treatment when compared to normal controls, but after the therapy, the elevation of IL-2 synthesis was observed in accordance with the elevation of serum AST in two cases. Nine patients who seroconverted to anti-HBe positive and/or HBe antigen negative showed the significantly lower levels of DNA polymerase before IFN-beta treatment than non-responder group. There were no other differences in sex, age, serum AST, histologic activities and cytokine production in vitro between two groups. These results indicate the presence of immunologic deficiencies in patients with HBe antigen positive chronic active hepatitis and give the rationales for the use of interferon treatment on immunologic basis.
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PMID:[In vitro cytokine production in patients with HBe antigen positive chronic active hepatitis receiving interferon-beta]. 250 83

The macrolide rapamycin arrests T lymphocytes stimulated by interleukin-2 (IL-2) at G1/S. We have recently found that IL-2 induced an increase in the binding of discrete transcription factors of the ATF/cAMP-responsive element binding factor (CREB) family at G1/S, and that this effect was inhibited by rapamycin (Feuerstein, N., Huang, D., Hinrichs, S. H., Orten, D. J., Aiyar, N., and Prystowsky, M. B. (1995) J. Immunol. 154, 68-79). We now show, by using high resolution two-dimensional gel electrophoresis, that rapamycin inhibited selectively the synthesis of three discrete IL-2-induced soluble proteins (35 kDa/pI approximately 5, 68 kDa/pI approximately 4, 110 kDa/pI approximately 4.3). Analysis of nuclear proteins demonstrated that rapamycin selectively blocked the expression of proliferating cell nuclear antigen (PCNA), an obligate cofactor of DNA polymerase-delta, an important component for DNA replication. Rapamycin inhibited the IL-2-induced PCNA mRNA, and the murine PCNA promoter activity in IL-2-stimulated cells. Inducible CRE-binding proteins were shown previously to be required for PCNA promoter activity in IL-2-stimulated T lymphocytes. Using DNA binding gel mobility shift assay we demonstrated that rapamycin potently inhibited the binding of CREB/ATF transcription factors to CRE elements in the murine proximal PCNA promoter. These results suggest that PCNA is a preferred target in a rapamycin-sensitive transduction pathway, and that the mechanism by which rampamycin inhibits PCNA gene expression may involve the inhibition of the interaction of CREB/ATF transcription factors with CRE elements in the proximal PCNA promoter.
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PMID:Rapamycin selectively blocks interleukin-2-induced proliferating cell nuclear antigen gene expression in T lymphocyte. Evidence for inhibition of CREB/ATF binding activities. 772 72

The purpose of this paper was to study the gamma delta T-cell receptor repertoire and target specificity following stimulation of peripheral T cells of BALB/c mice with autologous or bacterial ligands. The expression of V gamma and V delta chain families in T cells that had been expanded by stimulation in syngeneic mixed lymphocyte culture or with purified protein derivative (PPD) was determined by the semiquantitative DNA polymerase chain reaction (PCR) method. Responder T cells to either of these stimuli strongly expressed both V delta 5 and V delta 6 genes. However, addition of the ML 30 anti-murine heat shock protein (hsp) 60 monoclonal antibody (mAb) to the cell culture selectively inhibited only the expansion of V delta 5 T cells. A V delta 5 T-cell hybridoma (KMT-5), which recognized syngeneic splenic and fibrosarcoma Meth A cells but not allogeneic cells, was produced by cell fusion from autoreactive blast cells. Incubation of the KMT-5 hybridoma in the presence of ML 30 antibody blocked the stimulation of interleukin-2 (IL-2) secretion by syngeneic target cells. It was also found that the DNA of KMT-5 hybridoma and of the autoreactive gamma delta T cells contained the BALB/c invariant delta (BID) chain sequence. It is concluded from these results that BALB/c peripheral V delta 5 T cells recognize an autologous hsp 60 target specificity in a V delta-gene restricted manner. We also propose that T cells of this V gene family may be involved in the immune surveillance of certain tumours and intracellular infections.
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PMID:V delta 5+ T cells of BALB/c mice recognize the murine heat shock protein 60 target cell specificity. 790 91

We and others have described methods to label specific nucleic acid sequences in fixed cells by reverse in situ transcription (IST). They are simple alternatives to the tedious steps of in situ hybridization with labeled probes. We have favored use of thermostable DNA polymerases after heat denaturation of template secondary structure, accompanied by synthesis of cDNA from an annealed primer, but the approach has been limited by the low reverse transcriptase (RT) activity of Taq polymerase and delayed detection methods. We have improved the technique by the use of recombinant Thermus thermophilus (rTth) DNA polymerase and fluorescein-12-dUTP (FIST). Jurkat T lymphocytes were stimulated with ionomycin + phorbol myristate acetate to produce interleukin-2 (IL-2) mRNA in vitro overnight. They were cytospun onto slides and fixed in 70% ethanol + 30% DEPC-treated water, acetone, and air-dried. The slides were placed on a temperature-controlled heating block, and the cell spot was covered with a plastic coverslip. The temperature was raised to 95 degrees C, and 5-10 microliters of modified Perkin-Elmer/Cetus rTth RT reaction mix was injected under the edge of the coverslip. Each 10 microliters of mix in DEPC-water contained 10 mM Tris-HCl, pH 8.3, 90 mM KCl, 1 mM MnCl2, 1 mM dithiothreitol, 10 U placental ribonuclease inhibitor, 0.125 mM dA,C,GTPs, 0.1 mM fluorescein-12-dUTP, 2 U rTth DNA polymerase, and 4 pM 22-mer oligonucleotide primer, which spanned the second intron of IL-2. After 3 min at 95 degrees C, 1 min at 50 degrees C and 10 min at 72 degrees C, the slides were washed in 0.5 x phosphate-buffered saline, pH 7.0, at 42 degrees C, in 70% ethanol, 100% ethanol, and air-dried. The cells were mounted in antifade solution (2% n-propyl gallate in 70% glycerol), and could be viewed immediately by fluorescence microscopy. Image analysis showed that stimulated Jurkat cells were brighter than uninduced controls or those treated with RNase or without polymerase or primer. FIST appears to be useful for the detection of specific mRNAs in single cells.
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PMID:In situ transcription with Tth DNA polymerase and fluorescent nucleotides. 798 81

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

Purpose: Terminal deoxynucleotidyl transferase(TdT) is a DNA polymerase that is present in immature pre-B and pre-T cells. TdT inserts N-nucleotides to the V (D) J gene segment during rearrangements of genes, therefore, it plays a vital role in the development and variation of the immune system in vertebrates. Here we evaluated the relationship between cytokines like interleukin-2 (IL-2), interleukin-7 (IL-7), and interleukin-15 (IL-15) and TdT expression in cord blood mononuclear cells and also effect of inhibition in the expansion of B and T cells derived from cord blood. Methodes: The cord blood mononuclear cells were cultured with different combination of cytokines for 21days, which they were harvested in definite days (7, 14 and 21) and evaluated by flow cytometry. Results: Our data indicated that TdT expression increased in cord blood mononuclear cells using immune cell key cytokines without being dependent on the type of cytokines. TdT inhibition reduced both the expansion of B and T cells derived from cord blood and also declined the apoptosis and proliferation. Considered together, TdT played an important role in the control of the expansion of B and T cells derived from cord blood. Conclusion: considered together, it was observed that TdT expression was increased by cytokines and TdT inhibition not only reduced B and Tcells derived from cord blood, but it also affected the rate of apoptosis and proliferation.
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PMID:Terminal Deoxynucleotidyl Transferase (TdT) Inhibiti on of Cord Blood Derived B and T Cells Expansion. 2876 23