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)

In human long-term marrow cultures granulomonopoiesis is maintained for several weeks. Studies on granulomonocytic progenitors (CFU-GM) and their progeny have shown that survival, proliferation, differentiation and maturation of these cells are controlled by a set of glycoproteins, the colony-stimulating factors (CSFs) and the Steel factor. We have studied the expression of these factors using reverse transcriptase polymerase chain reaction (RT-PCR) in 17 adherent layers of normal bone marrow at 3, 5 or 7 weeks of culture. We have taken the 5637 bladder carcinoma cell line as a control for expression of GM-CSF, M-CSF, G-CSF and Steel factor, and PHA-activated T lymphocytes as a control for expression of multi-CSF (interleukin 3, IL-3). We have found that GM-CSF was expressed in the 17 adherent layers without induction by interleukin 1 beta (IL-1 beta). M-CSF was also detected in all cases, but in two early-stage (week 3 and week 5) cultures only after stimulation by IL-1 beta. G-CSF was detected in only 11 cases (three without IL-1 beta, and eight after addition of IL-1 beta). Steel factor was detected in 14 cases (ten without IL-1 beta, and four after addition of IL-1 beta). IL-3 was not detected even by means of nested RT-PCR. These data indicate in six late-stage (week 5 or week 7) cultures G-CSF messenger concentrations 10(3)-fold less than in 5637 control cells (for an identical amount of total cellular RNA). A similar conclusion may be drawn for Steel factor in three late-stage cultures. For IL-3 our negative results indicate a messenger concentration 10(5)-fold less than in activated T lymphocytes. These results suggest a crucial role for GM-CSF and M-CSF in the maintenance of granulomonopoiesis in human long-term cultures. The role of G-CSF and Steel factor may be more marginal. Eventually IL-2 may not be involved in the regulatory process.
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PMID:The detection of colony-stimulating factors and steel factor in adherent layers of human long-term marrow cultures using reverse-transcriptase polymerase chain reaction. 751 Mar 57

Azidothymidine (zidovudine, AZT) used for treatment of HIV infection blocks the viral reverse transcriptase after phosphorylation by cellular enzymes. The first step in this reaction is the formation of AZT monophosphate, primarily catalyzed by host cytoplasmatic thymidine kinase (TK1). The activity of TK1 was determined in extracts of PHA-stimulated peripheral blood mononuclear cells (PBMCs) from 20 healthy volunteers and 49 HIV-infected patients at different stages of disease. In both groups we found a large intra- and interindividual variation of TK activity. Because TK1 expression is cell cycle regulated the proportion of stimulated cells was determined in the samples and the median thymidine kinase activity calculated. It was 3.0 pmol/mg/min x % S phase in the HIV-seronegative group and 1.1 pmol/mg/min x % S phase in HIV-infected individuals. The difference in thymidine kinase activity is statistically significant (p = 0.0001). The concentration of TK1 protein in the same extracts was also determined by immunoblotting. A positive correlation (r = 0.74) was observed between TK activity and amount of TK1 protein. The reason for this downregulation of TK is still unknown but may be related to the anergy observed in lymphocytes from HIV-infected persons. The reduced capacity for intracellular phosphorylation of AZT in HIV-infected individuals may be an important factor in the emergence of clinical AZT resistance and should also be accounted for in testing AZT resistance in vitro with PBMCs from healthy blood donors.
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PMID:Decreased thymidine kinase levels in peripheral blood cells from HIV-seropositive individuals: implications for zidovudine metabolism. 754 7

We established an improved non-radioactive in situ hybridization (ISH) method to detect mRNA of cytokines in cell preparations and tissues. Via this method we could demonstrate various cytokines in stimulated peripheral blood mononuclear cells (PBMC), lymphoid cell lines and human lymphoid tissues. The probes for the in situ hybridization were made by labelling cytokine-specific PCR products with digoxigenin (Dig) in a repeated PCR. This resulted in an intrinsic labelling of the probe with several Dig-UTP molecules. Incorporation of Dig-11-dUTPs was shown on ethidium bromide-stained agarose gels by a higher molecular weight of the PCR products with incorporated Dig-dUTPs when compared to control PCR products without digoxigenin. Cytospin-centrifuged cells of PHA-stimulated PBMC or lymphoid cell lines and frozen sections of various human lymphoid tissues were hybridized with the Dig-labelled cytokine probes and the hybridized probes were detected immuno-histochemically. In this way, we detected and localized cytokine mRNAs (IL-2, IL-4, IL-6, IL-8, IL-10) in PBMC, in the human T-cell line Jurkat, in the follicular lymphoma cell line DoHH2, and in human lymph nodes and tonsils. The in situ hybridization had a high sensitivity as the results correlated closely with the detection of cytokine mRNA by reverse transcriptase-PCR (RT-PCR) data from the same samples. We showed that Jurkat and DoHH2 cells produce several cytokines constitutively and that, after activation with the phorbol ester PMA, expression of several cytokine mRNAS was enhanced.
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PMID:An improved, sensitive, non-radioactive in situ hybridization method for the detection of cytokine mRNAs. 765 59

The expression of insulin-like growth factors and their binding proteins in normal human peripheral lymphocytes was studied using the reverse transcriptase polymerase chain reaction method and Western ligand blotting. A quantitation of RT-PCR products was used to study the differences between normal and PHA stimulated lymphocytes. Normal freshly collected lymphocytes expressed mRNAs for both IGF-I receptor and IGF-II receptor but no expression of the corresponding growth factors was detectable. After stimulation with phytohemagglutinin the lymphocytes, however, expressed both IGF-I and IGF-II. Of the five IGFBPs examined, unstimulated lymphocytes expressed only IGFBP-2 and -3. Stimulated lymphocytes expressed IGFBP-4 and -5, in addition to IGFBP-2 and -3, whereas IGFBP-1 mRNA remained undetectable. The ligand blotting of lymphocyte conditioned media revealed production of 34K, 43K and 49K IGFBPs. The addition of estrogen, progesterone, IGF-I or growth hormone did not affect secretion of IGFBPs by lymphocytes.
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PMID:The expression of insulin-like growth factors and their binding proteins in normal human lymphocytes. 768 Aug 35

We present here thirteen patients (5 men and 8 women, aged 31 to 73, mean 55 years) with spastic paraparesis who showed clinical features similar to those of HTLV-1 associated myelopathy without HTLV-1 antibody, but with positive antibody to hepatitis B virus (HBV). All of these patients showed slowly progressive difficulty in walking. Five patients had previous histories of blood transfusion, of these one with history of B hepatitis. Neurologically, muscle weakness, spasticity and exaggerated deep tendon reflexes in the lower extremities were common to all the patients. Seven patients showed Babinski's reflex. Disturbance of micturition was noted in 3 patients. None showed organic changes of the spine on magnetic resonance image (MRI). None was serologically positive for syphilis and had cryoglobulin and hypergammaglobulinemia. Elevated levels of the liver enzymes were noted in two patients. All patients were positive for hepatitis B surface antibody (HBs-Ab) (EIA) but negative for hepatitis B surface antigen (HBs-Ag) (EIA). Five patients were seropositive for hepatitis C virus (HCV) (PHA). In 3 of them, reverse transcriptase polymerase chain reaction was performed but failed to detect HCV-RNA. All patients underwent spinal tap, and showed normal cell count and protein concentration in their cerebrospinal fluid (CSF). Atypical cells were not observed in all the patients. The CSFs from three patients were tested for HBs-Ag and HBs-Ab. HBs-Ag was negative in all three patients, but HBs-Ab was positive in two patients.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Hepatitis B virus antibody positive spastic paraparesis]. 795 26

T-cell responses have been reported to be impaired in cancer patients, and lymphocytes infiltrating human tumors (T-TIL) appear to be more affected than those in the peripheral blood. T-TIL display a poor proliferative response when compared to peripheral blood T (T-PBL) cells that show a strong response to all stimuli. Here we report that T-TIL from patients with renal cell carcinoma (RCC) also have a defect in transferrin receptor (TfR) expression that is not present in T-PBL cells. Immunocytometry studies (dual staining for CD3 epsilon and TfR) demonstrated that autologous T cells from the peripheral blood but not from the tumor expressed TfR following stimulation with IL2, anti-CD3 or PHA. Expression of TfR correlated with the capacity of T cells from the blood and tumor to proliferate. Gene expression studies using reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that TfR mRNA levels in T-TIL were undetectable or low relative to T-PBL following stimulation. The failure to detect TfR mRNA in T-TIL after stimulation was not due to a shift in kinetics of mRNA accumulation since TfR mRNA was not detectable at any of the times tested (4, 12, 24 and 36 hr). The defect in TfR gene expression is selective since IL2R alpha gene expression was induced in T-TIL. Because IL2 binding to its receptor results in TfR expression, the defect in TfR induction in T-TIL appears to be distal to IL2R alpha expression. Our studies illustrate another alteration in T-TIL that is not observed in T cells from the peripheral blood. The absence of TfR gene expression may contribute to the poor proliferative response of T cells from the tumor.
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PMID:T lymphocytes infiltrating renal cell carcinoma have a reduced expression of transferrin receptor. 805 Aug 20

IL-10, an immunoregulatory cytokine produced by T cells and monocytes, inhibits the expression of inflammatory and hemopoietic cytokines as well as its own expression. To evaluate the regulation of IL-10 production by T cells and monocytes, we measured IL-10 levels by ELISA in supernatants of PHA-stimulated PBMC following depletion of either T cells or monocytes. IL-10 production was significantly down-regulated in both T cell- and monocyte-depleted PBMC compared with undepleted PBMC, and IL-10 production could be restored by the addition of monocyte-conditioned medium (supernatant of PHA-stimulated, T cell-depleted PBMC), suggesting that IL-10 production by T cells is regulated by a monokine(s) produced by activated monocytes. To further clarify the monokine(s) responsible for IL-10 induction, we stimulated monocyte-depleted PBMC, purified CD4+, and CD8+ T cells with PHA and measured IL-10 production by ELISA and semiquantitative reverse transcriptase-PCR following monokine(s) addition. Addition of IL-6 and IL-12 enhanced IL-10 production in monocyte-depleted PBMC in a dose-dependent and additive manner. Furthermore, anti-IL-6 and anti-IL-12 Abs neutralized the IL-10-inductive effect of monocyte-conditioned medium. Similarly, IL-12 and IL-6 induced IL-10 production by purified CD4+ and CD8+ T cells. With respect to regulation of IL-10 produced by monocytes, TNF-alpha was found to induce IL-10 production by resting as well as by LPS-stimulated purified monocytes/macrophages. Taken together, these findings suggest that IL-10 production by human T cells and monocytes is differentially regulated. IL-12 and/or IL-6 can induce the expression of IL-10 by PHA-stimulated T cells, whereas TNF-alpha induces IL-10 production by monocytes. Since IL-10 inhibits the production of IL-6, IL-12, and TNF-alpha, these results may indicate a potential mechanism of negative feedback regulation of the immune response.
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PMID:IL-10 production is enhanced in human T cells by IL-12 and IL-6 and in monocytes by tumor necrosis factor-alpha. 868 5

To develop a rapid and sensitive means of detecting cell-associated human immunodeficiency virus (HIV), donor cells from HIV seropositive patients were treated with the potent viral activator sodium-n-butyrate (NaB) and subsequently assayed by both in situ RNA hybridization and a reverse transcriptase polymerase chain reaction (RT-PCR). The sensitivity of RT-PCR was estimated to be equivalent to 1 x 10(-16) grams (0.1 fg) or approximately 64 copies of the input standard viral RNA per reaction. The present study takes advantage of the ability of NaB to introduce changes in chromatin structure of latently infected cells, leading to increased HIV gene expression. Human ACH-2 and U1 cell lines were used as representatives of T-lymphocytic and monocytoid cells harboring latent inducible proviruses. HIV gene expression was readily detected when these cells were treated with NaB. Viral gag RNA was detected by both in situ and RT-PCR assays. When peripheral blood mononuclear cells (PBMCs) from acquired immunodeficiency syndrome (AIDS) patients, who were all negative for in situ hybridization and serum/plasma p24 assays, were used for detection of viral gene expression, four categories with distinct patterns of induction were observed. The first set of patients showed HIV-positive PBMCs by RT-PCR without any added NaB, and suppression by added NaB or PHA. The second set of samples showed induction of viral RNA by NaB alone. The third set could be induced with PHA, but not NaB, and the fourth set required both NaB and PHA for induction of HIV gene expression. Our results suggest that direct treatment of the cells with HIV activators may be useful in increasing sensitivity of the RT-PCR intended to be used for detection of cell-associated viral RNAs. This approach may be used to confirm true status of the HIV infection when p24 results are negative or HIV RNAs in serum/plasma are below the threshold of detection. Moreover, this method may identify the presence of latent proviral genomes possibly reflecting the true rate of cell-associated viral load in vivo and without possible mutations brought about by long-term co-cultivation assays with cells from seronegative donors.
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PMID:Rapid and sensitive detection of cell-associated HIV-1 in latently infected cell lines and in patient cells using sodium-n-butyrate induction and RT-PCR. 917 66

We investigated the role of endogenous or exogenous nitric oxide (NO) on human lymphocyte function. We used sodium nitroprusside, nitroglycerine, S-nitroso-N-acetylpenicillamine, sodium nitrite and S-nitroso-L-glutathione as NO-generating compounds. All agents were used at doses that do not produce direct cytotoxicity as measured by trypan blue exclusion as well as chromium-51 release assay. The immune responses examined were peripheral blood lymphocytes (PBL) proliferation and IL-2 production after activation with OKT3 and PHA; allogeneic mediated proliferation and cell mediated cytotoxicity (CML) in MLR; IgG and IgM production after PBL activation with Con-A; proliferation and expression of IFN-gamma and IL-4 mRNA after activation of allogeneic CD4+T cell clones. Cytokine mRNA expression was measured by reverse transcriptase PCR. Our results show that proliferating lymphocytes do not produce a detectable amount of NO as measured by the Griess reaction. In separate experiments, the addition of NG-monomethyl-L-arginine (L-NMMA) did not affect lymphocyte proliferation. Sodium nitroprusside and nitroglycerine exerted a dose dependent antimitogenic effect, inhibited cytokine production and expression, CML generation and antibody production. DNA gel electrophoresis showed no evidence for enhanced programmed cell death. The antimitogenic effect could not be blocked by the NO scavengers, hemoglobin or methylene blue. In contrast, the other nitric oxide generating compounds did not inhibit lymphocyte mitogenesis. The results suggest that human lymphocytes do not produce appreciable amounts of NO to affect lymphocyte mitogenesis. Sodium nitroprusside and nitroglycerine have a potent but nonspecific immunoinhibitory effect on human lymphocyte function by a mechanism other than NO production. In addition, pharmacological levels of NO do not inhibit human lymphocyte mitogenesis.
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PMID:Analysis of the in vitro effect of exogenous nitric oxide on human lymphocytes. 920 99

Immature postnatal thymocytes were shown to contain precursors which, under suitable culture conditions, give rise to phenotypically and functionally mature natural killer (NK) cells. Here, we analyzed the effect of different cytokines for their ability to induce the expression of HLA class I-specific inhibitory receptor(s) during the process of NK cell development from immature thymocytes. From thymocyte cell suspensions depleted of CD2+, CD3+, CD4+, CD8+, CD56+, and CD16+ cells, we further removed cells expressing HLA class I-specific inhibitory receptors including CD94/NKG2-A, p58.1, and p58.2 by immunomagnetic bead separation. The resulting cells did not contain any of the above NK receptors as determined by immunofluorescence and flow cytometric analysis, as well as by reverse transcriptase polymerase chain reaction (RT-PCR) amplification using appropriate sets of primers. Although different cytokines have been used, including interleukin (IL)-7, stem cell factor (SCF), IL-2, and IL-15, only IL-2 or IL-15 induced cell proliferation when used alone. Moreover, maturation towards CD3- CD56+ cells displaying cytolytic activity against the HLA class I- targets K562 or 221 was detectable in cultures containing IL-15 used alone or in combination with IL-7 or SCF. On the other hand, these CD3- CD56+ cell populations did not lyse HLA class I+ target cells, including autologous PHA blasts. Analysis of the expression of the various HLA class I-specific inhibitory NK receptors revealed the presence of high proportions of CD94/ NKG2-A+ cells, while the NK receptors belonging to the Ig superfamily were undetectable both by immunofluorescence and by RT-PCR analysis. The expression of CD94/NKG2-A appeared to be responsible for the inability of cells to lyse HLA class I+ target cells. Thus, addition of anti-CD94 monoclonal antibodies of IgM isotype resulted in lysis of autologous target cells. The use of 221 cells transfected with different HLA class I alleles as target cells confirmed the broad class I specificity of CD94/NKG2-A receptor. Our experiments indicate that IL-15 provides an appropriate stimulus to the expression of CD94/NKG2-A, but not of other class I-specific NK receptors in the process of maturation of NK cells from thymocyte precursors.
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PMID:Interleukin-15-induced maturation of human natural killer cells from early thymic precursors: selective expression of CD94/NKG2-A as the only HLA class I-specific inhibitory receptor. 920 87


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