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)

Interleukin 18 (IL-18) is a recently identified cytokine, originally called interferon gamma inducing factor, due to its capacity to induce interferon gamma production in Th1 type cells. IL-18 is expressed by a wide variety of cell types including mononuclear phagocytes, osteoblasts, keratinocytes and adrenal cortex cells. To quantify human IL-18 mRNA expression in small-scale cell samples the authors developed a competitive reverse transcriptase polymerase chain reaction using a competitive template as an internal standard. This assay was demonstrated as a valid, sensitive and precise tool to quantify human IL-18 mRNA expression. IL-18 mRNA expression of primary peripheral blood monocytes, CD4(+)T cells, CD8(+)T cells, B cells and NK cells was assessed by competitive RT-PCR. Basal IL-18 expression could be detected in all types of peripheral blood mononuclear cells (PBMC). The kinetics of IL-18 mRNA expression in PBMC from healthy donors was defined in vitro after monocyte-specific (lipopolysaccharide LPS), T-cell-specific (anti-CD3) and polyclonal-unspecific stimulation (phytohaemagglutinin PHA). Only LPS led to a strong increase of IL-18 mRNA expression peaking after 2 h. These results indicate that IL-18 is expressed constitutionally by all major PBMC subtypes. However, only monocyte specific stimulation resulted in a significant induction of IL-18 mRNA expression suggesting activated monocytes e.g. in inflammation as the main source of IL-18 expression.
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PMID:Quantification of human interleukin 18 mRNA expression by competitive reverse transcriptase polymerase chain reaction. 1034 85

The interaction of CD44 with its ligand hyaluronan (HA) plays a vital role in lymphopoiesis, lymphocyte homing, T cell activation, and metastasis. This study addresses the effect of cytokines involved in B cell growth on CD44-HA interactions in normal human B cells. Activation of B lymphocytes with LPS, pokeweed mitogen, or anti-IgM antibodies with or without IL-2 or IL-4 failed to induce HA adhesion. Stimulation of B cells with the phorbol ester PMA, however, induced strong HA recognition, which was inhibited by IFN-gamma and to some extent by IL-4. Investigation of the potential molecular mechanism involved revealed that PMA-induced HA adhesion correlated with enhanced expression of CD44-H- and V6-containing isoforms, as determined by flow cytometry, and the differential induction of V4- and V5-containing isoforms, as determined by reverse transcriptase-based polymerase chain reaction analysis. The inhibition of PMA-induced adhesion by IFN-gamma and IL-4 correlated with the downregulation of CD44 H expression and altered usage of exons V4 and V5. However, changes in the electrophoretic mobility of CD44 proteins, as a measure of posttranslational modifications, were not detected in response to PMA and IFN-gamma or PMA and IL-4. These results suggest that the inhibition of PMA-induced HA adhesion by IFN-gamma and IL-4 may influence B cell migration through their ability to downregulate CD44-HA interactions.
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PMID:Interferon-gamma inhibits CD44-hyaluronan interactions in normal human B lymphocytes. 1038 38

Gut-derived endotoxins (lipopolysaccharide, LPS) complexed to LPS-binding protein (LBP) activate liver Kupffer cells via their CD14 receptor. Pro-inflammatory cytokines are released and this is postulated to promote liver injury. We previously demonstrated enhanced expression of CD14 endotoxin receptor after 2 weeks of alcohol administration. A similar result, based on 6 weeks of ethanol treatment, was recently reported and suggested to correlate with alcohol-induced liver injury. To establish whether this occurs prior to or after the initiation of damage, we investigated the temporal effect of continuous ethanol exposure on the expression of CD14 and the associated LBP. In addition, we studied the effect of treatment with gadolinium chloride (GdCl3) that inactivates Kupffer cells and alleviates alcohol-induced liver damage. The amount of CD14 and LBP mRNA, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR), was unchanged 4-8 h after intragastric ethanol administration. However, after 24-48 h of repeated ethanol administration, CD14 and LBP mRNA both increased significantly and reached a level similar to that observed after 6 weeks of ethanol exposure by liquid diet. Immunostaining experiments with ED2 antibody demonstrated that GdCl3 efficiently inactivated Kupffer cells. However, there was no concomitant reduction in the expression of CD14 mRNA, suggesting that compensatory infiltration by ED2-negative, but CD14-positive, macrophages had occurred. Our results demonstrate that soon after the initiation of ethanol exposure, i.e. within 24-48 h, the hepatic expression of both the CD14 receptor and LBP is increased. This suggests that these increases could contribute to the initiation of alcoholic damage rather than being a consequence of the injury.
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PMID:Short-term ethanol exposure increases the expression of Kupffer cell CD14 receptor and lipopolysaccharide binding protein in rat liver. 1041 5

Several groups, including ours, have reported that chloroquine (CQ) or its analog hydroxychloroquine has anti-HIV-1 activity both in vitro and in vivo. We studied in vitro whether the addition of CQ to the combination of hydroxyurea (HU) plus didanosine (ddI) had an additive effect in inhibiting the replication of HIV-1. Therefore both the H-9 T lymphocytic cell line and the U-937 promonocytic cell line as well as primary T cells and monocytes were infected with HIV-1 and then treated with HU at 0.2 mM and ddI at 1 microM and varying concentrations of CQ. Addition of CQ resulted in an additional inhibition of HIV-1 replication, as assessed by reverse transcriptase (RT) activity, with a CQ EC50 of 0.4-0.9 microM for the cell lines and of 0.2-0.9 microM for the primary cells. Similarly, addition of CQ further inhibited HIV-1 replication in U-1 cells stimulated either with LPS or H2O2 and in ACH-2 cells stimulated either with PMA or H2O2, with CQ EC50 values of 0.1 and 1 microM, respectively. Under the experimental conditions used, CQ induced neither toxicity nor apoptosis in the H-9 and U-937 cells. This in vitro additive anti-HIV-1 activity of CQ, in combination with HU + ddI, supports the idea that this triple regimen should be studied in clinical trials. It may become of particular interest to HIV-1-infected individuals from the developing world, in view of the low cost of both CQ and HU.
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PMID:Chloroquine exerts an additive in vitro anti-HIV type 1 effect when associated with didanosine and hydroxyurea. 1050 72

1 The effects of nepalolide A on the expression of inducible nitric oxide synthase (iNOS) caused by incubation with lipopolysaccharide/interferon-gamma (LPS/IFN-gamma) or tumour necrosis factor-alpha/interleukin-1beta/IFN-gamma (TNF-alpha/IL-1beta/IFN-gamma, mixed cytokines) in C6 glioma cells and primary astrocytes of rat were investigated. The mechanisms by which nepalolide A confers its effect on iNOS expression were also elucidated. 2 Treatment with LPS/IFN-gamma and mixed cytokines for 24 h elicited the induction of iNOS activity as determined by nitrite accumulation in the culture medium and assay of enzyme activity. Nepalolide A at 10 microM abrogated the LPS/IFN-gamma- and mixed cytokines-mediated induction of iNOS by more than 90% in C6 glioma cells, and by 80% for mixed cytokines-induced induction of iNOS in primary astrocytes. The effect of nepalolide A (2-10 microM) was concentration-dependent. 3 The inhibition of iNOS induction by nepalolide A was attributed to decreases in the content of iNOS protein and the level of iNOS mRNA, as measured by immunoblotting and reverse transcriptase-polymerase chain reaction. 4 Electrophoretic mobility shift assay was used to evaluate the effect of nepalolide A on the activation of nuclear factor-kappaB (NF-kappaB). Results showed that nepalolide A diminished the LPS/IFN-gamma-mediated association of NF-kappaB with consensus oligonucleotide in a concentration-dependent manner. The activation of NF-kappaB by mixed cytokines was modulated both in the extent of activation and in its time-course by nepalolide A. 5 The ability of nepalolide A to inhibit NF-kappaB activation was further confirmed by studies on the degradation of the inhibitor of NF-kappaB, IkappaB, as measured by immunoblotting. 6 The present study demonstrates that the attenuation of NF-kappaB activation by nepalolide A was mediated by blockade of the degradation of IkappaB, leading to suppression of the expression of iNOS.
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PMID:Nepalolide A inhibits the expression of inducible nitric oxide synthase by modulating the degradation of IkappaB-alpha and IkappaB-beta in C6 glioma cells and rat primary astrocytes. 1051 Apr 44

In order to elucidate further the role of nitric oxide (NO) as an endogenous antiangiogenic mediator, mRNA expression of inducible nitric oxide synthase (iNOS), enzyme activity and production of NO were determined in the chick chorioallantoic membrane (CAM), an in vivo model of angiogenesis. In this model, maximum angiogenesis is reached between days 9 - 12 of chick embryo development. After that period, vascular density remains constant. Inducible NO synthase (iNOS) mRNA expression, determined by reverse transcriptase polymerase chain reaction (RT - PCR), increased from the 8th day reaching a maximum (70% increase) at days 10 - 11. NO synthase activity, determined as citrulline formation in the presence of calcium, also increased from day 8 reaching a maximum around day 10 (100% increase). Similar results were obtained in the absence of calcium suggesting that the NOS determined was the inducible form. Nitric oxide production, determined as nitrites, increased from day 8 reaching a maximum around day 10 (64% increase) and remaining stable at day 13. Finally, the bacterial lipopolysaccharide LPS (which activates transcriptionally iNOS), inhibited dose dependently angiogenesis in the CAM. These results in connection with previous findings from this laboratory, showing that NO inhibits angiogenesis in the CAM, suggest that increases in iNOS expression, enzyme activity and NO production closely parallel the progression of angiogenesis in the CAM, thus providing an endogenous brake to control this process. British Journal of Pharmacology (2000) 129, 207 - 213
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PMID:Nitric oxide synthase expression, enzyme activity and NO production during angiogenesis in the chick chorioallantoic membrane. 1069 22

Receptors for 1,25(OH)2vitaminD3 are found in most immune cells and important immunological effects have been described in vitro, reflected by its capacity to prevent autoimmunity and to prolong graft survival. The aim of this study was to examine the presence and nature of the enzyme responsible for final activation of the molecule, 1-alpha-hydroxylase, in murine macrophages and to analyse its regulation and possible role in the immune system. Peritoneal macrophages from C57Bl/6 mice were incubated with lipopolysaccharide (LPS; 100 microg/ml), interferon-gamma (IFN-gamma; 500 U/ml) or a combination of both. By quantitative reverse transcriptase-polymerase chain reaction, using primers based on the murine renal cDNA sequence, low levels of 1-alpha-hydroxylase mRNA were detected in freshly isolated cells (18 +/- 7 x 10-6 copies/beta-actin copies). Analysis of the cDNA sequence of the gene revealed identical coding sequences for the macrophage and renal enzymes. mRNA levels rose three-fold with LPS (NS), but a six-fold increase was seen after IFN-gamma stimulation (P < 0.05). Combining LPS and IFN-gamma did not result in a major additional increase, but addition of cyclosporin A further increased levels 2.5-fold both in IFN-gamma- and combination-stimulated cells (P < 0.05). Time course analysis revealed that up-regulation of 1-alpha-hydroxylase was a late phenomenon, preceded by the up-regulation of activating macrophage products such as IL-1 and tumour necrosis factor-alpha. Finally, a defect in 1-alpha-hydroxylase up-regulation by immune stimuli was found in autoimmune non-obese diabetic mice. In conclusion, we propose that the up-regulation of 1-alpha-hydroxylase in activated macrophages, resulting in the synthesis of 1,25(OH)2D3, might be a negative feedback loop in inflammation. A defect in this system might be an additional element in tipping the balance towards autoimmunity.
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PMID:Identification and immune regulation of 25-hydroxyvitamin D-1-alpha-hydroxylase in murine macrophages. 1075 75

During hemorrhagic shock there is a massive overproduction of nitric oxide (NO). In such conditions, the intravenous (i.v.) injection of melanocortin peptides in nanomolar amounts produces a long-lasting restoration of cardiovascular and respiratory functions associated with the normalization of NO blood levels. To clarify the mechanism of such melanocortin-induced inhibition of NO overproduction, the influence of the adrenocorticotropin fragment 1-24 [ACTH-(1-24)] on the NO synthesizing activity of rat macrophages was studied in vitro. Nitrite production, an indicator of NO synthesis, was measured in the supernatant of rat macrophages whose inducible NO synthase (NOS II, iNOS) had been stimulated by the addition of S. enteritidis lipopolysaccharide (LPS, 50 microg/ml). ACTH-(1-24) (25, 50 and 100 nM) inhibited nitrite production when incubated together with LPS, but had no effect when applied 6 h after LPS. Further, the effect of ACTH-(1-24) on the expression of iNOS mRNA in rat macrophages activated with LPS was studied by means of a reverse transcriptase-polymerase chain reaction assay. ACTH-(1-24) (25, 50 and 100 nM), applied together with LPS, dose-dependently suppressed iNOS gene activation. The present data suggest that the melanocortin-induced normalization of NO blood levels during hemorrhagic shock is due, at least in part, to a direct inhibition of iNOS induction, at the level of mRNA transcription.
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PMID:Adrenocorticotropin inhibits nitric oxide synthase II mRNA expression in rat macrophages. 1085 45

Regulation of IL-12 and IL-10 production in normal human monocytes infected with vaccinia virus (VV) was analysed. IL-12 and IL-10 mRNAs were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and IL-12 and IL-10 protein by ELISA. RT-PCR analysis revealed a marked-up regulation of IL-12 (p40) and IL-10 expression in virally infected cells compared with that from control (non-infected) cells at 24 h post-infection (p.i.). IL-12 transcripts occurred earlier (at 4 h p.i.) than IL-10 mRNA. A significant increase in IL-12 and IL-10 secretion into the medium was caused by the virus, and even a much more pronounced increase in both interleukins expression (mRNAs and proteins) followed LPS or Staphylococcus aureus treatment. Vaccinia virus infection did not alter IL-10 secretion and IL-10 mRNA content (or even cause a decrease) in a human monocytic cell line U937. Undetectable levels of IL-12 protein were found in the cell line although the transcripts were present in the cells at first hours p.i. It appears now that vaccinia virus transiently and sequentially induces IL-12 and IL-10 in human monocytes.
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PMID:Regulation of interleukin 12 and interleukin 10 expression in vaccinia virus-infected human monocytes and U-937 cell line. 1088 Feb 34

In this study we investigated the capacity of morphine to modulate expression of cytokines in peritoneal macrophages. Mice were implanted subcutaneously with a 75-mg morphine slow-release pellet, and 48 h later resident peritoneal macrophages were harvested. Control groups received placebo pellets, naltrexone pellets, or morphine plus naltrexone pellets. Adherent cells were stimulated with lipopolysaccharide (LPS: 10 microg/mL) plus interferon-gamma (IFN-gamma: 100 units/mL) to induce cytokine production. After 24 h RNA was extracted for analysis of cytokine mRNA levels by reverse transcriptase-polymerase chain reaction, or supernatants were collected after 48 h for determination of cytokine production by enzyme-linked immunosorbent assay (ELISA). Morphine enhanced mRNA expression of interleukin (IL)-12 p40 and tumor necrosis factor alpha (TNF-alpha) compared with controls, whereas IL-10 levels were unchanged by drug treatment. ELISA data showed that both IL-12 p40 and p70 were increased by morphine. The enhancement of IL-12 at both the mRNA and protein levels was antagonized by naltrexone, indicating that the modulation of this cytokine by morphine is via a classic opioid receptor. These results are particularly interesting in light of our previous observation that 48 h after morphine pellet implantation, the peritoneal cavity is colonized with gram-negative and other enteric bacteria. The enhancement of IL-12 by morphine might be related to morphine-induced sepsis.
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PMID:Morphine enhances interleukin-12 and the production of other pro-inflammatory cytokines in mouse peritoneal macrophages. 1107 13


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