Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Stable gene silencing by RNA interference (RNAi) can be achieved by expression of small hairpin RNAs (shRNAs) from RNA polymerase III promoters. We have tested lentiviral vectors expressing shRNAs targetting CCR5 in primary CD4 T cells from donors representing various CCR5 and CCR2 genetic backgrounds covering the full spectrum of CCR5 expression levels and permissiveness for HIV-1 infection. A linear decrease in CCR5 expression resulted in a logarithmic decrease in cellular infection, giving up to three logs protection from HIV-1 infection in vitro. Protection was maintained at very high multiplicity of infection. This and other recent reports on RNAi should open a debate about the use of RNAi gene therapy for HIV infection.
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PMID:Protection from HIV-1 infection of primary CD4 T cells by CCR5 silencing is effective for the full spectrum of CCR5 expression. 1464 Mar 83

The prevalence of transmitted resistance to antiretroviral drugs varies geographically, with little known about this effect in Asia. In Korea, zidovudine has been widely administered, without charge, through the National AIDS program since the early 1990s; with other potent antiretroviral agents also being introduced in the late 1990s. An analysis of the drug susceptibility to antiretroviral drugs was performed by genotyping of the drug-resistant mutations in HIV on plasma samples from 50 HIV-infected patients who had received no treatment, between February 1998 and November 2002, which was interpreted according to the consensus guidelines of the International AIDS Society-USA panel. The median CD4 cell count was 100 cells/mm3; the mean plasma RNA level was (5.19 +/- 0.56) log copies per milliliter. Of the 50 subjects tested 4 (8.0%) had one or more major drug-resistance mutation. The prevalence of resistance to multiple classes of drugs was 2.0%. No mutations, associated with resistance to non nucleoside reverse transcriptase inhibitors, were identified. Resistant mutations of the reverse-transcriptase gene were found at codons 67, 70, 118, 215, and 219, and a resistant mutation of the protease gene was found only at codon 46 (2.6%, 1 of 39). There was an 8.0% prevalence of primary drug resistance to antiretroviral drugs in Korean patients infected with HIV-1.
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PMID:Genotypic resistance of antiretroviral drugs among drug-naive HIV type 1 patients with the background of long-term access-easy zidovudine therapy. 1468 24

MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop systemic lupus erythematosus (SLE)-like disease. The natural history of the pulmonary involvement and the underlying mechanism of leukocyte infiltration into the lungs of MRL/lpr mice and SLE patients remains elusive. We aimed to investigate the expression profiles of chemokines and chemokine receptors in the lung of the SLE-prone mouse. We examined the correlation between lung inflammation and expression of IP-10 (interferon-gamma-inducible protein 10), a CXC chemokine, and TARC (thymus- and activation-regulated chemokine), a CC chemokine, in MRL/lpr mice, MRL/Mp-+/+ (MRL/+) mice, and C57BL/6 (B6) control mice. The extent of cell infiltration in the lung was assessed histopathologically. Reverse transcriptase PCR showed up-regulation of IP-10 mRNA expression in the lungs (P < 0.05) of MRL/lpr mice, in comparison with MRL/+ or B6 mice. The increase paralleled increased expression of a specific IP-10 receptor, CXCR3, and correlated with the degree of infiltration of mononuclear lymphocytes. In contrast, lung expression of TARC and its specific receptor, CCR4, were suppressed in MRL/lpr mice. Immunohistology showed that macrophage-like cells were the likely source of IP-10. Flow cytometric analyses revealed that the CXCR3-expressing cells were mainly infiltrating CD4 T cells and macrophages, which correlated with the degree of mononuclear lymphocyte infiltration. Recent data suggest that Th1 cells and Th1-derived cytokines play an important role in the development of SLE-like disease in MRL/lpr mice. Our results suggest that IP-10 expression in the lung is involved, through CXCR3, in the pathogenesis of pulmonary inflammation associated with migration of Th1 cells.
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PMID:Enhanced expression of interferon-inducible protein 10 associated with Th1 profiles of chemokine receptor in autoimmune pulmonary inflammation of MRL/lpr mice. 1497 41

The C terminus of the human immunodeficiency virus type 1 (HIV-1) accessory protein vpr acts in viral cell cycle arrest, nuclear localization, and apoptosis. Polymorphisms in this region are described in series of long-term nonprogression cases. We determined vpr sequences of archived baseline specimens from 96 participants in a historical trial of single- versus double-nucleoside reverse-transcriptase inhibitors. These sequences were then analyzed by study-entry and -outcome characteristics such as baseline absolute CD4(+) T cell count, prior treatment, CD4(+) T cell response, and clinical endpoints. Frequency of C-terminal mutations did not correlate to any measures of disease intensity. Changes in that portion of vpr did not attenuate disease.
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PMID:Alterations in the C-terminal region of the HIV-1 accessory gene vpr do not confer clinical advantage to subjects receiving nucleoside antiretroviral therapy. 1518 64

The aim of the present study was to evaluate the impact of highly active antiretroviral therapy (HAART) on HIV viral load of plasma and intraocular fluids in AIDS patients with ophthalmic opportunistic infections. We further compared the treatment effect of HAART on these patients. From June 1997 to July 2003, we examined and followed up the ophthalmic conditions of 49 patients receiving HAART with ophthalmic diseases during this period. The method of reverse-transcriptase polymerase chain reaction was used to detect and monitor HIV load in plasma and/or aqueous humor of AIDS patients. Before HAART, the HIV levels in the plasma and aqueous humor in 8 AIDS patients with ophthalmic opportunistic infections were significantly higher than those in 6 patients with HIV-related retinopathy (p < 0.05). Compared to the eye findings and clinical improvement, HIV loads of aqueous humor in 10 of 14 AIDS patients (6 with HIV-related retinopathy, 5 with cytomegalovirus retinitis, 2 with toxoplasmic retinitis and 1 with cryptococcal chorioretinitis) declined to undetectable levels (< 400 copies/ml) after 4-8 months of HAART. HIV virus levels in the plasma of AIDS patients were significantly decreased, and the CD4 counts of these patients were significantly increased (Wilcoxon test) after initiation of HAART.
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PMID:The HIV RNA Levels of Plasma and Ocular Fluids in AIDS Patients with Ophthalmic Infections. 1533 14

Intrathymic (IT) delivery of donor alloantigen is a potent strategy to induce operational tolerance. In this study we determined whether this effect was dependent on direct allorecognition of the tolerogen. Ten microgrammes of plasmid, encoding either the wildtype major histocompatibility complex (MHC) class I molecule K(b) or a truncated form in which the signal sequence for translocation into the endoplasmic reticulum was deleted, preventing cell surface expression and direct allorecognition of the tolerogen, was administered intrathymically to CBA.Ca (H2(k)) recipients. In addition, recipients were treated with anti-CD4 antibody (YTA3.1) at the time of IT injection and underwent transplantation 28 days later with a fully mismatched C57BL/10 (H2(b)) cardiac allograft. Wildtype, as well as truncated K(b) genes, were able to induce long-term survival of the cardiac allografts, in contrast to empty control plasmid. Reverse-transcriptase PCR showed expression of the K(b) genes for up to 28 days in thymus and spleen of pretreated recipients. These data show that direct allorecognition of the tolerogen was not required for the induction of long-term allograft survival following the introduction of plasmid-encoded MHC alloantigen into the thymus.
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PMID:Intrathymic delivery of plasmid-encoding endoplasmic reticulum signal-sequence-deleted MHC class I alloantigen can induce long-term allograft survival. 1537 45

The normal metabolism of mitochondria in T lymphocytes is unknown, as are the effects from nucleoside-analogue reverse-transcriptase inhibitors that impair mitochondrial polymerase- gamma . We isolated peripheral-blood CD4 and CD8 T lymphocytes from 6 healthy men and stimulated them with anti-CD3 and anti-CD28 antibodies, in the presence and in the absence of didanosine (ddI). In the absence of ddI, mitosis of T lymphocytes was paralleled by a transient up-regulation of both mtDNA and production of lactate. In CD4 lymphocytes, 10-day incubation with ddI at concentrations of 11.8 mu mol/L, 35.4 mu mol/L, 59.0 mu mol/L, and 118.0 mu mol/L induced (1) a concentration-dependent reduction of both mtDNA (to 73%, 29%, 24%, and 23%, respectively, of the levels in control samples) and subunit II of mtDNA-encoded cytochrome c oxidase (to 86%, 81%, 55%, and 31%, respectively, of the levels in control samples) and (2) a concentration-dependent increase in production of lactate (to 139%, 222%, 276%, and 312%, respectively, of the levels in control samples). Activation of lymphocytes (which was measured in terms of expression of CD25) was unaffected. Mitochondrial depolarization (assessed by staining with JC-1) was observed as early as day 7 of incubation. All changes were time dependent and also were observed in isolated CD8 lymphocytes. Electron microscopy revealed enlarged mitochondria with vacuoles, inclusions, and reduced electron density. ddI at a concentration of 11.8 mu mol/L induced changes that bordered statistical significance. After stimulation, there was a wide range in the change of mtDNA content in lymphocytes. Therefore, mtDNA measurements in blood are not necessarily a marker for the mitochondrial toxicity of ddI. Nevertheless, ddI does lead to depletion of mtDNA in lymphocytes and to functional impairment.
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PMID:Effects of of didanosine-related depletion of mtDNA in human T lymphocytes. 1571 58

Many viruses enter cells via an interaction of the viral envelope glycoprotein (Env) with receptor inducing fusion of viral and cellular membranes. These interactions are often evaluated in cell-cell fusion, gene-reporting systems with effector cells expressing Env and target cells expressing receptors. A common system utilizes vaccinia virus encoding T7 RNA polymerase (RNAP) in effector cells and a T7 promoted reporter plasmid in target cells. Fusion is quantified with expression of the reporter plasmid. However, direct activation of reporter plasmid from vaccinia virus can occur increasing background activity. We report here a modification of this assay in which T7 RNAP is expressed from a plasmid rather than vaccinia. This modification increased sensitivity with a ten-fold reduction in background. A novel dual T7/SP6 RNAP fusion assay was also developed to allow rapid screening for functional Env clones. Using these assays, we show that Envs from two CD4-independent HIV-2 isolates (VCP and ROD/B), which are able to fuse with chemokine receptor CXCR4 in a CD4-independent manner, are also able to fuse with alternative coreceptors GPR1 and GPR15 in the absence of CD4. The assay could also detect fusion of murine leukemia virus on target cells expressing the ecotropic MCAT-1 receptor showing its broad utility in other viral systems.
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PMID:Modification of a viral envelope glycoprotein cell-cell fusion assay by utilizing plasmid encoded bacteriophage RNA polymerase. 1594 97

A cell-free translation/glycosylation system derived from lepidopteran (Sf21) cells, which are widely used to express high yields of foreign active proteins that have post-translational modifications, was constructed. The insect cell extract was prepared using a Mini-Bomb cell disruption chamber by nitrogen pressure treatment, which stably retains translational and post-translational components. The gp120 mRNA was transcribed from the human immunodeficiency virus type-1 envelope glycoprotein gp120 gene with T7 RNA polymerase. When the gp120 mRNA was translated in the insect cell-free system, gp120 having a molecular mass of 100 kDa was detected by Western blot analysis. Synthesized gp120 and gp120 expressed in the intracellular fraction of recombinant-baculovirus-infected Sf21 cells had the same molecular mass, and they both had reduced mobility compared with gp120 secreted by recombinant baculovirus-infected Sf21 cells. In contrast, the 56-kDa gp120 protein, which corresponds to the polypeptide backbone of gp120, was synthesized in wheat germ and rabbit reticulocyte systems. The molecular mass of synthesized gp120 decreased from 100 kDa to 61 kDa after endoglycosidase H treatment, indicating that synthesized gp120 had been glycosylated with N-linked oligosaccharides. Furthermore, glycosylated gp120 was bound to human CD4 molecules expressed on the surface of quail cells. These results revealed that the insect cell-free system can synthesize gp120 that is folded in the proper conformation to provide a CD4-binding domain.
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PMID:A novel cell-free translation/glycosylation system prepared from insect cells. 1623

Runx1 binds the silencer and represses CD4 transcription in immature thymocytes. In this study, we found that Runx1 inhibits P-TEFb, which contains CycT1, CycT2, or CycK and Cdk9 and stimulates transcriptional elongation by RNA polymerase II (RNAPII) in eukaryotic cells. Indeed, its inhibitory domain, spanning positions 371 to 411, not only bound CycT1 but was required for silencing CD4 transcription in vivo. Our chromatin immunoprecipitation assays revealed that Runx1 inhibits the elongation but not initiation of transcription and that RNAPII is engaged at the CD4 promoter but is unable to elongate in CD4(-) CD8(+) thymoma cells. These results suggest that active repression by Runx1 occurs by blocking the elongation by RNAPII, which may contribute to CD4 silencing during T-cell development.
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PMID:Runx1 binds positive transcription elongation factor b and represses transcriptional elongation by RNA polymerase II: possible mechanism of CD4 silencing. 1631 94


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