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)

The CD8(+)-T-cell response to human immunodeficiency virus type 1 (HIV-1) is considered to be important in host control of infection and prevention of AIDS. We have developed a single-cell enzyme immunoassay (enzyme-linked immunospot assay) specific for gamma interferon (IFN-gamma) production stimulated by either autologous B-lymphoblastoid cell lines (B-LCL) infected with vaccinia virus vectors expressing HIV-1 proteins or synthetic peptides representing known HIV-1 CD8(+) cytotoxic T-lymphocyte (CTL) epitopes. Single-cell IFN-gamma production stimulated by HIV-1 Gag-, Pol-, and Env-expressing B-LCL was a reliable measure of HIV-1-specific T-cell immunity in peripheral blood CD8(+) T cells from HIV-1 infected individuals. This method was more sensitive than stimulation of IFN-gamma by direct infection of the cultures with HIV-1-vaccinia virus vectors. Comparable results were found for IFN-gamma production in CD8(+) T cells from HIV-1-negative, cytomegalovirus (CMV)-seropositive, healthy donors stimulated with B-LCL expressing the CMV pp65 lower matrix protein. HIV-1 peptides were immunodominant for both CD8(+) single-cell IFN-gamma production and CTL precursor frequencies. The number of cells producing IFN-gamma decreased in individuals with late-stage HIV-1 infection and was temporally enhanced during combination antiretroviral therapy with two reverse transcriptase nucleoside inhibitors and a protease inhibitor.
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PMID:CD8(+) T-cell gamma interferon production specific for human immunodeficiency virus type 1 (HIV-1) in HIV-1-infected subjects. 1070 5

The long-term efficacy of combination antiretroviral therapy may relate to augmentation of anti-human immunodeficiency virus type 1 (HIV-1) CD8(+) T-cell responses. We found that prolonged treatment of late-stage HIV-1-infected patients with a protease inhibitor and two nucleoside reverse transcriptase inhibitors failed to restore sustained, high levels of HIV-1-specific, HLA class I-restricted, cytotoxic-T-lymphocyte precursors and gamma interferon (IFN-gamma) production by CD8(+) T cells. In some patients, particularly those initiating three-drug combination therapy simultaneously rather than sequentially, there were early, transient increases in the frequency of anti-HIV-1 CD8(+) T cells that correlated with decreases in HIV-1 RNA and increases in T-cell counts. In the other patients, HIV-1-specific T-cell functions either failed to increase or declined from baseline during triple-drug therapy, even though some of these patients showed suppression of plasma HIV-1 RNA. These effects of combination therapy were not unique to HIV-1 specific T-cell responses, since similar effects were noted for CD8(+) T cells specific for the cytomegalovirus pp65 matrix protein. The level and breadth of CD8(+) cell reactivity to HLA A*02 HIV-1 epitopes, as determined by IFN-gamma production and HLA tetramer staining after combination therapy, were related to the corresponding responses prior to treatment. There was, however, a stable, residual population of potentially immunocompetent HIV-1-specific T cells remaining after therapy, as shown by tetramer staining of CD8(+) CD45RO(+) cells. These results indicate that new strategies will be needed to target residual, immunocompetent HIV-1-specific CD8(+) T cells to enhance the effectiveness of antiretroviral therapy in patients with advanced immunodeficiency.
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PMID:Anti-human immunodeficiency virus type 1 (HIV-1) CD8(+) T-lymphocyte reactivity during combination antiretroviral therapy in HIV-1-infected patients with advanced immunodeficiency. 1075 25

c-src deletion in mice leads to osteopetrosis as a result of reduced bone resorption due to an alteration of the osteoclast. We report that deletion/reduction of Src expression enhances osteoblast differentiation and bone formation, contributing to the increase in bone mass. Bone histomorphometry showed that bone formation was increased in Src null compared with wild-type mice. In vitro, alkaline phosphatase (ALP) activity and nodule mineralization were increased in primary calvarial cells and in SV40-immortalized osteoblasts from Src(-/-) relative to Src(+/+) mice. Src-antisense oligodeoxynucleotides (AS-src) reduced Src levels by approximately 60% and caused a similar increase in ALP activity and nodule mineralization in primary osteoblasts in vitro. Reduction in cell proliferation was observed in primary and immortalized Src(-/-) osteoblasts and in normal osteoblasts incubated with the AS-src. Semiquantitative reverse transcriptase-PCR revealed upregulation of ALP, Osf2/Cbfa1 transcription factor, PTH/PTHrP receptor, osteocalcin, and pro-alpha 2(I) collagen in Src-deficient osteoblasts. The expression of the bone matrix protein osteopontin remained unchanged. Based on these results, we conclude that the reduction of Src expression not only inhibits bone resorption, but also stimulates osteoblast differentiation and bone formation, suggesting that the osteogenic cells may contribute to the development of the osteopetrotic phenotype in Src-deficient mice.
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PMID:Decreased c-Src expression enhances osteoblast differentiation and bone formation. 1103 78

Long-term in vivo studies have highlighted smoking as a risk factor in postmenopausal osteoporosis, bone fracture incidence, and increased nonunion rates. In contrast, there are few data postulating the effects of smoking at the cellular level in human skeletal tissue. In this study, we present novel evidence demonstrating that the nicotinic receptor alpha4 subunit is present in human primary bone cells by using reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, we demonstrate direct cellular effects of nicotine on primary human bone cells and blockage of these effects with a nicotinic receptor antagonist, D-tubocurarine. Nicotine effects on cell proliferation were biphasic with toxic, antiproliferative effects at high levels of nicotine (>1 mmol/L) and stimulatory effects at very low levels (0.01-10 micromol/L) after 72 h. This nicotine-induced increase in cell proliferation was inhibited in a dose-dependent manner by the addition of D-tubocurarine. In addition, proliferation effects from low-level treatment correlated with an upregulation of expression of the AP-1 transcription factor, c-fos, within 1 h, which was blocked by incubation with D-tubocurarine. To determine in situ bone cell responses within their trabecular matrix, cores of human bone isolated from biopsies were perfused with 0.1 micromol/L nicotine for 24 h. Western analysis of proteins isolated from the cores highlighted an increase in osteopontin, a bone matrix protein implicated in regulating resorption, which was partially inhibited by the addition of D-tubocurarine. To conclude, our results suggest that nicotine has a direct effect on human bone cells in modulating proliferation, upregulation of the c-fos transcription factor, and the synthesis of the bone matrix protein, osteopontin.
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PMID:Nicotinic regulation of c-fos and osteopontin expression in human-derived osteoblast-like cells and human trabecular bone organ culture. 1142 48

Some dental implants are coated with hydroxyapatite (HA), which preferentially binds to bone. Several matrix proteins have an arginine-glycine-aspartic acid (RGD) sequence where cells attach via an integrin receptor. We hypothesized that coating an HA surface with an RGD-containing peptide might enhance the attachment and differentiation of osteoblasts. The HA disks (diameter 34 mm, thickness 1 mm) were treated with a solution (50 mM Tris/HCl and 150 mM NaCl, pH 7.4) containing the peptide EEEEEEEPRGDT, in which the E repetition exerts a high affinity to HA. After washing with phosphate-buffered saline, KUSA/A1 mouse osteoblastic cells were inoculated onto the HA surface and cultured. After 30 min, the number of cells attached to the surface was counted. The DNA content and alkaline phosphatase (ALP) activity were measured after 10 days in culture. Expression of bone matrix proteins was also examined by means of reverse transcriptase-polymerase chain reaction at 7 days; the mineralized area of the culture was also evaluated by staining with Alizarin Red S after 10 days. Treatment with the peptide stimulated cell attachment and increased DNA content and ALP activity. Furthermore, matrix protein expression and mineralized nodule formation were enhanced to a greater extent on the peptide-treated surface than on the nontreated surface. Our results indicate that coating an HA surface with RGD-containing peptide enhances osteoblast attachment and differentiation. This peptide treatment of HA-coated implants may stimulate the osseointegration of the implants.
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PMID:Enhancement of osteogenesis on hydroxyapatite surface coated with synthetic peptide (EEEEEEEPRGDT) in vitro. 1220 50

This study investigated the effects of mineral trioxide aggregate on cementoblast growth and osteocalcin production in tissue culture. For cellular morphology studies, cementoblasts on mineral trioxide aggregate, IRM, and amalgam were incubated for 48 h then fixed for scanning electron microscopic analysis. For gene expression on mineral trioxide aggregate and IRM, reverse transcriptase polymerase chain reaction was performed using primer sets for glyceraldehyde-3-phosphate dehydrogenase, type I collagen, alkaline phosphatase, osteocalcin, and bone sialoprotein after 3 and 5 days. In vitro matrix protein expression was evaluated by confocal microscopy for the presence of osteocalcin on MTA after 7 and 12 days. Images were compared with controls to assess qualitative differences. Results suggest that mineral trioxide aggregate permits cementoblast attachment and growth and the production of mineralized matrix gene and protein expression. Our data indicates that mineral trioxide aggregate can be considered cementoconductive.
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PMID:Cementoblasts maintain expression of osteocalcin in the presence of mineral trioxide aggregate. 1281 26

Collagen X is produced by hypertrophic cartilage undergoing endochondral ossification. Transgenic mice expressing defective collagen X under the control of 4.7- or 1.6-kb chicken collagen X regulatory sequences yielded skeleto-hematopoietic defects (Jacenko O, LuValle P, Olsen BR: Spondylometaphyseal dysplasia in mice carrying a dominant-negative mutation in a matrix protein specific for cartilage-to-bone transition. Nature 1993, 365:56-61; Jacenko O, Chan D, Franklin A, Ito S, Underhill CB, Bateman JF, Campbell MR: A dominant interference collagen X mutation disrupts hypertrophic chondrocyte pericellular matrix and glycosaminoglycan and proteoglycan distribution in transgenic mice. Am J Pathol 2001, 159:2257-2269; Jacenko O, Roberts DW, Campbell MR, McManus PM, Gress CJ, Tao Z: Linking hematopoiesis to endochondral ossification through analysis of mice transgenic for collagen X. Am J Pathol 2002, 160:2019-2034). Current data indicate that the hematopoietic abnormalities do not result from extraskeletal expression of endogenous collagen X or the transgene. Organs from mice carrying either promoter were screened by immunohistochemistry, in situ hybridization, and Northern blot; transgene and mouse collagen X proteins and messages were detected only in hypertrophic cartilage. Likewise, reverse transcriptase-polymerase chain reaction revealed both transgene and mouse collagen X amplicons only in the endochondral skeleton of mice with the 4.7-kb promoter; however, in mice with the 1.6-kb promoter, multiple organs were transgene-positive. Collagen X and transgene amplicons were also detected in marrow, but likely resulted from contaminating trabecular bone; this was supported by reverse transcriptase-polymerase chain reaction analysis of rat tibial zones free of trabeculae. Our data demonstrate that in mice, the 4.7-kb chicken collagen X promoter restricts transcription temporo-spatially to that of endogenous collagen X, and imply that murine skeleto-hematopoietic defects result from transgene co-expression with collagen X. Moreover, the 4.7-kb hypertrophic cartilage-specific promoter could be used for targeting transgenes to this tissue site in mice.
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PMID:Chicken collagen X regulatory sequences restrict transgene expression to hypertrophic cartilage in mice. 1474 55

The complete nucleotide sequence of the gene encoding the matrix protein (M) of the avian paramyxovirus, serotype 3b (APMV-3b), has been determined by means of the direct sequencing of viral RNA using reverse transcriptase reaction. The adjacent portions of the neighboring phosphoprotein (P) and fusion (F) protein genes were also sequenced that permitted to determine the consensus sequence of the viral genome, the poly(A) tract, downstream and upstream non-coding portions of the P and F genes, respectively, as well as the corresponding intergenic regions. The gene is 1478 nucleotides long with a protein-coding sequence of 1194 nucleotides. The deduced protein consists of 398 amino acids with a calculated MW 44,465. According to the multalignment and phylogenetic analyses, the APMV-3b M protein has shown the closest relatedness towards Newcastle disease virus (NDV) which has recently been suggested to be excluded from the Rubulavirus genus and assigned (together with APMV-6) to a new Avulavirus genus within the subfamily Paramyxovirinae of the Paramyxoviridae family. On the basis of the M protein genetic multalignment, phylogenetic relationships, bipartite nuclear localization signal identification in combination with the cysteine residues distribution, and by the degree of intrageneric heterogeneity, the APMV-3b is proposed to be another member (together with NDV and APMV-6) of the new genus.
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PMID:Nucleotide sequence of the matrix protein gene of avian paramyxovirus, serotype 3b: evidence on another member of the suggested new genus of the subfamily Paramyxovirinae. 1556 52

Although recombinant retroviruses are widely used in gene therapy and as gene transfer vehicles for basic biological studies, their titers are very low as compared to other recombinant viral systems, e.g., adenovirus. We investigated the rate-limiting steps in production of LacZ-encoding ecotropic (CRE BAG 2) and amphotropic (Psi-CRIP) retrovirus. We found that ecotropic retrovirus producer cells produced a large number of inactive viral particles because they were severely limited by the amount of mRNA that was packaged into viral capsids. Introduction of the gene for green fluorescence protein (GFP) increased retroviral titers 40-fold, without affecting the viral matrix protein, p30, or the activity of reverse transcriptase. Surprisingly, while transfer of GFP gene increased retrovirus production, beta-gal activity and X-gal titer decreased significantly. Quantitative real-time polymerase chain reaction (PCR) showed that although producer cells synthesized similar amounts of both mRNAs, retroviral supernatants contained significantly lower amount of LacZ mRNA, possibly due to competition between LacZ and GFP mRNAs for encapsidation into virions. In contrast to ecotropic producers, introduction of GFP gene copies into amphotropic producers resulted in a moderate twofold increase in retrovirus production. However, delivery of genes encoding for the viral proteins gp70 and p30 increased virus production by fivefold, suggesting that amphotropic producers may also be limited by synthesis of structural viral proteins. Our data show that in addition to the amount of viral genome or proteins, assembly of viral components into active viral particles may limit production of high titer retroviral preparations.
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PMID:Stoichiometric limitations in assembly of active recombinant retrovirus. 1581 99

Bovine bone marrow mesenchymal stem cells (MSCs) cultured in condensate culture, spontaneous and independent for any external biostimulants, undergo chondrogenic differentiation. In the present study, the bovine MSC chondrogenesis pathway was studied by analyzing stage-specific gene expression using quantitative "Real Time" reverse transcriptase polymerase chain reaction (qRT-PCR). Results showed that bovine MSCs underwent complete chondrogenesis; the initial stage was characterized by expression. of sox9 messenger ribonucleic acid (mRNA), followed by high transcription of chondrocyte specific genes, collagen type II and IX, biglycan and cartilage oligomeric matrix protein, and the final prehypertrophic and/or hypertrophic stage was distinguished by increased expression of collagen type X. From day 7 to day 14 of differentiation increased mRNA expression of the transforming growth factors beta1 and beta2, basic fibroblast growth factor (FGF 2), bone morphogenic protein 6 (BMP 6), insulin-like growth factors 1, parathyroid hormone related peptide and indian hedgehog (Ihh) were detected. These results suggest that these well know chondrogenic growth factors may play a role in bovine chondrogenesis in autocrine and/or paracrine manner. On day 21 of the culture, FGF 2, BMP 6 and Ihh were highly expressed, compared to cells cultured in monolayer manner, which suggests a possible function in maintaining the terminal stage of differentiation. This data extends our knowledge about the unusual species-specific bovine MSC chondrogenesis, allowing us to define the phenotype of the differentiated cells. Furthermore, this study contributes to our in understanding of known chondrogenic-growth factors in autocrine and/or paracrine manner playing a role in the spontaneous differentiation.
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PMID:Gene expression profile of bovine bone marrow mesenchymal stem cell during spontaneous chondrogenic differentiation in pellet culture system. 1654 33


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