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)

cDNA encoding the core protein of rat syndecan was cloned from a neonatal rat aortic cDNA library by polymerase chain reaction amplification. Expression of syndecan mRNA in rat aortic vascular smooth muscle (VSM) cells was demonstrated by reverse transcriptase-linked polymerase chain reaction amplification of syndecan sequences using total RNA from rat aortic VSM cells as templates. Polyclonal antibodies against rat syndecan core protein were produced by immunizing rabbits with a recombinant fusion protein containing a fragment of the extracellular domain. The anti-syndecan antibodies immunoprecipitated a large 35SO4-labeled molecule synthesized by cultured rat aortic VSM cells. The immunoprecipitated molecule was identified as a hybrid proteoglycan, based on results of alkaline, nitrous acid, and chondroitinase ABC digestions. On immunoblots the antibodies recognized a proteoglycan of greater than 200 kDa, with a core protein size after deglycosylation of approximately 50 kDa. The anti-syndecan antibodies stained cultured rat aortic VSM cells as well as tissue sections of neonatal and adult rat aortas in the medial, smooth muscle layer. On Northern blots of RNA isolated from cultured VSM cells, a syndecan cDNA probe hybridized to a major RNA species of 2.6 kilobases. Quantitative Northern blot analysis of total RNA isolated from VSM cells harvested at different cell densities revealed a decrease in syndecan mRNA levels with increased cell density. These results demonstrate the regulated synthesis of syndecan by rat VSM cells.
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PMID:Regulated expression of syndecan in vascular smooth muscle cells and cloning of rat syndecan core protein cDNA. 163 9

Proteoglycans metabolically labelled with [35S]sulphate and [3H]glucosamine or [3H]leucine were isolated from the incubation medium and cell layer of human adult mesangial cells and glomerular visceral epithelial cells using sequential DEAE chromatography purification steps followed by gel-filtration chromatography. The proteoglycan composition of each peak was analysed by treatment with HNO2, chondroitinase ABC or chondroitinase AC followed by chromatography on Sephadex G-50 columns. Heparan sulphate proteoglycan (HSPG) and dermatan sulphate proteoglycan were detected in both the culture medium and cell layer of mesangial cells. Culture medium of glomerular visceral epithelial cells contained HSPG and a second proteoglycan with the properties of a hybrid molecule containing HS and chondroitin sulphate (CS). The cell layer contained HSPG and CSPG. Detailed analysis of the hybrid molecule revealed that it had an apparent molecular mass of 400 kDa. SDS/PAGE of hybrid molecules, after treatment with heparitinase and chondroitinase ABC, revealed a core protein of 80 kDa. Using 1.8% polyacrylamide/0.6% agarose-gel electrophoresis, we deduced that the HS and CS were independently attached to one core protein. Because glomerular-basement-membrane HSPG is thought to be derived from mesangial cells and glomerular visceral epithelial cells and this molecule is involved in several kidney diseases, we investigated its synthesis in more detail. Anti-(rat glomerular-basement-membrane HSPG) monoclonal antibodies (JM403) and anti-(human glomerular-basement-membrane HSPG) polyclonal antibodies (both antibodies known to react with the large basement-membrane HSPG, perlecan) reacted strongly with HSPG obtained from both mesangial cells and glomerular visceral epithelial cells. However, the hybrid molecule did not react with these antibodies, suggesting that the HS side chain and the core protein were different from glomerular-basement-membrane HSPG. To quantify HS we performed an inhibition ELISA using mouse antibodies specific for glomerular-basement-membrane HS glycosaminoglycan side chains. Glomerular visceral epithelial cells produced significantly higher levels of HS (between 197.56 and 269.40 micrograms/72 h per 10(6) cells) than mesangial cells (between 29.8 and 45.5 micrograms/72 h per 10(6) cells) (three different cell lines; n = 3; P < 0.001). HS production by these cells was inhibited by cycloheximide, revealing that it was synthesized de novo. Expression of perlecan mRNA, demonstrated using reverse transcriptase PCR, was different in the two cell types. We conclude that glomerular visceral epithelial cells and mesangial cells have characteristic patterns of proteoglycan production. Glomerular visceral epithelial cells produced a hybrid proteoglycan containing CS and HS independently attached to its core protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proteoglycan production by human glomerular visceral epithelial cells and mesangial cells in vitro. 753 59

The role of cell-surface proteoglycans in human immunodeficiency virus (HIV) infection of T-cell lines was investigated. HIV-1-susceptible lymphoblastic T-cell lines, MT-4 and H9, were analyzed for proteoglycan synthesis and found to make heparan sulfate (HS) and chondroitin sulfate proteoglycans. Enzymatic treatment of these cells with heparitinase, but not chondroitinase, significantly prevented HIV-1(IIIB) infection as measured by inhibition of cytopathicity, reverse transcriptase production, and syncytia formation. Sulfation of glycosaminoglycans HS chains was critical to viral entry as shown by inhibition of viral infection with sodium chlorate and its specific reversal with exogenous sulfate addition. Quantitation of direct virus binding to cells showed that treatment of cells with heparitinase inhibited HIV-1 binding to the T-cell surface. Exogenous HS added to cultures inhibited virus infection in a manner analogous to dextran sulfate, further supporting a functional role for HS in HIV-1 binding. These results provide evidence for participation of cell-surface HS proteoglycans in HIV-cell attachment and virus entry.
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PMID:Cell-surface heparan sulfate proteoglycan mediates HIV-1 infection of T-cell lines. 809 45

Tissues must quickly recognize injury to respond to the rapid pace of microbial growth. In skin, dermal microvascular endothelial cells must also react to danger signals from the surrounding tissue and immediately participate by initiating the wound repair process. Components of the extracellular matrix such as hyaluronan are rapidly broken down into smaller molecular weight oligosaccharides in a wound, and these can activate a variety of biological processes. This study set out to determine if hyaluronan fragments released following injury can stimulate endothelial cells and what mechanism is responsible for this response. Using genechip microarray analysis, a response to hyaluronan fragments was detected in endothelial cells with the most significant increase observed for the chemokine IL-8. This observation was verified with qualitative reverse transcriptase-PCR and ELISA in human endothelial cell culture, and in a mouse model by observing serum levels of MIP-2 and KC following hyaluronan fragment administration in vivo. Activation was TLR4-dependent, as shown by use of TLR4 blocking antibody and TLR4-deficient mice, but not due to the presence of undetected contaminants as shown by inactivation following digestion with the hyaluronan-degrading enzyme chondroitinase ABC or incubation with the hyaluronan-specific blocking peptide Pep-1. Inactivation of LPS activity failed to diminish the action of hyaluronan fragments. These observations suggest that endogenous components of the extracellular matrix can stimulate endothelia to trigger recognition of injury in the initial stages of the wound defense and repair response.
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PMID:Hyaluronan fragments stimulate endothelial recognition of injury through TLR4. 1476 99

Cell surface proteoglycans play an important part in the functional and metabolic behaviour of leucocytes. We studied the expression of cell surface proteoglycans in human monocytes, in monocyte-derived immature and mature dendritic cells and in macrophages by metabolic labelling with [(35)S]-sulphate, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. Immature dendritic cells had the highest metabolic activity for the synthesis of cell surface proteoglycans. The major part of these proteoglycans was in phosphatidylinositol-anchored form and was released after treatment with phospholipase C. A minor part was released by trypsin. Digestion with chondroitinase ABC and mild HNO(2) treatment showed that cell surface proteoglycans had a higher proportion of chondroitin sulphate, both in the phospholipase C and trypsin fractions, suggesting that at least some glypicans contained chondroitin sulphate chains. RT-PCR detected the transcripts of glypicans 1, 3, 4 and 5 and all syndecans. Immature dendritic cells expressed a most complex spectrum of glypicans and syndecans, glypican-1 and syndecan-1 being expressed preferentially by this type of cells. Mature dendritic cells expressed glypican-3, which was not present in other lineages. These results suggest that different mononuclear cells synthesize cell surface proteoglycans actively with characteristic expression of different syndecans and glypicans genes, depending on the degree of cell differentiation and/or maturation.
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PMID:Cell surface proteoglycan expression during maturation of human monocytes-derived dendritic cells and macrophages. 1673 18