Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this study, we present a new method to obtain pure, viable, freshly isolated hepatic stellate cells. Stellate cells were purified by cell sorting using their high side scatter (SSC) of incident light. Purity of the cells was established by light and transmission electron microscopy (TEM). Starting from stellate cells that were 50% to 70% enriched by centrifugation in 11% Nycodenz, the cell purity after sorting was found to be 96.6% +/- 2.9%. Viability of the sorted cells was 90.8% +/- 2.2% as measured by the Trypan blue exclusion test and was confirmed by cell culturing. Per hour of sorting, 1.4 +/- 0.4 million stellate cells were obtained. Sorting runs of up to 4 hours were practically feasible, resulting in yields of 5 to 6 million cells per rat liver. Cells attached to plastic substratum within 24 hours. Subsequently, they spread and underwent spontaneous transition into myofibroblast-like cells. The purity of sorted cells was documented by reverse-transcriptase polymerase chain reaction (RT-PCR) experiments using specific primer pairs for messenger RNA (mRNA) species that were only present in parenchymal (preproalbumin), endothelial (endothelial cell nitric oxide synthase [eNOS]), stellate (desmin), or Kupffer cells (77- to 88-kd fucose receptor). Contaminating mRNA species were absent in sorted stellate cells. Next, we examined freshly sorted stellate cells by Western blotting to confirm the presence of relevant cytoskeletal proteins. Cells were positive for vimentin, desmin, and glial fibrillary acidic protein (GFAP), but negative for alpha-smooth muscle actin (alpha-SMA). Sorted and cultured cells were immunophenotyped for the presence of collagen types I, III, and IV, laminin, and the cytoskeletal proteins, alpha-SMA, desmin, vimentin, and GFAP. At 90 hours in culture, cells expressed all the investigated extracellular matrix proteins. Desmin was present in 82% +/- 1%, vimentin in 96% +/- 2.5%, and GFAP in 91% +/- 4.5% of cells. Alpha-SMA was present in 91% +/- 2% of cultured cells. We conclude that cell sorting based on SSC of incident light is a convenient method to obtain virtually pure stellate cells that can be used for direct analysis or for culturing. Although the yields obtained with this method are lower than with standard methods, and additional equipment is required, SSC-activated sorting offers the possibility of very pure cells when essential for analyses based on sensitive detection methods such as RT-PCR.
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PMID:Purification of rat hepatic stellate cells by side scatter-activated cell sorting. 946 62

Cholecyst-derived extracellular matrix (CEM) is a novel, proteinaceous biomaterial, derived from the porcine cholecyst, which may have potential applications as a scaffold in the area of heart valve tissue engineering. In this study the potential of CEM to support the proliferation of valvular endothelial cells (VECs) and valvular interstitial cells (VICs), while maintaining their phenotypic mRNA synthesis, protein expression and morphology was assessed by biochemical assays, electron microscopy, immunostaining and reverse-transcriptase polymerase chain reaction. VICs and VECs were isolated from the porcine aortic valve and techniques were developed for the isolation of CEM for cell culture. VECs and VICs cultured on CEM adhered and proliferated, maintaining their phenotypic morphology. VECs synthesised von Willebrand factor mRNA and endothelial nitric oxide synthase (eNOS) mRNA and expressed eNOS and VICs synthesised alpha-smooth muscle actin (alphaSMA) mRNA and expressed alphaSMA. Cellular area fraction of VICs expressing alphaSMA was 87.7+/-6.8% and cellular area fraction of VECs expressing eNOS was 93.8+/-9.3%. Findings of this study support the hypothesis that CEM is a potential biomaterial for tissue engineered heart valve scaffold design.
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PMID:The effect of cholecyst-derived extracellular matrix on the phenotypic behaviour of valvular endothelial and valvular interstitial cells. 1717 91

Bone marrow stromal cells (BMSCs) or mesenchymal stem cells (MSCs) are a heterogeneous population of cells that are multipotent. When rat BMSCs were seeded onto a 3-dimensional (3-D) tubular scaffold engineered from aligned type I collagen strands and cultured in osteogenic medium, they simultaneously matured and differentiated into osteoblastic and vascular cell lineages. In addition, these osteoblasts produced mineralized matricellular deposits. BMSCs were seeded at a density of 2 x 10(6) cells/15 mm tube and cultured in basal or osteogenic medium for 3, 6, and 9 days. These cells were subsequently processed for real-time reverse-transcriptase polymerase chain reaction (RT-qPCR), immunohistochemical, cytochemical, and biochemical analyses. Immunolocalization of lineage-specific proteins was visualized using confocal microscopy. In the present study, the expression pattern of key osteogenic markers significantly differed in response to basal and osteogenic media. Alkaline phosphatase activity and calcium content increased significantly over the observed period of time in osteogenic medium. The observed up-regulation of transcripts coding for osteoblastic phenotypic markers is reminiscent of in vivo expression patterns. Abundant sheets of Pecam (CD31) -, Flk-1 (vascular endothelial growth factor receptor-2) -, CD34-, tomato lectin-, and alpha-smooth muscle actin-positive cells were observed in these tube cultures. Moreover, nascent capillary-like vessels were also seen amid the osteoblasts in osteogenic cultures. Our 3-D culture system augmented the maturation and differentiation of BMSCs into osteoblasts. Thus, our in vitro model provides an excellent opportunity to study the concurrent temporal and spatial regulation of osteogenesis and vasculogenesis during bone development.
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PMID:A three-dimensional tubular scaffold that modulates the osteogenic and vasculogenic differentiation of rat bone marrow stromal cells. 1835 28

Thiazolidinediones (TZDs), synthetic peroxisome proliferator-activated receptor (PPAR)-gamma ligands, have a central role in insulin sensitization and adipogenesis. It has been reported that TZDs exert protective effects in both diabetic and nondiabetic models of renal disease, although the exact mechanism is not well understood. In particular, only a few studies have reported the renoprotective effects of TZDs in nondiabetic models of tubulointerstitial fibrosis and inflammation. Therefore, we investigated the anti-fibrotic and anti-inflammatory effects of the TZD troglitazone in the mouse model of unilateral ureteral obstruction (UUO). C57BL/6J mice underwent UUO and were studied after 3 and 7 days. Animals were divided into three groups and received control vehicle, troglitazone (150 mg/kg per day) or troglitazone (300 mg/kg per day) by gavage. Kidneys were harvested for morphological, mRNA and protein analysis. Reverse-transcriptase-PCR was used to assess the expression of transforming growth factor-beta1 (TGF-beta1) and the TGF-beta1 type I receptor (TGF beta R-I). Protein expression was assessed by western blotting (TGF beta R-I) and immunostaining (TGF beta R-I, alpha-smooth muscle actin (alpha-SMA), type I collagen (collagen I), F4/80, and proliferating cell nuclear antigen (PCNA)). The expression of alpha-SMA, collagen I, and F4/80 was decreased in mice treated with troglitazone compared with the control group. The numbers of PCNA-positive interstitial cells were decreased in mice treated with troglitazone. TGF-beta1 mRNA and TGF beta R-I mRNA and protein expression were decreased in the group treated with troglitazone compared with the control group. The beneficial effects of troglitazone treatment were also dose dependent. PPAR-gamma agonist significantly reduced TGF-beta and attenuated renal interstitial fibrosis and inflammation in the model of UUO.
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PMID:PPAR-gamma agonist attenuates renal interstitial fibrosis and inflammation through reduction of TGF-beta. 1900 5

Several studies have shown that hepatocyte growth factor (HGF) ameliorates renal interstitial fibrosis, but the mechanism is not fully clear. This study was designed to examine whether HGF can relieve renal interstitial injury in 5/6 nephrectomized rats, and to confirm whether this function was associated with decrease in alpha-smooth muscle actin (alpha-SMA) and transforming growth factor-betal (TGF-beta1) expression. The animals were randomized into 8 groups comprising 6 animals (n = 6) each: control (group I), PCI-neo (group II, 900 microg), sham-operation (group III, not nephrectomy), model or 5/6 nephrectomy group (group IV), lotensin group (an angiotensin converting enzyme inhibitor, group V, 0.6 mg/100 g/day for 5 weeks), low-dose PCI-neo-HGF group (group VI, 690 microg), high-dose PCI-neo-HGF group (group VII, 1380 microg) and lotensin + high-dose PCI-neo-HGF group (group VIII, 0.6 mg/100 g/day for 5 weeks, 1380 microg). The animals were sacrificed in the 5th week after 5/6 nephrectomy. The specimens of kidneys were used for pathological examination (hematoxylin-eosin staining), detection of alpha-SMA and TGF-beta1 mRNA (Reverse transcriptase-polymerase chain reaction) and protein (Western blot and immunohistochemistry) expression. The results showed that in 5/6 nephrectomized rats blood urea nitrogen (BUN), serum creatinine (CRE) and 24 h urinary albumin excretion (UAE) were increased, renal interstitium was injured seriously and alpha-SMA, TGF-beta1 mRNA and protein expression were elevated compared with those of control. The above changes were ameliorated and alpha-SMA and TGF-beta 1 expression was reduced by both PCI-neo-HGF and lotensin. The lotensin + high-dose PCI-neo-HGF group rats exhibited the most significant therapeutic effect both in decreasing the BUN, CRE and 24 h UAE and in relieving renal interstitial injury. In conclusion, the study demonstrated that HGF can relieve renal interstitial injury and this protection was associated with down-regulation of a-SMA and TGF-beta 1 expressions.
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PMID:Hepatocyte growth factor-induced amelioration in renal interstitial fibrosis is associated with reduced expression of alpha-smooth muscle actin and transforming growth factor-beta1. 2216 88