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)

Endothelin-1, a 21-amino acid peptide secreted by endothelial cells, has constrictor and mitogenic activity for vascular smooth muscle cells, and its mitogenic activity is synergistic with that of platelet-derived growth factor. Endothelial cell-derived endothelin-1 might therefore contribute to intimal hyperplasia in reendothelialized segments of vascular grafts or of endarterectomy and angioplasty sites. Because intimal hyperplasia occurs most often at sites with disordered flow patterns and lower fluid shear stress, we tested the effects of static culture versus high laminar shear stress (25 dyne/cm2) on endothelin-1 precursor (preproendothelin) gene mRNA transcript levels and endothelin-1 peptide release in cultured human endothelial cells. Primary cultures of human umbilical vein endothelial cells were subjected to controlled levels of shear stress in parallel plate flow chambers for 24 hours. To detect preproendothelin mRNA we applied a linked reverse transcriptase-polymerase chain reaction (RT/PCR) to RNA extracted from cultures. Southern blots of RT/PCR reaction products were hybridized with radioactive phosphorous (32P) labeled probes for the amplified preproendothelin complementary deoxyribonucleic acid (cDNA). Detection by RT/PCR of mRNA for glyceraldehyde 3-phosphate dehydrogenase was used to measure a constitutively expressed control signal. Endothelin-1 release into culture medium was measured by radioimmunoassay. Application of 25 dyne/cm2 of shear stress for 24 hours sharply reduced endothelial cell levels of precursor preproendothelin mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fluid flow decreases preproendothelin mRNA levels and suppresses endothelin-1 peptide release in cultured human endothelial cells. 206 49

Fluid shear stress can stimulate secretion of tissue plasminogen activator (tPA) by cultured human endothelial cells, while plasminogen activator inhibitor type-1 secretion remains unstimulated. To determine whether hemodynamically induced changes in tPA messenger RNA (mRNA) levels also occur, primary cultures from the same harvest of primary human umbilical vein endothelial cells were either maintained in stationary culture or exposed to arterial levels of shear stress (25 dynes/cm2) for 24 hours. Total cellular RNA was isolated from the shear stressed and stationary cultures and the relative levels of tPA mRNA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA were determined using a coupled reverse transcriptase/polymerase chain reaction method. As indicated by the amount of amplification product, tPA mRNA levels were many fold higher (greater than 10) in endothelial cells subjected to shear stress for 24 hours than in stationary controls. In contrast, mRNA levels for GAPDH were similar in control and shear stressed cells. The constancy of the measured GAPDH signal indicated that the tPA response was a selective effect of fluid shear stress. When a similar polymerase chain reaction method was used, the mRNA levels of basic fibroblast growth factor (bFGF) were found not to vary in comparison to GAPDH mRNA after 24 hours of shear stress. These results indicate that enhancement of the fibrinolytic potential of endothelial cells in response to hemodynamic forces could involve transcriptional events.
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PMID:Tissue plasminogen activator messenger RNA levels increase in cultured human endothelial cells exposed to laminar shear stress. 211 Jan 69

The effects of 3-methylcholanthrene (3-MC) and pyridine on rat renal cytochrome P450 (CYP) 1A1 and 1A2 mRNA expression have been examined by Northern-blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR), followed by Southern-blot analysis of the PCR products. Northern-blot hybridization and RT-PCR analyses of kidney poly(A)+ RNA revealed that 3-MC treatment produced a time-dependent increase in the renal CYP1A1 and 1A2 mRNA levels, with CYP1A1 and 1A2 mRNA levels maximally increased at 24 and 18 hr, respectively, after treatment. These data were confirmed via RT-PCR analysis using a subsaturating level of cDNA template and by Southern-blot analysis of the PCR products. This approach served as the foundation for examining the effects of pyridine on CYP1A1 and 1A2 expression in renal tissue. RT-PCR analysis of renal CYP1A1 and 1A2 poly(A)+ RNA levels after treatment with pyridine (200 mg/kg/day for 3 consecutive days) revealed that CYP1A1 mRNA levels were maximally elevated approximately 10-fold after pyridine treatment for 2 consecutive days, whereas CYP1A2 mRNA levels were maximally elevated approximately 3-fold at 24 hr after treatment. The mRNA levels of glyceraldehyde 3-phosphate dehydrogenase, which served as an internal control, remained constant after 3-MC or pyridine treatment. These results show that expression of CYP1A1 and 1A2 mRNAs is enhanced in renal tissue after exposure to 3-MC or pyridine, and that constitutive expression of CYP1A1 seems to be greater than that of CYP1A2 in renal tissue.
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PMID:3-Methylcholanthrene and pyridine effects on CYP1A1 and CYP1A2 expression in rat renal tissue. 749 48

To examine the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on thymic gene expression in vitro, freshly isolated rat thymocytes were incubated with 10 nM TCDD, and reverse transcriptase polymerase chain reaction experiments were performed using primers specific for prostaglandin G/H synthase (PGHS) and glyceraldehyde 3-phosphate dehydrogenase. TCDD selectively repressed PGHS gene expression, with maximal inhibition occurring within 60 min. Gel retardation assays demonstrated that dioxin transiently induced binding of the ubiquitous transcription factor NF kappa B to its cognate response element at early time points. However, TCDD had little ability to induce transformation of the Ah receptor to the xenobiotic responsive element in thymic cytosol. These results indicate that TCDD exerts changes in thymocyte gene expression prior to inducing toxicity.
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PMID:2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated gene expression in the immature rat thymus. 782 58

The reverse transcriptase polymerase chain reaction (RT-PCR) procedure is markedly inhibited in specimens of blood that contain commercial heparin as an anticoagulant or in cell preparations containing rat or mouse peritoneal mast cells. However, it was not known whether the levels of endogenous, mast cell-associated heparin that are present in some mammalian tissues are sufficient to interfere with the use of RT-PCR in these settings. We show that RT-PCR detects little or no mRNA transcripts for either mast cell-associated products, such as mouse mast cell-associated protease-2 or -4 (MMCP-2 or MMCP-4) or mast cell carboxypeptidase A, or for mast cell-nonspecific products, such as glyceraldehyde 3-phosphate dehydrogenase, in routinely prepared specimens of cells or tissues that include populations of heparin-containing mast cells. However, signals for mast cell-associated or mast cell-nonspecific transcripts can be readily detected in such specimens if they are treated with heparinase before RT-PCR. RT-PCR after heparinase treatment appears to represent an extremely sensitive method for detecting mast cell-associated transcripts in tissue specimens, permitting the identification of transcripts for mast cell-specific proteases in the skin of genetically mast cell-deficient WBB6F1-W/WV mice, a tissue that contains few or no mast cells according to histological analysis.
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PMID:Detection of mouse mast cell-associated protease mRNA. Heparinase treatment greatly improves RT-PCR of tissues containing mast cell heparin. 785 46

The amyloid precursor protein (APP), which is localized on both human chromosome 21 and its murine counterpart, chromosome 16 and which is involved in the formation of deposits in Alzheimer's disease, could be shown to bind effectively to a glytolytic enzyme: rat glyceraldehyde 3-phosphate dehydrogenase (Gapdh). We report here the isolation of a cDNA of murine Gapdh from mouse chromosome 16 (MMU16) originating from microclones of the distal part of MMU16 and the use of homologous genomic DNA sequences to further screen a cDNA phage library. The cDNA was sequenced, confirmed by polymerase chain reaction following reverse transcriptase (RT-PCR) and the open reading frame was expressed in vitro. The possible localization of Gapdh on MMU16--which may provide a mouse model for Down's syndrome and Alzheimer's disease--may lead to new insights into glycolysis and its role in the two syndromes.
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PMID:Isolation of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) cDNA from the distal half of mouse chromosome 16: further indication of a link between Alzheimer's disease and glycolysis. 789 98

Ferrochelatase (FC; heme synthetase, EC 4.99.1.1.) catalyses the synthesis of heme from protoporphyrin IX, the final step in the heme synthetic pathway. The hereditary deficiency of this enzyme gives rise to erythropoietic protoporphyria (EPP). We developed a rapid, non-radioactive means of measuring human FC mRNA levels in the EPP patients. It is based on the reverse transcriptase-polymerase chain reaction (RT-PCR) performed on the RNA obtained from peripheral blood. The amplified DNA was detected by agarose gel electrophoresis with ethidium bromide staining and the fluorescent intensity was measured by scanning densitometry applied directly to Polaroid 665 negative film. The relative expression level of FC mRNA, compared with that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, was estimated at several points in the exponential phase of PCR cycles or at a point in the exponential phase of PCR performed on serially diluted the cDNA samples. The estimate of the FC mRNA by this method correlated well with the level of the FC mRNA measured by Northern blotting in the EB virus-transformed lymphocytes of the same patients. The level of the FC mRNA appeared to vary among the patients in whom a decreased level of enzymatic activity was indicated.
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PMID:Quantitative analysis of ferrochelatase mRNA in blood cells of erythropoietic protoporphyria patients. 886 37

The purpose of this study was to develop a method by which endothelial cell nitric oxide synthase (ecNOS) mRNA expression could be measured in single coronary resistance arteries and to test the hypothesis that ecNOS gene expression is upregulated by exercise training. Yucatan miniature swine were randomly assigned to exercise-trained (ET; n = 5) or sedentary (Sed; n = 4) groups for 16 wk. Individual coronary resistance arteries (50-100 microns) were dissected, frozen in liquid nitrogen, and homogenized in a LiCl buffer, mRNA was isolated from each vessel, and ecNOS gene expression was assessed using reverse transcriptase (RT)-polymerase chain reaction (PCR) standardized by coamplifying ecNOS with glyceraldehyde 3-phosphate dehydrogenase (GAPHD). The ecNOS-to-GAPDH amplicon ratio was significantly greater in coronary resistance arteries isolated from ET pigs than in Sed controls. On the basis of these data, it is concluded that RT-PCR can be used on single coronary resistance arteries to assess cell-specific mRNA expression and that ecNOS gene expression is upregulated by exercise training in porcine coronary resistance arteries.
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PMID:Induction of nitric oxide synthase mRNA in coronary resistance arteries isolated from exercise-trained pigs. 943 89

The type XVII collagen alpha 1 chain has been identified as a component of the type I hemidesmosome, and is thus thought to play a role in extracellular matrix (ECM) maintenance and signal transduction between the cell and the ECM. We examined the expression of type XVII collagen alpha 1 chain mRNA in the mouse heart by Northern blot analysis and determined the sequential changes of its expression in different developmental stages of the heart using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Northern blotting: Total RNA was extracted from 10 adult mouse hearts by the guanidine/cesium method. Hybridization was performed with mouse cDNA for alpha 1 (XVII) collagen. RT-PCR: Total RNA was extracted from 7 embryos, 4 neonates and 8 adult mice. Reverse transcription was performed using oligo-dT primer and MMLV. Amplification was carried out in alpha 1 (XVII) collagen and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH served as an internal control. Northern blotting revealed a 5.6 kb signal that was identical to that of the alpha 1 (XVII) of skin and transformed keratinocyte reported previously. The sequences of the PCR products were also identical to those reported. The normalized expression ratios of alpha 1 (XVII) were 0.91 +/- 0.20 in the embryonic heart, 0.36 +/- 0.20 in the neonatal heart and 0.96 +/- 0.21 in the adult heart. In conclusion, we identified the expression of type XVII collagen alpha 1 chain mRNA in the mouse heart, suggesting that the type I hemidesmosome is located in the heart. The results of the RT-PCR at different developmental stages of the heart suggest that type XVII collagen contributes not only to cardiogenesis in the embryonic stage but also to maintenance of architecture and function in the adult heart.
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PMID:Expression of type XVII collagen alpha 1 chain mRNA in the mouse heart. 968 29

There is considerable interest in determining the role of prothrombin fragments, especially urinary prothrombin fragment 1 (UPTF1), in the pathogenesis of calcium oxalate (CaOx) urinary calculi. This fragment is present in abundance in the matrix of CaOx crystals generated in human urine in vitro and has also been detected in human urinary stones containing calcium. More recently, prothrombin gene expression has been reported in the human kidney. However, studies examining the renal biosynthesis of prothrombin or perhaps only its fragments during experimental lithogenesis, and in consequence, the role of UPTF1 in stone formation, cannot be carried out in humans. The aim of this investigation therefore was to determine whether prothrombin gene expression is present in the rat kidney. Total RNA was isolated from the kidneys and livers of 12 rats. Using reverse transcriptase PCR, mRNAs corresponding to the thrombin and fragment 1 + 2 (F1+2) regions of prothrombin were analysed by agarose gel electrophoresis. The expression of glyceraldehyde 3-phosphate dehydrogenase was also examined to determine whether the quality of the tissue mRNAs was adequate for analyses. The amplified products were identified by sequence analysis. All kidneys displayed evidence of expression of the thrombin and F1+2 domains of the prothrombin gene. Furthermore, the sequences of these PCR-derived products from kidney were identical to those from liver. This suggests that the prothrombins secreted by these two organs are identical. The fact that prothrombin biosynthesis occurs in both the human and rat kidney presents an opportunity for using established rat models of stone disease to evaluate the influence of lithogenic conditions on prothrombin gene expression, and the potential role of UPTF1 in vivo.
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PMID:The prothrombin gene is expressed in the rat kidney: Implications for urolithiasis research. 1060 51


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