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 fluid movements that arise during blastocyst formation (cavitation) are, at least in part, driven by the Na/K-ATPase. In this study, the reverse transcriptase-polymerase chain reaction (RT-PCR) was used to survey bovine pre-attachment embryos for transcripts encoding known isoforms of the Na/K-ATPase alpha- and beta-subunits, including isoforms not previously detected during the first week of mammalian development. Transcripts encoding the Na-K-ATPase alpha 1, alpha 2, alpha 3 and beta 2 isoforms were detected throughout bovine preattachment development. This is the first indication that alpha 2, alpha 3 and beta 2 mRNAs are expressed during this early developmental interval. As in the mouse, beta 1-subunit transcripts were not detected until the morula stage and were also present in blastocysts. Thus, in two mammalian species an increase in abundance of beta 1 isoform transcripts in the morula stage is coincident with the onset of cavitation. Transcripts encoding the recently characterized alpha 4 isoform were not detected. The sensitivity of bovine blastocysts to ouabain (a potent inhibitor of Na/K-ATPase) was determined by assessing the ability of bovine blastocysts to recover in ouabain supplemental culture medium following cytochalasin-induced blastocyst collapse. Re-expansion of bovine blastocysts was inhibited in all ouabain concentrations down to 10(-9) M. Mouse blastocysts, in contrast, were sensitive to ouabain at or above 10(-3)M. These results have established that transcripts encoding multiple isoforms of both the alpha and beta subunits of the Na/K-ATPase are expressed throughout early bovine development and that bovine blastocysts display a greater sensitivity to ouabain than murine blastocysts. Future analysis will determine the possible individual and collective roles of these isoforms during blastocyst formation.
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PMID:Ouabain sensitivity and expression of Na/K-ATPase alpha- and beta-subunit isoform genes during bovine early development. 902 43

Proximal tubular epithelial cells are the most abundant cells in the renal cortex, and recent studies suggest that they may play an important role in initiating pathological changes in renal disease. Transforming growth factor (TGF)-beta 1 has been implicated as a major factor controlling the development and progression of renal fibrosis in numerous diseases, including diabetic nephropathy. We have recently demonstrated that human proximal tubular epithelial cells synthesize and secrete TGF-beta 1 after the sequential addition of both 25 mmol/L D-glucose and platelet-derived growth factor (PDGF). The present study examines the control of this synthesis and in particular the polar requirements of the stimulation and the direction of release of the protein. A proximal tubular cell line (LLC-PK1) was cultured on porous tissue culture inserts. Confluent cells were exposed to 25 mmol/L D-glucose on either their apical or basolateral aspect. TGF-beta 1 mRNA induction (reverse transcriptase polymerase chain reaction) occurred only after basolateral exposure. Similarly, TGF-beta 1 synthesis and secretion was induced only by the subsequent addition of PDGF to the basolateral aspect of the cells. In contrast, TGF-beta 1 protein secretion was detected equally in the apical and basolateral compartments. This effect was maximal after 12-hour PDGF stimulation and represented a threefold increase over controls for TGF-beta 1 in both the apical and basolateral compartments (n = 3, P < 0.05 versus control). The glucose transporter inhibitors phlorizin and phloretin were used to investigate the role of specific D-glucose transport proteins. Application of either basolateral phlorizin or phloretin at the time of addition of 25 mmol/L D-glucose to the same compartment inhibited TGF-beta 1 synthesis in response to PDGF. Maximal inhibition was achieved at 0.5 mmol/L of either inhibitor (phlorizin percent inhibition of apical TGF-beta 1, 75%, P = 0.015, and of basolateral TGF-beta 1, 78%, P = 0.015; phloretin percent inhibition of apical TGF-beta 1, 68%, P = 0.03, and of basolateral TGF-beta 1, 79%, P = 0.001, n = 5, P versus control). No inhibition was seen with apical application of either inhibitor. These data demonstrate that the priming of proximal tubular cells for TGF-beta 1 synthesis occurs only after basolateral exposure of the cells to 25 mmol/L D-glucose. This mechanism is dependent on the activity of the basolateral D-glucose transporter GLUT-1. In another series of experiments, TGF-beta 1 synthesis in response to the addition of basolateral PDGF was also induced after basolateral pretreatment with D-galactose but not 2-deoxy-D-glucose. This priming effect demonstrates the dependence of this response on glucose metabolism by the cells, not simply the activity of the GLUT-1 transporter, as both 2-deoxy-D-glucose and D-galactose are transported by GLUT-1, although only the latter is metabolized. The extrapolation of these results to diabetic nephropathy would suggest that it is changes in the interstitial concentration of glucose rather than the urinary glucose level that likely modulate the synthesis of the profibrotic cytokine TGF-beta 1 and thereby influence the progression of interstitial fibrosis.
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PMID:Polarity of stimulation and secretion of transforming growth factor-beta 1 by cultured proximal tubular cells. 906 Aug 45

The differential expression of Rho family of low molecular weight GTP-binding proteins and protein kinase C (PKC) isozymes were examined during differentiation of rat C6 glial cells to astrocytic phenotypes induced by dibutyryl cAMP (dbcAMP)/theophylline. The cells showed rapid and distinct morphological changes, resembling stellate astrocytes at 12 h after the treatment. The treated cells had a round cell body that extended several long processes each with a beaded appearance. In addition to morphological changes, Western blot analysis revealed that S-100 protein, known as a glial cell differentiation marker, increased and reached the maximal level (approximately 6-fold increase) at 24 h following the addition of dbcAMP. In the control experiments with cells cultured in the absence of serum but also without dbcAMP/theophylline, morphological changes were marginal and apparent increases of S-100 protein were not observed by Western blotting. In response to dbcAMP/theophylline treatment, RhoA showed increases in the mRNA level followed by the protein level, as inferred by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Rac1 and Cdc42 proteins were undetectable by Western blot analyses. In PKC isozymes, increases were observed in PKC beta 1, epsilon, and zeta by RT-PCR, and in beta 1 and epsilon by Western blotting. Among them, PKC epsilon showed the most distinct changes. Its mRNA level transiently increased from 3 to 6 h and then decreased even below the basal level at 18 h after the treatment. In contrast, Western blot analysis revealed that PKC epsilon gradually increased time-dependently to 24 h (approximately 6-fold increase), and remained elevated until 48 h. These results suggested that RhoA and PKC epsilon, and probably also PKC beta 1 and PKC zeta, were closely implicated in C6 cell differentiation.
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PMID:Differential expression of Rho family GTP-binding proteins and protein kinase C isozymes during C6 glial cell differentiation. 910 74

We found that mice transgenic for native bovine growth hormone (bGH) gene had increased body size and rapidly progressive glomerulosclerosis, whereas mice transgenic for a mutated bGH gene (bGH-m11) had near normal body size and slowly progressive glomerulosclerosis. The aim of this study was to determine whether rapidly and slowly progressive glomerulosclerosis had distinct glomerular extracellular matrix (ECM) and growth factor mRNA levels. ECM and growth factors were quantitated by competitive reverse transcriptase-PCR in microdissected glomeruli from bGH, bGH-m11, and nontransgenic littermate control mice. In rapid progressors (bGH mice) at 2 to 3 months, the levels of mRNA-coding for some glomerular ECM and growth factors were increased (alpha 1(IV) collagen, 7.3-fold; laminin B1, 3.9-fold; tenascin, 8-fold; and tumor growth factor (TGF)-beta 1, 3.4-fold). These levels underwent a further 2.3-fold increase at 6 to 9 months. Platelet-derived growth factor (PDGF)-B mRNA was high at 2 to 3 months (7.4-fold) and 6 to 9 months (9.5-fold) and was associated with an increased [3H]-thymidine-labeling index and glomerular cell number. In slow progressors (bGH-m11 mice), the mRNA levels at 2 to 3 months were approximately one half that of rapid progressors (alpha 1(IV) collagen, 3.4-fold; laminin B1 1.9-fold; tenascin, 3-fold; TGF-beta 1, 2.2-fold). PDGF-B levels were normal. At 6 to 9 months, alpha 1(IV) collagen, TGF-beta 1, and PDGF-B mRNA levels doubled, whereas tenascin and laminin B1 levels remained stable. At 12 to 18 months, the alpha 1(IV) collagen, TGF-beta 1, and tenascin levels increased by nearly another 50%. The labeling index and PDGF levels were not increased at any time. The levels of expression of several glomerular ECM mRNA and growth factors of rapid progressors at 2 to 3 months of age was nearly double that of slow progressors, nearly doubling again by 6 to 9 months. In slow progressors, alpha 1(IV) collagen and TGF-beta 1 mRNA levels continued to increase at a slow rate, but tenascin and laminin mRNA levels were only further increased at 12 to 18 months. Thus, the initial levels of these mRNA and their rate of change correlated with the severity of glomerulosclerosis.
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PMID:Differential expression of glomerular extracellular matrix and growth factor mRNA in rapid and slowly progressive glomerulosclerosis: studies in mice transgenic for native or mutated growth hormone. 911 9

This article explores the clinical usefulness of reverse transcriptase-polymerase chain reaction in organ graft recipients. In this study, reverse transcriptase-polymerase chain reaction was used to identify intrarenal expression of cytotoxic attack molecules (granzyme B and perforin) and immunoregulatory cytokines (IL-2, IL-4, IL-10, IFN-gamma, and TGF-beta 1) in human renal allograft biopsies. The biopsies (n = 127) were classified using the Banff criteria, and intrarenal gene expression was correlated with the histologic diagnosis. Molecular analyses revealed that intragraft display of mRNA encoding granzyme B, IL-10, or IL-2 is a correlate of acute rejection, and intrarenal expression of TGF beta 1 mRNA is a correlate of chronic rejection. In addition to demonstrating differential and highly selective intragraft gene expression during rejection, these data suggest that therapeutic strategies directed at the molecular correlates of rejection might refine existing antirejection strategies.
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PMID:Clinical application of molecular biology: a study of allograft rejection with polymerase chain reaction. 914 34

Vascular smooth muscle cells (VSMC) contribute to the pathophysiology of hypertension through cell growth and contraction, and phospholipase C (PLC) is a critical effector enzyme in growth factor and vasoconstrictor signaling. There is indirect evidence that angiotensin II (ANG II) receptors are linked to the PLC-beta isoform signaling pathways. However, recent studies suggest that PLC-beta isoforms may not be expressed in VSMC. Our data demonstrate that in human aortic VSMC, PLC-beta 1 and PLC-gamma 1 proteins were detected by immunoblot analysis, and PLC-beta 1 mRNA was identified by reverse transcriptase-polymerase chain reaction in rat aortic VSMC. Incubation of permeabilized VSMC with anti-PLC-beta 1 or anti-Gq alpha antibodies inhibited ANG II-dependent inositol polyphosphate (IP) formation, while anti-PLC-gamma 1 antibodies did not inhibit ANG II-regulated IP formation. Conversely, anti-PLC-gamma 1 antibodies completely abolished platelet-derived growth factor (PDGF)-dependent IP generation, whereas anti-PLC-beta 1 antibodies had no effect on PDGF-induced PLC activation. Inhibition of tyrosine phosphorylation with genistein or herbimycin A did not diminish ANG II-stimulated IP formation or cytosolic free Ca2+ concentration transients, thereby confirming that ANG II signals via a PLC-gamma 1-independent mechanism. In summary, PLC-beta 1 and PLC-gamma 1 are expressed in human aortic VSMC, and PLC-beta 1 is the isoform that is critical for ANG II-regulated PLC signaling in these cells.
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PMID:Angiotensin II activates the beta 1 isoform of phospholipase C in vascular smooth muscle cells. 917 47

The beta 3-adrenoceptor (beta 3-AR) agonist SR-58611A {ethyl-[(7s)-7-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]5, 6,7,8-tetrahydronaphth-2-yl]oxyacetate hydrochloride} stimulated somatostatin and gastrin releases in isolated rat gastric antral epithelial cells. Stimulation was a concentration-dependent process with 50% effective concentrations of 2.7 +/- 1.1 and 3.8 +/- 1.9 nM compared with 209 +/- 71 and 230 +/- 51 nM for isoproterenol, respectively. It was inhibited by selective beta-AR antagonists with the following rank order of potency: SR-59230A 3-(2-ethylphenoxy)1-[(1S)-1,2,3,4-tetrahydronaphth- 1-ylamino]-(2S)-2-propranol oxalate; beta 3-AR antagonist > ICI-118551[erythro-(+/-)-1-(7-methylindan-4-yloxy)-3- isopropylaminobutan-2-ol-hydrochloride; beta 2-AR antagonist > CGP-20712A[(+/-)-[2-(3-carbarmoyl-4-hydroxyphenoxy)-et hyl- amino]-3-[4 (1-methyl-4-trifluoromethyl-2-imidazolyl)-phenoxy]- 2-propranol; beta 1-AR antagonist]. Furthermore, specific binding of 125I-cyanopindolol to the isolated cells was demonstrated and was displaced by the beta-AR antagonists according to the same rank order of potency and with apparent dissociation constants consistent with the 50% inhibitory concentrations for SR-58611A-stimulated somatostatin and gastrin releases. In addition, the presence of beta 3-AR mRNA was detected by reverse transcriptase polymerase chain reaction. These findings provide the first evidence for a gastric beta 3-AR mediating catecholamine stimulation of gastrin and somatostatin releases from antral cells.
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PMID:Characterization of a beta 3-adrenoceptor stimulating gastrin and somatostatin secretions in rat antrum. 917 7

We have used a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect the expression of mRNA for inflammatory cytokines, integrins and selectins in murine lung tissue, and T cells and eosinophils isolated from lung and bronchoalveolar lavage (BAL) fluid in an in vivo model of ovalbumin (OA)-induced airway inflammation. RNA was isolated from whole lung tissue at 1, 6, 24, 48, 72 h, and 7 days after OA inhalation. mRNA for the Th2 cytokines, IL-4, -5, -6, -10 and -13 in OA-sensitized mice were significantly elevated compared with non-sensitized mice. IL-2, TNF-beta, and eotaxin mRNA were also increased, but IFN-gamma mRNA was not. P- and E-selectin mRNA levels were also enhanced in lung tissue between 6 and 72 h after challenge. Lung T cells were isolated by cell sorting with a flow cytometer at 3, 12, 24, 48 and 72 h after challenge. mRNA levels for IL-5 and -10 were greater in T cells from OA-sensitized and -challenged mice than controls at 24 h. BAL fluid from OA-sensitized and -challenged mice also had significantly higher IL-5 levels than controls. BAL fluid T cells and eosinophils were obtained at 48 and 72 h after aerosol challenge and were purified by cell sorting. Messenger RNA for IL-1 alpha, -2, -4, -5, -10, IFN-gamma, and beta 1 were detected in T cells at both time points. Transcripts for IL-1 alpha, -4, -5, -13, TNF-alpha and beta, and alpha 4, beta 1 and beta 7 were also identified in BAL eosinophils. These data show that in addition to murine lung T cells, airway eosinophils may also contribute to the inflammatory response by their ability to express mRNA for cytokines and integrins.
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PMID:Identification of cytokine and adhesion molecule mRNA in murine lung tissue and isolated T cells and eosinophils by semi-quantitative reverse transcriptase-polymerase chain reaction. 919 71

We examined the relative expression of laminin alpha 1, alpha 2, alpha 4 and beta 1 chain genes in the heart of embryonic, neonatal and adult BALb/c mice. The reverse transcriptase-polymerase chain reaction was employed to determine the mRNA expression of these chains because of the relatively small amount of RNA extracted from rat embryonic hearts. Glyceraldehyde 3-phosphate dehydrogenase and beta-actin were used as internal controls. Among the hearts examined, a relatively high expression of laminin alpha 1 chain was observed in the embryonic hearts, while its expression was very weak in the neonatal and negligible in the adult hearts. Conversely, expression of laminin alpha 2 chain was virtually not observed in the embryonic hearts, but this chain was expressed weakly in the neonatal and substantially in the adult hearts. Similar to laminin alpha 1, laminin alpha 4 was expressed in the embryonic hearts, while its expression was relatively weak in the neonatal and adult hearts. Laminin beta 1 was expressed in the hearts of mice at all stages examined. These results demonstrate that the laminin chain gene expression changes in the different developmental stages of the hearts of BALb/c mice.
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PMID:Laminin alpha 1, alpha 2, alpha 4 and beta 1 chain mRNA expression in mouse embryonic, neonatal, and adult hearts. 920 Nov 15

The single-locus mutant mouse tottering (tg) is an established model for absence seizures. We have previously reported an impairment in GABA-induced chloride uptake in tg brain [Tehrani and Barnes, Epilepsy Res. 1995;22:13-21]. In order to determine if this alteration in GABAA receptor function can be related to specific receptor isoforms, we examined the radioligand binding properties of GABAA receptors and the expression of GABAA receptor subunit mRNAs in the cerebral cortex. Saturation binding of [3H]flunitrazepam revealed a significantly lower Kd value in tg cortical tissues (1.77 +/- 0.05 nM) in comparison to that for the background C57BL/6J strain (3.23 +/- 0.23 nM), while the Bmax values were indistinguishable. Biphasic displacement of [3H]flunitrazepam binding by 2-oxoquazepam showed that low affinity binding sites account for 36 +/- 7.6 and 51 +/- 7.5% of the total in control and tg, respectively. The level of [35S]-t-butylbicyclophosphorothionate (TBPS) binding to tg cortical membranes was 73.6 +/- 5.8% of that in controls. Paired measurements by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed no significant differences in the levels of GABAA receptor alpha 1, alpha 3, alpha 5, beta 2, beta 3, gamma 2 or gamma 3 subunit mRNAs between tg and control cortex. However, tg tissues showed elevated levels of alpha 2- and beta 1-subunit mRNAs, representing 256 and 177%, respectively, those of controls. For the tg cortex, the enhanced expression of GABAA receptor alpha 2 and beta 1 subunits correlates with recombinant subtypes known to have low affinity for 2-oxoquazepam and impaired binding of TBPS. These aberrant properties of GABAA receptors could influence the development or propagation of phenotypic seizures in the tottering mouse.
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PMID:Aberrant expression of GABAA receptor subunits in the tottering mouse: an animal model for absence seizures. 933 86


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