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)

While there is considerable evidence for phosphate (Pi) reabsorption in the distal tubule, Pi transport and its regulation have not been well characterized in this segment of the nephron. In the present study, we examined Na+-dependent Pi transport in immortalized mouse distal convoluted tubule (MDCT) cells. Pi uptake by MDCT cells is Na+-dependent and, under initial rate conditions, is inhibited by phosphonoformic acid (41 +/- 3% of control), a competitive inhibitor of Na+-Pi cotransport. The transport system has a high affinity for Pi (Km = 0.46 mM) and is stimulated by lowering the extracellular pH from 7.4 to 6.4 and inhibited by raising the pH from 7.4 to 8.4. Exposure to Pi-free medium for 21 h increased Na+-Pi cotransport from 2.1 to 5.5 nmol/mg of protein/5 minutes (p < 0.05) while parathyroid hormone, forskolin, and phorbol 12-myristate 13-acetate failed to alter Pi uptake in MDCT cells. Reverse transcriptase polymerase chain reaction of MDCT cell RNA provided evidence for the expression of the Npt1 but not the Npt2 Na+-Pi cotransporter gene. However, preincubation of MDCT cells with Npt1 antisense oligonucleotide led to only 20% inhibition of Na+-Pi cotransport, suggesting that other Na+-Pi cotransporters are operative in MDCT cells. Indeed, we showed, by ribonuclease protection assay, that MDCT cells express the ubiquitous cell surface receptors for gibbon ape leukemia virus (Glvr-1) and amphoteric murine retrovirus (Ram-1) that also function as Na+-Pi cotransporters. In summary, we demonstrate that the pH dependence and regulation of Na+-Pi cotransport in MDCT cells is distinct from that in the proximal tubule and suggest that different gene products mediate Na+-Pi cotransport in the proximal and distal segments of the nephron.
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PMID:Na+ -phosphate cotransport in mouse distal convoluted tubule cells: evidence for Glvr-1 and Ram-1 gene expression. 955 59

Chlamydiae are obligate intracellular bacteria that replicate within a non-acidified vacuole, termed an inclusion. To identify chlamydial proteins that are unique to the intracellular phase of the life cycle, a lambda expression library of Chlamydia psittaci DNA was differentially screened with convalescent antisera from infected guinea pigs and antisera directed at formalin-fixed purified chlamydial elementary bodies (EBs). One library clone was identified that harboured two open reading frames (ORFs) with coding potential for similar-sized proteins of approximately 20 kDa. These proteins were subsequently termed IncB and IncC. Sequencing of the cloned insert revealed a strong Escherichia coli-like promoter sequence immediately upstream of incB and a 36nt intergenic region between the ORFs. Sequence analysis of the region upstream of incB and incC revealed two ORFs that had strong homologies to an amino acid transporter and a sodium-dependent transporter. Immunoblotting with antisera directed at IncB or IncC demonstrated that these proteins are present in C. psittaci-infected HeLa cells but are absent or below the level of detection in purified EBs. Reverse transcriptase-polymerase chain reactions provided evidence that incB and incC are transcribed in an operon. Immunofluorescence microscopy demonstrated that IncB and IncC are each localized to the inclusion membrane of infected cells. No primary sequence similarity is evident between IncA, IncB or IncC, but each contains a large hydrophobic domain of similar size and character as in IncA. Analysis of the recently completed C. trachomatis serovar D genome database has revealed C. trachomatis ORFs encoding homologues to incB and incC, indicating that these genes are conserved among the chlamydiae.
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PMID:Tandem genes of Chlamydia psittaci that encode proteins localized to the inclusion membrane. 966 87

Cultured astrocytes derived from neonatal rat brain exhibited high affinity, Na+-dependent, paroxetine and fluoxetine sensitive [3H]5-HT uptake. Reverse transcriptase-PCR demonstrated that astrocytes in culture expressed messenger RNA for the cloned serotonin transporter protein which has been characterised as the neuronal serotonin transporter. Although the serotonin transporter in cultured astrocytes displayed a Km value approximately 10 times greater than found in adult brain synaptosomes, these observations indicated that astrocytes in vitro may express the same serotonin transporter as neurons. Reverse transcriptase-PCR demonstrated the presence of serotonin transporter mRNA in the adult rat cerebral cortex, suggesting that astrocytes in vivo may express low levels of this mRNA. To investigate whether astrocytes in the adult CNS express functional serotonin transporters, glial plasmalemmal vesicles were prepared from cerebral cortex, representing a subcellular fraction composed primarily of vesicles derived from astrocytes. These vesicles were characterised by [3H]-glutamate and [3H]-dopamine uptake and by immunoblot analysis, using glial and synaptic markers: glutamate synthase, SNAP-25 and synaptobrevin. [3H]5-HT was taken up into glial plasmalemmal vesicles in a high affinity (Km approximately 40 nM), Na+ dependent, paroxetine-sensitive manner. The [3H]5-HT uptake capacity (Vmax) in these vesicles was approximately one quarter of that observed in synaptosomes. These data indicate that astrocytes in culture and in vivo are capable of 5-HT uptake via the previously characterised 'neuronal' serotonin transporter.
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PMID:Serotonin transporters in adult rat brain astrocytes revealed by [3H]5-HT uptake into glial plasmalemmal vesicles. 969 37

The aim of this study was to investigate the involvement of endothelins (ET) in brain injury. The effect of ET was studied in the isolated basilar artery (BA) taken from control, sham-operated, and cold-lesioned rats. Cold lesion was induced by application of a precooled (-78 degrees C) copper cylinder (outer diameter 5 mm) for 60 seconds to the intact dura over the parietal cortex. After precontraction with prostaglandin (PG) F2alpha, ET-3 (10(-10) to 10(-8) mol/L) dilated BA with a pD2 (negative log of the half-maximal concentration) of 9.06+/-0.031 (mean +/- SD) and a maximal effect (Emax) of 1.64+/-1.0 mN at 3 x 10(-9) mol/L in sham-operated animals. This dilation was reduced 24 and 48 hours after cold lesion by 33% and 73%, respectively, at 3 x 10(-9) mol/L. The effects of acetylcholine (10(-8) to 10(-4) mol/L) and sodium nitroprusside (10(-3) mol/L) were unaltered. Activation of the ETB receptor in thoracic aorta by the specific agonist IRL 1620 also resulted in a reduced dilation (51% by 48 hours after cold lesion). Reverse transcriptase-polymerase chain reaction of the BA showed unaltered expression of mRNA for the ETB receptor after cold lesion whereas ETB immunoreactivity in BA and in its intraparenchymal arteries was reduced at 24 and 48 hours. In contrast to the reduction of ET-3-induced dilation, the constrictor effects of ET-1 and ET-3 were retained after cold lesion. Endothelin-1 (10(-12) to 10(-6) mol/L) dose-dependently contracted segments of untreated control BA segments under resting conditions with a pD2 of 8.03+/-0.22 and an Emax of 6.35+/-0.70 mN. Further evidence that the constrictor ability of BA was not influenced by cold lesion is given by the unaltered response to 124 mmol/L K+ and 10(-6) mol/L serotonin. We conclude that the ETB receptor of BA after cold lesion is downregulated specifically, apparently at the posttranscriptional level. Because the ETB-mediated dilation in thoracic aorta was also reduced, downregulation of the ETB receptor apparently is not restricted to cerebral arteries. The nitric oxide-cyclic guanosine monophosphate system in BA is, however, intact.
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PMID:Delayed loss of ETB receptor-mediated vasorelaxation after cold lesion of the rat parietal cortex. 985 Jan 48

Epidermal cytokines such as interleukin (IL)-1beta, tumor necrosis factor-alpha, and IL-12 have been described to play a crucial role in the induction and elicitation phase of allergic contact dermatitis upon exposure to haptens. In this study we asked whether these cytokines may also play a role in the epidermis of patients with atopic dermatitis after the application of house dust mite antigens (HDM) to their skin. Epidermal samples were collected by scraping healthy appearing skin of atopic patients and healthy individuals 8 h after the application of an extract of HDM. Sodium lauryl sulfate and saline served as controls. Reverse transcriptase-polymerase chain reaction was performed for IL-1beta, tumor necrosis factor-alpha, IL-12 p35, and IL-12 p40. Exposure to HDM led to a significant upregulation of mRNA of these cytokines in atopic patients only. Whereas IL-1beta and tumor necrosis factor-alpha also showed an upregulation in part of these patients after exposure to the irritant sodium lauryl sulfate, IL-12 p40 mRNA was exclusively enhanced by the application of the allergen. In contrast to IL-12 p40, IL-12 p35 mRNA was not detectable in significant amounts. Interestingly, also in untreated, normal appearing skin of atopic individuals (n = 16), the levels of these cytokines were higher than in normal individuals (n = 8), possibly explaining the increased skin irritability of atopic individuals. Finally, comparing epidermal cytokines in the skin of patients who developed a positive allergen patch test to those who stayed negative, suggests that only expression of IL-1beta mRNA may be a predictive marker for the development of a positive patch test reaction to HDM.
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PMID:Epidermal cytokines IL-1beta, TNF-alpha, and IL-12 in patients with atopic dermatitis: response to application of house dust mite antigens. 985 37

The NaCl-reabsorbing collecting duct epithelium develops by budding and branching of the embryonic ureter. The expression of Na+ channels during this branching morphogenesis was studied in the outermost branches of rat ureteric buds (UB; embryonic day E15 to postnatal day P6) and in cortical collecting ducts (CCD; days P7-P28) in primary monolayer culture. Expression of both Na+ channel mRNA and of Na+-selective membrane conductance were estimated by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and by patch-clamp recording, respectively. UB and CCD uniformly represented a principal-like cell type in culture. Messenger RNA encoding the alpha-ENaC subunit was detected in oligo-dT primed cDNA (5 ng) of embryonic UB cells (E15-17) after 30 PCR cycles. The abundance of alpha-ENaC mRNA, when normalized by reference to beta-actin, was higher by a factor of 2 in postnatal (P1-6) UB and by a factor of 5 in CCD cells (P7-14) compared with the embryonic stage. Highly Na+-selective, low-conductance channels were identified in apical patches from both UB and CCD monolayers, but only CCD cells exhibited macroscopic, amiloride-sensitive Na+ currents in whole-cell patch-clamp recordings. We conclude that alpha-ENaC mRNA and functional Na+ channel protein are expressed already before morphogenesis of the CCD is completed and prior to the onset of epithelial NaCl reabsorption.
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PMID:Expression of the epithelial sodium channel (ENaC) during ontogenic differentiation of the renal cortical collecting duct epithelium. 991 8

The molecular mechanisms that link acute pancreatitis (AP) and multiple organ failure remain unknown. To clarify the role of endothelial activation, we examined the effects of ascitic fluids from rats with experimental pancreatitis on the expression of adhesion molecules in human umbilical vein endothelial cells (HUVECs). Necrotizing hemorrhagic pancreatitis was induced with sodium taurocholate. Six and 24 h later, peritoneal exudates were collected, centrifuged and HUVECs were treated with the supernatants. The expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was quantified by enzyme-linked immunosorbent assay. Induction of mRNA was assessed by reverse-transcriptase polymerase chain reaction. The activation of transcription factors was examined by electrophoretic mobility shift assay. The expression of ICAM-1 in the tissues was examined immunohistochemically. ICAM-1 and VCAM-1, but not E-selectin expression was upregulated with comparable mRNA induction. Nuclear factor kappaB was activated, while activator protein-1 binding activity was not altered. Immunohistochemically, enhanced ICAM-1 expression was observed in the pancreas and lung, but not in the liver. Ascitic fluids may contain soluble factors responsible for the transcriptional activation of endothelial adhesion molecules, and ICAM-1 may play roles in the pathogenesis of complicated AP.
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PMID:Specific induction of adhesion molecules in human vascular endothelial cells by rat experimental pancreatitis-associated ascitic fluids. 1009 Apr 11

In the present investigation of rat thyroid FRTL-5 cells, we show using reverse-transcriptase PCR that these cells express both Edg-1 and Edg-5. We show using a [35S]GTPgammaS-binding assay that sphingosylphosphorylcholine (SPC), which binds to both Edg-1 and EDG-5, activates Gq, Gi-2, and Gi-3 proteins. SPC potently increases intracellular free calcium concentrations ([Ca2+]i). This effect is mediated through both Gq and Gi proteins, as the mobilization of sequestered calcium was insensitive to pertussis toxin (i.e., mediated by Gq), while the SPC-evoked calcium entry was inhibited by pretreatment with pertussis toxin (i.e., mediated by Gi). Furthermore, SPC in a concentration-dependent manner increases intracellular pH in acidified cells via a Na+-H+ exchange mechanism. The enhanced activation of Na+-H+ exchange is independent of both an increase in [Ca2+]i and an activation of protein kinase C. The effect of SPC on Na+-H+ exchange is insensitive to pertussis toxin, suggesting an effect mediated via Gq.
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PMID:Sphingosylphosphorylcholine activates Gq, Gi-2, and Gi-3 in thyroid FRTL-5 cells: implications for the activation of calcium fluxes and Na+-H+ exchange. 1032 69

Vascular endothelial growth factor (VEGF) is a potent inducer of angiogenesis and is constitutively expressed in the synovium of rheumatoid arthritis (RA). Over-expression of VEGF may play an important role in pathogenic vascularization and synovial hyperplasia of RA. In the present study, we examined whether disease-modifying anti-rheumatic drugs (DMARDs), including bucillamine (BUC), gold sodium thiomalate (GST), methotrexate (MTX) and salazosulfapiridine (SASP), act by inhibiting the production of VEGF by cultured synovial cells of patients with RA. Treatment of cultured synoviocytes with lipopolysaccharide (LPS) significantly increased VEGF production by cultured synovial cells. BUC significantly inhibited LPS-induced VEGF production, while GST tended to inhibit the production of VEGF. The inhibitory effects on VEGF production were dose-dependent. In contrast, MTX and SASP did not affect VEGF production. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that BUC also inhibited LPS-induced VEGF mRNA expression in RA synovial cells. The present study provides the first evidence that BUC inhibits VEGF production and the expression of its mRNA in synovial cells of RA patients. Our results indicate that the anti-rheumatic effects of BUC are mediated by suppression of angiogenesis and synovial proliferation in the RA synovium through the inhibition of VEGF production by synovial cells.
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PMID:Inhibitory effects of anti-rheumatic drugs on vascular endothelial growth factor in cultured rheumatoid synovial cells. 1033 31

Natriuretic peptide binding sites were examined in the gills of the hagfish Eptatretus cirrhatus (Class Agnatha, subfamily Eptatretinae) using radio-ligand binding techniques, molecular cloning and guanylyl cyclase assays. Iodinated rat atrial natriuretic peptide ((125)I-rANP) and iodinated porcine C-type natriuretic peptide ((125)I-pCNP) bound specifically to the lamellar folds and cavernous tissue of E. cirrhatus gills, and 0.3 nmol l(-1) rat ANP competed for 50 % of specific (125)I-rANP binding sites. Affinity cross-linking of (125)I-rANP to gill membranes followed by sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed a single binding site of 150 kDa. In the presence of Mn(2+), 0.1 nmol l(-1) rANP inhibited cGMP production, whereas 1 micromol l(-1) rANP stimulated cGMP production rates. At 1 micromol l(-1), pCNP also stimulated cGMP production. The production of cGMP was also measured in the presence and absence of ATP with either Mn(2+) or Mg(2+). Reverse transcriptase polymerase chain reaction (RT-PCR) of hagfish gill RNA, followed by cloning and sequencing of PCR products, produced a partial cDNA sequence of a natriuretic peptide guanylyl cyclase receptor. The deduced amino acid sequence indicated 87-91 % homology with other natriuretic peptide guanylyl cyclase receptors. This study indicates the presence of a natriuretic peptide guanylyl cyclase receptor in the gills of E. cirrhatus that is similar to the natriuretic peptide guanylyl cyclase receptors in higher vertebrates. These observations demonstrate that the coupling of natriuretic peptide receptors with guanylyl cyclase has a long evolutionary history.
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PMID:Evidence of a guanylyl cyclase natriuretic peptide receptor in the gills of the new zealand hagfish Eptatretus cirrhatus (Class Agnatha). 1093 96


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