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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although bile salts are toxic to the liver at high plasma concentrations, the effects of physiological concentrations of bile salts on normal hepatic function are poorly understood. We examined the effect of taurocholate (TC) on the basolateral uptake of [3H]TC in WIF-B cells, a hybrid cell line stably exhibiting in vitro the structural and functional polarity of hepatocytes. Cells were grown in the absence or presence of TC (50 micromol/L) over 12 days, and then incubated with [3H]TC concentrations ranging from 1 to 250 micromol/L. For both control and TC-grown cells, uptake of [3H]TC was linear over 2 minutes. In control cells, the Km for [3H]TC Na+-dependent uptake over 1 minute was 6 +/- 5 micromol/L, and the Vmax was 45 +/- 6 pmol TC/mg protein/min (+/- SEM). TC-grown cells exhibited no significant change in Km but showed a doubling of Vmax to 87 +/- 6 pmol TC/mg protein/min (P < .005). In both control and TC-grown cells, maximal uptake of [3H]TC occurred following 10 to 12 days in culture, with TC-grown cells consistently showing greater rates of [3H]TC uptake from 4 to 14 days in culture. Western blots immunostained for the basolateral Na+-dependent plasma membrane protein, ntcp, revealed the appropriate approximately 50-kd band in control and TC-grown cells, and confocal immunofluorescence microscopy demonstrated staining along the basolateral plasma membrane. Northern blots hybridized with a cDNA probe directed against ntcp indicated a modest TC-induced increase in mRNA levels. Reverse-transcriptase polymerase chain reaction (RT-PCR) using RNA isolated from WIF-B cells and oligonucleotide primers specific for rat ntcp or human NTCP transcripts revealed only the presence of the rat ntcp transcript. We conclude that bile salts, at concentrations normally found in mammalian portal blood, may be capable of promoting enhanced hepatocellular bile salt uptake via an increase in basolateral Na+-dependent plasma membrane transport capacity.
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PMID:Enhanced Na+-dependent bile salt uptake by WIF-B cells, a rat hepatoma hybrid cell line, following growth in the presence of a physiological bile salt. 942 37

Human aortic extracts contain significant cholesteryl ester hydrolytic activity. The enzymic activity was shown to be activated by trihydroxylated bile salt, but not by dihydroxylated bile salt. Monospecific antibodies prepared against rat pancreatic carboxyl ester lipase (CEL, cholesterol esterase) immunoprecipitated cholesteryl ester hydrolytic activity from human aorta, demonstrating that the neutral CEL in aorta is highly similar to and probably identical with the pancreatic enzyme. Reverse transcriptase PCR amplification of mRNA from human aortic endothelial cells revealed de novo synthesis of the pancreatic-type CEL by these cells. Preincubating human aortic endothelial cells with oxidized or native low-density lipoprotein resulted in an 8- and 3-fold increase in CEL activity secreted into the culture medium respectively. A potential physiological role for the endothelial CEL was demonstrated by studies showing its ability to confer partial protection against the cytotoxic effects of lysophosphatidylcholine. The protective effect of CEL is related to its bile-salt-independent lysophospholipase activity. However, CEL hydrolysis of lysophosphatidylcholine can be inhibited by excess cholesterol. Taken together, these results indicate that pancreatic-type CEL is synthesized by cells lining the vessel wall. Moreover, vascular CEL may interact with cholesterol and oxidized lipoproteins to modulate the progression of atherosclerosis.
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PMID:Synthesis and secretion of the pancreatic-type carboxyl ester lipase by human endothelial cells. 944 98

Reverse transcriptase-mediated PCR has been used to isolate two distinct metallothionein (MT) cDNA species from RNA extracted from icefish liver, namely MT-I and MT-II. Northern blot analysis with these cDNA species revealed that significant endogenous levels of MT mRNA were present in liver tissues of normal animals despite the fact that no MT protein could be found accumulating in the same tissue. However, multiple injections of CdCl2 induced high levels of both MT mRNA and MT protein. Sequence analysis of the cDNA species that were present after cadmium injection revealed the presence of both isoforms. Quantification of the MT-I and MT-II transcripts from normal and heavy-metal-treated fish showed an alteration in the ratio of the MT isoform transcripts. Endogenous transcripts consisted mostly of MT-II, whereas the MT-I transcript was preferentially accumulated only in response to the cadmium salt. The protein encoded by each cDNA isoform was isolated from the heavy-metal-treated fish and the availability of the specific MT mRNA for translation was demonstrated by translation in vitro. These results show that: (1) there is a discrepancy between the significant endogenous levels of MT mRNA and the absence of MT protein; (2) the accumulation of MT in icefish liver can be triggered by heavy metals; (3) genes encoding distinct MT isoforms are differentially regulated by heavy metals.
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PMID:Cadmium-induced differential accumulation of metallothionein isoforms in the Antarctic icefish, which exhibits no basal metallothionein protein but high endogenous mRNA levels. 960 Oct 77

Endotoxin-induced cholestasis is mainly caused by an impaired canalicular secretion. Mrp2, the canalicular multispecific organic anion transporter, is strongly down-regulated in this situation, and canalicular bile salt secretion is also reduced. We hypothesized that other adenosine triphosphate-binding cassette (ABC) transporters may compensate for the decreased transport activity to protect the cell from cytokine-induced oxidative damage. Therefore, we examined the expression of ABC-transport proteins in membrane fractions of whole liver and of isolated hepatocytes of endotoxin-treated rats and performed reverse-transcriptase polymerase chain reaction (RT-PCR) on mRNA isolated from these livers. In addition, the localization of these transporters was examined using confocal scanning laser microscopy. By 6 hours after endotoxin administration, we found a clear increase of mrp1 mRNA and protein, whereas mrp2 mRNA and protein were decreased. This was confirmed in isolated hepatocytes. In addition, mdr1b mRNA was strongly increased, whereas mdr1a and mdr2 mRNA did not change significantly. Both the mRNA and protein levels of the sister of P-glycoprotein (spgp), the recently cloned bile salt transporter, decreased. After endotoxin treatment, the normally sharply delineated canalicular staining of mrp2 and spgp had changed to a fuzzy pattern, suggesting localization in a subapical compartment. We conclude that endotoxin-induced cholestasis is caused by decreased mrp2 and spgp levels, as well as an abnormal localization of these proteins. The simultaneous up-regulation of mrp1 and mdr1b may confer resistance to hepatocytes against cytokine-induced metabolic stress.
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PMID:Up-regulation of the multidrug resistance genes, Mrp1 and Mdr1b, and down-regulation of the organic anion transporter, Mrp2, and the bile salt transporter, Spgp, in endotoxemic rat liver. 982 29

RNA-dependent RNA polymerase (RDRP) activity was identified in lysates of Eimeria maxima sporozoites and E. necatrix sporozoites and merozoites. Pretreatment of cell lysates with DNase I, RNase A, proteinase K and actinomycin D prior to RDRP assay was employed to characterize RDRP activity. DNase I and actinomycin D had little effect, while proteinase K abolished RDRP activity in both species. RNase A at a concentration of 1 mg/ml also reduced the polymerase activity in E. maxima and E. necatrix sporozoite lysates to 2% and 0%, respectively. Gel electrophoresis of RDRP products revealed that while most migrated at sizes less than 3 kb, a proportion of labelled products of E. necatrix and E. maxima also migrated to the sizes of their respective putative viral genomes. The RDRP products of E. necatrix were shown to be single-stranded by digestion with RNase in both low- and high-salt solutions and by methylmercuric hydroxide treatment. Moreover, the RDRP products of E. necatrix only hybridized to the 5.6-kb dsRNA of E. necatrix but not to the 4.5-kb dsRNAs of E. necatrix or E. maxima.
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PMID:RNA-dependent RNA polymerase activity associated with virus-like dsRNA in Eimeria maxima and E. necatrix of the domestic fowl. 995 Feb 24

Single-stranded (ss) RNA containing and double-stranded (ds) RNA containing virus particles of Eimeria necatrix were isolated by centrifugation through a CsCl gradient. RNA from the gradient fractions was identified as single-stranded or double-stranded by probing northern blots with digoxigenin-labeled riboprobes. These probes were generated with SP6 and T7 RNA polymerases from a partial cDNA clone derived from 5.6-kb viral dsRNA of E. necatrix. RNA-dependent RNA polymerase (RDRP) activity was identified in these CsCl-purified virus particles. The polymerase products of the ssRNA particles consisted of dsRNA indicating replicase activity, whereas the polymerase products of the dsRNA particles consisted of ssRNA indicating transcriptase. activity. RNase treatment in high salt solution (0.3 M NaCl) of the pooled RDRP products revealed that the products consisted of both RNase-resistant dsRNA and RNase-sensitive ssRNA. These results show that both replicase and transcriptase activities were present in the purified virus. The digoxigenin-labeled products hybridized to both SP6 and T7 transcripts confirming the presence of both activities.
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PMID:RNA-dependent RNA polymerase activity associated with Eimeria necatrix virus particles containing either double-stranded or single-stranded RNA. 1021 97

Reverse transcriptase-polymerase chain reaction has been used to isolate one metallothionein isoform (MT-20) complementary DNA from RNA extracted from mussel gill. Another cDNA, isolated by screening a Mytilus edulis cDNA mantle library using the first cDNA as probe, codes for the MT-10 IV isoform. Northern blot analysis using these cDNAs revealed different expression of these isoforms. Induction with CdC1(2) caused high levels of both MT messenger RNAs, especially the MT-20, which was induced by cadmium salt but not by zinc and copper salts. An induction of MT-10 was detected with ZnCl(2). These results show that genes encoding distinct MT isoforms are differentially regulated by heavy metals.
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PMID:Metallothionein Isoforms in Mytilus edulis (Mollusca, Bivalvia): Complementary DNA Characterization and Quantification of Expression in Different Organs after Exposure to Cadmium, Zinc, and Copper. 1081 60

The replicase activity of rotavirus open cores has been used to study the synthesis of (-) strand RNA from viral (+) strand RNA in a cell-free replication system. The last 7 nt of the (+) strand RNA, 5'-UGUGACC-3', are highly conserved and are necessary for efficient (-) strand synthesis in vitro. Characterization of the cell-free replication system revealed that the addition of NaCl inhibited (-) strand synthesis. By preincubating open cores with (+) strand RNA and ATP, CTP, and GTP prior to the addition of NaCl and UTP, the salt-sensitive step was overcome. Thus, (-) strand initiation, but not elongation, was a salt-sensitive process in the cell-free system. Further analysis of the requirements for initiation showed that preincubating open cores and the (+) strand RNA with GTP or UTP, but not with ATP or CTP, allowed (-) strand synthesis to occur in the presence of NaCl. Mutagenesis suggested that in the presence of GTP, (-) strand synthesis initiated at the 3'-terminal C residue of the (+) strand template, whereas in the absence of GTP, an aberrant initiation event occurred at the third residue upstream from the 3' end of the (+) strand RNA. During preincubation with GTP, formation of the dinucleotides pGpG and ppGpG was detected; however, no such products were made during preincubation with ATP, CTP, or UTP. Replication assays showed that pGpG, but not GpG, pApG, or ApG, served as a specific primer for (-) strand synthesis and that the synthesis of pGpG may occur by a template-independent process. From these data, we conclude that initiation of rotavirus (-) strand synthesis involves the formation of a ternary complex consisting of the viral RNA-dependent RNA polymerase, viral (+) strand RNA, and possibly a 5'-phosphorylated dinucleotide, that is, pGpG or ppGpG.
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PMID:De novo synthesis of minus strand RNA by the rotavirus RNA polymerase in a cell-free system involves a novel mechanism of initiation. 1107 21

We previously reported increased aortic reactive oxygen species (ROS) production in mineralocorticoid (deoxycorticosterone acetate [DOCA]-salt) hypertensive rats. In the present study, we tested the hypothesis that NADH/NADPH oxidase is responsible for increased ROS production, namely superoxide (O(2-)), in aorta from the DOCA-salt rat. Treatment of aortic rings from DOCA-salt rats with the NO synthase inhibitor N-nitro-L-arginine and the xanthine oxidase inhibitor allopurinol did not significantly change O(2-) production. Furthermore, de-endothelialization of aorta from DOCA-salt rats did not affect O(2-) production compared with that of sham-operated rats. Thus, xanthine oxidase and uncoupled endothelial NO synthase were not responsible for increased O(2-) production in the DOCA-salt rats. In contrast, treatment with the NADPH oxidase inhibitor apocynin significantly decreased O(2-) production in aortic rings from DOCA-salt rats compared with sham-operated rats. Moreover, long-term administration of apocynin (in drinking water, 1.5 mmol/L, 28 days) to DOCA-salt rats significantly decreased systolic blood pressure compared with that of rats treated with DOCA-salt alone. Furthermore, O(2-) production in aortic rings from DOCA-salt rats treated with apocynin for 28 days was reduced compared with that of untreated DOCA-salt rats. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that DOCA-salt rats have significantly greater mRNA levels of the NADPH oxidase subunit p22phox than do sham-operated rats. These findings suggest that NADPH oxidase is increased and is responsible for increased O(2-) production and possibly contributes to increased blood pressure in the DOCA-salt hypertensive rat.
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PMID:NADH/NADPH oxidase and enhanced superoxide production in the mineralocorticoid hypertensive rat. 1171 6

Experiments were performed to study angiotensin (Ang) AT1a and AT1b mRNA expression in mice, including, examination of brain distribution and the effect of salt loading. In situ hybridization (ISH) methods showed that the pattern of mRNA expression was identical for AT1a and AT1b, with cellular labeling in rostral forebrain, hypothalamus and brainstem. Receptor mRNAs were concentrated in brain regions involved in the regulation of electrolyte and cardiovascular balance. Immunocytochemistry with AT1 specific antisera showed a pattern that was consistent with the ISH. Reverse transcriptase-polymerase chain reaction (RT-PCR) of hypothalamus and pituitary verified the presence of both AT1a and AT1b mRNA. Using quantitative ISH, we found that AT1a mRNA expression was significantly increased after 5 days of 2% NaCl consumption in anterior third ventricle (AV3V), paraventricular hypothalamus (PVN) and subfornical organ (SFO), but unchanged in anterior pituitary. There were no significant changes in AT1b mRNA. These results document the utility of ISH coupled with quantitative imaging techniques for the study of subtype specific expression. Using ISH and RT-PCR, we verified that AT1a and AT1b receptors are expressed in mouse brain and pituitary and show a similar pattern of distribution. Salt loading produced a specific increase in AT1a mRNA in osmosensitive regions, suggesting that this receptor subtype is regulated by sodium/osmolar input.
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PMID:Osmotic regulation of angiotensin AT1 receptor subtypes in mouse brain. 1259 Nov 17


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