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)

We have characterized the interactions between mutant or wild-type M protein and nucleocapsids of vesicular stomatitis virus (VSV) by assaying for inhibition of in vitro transcriptase activity. The interactions are primarily electrostatic in nature: high concentrations of NaCl or poly(L-glutamic acid) reverse the inhibition. These interactions are much weaker in each of the four M protein mutants (complementation group III) tested than in wild-type VSV. Temperature-sensitive revertants were selected from each of the M protein mutants studied. The salt-dependent inhibitory profiles of all the revertants resemble that of wild-type VSV, suggesting that M-nucleocapsid interactions are integrally related to the temperature-sensitive phenotype of group III mutants. These results are discussed in relation to the accompanying paper [Reidler, J.A., Keller, P.M., Elson, E.L., & Lenard, J. (1981) Biochemistry (preceding paper in this issue)] which shows that interaction between M protein and infected cell membranes is increased in all group III mutants studied.
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PMID:Interaction of wild-type and mutant M protein vesicular stomatitis virus with nucleocapsids in vitro. 626 91

A host cell protein required for poliovirus RNA-dependent RNA replicase activity in vitro has been purified several thousand-fold from an uninfected HeLa cell postmitochondrial supernatant. A single protein of apparent Mr = approximately 67,000 daltons and pI 6.3 is associated with this "host factor" activity. Poly(U)-Sepharose chromatography of the template-dependent replicase isolated from poliovirus-infected cells results in the complete loss of replicase activity if a salt gradient is used to develop the column. Host factor elutes early in the salt gradient and restores replicase activity to protein fractions eluted later in the gradient. The host factor, estimated to be present at 50,000-100,000 copies/cell, interacts physically with replicase.
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PMID:Purification and properties of a host cell protein required for poliovirus replication in vitro. 628 18

Virions of Newcastle disease virus (NDV) were disrupted with Triton X-100 in the presence of high salt and nucleocapsids were isolated by ultracentrifugation. The nucleocapsids had very low transcriptase activity and contained only NP as a prominent protein constituent, the bulk of L and P proteins not being retained. The L and P proteins were isolated by sequential treatment of the virions with low- and high-salt detergent followed twice by successive chromatography on phosphocellulose column and examined for their effect on RNA synthesis in a standard transcriptase system using the nucleocapsids as template. When both L and P proteins were added to the template, the RNA synthetic activity was greatly stimulated. P protein alone could not enhance but rather suppressed the activity. L protein exhibited stimulation to some extent but due to residual small amount of P protein in both L protein fraction and the template it has not been elucidated whether L protein could function as a polymerase by itself. These results indicate that both L and P proteins are required to reconstitute a fully active transcriptive complex with a functional template. Attempts have been made to isolate intracellular transcriptive complex from NDV-infected MDBK cells and to determine the protein species involved. The active complex has been recovered neither from cytoplasmic extract obtained by hypotonic disruption nor from Triton X-100 soluble fraction of the cells. However, we could isolate the complex from an extract by double detergents (Tween 40 and deoxycholate) solubilization. The complex contained L, P, and NP as virus specific proteins and several cellular proteins. These results support the concept that both L and P proteins are required for NDV-RNA synthesis and suggest further that the intracellular transcriptive complex may be associated with some cellular structure resistant to Triton X-100 but sensitive to the double detergents, presumably cytoskeletal frame work.
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PMID:Transcriptive complex of Newcastle disease virus. I. Both L and P proteins are required to constitute an active complex. 668 7

An RNA-dependent RNA polymerase activity was found associated with virions of tomato spotted wilt virus (TSWV), a plant- and insect-infecting member of the family Bunyaviridae. Radiolabeled nucleoside triphosphates were incorporated into trichloroacetic acid-precipitable products by detergent-disrupted, purified TSWV virions. Incorporation was reduced to near-background levels when RNase was present in the reaction mixture. The predominantly double-stranded RNA products were RNase-resistant at high but not low salt concentrations. The activity required manganese and was independent of a DNA template. Discrete products of approximately 3.0 kb and heterogeneous smaller products were synthesized that hybridized to purified TSWV RNA and transcripts of cDNA clones encompassing parts of each of the three genomic RNAs. The predominant products were viral sense although significant amounts of viral complementary sense S RNA products were also synthesized.
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PMID:An RNA-dependent RNA polymerase activity associated with virions of tomato spotted wilt virus, a plant- and insect-infecting bunyavirus. 787 44

Replication of the influenza virus genome involves two discrete step reactions: vRNA-directed primer-independent (unprimed) synthesis of cRNA; and cRNA-directed unprimed synthesis of vRNA. Nuclear extracts from both MDCK and HeLa cells infected with influenza virus A/PR8/34 exhibited unprimed synthesis of both cRNA and vRNA strands (a parameter of RNA replication). Ribonucleoprotein (RNP) complexes with the replication activity were isolated from these nuclear extracts by glycerol gradient centrifugation in the presence of 0.1 M KCl. At 0.5 M KCl, however, these complexes were dissociated into stripped RNP and soluble protein fractions. The soluble fraction contained the activity of exogenous template-dependent unprimed RNA synthesis, indicating that the RNA replicase is dissociated from RNP upon exposure to high salt concentrations. On the other hand, the high salt-treated RNP catalyzed only primer-dependent RNA synthesis, but regained a low level activity of exogenous template-dependent unprimed RNA synthesis by adding nuclear extracts from uninfected cells, suggesting that host factor(s) is involved in the functional interconversion of influenza virus RNA polymerase.
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PMID:Replication in vitro of the influenza virus genome: selective dissociation of RNA replicase from virus-infected cell ribonucleoprotein complexes. 803 Dec 33

A method was developed for fast and efficient isolation of RNA from paraffin-embedded tissue sections for subsequent PCR analysis. This method is based on the binding of RNA to acid-treated glass beads in the presence of a high molarity of guanidinium salt. It can be completed within an hour, and obviates the need for dewaxing and phenol/chloroform extractions. The effect of various fixatives and fixation times was tested and the amplification of actin mRNA fragments ranging in length from 82 to 507 bp was used to demonstrate the presence of RNA in the extracts. The method was compared to existing extraction techniques by studying the quality of the templates for reverse-transcriptase polymerase chain reaction amplification (RT-PCR), using virus-infected and mock-infected paraffin-embedded cell pellets as a model. PCR amplification of cellular and viral RNA was successful for RNA isolated by use of all extraction techniques, although the glass bead method was preferred for its simplicity and rapidity. Specimens fixed with formalin were found to be suitable for PCR, but the best results were obtained with acetone-fixed paraffin-embedded material. Dewaxing of tissue sections had no effect on the yield and quality of RNA extractions, and further purification of the extracts using gel filtration did not improve the results. After the protocols were optimized, rotavirus-infected cell pellets were used to demonstrate that extraction and amplification of dsRNA was possible. The information obtained from the studies with the model system was used for extraction of toroviral and rotaviral RNA from archival intestinal material. These data indicate that paraffin-embedded archival tissue can be used for RT-PCR analysis, adding an important technique to diagnostic pathology and retrospective studies.
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PMID:Optimization of extraction and PCR amplification of RNA extracts from paraffin-embedded tissue in different fixatives. 839 55

Mesotocin is the oxytocin-like hormone found in most terrestrial vertebrates from lungfishes to marsupials, which includes all non-mammalian tetrapods (amphibians, reptiles, and birds). It has the largest distribution in vertebrates after vasotocin found in all non-mammalian vertebrates and isotocin identified in bony fishes. In this study, we report the cloning and functional characterization of the cDNA for the mesotocin receptor (MTR) from the urinary bladder of the toad Bufo marinus. The cloned cDNA encodes a polypeptide of 389 amino acids that shows the greatest similarity to the teleost fish isotocin receptor and to mammalian oxytocin receptors with mutations in extracellular loops which are involved in ligand binding. When expressed in COSM6 cells, MTR exhibits the following relative order of ligand affinity: mesotocin > vasotocin = oxytocin > vasopressin > hydrin 1, isotocin, hydrin 2. Injection of MTR cRNA into Xenopus laevis oocytes induces membrane chloride currents in response to mesotocin, which indicates the coupling of the mesotocin receptor to the inositol phosphate/calcium pathway. This response is inhibited by an oxytocin antagonist, but not by a vasopressin antagonist specific for V2 vasopressin receptors. MTR mRNA is not only found in toad urinary bladder, but also in kidney, muscle, and brain tissue of the toad as revealed by northern blot analysis and reverse-transcriptase PCR. The results suggest a variety of function for mesotocin and its receptor including, in particular, an involvement in the regulation of water and salt transport.
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PMID:Cloning and functional characterization of the amphibian mesotocin receptor, a member of the oxytocin/vasopressin receptor superfamily. 864 23

Chronic hypoxia has recently been shown to upregulate inducible nitric oxide synthase (iNOS) gene expression in rat lung. In the present study, we questioned whether induction of NO synthesis could alter the reactivity of pulmonary arteries (PA) from chronically hypoxic (CH) rats. Dose-response curves to phenylephrine (PE) 10(-9) to 5 x 10(-6) M) were examined in PA rings as well as response to L-arginine analogues in isolated lungs from CH or normoxic (N) rats after various incubation times. Although maximal contraction to PE did not differ in PA from CH rats compared to N rats at time 0 (361 +/- 53 vs 506 +/- 52 mg, respectively), it was markedly decreased after prolonged incubation (149 +/- 28 vs 386 +/- 47 mg, respectively, at 4 h; p < 0.001). This phenomenon persisted after endothelial-denudation, but was reversed by NG-monomethyl-L-arginine (L-NMMA) (5 x 10(-4) M) and prevented by actinomycin D (2 x 10(-6) M). In contrast, maximal contraction to PE in aorta from CH rats was similar at time 0 and 4 h. After a short incubation, PA contraction to L-NMMA was greater in CH than in N rats (96 +/- 17 vs 33 +/- 9 mg at 90 min; p < 0.05), was abolished after endothelial denudation, but persisted in CH rats in the presence of calmidazolium (5 x 10(-4) M). At 4 h, contraction to L-NMMA was abolished in endothelium-denuded PA from N rats but only attenuated in those from CH rats. In salt solution perfused lungs, L-NMMA added 30 or 90 min after isolation did not alter baseline pressure in N rats but caused its increase in CH rats. Whereas iNOS messenger ribonucleic acid (mRNA) was detectable by reverse-transcriptase polymerase chain reaction in the PA wall of N or CH rats after 4 h of incubation, it was absent in both at the time of isolation. In contrast, there was evidence of iNOS mRNA in lungs from CH rats at the time of isolation but no signal in those from N rats. In conclusion, there is induction of nitric oxide synthase activity in pulmonary arteries from normoxic and chronically hypoxic rats after prolonged incubation, but this effect is more pronounced in pulmonary arteries from chronically hypoxic rats.
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PMID:Induction of nitric oxide synthase activity in pulmonary arteries from normoxic and chronically hypoxic rats. 904 46

The aim of the study was to investigate whether the adrenal renin-angiotensin system plays an independent role in the regulation of mineralocorticoid biosynthesis in the adrenal gland and to explore the mechanisms of this action. Twelve-week-old male Sprague-Dawley rats were studied: 22 rats were maintained on a regular diet; 27 and 22 rats received a low salt diet with and without treatment, respectively, with the angiotensin II (Ang II) AT1-subtype receptor antagonist losartan (10 mg/kg per day). A fraction of each group of rats underwent bilateral nephrectomy (n = 12, 15, and 10, respectively) and was killed 48 hours later. In an additional group of 24 (12 intact and 12 nephrectomized) rats, the effects of the Ang II AT2-subtype receptor antagonist PD123319 were investigated. In intact rats, plasma renin activity (PRA) and adrenal renin activity and expression were progressively raised by salt restriction and losartan, whereas aldosterone synthase mRNA and plasma aldosterone (PA) levels were increased by salt restriction and reduced by losartan. Forty-eight hours after nephrectomy, PRA fell to undetectable levels; in contrast, adrenal renin expression, assessed by semiquantitative reverse-transcriptase polymerase chain reaction (using GAPDH as a standard for gene expression), showed an 18-fold increase and was further increased after salt restriction and losartan (all P < .05). Also, adrenal renin activity was raised after nephrectomy and further increased after salt restriction (P < .05) and losartan. Cytochrome P450 aldosterone synthase expression in the adrenal cortex was stimulated by nephrectomy alone and by nephrectomy combined with low salt intake (P < .05), with consequent increases in PA concentrations. In losartan-treated salt-restricted nephrectomized rats, cytochrome P450 aldosterone synthase expression (P < .05 versus nephrectomy alone and nephrectomy plus salt restriction) and PA concentrations were diminished (P < .05) in spite of the observed increases of adrenal renin expression. The AT2-receptor antagonism did not significantly affect PRA, adrenal renin, and aldosterone biosynthesis and production in either intact or nephrectomized salt-restricted rats. These results demonstrate that the adrenal renin-angiotensin system plays an independent role in the regulation of mineralocorticoid biosynthesis in vivo. This action is mediated primarily via the Ang II AT1-subtype receptors.
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PMID:Role of tissue renin in the regulation of aldosterone biosynthesis in the adrenal cortex of nephrectomized rats. 935 60

Reverse transcriptase-polymerase chain reaction was used to study the biosynthesis of two different cholesteryl ester hydrolases by human and mouse macrophages. Oligonucleotide primers for bile salt-stimulated cholesterol esterase yielded positive reactions with RNA isolated from human peripheral blood monocytes, monocyte-derived macrophages, the human monocytic THP-1 cells, and phorbol ester-induced THP-1 macrophages. In contrast, oligonucleotide primers for hormone-sensitive lipase yielded positive reactions only with RNA isolated from non-differentiated human THP-1 monocytic cells and peripheral blood monocytes, but not those obtained from differentiated THP-1 macrophages or monocyte-derived macrophages. Thus, while human monocytes were capable of synthesizing both enzymes, human macrophages synthesized only bile salt-stimulated cholesterol esterase and not the hormone-sensitive lipase. The synthesis of bile salt-stimulated cholesterol esterase by human macrophages was confirmed by detection of bile salt-stimulated cholesteryl ester hydrolytic activity in conditioned media of differentiated THP-1 cells and human peripheral blood monocyte-derived macrophages. Moreover, incubating human macrophages with oxidized low density lipoprotein (LDL) or acetylated LDL increased bile salt-stimulated cholesterol esterase activity in the conditioned media of these cells. These results with human macrophages were contrasted with results of studies with mouse macrophages, which showed the presence of hormone-sensitive lipase mRNA but not the bile salt-stimulated cholesterol esterase mRNA. Taken together, these results demonstrated species-specific differences in expression of cholesteryl ester hydrolytic enzymes in macrophages. The expression of bile salt-stimulated cholesterol esterase by human macrophages, in a process inducible by modified LDL, suggests a role of this protein in atherogenesis.
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PMID:Modified low density lipoprotein enhances the secretion of bile salt-stimulated cholesterol esterase by human monocyte-macrophages. species-specific difference in macrophage cholesteryl ester hydrolase. 935 34


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