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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It has been shown previously that the anticonvulsant agent, sodium valproate, induces certain cytochrome P-450 monooxygenase activities and decreases glutathione S-transferase activity. We have used Western blotting, RNase protection assays and Northern blot hybridization to determine the effects of valproate on the abundance of individual components of the cytochrome P-450 monooxygenase and of glutathione S-transferase subunits. Due to the short half-life of the drug in rats we have used an in vitro experimental system comprised of rat hepatocytes co-cultured with rat primitive biliary epithelial cells. Valproate was shown to be a potent inducer of two members of the cytochrome P-450 (CYP)2B subfamily, CYP2B1 and 2B2. The induction of the proteins was mediated at the level of the mRNAs, with the mRNA for CYP2B1 being more highly induced than that for CYP2B2. The drug also induced, but to a much lesser extent, two important components of the cytochrome-P-450-mediated monooxygenase system, NADPH-dependent cytochrome P-450 reductase and cytochrome b5, and their corresponding mRNAs. Thus, the effects of valproate on cytochromes P-450 and other components of the cytochrome-P450-mediated monooxygenase system mimic those of another, structurally diverse, antiepileptic drug, phenobarbital. However, in contrast to phenobarbital, which induces glutathione S-transferase subunits 1, 2, 3, 4 and 7, valproate selectively decreases the abundance of subunits 3 and/or 4. It has been shown previously that CYP2B1 is involved in the production of metabolites of valproate implicated in hepatotoxicity. The induction of this protein by valproate would thus contribute substantially to the hepatotoxic effects associated with the drug.
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PMID:Effects of the anticonvulsant, valproate, on the expression of components of the cytochrome-P-450-mediated monooxygenase system and glutathione S-transferases. 763 45

The selective encapsidation of retroviral RNA requires sequences in the Gag protein, as well as a cis-acting RNA packaging signal (psi site) near the 5' end of the genomic transcript. Gag protein of human immunodeficiency virus type 1 (HIV-1) has recently been found to bind specifically to the HIV-1 psi element in vitro. Here we report studies aimed at mapping features within the genetically defined psi locus that are required for binding of HIV-1 Gag or of its processed nucleocapsid derivative. The full-length HIV-1 Gag (p55) and nucleocapsid (p15) sequences were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. In a gel shift assay containing excess competitor tRNA, affinity-purified GST-p15 and GST-p55 proteins bound to a 206-nucleotide psi RNA element spanning the major splice donor and gag start codons but did not bind to antisense psi transcripts. Quantitative filter-binding assays revealed that both GST-p55 and GST-p15 bound to this RNA sequence with identical affinities (apparent Kd congruent to 5 x 10(-8) M), indicating that all major determinants of psi binding affinity reside within the nucleocapsid portion of Gag. Chemical and RNase accessibility mapping, coupled with computerized sequence analysis, suggested a model for psi RNA structure comprising four independent stem-loops. Filter-binding studies revealed that RNAs corresponding to three of these hypothetical stem-loops can each function as a independent Gag binding site and that each is bound with approximately fourfold-lower apparent affinity than the full-length psi locus. Interaction of Gag with these regions is likely to play a major role in directing HIV-1 RNA encapsidation in vivo.
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PMID:RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1. 788 56

A cDNA library was constructed from poly(A)+ RNA fractions obtained from a Marek's disease (MD) lymphoblastoid cell line, MDCC-CU41, in which viral gene expression is very limited. Three independent groups (1, 2, and 3) of MD virus (MDV)-specific clones were obtained, which were mapped in the inverted repeat region of the BamHI-A fragment of the MDV genome. Northern blot analysis showed that probes prepared from these cDNA clones hybridized with several transcripts of different sizes in poly(A)+ RNA of MDCC-CU41, although the amounts of these transcripts were relatively small compared to those in MDV lytically infected cells. Moreover, a small open reading frame, which can encode a 94-amino-acid protein, was identified in the A41 cDNA clone (Group 3). By RNase protection assays, the 1.2-kb Group 3 transcriptional unit has been defined. In indirect immunofluorescent antibody assays, antiserum against the bacterially expressed fusion protein, glutathione S-transferase-A41, reacted specifically with the cytoplasmic regions of MDV (strain RB1B)-infected chick kidney cells. However, MDCC-CU41 did not contain a detectable level of the protein determined by these methods.
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PMID:Characterization of Marek's disease virus BamHI-A-specific cDNA clones obtained from a Marek's disease lymphoblastoid cell line. 812 61

EAP (EBER-associated protein) is an abundant, 15-kDa cellular RNA-binding protein which associates with certain herpesvirus small RNAs. We have raised polyclonal anti-EAP antibodies against a glutathione S-transferase-EAP fusion protein. Analysis of the RNA precipitated by these antibodies from Epstein-Barr virus (EBV)- or herpesvirus papio (HVP)-infected cells shows that > 95% of EBER 1 (EBV-encoded RNA 1) and the majority of HVP 1 (an HVP small RNA homologous to EBER 1) are associated with EAP. RNase protection experiments performed on native EBER 1 particles with affinity-purified anti-EAP antibodies demonstrate that EAP binds a stem-loop structure (stem-loop 3) of EBER 1. Since bacterially expressed glutathione S-transferase-EAP fusion protein binds EBER 1, we conclude that EAP binding is independent of any other cellular or viral protein. Detailed mutational analyses of stem-loop 3 suggest that EAP recognizes the majority of the nucleotides in this hairpin, interacting with both single-stranded and double-stranded regions in a sequence-specific manner. Binding studies utilizing EBER 1 deletion mutants suggest that there may also be a second, weaker EAP-binding site on stem-loop 4 of EBER 1. These data and the fact that stem-loop 3 represents the most highly conserved region between EBER 1 and HVP 1 suggest that EAP binding is a critical aspect of EBER 1 and HVP 1 function.
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PMID:The cellular RNA-binding protein EAP recognizes a conserved stem-loop in the Epstein-Barr virus small RNA EBER 1. 838 Feb 32

The maize Bronze2 (Bz2) gene encodes a glutathione S-transferase that is required for anthocyanin pigment accumulation. Two classes of regulatory proteins, R and C1, are required for transcriptional activation of Bz2 and several additional structural genes. Functional domains of the Bz2 promoter were identified using Bz2 promoter-driven luciferase reporter genes electroporated into maize protoplasts together with R and C1 expression plasmids. Complete regulation was conferred by 224 nt of the Bz2 promoter. Within this region at least two separable regions are independently capable of conferring regulation by R and C1. Predicted regulatory elements corresponding to two classes of sequence motifs, the Myb-box homologous 'C1-motif', TAACTG/CAGTTA, and the G-box and E-box homologous 'R-motif', CACGTG, were shown to be important for full R and C1 activation of the Bz2 promoter. Expression of reconstructed Bz2 genes with mutated promoters was quantified using RNase protection, and this analysis confirmed results obtained using reporter genes.
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PMID:Structure and regulation of the maize Bronze2 promoter. 898 May 12

The 322 amino acid cellular protein, Puralpha, is a sequence-specific single-stranded DNA-binding protein implicated in control of transcription and replication. Previous studies have demonstrated that the interaction between Puralpha and its target DNA sequence results in the formation of multimeric complexes. In this study, we demonstrate that Puralpha can self-associate in the absence of DNA. This self-association, while independent of DNA, is mediated by RNA. Through in vitro studies with bacterially expressed glutathione S-transferase fusion proteins, and the synthetic peptides corresponding to various central regions of Puralpha, the domain which is important for the self-association of Puralpha is localized to acidic leucine-rich repeats. Interestingly, these repeats have previously been shown to interact with the human immunodeficiency virus 1 (HIV-1) Tat protein and in this study we demonstrate that Tat is able to disrupt the self- association of Puralpha. We have recently cloned a Puralpha associated-RNA, PU-RNA, and here we show that PU-RNA can specifically reconstitute the self-association of Puralpha. RNA not only mediates the self-association of Puralpha, but also modulates the ability of Puralpha to interact with its target DNA sequence. Electrophoretic mobility shift assays performed with and without RNase treatment demonstrate that RNA inhibits the interaction between Puralpha and its target DNA sequence. Moreover, we demonstrate that the self-association of Puralpha can be reconstituted by a specific oligonucleotide encompassing the Puralpha binding site. The implications of these findings with respect to Puralpha's role in transcription and replication are discussed.
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PMID:Self-association of Puralpha is mediated by RNA. 1041 36

A procedure for the enzymatic synthesis of neoglycoenzymes is described. The gene encoding endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was overexpressed in Escherichia coli as a fusion protein linked to glutathione S-transferase (GST). GST-Endo-A fusion was extracted as a soluble protein. The fusion protein was purified to homogeneity with glutathione-Sepharose 4B and showed transglycosylation activity toward high-mannose-type glycopeptides without removing the GST moiety. The GST-Endo-A immobilized on glutathione-Sepharose 4B retained its transglycosylation activity. The immobilized enzyme could transfer (Man)(6)GlcNAc en bloc to partially deglycosylated ribonuclease B without damaging its enzyme activity. The immobilized GST-Endo-A should be very useful for synthesizing active neoglycoenzymes attached with homogeneous N-linked oligosaccharides.
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PMID:Synthesis of neoglycoenzymes with homogeneous N-linked oligosaccharides using immobilized endo-beta-N-acetylglucosaminidase A. 1062 87

Accumulating evidence from human and experimental animal studies indicates that consumption of heterocyclic amines (HA), derived from cooked meat and fish, may be associated with an increased incidence of cancer. Experiments were initiated to assess the role of one of these compounds, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), as a potential transplacental carcinogen, as well as to evaluate whether in utero exposure to IQ results in the induction of fetal cytochrome P4501A1 (Cyp1a1), P4501B1 (Cyp1b1), and/or glutathione S-transferase (GST). Inducible, or responsive, backcrossed fetuses resulting from a cross between congenic C57BL/6 (Ah(d)Ah(d)) nonresponsive female mice and C57BL/6 (Ah(b)Ah(b)) responsive male mice were transplacentally exposed to olive oil or 6.25, 12.5, or 25 mg/kg of IQ on day 17 of gestation. No macroscopically or microscopically visible liver, lung, or colon tumors were found in the transplacentally treated offspring by one year after birth. Ethoxyresorufin O-deethylase (EROD) and 1-chloro-2,4-dinitrobenzene assays were performed to evaluate whether transplacental exposure to IQ results in the induction of fetal Cyp1a1 and GST, respectively, in lung and liver tissues. Results showed levels of EROD and GST activity in tissues of IQ-treated mice to be very close, if not identical, to those of mice treated with olive oil. Similarly, ribonuclease protection assay data showed that the levels of Cyp1a1 and Cyp1b1 RNA in tissues of IQ-treated mice were not significantly different from those of oil-treated controls. Previous studies have shown that the developing organism expresses very low levels of Cyp1a2. Thus, in utero exposure to IQ does not lead to induction of Cyp1a1, Cyp1a2, or Cyp1b1 in the fetal compartment, thereby maintaining the low levels of these activating enzymes in the developing organism. Taken together, these data imply that, at least under the conditions employed for these experiments, IQ may not play an important role in transplacentally induced tumorigenesis.
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PMID:Prenatal toxicity and lack of carcinogenicity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) following transplacental exposure. 1082 74

Era, an essential GTPase, appears to play an important role in the regulation of the cell cycle and protein synthesis of bacteria and mycoplasmas. In this study, native Era, His-tagged Era (His-Era) and glutathione S-transferase (GST)-fusion Era (GST-Era) proteins from Escherichia coli were expressed and purified. It was shown that the GST-Era and His-Era proteins purified by 1-step affinity column chromatographic methods were associated with RNA and exhibited a higher GTPase activity. However, the native Era protein purified by a 3-step column chromatographic method had a much lower GTPase activity and was not associated with RNA which had been removed during purification. Purified GST-Era protein was shown to be present as a high- and a low-molecular-mass forms. The high-molecular-mass form of GST-Era was associated with RNA and exhibited a much higher GTPase activity. Removal of the RNA associated with GST-Era resulted in a significant reduction in the GTPase activity. The RNA associated with GST-Era was shown to be primarily 16S rRNA. A purified native Era protein preparation, when mixed with total cellular RNA, was found to bind to some of the RNA. The native Era protein isolated directly from the cells of a wild-type E. coli strain was also present as a high-molecular-mass form complexed with RNA and RNase treatment converted the high-molecular-mass form into a 32 kDa low-molecular-mass form, a monomer of Era. Furthermore, a C-terminally truncated Era protein, when expressed in E. coli, did not bind RNA. Finally, the GTPase activity of the Era protein free of RNA, but not the Era protein associated with the RNA, was stimulated by acetate and 3-phosphoglycerate. These carbohydrates, however, failed to activate the GTPase activity of the C-terminally truncated Era protein. Thus, the results of this study establish that the C-terminus of Era is essential for the RNA-binding activity and that the RNA and carbohydrates modulate the GTPase activity of Era possibly through a similar mechanism.
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PMID:Era GTPase of Escherichia coli: binding to 16S rRNA and modulation of GTPase activity by RNA and carbohydrates. 1083 34

AGS3, a 650-amino acid protein encoded by an approximately 4-kilobase (kb) mRNA enriched in rat brain, is a Galpha(i)/Galpha(t)-binding protein that competes with Gbetagamma for interaction with Galpha(GDP) and acts as a guanine nucleotide dissociation inhibitor for heterotrimeric G-proteins. An approximately 2-kb AGS3 mRNA (AGS3-SHORT) is enriched in rat and human heart. We characterized the heart-enriched mRNA, identified the encoded protein, and determined its ability to interact with and regulate the guanine nucleotide-binding properties of G-proteins. Screening of a rat heart cDNA library, 5'-rapid amplification of cDNA ends, and RNase protection assays identified two populations of cDNAs (1979 and 2134 nucleotides plus the polyadenylation site) that diverged from the larger 4-kb mRNA (AGS3-LONG) in the middle of the protein coding region. Transfection of COS-7 cells with AGS3-SHORT cDNAs resulted in the expression of a major immunoreactive AGS3 polypeptide (M(r) approximately 23,000) with a translational start site at Met(495) of AGS3-LONG. Immunoblots indicated the expression of the M(r) approximately 23,000 polypeptide in rat heart. Glutathione S-transferase-AGS3-SHORT selectively interacted with the GDP-bound versus guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-bound conformation of Galpha(i2) and inhibited GTPgammaS binding to Galpha(i2). Protein interaction assays with glutathione S-transferase-AGS3-SHORT and heart lysates indicated interaction of AGS3-SHORT with Galpha(i1/2) and Galpha(i3), but not Galpha(s) or Galpha(q). Immunofluorescent imaging and subcellular fractionation following transient expression of AGS3-SHORT and AGS3-LONG in COS-7 and Chinese hamster ovary cells indicated distinct subcellular distributions of the two forms of AGS3. Thus, AGS3 exists as a short and long form, both of which apparently stabilize the GDP-bound conformation of Galpha(i), but which differ in their tissue distribution and trafficking within the cell.
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PMID:Identification of a truncated form of the G-protein regulator AGS3 in heart that lacks the tetratricopeptide repeat domains. 1127 52


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