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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human selenium-dependent glutathione peroxidase (hGPx1) (EC 1.11.1.9) is thought to be involved in many critical cellular functions as a result of its role in glutathione-mediated reduction of toxic peroxides, and it is implicated as a mechanism of resistance against oxygen free radicals. Previous studies have demonstrated that the gene encoding hGPx1 (hgpx1) is more highly expressed in multidrug-resistant AdrR MCF-7 human breast cancer cells than in the parental WT MCF-7 cell line. In order to further study the transcriptional regulation of hgpx1, we have cloned the genomic hgpx1 gene and determined its nucleotide sequence. The 2550-base pair (bp) 5'-flanking sequence of hgpx1 contained the terminal 511 bp of the 3' end of a previously reported rhoH12 cDNA (Yeramian, P., Chardin, P., Madaule, P., and Tavitian, A. (1987) Nucleic Acids Res. 15, 1989), a ras-related oncogene. Further downstream from rhoH12, but before the start of transcription of hgpx1, RNase protection analysis revealed a transcribed sequence of at least 270 bp which we have called mid. RNA transcripts homologous to both rhoH12 (1.8 and 1.5 kilobase pairs (kb)) and mid (1.8 kb) are also more highly expressed in AdrR MCF-7 cells than in WT MCF-7 cells. We screened an AdrR MCF-7 cDNA library with the mid sequence and isolated a partial cDNA clone which contains both mid and rhoH12 sequences and is colinear with the genomic sequence which extends from 10 bp 3' to the rhoH12 stop codon to 810 bp 5' to the start of transcription of hgpx1. The start of transcription of hgpx1 in AdrR MCF-7 cells was determined by primer extension analysis. The promoter and 2 kb of the 5'-flanking sequence of hgpx1 was fused to the bacterial chloramphenicol acetyltransferase gene (hGPx1-CAT1). Analysis of deletion constructs of hGPx1-CAT1 revealed three possible cis-acting regulatory regions. The transcriptional regulation of hgpx1 was examined using the hGPx1-CAT hybrid genes and nuclear run-on studies. We found no evidence that increased mRNA transcript formation could account for different levels of hgpx1 RNA either in different breast cancer cell lines or in response to selenium.
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PMID:Structure and function of the 5'-flanking sequence of the human cytosolic selenium-dependent glutathione peroxidase gene (hgpx1). 155 8

A 77Se-containing moiety has been attached to cysteine residues in bovine hemoglobin, reduced ribonuclease A, and glutathione by reaction with [77Se]6,6'-diselenobis(3-nitrobenzoic acid). The resultant species contain Se-S linkages that have 77Se NMR absorptions in the range range of 568-580 ppm. Spectra have been recorded at 4.7 and 9.7 tesla (T). For labeled hemoglobin a line width of 250 Hz is seen at 4.7 T and 1000 Hz at 9.4 T. This quadrupling of line width with doubling of observational field strength is consistent with exclusive relaxation by the chemical shift anisotropy (CSA) mechanism. These line widths are greater than expected for a molecule the size of hemoglobin and indicate some aggregation at the high concentrations used. Upon dissociation and partial unfolding of the hemoglobin subunits, the line widths of the selenium resonance decrease to 35 and 120 Hz at 4.7 and 9.4 T, respectively. The spin-lattice relaxation time (T1) for the dissociated hemoglobin at 9.4 T was found to be 220 ms. Together with a value of 377 ms for the spin-spin relaxation time (T2), determined from the line width, an estimate of the CSA was made. This gave a value of 890 ppm, which is in accord with other values for Se(II) linked only by single bonds. When this value for the CSA is used, together with the CSA contribution to the line width, in estimating a correlation time for seleno(3-nitrobenzoic acid) (SeNB)-labeled glutathione, a value of 4 x 10(-11) s is obtained. For SeNB-labeled denatured ribonuclease, four distinct resonances are resolvable at 4.7 T and five resonances at 9.4 T. From T1 values for these resonances and the value of 890 ppm for the CSA, an appropriate correlation time of 0.1 ns was determined, which should result in 77Se resonances of 0.2-1.0 Hz at 4.7 and 9.4 T, respectively. Much greater apparent line widths are observed, which are attributed to microheterogeneity resulting from formation of inter- and intramolecular disulfide linkages. It is concluded that when there are no complications from protein aggregation or chemical exchange, the CSA values anticipated to exist in glutathione peroxidase or other selenoproteins should result in resonances with line widths in the range from 27 to 170 Hz, depending on field strength. These resonances should therefore be observable in the intact protein, if 77Se-enriched material is available.
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PMID:NMR relaxation properties of 77Se-labeled proteins. 199 5

Presence of carbonate anions increases the oxidation of luminol in different chemical systems. Lysis of human erythrocytes due to the action of dihydroxyfumaric acid or of perborate is also stimulated by carbonate ions. These anions also change considerably the loss of activity of different enzymes treated with superoxide, hydroxyl or formate radicals and can increase or decrease the effect as a function of the nature of the active centre of the enzyme. The relative effects of superoxide, hydroxyl, formate and carbonate radicals for the inactivation of various enzymes (superoxide dismutases, catalase, ribonuclease, glucose oxidase and glutathione peroxidase) have been examined. Three systems were used: gamma-irradiation under different conditions, photoproduction of radicals and sonication. Inactivation of the enzymes is a function not only of the radical used but also of the nature of the active site. Thus glutathione peroxidase is remarkably resistant to hydroxyl radicals while the superoxide dismutases are rapidly inactivated by carbonate radicals. All of the results combine to show that the presence or absence of carbonate anions must be considered in all studies of oxygen containing free radicals whether chemical, biochemical or biological or high energy irradiation.
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PMID:Carbonate anions; effects on the oxidation of luminol, oxidative hemolysis, gamma-irradiation and the reaction of activated oxygen species with enzymes containing various active centres. 630 56

Oxidative mechanisms are thought to play a major role in several biological phenomena, including cataract formation. In the following studies we determined the relative levels of expression of the genes for the mRNAs for glutathione peroxidase (GPx), glutathione reductase (GR), CuZn-superoxide dismutase (CuZn-SOD) and catalase, in both the rat lens and liver. Northern blot hybridization methods were used to determine the mRNA size. The RNase protection method was used to determine levels of expression for these mRNAs plus levels of expression for alpha A-crystallin and gamma-crystallin mRNAs in the lens, and gamma-actin mRNAs in both the lens and the liver; using [32P]-labeled specific cRNA probes transcribed from the various cDNA clones for the mRNAs being studied. The data was normalized relative to the level of expression of alpha A-crystallin and gamma-actin mRNAs in the lens, and to gamma-actin mRNA in the liver. We find the levels of the mRNAs in the lens fall in the following descending order: GPx > GR > CuZn-SOD > catalase, in the same order as has been reported for the activities of the enzymes in the lens. In the liver, levels of these mRNAs were as follows: GPx > CuZn-SOD > GR > catalase. In the liver, CuZn-SOD mRNA was expressed at about four times the level found in the lens, GPx at three times, catalase at three times and GR at about the same level.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Levels of expression of the genes for glutathione reductase, glutathione peroxidase, catalase and CuZn-superoxide dismutase in rat lens and liver. 783 6

In eukaryotes, co-translational insertion of selenocysteine into selenoproteins necessitates the participation of the selenocysteine insertion sequence (SECIS), an element lying in the 3'-untranslated region of selenoprotein mRNAs. We report a detailed experimental study of the secondary structures of the SECIS elements of three selenoprotein mRNAs, the rat and human type I iodothyronine deiodinase (5'DI) and rat glutathione peroxidase (GPx). Based on RNase and chemical probing, a new secondary structure model is established. It is characterized by a stem-loop structure, comprising two helices (I and II) separated by an internal loop, with an apical loop surmounting helix II. Sequence comparisons of 20 SECIS elements, arising from 2 5'DI, 13 GPx, 2 selenoprotein P, and 1 selenoprotein W mRNAs, confirm the secondary structure model. The most striking finding of the experimental study concerns a set of conserved sequences in helix II that interact to form a novel RNA structural motif consisting of a quartet composed of non-Watson-Crick base pairs 5'UGAY3': 5'UGAU3'. The potential for forming the quartet is preserved in 15 SECIS elements, but three consecutive non-Watson-Crick base pairs can nevertheless form in the other five SECIS, the central G.A tandem being invariant in all cases. A 3D model, derived by computer modeling with the use of the solution data, suggests that the base pairing interactions in the G.A tandem are of the type found in GNRA loops. The 3D model displays the quartet lying in an accessible position at the foot of helix II, which is bent at the internal loop, suggesting that the non-Watson-Crick base pair arrangement provides an unusual pattern of chemical groups for putative ligand interaction.
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PMID:A novel RNA structural motif in the selenocysteine insertion element of eukaryotic selenoprotein mRNAs. 863 17

Classical glutathione peroxidase (GPX1) mRNA levels can decrease to less than 10% in selenium (Se)-deficient rat liver. The cis-acting nucleic acid sequence requirements for Se regulation of GPX1 mRNA levels were studied by transfecting Chinese hamster ovary (CHO) cells with GPX1 DNA constructs in which specific regions of the GPX1 gene were mutated, deleted, or replaced by comparable regions from unregulated genes such as phospholipid hydroperoxide glutathione peroxidase (GPX4). For each construct, stable transfectants were pooled two weeks after transfection, divided into Se-deficient (2 nM Se) or Se-adequate (200 nM Se) medium, and grown for an additional four days. On day of harvest, Se-deficient GPX1 and GPX4 activities averaged 13 +/- 2% and 15 +/- 2% of Se adequate levels, confirming that cellular Se status was dramatically altered by Se supplementation. RNA was isolated from replicate plates of cells and transfected mRNA levels were specifically determined by RNase protection assay. Analysis of chimeric GPX1/GPX4 constructs showed that the GPX4 3'-UTR can completely replace the GPX1 3'-UTR in Se regulation of GPX1 mRNA. We did not find any GPX1 coding regions that could be replaced by the corresponding GPX4 coding regions without diminishing or eliminating Se regulation of the transfected GPX1 mRNA. Further analysis of the GPX1 coding region demonstrated that the GPX1 Sec codon (UGA) and the GPX1 intron sequences are required for full Se regulation of transfected GPX1 mRNA levels. Mutations that moved the GPX1 Sec codon to three different positions within the GPX1 coding region suggest that the mechanism for Se regulation of GPX1 mRNA requires a Sec codon within exon 1. Lastly, we found that addition of the GPX1 3'-UTR to beta-globin mRNA can convey significant Se regulation to beta-globin mRNA levels when a UGA codon is placed within exon 1. We conclude that Se regulation of GPX1 mRNA requires a functional selenocysteine insertion sequence (SECIS) in the 3'-UTR and a Sec codon followed by an intron.
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PMID:Cis-acting elements are required for selenium regulation of glutathione peroxidase-1 mRNA levels. 967 Oct 54

Our previous studies have shown that exercise caused changes in the tissue activities of the antioxidant enzymes glutathione peroxidase, superoxide dismutase, and catalase in spontaneously hypertensive (SH) and Wistar-Kyoto (WKY) rats. To determine whether the changes observed were due to changes in mRNA levels of the enzymes, levels of tissue mRNA were determined by quantitative RNase protection assay. Comparisons of tissue enzyme activities and mRNA levels in sedentary and exercised animals showed that, in some cases (e.g., glutathione peroxidase in SH and WKY myocardium), parallel changes in enzyme activity and mRNA levels occurred, whereas in other cases (e.g., catalase in SH and WKY liver), nonparallel changes were found. Exercise of hypertensive rats altered antioxidant enzyme mRNA levels to those seen in normotensive animals in some, but not all, cases. The results suggest that transcriptional control over changes in exercise-related antioxidant enzyme activities is operative in some cases, although in other cases posttranscriptional regulatory mechanisms may exist.
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PMID:Exercise modulates antioxidant enzyme gene expression in rat myocardium and liver. 1079 43

Mammalian thioredoxin reductase (TRR; NADPH(2):oxidized thioredoxin oxidoreductase, E.C. 1.6.4.5) is a new member of the family of selenocysteine-containing proteins. TRR activity in Se-deficient rat liver is reported to decrease to 4.5 to 15% of the activity in Se-adequate rat liver, similar to the fall in Se-dependent glutathione peroxidase-1 activity. Both glutathione peroxidase-1 enzyme activity and mRNA levels decrease dramatically in Se deficiency, whereas glutathione peroxidase-4 activity only decreases to 40% of Se-adequate levels and mRNA level is little affected by Se deficiency. The purpose of these experiments is to study the effect of Se status on TRR mRNA levels and enzyme activity in our well-characterized rat model, and to compare this regulation directly to the regulation of other Se-dependent proteins in male weanling rats fed Se-deficient diets or supplemented with dietary Se for 28 days. In two experiments, TRR activity in Se-deficient liver decreased to 15% of Se-adequate activity as compared to 2% and 40% of Se-adequate levels for GPX1 and GPX4, respectively. Using ribonuclease protection analysis, we found that TRR mRNA levels in Se-deficient rat liver decreased to 70% of Se-adequate levels. This decrease in TRR mRNA was similar to the GPX4 mRNA decrease in Se-deficient liver in these experiments, whereas GPX1 mRNA levels decreased to 23% of Se-adequate levels. This study clearly shows that TRR represents a third pattern of Se regulation with dramatic down-regulation of enzyme activity in Se deficiency but with only a modest decrease in mRNA level. The conservation of TRR mRNA in Se deficiency suggests that this is a valued enzyme; the loss of TRR activity in Se deficiency may be the cause of some signs of Se deficiency.
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PMID:Selenium regulation of thioredoxin reductase activity and mRNA levels in rat liver. 1203 Dec 52

Liver glutathione peroxidase-1 (GPX1) mRNA is highly regulated by Se status relative to other parameters, but is of limited use for determining Se requirements in humans. To examine the efficacy of using blood for Se status assessment using molecular biology markers, we used a ribonuclease protection assay (RPA) to study mRNA levels in whole blood relative to 16 other rat tissues. Significant amounts of total RNA (>50 microg) were obtained from 1 mL of whole blood. Total RNA from 28-d postweaning Se-adequate (0.2 microg Se/g diet) male rats was analyzed for GPX1, GPX4, GPX3, thioredoxin reductase-1 (TRR1), and selenoprotein-P (SelP). RPA detected significant mRNA expression for at least 1 selenoprotein in all tissues except pancreas. GPX1 mRNA expression using this mix of RPA probes yielded the highest signal for GPX1 relative to the other selenoprotein signals in all tissues except testis; GPX1 expression was 4th highest in blood and similar to the major organs (liver, 1st; heart, 5th; kidney, 6th). Kidney was highest for GPX3, and testes was highest for GPX4, TRR1, and SelP. This study is the first to report the gene expression pattern for a number of selenoproteins and across a comprehensive set of tissues. The mRNA levels for all selenoproteins in blood were comparable to levels in the major organs, and decreases in blood and liver GPX1 mRNA levels in Se deficiency were similar, supporting potential use of whole blood for assessing Se status using molecular biology markers.
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PMID:Selenoprotein mRNA is expressed in blood at levels comparable to major tissues in rats. 1546 60

After experimental traumatic brain injury (TBI), widespread neuronal loss is progressive and continues in selectively vulnerable brain regions, such as the hippocampus, for months to years after the initial insult. To clarify the molecular mechanisms underlying secondary or delayed cell death in hippocampal neurons after TBI, we compared long-term changes in gene expression in the CA1, CA3 and dentate gyrus (DG) subfields of the rat hippocampus at 24 h and 3, 6, and 12 months after TBI with changes in gene expression in sham-operated rats. We used laser capture microdissection to collect several hundred hippocampal neurons from the CA1, CA3, and DG subfields and linearly amplified the nanogram samples of neuronal RNA with T7 RNA polymerase. Subsequent quantitative analysis of gene expression using ribonuclease protection assay revealed that mRNA expression of the anti-apoptotic gene, Bcl-2, and the chaperone heat shock protein 70 was significantly downregulated at 3, 6 (Bcl-2 only), and 12 months after TBI. Interestingly, the expression of the pro-apoptotic genes caspase-3 and caspase-9 was also significantly decreased at 3, 6 (caspase-9 only), and 12 months after TBI, suggesting that long-term neuronal loss after TBI is not mediated by increased expression of pro-apoptotic genes. The expression of two aging-related genes, p21 and integrin beta3 (ITbeta3), transiently increased 24 h after TBI, returned to baseline levels at 3 months and significantly decreased below sham levels at 12 months (ITbeta3 only). Expression of the gene for the antioxidant glutathione peroxidase-1 also significantly increased 6 months after TBI. These results suggest that decreased levels of neuroprotective genes may contribute to long-term neurodegeneration in animals and human patients after TBI. Conversely, long-term increases in antioxidant gene expression after TBI may be an endogenous neuroprotective response that compensates for the decrease in expression of other neuroprotective genes.
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PMID:Analysis of long-term gene expression in neurons of the hippocampal subfields following traumatic brain injury in rats. 1568 Jun 94


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