Haruka Toyoda

Regulation of glutathione peroxidase mRNA level by dietary selenium manipulation

Glutathione peroxidase (GSH-Px) contains selenium at its active site as a selenocysteine moiety. We have shown that feeding mice a selenium-deficient diet for a long period caused a large decrease in the GSH-Px mRNA level as well as in GSH-Px activity both in the liver and kidneys (Toyoda, H., Himeno, S. and Imura, N. (1989) Biochim. Biophys. Acta 1008, 301-308). In the present study, the transcription rate of the GSH-Px gene was determined by a nuclear run-on assay using liver nuclei of mice fed a selenium-deficient or selenium-adequate diet. The results clearly demonstrate that the transcription rate of the GSH-Px gene was not changed by dietary selenium manipulation, indicating that dietary selenium regulates the level of GSH-Px mRNA in the post-transcriptional step.

LOWER CONCENTRATION OF LA PROTEIN REQUIRED FOR INTERNAL RIBOSOME ENTRY ON HEPATITIS C VIRUS RNA THAN ON POLIOVIRUS RNA

Translation initiation of poliovirus and hepatitis C virus (HCV) RNA occurs by entry of ribosomes to the internal RNA sequence, called the internal ribosomal entry site (IRES). Both IRES bind to the La protein and are thought to require the protein for their translation initiation activity, although they are greatly different in both the primary and predicted secondary structures. To compare the La protein requirement for these IRES, we took advantage of I-RNA from the yeast Saccharomyces cerevisiae, which has been reported to bind to La protein and block poliovirus IRES-mediated translation initiation. In a cell-free translation system prepared from HeLa cells, yeast I-RNA inhibited translation initiation on poliovirus RNA as expected, but did not significantly inhibit translation initiation on HCV RNA. However, the translation initiation directed by either IRES was apparently inhibited by I-RNA in rabbit reticulocyte lysates, in which La protein is limiting. I-RNA-mediated inhibition of HCV IRES-dependent translation in rabbit reticulocyte lysates was reversed by exogenous addition of purified recombinant La protein of smaller amounts than necessary to reverse poliovirus IRES-dependent translation. These results suggest that HCV IRES requires lower concentrations of La protein for its function than does poliovirus IRES. Immunofluorescence studies showed that HCV infection appeared not to affect the subcellular localization of La protein, which exists mainly in the nucleus, although La protein redistributed to the cytoplasm after poliovirus infection. The data are compatible with the low requirement of La protein for HCV IRES activity.

The regulation of glutathione peroxidase gene expression relevant to species difference and the effects of dietary selenium manipulation

Glutathione peroxidase (GSH-Px) contains selenium (Se) as selenocysteine in the active site of the enzyme. GSH-Px activities in the cytosol of all guinea-pig tissues examined were extremely low compared with those in mice and rats, while Se concentrations in tissues were almost the same among three animal species. In addition, no GSH-Px mRNA was detectable in any tissues of guinea-pigs, although the guinea-pig had the same copy number (probably a single copy) of the GSH-Px gene in its genomic DNA as that of the mouse and rat, suggesting that the species difference of GSH-Px activity observed in rodents might be due to incapability of gene transcription. On the other hand, feeding of mice with Se-deficient diet for 6 weeks resulted in a remarkable decrease in GSH-Px mRNA as well as GSH-Px activity both in the liver and kidneys. The detailed time-course experiment revealed that the drop in GSH-Px activity preceded the decrease in the mRNA level in Se-depleted mice and the mRNA level recovered rapidly in contrast to the slow rate of increase in the enzyme activity in Se-repleted mice. These results suggested that the alteration in GSH-Px activity in mice subjected to dietary Se manipulation is attributable not only to transcriptional but also to post-transcriptional regulation.

Cleavage of the cap binding protein complex polypeptide p220 is not effected by the second poliovirus protease 2A

Poliovirus protein 2A contains a short amino acid sequence that also occurs in the putative active site of the known viral proteinase, 3C, previously shown to be responsible for glutamine/glycine cleavages in the poliovirus polyprotein precursor. Experimental evidence indicates that 2A is a second viral proteinase that mediates the cleavage of two tyrosine/glycine cleavages in the generation of virus-specific proteins. Since poliovirus inhibition of host cell protein synthesis correlates with the specific cleavage of the 220,000-Da component of the cap binding protein complex, we have tested whether viral protein 2A contains the p220 cleavage activity. The results show that 2A does not copurify with p220 cleavage activity, partially purified fractions containing high p220 cleavage activity contain no detectable 2A sequences in the form of either mature or precursor protein, and anti-2A serum or IgG does not inhibit p220 cleavage in vitro.

Overexpression of manganese-superoxide dismutase prevents methylmercury tox1city in hela cells

HeLa cells were stably transformed with plasmid constructs that allowed constitutive expression of antioxidant enzymes such as catalase, glutathione peroxidase (GSH-Px), Cu,Zn-superoxide dismutase (Cu,Zn-SOD) or Mn-superoxide dismutase (Mn-SOD) to examine the involvement of reactive oxygen generation in methylmercury toxicity. Overexpression of catalase, GSH-Px or Cu,Zn-SOD did not affect the sensitivity of HeLa cells against methylmercury. However, the sensitivity of HeLa cells against methylmercury was decreased by overexpression of Mn-SOD, an enzyme localized in matrix of mitochondria and which decomposes superoxide anions. These results suggest that formation of superoxide anions in the mitochondria might be involved in the mechanism of the cytotoxicity of methylmercury.

The Genome-Linked Protein of Picornaviruses

Digestion of poliovirus type 2 [32P]-RNA with enzymes and analysis of the products by column chromatography and paper electrophoresis provides evidence that the RNA is covalently linked to

HeLa Cell Transformants Overproducing Mouse Metallothionein Show in vivo Resistance to cis‐Platinum in Nude Mice

Plasmid pSV2MT‐I encoding mouse metallothionein‐I (MT‐I) designed to be expressed under the control of an SV40 promoter was introduced into human HeLa S3 cells. Several transformants (HeLa/MTH) carrying multi‐copies of mouse MT‐I cDNA in their genomes were isolated. These transformants produced 4 to 20‐fold larger amounts of MT than their parent cells. The MT levels in HeLa/MTH were well correlated with the extent of resistance to cadmium, but not with that to cis‐platinum (cis‐DDP) in vitro. To study the role of MT in resistance to cis‐DDP in vivo, nude mice were inoculated subcutaneously with two independent HeLa/MTH clones. MT levels in these tumors were about 3‐fold higher than those in the parental cells. The growth of tumors derived from either HeLa/MTH clone was not inhibited in the presence of 15 μmol/kg of cis‐DDP, which completely inhibited the growth of tumors derived from the parental HeLa cells. These data strongly suggest that the elevated level of MT confers resistance to cis‐DDP in vivo but not in vitro. Thus, the results of this study indicate that in vitro determinations of the influence of MT on cis‐DDP resistance may underestimate its importance in in vivo situations.

Tissue-specific expression of glutathione peroxidase gene in guinea pigs

Glutathione peroxidase (GSH-Px), a selenocysteine-containing enzyme, is generally considered to be important in protecting animals from oxidative injury. However, guinea pigs have very low GSH-Px activity in major tissues such as liver and kidney, while the activity in the erythrocytes is as high as that of mice or rats. The present study attempted to clarify which step in the gene expression of GSH-Px is responsible for the tissue specific regulation of GSH-Px activity in guinea pigs. Northern blot analysis showed clear signals of GSH-Px mRNA in the reticulocytes and erythroblast-enriched bone marrow cells of guinea pigs, while it was barely detectable in the liver, kidney and heart. Using the nuclear run-on assay, we confirmed that the difference in GSH-Px mRNA levels among tissues of guinea pigs results primarily from the difference in the transcription rate of the GSH-Px gene. Thus, the guinea pig may be a good model for studying the factors regulating the tissue-specific gene expression of this selenoenzyme as well as its essential role.

Comparative sequence analysis of the 5′-terminal noncoding regions of poliovirus vaccine strain sabin 1, Sabin 2, and Sabin 3 genomes

Abstract Complementary DNAs (cDNAs) were synthesized with virion RNAS from immunologically distinct vaccine strains of poliovirus, that is, Sabin 1 (LSc, 2ab), Sabin 2 (p712, Ch, 2ab), and Sabin 3 (Leon 12a 1 b). Restriction endonuclease Hae III cleavage patterns of these cDNAs suggested striking difference in RNA sequences among these poliovirus serotypes. The virion RNA of Sabin 3 was shown to have a genome-linked protein (VPg) and physical properties of the VPg were identical to those of VPgs of the other poliovirus strains. Virion RNA from each serotype was labeled with 125 I-Bolton-Hunter reagent, after proteinase K treatment to remove all but a small peptide of the VPg which covalently linked to the 5′ terminus. The specifically 5′-end-labeled RNAs were analyzed by rapid RNA sequencing techniques. Sequences of the first 20 nucleotides were identical in Sabin 1, Sabin 2, and Sabin 3 strains, that is, VPg-pU-U-A-A-A-A-C-A-G-C-U-C-U-G-GG-G-U-U-G. Complete sequence homology was observed between virion RNAs of Sabin 1 and Sabin 2 strains up to the 43rd nucleotide from the 5′ end except for the 25th nucleotide by a “wandering spot” analysis of 125 I-labeled RNA mixture of these two strains. These results suggested that serotypes of poliovirus were derived by numerous mutations from a putative prototype poliovirus, and that the homologous sequence at 5′ termini, conserved through a long evolutional process, may have essential roles for replication and viral function of poliovirus.

Reduced sensitivity of HeLa cells to cis-platinum by simultaneous overexpression of copper, zinc-superoxide dismutase and catalase

The overexpression of catalase or Cu,Zn-superoxide dismutase (Cu,Zn-SOD) did not affect the sensitivity of HeLa cells to cis-platinum. However, the cytotoxicity of cis-platinum was depressed significantly by the simultaneous overexpression of catalase and Cu,Zn-SOD. We concluded that cis-platinum accelerated the generation of superoxide anion in the cells, and the superoxide anion produced was converted into H2O by the cooperative roles of catalase and Cu,Zn-SOD.