Nobuko Shindo-Okada

Bio: Nobuko Shindo-Okada is an academic researcher from National Cancer Research Institute. The author has contributed to research in topics: Queuine & Transfer RNA. The author has an hindex of 13, co-authored 24 publications receiving 674 citations.

Transfer ribonucleic acid guanine transglycosylase isolated from rat liver.

Abstract: Transfer ribonucleic acid (tRNA) guanine transglycosylase (guanine insertion enzyme) was isolated from rat liver and extensively purified. The enzyme catalyzes an exchange of queuine (the base of queuosine, Q) as well as its precursors and guanine for guanine originally located in the first position of the anticodon of "undermodified" tRNATyr, tRNAHis, tRNAAsn, and tRNAAsp from an Escherichia coli mutant or rat ascites hepatoma cells. This is in contrast to the previous observation that E. coli tRNA-guanine transglycosylase catalyzes the exchange of queuine precursors, such as 7-(aminoethyl)-7-deazaguanine and 7-cyano-7-deazaguanine, but not of queuine itself [Okada, N., Noguchi, S. Kasai, H., Shindo-Okada, N., Ohgi, T., Goto, T., & Nishimura, S. (1979) J. Biol. Chem. 254, 3067-3073]. The Km value for queuine of the rat liver enzyme is 9.2 X 10(-7) M, much lower than the values for the bases of queuosine precursors or guanine. Thus, the actual substrate for tRNA-guanine transglycosylase in queuosine biosynthesis in vivo in rat liver may not be 7-(aminomethyl)-7-deazaguanine, which is thought to be an actual substrate guanine, the E. coli system. Queuine or some queuine derivative may be the actual substrate for the tRNA-guanine transglycosylase reaction in the biosynthesis of Q in tRNA of mammalian cells. 6-Thioguanine and 8-azaguanine are also found to be good substrates.

Detection of unique tRNA species in tumor tissues by Escherichia coli guanine insertion enzyme.

Abstract: The guanine insertion enzyme from Escherichia coli catalyzes exchange of guanine located at the first position of the anticodon of tRNA with radioactive guanine (N. Okada and S. Nishimura, unpublished data). tRNA isolated from various tumors, including slowly growing Morris hepatoma 7794A, incorporated considerable guanine with E. coli guanine insertion enzyme, whereas tRNA isolated from all normal tissues so far tested, except regenerating rat liver, incorporated scarcely any. In the rat ascites hepatoma AH7974, the guanine was mostly incorporated into minor isoaccepting species of tRNAAsp that contained the guanine residue instead of Q base in the first position of the anticodon. This is a sensitive and easy method for identifying unique tRNA species in tumor tissues.

Identification of Genes Differentially Expressed in Association with Metastatic Potential of K-1735 Murine Melanoma by Messenger RNA Differential Display

Abstract: To identify genes differentially expressed in association with the metastatic potential of K-1735 mouse melanoma cells, the mRNA differential display method was applied to compare mRNAs from high- and low-metastatic K-1735-derived cells. Three of the high- and three of the low-metastatic clones were used to reduce the false positives in the initial screening, and Southern blot screening against reverse transcription-PCR products was used to confirm that cDNA fragments detect differential expression between high- and low-metastatic cells. By using 256 different combinations of modified long arbitrary primers which provide broad screening of expressed genes, approximately 12,000 cDNA fragments were amplified from mRNA of each cell line. Among them, eight genes were identified as being expressed in either high- or low-metastatic cells using Northern blot analysis. Integrin alpha6 and two unknown genes were expressed in high-metastatic cells, whereas beta-tropomyosin, macrophage colony-stimulating factor, inhibin/activin betaB subunit, and two unknown genes were expressed in low-metastatic cells. These results indicate that the acquisition of metastatic potential in tumor cells was regulated by activation and/or inactivation of several specific genes, such as those for cell adhesion molecule, cytoskeletal protein, and growth factors.

Isolation of mammalian tRNAAsp and tRNATyr by lectin-Sepharose affinity column chromatography

Abstract: tRNAAsp from rabbit liver, rat liver and rat ascites hepatoma was readily isolated by concanavalin A-Sepharose (Con A-Sepharose) affinity column chromatography. tRNATyr from these sources was extensively purified by Ricinus communis lectin-Sepharose column chromatography. These results, together with the chromatographic behaviour of four tRNAs (tRNATyr, tRNAHis, tRNAAsn and tRNAAsp) on acetylated DBAE-cellulose column chromatography suggested that tRNAAsp contains a Q nucleoside species having a mannose moiety while tRNATyr contains Q nucleoside with galactose. The sugars attached in 4-position of cyclopentene diol in the Q molecule are therefore not present at random in the four tRNAs, but present only in each specific tRNA. This is the first case which shows that plant agglutinin interacts with nucleic Acid as well as polysaccharide and glycoproteins.

Changes in Amount of Hypo‐Modified tRNA Having Guanine in Place of Queuine during Erythroid Differentiation of Murine Erythroleukemia Cells

Abstract: The amounts of hypo-modified tRNAs having guanine in place of queuine in murine erythroleukemic cells decreased markedly when the cells differentiated into mature erythroid cells. The amounts of these hypo-modified tRNAs can be determined easily by measuring incorporation of labeled guanine into tRNA with Escherichia coli tRNA-guanine transglycosylase. The decrease was detected at on early stage of erythroid differentiation: namely, before any detectable increase in the percentage of cells containing hemoglobin. The amount of guanine-accepting tRNA species was nearly proportional to the percentage of undifferentiated cells in the population, regardless of the type of inducer used. Decrease in the amounts of hypo-modified tRNAs in the cells was effectively blocked by 12-O-tetradecartoylphorbol 13-acetate, which inhibits differentiation of these cells. 8-Azaguanine, which is known to be substrate of tRNA—guanine transglycosylase. was incorporated almost exclusively into the first position of hypo-modified tRNA in murine erythroleukemic cells when they were pulselabeled in culture with 8-azaguanine, suggesting strongly that tRNA-guanine transglycosylase in the cells is actually involved in incorporation of 8-azaguanine into tRNA in vivo. The amount of 8-azaguanine incorporated into tRNA in differentiated cells was one third of that in undifferentiated cells, the decrease being parallel with that in the amount of guanine-accepting tRNA in these cells. The results suggest that the appearance of hypomodified tRNAs in the transformed cells was due to lack of substrate for queuosine biosynthesis in tRNA.

Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels

Abstract: A new modification of silver staining is presented which utilizes two chemical properties of thiosulfate: image enhancement by pretreatment of fixed gels, and formation of soluble silver complexes which prevents unspecific background staining during image development. This procedure provides high sensitivity for proteins, RNA and DNA in the nanogram range on a colorless, transparent background. The performance of this method is documented by staining one-and two-dimensional patterns of plant leaf proteins. Moreover, we achieved, for the first time, the detection of the non-structural, tobacco mosaic virus-specific 126 kDa protein directly in the one-dimensional protein pattern of infected protoplasts by a staining procedure.

From head to toes: The multiple facets of Sox proteins

Abstract: Sox proteins belong to the HMG box superfamily of DNA-binding proteins and are found throughout the animal kingdom. They are involved in the regulation of such diverse developmental processes as germ layer formation, organ development and cell type specifi-cation. Hence, deletion or mutation of Sox proteins often results in developmental defects and congenital disease in humans. Sox proteins perform their function in a complex interplay with other transcription factors in a manner highly dependent on cell type and promoter context. They exhibit a remarkable crosstalk and functional redundancy among each other.

Sox10 mutation disrupts neural crest development in Dom Hirschsprung mouse model.

Abstract: Hirschsprung disease (HSCR, MIM #142623) is a multigenic neurocristopathy (neural crest disorder) characterized by absence of enteric ganglia in a variable portion of the distal colon. Subsets of HSCR individuals also present with neural crest-derived melanocyte deficiencies (Hirschsprung-Waardenburg, HSCR-WS, MIM #277580). Murine models have been instrumental in the identification and analysis of HSCR disease genes. These include mice with deficiencies of endothelin B receptor (Ednrb(s-l); refs 1,2) endothelin 3 (Edn3(ls): refs 1,3) the tyrosine kinase receptor cRet and glial-derived neurotrophic factor. Another mouse model of HSCR disease, Dom, arose spontaneously at the Jackson Laboratory. While Dom/+ heterozygous mice display regional deficiencies of neural crest-derived enteric ganglia in the distal colon, Dom/Dom homozygous animals are embryonic lethal. We have determined that premature termination of Sox10, a member of the SRY-like HMG box family of transcription factors, is responsible for absence of the neural crest derivatives in Dom mice. We demonstrate expression of Sox10 in normal neural crest cells, disrupted expression of both Sox10 and the HSCR disease gene Ednrb in Dom mutant embryos, and loss of neural crest derivatives due to apoptosis. Our studies suggest that Sox10 is essential for proper peripheral nervous system development. We propose SOX10 as a candidate disease gene for individuals with HSCR whose disease does not have an identified genetic origin.