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Biomedical subjects

Y Nishina

Publications and source records attributed to Y Nishina.

At least 55 records · Page 3Linked to original sources
PubMed1995

Structures of charge-transfer complexes of flavoenzyme D-amino acid oxidase: a study by resonance Raman spectroscopy and extended Hückel molecular orbital method.

The structure of the ligand in a charge-transfer complex of oxidized D-amino acid oxidase (DAO) formed upon aerobic addition of chloropyruvate and ammonium sulfate was determined by resonance Raman spectroscopy. The ligand in the complex is in the enamine form, 2-amino-3-chloracrylate, not in the imine form, 2-imino-3-chloropropionate. This conclusion is consistent with our hypothesis that the ligands in the charge-transfer complexes of oxidized DAO and reduced DAO are generally in the enamine and imine forms, respectively. The calculation of HOMO and LUMO of some enamine and imine forms by the extended Hückel molecular orbital method indicated that the enamine form is a better electron donor than the imine form and the imine form is a better electron acceptor than the enamine form. These results, as well as the information about the structure of enzyme-bound ligand, support the following ideas. (i) In the charge-transfer complex of reduced DAO, the reduced flavin is an electron donor. (ii) In the charge-transfer complex of reduced DAO, the reduced flavin is an electron donor and the ligand is an electron acceptor. Resonance Raman studies on the charge-transfer complexes of oxidized DAO and those of reduced DAO, and calculated results of HOMO and LUMO for some enamine and imine forms, corroborated the structure of the stacking form between the flavin ring and the ligand in the active site of DAO.

Ammonium Sulfate↗
PubMed1995

Characterization of development-specific, cell type-specific mouse testicular antigens using testis-specific polyclonal antibodies.

Antisera were raised by immunizing rabbits with mouse testicular germ cells, and absorbed in vivo by injection into castrated male whole mice to obtain a specific antiserum which reacted with mouse germ cells. The expression of mouse testis-specific antigenic macromolecules was then studied immunochemically with the antiserum. Approximately 20 antigenic macromolecules with molecular weights ranging from 26 to 110 kD were detected in the normal adult testis. At least 12 of these were differentiation-specific antigens appearing during development of germ cells, while others were expressed in testicular germ and/or somatic cells or detected only in mature spermatozoa. This technique for raising testis-specific antisera could be useful for isolation of cDNA clones encoding their antigens, as well as for investigation of the physiological roles of these molecules in germ cell differentiation at the molecular level.

Animals↗
PubMed1995

Induction of differentiation of embryonal carcinoma F9 cells by iron chelators.

The effects of several chelators, including hinokitiol, on embryonal carcinoma F9 cell differentiation were assessed by assaying the production of plasminogen activator (PA) as a differentiation marker protein. Hinokitiol and tropolone which were potent differentiation inducers lost their activity following preincubation with Fe2+ and Fe3+ ions. Other metal ions had no or little effect on the hinokitiol-induced differentiation. Of several chelators examined, dithizone induced differentiation as effectively as did hinokitiol and tropolone. Dithizone-induced differentiation was also inhibited by preincubation with Fe3+ ions. It was concluded that some potent iron chelators could trigger the teratocarcinoma F9 cells to differentiate through the chelation with intracellular iron ions.

Animals↗
PubMed1994

Isolation and characterization of cDNA clones specifically expressed in testicular germ cells.

We have cloned cDNAs involved in germ cell-specific expression. For this, a subtracted cDNA library was generated by subtracting cDNAs derived from supporting cells of mutant testis from wild-type testis cDNAs. Detailed analyses of mRNA expression revealed that the genes corresponding to the cloned cDNAs were exclusively expressed in testes and were developmentally controlled.

Actin Depolymerizing Factors↗
PubMed1994

Molecular cloning of a novel Ca(2+)-binding protein (calmegin) specifically expressed during male meiotic germ cell development.

During mammalian spermatogenesis, many specific molecules are expressed. We have recently identified a 93-kDa male meiotic germ cell-specific antigen (Meg 1) exclusively expressed in germ cells from the pachytene spermatocyte to the spermatid stage using the monoclonal antibody TRA 369 (Watanabe, D., Sawada, K., Koshimizu, U., Kagawa, T., and Nishimune, Y. (1992) Mol. Reprod. Dev. 33, 307-312). In this study, we cloned a cDNA representing this antigen from a mouse testis cDNA expression library, using the monoclonal antibody TRA 369. Northern blotting showed that this transcript was 2.3 kilobases in length and was expressed only in the testis and not in other somatic tissues or in the ovary. The expression of the mRNA was first detected at the pachytene spermatocyte stage of male germ cell development, and this expression was correlated with the expression of the protein. Sequence analysis of the cDNA revealed that the predicted protein consists of 611 amino acids, including a hydrophobic NH2 terminus characteristic of a signal peptide, two sets of internal repetitive sequences (four repeats of IPDPSAVKPEDWDD and GEWXPPMIPNPXYQ), and a hydrophilic COOH terminus. The deduced amino acid sequence has 58% homology with dog calnexin (the ER membrane phosphoprotein of pancreatic cells) and significant partial homology with calreticulin (high affinity Ca(2+)-binding protein of the ER membrane) at the repetitive sequence. Furthermore, we demonstrated the 45Ca2+ binding ability of this antigen by a 45Ca2+ overlay assay, and the name calmegin is proposed for this antigen. Calmegin is a novel Ca(2+)-binding protein that is specifically expressed in spermatogenesis. The highly regulated, specific, and abundant expression of calmegin suggests that it has important roles in spermatogenesis.

Amino Acid Sequence↗
PubMed1994

Preparation of separated alpha and beta subunits of electron-transferring flavoprotein in unfolded forms and their restoration to the native holoprotein form.

Electron-transferring flavoprotein (ETF) isolated from pig kidney is a heterodimer containing one FAD and one AMP [Sato, K. et al. (1993) J. Biochem. 114, 215-222]. This paper presents a method for separation of the alpha and beta subunits on a preparative scale. The subunits were separated by cation-exchange chromatography in the presence of 6 M urea at pH 7.6. The subunits were obtained in unfolded forms with denaturant. The unfolded subunits were restored to the heterodimeric form having FAD and AMP with high yield after incubation with FAD and AMP under non-denaturing conditions. The far-UV CD spectrum, the fluorescence and absorption spectra of the bound FAD, and the enzyme activity of the reconstituted ETF were all identical to those of the ETF isolated from pig kidney. Thus, an experimental system was established for the reconstitution of ETF from the four components, alpha, beta, FAD, and AMP. Analyses of the absorption spectra of the separated subunits suggested that the contents of aromatic residues were one tryptophan and six tyrosines in alpha and one tryptophan and one tyrosine in beta. The possibility that one subunit binds with FAD or AMP in the absence of the other subunit was examined by measuring the flavin and protein fluorescence spectra, but no spectral changes reflecting such binding were detected. This result suggests that the coexistence of both the subunits is necessary for the binding of FAD and AMP with the protein.

Adenosine Monophosphate↗
PubMed1993

Suppression of tumorigenicity in temperature-sensitive mutants of embryonal carcinoma cells by induction of cell differentiation at non-permissive temperature.

The temperature-sensitive (ts) mutants of cell differentiation derived from the mouse teratocarcinoma cell line F9, when exposed to non-permissive temperature, undergo stem-cell differentiation concomitant with a transient retardation of cell-cycle progression. By incubating these mutant strains at non-permissive temperature, we were able to study the relationship between cell differentiation and tumorigenicity. Upon exposure to non-permissive temperature, the mutant cells undergo extensive differentiation and lose their ability to initiate tumors in vivo, but retain their in vitro proliferative potential. Our data suggest that the loss of tumorigenicity is not caused by altered proliferative potential, but rather by cell differentiation. We therefore suggest that the loss of proliferative potential and the onset of cell differentiation in teratocarcinoma F9 cells are 2 independent events which can be genetically dissected, and that there is (a) crucial step(s) in the differentiation pathway at which these ts mutant cells lose their tumorigenicity.

Alkaline Phosphatase↗
PubMed1993

Changes in Hox1.6, c-jun, and Oct-3 gene expressions are associated with teratocarcinoma F9 cell differentiation in three different ways of induction.

To obtain information on the mechanism of teratocarcinoma cell differentiation, the expression of genes having a specific role in gene regulation was examined using three different methods of inducing differentiation: drug treatments with retinoic acid (RA) and sodium butyrate, and a genetic approach using ts mutants. The RAR-beta and MK genes were induced specifically by RA treatment, which indicates that these two genes are solely RA responsive. By contrast, a change in c-jun, Hox1.6, and Oct-3 gene expression was observed in all three methods of F9 cell differentiation. These findings suggest that these three genes, whether down-or up-regulated, play an important and key role during teratocarcinoma cell differentiation.

Animals↗
PubMed1993

The induction of jun genes during the reversible changes induced with sodium butyrate on the differentiation of F9 cells.

Sodium butyrate (NaB) induces the phenotypic changes and differentiation markers in embryonal carcinoma F9 cells (Kosaka et al., Exp. Cell Res. 192, 46, 1991). However, the effects are rapid and reversible and different from those induced with retinoic acid (RA). We examined the expression of jun genes during differentiation induced with NaB and compared it with that of RA induction. Although c-jun expression was induced with RA, rapid and predominant induction of junB expression was specifically observed with NaB, which is regulated by both transcriptional and post-transcriptional mechanisms. These results suggest that the mechanism of differentiation induced with NaB differs from that of RA; the former may be mediated by the junB gene and the latter by c-jun.

Animals↗
PubMed1993

Degeneration of skeletal and cardiac muscles in c-myb transgenic mice.

In order to reveal cellular processes sensitive to abnormal c-myb expression in vivo, transgenic mice were produced by introducing the c-myb nuclear proto-oncogene under the ubiquitous transcriptional regulatory unit of the cytoplasmic beta-actin gene. Expression of c-myb in thymus did not cause apparent abnormality, but the mice unexpectedly developed degenerative abnormalities in skeletal and cardiac muscles; this occurred predominantly in males. Expression of c-myb in skeletal muscle was correlated with an inflammation of muscle and was accompanied by vacuolar degeneration of muscle fibres, their regeneration, and lymphocyte infiltration. The identical pathological progression in cardiac muscle was associated with cardiomegaly.

Actins↗
PubMed1993

13C- and 15N-NMR studies on medium-chain acyl-CoA dehydrogenase reconstituted with 13C- and 15N-enriched flavin adenine dinucleotide.

The 13C- and 15N-NMR spectra of porcine kidney medium-chain acyl-CoA dehydrogenase (MCAD) reconstituted with 13C- and 15N-enriched FADs were measured. The positions of selective enrichment were C(2), C(4), C(4 alpha), C(10 alpha), N(1), N(3), and N(5) of the isoalloxazine nucleus of FAD. The NMR signals of the labeled atoms were observed as broad but distinct peaks in each NMR spectrum. The chemical shift values of the 2-, 4-, 4 alpha-, and 10 alpha-13C for the oxidized form of MCAD were 159.5, 166.8, 141.1, and 155.5 ppm, respectively, relative to the methyl resonance of 3-(trimethylsilyl)propionic acid-d4, while those of 1-, 3-, and 5-15N for the oxidized form were 183.6, 161.1, and 334.7 ppm, relative to liquid ammonia, respectively. The upfield shift of 2-13C of MCAD relative to that of FMN in the aqueous medium and its downfield shift relative to that of tetraacetylriboflavin in an apolar medium imply that a weaker hydrogen bond exists between C(2) = O and apoMCAD or a water molecule than that of free FMN with a water molecule. That the 4-13C resonance was observed downfield-shifted relative to that of free FMN in aqueous solution suggests a strong hydrogen bond between C(4) = O and apoMCAD. The chemical shift for 4 alpha-13C in oxidized MCAD is considerably downfield-shifted from that of FMN or any other flavoprotein observed thus far, indicating a unique environment around this position in MCAD. The 1-15N resonance of MCAD was most upfield-shifted among the flavoproteins observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl Coenzyme A↗
PubMed1993

Electron-transferring flavoprotein has an AMP-binding site in addition to the FAD-binding site.

Mammalian electron-transferring flavoprotein (ETF) has been reported to consist of two non-identical subunits and one FAD. The present paper shows that ETF purified from pig kidney contains one more molecule, an AMP. ETF was denatured by guanidine hydrochloride and ultrafiltered for the purpose of removing proteins. The filtrate was analyzed by reverse-phase chromatography. Two peaks appeared on the chromatogram: they were identified as FAD and AMP, and their molar amounts were identical, indicating that ETF contains one AMP molecule. ApoETF, which was prepared by KBr treatment of ETF, also contains one AMP molecule. ApoETF, which was prepared by KBr treatment of ETF, also contain one AMP molecule. These results clearly demonstrate that ETF has an AMP-binding site in addition to the FAD-binding site. AMP-free apoETF was prepared by guanidine treatment of ETF. Mixing AMP-free apoETF, FAD, and AMP produced reconstituted ETF, which showed the same properties as native ETF. Mixing AMP-free apoETF and FAD produced AMP-free ETF, regardless of the coexistence of ATP or ADP: the AMP-binding site cannot bind FAD, ADP, or ATP. The enzymatic activity of the AMP-free ETF for electron transfer from substrate-reduced medium-chain acyl-CoA dehydrogenase to 2,6-dichlorophenolindophenol was identical to that of native ETF. This indicates that the AMP contained in holoETF has no apparent influence on this enzymatic activity. A role of AMP recognized in this study is that AMP facilitates the formation of holoETF from AMP-free apoETF, FAD, and AMP.

Adenosine Monophosphate↗