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Biomedical subjects

Y Nishina

Publications and source records attributed to Y Nishina.

At least 91 records · Page 5Linked to original sources
PubMed1989

Isolation of mutants showing temperature-sensitive cell growth from embryonal carcinoma cells: control of stem cell differentiation by incubation temperatures.

Embryonal carcinoma(EC) cells, the undifferentiated stem cells of teratocarcinomas, have many properties in common with pluripotent embryonic cells, and thus provide an excellent system for studying the early events involved in embryonic development and stem cell differentiation. We have isolated three novel mutants with temperature-sensitive(ts) cell growth that were able to differentiate at a non-permissive temperature for cell growth. These mutations affect the progression of the cell cycle, leading to the transient accumulation of cells in a specific phase, the S phase, of the cell cycle, which is likely to be the primary cause of stem cell differentiation of EC cells at non-permissive temperature. Isolation of these mutants strongly supports the notion that there is a close association between the inhibition of DNA synthesis and EC cell differentiation.

Animals↗
PubMed1989

Inhibition of DNA synthesis causes stem cell differentiation: induction of teratocarcinoma F9 cell differentiation with nucleoside analogues of DNA-synthesis inhibitors and their inducing abilities counterbalanced specifically by normal nucleosides.

Nucleoside analogues inhibiting DNA synthesis can induce cell differentiation in teratocarcinoma cells. We have examined how their abilities to induce F9 cell differentiation were specifically counterbalanced by their corresponding normal nucleosides. We have also compared the differentiation inducing ability of the wild type F9 cells with that of its thymidine kinase-less mutant using plasminogen activator, as a differentiation marker, which is expressed at a very early stage of endodermal cell differentiation and can be assayed quantitatively. The results obtained were clearly explainable by the conventionally accepted action mechanisms of the nucleoside analogues, thus strongly suggesting that their abilities to induce cell differentiation were direct consequences of the inhibition of DNA synthesis; thus this confirms the notion that a close association exists between the inhibition of DNA synthesis and the induction of teratocarcinoma stem cell differentiation.

Animals↗
PubMed1989

Trans-activation by the c-myb proto-oncogene.

We present evidence that the mouse c-myb proto-oncogene encodes a transcriptional trans-activator. Trans-activation was assayed by cotransfection into CV1 monkey kidney cells of a c-myb cDNA expression plasmid together with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under the control of a test promoter and enhancer. Cotransfection with the c-myb cDNA plasmid caused a 20-fold stimulation of transcription from the promoter of the mouse alpha 2(I) collagen gene linked to tandem repeats of the simian virus 40 (SV40) enhancer element. Using different promoters in combination with varying numbers of repeats of the SV40 enhancer element, it was shown that tandem repeats of the SV40 enhancer mediated the c-myb-induced activation of transcription. These results show that the mouse c-myb gene product either is itself or induces, an activator of transcription that recognizes specific sequences in the SV40 enhancer.

Animals↗
PubMed1989

Effects of pH and ionic strength on the binding of egg white riboflavin binding protein with flavins.

The effects of pH and ionic strength on the equilibrium constants and rate constants (binding and dissociation rate constants) between riboflavin binding protein (RBP) and flavins (riboflavin, 3-carboxymethylriboflavin [CMRF], and FMN) were studied by fluorometry. The equilibrium constant and the binding rate constant between RBP and riboflavin were pH-independent between pH 6 and 9, and both constants were also independent of the ionic strength, while the constants between RBP and CMRF or FMN were dependent on both pH and ionic strength. The dissociation rate constants between RBP and the flavins used here were not so dependent on pH and ionic strength in the pH region 6 to 9, and the patterns of pH profiles as a whole were similar to each other, although the constants for FMN were about 30-60 times larger than those for CMRF or riboflavin. RBP had lower affinity for FMN than for riboflavin in the neutral pH region, which is based on the small binding rate constant and the large dissociation rate constant for FMN. The former is due to an electrostatic repulsion force between negative net charges of RBP and the phosphate group of FMN, and the latter is due to steric interference by the phosphate group of FMN.

Animals↗
PubMed1989

Characterization of alternate and truncated forms of murine c-myb proteins.

The expression of c-myb proteins in transformed and normal murine cells was investigated. Two c-myb proteins, p75c-myb and p90c-myb, were detected in normal thymocytes and cell lines with intact c-myb genes. These most likely differ by the inclusion of additional amino acids encoded by an alternatively spliced c-myb mRNA. The use of this alternative exon is therefore not a feature exclusively of those cells with viral integrations in their c-myb gene. Smaller c-myb proteins in myeloid leukemic cell lines with rearranged c-myb genes were also characterized. Viral integration into the 5' region of the c-myb gene in the W265 and W274 cell lines leads to the synthesis in each case of two amino-terminally truncated c-myb proteins. By contrast, in NFS60 cells, viral integration into a more 3' region of the c-myb locus (but upstream of an alternate exon) leads to the production of a single p50c-myb protein.

Animals↗
PubMed1988

Resonance Raman spectra of anionic semiquinoid form of a flavoenzyme, D-amino acid oxidase.

Resonance Raman (RR) spectra of the complex of anionic semiquinoid D-amino acid oxidase (DAO) with picolinate in H2O and D2O were observed in the 300-1,750 cm-1 region. RR spectra were also measured for the complex of the semiquinoid enzyme reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]-FAD. On the basis of the isotope effects, tentative assignments of the observed bands of the anionic semiquinoid flavin were made. The spectra differ from those of oxidized, neutral semiquinoid, and anionic reduced flavins previously reported. The 1,602 cm-1 band was not shifted for any FAD labeled in ring II and/or ring III and was assigned to a ring I mode. The 1,516 cm-1 band underwent an isotopic shift upon [4a-13C]- or [4,10a-13C2]-labeling. The band was assigned to the mode containing C(4a)-C(10a) stretching. The 1,331 and 1,292 cm-1 bands shifted upon [4a-13C]- or [5-15N]-labeling and were assigned to the modes containing C(4a)-N(5) stretching. The 1,217 and 1,188 cm-1 bands were assigned to the skeletal vibrations of ring III coupled with the N(3)-H bending mode. The RR spectrum of the complex of anionic semiquinoid DAO with alpha-iminopropionate or N-methyl-alpha-iminopropionate was essentially identical with that of the complex with picolinate.

Animals↗
PubMed1988

Complex formation between anionic semiquinoid form of a flavoenzyme D-amino acid oxidase and ligands. Stabilizing mechanism of anionic semiquinoid flavoenzyme.

Picolinate binds to the anionic semiquinoid form of D-amino acid oxidase (DAO), and the complex formed has a broad absorption band in the long-wavelength region extending beyond 800 nm, which is reminiscent of a charge transfer interaction. The binding has a stoichiometry of 1:1 with respect to the enzyme. The dissociation constant at 25 degrees C was 30 microM at pH 7.0. The pH dependence (pH 7.0-8.3) of the dissociation constant indicates that one proton is associated with the complex formation, and suggests that picolinate able to bind to the anionic semiquinoid enzyme is in the cationic form protonated at the nitrogen atom. By adding dithionite to the oxidized DAO solution containing pyruvate and various amines, a similar anionic semiquinoid DAO complex having a broad long-wavelength absorption band, appeared. Resonance Raman spectra with excitation at 623.8 nm of the anionic semiquinoid DAO complex formed in the presence of pyruvate and methylamine indicate that the complex consists of the anionic semiquinoid DAO and N-methyl-alpha-iminopropionate produced from pyruvate and methylamine, and that the imino group must be protonated. This supports the proposal that the presence of a positively charged group in the vicinity of flavin is required for the stabilization of the anionic semiquinoid flavin. The results also suggest that the broad absorption band is derived from the charge transfer interaction between the anionic semiquinoid flavin and the imino acid, in which the flavin C(4a)-N(5) locus and the locus containing (Formula: see text) of the amino acid are important for the interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed1987

Kinetic and equilibrium studies on the interaction of reduced flavoprotein D-amino acid oxidase with pyridine carboxylates.

The equilibrium constants and the rate constants (binding and dissociation constants) between reduced D-amino acid oxidase and pyridine carboxylates were obtained at various pH values (from pH 6.0 to 8.3). The pH dependence of the constants is consistent with the previous conclusion from a resonance Raman study that pyridine carboxylates in the form of a cation protonated at the N atom can bind to the reduced enzyme, but those in the neutral form cannot bind, showing that the positive charge of cationic pyridine carboxylates interacts with the negative charge of the anionic reduced flavin in the reduced enzyme. The binding rate constants of picolinate and nicotinate in the cationic form for the reduced enzyme were quite similar to each other, but the dissociation rate constant of picolinate is several times smaller than that of nicotinate. Thus, it is concluded that the difference in affinity of picolinate and nicotinate for the reduced enzyme is derived from the difference of the dissociation rate constants.

Animals↗
PubMed1986

A resonance Raman study on the structures of complexes of flavoprotein D-amino acid oxidase.

Resonance Raman (RR) spectra were obtained for the purple complexes of D-amino acid oxidase (DAO) with D-lysine or N-methylalanine. RR spectra of a complex of oxidized DAO with the oxidation product of D-lysine or D-proline were also measured. The isotope shifts of the observed bands of the purple complex with D-lysine upon 13C- or 15N-substitution of lysine indicate that the ligand is delta 1-piperideine-2-carboxylate. That the band at 1671 cm-1 for the purple intermediate with N-methylalanine shifts to 1666 cm-1 in D2O solution indicates that the imino acid, N-methyl-alpha-iminopropionate, has a protonated imino group. Many bands due to a ligand in the RR spectra of the complex of oxidized DAO with an oxidation product can be observed below 1000 cm-1, but no band for the purple complex is seen in this frequency region. The band associated with the CO2-symmetric stretching mode of the product, such as delta 1-piperideine-2-carboxylate or delta 1-pyrrolidine-2-carboxylate, complexed with the oxidized DAO shifts in D2O solution. This suggests that the product imino acid interacts with the enzyme through some proton(s).

Alanine↗
PubMed1986

Complex formation between reduced D-amino acid oxidase and pyridine carboxylates.

Picolinate binds to a reduced form of D-amino acid oxidase, and the complex formed has a broad absorption band around 600 nm as in the case of the purple intermediate of the enzyme with a substrate. The dissociation constant at 25 degrees C was 35 microns at pH 7.0. The pH dependence (pH 8.3-pH 6.4) of the dissociation constant indicates that one proton is associated with the complex formation, and picolinate protonated at the N atom binds to the reduced enzyme. Resonance Raman spectra of the complex support that picolinate in the complex is a cationic form protonated at the N atom. Nicotinate also binds to the reduced enzyme, but isonicotinate does not.

Animals↗
PubMed1985

Self-association mode of a flavoenzyme D-amino acid oxidase from hog kidney. I. Analysis of apparent weight-average molecular weight data for the apoenzyme in terms of models.

The self-association of D-amino acid oxidase apoenzyme in 0.1 M sodium pyrophosphate, pH 8.3, at 25 degrees C was studied by low-angle laser light scattering. The concentration (c) dependence of the apparent weight-average molecular weight (Mwapp) was determined over a wide concentration range of 0.04 to 6.1 mg/ml. The extrapolated Mwapp value, to zero enzyme concentration, corresponded to the Mr value of the monomer. The self-association mode of the apoenzyme was systematically explored with nonlinear least-squares analysis of the Mwapp versus c data. The simplest model that fitted the data well was a model of isodesmic indefinite self-association of the monomer with the isodesmic association constant of 0.467 +/- 0.034 liter/g. The monomer-dimer model proposed previously, but only in a low enzyme concentration range of less than 0.9 mg/ml at 5-20 degrees C (Henn, S. W., and Ackers, G. K. (1969) Biochemistry 8, 3829-3838), did not fit the Mwapp versus c data either in the limited low concentration range or in the whole concentration range examined at 25 degrees C. To test the validity of the chosen model, the observed sedimentation boundary profiles were compared with the idealized boundary profiles calculated for the better-fit models. The profile calculated with the model of the isodesmic indefinite self-association mechanism was qualitatively consistent with the observed ones. The utility of the nonlinear least-squares procedure for analyzing self-associating systems was demonstrated.

Animals↗
PubMed1985

Self-association mode of a flavoenzyme D-amino acid oxidase from hog kidney. II. Stoichiometry of holoenzyme association and energetics of subunit association.

The self-association pattern of D-amino acid oxidase holoenzyme in 0.1 M sodium pyrophosphate, pH 8.3, at 25 degrees C was examined by the low-angle laser light-scattering method. As to the results of nonlinear least-squares analysis of the apparent weight-average molecular weight (Mwapp) versus protein concentration (c) data, the following three models fitted equally well the data over the concentration range of 0.03-11.4 mg/ml: 1) the model of isodesmic indefinite self-association of the monomer where the dimerization constant differs from the isodesmic association constant, 2) the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, and 3) the model which involves the trimerization of the monomer and isodesmic indefinite self-association of the trimer. In a more limited concentration range (0.3-11.4 mg/ml), a model of isodesmic indefinite self-association of the stable dimer where the dimer does not dissociate into the monomers cannot be excluded from the above three models. Measurements with the concentration range lowered to 0.03 mg/ml enabled us to exclude unequivocally the model involving such a stable dimer and to extrapolate the Mwapp data to the Mr of the monomer at infinite dilution as in the case of the apoenzyme. The observed sedimentation boundary profiles were qualitatively consistent with the idealized boundary profiles calculated with the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, so this model is the most probable of the models examined. These results provide the first evidence that the association mode of the holoenzyme is different from that of the apoenzyme, i.e. isodesmic indefinite self-association of the monomer (Tojo, H., Horiike, K., Shiga, K., Nishina, Y., Watari, H., and Yamano, T. (1985) J. Biol. Chem. 260, 12607-12614). The overall linkage scheme, between binding of coenzyme FAD and subunit association, was considered, and the overall free energy change in each process in the scheme was calculated. The total stabilization energies of the intersubunit interaction in the holoenzyme relative to the apoenzyme were found to be -2.2 kcal/mol at the dimerization step and -0.5 kcal/mol at the step of the addition of the dimer to any 2i-mer (i = 1,2, ...).

Animals↗