Biomedical subjects

Hiroshi Ooshima

Publications and source records attributed to Hiroshi Ooshima.

8 recordsLinked to original sources

PubMed2006

Relationship between cell morphology and intracellular potassium concentration in Candida albicans.

Previously we reported that valinomycin inhibited hyphal growth and induced growth as a chain of yeast cells under hyphal growth induction conditions in Candida albicans. To elucidate the hyphal growth inhibition by valinomycin, we examined the effect of various chemicals on the morphology and found that miconazole inhibited hyphal growth as well as valinomycin: both compounds promoted the leakage of potassium from cells. Analysis of intracellular potassium suggested that hyphal cells contain potassium at high concentrations in comparison with yeast cells. Hyphal growth inhibition by valinomycin was obstructed by the addition of serum. Potassium measurement showed that the addition of serum causes an increase in intracellular potassium, suggesting that the obstruction by serum might be due to an increase in intracellular potassium. The above-mentioned results strongly suggest that the addition of valinomycin and miconazole decreased interacellular potassium and this decrease inhibited hyphal transition.

Acetylglucosamine↗

PubMed2006

Purification and characterization of enzyme responsible for N-myristoylation of octapeptide in aqueous solution without ATP and coenzyme a from Pseudomonas aeruginosa.

The enzyme that catalyzes N-acyl linkage between myristic acid and the NH(2)-terminal glycine residue of the octapeptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg-NH(2) in aqueous solution without ATP and coenzyme A was found in Pseudomonas aeruginosa. The enzyme was purified from cell-free crude extract using DEAE-Cellulose, Sephadex G-200, CM-Sephadex C-50, and hydroxyapatite column chromatographies, and then purified approximately 1900-fold with about 1.5% recovery of enzyme activity from the crude extract. Finally, the purified enzyme showed a main band on SDS polyacrylamide gel electrophoresis after staining with Coomassie Brilliant Blue. The band corresponded to a molecular mass of approximately 60 kDa. The K(m)s of the purified enzyme for the substrate myristic acid and the octapeptide were 0.36 and 2.6 mM, respectively. When myristoyl-CoA instead of myristic acid was used as the substrate for the enzyme reaction, myristoyl octapeptide could be synthesized as observed in the case of myristic acid. The K(m) of myristoyl-CoA was 0.17 mM.

Adenosine Triphosphate↗

PubMed2006

New detection method for hydrogen gas for screening hydrogen-producing microorganisms using water-soluble wilkinson's catalyst derivative.

A water-soluble color indicator was developed for the effective screening of hydrogen-producing microorganisms. This indicator consists of a coloring agent and a water-soluble derivative of Wilkinson's catalyst. Wilkinson's catalyst, Tris(triphenylphosphine) rhodium chloride, had been developed as a catalyst for the hydrogenation of olefins. We used a sulfonate of the catalyst for the hydrogenation of coloring agent in an aqueous medium. Several coloring agents, such as methyl orange, methyl red sodium, neutral red and Evan's blue, dissolved in water together with the sulfonated catalyst showed a change in color when hydrogen gas was fed into the solution by sparging at room temperature. We confirmed that methyl orange was decolorized by biologically produced hydrogen, when the photosynthetic bacterial strain Rhodobacter capsulatus ST-410 was grown in a medium containing 0.6 mM catalyst and 0.075 mM methyl orange in test tubes of 5 ml working volume.

Catalysis↗

PubMed2005

Valinomycin affects the morphology of Candida albicans.

Microbial metabolites were screened for inhibitors of hyphal growth in Candida albicans. Inhibitory activity was found among metabolites of a culture of an actinomycete, which had been isolated from soil. The active substance inhibited hyphal growth and induced growth as a chain of yeast cells under hyphal growth induction conditions. The active substance was purified and analyzed with 1H-NMR, 13C-NMR and mass spectra. The substance was identified as valinomycin, and commercial valinomycin inhibited hyphal growth as effectively as the purified metabolite. The effective concentration was from 0.49 to 62.5 microg/ml. Valinomycin also inhibited hyphal growth in other dimorphic fungi, Candida tropicalis and Aureobasidium pullulans. These results suggest that valinomycin may be a useful tool for understanding the morphological transition of dimorphic fungi.

Anti-Bacterial Agents↗

PubMed2005

Deletion of MCD 4 involved in glycosylphosphatidylinositol (GPI) anchor synthesis leads to an increase in beta-1,6-glucan level and a decrease in GPI-anchored protein and mannan levels in the cell wall of Saccharomyces cerevisiae.

Most proteins involved in the synthesis of the GPI core structure of Saccharomyces cerevisiae are essential for growth. To explore the relationship between the GPI anchor structure and beta-1,6-glucan synthesis, we screened deletion mutants in genes involved in GPI synthesis for osmotic remedial growth. Heterozygous diploid strains were dissected on medium with osmotic support and slow growth of the mcd 4 deletion mutant was observed. The mcd 4 mutant showed abnormal morphology and cell aggregation, and was hypersensitive to SDS, hygromycin B and K1 killer toxin. Incorporation of GPI cell wall proteins was examined using a GPI-Flo 1 fusion protein. The result suggested that the mcd 4 deletion causes a decrease in GPI cell wall proteins levels. The mutation also caused a decrease in mannan levels and an increase in alkali-insoluble beta-1,6-glucan and chitin levels in the cell wall.

Cell Membrane↗

PubMed2005

Analysis of chitin at the hyphal tip of Candida albicans using calcofluor white.

Staining with calcofluor white (CFW), which is known to bind chitin-rich areas of the cell wall, revealed a difference in the fluorescence intensity at the hyphal tip between Candida albicans hyphal cells that were grown in modified Lee (M-Lee) and SPG media. The fluorescence intensity at the tip increased with the addition of salts and sugar to SPG. The chitin levels per dry cell weight in cells grown in modified Lee and SPG with 1.0 M NaCl were also higher than in SPG. These results suggest that chitin synthesis at the tip of C. albicans might be activated by the addition of salts and sugar to a medium.

Benzenesulfonates↗

PubMed2004

Rot1p of Saccharomyces cerevisiae is a putative membrane protein required for normal levels of the cell wall 1,6-beta-glucan.

Although ROT1 is essential for growth of Saccharomyces cerevisiae strain BY4741, the growth of a rot1Delta haploid was partially restored by the addition of 0.6 M sorbitol to the growth medium. Rot1p is predicted to contain 256 amino acids, to have a molecular mass of 29 kDa, and to possess a transmembrane domain near its C-terminus. Candida albicans and Schizosaccharomyces pombe have Rot1p homologues with high identity that also have predicted transmembrane domains. To explore the role of Rot1p, the phenotypes of the rot1Delta haploid were analysed. Deletion of ROT1 caused cell aggregation and an abnormal morphology. Analysis of the cell cycle showed that rot1Delta cells are delayed at the G2/M phase. The rot1Delta cells were resistant to K1 killer toxin and hypersensitive to SDS and hygromycin B, suggesting that they had cell wall defects. Indeed, greatly reduced levels of alkali-soluble and -insoluble 1,6-beta-glucan, and increased levels of chitin and 1,3-beta-glucan, were found in rot1Delta cells. Furthermore, the phenotypes of rot1Delta cells resemble those of disruption mutants of the KRE5 and BIG1 genes, which show greatly reduced levels of cell wall 1,6-beta-glucan. Incorporation of glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in big1Delta and rot1Delta cells was examined using a GFP-Flo1 fusion protein. GFP fluorescence was detected both on the cell surface and in the culture medium, suggesting that, in these mutants, mannoproteins may become only weakly bound to the cell wall and some of these proteins are released into the medium. Electron microscopic analyses of rot1Delta and big1Delta cells showed that the electron-dense mannoprotein rim staining was more diffuse and paler than that in the wild-type, and that the outer boundary of the cell wall was irregular. A big1Deltarot1Delta double mutant had a growth rate similar to the corresponding single mutants, suggesting that Rot1p and Big1p have related functions in 1,6-beta-glucan synthesis.

Cell Wall↗

PubMed2003

Characterization of a Saccharomyces cerevisiae mutant with pseudohyphae and cloning of a gene complementing the mutation.

Screening for morphological mutants of a haploid strain of Saccharomyces cerevisiae was done on the basis of their cell-shape on a solid medium containing isoamyl alcohol, which causes cell elongation, to obtain information on the morphogenesis. Mutant J19, which had pseudohyphae in liquid medium even in the absence of isoamyl alcohol, had many elongated cells. Few reports exist of haploid cells growing as pseudohyphae in liquid culture. Cell-wall analysis showed that J19 had ordinary amounts of alkali-insoluble glucan and chitin, but that isoamyl alcohol in the medium caused structural changes in the cell wall. Addition of a DNA fragment that included the wild-type SCL1 gene to J19 complemented its morphological phenotype. Sequencing of J19 SCL1 showed that the glycine at position 226 in the Scl1 protein had been replaced by asparatic acid, suggesting that this mutation in the protein, a subunit of proteasomes, may be involved in the morphological change.

Cell Wall↗