Biomedical subjects

Masayuki Azuma

Publications and source records attributed to Masayuki Azuma.

17 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

Deletion of the CaBIG1 gene reduces beta-1,6-glucan synthesis, filamentation, adhesion, and virulence in Candida albicans.

The human fungal pathogen Candida albicans is able to change its shape in response to various environmental signals. We analyzed the C. albicans BIG1 homolog, which might be involved in beta-1,6-glucan biosynthesis in Saccharomyces cerevisiae. C. albicans BIG1 is a functional homolog of an S. cerevisiae BIG1 gene, because the slow growth of an S. cerevisiae big1 mutant was restored by introduction of C. albicans BIG1. CaBig1p was expressed constitutively in both the yeast and hyphal forms. A specific localization of CaBig1p at the endoplasmic reticulum or plasma membrane similar to the subcellular localization of S. cerevisiae Big1p was observed in yeast form. The content of beta-1,6-glucan in the cell wall was decreased in the Cabig1Delta strain in comparison with the wild-type or reconstituted strain. The C. albicans BIG1 disruptant showed reduced filamentation on a solid agar medium and in a liquid medium. The Cabig1Delta mutant showed markedly attenuated virulence in a mouse model of systemic candidiasis. Adherence to human epithelial HeLa cells and fungal burden in kidneys of infected mice were reduced in the Cabig1Delta mutant. Deletion of CaBIG1 abolished hyphal growth and invasiveness in the kidneys of infected mice. Our results indicate that adhesion failure and morphological abnormality contribute to the attenuated virulence of the Cabig1Delta mutant.

Animals↗

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↗

PubMed2006

Cepharanthin, a biscoclaurine alkaloid, prevents destruction of acinar tissues in murine Sjögren's syndrome.

OBJECTIVE: Our previous study suggested that suppression by cepharanthin of tumor necrosis factor-a (TNF-a)-induced matrix metalloproteinase-9 (MMP-9) could prevent destruction of the acinar structure in the salivary glands of patients with Sjögren's syndrome (SS). In this study, we observed that in vivo administration of cepharanthin prevented severe damage to acinar tissues in the murine model of human SS. METHODS: Cepharanthin was intraperitoneally administered to thymectomized female NFS/sld mice. Inflammatory lesions in the salivary and lacrimal glands were then examined histologically. Expression of phosphorylated IkB-a, MMP-9, and type IV collagen was analyzed immunohistochemically. The apoptotic cell death of acinar cells was determined. RESULTS: Although extensive mononuclear cell infiltration and destruction of acinar tissue in salivary and lacrimal glands were observed in control mice, significant improvement of these lesions was evident in mice treated with cepharanthin. Immunohistochemical analysis revealed that p65, phosphorylated IkB-a, and MMP-9 were more strongly stained in the acinar cells of control mice than in cepharanthin-treated mice. Although no staining for type IV collagen was observed in the acinar tissues of control mice, continuity of staining for type IV collagen was observed in acinar tissues of cepharanthin-treated mice. Destruction of acinar tissues was attributed to the induction of apoptosis, suggesting that cepharanthin inhibits apoptosis by suppressing phosphorylation of IkB-a, followed by prevention of MMP-9 activation. CONCLUSION: Our findings suggest that cepharanthin may be a promising agent for use in preventing destruction of acinar tissues in murine SS.

Alkaloids↗

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

Expression of aquaporin-5 in and fluid secretion from immortalized human salivary gland ductal cells by treatment with 5-aza-2'-deoxycytidine: a possibility for improvement of xerostomia in patients with Sjögren's syndrome.

The aim of the present study was to investigate the possibility that ductal cells, which preferentially survive and/or proliferate in Sjögren's syndrome (SS) salivary glands of patients with SS, could acquire the functional expression of membrane water channel aquaporin-5 (AQP5). Thus, in this study, we demonstrate that an immortalized normal human salivary gland ductal cell (NS-SV-DC) line, lacking the expression of AQP5, acquires AQP5 gene expression in response to treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA demethylating agent. Confocal microscopic analysis revealed the localization of AQP5 expression mainly at the apical and lateral sides of the plasma membrane. The expressed AQP5 protein was functionally active because AQP5 expression resulted in a significant increase in the osmotically directed net fluid rate across monolayers of NS-SV-DC cells. By the analysis of bisulfite sequencing of CpG islands in the AQP5 promoter, hypermethylation within the consensus Sp1-binding sites was commonly observed in parental cell clones, whereas demethylation at the CGs, one in the second consensus Sp1 element and the other outside of the third consensus Sp1 element in the AQP5 promoter, was detected in NS-SV-DC cells after treatment with 5-Aza-CdR. By analyzing the luciferase activity of transfected AQP5 promoter vectors, it became evident that demethylation at the CGs cooperatively functions between these two sites to induce AQP5 expression. Our data, therefore, suggest that treatment of ductal cells with 5-Aza-CdR could result in the expression of the AQP5 gene, thereby leading to increased fluid secretion from ductal cells in SS salivary glands.

Aquaporin 5↗

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

Enhanced radiosensitization and chemosensitization in NF-kappaB-suppressed human oral cancer cells via the inhibition of gamma-irradiation- and 5-FU-induced production of IL-6 and IL-8.

We examined the mechanisms underlying the enhancement of radiosensitivity and chemosensitivity to gamma-irradiation (IR) and 5-Fluorouracil (5-FU) in human oral carcinoma cells (B88) in which NF-kappaB activity was constitutively suppressed. Three super-repressor form of IkappaBalpha cDNA-transfected cell (B88mI) clones and 1 empty vector-transfected cell clone (B88neo) have been established. We found that the tumor-forming ability in nude mice of B88mI clones was significantly lower than that of B88 or B88neo. This suppressed ability in tumorigenicity was attributed to the down-regulation of the expression of interleukin (IL)-1alpha, IL-6, IL-8, vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 in B88mI cell clones as compared to that in B88 or B88neo. IR and 5-FU induced a much greater degree of apoptosis, as evidenced by flow cytometry analysis and annexin V staining, in B88mI cell clones than in B88 or B88neo. When tumor-bearing nude mice were treated with IR or 5-FU, the suppression of tumor growth was significantly augmented in B88mI cell clones as compared to that in B88 or B88neo. ELISA analysis indicated that although a remarkable increase in production of IL-6 and IL-8 was observed in B88 and B88neo after in vitro exposure to IR or treatment with 5-FU, radiotherapy and chemotherapy-induced production of these cytokines was significantly suppressed in B88mI cell clones. These findings suggest that production of angiogenic factors and growth factors in response to radiotherapy and chemotherapy is a principal mechanism of inducible radioresistance and chemoresistance in human oral cancers, and establish the inhibition of NF-kappaB as a rational approach to improve conventional radiotherapy and chemotherapy outcomes.

Animals↗

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↗

PubMed2004

Potentiation of induction of apoptosis by sequential treatment with cisplatin followed by 5-fluorouracil in human oral cancer cells.

We examined the mechanism involved in the induction of apoptosis in human oral cancer (B88) cells with 5-fluorouracil (5-FU) and cisplatin (CDDP) combination. Three different combination treatment sequences were evaluated: i) 5-FU administered simultaneously with CDDP for 72 h (sequence I); ii) CDDP administered for 24 h before 5-FU treatment for 48 h (sequence II); and iii) 5-FU administered for 24 h before CDDP treatment for 48 h (sequence III). When combining the two drugs at doses 40% of their respective cytotoxicity, the growth-suppressing ratios were 49, 55, and 40% in sequences I, II, and III, respectively. Caspase 3 was significantly activated in sequence II as compared to its level of activation in sequence I or III. The mitochondrial release of cytochrome c was much greater in sequence II than in sequence III. In addition, activation of caspase 8 was most strongly activated in sequence II as compared with sequences I and III. Activation of caspase-9 was also observed in sequence II, and, to a lesser extent, in sequence III. Finally, although Bcl-2 was reduced in sequence II, no significant change was observed in sequence III. Accordingly, the data presented here demonstrate that sequence II showing a significant increase in the induction of apoptosis of cancer cells, is superior to sequences I and III.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2003

A Saccharomyces cerevisiae genome-wide mutant screen for altered sensitivity to K1 killer toxin.

Using the set of Saccharomyces cerevisiae mutants individually deleted for 5718 yeast genes, we screened for altered sensitivity to the antifungal protein, K1 killer toxin, that binds to a cell wall beta-glucan receptor and subsequently forms lethal pores in the plasma membrane. Mutations in 268 genes, including 42 in genes of unknown function, had a phenotype, often mild, with 186 showing resistance and 82 hypersensitivity compared to wild type. Only 15 of these genes were previously known to cause a toxin phenotype when mutated. Mutants for 144 genes were analyzed for alkali-soluble beta-glucan levels; 63 showed alterations. Further, mutants for 118 genes with altered toxin sensitivity were screened for SDS, hygromycin B, and calcofluor white sensitivity as indicators of cell surface defects; 88 showed some additional defect. There is a markedly nonrandom functional distribution of the mutants. Many genes affect specific areas of cellular activity, including cell wall glucan and mannoprotein synthesis, secretory pathway trafficking, lipid and sterol biosynthesis, and cell surface signal transduction, and offer new insights into these processes and their integration.

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↗

PubMed2002

Saccharomyces cerevisiae Big1p, a putative endoplasmic reticulum membrane protein required for normal levels of cell wall beta-1,6-glucan.

Deletion of Saccharomyces cerevisiae BIG1 causes an approximately 95% reduction in cell wall beta-1,6-glucan, an essential polymer involved in the cell wall attachment of many surface mannoproteins. The big1 deletion mutant grows very slowly, but growth can be enhanced if cells are given osmotic support. We have begun a cell biological and genetic analysis of its product. We demonstrate, using a Big1p-GFP fusion construct, that Big1p is an N-glycosylated integral membrane protein with a Type I topology that is located in the endoplasmic reticulum (ER). Some phenotypes of a big1Delta mutant resemble those of strains disrupted for KRE5, which encodes another ER protein affecting beta-l,6-glucan levels to a similar extent. In a big1Deltakre5Delta double mutant, both the growth and alkali-soluble beta-l,6-glucan levels were reduced as compared to either single mutant. Thus, while Big1p and Kre5p may have similar effects on beta-l,6-glucan synthesis, these effects are at least partially distinct. Residual beta-l,6-glucan levels in the big1Deltakre5Delta double mutant indicate that these gene products are unlikely to be beta-l,6-glucan synthase subunits, but rather may play some ancillary roles in beta-l,6-glucan synthase assembly or function, or in modifying proteins for attachment of beta-l,6-glucan.

Amino Acid Sequence↗

PubMed2002

Stable inhibition of NF-kappa B in salivary gland cells does not enhance sensitivity to TNF-alpha-induced apoptosis due to upregulation of TRAF-1 expression.

The transcription factor NF-kappa B inhibits the apoptotic response induced by TNF-alpha. However, in salivary gland cell clones (ACMT-6 and ACMT-7) in which NF-kappa B activation was suppressed by introduction of a super-repressor form of I kappa B-alpha cDNA, TNF-alpha did not cause apoptosis. Thus, to investigate the molecular mechanism involved in the unresponsiveness of these cell clones to TNF-alpha-induced apoptosis, we examined the effect of TNF-alpha on the expression of antiapoptotic proteins, including TNF receptor-associated factor (TRAF)-1, TRAF-2, cellular inhibitor of apoptosis protein (cIAP)-1, and cIAP-2. Here we show that expression of TRAF-1 was commonly detected by treatment with TNF-alpha in ACMT-6, ACMT-7, and an empty vector-transfected cell clone (ACpRc-1) and that downregulation of TRAF-1 protein by either treatment with an antisense oligonucleotide or introduction of an antisense plasmid resulted in the induction of apoptosis in these cell clones. Our results, therefore, suggest that one of the mechanisms by which cells acquire resistance to TNF-alpha-induced apoptosis is a TNF-alpha induction of TRAF-1.

Apoptosis↗

PubMed2002

Suppression of tumor necrosis factor alpha-induced matrix metalloproteinase 9 production in human salivary gland acinar cells by cepharanthine occurs via down-regulation of nuclear factor kappaB: a possible therapeutic agent for preventing the destruction of the acinar structure in the salivary glands of Sjögren's syndrome patients.

OBJECTIVE: Our previous results suggested that suppression of tumor necrosis factor alpha (TNFalpha)-induced matrix metalloproteinase 9 (MMP-9) could prevent the destruction of acinar tissue in the salivary glands of patients with Sjögren's syndrome (SS). The present study was undertaken to investigate the effect of cepharanthine on the suppression of TNFalpha-induced MMP-9 production in NS-SV-AC, an SV40-immortalized normal human acinar cell clone. METHODS: After pretreatment with or without cepharanthine, NS-SV-AC cells were treated with TNFalpha alone or with a combination of TNFalpha and cepharanthine. The expression of MMP-9 was then examined at the protein and messenger RNA levels. In addition, the effect of cepharanthine on the morphogenetic behavior of NS-SV-AC cells cultured on type IV collagen-coated dishes in the presence of TNFalpha was examined. RESULTS: Although TNFalpha induced the production of MMP-9 in NS-SV-AC cells, this production was greatly suppressed when cells were pretreated with cepharanthine, followed by treatment with both TNFalpha and cepharanthine. In addition, cepharanthine suppressed the TNFalpha-stimulated NF-kappaB activity by partly preventing the degradation of IkappaBalpha protein in NS-SV-AC cells. When NS-SV-AC cells were seeded on type IV collagen-coated dishes in the presence of both TNFalpha and plasmin, type IV collagen interaction with the cells was lost and the cells entered apoptosis. However, pretreatment with cepharanthine restored the aberrant in vitro morphogenesis of the NS-SV-AC cells. CONCLUSION: These results may indicate a molecular mechanism by which cepharanthine is able to protect against the destruction of the acinar structure in salivary glands from patients with SS.

Alkaloids↗