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M Osumi

Publications and source records attributed to M Osumi.

At least 19 recordsLinked to original sources
PubMed2001

In situ localization of beta-glucans in the cell wall of Schizosaccharomyces pombe.

The chemical composition of the cell wall of Sz. pombe is known as beta-1,3-glucan, beta-1,6-glucan, alpha-1,3-glucan and alpha-galactomannan; however, the three-dimensional interactions of those macromolecules have not yet been clarified. Transmission electron microscopy reveals a three-layered structure: the outer layer is electron-dense, the adjacent layer is less dense, and the third layer bordering the cell membrane is dense. In intact cells of Sz. pombe, the high-resolution scanning electron microscope reveals a surface completely filled with alpha-galactomannan particles. To better understand the organization of the cell wall and to complement our previous studies, we set out to locate the three different types of beta-glucan by immuno-electron microscopy. Our results suggest that the less dense layer of the cell wall contains mainly beta-1,6-branched beta-1,3-glucan. Occasionally a line of gold particles can be seen, labelling fine filaments radiating from the cell membrane to the alpha-galactomannan layer, suggesting that some of the radial filaments contain beta-1,6-branched beta-1,3-glucan. beta-1,6-glucan is preferentially located underneath the alpha-galactomannan layer. Linear beta-1,3-glucan is exclusively located in the primary septum of dividing cells. beta-1,6-glucan only labels the secondary septum and does not co-localize with linear beta-1,3-glucan, while beta-1,6-branched beta-1,3-glucan is present in both septa. Linear beta-1,3-glucan is present from early stages of septum formation and persists until the septum is completely formed; then just before cell division the label disappears. From these results we suggest that linear beta-1,3-glucan is involved in septum formation and perhaps the separation of the two daughter cells. In addition, we frequently found beta-1,6-glucan label on the Golgi apparatus, on small vesicles and underneath the cell membrane. These results give fresh evidence for the hypothesis that beta-1,6-glucan is synthesized in the endoplasmic reticulum-Golgi system and exported to the cell membrane.

Cell Wall↗
PubMed2001

Characterization of a Schizosaccharomyces pombe mutant deficient in UDP-galactose transport activity.

In fission yeast, Schizosaccharomyces pombe, the carbohydrate components of the cell wall consist of galactomannan, unlike in Saccharomyces cerevisiae. We previously found that the disruption of gms1+, a gene encoding the UDP-galactose transporter required for the synthesis of galactomannan, led to the complete defect of cell surface galactosylation in Sz. pombe. The Deltagms1 strain is therefore useful for the analysis of physiological properties of galactose residues in Sz. pombe. The deletion strain of gms1+ was viable; however, itshowed an aberrant cell morphology and increased sensitivities to digestion with beta-glucanase and to various drugs, such as hygromycin B, sodium orthovanadate and Calcofluor white. A reduction of galactomannan layers of the cell wall in the Deltagms1 strain was observed by scanning and transmission electron microscopic analyses. The addition of osmotic stabilizer suppressed the morphologic defect of the Deltagms1 cells, while other phenotypes were weakly suppressed. The Deltagms1 (h90) strain was incapable of sexual conjugation during nutritional starvation. These results suggest that the cell surface galactosylation is required not only for non-sexual flocculation but also for sexual conjugation in Sz. pombe.

Biological Transport↗
PubMed2001

The formation of actin rods composed of actin tubules in Dictyostelium discoideum spores.

A new type of actin rod formed in both the nucleus and the cytoplasm, as well as tyrosine phosphorylation of actin, is implicated in the maintenance of dormancy and viability of Dictyostelium discoideum spores. Here the ultrastructure of the rods and their relationship to the phosphorylation of actin were examined. The rods first appeared in premature spores at the midculmination stage as bundles composed of actin tubules hexagonally cross-linked. The 13-nm-diameter bundles were composed of three actin filaments. Formation of the actin rods begins during the late culmination stage and proceeds until 2 days after completion of fruiting bodies. The physical events occur in the following order; association of several modules of bundles, close packing and decrease in diameter of actin tubules, elongation of rods across the nucleus or the cytoplasm. Actin phosphorylation levels increased at the late culmination stage and reached a maximum level 12 h later. Immediately following activation of spore germination, actin was rapidly dephosphorylated, followed shortly thereafter by the disappearance of rods. Shortened actin tubules once again became arranged in a hexagonal pattern. This hexagonal arrangement of actin tubules is possibly involved in rod formation and disappearance and does not depend upon actin phosphorylation. In contrast, rod-maturation processes may correlate with actin phosphorylation.

Actin Cytoskeleton↗
PubMed2001

Quantitative evaluation of the enhanced green fluorescent protein displayed on the cell surface of Saccharomyces cerevisiae by fluorometric and confocal laser scanning microscopic analyses.

The number of foreign protein molecules expressed on the cell surface of the budding yeast Saccharomyces cerevisiae by cell surface engineering was quantitatively evaluated using enhanced green fluorescent protein (EGFP). The emission from EGFP on the cell surface was affected by changes in pH. The amount of EGFP on the cell surface, displayed as alpha-agglutinin-fusion protein under control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, was determined at the optimum pH of 7.0. The fluorometric analysis and the image analysis by confocal laser scanning microscopy (CLSM) showed a similar number of molecules displayed on the cell surface, demonstrating that 10(4)-10(5) molecules of alpha-agglutinin-fused molecules per cell were expressed. Furthermore, the amount of fluorescent protein expressed on cells harboring a multicopy plasmid was three to four times higher than that on cells harboring the gene integrated into the genome.

Arabidopsis↗
PubMed2001

Creation of cell surface-engineered yeast that display different fluorescent proteins in response to the glucose concentration.

We have successfully created a novel yeast strain able to monitor changes in environmental conditions by displaying either green fluorescent protein (GFP) from Aequorea victoria or blue fluorescent protein (BFP), a variant of GFP, on its cell surface as a visible reporter. For the display of these fluorescent proteins on the cell surface of Saccharomyces cerevisiase, our cell-surface-engineering system was utilized. The GAPDH promoter, which is active in the presence of glucose, and the UPR-ICL promoter from Candida tropicalis, which starts to function in the presence of a reduced level of glucose, were employed simultaneously to express the GFP-encoding gene and the BFP-encoding gene, respectively. This cell-surface-engineered yeast strain emitted green fluorescence from the cell surface when sufficient glucose was present in the medium, and blue fluorescence from the same cell surface when the glucose in the medium was consumed. The fluorescent proteins displayed on the cell surface using the different promoters enabled us to monitor the concentrations of intra- and/or extracellular glucose that regulated activation or inactivation of the promoters. This novel yeast strain could facilitate the computerized control of various bioprocesses measuring emitted fluorescence.

Genetic Engineering↗
PubMed2000

Cytochemical evaluation of localization and secretion of a heterologous enzyme displayed on yeast cell surface.

A starch-utilizing Saccharomyces cerevisiae strain was constructed by cell surface engineering. Distribution of the heterologous glucoamylase-alpha-agglutinin fusion protein on the yeast cell was analyzed by indirect fluorescence microscopy using an anti-glucoamylase antibody. Most of the intense fluorescence was first localized in the small bud, then observed on the entire cell wall of the daughter and mother cells. Fluorescence also accumulated at the neck region. These observations suggest that the display of the heterologous protein on the cell surface is carried with other cell wall components to the areas in which the cell wall is newly synthesized; the distribution is controlled by the cell cycle. Then, the heterologous protein-encoding gene was expressed in a sec1 mutant, in which secretory vesicles accumulate under restrictive temperature, and the produced protein was detected by immunoelectron microscopy. Most of the gold particles that reacted with the fusion protein were not localized in vesicles but in expanding endoplasmic reticulum. This phenomenon may be due to overproduction of the heterologous protein which was designed to be displayed on the cell wall. Artificial production of heterologous protein may have caused a relative shortage of glycosyl phosphatidylinositol anchors.

Agglutinins↗
PubMed2000

Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface.

An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The GFP gene was fused with the gene encoding the C-terminal half of alpha-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase precursor protein was connected to the N-terminal of GFP. This designed gene was integrated into the TRP1 locus of the chromosome of S. cerevisiae with homologous recombination. Fluorescence microscopy demonstrated that the transformant cells emitted green fluorescence derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further verified by immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine Red-X-conjugated goat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each cell fraction obtained after the subcellular fractionation. As GFP was proved to be displayed as an active form on the cell surface, selection of promoters will endow yeast cells with abilities to respond to changes in environmental conditions, including nutrient concentrations in the media, through the emission of fluorescence.

Base Sequence↗
PubMed2000

Aberrant chloroplasts in transgenic rice plants expressing a high level of maize NADP-dependent malic enzyme.

NADP-dependent malic enzyme (NADP-ME) is a major decarboxylating enzyme in NADP-ME-type C4 species such as maize and Flaveria. In this study, chloroplastic NADP-ME was transferred to rice (Oryza sativa L.) using a chimeric gene composed of maize NADP-ME cDNA under the control of rice light-harvesting chlorophyll-a/b-binding protein (Cab) promoter. There was a 20- to 70-fold increase in the NADP-ME activity in leaves of transgenic rice compared to that in wild-type rice plants. Immunocytochemical studies by electron microscopy showed that maize NADP-ME was mostly localized in chloroplasts in transgenic rice plants, and that the chloroplasts were agranal without thylakoid stacking. Chlorophyll content and photosystem II activity were inversely correlated with the level of NADP-ME activity. These results suggest that aberrant chloroplasts in transgenic plants may be caused by excessive NADP-ME activity. Based on these results and the known fact that only bundle sheath cells of NADP-ME species, among all three C4 subgroups, have agranal chloroplasts, we postulate that a high level of chloroplastic NADP-ME activity could strongly affect the development of chloroplasts.

Chlorophyll↗
PubMed2000

Characteristic biological effects of itraconazole on L929 fibroblasts and their cell membrane.

Itraconazole (ITCZ), a triazole antifungal agent, was studied for its effects on the morphology and function of L929 fibroblasts. L929 fibroblasts were cultured for 20 h with ITCZ or one of several other triazoles (fluconazole, ketoconazole, and hydroxy-itraconazole [ITCZ-OH]) at the concentration of 0.5 microg/ml. Among these agents, only ITCZ and its metabolite ITCZ-OH markedly elongated the cells bidirectionally. Scanning electron microscopy studies showed that the surface of the elongated cells was smoother than that of the untreated cells. The viability of L929 cells cultured with 0.5 microg/ml of ITCZ for 20 h was not lowered. However, after treatment with 0.0375% sodium deoxycholate (DOC) solution, the viability of the cells treated with ITCZ, as evaluated by the 3-(4,5-dimethyl-2thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) proliferation assay or the release of lactic dehydrogenase from cytoplasm, was decreased. When L929 cells were cultured in the presence of a combination of ITCZ and vincristine, their growth was synergistically inhibited. This synergism was also observed when ITCZ was replaced by ITCZ-OH, but not by the other azoles. These findings suggest that the exposure of L929 fibroblasts to low ITCZ concentrations affects the physiological nature of their cell membrane.

Animals↗
PubMed2000

Novel actin cytoskeleton: actin tubules.

In spores of Dictyostelium discoideum three actin filaments are bundled to form a novel tubular structure and the tubules are then organized into rods. These tubular structures we will term actin tubules. Actin tubules are reconstructed from the supernatant of spore homogenates, while the usual actin filaments were bundled after incubation of supernatants from growing cells. Alpha-actinin, ABP-120 and EF-1alpha are not essential for rod formation. Cofilin is a component of the cytoplasmic rods but few cofilin molecules are included in the nuclear rods. The viability of spores lacking actin rods is very low, and the spore shape is round instead of capsular. The rods can be fragmented by pressure, indicating that the rods may be effective in absorbing physical pressure. The complex organization of actin filaments, actin tubules and rods may be required for spores to achieve complete dormancy and maintain viability.

Actin Cytoskeleton↗
PubMed1999

Ultrastructure of cell wall of the cps8 actin mutant cell in Schizosaccharomyces pombe.

A Schizosaccharomyces pombe cps8 mutant, of which the gene encodes a mutant actin with an amino acid substitution of Asp for Gly(273) [J. Ishiguro and W. Kobayashi (1996) FEBS Lett. 392, 237-241], was used to determine the role of the actin cytoskeleton in cell wall formation. In the cps8 mutant cells, atomic force microscopic and scanning electron microscopic images showed abnormal depolarized and branched morphology. Fibrous material covered a part of the surface of growing cps8 cells. Transmission electron microscopic images showed variable thickness of the cell wall due to multilayering of cell wall materials, and aberrant multisepta due to diagonal growth of the primary septum, whereas the normal primary septum grows at a right angle from the cortex. This abnormal septum formation may induce abnormality of the cell with multinuclei and/or multisepta, caused by non-separation of daughter cells. These results indicate that actin plays an important role in cell wall and septum formation.

Actins↗
PubMed1999

Effects of pressure stress on the fission yeast Schizosaccharomyces pombe cold-sensitive mutant nda3.

To investigate the influence of pressure stress on the cell cycle of Schizosaccharomyces pombe, we used a cold-sensitive nda3-KM311 mutant which arrests cell division at a step similar to the mitotic prophase, proposed by Hiraoka and colleagues (Cell 39 (1984) 349-358), under the restrictive temperature, 20 degrees C. The nda3-KM311 cells were first aerobically grown at 30 degrees C, transferred to 20 degrees C for 4 h and shifted to a permissive temperature of 36 degrees C for 15 min. The cells were treated with 100-200 MPa pressure and studied by electron and fluorescence microscopy. At 100 MPa, the nuclear membrane was damaged and the matrix of mitochondria had an electron-dense area. At 150 MPa, the nuclear membrane was broken over broad areas; numerous small vacuoles had fused into large pieces. Actin patches were concentrated in the central region and actin rings were seen in the 20 degrees C-grown cells. Even at 100 MPa, specific actin distribution was lost. Although at 100 MPa, long and fine actin cables were seen all over the cells, large actin patches and the actin rings remained in the center of the cell. They changed into thick and short cables at 150 MPa and above 200 MPa they decomposed but the actin ring was visible even with faint fluorescence. Immunoelectron microscopic observation confirmed this phenomenon.

Actins↗
PubMed1999

Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface.

The construction of a whole-cell biocatalyst with its sequential reaction has been performed by the genetic immobilization of two amylolytic enzymes on the yeast cell surface. A recombinant strain of Saccharomyces cerevisiae that displays glucoamylase and alpha-amylase on its cell surface was constructed and its starch-utilizing ability was evaluated. The gene encoding Rhizopus oryzae glucoamylase, with its own secretion signal peptide, and a truncated fragment of the alpha-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast alpha factor, respectively, were fused with the gene encoding the C-terminal half of the yeast alpha-agglutinin. The constructed fusion genes were introduced into the different loci of chromosomes of S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The glucoamylase and alpha-amylase activities were not detected in the culture medium, but in the cell pellet fraction. The transformant strain co-displaying glucoamylase and alpha-amylase could grow faster on starch as the sole carbon source than the transformant strain displaying only glucoamylase.

Agglutinins↗
PubMed1999

Amylose-like polysaccharide accumulation and hyphal cell-surface structure in relation to citric acid production by Aspergillus niger in shake culture.

When 120 mg glucose/ml was used as a carbon source, in shake culture Aspergillus niger Yang no. 2 maximally produced only 15.4 mg citric acid/ml but accumulated 3.0 mg extracellular polysaccharide/ml. The polysaccharide secreted by mycelia of Yang no. 2 in shake culture was confirmed to be an amylose-like alpha-1,4-glucan by hydrolysis analysis with acid, amylase and glucoamylase. However, in static cultures, such as semisolid and surface cultures free from physical stresses caused by shaking damage, Yang no. 2 produced more citric acid but did not accumulate the polysaccharide. With cultivation time in shake culture, the amount of extracellular polysaccharide and the viscosity of the culture broth increased. The increase of shaking speed caused a remarkable increase in the accumulation of extracellular polysaccharide, e.g. 11.2 mg extracellular polysaccharide/ml was accumulated in the medium at a shaking speed of 200 rpm. The addition of 2.0 mg carboxymethylcellulose (CMC)/ml as a viscous additive to the medium reduced drastically the amount of extracellular polysaccharide accumulated to 1.5 mg/ml, but increased the citric acid produced to 52.0 mg/ml. However, intracellular polysaccharide accumulation kept up a steady rate of 0.26 microgram/mg dried mycelium through the entire period of cultivation. The addition of 3.0 mg polysaccharide/ml purified from the culture broth to the medium at the start of a culture resulted in a decrease of extracellular polysaccharide accumulation but an increase of citric acid accumulation. From electronmicroscopic observation, cell surfaces of hyphae cultivated with CMC were smooth, while hyphae cultivated without CMC had fibrous and granular polysaccharide on the cell surface. These results suggested that Yang no. 2 secreted the polysaccharide on the cell surface as a viscous substance and/or a shock absorber to protect itself from physical stresses caused by shaking damage in shake culture.

Adaptation, Physiological↗
PubMed1999

Three-dimensional analysis of protein aggregate body in Saccharomyces cerevisiae cells.

Modified genes of peroxisomal isocitrate lyase of Candida tropicalis (CT-ICL) were constructed and expressed in Saccharomyces cerevisiae cells. We observed subcellular localization of expressed products of the mutant CT-ICL genes by immunoelectron microscopy. An unknown structure termed a protein aggregate body (PAB) storing the expressed product was observed in cytoplasm in various mutants (Kamasawa et al. (1996) J. Electron Microsc. 45: 491-497). We chose two typical cells harbouring the mutant ICL genes delta 550 and delta 237-339 to analyse the ultrastructure and three-dimensional (3D) structure of PABs. The PABs had a homogeneous matrix with a wavy periphery in the cell image using a high-pressure freezing fixation method. Although PABs could not be separated from the cytoplasm or mitochondria under a confocal fluorescence microscope, 3D reconstruction of serial electron micrographs clearly showed the PAB was an independent structure of varying size and had the shape of an incomplete sphere. A cell was sometimes observed to have multiple PABs.

Candida↗
PubMed1999

The effects of various GTP analogues on microtubule assembly.

We synthesized 27 GTP analogues with modification or substitution at positions C2, C6, C8 and ribose moiety to investigate their effect on microtubule (Mt) assembly. It was found that C2 and C6 are both functional for the analogues supporting Mt assembly. It was surprising to find that 2-amino- ATP (n2ATP) substantially supports assembly, and that the appearance of the assembled Mts was indistinguishable from those assembled in the standard GTP assembly buffer solution. Furthermore, 2-amino dATP and dGTP are even more potent than GTP in supporting assembly. The substitution of oxo group at C6 with reactive thiol largely reduced the activity of the analogue to support assembly. When free rotation of the glycosidic linkage of GTP was blocked by the introduction of sulfur atom between C8 and C2' of ribose moiety, it resulted in total suppression of assembly. Purine nucleoside triphosphate was found to support assembly better than GTP, and even more efficient was 2-amino purine nucleoside triphosphate. Interestingly, their deoxy-type analogues were totally inhibitory. Although 2-amino 8-hydroxy ATP and other analogues supported assembly much better than did GTP, their diphosphate analogues were totally incapable of supporting assembly. Finally, bulky fluorescent probes were introduced at C3' of ribose moiety (Mant-8-Br-GTP or Mant-GTP) to visualize the fluorescent signal in assembled Mts. Even in this case, the number of most protofilaments was found to be 14, consistent with that found in Mts assembled in GTP standard buffer solution.

Adenine Nucleotides↗
PubMed1999

[Comparison of the newly developed MB redox system with mycobacteria growth indicator tube (MGIT) and 2% Ogawa egg media for recovery of mycobacteria in clinical specimens].

The rate of recovery and the mean time to detection of mycobacteria in clinical specimens were determined in a newly-developed MB Redox system based on liquid medium, and the results were compared with those of MGIT and 2% Ogawa egg media. From 587 sputum specimens processed, totally 203 mycobacterial isolates were detected, of which 177 (87.2%) with MB Redox, 185 (91.1%) with MGIT and 133 (65.6%) with 2% Ogawa medium. The difference in the percentages of positive cultures between either of the two liquid media and 2% Ogawa medium was significant (p < 0.0001). The mean time to detection of the Mycobacterium tuberculosis complex was 17.5 days with MB Redox, 18.7 days with MGIT, and 26.2 days with 2% Ogawa medium. The contamination rates were 1.5, 1.7, and 4.1% for MB Redox, MGIT, and 2% Ogawa medium, respectively. In conclusion, both MB Redox and MGIT systems, based on liquid medium, are more efficient than 2% Ogawa medium for the recovery of mycobacteria in clinical specimens.

Bacteriological Techniques↗