Search PubMed⌕ Search

Biomedical subjects

A Takeoka

Publications and source records attributed to A Takeoka.

At least 19 recordsLinked to original sources
PubMed2000

Identification of an alpha-tubulin mutant of fission yeast from gamma-tubulin-interacting protein screening: genetic evidence for alpha-/gamma-tubulin interaction.

gamma-Tubulin has been determined to be a central element of microtubule nucleation and, thus, indispensable for cellular organization of the microtubule. Utilizing the fact that human gamma-tubulin can function in the fission yeast Schizosaccharomyces pombe, we have generated a unique mutant screening procedure which can specifically select mutants of genes encoding gamma-tubulin-interacting proteins. One of the isolated mutants, cs76, turned out to carry a mutation in the alpha 1-tubulin gene (nda2(+)). This result suggests a direct interaction between the alpha- and gamma-tubulins. We located the mutation site in the nda2 gene and characterized the mutant phenotype. Our results demonstrate the importance of the alpha-/gamma-tubulin interaction in microtubule nucleation and should complement previous knowledge.

Alleles↗
PubMed1999

Lethal level overexpression of gamma-tubulin in fission yeast causes mitotic arrest.

gamma-Tubulin is a member of the tubulin superfamily and plays essential roles in microtubule nucleation. While the level of other tubulins, alpha- and beta-tubulin, is strictly regulated in higher eukaryotes and overexpression of beta-tubulin is toxic in yeasts, gamma-tubulin can be overexpressed by fivefold in fission yeast without any obvious defect in growth. Extreme overexpression of gamma-tubulin in mammalian cells caused growth arrest; however, the exact level of gamma-tubulin and the critical level of gamma-tubulin necessary for growth defect were undetermined. We have constructed strains that over- or underexpress gamma-tubulin by placing the gamma-tubulin gene under the control of the inducible nmt1 promoter and its variants. Among these, the weakest promoter was able to produce enough gamma-tubulin to support normal growth when its expression was induced. A strain in which the gamma-tubulin gene was placed under the control of the strongest inducible promoter achieved 160-fold overexpression of gamma-tubulin and its growth was suppressed. Normal cytoplasmic microtubules were mostly lost in gamma-tubulin overexpressing cells and gamma-tubulin was accumulated around the periphery of nuclei. Many of the cells were arrested in mitosis. A small fraction of cells did proceed to undergo nuclear division; however, its process looked either significantly deterred or abnormal. Our results presented here suggest that excess gamma-tubulin disrupts the microtubule array and significantly deters the formation of the mitotic spindle, most likely because of random nucleation of microtubules from excess gamma-tubulin in the cytoplasm.

Blotting, Western↗
PubMed1999

Acknowledgment

Explore the source record for details and available documents.

Journal Article↗
PubMed1991

Purification and characterization of S layer proteins from Clostridium difficile GAI 0714.

The S layer of Clostridium difficile GAI0714 was shown to be composed of two proteins, of 32 kDa and 45 kDa, as determined by SDS-PAGE. The two proteins were extracted with 8 M-urea (pH 8.3) from a cell wall preparation and purified by DEAE-Sepharose CL-6B chromatography followed by HPLC gel filtration. When solubilized in 0.1 M-urea, both proteins appeared to exhibit dimeric forms, with respective molecular masses of about 61 kDa and 99 kDa, upon HPLC. Although the amino acid compositions of the two proteins differed from each other, both proteins had a high content of acidic amino acids, very low contents of histidine and methionine, and no cysteine. The 32 kDa protein exhibited multiple isoelectric forms (pI 3.7-3.9), whereas the 45 kDa protein had a single form (pI 3.3). Radioiodination and immunogold labelling revealed that both proteins were exposed evenly over the entire cell surface. Based on immunodiffusion analysis using monospecific antiserum raised to the individual proteins, there was no antigenic relationship between the two proteins. Furthermore, immunoblot analysis showed that the antigenicity of the 32 kDa protein appeared to be strain specific, whereas that of the 45 kDa protein appeared to be group specific.

Amino Acids↗
PubMed1991

Demonstration and characterization of the cell wall carbohydrate and protein antigens from Clostridium botulinum type E Saroma.

Two different cell wall antigens, carbohydrate (CHO) and protein (P), from Clostridium botulinum type E Saroma were extracted with sodium dodecyl sulfate (SDS) and purified by chromatography on DEAE-Sepharose CL-6B and Sephadex G-75 or G-100. The CHO antigen was composed of glucose, galactose, glucosamine, galactosamine, alanine and phosphorus with a molar ratio of 1.5:1.5:0.25:0.25:1:1. The P antigen was an acidic protein with a molecular weight of 60 kDa, in which the major amino acids were aspartate, glutamate and serine, while the minor ones were cysteine and methionine. Thin sections of the intact or SDS-extracted cells of the organism demonstrated that the cell wall was composed of a two-layered structure, an inner layer about 20 nm thick and an outer layer about 10 nm, and by the extraction with SDS, the outer layer disappeared from the cell surface, leaving the inner layer. Immunogel diffusion tests demonstrated that either CHO antigen or P antigen was common among the nonproteolytic strains of C. botulinum.

Amino Acids↗
PubMed1991

Purification and partial characterization of a soluble hemagglutinin from Yersinia pseudotuberculosis.

A soluble hemagglutinin (HA) produced by Yersinia pseudotuberculosis strain Inoue, serotype 5b, was purified by ammonium sulfate precipitation, gel filtration on Sepharose CL-6B and high performance liquid chromatography on a DEAE-5PW anion-exchange column. The purified HA was a 14.5 kDa protein with an isoelectric point of 4.5. Amino acid analysis indicated that the HA consisted of 133 residues, corresponding to the molecular weight of 14,100. The amino acid sequence of N-terminal 38 amino acid residues showed no homology with that of several fimbrial proteins from Escherichia coli.

Amino Acid Sequence↗
PubMed1991

S layer protein of Clostridium tetani: purification and properties.

S layer protein of Clostridium tetani strain AO 174, a nontoxigenic derivative of strain Harvard A 47, was prepared from the cell walls by 4 M urea extraction and purified by DEAE-Sepharose CL-6B chromatography followed by a combination of anion-exchange chromatography and reverse-phase chromatography using an HPLC system. The molecular weight of the S layer protein was estimated to be 140 kilodaltons (kDa) by SDS-PAGE. The amino acid composition of the 140 kDa protein was very similar to those of S layer proteins from the other bacterial species: it was rich in acidic amino acid and lacked cysteine. Also, the protein was unique in its extremely low content of proline (0.02 to 0.03 mol%). Multiple isoelectric forms ranging from pH 4.0 to 4.5 were observed in the purified preparation. Immunodiffusion analysis showed that the 140 kDa protein was a common antigen to the three strains of C. tetani tested.

Amino Acids↗
PubMed1988

A nasal allergy model developed in the guinea pig by intranasal application of 2,4-toluene diisocyanate.

An experimental model of nasal allergy has been developed in guinea pigs by intranasal application of 2,4-toluene diisocyanate (TDI). A 10% TDI solution in ethyl acetate was painted onto the nasal vestibuli of the animals once a day for 5-10 days. During the course of repeated application of TDI, the number of animals which secreted rhinorrhea containing eosinophils increased. Morphological survey of the nasal mucosa showed infiltration of eosinophils and some other changes indicative of acute inflammation. Moreover, mast cells were found not only in the subepithelial connective tissue but also in the epithelial layer. Nasal mucus obtained from the mucosa has been found to be an effective test material for studies of nasal allergy. A striking decrease of specific granules was found in some mast cells contained in the mucus. In parallel with the symptomatology, biochemical and serological studies suggested the involvement of type I allergy in the experimental system; TDI-specific histamine release from the nasal mucosa and positive passive cutaneous anaphylaxis were found 3 weeks after the application of TDI.

Administration, Intranasal↗
PubMed1987

Purification and immunochemical properties of a wall protein antigen from Clostridium difficile ATCC 11011.

A wall-surface protein antigen, designated 32K antigen, was extracted from whole cells of Clostridium difficile strain ATCC 11011 with phosphate buffered saline and purified by ion-exchange chromatography, gel filtration, and chromatofocusing. The 32K antigen preparation was determined to be highly homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of the antigen was characteristic in the predominance of the acidic amino acids, the very low contents of methionine and histidine, and the lack of cysteine. A monomeric molecular weight of the 32K antigen was estimated to be 32,000 by SDS-PAGE and 30,200 by sedimentation equilibrium. The antigen exhibited two isoelectric forms (IP, 4.12 and 3.96). Neither carbohydrate nor phosphorus was detectable in the antigen. The antigen was relatively resistant to trypsin but sensitive to pepsin. Immunoblot analysis of the wall proteins isolated from other strains of C. difficile probed with monospecific antiserum against the antigen from ATCC 11011 showed that the antigenicity of 32K wall protein was common among some of the strains containing 32K wall proteins.

Amino Acids↗
PubMed1987

Contact sensitivity induced in mice by methylene bisphenyl diisocyanate.

An experimental model of contact sensitivity has been developed in C57BL/6 mice using methylene bisphenyl diisocyanate (MDI), a raw material of polyurethane resins. Sensitization through a single epidermal application of 1% MDI solution in ethyl acetate to the backs of the mice resulted in marked ear swelling. The time course of the swelling was characteristic of delayed-type hypersensitivity and the increment of the ear thickness was compatible with that induced by toluene diisocyanate (TDI). Passive transfer of the MDI-induced contact sensitivity was successfully achieved using lymphocytes from the lymph nodes of MDI-sensitized syngeneic mice, and the effector cells were found to be T cells. Cross reaction between MDI and TDI has shown that MDI is not only a potent contact sensitizer but also can form a contact sensitizer group together with TDI.

Administration, Cutaneous↗
PubMed1985

Solubilization and partial properties of receptor substance for bacteriophage alpha 2 induced from Clostridium botulinum type A 190L.

Bacteriophage alpha 2, one of the two inducible phages from Clostridium botulinum type A 190L, had a latent period of 55 min and an average burst size of 75 in C. botulinum type A Hall used as the host bacterium. The phage particles were adsorbed on the cell walls extracted with hot trichloroacetic acid (TCA-walls). The receptor substance for the phage was solubilized from the TCA-walls with Achromopeptidase and fractionated by gel filtration on Sephadex G-150. The fraction having the highest level of receptor activity for the phage contained large amounts of muramic acid and glucosamine. Both authentic muramic acid and glucosamine significantly inactivated the phage, whereas glucose, galactose, L-and D-alanine, diaminopimeric acid, or D-glutamic acid did not exhibit similar activity. There results strongly suggest that the receptor site for phage alpha 2 is closely associated with glycan moieties of the cell wall peptidoglycan.

Bacteriophages↗
PubMed1984

Leiomyosarcoma of the breast--a case report and an electron microscopic study.

We treated a fifty six-year-old woman with leiomyosarcoma of the breast. Light microscopy showed typical findings of leiomyosarcoma and electron microscopy confirmed the smooth muscle origin of the tumor. The patient is well without evidence of metastases or local recurrence of the tumor fifty-five months after radical mastectomy.

Basement Membrane↗
PubMed1983

Purification and characterization of a wall protein antigen from Clostridium botulinum type A.

A wall surface protein, designated antigen S, was extracted from Clostridium botulinum type A strain 190L with 0.1% Brij 58-2 M LiCl and purified sequentially by acetone pecipitation, ion-exchange chromatography, hydroxyapatite chromatography, chromatofocusing, and gel filtration. Crossed immunoelectrophoresis of the purified antigen S preparation against homologous multispecific antiserum to whole cells revealed only a single precipitin line. Antigen S had an apparent molecular weight of about 195,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigen was composed predominantly of acidic amino acid residues, and the isoelectric point was estimated to be at pH 4.75. Tryptic digestion of antigen S destroyed antigenic activity and produced one major polypeptide fragment with a molecular weight of about 44,000. Indirect immunoferritin labeling showed that antigen S was located on the outer layer of the cell wall.

Amino Acids↗