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Biomedical subjects

K Shishido

Publications and source records attributed to K Shishido.

At least 73 records · Page 4Linked to original sources
PubMed1988

Alpha-factor-directed synthesis of Bacillus stearothermophilus alpha-amylase in Saccharomyces cerevisiae.

Promoter and leader sequence of Bacillus stearothermophilus alpha-amylase gene were removed and the gene was joined in-frame to sequences encoding the leader region of Saccharomyces cerevisiae mating pheromone alpha-factor on plasmid p69A (a hybrid of pBR322 and S. cerevisiae 2-microns plasmid). S. cerevisiae cells were transformed with plasmids containing the hybrid genes, obtaining yeast transformants which exhibit a significant extra-cellular amylolytic activity in solid medium, but not in liquid medium. Levels of alpha-amylase activity in solid medium were found to depend on the mode of fusion of the alpha-amylase gene to the alpha-factor leader region.

Amino Acid Sequence↗
PubMed1988

Molecular cloning of a tetracycline-resistance determinant from Bacillus subtilis chromosomal DNA and its expression in Escherichia coli and B. subtilis.

Bacillus subtilis GSY908 DNA fragments (5.1 and 4.4 kilobase pairs (kb)) containing a tetracycline-resistance determinant were cloned in Escherichia coli using a shuttle plasmid vector pLS353. Restriction endonucelase analysis showed that the 4.4 kb fragment is a spontaneous deletion derivative of the 5.1 kb fragment. E. coli tetracycline-resistance transformants carrying pLS353 with the 5.1 kb fragment (named pTBS1) and that with 4.4 kb fragment (pTBS1.1) could grow at tetracycline concentrations up to 80 and 50 micrograms per ml, respectively. B. subtilis MI112 and RM125 were transformed by pTBS1, resulting in isolation of transformants of MI112 maintaining pTBS1 and RM125 maintaining either pTBS1 or pTBS1.1. Maximum tetracycline concentrations permitting growth of plasmidless MI112 and MI112 with pTBS1 were 4 and 10 micrograms per ml, respectively, while those of plasmidless RM125, RM125 with pTBS1 and RM125 with pTBS1.1 were 7, 50 and 80 micrograms per ml, respectively. It was interesting to note that the tetracycline-resistance level in E. coli conferred by the 5.1 kb fragment is higher than that conferred by the 4.4 kb fragment, but in B. subtilis the 4.4 kb fragment, in contrast, confers a higher level of tetracycline resistance. The level of tetracycline resistance in B. subtilis conferred by the cloned determinant clearly depends on the host strain. The tetracycline resistance conferred by the cloned determinant was associated with decreased accumulation of the drug into the cells. However, it was constitutive in E. coli, but inducible in B. subtilis. The cloned tetracycline-resistance determinant was detected specifically on the chromosome of B. subtilis Marburg 168 derivatives.

Bacillus subtilis↗
PubMed1988

Reevaluation of analytical procedures on quantitation of urinary protein in rats.

In this study, the original method (Lowry method) of Lowry et al. (1951), its modified method (Lowry-TCA method) with protein precipitation by trichloroacetic acid (TCA) and the new method (BCA method) with bicinchoninic acid reaction, were reexamined as to whether these three methods were applicable to urinary protein quantitation of rats. Because of the presence of the urinary interfering substances, the quantitations in the Lowry- and BCA methods were found to be overestimated more than 3 times as large as in the Lowry-TCA method. When the urine sample was dialyzed against water and determined by the Lowry- and BCA methods, more than 24 hours were found to be needed in order to completely exclude the interfering substances from the urine. From these findings, the Lowry-TCA method is recommended for quantitation of urinary protein in rats.

Animals↗
PubMed1988

Clinical study of sudden deafness with special reference to onset.

In a comparative study, patients with sudden deafness were classified into four groups according to type of onset. The results showed that those who noticed deafness occurring when awakening had a better prognosis than others, particularly those who found themselves already suffering from deafness on awakening. These findings suggest that among patients who have deafness on awaking, the disorder may not be sudden deafness in sensu strictu.

Adult↗
PubMed1987

Increased production of a knotted form of plasmid pBR322 DNA in Escherichia coli DNA topoisomerase mutants.

Plasmid pBR322 prepared from Escherichia coli strains carrying deletion of the DNA topoisomerase I gene (delta topA) with a compensatory mutation of the DNA gyrase gene (gyrA or gyrB) and from their TopA+ transductants was analyzed by agarose gel electrophoresis followed by electron microscopy, and compared with that from isogenic wild-type strains. It was found that about 1% of the plasmid DNA molecules was a knotted species in the topA+ gyr+ strains W3110 and DM4100, while strains DM750 (delta topA gyrA224), DM800 (delta topA gyrB225), SD275 (topA+ gyrA224) and SD108 (topA+ gyrB225) produced six to ten times as much knotted DNA as the topA+ gyr+ controls. The results suggest that the increased production of knotted pBR322 DNA is closely related to mutations of the gyrase genes.

DNA Topoisomerases, Type I↗
PubMed1986

Purification of S1 nuclease to homogeneity and its chemical, physical and catalytic properties.

An S1 nuclease preparation was used to purify the enzyme to homogeneity. The enzyme had an isoelectric point of 4.2, and a high content of hydrophobic amino acids, especially tyrosine. It exhibited low 3'-ribonucleotidase activity. Circular dichroism analysis suggested that the contents of alpha-helix, beta-structure and random coil are 25%, 31% and 44%, respectively. The enzyme contained about 3 g atoms Zn/mol and the removal of Zn from the enzyme by addition of EDTA resulted in disruption of its secondary structure with resultant inactivation. From Con A-Sepharose chromatography, we suggest that the enzyme is a high-mannose glycoprotein. After treatment with endo-beta-N-acetylglucosaminidase H under moderate conditions, a small part of the enzyme was converted to a form lacking the sugar side chain. This form of the enzyme was as thermostable as the parent enzyme, suggesting that the sugar side chain may not be involved in thermostability of the enzyme.

Amino Acids↗
PubMed1986

Cloning of Lentinus edodes mitochondrial DNA fragment capable of autonomous replication in Saccharomyces cerevisiae.

Mitochondrial (mt) DNA of the higher basidiomycetes Lentinus edodes with a molecular weight of about 69 kb was partially digested with Sau3AI, cloned with plasmid YIp32 (a hybrid of pBR322 and the yeast leu2 gene) and analyzed for sequences capable of autonomous replication (ARSs) in the eukaryote Saccharomyces cerevisiae. One recombinant plasmid was isolated which contained 3.2 kb fragment of the mtDNA with ARS activity. This plasmid (named pSK52) exhibited a high-frequency yeast transformation and was found to be maintained within the cell as an extrachromosomal element. The stability and copy number properties of pSK52 were similar to those of the recombinant plasmid of YIp32 and S. cerevisiae mt-ARS constructed as a reference. Subcloning experiments were carried out to assess the localization of ARS on the above 3.2 kb fragment, revealing that the fragment contains at least two ARSs.

Basidiomycota↗
PubMed1986

Studies on binding of toromycin, an antitumor antibiotic, to DNA.

Toromycin, an antitumor, bactericidal and antiviral compound, was found to bind to DNA in such a way as to interfere with the dissociation of double helix at an elevated temperature. The antibiotic did not introduce strand scission into DNA. Single-strand-specific nuclease S1-susceptibility of negatively supercoiled DNA was not influenced by its binding. The antibiotic was shown to bind to both of the alternating purine-pyrimidine copolymers, poly(dG-dC):poly(dG-dC) and poly(dA-dT):poly(dA-dT). The unique C-glycoside molecule of toromycin interacted with single-stranded DNA, but was found to have no affinity for RNA.

Aminoglycosides↗
PubMed1985

Effect of spermine on cleavage of plasmid DNA by nucleases S1 and Bal 31.

S1, a single-strand-specific nuclease, cleaves both strands of supercoiled DNA mostly once at unpaired sites. However, all the sites are not cleaved with the same frequency by the enzyme, there being sites of preferential cleavage and infrequent ones. Spermine was found to reduce this cleavage specificity of S1 with supercoiled plasmid DNA. A similar effect of spermine was observed with Bal 31 nuclease. Bal 31 contains in addition to S1-like activity a quasi-processive exonuclease activity that simultaneously degrades both 3'- and 5'-termini of linearized duplex DNA. Spermine stimulated the exonuclease activity. The above results were obtained within the concentration range of spermine that did not induce DNA aggregation.

DNA, Bacterial↗
PubMed1985

Partial purification and characterization of type I DNA topoisomerase from Bacillus stearothermophilus.

Type I DNA topoisomerase was partially purified from Bacillus stearothermophilus by ammonium sulfate precipitation and column chromatographies on phosphocellulose, DEAE-cellulose and heparin-agarose. On heparin-agarose chromatography, topoisomerase I activity was separated into three fractions (designated Fractions A, B, and C). Each fraction was further subjected to gel filtration on Sephacryl S-200. From electrophoretic analysis on polyacrylamide gel, Fraction A was found to contain two enzyme species having molecular weights of 110,000 and 100,000, and Fraction B one enzyme species with a molecular weight of 80,000. The molecular weight of the enzyme in Fraction C was estimated to be around 150,000 by gel filtration. The enzymes in Fractions A and B exhibited little activity in the presence of Mg2+, while the activity was increased remarkably by NaCl with Mg2+. No activity was observed in the presence of NaCl alone. The enzyme in Fraction C required only Mg2+ for full activity. With Fraction A, the topoisomerase I-induced cleavage sites on tetracycline-resistant plasmid pNS1 (2.55 megadaltons) were mapped. Fraction A cleaved the DNA at ten specific sites. These sites were compared to those of the Haemophilus gallinarum enzyme, which have already been mapped (Shishido et al. (1983) Biochem. Biophys. Acta 740, 108). The results showed that there is a remarkably coincidence between the cleavage sites induced by the B. stearothermophilus and H. gallinarum enzymes.

Binding Sites↗
PubMed1985

[S1 nuclease].

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Chromatography↗
PubMed1985

[BAL 31 nuclease].

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Endodeoxyribonucleases↗