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Biomedical subjects

H Otsuka

Publications and source records attributed to H Otsuka.

At least 325 records · Page 18Linked to original sources
PubMed1981

Functional expression of the Herpes simplex virus thymidine kinase gene in Escherichia coli K-12.

The recombinant plasmid pAGO contains the Herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene and consists of a 2-kb PvuII fragment of HSV-1 DNA inserted into the PvuII site of pBR322. A deletion mutant of pAGO, designated pMH110, has been isolated which removes the normal HSV-1 TK gene promoter but places the promoter of the pBR322 tetracycline-resistance (tetr) gene only about 400 bp from the translational start codon of the HSV-1 TK polypeptide. In contrast to pAGO, which transforms mouse LM(TK-) cells to TK+ but is only weakly expressed in TK- bacteria, pMH110 not only efficiently transforms LM(TK-) cells to TK+ but also enables TK- Escherichia coli K-12 cells to form colonies on selective plates containing 5-fluorodeoxyuridine (FdUrd) plus thymidine (dThd) and to exhibit fully restored ability to incorporate [3H]dThd into DNA. The levels of TK activity expressed by bacteria harboring pMH110 were about as high as those expressed by bacteria harboring plasmid pTK3, which contains the wild-type E. coli TK gene. The TK activity expressed in bacteria harboring pMH110 was partially purified and shown to be HSV-1-specific by serological and disc PAGE analyses and by experiments demonstrating that this enzyme phosphorylated [125I]deoxycytidine.

Animals↗
PubMed1981

Herpes simplex virus thymidine kinase activity of thymidine kinase-deficient Escherichia coli K-12 mutant transformed by hybrid plasmids.

A hybrid plasmid (pAGO) that contains the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in the form of a 2-kilobase-pair (kbp) Pvu II fragment inserted at the Pvu II site of plasmid pBR322 was used to transform TK- Escherichia coli K-12 strain KY895. pAGO-transformed KY895 cells exhibited partially restored ability to incorporate [3H]dThd into DNA and an HSv-1-specific TK activity. Bacteria cured of plasmid pAGO (or transformed by plasmid pBR322) did not show enhanced incorporation of [3H]dThd into DNA or HSV-1 TK activity. Plasmid pMH1A was derived from pAGO by deletion of 2067 bp of DNA sequence from pBR322 and 105 bp from the HSV-1 TK gene. E. coli K-12 strain KY895 cells transformed by pMH1A did not show enhanced incorporation of [3H]dThd into bacterial DNA, although pMH1A DNA isolated from transformed KY895 cells, like pAGO DNA, did transform TK- mouse fibroblast [LM(TK-)] cells to the TK+ phenotype. The expression of HSV-1 TK activity by E. coli K-12 suggests that intervening sequences may be absent from the coding region of HSV-1 tk or that the coding region of the gene possesses short intervening sequences which do not disrupt the translational reading frame.

Animals↗
PubMed1981

Identification of alkyldiacylglycerols containing saturated methyl branched chains in the Harderian gland of guinea pig.

More than 90% of total lipids in the Harderian gland of guinea pig was identified as 1-alkyl-2,3-diacylglycerol. The alkyl and acyl moieties consisted of saturated aliphatic chains ranging from 14 to 21 carbon atoms and from 15 to 26 carbon atoms, respectively. About 60 mol% of them had methyl branches, which were located at the even-numbered carbon atoms. Mono- and di-methyl branched chains accounted for most of the branched chains. The aliphatic chains contained methyl branches especially in the alkyl chains at the 1-position and the acyl chains at the 2-position

Animals↗
PubMed1981

Simplified determination of cholestanol in serum by gas-liquid chromatography: biochemical diagnosis of cerebrotendinous xanthomatosis.

Cholestanol (5 alpha-cholestan-3 beta-ol) in human serum was determined by gas-liquid chromatography after removal by epoxidation of cholesterol which is present in amounts about 400 times those of cholestanol and disturbs the accurate determination of minor sterols in serum. Epicholestanol (5 alpha-cholestan-3 alpha-ol) was used as an internal standard. By this method the cholestanol content in sera of 7 patients with cerebrotendinous xanthomatosis was determined. The cholestanol levels in those sera were significantly higher than those of normal subjects and the present method proved to be useful in a biochemical diagnosis of cerebrotendinous xanthomatosis.

Cholestanols↗
PubMed1981

The application of droplet counter-current chromatography (DCC) for the separation of acidic glycolipids.

Droplet counter-current chromatography (DCC) a novel liquid-liquid partition technique, is a useful method for the separation of glycolipids. After a series of investigations on suitable experimental conditions for DDC, mouse brain chloroform-methanol-0.5% CaCl2-n-propanol (50:60:40:6) by the ascending method with 500 standard columns. GM1 and cerebroside sulfuric ester (CSE) from whale brain glycolipid fraction were separated by DCC into two peaks and three peaks, respectively, with chloroform-methanol-water-n-propanol (50:60:40:2) and 500 standard columns. The occurrence of two peaks of GM1 was due to the differences of sphingenine and fatty acid compositions. In the former peak of GM1, the majority of long chain base and fatty acid were 18:1 sphingenine and stearic acid, respectively, while the latter contained 18:1 (32%), 20:1 (68%) sphingenine and 16:0 (14.5%), 18:0 (61.4%), and 20:0 (21.0%) fatty acids. The three peaks of CSE were due to the difference of carbon chain length and to the presence or absence of a hydroxyl group in fatty acids. The first polar fraction (CSE-I) had hydroxy 23:0 (34%) and 24.1 (53.4%) fatty acids and the second fraction (CSE-II) contained hydroxy 24:0 (70.5%) and 25:1 (12.8%) fatty acids, while the third fraction (CSE-III) had normal 23:0 (11.9%), 24:1 (56.4%), hydroxy 25:0 (11.4%) and 26:1 (9.7%) fatty acids. Preparative DCC was also applied to the glycolipid fraction from Tay-Sachs' brain to give 242 mg of practically pure GM2 from 700 mg of crude brain extract without any preliminary purification.

Animals↗
PubMed1981

Fatty acid synthetase from the Harderian gland of guinea pig: biosynthesis of methyl-branched fatty acids.

Fatty acid synthetase was isolated from guinea pig Harderian gland. This enzyme complex exhibited a unique character as compared with the fatty acid synthetase from the liver of the same animal. The former enzyme produced many odd-numbered and methyl-branched fatty acids in the presence of methylmalonyl-CoA. These fatty acids are characteristic components of the lipid secreted from this gland. The chemical structure of this lipid has been identified as 1-O-alkyl-2,3-diacylglycerol by previous work from this laboratory (Yamazaki, T., Seyama, Y., Otsuka, H., Ogawa, H., & Yamakawa, T. (1981) J. Biochem. 89, 683-691). Apparent Km values (5 X 10(-6) M) for acetyl-CoA and propionyl-CoA were the same, but the Vmax for propionyl-CoA was much higher than that for acetyl-CoA. The pI value of the fatty acid synthetase from Harderian gland was 5.3, and the molecular weight of the enzyme was 9 X 10(5) daltons. The beta-ketoacyl reductase had pro-S stereospecificity and the enoly reductase had pro-R stereospecificity for NADPH. The results presented in this paper indicate that the fatty acid synthetase from guinea pig Harderian gland can produce a set of fatty acids needed for the synthesis of the lipid secreted from this gland, and that the fatty acid synthetase has a characteristic organ specificity.

Acetyl Coenzyme A↗
PubMed1981

Studies on isolated smooth muscle cells. VII. Response to agonists and the contraction velocity of taenia coli of guinea pig and the single smooth muscle cells.

Single smooth muscle cells were isolated from taenia coli of guinea pigs and degrees of responses of acetylcholine, histamine and prostaglandin E2, and velocity of acetylcholine-contraction were compared with those of whole tissue. The contraction of single cells by these agonists were dose-dependent and ED50 of acetylcholine, histamine and prostaglandin E2 were 0.3 - 1.1 x 10(-6), 5-11 x 10(-7) and 1-3 x 10(-9) M, respectively. These values were quite similar to those obtained with whole tissue. It took 2.0 +/- 0.2 s for the single cells to be contracted completely by 10(-4) M acetylcholine while it took 6.9 +/- 0.2 s for the isotonic contraction of whole tissue. Times for a half maximum contraction, assuming that the whole tissue and the single cells were contracted at the maximum velocity, were 1.43 +/- 0.05 and 0.92 +/- 0.12 s, respectively when 10(-4) M acetylcholine was applied. These results revealed the response of muscle cells in the tissue to agonists was reflected on magnitude of the contraction of whole tissue and suggested that pharmacological properties of smooth muscle cells of taenia coli were not changed during the isolation procedure, whereas velocity of contraction of single cells was faster than that of whole tissue. Reasons for the difference in velocity were discussed.

Acetylcholine↗
PubMed1981

Water-soluble siomycin-A derivatives. Preparation, chemical structures and biological properties of half-esters of the peptide antibiotic.

The peptide antibiotic siomycin-A was transformed into half-esters with dicarboxylic acids with the intention of making siomycin-A soluble in water. Sodium salts of the half-esters were also prepared. Some of the salts showed antibacterial activities comparable to siomycin-A against Gram-positive bacteria in vitro and exhibited better therapeutic effects in infected mice than siomycin-A. The chemical structures of siomycin-A hemiadipate-II and -III were elucidated by comparing their 13C and 1H NMR spectra with those of siomycin-A. Their physicochemical properties are described.

Animals↗