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Biomedical subjects

H Otsuka

Publications and source records attributed to H Otsuka.

At least 289 records · Page 16Linked to original sources
PubMed1984

Effects of 12-O-tetradecanoylphorbol-13-acetate on carcinogenesis in the heterotopically transplanted rat urinary bladder.

12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent promoter of mouse skin carcinogenesis, was tested for possible tumor-enhancing effects on urinary bladder carcinogenesis using the heterotopically transplanted bladder (HTB) model. Weekly administration of TPA at 1.0 microgram/week to N-methyl-N-nitrosourea-initiated HTBs did not increase tumor incidence, but instead, resulted in a significantly high incidence of nodulopapillary hyperplasia, an early neoplastic lesion, suggesting possible tumor enhancement by TPA. In addition, administration of a high dose of TPA with or without a carcinogen treatment led to the development of numerous finger-like epithelial projections on the luminal surface of the HTBs. Evidence indicates that epithelial projections are formed as a result of proliferation of intermediate cells. Whether these structures evolve into true neoplastic lesions is at present unknown.

Animals↗
PubMed1983

Nucleotide sequence of the herpes simplex virus type 2 (HSV-2) thymidine kinase gene and predicted amino acid sequence of thymidine kinase polypeptide and its comparison with the HSV-1 thymidine kinase gene.

To analyze the boundaries of the functional coding region of the HSV-2(333) thymidine kinase gene (TK gene), deletion mutants of hybrid plasmid pMAR401 H2G, which contains the 17.5 kbp BglII-G fragment of HSV-2 DNA, were prepared and tested for capacity to transform LM(TK-) cells to the thymidine kinase-positive phenotype. These studies showed that hybrid plasmids containing 2.2-2.4 kbp subfragments of HSV-2 BglII-G DNA transformed LM(TK-) cells to the thymidine kinase-positive phenotype and suggested that the region critical for transformation might be less than 2 kbp. That the activity expressed in the transformants was HSV-2 thymidine kinase was shown by experiments with type-specific enzyme-inhibiting rabbit antisera and by disc-polyacrylamide gel electrophoresis analyses. DNA fragments of the HSV-2 TK gene were subcloned in phage M13mp9 and M13mp8. A sequence of 1656 bp containing the entire coding region of the TK gene and the flanking sequences was determined by the dideoxynucleotide chain termination method. Comparisons with the HSV-1(Cl 101) TK gene revealed that PstI, PvuII, and EcoRI cleavage sites had homologous locations as did promoter, translational start and stop, and polyadenylation signals. Extensive homology was observed in the nucleotide sequence preceding the ATG translational start signal and in portions of the coding region of the genes. Comparisons of the predicted amino acid sequences of the HSV-1 and HSV-2 thymidine kinase polypeptides revealed that both were enriched in alanine, arginine, glycine, leucine, and proline residues and that clear, but interrupted homology existed within several regions of the polypeptide chains. Stretches of 15-30 amino acid residues were identical in conserved regions. The possibility is suggested that domains containing some of the conserved amino acid sequences might have a role in substrate binding and as major antigenic determinants.

Amino Acid Sequence↗
PubMed1983

Mapping thymidine kinase-deficient mutants of vaccinia virus by marker rescue with hybrid plasmid DNAs containing portions of the HindIII-J fragment of virus DNA.

Five hybrid plasmids were constructed, each containing a portion of the vaccinia virus DNA HindIII-J fragment. These plasmid DNAs were used in marker rescue experiments to map the mutations in the thymidine kinase (TK) gene of three TK- vaccinia virus mutants. The TK gene of each of the three mutants was rescued by DNA from plasmid pPJ701, which contained about one-half of the HindIII-J fragment. Two mutants, 1004B and 1017-1, but not the third, 1016-1, were rescued by DNA of two plasmids, pPJ702 and pPJ703, which contained 16 and 18%, respectively, of one end of the J fragment. Mutant 1016-1 could be rescued by plasmid pPJ705 containing a 1.69-kb fragment of the HindIII-J fragment. The J fragment DNA in plasmid pPJ705 is located adjacent to that and separated by an EcoRI site from pPJ703 in the vaccinia virus genome. These results indicate that the mutation site in the TK gene of 1016-1 differs from that in 1004B or 1017-1 and suggests that the structural gene for the vaccinia virus TK lies near one end of the HindIII-J fragment and spans the EcoRI site.

DNA Restriction Enzymes↗
PubMed1983

Purification, characterization and identification of rat liver mitochondrial kynurenine aminotransferase with alpha-aminoadipate aminotransferase.

Kynurenine aminotransferase (L-kynurenine:2-oxoglutarate aminotransferase (cyclizing), EC 2.6.1.7) was purified 378-fold from rat liver mitochondria by digitonin solubilization, heat treatment, DEAE-Sepharose CL-6B chromatography, Sephadex G-100 gel filtration, hydroxyapatite chromatography and chromatofocusing. Elution patterns of alpha-aminoadipate aminotransferase (EC 2.6.1.39) activity were identical with those of kynurenine aminotransferase activity on all column chromatographies. The ratios of the two specific activities were constant throughout the purification. On polyacrylamide gel electrophoresis both activities were detected at the same position. Both enzymatic activities showed the same inactivation curves upon heat inactivation at various temperatures. alpha-Aminoadipate showed competitive inhibiton against kynurenine or 3-hydroxykynurenine. alpha-Ketoadipate was utilized in the kynurenine aminotransferase reaction as an amino acceptor in place of alpha-ketoglutarate. The Km value for alpha-ketoadipate was 10 microM, lower than for alpha-ketoglutarate. These observations indicate that kynurenine aminotransferase is identical with alpha-aminoadipate aminotransferase. The Km values of purified kynurenine aminotransferase were determined at pH 6.5 as: kynurenine, 4.3 mM; pyridoxal 5'-phosphate, 4.2 microM; alpha-ketoglutarate, 20 microM (kynurenine substrate), and 3-hydroxykynurenine, 5.7 mM; pyridoxal 5'-phosphate, 1.7 microM; alpha-ketoglutarate, 13 microM (3-hydroxy-kynurenine substrate). The enzyme was strongly inhibited by Hg2+ and p-chloromercuribenzoate.

2-Aminoadipate Transaminase↗
PubMed1983

Attenuated marmoset herpesvirus isolated from recombinants of virulent marmoset herpesvirus and hybrid plasmids.

Marmoset herpesvirus (MarHV) deletion mutants in the thymidine kinase (TK) gene were isolated after infection of OMK cells with TK+ MarHV DNA and the hybrid plasmid, pMAR401, which lacks a 2.6-kb KpnI-M fragment in the coding region of the MarHV TK gene. After plaque purification in TK- HeLa(BU25) cells, the DNA's from five recombinant araT-resistant MarHV clones were analyzed with restriction nucleases to verify that the 2.6-kb KpnI-M fragment (and a 0.9-kb BglII-Q fragment) were deleted from the viral DNA's. Molecular hybridization experiments using 32P-labeled pMAR4 probes and viral DNA fragments also showed that the recombinant viral DNA's lacked the KpnI-M and BglII-Q fragments, that BamHI-I of parental virus was shortened, and that three new HindIII fragments replaced the parental virus HindIII-G fragment. The recombinants did not induce TK activity in LM (TK-) cells. To study the relative virulence of the recombinants, 3-week-old Swiss mice were injected intracerebrally (Ic) or subcutaneously (Sc) in the sacro-lumbar region with either parental or recombinant viruses. The LD50 for the parental virus was 3 p.f.u. (Ic) and 7,600 p.f.u. (Sc). The recombinant viruses were significantly less virulent than TK+ MarHV after Ic inoculation (LD50 of 62,000 and 32,000 p.f.u., respectively, for viruses 5D-6B and 5D-4B) and gave no fatalities after Sc inoculation. Mice surviving TK- MarHV infections were protected from challenge with TK+ parental MarHV. Recombinant TK- MarHV's may be useful as vectors for the expression of foreign genes in animal cells and as the starting material for the design of vaccines.

Animals↗
PubMed1983

Determination of sulfated and nonsulfated bile acids in serum by mass fragmentography.

A Sep-Pak C18 cartridge was used for purification of bile acids from serum. Three kinds of deuterium labeled internal standards were required for accurate measurement of individual sulfated and nonsulfated bile acids. These internal standards were added to the serum before its application to the cartridge. Separation of sulfated and nonsulfated bile acids was performed on piperidinohydroxypropyl Sephadex LH-20 column chromatography. The nonsulfate fraction was submitted to alkaline hydrolysis, and the sulfate fraction to solvolysis followed by alkaline hydrolysis. Each fraction was converted to the hexafluoroisopropyl-trifluoroacetyl derivatives and quantitated by mass fragmentography. The recovery of each bile acid sulfate was quite satisfactory. In fasting healthy subjects the mean of total nonsulfated bile acids in serum was 1.324 micrograms/ml, and that of total sulfated bile acids was 0.450 micrograms/ml. Sulfated lithocholic acid comprised a large part of sulfated bile acids in healthy subjects.

Bile Acids and Salts↗
PubMed1983

Genetic regulation of GM2 expression in liver of mouse.

GM2 containing NeuGc was found to be a major ganglioside in the liver of most inbred strains of mouse we have examined so far. They were DBA/2, C3H/He, C57BL/6, BALB/c, and CBA mice. However, WHT/Ht mouse does not contain a detectable amount of GM2 (NeuGc) but has GM3 containing NeuGc as the major ganglioside in the liver. The positive expression of GM2 (NeuGc) was demonstrated in the liver of F1 hybrids between WHT/Ht and DBA/2 mice, as one of the possible combinations. Moreover, the expression of GM2 (NeuGc) in the liver was proved to be an autosomal dominant trait by ganglioside analysis of 34 individual mice of the F2 and backcross generations. WHT/Ht mouse was demonstrated to be a recessive homozygote lacking GM2 (NeuGc) expression. During the analysis, interestingly enough, the enhanced elongation of the sugar chain in the gangliosides from GM2 (NeuGc) to GM1 (NeuGc) and GD1 was observed in the liver of F1 mice and this elongation was segregated only in half members of F2 and backcross generations.

Animals↗
PubMed1983

Branched chain fatty acids in phospholipids of guinea pig Harderian gland.

Fatty acid compositions of phosphatidyl ethanolamine and phosphatidyl choline extracted from the guinea pig Harderian gland were examined by gas chromatography and gas chromatography-mass spectrometry. Oleic acid was the major component of both phospholipids, but a large amount of saturated branched chain fatty acids was found: 34.5% in phosphatidyl ethanolamine, and 42.9% in phosphatidyl choline. On the other hand, linolenic and arachidonic acids were not found in these phospholipids. At the 1-position of phosphatidyl choline and phosphatidyl ethanolamine, 54.3% and 40.9% of fatty acids, respectively, had methyl branches. These methyl branches were located at the even-numbered carbon atoms. Branched chain fatty acids were also found at the 2-position of both lipids: 36.2% in phosphatidyl choline and 31.2% in phosphatidyl ethanolamine. The fatty acids of phosphatidyl ethanolamine and phosphatidyl choline from liver, cerebrum, cerebellum, erythrocytes, and plasma of the same animal were also analyzed. Saturated and unsaturated fatty acids, including arachidonic acid, were the major components. Branched chain fatty acids were also found in these lipids, but in very small amounts.

Animals↗
PubMed1983

The separation of neutral glycosphingolipids from mammalian erythrocytes by droplet counter-current chromatography (DCC).

In a previous paper (Otsuka, H. & Yamakawa, T. (1981) J. Biochem. 90, 247-254), we reported the separation of acidic glycolipids by droplet counter-current chromatography using 500 columns with a commercially available DCC apparatus and described the precise conditions of the separation. In this paper, separation of neutral glycolipids from rabbit and human erythrocytes by DCC is described. For efficient separation of neutral glycolipids, addition of a definite amount of benzene was required. The solvent system of chloroform: benzene: methanol: water = 50: 50: 70: 20 gave clear separation of these glycolipids and further modification of this solvent system to chloroform: benzene: methanol: water = 50: 25: 65: 30 gave satisfactory results for the separation of Globoside I from human erythrocytes due to the difference between the amide linked fatty acids. Also under the same conditions, the clear separation of two peaks with blood group A activity was demonstrated by a hemagglutination inhibition test.

ABO Blood-Group System↗
PubMed1983

The characterization of 2,3-alkanediol diacyl esters obtained from the Harderian glands of Mongolian gerbil (Meriones unguiculatus).

A major lipid class in the Harderian glands of the Mongolian gerbil was investigated. The IR and 1H-NMR spectra suggested that it was a wax-like compound. Fatty acids present were capric, lauric, myristic, palmitic, stearic, arachidic, and behenic acids in ratios of 3.0%, 16.3%, 16.5%, 29.5%, 5.1%, 16.4%, and 9.8%, respectively. Odd-numbered, branched chain and unsaturated fatty acids were not present in large amounts. The structure of the alcohol moiety was elucidated to be 2,3-alkanediol with carbon chain lengths from C12 to C22 by GC-MS of the TMS and isopropylidine derivatives. Lemieux-von Rudloff oxidation of these alcohols confirmed the 2,3-diol structure, giving fatty acids two carbon units shorter.

Animals↗
PubMed1983

Monocyte-mediated suppression of mitogen responses of lymphocytes in uremic patients.

In the present experiment, we investigated the mechanism of the suppressed mitogen responses of peripheral blood mononuclear cells (PBMC) from uremic patients. We used phytohemagglutinin (PHA) and concanavalin A (Con A) as T cell mitogens, pokeweed mitogen (PWM) as a T cell-dependent B cell mitogen, and Staphylococcus aureus Cowan I (STA) as a T cell-independent B cell mitogen. PBMC from uremic patients showed significantly suppressed responses to PHA (p less than 0.05), Con A (p less than 0.05) and STA (p less tha 0.01) compared with those from healthy controls, but there was no significant difference in PWM response. However, these suppressed responses to PHA and Con A were markedly restored by depletion of phagocytic cells from PBMC. Although STA responses were also restored markedly in uremic patients, some patients still showed lower responsiveness to STA indicating the possibility of functional B cell defects. To further clarify the mechanism of the suppressed responses to mitogens, PBMC or nonphagocytic cells from uremic patients were cocultured with control T cells in the presence of PHA, or the effects of adherent cells from uremic patients on PHA responses of autologous or allogeneic control T cells were studied. From these experiments, it was suggested that the suppressed responses of PBMC to mitogens in uremia were mediated by monocytes.

Adult↗
PubMed1983

Serum concentrations of bile acid glucuronides in hepatobiliary diseases.

Bile acid glucuronides in the serum in various hepatobiliary diseases (36 cases) were quantitated by mass fragmentography and their clinical significance was discussed. Serum was added to defined amounts of deuterium-labeled bile acids and their glucuronide and sulfate derivatives, and the bile acids were separated into unconjugated, glucuronidated and sulfated groups after enzymatic cleavage of amide bonds. The liberated bile acids were quantitated by mass fragmentography. Bile acid glucuronides comprised about 7-8% of the total bile acids in the serum of various patients. Chenodeoxycholic acid was the major glucuronidated bile acid while cholic acid was mostly unconjugated. Lithocholic acid was almost all either sulfated or glucuronidated. In patients with obstructive jaundice, glucuronidated bile acids also comprised about 5%, although their absolute amounts were increased. In patients with liver cirrhosis, bile acid glucuronides were decreased, especially in decompensated cases, possibly as a result of hepatocellular dysfunction.

Adult↗
PubMed1983

Carcinogenicity of dipyrone in (C57BL/6 X C3H)F1 mice.

The carcinogenicity of dipyrone (sulpyrin)--[(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4 -yl) methylamino]methanesulfonic acid sodium salt monohydrate--which is widely used as an antipyretic anodyne in Japan and in some European countries, was examined in 314 (C57BL/6 X C3H)F1 mice. Male animals were given 0.5% (group I-a) or 0.125% (group I-b) dipyrone in their drinking water for 78 weeks, and female animals were given 1.0% (group II-a) or 0.25% (group II-b) dipyrone in their drinking water for 78 weeks; both males and females were observed for 86 weeks. Twenty-seven of 48 (56%) group I-a animals and 36 of 44 (82%) group II-a animals developed hepatic tumors, and the tumors in group II-a mice developed earlier than those in the control animals. The tumor incidences were significantly higher than those of 8 of 44 (18%) and 3 of 51 (6%) in the respective control groups. The multiplicity of the hepatic tumors was also significantly increased in groups I-a, I-b, and II-a. Hepatic adenoma incidence was related to the dose of dipyrone in the males. These results show that dipyrone enhances the development of hepatic tumors in mice.

Aminopyrine↗