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Biomedical subjects

K Matsubara

Publications and source records attributed to K Matsubara.

At least 487 records · Page 27Linked to original sources
PubMed1984

Molecular cloning of cDNA sequences for rat M2-type pyruvate kinase and regulation of its mRNA.

Rat M2-type pyruvate kinase mRNA was enriched from total polysomes isolated from AH-130 Yoshida ascites hepatoma cells, which contain a very high concentration of the M2-type enzyme, by immunoprecipitation with a specific antibody and Staphylococcus aureus cells. Double-stranded cDNA synthesized from the enriched mRNA was inserted into the PstI site of pBR322, and the resultant recombinant DNA molecules were used to transform Escherichia coli. Three clones containing DNA complementary to M2-type pyruvate kinase mRNA were identified by colony hybridization, hybrid-arrested translation, and hybrid-selected translation. A partial restriction map was constructed covering about 1.44 kilobase pairs. The cloned region of the M2-type mRNA showed a high degree of sequence homology with the M1-type mRNA and some homology with the L-type mRNA as determined by dot blot hybridization. The molecular size of the M2-type mRNA, which was estimated to be 2.35-2.65 kilobases on denaturing gel, was the same as that of the M1-type mRNA. The level of hepatic M2-type pyruvate kinase mRNA measured by hybridization assay using cloned cDNA as a probe was increased 2.5-fold 1 day and 3.9-fold 2 days after partial hepatectomy and then started to decrease. This induction was followed by similar changes in the enzyme activity. AH-130 hepatoma cells contained 100-150 times more M2-type isozyme mRNA than regenerating liver. These results suggest that the increased levels of M2-type isozyme in regenerating liver and hepatoma cells are primarily due to elevation of hybridizable M2-type mRNA concentration.

Animals↗
PubMed1984

Identification of the minimal essential region for the replication origin of miniF plasmid.

The minimal replication origin of miniF plasmid was found to lie within a region of 217 bp in length. This region contains an AT cluster and the four 19 bp direct repeats responsible for incompatibility, termed incB. Its location coincides with hat of ori2 of plasmid F, previously inferred to be the replication starting point by electron microscopic analysis (Eichenlaub et al. 1981).

Base Sequence↗
PubMed1984

Plasmid vectors designed for high-efficiency expression controlled by the portable recA promoter-operator of Escherichia coli.

A novel expression vector using the 236-bp promoter-operator fragment of the recA gene (recApo) of Escherichia coli has been constructed. This DNA fragment contains complete signals for the initiation of RNA synthesis, as well as for regulation by the lexA product, but lacks the coding sequence for the RecA protein. The strength of the recA promoter was examined by assaying beta-galactosidase activity expressed from a cro-lacZ fused gene placed downstream of the promoter. Under noninducing conditions, the promoter was regulated by the LexA protein, and the fused gene was expressed only weakly. Upon induction by nalidixic acid in a recA+ strain, high expression was observed for an extended period. After 5 h under inducing conditions, as much as 11% of the total cellular protein was cro-lacZ product. The expression level was higher than that from promoters of lac, trp, and lambda early genes.

Bacterial Proteins↗
PubMed1984

Corrected sequences of cDNAs for human salivary and pancreatic alpha-amylases [corrected].

The nucleotide sequences of the cloned human salivary and pancreatic alpha-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5'-noncoding region and 32 in the 3'-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5'- and 3'-noncoding regions, respectively. The nucleotide sequence homology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous. Comparison of the sequences of human alpha-amylase cDNAs with those previously obtained for mouse alpha-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human alpha-amylase.

Amino Acid Sequence↗
PubMed1984

In vivo and in vitro studies on cocaine metabolism: ecgonine methyl ester as a major metabolite of cocaine.

The levels of cocaine, benzoylecgonine and ecgonine methyl ester were measured periodically in the urine of dogs and rabbits by gas chromatography-chemical ionization selected ion monitoring mass spectrometry after subcutaneous injection of 2.9 mumol/kg (1.0 mg/kg) cocaine--HCl. Ecgonine methyl ester persisted much longer than benzoylecgonine. Urinary ecgonine methyl ester could be identified for 72 h in all the experimental dogs. The ester accounted for 6.6-27.1% of a dose of cocaine in dogs and 8.8-31.9% in rabbits 24 h after administration. Excretion of benzoylecgonine in urine was 6.3-19.2% in dogs and 7.5-32.9% in rabbits. This confirms that ecgonine methyl ester is one of the major metabolites of cocaine as well as benzoylecgonine. Excretion of the parent drug and its two hydrolyzed metabolites in faeces was very small, less than 1% of administered dose. Tri-o-tolylphosphate and eserine significantly inhibited cocaine hydrolysis to benzoylecgonine and ecgonine methyl ester, respectively. Parathion and EDTA, however, had no effects on cocaine hydrolysis in vivo. In vitro demethylation of cocaine to benzoylecgonine was demonstrated in the plasma from dogs and this production of benzoylecgonine was much more than that of non-enzymatic formation in buffer at physiological pH. It was concluded that a large part of the benzoylecgonine excreted in urine is an in vivo enzymatic product of cocaine. On the other hand, plasma cholinesterase and liver esterase mediated ecgonine methyl ester formation. This liver esterase was abundant in the soluble fraction and could be found in both of microsomal and mitochondrial fractions.

Animals↗
PubMed1984

Quantitation of cocaine, benzoylecgonine and ecgonine methyl ester by GC-CI-SIM after Extrelut extraction.

A method of gas chromatography-chemical ionization selected ion monitoring (GC-CI-SIM) is described for the determination of cocaine, benzoylecgonine and ecgonine methyl ester in biological materials using an Extrelut extraction technique. Recoveries of cocaine, benzoylecgonine and ecgonine methyl ester by this technique were 95, 81 and 97%, respectively. The method uses cocaine-d5, benzoylecgonine-d5 and lidocaine as internal standards, and isobutane as reagent gas for chemical ionization. Sensitivity of the method proved to be 1 ng/ml for cocaine and benzoylecgonine, and 10 ng/ml for ecgonine methyl ester when used in a 10-ml urine sample. With animal experiments, ecgonine methyl ester as well as benzoylecgonine was confirmed as a major metabolite of cocaine.

Animals↗
PubMed1984

Multiple initiation sites of DNA replication flanking the origin region of lambda dv genome.

Early replicative intermediates of lambda dv plasmid were prepared by an in vitro replication system in the presence of 2',3'-dideoxycytidine 5'-triphosphate, an inhibitor of DNA chain elongation. Short-chain DNAs produced from regions near the replication origin were purified from the intermediates. A fraction of the DNAs was covalently linked to primer RNA. The transition sites from primer RNA to DNA synthesis were mapped along the nucleotide sequence of the genome, by eliminating the RNA by alkaline hydrolysis and labeling the freshly exposed 5' ends of DNA with 32P. The transition sites were found to be located on both sides of the ori region, which includes four 19-base-pair repeats where one of the lambda specific initiator proteins, O, binds. No transition arose within the ori region. The transition sites are multiple on both sides of the ori region and are clustered in one of the two strands in such a way that DNA syntheses from the two sides converge. The frequency of the "leftward" DNA synthesis is several times higher than that of "rightward" synthesis, reflecting the asymmetric bidirectional replication of lambda dv DNA.

Bacteriophage lambda↗
PubMed1984

Expression of poliovirus complementary DNA coding for viral antigenic determinants in Escherichia coli.

DNA sequences coding for the immunogenic capsid protein VP1 and/or VP3 of poliovirus strain LSc-2ab (Sabin 1) were prepared by digesting the cloned complementary DNA with restriction endonuclease PstI. The DNA fragments were inserted into the unique PstI site of Escherichia coli plasmid vectors pBR322, pKT 280 and/or pKT 287 that lay in the region expressed under control of the penicillinase promoter system. In the expression vectors, poliovirus sequences were designed to be read in phase and therefore to be expressed as fusion proteins with the bacterial peptides. In addition, the Escherichia coli tryptophan operon promoter-operator system was inserted upstream of the penicillinase system to obtain stronger expression of the poliovirus sequences. Escherichia coli transformed with these plasmids appeared to produce the antigenic polypeptides, which were detected by immunoprecipitation with antibodies to capsid proteins VP1 and/or VP3 followed by SDS-polyacrylamide gel electrophoresis.

Amino Acid Sequence↗
PubMed1984

Metabolic disposition of 8 alpha, 9 alpha- and 8 beta, 9 beta-epoxyhexahydrocannabinols in the mouse.

Metabolic disposition of 8 alpha, 9 alpha- and 8 beta, 9 beta-epoxyhexahydrocannabinols (EHHCs) was studied using mice to clarify mechanisms which cause a difference in their pharmacological activities. At given time intervals from 0.5 to 60 min after intravenous injections of 8, 9-EHHCs (10 mg/kg), levels of unchanged epoxides extracted from blood, liver and brain of mice were determined by gas chromatography. Blood levels of both epoxides declined biphasically, and the concentrations of 8 alpha, 9 alpha-EHHC were higher than those of 8 beta, 9 beta-EHHC at all the time intervals determined. Biological half-lives in the slower phase were 17 and 13 min, respectively, for 8 alpha, 9 alpha-and 8 beta 9 beta-EHHCs. A similar result was obtained for 8,9-EHHCs concentrations in the liver. However, no significant difference in the brain levels was found between 8 alpha, 9 alpha-and 8 beta, 9 beta-EHHCs. Concentrations of 8 alpha, 9 beta-and 8 beta, 9 alpha-dihydroxyhexahydrocannabinols as well as unchanged epoxides 15 min after 8, 9-EHHCs injections increased significantly in the liver of mice pretreated with SKF 525-A (25 mg/kg, i.p.) comparing with the control. When delta 8-tetrahydrocannabinol (delta 8-THC), 8 alpha, 9 alpha- or 8 beta, 9 beta-EHHC was injected into mice intracerebroventricularly (25 micrograms/head), pentobarbital (40 mg/kg, i.p.)-induced sleep prolonging effect was ranked in the following order, 8 beta, 9 beta-EHHC greater than delta 8-THC greater than 8 alpha, 9 alpha-EHHC. These results suggest that monooxygenase system involving cytochrome P-450 and epoxide hydrolase together play important roles in the epoxides metabolism. In addition, different activities of 8 alpha, 9 alpha-and 8 beta, 9 beta-EHHCs to the central nervous system may cause a difference in their pharmacological effects rather than metabolic factors.

Animals↗
PubMed1984

Virological classification of autopsy livers with hepatic disorders.

Forty six selected autopsy livers with hepatic disorders were classified histologically into three groups, i.e., hepatitis 8, liver cirrhosis 16 and hepatocellular carcinoma 22, chiefly by histological findings. These groups were subdivided into two categories after determining the presence or absence of three HBV associated markers, i.e., the hepatitis B surface antigen (HBsAg), the antibody to hepatitis B surface antigen (anti-HBs) and the antibody to hepatitis B core antigen (anti-HBc) in the sera and liver homogenates. At least one of these markers was found to be present in the sera and homogenates of 38% of the livers with hepatitis, 69% of the livers with liver cirrhosis and 77% of the livers with hepatocellular carcinoma. When the liver tissues were examined for the presence of HBV DNA using Southern blot technique, the majority of them (10 out of 11) which proved to be positive for at least one HBV associated marker were also positive for HBV DNA. However, HBV DNA could not be detected in the other 10 livers which contained no HBV associated markers. These results showed that a HBV associated serological marker was always expressed, when liver tissue HBV DNA was demonstrable. The results of the two detection methods we used in this study were found to be almost equivalent. These results showed no evidence of nucleic acid homology between DNA from the liver of patients with non-B type liver disorders and HBV DNA.

Autopsy↗
PubMed1984

[Pharmacological studies of a new sleep-inducer, 1H-1,2,4-triazolyl benzophenone derivatives (450191-S) (I). Behavioral analysis].

The behavioral effects of 450191-S and its metabolites were investigated in mice, rats, cats and rhesus monkeys, and they were compared with those of related benzodiazepines (BDZ) such as diazepam and nitrazepam. Oral administration of 450191-S consistently caused sedation without excitability in mice and rats, and it was only 1/2 to 1/266 as potent as the BDZ in producing motor incoordination as assessed by traction, rotarod performance and inclined screen tests in mice, induced much less ataxia in cats and monkeys, and inhibited respiration in anesthetized cats. The locomotor activities of mice and rats measured by Animex and the open field test were not affected by 450191-S, but rearing and preening decreased with 450191-S as with the BDZ. 450191-S was equipotent with nitrazepam and 2 to 6 times more potent than diazepam and estazolam in potentiating chlorprothixene-induced hypnosis and thiopental-Na-induced anesthesia. These effects were not different with successive 14-day administration of 450191-S. Anti-pentylenetetrazol, picrotoxin and bicuculline convulsions of 450191-S had the same potency as nitrazepam, but caused much less anti-electroshock convulsion than the BDZ. 450191-S had potent antianxiety activity as observed by anti-aggressive and anti-conflict activities and had almost the same effect as diazepam on operant behavior. The metabolites M-1, M-2, M-A and M-3 showed approximately the same potency as 450191-S in inducing anesthetic potentiation and antianxiety activity, but they were much more potent in causing disturbance of the somatic functions. These results indicate that 450191-S possesses inhibitory effects on the central nervous system, including a potent sleep-inducing effect, and is characterized by markedly weak muscle relaxant activity and motor incoordination.

Aggression↗
PubMed1984

Quantification of individual serum bile acids in patients with liver diseases using high-performance liquid chromatography.

Using high-performance liquid chromatography combined with a column of immobilized 3 alpha-hydroxysteroid dehydrogenase, 15 bile acids, i.e., cholate, chenodeoxycholate, deoxycholate, lithocholate, ursodeoxycholate, and their five taurine conjugates and five glycine conjugates, were clearly separated and quantified in 38 patients with various hepatobiliary diseases and 9 normal controls. The serum levels of bile acids, both primary and total, were elevated in patients with liver disease, but did not differentiate between parenchymal disease and cholestasis. The ratio of cholate/chenodeoxycholate was significantly increased in cholestasis as compared with parenchymal liver injury. In primary biliary cirrhosis, the ratio of total glycine/taurine conjugates was decreased, with a marked increase of taurochenodeoxycholate. The bile acid pattern was distinctly different between extrahepatic cholestasis and primary biliary cirrhosis, which mainly reflects intrahepatic cholestasis. In acute hepatitis, there was a quick normalization of major taurine and glycine conjugates in the convalescent stage. Most of the major taurine and glycine conjugated bile salts were significantly elevated in cirrhosis, and the elevation of taurochenodeoxycholate was particularly marked in the decompensated state. Furthermore, the quantitative relationship between taurocholate and taurochenodeoxycholate in cirrhosis was reversed from that in acute hepatitis. These changes in absolute and relative concentrations of bile acids in various liver diseases perhaps reflect differing pathology and metabolism, and may prove diagnostic. Measurement of individual bile salts is easily and quickly done with this method, and may lend itself clinical application.

Adolescent↗
PubMed1983

Molecular cloning of DNA complementary to rat L-type pyruvate kinase mRNA. Nutritional and hormonal regulation of L-type pyruvate kinase mRNA concentration.

Rat liver L-type pyruvate kinase mRNA was enriched from total polysomes by immunoprecipitation with a specific antibody and Staphylococcus aureus cells. Double-stranded cDNA synthesized from the enriched mRNA was inserted into the PstI site of pBR322, and the resultant recombinant DNA molecules were used to transform Escherichia coli. Three clones containing DNA complementary to L-type pyruvate kinase mRNA were identified by colony hybridization, hybrid-selected translation, and dot blot hybridization. A partial restriction endonuclease map of cDNA inserts was constructed covering about 1.86 kilobase pairs. The cDNA insert of recombinant plasmid pLPK-14 was used as a hybridization probe to quantitate L-type pyruvate kinase mRNA in rat liver after various treatments. The level of hybridizable L-type enzyme mRNA was markedly increased by a high carbohydrate diet. Diabetes greatly reduced the mRNA level in the liver of rats maintained on a high carbohydrate diet, but insulin administration resulted in restoration of the mRNA level to normal within 24 h. These changes were approximately proportional to the changes in the level of translatable L-type pyruvate kinase mRNA. Thus, we conclude that nutritional and hormonal regulation of synthesis of hepatic L-type pyruvate kinase occurs at the pretranslational level.

Animals↗
PubMed1983

Molecular cloning of the human gastrin gene.

A genomic clone that contains the human gastrin gene was isolated from a human gene library. Restriction endonuclease mapping and DNA sequencing analysis revealed that this gene is about 0.7 kb long, and has an intron. The intron is located at a position that separates the coding region into the peptide region essential for biological activities of gastrin and the non-essential, N-terminal peptide region.

Amino Acid Sequence↗