Biomedical subjects

K Miyazaki

Publications and source records attributed to K Miyazaki.

At least 631 records · Page 35Linked to original sources

PubMed1987

Bile acid metabolism in isolated rat hepatocytes: studied by gas-liquid chromatography-mass spectrometry-selected ion monitoring.

Bile acid contents in isolated rat hepatocytes were determined by gas-liquid chromatography-mass spectrometry-selected ion monitoring with the use of deuterium-labeled internal standards. This allowed us first to monitor the actual amounts of not only major but also minor bile acid components present with sufficient sensitivity and specificity and to follow the changes of individual bile acids in cultured rat hepatocytes simultaneously. In freshly isolated rat hepatocytes, cholic and beta-muricholic acids were the major components, comprising 35 and 46% of the total bile acids, respectively. These two bile acids were found to be most actively synthesized during the first 2 h of incubation and continued to increase thereafter for up to 6 h (the end of the period studied). In contrast, chenodeoxycholic and alpha-muricholic acids, which are the precursors of beta-muricholic acid, showed slight increases only in the first hour of incubation and decreased thereafter. These results suggested that the conversion to beta-muricholic acid from chenodeoxycholic acid via alpha-muricholic acid occurred rapidly in cultured rat hepatocytes. The secondary bile acids such as deoxycholic, hyodeoxycholic, and 3 alpha, 12 beta-dihydroxy-5 beta-cholanoic acids declined steadily from the start of incubation, which supported the findings that further hydroxylation of these dihydroxy bile acids occurs in rat liver.

Animals↗

PubMed1987

Transformation of rat liver cell line by Rous sarcoma virus causes loss of cell surface fibronectin, accompanied with secretion of metallo-proteinase that preferentially digests the fibronectin.

An epithelial cell line derived from the liver of a normal Buffalo rat (BRL) was transformed by Rous sarcoma virus (RSV). The RSV-transformed cells were separated into five clones (RSV-BRL1 through 5), which were morphologically different. RSV-BRL cells exhibited the following characteristics distinct from those of BRL cells: tumorigenicity, irregular cell arrangement, loose intercellular junction, growth in soft agar (anchorage-independent growth) except for RSV-BRL3 and 5, and loss of cell surface fibronectin. When BRL cells were cultured in the standard medium supplemented with the serum-free conditioned medium of RSV-BRL cells, the amount of the cell surface fibronectin decreased significantly. It was found that RSV-BRL cells secreted a proteinase capable of hydrolyzing the fibronectin, whereas BRL cells secreted hardly any of this proteinase. The fibronectin-hydrolyzing proteinase (FNase) could also hydrolyze plasma fibronectin added as an exogenous substrate. The hydrolysis of plasma fibronectin was inhibited by ethylenediamine tetraacetate, but stimulated by rho-chloromercuribenzoate and calcium ion. This indicates that FNase is a metallo-enzyme, but not a serine or thiol enzyme. In addition to the proteinase, RSV-BRL cells secreted plasminogen activator and a proteinase inhibitor which inhibited the activity of plasmin but not FNase.

Animals↗

PubMed1987

DNA polymerase A and its stimulative factor of baker's yeast: purification and characterization.

DNA polymerase A (I or major) and its stimulative factor were purified from 15-20 kg wet weight of baker's yeast by several procedures, which were varied in order to examine the possible occurrence of proteolysis. The extraction was carried out in the presence of 10 or 3 mM phenylmethylsulfonyl fluoride (PMSF), followed by either batchwise adsorption-elution or column chromatography on DEAE-Sepharose (rapid or time-consuming, respectively). These early steps were followed by column chromatographies on DEAE-, CM-, and heparin-Sepharoses, phosphocellulose, and Sephacryl S-300. Preparations of the polymerase obtained by all the procedures described above showed a single protein band at Mr of about 145,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), unless they had been treated with 2-mercaptoethanol (ME). After ME treatment, however, they showed two protein bands at Mr of about 145,000 and 75,000 in SDS-PAGE, except for those obtained by the procedure involving 10 mM PMSF and the batchwise adsorption-elution. All the preparations described above showed practically the same specific activity. This indicates that in intact cells, the polymerase consisted of a single peptide with Mr of about 145,000, and that after cell disruption, it was artificially hydrolyzed in a limited fashion into two peptides with Mr of about 75,000, which were still active and were linked to each other through a disulfide bond. Preparations of the factor obtained by all the procedures described above showed a single protein band at Mr of about 20,000 in SDS-PAGE before and after ME treatment. The relative activities of the purified polymerase were (100%), 123, 21, 37, 196, and 38% with native and denatured salmon sperm DNA, native and denatured calf thymus DNA, poly(dA-dT), and poly(dA).oligo(dT)10, respectively. With the addition of the purified factor, they were 173, 272, 173, 217, 173, and 247%, respectively, i.e., significantly stimulated. The purified factor also stimulated the activity of calf thymus DNA polymerase alpha by 150% with denatured salmon sperm DNA; Km was about 5 X 10(-10)M, practically the same as that of yeast DNA polymerase A. However, it hardly influenced the activities of Escherichia coli enzyme I or Micrococcus luteus enzyme.

DNA Polymerase I↗

PubMed1987

A boy with Down's syndrome having recombinant chromosome 21 but no SOD-1 excess.

A 5-year-old boy with Down's syndrome of mild phenotype is described. Chromosome studies revealed that the karyotype of the proband was 46,XY,rec(21),dup q,inv(21) (p11.2q22.1)mat, and the segment 21q22.1----21qter was trisomic. The erythrocyte superoxide dismutase-1 (SOD-1) was found to be normal, and so we conclude that SOD-1 excess is not necessarily observed in patients with Down's syndrome caused by partial 21 trisomy. It is suggested that the gene for SOD-1 is located on the more proximal segment of the sub-band 21q22.1.

Child, Preschool↗

PubMed1987

A highly sensitive HPLC method for the assay of propantheline used to measure its uptake by rat intestinal brush border membrane vesicles.

A simple, sensitive, high-performance liquid chromatographic method for propantheline has been developed. Propantheline was quantitatively hydrolysed into xanthene-9-carboxylic acid in neutral or alkaline medium and the hydrolysate assayed by reversed phase high-performance liquid chromatography. This method measured to 2 pmol per injection and was used to investigate the uptake of the drug by rat intestinal brush border membrane vesicles. Propantheline was highly bound to the membrane and this binding was inhibited to varying extents by several quaternary ammonium compounds. Mepenzolate and methylbenactyzium inhibited it significantly, and neostigmine, butylscopolamine and N-methylnicotinamide inhibited it moderately. Choline, acetylcholine and thiamine had no effect.

Animals↗

PubMed1987

Lack of effect of ofloxacin on theophylline pharmacokinetics in rats.

Effect of ofloxacin, a new quinolone antibacterial agent, on the pharmacokinetics of theophylline was studied in rats in comparison with that of enoxacin and cimetidine. Ofloxacin by pretreatment with five oral doses of 50 mg/kg did not increase serum concentrations of theophylline (5 mg/kg, i.v. single) and showed no significant effect on total body clearance, serum half-life (T1/2) and AUC of theophylline, while enoxacin by the same pretreatment increased significantly serum theophylline concentrations and resulted in significant effect on all the pharmacokinetic parameters. Coadministration of ofloxacin (80 mg/kg, p.o. twice) did not induce a significant effect on the pharmacokinetic parameters of theophylline at repeated doses (50 mg/kg, i.v., twice daily for 3 days). On the contrary, coadministration of enoxacin and cimetidine at the same dose as ofloxacin remarkably increased serum concentrations of theophylline at the same repeated doses, and caused a significant decrease in clearance and an increase in T1/2 and AUC. The three drugs had no influence on rat serum protein binding of theophylline. Ofloxacin exhibited a weak inhibitory effect on rat hepatic microsomal cytochrome P-450-dependent monooxygenases, whereas enoxacin and cimetidine induced a significant inhibition of the enzymes. Thus, it is concluded that ofloxacin has no significant effect on the pharmacokinetics of theophylline in rats, and that enoxacin raises serum theophylline concentrations and results in a significant effect on the theophylline pharmacokinetics by inhibition of the hepatic microsomal monooxygenases in rats.

Animals↗

PubMed1987

In vitro study of cultured human insulinoma cells: evidence of abnormal sensitivity to glucose.

Human insulinoma cells were isolated and cultured in vitro, and their functional and morphological characteristics were determined. The cells, isolated as single cells or small cell clusters, reaggregated to almost the size of islets by the fifth culture day and were maintained in vitro for more than 1 month. Morphologically (light and electron microscopies) they were intact throughout the culture period. Immunohistochemically more than 50% of the cells in each reaggregate contained insulin. Incubation experiments revealed that a low glucose concentration (15 mg/dL) was sufficient to produce maximal insulin release. In the absence of glucose, 1 microgram/mL glibenclamide increased insulin release. On the other hand, 5 mM theophylline and 10 mM arginine did not alter insulin release significantly. Theophylline, arginine, and glibenclamide did not have any stimulatory effect on insulin release in the presence of 50 mg/dL glucose. Perifusion experiments with 50 mg/dL glucose disclosed a biphasic pattern of insulin release, and no significant change in insulin release occurred when the glucose concentration in the perifusate was switched from 50 to 150 and then back to 50 mg/dL. These findings demonstrate that human insulinoma cells can be isolated and maintained in vitro and that the cells have abnormal sensitivity to glucose.

Adenoma, Islet Cell↗

PubMed1987