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Biomedical subjects

Y Miyake

Publications and source records attributed to Y Miyake.

At least 19 recordsLinked to original sources
PubMed1992

Molecular cloning and chromosomal localization of a human gene encoding D-amino-acid oxidase.

Genomic clones covering the entire sequence of the gene encoding human D-amino-acid oxidase (DAO) (EC 1.4.3.3), one of the principal and characteristic flavoenzymes of peroxisomes, were isolated from human placental genomic libraries with the aid of a previously cloned cDNA for human DAO as a probe. Nucleotide sequence analysis revealed that the gene, present as a single copy in the human genome, comprises 11 exons and spans 20 kilobase pairs. The protein sequences containing the catalytically important residues, Tyr-228 and His-307, are coded for by separate exons. Heterologous transcription initiation sites were identified by primer extension analysis, and the sequence of the 5'-flanking region of the DAO gene was found to show some features common to other mammalian genes, such as those of glucocorticoid and the cAMP-responsive element. An additional noteworthy feature is the presence of promoter-like sequences in the first intron of the gene. In addition, two sequences of alternating pyrimidine and purine nucleotides, (CA)20 and (CA)17, are also present in the first intron. Such sequences may play some role in the expression of the DAO gene in human tissues. With the use of genomic DNAs prepared from human and Chinese hamster somatic hybrid cells as templates for the polymerase chain reaction, the gene for DAO was localized to human chromosome 12.

Animals↗
PubMed1992

Simple method for measuring the antibiotic concentration required to kill adherent bacteria.

A simple method was developed for measuring the antibiotic susceptibility of bacteria adherent to plastic surfaces. Staphylococcus aureus cells adhered to the bottom of a 96-well plastic tissue culture plate were incubated in serially diluted antibiotic solutions. After a 24-hour-incubation the solutions were removed, and fresh medium without antibiotics was added to each well. The viability of the cells was judged by their growth after a further 24-hour-incubation. The antibiotic concentration required to kill adherent bacteria was far higher than that required for planktonic cells, although we used bactericidal drugs; beta-lactam, quinolone, and aminoglycoside antibiotics. The tolerance demonstrated by adherent cells is likely to play a role in the difficulties encountered in the chemotherapy of biofilm infections.

Anti-Bacterial Agents↗
PubMed1992

Entrainment to external Ca2+ oscillation in ionophore-treated Physarum plasmodium.

To elucidate the mechanism of mutual interaction between intracellular chemical rhythms in the Physarum plasmodium, external Ca2+ oscillation was applied to the ionophore-treated plasmodial strand and its response was measured as tension oscillation. (i) Tension oscillation is entrained and phase locked to the externally applied Ca2+ oscillation. (ii) Two kinds of stable phase relationship, in-phasic and anti-phasic ones, are observed between them. (iii) Transition between the two stable phase relationships is also observed. These results suggest that intracellular rhythms with control tension generation are mutually entrained by means of cytosolic Ca2+ oscillation in the organism and that their interactions have two kinds of stable phase relationships.

Animals↗
PubMed1992

[Effects of ifenprodil on the adenosine triphosphatase of guinea pig liver mitochondria].

The effects of ifenprodil on adenosine triphosphatase (ATPase) activity were examined using guinea pig liver mitochondria. 1) Intact mitochondrial ATPase activity was stimulated by ifenprodil in a concentration-dependent manner, this effect being further potentiated with dinitrophenol. The stimulation by ifenprodil appeared with only ATP among four nucleotides as substrate. Mg2+ and Ca2+ attenuated the effect of ifenprodil. Ifenprodil abolished the KCN-induced inhibition. 2) Heat-treated mitochondrial ATPase activity, kept for 60 min at 50 degrees C, was decreased in a concentration-dependent manner by ifenprodil. The inhibitory effect of ifenprodil was abolished by Mg2+ and Ca2+. These results indicate that ifenprodil has two behaviors, acceleration of a latent ATPase and inhibition of an activated ATPase. These findings, together with our previous data, suggest that ifenprodil seems to affect the actions of Mg2+ and Ca2+ on mitochondrial ATPase by directly affecting the membrane, and these mechanisms may be involved in its anti-cyanide effect.

Adenosine Triphosphatases↗
PubMed1992

[A case of Fabry's disease associated with lupus nephritis].

A 36-year-old woman was hospitalized because of nephrotic syndrome. On admission, laboratory studies revealed total protein 5.9g/dl, total cholesterol 381mg/dl, urine protein 2-4g/day, C3 68mg/dl(90-185mg/dl) and the immunological tests showed that antinuclear factor, anti-DNA antibodies and the LE cell phenomenon were positive. Renal function was within normal range. After admission, renal biopsy was done. Light microscopic finding showed diffuse membranous glomerulonephritis, and vacuolization of epithelial cells. Immunofluorescent microscopic finding showed a granular specific staining for IgG, IgM, C3 and C1q along the capillary loops. Electron microscopic finding showed subepithelial and subendothelial dense deposits, and visceral epithelial cell cytoplasm containing osmiophilic multilamellar lipoid bodies. In the studies of the enzyme activities, the patient's fibroblast extract demonstrated a partial deficiency of alpha-galactosidase, and urine ceramide trihexoside was positive. But the patient's leukocyte extract did not demonstrate a deficiency of alpha-galactosidase. So Fabry's disease associated with lupus nephritis was diagnosed. It seems that the case of Fabry's disease which is an X-linked disorder caused by deficiency of the lysosomal enzyme alpha-galactosidase, associated with lupus nephritis, is extremely rare.

Adult↗
PubMed1979

Identification of the covalently bound flavin prosthetic group of cholesterol oxidase.

Highly purified preparations of cholesterol oxidase from Schizophyllum commune contain a covalently bound flavin component. A flavin peptide has been obtained by digestion with trypsin-chymotrypsin and purification on a column of phosphocellulose. Digestion with nucleotide pyrophosphatase results in increased fluorescence at pH 3.4 and release of 5'-adenylate, showing that the flavin is in the dinucleotide form. The absorption spectrum of the flavin peptide shows the hypsochromic shift of the second absorption band characteristic of 8 alpha-substituted flavins. The fluorescence at pH 7 is extensively quenched even in the mononucleotide form, with a pKa at pH 5.8 in the flavin peptide and at 5.05 following acid hydrolysis to the aminoacyl flavin level. This suggests that histidine is the amino acid substituted at the 8 alpha position of the flavin and that N(1) of the imidazole ring is the site of attachment. These data, the reduction of the flavin by borohydride, and comparison of the mobilities in high voltage electrophoresis at two pH values with N(1)- and N(3)-histidyl riboflavin and their 2',5'-anhydro forms shows that the prosthetic group of cholesterol oxidase is 8 alpha-[N(1)-histidyl]-FAD.

3-Hydroxysteroid Dehydrogenases↗
PubMed1979

Purification and characterization of a chymotrypsin-like enzyme (protease-S) in thermally injured rat skin.

Protease-S was extracted from thermally injured rat skin, and partially purified by column chromatography using Sephadex G-50, CM-Sephadex (A-50), Sephadex G-75 gel filtration. The optimum pH of this enzyme was 8.6--8.8, and the molecular weight determined by Sephadex G-75 gel filtration was approximately 30 000. This enzyme is active on the N-acetyl-L-tyrosine ethyl ester, N-succinyl-L-phenylalanine-p-nitroanilide (of chymotrypsin substrate) but not N-tosyl-L-arginine methyl ester, N-benzoyl-L-arginine-p-nitroanilide. Also, protease-S was completely inhibited by diisopropylfluorophosphate (1 mM) or phenylmethylsulfonylfluoride (10 micrometer), and N-tosyl-L-phenylalanine chloromethylketone (1 mM). These results are very similar to those obtained with bovine chymotrypsin. But the enzyme is not identical with the chymotrypsin-like proteases in mast cells and leukocyte granules. When proteases-S was measured during the inflammatory reaction in vivo, maximal activity was found after 8 h, at the end of inflammation.

Animals↗
PubMed1979

Reconstitution of lipid vesicles associated with HVJ (Sendai virus) sikes. Purification and some properties of vesicles containing nontoxic fragment A of diphtheria toxin.

A mixture of HVJ (Sendai virus) spike proteins, the nontoxic fragment A of diphtheria toxin, lecithin, and cholesterol was solubilized in sucrose solution containing a nonionic neutral detergent. The liposomal vesicles which formed on removal of the detergent by dialysis were purified by gel filtration and centrifugation on a sucrose gradient. The resulting purified vesicles had hemagglutinating activity, hemolytic activity and, after solubilization, the enzymic activity of fragment A. The vesicles had no cell fusion activity. Electron microscopy showed that both the outside and inside of membranes of the vesicles were associated with the spikes. When the vesicles were freeze-fractured, no large aggregates of particles were seen on either face. Such fragment A-containing lipid vesicles (liposomes) with HVJ spikes bound to mamalian cell membrane and released their fragment A into the cytoplasm causing cell death. Neither fragment A-containing liposomes without spikes nor empty liposomes with spikes were toxic.

Cell Fusion↗
PubMed1979

Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.

3-Hydroxysteroid Dehydrogenases↗
PubMed1979

Pigmentary paravenous chorioretinal degeneration: fundus appearance and retinal functions.

A case of pigmentary paravenous chorioretinal degeneration initially diagnosed as retinitis pigmentosa is presented. The focal macular visual evoked response (VER) was normal. Both rod and cone components in the electroretinogram showed reduced amplitude with normal peak time. The psychophysical threshold in the dark adaptation was nearly normal in some parts of the retina and extremely elevated in other parts of the retina. Fundus appearance and retinal function studies suggest that the impaired area in the retina and choroid is localized in this disease.

Adult↗