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Y Shirakura

Publications and source records attributed to Y Shirakura.

11 recordsLinked to original sources
PubMed2000

Hyperleptinemia in female patients with ossification of spinal ligaments.

In order to examine the involvement of leptin in the ossification of spinal ligaments (OSL), the present study examined (i) serum levels of leptin and insulin in OSL patients and controls, (ii) serum leptin levels in children of OSL females with severe obesity, (iii) the expression of leptin receptor mRNA in human spinal ligaments, and (iv) effects of leptin on cultured human ligament cells. In the OSL females, serum leptin levels were significantly higher than those of the control females, and the levels were positively correlated to the serum insulin levels, while in the control females, there was a tendency of inverse correlation. The daughters of OSL females with severe obesity also had high serum leptin levels, although they had not developed OSL. The expression of leptin receptor mRNA was confirmed in the ligaments, but leptin did not influence the alkaline phosphatase activity nor procollagen type I carboxyl-terminal peptide content of the ligament cells. These findings suggest that leptin is involved genetically and indirectly with the pathogenesis of OSL in female patients.

Adolescent↗
PubMed1989

Porcine luteinizing hormone isoform(s): relationship between their molecular structures, and renotropic versus gonadotropic activities.

We recently demonstrated the renotropic activity in ovine LH isoform(s). In this study we purified porcine (p) LH isoforms and analyzed their structures and bioactivities. Purified pLH preparation (G-100 fraction 3) was dissociated into alpha- and beta-subunits, followed by isolation with reverse phase HPLC. Six components were isolated and analyzed for their amino acid compositions and amino-terminal amino acid sequences. alpha-Subunits were found to have heterogeneous N-terminal sequences, which started at Phe-1, Gly-4, Phe-6, or Thr-7. This N-terminal heterogeneity and the presence of the initial 6 amino acids has not been reported previously to the best of our knowledge. Moreover, the first 10 residues, Phe-Pro-Asp-Gly-Glu-Phe-Thr-Met-Gln-Gly, were identical with those of the ovine LH alpha subunit. At least 3 different beta-subunits were identified as heterogeneous in their carboxyl-terminal amino acids. The pLH preparation (G-100-fraction 3) was then chromatographed by an isoelectrofocusing (IEF) column. Four IEF fractions were obtained. Their amino acid structures appeared to be identical, as judged by the identical elution profiles on reverse phase HPLC, but their carbohydrate compositions were slightly different, especially in N-acetylgalactosamine in alpha-subunits. Thus, fractionation on IEF depended on the heterogeneity in carbohydrate structures. Each IEF fraction had different potencies in terms of its gonadotropic activity (in vitro) and renotropic activity (in vivo and in vitro). The discrepancy was observed not only between gonadotropic activity and renotropic activity, but also between in vivo and in vitro renotropic activity. This study identified the structural heterogeneity of pLH isoforms and demonstrated their biological heterogeneity. We concluded that the carbohydrate structure of the pLH isoform is important for expressing its biological heterogeneity (gonadotropic and renotropic activity.

Amino Acid Sequence↗
PubMed1988

Effect of limited tryptic modification of a bacterial poly(3-hydroxybutyrate) depolymerase on its catalytic activity.

The extracellular poly(3-hydroxybutyrate) depolymerase of Alcaligenes faecalis T1, which hydrolyzes both hydrophobic poly(3-hydroxybutyrate) and water-soluble oligomers of D(-)-3-hydroxybutyrate, lost its hydrolyzing activity toward the hydrophobic substrate on mile trypsin treatment, but retained its activity toward water-soluble oligomers. The molecular mass of the trypsin-treated enzyme was 44 kDa, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, which was 6 kDa smaller than that of the native enzyme (50 kDa). The trypsin-treated enzyme seemed to be less hydrophobic than the native one, because it was rather weakly adsorbed to a hydrophobic butyl-Toyopearl column compared with the native enzyme, and showed no ability to bind to poly(3-hydroxybutyrate), to which the native enzyme tightly bound. These results suggest that, in addition to a catalytic site, the enzyme has a hydrophobic site, which is not essential for the hydrolysis of water-soluble oligomers, but is necessary for the hydrolysis of hydrophobic substrates, and this hydrophobic site is removed from the enzyme by the action of trypsin.

Alcaligenes↗
PubMed1986

Degradation of poly(3-hydroxybutyrate) by poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1.

The extracellular poly(3-hydroxybutyrate) depolymerase purified from Alcaligenes faecalis T1 has two disulfide bonds, one of which appears to be necessary for the full enzyme activity. This depolymerase hydrolyzed not only hydrophobic poly(3-hydroxybutyrate) but also water-soluble trimer and larger oligomers of D-(-)-3-hydroxybutyrate, regardless of their solubilities in water. Kinetic analyses with oligomers of various sizes indicated that the substrate cleaving site of the enzyme consisted of four subsites with individual affinities for monomer units of the substrate. Analyses of the hydrolytic products of oligomers, which had labeled D-(-)-3-hydroxybutyrate at the hydroxy terminus, showed that the enzyme cleaved only the second ester linkage from the hydroxy terminus of the trimer and tetramer, and acted as an endo-type hydrolase toward the pentamer and higher oligomers. The enzyme appeared to have a hydrophobic site which interacted with poly(3-hydroxybutyrate) and determined the affinity of the enzyme toward the hydrophobic substrate.

Alcaligenes↗
PubMed1986

[Studies on the diuretic effect of haloperidol in adult rats].

An increase in urinary flow has been observed in rats during cataleptic response to haloperidol. The present experiment was carried out to study the mechanism of haloperidol-induced diuresis. Wistar-Imamichi adult female rats were injected i.p. with haloperidol in a dose of 0.1, 1 or 10 mg/kg, and the time course of changes in urine volume was observed. The dose-dependent diuretic effect of 1 or 10 mg/kg haloperidol was significant from 4 hr afterward, and the haloperidol-induced diuresis was prevented by pretreatment with phenoxybenzamine, prazosin or yohimbine. Chlorpromazine but not spiperone and pimozide induced a significant increase in urine volume, though the effect of chlorpromazine was less marked as compared with that of haloperidol. Clonidine in a dose of 0.125-1.0 mg/kg enhanced urine flow markedly from 30 min, and the same alpha-adrenergic blockers were also effective in blocking the diuretic effect of clonidine. Urinary osmolarity in 1 mg/kg haloperidol- and 0.125 mg/kg clonidine-treated rats decreased significantly, whereas only clonidine stimulated urinary Na and K excretion. Plasma osmolarity and negative free water clearance did not change in both haloperidol- and clonidine-treated rats. The present results suggest that the haloperidol-induced diuretic effect could be due to the central alpha-adrenoceptor blocking action of haloperidol.

Animals↗
PubMed1985

Purification and properties of extracellular poly(3-hydroxybutyrate) depolymerases from Pseudomonas lemoignei.

Extracellular poly(3-hydroxybutyrate) depolymerase was purified from the culture medium of Peudomonas lemoignei and separated into four isozymes (A1, A2, B1 and B2) by CM-Sepharose CL-6B chromatography. The molecular weights of A1 and A2 and those of B1 and B2 were estimated to be 54 000 and 58 000, respectively, by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric points of A1, A2, B1 and B2 were found to be approximately pH 9.7, 10.0, 10.0 and 10.6, respectively, by isoelectric focusing. All four enzymes hydrolyzed poly(3-hydroxybutyrate) and oligomeric esters of D-(-)-3-hydroxybutyrate, but showed no activity toward the dimeric ester. Analysis of hydrolytic products of the oligomeric esters with A1 and B2 suggested that the enzymes hydrolyzed mainly the second and third ester bonds from the free hydroxy terminus at different frequencies, depending upon the chain length of the substrates.

Carboxylic Ester Hydrolases↗
PubMed1983

An extracellular D(-)-3-hydroxybutyrate oligomer hydrolase from Alcaligenes faecalis.

A strain of Alcaligenes faecalis secretes an extracellular D(-)-3-hydroxybutyrate oligomer hydrolase, in addition to poly(3-hydroxybutyrate) depolymerase, when it is grown in a medium containing poly(3-hydroxybutyrate) as the sole carbon source. The oligomer hydrolase (EC 3.1.1.22), which has been purified to electrophoretic homogeneity, has a molecular weight of 68 000, as estimated by Sephadex G-100 gel filtration, and of 74 000, by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric point of the enzyme is approx. 6.0 and the pH optimum for the enzyme reaction is 8.5. The purified oligomer hydrolase has high affinity for oligomeric esters (apparent Km for the D(-)-3-hydroxybutyrate dimer = 32.8 microM; for the dodecamer = 1.3 microM), but does not attack poly(3-hydroxybutyrate) (average molecular weight, 32 500) at all. Analysis of hydrolysates of the oligomeric esters suggests that the enzyme hydrolyzes these substrates from the carboxyl terminus, releasing D(-)-3-hydroxybutyrate units one by one.

Alcaligenes↗
PubMed1982

An extracellular poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis.

A strain of Alcaligenes faecalis T1, which was isolated from activated sludge, excreted an extracellular poly(3-hydroxybutyrate) depolymerase as it grew in a medium containing poly(3-hydroxybutyrate) as the sole carbon source. The molecular weight of the enzyme, purified from the culture medium to electrophoretic homogeneity, was 48 000 as determined by Sephadex G-100 filtration, and 50 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The pH optimum for the enzyme reaction was 7.5. The purified enzyme depolymerized poly(3-hydroxybutyrate) purified from Zoogloea ramigera 1-16-M, but did not attack the bacterial native poly(3-hydroxybutyrate)-containing granules. Km values were 13.3 micrograms/ml (= 0.78 microM, based on an estimated average molecular weight of 17 000) for poly(3-hydroxybutyrate) and 5.4 mM for the trimeric ester of D(--)-3-hydroxybutyric acid. Analysis of hydrolytic products of poly(3-hydroxybutyrate), several oligomeric esters of D(--)-3-hydroxybutyric acid, and the methyl ester of the trimeric ester indicated that the enzyme hydrolyzed these substrates from the free hydroxyl terminus, releasing D(--)-3-hydroxybutyrate dimer units one at a time.

Alcaligenes↗
PubMed1981

An NAD-linked acetoacetyl-CoA reductase from Zoogloea ramigera I-16-M.

An NAD-linked acetoacetyl-CoA reductase of Zoolgoea ramigera I-16-M was purified to electrophoretic homogeneity. In contrast to the D(-)-3-hydroxybutyryl-CoA-specific NADP-linked acetoacetyl-CoA reductase from the same bacterium [Saito, T. et al (1977) Arch. Microbiol. 114, 211 - 217], the purified enzyme was strictly stereospecific to L(+)-3-hydroxybutyryl-CoA, and was active not only with NAD+ but also with NADP+, although NADP+ was less effective than NAD+ as coenzyme. The enzyme showed a pH optimum at 6.3 for the reduction of acetoacetyl-CoA and at 8.0 for the oxidation of L(+)-3-hydroxybutyryl-CoA. In the reduction reaction, Km values for acetoacetyl-Coa and NADH were 8.8 microM and 6.5 microM, respectively, and in the oxidation reaction, Km values for L(+)-3-hydroxybutyryl-CoA and DNA+ were 7.0 microM and 32 microM, respectively. Among various 3-hydroxyacyl-CoAs tested, L(+)-3-hydroxybutyryl-CoA and L(+)-3-hydroxyvaleryl-CoA were the most active substrates. Poly(3-hydroxybutyrate) synthesis from acetyl-CoA, by a system reconstituted from purified preparations of 3-oxothiolase, acetoacetyl-CoA reductase and poly(3-hydroxybutyrate) synthase, was observed when the NADP-linked but not the NAD-linked reductase was used. These findings indicate that the NAD-linked acetoacetyl-CoA reductase is not directly involved in the biosynthesis of poly(3-hydroxybutyrate).

Acetoacetates↗