Biomedical subjects

Y Doi

Publications and source records attributed to Y Doi.

At least 37 records · Page 2Linked to original sources

PubMed2001

PhaR, a protein of unknown function conserved among short-chain-length polyhydroxyalkanoic acids producing bacteria, is a DNA-binding protein and represses Paracoccus denitrificans phaP expression in vitro.

A putative regulatory protein, PhaR, which was identified in the polyhydroxyalkanoic acid synthetic locus (phaZCPR) in Paracoccus denitrificans, was investigated. The PhaR protein purified from a recombinant Escherichia coli was estimated to be 22 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being consistent with the mass calculated from the nucleotide sequence. The molecular mass was determined to be 93 kDa by size-exclusion chromatography, suggesting that the protein formed a tetramer. A gel mobility shift assay showed that PhaR specifically bound to the intergenic region of phaC--phaP. In a cell-free protein synthesis system using E. coli S30 extract, the expression of the phaP gene was repressed by the addition of purified PhaR. These results suggest that PhaR is a DNA-binding protein and may play a role in the regulation of phaP gene expression.

Bacterial Proteins↗

PubMed2001

Analysis of mutational effects of a polyhydroxybutyrate (PHB) polymerase on bacterial PHB accumulation using an in vivo assay system.

Polymerase is a central enzyme involved in the biosynthesis of polyhydroxybutyrate (PHB), a well-known bacterial biodegradable polyester. In this study, we have established an in vivo assay system to analyze mutational effects of Ralstonia eutropha polymerase (termed PhbC(Re)) on the level of PHB accumulation in recombinant strains of Escherichia coli. This in vitro evolution system consists of a polymerase chain reaction-mediated random mutagenesis and two assay procedures, a plate assay using a PHB-staining dye and a high-pressure liquid chromatographic assay based on the converting reaction from PHB to crotonic acid. The distribution pattern of the PHB accumulation level of the mutant population using 378 clones arbitrarily selected, suggested that the present level of PhbC(Re) is high and well-optimized. It is noteworthy that many of the amino acid substitutions affecting the PHB accumulation occurred in the conserved positions or regions within an 'alpha/beta hydrolase fold' which is commonly found among hydrolytic enzymes. From a good correlation with the level of PHB accumulation, an activity estimation of the PhbC(Re) would be efficiently achieved by monitoring the level of PHB accumulation using the in vivo assay system established here.

Acyltransferases↗

PubMed2001

Measurement of B(0)(d)-B_(0)(d) mixing rate from the time evolution of dilepton events at the upsilon(4S).

We report a determination of the B(0)(d)-&B_(0)(d) mixing parameter Deltam(d) based on the time evolution of dilepton yields in Upsilon(4S) decays. The measurement is based on a 5.9 fb(-1) data sample collected by the Belle detector at KEKB. The proper-time difference distributions for same-sign and opposite-sign dilepton events are simultaneously fitted to an expression containing Deltam(d) as a free parameter. Using both muons and electrons, we obtain Deltam(d) = 0.463+/-0.008 (stat)+/-0.016 (syst) ps(-1). This is the first determination of Deltam(d) from time evolution measurements at the Upsilon(4S). We also place limits on possible CPT violations.

Journal Article↗

PubMed2001

Measurement of the CP violation parameter sin2 phi(1) in B(0)(d) meson decays.

We present a measurement of the standard model CP violation parameter sin2 phi(1) (also known as sin2beta) based on a 10.5 fb(-1) data sample collected at the Upsilon(4S) resonance with the Belle detector at the KEKB asymmetric e(+)e(-) collider. One neutral B meson is reconstructed in the J/psiK(S), psi(2S)K(S), chi(c1)K(S), eta(c)K(S), J/psiK(L), or J/psipi(0) CP-eigenstate decay channel and the flavor of the accompanying B meson is identified from its charged particle decay products. From the asymmetry in the distribution of the time interval between the two B-meson decay points, we determine sin2 phi(1) = 0.58(+0.32)(-0.34)(stat)+0.09-0.10(syst).

Journal Article↗

PubMed2001

Analysis of the accumulation of mutants in Sabin attenuated polio vaccine viruses passaged in Vero cells.

To confirm the safety of oral poliomyelitis vaccine (OPV) cultured in Vero cells, the genetic stability of cultured polio vaccine viruses was analysed by MAPREC (mutant analysis by PCR and restriction enzyme cleavage). The rates of mutant accumulation of the viruses passaged in Vero cells under a low multiplicity of infection (MOI) condition (approximately 10(-3.5)CCID50/cell; the same as under usual OPV production conditions) were higher than those passaged in secondary cultured monkey kidney cells. However, the rates of mutant accumulation were restrained when the viruses were cultured under a high MOI condition (approximately 10(-1.5)CCID50/cell) in Vero cells. Furthermore, neurovirulence of the passaged viruses in pollovirus susceptible transgenic mice PVR-Tg21 was shown to correlate highly with the results of MAPREC. It is expected that our results will contribute to the large scale preparation of safe and effective OPV using Vero cells.

Animals↗

PubMed2001

Application of (19)F chemical shift imaging in studies of mice with orally administered 5-fluorouracil.

In vivo quantitative metabolic mapping is an ideal tool for pharmacokinetic studies. Oral 5-fluorouracil (5-FU) and its metabolites in mice were imaged simultaneously by the (19)F fast spin echo (FSE) sequence using interleaved frequency selection at 9.4T. However, 5-FU images in the small intestine have never been obtained regardless of concentration. The reason for the discrepancy between image intensity and concentration was T(2). At a pH above 6, a dramatic decrease in T(2) of a (19)F 5-FU signal in an aqueous solution was found; T(2) was shorter in the small intestine (14 ms) than in the stomach (52 ms). The (19)F CSI sequence in FID sampling mode was employed for detecting short T(2) signals. With a 13-min resolution time, the detection of the 5-FU signals in the region of the small intestine (0.6 mmol/kg) was successful with a 5 x 5 mm(2) in-plane resolution. Furthermore, two signals separated by 2 ppm were clearly distinguishable, but failed to be separately detectable with the (19)F FSE sequence. For quantitative simultaneous monitoring of 5-FU and its metabolites of varying T(2), the (19)F CSI sequence in FID sampling mode was found to be superior to the (19)F FSE sequence.

Administration, Oral↗

PubMed2001

Effect of tranexamic acid on blood loss reduction after cardiopulmonary bypass.

OBJECTIVE: We evaluated the effect of tranexamic acid on blood loss in patients undergoing elective cardiopulmonary bypass for coronary artery bypass surgery. METHODS: We randomly assigned 7 of 14 patients to a group receiving 50 mg/kg tranexamic acid before skin incision and after the start of cardiopulmonary bypass and the other 7 as controls. RESULTS: Intraoperative and postoperative blood loss was significantly (p = 0.025) reduced in the tranexamic acid group. A similar decrease in platelet count was observed during cardiopulmonary bypass in both groups. Antithrombin III was significantly (p = 0.013) decreased in both groups during cardiopulmonary bypass. Antithrombin III and thrombin-antithrombin III complexes were significantly (p = 0.001) increased after protamine administration. A significant (p = 0.010) decrease in alpha 2-plasmin inhibitor was noted at 5 and 60 minutes after the start of cardiopulmonary bypass in the tranexamic acid group. alpha 2-plasmin inhibitor-plasmin complexes were significantly (p = 0.001) increased after the start of cardiopulmonary bypass in both groups and were significantly (p = 0.012) decreased after protamine administration. alpha 2-plasmin inhibitor-plasmin complexes in the tranexamic acid group were significantly (p = 0.030) lower than in controls 60 minutes after the start of cardiopulmonary bypass, just prior to the end of cardiopulmonary bypass, and after protamine administration. CONCLUSIONS: These findings showed that tranexamic acid administration effectively prevented perioperative blood loss without thromboembolic complications and that tranexamic acid during cardiopulmonary bypass coordinates the anticoagulative effect of heparin and the antifibrinolytic effect of tranexamic acid.

Aged↗

PubMed2001

Glucagon attenuates the action of insulin on glucose output in the liver of the Goto-Kakizaki rat perfused in situ.

The effects of glucagon and insulin on glucose production were explored directly using the isolated perfused liver of the Goto-Kakizaki (GK) rat, an animal model of type-2 diabetes. In the perfused liver of control rats, infusion of glucagon (0.06-1.0 nM) into the portal vein dose-dependently increased glucose output. In the GK rat liver, in which the intracellular distribution of glycogen was heterogeneous, basal glucose output during perfusion was significantly higher than in control, whereas the effect of glucagon on the maximum glucose output was not different. Infusion of insulin inhibited the glucagon-induced hepatic glucose output by 30-40% in control livers, but had no effect on that from the GK rat liver. The increase in hepatic cAMP content after glucagon infusion was antagonized by insulin in control livers, but not in the livers of GK rats. These results indicate that the antagonistic effect of insulin on glucagon-induced hepatic glucose production was attenuated in the isolated liver of the GK rat and suggest that this insulin resistance appeared in the signal transduction process of glucagon upstream from cAMP production.

Animals↗

PubMed2001

Expressions of endothelin-1, fibronectin, and interleukin-1alpha of human umbilical vein endothelial cells under prolonged culture.

We examined human umbilical vein endothelial cells (HUVECs) under prolonged culture by electron microscopy and by light and electron immunocytochemistry including double immunolabeling. Based on the cell area of HUVECs through multiple passages, we divided the cells into first, second, and third stages, which exhibited distinct morphological and immunocytochemical characteristics. During the first stage, HUVECs were polygonal in shape and had already formed the monolayer confluence. During the second stage, they were characterized by an increased number of Weibel-Palade (WP) bodies, which were actively segregated from Golgi cisterns. Endothelin (ET)-1 and von Willebrand factor, an endothelial cell marker, were occasionally colocalized in WP bodies. The increase in WP bodies correlated with high ET-1 concentration in the cultured medium, suggesting that these inclusions are involved in storage and release of ET-1 in a manner indicating a regulatory pathway. During the third stage, fibronectin and interleukin (IL)-1alpha were expressed in HUVECs as well as in multinucleated giant cells, which originated from HUVECs, but WP bodies decreased in number in association with a decrease in ET-1 immunoreactivity and concentration. The foregoing changes in immunoreactivities for ET-1, fibronectin, and IL-1alpha affecting HUVECs under prolonged culture may reflect a senescent process of these cells.

Apoptosis↗

PubMed2001

Alcohol consumption and severity of angiographically determined coronary artery disease in Japanese men and women.

The relation of alcohol consumption to the severity of coronary atherosclerosis was examined among 323 men and 220 women who underwent coronary arteriography. Severity of coronary atherosclerosis was assessed by the number of vessels obstructed > or =75% in diameter and Gensini's severity score. Alcohol consumption was divided into 5 categories in men (never, past, 1-24, 25-49, and > or =50 ml per day) and 3 categories in women (never, past, and current). Among men, odds ratios of severe stenosis (multiple-vessel disease or Gensini's score >15) decreased substantially and significantly in all current drinking categories but without dose-response effect. There was a weak, inverse association of current alcohol consumption with one-vessel disease, but not with moderate stenosis in terms of Gensini's score (< or =15). Past drinkers showed a fairly large, but statistically nonsignificant, decrease in the odds ratios of not only severe stenosis but also of moderate stenosis. Among women, current drinkers showed a small, statistically nonsignificant decrease in the risk of severe stenosis in terms of Gensini's score. These associations with alcohol use did not change after adjustment for known coronary risk factors. The present findings add to evidence that alcohol drinking confers protection against coronary atherosclerosis.

Adult↗

PubMed2001

Development of a new calcium phosphate cement that contains sodium calcium phosphate.

A cement powder consisting of sodium calcium phosphate, Na3Ca6(PO4)5, in addition to tetracalcium phosphate and beta-tricalcium phosphate was prepared by pulverizing blocks of 4 wt% sodium-, 11 wt% carbonate-containing apatite samples that were heated at 1700 degrees C for 5 h. When mixed with 30 wt% malic acid or citric acid at a powder liquid ratio of 3:1, the cement set in 3 or 7 min at room temperature with compressive strength being around 52 or 27 MPa. In HeLa-cell cultures, the cement mixed with malic acid was less cytotoxic than the cement mixed with citric acid, which was far less cytotoxic than a commercial carboxylate cement used as a negative control, suggesting malic acid to be superior to citric acid as a liquid in this regard. Similar findings were also obtained with osteoclasts, of which culture experiments clearly suggested that the number of osteoclasts on the cement mixed with malic acid was significantly greater than that on the cement mixed with citric acid. Since osteoclastic response to substrates could be used as a maker in evaluating their bioresorbability associated with osteoclasts, the above finding may suggest that the cement that is to be mixed with malic acid would be more useful as bone substitutes.

Animals↗

PubMed2001

Synthesis of variously oxidized abietane diterpenes and their antibacterial activities against MRSA and VRE.

Variously oxidized 12 natural abietanes, 6,7-dehydroferruginol methyl ether (3), ferruginol (5), 11-hydroxy-12-oxo-7,9(11),13-abietatriene (7), royleanone (9), demethyl cryptojaponol (12), salvinolone (14), sugiol methyl ether (16), sugiol (17), 5,6-dehydrosugiol methyl ether (19), 5,6-dehydrosugiol (20), 6beta-hydroxyferruginol (23), and taxodione (25) were synthesized. Antimicrobial activities of synthesized phenolic diterpenes and their related compounds against MRSA and VRE were evaluated. Phenols (12-hydroxyabieta-8,11,13-trien-6-one 22 and 23), catechols (12 and 14) and taxodione 25 showed potent activity with 4-10 microg/mL of MIC against MRSA and 4-16 microg/mL of MIC against VRE. (-)-Ferruginol showed more potent activity than natural type (+)-ferruginol. Quinone methide 7 showed the most potent activity with 0.5-1 microg/mL of MIC against both MRSA and VRE.

Anti-Bacterial Agents↗

PubMed2001

Analysis of adsorption function of polyhydroxybutyrate depolymerase from Alcaligenes faecalis T1 by using a quartz crystal microbalance.

Enzymatic adsorption and degradation of three types of aliphatic polyester films and two types of polyolefin films by the extracellular PHB depolymerase from Alcaligenes faecalis T1 have been studied by using a quartz crystal microbalance (QCM) technique. Hydrolysis of poly[(R)-3-hydroxybutyrate] was quantitatively followed by the QCM technique. Adsorption of the enzymes to films was also quantitatively detected by the QCM. Kinetic study on enzymatic adsorption suggests that the PHB depolymerase binds to the substrates not only by hydrophobic interaction but also by specific interaction between the ester bonds of polyesters and the binding domain of the enzyme. The results show that the QCM technique is a sensitive tool to study enzymatic degradation kinetics of biodegradable polyesters.

Adsorption↗

PubMed2001

Cloning and characterization of the Pseudomonas sp. 61-3 phaG gene involved in polyhydroxyalkanoate biosynthesis.

Pseudomonas sp. 61-3 produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) [P(3HB-co-3HA)] random copolymer consisting of monomeric units of 4-12 carbon atoms from sugars. The phaG(Ps) gene encoding (R)-3-hydroxyacyl-acyl carrier protein coenzyme A transferase was cloned from this strain, and homologous expression of this gene under the control of the lac or the native promoter was investigated. Additional copies of the phaG(Ps) gene in Pseudomonas sp. 61-3 led to an increase in both the polyhydroxyalkanoate (PHA) content in the cells and the fraction of medium-chain-length 3HA units in PHA. Disruption of the chromosomal phaG(Ps) gene resulted in an increase in the fraction of the 3HB unit in PHA. The site-directed mutagenesis of the phaG(Ps) gene was carried out to investigate the role of a HX(4)D motif which has been proposed to be related to PhaG activity.

Acyltransferases↗

PubMed2001

Characterization of 13 kDa granule-associated protein in Aeromonas caviae and biosynthesis of polyhydroxyalkanoates with altered molar composition by recombinant bacteria.

Analysis of native poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] inclusions from Aeromonas caviae FA440 revealed that ORF1 (a 348-bp gene located immediately upstream of phaC(Ac)) encodes a 13-kDa granule-associated protein, which was referred to as phaP(Ac). Several recombinant strains of A. caviae were constructed and conducted to analyze their PHA-producing abilities. A transconjugant of FA440 harboring additional copies of phaPCJ(Ac) genes accumulated P(3HB-co-3HHx) copolyesters with much higher 3HHx composition (46-63 mol %) than wild-type strain from alkanoates or olive oil. Deletion analysis revealed that overexpression of phaJ(Ac) encoding monomer-supplying (R)-hydratase was not a reason for the compositional change in the recombinant strains. PHA synthase activity in PHA inclusion fraction from the transconjugant composed of 60 mol % of 3HHx was 10-fold higher than that from the strain FA440 with 13 mol % of 3HHx, suggesting an importance of the level of PHA synthase activity for controlling the PHA composition in vivo.

Acyltransferases↗

PubMed2001

Crystallization, melting, and enzymatic degradation of biodegradable poly(butylene succinate-co-14 mol % ethylene succinate) copolyester.

The crystal structure and growth kinetics of melt-crystallized poly(butylene succinate-co-14 mol % ethylene succinate) [P(BS-co-14 mol % ES)] copolyester have been investigated at a wide crystallization temperature range of 30 to 90 degrees C. By means of wide-angle X-ray diffraction (WAXD), the copolyester composed of BS and ES units has been identified to have the same crystal structure with that of poly(butylene succinate) (PBS) homopolymer, suggesting that only PBS sequences crystallize while that the ES units are in an amorphous form. On the basis of secondary nucleation theory, two regimes of II and III have been observed and their transition temperature is around 80 degrees C. The spherulitic morphologies of P(BS-co-14 mol % ES) copolyester developed from banded spherulites to the normal ones without banding extinction patterns as the crystallization temperature increased. The melting behavior of P(BS-co-14 mol % ES) copolyester under different conditions has been studied by differential scanning calorimetry (DSC). Four melting peaks and one exothermal peak on the melting curves were observed during heating process, and their origination is discussed. The enzymatic degradation was carried out on the melt-crystallized P(BS-co-14 mol % ES) thin film by an extracellar PHB depolymerase from Pseudomonas stutzeri and the morphologies of lamellar crystals before and after degradation have been examined by atomic force microscopy (AFM). The results have indicated that enzymatically degradable ES units exist on the surface of lamellar crystals and are hydrolyzed by the enzyme, while that the crystalline cores composed of PBS chains are not degraded.

Absorbable Implants↗

PubMed2001

Enzymatic degradation of atactic poly(R,S-3-hydroxybutyrate) induced by amorphous polymers and the enzymatic degradation temperature window of an amorphous polymer system.

The phase structure and biodegradability were investigated for amorphous blends of chemosynthetic fully amorphous atactic poly(R,S-3-hydroxybutyrate) (a-PHB) with atactic poly(methyl methacrylate) (PMMA) and atactic poly(R,S-lactide) (a-PLA). The differential scanning calorimetry thermal analysis indicated that a-PHB/PMMA blends were partially miscible while a-PHB/a-PLA blends were miscible in the studied composition range. The biodegradations of the blends were carried out in phosphate buffer solution in the presence of bacterial poly(R-3-hydroxybutyrate) extracellular depolymerases purified from Alcaligenes faecalis T1 and P. stutzeri. Although a-PHB in the pure state was not degraded by these depolymerase, it was degraded by blending with PMMA and a-PLA. The results demonstrated that the enzymatic degradation of a-PHB can be induced by amorphous polymers such as PMMA and a-PLA. Also, the biodegradation rate of a-PHB in the blends decreased drastically when the degradation temperature is too much away from the polymer glass transition temperatures. On the basis of these results, a temperature window of the enzymatic degradation was first proposed for the blend and the essence of induced degradation was discussed.

Alcaligenes↗

PubMed2001

Involvement of catalytic amino acid residues in enzyme-catalyzed polymerization for the synthesis of polyesters.

Recently, a variety of aliphatic polyesters have been synthesized using hydrolases such as lipases and PHB depolymerases, and the reaction mechanism for these enzyme-catalyzed polymerization has been discussed. In this paper, we have studied the involvement of the catalytic amino acid residues of the hydrolase in enzyme-catalyzed polymerization with an extracellular PHB depolymerase from Alcaligenes faecalis T1. A wild-type PHB depolymerase and three kinds of site-specific mutants (catalytic amino acids were substituted) were prepared and their polymerization activities for the ring-opening polymerization of (R)-beta-butyrolactone (BL) were compared. BL was polymerized at 80 degrees C in bulk by the wild-type enzyme to yield polymers consisting of cyclic and linear structures in a high monomer conversion. In contrast, none of the mutant enzymes showed obvious polymerization activity. These results have clearly demonstrated that the catalytic triad is indeed responsible for the enzyme-catalyzed polymerization of BL.

4-Butyrolactone↗