PubMed Health⌕ Search

Biomedical subjects

Y Yoshida

Publications and source records attributed to Y Yoshida.

At least 19 recordsLinked to original sources
PubMed2007

Improving the thermal stability of lactate oxidase by directed evolution.

Lactate oxidase is used in biosensors to measure the concentration of lactate in the blood and other body fluids. Increasing the thermostability of lactate oxidase can significantly prolong the lifetime of these biosensors. We have previously obtained a variant of lactate oxidase from Aerococcus viridans with two mutations (E160G/V198I) that is significantly more thermostable than the wild-type enzyme. Here we have attempted to further improve the thermostability of E160G/V198I lactate oxidase using directed evolution. We made a mutant lactate oxidase gene library by applying error-prone PCR and DNA shuffling, and screened for thermostable mutant lactate oxidase using a plate-based assay. After three rounds of screening we obtained a thermostable mutant lactate oxidase, which has six mutations (E160G/V198I/G36S/T103S/A232S/F277Y). The half-life of this lactate oxidase at 70 degrees C was about 2 times that of E160G/V198I and about 36 times that of the wild-type enzyme. The amino acid mutation process suggests that the combined neutral mutations are important in protein evolution.

Amino Acid Substitution↗
PubMed2006

Restoring cervical lesions with flexible composites.

OBJECTIVES: Flexure at the tooth cervix has been suggested to dislodge cervical restorations more rapidly. The objective of this randomized controlled clinical trial was to test the hypothesis that a higher composite flexibility better withstands tooth flexure stress. METHODS: One hundred and forty-two non-carious cervical lesions were restored with composites with contrasting stiffness. Seventy-one patients randomly received two cervical restorations placed following two out of the three experimental groups: (1) the three-step etch-and-rinse adhesive Permaquick applied with the stiff micro-hybrid composite Amelogen Hybrid (PMQ/A-Hy, Ultradent), (2) Permaquick applied with the more flexible micro-filled composite Amelogen Microfill (PMQ/A-Mi, Ultradent), or (3) the 'golden standard' three-step etch-and-rinse adhesive Optibond FL applied with the micro-hybrid composite Prodigy (O-FL/Pro, Kerr). RESULTS: The recall rate at 7 years was 80%. No statistically significant difference was found for any of the parameters evaluated neither between both adhesives, nor between both composites (McNemar, p>0.05). Eleven percent of the O-FL/Pro restorations were clinically unacceptable due to loss of retention (5.5%) and severe marginal discoloration (5.5%). In the PMQ-group, 22% of the PMQ/A-Mi restorations (8% loss of retention, 5% severe enamel margin defects, 3% severe dentin margin defects, 6% severe marginal discoloration, 3% extreme sensitivity) and 19% of the PMQ/A-Hy restorations (13% loss of retention, 3% severe enamel margin defects, 3% severe marginal discoloration) needed repair or replacement. SIGNIFICANCE: The clinical performance of the three adhesive/composite combinations was good and reliable during the 7-year clinical trial. It was concluded that the composite stiffness does not affect the clinical longevity of cervical composite restorations.

Composite Resins↗
PubMed2006

Differences in circadian variation of cerebral infarction, intracerebral haemorrhage and subarachnoid haemorrhage by situation at onset.

BACKGROUND: The precise time of stroke onset during sleep is difficult to specify, but this has a considerable influence on circadian variations of stroke onset. AIM: To investigate circadian variations in situations at stroke onset--that is, in the waking state or during sleep--and their differences among subtypes. METHODS: 12,957 cases of first-ever stroke onset diagnosed from the Iwate Stroke Registry between 1991 and 1996 by computed tomography or magnetic resonance imaging were analysed. Circadian variations were compared using onset number in 2-h periods with relative risk for the expected number of the average of 12 2-h intervals in the waking state or during sleep in cerebral infarction (CIF), intracerebral haemorrhage (ICH) and subarachnoid haemorrhage (SAH). RESULTS: ICH and SAH showed bimodal circadian variations and CIF had a single peak in all situations at onset, whereas all three subtypes showed bimodal circadian variations of stroke onset in the waking state only. These variations were different in that CIF showed a bimodal pattern with a higher peak in the morning and a lower peak in the afternoon, whereas ICH and SAH had the same bimodal pattern with lower and higher peaks in the morning and afternoon, respectively. CONCLUSIONS: Sleep or status in sleep tends to promote ischaemic stroke and suppress haemorrhagic stroke. Some triggers or factors that promote ischaemic stroke and prevent haemorrhagic stroke in the morning cause different variations in the waking state between ischaemic and haemorrhagic stroke.

Aged↗
PubMed2006

Decrease in tetrahydrobiopterin as a possible cause of nephropathy in type II diabetic rats.

A decrease in renal synthesis of nitric oxide (NO) in the progression of diabetic nephropathy has been documented. As (6R)-5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor of NO synthase, we investigated whether BH4 deficiency is involved in the pathogenesis of nephropathy. Ten-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats were used as a type II diabetic model, and Long-Evans Tokushima Otsuka (LETO) rats as the healthy controls. OLETF rats were orally treated with BH4 (10 mg/kg daily) or with water from 10 to 61 weeks of age. In another experiment, OLETF rats were treated orally with a calcium channel blocker, benidipine (5 mg/kg daily), or with 0.3% carboxymethyl cellulose (nontreated) from 10 to 52 weeks of age. Proteinuria was observed periodically, and at the end of the study, BH4 level and GTP cyclohydrolase I (GTPCH) activity in the kidney were measured. Proteinuria was observed at 13 weeks of age in the OLETF rats, and deteriorated until 61 weeks of age. Supplemental BH4 reduced the proteinuria. At 52 weeks of age, GTPCH activity and the BH4 level were decreased in the plasma and kidneys of OLETF rats, whereas they were significantly higher in the benidipine group than in the nontreated group. Proteinuria was milder in the benidipine group than in the nontreated group, without a concomitant decrease in blood pressure. Histologically observed glomerulosclerosis was mild in the BH4 and benidipine groups. In type II diabetic rats, renal BH4 is considered to play a crucial role in the pathogenesis of diabetic nephropathy. Benidipine was found to preserve BH4 levels, suggesting therapeutic renoprotective effects.

Animals↗
PubMed2006

1-BP inhibits NF-kappaB activity and Bcl-xL expression in astrocytes in vitro and reduces Bcl-xL expression in the brains of rats in vivo.

1-Bromopropane (1-BP) has been widely used as a substitute for chlorofluorocarbon that destroys the ozone layer. Although the central neurotoxicity of 1-BP has been recently reported, a molecular mechanism is not clear. In particular, the effects on cells in brain have not been fully analyzed. Here, we studied the effects of 1-BP on the activation of transcription factors involved in anti-apoptotic function or cell survival in astrocytes. Astrocytoma cell lines, U251, U373 and VM, or murine primary astrocytes were used for in vitro assay. DNA binding activities of NF-kappaB in these cells induced by interleukin (IL)-1 or LPS were inhibited by 1-BP. Consequently, the treatment of U251 cells with 1-BP resulted in suppression of NF-kappaB reporter activity. Furthermore, 1-BP blocked IkappaBalpha degradation, which is important for NF-kappaB activation. In addition, the level of Bcl-xL mRNA, which is known as an anti-apoptotic gene, were reduced in U251 treated with 1-BP or in the brain from rat exposed to 1-BP (400 ppm, 12 weeks). These results suggest that subchronic inhalation exposure to 1-BP vapor may affect the Bcl-xL expression in astrocytes.

Animals↗
PubMed2006

Structural and magnetic investigations for the doping effect of nonmagnetic impurity on the spin-Peierls-like transition in a quasi-one-dimensional magnet: 1-(4'-nitrobenzyl)pyridinium bis(maleonitriledithiolato)nickelate.

A nonmagnetic compound, [NO(2)BzPy][Au(mnt)(2)] (NO(2)BzPy(+) = 1-(4'-nitrobenzyl)pyridinium; mnt(2-) = maleonitriledithiolate), was synthesized and characterized structurally, which is isostructural with [NO(2)BzPy][Ni(mnt)(2)] that is a quasi-one-dimensional magnet and possesses a spin-Peierls-like transition with J = 192 K in the gapless state and spin energy gap = 738 K in the dimerization state, respectively. Further, ten nonmagnetic impurity doped compounds with a formula [NO(2)BzPy][Au(x)Ni(1-x)(mnt)(2)] (x = 0.01-0.73) were prepared and investigated by crystal structural determinations and magnetic susceptibility measurements. The nonmagnetic doping causes the suppression of the spin transition with an average rate of 221(12) K/percentage of dopant concentration. From the plots of chi(m)-T, the transition collapse (the characteristic of the transition is the sudden drop of chi(m) upon cooling, and the disappearance of this characteristic is considered as the criterion for the transition collapse) is estimated at around x > 0.27. In heavier doped system x = 0.49, the spin gap vanishes and a gapless phase is achieved again.

Journal Article↗
PubMed2006

An improved method for primary culture of rat podocytes.

A gentle method to isolate glomeruli simply by cutting renal cortices without forced sieving was devised in our previous study of primary podocyte culture. Yields of glomeruli isolated by this method, however, were too small to perform subculture or biological assays. In the present study, we tried an isolation method with magnetic beads and collagenase to increase the yields. Rat kidneys were perfused with magnetic particles. Renal cortices were digested with collagenase and filtered. Utilizing magnetic particles trapped within glomeruli, glomeruli were collected by attractive power of a magnet and cultured. The number of glomeruli isolated from one adult rat was more than 20,000 and the purity was more than 97%. About half of them were attached to culture dishes and exhibited cellular outgrowths, which were identified as podocytes by their distinct staining for podocyte markers. After 3 days of primary culture, the cellular outgrowths were subcultured. Approximately 60 podocytes were obtained per attached glomerulus. Their significant expression of podocytes markers was demonstrated by immunostaining and quantitative reverse transcriptase-polymerase chain reaction. The isolation method with magnetic beads and collagenase provides a number of glomeruli suitable for primary podocyte culture.

Animals↗
PubMed2006

Development of a self-etch adhesive for resin-modified glass ionomers.

The favorable self-adhesiveness of resin-modified glass ionomers (RMGIs) might be even further improved if the time-consuming and technically sensitive etch-and-rinse pre-treatment step with polyalkenoic acids could be avoided. We undertook this study to assess the effectiveness of an experimental self-etch adhesive for RMGIs that does not need to be rinsed off. Ultrastructural analysis and micro-tensile bond strength testing to enamel and dentin of a RMGI restorative material and a RMGI adhesive were performed after 4 different surface pre-treatments: no conditioning; 25% polyalkenoic acid; an experimental self-etch adhesive; and 37.5% phosphoric acid followed by the experimental self-etch adhesive. The use of an experimental self-etch adhesive increased the bond strength of RMGIs, especially after an additional conditioning step. Interfacial analysis showed the formation of a thin hydroxyapatite-containing hybrid layer. The self-etch technique enhances the user-friendliness of RMGIs and lowers their technique-sensitivity, while maintaining desirable characteristics of the conventional etch-and-rinse approach with polyalkenoic acids.

Acid Etching, Dental↗
PubMed2006

Chemical interaction of phosphoric acid ester with hydroxyapatite.

Among functional monomers used in contemporary dental adhesives, 10-methacryloyloxydecyl dihydrogen phosphate (MDP) has been found to interact chemically with hydroxyapatite (HAp) most intensively and stably. This effect was thought to be the basis of the superior bonding effectiveness of MDP-based self-etch adhesives to enamel/dentin. To elucidate fully the chemical interaction and reactivity of MDP with HAp, we used (31)P CP-MAS NMR spectroscopy and powder x-ray diffraction. In an aqueous ethanol solution, Ca ions were leached from HAp to form, at short term, a MDP-calcium salt (CaMHP(2)) layered structure on the HAp surface. When MDP was allowed to interact for longer time (< 24 hrs), CaHPO(4).2H(2)O precipitated on top of this MDP-calcium salt layered structure. In conclusion, the intense chemical interaction of MDP with HAp must be ascribed to superficial dissolution of HAp induced by the MDP adsorption and subsequent deposition of MDP-calcium salt with a solubility lower than that of CaHPO(4).2H(2)O.

Adsorption↗
PubMed2006

The effect of nitrite on aerobic phosphate uptake and denitrifying activity of phosphate-accumulating organisms.

An anaerobic/aerobic/anoxic/aerobic sequencing batch reactor (SBR) was operated with municipal wastewater to investigate the effect of nitrite on biological phosphorus removal (BPR). When nitrite accumulated, aerobic phosphate uptake activity significantly decreased and, in case of hard exposure to nitrite, BPR severely deteriorated. The interesting observation was that the relative anoxic activity of phosphate accumulating organisms (PAOs) increased after nitrite exposure. Moreover batch tests of aerobic phosphate uptake in the presence/absence of nitrite indicated that PAOs with the higher relative anoxic activity are less sensitive to nitrite exposure. From these results, we concluded that BPR is sensitive to nitrite exposure, but BPR containing PAOs with the higher relative anoxic activity is possibly more stable against nitrite than BPR containing PAOs with the lower relative anoxic activity.

Bacteria, Anaerobic↗
PubMed2006

[Airway injuries due to blunt chest trauma].

Although the incidence of blunt chest trauma is very high, the mediastinal tracheobronchial injuries are quite rare. The airway injuries are thought to be one of the most urgent clinical conditions in thoracic surgery, and we are requested to make not only a rapid and sharp diagnosis but also an appropriate treatment plan considering combined injuries. We present 9 cases of tracheobronchial injuries due to blunt chest trauma in recent years. The average age of these patients is 26.1 years, and they are consisted of 6 male and 3 female. The cause of trauma is traffic accident in 7, and occupational crane accident in 2. Bronchoplasty were done in 5 cases (right main bronchus in 2, left main bronchus in 1, trunks intermediate bronchus in 1, and the spur between middle and lower lobe in 1), membranous-tracheoplasty with right pneumonectomy in 1, left pneumonectomy in 1, conservative treatment in 2. Postoperative mortality is occurred in 1 case who was suffering from multiple injuries including severe head injury and contralateral lung contusion. Tracheobronchial plasties should be chosen if possible to preserve lung function for the patient suffering from airway injuries.

Adolescent↗
PubMed2005

Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). II. An inter-laboratory study of the h-CLAT.

Recent regulatory changes have placed a major emphasis on in vitro safety testing and alternative models. In regard to skin sensitization tests, dendritic cells (DCs) derived from human peripheral blood have been considered in the development of new in vitro alternatives. Human cell lines have been also reported recently. In our previous study, we suggested that measuring CD86 and/or CD54 expression on THP-1 cells (human monocytic leukemia cell line) could be used as an in vitro skin sensitization method. An inter-laboratory study among two laboratories was undertaken in Japan in order to further develop an in vitro skin sensitization model. In the present study, we used two human cell lines: THP-1 and U-937 (human histiocytic lymphoma cell line). First we optimized our test protocol (refer to the related paper entitled "optimization of the h-CLAT protocol" within this journal) and then we did an inter-laboratory validation with nine chemicals using the optimized protocol. We measured the expression of CD86 and CD54 on the above cells using flow cytometry after a 24h and 48h exposure to six known allergens (e.g., DNCB, pPD, NiSO(4)) and three non-allergens (e.g., SLS, tween 80). For the sample test concentration, four doses (0.1x, 0.5x, 1x, and 2x of the 50% inhibitory concentration (IC(50))) were evaluated. IC(50) was calculated using MTT assay. We found that allergens/non-allergens were better predicted using THP-1 cells compared to U-937 cells following a 24 h and a 48 h exposure. We also found that the 24h treatment time tended to have a better accuracy than the 48 h treatment time for THP-1 cells. Expression of CD86 and CD54 were good predictive markers for THP-1 cells, but for U-937 cells, expression of CD86 was a better predictor than CD54, at the 24h and the 48 h treatment time. The accuracy also improved when both markers (CD86 and CD54) were used as compared with a single marker for THP-1 cells. Both laboratories gave a good prediction of allergen/non-allergen, especially using THP-1 cells. These results suggest that our method, human Cell Line Activation Test (h-CLAT), using human cell lines THP-1 and U-937, but especially THP-1 cells at 24h treatment, may be a useful in vitro skin sensitization model to predict various contact allergens.

Allergens↗
PubMed2005

Development of an in vitro skin sensitization test using human cell lines: the human Cell Line Activation Test (h-CLAT). I. Optimization of the h-CLAT protocol.

The aim of this study is to optimize the experimental conditions for an in vitro skin sensitization test using the human cell lines THP-1 and U-937. As regards pre-culturing time, the expression of CD86 on DNCB-treated THP-1 cells tended to be higher after 48h and 72h pre-culture compared with other time points evaluated. Next, we investigated the effect of chemical treatment time, and found that induction of CD86 expression on THP-1 cells by DNCB reached a plateau after 24h. Augmentation of CD86 expression is often observed when cells are treated with a subtoxic dose of allergens. To determine the appropriate dose of test samples, the cytotoxicity of test samples to THP-1 and U-937 cells was assessed with MTT assay, and the 50% inhibitory concentration (IC50) of each test sample was calculated. Based on the cytotoxicity assay data, four concentrations in the range between toxic and non-toxic were selected (0.1x, 0.5x, 1x and 2x IC50). Several kinds of antibodies were tested for staining THP-1 and U-937 cells treated with allergens/non-allergens (e.g., DNCB, Ni/SLS), and suitable antibodies for staining CD86 and CD54 were selected. We confirmed that the working dilutions of the selected CD86 and CD54 antibodies were appropriate for use in our method. The effect of an FcR blocking procedure was also evaluated. The mean fluorescence intensity (MFI value) was decreased by the FcR blocking procedure, which indicated that non-specific staining was blocked. Therefore, this procedure should be included in the method. Based on our findings, the protocol for this assay was optimized and the experimental conditions to be used in a future validation study were identified. We propose to call this kind of in vitro skin sensitization test h-CLAT, which is short for human Cell Line Activation Test.

Allergens↗
PubMed2005

Parylene flexible neural probes integrated with microfluidic channels.

The fluidic channel in the flexible probe has three functions: (i) to inject chemicals into the tissues, (ii) to measure the neural activities from the tissues, and (iii) to improve the mechanical stiffness of the probe by filling the channel with a solid material. A 10-microm-thick microfluidic channel was embedded into the probe by using sacrificial photoresist patterns. Polyethylene glycol (PEG), which is solid at room temperature and dissolves when in contact with water, was used to fill the channel and increase the stiffness of the probe before insertion into the tissue. The impedance of the electrode inside the fluidic channel was around 100 kOmega at 1 kHz when the channel was filled with saline solution. We were able to insert the probe into a rat's brain and measure the neural signals with the electrode.

Animals↗
PubMed2005

Specific heat study of an S = 1/2 alternating Heisenberg chain system: F5PNN in a magnetic field.

We have measured the specific heat of an S = 1/2 antiferromagnetic alternating Heisenberg chain pentafulorophenyl nitronyl nitroxide in magnetic fields up to H > H(C2). This compound has a field-induced magnetic ordered (FIMO) phase between H(C1) and H(C2). Characteristic behaviors are observed depending on the magnetic field up to above H(C2) outside of the H-T boundary for the FIMO. The temperature and field dependence of the specific heat are qualitatively in good agreement with the theoretical calculation on an S = 1/2 two-leg ladder [Wang et al., Phys. Rev. Lett. 84, 5399 (2000)]]. This agreement suggests that the observed behaviors are related with the low-energy excitation in the Tomonaga-Luttinger liquid.

Journal Article↗