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Biomedical subjects

K Yoshii

Publications and source records attributed to K Yoshii.

At least 19 recordsLinked to original sources

Sub-genomic replicons of Tick-borne encephalitis virus.

We constructed three sub-genomic replicons of Tick-borne encephalitis virus (TBEV) (Oshima REP, Oshima REP-GFP and Oshima REP-Neo) by deleting genes coding for structural proteins without or with insertion of green fluorescent protein (GFP) or Neo genes, respectively. BHK cells transfected with Oshima REP expressed the viral non-structural antigens in immunofluorescent and western blot analyses. GFP and viral antigens were co-expressed in the transfected cells with Oshima REP-GFP. G418-resistant cells harboring Oshima REP-Neo consistently expressed the antigens without showing any apparent CPE. These replicons constructed in this study will be useful in studies on the replication, assembly and packaging of TBEV, and to develop vaccines and gene-delivering systems.

Animals↗

Energetic protons from a few-micron metallic foil evaporated by an intense laser pulse.

With detailed experimental studies and hydrodynamics and particle-in-cell simulations we investigate the role of the prepulse in laser proton acceleration. The prepulse or pedestal (amplified spontaneous emission) can completely evaporate the irradiated region of a sufficiently thin foil; therefore, the main part of the laser pulse interacts with an underdense plasma. The multiparametric particle-in-cell simulations demonstrate that the main pulse generates the quasistatic magnetic field, which in its turn produces the long-lived charge separation electrostatic field, accelerating the ions.

Journal Article↗

Evaluation of European tick-borne encephalitis virus vaccine against recent Siberian and far-eastern subtype strains.

To evaluate the efficacy of the European TBE vaccine in east-Siberian and far-eastern regions of Russia, we examined the immune responses of the vaccine against recent TBE virus Siberian (Irkutsk) and far-eastern (Khabarovsk and Vladivostok) isolates. The sera of vaccinated humans showed efficient neutralizing antibody titers (> or =20) against Siberian and far-eastern strains. To evaluate the efficacy of the vaccine in vivo, mice were vaccinated and challenged with lethal doses of the viruses. All vaccinated mice survived each virus challenge. These results suggest that the European vaccine can prevent the TBE virus infection in east-Siberian and far-eastern regions of Russia.

Adult↗

Comparison of neuropathogenicity of poliovirus type 3 in transgenic mice bearing the poliovirus receptor gene and cynomolgus monkeys.

To clarify the similarities of poliovirus infection in cynomolgus monkeys and transgenic mice bearing the poliovirus receptor, TgPVR21, we compared the pathological changes of these animals following intraspinal inoculation of two strains of poliovirus type 3 using immunohistochemical detection of the capsid antigen. All of the monkeys inoculated with 10(6) TCID(50) viruses showed flaccid paralysis 2 or 3 days post-inoculation (p.i.). TgPVR21 mice showed paralysis starting from 2 to 3 days p.i. Histologically, neurons having pyknotic nuclei and eosinophilic cytoplasm and neuronophagia were characteristically observed in both animals, but central chromatolysis was not observed in infected TgPVR21. The median lesion scores in the monkeys and TgPVR21 were well correlated, though the distribution of poliovirus-infected lesions in the central nervous system was different. In both animals the motor neurons and the brainstem nuclei responsible for flaccid paralysis were infected by the virus, while the cerebral cortex and thalamus were infected in the monkeys but not in TgPVR21. These results confirmed the reliability of neurovirulence tests using TgPVR21 as a substitute for monkeys, in respect to the spinal and brainstem lesions of poliovirus type 3.

Animals↗

XAS and MCD studies in Eu0.6Sr0.4MnO3.

Eu(1-x)Sr(x)MnO3 system is one of the perovskite manganites that exhibit the giant magneto resistance effect and the transition from the insulator phase to the metal. Eu(0.6)Sr(0.4)MnO3 behaves as a ferromagnetic semiconductor and has an Insulator-Metal (IM) transition induced by external magnetic field, which could be found only in this system among the perovskite manganites group. The mechanism of the transition was investigated by X-ray Magnetic Circular Dichroism (XMCD) at the Mn L2,3-edges of and the Eu M4,5-edges in Eu(0.6)Sr(0.4)MnO3.

Journal Article↗

Di(2-ethylhexyl) phthalate contamination of retail packed lunches caused by PVC gloves used in the preparation of foods.

Plasticizer contamination of foods sold in retail packed lunches and set lunches in restaurants was determined by GC/MS. The phthalate esters were as follows: diethyl, dipropyl, dibutyl, dipentyl, dihexyl, butylbenzyl, dicyclohexyl, di(2-ethylhexyl), dioctyl, diisooctyl (mixture of isomers) and diisononyl (mixture). Di(2-ethylhexyl) adipate was also determined. Sixteen packed lunches and ten set lunches were analysed, and in all samples the concentration of di(2-ethylhexyl) phthalate (DEHP) was the highest, at 0.80-11.8 mg/kg in packed lunches and 0.012-0.30 mg/kg in set lunches. The DEHP content of five packed lunches exceeded 1.85 mg, which is the EU tolerable daily intake (TDI) for a person of 50 kg body weight. Foodstuffs that were components of the packed lunches were taken from the factory at each step of preparation and phthalates were determined. For example, chicken contained 0.08 mg/kg DEHP when uncooked, 13.1 mg/kg after frying and 16.9 mg/kg after packing. Disposable PVC gloves used in the preparation of foods were apparently the source of high DEHP concentrations. The gloves used during cooking or packaging were sprayed with 68% (w/w) ethanol to sterilize them. PVC gloves from the factory contained 22 or 41% by weight of DEHP. To confirm the link with the contamination problem, samples of boiled rice, croquette and boiled dry radish were handled in the laboratory with PVC gloves containing 30% (w/w) DEHP. DEHP migration levels of 0.05 mg/kg in rice or 0.33 mg/kg in croquette, and 11.1 mg/kg in radish were found. The alcohol sprayed onto the gloves increased the migration of DEHP to 2.03 mg/kg in rice, 2.45 mg, kg in croquette, and 18.4 mg/kg in radish.

Diethylhexyl Phthalate↗

(1S,2R)-1-Phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC), a new class of NMDA-receptor antagonist: molecular design by a novel conformational restriction strategy.

We have found that milnacipran, a clinically useful antidepressant due to its inhibition of the re-uptake of serotonin (5-HT) and noradrenaline, is also a non-competitive NMDA-receptor antagonist. Based on the cyclopropane structure of milnacipran, conformationally restricted analogs were designed and synthesized. Of these analogs, (1S,2R)-1-phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC) is 30-fold stronger than milnacipran as an NMDA-receptor antagonist with virtually no inhibitory effect on the neurotransmitter re-uptake. PPDC was identified as a new class of NMDA-receptor antagonist because it has a mode of action different from that of the previous antagonists; it selectivly binds the GluRepsilon3/GluRzeta1 and GluRepsilon4/GluRzeta1 subtype receptors in an agonist-independent allosteric manner. Functional assays of PPDC with the Xenopus oocytes system and cultured mouse neurons under voltage-clamp conditions confirmed that it acts as a potent NMDA-receptor antagonist. PPDC effectively protected against NMDA-induced neurotoxicity in both cultured mouse cerebral cortex and delayed neuronal death in a gerbil ischemic model. It was also active in a reserpine-treated mouse Parkinsons disease model. Thus, PPDC may be a candidate for a clinically useful NMDA-receptor antagonist, since the development of previous NMDA-receptor antagonists as drugs has been hindered by various undesirable side effects.

Animals↗

[Contents of eleven phthalates and di(2-ethylhexyl) adipate in retail packed lunches after prohibition of DEHP-containing PVC gloves for cooking purposes].

Ten samples of retail packed lunches purchased from convenience stores were determined for 11 phthalates and di(2-ethylhexyl) adipate (DEHA) in August 2000, 2 months after the prohibition of DEHP-containing PVC gloves in Japan. Each homogenized sample was extracted with acetonitrile, partitioned with n-hexane, and cleaned up using Florisil and PSA columns. Phthalates in the extract were determined by GC/MS (SIM). The limits of detection were 14.9 ng/g for di(2-ethylhexyl) phthalate (DEHP) and 18.6 ng/g for dibutyl phthalate (DBP). Levels of phthalates in packed lunch samples were 45 to 517 ng DEHP/g (198 ng/g, average), ND to 90 ng DEHA/g, and ND to 10.0 ng BBP/g. Diisononyl phthalate (DINP) was detected in one sample at 76 ng/g. Average DEHP level in ten samples was 4% of that in 1999. The contents of other phthalates were also reduced. DBP was not detected in any sample. Recovery of deuterated isomers added as surrogates was 27.9% for DNP-d4, and 40.6 to 101.5% for the other phthalates.

Adipates↗

[Clean-up procedure with ion-exchange mini column in the analysis of flusulfamide in agricultural products by HPLC].

A clean-up procedure with an ion-exchange column in the analysis of flusulfamide by HPLC was examined. Pesticide in the sample was extracted with methanol following liquid-liquid partition with ethyl acetate. The ethyl acetate fraction was cleaned up with silica gel column chromatography. The eluate from the silica gel column was further cleaned up with SAX + PSA mini column, then determined by HPLC. Carotenoids and interfering peaks were removed by washing the combined mini columns with 10 mL of 20% acetone-containing n-hexane and 5 mL of acetone, and flusulfamide was eluted with 35 mL of acetone.

Chromatography, High Pressure Liquid↗

Kinetics of testosterone 6beta-hydroxylation in the reconstituted system with similar ratios of purified CYP3A4, NADPH-cytochrome p450 oxidoreductase and cytochrome B5 to human liver microsomes.

Kinetics of testosterone 6beta-hydroxylation were determined using a reconstituted system that consisted of CYP3A4, cytochrome b5 and NADPH-cytochrome P450 oxidoreductase (OR) with similar ratios as those seen in human liver microsomes and compared with those determined using human liver microsomes. Two reconstituted systems were constructed in accordance with two human liver microsomal samples that showed extremely high and low ratios of OR/CYP3A4. The Km values of testosterone 6beta-hydroxylation obtained from the reconstituted systems with high and low OR/CYP3A4 ratios were 29.3 and 35.2 microM, respectively, which were similar to that of the corresponding human liver microsomal samples (23.2 and 40.0 microM, respectively). However, Vmax values obtained from the reconstituted systems (3.7 and 0.8 pmol/min/pmol CYP3A4) were much lower than those from the human liver microsomes (44.2 and 31.1 pmol/min/pmol CYP3A4). The results suggest that the interaction between substrate and CYP3A4 in the reconstituted systems appear to be similar to human liver microsomes but that the velocity of the substrate metabolism in the reconstituted systems is different from that in human liver microsomes. In conclusion, our reconstituted systems could be used for the determination of affinity but not for the determination of the maximum velocity of substrate metabolism. Further studies on the protein-protein interactions between CYP3A4, OR, cytochrome b5 and/or a specific lipid environment are required to establish a reconstituted system showing similar kinetic properties to those of human liver microsomes.

Cytochrome P-450 CYP3A↗

Neurovirulence of Sabin 1-derived polioviruses isolated from an immunodeficient patient with prolonged viral excretion.

We have analysed the neurovirulence of Sabin 1-derived isolates which persisted more than nine years in an immunodeficient patient in the U.S.A. Samples were collected from stool specimens at days 11 (St1), 23 (St2), 48 (St3), 126 (St5), and 200 (St7) after the onset of paralysis. Critical nucleotides associated with the reversion of virulence were examined. All the isolates had the substitutions at nucleotide positions 480 (G to A) in the 5'-non-coding region (NCR), 2438 (A to U) in VP3, 2795 (A to G) in VP1, and 6203 (C to U) in 3D. Serially diluted samples were injected intracerebrally to transgenic mice harbouring the human poliovirus receptor gene. Samples St2, 3, 5 and 7 showed typical virulent characters in transgenic mice, whereas the sample ST1 showed intermediate neurovirulence. It seemed that there were two variant viruses providing for a major (M) and a minor (m) populations. After disappearance of the m-variant, samples obtained at the later stages showed neurovirulence almost equivalent to that of the Mahoney strain. Thus, the Sabin 1 strain evolved towards full neuropathogenicity after long-term replication in humans by accumulating mutations. Therefore, OPV-vaccination of immunodeficient persons should be avoided.

Animals↗

Liquid chromatographic determination of emamectin, milbemectin, ivermectin and abamectin in crops and confirmation by liquid chromatography-mass spectrometry.

Emamectin, milbemectin, ivermectin and abamectin are similar macrocyclic lactone chemicals used as an acaricides or parasiticides. We developed a simultaneous analytical method for determining the residual amounts of these compounds and emamectin metabolites in crops. A sample extracted with acetone was cleaned up with Bond Elut C18 and NH2. The sample was then fluorescence-derivatized with trifluoroacetic anhydride and 1-methylimidazole in acetonitrile. The analyte was measured by HPLC with fluorescence detection using an octadecylsilyl column with 3 microm particle size and gradient elution. In most crops, their recoveries by the developed method were ca. 80-110%. The detection limits of the analytes in vegetables were 0.1-0.3 ppt. Using the developed method, we surveyed the residues of these compounds in 20 commercial crops in Osaka, Japan. The result of the surveillance was that emamectin benzoate of 0.2-6.7 ppb was detected in nine cases and milbemectin of 16.7-279.3 ppb was detected in four cases. The detected samples were confirmed by LC-electrospray ionization (ESI) MS. The limit of detection by LC-ESI-MS was similar to the fluorescence detection level of 0.1-0.3 ppt in vegetables except for milbemectin.

Antinematodal Agents↗

Identification of human cytochrome P450 isoforms involved in the 7-hydroxylation of chlorpromazine by human liver microsomes.

Studies to identify the cytochrome P450 (CYP) isoform(s) involved in chlorpromazine 7-hydroxylation were performed using human liver microsomes and cDNA-expressed human CYPs. The kinetics of chlorpromazine 7-hydroxylation in human liver microsomes showed a simple Michaelis-Menten behavior. The apparent Km and Vmax values were 3.4+/-1.0 microM and 200.5+/-83.7 pmol/min/mg, respectively. The chlorpromazine 7-hydroxylase activity in human liver microsomes showed good correlations with desipramine 2-hydroxylase activity (r = 0.763, p < 0.05), a marker activity for CYP2D6, and phenacetin O-deethylase activity (r = 0.638, p < 0.05), a marker activity for CYP1A2. Quinidine (an inhibitor of CYP2D6) completely inhibited while alpha-naphthoflavone (an inhibitor of CYP1A2) marginally inhibited the chlorpromazine 7-hydroxylase activity in a human liver microsomal sample showing high CYP2D6 activity. On the other hand, alpha-naphthoflavone inhibited the chlorpromazine 7-hydroxylase activity to 55-65% of control in a human liver microsomal sample showing low CYP2D6 activity. Among eleven cDNA-expressed CYPs studied, CYP2D6 and CYP1A2 exhibited significant activity for the chlorpromazine 7-hydroxylation. The Km values for the chlorpromazine 7-hydroxylation of both cDNA-expressed CYP2D6 and CYP1A2 were in agreement with the Km values of human liver microsomes. These results suggest that chlorpromazine 7-hydroxylation is catalyzed mainly by CYP2D6 and partially by CYP1A2.

Antipsychotic Agents↗

Degradation of malathion and phenthoate by glutathione reductase in wheat germ.

Residual malathion in wheat was estimated at a lower value when analysis was performed by extraction with acetone after addition of water to swell the wheat, according to the Japanese Bulletin Method. The supernatant of the wheat homogenate showed degradation not only of malathion but also of phenthoate. Malathion and phenthoate were not degraded by the boiled supernatant of the wheat homogenate. It was presumed for this reason that glutathione reductase (GR; EC 1.6. 4.2) in the wheat degraded malathion. The following results were obtained: (1) GR originating in wheat could degrade malathion and phenthoate. (2) The degradation of malathion by the GR was inhibited by excessive GSSG. (3) There was a high correlation between GR activity and malathion degradation activity of the supernatant of wheat homogenates. It is likely that GR acted on the specific structure of malathion and phenthoate, the S=P-S bond, and the blanch structure bonding with the sulfur atom. Following the above, extraction with acetone after addition of water (the Japanese Bulletin Method) should be replaced by extraction with pure organic solvent and without addition of water for swelling.

Biodegradation, Environmental↗

Mutations in the 2C region of poliovirus responsible for altered sensitivity to benzimidazole derivatives.

MRL-1237, [1-(4-fluorophenyl)-2-(4-imino-1,4-dihydropyridin-1-yl) methylbenzimidazole hydrochloride], is a potent and selective inhibitor of the replication of enteroviruses. To reveal the target molecule of MRL-1237 in viral replication, we selected spontaneous MRL-1237-resistant poliovirus mutants. Of 15 MRL-1237-resistant mutants obtained, 14 were cross-resistant to guanidine hydrochloride (mrgr), while 1 was susceptible (mrgs). Sequence analysis of the 2C region revealed that the 14 mrgr mutants contained a single nucleotide substitution that altered an amino acid residue from Phe-164 to Tyr. The mrgs mutant, on the other hand, contained a substitution of Ile-120 to Val. Through the construction of a cDNA-derived mutant, we confirmed that the single mutation at Phe-164 was really responsible for the reduced susceptibility to MRL-1237. MRL-1237 inhibited poliovirus-specific RNA synthesis in HeLa cells infected with a wild strain but not with an F164Y mutant. We furthermore examined the effect of mutations of the 2C region on the drug sensitivity of cDNA-derived guanidine-resistant and -dependent mutants. Two guanidine-resistant mutants were cross-resistant to MRL-1237 but remained susceptible to another benzimidazole, enviroxime. Either MRL-1237 or guanidine stimulated the viral replication of two guanidine-dependent mutants, but enviroxime did not. These results indicate that MRL-1237, like guanidine, targets the 2C protein of poliovirus for its antiviral effect.

Amino Acid Sequence↗

Purification, identification and phosphorylation of annexin I from rat liver mitochondria.

Annexin was purified from rat liver mitochondria to an apparent homogeneity with a molecular weight of 35 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified mitochondrial annexin (AXmito) was identified as annexin I by an immunoblot analysis using anti-annexin I antibody. The inhibitory effect of AXmito I on porcine pancreatic phospholipase A2 activity was as potent as that of bovine lung annexin I. The presence of annexin I in mitochondria was confirmed by an electron-microscopic study. AXmito I was shown to be phosphorylated by intrinsic protein tyrosine kinases on its tyrosine residues. This annexin was also phosphorylated by protein kinase C.

Animals↗