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Biomedical subjects

N Okamura

Publications and source records attributed to N Okamura.

At least 55 records · Page 3Linked to original sources
PubMed1999

Purification and properties of major alpha-D-mannosidase in the luminal fluid of porcine epididymis.

A lysosomal type alpha-D-mannosidase was successfully purified by DEAE-Sephacel, Red-Amicon and Superdex 200 column chromatographies from porcine cauda epididymal fluid. The purified enzyme consisted of 63 and 51 kDa subunits at equimolar amounts. It cleaved alpha1-2 linked mannosyl residues and less but significantly cleaved alpha1-3 and alpha1-6 linked mannosyl residues in the high-mannose oligosaccharides. The optimal pH to hydrolyze oligosaccharide was in the acidic pH range (pH 3.5 approximately 4.0). Total alpha-D-mannosidase activities in the porcine epididymal fluid increased from proximal to distal caput epididymis, which maintained to cauda epididymis. At least two kinds of alpha-D-mannosidase (lysosomal type enzyme and 135 kDa alpha-D-mannosidase (MAN2B2)) were contained in the porcine epididymal fluid. The activity of the lysosomal type enzyme is much higher than MAN2B2 at the physiological pH. These results suggest that the lysosomal type alpha-D-mannosidase is the predominantly active enzyme in the luminal fluid of porcine epididymis and that it participates in the glycoprotein modification on the sperm surface during epididymal transit.

Amino Acid Sequence↗
PubMed1999

A porcine homolog of the major secretory protein of human epididymis, HE1, specifically binds cholesterol.

A porcine homolog of the major secretory protein of human epididymis, HE1, was for the first time purified from the porcine cauda epididymal fluid. The HE1 homolog was secreted into the epididymal fluid as a 19-kDa glycoprotein, whose sugar moiety was gradually processed to form a 16-kDa protein during transit through the epididymis. The HE1 homolog mRNA was detected only in the caput and corpus epididymis among the porcine tissues examined. The purified HE1 homolog specifically bound cholesterol with high affinity (Kd=2. 3 microM). The binding stoichiometry was determined to be 0.94 mol/mol, suggesting that 1 mol of cholesterol binds to 1 mol of the protein. It was also found that the HE1 homolog is a major cholesterol-binding protein in the porcine epididymal fluid. The possibility that the HE1 homolog is involved in the regulation of the lipid composition of the sperm membranes during the maturation in epididymis is discussed.

Amino Acid Sequence↗
PubMed1999

Pervanadate activates NADPH oxidase via protein kinase C-independent phosphorylation of p47-phox.

We studied differences between the NADPH oxidase activation pathways triggered by pervanadate, a protein tyrosine phosphatase inhibitor, and phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, in guinea pig neutrophils. Previously, pervanadate has been shown to activate NADPH oxidase via the tyrosine kinase-dependent pathway (Yamaguchi et al. Arch. Biochem. Biophys. 323, 382-386, 1995). Both pervanadate and PMA induced superoxide anion (O-2) production, translocation of the 47-kDa protein component of the phagocyte oxidase (p47-phox) to the plasma membrane, and phosphorylation of p47-phox in the membrane. A selective protein kinase C inhibitor, GF 109203X, markedly inhibited PMA-induced O-2 production, p47-phox translocation, and p47-phox phosphorylation, but did not inhibit pervanadate-induced O-2 production and only slightly suppressed pervanadate-induced translocation and phosphorylation. These results demonstrate that pervanadate activates NADPH oxidase independently of protein kinase C. Phosphopeptide mapping of p47-phox revealed differences in the mechanism between pervanadate-induced and PMA-induced phosphorylation. Furthermore, some protein kinases which phosphorylate p47-phox-derived peptide are activated by pervanadate. These results suggest the existence of novel protein kinases responsible for the phosphorylation of p47-phox and the activation of these protein kinases by tyrosine kinase.

Amino Acid Sequence↗
PubMed1999

Simultaneous high-performance liquid chromatographic determination of puerarin, daidzin, paeoniflorin, liquiritin, cinnamic acid, cinnamaldehyde and glycyrrhizin in Kampo medicines.

We report a high-performance liquid chromatographic method to determine the quantities of puerarin, daidzin, paeoniflorin, liquiritin, cinnamic acid, cinnamaldehyde and glycyrrhizin in Kampo medicine. All seven compounds were separated in less than 30 min with a Wakosil-II 5C18 AR column by linear gradient elution using 0.01% (v/v) phosphoric acid acetonitrile (0 min 90:10, 10 min 88:12, 22 min 70:30, 30 min 30:70) as the mobile phase at a flow-rate of 1.0 ml/min(-1), and detection at 250 nm. The detection limits of these compounds are 0.15-0.3 microM with response linearity. This method was applied to determine the quantities in eight Kampo decoctions; Mao-to, Makyo-yokukan-to, Makyo-kanseki-to, Yokuinin-to, Sho-seiryu-to, Keima-kakuhan-to, Kakkon-to and Kakkon-to-ka-senkyu-sin'i. Glycyrrhizin content was lower in both the decoction and the methanol-diluted decoction of Sho-seiryu-to compared with the others. Low pH due to organic acids of Schisandrae fructus in the decoction caused inhibition for glycyrrhizin dissolution in Sho-seiryu-to.

Acrolein↗
PubMed1999

Simultaneous determination of ephedrine, pseudoephedrine, norephedrine and methylephedrine in Kampo medicines by high-performance liquid chromatography.

A simultaneous high-performance liquid chromatographic method for the determination of ephedrine, pseudoephedrine, norephedrine and methylephedrine (ephedrine alkaloids) in Kampo medicines which contain Ephedrae Herba was established. The analysis can be accomplished within 25 min with a Wakosil-II 5C18 HG column by isocratic elution using a mixture of water, acetonitrile and sodium dodecyl sulfate (65:35:0.4) as the mobile phase at a flow-rate of 1.0 ml min(-1), and detection at 210 nm. The detection limits of ephedrine alkaloids are 0.37-1.06 microM per injection (5 microl). This method was applied to analyze the quantities in eight Kampo decoctions; Mao-to, Makyo-yokukan-to, Makyo-kanseki-to, Yokuinin-to, Sho-seiryu-to, Keima-kakuhan-to, Kakkon-to and Kakkon-to-ka-senkyu-sin'i. The concentration (per Ephedrae Herba gram) of ephedrine alkaloids was higher in the Makyo-kanseki-to decoction than in the others. Calcium sulfate from Gypsum Fibrosum raised ephedrine alkaloids dissolution in the Makyo-kanseki-to decoction.

Central Nervous System Stimulants↗
PubMed1999

Molecular typing of methicillin-resistant Staphylococcus aureus in a university teaching hospital.

Plasmid analysis and pulsed-field gel electrophoresis (PFGE) were used to study the epidemiologic relationship among methicillin-resistant Staphylococcus aureus (MRSA) strains isolated at Tokyo Medical and Dental University Hospital. We found that 263 of 276 MRSA isolates had plasmids, which could be classified into 30 different patterns according to the number and plasmid molecular weight. Strains which harboured a single plasmid of approximately 13.4 Mds in molecular weight were the most numerous (55.7% of the isolates). These strains were isolated from 14 of 17 hospital wards. The largest number of strains with this plasmid pattern (33 strains) were isolated from a single ward. PFGE typing was then performed to further confirm the relationships among these 33 strains. The PFGE banding patterns of these strains were highly similar. The antibiogram profiles of these strains were also correlated with the PFGE pattern. Thus, the results suggest that these strains are epidemiologically related and spread throughout the ward. Combined plasmid analysis and PFGE were effective for discriminating the various MRSA isolates.

DNA, Bacterial↗
PubMed1999

Simultaneous determination of baicalin, wogonoside, baicalein, wogonin, berberine, coptisine, palmatine, jateorrhizine and glycyrrhizin in Kampo medicines by ion-pair high-performance liquid chromatography.

An ion-pair high-performance liquid chromatographic method for the simultaneous determination of four flavonoids, namely baicalin, wogonoside, baicalein and wogonin, and four berberine-type alkaloids, namely berberine, coptisine, palmatine and jateorrhizine, and glycyrrhizin in Kampo medicines is described. The analysis can be accomplished within 30 min with a Wakosil-II 5C18 HG column by linear gradient elution using a mobile phase containing aqueous phosphoric acid, sodium dodecyl sulfate and acetonitrile at a flow-rate of 1.0 ml x min(-1), a thermostatic oven at 45 degrees C, and detection at 265 nm. The method was applied to quantifying these components in three Kampo decoctions: Oren-gedoku-to, San'o-shashin-to and Hange-shashin-to. The decoctions were diluted with 65% methanol at the final stage because a large quantity of precipitate, mainly from baicalin and berberine, was formed. The within-day relative standard deviations were less than 2.02% (n=10). The recoveries of these compounds were 90.3-102%. The detection limits of these compounds were 0.02-1.96 microM per injection (5 microl).

Alkaloids↗
PubMed1999

Participation of cytosolic protein phosphatase in regulation of NADPH oxidase in polymorphonuclear leukocytes.

Calyculin A, a protein phosphatase inhibitor, enhanced phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2-) production and translocation of the cytosolic NADPH oxidase factor, p47phox, to the plasma membrane in guinea pig polymorphonuclear leukocytes (PMNs). When PMNs were treated with t-(5-isoquino-line-sulfonyl)-3-methyl-piperazine (H-7), a protein kinase C (PKC) inhibitor, after exposure to PMA, inhibition of O2- production and of translocation of p47phox to the membrane fraction in PMA-stimulated PMNs were observed. When calyculin A was added to the PMA-stimulated PMNs after the addition of H-7, O2- production was again observed, and translocation of p47phox to the membrane fraction also occurred. The activity of NADPH oxidase, the amount of p47phox and the level of phosphorylation of p47phox in the membrane fraction prepared from PMA-stimulated PMNs, were reduced by the addition of the cytosol fraction from unstimulated PMNs. These reductions were attenuated by calyculin A. These results indicate that the active form of NADPH oxidase in PMNs can be reconstituted after the active complex of the enzyme has disappeared once, and that one of the mechanisms of regulation of this enzyme activity involves the phosphorylation of p47phox in the cyotosol and dephosphorylation of phosphorylated p47phox in the NADPH oxidase complex by protein kinase and protein phosphatase, respectively.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1999

Decavanadate inhibits the cell-free activation of neutrophil NADPH oxidase without affecting tyrosine phosphorylation.

NADPH oxidase was activated by arachidonate in a cell-free system consisting of membrane and cytosol fractions prepared from guinea pig neutrophils. Vanadate apparently inhibited the NADPH oxidase activity in the cell-free system (IC50=2 microM) without phosphotyrosine accumulation. The pH dependency and stability of the inhibitory effect observed for vanadate solution indicated that decavanadate, an isopolyanion of vanadate, was responsible for the inhibition. Pervanadate (vanadyl hydroperoxide) also inhibited the oxidase activity but at a higher concentration (IC50=0.2 mM). Decavanadate lowered the Vmax but did not affect the Km value of NADPH oxidase for NADPH. Decavanadate inhibited the activation process of NADPH oxidase but not the oxidase activity itself. Decavanadate-pretreatment of membrane and cytosol fractions irreversibly decreased the abilities of both fractions to activate NADPH oxidase in the cell-free system. Translocation of p47-phox, one of the cytosolic activation factors of NADPH oxidase, from cytosol to membrane, was little affected by decavanadate. These results suggest that decavanadate inhibits the activation of NADPH oxidase in the cell-free system without affecting the phosphotyrosine phosphatase, and that decavanadate can bind to both the membrane and cytosolic activation factors when they are in a dormant state, but not to the active oxidase complex.

Amino Acid Sequence↗
PubMed1999

Cerebrospinal fluid tau protein levels in demented and nondemented alcoholics.

The tau protein levels in cerebrospinal fluid (CSF-tau) were examined in 27 patients with alcohol dependence (20 demented and 7 nondemented), 36 age and dementia severity-matched patients with Alzheimer's disease (AD), and 23 age-matched normal control subjects. The CSF-tau levels in the demented alcoholic group (alcohol-induced organic brain disorders, 25.4 +/- 10.2 pg/ml) was significantly lower (p < 0.0001) than that in the AD group (96.1 +/- 53.3 pg/ml), but not significantly different from that in the nondemented alcoholics (18.1 +/- 10.2 pg/ml) or the controls (19.2 +/- 12.9 pg/ml). Using a 44.9 pg/ml as a cut-off value (mean + 2 SD of the normal control group), only one patient with alcohol-induced organic brain disorders exceeded the value, whereas 3 of 36 of the AD group showed CSF-tau levels less than this level. These findings suggest that alcohol-induced organic brain disorders are a group of dementias that are characterized by normal CSF-tau levels, and that the CSF examination for tau in combination with other clinical findings may help in differentiating alcohol-induced organic brain disorders from AD.

Adolescent↗
PubMed1999

New findings in pharmacological effects induced by antihistamines: from PET studies to knock-out mice.

Antihistamines are efficacious drugs to be used for the symptomatic relief of allergic diseases. The safety issue of antihistamines is of central importance because of their widespread use in current medical practice. To better understand the pharmacological effects of antihistamines on the central nervous system (CNS), we used two kinds of new methods, positron emission tomography (PET) and gene targeting regarding on histamine H1 receptors. The histamine H1 receptor occupancy was examined in young male volunteers with[11C]-doxepin (a potent H1 antagonist) after the oral or intravenous administration of antihistamines. In other studies, the cognitive performance was also measured tachistoscopically before and after taking antihistamines. The mutant mice lacking H1 receptors were used in the behavioural and neurochemical experiments to re-evaluate the role of H1 receptors. The H1-receptor occupancy in the human frontal cortex caused by antihistamines is significantly correlated with the reported values of incidence of sleepiness in clinical trials, and the occupancy is well proportional to the impaired cognitive performance. The behavioural studies of the H1-receptor knock-out mice confirmed the role of H1 receptors in arousal, the sleep-wake cycle, locomotion, nociception and aggressive behaviour. The pharmacological effects induced by H1 antagonism were re-evaluated by the PET and gene-targetting. Although any serious effects could not be observed in mice by the destruction of the H1-receptor gene, the cognitive performance was impaired in humans after taking first generation antihistamines in recommended doses.

Animals↗
PubMed1999

Additional O antigens of Vibrio fluvialis and Vibrio furnissii.

A total of 297 strains of Vibrio fluvialis and Vibrio furrnissii, which were collected from various countries for the past 15-year period of 1984-1998, were serogrouped. Of those examined, 239 strains of V. fluvialis and V. furnissii were classified into 29 known O serogroups; 9 strains were found to belong to R-form cultures, and the rest of the 49 strains could not be serogrouped. Of those serologically untypable strains, 26 novel O serogroups (O36 to O61) were established and added to our reference of the V. fluvialis and V. furnissii antigenic scheme. As all antisera against the O reference strains of the organisms contained some amount of antibody to the rough (R) antigen, all diagnostic O antisera were absorbed with the reference rough strain, V. fluvialis GF25.

Agglutination Tests↗
PubMed1998

Expression of human placental-type 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase in various cells and cell lines.

The expression of the human placental-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (HP2K) in various human cells and cell lines was investigated at the levels of transcription and translation. Analyses by both Northern blotting and a reverse transcription-polymerase chain reaction (RT-PCR) showed that BeWo, U-937, SupT1, H9, HeLa, HepG2, and human mononuclear cells, as well as human placental chorionic cells, expressed HP2K mRNA. All the nucleotide sequences of RT-PCR products from these cell lines were identical to that of HP2K. The expression of HP2K protein was determined by Western blot analysis of fractions from POROS-HQ column chromatography of the cell extracts from U-937 cells, which was used as an example of HP2K-mRNA positive cell lines. As with the 6-phosphofructo-2-kinase activity of HP2K, the activity of 6-phosphofructo-2-kinase in extracts of U-937 cells was not inhibited by glycerol 3-phosphate, a known 6-phosphofructo-2-kinase inhibitor of liver- and testis-type isozymes. These results strongly suggested that various cell lines, in particular U-937 cells, express functional HP2K enzyme. Furthermore, 6-phosphofructo-2-kinase in U-937 cells was found to be activated by treatments with isoproterenol and phorbol 12-myristate 13-acetate, indicating regulation of 6-phosphofructo-2-kinase activity in U-937 cells by protein kinases A and C.

Blotting, Northern↗
PubMed1998

High concentration of glucose causes impairment of the function of the glutathione redox cycle in human vascular smooth muscle cells.

We demonstrated that high glucose reduced H2O2 scavenge activity in human vascular smooth muscle cells. In the cells exposed to high glucose, the intracellular glutathione content decreased, although the NADPH content was unchanged. The rate of uptake of cystine, which is a rate-limiting precursor of the glutathione synthesis, decreased in the high glucose group compared with the control group. These decreases were shown to be dependent on glucose concentration. It was suggested that high glucose causes impairment of the function of the glutathione redox cycle in human vascular smooth muscle cells, resulting in reduced H2O2 scavenge activity.

Aorta↗
PubMed1998

Post-meiotic expression of the mouse dihydropyrimidinase-related protein 3 (DRP-3) gene during spermiogenesis.

The dihydropyrimidinase-related protein (DRP) family, originally identified in humans by their homology to dihydropyrimidinase, contains at least four members. Genes of this family, and their counterparts in other mammals and chickens, are expressed mainly in fetal and neonatal brain, suggesting that the encoded proteins have a physiological role in the development of the central nervous system. In addition, the DRP-3 gene is expressed in testis as a shorter mRNA than the brain form. As a first step in understanding the extra-neuronal function of DRP-3, the structure and expression of testis DRP-3 were examined. Testis DRP-3 cDNA showed the same sequence as brain DRP-3 cDNA, except for the 5'-terminal end, which encodes a 5'-untranslated region and the 11 N-terminal amino acid residues, indicating that the two forms of DRP-3 mRNA were transcribed from a single copy gene. Northern blotting analysis detected DRP-3 mRNA in 30-, 40- and 70-day-old, but not in 10- and 20-day-old testes. In situ hybridization analysis indicated that the expression of DRP-3 in testis is restricted to post-meiotic round spermatids. This is the first report of the expression of DRP genes in extra-neuronal cells.

Amino Acid Sequence↗
PubMed1998

An inhibitor of aldose reductase and sorbitol accumulation from Anthocepharus chinensis.

A flavonoid glycoside was isolated from Anthocepharus chinensis. Its structure was elucidated by spectral data and determined to be myricetin 3-O-(4"-acetyl)-alpha-fucoside. This flavonoid glycoside and its aglycone showed potent inhibition against rat and porcine lens aldose reductase. The flavonoid aglycone also inhibited sorbitol accumulation in human red blood cells.

Aldehyde Reductase↗
PubMed1998

[Functional neuropharmacology in the human brain using positron emission tomography: PET imaging of impaired cognitive performance induced by sedative antihistamines].

Antihistamines are the efficacious drugs to be used for the symptomatic relief of allergic diseases. The safety issue of antihistamines is of central importance because of their widespread use in current medical practice. Positron emission tomography (PET) was used to better understand the pharmacological effects of antihistamines on the central nervous system. The H1 receptor occupancy was examined in young male volunteers with [11C]-doxepin after the oral or intravenous administration of antihistamines. In other studies, the cognitive performance was also measured tachistoscopically before and after taking antihistamines. The H1 receptor occupancy in the human cortex caused by antihistamines is significantly correlated with the reported values of incidence of sleepiness in clinical trials, and the occupancy is well proportional to the impaired cognitive performance. To understand the brain mechanism of antihistamine-induced "sleepiness and impaired cognition", the regional cerebral blood flow (rCBF) during the task was measured using 3D-PET and H2(15)O before and after administration of d-chlorpheniramine. After its administration, the rCBF was significantly decreased on the bilateral middle temporal gyrus, midbrain and anterior cingulate. These findings suggest that H1 receptor blockade would be affected on the activity of the attention and cognitive system in the brain.

Animals↗