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Biomedical subjects

M Nakano

Publications and source records attributed to M Nakano.

At least 721 records · Page 40Linked to original sources
PubMed1991

Oxidative modification of glycated low density lipoprotein in the presence of iron.

This is the first observation for contributing to the glycation of low density lipoprotein (LDL) to oxidative modification of its own lipids and protein. Human plasma LDL was glycated by incubation with glucose (G-LDL). Glucose incorporated into apoprotein B was approximately 10 mol/mol of apoprotein (2.8% modification of lysine residues) and 84% of G-LDL was adsorbed on phenylboronate affinity column. G-LDL incubated with Fe3+ for 4 h caused a significantly higher level of lipid peroxidation than U-LDL (untreated with glucose), and a higher molecular weight protein was observed in apoprotein B on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), increasing with incubation period. Corresponding to change on SDS-PAGE, G-LDL exposed to Fe3+ moved faster than G-LDL per se or U-LDL to anode on agarose gel electrophoresis. The higher the Fe3+ concentration, the more lipid peroxidation was caused. Alpha-tocopherol or probucol suppressed the lipid peroxidation of G-LDL exposed to Fe3+.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

Purification and characterization of the 65-kDa protein phosphorylated in murine macrophages by stimulation with bacterial lipopolysaccharide.

Modification of cellular proteins via phosphorylation is known to be a major regulatory mechanism whereby external stimuli control intracellular events. We demonstrated that bacterial LPS induced a distinct set of phosphorylated protein (pp) in murine peritoneal macrophages, and that the LPS-induced pp were specifically located in cytosol and/or membrane fractions. One of the most heavily phosphorylated substrate proteins with a molecular mass of 65 kDa (pp65) was purified to homogeneity via SDS-PAGE analysis and autoradiography by sequential chromatography on Sephacryl S-200, HPLC anion exchange, and hydroxyapatite HPLC. Our pp65 is apparently the first purified LPS-induced pp, and is thought to be a novel protein. Serine residues on pp65 were found to be exclusively phosphorylated, indicating a contribution by LPS-inducible serine kinase. Interestingly, LPS-induced phosphorylation of pp65 was not observed in macrophages from a LPS-nonresponsive C3H/HeJ strain of mice, although their macrophages had about the same amounts of unphosphorylated p65 as normal macrophages when detected under Western blot analysis by using polyclonal anti-pp65 antibodies. This suggests that the functional defect of C3H/HeJ macrophages exists somewhere in the process before the pp65 phosphorylation. Moreover, the degree of the pp65 phosphorylation in macrophages stimulated with LPS or lipd A correlated well to that of cellular responses such as IL-1 production in the same macrophages. Considering these observations, the pp65 seems to play a crucial role in macrophage activation, and the studies on the structure and function of the pp65 should lead to progress in our understanding of the mechanisms of macrophage activation by LPS.

Amino Acids↗
PubMed1991

Detection of heterogeneity of Cu, Zn-superoxide dismutase with monoclonal antibodies and the establishment of a highly sensitive fluorescence sandwich enzyme-linked immunosorbent assay.

Murine monoclonal antibodies directed against a native form of Cu, Zn-superoxide dismutase (SOD) were produced by immunizing SOD purified from human erythrocytes. The monoclonal antibodies able to bind SOD were further screened for their ability to absorb SOD activity using anti-mouse IgG conjugated iron beads as solid supports in magnetic separation. This new screening method revealed the heterogeneity of native SOD in the reactivity with the antibodies. One monoclonal antibody successfully absorbed the entire activity of SOD detected by an inhibition assay of cypridina luciferin analog (MCLA)-dependent chemiluminescence induced by superoxide anion production, while other absorbed only a part of the SOD activity. The evidence that all of the latter antibodies failed to react with recombinant artificial SOD free of charge isomers suggested correlation of the heterogeneity with the presence of charge isomeric forms. The former antibody was further used to establish a fluorescence sandwich enzyme immunoassay, and this assay provided a very sensitive detection limit as low as 100 pg/ml.

Animals↗
PubMed1991

High-performance liquid chromatographic method for simultaneous determination of enantiomers of 5-dimethylsulphamoyl-6,7-dichloro-2-dihydrobenzofuran-2, carboxylic acid and its N-monodemethyl metabolite in monkey plasma and urine after chiral derivatization.

A quantitative method for the simultaneous high-performance liquid chromatographic (HPLC) resolution and determination of the enantiomers of 5-dimethylsulphamoyl-6,7-dichloro-2,3-dihydrobenzofuran-2-carboxyl ic acid, a new diuretic, and its N-monodemethylated metabolite in monkey plasma and urine is described. The method includes diethyl ether extraction of the samples and S-(-)-alpha-methylbenzylamide derivatization of the extract, followed by reversed-phase solid-phase extraction and injection of the resulting diastereoisomers onto a reversed-phase HPLC column. Baseline separation was obtained. The assay showed linearity over the range 0.1-50 micrograms/ml of plasma and 0.25-500 microliters of urine, with a lower limit of detection of ca. 0.01 micrograms/ml for each of the enantiomers. The method is adequate for pharmacokinetic and enantioselective disposition studies of both the diuretic and its metabolite.

Animals↗
PubMed1991

Circulating thrombomodulin in thrombotic thrombocytopenic purpura.

Endothelial cell injury is thought to be one of the causative factors in thrombotic thrombocytopenic purpura (TTP). A novel index of endothelial injury, plasma thrombomodulin, was measured in 13 patients with acute TTP. The mean plasma concentration of thrombomodulin was elevated in patients with TTP (34.23 +/- 19.08 ng/ml) as compared with healthy subjects (16.99 +/- 2.63 ng/ml, P less than 0.001). Eight (61.5%) of 13 patients had high thrombomodulin values. Markedly elevated thrombomodulin levels were observed in TTP patients who had suffered from systemic lupus erythematosus, in whom plasma thrombomodulin was still elevated when they achieved remission. Five of these 13 patients with TTP had normal plasma levels of thrombomodulin. In addition, the plasma thrombomodulin concentrations were correlated well with von Willebrand factor antigen and tissue-type plasminogen activator antigen levels, both of which are released from stimulated or damaged endothelial cells. No difference was found in plasma thrombomodulin levels between patients who achieved remission and who did not. These findings suggest that the magnitude of the endothelial damage in TTP is variable among patients and that plasma thrombomodulin has limited clinical relevance to the severity of TTP.

Adult↗
PubMed1991

Establishment of a variety of human bone marrow stromal cell lines by the recombinant SV40-adenovirus vector.

Various human bone marrow stromal cell lines were established from the adherent cell populations by introduction of the recombinant SV40-adenovirus vector with an infection or electric poration procedure. As compared with DNA transfection, the vector introduction was able to immortalize the cells with more than 100 times higher efficiency. Morphological and cytochemical analyses indicated that various cloned cell lines with different properties were isolated by the vector introduction. All the established cell lines expressed SV40 large T antigen. These results provided the evidence indicating that the recombinant SV40-adenovirus vector was a useful tool to establish a variety of cell lines with different biological activities from human bone marrow stroma.

Adenoviridae↗
PubMed1991

Highly sensitive chemiluminescence method for determining myeloperoxidase in human polymorphonuclear leukocytes.

Myeloperoxidase activity was assayed by a chemiluminescence method, using a cypridina luciferin analog as a chemiluminescence probe, after extraction from peripheral human polymorphonuclear leukocytes. The chemiluminescence method was based on the detection of 1O2 generated by myeloperoxidase-catalyzed HOBr formation followed by the interaction of HOBr with H2O2 at pH 4.5. With this method, myeloperoxidase in less than 100 polymorphonuclear leukocytes could be detected and myeloperoxidase in 10(6) polymorphonuclear leukocytes would be calculated to be 14.4 pmol. Eosinophil extract, which contains eosinophil peroxidase, catalyzed 1O2 generation to a great extent, compared with the polymorphonuclear leukocyte extract at pH 4.5. Myeloperoxidase activity in extract of neutrophil fraction could be greatly influenced by eosinophil contamination.

Deferoxamine↗
PubMed1991

Mechanism for the generation of superoxide anion and singlet oxygen during heme compound-catalyzed linoleic acid hydroperoxide decomposition.

Heme compound, hematin or cytochrome c, catalyzes the decomposition of 13-hydroperoxy linoleic acid yielding both O2- and 1O2 under aerobic conditions. No 1O2 is produced when hydrogen peroxide and cumene hydroperoxide are used as substrates. In these experiments, both O2- and 1O2 could be precisely detected by a chemiluminescence method using a cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin++ +-3-one, as a chemiluminescence probe, in the absence and presence of Cu-Zn superoxide dismutase in catalytic amounts. The reduction and oxidation cycle of ferric heme compound and the bimolecular reaction of peroxyl radicals are plausible reaction mechanisms for O2- and 1O2 production, respectively, in the systems studied.

Catalysis↗
PubMed1991

Effectiveness of an intussusceptive antireflux valve to prevent ascending cholangitis after hepatic portojejunostomy in biliary atresia.

An intussusceptive antireflux valve was created in the Roux-en-Y loop at the same time as the hepatic portojejunostomy (Kasai procedure) for the purpose of preventing ascending cholangitis in 14 new cases of biliary atresia and in 6 long-term survivors of the Kasai operation after episodes of ascending cholangitis. Ascending cholangitis did not develop in any of the 13 new patients who showed bile outflow following a modified Kasai procedure with antireflux valve nor in 5 of 6 patients who had secondary valve implementation. Postmortem examination of a patient with a valve who died 2 years 3 months after surgery showed that the structure of the valve was intact and that the valve still was quite effective as an antireflux mechanism.

Anastomosis, Roux-en-Y↗
PubMed1991

The relationship between lateral border movements of the mandible and the determinants of occlusion.

Border movements of the mandible were measured with a jaw movement analyzer in nine subjects. This instrument records movement with 6 degrees of freedom and has the ability to accurately and linearly record the full range of jaw movement. The result of this study showed a relationship between the presence of balancing-side molar contacts and restricted contralateral jaw border movements. This restriction is probably due to aberrant external pterygoid muscle activity.

Adult↗
PubMed1991

Morphological and biochemical analyses of trichloroethylene hepatotoxicity: differences in ethanol- and phenobarbital-pretreated rats.

The histological and biochemical differences between ethanol- and phenobarbital (PB)-potentiated hepatotoxicity of trichloroethylene (TRI) in Wistar strain male rats were investigated. Both ethanol (2 g in daily liquid diet for 3 weeks) and PB (80 mg/kg/day for 4 days, ip) pretreatments enhanced TRI (inhalation exposures of 500 ppm for 8 hr, 2000 ppm for 2 or 8 hr, and 8000 ppm for 2 hr)-induced hepatic damage as judged by increases in plasma transaminase activities. Livers from PB-treated rats exposed to TRI displayed centrilobular necrosis, whereas livers from ethanol-treated rats exposed to TRI were characterized by ballooning degeneration mainly in midzonal areas. TRI exposure decreased the in vitro metabolism of TRI, high-Km benzene aromatic hydroxylase (BAH) activity, and cytochrome P450 content in livers of PB-treated rats with severe hepatic damage. In ethanol-treated rats, TRI exposure increased both the in vitro metabolism of TRI and the low-Km BAH activity but did not cause an apparent decrease in cytochrome P450 content even in animals with severe hepatic damage. These results suggest that TRI caused necrosis of centrilobular hepatocytes in PB-pretreated rats, which was accompanied by loss of xenobiotic metabolizing functions, whereas ballooning degeneration of hepatocytes mainly in midzonal areas occurred in ethanol-pretreated rats without loss of xenobiotic metabolizing functions.

Animals↗
PubMed1991

Isolation of two toxic heptapeptide microcystins from an axenic strain of Microcystis aeruginosa, K-139.

Two toxic heptapeptides were isolated from an axenic Microcystis aeruginosa strain, K-139. Using mainly a non-destructive NMR method, we determined the structure of the major toxin to be 7-desmethylmicrocystin LR which lacks an N-methyl group of the dehydroalanine moiety of microcystin LR. The minor toxin was deduced to be 3,7-didesmethylmicrocystin LR. The chromatographic and NMR analyses of 7-desmethylmicrocystin LR were compared with those of 3-desmethylmicrocystin LR.

Amino Acid Sequence↗
PubMed1991

The effects of anticonvulsant drugs on vitamin D3-activating cytochrome P-450-linked monooxygenase systems.

The effects of two anticonvulsant drugs, phenytoin and sodium valproate, on the bioactivation of vitamin D3 have been studied with respect to the microsomal and mitochondrial cytochrome P-450-linked monooxygenase systems that contribute to 25-hydroxylation of vitamin D3 in rabbit liver, and the mitochondrial cytochrome P-450-linked monooxygenase system that catalyzes 1 alpha-hydroxylation of 25-hydroxyvitamin D3 in rabbit kidney. These anticonvulsant drugs were found to inhibit the 25-hydroxylase activity on vitamin D3 in liver microsomes and mitochondria, respectively, but not to inhibit the 1 alpha-hydroxylation of 25-hydroxyvitamin D3, even over a wide concentration range. Moreover, the activities of the components of the cytochrome P-450-linked monooxygenase systems: NADPH-cytochrome P-450 reductase, NADPH-ferredoxin reductase and ferredoxin, were never inhibited by these drugs. It is possible that the inhibition of bioactivation of vitamin D3 by these anticonvulsant drugs causes rickets and osteomalacia, and the site of inhibition is expected to be the cytochrome P-450 mediated reactions in liver mitochondria.

Animals↗
PubMed1991

Sensitivity of bacteria to NaNO2 and to L-arginine-dependent system in murine macrophages.

Sensitivity of bacteria to NO2-/NO and to L-arginine-dependent system in murine macrophages was tested. The growth of all bacteria tested, Salmonella typhimurium, Pseudomonas aeruginosa and Staphylococcus epidermidis, was significantly inhibited by NaNO2 at the concentration of 5 to 10 mM. However, L-arginine-dependent effector mechanism of LPS-activated murine macrophages was ineffective in killing these bacteria. It seems that the concentration of NO2-/NO in phagolysosome in the activated macrophages is insufficient to kill the bacteria in situ. We concluded that L-arginine-dependent effector mechanism can hardly work on bacteria killing in activated macrophages.

Animals↗
PubMed1991

Failure of inhibitory effects by platelets on super-oxide anion generation from stimulated neutrophils in a severe bronchial asthmatic.

Blood was drawn from healthy human volunteers and a 30-year-old woman with severe bronchial asthma (atopic and perennial type), whose peripheral blood platelets counts were continuously higher than 60 x 10(4)/mm3. Neutrophils were purified on a Conray-Ficoll gradient and platelets were isolated by a gel filtration method with Sepharose 2B. Superoxide anion (O2-) generation from the cells was measured by 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo [1,2-a]purazin-3-one (MCLA)-dependent luminescence. Addition of 0.5 microM MCLA and 10(-8) M N-formyl-methionyl-leucyl-phenylalanine (fMLP) or 10 ng/ml phorbol myristate acetate (PMA) to a suspension of platelets obtained from the healthy controls and the patient caused no significant MCLA-dependent luminescence, 02- generation by neutrophils from healthy human volunteers, activated by 10(-8) M fMLP or 10 ng/ml PMA, was inhibited by their platelets in a concentration-dependent manner. Platelets from the patient failed to inhibit O2- generation from neutrophils activated by 10(-8)M fMLP. Cu-Zn superoxide dismutase concentrations in platelets from healthy human volunteers were not higher than the patient's. These data suggest that the absence of inhibition of superoxide generation from activated neutrophils by platelets might be involved in the pathogenesis of bronchial asthma.

Adult↗
PubMed1991

Neutralization of microcystin shock in mice by tumor necrosis factor alpha antiserum.

Microcystis aeruginosa is a common cyanobacterium in water blooms that appear widely in nutrient-rich, fresh, and brackish waters, and its toxic blooms cause the death of domestic animals. The administration of a crude toxic cell extract of M. aeruginosa K-139 to mice can produce tumor necrosis factor (TNF) and prompt severe physiological disturbances, especially liver damage, which can lead to death. The in vitro production of TNF-alpha by peritoneal macrophages was observed after stimulation with the cell extract or the purified toxin from K-139 cells. The expression of a TNF-alpha mRNA was also detected in spleen cells and peritoneal macrophages after stimulation with the cell extract. However, a previous injection of rabbit anti-murine TNF-alpha serum could prevent the liver damage to some extent and protect the mice from death. These findings indicate the involvement of TNF in microcystin shock.

Animals↗
PubMed1991

Ability of polymorphonuclear leukocytes to generate active oxygen species in children with bronchial asthma. Use of chemiluminescence probes with a Cypridina luciferin analog and luminol.

Airway inflammation with polymorphonuclear leukocytes (PMN) may play an important role in bronchial hyperresponsiveness (BHR). PMN generate superoxide anion (O2-) and other oxygen radicals that can damage lung tissue. We investigated the ability of peripheral PMN of children with bronchial asthma and control subjects to generate O2- and other active oxygen species using a 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin++ +-3-one, a highly sensitive and specific chemiluminescence (CL) probe for O2-, and luminol-dependent CL. The ability of PMN of subjects with asthma to generate O2- and other active oxygen species was significantly greater than that of PMN of control subjects when stimulated with opsonized zymosan (OZ), phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine. Furthermore, in the same asthmatic children, the generation of O2- and other active oxygen species was significantly higher with attacks than without attacks when PMN were stimulated with OZ. We also demonstrated that O2- generation correlated with the degree of BHR to inhaled histamine. These results suggest that PMN of asthmatic children, especially those with attacks, generate more active oxygen species than that of control subjects and that airway inflammation caused by O2- may be closely related to BHR in subjects with bronchial asthma.

Adolescent↗