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K Takeshige

Publications and source records attributed to K Takeshige.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cardioprotective effect of succinate against ischemia/reperfusion injury.

We investigated the protective effects of succinate, which is a respiratory substrate and a potential antioxidant, on myocardial ischemia/reperfusion injury with the whole heart. Isolated rat hearts were loaded with 25-min normothermic global ischemia followed by 30-min reperfusion in a working heart model. Succinate administered either before reperfusion or added to the cardioplegic solution improved the postischemic cardiac function significantly. The hearts arrested with succinate-supplemented cardioplegic solution replenished high-energy phosphates and maintained the total adenine nucleotides during the reperfusion period, whereas those arrested with succinate-nonsupplemented cardioplegic solution replenished the high-energy phosphates less, and also lost total adenine nucleotides during that period. We thus conclude that succinate administered before reperfusion may decrease the degree of mitochondrial damage during reperfusion and thereby reduce the amount of myocardial ischemia/reperfusion injury.

Animals↗
PubMed1997

Roles for proline-rich regions of p47phox and p67phox in the phagocyte NADPH oxidase activation in vitro.

The cytosolic proteins p47phox and p67phox, each containing two SH3 domains, are required for activation of the superoxide-producing phagocyte NADPH oxidase in a cell-free system with human neutrophil membrane and the small GTPase Rac. Here we focus on roles of proline-rich regions (PRRs) that reside in p47phox and p67phox. Deletion of the p47phox PRR, to which the C-terminal SH3 domain of p67phox binds, results in three-fold decreased activation of the enzyme in the cell-free system with the full-length p67phox, suggesting a modulatory role of the p47phox PRR. The modulation is likely mediated via the C-terminal region of p67phox, since the p47phox mutant protein fully activates the oxidase in combination with the N-terminus of p67phox. Neither deletion of the p67phox PRR nor substitutions for prolines in the region affects the ability to support superoxide production under the cell-free conditions, indicating that the PRR of p67phox has no primary function in the oxidase activation.

Amino Acid Sequence↗
PubMed1997

In vivo determination of replication origins of human mitochondrial DNA by ligation-mediated polymerase chain reaction.

A large part of replication is aborted in human mitochondria, the result being a D-loop. As few attempts have been made to distinguish free 5' ends of true replicate from those of abortive ones, we examined the 5' ends of true replicate of human mitochondrial DNA at one nucleotide resolution in vivo by making use of ligation-mediated polymerase chain reaction. The distribution and relative amounts of origins of the true replicate are exactly the same as those of total newly synthesized heavy strands, which means that the abortion of replication is independent of 5' ends. Treatment of DNA with RNase H frees 5' ends on both heavy and light strands. This is the first in vivo evidence for covalently attached primer RNA to nascent strand in human mitochondrial DNA.

DNA Replication↗
PubMed1997

The content of intracellular mitochondrial DNA is decreased by 1-methyl-4-phenylpyridinium ion (MPP+).

1-Methyl-4-phenylpyridinium ion (MPP+), an oxidative metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is considered to be directly responsible for MPTP-induced Parkinson's disease-like symptoms by inhibiting NADH-ubiquinone oxidoreductase (complex I) in the mitochondrial respiratory chain. Here we demonstrate that 25 microM MPP+ decreases the content of mitochondrial DNA to about one-third in HeLa S3 cells. On the contrary, 0.1 microM rotenone, which inhibits complex I to the same extent as 25 microM MPP+ in the cells, increases the content of mitochondrial DNA about 2-fold. Hence, the effect of MPP+ on mitochondrial DNA is not mediated by the inhibition of complex I. To examine the replication state of mitochondrial DNA, we measured the amount of nascent strands of mitochondrial DNA. The amount is decreased by MPP+ but increased by rotenone, suggesting that the replication of mitochondrial DNA is inhibited by MPP+. Because the proper amount of mitochondrial DNA is essential to maintain components of the respiratory chain, the decrease of mitochondrial DNA may play a role in the progression of MPTP-induced Parkinson's disease-like symptoms caused by the mitochondrial respiratory failure.

1-Methyl-4-phenylpyridinium↗
PubMed1997

A dual effect of 1-methyl-4-phenylpyridinium (MPP+)-analogs on the respiratory chain of bovine heart mitochondria.

We examined effects of several compounds, structurally related to 1-methyl-4-phenylpyridinium (MPP+), on the NADH-dependent respiration of bovine heart submitochondrial particles. 1-Methyl-4-(3 '-trimethylammoniophenyl)pyridinium (analog 8) as well as MPP+ completely inhibited O2 consumption, reduction of ubiquinone-10, and reduction of cytochrome b in a dose-dependent manner. The production of superoxide (O2-) induced by MPP+ or analog 8 was to the same extent as that by rotenone, an inhibitor of complex I of the mitochondrial respiratory chain. Rotenone had no additive effect on the maximal production of O2- induced by MPP+ or analog 8, suggesting that the production was mediated by the same way as rotenone. 1-Methyl-4-(4'-nitrophenyl) pyridinium (analog 1) induced about 20-fold more production of O2 than MPP+ and the production was additively increased by rotenone. Analog 1 only partially inhibited rotenone-sensitive O2 consumption. Paraquat induced the production of O2- as much as analog 1. Paraquat, however, did not inhibit rotenone-sensitive O2 consumption or reduction of cytochrome b. These results suggest that MPP+ and its analogs interact with the mitochondrial respiratory chain at two sites, the substrate side of the rotenone-binding site and the rotenone-binding site. The analogs may be reduced to produce O2- at the former site and inhibit the respiratory chain at the latter site.

Animals↗
PubMed1997

Changes of energy metabolism induced by 1-methyl-4-phenylpyridinium (MPP+)-related compounds in rat pheochromocytoma PC12 cells.

We examined effects of three structurally related pyridinium compounds, 1-methyl-4-phenylpyridinium (MPP+), paraquat, and 1-methyl-4-(4'-nitrophenyl) pyridinium (analog 1), on the energy metabolism in pheochromocytoma PC12 cells. MPP+ inhibited the intracellular NADH oxidation by the mitochondrial respiratory chain, judging from the decrease of the cytosolic NAD+/NADH ratio. Paraquat enhanced the oxidation of NADH and decreased intracellular ATP more than MPP+. The inhibition of the mitochondrial respiration by MPP+ was partially compensated by enhanced glycolysis, while paraquat inhibited glycolysis at the level of hexokinase probably due to the intracellular production of oxygen radicals. Analog 1 moderately enhanced glycolysis, moderately increased a cytosolic ratio of NAD+/NADH, and caused only a slight decline of intracellular ATP. Paraquat was the most cytotoxic of the three compounds. Thus, the three structurally related compounds, MPP+, paraquat, and analog 1, showed different effects on the mitochondrial respiratory chain and the glycolytic pathway in PC 12 cells. Their properties found in the cells well reflected those obtained by using bovine heart submitochondrial particles.

Adenosine Triphosphate↗
PubMed1996

Assembly and activation of the phagocyte NADPH oxidase. Specific interaction of the N-terminal Src homology 3 domain of p47phox with p22phox is required for activation of the NADPH oxidase.

The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activation involves assembly of membrane-integrated cytochrome b558 comprising gp91(phox) and p22(phox), two specialized cytosolic proteins (p47(phox) and p67(phox)), each containing two Src homology 3 (SH3) domains, and the small G protein Rac. In the present study, we show that the N-terminal SH3 domain of p47(phox) binds to the C-terminal cytoplasmic tail of p22(phox) with high affinity (KD = 0.34 microM). The binding is specific to this domain among several SH3 domains including the C-terminal one of p47(phox) and the two of p67(phox) and requires the Pro156-containing proline-rich sequence but not other putative SH3 domain-binding sites of p22(phox). Replacement of Trp193 by Arg in the N-terminal SH3 domain completely abrogates the association with p22(phox). A mutant p47(phox) with this substitution is incapable of supporting superoxide production under cell-free activation conditions. These findings provide direct evidence that the interaction between the N-terminal SH3 domain of p47(phox) and the proline-rich region of p22(phox) is essential for activation of the NADPH oxidase.

Cytochrome b Group↗
PubMed1996

An SH3 domain-mediated interaction between the phagocyte NADPH oxidase factors p40phox and p47phox.

The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, following assembly of a membrane-integrated cytochrome b558 with cytosolic proteins, p47phox, p67phox and p40phox, each containing Src homology 3 (SH3) domains. While both p47phox and p67phox are indispensable for the oxidase activity, role of p40phox remains obscure. Here we study interaction between p40phox and p47phox by two independent methods, a two-hybrid system in the yeast and an in vitro binding assay using purified proteins. The present results show that the interaction is mediated via binding of the SH3 domain of p40phox to a C-terminal proline-rich region of p47phox. This proline-rich region is also the target for binding of p67phox, and the SH3 domain of p40phox can inhibit the binding of the C-terminal one of p67phox to p47phox.

Amino Acid Sequence↗
PubMed1995

Quantitative contribution of the acid production to the intracellular acidification in human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine.

A chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP), induced an acidification of cytosol by about 0.05 pH units in 30 sec followed by an alkalinization in human neutrophils. The quantitative contribution of acid production to the acidification was studied. The superoxide (O2-) production stimulated by fMLP was not involved in the acidification because the production of acids in neutrophils from patients with chronic granulomatous disease who do not produce O2-, was the same as that in normal neutrophils. The intracellular acidification was completely inhibited by deoxyglucose, suggesting that energy metabolism enhanced upon stimulation by fMLP might be the main source of the acidification. Although enhancement of the lactate formation by fMLP was 0.8 nmol/10(6) cells, which could lower intracellular pH by 0.08 pH units, the lactate production could not explain the initial acidification because the production of lactate started at 1 min after the stimulation while the intracellular acidification began immediately after the stimulation. Mitochondrial respiratory inhibitors such as KCN and rotenone had no effects on the fMLP-induced intracellular acidification. The fMLP-induced production of CO2 in 30 sec through the hexose monophosphate shunt was only 2.6 pmol/10(6) cells, which was calculated to decrease intracellular pH by only 0.0014. Thus, changes of energy metabolism induced by fMLP does not explain the acidification.

Acids↗
PubMed1995

Intracellular localization of 8-oxo-dGTPase in human cells, with special reference to the role of the enzyme in mitochondria.

We examined the intracellular distribution of 8-oxo-dGTPase (8-oxo-7,8-dihydrodeoxyguanosine triphosphatase) encoded by the MTH1 gene, a human mutator homologue. The activity of 8-oxo-dGTPase mainly located in cytosolic and mitochondrial soluble fractions of Jurkat cells, a human T-cell leukemia line. Electron microscopic immunocytochemistry, using a specific antibody against MTH1 protein, showed localization of MTH1 protein in the mitochondrial matrix. Activity in the mitochondria accounted for about 4% of the total activity. The specific activity in the mitochondrial soluble fraction (8093 units/mg protein) was as high as that in the cytosolic fraction (8111 unit/mg protein). The 8-oxo-dGTPase activities in cytosolic and mitochondrial soluble fractions co-eluted with MTH1 protein by anion-exchange chromatography, and the molecular mass of the mitochondrial MTH1 protein was much the same as that of the cytosolic MTH1 protein (about 18 kDa). HeLa cells expressing MTH1 cDNA showed an increased cytoplasmic signal together with a weak signal in the nucleus in in situ immunostaining of MTH1 protein, and the overexpressed MTH1 protein was recovered from both cytosolic and mitochondrial fractions. Thus, the 8-oxo-dGTPase encoded by MTH1 gene is localized in mitochondrial and cytosol.

Blotting, Western↗
PubMed1995

An inhibitor of cyclic AMP-dependent protein kinase enhances the superoxide production of human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine.

Intact human neutrophils produced superoxide (O2-) by the stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) even when the extracellular Ca2+ was absent (0.56 +/- 0.13 nmol/min per 10(6) cells). The production by fMLP was enhanced more than twice in the presence of the extracellular Ca2+. Moreover, the O2- production by fMLP in the presence of extracellular Ca2+ was enhanced nearly three times by the treatment of cells with H-89, an inhibitor of cyclic AMP-dependent protein kinase (PKA). The enhancement was not observed when the extracellular Ca2+ was depleted from the reaction mixture. In addition, H-89 did not enhance fMLP-induced O2- production of electropermeabilized neutrophils in which the intracellular Ca2+ concentration was fixed to about 100 nM. These observations suggest that not only Ca2+ influx but the inhibition of PKA is necessary for the maximum O2- production by fMLP and that the O2- production is partially suppressed by the activation of PKA induced by fMLP.

Bucladesine↗
PubMed1995

Effects of L-arginine analogues on vasomotion of isolated porcine coronary arteries.

L-Arginine analogues have been widely used to examine the role of endothelium-derived nitric oxide (NO) in vascular responses; however, the effects of the agents on coronary vasomotion are not fully understood. In this study, we examined the effects of the analogues on vasomotion of isolated porcine coronary arteries. Strips of the porcine coronary artery were suspended for isometric tension recording in Krebs-Henseleit solution. L-Arginine analogues, N omega-nitro-L-arginine methyl ester (L-NAME, 10(-9)-10(-3) M), NG-monomethyl-L-arginine (L-NMMA, 10(-9)-10(-3) M), and NG-nitro-L-arginine (L-NNA, 10(-9)-10(-3) M), caused dose-dependent contractions, which were greater in strips with than in those without endothelium. Those endothelium-dependent contractions were almost abolished by indomethacin (10(-5) M) and FeCl2 (10(-3) M). The latter reduces prostaglandin H2 to 12-heptadecatrienoic acid, which has no vasoconstrictor effect. These results indicate that the L-arginine analogues cause endothelium-dependent contractions that are mediated by prostaglandin endoperoxides and suggest that they have properties other than simple inhibition of NO synthesis in porcine coronary arteries.

Animals↗
PubMed1995

1-Methyl-4-phenylpyridinium (MPP+) inhibits mitochondrial oxygen consumption mediated by succinate as well as malate in rat pheochromocytoma PC12 cells.

When rat pheochromocytoma PC12 cells are cultured with 1 mM 1-methyl-4-phenylpyridinium (MPP+), the number of viable cells decreases to one third in 4 days while the number increases ten-fold without MPP+. Oxygen consumption by mitochondria in the presence of malate is inhibited about 80% by the treatment of the cells with MPP+ for 4 days. Unexpectedly, succinate-dependent oxygen consumption is also inhibited to essentially the same extent as malate-dependent one. These results suggest that the impairment of the respiration mediated by succinate as well as malate is important as a mechanism of MPP(+)-induced cell death.

1-Methyl-4-phenylpyridinium↗
PubMed1994

omega-Oxidation of lipoxin B4 by rat liver. Identification of an omega-carboxy metabolite of lipoxin B4.

Lipoxin B4 (LXB4) is metabolized to 20-hydroxy-LXB4 by rat liver microsomes. The omega-hydroxylation requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide, indicating involvement of a cytochrome P-450 (P-450). This is supported by inhibition of the reaction by antibodies raised against NADPH-P-450 reductase. The P-450 appears to be the one responsible for leukotriene B4 omega-hydroxylation, because leukotriene B4 inhibits the formation of 20-hydroxy-LXB4 and LXB4 blocks the leukotriene B4 omega-hydroxylase activity in microsomes. Incubation of 20-hydroxy-LXB4 with both rat liver cytosol and NAD+ leads to formation of a more polar metabolite on high-performance liquid chromatography. The metabolite is identified as 20-carboxy-LXB4, a novel metabolite of LXB4, based on analyses by ultraviolet spectrometry and by gas chromatography/mass spectrometry. The 20-carboxy-LXB4-forming activity is localized in cytosol, with an optimal pH of 8.5. The activity is dependent on NAD+, but NADP+ can not replace NAD+. The reaction is inhibited by pyrazole and 4-methylpyrazole, inhibitors of alcohol dehydrogenase, and by substrates of the enzyme such as ethanol and 20-hydroxy-leukotriene B4. Disulfiram, an inhibitor of aldehyde dehydrogenase, also blocks the 20-carboxy-LXB4 formation. These observations suggest that both alcohol dehydrogenase and aldehyde dehydrogenase participate in the oxidation of 20-hydroxy-LXB4 to 20-carboxy-LXB4.

Animals↗
PubMed1994

Preferential heme transport through endoplasmic reticulum associated with mitochondria in rat liver.

The transport of de novo synthesized protoheme into the conventional microsomal fraction and endoplasmic reticulum associated with mitochondria (MAER) was studied by injecting amino[14C]levulinic acid (ALA) into phenobarbital-treated rats to evaluate the role of MAER in the trafficking of heme between mitochondria and endoplasmic reticulum. In mitochondria, the specific radioactivity of the radiolabeled heme reached a maximum level at 4 min after the injection of 14C-ALA. The specific radioactivity in cytosol was about 2-fold lower than that in microsomes, suggesting that the cytosolic pathway of the heme transport from mitochondria to endoplasmic reticulum is not predominant, because the specific radioactivity of heme in cytosol should be higher than that in microsomes if heme is transported mainly through cytosol. MAER showed higher specific radioactivity than the conventional microsomal fraction up to 4 min and thereafter the specific radioactivities in MAER and the conventional microsomal fraction became nearly the same. The extents of decrease in cytochrome P-450 and the radioactivity in microsomes by the treatment with allylisopropylacetamide which destroyed cytochrome P-450 but not cytochrome b5, were essentially the same, suggesting that most of the radiolabeled heme in microsomes was incorporated into cytochrome P-450. These results suggest that MAER is a preferential site for the protoheme transport from mitochondria to endoplasmic reticulum.

Animals↗
PubMed1994

Cloning and sequencing of the cDNA encoding human glutaredoxin.

Glutaredoxin (thioltransferase) is a small, heat-stable protein, which is involved in thiol/disulfide exchange reactions. We have isolated a cDNA that encodes glutaredoxin from a human brain cDNA library. The encoded protein contains 106 amino acids with a calculated molecular mass of 11.76 kDa and an isoelectric point of 8.09. The amino acid sequence deduced from the cDNA is more than 80% identical to those of other mammalian glutaredoxins.

Amino Acid Sequence↗