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K Takeshige

Publications and source records attributed to K Takeshige.

At least 19 recordsLinked to original sources
PubMed1998

Anionic amphiphile-independent activation of the phagocyte NADPH oxidase in a cell-free system by p47phox and p67phox, both in C terminally truncated forms. Implication for regulatory Src homology 3 domain-mediated interactions.

Anionic amphiphiles, such as arachidonate, activate the superoxide-producing phagocyte NADPH oxidase in a cell-free system with human neutrophil membrane, which contains cytochrome b558 comprising gp91(phox) and p22(phox), and three cytosolic proteins: p47(phox) and p67(phox), each harboring two SH3 domains, and the small GTPase Rac. Here we show that, even without the amphiphiles, the oxidase is activated in vitro by a C terminally truncated p47(phox), retaining the N-terminal and the two SH3 domains, and the N terminus of p67(phox). When either truncated p47(phox) or p67(phox) is replaced by the respective full-length one, the activation absolutely requires the amphiphiles. The results indicate that both p47(phox) and p67(phox) are the primary targets of the amphiphiles, and that their C-terminal regions play negative regulatory roles. We also find that the truncated p47(phox), but not the full-length one, can bind to p22(phox), a binding required for the oxidase activation. The N-terminal SH3 domain of p47(phox) is responsible for the binding not only to p22(phox), but also to the p47(phox) C terminus. Thus the SH3 domain is accessible in the active p47(phox), but is normally masked in the full-length one probably via intramolecularly interacting with the C terminus. The present findings support our previous proposal of regulatory SH3 domain-mediated interactions.

Cell Membrane↗
PubMed1998

Functional modules and expression of mouse p40(phox) and p67(phox), SH3-domain-containing proteins involved in the phagocyte NADPH oxidase complex.

The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The formation of the active oxidase complex at the membrane requires translocation of the Rac GTPase and two specialized cytosolic proteins that harbor SH3 domains, p67phox and p47phox. Another SH3-domain-containing protein p40phox, which is constitutively associated with p67phox in phagocytes, also enters the complex upon cell stimulation. Here we describe how we cloned mouse cDNAs encoding p40phox and its partner in phagocytes, p67phox. Both p40phox and p67phox comprise several protein-binding modules that are structurally and functionally well conserved between mouse and human, indicating their nature as adaptor proteins. We have also systematically investigated expression of the gene for p40phox in comparison with those for p67phox and p47phox. Distributions of the mRNAs for the three proteins among tissues are similar, with the most abundant expression in the spleen. The messages are abundant not only in phagocytic cells, but also in B cell lineage. The p40phox gene, but not the other two, is expressed in some types of cells such as plasma cells and T lymphocytes. Furthermore, in situ hybridization analysis shows that the p40phox mRNA is distributed in neuronal cells of mouse brain, providing evidence that one of the genes for the specialized oxidase factors is expressed in neurons. These observations raise the possibility that the adaptor protein p40phox plays a heretofore unsuspected role via interacting with other proteins in the cells that do not express p67phox or p47phox.

Amino Acid Sequence↗
PubMed1998

The PC motif: a novel and evolutionarily conserved sequence involved in interaction between p40phox and p67phox, SH3 domain-containing cytosolic factors of the phagocyte NADPH oxidase.

The superoxide-generating NADPH oxidase, dormant in resting phagocytes, is activated during phagocytosis following assembly of the membrane-integrated protein cytochrome b558 and cytosolic factors. Among the latter are the three proteins containing Src homology 3 (SH3) domains, p67phox, p47phox and p40phox. While the first two factors are indispensable for the activity, p40phox is tightly associated with p67phox in resting cells and is suggested to have some modulatory role. Here we describe a systematic analysis of the interaction between p40phox and p67phox using the yeast two-hybrid system and in vitro binding assays with recombinant proteins. Both methods unequivocally showed that the minimum requirements for stable interaction are the C-terminal region of p40phox and the region between the two SH3 domains of p67phox. This interaction is maintained even in the presence of anionic amphiphiles used for the activation of the NADPH oxidase, raising a possibility that it mediates constitutive association of the two factors in both resting and activated cells. The C-terminal region of p40phox responsible for the interaction contains a characteristic stretch of amino acids designated as the PC motif, that also exists in other signal-transducing proteins from yeast to human. Intensive site-directed mutagenesis to the motif in p40phox revealed that it plays a critical role in the binding to p67phox. Thus the PC motif appears to represent a novel module for protein-protein interaction used in a variety of signaling pathways.

Amino Acid Sequence↗
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↗