Masaharu Miyake

Publications of Masaharu Miyake

• β-Citryl-L-glutamate acts as an iron carrier to activate aconitase activity.

  Authors: Michiko Hamada-Kanazawa, Masanori Narahara, Masaoki Takano, Kyong Son Min, Keiichi Tanaka, Masaharu Miyake

  Biological & pharmaceutical bulletin. 01/2011; 34(9):1455-64.

  The compound β-citryl-L-glutamate (β-CG) was initially isolated from developing brains, though its functional roles remain unclear. In in vitro experiments, the [Fe(II)(β-CG)] complex activatedThe compound β-citryl-L-glutamate (β-CG) was initially isolated from developing brains, though its functional roles remain unclear. In in vitro experiments, the [Fe(II)(β-CG)] complex activated aconitase in the presence of reducing reagents, whereas no Fe complex with citrate, glutamate, or deferoxamine displayed such an effect. β-CG and [Fe(II)(β-CG)] both bound to the fourth labile Fe atom (Fe(a)) in the [4Fe-4S] cluster of aconitase. Furthermore, [Fe(II)(β-CG)] reactivated aconitase damaged by ammonium peroxodisulfate (APS), while β-CG and citrate had no effect. These findings suggest that [Fe(II)(β-CG)] can transfer Fe to aconitase disassembled by APS. In intact mitochondria, both β-CG and [Fe(II)(β-CG)] bound to Fe(a) of aconitase, whereas only [Fe(II)(β-CG)] reactivated the enzyme disassembled by APS. In cultured neuronal cells, β-CG significantly enhanced cell viability by accelerating mitochondrial activity in primary cultures of neurons from newborn mouse cerebrum tissues. Thus, the β-CG plays a role as an Fe-carrier for mitochondrial aconitase, and then activates it. Taken together, these findings suggest that β-CG is an endogenous low molecular weight Fe chaperone for aconitase.

• Superoxide scavenging and xanthine oxidase inhibiting activities of copper-β-citryl-L-glutamate complex.

  Authors: Masanori Narahara, Michiko Hamada-Kanazawa, Makiko Kouda, Akira Odani, Masaharu Miyake

  Biological & pharmaceutical bulletin. 01/2010; 33(12):1938-43.

  β-Citryl-L-glutamate (β-CG) is a unique compound initially isolated from developing brains, which also appears in high concentrations during the period characterized by growth and differentiation ofβ-Citryl-L-glutamate (β-CG) is a unique compound initially isolated from developing brains, which also appears in high concentrations during the period characterized by growth and differentiation of neurons in developing animals, and then decreases with maturation. However, its functional roles remain unclear. The stability constant obtained in our previous pH titration studies showed that β-CG forms relatively strong complexes with copper. Reactive oxygen species (ROS) and nitric oxide (NO) have been suggested to act as mediators of the cell death that occurs in neurons during development of the nervous system. However, regulation of ROS and NO formation by Cu in the developing brain remains poorly understood. The activity of superoxide dismutase (SOD), a key superoxide scavenging enzyme, is low in the developing brain. Furthermore, xanthine oxidase (XO) has been implicated in diverse pathological situations due to its capability of generating both ROS and NO. Therefore, we examined the effects of β-CG and its Cu-complex on SOD and XO activities. We found that the [Cu(II)(β-CG)] complex had SOD activity and a strong competitive inhibition of XO, while reduced glutathione caused concentration-dependent decreases of the XO inhibitory activities in the [Cu(II)(β-CG)] complex.

• beta-Citryl-L-glutamate is an endogenous iron chelator that occurs naturally in the developing brain.

  Authors: Michiko Hamada-Kanazawa, Makiko Kouda, Akira Odani, Kaori Matsuyama, Kiyoka Kanazawa, Tatsuya Hasegawa, Masanori Narahara, Masaharu Miyake

  Biological & pharmaceutical bulletin. 01/2010; 33(5):729-37.

  The compound beta-citryl-L-glutamate (beta-CG) was initially isolated from developing brains, while it has also been found in high concentrations in testes and eyes. However, its functional roles areThe compound beta-citryl-L-glutamate (beta-CG) was initially isolated from developing brains, while it has also been found in high concentrations in testes and eyes. However, its functional roles are unclear. To evaluate its coordination with metal ions, we performed pH titration experiments. The stability constant, logbeta(pqr) for M(p)(beta-CG)(q)H(r) was calculated from pH titration data, which showed that beta-CG forms relatively strong complexes with Fe(III), Cu(II), Fe(II) and Zn(II). beta-CG was also found able to solubilize Fe more effectively from Fe(OH)(2) than from Fe(OH)(3). Therefore, we examined the effects of beta-CG on Fe-dependent reactive oxygen species (ROS)-generating systems, as well as the potential ROS-scavenging activities of beta-CG and metal ion-(beta-CG) complexes. beta-CG inhibited the Fe-dependent degradation of deoxyribose and Fe-dependent damage to DNA or plasmid DNA in a dose-dependent manner, whereas it had no effect on Cu-mediated DNA damage. In addition, thermodynamic data showed that beta-CG in a physiological pH solution is an Fe(II) chelator rather than an Fe(III) chelator. Taken together, these findings suggest that beta-CG is an endogenous low molecular weight Fe chelator.

• Lipopolysaccharide activates the kallikrein-kinin system in mouse choroid plexus cell line ECPC4.

  Authors: Masaoki Takano, Chieko Satoh, Naomi Kunimatsu, Mieko Otani, Michiko Hamada-Kanazawa, Masaharu Miyake, Kyongsong Ming, Katsutoshi Yayama, Hiroshi Okamoto

  Neuroscience letters. 05/2008; 434(3):310-4.

  Regulation of the kallikrein-kinin system in cerebral inflammation is still unclear. Here, we used reverse-transcription polymerase chain reaction (RT-PCR) techniques to show that lipopolysaccharideRegulation of the kallikrein-kinin system in cerebral inflammation is still unclear. Here, we used reverse-transcription polymerase chain reaction (RT-PCR) techniques to show that lipopolysaccharide (LPS) activates the kallikrein-kinin system by enhancing liberation of bradykinin (BK), and alters mRNA levels of kallikrein-kinin system components, including high molecular weight (H-) and low molecular weight (L-) kininogens, in ECPC4 cells, a cell line of mouse choroid plexus epithelium. LPS treatment increased liberation of immunoreactive bradykinin in the supernatant of ECPC4 cells, and addition of LPS (500 ng/ml) to cultures resulted in elevation of H- and L-kininogen mRNA levels in ECPC4 cells within 24-48 h. Furthermore, LPS treatment elevated bradykinin type 2 and type 1 receptor mRNA levels within 4h, but did not change tissue kallikrein or plasma kallikrein mRNA levels. On the other hand, expression of pro-inflammatory mediators interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and cyclooxygenase-2 mRNA increased within 4-8h after addition of LPS to ECPC4 cells. The addition of IL-1beta and TNF-alpha to investigate the major mediator for kininogen expression in ECPC4 cells remarkably induced expression of H- and L-kininogen mRNAs in ECPC4 cells. These results suggest that LPS activates the kallikrein-kinin system in the choroid plexus via autocrine induction of IL-1beta and TNF-alpha.

• Triglyceride accumulation by peroxisome proliferators in rat hepatocytes.

  Authors: Hiroko Kawano, Tomomi Nagata, Masanori Narahara, Michiko Kanazawa, Masaharu Miyake

  Biological & pharmaceutical bulletin. 05/2007; 30(4):627-32.

  Peroxisome proliferators (PxPs) induce peroxisomal beta-oxidation (Px-ox) in the liver of rodents and have a hypolipidemic function. To investigate hypolipidemic effect of PxPs, the relationshipPeroxisome proliferators (PxPs) induce peroxisomal beta-oxidation (Px-ox) in the liver of rodents and have a hypolipidemic function. To investigate hypolipidemic effect of PxPs, the relationship between TG fluctuation and Px-ox activity, as an indicator of the function of PxPs, was studied in primary cultured rat hepatocytes. Nafenopin (Nf) treatment of hepatocytes caused an increase in Px-ox activity in association with cellular TG accumulation in a time-dependent manner with a coefficient of r=0.918. This relationship between the activity and cellular TG were obtained using structurally diverse PxPs with a correlation coefficient of r=0.747. Treatment of the hypolipidemic drug, but non-PxP Pravastatin, decreased TG in the medium, but did not have the effects on cellular TG and Px-ox activity. The total amount of TG and diacylglycerol acyltransferase activity, the last enzyme in the TG de novo synthesis pathway, were not affected by Nf treatment. When hepatocytes were cultured with Brefeldin A, cellular TG was accumulated, the same as with Nf, however, Px-ox activity was not enhanced. Nf treatment markedly decreased the level of apolipoprotein B (apo B) in very low density lipoprotein (VLDL) fractions prepared from conditioned media and increased that of cellular apoB by Western blot analysis. Microsomal triglyceride transfer protein activity was not influenced by Nf. Together, with regards to TG lowering effect of PxPs, it is suggested that PxPs cause hepatocellular accumulation of TG without effects on TG biosynthesis and VLDL construction, and they might have inhibitory effect on VLDL secretion process.

• Suppression of Sox6 in P19 cells leads to failure of neuronal differentiation by retinoic acid and induces retinoic acid-dependent apoptosis.

  Authors: Michiko Hamada-Kanazawa, Kyoko Ishikawa, Daisuke Ogawa, Miyuki Kanai, Yuichi Kawai, Masanori Narahara, Masaharu Miyake

  FEBS letters. 12/2004; 577(1-2):60-6.

  The Sox6 gene is a member of the Sox gene family, which encodes transcription factors, and previous studies have suggested that it plays an important role in the development of the central nervousThe Sox6 gene is a member of the Sox gene family, which encodes transcription factors, and previous studies have suggested that it plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of neurons, glia, and fibroblast-like cells. Sox6 mRNA increases rapidly in P19 cells during RA induction and then decreases during differentiation into neuronal cells. To investigate whether Sox6 expression is essential for neuronal differentiation, we established Sox6-suppressed P19 (P19[anti-Sox6]) cells by transfection of antisense-Sox6 cDNA. Most of the P19[anti-Sox6] cells showed no neurites and were not stained by the anti-MAP 2 antibody, while the suppression of Sox6 expression nearly totally blocked neuronal differentiation in P19 cells. Further, Sox6 suppression caused RA-dependent apoptosis by P19[anti-Sox6] cells: RA-treated P19[anti-Sox6] cells showed chromatin condensation, DNA fragmentation, and an increase in caspase-3-like activity. Thus, Sox6 is considered essential for neuronal differentiation and may play an important role in the early stages of neuronal differentiation or apoptosis.

• Sox6 overexpression causes cellular aggregation and the neuronal differentiation of P19 embryonic carcinoma cells in the absence of retinoic acid.

  Authors: Michiko Hamada-Kanazawa, Kyoko Ishikawa, Kaori Nomoto, Takako Uozumi, Yuichi Kawai, Masanori Narahara, Masaharu Miyake

  FEBS letters. 03/2004; 560(1-3):192-8.

  The Sox6 gene is a member of the Sox gene family that encodes transcription factors. Previous studies have suggested that Sox6 plays an important role in the development of the central nervousThe Sox6 gene is a member of the Sox gene family that encodes transcription factors. Previous studies have suggested that Sox6 plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of neurons, glia and fibroblast-like cells. In this report, we have shown that Sox6 mRNA increased rapidly in P19 cells during RA induction and then decreased during the differentiation of P19 into neuronal cells. To explore the possible roles of Sox6 during this process, stably Sox6-overexpressing P19 cell lines (P19[Sox6]) were established. These P19[Sox6] had acquired both characteristics of the wild-type P19 induced by RA. First, P19[Sox6] cells showed a marked cellular aggregation in the absence of RA. Second, P19[Sox6] could differentiate into microtubule-associated protein 2 (MAP2)-expressing neuronal cells in the absence of RA. Sox6 expression could cause the activation of endogenous genes including the neuronal transcription factor Mash-1, the neuronal development-related gene Wnt-1, the neuron-specific cell adhesion molecule N-cadherin, and the neuron-specific protein MAP2, resulting in neurogenesis. Moreover, E-cadherin, a major cell adhesion molecule of wild-type P19, was strongly induced by Sox6, resulting in cellular aggregation without RA. Thus Sox6 may play a critical role in cellular aggregation and neuronal differentiation of P19 cells.

• cDNA cloning of the Sry-related gene Sox6 from rat with tissue-specific expression.

  Authors: Masanori Narahara, Akihisa Yamada, Michiko Hamada-Kanazawa, Yuichi Kawai, Masaharu Miyake

  Biological & pharmaceutical bulletin. 07/2002; 25(6):705-9.

  A cDNA encoding rat homologue of the previously characterized mouse Sox6 was isolated by a polymerase chain reaction (PCR) cloning strategy. Comparison of this eDNA with homologous mouse, human andA cDNA encoding rat homologue of the previously characterized mouse Sox6 was isolated by a polymerase chain reaction (PCR) cloning strategy. Comparison of this eDNA with homologous mouse, human and rainbow trout cDNA exhibited an overall amino acid sequence identity of 99.6, 89.3 and 76.3% respectively. The leucine-zipper and HMG-box motif were almost completely conserved between these homologues. The expression of Sox6 was determined in rat by Northern hybridization and Real-time quantitative reverse transcription (RT)-PCR. rSox6 (rat Sox6) was specifically expressed in the neonatal brain and adult testis with Northern blotting. Real-time quantitative RT-PCR for the determination of Sox6 mRNA was examined. The rSox6 was expressed in the neonatal brain and adult testis as well as by Northern blotting and also expressed in the adult eyeball and slightly in the ovary.

• Effect of protein phosphatase inhibitors on the development of mouse embryos: protein phosphorylation is involved in the E-cadherin distribution in mouse two-cell embryos.

  Authors: Yuichi Kawai, Takaomi Yamaguchi, Takahiro Yoden, Motoki Hanada, Masaharu Miyake

  Biological & pharmaceutical bulletin. 03/2002; 25(2):179-83.

  Protein phosphorylation plays many important roles in cell functions and cell differentiation. To clarify the roles of protein phosphorylation in early embryonic development in mice, 2-cell embryosProtein phosphorylation plays many important roles in cell functions and cell differentiation. To clarify the roles of protein phosphorylation in early embryonic development in mice, 2-cell embryos were cultured in the presence of various protein phosphatase inhibitors such as calyculin A, okadaic acid, cyclosporin A, tacrolimus (FK506) and benzyl-phosphonic acid. Calyculin A potently inhibited the 2-cell cleavage to the 4-cell stage. The concentration for 50% inhibition was 0.26 nM. At the same time, we found that calyculin A-treated 2-cell embryos showed a morula-like shape at a concentration of 2 nM in 24 h. It is well known that E-cadherin plays a key role in the compaction of late 8-cell stage embryos. In this report, we observed the distribution of E-cadherin protein using anti-E-cadherin antibody with a fluorescence microscope, and also evaluated the relative E-cadherin mRNA content at various stages of embryos by RT-PCR and ABI PRISM 7700 System (a real time PCR apparatus). The fluorescence intensity of E-cadherin increased along with the embryonic development. During the embryonic development from the 2-cell stage to the blastocyst stage, the relative E-cadherin mRNA content greatly increased in a time-dependent manner, while the mRNA did not increase with the addition of calyculin A at the 2-cell stage. Therefore, we observed the localization of the E-cadherin protein in calyculin A-treated embryos with a laser microscope. The distribution pattern of E-cadherin was altered by the addition of calyculin A from a scattered pattern throughout the embryos to a localized pattern at the cell-cell boundary region. These results strongly suggest that the distribution of E-cadherin protein is regulated by protein phosphorylation and/or dephosphorylation.

• Expression of kininogen mRNAs and plasma kallikrein mRNA by cultured neurons, astrocytes and meningeal cells in the rat brain

  Authors: Masaoki Takano, Masato Horie, Masanori Narahara, Masaharu Miyake, Hiroshi Okamoto

  Immunopharmacology.

  Expression of kininogen mRNAs has been studied in cultures of three different types of cells in rat brain, including neurons and astrocytes from cerebral cortex and meningeal cells from theExpression of kininogen mRNAs has been studied in cultures of three different types of cells in rat brain, including neurons and astrocytes from cerebral cortex and meningeal cells from the leptomeninges/choroid plexus. T-kininogen mRNA was expressed by meningeal cells, but not by neurons and astrocytes, and the expression in meningeal cells was enhanced by culture with prostaglandin E2 (PGE2) or dibutyryl cAMP (Bt2cAMP). Low-molecular-weight kininogen mRNA was not detected in these cultures of cells, even after treatment with PGE2. Although expression of high-molecular-weight kininogen mRNA was very low in these cultures of cells, PGE2 or Bt2cAMP markedly stimulated its expression in cultures of meningeal cells and slightly in neurons, but not in astrocytes. We also found that expression of plasma kallikrein mRNA was strong in cultures of meningeal cells and slight in astrocytes, but absent in neurons. These results suggest that cells in the leptomeninges/choroid plexus are major sources of kininogens in rat brain which may function as precursor proteins for kinins and/or potent cysteine proteinase inhibitors during cerebral inflammation.

• Sox6 overexpression causes cellular aggregation and the neuronal differentiation of P19 embryonic carcinoma cells in the absence of retinoic acid

  Authors: Michiko Hamada-Kanazawa, Kyoko Ishikawa, Kaori Nomoto, Takako Uozumi, Yuichi Kawai, Masanori Narahara, Masaharu Miyake

  FEBS Letters.

  The Sox6 gene is a member of the Sox gene family that encodes transcription factors. Previous studies have suggested that Sox6 plays an important role in the development of the central nervousThe Sox6 gene is a member of the Sox gene family that encodes transcription factors. Previous studies have suggested that Sox6 plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of neurons, glia and fibroblast-like cells. In this report, we have shown that Sox6 mRNA increased rapidly in P19 cells during RA induction and then decreased during the differentiation of P19 into neuronal cells. To explore the possible roles of Sox6 during this process, stably Sox6-overexpressing P19 cell lines (P19[Sox6]) were established. These P19[Sox6] had acquired both characteristics of the wild-type P19 induced by RA. First, P19[Sox6] cells showed a marked cellular aggregation in the absence of RA. Second, P19[Sox6] could differentiate into microtubule-associated protein 2 (MAP2)-expressing neuronal cells in the absence of RA. Sox6 expression could cause the activation of endogenous genes including the neuronal transcription factor Mash-1, the neuronal development-related gene Wnt-1, the neuron-specific cell adhesion molecule N-cadherin, and the neuron-specific protein MAP2, resulting in neurogenesis. Moreover, E-cadherin, a major cell adhesion molecule of wild-type P19, was strongly induced by Sox6, resulting in cellular aggregation without RA. Thus Sox6 may play a critical role in cellular aggregation and neuronal differentiation of P19 cells.