DNA鎖間架橋修復に関与するヌクレアーゼの遺伝学的解析
塚田耕太郎; 吉原亮平; 畠山晋; 田中秀逸
Volume：92nd, 2020
J-Global ID：202002243003078261
複製ストレス応答におけるヒストンH3K4メチル化の重要性
柳澤健斗; 吉原亮平; 畠山晋; 田中秀逸
Volume：92nd, 2020
J-Global ID：202002259315982936
アカパンカビにおけるDNA損傷に応答したアポトーシス機構活性化の可視化に関する研究
田中秀逸
Volume：第6号(平成19年度), 2008
アカパンカビにおけるDNA損傷に応答したアポトーシス機構活性化の可視化に関する研究
田中秀逸
総合研究機構研究プロジェクト研究成果報告書, Volume：第6号(平成19年度), 2008
高速分子進化による高機能バイオ分子の創出 : 相同組換え機能を使っての遺伝子ターゲッティング法
井上弘一; 田中秀逸; 畠山晋
Volume：8, First page：24, Last page：26, 2007
アカパンカビにおけるDNA損傷応答に関する転写レベルのプロファイリング
田中秀逸
Volume：第5号(18年度), First page：209, Last page：210, 2007
高速分子進化による高機能バイオ分子の創出 : 相同組換え機能を使っての遺伝子ターゲッティング法
井上弘一; 田中秀逸; 畠山晋
埼玉大学地域共同研究センター紀要, Volume：8, Number：8, First page：24, Last page：26, 2007
DNA double strand breaks are mainly repaired by homologous recombination (HR) and nonhomologous end joining (NHEJ). In this article, we describe gene targeting efficiency in HR mutants and NHEJ mutants of the filamentous fungus Neurospora crassa. And we conclude that NHEJ-defective mutant is a most ideal host for gene targeting.
Japanese
ISSN：1347-4758, CiNii Articles ID：120001371294, CiNii Books ID：AA11808968
アカパンカビにおけるDNA損傷応答に関する転写レベルのプロファイリング
田中秀逸
総合研究機構研究プロジェクト研究成果報告書, Volume：第5号(18年度), First page：209, Last page：210, 2007
非相同組換え修復欠損細胞における標的遺伝子特異的な遺伝子破壊法の開発
田中秀逸
Volume：第4号(17年度), 2006
非相同組換え修復欠損細胞における標的遺伝子特異的な遺伝子破壊法の開発
田中秀逸
総合研究機構研究プロジェクト研究成果報告書, Volume：第4号(17年度), 2006
Selective inflammatory stimulations enhance release of microglial response factor (MRF)-1 from cultured microglia S Tanaka; T Koike
GLIA,
Volume：40,
Number：3,
First page：360,
Last page：371, Dec. 2002
The mrf-1 gene has been isolated from microglia exposed to cultured cerebellar granule neurons undergoing apoptosis. We have shown that mrf-1 is upregulated in response to neuronal death and degeneration both in vitro and in vivo. However, the exact role of MRF-1 remains unknown. Here we show that MRF-1 is released from cultured rat microglia, and its release is greatly enhanced under inflammatory conditions. When microglia were treated with ATP, the amount of MRF-1 that was released increased 10-fold compared to the basal level of release. Enhanced MRF-1 release was induced within 10 min and peaked within 1 h; after similar to 4 h, the MRF-1 release had returned to normal. MRF-1 release was stimulated by 2-methyl-thio-ATP (five-fold) and a P2X(7) selective agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (ten-fold). Moreover, the ATP-stimulated MRF-1 release was inhibited by a P2X(7) selective antagonist, oxidized ATP (oATP), and also under a Ca2+-free condition. These results indicate that the effects of ATP are dependent on Ca2+ influx through P2X(7) receptors. MRF-1 release was enhanced by Ca2+-ionophore A23187 (sixfold), thapsigargin (threefold); however, it was not enhanced by glutamate or lipopolysaccharide. Moreover, a platelet-activating factor enhanced microglial MRF-1 release in a dose-dependent manner. We also showed that a conditioned medium from cerebellar granule neurons undergoing apoptosis markedly increased MRF-1 release from microglia; that effect was significantly inhibited by oATP. These results indicate that selective inflammatory stimulations, including ATP and PAF, enhance MRF-1 release from microglia through a Ca2+-dependent mechanism and suggest that MRF-1 may play a role in cell-cell interactions under inflammatory conditions. (C) 2002 Wiley-Liss, Inc.
WILEY, English
DOI：https://doi.org/10.1002/glia.10142DOI ID：10.1002/glia.10142,
ISSN：0894-1491,
eISSN：1098-1136,
Web of Science ID：WOS:000179539700008 Activation of protein kinase C delays apoptosis of nerve growth factor-deprived rat sympathetic neurons through a Ca2+-influx dependent mechanism S Tanaka; T Koike
NEUROSCIENCE LETTERS,
Volume：313,
Number：1-2,
First page：9,
Last page：12, Nov. 2001
The effects of protein kinase C (PKC) activation on apoptosis depend on the cell type and on the isoenzymes activated. We show that the apoptosis of nerve growth factor (NGF)-deprived rat sympathetic neurons is delayed for about 24 h by treatment with O-tetradecanoylphorbol 13-acetate (TPA). The cell death was estimated by both morphological changes and the release of a cytoplasmic enzyme into the medium. The PKC inhibitor bisindolylmaleimide inhibited the TPA-mediated delay of neuronal death. The effect of TPA was abolished in conditions of Ca2+-free or in the presence of both Ca2+/calmodulin-dependent protein kinase II and phosphatidylinositol 3-kinase inhibitors, but was not blocked by either an L- or N-type Ca2+ channel inhibitor. These results suggest that the survival of the NGF-deprived neurons may be supported by PKC activation followed by Ca2+ influx. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.
ELSEVIER SCI IRELAND LTD, English
DOI：https://doi.org/10.1016/S0304-3940(01)02193-0DOI ID：10.1016/S0304-3940(01)02193-0,
ISSN：0304-3940,
Web of Science ID：WOS:000172059000003 Microglial response factor (MRF)-1: Constitutive expression in ramified microglia and upregulation upon neuronal death induced by ischemia or glutamate exposure S Tanaka; H Kato; T Koike
ZOOLOGICAL SCIENCE,
Volume：17,
Number：5,
First page：571,
Last page：578, Jul. 2000
We have isolated a new microglial gene, mrf-1,which is upregulated on microglia in response to apoptosis of granule neurons in cerebellar cell cultures. We examined whether or not upregulation of MRF-1 is observed in response to necrotic neuronal death both in vivo and in vitro. Though MRF-1 was detected on ramified/resting microglia in the brain of normal adult rats, activated microglia in the region of the brain where neuronal damage was induced by ischemia were strongly immunostained with anti-MRF-1 antibody, In the in vitro system, we confirmed, with immunocytochemistry or RT-PCR, that MRF-1 or mrf-1 mRNA were constitutively expressed in ramified microglia at significant but lower levels than in amoeboid one. Moreover, by Northern blot, it was ascertained that expression level of mrf-1 mRNA on microglia was markedly upregulated in response to glutamate-induced death of granule cells in a cerebellar cell culture. These results indicate the following: 1) expression of mrf-1 in microglia may be markedly enhanced upon not only apoptotic but also necrotic neuronal death, and 2) MRF-1 is, thus, an useful marker for identifying all types of microglia in vivo and in vitro.
ZOOLOGICAL SOC JAPAN, English
DOI：https://doi.org/10.2108/zsj.17.571DOI ID：10.2108/zsj.17.571,
ISSN：0289-0003,
CiNii Articles ID：110003371170,
PubMed ID：8940912,
Web of Science ID：WOS:000088872800002 Upregulation of a new microglial gene, mrf-1, in response to programmed neuronal cell death and degeneration
S Tanaka; K Suzuki; M Watanabe; A Matsuda; S Tone; T Koike
JOURNAL OF NEUROSCIENCE, Volume：18, Number：16, First page：6358, Last page：6369, Aug. 1998
Cerebellar granule neurons isolated from postnatal day 7 (P7) rats and grown in normal K(+)medium begin to degenerate at approximately 4 d in vitro (DIV) and die. To search for genes upregulated in the process of neuronal cell death, differential hybridization was performed with subtracted cDNA probes and a cDNA library from 5 DIV. One of the genes isolated was microglial response factor-1 (mrf-1), which encoded a sequence of 177 amino acids with a single EF-hand calcium-binding motif. By Northern blots, the transcript was upregulated in cerebellar culture at 4 DIV, peaked at 6 DIV, and decreased at 7 DIV. Upregulation was also found when the apoptosis of granule cells was induced by replacing high K+ medium with normal K+ medium. However, when non-neuronal cells were thoroughly eliminated with aphidicolin, an antimitotic agent, the upregulation at 4-7 DIV did not occur. By immunocytochemistry, MRF-I was detected at 5 DIV in OX-42-positive cells (microglia), and it exhibited an increase in response to granule cell death. MRF-1 levels in microglia purified from cerebral cortex also upregulated in the presence of 5 DIV granule cells. In the developing cerebellum in vivo, levels of mrf-1 mRNA transiently increased in the early postnatal stages, reaching a peak at P7 when cerebellar neurons and astrocytes undergo extensive apoptosis. In adult brain sections, MRF-1 was detected in the perikarya and processes of ramified/resting microglia, and peripheral motor nerve dissection prominently increased the expression in activated microglia surrounding injured central motoneurons. Therefore, mrf-1 appears to be one of the microglial genes that respond to neuronal cell death and degeneration.
SOC NEUROSCIENCE, English
ISSN：0270-6474, Web of Science ID：WOS:000075246500028
Veratridine delays apoptotic neuronal death induced by NGF deprivation through a Na+-dependent mechanism in cultured rat sympathetic neurons S. Tanaka; T. Koike
International Journal of Developmental Neuroscience,
Volume：15,
Number：1,
First page：15,
Last page：27, 31 Jul. 1997
Superior-cervical ganglion (SCG) cells dissociated from newborn rats depend on nerve growth factor (NGF) for survival. Membrane depolarization with elevated K+ is known to prevent neuronal death following NGF deprivation and/or to promote survival via a Ca2+-dependent mechanism. Here we have exploited the possibility of whether or not a Na+-dependent pathway for neuronal survival is present in these cells. Veratridine (EC50 = 40 nM), a voltage-dependent Na+ channel activator, significantly delayed the onset of apoptotic cell death in NGF-deprived SCG neurons that had been cultured for 7 days in the presence of NGF. This effect was blocked completely by Na+ channel blockers including tetrodotoxin (TTX, 1 μM), benzamil (25 μM) and flunarizine (1 μM), but was not attenuated by nimodipine (1 μM), an L-type Ca2+ channel blocker. The saving effect of veratridine on cultured neurons was observed even in low Ca2+ media (0-1.0 mM), but was completely abolished in a low Na+ medium (38 mM). Sodium-binding benzofuran isophthalate was employed as a fluorescent probe for monitoring the level of cytoplasmic free Na+, which revealed a sustained increase in its level (12.9 mM, 307% of that of control) in response to veratridine (0.75 μM). The TTX or flunarizine completely blocked veratridine-induced Na+ influx in these cultured neurons. Moreover, no appreciable increase in intracellular Ca2+ was detected under these conditions. Though Na+ channels were effectual in SCG neurons which were freshly isolated from newborn rats, the Na+-dependent saving effect of veratridine was not observed in these young neurons. These lines of evidence suggest that the death-suppressing effect of veratridine on cultured SCG neurons depends on the Na+ influx via voltage-dependent Na+ channels, and suggests the presence of Na+-dependent regulatory mechanism(s) in neuronal survival.
English
DOI：https://doi.org/10.1016/S0736-5748(96)00082-2DOI ID：10.1016/S0736-5748(96)00082-2,
ISSN：0736-5748,
PubMed ID：9099612,
SCOPUS ID：0030887258 Veratridine delays apoptotic neuronal death induced by NGF deprivation through a Na+ dependent mechanism in cultured rat sympathetic neurons. TANAKA S; KOIKE T
Volume：15,
Number：1,
First page：15,
Last page：27, 1996
DOI：https://doi.org/10.1016/S0736-5748(96)00082-2DOI ID：10.1016/S0736-5748(96)00082-2,
ISSN：0736-5748,
PubMed ID：9099612 UP-REGULATION OF L-TYPE CA2+ CHANNEL ASSOCIATED WITH THE DEVELOPMENT OF ELEVATED K+-MEDIATED SURVIVAL OF SUPERIOR CERVICAL-GANGLION CELLS IN-VITRO S TANAKA; T KOIKE
DEVELOPMENTAL BIOLOGY,
Volume：168,
Number：1,
First page：166,
Last page：178, Mar. 1995
We have previously shown that elevated K+-mediated neuronal survival correlates well with a sustained increase in cytoplasmic Ca2+ ([Ca2+](i)) through the opening of L-type Ca2+ channels. Elevated K+ (40 mM), however, failed to support the survival of superior cervical ganglion (SCG) cells freshly isolated from newborn rats, while another depolarizing agent, veratridine, was able to do so, suggesting that elevated K+-mediated Ca2+ influx occurs in a stage-dependent manner. Indeed, sustained levels of [Ca2+](i) in response to high K+ remained low (91 nM) in these neurons, but became elevated (238 nM) in SCG neurons which had been exposed to nerve growth factor (NGF) for 5-7 days and were capable of responding to elevated K+ by survival. Semiquantitative PCR measurements of L-type Ca2+ channel mRNA revealed that this transcript increased dramatically during incubation with NGF for 3 days and reached a plateau level at Day 5 (seven-fold per surface area or nine-fold per total volume compared to that at Day 1). These findings show that NGF-mediated up-regulation of the expression of L-type Ca2+ channel mRNA correlates with the development of elevated K+-mediated neuronal survival in vitro and suggest its involvement in coordinate strengthening of pre- and postganglionic synapses in rat SCG neurons. (C) 1995 Academic Press,Inc.
ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, English
DOI：https://doi.org/10.1006/dbio.1995.1069DOI ID：10.1006/dbio.1995.1069,
ISSN：0012-1606,
CiNii Articles ID：30018554932,
PubMed ID：7883071,
Web of Science ID：WOS:A1995QK99100014 PROGRAMMED NEURONAL CELL-DEATH IN PC12 CELLS PRIMED WITH NERVE GROWTH-FACTOR
T KOIKE; S TANAKA
JOURNAL OF CELLULAR BIOCHEMISTRY, First page：150, Last page：150, Mar. 1993
WILEY-LISS, English, Summary international conference
ISSN：0730-2312, Web of Science ID：WOS:A1993KV88000518
High throughput gene-targeting with NHEJ deficient cells in plant
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), 01 Apr. 2012 - 31 Mar. 2015
TANAKA Shuuitsu, Saitama University
Grant amount(Total)：5590000, Direct funding：4300000, Indirect funding：1290000
In the research, we will show the sure availability of a new system, which we had published in 2010, of gene targeting with NHEJ deficient cells in plant for analysis of gene function and plant bleeding.
We had tried that a target gene tagged with GFP gene is expressed from LIG4 deficient A. thaliana, and gene targeting is performed in either KU80 deficient or KU70 knocked-down plants. Because of problems, may be, from either condition of calli culture or constructs for gene manipulation, neither their transformed plants nor results have not yet been obtained. But, we observed shoot formation from our cultured calli. These experiments will do continuously after addition of both some checks and modifications.
Grant number：24570004
非相同末端結合能破壊株を用いた高効率な遺伝子組換え作物作製技術の新規基盤開発
2009 - 2010
Grant amount(Total)：3200000, Direct funding：3200000
Grant number：21658003
Mechanism of mtDNA deletion in short lived mutant of Neurospora.
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), 2008 - 2010
INOUE Hirokazu; TANAKA Shuuitsu; HATAKEYAMA Shin, Saitama University
Grant amount(Total)：4810000, Direct funding：3700000, Indirect funding：1110000
The mus-10 mutant was isolated which showed highly sensitivity to alkylating agent methylmethane sulfonate (MMS). It had been forecasted that mus-10 gene belonged to the some DNA repair pathway, because of it sensitivity to mutagen. This mutant have other unique characteristics; unable to grow after several times sequential inoculation, or stop growing after 2 to 3 weeks culture. Furthermore, these phenotypes are accompanied the deletion of mitochondrial DNA and fragmented mitochondrial feature comparing to the normal (tubular) shape in wild type strain. The responsible gene of mus-10 was cloned by complementation of its MMS sensitivity. This gene encodes the F-box domain containing polypeptide, and deletion of F-box domain showed identical phenotype with mus-10 mutant. Since F-box protein is known as a counterpart of SCF (Skp-Cullin-F-box) comlex, which have a role for the degradation of some target protein via ubiquitination following degrading in The mus-10 mutant was isolated which showed highly sensitivity to alkylating agent methylmethane sulfonate (MMS). It had been forecasted that mus-10 gene belonged to the some DNA repair pathway, because of it sensitivity to mutagen. This mutant have other unique characteristics ; unable to grow after several times sequential inoculation, or stop growing after 2 to 3 weeks culture. Furthermore, these phenotypes are accompanied the deletion of mitochondrial DNA and fragmented mitochondrial feature comparing to the normal (tubular) shape in wild type strain. The responsible gene of mus-10 was cloned by complementation of its MMS sensitivity. This gene encodes the F-box domain containing polypeptide, and deletion of F-box domain showed identical phenotype with mus-10 mutant. Since F-box protein is known as a counterpart of SCF (Skp-Cullin-F-box) comlex, which have a role for the degradation of some target protein via ubiquitination following degrading in proteasome.
To uncover the mus-10 gene function, we focused the feature of mitochondria. We examined whether 1) mitochondrial fusion is inhibited, or 2) mitochondrial fission is stimulated in mus-10 mutant. Double mutation of mus-10 and fis-1, which was essential for mitochondrial fission, showed quite resemble feature of mitochondria with wild type strain. And also this double mutant suppressed sensitivity to mutagen and short life span. These results suggested that MUS-10 protein prevent from the mutagen sensitivity and short life span according to maintain a mitochondrial feature. MUS-10 protein was considered to have a function of degradation of some target protein. So MUS-10 protein should be bound to that protein. Considering MUS-10 protein was correlated to maintenance of mitochondria feature, one candidate FZO-1 arose which had functions in the mitochondrial fusion. Using immunoprecipitation mthod, we could show that MUS-10 protein bound to FZO-1. Next, we tried to make fzo-1 knock out strain, but couldn't. The fzo-1 gene thought to be essential gene and fusion of mitochondria was important for maintenance of life span in Neurospora. Further, we forecasted that constitutive expression of FZO-1 protein in mus-10 mutant might show eviler phenotype than the mus-10 single mutant, because FZO-1, target of MUS-10, might be accumulated in that strain by escaping degradation. However, any evil phenotypes were not observed. Above these results, it was suggested that there were complex mechanism to maintain the mitochondrial feature.
Grant number：20570001
ミトコンドリアゲノムの安定性維持機構
2008 - 2010
Grant amount(Total)：2000000, Direct funding：2000000
Grant number：08F08756
Host for highly efficient gene targeting in filamentous fungi
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), 2006 - 2007
INOUE Hirokazu; TANAKA Syuuitu; HATAKEYAMA Shin; GOMI Katsuya; ABE Keietsu, Saitama University
Grant amount(Total)：9420000, Direct funding：8400000, Indirect funding：1020000
To investigate gene function, most desirable method is gene targeting. However, gene targeting frequency is extremely low in many organisms. To raise the frequency, some trials have been carried out, but conclusive technique was not found.
Double strand DNA breaks are repaired by two different recombination mechanisms: homologous recombination and nonhomologous end joining. From our study in Neurospora crassa, we speculated that homologous recombination frequency is increased if function of nonhomologous end joining is blocked. We disrupted ku70 and ku80 homolog genes of Neurospora and used them as a host in transformation experiments. In this study, high homologous integration of DNA was observed (Ninomiya, et. al. 2004).
To develop more convenient host, we disrupted other Neurospora genes involving in nonhomologous end joining; Ligase IV and XRCC4 homolog genes. These mutants showed high targeting frequency even if homologous length of introduced DNA is short. We also tested targeting frequency in MRX-defective strains. MRX is complex of Mre11-Rad50-Xrs2 and functions in double strand breaks repair. Many transformants were from homologous integration, though transformation frequency was low.
To know whether this system works in other organisms, Aspergillus oryzae was selected for test. Mutant of LigD ( human Lig4 homolog) was constructed in A. oryzae. Targeting experiments using ligD mutant as a host indicated high homologous integration.
In many other fungi, similar experiments have been coducted by researchers all over the world and they confirmed high targeting efficiency by using ku mutants. as we presented.
However, our trials in basidiomycetes did not reached to final step since there were unexpected problems in transformation, selection marker, so on. We will continue these experiments for construction of more convenient targeting system.
Grant number：18370001
神経突起変性により誘導されるカスケードとその神経変性疾患モデルにおける解析
2001 - 2001
Grant amount(Total)：5400000, Direct funding：5400000
Competitive research funding, Grant number：13210002
神経突起変性により誘発される細胞内カスケードの解明
2000 - 2000
Competitive research funding, Grant number：12210023
BDNFは小脳外顆粒層由来顆粒細胞のアポトーシスを誘導するか?
1998 - 1999
Grant amount(Total)：2100000, Direct funding：2100000
Grant number：10878145
Identification of novel microglial genes up-regulated during neuronal apoptosis
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), 1997 - 1999
KOIKE Tatsuro; TANAKA Shuuitsu; WATANABE Masahiko, HOKKAIDO UNIVERSITY
Grant amount(Total)：13400000, Direct funding：13400000
To searach for genes up-regulated during neuronal cell death, we have employed cerebellar cell cultures, in which glial cells respond to degeneration and cell death of granule neurons that begins to occur at 4DIV. Differential hybridization was performed with subtracted cDNA probes and a cDNA library from 5DIV. One of the genes up-regulated during cell death is a novel gene, mrf-1, in microglia. Another gene is highly homologous to the mouse genes with hematopoietic cell origin which may be involved in microglial activation associated with enhanced functions of lysosomes
Grant number：09480217
神経細胞のアポトーシス抑制へのNF-KBカスケードの関与
1997 - 1998
Grant amount(Total)：2000000, Direct funding：2000000
Grant number：09780715
小胞体ストレスによるニューロン死制御
1997 - 1997
Grant amount(Total)：1500000, Direct funding：1500000
Grant number：09280202
神経細胞のアポトーシス初期過程で活性化する遺伝子の探索
1996 - 1996
Grant amount(Total)：1100000, Direct funding：1100000
Grant number：08780693
膜脱分極による遅延性遺伝子発現機構
1996 - 1996
Grant amount(Total)：2000000, Direct funding：2000000
Grant number：08271201
培養細胞モデル系によるニューロナルアポトーシスの分子機構
1996 - 1996
Grant amount(Total)：1500000, Direct funding：1500000
Grant number：08256202
Studies on Regulatory Mechanisms of Developmental Neuronal Cell Death
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for General Scientific Research (B), 1994 - 1995
KOIKE Tatsuro, HOKKAIDO UNIVERSITY
Grant amount(Total)：5700000, Direct funding：5700000
We have shown previously that neuronal survival under depolarizing conditions correlates well with sustained levels of cytoplasmic free calcium ([Ca^<2+>]_i) in rat superior cervical ganglion (SCG) cells and cerebellar granule neurons. Moreover, developmental acquisition of trophic factor-independent survival is coincident with a gradual elevation of[Ca^<2+>]_i in SCG and sensory neurons. In contrast, when basal levels of[Ca^<2+>]_i in these neurons were decreased by treatment with an excessive dose of the Ca^<2+> chelator BAPATAAM,these neurons died following fragmentation of chromatin as visualized by fluorescent staining with bis-benzamide, a hallmark of apoptosis. Indeed, our Ca^<2+> set-point hypothesis predicts that a decrease in the level of cytoplasmic Ca^<2+> occurs during neuronal death following trophic factor withdrawal. In support of this, when SCG neurons were treated with 10-20 muM BAPTA-AM thus chelating the level of intracellular Ca^<2+>, they became resistant to NGF deprivation. Fura-2 measurements of[Ca^<2+>]_i also indicated a decrease in basal Ca^<2+> levels. Since there is evidence suggesting that NGF facilitates Ca^<2+>-dependent processes in these neurons, NGF deprivation causes a disruption of Ca^<2+> homeostasis in these neurons thus leading to the activation of neuronal apoptosis cascades.
Grant number：06454686
過剰なNa^+流入による神経細死の機構
1994 - 1994
Grant amount(Total)：2200000, Direct funding：2200000
Grant number：06272225
神経栄養因子欠乏による神経細胞死の機序
1992 - 1992
Grant amount(Total)：2200000, Direct funding：2200000
Grant number：04258216
Neuronal Cell Degeneration Cascade
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for General Scientific Research (C), 1991 - 1992
KOEKI Tatsuro; TANAKA Shuuitsu, SAGA MEDICAL SCHOOL
Grant amount(Total)：1700000, Direct funding：1700000
PC12 cells undergo degeneration after nerve growth factor (NGF) withdrawal only when they have been treated with NGF Actinomycin D or cordycepin blocked the release of lactate dehyrogenase (LDH) activity up to 85-90%; the residual release was due to neurite degeneration. Thus, NGF deprivation-induced cell death of PC12 cells occurs in a transcription-dependent manner, while serum deprivation-induced PC12 cell death has been reported to be a transcription-independent process. The cell death was completely prevented by chronic depolarization with high potassium (>35mM), CPT-cAMP(>0.1mM), acidic and basic FGF(10ng/ml). Upon NGF deprivation, rapid dephosphorylation of major tyrosine-phosphorylated proteins, p120, p80, p70 as well as p140 trk occurred followed by gradual down-regulation of kinase activities (Ser/Thr) which appears to require RNA and protein synthesis. These results suggest that a dephosphorylation cascade initiated by trophic factor deprivation is highly reulated, and may constitute a part of programmed neuronal death cascade(s).
Grant number：03833026
神経栄養因子欠乏による神経細胞死の機序
1991 - 1991
Grant amount(Total)：2000000, Direct funding：2000000
Grant number：03263217
栄養因子欠如による神経細胞死の分子生物学的研究
1990 - 1990
Grant amount(Total)：1400000, Direct funding：1400000
Grant number：02807012
交感神経節細胞の神経成長因子依存性と細胞内カルシウム
1989 - 1989
Grant amount(Total)：1700000, Direct funding：1700000
Grant number：01641533
Regulatory Mechanisms of Cellular Responses to Nerve Growth Factor(NGF) --Studies on the Interaction between NGF and NGF Receptors--
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for General Scientific Research (C), 1987 - 1988
TANAKA Shuitsu; TAKASHIMA Akihiko; KOIKE Tatsuro, Saga Medical School
Grant amount(Total)：1900000, Direct funding：1900000
1. Response to NGF by PC12 cells is regulated by modulation of NGF receptors. Fractions of the binding and internalization of ^<125>I-NGF by the cells increased with an increase in extracellular potassium concentration. This high K^+ effect was also induced by treatment of the cells with veratridine. Neurite outgrowth of the cells in the presence of low concentrations of NGF was potentiated in high K^+ medium. The fractions of ^<125>I-NGF binding and internalization by the cells were also influenced by treatent of the cells with catecholamines.
2. A regulatory mechanism of dopamine release in PC12 cells. Pre-treatment of the cells with phorbol ester(TPA) resulted in enhanced dopamine release, which could be further stimulated by high K^+. This enhanced dopamine release was associated with concomitant reduction of the high K^+-induced increase in intracellular Ca^<2+> concentration. Moreover, in TPA-treated cells, the high K^+-evoked dopamine release was completely abolished by the presence of nicardipine, Cd^<2+>, or Co^<2+>, but only partially inhibited by the presence of verapamil. These findings suggest possible involvement of protein kinase C in regulating the efficiency of high K^+-evoked dopamine release through the modification of nicardipine-sensitive Ca^<2+> channels.
3. Enhancement of the NGF action by intracellular application of GTP analougues. Pre-treatment of PC12 cells with NGF induced an increase in the fraction of ^<35>S-GTP S bound to the cell membranes. The fractions of the cell-surface binding of ^<125>I-NGF were measured after introduction of some nucleotides into the cytoplasm by electric pulses. Introduction of GTP s or GTP into the cytoplasm caused an increase in the fraction of ^<125>I-NGF binding. Moreover, the number of cells which grew neurite increased when GTP s or GTP was introduced into theic cytoplasm before a treatment of the cell with NGF for 1-2 hours. These results indicate that GTP plays a role in the process of the binding of NGF to NGF receptors.
Grant number：62540552
アカパンカビの遺伝学的研究－DNA損傷後のDNA修復と細胞死との選択制御－
Competitive research funding
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Competitive research funding
