研究者総覧

遠藤 仁司 (エンドウ ヒトシ)

  • 生化学講座(機能生化学部門) 教授
Last Updated :2021/12/04

研究者情報

学位

  • 医学博士

ホームページURL

J-Global ID

研究キーワード

  • 遺伝子発現調節   生体エネルギー学   生化学   Gene expression regulation   Energy transduction   Biochemistry   

研究分野

  • ライフサイエンス / 医化学

所属学協会

  • 日本RNA学会   The RNA Society   日本分子生物学会   日本生化学会   

研究活動情報

論文

  • Rintaro Kuroda, Kaoru Tominaga, Katsumi Kasashima, Kenji Kuroiwa, Eiji Sakashita, Hiroko Hayakawa, Tom Kouki, Nobuhiko Ohno, Kensuke Kawai, Hitoshi Endo
    PLOS ONE 16 7 e0255355 - e0255355 2021年07月 
    Mitochondrial dysfunction is significantly associated with neurological deficits and age-related neurological diseases. While mitochondria are dynamically regulated and properly maintained during neurogenesis, the manner in which mitochondrial activities are controlled and contribute to these processes is not fully understood. Mitochondrial transcription factor A (TFAM) contributes to mitochondrial function by maintaining mitochondrial DNA (mtDNA). To clarify how mitochondrial dysfunction affects neurogenesis, we induced mitochondrial dysfunction specifically in murine neural stem cells (NSCs) by inactivating Tfam. Tfam inactivation in NSCs resulted in mitochondrial dysfunction by reducing respiratory chain activities and causing a severe deficit in neural differentiation and maturation both in vivo and in vitro. Brain tissue from Tfam-deficient mice exhibited neuronal cell death primarily at layer V and microglia were activated prior to cell death. Cultured Tfam-deficient NSCs showed a reduction in reactive oxygen species produced by the mitochondria. Tfam inactivation during neurogenesis resulted in the accumulation of ATF4 and activation of target gene expression. Therefore, we propose that the integrated stress response (ISR) induced by mitochondrial dysfunction in neurogenesis is activated to protect the progression of neurodegenerative diseases.
  • Satsuki Miyata, Kaoru Tominaga, Eiji Sakashita, Masashi Urabe, Yoshiyuki Onuki, Akira Gomi, Takashi Yamaguchi, Makiko Mieno, Hiroaki Mizukami, Akihiro Kume, Keiya Ozawa, Eiju Watanabe, Kensuke Kawai, Hitoshi Endo
    Scientific reports 9 1 9787 - 9787 2019年07月 [査読有り][通常論文]
     
    Gliomas with Isocitrate dehydrogenase 1 (IDH1) mutation have alterations in several enzyme activities, resulting in various metabolic changes. The aim of this study was to determine a mechanism for the better prognosis of gliomas with IDH mutation by performing metabolomic analysis. To understand the metabolic state of human gliomas, we analyzed clinical samples obtained from surgical resection of glioma patients (grades II-IV) with or without the IDH1 mutation, and compared the results with U87 glioblastoma cells overexpressing IDH1 or IDH1R132H. In clinical samples of gliomas with IDH1 mutation, levels of D-2-hydroxyglutarate (D-2HG) were increased significantly compared with gliomas without IDH mutation. Gliomas with IDH mutation also showed decreased intermediates in the tricarboxylic acid cycle and pathways involved in the production of energy, amino acids, and nucleic acids. The marked difference in the metabolic profile in IDH mutant clinical glioma samples compared with that of mutant IDH expressing cells includes a decrease in β-oxidation due to acyl-carnitine and carnitine deficiencies. These metabolic changes may explain the lower cell division rate observed in IDH mutant gliomas and may provide a better prognosis in IDH mutant gliomas.
  • Takei H, Edahiro Y, Mano S, Masubuchi N, Mizukami Y, Imai M, Morishita S, Misawa K, Ochiai T, Tsuneda S, Endo H, Nakamura S, Eto K, Ohsaka A, Araki M, Komatsu N
    British journal of haematology 181 6 791 - 802 2018年06月 [査読有り][通常論文]
     
    Somatic mutations in the calreticulin (CALR) gene have been found in most patients with JAK2- and MPL-unmutated Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). It has recently been shown that mutant CALR constitutively activates the thrombopoietin receptor MPL and, thus, plays a causal role in the development of MPNs. However, the roles of mutant CALR in human haematopoietic cell differentiation remain predominantly elusive. To examine the impact of the 5-base insertion mutant CALR gene (Ins5) on haematopoietic cell differentiation, we generated induced pluripotent stem cells from an essential thrombocythaemia (ET) patient harbouring a CALR-Ins5 mutation and from a healthy individual (WT). Megakaryopoiesis was more prominent in Ins5-haematopoietic progenitor cells (Ins5-HPCs) than in WT-HPCs, implying that the system recapitulates megakaryocytosis observed in the bone marrow of CALR-mutant ET patients. Ins5-HPCs exhibited elevated expression levels of GATA1 and GATA2, suggesting a premature commitment to megakaryocytic differentiation in progenitor cells. We also demonstrated that 3-hydroxy anagrelide markedly perturbed megakaryopoiesis, but not erythropoiesis. Collectively, we established an in vitro model system that recapitulates megakaryopoiesis caused by mutant CALR. This system can be used to validate therapeutic compounds for MPN patients harbouring CALR mutations and in detailed studies on mutant CALR in human haematological cell differentiation.
  • Megumi Sumitani, Mari Kondo, Katsumi Kasashima, Hitoshi Endo, Kaoru Nakamura, Toshihiko Misawa, Hiromitsu Tanaka, Hideki Sezutsu
    Gene 608 103 - 113 2017年04月 [査読有り][通常論文]
     
    In the present study, we initially cloned and characterized a mitochondrial transcription factor A (Tfam) homologue in the silkworm, Bombyx mori. Bombyx mori TFAM (BmTFAM) localized to mitochondria in cultured silkworm and human cells, and co-localized with mtDNA nucleoids in human HeLa cells. In an immunoprecipitation analysis, BmTFAM was found to associate with human mtDNA in mitochondria, indicating its feature as a non-specific DNA-binding protein. In spite of the low identity between BmTFAM and human TFAM (26.5%), the expression of BmTFAM rescued mtDNA copy number reductions and enlarged mtDNA nucleoids in HeLa cells, which were induced by human Tfam knockdown. Thus, BmTFAM compensates for the function of human TFAM in HeLa cells, demonstrating that the mitochondrial function of TFAM is highly conserved between silkworms and humans. BmTfam mRNA was strongly expressed in early embryos. Through double-stranded RNA (dsRNA)-based RNA interference (RNAi) in silkworm embryos, we found that the knockdown of BmTFAM reduced the amount of mtDNA and induced growth retardation at the larval stage. Collectively, these results demonstrate that BmTFAM is a highly conserved mtDNA regulator and may be a good candidate for investigating and modulating mtDNA metabolism in this model organism.
  • Takafumi Mashiko, Eiji Sakashita, Katsumi Kasashima, Kaoru Tominaga, Kenji Kuroiwa, Yasuyuki Nozaki, Tohru Matsuura, Toshiro Hamamoto, Hitoshi Endo
    The Journal of biological chemistry 291 29 14996 - 5007 2016年07月 [査読有り][通常論文]
     
    Cytoplasmic protein aggregates are one of the pathological hallmarks of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Several RNA-binding proteins have been identified as components of inclusion bodies. Developmentally regulated RNA-binding protein 1 (Drb1)/RNA-binding motif protein 45 is an RNA-binding protein that was recently described as a component in ALS- and FTLD-related inclusion bodies. However, the molecular mechanism underlying cytoplasmic Drb1 aggregation remains unclear. Here, using an in vitro cellular model, we demonstrated that Drb1 co-localizes with cytoplasmic aggregates mediated by TAR DNA-binding protein 43, a major component of ALS and FTLD-related inclusion bodies. We also defined the domains involved in the subcellular localization of Drb1 to clarify the role of Drb1 in the formation of cytoplasmic aggregates in ALS and FTLD. Drb1 predominantly localized in the nucleus via a classical nuclear localization signal in its carboxyl terminus and is a shuttling protein between the nucleus and cytoplasm. Furthermore, we identify a double leucine motif serving as a nuclear export signal. The Drb1 mutant, presenting mutations in both nuclear localization signal and nuclear export signal, is prone to aggregate in the cytoplasm. The mutant Drb1-induced cytoplasmic aggregates not only recruit TAR DNA-binding protein 43 but also decrease the mitochondrial membrane potential. Taken together, these results indicate that perturbation of Drb1 nuclear-cytoplasmic trafficking induces toxic cytoplasmic aggregates, suggesting that mislocalization of Drb1 is involved in the cause of cytotoxicity in neuronal cells.
  • Katsumi Kasashima, Hitoshi Endo
    Genes to cells : devoted to molecular & cellular mechanisms 20 12 1017 - 27 2015年12月 [査読有り][通常論文]
     
    Mitochondrial transcription factor A (TFAM) is a key regulator of mitochondrial DNA (mtDNA). TFAM interacts with itself and forms dimers; however, the precise interaction domain in vivo has not yet been determined. We herein showed that human TFAM formed oligomers in mitochondria by in situ chemical cross-linking. We used the separated fluorescent protein, monomeric Kusabira-Green, as a reporter to monitor their self-association in mitochondria. This reporter successfully detected the TFAM-TFAM interaction in cells as fluorescent signals on mitochondria. We also found that the N-terminal high-mobility group box domain was sufficient for this interaction. The expression of the dimer-defective mutant induced enlarged mtDNA nucleoids, suggesting the importance of dimerization in the distribution of mtDNA. The reporter system also supported the association and mixture between independent nucleoids through TFAM by a cell fusion assay using hemagglutinating virus of Japan. We here, for the first time, visualized the interaction of TFAM molecules in mitochondria and proposed its implications for the dynamics of mtDNA nucleoids.
  • Satoshi Yamamoto, Yasumitsu Nagao, Kenji Kuroiwa, Yoji Hakamata, Masaru Ichida, Fumiko Saito-Ohara, Kaoru Tominaga, Hitoshi Endo
    Transgenic research 23 5 757 - 65 2014年10月 [査読有り][通常論文]
     
    We developed a transgenic mouse line with Y chromosome-linked green fluorescent protein expressing transgenes (Y-GFP) by the conventional microinjection into the pronucleus of C57BL/6J fertilized oocytes. Embryonic stem (ES) cells derived from Y-GFP mice enabled not only sexing but also the identification of 39, XO karyotype by the lack of Y chromosome. Actually, when fluorescence activated cell sorting (FACS) was applied to Y-GFP ES cells, non-fluorescent ES cells were conveniently collected and showed the lack of Y chromosome by PCR genotyping and Southern blot analysis. FACS analysis revealed Y chromosome loss occurred at 2.9 % of 40, XY ES cells after five passages. These Y-GFP ES cells are potentially applicable to reduce the time, cost and effort needed to generate the gene-targeted mice by the production of male and female mice derived from the same ES cell clone.
  • Katsumi Kasashima, Yasumitsu Nagao, Hitoshi Endo
    Reproductive medicine and biology 13 11 - 20 2014年 [査読有り][通常論文]
     
    Mitochondria play a crucial role in the development and function of germ cells. Mitochondria contain a maternally inherited genome that should be transmitted to offspring without reactive oxygen species-induced damage during germ line development. Germ cells are also involved in the mitochondrial DNA (mtDNA) bottleneck; thus, the appropriate regulation of mtDNA in these cells is very important for this characteristic transmission. In this review, we focused on unique regulation of the mitochondrial genome in animal germ cells; paternal elimination and the mtDNA bottleneck in females. We also summarized the mitochondrial nucleoid factors involved in various mtDNA regulation pathways. Among them, mitochondrial transcription factor A (TFAM), which has pleiotropic and essential roles in mtDNA maintenance, appears to have putative roles in germ cell regulation.
  • Syuichi Tetsuka, Kaoru Tominaga, Eriko Ohta, Kenji Kuroiwa, Eiji Sakashita, Katsumi Kasashima, Toshiro Hamamoto, Michito Namekawa, Mitsuya Morita, Shinsuke Natsui, Tatsuo Morita, Keiko Tanaka, Yoshihisa Takiyama, Imaharu Nakano, Hitoshi Endo
    Journal of the neurological sciences 335 1-2 48 - 57 2013年12月 [査読有り][通常論文]
     
    Onconeural immunity, a cancer-stimulated immune reaction that cross-reacts with neural tissues, is considered to be the principal pathological mechanism for paraneoplastic neurological syndromes (PNS). A common PNS is paraneoplastic cerebellar degeneration (PCD). We had encountered a PCD patient with urothelial carcinomas (UC) of the urinary bladder who was negative for the well-characterized PNS-related onconeural antibodies. In the present study, we aimed to identify a new PCD-related onconeural antibody, capable of recognizing both cerebellar neurons and cancer tissues from the patient, and applied a proteomic approach using mass spectrometry. We identified anti-creatine kinase, brain-type (CKB) antibody as a new autoantibody in the serum and cerebrospinal fluid from the patient. Immunohistochemistry indicated that anti-CKB antibody reacted with both cerebellar neurons and UC of the urinary bladder tissues. However, anti-CKB antibody was not detected in sera from over 30 donors, including bladder cancer patients without PCD, indicating that anti-CKB antibody is required for onset of PCD. We also detected anti-CKB antibody in sera from three other PCD patients. Our study demonstrated that anti-CKB antibody may be added to the list of PCD-related autoantibodies and may be useful for diagnosis of PCD.
  • Kotaro Kawanishi, Mitsuya Morita, Keiichi Nakahara, Syuichi Tetsuka, Tom Kouki, Kaoru Tominaga, Hitoshi Endo, Takashi Yashiro, Keiko Tanaka, Imaharu Nakano
    Brain and nerve = Shinkei kenkyu no shinpo 65 11 1401 - 5 2013年11月 [査読有り][通常論文]
     
    A 66-year-old man was diagnosed with bladder cancer at our urology department. Three months later, he developed subacute progressive cerebellar limb ataxia and truncal oscillation. Analysis of cerebrospinal fluid showed pleocytosis and increased concentrations of protein, while brain magnetic resonance imaging revealed no abnormalities. Based on the presence of the bladder cancer, the etiology of subacute cerebellar ataxia could be a paraneoplastic neurological syndrome. Four months later, the patient underwent transurethral resection of the bladder tumor, which was identified as urothelial cancer on the basis of pathological examinations. However, this procedure failed to improve his neurological symptoms. Serum paraneoplastic markers such as anti-Yo, anti-Hu, anti-Tr, and other antibodies were not detected. Immunohistochemical staining of mouse cerebellum using the patient's serum revealed coarse granular staining in the cytoplasm of Purkinje cells and diffuse staining in the neuropil of the molecular layer, suggesting the presence of an unknown antibody. Subsequently, one-dimensional electrophoresis western blotting using the patient's serum revealed several bands including a strong positive band of approximately 45 kDa in mouse cerebellum lysates but not in liver lysates. These bands have never been detected in sera derived from healthy donors. These results suggested the presence of a novel antibody in the patient's serum that might recognize the approximately 45 kDa protein related to paraneoplastic cerebellar degeneration. Cases of paraneoplastic neurological syndrome associated with bladder cancer have rarely been reported. We concluded that the present case may be categorized as paraneoplastic neurological syndrome caused by an unknown antibody.
  • Chizuru Akimoto, Eiji Sakashita, Katsumi Kasashima, Kenji Kuroiwa, Kaoru Tominaga, Toshiro Hamamoto, Hitoshi Endo
    Biochimica et biophysica acta 1830 3 2728 - 38 2013年03月 [査読有り][通常論文]
     
    BACKGROUND: Upstream open reading frames (uORFs) are commonly found in the 5'-untranslated region (UTR) of many genes and function in translational control. However, little is known about the existence of the proteins encoded by uORFs, and the role of the proteins except translational control. There was no report about uORFs of the McKusick-Kaufman syndrome (MKKS) gene that causes a genetic disorder. METHODS: Northern blotting, 3'-RACE, and bioinformatics were used for determining the length of transcripts and their 3' ends. Luciferase assay and in vitro translation were used for evaluation of translational regulatory activity of uORFs. Immunoblotting and immunocytochemical analyses were used for detection of uORF-derived protein products and their subcellular localization. RESULTS: The MKKS gene generates two types of transcripts: a canonical long transcript that encodes both uORFs and MKKS, and a short transcript that encodes only uORFs by using alternative polyadenylation sites at the 5'-UTR. The simultaneous disruption of the uORF initiation codons increased the translation of the downstream ORF. Furthermore, both protein products from the two longest uORFs were detected in the mitochondrial membrane fraction of HeLa cells. Database searches indicated that such uORFs with active alternative polyadenylation sites at the 5'-UTR are atypical but surely exist in human transcripts. CONCLUSIONS: Multiple uORFs at the 5'-UTR of the MKKS long transcript function as translational repressor for MKKS. Two uORFs are translated in vivo and imported onto the mitochondrial membrane. GENERAL SIGNIFICANCE: Our findings provide unique insights into production of uORF-derived peptides and functions of uORFs.
  • Elena J Tucker, Bas F J Wanschers, Radek Szklarczyk, Hayley S Mountford, Xiaonan W Wijeyeratne, Mariël A M van den Brand, Anne M Leenders, Richard J Rodenburg, Boris Reljić, Alison G Compton, Ann E Frazier, Damien L Bruno, John Christodoulou, Hitoshi Endo, Michael T Ryan, Leo G Nijtmans, Martijn A Huynen, David R Thorburn
    PLoS genetics 9 12 e1004034  2013年 [査読有り][通常論文]
     
    Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis.
  • 抗神経抗体の存在が確認できた膀胱癌を伴う傍腫瘍性小脳変性症の1例
    川西康太郎, 森田光哉, 中原圭一, 手塚修一, 幸喜 富, 冨永 薫, 遠藤仁司, 屋代 隆, 田中惠子, 中野今治
    Brain Nerve 65 11 1401 - 1405 2013年 [査読有り][通常論文]
  • Katsumi Kasashima, Megumi Sumitani, Hitoshi Endo
    Experimental cell research 318 18 2335 - 43 2012年11月 [査読有り][通常論文]
     
    The segregation of mitochondrial DNA (mtDNA) is important for the maintenance and transmission of the genome between generations. Recently, we clarified that human mitochondrial transcription factor A (TFAM) is required for equal distribution and symmetric segregation of mtDNA in cultured cells; however, the molecular mechanism involved is largely unknown. ClpX is an ATPase associated with various cellular activities (AAA+) proteins that localize to the mitochondrial matrix and is suggested to associate with mtDNA. In this study, we found that RNAi-mediated knockdown of ClpX in HeLa cells resulted in enlarged mtDNA nucleoids, which is very similar to that observed in TFAM-knockdown cells in several properties. The expression of TFAM protein was not significantly reduced in ClpX-knockdown cells. However, the enlarged mtDNA nucleoids caused by ClpX-knockdown were suppressed by overexpression of recombinant TFAM and the phenotype was not observed in knockdown with ClpP, a protease subunit of ClpXP. Endogenous ClpX and TFAM exist in close vicinity, and ClpX enhanced DNA-binding activity of TFAM in vitro. These results suggest that human ClpX, a novel mtDNA regulator, maintains mtDNA nucleoid distribution through TFAM function as a chaperone rather than as a protease and its involvement in mtDNA segregation.
  • Kenji Kuroiwa, Yusuke Torikai, Mafumi Osawa, Takaaki Nakashima, Mitsuhiro Nakashima, Hitoshi Endo, Takao Arai
    Hybridoma (2005) 31 4 225 - 32 2012年08月 [査読有り][通常論文]
     
    Glycosylphosphatidylinositol-anchored protein Thy-1 is abundantly expressed on the cell surface of neurons and T lymphocytes in rodents. Although Thy-1 is known to bind integrins as a ligand and to mediate neurite outgrowth and immune responses, its precise function is not fully understood. Previously we produced several anti rat Thy-1 monoclonal antibodies and identified one, 2E11, which induces PC12 cell neurite outgrowth. Here we screened antibodies that inhibit 2E11-induced neurite outgrowth and stimulate or inhibit rat thymocyte aggregation. Since Thy-1 lacks an intracellular region, it requires other membrane-bound molecules for the signal transduction. Hence these antibodies are hypothesized to play key roles in the interaction between Thy-1 and signaling molecules. To elucidate the mechanisms of antibody-induced Thy-1 functions, antibody characterization and epitope determination were carried out. Thy-1 cleavage and mutation revealed that the antibodies recognize not only amino acid sequences, but also the three-dimensional structures consisting of immunoglobulin-like domains. Two antibodies were suggested to bind spatially close to the integrin binding site and crosslink Thy-1 molecules, while a third antibody is believed to inhibit Thy-1 crosslinking and subsequent Thy-1 signaling. The antibodies reported here may therefore function as crosslinkers, agonists, or antagonists that modify Thy-1 signaling.
  • Katsumi Kasashima, Megumi Sumitani, Hitoshi Endo
    Experimental cell research 317 2 210 - 20 2011年01月 [査読有り][通常論文]
     
    The segregation and transmission of the mitochondrial genome in humans are complicated processes but are particularly important for understanding the inheritance and clinical abnormalities of mitochondrial disorders. However, the molecular mechanism of the segregation of mitochondrial DNA (mtDNA) is largely unclear. In this study, we demonstrated that human mitochondrial transcription factor A (TFAM) is required for the segregation of mtDNA in cultured cells. RNAi-mediated knockdown of TFAM in HeLa cells resulted in the enlarged mtDNA, as indicated by the assembly of fluorescent signals stained with PicoGreen. Fluorescent in situ hybridization confirmed the enlarged mtDNA and further showed the existence of increased numbers of mitochondria lacking mtDNA signals in TFAM knockdown cells. By complementation analysis, the C-terminal tail of TFAM, which enhances its affinity with DNA, was found to be required for the appropriate distribution of mtDNA. Furthermore, we found that TFAM knockdown induced asymmetric segregation of mtDNA between dividing daughter cells. These results suggest an essential role for human TFAM in symmetric segregation of mtDNA.
  • Megumi Sumitani, Katsumi Kasashima, Eriko Ohta, Dongchon Kang, Hitoshi Endo
    Journal of biochemistry 146 5 725 - 32 2009年11月 [査読有り][通常論文]
     
    We have identified a novel mitochondrial protein, termed M19, by proteomic analysis of mitochondrial membrane proteins from HeLa cells. M19 is highly conserved among vertebrates, and possesses no homologous domains with other known proteins. By northern and western blotting, mouse M19 was shown to be expressed in various tissues, and to be especially abundant in the brain. Human M19 (hM19) is present in mitochondria, and protease-protection experiment showed it to be sublocalized in the matrix space. Carboxy-terminally tagged hM19 appeared as spotted signals within mitochondria and co-localized with signals arising from mitochondrial DNA (mtDNA), suggesting the inclusion of M19 in the mtDNA-protein complex (mitochondrial nucleoids). Fractionation of mitochondrial nucleoids from HeLa cells revealed that hM19 has a similar distribution pattern like that of known nucleoid components, such as mtSSB and PHBs, and surely exists in the nucleoid fraction. Furthermore, expression of M19 is closely related to the amount of mtDNA, because it was down-regulated in mtDNA-depleted rho(0) HeLa cells. These results indicate that M19 associates with the nucleoid and likely regulates the organization and metabolism of mtDNA.
  • Katsumi Kasashima, Megumi Sumitani, Masaaki Satoh, Hitoshi Endo
    Experimental cell research 314 5 988 - 96 2008年03月 [査読有り][通常論文]
     
    Mitochondrial prohibitin (PHB) proteins have diverse functions, such as the regulation of apoptosis and the maintenance of mitochondrial morphology. In this study, we clarified a novel mitochondrial function of PHB1 that regulates the organization and maintenance of mitochondrial DNA (mtDNA). In PHB1-knockdown cells, we found that mtDNA is not stained by fluorescent dyes, such as ethidium bromide and PicoGreen, although the mitochondrial membrane potential still maintains. We also demonstrated that mtDNA, which is predominantly found in the NP-40-insoluble fraction when isolated from normal mitochondria, is partially released into the soluble fraction when isolated from PHB1-knockdown cells, indicating that the organization of the mitochondrial nucleoids has been altered. Furthermore, we found that PHB1 regulates copy number of mtDNA by stabilizing TFAM protein, a known protein component of the mitochondrial nucleoids. However, TFAM does not affect the organization of mtDNA as observed in PHB1-knockdown cells. Taken together, these results demonstrate that PHB1 maintains the organization and copy number of the mtDNA through both TFAM-independent and -dependent pathways.
  • Mie Uchida, Keita Kirito, Hitoshi Endo, Keiya Ozawa, Norio Komatsu
    International journal of hematology 86 4 315 - 24 2007年11月 [査読有り][通常論文]
     
    FKHRL1 is one of the human homologues of DAF-16, which is concerned with longevity in Caenorhabditis elegans. Previously, we demonstrated that FKHRL1 functions downstream of Akt in erythropoietin (EPO) signaling and that it is directly phosphorylated by activated Akt. Because phosphorylated FKHRL1 loses its transcriptional activity and translocates into the cytoplasm, FKHRL1 appears to be nonfunctional in the presence of EPO. Conversely, EPO deprivation leads to FKHRL1 dephosphorylation and its translocation into the nucleus, suggesting that FKHRL1 becomes active as a transcription factor in the absence of EPO. On the basis of these findings, we hypothesized, by analogy with C elegans, that erythroid cells possess self-defense machinery against life-threatening surroundings. We prepared a dominant-negative mutant of FKHRL1 (FKHRL1-DN) lacking the transactivation domain and prepared FKHRL1 small interfering RNA (siRNA), and we used constructs to transfect a human EPO-dependent cell line, UT-7/EPO. In the parental cells, 24-hour EPO deprivation induced transient cell cycle arrest without apoptosis. On the other hand, stable transfectants expressing FKHRL1-DN or FKHRL1 siRNA underwent rapid apoptosis after EPO deprivation in the UT-7/EPO cells. In conclusion, FKHRL1 activation plays an important role in the extension of survival of erythroid cells after EPO deprivation. This phenomenon appears to correspond to dauer formation in C elegans. Thus, the mechanism of lifespan extension may be broadly conserved from C elegans to humans.
  • Katsumi Kasashima, Eriko Ohta, Yasuo Kagawa, Hitoshi Endo
    The Journal of biological chemistry 281 47 36401 - 10 2006年11月 [査読有り][通常論文]
     
    Proteins with multiple cellular functions provide biological diversity to eukaryotic cells. In the current studies, we identified the mitochondrial functions of human prohibitin 2 (PHB2), which was initially identified as a repressor of estrogen-dependent transcriptional activity. The mitochondrial complex of PHB2 consists of PHB1, voltage-dependent anion channel 2, adenine nucleotide translocator 2, and the anti-apoptotic Hax-1, which is a novel binding partner for PHB2. RNA interference-mediated knockdown of PHB2 in HeLa cells resulted in caspase-dependent apoptosis through down-regulation of Hax-1 and fragmentation of mitochondria. We also found that, although PHB2 is predominantly expressed in the mitochondria of HeLa cells, it translocates to nucleus in the presence of estrogen receptor alpha and estradiol. Here, we first demonstrated the roles of mammalian PHB2 in mitochondria and the molecular mechanism of its nuclear targeting and showed that PHB2 is a possible molecule directly coupling nuclear-mitochondrial interaction.
  • Atsuko Kasahara, Kaori Ishikawa, Makiko Yamaoka, Masahito Ito, Naoki Watanabe, Miho Akimoto, Akitsugu Sato, Kazuto Nakada, Hitoshi Endo, Yoko Suda, Shinichi Aizawa, Jun-Ichi Hayashi
    Human molecular genetics 15 6 871 - 81 2006年03月 [査読有り][通常論文]
     
    Generation of various kinds of trans-mitochondrial mice, mito-mice, each carrying mtDNAs with a different pathogenic mutation, is required for precise investigation of the pathogenesis of mitochondrial diseases. This study used two respiration-deficient mouse cell lines as donors of mtDNAs with possible pathogenic mutations. One cell line expressed 45-50% respiratory activity due to mouse mtDNAs with a T6589C missense mutation in the COI gene (T6589C mtDNA) and the other expressed 40% respiratory activity due to rat (Rattus norvegicus) mtDNAs in mouse cells. By cytoplasmic transfer of these mtDNAs to mouse ES cells, we isolated respiration-deficient ES cells. We obtained chimeric mice and generated their F(6) progeny carrying mouse T6589C mtDNAs by its female germ line transmission. They were respiration-deficient and thus could be used as models of mitochondrial diseases caused by point mutations in mtDNA structural genes. However, chimeric mice and mito-mice carrying rat mtDNAs were not obtained, suggesting that significant respiration defects or some deficits induced by rat mtDNAs in mouse ES cells prevented their differentiation to generate mice carrying rat mtDNAs.
  • Naoki Takezako, Morisada Hayakawa, Hiroko Hayakawa, Shinsuke Aoki, Ken Yanagisawa, Hitoshi Endo, Shin-ichi Tominaga
    Biochemical and biophysical research communications 341 2 425 - 32 2006年03月 [査読有り][通常論文]
     
    LPS induces the production of inflammatory cytokines via the stimulation of Toll-like receptors. In this study, we demonstrated that a soluble secreted form of the ST2 gene product (ST2), a member of the interleukin-1 receptor family, suppressed the production of IL-6 in an LPS-stimulated human monocytic leukemia cell line, THP-1. Immunofluorescence confocal microscopy revealed the binding of ST2 to the surface of the THP-1 cells, in which ST2 led to decreased binding of nuclear factor-kappaB to the IL-6 promoter. Furthermore, the degradation of IkappaB in the cytoplasm after LPS stimulation was reduced by pretreatment with ST2. These results demonstrated that ST2 negatively regulates LPS-induced IL-6 production via the inhibition of IkappaB degradation in THP-1 cells.
  • Yasuyuki Fukuhara, Xiao-Kang Li, Yusuke Kitazawa, Masumi Inagaki, Kentaro Matsuoka, Motomichi Kosuga, Rika Kosaki, Takuya Shimazaki, Hitoshi Endo, Akihiro Umezawa, Hideyuki Okano, Takao Takahashi, Torayuki Okuyama
    Molecular therapy : the journal of the American Society of Gene Therapy 13 3 548 - 55 2006年03月 [査読有り][通常論文]
     
    The therapeutic efficacy of neural stem cell transplantation for central nervous system (CNS) lesions in lysosomal storage disorders was explored using a murine model of mucopolysaccharidosis type VII (MPS VII). We used fetal neural stem cells derived from embryonic mouse striata and expanded in vitro by neurosphere formation as the source of graft materials. We transplanted neurospheres into the lateral ventricles of newborn MPS VII mice and found that donor cells migrated far beyond the site of injection within 24 h, and some of them could reach the olfactory bulb. A quantitative measurement indicated that the GUSB activity in the brain was 12.5 to 42.3% and 5.5 to 6.3% of normal activity at 24 h and 3 weeks after transplantation. In addition, histological analysis revealed a widespread decrease in lysosomal storage in the recipient's hippocampus, cortex, and ependyma. A functional assessment with novel-object recognition tests confirmed improvements in behavioral patterns. These results suggest that intracerebral transplantation of neural stem cells is feasible for treatment of CNS lesions associated with lysosomal storage disorders.
  • Masanori Ito, Kou Yokouchi, Kunihiko Naito, Hitoshi Endo, Yoji Hakamata, Jun-Ichi Miyazaki, Hideaki Tojo
    Biochemical and biophysical research communications 337 1 264 - 70 2005年11月 [査読有り][通常論文]
     
    We have successfully specified essential sequences of the 5' upstream region for the stage- and tissue-specific expression of mouse Sry by using an in vitro Cre/loxP system. Sry/Cre plasmids carrying Sry 5' sequences of various sizes were transfected into the primary cultured cells from different tissues of CAG/loxP/CAT/loxP/LacZ transgenic fetuses on 11.5-day post coitus (dpc) or 13.5-dpc. Stage- and tissue-specific regulation of Sry expression was disrupted by the deletion of positions 7549-7660 (from -0.4 to -0.5 kb region). In vitro transcription assay also suggested that the region contains element(s) responsible for stage- and tissue-specific expression of mouse Sry. SRY promoter of Shiba goat (Capra hircus var Shiba), a native Japanese miniature goat, showed the tissue-specific activity in the cells from urogenital ridges of the male mouse, but not in the cells from female mice, indicating a possibly different mechanism among species in the regulation of Sry expression.
  • Janeen H Trembley, Sawako Tatsumi, Eiji Sakashita, Pascal Loyer, Clive A Slaughter, Hitoshi Suzuki, Hitoshi Endo, Vincent J Kidd, Akila Mayeda
    Molecular and cellular biology 25 4 1446 - 57 2005年02月 [査読有り][通常論文]
     
    Human RNPS1 was originally characterized as a pre-mRNA splicing activator in vitro and was shown to regulate alternative splicing in vivo. RNPS1 was also identified as a protein component of the splicing-dependent mRNP complex, or exon-exon junction complex (EJC), and a role for RNPS1 in postsplicing processes has been proposed. Here we demonstrate that RNPS1 incorporates into active spliceosomes, enhances the formation of the ATP-dependent A complex, and promotes the generation of both intermediate and final spliced products. RNPS1 is phosphorylated in vivo and interacts with the CK2 (casein kinase II) protein kinase. Serine 53 (Ser-53) of RNPS1 was identified as the major phosphorylation site for CK2 in vitro, and the same site is also phosphorylated in vivo. The phosphorylation status of Ser-53 significantly affects splicing activation in vitro, but it does not perturb the nuclear localization of RNPS1. In vivo experiments indicated that the phosphorylation of RNPS1 at Ser-53 influences the efficiencies of both splicing and translation. We propose that RNPS1 is a splicing regulator whose activator function is controlled in part by CK2 phosphorylation.
  • Taeko Inoki, Hitoshi Endo, Yutaka Inoki, Toshiro Hamamoto, Tadahiko Tsuru, Toshio Mori, Kazunori Miyata, Shiro Amano, Satoru Yamagami
    Experimental eye research 79 3 367 - 76 2004年09月 [査読有り][通常論文]
     
    PURPOSE: To determine damaged DNA-binding protein 2-gene expression levels in vitro and ex vivo, and the degree of DNA repair in damaged DNA-binding protein 2-overexpressing cultured human corneal endothelium after ultraviolet irradiation. METHODS: Constitutive damaged DNA-binding protein 2-gene expression levels in various human tissues were determined by semi-quantitative reverse transcription-polymerase chain reactions. The dynamics of nucleotide excision repair-related gene expression in cultured human corneal endothelium were investigated in a ribonuclease protection assay after ultraviolet-irradiation. The effect of damaged DNA-binding protein 2 on DNA repair was studied after ultraviolet-irradiation in cultured human corneal endothelium infected with adenovirus carrying damaged DNA-binding protein 2. RESULTS: Human corneal endothelium and epithelium in the donor cornea had the highest constitutive damaged DNA-binding protein 2-gene expression of the various human tissues studied. Gene expression level dynamics associated with nucleotide excision repair factors after ultraviolet-irradiation showed that the increase in the rate of damaged DNA-binding protein 2-gene expression in cultured human corneal endothelium was highest of the nucleotide excision repair-related genes studied. An in vivo DNA repair assay showed that DNA repair efficiency in damaged DNA-binding protein 2-overexpressing cultured human corneal endothelium after ultraviolet-irradiation was significantly improved as compared with that in the control human corneal endothelium. CONCLUSION: The human corneal endothelium abundantly expresses the damaged DNA-binding protein 2-gene that is produced efficiently on ultraviolet exposure. This overexpressed damaged DNA-binding protein 2 in the human corneal endothelium contributes to the protection system against DNA damage after ultraviolet-irradiation. Our findings show a critical role for damaged DNA-binding protein 2 in DNA repair to maintain the human corneal endothelium function.
  • Yuki Sato, Hitoshi Endo, Takashi Ajiki, Yoji Hakamata, Takashi Okada, Takashi Murakami, Eiji Kobayashi
    Biochemical and biophysical research communications 319 4 1197 - 202 2004年07月 [査読有り][通常論文]
     
    The rat has offered an important animal model in biomedical research including surgical procedure. However, advanced genetic manipulation has progressed less far in the rat than in the mouse. Here we report the Cre/LoxP transgenic rat system, demonstrating conditional chromosomal translocation both in the fertilization and adult stage, spatio-temporal gene controlling by catheter-based adenoviral gene transfer, and muscular fusion events in the limb transplant. Taking advantage of the larger body size of the rat than the mouse, this rat system provides a potential value to evaluate biomedical and therapeutic significance for gene therapy and regenerative medicine.
  • Taeko Inoki, Satoru Yamagami, Yutaka Inoki, Tadahiko Tsuru, Toshiro Hamamoto, Yasuo Kagawa, Toshio Mori, Hitoshi Endo
    Biochemical and biophysical research communications 314 4 1036 - 43 2004年02月 [査読有り][通常論文]
     
    Damaged DNA-binding protein (DDB) is a heterodimer (DDB1 and DDB2), which is implicated in the repair of UV-irradiated DNA damage. Here we have identified four DDB2 variants from HeLa cells (D1-D4) that are generated by alternative splicing. Analysis of tissue distribution by RT-PCR indicates that D1 is the most highly expressed in human brain and heart. A DNA repair assay revealed that both D1 and D2 are dominant negative inhibitors. Electrophoresis mobility shift assays indicated that D1 and D2 are not part of the damaged DNA-protein complex. Co-immunoprecipitation studies show that DDB2-WT interacts with D1 and itself. Nuclear import of DDB1 was less induced by transfection with D1 than WT. Based on these results, D1 and D2 are dominant negative inhibitors of DNA repair, which is probably due to disruption of complex formation between DDB1 and DDB2-WT and of DDB1 nuclear import.
  • Eiji Sakashita, Sawako Tatsumi, Dieter Werner, Hitoshi Endo, Akila Mayeda
    Molecular and cellular biology 24 3 1174 - 87 2004年02月 [査読有り][通常論文]
     
    Human RNPS1 was originally purified and characterized as a pre-mRNA splicing activator, and its role in the postsplicing process has also been proposed recently. To search for factors that functionally interact with RNPS1, we performed a yeast two-hybrid screen with a human cDNA library. Four factors were identified: p54 (also called SRp54; a member of the SR protein family), human transformer 2 beta (hTra2 beta; an exonic splicing enhancer-binding protein), hLucA (a potential component of U1 snRNP), and pinin (also called DRS and MemA; a protein localized in nuclear speckles). The N-terminal region containing the serine-rich (S) domain, the central RNA recognition motif (RRM), and the C-terminal arginine/serine/proline-rich (RS/P) domain of RNPS1 interact with p54, pinin, and hTra2 beta, respectively. Protein-protein binding between RNPS1 and these factors was verified in vitro and in vivo. Overexpression of RNPS1 in HeLa cells induced exon skipping in a model beta-globin pre-mRNA and a human tra-2 beta pre-mRNA. Coexpression of RNPS1 with p54 cooperatively stimulated exon inclusion in an ATP synthase gamma-subunit pre-mRNA. The RS/P domain and RRM are necessary for the exon-skipping activity, whereas the S domain is important for the cooperative effect with p54. RNPS1 appears to be a versatile factor that regulates alternative splicing of a variety of pre-mRNAs.
  • Masao Naito, Jun Mimuro, Hitoshi Endo, Seiji Madoiwa, Kyo-ichi Ogata, Jiro Kikuchi, Teruko Sugo, Takanori Yasu, Yusei Kariya, Yuichi Hoshino, Yoichi Sakata
    Circulation research 92 8 865 - 72 2003年05月 [査読有り][通常論文]
     
    Three thrombophilic patients with protein C (PC) deficiency were found to have independent mutations in the PC gene. These mutations resulted in single amino acid substitutions of R169W, R352W, and G376D in the affected PC molecules. These abnormal PC molecules were expressed in CHO-K1 cells in the presence or absence of vitamin K, and their synthesis, posttranslational modification, and secretion were studied. PC G376D was not secreted from the cells and was gradually degraded inside the cells. There was partial secretion of PC R169W and PC R352W, but most of these molecules were not secreted but were degraded intracellularly. On the basis of pulse-chase, immunofluorescence, and endo-beta-N-acetylglucosaminidase H digestion experiments, the majority of wild-type PC molecules localize not in the Golgi apparatus but in the rough endoplasmic reticulum inside the cells. This suggests that wild-type PC molecules are secreted immediately after gamma-carboxylation and modification at the Golgi apparatus. In contrast, the mutant PC molecules were retained inside the cells even after modification of oligosaccharides at the trans-Golgi apparatus, which was probably due to impaired conformation of the abnormal molecules. Data suggest that these abnormal PC molecules were not sorted to secretory vesicles in the trans-Golgi network because of conformational defects in addition to the transport defect from the rough endoplasmic reticulum to the Golgi apparatus and were degraded inside the cells, thereby resulting in a PC deficiency in the affected patients.
  • Kou Yokouchi, Masanori Ito, Koichiro Nishino, Keitaro Yamanouchi, Kunihiko Naito, Miyuki Suzawa, Shigeaki Kato, Yoji Hakamata, Hitoshi Endo, Hideaki Tojo
    Molecular reproduction and development 64 4 389 - 96 2003年04月 [査読有り][通常論文]
     
    Sry expression is essential for initiating male sex differentiation, and the expression occurs only during a restricted period in the developing gonad. It is thought that Sry is part of a pathway of genes that regulate sex determination. Although the interactions of several genes with Sry expression have been suggested, the exact cascade of gene expression regulating Sry transcription is entirely obscure because there is no available cell line expressing Sry and reflecting an in vivo condition. The present study was carried out to investigate the cis-acting element of the mouse Sry that responds stage specifically to its expression, in part, using transgenic mice expressing GFP on the Y chromosome. Ten DNA fragments were generated by digesting the 5' upstream region (positions 5491-8039; 2,549 bp) of mouse Sry with appropriate restriction enzymes. In an electrophoretic mobility assay with these fragments, the region from position 5491 to position 5799 (309 bp) was identified as forming specific protein-DNA complexes with nuclear extracts from 11.5 days post coitus (dpc) gonads, but not from 12.5 and 13.5-dpc gonads. This region also formed specific protein-DNA complexes with the nuclear extracts from adult testicular germ cells that generate only a circular form from Sry. This stage-specific responsive region was narrowed down to positions 5559-5616 by DNase I footprinting analysis. The assay of DNase I hypersensitive (HS) using the nuclear lysates from the 11.5-dpc urogenital ridges demonstrated that the novel HS site was located in the proximity of position 5600. This region DNase I HS was also detected at the same position when the lysates from adult testicular germ cells were applied. The results indicate that the present HS site may be involved in the transcriptional regulation of the linear and/or circular molecule transcripts from mouse Sry gene.
  • Norio Komatsu, Tomoko Watanabe, Mie Uchida, Masaki Mori, Keita Kirito, Satoru Kikuchi, Qifa Liu, Tetsuzo Tauchi, Keisuke Miyazawa, Hitoshi Endo, Tadashi Nagai, Keiya Ozawa
    The Journal of biological chemistry 278 8 6411 - 9 2003年02月 [査読有り][通常論文]
     
    A member of the Forkhead transcription factor family, FKHRL1, lies downstream of the phosphatidylinositol 3-kinase-Akt activation pathway in cytokine signaling. Because the phosphatidylinositol 3-kinase-Akt activation pathway is required for BCR-ABL-mediated transformation and survival signaling in chronic myelogenous leukemia (CML), in this study we examined the involvement of FKHRL1 in the BCR-ABL-mediated signaling pathway. FKHRL1 was constitutively phosphorylated in BCR-ABL-expressing cell lines KCL22 and KU812, and its phosphorylation was inhibited by treatment with STI571, a specific inhibitor of BCR-ABL tyrosine kinase. Concomitantly, STI571 induced cell cycle arrest at the G(0)/G(1) phase, accompanied by up-regulation of a cyclin-dependent kinase inhibitor p27/Kip1 in KCL22 cells. In addition, FKHRL1 was constitutively phosphorylated in the TF-1/bcr-abl cell line ectopically expressing BCR-ABL but not in the parent TF-1 cell line. Considering several lines of evidence that phosphorylated FKHRL1 has lost transcriptional activity and that p27/Kip1 expression is positively regulated by dephosphorylated "active" FKHRL1, BCR-ABL may down-regulate p27/Kip1 expression via the loss of FKHRL1 function as a transcription factor. To demonstrate this hypothesis, we generated a tamoxifen-inducible "active FKHRL1" FKHRL1-TM (a triple mutant of FKHRL1, in which all three Akt phosphorylation sites have been mutated), estrogen receptor system in the KCL22 cell line. The addition of tamoxifen inhibited the cell growth indicating that overexpression of FKHRL1 in the nucleus antagonized deregulated proliferation of CML cells. Collectively, FKHRL1 regulates the expression of p27/Kip1 as a downstream molecule of BCR-ABL signaling in CML cells. BCR-ABL-induced loss of FKHRL1 function may be involved in oncogenic transformation of CML partially via the down-regulation of p27/Kip1 proteins.
  • Yui Jin, Hitoshi Suzuki, Shingo Maegawa, Hitoshi Endo, Sumio Sugano, Katsuyuki Hashimoto, Kunio Yasuda, Kunio Inoue
    The EMBO journal 22 4 905 - 12 2003年02月 [査読有り][通常論文]
     
    Alternative splicing is one of the central mechanisms that regulate eukaryotic gene expression. Here we report a tissue-specific RNA-binding protein, Fox-1, which regulates alternative splicing in vertebrates. Fox-1 bound specifically to a pentanucleotide GCAUG in vitro. In zebrafish and mouse, fox-1 is expressed in heart and skeletal muscles. As candidates for muscle-specific targets of Fox-1, we considered two genes, the human mitochondrial ATP synthase gamma-subunit gene (F1gamma) and the rat alpha-actinin gene, because their primary transcripts contain several copies of GCAUG. In transfection experiments, Fox-1 induced muscle-specific exon skipping of the F1gamma gene via binding to GCAUG sequences upstream of the regulated exon. Fox-1 also regulated mutually exclusive splicing of the alpha-actinin gene, antagonizing the repressive effect of polypyrimidine tract-binding protein (PTB). It has been reported that GCAUG is essential for the alternative splicing regulation of several genes including fibronectin. We found that Fox-1 promoted inclusion of the fibronectin EIIIB exon. Thus, we conclude that Fox-1 plays key roles in both positive and negative regulation of tissue-specific splicing via GCAUG.
  • Masaaki Satoh, Toshiro Hamamoto, Norimasa Seo, Yasuo Kagawa, Hitoshi Endo
    Biochemical and biophysical research communications 300 2 482 - 93 2003年01月 [査読有り][通常論文]
     
    OPA1 is a cause gene for autosomal dominant optic atrophy and possesses eight alternative splicing variants. Here, we identified two isoforms of OPA1 proteins in HeLa cells and examined their submitochondrial localization and complex formations. RT-PCR shows that HeLa cells mainly express isoforms 7 and 1 of OPA1. Since the third cleavage site is mainly utilized in HeLa cells, the predicted molecular masses of their processed proteins are consistent with the 93- and 88-kDa proteins. Biochemical examinations indicate that both of the OPA1 isoforms are present in the intermembrane space. Submitochondrial fractionation by sucrose density-gradient centrifugation shows that the 88-kDa protein predominantly associates with the mitochondrial outer membrane, on the contrary, the 93-kDa protein associates with the inner membrane. Gel filtration analysis indicates that they compose the different molecular mass complexes in mitochondria. These differences between two isoforms of OPA1 would suggest their crucial role involved in the mitochondrial membrane formation.
  • Masanori Ito, Kou Yokouchi, Kunihiko Naito, Hitoshi Endo, Yoji Hakamata, Jun-Ichi Miyazaki, Hideaki Tojo
    Development, growth & differentiation 44 6 549 - 57 2002年12月 [査読有り][通常論文]
     
    There have been few studies on the regulatory elements of the Sry gene, mainly because no Sry-expressing cell lines have yet been established. This paper describes a useful tool for investigating the regulation and upstream region of Sry by means of the in vitro Cre/loxP system. Using plasmids containing the 9.9 kb mouse genomic Sry previously shown to induce testis development in XX transgenic mice, we constructed a Sry/Cre fusion gene plasmid in which Cre expression is controlled by the 5' and 3' untranslated regions of mouse Sry. To distinguish between male and female gonads of 11.5 days post-coitus (d.p.c.) fetuses, double transgenic fetuses carrying both the CAG (cytomegalovirus enhancer and beta-actin promoter)/loxP/lacZ transgene on the autosome and the green fluorescent protein transgene ubiquitously expressed on the Y chromosome were produced by crossing between two transgenic mouse lines. When Sry/Cre plasmids were transfected into the cells that had been prepared from the gonads, brains and livers of double transgenic fetuses, only a small number of X-gal-stained cells were detected among the primary cultured cells from male and female gonads, and none were detected among the cells from the other tissues. The X-gal-positive cells were negative for alkaline phosphatase, indicating that these cells were somatic cells expressing Sry. The Sry/Cre plasmids with a 0.4 kb upstream region of Sry yielded a large number of X-gal-positive cells in the cells from gonads, including various tissues of 11.5 d.p.c. fetuses, indicating the loss of the tissue-specific expression of Sry. The Sry/Cre with a 1.4 kb upstream region maintained tissue-specific activity of Sry. The results indicate that the present in vitro Cre/loxP system using transgenic mice is a simple and useful system for investigating the regulatory element of sex determination-related genes, including Sry.
  • Yusuke Furukawa, Noriko Nishimura, Yutaka Furukawa, Masaaki Satoh, Hitoshi Endo, Satsuki Iwase, Hisashi Yamada, Michio Matsuda, Yasuhiko Kano, Mitsuru Nakamura
    The Journal of biological chemistry 277 42 39760 - 8 2002年10月 [査読有り][通常論文]
     
    E2F-1 is capable of promoting both cell cycle progression and apoptosis. The latter is important for suppressing untoward expansion of proliferating cells. In this study, we investigated its underlying mechanisms. E2F-1-induced apoptosis was accompanied by caspase-9 activation and inhibited by a specific inhibitor of caspase-9 in K562 sublines overexpressing E2F-1. E2F-1 enhanced the expression of Apaf-1 without the cytosolic accumulation of cytochrome c. Apaf-1-deficient melanoma cell lines were resistant to E2F-1, indicating that Apaf-1 is an essential element of E2F-1-mediated apoptosis. Finally, we isolated the promoter region of the Apaf-1 gene and found a putative binding site for E2F. A chromatin immunoprecipitation assay revealed that E2F-1 bound to Apaf-1 promoter upon E2F-1 overexpression, suggesting that Apaf-1 is under transcriptional regulation of E2F-1. These data demonstrate a novel mechanism of apoptosis in which an increase in Apaf-1 levels results in direct activation of caspase-9 without mitochondrial damage, leading to the initiation of a caspase cascade.
  • Hiroshi Tamada, Eiji Sakashita, Kuniko Shimazaki, Eriko Ueno, Toshiro Hamamoto, Yasuo Kagawa, Hitoshi Endo
    Biochemical and biophysical research communications 297 1 96 - 104 2002年09月 [査読有り][通常論文]
     
    Neural RNA recognition motif (RRM)-type RNA-binding proteins play essential roles in neural development. To search for a new member of neural RRM-type RNA-binding protein, we screened rat cerebral expression library with polyclonal antibody against consensus RRM sequences. We have cloned and characterized a rat cDNA that belongs to RRM-type RNA-binding protein family, which we designate as drb1. Orthologs of drb1 exist in human and mouse. The predicted amino acid sequence reveals an open reading frame of 476 residues with a corresponding molecular mass of 53kDa and consists of four RNA-binding domains. drb1 gene is specifically expressed in fetal (E12, E16) rat brain and gradually reduced during development. In situ hybridization demonstrated neuron-specific signals in fetal rat brain. RNA-binding assay indicated that human Drb1 protein possesses binding preference on poly(C)RNA. These results indicate that Drb1 is a new member of neural RNA-binding proteins, which expresses under spatiotemporal control.
  • Shoji Yamazaki, Tatuo Morita, Hitoshi Endo, Toshiro Hamamoto, Masaki Baba, Yoshiko Joichi, Sanae Kaneko, Yoshihito Okada, Toru Okuyama, Hoyoku Nishino, Akihiko Tokue
    Cancer letters 183 1 23 - 30 2002年09月 [査読有り][通常論文]
     
    Isoliquiritigenin is a chalcone isolated from licorice and shallots. The ability of isoliquiritigenin to suppress metastasis was examined in a pulmonary metastasis model of mouse renal cell carcinoma. Isoliquiritigenin significantly reduced pulmonary metastasis, without any weight loss or leukocytopenia. Isoliquiritigenin suppressed in vitro proliferation of carcinoma cells, potentiated nitric oxide production by lipopolysaccharide-stimulated macrophages, and facilitated cytotoxicity of splenic lymphocytes in vitro. These findings suggest activation of macrophages, activation of cytotoxicity of lymphocytes, and direct cytotoxicity as possible mechanisms of metastasis suppression by isoliquiritigenin. In addition, isoliquiritigenin prevented severe leukocytopenia caused by administration of 5-fluorouracil.
  • Keita Kirito, Tomoko Watanabe, Ken-ichi Sawada, Hitoshi Endo, Keiya Ozawa, Norio Komatsu
    The Journal of biological chemistry 277 10 8329 - 37 2002年03月 [査読有り][通常論文]
     
    Thrombopoietin (TPO), an essential factor for megakaryopoiesis and thrombopoiesis, works as a survival factor for megakaryocytic lineage cells. However, little is known about the molecular mechanism in detail. We show here that TPO supports the survival of TPO-dependent leukemia cell line UT-7/TPO and normal megakaryocytic progenitors via the induction of Bcl-xL, an anti-apoptotic member of the Bcl-2 family. We further analyzed the signal transduction pathways required for TPO-induced Bcl-xL gene expression. A reporter assay with various lengths of Bcl-x gene promoter revealed that both Stat- and nuclear factor kappa B-binding sites are prerequisites for TPO-induced promoter activity. Consistent with these results, TPO induced the binding of Stat5 and subunits of nuclear factor kappa B, p50, and c-Rel to the Bcl-x gene promoter. AG490, a specific inhibitor for Jak2, and LY294002, a specific inhibitor for phosphatidylinositol (PI) 3-kinase, reduced the protein level of Bcl-xL in UT-7/TPO cells, accompanied by an increase in the ratio of apoptotic cells. Interestingly, LY294002 enhanced the TPO-induced DNA binding activity of Stat5 without affecting the Jak2 activation and tyrosine phosphorylation of Stat5. Concomitantly, confocal microscopy revealed that LY294002 clearly inhibited the nuclear export of Stat5, suggesting that PI 3-kinase regulates the subcellular localization of Stat5. Taken together, our results suggest that both Jak-Stat and PI 3-kinase activation pathways regulate the TPO-induced survival of megakaryocytic cells via Bcl-xL gene expression. In addition, our data suggest possible cross-talk between these two signaling pathways.
  • Morisada Hayakawa, Eiji Sakashita, Eriko Ueno, Shin-ichi Tominaga, Toshiro Hamamoto, Yasuo Kagawa, Hitoshi Endo
    The Journal of biological chemistry 277 9 6974 - 84 2002年03月 [査読有り][通常論文]
     
    Mitochondrial ATP synthase gamma-subunit (F(1)gamma) pre-mRNA undergoes alternative splicing in a tissue- or cell type-specific manner. Exon 9 of F(1)gamma pre-mRNA is specifically excluded in heart and skeletal muscle tissues and in acid-stimulated human fibrosarcoma HT1080 cells, rhabdomyosarcoma KYM-1 cells, and mouse myoblast C2C12 cells. Recently, we found a purine-rich exonic splicing enhancer (ESE) element on exon 9 via transgenic mice bearing F(1)gamma mutant minigenes and demonstrated that this ESE functions ubiquitously with exception of muscle tissue (Ichida, M., Hakamata, Y., Hayakawa, M., Ueno E., Ikeda, U., Shimada, K., Hamamoto, T., Kagawa, Y., Endo, H. (2000) J. Biol. Chem. 275, 15992-16001). Here, we identified an exonic negative regulatory element responsible for muscle-specific exclusion of exon 9 using both in vitro and in vivo splicing systems. A supplementation assay with nuclear extracts from HeLa cells and acid-stimulated HT1080 cells was performed for an in vitro reaction of muscle-specific alternative splicing of F(1)gamma minigene and revealed that the splicing reaction between exons 8 and 9 was the key step for regulation of muscle-specific exon exclusion. Polypyrimidine tract in intron 8 requires ESE on exon 9 for constitutive splice site selection. Mutation analyses on the F(1)gammaEx8-9 minigene using a supplementation assay demonstrated that the muscle-specific negative regulatory element is positioned in the middle region of exon 9, immediately downstream from ESE. Detailed mutation analyses identified seven nucleotides (5'-AGUUCCA-3') as a negative regulatory element responsible for muscle-specific exon exclusion. This element was shown to cause exon skipping in in vivo splicing systems using acid-stimulated HT1080 cells after transient transfection of several mutant F(1)gammaEx8-9-10 minigenes. These results demonstrated that the 5'-AGUUCCA-3' immediately downstream from ESE is a muscle-specific exonic splicing silencer (MS-ESS) responsible for exclusion of exon 9 in vivo and in vitro.
  • Hayakawa M, Endo H, Hamamoto T, Kagawa Y
    Biochemical and biophysical research communications 251 603 - 608 2 1998年10月 [査読有り][通常論文]
  • Y Yoshida, E Kobayashi, H Endo, T Hamamoto, T Yamanaka, A Fujimura, Y Kagawa
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 234 3 695 - 700 1997年05月 [査読有り][通常論文]
     
    DNA-coated Au particles were accelerated by pressurized He gas to supersonic velocities for introduction of a gene into cells. Experimental and theoretical analyses both revealed a heterogeneous distribution of the particles per shot (1 mg Au = 2.4 x 10(7) particles with 2 mu g [P-32] DNA = 2.5 x 10(11) moles). For introduction of genes into the liver of living rats, the best results were obtained with a newly developed hand-held gene delivery system. The beta-galactosidase gene introduced into rat liver with Au particles by He at 250 psi was expressed (1.2 mu units/mu g protein) in a limited area of the liver surface (8 x 8 mm, depth 0.5 mm). When the same gene gun was used on a monolayer of cultured COS7 cells (about 5 mu m thick), cells were lost in the central area of heavy bombardment. Cell death caused by influx of Ca2+ was prevented by the use of the cytosol-type culture medium. (C) 1997 Academic Press.
  • S OHTA, K TOMINAGA, Y KAGAWA, H TOMURA, H ENDO
    MITOCHONDRIAL ENCEPHALOMYOPATHIES 7 103 - 112 1991年 [査読有り][通常論文]

書籍

  • Heart/muscle-specific alternative splicing in ATP Synthase r-subunit pre-mRNA during muscle development
    ()
    Cardiac Development and Gene Regulation (Ed. by Y. Yazaki) Excerpta Medica. Tokyo. Japan 1995年

MISC

  • 長尾恭光, 冨永薫, 坂下英司, 阿部朋行, 秋本千鶴, 花園豊, 遠藤仁司 日本生化学会大会(Web) 90th ROMBUNNO.3P‐0857 (WEB ONLY) -0857] 2017年12月 [査読無し][通常論文]
  • 松儀 実広, 原田 三男, 菊地 元史, 黒岩 憲二, 淺田 義和, 輿水 崇鏡, 奥田 浩, 遠藤 仁司, 野田 泰子, 岡崎 仁昭 医学教育 45 (Suppl.) 177 2014年07月 [査読無し][通常論文]
  • 手塚修一, 黒岩憲二, 滑川道人, 森田光哉, 冨永薫, 田中惠子, 瀧山嘉久, 遠藤仁司, 中野今治 日本神経学会学術大会プログラム・抄録集 54th 430 2013年 [査読無し][通常論文]
  • M Tanaka, K Kirito, Y Kashii, M Uchida, T Watanabe, H Endo, T Endoh, K Sawada, K Ozawa, N Komatsu JOURNAL OF BIOLOGICAL CHEMISTRY 276 (18) 15082 -15089 2001年05月 [査読無し][通常論文]
     
    FKHRL1, a member of the Forkhead transcription factor family, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt. This molecule is a mammalian homolog of DAF-16, which plays an important role in the longevity of Caenorhabditis elegans. In this study we found that Akt and FKHRL1 proteins were detectable in highly purified normal human megakaryocytes and that these molecules were actually phosphorylated by thrombopoietin (TPO). To clarify the functional role of FKHRL1 in TPO signaling, we established a tetracycline-inducible system in the human TPO-dependent leukemia cell line UT-7/TPO. Induced expression of active FKHRL1 led to cell cycle arrest at G(0)/G(1) phase in this cell line. These results suggest that FKHRL1 plays an important role in the cell cycle of megakaryocytic cells as one of the downstream target molecules of phosphatidylinositol 3-kinase-Akt, presumably mediated through the activation or inactivation of cell cycle-associated gene(s).
  • S Yoshida, D Ioka, H Matsuoka, H Endo, A Ishii MOLECULAR AND BIOCHEMICAL PARASITOLOGY 113 (1) 89 -96 2001年03月 [査読無し][通常論文]
     
    Single-chain immunotoxins are ideal tools to selectively kill infectious agents. In applying this technology to block transmission of malaria parasites in the mosquito vector, we have constructed a single-chain immunotoxin composed of a single-chain antibody fragment (scFv) directed to Pbs21 on the surface of Plasmodium berghei ookinetes linked to a lytic peptide, Shiva-l. The single-chain immunotoxin was expressed in Escherichia coli, and the protein was purified by a NI-NTA column. The single-chain immunotoxin was initially shown to exhibit greater killing properties for P. berghei ookinetes in vitro compared with the scFv or synthetic Shiva-l peptide alone. In an attempt to block malaria transmission by genetically engineered bacteria, recombinant E. coli harboring the single-chain immunotoxin gene were introduced into the mosquito midgut by membrane feeding. The number of infected mosquitoes and their oocyst densities were significantly reduced when the mosquitoes were subsequently allowed to feed on P. berghei-infected mice. These results indicate not only that a single-chain immunotoxin with enhanced parasiticidal activity could form a basis for the development of more effective malaria therapeutic agents, but also that introduction of genetically engineered bacteria into anopheline mosquitoes may offer a practical approach to the regulation of malaria transmission. (C) 2001 Elsevier Science B.V. All rights reserved.
  • T Sugo, O Sekine, C Nakamikawa, H Endo, CL Arocha-Pinango, M Matsuda FIBRINOGEN 936 223 -225 2001年 [査読無し][通常論文]
     
    Steric hindrance by the backbone of extra oligosaccharides at gamma -Asn 308 may cause the repulsive force to widen the junction at the D:D interface, and thus, interfere with the longitudinal elongation and lateral association of protofibrils.
  • Y Inoki, Y Hakamata, T Hamamoto, T Kinouchi, S Yamazaki, Y Kagawa, H Endo BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 278 (1) 183 -191 2000年11月 [査読無し][通常論文]
     
    Colocalization of mitochondria is the first step of intermitochondrial interaction or fusion in a cell. Here, we showed colocalization between exogenous mitochondria and endogenous ones or between exogenous proteoliposomes and endogenous mitochondria in mouse fertilized eggs by confocal laser microscopy. Isolated mitochondria from mouse liver and proteoliposomes containing mitochondrial membrane were directly labeled with red fluorescent aliphatic marker, PKH26, which is incorporated into lipid membrane, and then were microinjected into fertilized mouse eggs. Exogenous mitochondria appeared to be almost colocalized with endogenous mitochondria at the 4- and 8-cell stages, when mitochondria were stained with Rhodamine 123 (green fluorescent marker). On the contrary, when liposomes consisted of soy bean phospholipid were microinjected into the eggs as a control, their localization was different from that of endogenous mitochondria. Next, the submitochondrial particles and proteoliposomes were microinjected. Both the proteoliposomes and the submitochondrial particles appeared to colocalize with endogenous mitochondria at the 4-cell stage. These results suggest the existence of a factor that makes liposomes colocalize with mitochondria. Such a proteoliposome would be useful for the development of mitochondrial gene transfer techniques. (C) 2000 Academic Press.
  • Y Kagawa, T Hamamoto, H Endo JOURNAL OF BIOENERGETICS AND BIOMEMBRANES 32 (5) 471 -484 2000年10月 [査読無し][通常論文]
     
    ATP synthase (FoF1) consists of F-1 (ATP-driven motor) and F-o (H+-driven motor). Fl is a complex of alpha (3)beta (3)gamma delta epsilon subunits, and gamma is the rotating cam in alpha (3)beta (3) Thermophilic F-1 (TF1) is exceptional in that it can be crystallized as a beta monomer and an alpha (3)beta (3) oligomer, and it is sufficiently stable to allow alpha beta refolding and reassembly of hybrid complexes containing 1, 2, and 3 modified ct or beta. The nucleotide dependent open-close conversion of conformation is an inherent property of an isolated beta and energy and signals are transferred through alpha/beta interfaces. The catalytic and noncatalytic interfaces of both mitochondrial F-1 (MFI) and TF1 were analyzed by an atom search within the limits of 0.40 nm across the alpha beta interfaces. Seven (plus thermophilic loop in TF1) contact areas are located at both the catalytic and noncatalytic interfaces on the open beta form. The number of contact areas on closed beta increased to 11 and 9, respectively, in the catalytic and noncatalytic interfaces. The interfaces in the barrel domain are immobile. The torsional elastic strain applied through the mobile areas is concentrated in hinge residues and the P-loop in beta. The notion of elastic energy in FoF1 has been revised. X-ray crystallography of F-1 is a static snap shot of one state and the elastic hypotheses are still inconsistent with the structure, dyamics, and kinetics of FoF1, The domain motion and elastic energy in FoF1 will be elucidated by time-resolved crystallography.
  • Y Inoki, T Miura, T Kajimoto, M Kawase, Y Kawase, Y Yoshida, S Tsuji, T Kinouchi, H Endo, Y Kagawa, T Hamamoto BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 276 (3) 1210 -1216 2000年10月 [査読無し][通常論文]
     
    Ganglioside GD3 induced the release of cytochrome c from isolated rat liver mitochondria. This process was completely prevented by cyclosporin A and partially prevented by a cysteine protease inhibitor, n-acetyl-leu-leu-norleucinal. Cyclosporin A is a potent inhibitor of the permeability transition pore, whereas n-acetyl-leu-leu-norleucinal has no effect on this pore. These results indicate that the release of cytochrome c from mitochondria requires both the opening of the permeability transition pore and a cysteine protease inhibitor-sensitive mechanism. Gangliosides GD1a, GD1b, GT1b, and GQ1b along with the synthetic GD3 mimetics TMS-42 and CI-22, which are glycerophospholipids carrying a disialo residue, also induced cytochrome c release. In contrast, gangliosides GM1, GM2, and GM3 did not induce cytochrome c release. These results indicate that two sialo residues must play an important role in the induction of cytochrome c release by gangliosides. (C) 2000 Academic Press.
  • Hepatic Mononucler Cells(HMCs)におけるGreen fluorescent protein(GFP)発現 GFP-transgenic Rat vs Mouse
    中村 正彦, 高野 靖悟, 岩井 重富, 袴田 陽二, 遠藤 仁司, 小林 英司, 平林 真澄, 高橋 利一, 上田 正次 移植 35 (総会臨時) 209 -209 2000年09月 [査読無し][通常論文]
  • M Ichida, Y Hakamata, M Hayakawa, E Ueno, U Ikeda, K Shimada, T Hamamoto, Y Kagawa, H Endo JOURNAL OF BIOLOGICAL CHEMISTRY 275 (21) 15992 -16001 2000年05月 [査読無し][通常論文]
     
    Muscle-specific isoform of the mitochondrial ATP synthase gamma subunit (F(1)gamma) was generated by alternative splicing, and exon 9 of the gene was found to be lacking particularly in skeletal muscle and heart tissue. Recently, we reported that alternative splicing of exon 9 was induced by low serum or acidic media in mouse myoblasts, and that this splicing required de novo protein synthesis of a negative regulatory factor (Ichida, M., Endo, H., Ikeda, U., Matsuda, C., Ueno, E., Shimada, K., and Kagawa, Y. (1998) J. Biol. Chem. 273, 8492-8501; Hayakawa, M, Endo, H., Hamamoto, T., and Kagawa, Y. (1998) Biochem Biophys. Res. Commun. 251, 603-608). In the present report, we identified a cis-acting element on the muscle-specific alternatively spliced exon of F(1)gamma gene by an in vivo splicing system using cultured cells and transgenic mice. We constructed a F(1)gamma wild-type minigene, containing the full-length gene from exon 8 to exon 10, and two mutants; one mutant involved a pyrimidine-rich substitution on exon 9, whereas the other was a purine-rich substitution, abbreviated as F(1)gamma Pudel and F(1)gamma Pu-rich mutants, respectively. Based on an in vivo splicing assay using low serum- or acid-stimulated splicing induction system in mouse myoblasts, Pudel mutation inhibited exon inclusion, indicating that a Pu-del mutation would disrupt an exonic splicing enhancer. On the other hand, the Pu-rich mutation blocked muscle-specific exon exclusion following both inductions. Next, we produced transgenic mice bearing both mutant minigenes and analyzed their splicing patterns in tissues. Based on an analysis of F(1)gamma Pu-del mini-gene transgenic mice, the purine nucleotide of this element was shown to be necessary for exon inclusion in non-muscle tissue. In contrast, analysis of F(1)gamma Pu-rich minigene mice revealed that the F(1)gamma Pu-rich mutant exon had been excluded from heart and skeletal muscle of these transgenic mice, despite the fact mutation of the exon inhibited muscle-specific exon exclusion in myotubes of early embryonic stage. These results suggested that the splicing regulatory mechanism underlying F(1)gamma pre-mRNA differed between myotubes and myofibers during myogenesis and cardiogenesis.
  • Y Kagawa, SH Cha, K Hasegawa, T Hamamoto, H Endo BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 266 (3) 662 -676 1999年12月 [査読無し][通常論文]
     
    Recent advances in bioenergetics consist of discoveries related to rotational coupling in ATP synthase (FoF(1)), uncoupling proteins (UGP), reactive oxygen species (ROS) and mitochondrial DNA (mtDNA). As shown in cloned sheep, mammalian genomes are composed of both nuclear DNA (nDNA) and maternal mtDNA. Oxidative phosphorylation (oxphos) varies greatly depending on cellular activities, and is regulated by both gene expression and the electrochemical potential difference of H+ (Delta mu H+) The expression of both mtDNA (by mtTFA) and nDNA for oxphos and UCP (by NRFs, etc.) is coordinated by a factor called PGC-1. The Delta mu H+ rotates an axis in FoF(1) that is regulated by inhibitors and ATP-sensitive K+-channels.
  • Y Kagawa, M Hayakawa, SH Cha, H Endo FASEB JOURNAL 13 (7) A1553 -A1553 1999年04月 [査読無し][通常論文]
  • S Yamagami, T Tsuru, T Ohkawa, H Endo, M Isobe TRANSPLANTATION 67 (4) 600 -604 1999年02月 [査読無し][通常論文]
     
    Background. Anti-alpha beta T cell receptor monoclonal antibody (R73) has been reported to be a potent immunosuppressant. The suppressive effects of this antibody on allograft rejection after corneal transplantation are unknown. Methods. Orthotopic rat penetrating keratoplasty was performed using Lewis rats as recipients and Brown Norway and Fisher rats as donors. The treated groups received R73 intraperitoneally until day 12 after the transplantation. In grafted rats with or without R73 treatment, cytokine expression of the aqueous humor, corneal-infiltrating cells, draining lymph nodes, and splenocytes was determined. Delayed-type hypersensitivity (DTH) responses were compared. Results. All allografts in the untreated controls of Fisher-to-Lewis or BN-to-Lewis rat combinations were rejected within 14 days. In contrast, indefinite survival rates of the postoperative R73-treated group increased to 86% in the Fisher-to-Lewis and 23% in the Brown Norway-to-Lewis combinations, respectively. Interferon-gamma, interleukin (IL)-2 (T helper [Th]1), and IL-10 (Th2), but not IL-4 (Th2), expression of the eye and DTH responses in the control group were suppressed in the R73-treated group. Both IL-2 and IL-10 expression after mixed lymphocyte culture in the R73-treated group were significantly lower than those of the naive and untreated control group. Conclusions. alpha beta T cell receptor-targeted therapy prevents allograft rejection in rat corneal transplantation as evidenced by suppression of DTH responses. The cytokine profile after R73 treatment was characterized by low interferon-gamma, IL-2, and IL-10, and high IL-4 expression.
  • S Yamagami, H Kawashima, H Endo, T Tsuru, H Shibui, Y Kagawa, J Hori, H Yamagami, M Isobe TRANSPLANTATION 66 (11) 1504 -1510 1998年12月 [査読無し][通常論文]
     
    Background. Cytokine profile is a key in understanding the mechanisms of allograft rejection. Cytokine expression in the aqueous humor and the correlation between the aqueous humor cells and corneal infiltrating cells are not fully understood in corneal transplantation. Methods. Orthotopic mouse corneal transplantation was performed using BALB/c (H2(d)) mice as recipients, and C3H/He (H2(k)) and BALB/c mice as donors for allografts and isografts, respectively. Immunocytochemistry was performed on aqueous humor cells. Corneal graft was studied immunohistochemically. Cytokine gene expressions of the cells infiltrating the aqueous hunter and corneal grafts were determined by the semiquantitative reverse transcription and polymerase chain reaction method. Results Interferon-gamma, interleukin (IL)-2, IL-4, and IL-10 were detected in the cells infiltrating the aqueous humor and corneal grafts at both the protein and gene expression levels. T helper 1 (Th1) cytokine expressions at the protein level, however, were consistently predominant in the rejected allografts compared to those of Th2 cytokines. The cytokine and surface marker profiles of the cells in the aqueous humor corresponded well to those of the cells infiltrating the corneal grafts. Cytokine protein and mRNA expression levels in the aqueous humor decreased rapidly. Conclusions. Allorejection in corneal transplantation is Th1 cytokine-predominant. Infiltrating cells do not express Th2 cytokine so much in allograft rejection,as compared with Th1 cytokine, The cell infiltration patterns of the aqueous humor were well correlated with those of the cornea.
  • Y Kagawa, T Hamamoto, H Endo, M Ichida, H Shibui, M Hayakawa BIOSCIENCE REPORTS 17 (2) 115 -146 1997年04月 [査読無し][通常論文]
     
    The reaction of ATP synthase (F0F1) is the final step in oxidative phosphorylation (OXPHOS). Although OXPHOS has been studied extensively in bacteria, no tissue-specific functions nor bioenergetic disease, such as mitochondrial encephalomyopathy and aging occur in these organisms. Recent developments of the Human Genome Project will become an important factor in the study of mammalian bioenergetics. To elucidate the physiological roles of human F0F1, genes encoding the subunits of F0F1 were sequenced, and their expression in human cells was analyzed. The following results were obtained: A. The roles of the residues in F0F1 are not only to transform the energy of the electrochemical potential (Delta mu H+) across the membrane, but also to respond rapidly to the changes in the energy demand by regulating the intramolecular rotation of F0F1 with the Delta mu H+ and the inhibitors of the ATPase. B. The roles of the control regions of the F0F1 genes, are to coordinate both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) depending on the energy demand of the cells, especially in muscle. C. The cause of the age-dependent decline of ATP synthesis has been attributed to the accumulation of mutations in mtDNA. However. the involvement of nDNA in the decline is also important because of telomere shortening in somatic cells, and age-dependent mtDNA expression analyzed with rho degrees cells (cells without mtDNA).
  • 筋特異的選択的RNAスプライシングの調節機構
    最新医学 51 1689 -1696 1996年 [査読無し][通常論文]
  • Y KAGAWA, T OHTA, Y ABE, H ENDO, M YOHDA, N KATO, ENDO, I, T HAMAMOTO, M ICHIDA, T HOAKI, T MARUYAMA BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 214 (2) 730 -736 1995年09月 [査読無し][通常論文]
     
    To elucidate thermoresistance, a gene of a hyperthermophilic heat shock protein (HHSP) was isolated from the hyperthermophile Desulfurococcus strain SY which grows at 95 degrees C. The molecular weight of HHSP deduced from the open reading frame was 59,137 (545 amino acid residues). Sequence alignments of peptides reveal similarities (evolutionary distances) to the alpha (0.279) and beta (0.296) subunits of thermosome, TF55 (0.343) and human t-complex polypeptide 1. The structure of a thermophilic heat shock protein TGroEL (Tamada et al. (1991) Biochem. Biophys. Res. Commun. 179, 565) was quite different from that of HHSP. TGroEL and HSP60 have sequences identical to HHSP at its equatorial domain, while those identical to the alpha subunit of F-type ATPase are at its apical domain. (C) 1995 Academic Press, Inc.
  • S AKIYAMA, H ENDO, N INOHARA, S OHTA, Y KAGAWA BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION 1219 (1) 129 -140 1994年09月 [査読無し][通常論文]
     
    The gene structure of the human ATP synthase alpha subunit (hATP1) was determined by cloning and sequencing. This gene is approximately 14 kbp in length and contains 12 exons interrupted by 11 introns. Mapping of the clones of hATP1 and Southern blot analysis of the genomic gene showed that there were a single copy of bona fide hATP1 gene and two pseudogenes. Primer extension and S1 mapping analysis showed the presence of multiple transcription initiation sites of the hATP1 gene. No TATA box or CAAT box was found near the transcription initiation sites. Comparison with the bovine gene showed that the 5'-flanking region of the hATP1 gene has an unconserved guanine-cytosine (GC) rich region, including several binding motifs of transcriptional factors, such as Sp1, AP-2, and GCF. By functional assay of gene expression, the basal promoter activity was located near the GC rich region. Comparison of the 5'-upstream region of the hATP1 gene with those of the genes for bovine ATP synthase alpha, human beta, and human gamma subunits indicated three common sequences, suggesting that putative cis-elements coordinate the expressions of the three subunit genes for the ATP synthase. The enhancer activities derived from the 5'-deletion mutants of a hATP1-CAT chimeric gene were different in cell lines from four different human tissues, suggesting the existence of cell type-specific gene regulation.
  • C MATSUDA, E MUNEYUKI, H ENDO, M YOSHIDA, Y KAGAWA BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 200 (2) 671 -678 1994年04月 [査読無し][通常論文]
     
    The kinetics of heart and liver mitochondrial ATPase (FoF(1)) were examined using submitochondrial particles (SMPs) purified from the two tissues to obtain information on the role of gamma subunit isoforms. The F-1 portion is mainly composed of the catalytic, common alpha beta subunits and tissue-specific gamma subunits. In contrast to the previous reports on the kinetics and crystallography of various F-1's, the Vmax and Km of the two isoforms of FoF(1) were identical although the SMPs were prepared from different tissues. Moreover sodium azide inhibited the two equally. The ATPase activity of liver SMP showed slightly steeper pH-dependency than that of heart SMP but the pH optima of the two were the same (pH 8). (C) 1994 Academic Press, Inc.
  • H ENDO, C MATSUDA, Y KAGAWA JOURNAL OF BIOLOGICAL CHEMISTRY 269 (17) 12488 -12493 1994年04月 [査読無し][通常論文]
     
    Tissue-specific isoforms of the mitochondrial ATP synthase gamma-subunit are generated by alternative splicing, and the heart/skeletal muscle specific transcript lacks exon 9 in a cassette fashion (Matsuda, C., Endo, H., Hirata, H., Morosawa, H., Nakanishi, M., and Kagawa, Y. (1993) FEBS Lett. 325, 281-284; and Matsuda, C., Endo, H., Ohta, S., and Kagawa, Y. (1993) J. Biol. Chem. 268, 24950-24958). Here, we show that the endogenous heart-type mRNA is cell-specifically induced by the extracellular pH value in the HT1080 (human fibrosarcoma) and KYM-1 (human rhabdomyosarcoma) cell lines. In these cells, a low extracellular pH value induced exclusion of exon 9, and this induction was inhibited by cycloheximide treatment. In contrast, a high extracellular pH value resulted in mRNA transcription of the liver type, including exon 9, and did not require de novo protein synthesis. These results suggest that alternative splicing in the gamma-subunit pre-mRNA is regulated by on-off switching of protein synthesis of a trans-acting factor involved in this exon-excluding step. The signal of low pH value was blocked by the protein kinase inhibitor H-7 or Calphostin C (protein kinase C inhibitor), indicating the involvement of protein kinase C in the alternative splicing. This is a good model system for studies on the induction mechanism of alternative splicing in cultured mammalian cells, in which intracellular factors play a pivotal role for the exon-excluding step in the tissue-specific alternative splicing mechanism.
  • C MATSUDA, H ENDO, S OHTA, Y KAGAWA JOURNAL OF BIOLOGICAL CHEMISTRY 268 (33) 24950 -24958 1993年11月 [査読無し][通常論文]
     
    We completely sequenced the human gene for ATP synthase gamma-subunit, which was approximately 23 kilobases long and was composed of 10 exons. Exons 1 and 2 encoded the N-terminal presequence required for mitochondrial import, while exons 9 and 10 encoded the C-terminal portions of mature protein. Enzymatic amplification of human heart and liver cDNAs using the polymerase chain reaction revealed two mRNA transcripts that were predicted to encode two 30-kDa isoforms of the gamma-subunit, which differed by the addition of a single amino acid (Asp273) at the C terminus of the liver type isoform. These two mRNA transcripts of the heart (H) type and liver (L) type were generated by alternative splicing of an exon. The same alternative splicing event was observed in bovine tissue. In human tissues, the H type mRNA devoid of exon 9 was expressed specifically in the heart and skeletal muscle, which require rapid energy supply. The L type mRNA was expressed in the brain, liver, kidney etc. Both transcripts were expressed in the skin, intestine, stomach, and aorta. This tissue specificity of transcript heterogeneity suggests the distinct functional or regulatory roles of the gamma-subunit isoforms in the catalysis of ATP synthase. This is the first report on tissue-specific isoforms generated by alternative splicing in an energy transducing mitochondrial protein.
  • C MATSUDA, H ENDO, H HIRATA, H MOROSAWA, M NAKANISHI, Y KAGAWA FEBS LETTERS 325 (3) 281 -284 1993年07月 [査読無し][通常論文]
     
    Tissue-specific isoforms of the gamma-subunit of the bovine F(o)F1-ATP synthase were identified for the first time. The two isoforms, heart and liver type, were generated by alternative splicing, the liver-type RNA transcript containing a 37-nucleotide sequence as a cassette exon. Protein sequencing of the C-terminal fragments of the two isoforms of the F1gamma-subunit indicated that the liver-type isoform had an additional aspartate residue at the C terminus, not present in the heart type one.
  • Y MATSUBARA, H IKEDA, H ENDO, K NARISAWA NUCLEIC ACIDS RESEARCH 20 (8) 1998 -1998 1992年04月 [査読無し][通常論文]

共同研究・競争的資金等の研究課題

  • 悪性神経膠腫における脂肪酸代謝を標的とした新規治療戦略の確立
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 遠藤 仁司
  • 脂肪酸によるアロ反応性T細胞のヒストン修飾及びエフェクター活性の制御
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 佐藤 一也, 森田 薫, 神田 善伸, 遠藤 仁司
  • ミトコンドリアの適合性の機構解析と治療法開発に関する基盤研究
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 遠藤 仁司
     
    ミトコンドリアDNA(mtDNA)は正常の個体についても個々に多型が存在する。近年、生殖医療においてミトコンドリア遺伝子異常症の受精卵に対して前核置換法を用いた遺伝子治療法が実施されており、その過程で第三者のミトコンドリアとほぼ置換されること、一部では二者のmtDNAが混在するヘテロプラスミーの状態であることが報告されている。電子伝達系を構成する複合体はmtDNA由来のタンパク質と核由来のタンパク質から構成されているが、mtDNAと核遺伝子との適合性に関する研究は極めて少ない。そこで、本研究では①同一核型に亜種mtDNAを置換したコンプラスティックマウス(conplastic mouse)の独自作製によって表現形が変化するマウスモデルを確立すること、②これらのコンプラスティックマウスから前核置換法を用いてmtDNAが混在するヘテロプラスミーのES細胞を樹立すること、③TALEN等のヌクレアーゼのミトコンドリア標的への最適化を試み、特定のmtDNAを除去する治療基盤を確立することを目的としている。本年度までは①ミトコンドリアと核の適合性が糖尿病の症状の増悪に関することを示し、核-ミトコンドリアの不適合性が、病態に影響を与えることを初めて明らかにした。②B6マウスと亜種マウスミトコンドリアのヘテロプラスミーのES細胞を作製した。ミトコンドリアを標的する複数のミトコンドリアタンパク質を連結したTALEN改変ベクターを作製して、ミトコンドリア遺伝子治療の基盤実験を行なったところ、一方のmtDNAを特異的に切断し除去するゲノム編集の効果が認められた。特異性や除去効率などを詳細な検討など、来年度はより詳細な検討を加え、論文化を実施する予定である。
  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2010年 -2012年 
    代表者 : 遠藤 仁司
     
    ミトコンドリア内膜に存在するプロヒビチン2(PHB2)はエストロゲン(E2)依存的に核に移行する。一方、E2の減少は閉経早期の閉経後肥満の原因となる。本研究では、E2によって核へ移行したPHB2が、PGC-1αを介してPPARγの転写活性を抑制することにより脂肪分化を負に制御することを示した。E2は脂肪前駆細胞において内在性のPHB2を核に移行させ、PHB2はPGC-1αに直接結合してPPARγの転写活性を抑制する。ミトコンドリア移行シグナルを欠失したPHB2変異体(PHB2C)を脂肪前駆細胞に発現させる
  • 神経性RNA結合タンパク質に着目した吸入麻酔薬の中枢性麻酔薬作用の解析
    日本学術振興会:科学研究費助成事業 萌芽研究
    研究期間 : 2006年 -2007年 
    代表者 : 佐藤 正章, 遠藤 仁司, 笠島 克己, 蛭田 昌宏
     
    本研究の目的は,神経性RNA結合タンパク質の機能に着目し,吸入麻酔薬の機能を解析することであり,その概要は麻酔薬が発現に影響する神経性RNA結合タンパク質を同定して,それらに調節されるRNAを網羅的に解析することであった. 本研究では,吸入麻酔薬セボフルランの投与による代表的な神経性RNA結合タンパク質(Musashi,Hu,NOVA1,Drb1)のRNAレベルおよびタンパク質レベルで発現量の変化を成人マウスおよび仔胎ラットで確認した.成人マウスでHuタンパク質の発現量に軽度の変化を確認できた.RNAの発現量の変化の確認に難渋し,結果的に有意な変化を確認するまでには至らなかった. うまくいかなかった理由として脳組織以外の組織片や血液の混入が一因と考えた.そこで培養細胞を用いた実験系で成体のデータの再現を試みた.しかし当研究室では培養細胞に吸入麻酔薬を使用する実験システムがなく,まずそれらを構築に着手した.現在,それらの実験系を用いて,いままで得られた結果の再現性を確認している. 本研究は,吸入麻酔薬の作用機序の解明をいままでに着手されていない側面で行っている.萌芽的ではあるが,吸入麻酔薬の中枢性麻酔作用機序の解明のきっかけをうる研究である.残りの試薬等を用いて現在も実験を行っている.
  • ヒトミトコンドリア融合因子を用いたミトコンドリア標的リポソームの研究
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2002年 -2004年 
    代表者 : 遠藤 仁司
     
    細胞内小器官であるミトコンドリアには核と異なる独自の遺伝子が存在する。ミトコンドリアDNA異常に基づくミトコンドリア病の根本的治療には、従来の核遺伝子への遺伝子治療法とは異なる、ミトコンドリアを標的とした新たな遺伝子導入法の開発が必要である。本研究では、ミトコンドリア融合因子を有効構成成分とするミトコンドリアを特異的に標的する新規なリポソームベクターの基礎的開発を目的としている。このようなベクターを開発することによって、正常なミトコンドリアDNAだけでなく、リボザイムなどの任意の外来性遺伝子やミトコンドリアの機能を調節する薬剤などをミトコンドリア内に導入することが可能となり、将来的にはミトコンドリアの機能が関わる多くの疾患を対象とした新たなドラッグデリバリーシステムとして利用が可能になると考えられる。 我々はミトコンドリア融合因子であるヒトFzoタンパク質を単離し、膜融合活性の必須ドメインを同定した。またこのドメインの組換えタンパク質を作製し、試験管内で作成したプロテオリポソームを用いて膜融合活性を証明した。さらにミトコンドリア融合装置のひとつであるOPA1タンパク質を検討し、ヒト細胞にアイソフォームが存在しミトコンドリア内の局在が異なることを明らかにした。本因子はミトコンドリア融合装置の補助因子のひとつである。このように、ミトコンドリア融合装置の全容を明らかにすることはミトコンドリアを標的するリポソームベクターのミトコンドリア融合効率を高める上で重要である。今後はミトコンドリアDNAを抱埋した本プロテオリポソームを細胞にマイクロインジェクションすることにより、実際に細胞内でミトコンドリアを標的するかを検討することが重要である。本研究はミトコンドリア病の遺伝子治療の基礎的技術を確立する第一歩である。
  • 血管修復の分子機序:血管壁再構築における血栓の役割
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2002年 -2003年 
    代表者 : 諏合 輝子, 窓岩 清治, 遠藤 仁司
     
    血栓は血管損傷部位を血流などの外部環境から保護するという役割以外に、各種炎症性細胞群との相互作用の場を提供して、損傷部位の組織再構築にも深く関わっていると考えられる。この仮定を証明するためには、長期に血管内皮細胞(EC)が生育できる培養環境を整える必要があり、まずこれに着手した。そこでこれまで疑似血管壁として検討してきたコラーゲンゲルに、様々な因子を混在させ、この上にECの培養を行った。その結果、ヘパリン硫酸を含むペプシン非処理Type IとIVのcollagenからなるコラーゲンゲル上に、低seedinddensity(1~4 x104)でECを蒔くと10日でconfluentに達し、以後週1度の培養液交換でconfluencyを保ったまま2ヶ月以上生存し、長期培養が可能となった。この培養条件下で、ECに機械的および酵素処理による細胞損傷を加え、その回復過程を走査電子顕微鏡で観察した結果、ECは損傷部位や自身の周囲の環境を"認知"して、形態を保持あるいは変化させ、一定方向に配向して分裂・増殖を行うことが判明した(投稿準備中)。現在、この"認知"現象の物質的裏付けを遺伝子工学的手法を用いてどのような遺伝子が関与しているのか検討中である。また、この認知を行う細胞内構造体を電子顕微鏡観察で追求している。一方、こうした損傷モデル系を用いて、損傷細胞上にfibrinが存在すると細胞の損傷回復が早まる傾向が認められたため、現在、種々のfibrin構造を形成する異常フィブリノゲンを利用して、どのようなfibrin構造が、損傷からの回復に有効であるか検討中である。 こうした研究と平行して、当教室でこれまで研究してきた30数種の異常Fbgの電子顕微鏡によるFbn立体構造の解析などを行い、出血や血栓傾向などの臨床症状を示す異常フィブリノゲン(Fbg)患者の異常分子は、すべて正常Fbnとは構造も性質もかなり異なるFbnを形成することが判明した。また、強いプラスミン抵抗性を持った細いFbn線維からなるメッシュ状の異常Fbnを形成する異常分子を持つ患者は、血栓傾向が高く血管修復や創傷治癒不全を示すことが明かとなった。しかし、細い線維からなるメッシュ構造を形成しても、線維の強度が低い場合やプラスミン抵抗性を持たないFbn線維である場合は、創傷治癒遅延を引き起こさないことから、プラスミンによるFbn網の分解が、血管修復に必須であることが明かとなった。(投稿準備中)
  • 筋分化システムに働くスプライシング因子の同定とその調節メカニズムの研究
    日本学術振興会:科学研究費助成事業 特定領域研究
    研究期間 : 2002年 -2002年 
    代表者 : 遠藤 仁司, 坂下 英司
     
    筋分化などの臓器の最終分化過程における劇的な細胞構造の変換には、一般に多数の分子種を効率的に変換する制御機構が必要である。本研究の目的は、筋分化システムにおける選択的スプライシングを調節する具体的なスプライシング調節因子を単離しその機能を解析することである。我々はこれまで、ATP合成酵素γサブユニット遺伝子の筋特異的な選択的スプライシングを再現するin vitro系を構築し、筋特異的なエキソン除外に働くシス配列(muscle-specific exonic splicing silencer)を同定した。 本年度は、ATP合成酵素γサブユニットmRNA前駆体を基質としてin vitro splicing系やシスエレメント(MS-ESS)をプローブとしたノースウエスタン法、アフィニティカラム法などを用いて、HeLa細胞核抽出液から構成的スプライシング因子を、また筋特異的スプライシング誘導を示す酸性刺激後のHT1080細胞核抽出液から選択的スプライシング因子を精製した。さらに、これらのサンプルを二次元電気泳動法や質量分析法(TOF-MS Finger Print法)によりタンパク質の一次配列を決定し、スプライシング調節の候補トランス因子を複数同定した。それらはいずれもRNA結合モチーフを持ち、RNA結合蛋白質と予測される。現在、これらのcDNAのクローン化を試みており、これらのスプライシング調節活性をin vivo splicing系にて検定する予定である。一部cDNAの結果では、シスエレメント依存的に働く因子群と、非依存的に働く因子群とに分類できている。今後、得られたタンパク質を網羅的に解析し、筋特異的なスプライシング制御の全体像の検討を試みる予定である。
  • 無精子症の発現に関する新しい候補遺伝子の解明
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2000年 -2002年 
    代表者 : 出居 貞義, 柴原 浩章, 遠藤 仁司, 小池 俊光
     
    射出精液に精子がいない無精子症の患者の中で不妊症治療の診断と治療をかねた精製生検にて、精巣の造精機能障害がMaturation arrestまたはSeltolli cell onlyと病理診断された患者からインフォームドコンセントを得て、永久病理組織標本50検体より、固定超薄切片を取り出した。その固定超薄切片をDNAの鋳型にして、我々が新たに改良した[無精子症の責任遺伝子として知られているDAZ(Deleted in Azoospermia)やYRRM(Y chromosome ribonucleic acid recognition motif)のアミノ酸配列にはRNA-binding domainが存在し、種によらずRNP-1,RNP-2の領域も良く保存され、この領域は無精子症と深い関りがあることを示唆している。この領域を増幅できるように考案した]degenerate Primerを用いたRT-PCR法にて睾丸組織特異的に発現しているmRNAsのcDNAsを作成した。この得られたcDNAs遺伝子を電気泳動して正常精巣組織では発現していない遺伝子や正常では発現しているが患者検体においては発現していない遺伝子を選び出して(differential display法)無精子症患者に特異的に欠失または変異している可能性のある候補遺伝子が960個ほど得られた。得られた遺伝子を大陽菌を用いてsubcloningし、現在約300程その遺伝子配列を決めた。コドンに矛盾が無くRRM/RNP Consensusを有する遺伝子をその中より選別した。求めた遺伝子配列をインターネット上のGenome Netの中のBLASTにてホモロジー解析し、どのくらいの変異が起きているか、また現在までに分かっており精巣特異的に発現している正常などの遺伝子に最も近いか、解析の最中です。
  • 筋特異的選択的RNAスプライシングの分子メカニズムの研究
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 1999年 -2001年 
    代表者 : 遠藤 仁司
     
    真核生物ではミトコンドリアのATP合成酵素がほとんどのATPを供給している。本酵素ではγサブユニットの筋特異的なアイソフォームが選択的スプライシングにて生成される。本研究ではヒトATP合成酵素γサブユニットの筋特異的スプライシング調節のシス領域およびトランス因子を同定し、筋特異的な選択的スプライシングの分子メカニズムを解明することを目的とした。申請者は培養細胞を用いて筋特異的なスプライシングの誘導系を確立し、これを用いてin vitro系で筋特異的なスプライシング反応を構築した。この系を用いて、本遺伝子のカセット型エキソン上に構成的なスプライシングの条件で働くスプライシングエンハンサー配列(Exonic Splicing Enhancer, ESE)、筋特異的に働くエキソン上のスプライシングサイレンサー(Muscle-Specific Exonic Splicing Silencer, MS-ESS)配列を明らかにした。これらの各配列に結合するトランス因子をノースウエスタン法やUVクロスリンク法にて検討したところ、各々約48kDaと42kDaのRNA結合蛋白が検出された。現在精製中である。次に、in vivoの筋分化過程におけるスプライシング調節過程を検討した。ESEやMS-ESSのシス配列に変異を導入したミニ遺伝子を用いてトランスジェニックマウスを作成し、各臓器におけるスプライシングパターンを検討したところ、筋管細胞と成熟筋繊維ではスプライシング調節メカニズムが異なることが示された。今後、筋特異的なスプライシング調節因子を同定することが、これらの分子メカニズムを詳細に解明できると思われる。
  • 核孔を介したmRNA核外輸送に関与するRNA結合蛋白の解析
    日本学術振興会:科学研究費助成事業 重点領域研究
    研究期間 : 1996年 -1996年 
    代表者 : 遠藤 仁司, 香川 靖雄
     
    我々はこれまで、ATP合成酵素γサブユニット遺伝子が筋特異的にエクソンを除外するカセット型の選択的RNAスプライシングをうけて、アイソフォームを形成することを示した。我々は、この選択的スプライシングの調節を司る因子を明らかにするために、degenerate primerを用いたポリメラーゼ連鎖反応による、新規な筋特異的なRNA結合蛋白のクローニングを目的とした。RRM(RNA recognition motif)型RNA結合ドメインの保存配列から作成したdegenerate primerを用いて、筋特異的なスプライシングを誘導する前後の細胞からRNAを抽出し、デファレンシャルディスプレイ変法を用いて誘導後に特異的に発現する新規なRRM型RNA結合蛋白のcDNA断片を得た。この遺伝子断片をプローブとして、ヒト骨格筋cDNAライブラリーを用いて、プラークハイブリダイゼーション法にてcDNAのクローン化を試みた。その結果、少なくとも2種類のcDNAを得た。いずれもcDNA前半はポリA結合蛋白質と非常によく似ており、そのRNA結合部位に関してはアミノ酸レベルで94%の一致、98%のホモロジーが存在した。一方のcDNAは全長がポリA結合蛋白と似ていた。このcDNAは、筋組織で優位に発現していた。もう一方のcDNAは、後半がRan結合ドメインをコードしていた。RACE法にて全長cDNAをクローン化したところ、4個のRan結合ドメインと12個のXFXFGモチーフを有しており、核孔に存在する蛋白と考えられた。この後半部分の遺伝子はすでに報告されてるRanBP2/Nup358と一致していた。RNA結合ドメインとRan結合ドメインの間の遺伝子断片を用いてRNAブロット法にて発現量を検討したところ、約10kbのRanBP2の遺伝子の下方、約8kbの位置に発現量の少ないバンドが認められた。その構造から、このcDNAは核孔を介したRNA核外輸送に関与するのではないかと考えられた。このように、RNA結合領域とRan結合領域、およびXFXFGモチーフを持つ遺伝子は、酵母においても報告がないため、慎重にこの遺伝子の存在を検討中である。
  • ATP合成酵素RNA前駆体における筋特異的スプライシング調節因子の研究
    日本学術振興会:科学研究費助成事業 重点領域研究
    研究期間 : 1995年 -1995年 
    代表者 : 遠藤 仁司, 香川 靖雄
     
    申請者は本研究にて筋特異的なエクソン選択の制御機構を解明するために、ミトコンドリアATP合成酵素γサブユニット遺伝子を用いて、心筋・骨格筋特異的な選択的スプライシングを制御するシス領域およびトランス因子を解明することを目的とした。 1)選択的スプライシングを制御するシス領域の解析:申請者はヒト、ウシ、マウスの本遺伝子における筋特異的なアイソフォームの存在を初めて示し、それが選択的RNAスプライシングで生成されることを示した。それぞれのゲノム遺伝子の塩基配列を比較すると、エクソン選択を受ける第9エクソンの約400塩基上流に保存領域が存在した。またミニ遺伝子を作成し、このミニ遺伝子はエクソン選択の調節に必要なシス領域をすべて含むことを証明した。今後、保存領域に塩基変異を導入するなどして、調節領域を決定する予定である。 2)無細胞系を用いた筋特異的スプライシング調節因子の解析:無細胞系にてスプライシング調節因子を解析するために、まず核抽出液を精製した。核抽出液はエクソンの陰性選択が行われない誘導前の細胞および低pHによりエクソン除外が行われた誘導後の細胞の各々から抽出した。保存配列を含むエクソン9の周辺領域に相当するリボプローブを用い、各々の核抽出液にてゲルシフトおよびUVクロスリンクアッセイを行った結果、誘導前の核抽出液では分子量約60kD及び100kDの蛋白がRNAに結合するが、誘導後の核抽出液をそれに加えると二つの蛋白の結合が阻害された。 3)既知スプライシング因子の関与に対する検討:既知のスプライシング因子であるSF2/ASF, U2AF, PTB, SR蛋白などに対する抗体を用いてウエスタンブロット法にて、各々の核抽出液を検討した。その結果、蛋白量に大きな変化は認められなかったので、これらのスプライシング因子は、本遺伝子の選択的スプライシングの調節蛋白ではないと考えられた。 4)筋特異的RNA結合蛋白の部分cDNAクローニング:またdegenerate primerを用いたPCRにて筋特異的なRRM型RNA結合蛋白の遺伝子断片をクローニングした。現在、全長cDNAをクローニング中である。
  • 新たな方法によるヒトRNA結合蛋白のcDNAクローニングの研究
    日本学術振興会:科学研究費助成事業 奨励研究(A)
    研究期間 : 1995年 -1995年 
    代表者 : 遠藤 仁司
     
    申請者はヒト培養細胞においてATP合成酵素γサブユニットの筋組織特異的選択的スプライシングが可逆的に誘導される系を確立し、この筋特異的なエクソン選択には細胞内因子の蛋白合成が必須であることを示した(J. Biol.Chem.269, 12488-12493, 1994)。そのスプライシング調整因子としてRNA結合蛋白が可能性として考えやすい。申請者は選択的スプライシングの誘導前後で遺伝子発現量の変化するRNA結合蛋白遺伝子群のcDNAクローニングを目的とした。 申請者は、まずスプライシング誘導前後の細胞からmRNAを精製し、cDNAを作成した。つぎにショウジョウバエからヒトまでのRNA結合蛋白のRNA結合保存領域に対応するdegenerate primerを用いて、PCRを行いdifferential displayにて誘導後に特異的に存在するRNA結合蛋白cDNA断片をクローン化した。この方法は通常のdifferential displayと異なり、特定のdegenerate primerを用いることにより、より特異的な発現を有する遺伝子群のcDNAクローニングを行なうことが可能である。誘導後に特異的に生じるPCR産物の塩基配列を決定し、アミノ酸の一次配列をコンピュータを用いてデータベースと比較した結果、未知のRNA結合蛋白のであった。RNAブロット法による遺伝子発現量の変化を誘導の前後で検討した結果、得られた遺伝子は誘導後にのみ発現していた。さらに各臓器では、心臓・骨格筋に臓器特異的に発現していた。現在、完全長cDNAをクローニング中である。 RNA結合蛋白
  • 心筋分化におけるATP合成酵素の筋特異的選択的スプライシングの調節機構とその機能
    日本学術振興会:科学研究費助成事業 重点領域研究
    研究期間 : 1994年 -1994年 
    代表者 : 遠藤 仁司, 香川 靖雄
     
    本年度の研究は三項目に分かれている。 1)心筋型ATP合成酵素の活性調節の検討:申請者はウシ心筋および肝より亜ミトコンドリア粒子を調整しさまざまな条件でオリゴマイシン感受性ATPase活性を測定した。その結果、それぞれのVmax,Kmには特に差を認めなかったが、pH依存性にわずかながら差が認められ、心筋におけるpH依存性は低かった。現在のところATP合成酵素におけるアイソフォームの存在はγサブユニットしか報告されていないため、このサブユニットにより活性の差が生じると考えやすい。 2)筋分化過程における選択的スプライシングの検討と筋芽細胞を用いた選択的スプライシングの可逆的誘導系の確率:マウスにおいて、MyoDを用いることにより線維芽細胞から筋芽細胞さらに筋線維に分化する系が確立されている。この系を用いてスプライシングの誘導過程を検討するために、まず申請者は新たにマウスATP合成酵素γサブユニットcDNAをクローン化した。次に筋芽細胞の段階で筋特異的な選択的スプライシングが分化培地で誘導されることを確認した。さらに筋芽細胞では低pH培地にて筋型スプライシングの誘導が、筋繊維への形態変化を伴わずに可逆的に生じることを示した。筋型スプライシングの誘導は蛋白合成阻害剤およびプロテインキナーゼC阻害剤にて阻害された。また低pH培地の誘導により、他に筋特異的選択的スプライシングを生じるNCAMとの協調的スプライシング機構が存在することを示した。その調節機構については現在検討中である。 3)筋特異的スプライシング調節因子の同定と精製:申請者はヒト培養細胞にて心筋型RNA転写物と肝型RNA転写物の各々が環境pHによって別々に発現することを見いだした。また、心筋型転写物が誘導される際に新たな細胞内因子の合成が不可欠であることを明らかにした。申請者はこの筋特異的な選択的スプライシングを調節する分子機構を解明するために、ヒト培養細胞から核抽出液を粗精製し、ゲルシフトアッセイやUVクロスリング法を用いて解析した。その結果、選択的スプライシングを受ける第9エキソンの上流にある第8イントロンに結合するRNA結合蛋白が恒常的に存在し、低pH培地にて誘導後の核抽出液にはこの恒常的なRNA結合蛋白のRNAに対する結合を阻害する特異的な核内蛋白が存在することを明らかにした。現在まずこの恒常的なRNA結合蛋白がPTB(polypyrimidine tract-binding protein)であるかどうかを検討中である。つぎに心筋型RNA転写物のみを発現する培養細胞から核抽出液を抽出し、筋特異的な陰性調節を示すスプライシング因子をカラム等を用いて精製する予定である。
  • 変異ミトコンドリアDNAを導入したトランスジェニックマウスの作製法の開発
    日本学術振興会:科学研究費助成事業 試験研究(B)
    研究期間 : 1992年 -1994年 
    代表者 : 太田 成男, 林 純一, 香川 靖雄, 猪原 直弘, 遠藤 仁司
     
    ミトコンドリア(mtDNA)を細胞に導入する方法の開発で試みたのは、mtDNAを完全に除去した細胞EB8と核を除去した細胞質を融合させ細胞質のミトコンドリアをEB8に導入するという方法である。この方法によって、ミトコンドリア病の原因遺伝子と個人差である多型と区別することが可能になった。この方法の応用が可能になり、実用化できるようになったのは本研究の大きな進歩であった。この研究では、ヒト細胞を用いているためトランスジェニックマウスの作製はできないので限界があった。 もうひとつの試みは、mtDNA自身を細胞に導入する方法の開発である。この目的では金属粒子にmtDNAを結合させ、音速以上の速度で細胞内に打ち込む方法を試みた。EB8は酸化的リン酸化が欠損しているので、解糖系の基質であるグルコースなしでは生育できない。金属粒子を打ち込んで、グルコースなしで生育する細胞の出現を待つと、わずかながら生育してくる細胞が出現し、mtDNAが検出された。従って、新たにmtDなを導入する方法が発見された。しかしながら、再現性に問題があり、方法の確立という点では問題が残された。 トランスジェニックマウスを作製するには、mtDNAを完全に失った細胞を分離することが必要であるが、結果的には成功しなかった。このようなマウス細胞は世界の多くの研究室で試みられたが成功していない。
  • ミトコンドリア異常増殖腫瘍に関する分子遺伝子学的解析
    日本学術振興会:科学研究費助成事業 奨励研究(A)
    研究期間 : 1993年 -1993年 
    代表者 : 遠藤 仁司
     
    現在、ミトコンドリア形成制御に関してのin vitroのモデル系は非常に少ない。本研究では電子顕微鏡的には細胞質内に充満するミトコンドリア像がこの腫瘍の特徴であるオンコサイトーマの初代培養系を確立しその分子生物学的特徴を明らかにし、更にはその病因遺伝子を同定することにより、ミトコンドリア形成制御の機構の一端を解明する端緒とすることを目的とした。 先ず、Hurtle細胞陽性の甲状腺腫の一部を手術検体から得(倫理委員会許可済)、電子顕微鏡にてミトコンドリア様構造物の充満像を確かめた。本腫瘍組織からDNAを抽出し、ミトコンドリアDNAの増加の有無をサザンブロット法にて確かめた結果、コントロールとして用いた甲状腺機能亢進症や甲状腺腫のDNAに較べ2-5倍ミトコンドリアDNA量が増加していた。またチトロームCオキシダーゼの電子顕微鏡を用いた活性染色の結果、細胞質内のミトコンドリア様構造物はコントロールに較べ強く活性が認められた。その結果、本腫瘍では生理活性およびミトコンドリアDNAを有するミトコンドリアが細胞質内に増加していることを直接証明した(日本組織細胞化学会に発表)。 次に、この検体から腫瘍細胞の初代培養を、グルコース添加培地、グルコース除去培地の二通りの培養条件下で試みた。その結果、継代を重ねるにつれ線維芽細胞の混入が激しく腫瘍細胞のクローン化は困難であった。また初代培養細胞に複製開始点欠失SV40DNAを遺伝子導入し細胞のクローン化を試み、約20個のクローンを得た。得られた細胞からDNAを抽出し、サザンブロット法にてミトコンドリアDNAの増加を検討した結果約20倍前後の変動しかなく、またロダミン123を用いてミトコンドリアの生染色を行いフローサイトメトリーにてミトコンドリアが増加しているか否かを検討した結果、コントロールとの間に有意の差は認められなかった。クローン化した細胞が上皮由来でなく繊維芽細胞由来である可能性や、また上皮由来であってもSV40DNAを導入したためにミトコンドリア異常増加の形質を落としてしまった可能性もあり現在検討中である。また培養条件によってもミトコンドリア量は影響を受ける可能性があり、今後の検討が必要である。
  • ピルビン酸脱水素酵素のTPP結合部位に関する分子生物学的研究
    日本学術振興会:科学研究費助成事業 奨励研究(A)
    研究期間 : 1992年 -1992年 
    代表者 : 遠藤 仁司
  • 中鎖アシルCoA脱水素酵素欠損症の分子遺伝学的研究
    日本学術振興会:科学研究費助成事業 一般研究(C)
    研究期間 : 1991年 -1991年 
    代表者 : 松原 洋一, 遠藤 仁司
     
    中鎖アシルCoA脱水素酵素(MCAD)欠損症は,その臨床像がライ症候群や乳児突然死症候群に酷似する脂肪酸代謝異常症である。これまでに、本研究代表者によって、MCAD欠損症の90%を占める遺伝子変異(K329E変更)が明らかにされた。今年度の研究により、さらにこの遺伝子変異は、MCAD遺伝子の制限酵素消化断片長多型(RFLP)のハプロタイプ1型と連鎖していることが判明した。このことより、K329E変異は創始者効果によって欧米白人に伝幡した可能性が高いと考えられた。 次にわれわれは、英国、米国、オ-ストラリア、日本のマススクリ-ニング施設より得られた乾燥瀘紙血を用いて一般新生児におけるK329E変異の保因者頻度調査を行った。その結果、英、米、豪の新生児計1368人から、22人の保因者が同定されたが、日本人新生児500人には、この変異は認められなかった。今回の検索によれば、MCAD欠損症のK329E変異の保因者頻度は英国では40人に1人、米国では100人に1人、オ-ストラリアでは70人に1人で、平均すると60人に1人と推定される。本症は常染色体劣性遺伝形式をとるため、その患者頻度は約1万5千人に1人と考えられる。これは,現在新生児マススクリ-ニングの対象と成っている種々の代謝異常症の頻度に匹敵する数字である。MCAD欠損症は,早期発見と適切な食事療法が、患者を不幸な転帰から予防するために重要である。従って、将来、英・米・豪において新生児マススクリ-ニングにMCAD欠損症を組みこむことが必要だと考えられる。またそのためには、マススクリ-ニングに適した、K329E変異のDNA診断法の開発が必須であろう。
  • ピルビン酸脱水素酵素欠損症患者の変異遺伝子の同定及び病態に関する研究
    日本学術振興会:科学研究費助成事業 一般研究(C)
    研究期間 : 1990年 -1991年 
    代表者 : 宮林 重明, 花水 啓, 遠藤 仁司
     
    E1欠損症5例の分子遺伝学的解析を行った。蛋白ブロット法ではE1α蛋白、E1β蛋白の低下している例1例、E1β蛋白のみ低下している例1例、E1α蛋白、E1β蛋白とも正常対象と差を認めない2例である。まず5例の患者線維芽細胞よりmRNAを抽出し、RNAブロットを施行した。その結果5例の患者線維芽細胞では各mRNAともその大きさ及び発現量に異常は認められなかった。次に抽出したポリA+RNAから逆転写酵素を用いてcDNAを合成し、PCRにより各々のE1α及びE1βcDNAの遺伝子増幅をおこない、この増幅産物を用いて直接塩基決定法にて塩基の一次構造を決定し、塩基変異を同定した。E1α蛋白のサイズの異常を認めた例はE1αcDNA上に4塩基欠失を、E1α蛋白、E1β蛋白の低下している症例ではE1αcDNA上で4塩基挿入が認められた。また蛋白ブロットにてE1β蛋白のみ低下した例でも、E1αcDNAにてC214→T(Arg72→Cys)の置換が認められたが、E1βcDNAの塩基異常は認められなかった。これらの結果よりE1α蛋白の異常がE1β蛋白を不安定化させたもので、ピルビン酸脱水素酵素ではE1α蛋白がE1β蛋白の安定性に深く関与している可能性が示唆された。女児での発症の検討は、cDNAとゲノムDNAで変異遺伝子周囲をPCRで増幅し、ASOハイブリダイゼ-ションをおこなった。cDNA上は変異遺伝子のホモ接合体で、ゲノムDNAは正常とのヘテロ接合体を示した例と、両者でヘテロ接合体をとった例があったが、培養線維芽細胞をSV40でライン化した後、選択メジュウムでクロ-ン化したところ、正常活性を持つ細胞と、活性のない細胞とが得られた。しかも、正常活性を持つ細胞のcDNAは正常アレルのみで、活性のない細胞のcDNAは変異アレルのみであった。以上のことから女児での発症病態はX染色体の不規則な不活性化によることがわかった。また我々の経験したいずれの例でも、両親には同変異を認めず突然変異と考えられ、本疾患の変異発生の特徴と考えられた。
  • ピルビン酸脱水素酵素複合体欠損症の分子生物学的研究
    日本学術振興会:科学研究費助成事業 奨励研究(特別研究員)
    研究期間 : 1990年 -1990年 
    代表者 : 遠藤 仁司
  • ミトコンドリア膜タンパク質の網羅的プロテオーム解析
    その他の研究制度
  • RNAプロセシングの分子メカニズムの研究
    科学研究費補助金
    ヒトゲノム研究により2万種強の遺伝子の約50%が選択的RNAスプライシングの調節を受け、タンパク質分子の多様性を生み出すことが知られている。本研究は組織特異的および発達段階特異的なRNAプロセシングの分子メカニズムを明らかにすることを課題とする。
  • Study for molecular mechanism of RNA processing
    Grant-in-Aid for Scientific Research
  • Study for comprehensive proteomic analysis of mitochondrial membrane proteins
    The Other Research Programs


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