Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Mizuki Ohno Last modified date:2021.09.14

Assistant Professor / Department of Medical Biophysics & Radiation Biology, / Department of Basic Medicine / Faculty of Medical Sciences


Papers
1. Yasunobu Aoki, Mizuki Ohno, Michiyo Matsumoto, Michi Matsumoto, Kenichi Masumura, Takehiko Nohmi, Teruhisa Tsuzuki , Characteristic mutations induced in the small intestine of Msh2-knockout gpt delta mice , Genes and Environment , 10.1186/s41021-021-00196-0. , 2021.07.
2. Yasunobu Aoki, YosukeTaniguchi, MichiyoMatsumoto, Michi Matsumoto, Mizuki Ohno, Kenichi Masumura, Shigeki Sasaki, Teruhisa Tsuzuki, Masayuki Yamamoto, Takehiko Nohmi, Oxidative-stress-driven in vivo mutagenesis induced by oral administration of potassium bromate in the small intestines of gpt delta mice, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, https://doi.org/10.1016/j.mrgentox.2020.503136, 2020.01.
3. Mizuki Ohno, Toward understanding de novo germline mutations in mammals, Genes and Genetic Systems, 10.1266/ggs.94.1, 94, 1, 1, 2019.01.
4. Mizuki Ohno, Spontaneous de novo germline mutations in humans and mice
rates, spectra, causes and consequences, Genes and Genetic Systems, 10.1266/ggs.18-00015, 94, 1, 13-22, 2019.01, Germline mutations are the origin of genetic variation and are widely considered to be the driving force of genome evolution. The rates and spectra of de novo mutations (DNMs) directly affect evolutionary speed and direction and thereby establish species-specific genomic futures in the long term. This has resulted in a keen interest in understanding the origin of germline mutations in mammals. Accumulating evidence from next-generation sequencing and family-based analysis indicates that the frequency of human DNMs varies according to sex, age and local genomic context. Thus, it is likely that there are multiple causes and drivers of mutagenesis, including spontaneous DNA lesions, DNA repair status and DNA polymerase errors. In this review, recent studies of human and mouse germline DNMs are discussed, and the rates and spectra of spontaneous germline DNMs in the mouse mutator lines Pold1exo/exo and TOY-KO (Mth1−/−/Ogg1−/−/ Mutyh−/− triple knockout) are summarized in the context of endogenous causes and mechanisms..
5. Arikuni Uchimura, Mayumi Higuchi, Yohei Minakuchi, Ohno Mizuki, Atsushi Toyoda, Asao Fujiyama, Ikuo Miura, Shigeharu Wakana, Jo Nishino, Takeshi Yagi, Germline mutation rates and the long-term phenotypic effects of mutation accumulation in wild-type laboratory mice and mutator mice, Genome Research, 10.1101/gr.186148.114, 25, 8, 1125-1134, 2015.08, The germline mutation rate is an important parameter that affects the amount of genetic variation and the rate of evolution. However, neither the rate of germline mutations in laboratory mice nor the biological significance of the mutation rate in mammalian populations is clear. Here we studied genome-wide mutation rates and the long-term effects of mutation accumulation on phenotype in more than 20 generations of wild-type C57BL/6 mice and mutator mice, which have high DNA replication error rates. We estimated the base-substitution mutation rate to be 5.4 × 10-9 (95% confidence interval = 4.6 × 10-9-6.5 × 10-9) per nucleotide per generation in C57BL/6 laboratory mice, about half the rate reported in humans. The mutation rate in mutator mice was 17 times that in wild-type mice. Abnormal phenotypes were 4.1-fold more frequent in the mutator lines than in the wild-type lines. After several generations, the mutator mice reproduced at substantially lower rates than the controls, exhibiting low pregnancy rates, lower survival rates, and smaller litter sizes, and many of the breeding lines died out. These results provide fundamental information about mouse genetics and reveal the impact of germline mutation rates on phenotypes in a mammalian population..
6. Yoko Ohnishi, Yoshinori N. Ohnishi, Takanori Nakamura, Ohno Mizuki, Pamela J. Kennedy, Ohkawa Yasuyuki, Akinori Nishi, Rachael Neve, Teruhisa Tsuzuki, Eric J. Nestler, PSMC5, a 19S Proteasomal ATPase, Regulates Cocaine Action in the Nucleus Accumbens, PLOS ONE, 2015.07.
7. Yoko H. Ohnishi, Yoshinori N. Ohnishi, Takanori Nakamura, Ohno Mizuki, Pamela J. Kennedy, Yasuyuki Ohkawa, Akinori Nishi, Rachael Neve, Teruhisa Tsuzuki, Eric J. Nestler, Erratum
PSMC5, a 19S proteasomal ATPase, regulates cocaine action in the nucleus accumbens (PLoS ONE (2015) 10:5 (e0126710) DOI: 10.1371/journal.pone.0126710), PLoS One, 10.1371/journal.pone.0126710, 10, 6, 2015.06.
8. Yoko H. Ohnishi, Yoshinori N. Ohnishi, Takanori Nakamura, Ohno Mizuki, Pamela J. Kennedy, Yasuyuki Ohkawa, Akinori Nishi, Rachael Neve, Teruhisa Tsuzuki, Eric J. Nestler, PSMC5, a 19S proteasomal ATPase, regulates cocaine action in the nucleus accumbens, PLoS One, 10.1371/journal.pone.0131263, 10, 5, 2015.05, ΔFosB is a stable transcription factor which accumulates in the nucleus accumbens (NAc), a key part of the brain's reward circuitry, in response to chronic exposure to cocaine or other drugs of abuse. While ΔFosB is known to heterodimerize with a Jun family member to form an active transcription factor complex, there has not to date been an open-ended exploration of other possible binding partners for ΔFosB in the brain. Here, by use of yeast two-hybrid assays, we identify PSMC5 - also known as SUG1, an ATPase-containing subunit of the 19S proteasomal complex - as a novel interacting protein with ΔFosB. We verify such interactions between endogenous ΔFosB and PSMC5 in the NAc and demonstrate that both proteins also form complexes with other chromatin regulatory proteins associated with gene activation. We go on to show that chronic cocaine increases nuclear, but not cytoplasmic, levels of PSMC5 in the NAc and that overexpression of PSMC5 in this brain region promotes the locomotor responses to cocaine. Together, these findings describe a novel mechanism that contributes to the actions of ΔFosB and, for the first time, implicates PSMC5 in cocaine-induced molecular and behavioral plasticity..
9. Ohno Mizuki, SAKUMI Kunihiko, Fukumura Ryutaro, Masato Furuichi, Iwasaki Yuki, Hokama Masaaki, Ikemura Toshimichi, Teruhisa Tsuzuki, Gondo Yoichi, Yusaku Nakabeppu, 8-oxoguanine causes spontaneous de novo germline mutations in mice , Scientific reports, 10.1038/srep04689, 4:4689, 2014.04.
10. Jingshu Piao, Yoshimichi Nakatsu, Ohno Mizuki, Taguchi Kennichi, Teruhisa Tsuzuki, Mismatch repair deficient mice show susceptibility to oxidative stress-induced intestinal carcinogenesis, International Journal of Biological Sciences, 10.7150/ijbs.5750, 10, 1, 73-79, 2014.01.
11. Hideo Tsuji, Hiroko Ishii-Ohba, Tadahiro Shiomi, Naoko Shiomi, Takanori Katsube, Masahiko Mori, Mitsuru Nenoi, Ohno Mizuki, Daisuke Yoshimura, Sugako Oka, Yusaku Nakabeppu, Kouichi Tatsumi, Masahiro Muto, Toshihiko Sado, Nature of nontargeted radiation effects observed during fractionated irradiation-induced thymic lymphomagenesis in mice, Journal of Radiation Research, 10.1093/jrr/rrs128, 54, 3, 453-466, 2013.05, Changes in the thymic microenvironment lead to radiation-induced thymic lymphomagenesis, but the phenomena are not fully understood. Here we show that radiation-induced chromosomal instability and bystander effects occur in thymocytes and are involved in lymphomagenesis in C57BL/6 mice that have been irradiated four times with 1.8-Gy γ-rays. Reactive oxygen species (ROS) were generated in descendants of irradiated thymocytes during recovery from radiation-induced thymic atrophy. Concomitantly, descendants of irradiated thymocytes manifested DNA lesions as revealed by γ-H2AX foci, chromosomal instability, aneuploidy with trisomy 15 and bystander effects on chromosomal aberration induction in co-cultured ROS-sensitive mutant cells, suggesting that the delayed generation of ROS is a primary cause of these phenomena. Abolishing the bystander effect of post-irradiation thymocytes by superoxide dismutase and catalase supports ROS involvement. Chromosomal instability in thymocytes resulted in the generation of abnormal cell clones bearing trisomy 15 and aberrant karyotypes in the thymus. The emergence of thymic lymphomas from the thymocyte population containing abnormal cell clones indicated that clones with trisomy 15 and altered karyotypes were prelymphoma cells with the potential to develop into thymic lymphomas. The oncogene Notch1 was rearranged after the prelymphoma cells were established. Thus, delayed nontargeted radiation effects drive thymic lymphomagenesis through the induction of characteristic changes in intrathymic immature T cells and the generation of prelymphoma cells..
12. Hideo Tsuji, Hiroko Ishii-Ohba, Tadahiro Shiomi, Naoko Shiomi, Takanori Katsube, Masahiko Mori, Mitsuru Nenoi, Ohno Mizuki, Daisuke Yoshimura, Sugako Oka, Yusaku Nakabeppu, Kouichi Tatsumi, Masahiro Muto, Toshihiko Sado, Nature of nontargeted radiation effects observed during fractionated irradiation-induced thymic lymphomagenesis in mice., Journal of Radiation Research, 10.1093/jrr/rrs128 , 54, 3, 453-466, 2013.01.
13. Abolhassani, N, Iyama, T, Tsuchimoto, D, Sakumi, K, Ohno, M, Behmanesh, M, Nakabeppu, Y., NUDT16 and ITPA play a dual protective role in maintaining chromosome stability and cell growth by eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals, Nucleic Acids Research, 38, 9, 2891-2903, 2010.05.
14. Ohno Mizuki, The influence of oxidized DNA on mammalian genome, Fukuoka Acta Medica, 101, 4, 61-68, 2010.04.
15. Nona Abolhassani, Teruaki Iyama, Daisuke Tsuchimoto, Sakumi Kunihiko, Ohno Mizuki, Mehrdad Behmanesh, Yusaku Nakabeppu, NUDT16 and ITPA play a dual protective role in maintaining chromosome stability and cell growth by eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals, Nucleic Acids Research, 10.1093/nar/gkp1250, 38, 9, 2891-2903, 2010.01, Mammalian inosine triphosphatase encoded by ITPA gene hydrolyzes ITP and dITP to monophosphates, avoiding their deleterious effects. Itpa- mice exhibited perinatal lethality, and significantly higher levels of inosine in cellular RNA and deoxyinosine in nuclear DNA were detected in Itpa- embryos than in wild-type embryos. Therefore, we examined the effects of ITPA deficiency on mouse embryonic fibroblasts (MEFs). Itpa- primary MEFs lacking ITP-hydrolyzing activity exhibited a prolonged doubling time, increased chromosome abnormalities and accumulation of single-strand breaks in nuclear DNA, compared with primary MEFs prepared from wild-type embryos. However, immortalized Itpa- MEFs had neither of these phenotypes and had a significantly higher ITP/IDP-hydrolyzing activity than Itpa- embryos or primary MEFs. Mammalian NUDT16 proteins exhibit strong dIDP/IDP-hydrolyzing activity and similarly low levels of Nudt16 mRNA and protein were detected in primary MEFs derived from both wild-type and Itpa- embryos. However, immortalized Itpa- MEFs expressed significantly higher levels of Nudt16 than the wild type. Moreover, introduction of silencing RNAs against Nudt16 into immortalized Itpa- MEFs reproduced ITPA-deficient phenotypes. We thus conclude that NUDT16 and ITPA play a dual protective role for eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals..
16. Sugako Oka, Ohno Mizuki, Yusaku Nakabeppu, Construction and characterization of a cell line deficient in repair of mitochondrial, but not nuclear, oxidative DNA damage., Methods in Molecular Biology, 10.1007/978-1-59745-521-3_16, 554, 251-264, 2009.10, Oxidative base lesions, such as 8-oxoguanine, accumulate in nuclear and mitochondrial DNAs under oxidative stress, resulting in cell death. However, it is not known whether only oxidative lesion accumulated in mitochondrial DNA is involved in such cell death. By introducing human cDNA encoding a nuclear form of 8-oxoG DNA glycosylase (hOGG1-1a) into immortalized mouse embryo fibroblasts lacking Ogg1 gene, we established a cell line which selectively accumulates 8-oxoguanine in mitochondrial DNA under oxidative stress. Selective accumulation of 8-oxoguanine in mitochondrial DNA in this cell line causes degradation of mitochondrial DNA followed by ATP depletion, mitochondrial membrane permeability transition, and Ca(2+) efflux, which in turn activates calpains to execute cell death. Knockdown of MUTYH which excises adenine opposite 8-oxoG in DNA prevents degradation of mitochondrial DNA and activation of calpain, thus suppressing the cell death induced by menadione..
17. Ohno Mizuki, Sugako Oka, Yusaku Nakabeppu, Quantitative analysis of oxidized guanine, 8-oxoguanine, in mitochondrial DNA by immunofluorescence method., Methods in Molecular Biology, 10.1007/978-1-59745-521-3_13, 554, 199-212, 2009.10, 8-Oxoguanine (8-oxoG), an oxidized form of guanine, is one of the major mutagenic lesions generated under oxidative stress. Oxidative damage in mitochondrial DNA has been implicated as a causative factor for a wide variety of degenerative diseases as well as for cancer during aging. We established a quantitative method for in situ detection of 8-oxoG in mitochondrial DNA in a single-cell level using a monoclonal antibody. Specific detection of 8-oxoG in mitochondrial DNA was confirmed by pre-treatment of samples with DNase I or MutM, the latter excising 8-oxoG opposite C in DNA. We then analyzed 8-oxoG dynamics in mitochondrial DNA of the wild-type and 8-oxoG DNA glycosylase (OGG1)-deficient mouse cells after exposure to hydrogen peroxide. Intensities for the 8-oxoG immunoreactivity in mitochondrial DNA were increased immediately after the exposure to hydrogen peroxide in both types of cells. The increased intensities returned to basal levels within a few hours only in wild-type cells, but not in OGG1-deficient cells which exhibited the increased intensities even 24 h after the exposure. These results indicate that OGG1 is a major enzyme for excision repair of 8-oxoG in mitochondrial DNA in mouse cells, and that our method described here is appropriate to study 8-oxoG dynamics in mitochondrial DNA..
18. Kyota Fujita, Toshihiro Seike, Noriko Yutsudo, Mizuki Ohno, Hidetaka Yamada, Hiroo Yamaguchi, Kunihiko Sakumi, Yukiko Yamakawa, Mizuho A. Kido, Atsushi Takaki, Toshihiko Katafuchi, Yoshinori Tanaka, Yusaku Nakabeppu, Mami Noda, Hydrogen in Drinking Water Reduces Dopaminergic Neuronal Loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Mouse Model of Parkinson’s Disease
, PLoS ONE, 4(9) , e7247, 2009.09.
19. Kyota Fujita, Toshihiro Seike, Noriko Yutsudo, Ohno Mizuki, Hidetaka Yamada, hiroo yamaguchi, Sakumi Kunihiko, Yukiko Yamakawa, Mizuho A. Kido, Atsushi Takaki, Toshihiko Katafuchi, Yoshinori Tanaka, Yusaku Nakabeppu, Mami Noda, Hydrogen in drinking water reduces dopaminergic neuronal loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease, PLoS One, 10.1371/journal.pone.0007247, 4, 9, 2009.09, It has been shown that molecular hydrogen (H2) acts as a therapeutic antioxidant and suppresses brain injury by buffering the effects of oxidative stress. Chronic oxidative stress causes neurodegenerative diseases such as Parkinson's disease (PD). Here, we show that drinking H2-containing water significantly reduced the loss of dopaminergic neurons in PD model mice using both acute and chronic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The concentration-dependency of H2 showed that H2 as low as 0.08 ppm had almost the same effect as saturated H2 water (1.5 ppm). MPTP-induced accumulation of cellular 8-oxoguanine (8-oxoG), a marker of DNA damage, and 4-hydroxynonenal (4-HNE), a marker of lipid peroxidation were significantly decreased in the nigro-striatal dopaminergic pathway in mice drinking H2-containing water, whereas production of superoxide (O2·-) detected by intravascular injection of dihydroethidium (DHE) was not reduced significantly. Our results indicated that low concentration of H2 in drinking water can reduce oxidative stress in the brain. Thus, drinking H2-containing water may be useful in daily life to prevent or minimize the risk of life style-related oxidative stress and neurodegeneration..
20. Dan Y, Ohta Y, Tsuchimoto D, Ohno M, Ide Y, Sami M, Kanda T, Sakumi K, Nakabeppu Y., Altered gene expression profiles and higher frequency of spontaneous DNA strand breaks in APEX2-null thymus., DNA Repair, 7 (9): 1437-1454, 2008.09.
21. Yukihiko Dan, Yutaka Ohta, Daisuke Tsuchimoto, Ohno Mizuki, Yasuhito Ide, Manabu Sami, Tomomasa Kanda, Sakumi Kunihiko, Yusaku Nakabeppu, Altered gene expression profiles and higher frequency of spontaneous DNA strand breaks in APEX2-null thymus, DNA Repair, 10.1016/j.dnarep.2008.05.003, 7, 9, 1437-1454, 2008.09, A second class II AP endonuclease, APEX2, possesses strong 3′-5′ exonuclease and 3′-phosphodiesterase activities but only very weak AP-endonuclease activity. APEX2 associates with proliferating cell nuclear antigen (PCNA), and the progression of S phase of the cell cycle is accompanied by its expression. APEX2-null mice exhibit severe dyslymphopoiesis in thymus as well as moderate dyshematopoiesis and growth retardation. Comparative gene expression profiling of wild-type and APEX2-null mice using an oligonucleotide microarray revealed that APEX2-null thymus has significantly altered gene expression profiles, reflecting its altered populations of thymocytes. Beyond these altered populations, APEX2-null thymus exhibits significant alterations in expression of genes involved in DNA replication, recombination and repair, including Apex1, Exo1 and Fen1 as well as master genes for the DNA damage response, such as E2f1, Chek1, and proapoptotic genes. We therefore examined the extent of DNA strand breakage, and found that both of single-strand breaks detected as comets and double-strand breaks detected as γH2AX foci were significantly higher in frequency in most APEX2-null thymocytes compared to wild-type thymocytes. This higher frequency of DNA breaks was accompanied by increased expression of PCNA and increased phosphorylation of p53 at Ser23 and to a lesser extent, at Ser18. The present study clearly demonstrates that APEX2-null lymphocytes have a higher frequency of DNA breaks, indicating that APEX2 may play an important role(s) during their generation and/or repair..
22. Ichikawa J, Tsuchimoto D, Oka S, Ohno M, Furuichi M, Sakumi K, Nakabeppu Y., Oxidation of mitochondrial deoxynucleotide pools by exposure to sodium nitroprusside induces cell death., DNA Repair, 7(3):418-430, 2008.03.
23. Junji Ichikawa, Daisuke Tsuchimoto, Sugako Oka, Ohno Mizuki, Masato Furuichi, Sakumi Kunihiko, Yusaku Nakabeppu, Oxidation of mitochondrial deoxynucleotide pools by exposure to sodium nitroprusside induces cell death, DNA Repair, 10.1016/j.dnarep.2007.11.007, 7, 3, 418-430, 2008.03, Human MutT homolog (hMTH1) hydrolyzes oxidized purine nucleoside triphosphates to monophosphates, thereby avoiding incorporation of such oxidized purines into DNA or RNA. We examined whether hMTH1 prevents cellular dysfunction induced by sodium nitroprusside, a spontaneous NO donor. Exposure to sodium nitroprusside caused an 8-oxoguanine (8-oxoG) buildup in DNA of proliferating MTH1-null cells which underwent mitochondrial degeneration and subsequently died. Quiescent MTH1-null cells also died with 8-oxoG buildup but only when the buildup affected mitochondrial and not nuclear DNA. In both proliferative and quiescent conditions, the accumulation of 8-oxoG in DNA and cell death was effectively prevented by hMTH1. Knockdown of MUTYH in quiescent MTH1-null cells significantly prevented the cell death, suggesting that 8-oxoG incorporated into mitochondrial DNA is a main cause of this form of cell death. To verify this possibility, an artificially modified hMTH1, namely mTP-EGFP-hMTH1, which localizes exclusively in mitochondria, was expressed in MTH1-null cells. mTP-EGFP-hMTH1 selectively prevented buildup of 8-oxoG in mitochondrial but not nuclear DNA after exposure of proliferating cells to sodium nitroprusside, and also efficiently prevented cell death. We thus concluded that exposure of cells to sodium nitroprusside causes oxidation of mitochondrial deoxynucleotide pools, and that buildup of oxidized bases in mitochondrial DNA initiates cell death..
24. Oka S, Ohno M, Tsuchimoto D, Sakumi K, Furuichi M, Nakabeppu Y., Two distinct pathways of cell death triggered by oxidative damage to nuclear and mitochondrial DNAs., EMBO Journal.,27(2):421-432., 2008.01.
25. Sugako Oka, Ohno Mizuki, Daisuke Tsuchimoto, Sakumi Kunihiko, Masato Furuichi, Yusaku Nakabeppu, Two distinct pathways of cell death triggered by oxidative damage to nuclear and mitochondrial DNAs, EMBO Journal, 10.1038/sj.emboj.7601975, 27, 2, 421-432, 2008.01, Oxidative base lesions, such as 8-oxoguanine (8-oxoG), accumulate in nuclear and mitochondrial DNAs under oxidative stress, resulting in cell death. However, it is not known which form of DNA is involved, whether nuclear or mitochondrial, nor is it known how the death order is executed. We established cells which selectively accumulate 8-oxoG in either type of DNA by expression of a nuclear or mitochondrial form of human 8-oxoG DNA glycosylase in OGG1-null mouse cells. The accumulation of 8-oxoG in nuclear DNA caused poly-ADP-ribose polymerase (PARP)-dependent nuclear translocation of apoptosis-inducing factor, whereas that in mitochondrial DNA caused mitochondrial dysfunction and Ca 2+ release, thereby activating calpain. Both cell deaths were triggered by single-strand breaks (SSBs) that had accumulated in the respective DNAs, and were suppressed by knockdown of adenine DNA glycosylase encoded by MutY homolog, thus indicating that excision of adenine opposite 8-oxoG lead to the accumulation of SSBs in each type of DNA. SSBs in nuclear DNA activated PARP, whereas those in mitochondrial DNA caused their depletion, thereby initiating the two distinct pathways of cell death..
26. Ohno M, Miura T, Furuichi M, Tominaga Y, Tsuchimoto D, Sakumi K, Nakabeppu Y., A genome-wide distribution of 8-oxoguanine correlates with the preferred regions for recombination and single nucleotide polymorphism in the human genome., Genome Res.,6(5):567-575, 2006.05.
27. Ohno Mizuki, Tomofumi Miura, Masato Furuichi, Yohei Tominaga, Daisuke Tsuchimoto, Sakumi Kunihiko, Yusaku Nakabeppu, A genome-wide distribution of 8-oxoguanine correlates with the preferred regions for recombination and single nucleotide polymorphism in the human genome, Genome Research, 10.1101/gr.4769606, 16, 5, 567-575, 2006.05, 8-Oxoguanine (8-oxoG), a major spontaneous form of oxidative DNA damage, is considered to be a natural cause of genomic diversity in organisms because of its mutagenic potential. The steady-state level of 8-oxoG in the nuclear genome of a human cell has been estimated to be several residues per 106 guanines. In the present study, to clarify the genome-wide distribution of 8-oxoG in the steady state, we performed fluorescence in situ detection of 8-oxoG on human metaphase chromosomes using a monoclonal antibody. Multiple dot-like signals were observed on each metaphase chromosome. We then mapped the position of the signal at megabase resolution referring to the cytogenetically identified chromosomal band, and demonstrated that 8-oxoG is unevenly distributed in the normal human genome and that the distribution pattern is conserved among different individuals. Moreover, we found that regions with a high frequency of recombination and single nucleotide polymorphisms (SNPs) are preferentially located within chromosomal regions with a high density of 8-oxoG. Our findings suggest that 8-oxoG is one of the main causes of frequent recombinations and SNPs in the human genome, which largely contribute to the genomic diversity in human beings..
28. Yamaguchi, H., Kajitani K., Dan Y., Furuichi M., Ohno M., Sakumi K., Kang D. and Nakabeppu Y., MTH1, an oxidized purine nucleoside triphosphatase, protects the dopamine neurons from oxidative damage in nucleic acids caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine., Cell Death Differ,13(4):551-563, 2006.04.
29. hiroo yamaguchi, Kosuke Kajitani, Y. Dan, Masato Furuichi, Ohno Mizuki, Sakumi Kunihiko, Dongchon Kang, Yusaku Nakabeppu, MTH1, an oxidized purine nucleoside triphosphatase, protects the dopamine neurons from oxidative damage in nucleic acids caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, Cell Death and Differentiation, 10.1038/sj.cdd.4401788, 13, 4, 551-563, 2006.04, We previously reported that 8-oxoguanine (8-oxoG) accumulates in the cytoplasm of dopamine neurons in the substantia nigra of patients with Parkinson's disease and the expression of MTH1 carrying an oxidized purine nucleoside triphosphatase activity increases in these neurons, thus suggesting that oxidative damage in nucleic acids is involved in dopamine neuron loss. In the present study, we found that levels of 8-oxoG in cellular DNA and RNA increased in the mouse nigrostriatal system during the tyrosine hydroxylase (TH)-positive dopamine neuron loss induced by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MTH1-null mice exhibited a greater accumulation of 8-oxoG in mitochondrial DNA accompanied by a more significant decrease in TH and dopamine transporter immunoreactivities in the striatum after MPTP administration, than in wild-type mice. We thus demonstrated that MTH1 protects the dopamine neurons from oxidative damage in the nucleic acids, especially in the mitochondrial DNA of striatal nerve terminals of dopamine neurons..
30. Kurushima, H., Ohno M., Miura T., Nakamura T. Y., Horie H., Kadoya T., Ooboshi H., Kitazono T., Ibayashi S., Iida M. and Nakabeppu Y., Selective induction of ΔFosB in the brain after transient forebrain ischemia accompanied by an increased expression of galectin-1, and the implication of ΔFosB and galectin-1 in neuroprotection and neurogenesis., Cell Death Differ, 10.1038/sj.cdd.4401648, 12, 8, 1078-1096, 12 (8):1078-1096., 2005.08.
31. Ide, Y., Tsuchimoto, D., Tominaga, Y., Nakashima, M., Watanabe, T., Sakumi, K., Ohno, M. and Nakabeppu, Y.,, Growth retardation and dyslymphopoiesis accompanied by G2/M arrest in APEX2-null mice., Blood, 10.1182/blood-2004-04-1476, 104, 13, 4097-4103, 104(13):4097-4103,2004, 2004.12.
32. Yusaku Nakabeppu, Daisuke Tsuchimoto, Akimasa Ichinoe, Ohno Mizuki, Yasuhito Ide, Seiki Hirano, Daisuke Yoshimura, Yohei Tominaga, Masato Furuichi, Sakumi Kunihiko, Biological significance of the defense mechanisms against oxidative damage in nucleic acids caused by reactive oxygen species
From mitochondria to nuclei, Annals of the New York Academy of Sciences, 10.1196/annals.1293.011, 1011, 101-111, 2004, In mammalian cells, more than one genome in a single cell has to be maintained throughout the entire life of the cell, namely, one in the nucleus and the other in the mitochondria. The genomes and their precursor nucleotides are highly exposed to reactive oxygen species, which are inevitably generated as a result of the respiratory function in mitochondria. To counteract such oxidative damage in nucleic acids, cells are equipped with several defense mechanisms. Modified nucleotides in the nucleotide pools are hydrolyzed, thus avoiding their incorporation into DNA or RNA. Damaged bases in DNA with relatively small chemical alterations are mainly repaired by the base excision repair (BER) system, which is initiated by the excision of damaged bases by specific DNA glycosylases. MTH1 protein hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP, 8-oxo-dATP, and 2-hydroxy (OH)-dATP to the monophosphates, and MTH1 are located in the cytoplasm, mitochondria, and nucleus. We observed an increased susceptibility to spontaneous carcinogenesis in Mth1-deficient mice and an alteration of MTH1 expression along with the accumulation of 8-oxo-dG in patients with various neurodegenerative diseases. Enzymes for the BER pathway, namely, 8-oxoG DNA glycosylase (OGG1), 2-OH-A/adenine DNA glycosylase (MUTYH), and AP endonuclease (APEX2) are also located both in the mitochondria and in the nuclei, and the expression of mitochondrial OGG1 is altered in patients with various neurodegenerative diseases. We also observed increased susceptibilities to spontaneous cardiogenesis in OGG1 and MUTYH-deficient mice. The increased occurrence of lung tumor in OGG1-deficient mice was completely abolished by the concomitant disruption of the Mth1 gene..
33. Yoshimura, D., K. Sakumi, M. Ohno, Y. Sakai, M. Furuichi, S. Iwai, and Y. Nakabeppu.,, An oxidized purine nucleoside triphosphatase, MTH1 suppresses cell death caused by oxidative stress., J. Biol. Chem.,278:37965-37973, 2003.09.
34. Daisuke Yoshimura, Sakumi Kunihiko, Ohno Mizuki, Yasunari Sakai, Masato Furuichi, Shigenori Iwai, Yusaku Nakabeppu, An oxidized purine nucleoside triphosphatase, MTH1, suppresses cell death caused by oxidative stress, Journal of Biological Chemistry, 10.1074/jbc.M306201200, 278, 39, 37965-37973, 2003.09, MTH1 hydrolyzes oxidized purine nucleoside triphosphates such as 8-oxo-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP) and 2-hydroxy-2′-deoxyadenosine 5′-triphosphate (2-OH-dATP) and thus protects cells from damage caused by their misincorporation into DNA. In the present study, we established MTH1-null mouse embryo fibroblasts that were highly susceptible to cell dysfunction and death caused by exposure to H 2O2, with morphological features of pyknosis and electron-dense deposits accumulated in mitochondria. The cell death observed was independent of both poly(ADP-ribose) polymerase and caspases. A high performance liquid chromatography tandem mass spectrometry analysis and immunofluorescence microscopy revealed a continuous accumulation of 8-oxo-guanine both in nuclear and mitochondrial DNA after exposure to H 2O2. All of the H2O2-induced alterations observed in MTH1-null mouse embryo fibroblasts were effectively suppressed by the expression of wild type human MTH1 (hMTH1), whereas they were only partially suppressed by the expression of mutant hMTH1 defective in either 8-oxo-dGTPase or 2-OH-dATPase activity. Human MTH1 thus protects cells from H2O2-induced cell dysfunction and death by hydrolyzing oxidized purine nucleotides including 8-oxo-dGTP and 2-OH-dATP, and these alterations may be partly attributed to a mitochondrial dysfunction..
35. Yamazaki, K., T. Aso, Y. Ohnishi, M. Ohno, K. Tamura, T. Shuin, S. Kitajima, and Y. Nakabeppu., Mammalian elongin A is not essential for cell viability but required for proper cell-cycle progression with limited alteration of gene expression., J. Biol. Chem.,278:13585-13589,, 10.1074/jbc.C300047200, 278, 15, 13585-13589, 2003.04.
36. Ohno M, Fukagawa T, Lee JS, Ikemura T., Triplex-forming DNAs in the human interphase nucleus visualized in situ by polypurine/polypyrimidine DNA probes and antitriplex antibodies., Chromosoma. 2002 Sep;111(3):201-13. Epub 2002 Jul 16., 2002.09.
37. Mizuki Ohno, Tenzen T., Watanabe Y., Yamagata T., Kanaya S., Ikemura T., Non-B DNA structures spatially and sequence-specifically associated with individual cetromeres in the human interphase nucleus, Chromosome Today. Vol.13, 2000.10.
38. Ohno M, Aoki N, Sasaki H., Allele-specific detection of nascent transcripts by fluorescence in situ hybridization reveals temporal and culture-induced changes in Igf2 imprinting during pre-implantation mouse development., Genes Cells. 2001 Mar;6(3):249-59., 10.1046/j.1365-2443.2001.00417.x, 6, 3, 249-259, 2001.03.
39. Ohno Mizuki, Tatsuo Fukagawa, Jeremy S. Lee, Toshimichi Ikemura, Triplex-forming DNAs in the human interphase nucleus visualized in situ by polypurine/polypyrimidine DNA probes and antitriplex antibodies, Chromosoma, 10.1007/s00412-002-0198-0, 111, 3, 201-213, 2002.01, The polypurine/polypyrimidine (PuPy) tracts present in the human genome are known to be scattered among and within chromosomes. In PuPy tract sequences, triplex formation occurs readily under physiological conditions, leaving single-stranded DNAs capable of hybridization with complementary single-stranded DNAs and RNAs. The formation of single-strands and transmolecular triplexes is thought to enable sequences spaced distantly along the genome to associate with each other and organize nuclear DNA into ordered configurations. Triplex-forming DNAs in the human interphase nucleus were analyzed by combining fluorescence in situ "non-denaturing" hybridization employing PuPy tract probes and immunodetection by antitriplex antibodies. The non-denaturing hybridization technique, which has been used to detect RNA, may detect single-stranded DNAs in non-denatured nuclei, if present. Probes such as (GA/TC)n and (GAA/TTC)n sequences gave sequence-specific signals that overlapped with or were closely associated with triplexes immunolocalized by using known antitriplex antibodies. Pretreatment of nuclei with antitriplex antibodies blocked probe signal formation. Signal formation was resistant to pretreatment of nuclei with RNases but sensitive to single strand-specific nucleases. Triplexes visualized differentially with distinct PuPy tract probes were associated spatially with centromeric sequences in the interphase nucleus in a sequence-specific manner..
40. Takahito Inoue, Haruyoshi Yamaza, Yasuyoshi Sakai, Shin Ichi Mizuno, Ohno Mizuki, Naotaka Hamasaki, Yasuyuki Fukumaki, Position-independent human β-globin gene expression mediated by a recombinant adeno-associated virus vector carrying the chicken β-globin insulator, Journal of Human Genetics, 10.1007/s100380050133, 44, 3, 152-162, 1999.01, The position-independent expression of transgenes in target cells is an essential subject for determining effective gene therapies. The chicken β- globin insulator blocks the effects of regulatory sequences on transcriptional units at differential domains. We prepared a recombinant adeno-associated virus (rAAV) containing various combinations of the DNase I- hypersensitive site 2 (HS2), 3 (HS3), and 4 (HS4) core elements from the human β-globin locus control region (LCR), the human β-globin gene, and the herpes virus thymidine kinase promoter driven neomycin-resistant gene (neo(R)) (rHS432, rHS43, rHS42, rHS32, and rHS2), and also rAAV containing two copies of the 250-bp core sequence of the chicken β-globin insulator on both sides of the rHS2 (rIns/HS2/2Ins). After isolating neomycin-resistant mouse erythroleukemia (MEL) cells infected with each rAAV, we analyzed the rAAV genome by Southern blots and polymerase chain reaction (PCR), using primers specific for HS core elements and the human β-globin gene. All clones contained a single copy of the rAAV genome in the chromosome, however, some of them had a rearranged proviral genome. In five clones with a single unrearranged rAAV genome for each rAAV construct, we assayed the expression of the human b-globin gene relative to the endogenous mouse β(maj)-globin gene, using quantitative reverse transcriptase (RT)-PCR. In clones infected with rHS432, the expression level of the human β-globin gene ranged from 51.6% to 765.6% of that in the mouse β(maj)globin gene. Likewise, in rHS43, the expression level ranged from 36.7% to 259.0%; in rHS42, from 47.8% to 207.0%; in rHS32, from 47.9% to 105.4%; and in rHS2, from 6.1% to 172.1%, indicating a high variability of expression level in clones infected with recombinant virus lacking the insulator. In contrast, in clones infected with rIns/HS2/Ins, the range of expression of the human β-globin gene ranged from 52.8% to 58.3% of that in the mouse β(maj)globin gene. These results indicate that the insulator functioned dramatically to reduce the variability of transgene expression due to the position effect. This insulator-rAAV vector system holds promise to provide a constant level of transgene expression for gene therapy, regardless of the insertion sites on the chromosome..
41. Ohno Mizuki, T. Ikemura, Triplex DNA in human interphase nuclei and its function, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 44, 12 Suppl, 1757-1763, 1999.
42. T. Fukagawa, Ohno Mizuki, T. Ikemura, Biological significance of human chromosome bands, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 41, 15 Suppl, 2277-2287, 1996.11.