研究者データベース

大野 円実(オオノ マルミ)
人獣共通感染症国際共同研究所 生物製剤研究開発部門
助教

基本情報

所属

  • 人獣共通感染症国際共同研究所 生物製剤研究開発部門

職名

  • 助教

学位

  • 獣医学博士(2012年06月 北海道大学)

科研費研究者番号

  • 50794202

ORCID ID

J-Global ID

職歴

  • 2021年04月 - 現在 北海道大学 人獣共通感染症国際共同研究所 助教
  • 2020年07月 - 現在 北海道大学 人獣共通感染症リサーチセンター 助教
  • 2016年04月 - 2020年06月 北海道大学 人獣共通感染症リサーチセンター 博士研究員
  • 2012年08月 - 2016年04月 アメリカ国立衛生研究所
  • 2012年06月 - 2012年08月 北海道大学 大学院獣医学研究科 学術振興会特別研究員(PD)
  • 2010年04月 - 2012年06月 北海道大学 大学院獣医学研究科 学術振興会特別研究員(DC1)

学歴

  • 2009年04月 - 2012年06月   北海道大学   大学院獣医学研究科
  • 2003年04月 - 2009年03月   北海道大学   獣医学部   獣医学科

研究活動情報

論文

  • Divyavani Gowda, Yonghan Li, Siddabasave Gowda B Gowda, Marumi Ohno, Hitoshi Chiba, Shu-Ping Hui
    Analytical and bioanalytical chemistry 2022年07月16日 
    Short-chain fatty acids (SCFAs) are the end products of the fermentation of complex carbohydrates by the gut microbiota. Although SCFAs are recognized as important markers to elucidate the link between gut health and disease, it has been difficult to analyze SCFAs with mass spectrometry technologies due to their poor ionization efficiency and high volatility. Here, we present a novel and sensitive method for the quantification of SCFAs, including C2-C6 SCFAs and their hydroxy derivatives, by liquid chromatography/tandem mass spectrometry (LC-MS/MS) upon N,N-dimethylethylenediamine (DMED) derivatization with a run time of 10 min. Moreover, the quantification method of DMED-derivatized SCFAs in intestinal contents using isotope-labeled internal standards was also established. The method validation was performed by analyzing spiked intestinal samples; the limits of detection and quantification of SCFAs with this method were found to be 0.5 and 5 fmol, respectively; the recovery was greater than 80% and good linearity (0.9932 to 0.9979) of calibration curves was obtained over the range from 0.005 to 5000 pmol/μL; the intraday and interday precisions were achieved in the range of 1-5%. Furthermore, the validated method was applied to analyze SCFAs in the cecum and colon contents of mice infected with the influenza virus. The results showed that the concentration of most of the SCFAs tested here decreased significantly in a time-dependent manner after the infection, suggesting a possibility that SCFAs in intestinal samples could be used as severe disease markers. Overall, we here successfully developed a simple, fast, and sensitive method for SCFA analysis by LC-MS/MS combined with DMED derivatization. The method for the quantification of SCFAs will be a useful tool for both basic research and clinical studies.
  • Masanori Shiohara, Saori Suzuki, Shintaro Shichinohe, Hirohito Ishigaki, Misako Nakayama, Naoki Nomura, Masashi Shingai, Toshiki Sekiya, Marumi Ohno, Sayaka Iida, Naoko Kawai, Mamiko Kawahara, Junya Yamagishi, Kimihito Ito, Ryotarou Mitsumata, Tomio Ikeda, Kenji Motokawa, Tomoyoshi Sobue, Hiroshi Kida, Kazumasa Ogasawara, Yasushi Itoh
    Vaccine 40 30 4026 - 4037 2022年06月26日 [査読有り][通常論文]
     
    The All-Japan Influenza Vaccine Study Group has been developing a more effective vaccine than the current split vaccines for seasonal influenza virus infection. In the present study, the efficacy of formalin- and/or β-propiolactone-inactivated whole virus particle vaccines for seasonal influenza was compared to that of the current ether-treated split vaccines in a nonhuman primate model. The monovalent whole virus particle vaccines or split vaccines of influenza A virus (H1N1) and influenza B virus (Victoria lineage) were injected subcutaneously into naïve cynomolgus macaques twice. The whole virus particle vaccines induced higher titers of neutralizing antibodies against H1N1 influenza A virus and influenza B virus in the plasma of macaques than did the split vaccines. At challenge with H1N1 influenza A virus or influenza B virus, the virus titers in nasal swabs and the increases in body temperatures were lower in the macaques immunized with the whole virus particle vaccine than in those immunized with the split vaccine. Repertoire analyses of immunoglobulin heavy chain genes demonstrated that the number of B-lymphocyte subclones was increased in macaques after the 1st vaccination with the whole virus particle vaccine, but not with the split vaccine, indicating that the whole virus particle vaccine induced the activation of vaccine antigen-specific B-lymphocytes more vigorously than did the split vaccine at priming. Thus, the present findings suggest that the superior antibody induction ability of the whole virus particle vaccine as compared to the split vaccine is attributable to its stimulatory properties on the subclonal differentiation of antigen-specific B-lymphocytes.
  • Chimuka Handabile, Toshiki Sekiya, Naoki Nomura, Marumi Ohno, Tomomi Kawakita, Masashi Shingai, Hiroshi Kida
    Vaccines 10 5 2022年05月19日 
    Despite the use of vaccines, seasonal influenza remains a risk to public health. We previously proposed the inactivated whole virus particle vaccine (WPV) as an alternative to the widely used split vaccine (SV) for the control of seasonal and pandemic influenza based on the superior priming potency of WPV to that of SV. In this study, we further examined and compared the immunological potency of monovalent WPV and SV of A/California/7/2009 (X-179A) (H1N1) pdm09 (CA/09) to generate immune responses against heterologous viruses, A/Singapore/GP1908/2015 (IVR-180) (H1N1) pdm09 (SG/15), and A/duck/Hokkaido/Vac-3/2007 (H5N1) (DH/07) in mice. Following challenge with a lethal dose of heterologous SG/15, lower virus titer in the lungs and milder weight loss were observed in WPV-vaccinated mice than in SV-vaccinated ones. To investigate the factors responsible for the differences in the protective effect against SG/15, the sera of vaccinated mice were analyzed by hemagglutination-inhibition (HI) and neuraminidase-inhibition (NI) assays to evaluate the antibodies induced against viral hemagglutinin (HA) and neuraminidase (NA), respectively. While the two vaccines induced similar levels of HI antibodies against SG/15 after the second vaccination, only WPV-vaccinated mice induced significantly higher titers of NI antibodies against the strain. Furthermore, given the significant elevation of NI antibody titers against DH/07, an H5N1 avian influenza virus, WPV was also demonstrated to induce NA-inhibiting antibodies that recognize NA of divergent strains. This could be explained by the higher conservation of epitopes of NA among strains than for HA. Taking these findings together, NA-specific antibodies induced by WPV may have contributed to better protection from infection with heterologous influenza virus SG/15, compared with SV. The present results indicate that WPV is an effective vaccine for inducing antibodies against both HA and NA of heterologous viruses and may be a useful vaccine to conquer vaccine strain mismatch.
  • Yasutake Yanagihara, Sharon Y A M Villanueva, Naoki Nomura, Marumi Ohno, Toshiki Sekiya, Chimuka Handabile, Masashi Shingai, Hideaki Higashi, Shin-Ichi Yoshida, Toshiyuki Masuzawa, Nina G Gloriani, Mitsumasa Saito, Hiroshi Kida
    Microbiology spectrum 10 2 e0215721  2022年03月15日 
    Leptospirosis is a zoonotic disease caused by infection with pathogenic leptospires. Consistent with recent studies by other groups, leptospires were isolated from 89 out of 110 (80.9%) soil or water samples from varied locations in the Philippines in our surveillance study, indicating that leptospires might have a life cycle that does not involve animal hosts. However, despite previous work, it has not been confirmed whether leptospires multiply in the soil environment under various experimental conditions. Given the fact that the case number of leptospirosis is increased after flood, we hypothesized that waterlogged soil, which mimics the postflooding environment, could be a suitable condition for growing leptospires. To verify this hypothesis, pathogenic and saprophytic leptospires were seeded in the bottles containing 2.5 times as much water as soil, and bacterial counts in the bottles were measured over time. Pathogenic and saprophytic leptospires were found to increase their number in waterlogged soil but not in water or soil alone. In addition, leptospires were reisolated from soil in closed tubes for as long as 379 days. These results indicate that leptospires are in a resting state in the soil and are able to proliferate with increased water content in the environment. This notion is strongly supported by observations that the case number of leptospirosis is significantly higher in rainy seasons and increased after flood. Therefore, we reached the following conclusion: environmental soil is a potential reservoir of leptospires. IMPORTANCE Since research on Leptospira has focused on pathogenic leptospires, which are supposed to multiply only in animal hosts, the life cycle of saprophytic leptospires has long been a mystery. This study demonstrates that both pathogenic and saprophytic leptospires multiply in the waterlogged soil, which mimics the postflooding environment. The present results potentially explain why leptospirosis frequently occurs after floods. Therefore, environmental soil is a potential reservoir of leptospires and leptospirosis is considered an environment-borne as well as a zoonotic disease. This is a significant report to reveal that leptospires multiply under environmental conditions, and this finding leads us to reconsider the ecology of leptospires.
  • Marumi Ohno, Michihito Sasaki, Yasuko Orba, Toshiki Sekiya, Md Abdul Masum, Osamu Ichii, Tatsuya Sawamura, Akemi Kakino, Yasuhiko Suzuki, Hiroshi Kida, Hirofumi Sawa, Masashi Shingai
    Viruses 13 11 2021年11月 
    Systemic symptoms have often been observed in patients with coronavirus disease 2019 (COVID-19) in addition to pneumonia, however, the details are still unclear due to the lack of an appropriate animal model. In this study, we investigated and compared blood coagulation abnormalities and tissue damage between male Syrian hamsters of 9 (young) and over 36 (aged) weeks old after intranasal infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite similar levels of viral replication and inflammatory responses in the lungs of both age groups, aged but not young hamsters showed significant prolongation of prothrombin time and prominent acute kidney damage. Moreover, aged hamsters demonstrated increased intravascular coagulation time-dependently in the lungs, suggesting that consumption of coagulation factors causes prothrombin time prolongation. Furthermore, proximal urinary tract damage and mesangial matrix expansion were observed in the kidneys of the aged hamsters at early and later disease stages, respectively. Given that the severity and mortality of COVID-19 are higher in elderly human patients, the effect of aging on pathogenesis needs to be understood and should be considered for the selection of animal models. We, thus, propose that the aged hamster is a good small animal model for COVID-19 research.
  • Divyavani Gowda, Marumi Ohno, Siddabasave Gowda B Gowda, Hitoshi Chiba, Masashi Shingai, Hiroshi Kida, Shu-Ping Hui
    Scientific reports 11 1 20161 - 20161 2021年10月11日 
    Influenza remains a world-wide health concern, causing 290,000-600,000 deaths and up to 5 million cases of severe illnesses annually. Noticing the host factors that control biological responses, such as inflammatory cytokine secretion, to influenza virus infection is important for the development of novel drugs. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite and has essential biological functions in inflammation. However, the kinetic effects of influenza virus infection on physiological S1P levels and their signaling in multiple tissues remain unknown. In this study, we utilized a mouse model intranasally infected with 50 or 500 plaque forming units (PFU) of A/Puerto Rico/8/34 (H1N1; PR8) virus to investigate how S1P levels and expression of its regulating factors are affected by influenza virus infection by the liquid-chromatography/mass spectrometry and real-time PCR, respectively. The S1P level was significantly high in the plasma of mice infected with 500 PFU of the virus than that in control mice at 6 day-post-infection (dpi). Elevated gene expression of sphingosine kinase-1 (Sphk1), an S1P synthase, was observed in the liver, lung, white adipose tissue, heart, and aorta of infected mice. This could be responsible for the increased plasma S1P levels as well as the decrease in the hepatic S1P lyase (Sgpl1) gene in the infected mice. These results indicate modulation of S1P-signaling by influenza virus infection. Since S1P regulates inflammation and leukocyte migration, it must be worth trying to target this signaling to control influenza-associated symptoms.
  • Marumi Ohno, Akemi Kakino, Toshiki Sekiya, Naoki Nomura, Masashi Shingai, Tatsuya Sawamura, Hiroshi Kida
    Scientific reports 11 1 15675 - 15675 2021年08月03日 
    Although coagulation abnormalities, including microvascular thrombosis, are thought to contribute to tissue injury and single- or multiple-organ dysfunction in severe influenza, the detailed mechanisms have yet been clarified. This study evaluated influenza-associated abnormal blood coagulation utilizing a severe influenza mouse model. After infecting C57BL/6 male mice with intranasal applications of 500 plaque-forming units of influenza virus A/Puerto Rico/8/34 (H1N1; PR8), an elevated serum level of prothrombin fragment 1 + 2, an indicator for activated thrombin generation, was observed. Also, an increased gene expression of oxidized low-density lipoprotein (LDL) receptor-1 (Olr1), a key molecule in endothelial dysfunction in the progression of atherosclerosis, was detected in the aorta of infected mice. Body weight decrease, serum levels of cytokines and chemokines, viral load, and inflammation in the lungs of infected animals were similar between wild-type and Olr1 knockout (KO) mice. In contrast, the elevation of prothrombin fragment 1 + 2 levels in the sera and intravascular thrombosis in the lungs by PR8 virus infection were not induced in KO mice. Collectively, the results indicated that OLR1 is a critical host factor in intravascular thrombosis as a pathogeny of severe influenza. Thus, OLR1 is a promising novel therapeutic target for thrombosis during severe influenza.
  • Siddabasave Gowda B Gowda, Divyavani Gowda, Marumi Ohno, Chongsheng Liang, Hitoshi Chiba, Shu-Ping Hui
    Journal of the American Society for Mass Spectrometry 32 8 2196 - 2205 2021年06月25日 
    Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of endogenous lipids with promising physiological functions in mammals. We previously introduced a new type of lipids to this family called short-chain fatty acid esters of hydroxy fatty acids (SFAHFAs), branching specific to the C2 carbon of a long-chain fatty acid (≥C20). In this study, we discovered a homologous series of SFAHFAs comprising C16-C26 hydroxy fatty acids esterified with short-chain fatty acids (C2-C5) in mouse colon contents. The detected SFAHFAs were characterized by high-resolution mass spectrometry with MSn analysis. The double-bond position of monounsaturated SFAHFAs was determined by the epoxidation reaction of samples with m-chloroperoxybenzoic acid and their MSn analysis. Further, the measurement of SFAHFA concentration in the colon contents of mice infected with influenza A/Puerto Rico/8/34 (H1N1; PR8) virus revealed a significant increase in their levels compared to native control. A strong correlation was observed between hydroxy fatty acid and SFAHFAs. Detection, characterization, and profiling of these new SFAHFA levels in relation with pandemic H1N1; PR8 influenza virus will contribute to the in-depth study of their function and metabolism.
  • Masashi Shingai, Naoki Nomura, Toshiki Sekiya, Marumi Ohno, Daisuke Fujikura, Chimuka Handabile, Ryosuke Omori, Yuki Ohara, Tomohiro Nishimura, Masafumi Endo, Kazuhiko Kimachi, Ryotarou Mitsumata, Tomio Ikeda, Hiroki Kitayama, Hironori Hatanaka, Tomoyoshi Sobue, Fumihito Muro, Saori Suzuki, Cong Thanh Nguyen, Hirohito Ishigaki, Misako Nakayama, Yuya Mori, Yasushi Itoh, Marios Koutsakos, Brendon Y Chua, Katherine Kedzierska, Lorena E Brown, David C Jackson, Kazumasa Ogasawara, Yoichiro Kino, Hiroshi Kida
    Vaccine 39 29 3940 - 3951 2021年06月 [査読有り]
     
    Current detergent or ether-disrupted split vaccines (SVs) for influenza do not always induce adequate immune responses, especially in young children. This contrasts with the whole virus particle vaccines (WPVs) originally used against influenza that were immunogenic in both adults and children but were replaced by SV in the 1970s due to concerns with reactogenicity. In this study, we re-evaluated the immunogenicity of WPV and SV, prepared from the same batch of purified influenza virus, in cynomolgus macaques and confirmed that WPV is superior to SV in priming potency. In addition, we compared the ability of WPV and SV to induce innate immune responses, including the maturation of dendritic cells (DCs) in vitro. WPV stimulated greater production of inflammatory cytokines and type-I interferon in immune cells from mice and macaques compared to SV. Since these innate responses are likely triggered by the activation of pattern recognition receptors (PRRs) by viral RNA, the quantity and quality of viral RNA in each vaccine were assessed. Although the quantity of viral RNA was similar in the two vaccines, the amount of viral RNA of a length that can be recognized by PRRs was over 100-fold greater in WPV than in SV. More importantly, 1000-fold more viral RNA was delivered to DCs by WPV than by SV when exposed to preparations containing the same amount of HA protein. Furthermore, WPV induced up-regulation of the DC maturation marker CD86 on murine DCs, while SV did not. The present results suggest that the activation of antigen-presenting DCs, by PRR-recognizable viral RNA contained in WPV is responsible for the effective priming potency of WPV observed in naïve mice and macaques. WPV is thus recommended as an alternative option for seasonal influenza vaccines, especially for children.
  • Naoki Nomura, Keita Matsuno, Masashi Shingai, Marumi Ohno, Toshiki Sekiya, Ryosuke Omori, Yoshihiro Sakoda, Robert G. Webster, Hiroshi Kida
    Virology 557 55 - 61 2021年05月 [査読有り]
     
    Genetic reassortment of influenza A viruses through cross-species transmission contributes to the generation of pandemic influenza viruses. To provide information on the ecology of influenza viruses, we have been conducting a global surveillance of zoonotic influenza and establishing an influenza virus library. Of 4580 influenza virus strains in the library, 3891 have been isolated from over 70 different bird species. The remaining 689 strains were isolated from humans, pigs, horses, seal, whale, and the environment. Phylogenetic analyses of the HA genes of the library isolates demonstrate that the library strains are distributed to all major known clusters of the H1, H2 and H3 subtypes of HA genes that are prevalent in humans. Since past pandemic influenza viruses are most likely genetic reassortants of zoonotic and seasonal influenza viruses, a vast collection of influenza A virus strains from various hosts should be useful for vaccine preparation and diagnosis for future pandemics.
  • Marios Koutsakos, Toshiki Sekiya, Brendon Y Chua, Thi Hoang Oanh Nguyen, Adam K Wheatley, Jennifer A Juno, Marumi Ohno, Naoki Nomura, Yuki Ohara, Tomohiro Nishimura, Masafumi Endo, Saori Suzuki, Hirohito Ishigaki, Misako Nakayama, Cong T Nguyen, Yasushi Itoh, Masashi Shingai, Kazumasa Ogasawara, Yoichiro Kino, Stephen J Kent, David C Jackson, Lorena E Brown, Hiroshi Kida, Katherine Kedzierska
    Immunology & Cell Biology 99 1 97 - 106 2020年09月07日 [査読有り][通常論文]
     
    Influenza remains a significant global public health burden, despite substantial annual vaccination efforts against circulating virus strains. As a result, novel vaccine approaches are needed to generate long-lasting and universal broadly cross-reactive immunity against distinct influenza virus strains and subtypes. Several new vaccine candidates are currently under development and/or in clinical trials. The successful development of new vaccines requires testing in animal models, other than mice, which capture the complexity of the human immune system. Importantly, following vaccination or challenge, the assessment of adaptive immunity at the antigen-specific level is particularly informative. In this study, using peripheral blood mononuclear cells (PBMCs) from cynomolgus macaques, we describe detection methods and in-depth analyses of influenza virus-specific B cells by recombinant hemagglutinin probes and flow cytometry, as well as the detection of influenza virus-specific CD8+ and CD4+ T cells by stimulation with live influenza A virus and intracellular cytokine staining. We highlight the potential of these assays to be used with PBMCs from other macaque species, including rhesus macaques, pigtail macaques and African green monkeys. We also demonstrate the use of a human cytometric bead array kit in detecting inflammatory cytokines and chemokines from cynomolgus macaques to assess cytokine/chemokine milieu. Overall, the detection of influenza virus-specific B and T cells, together with inflammatory responses, as described in our study, provides useful insights for evaluating novel influenza vaccines. Our data deciphering immune responses toward influenza viruses can be also adapted to understanding immunity to other infections or vaccination approaches in macaque models.
  • Marumi Ohno, Toshiki Sekiya, Naoki Nomura, Taku ji Daito, Masashi Shingai, Hiroshi Kida
    Scientific Reports 10 1 10879 - 10879 2020年06月 [査読有り][通常論文]
     
    Although the severity of influenza virus infections has been associated with host energy metabolism, the related mechanisms have not yet been clarified. Here we examined the effects of influenza virus infection on host energy metabolism in mice. After infecting mice with intranasal applications of 500 plaque-forming units of A/Puerto Rico/8/34 (H1N1; PR8) virus, the serum levels of most intermediates in the tricarboxylic acid (TCA) cycle and related metabolic pathways were significantly reduced. These data suggest that substrate supply to the TCA cycle is reduced under these conditions, rather than specific metabolic reactions being inhibited. Then, we focused on glucose and fatty acid metabolism that supply substrates to the TCA cycle. Akt phosphorylation following insulin injections was attenuated in the livers of PR8 virus-infected mice. Furthermore, glucose tolerance tests revealed that the PR8 virus-infected mice showed higher blood glucose levels than the vehicle-inoculated control mice. These results suggest that influenza virus infection impairs insulin signaling, which regulates glucose uptake. However, increases in the hepatic expressions of fatty acid-metabolizing enzymes suggest that fatty acids accumulate in liver cells of infected mice. Collectively, our data indicate that influenza virus infection dysregulates host energy metabolism. This line of investigation provides novel insights into the pathogenesis of influenza.
  • Toshiki Sekiya, Edin J Mifsud, Marumi Ohno, Naoki Nomura, Mayumi Sasada, Daisuke Fujikura, Takuji Daito, Masashi Shingai, Yuki Ohara, Tomohiro Nishimura, Masafumi Endo, Ryotarou Mitsumata, Tomio Ikeda, Hironori Hatanaka, Hiroki Kitayama, Kenji Motokawa, Tomoyoshi Sobue, Saori Suzuki, Yasushi Itoh, Lorena E Brown, Kazumasa Ogasawara, Yoichiro Kino, Hiroshi Kida
    Vaccine 37 15 2158 - 2166 2019年04月 [査読有り][通常論文]
     
    In contrast to current ether- or detergent-disrupted “split” vaccines (SVs) for influenza, inactivated whole influenza virus particle vaccines (WPVs) retain the original virus structure and components and as such may confer similar immunity to natural infection. In a collaboration between academia and industry, the potential of WPV as a new seasonal influenza vaccine was investigated. Each of the four seasonal influenza vaccine manufacturers in Japan prepared WPVs and SVs from the same batches of purified influenza virus. Both mice and monkeys vaccinated with the WPVs exhibited superior immune responses to those vaccinated with the corresponding SVs. Vaccination with A/California/07/2009 (H1N1) WPV enabled mice to survive a lethal challenge dose of homologous virus whereas those vaccinated with SV succumbed to infection within 6 days. Furthermore, mice vaccinated with WPV induced substantial numbers of multifunctional CD8 + T cells, important for control of antigenically drifted influenza virus strains. In addition, cytokines and chemokines were detected at early time points in the sera of mice vaccinated with WPV but not in those animals vaccinated with SV. These results indicate that WPVs induce enhanced innate and adaptive immune responses compared to equivalent doses of SVs. Notably, WPV at one fifth of the dose of SV was able to induce potent immunity with limited production of IL-6, one of the pyrogenic cytokines. We thus propose that WPVs with balanced immunogenicity and safety may set a new global standard for seasonal influenza vaccines.
  • Marumi Ohno, Rick Moore, Page Myers, Masahiko Negishi
    SHOCK 50 2 248 - 254 2018年08月 [査読有り][通常論文]
     
    Co-chaperone cytoplasmic constitutive active/androstane receptor retention protein (CCRP), a member of heat shock protein (HSP) 40, was first characterized to retain a nuclear-destined protein in the cytoplasm. Here we have used CCRP KO mice and demonstrated that CCRP suppresses lipopolysaccharide (LPS)-induced cardiac toxicity in mice. LPS treatment decreased heart rates in CCRP KO mice, but not in wild-type (WT) mice. In addition, LPS-treated KO mice showed reduced fraction shortening, an indicator of ventricular contractile function, to a greater degree than WT mice did. Rat cardiomyocyte-derived H9c2 cells, in which CCRP is not expressed, were used to examine a cell signal through which CCRP suppressed LPS-induced cardiac toxicity. Overexpression of CCRP prevented p65, a nuclear factor κB (NFκB) subunit, from accumulating in the nucleus after LPS treatment. As observed with H9c2 cells, nuclear accumulation of p65 was found to be higher in the hearts of KO mice than WT mice after LPS treatment. Furthermore, induction of TNFα by LPS was markedly suppressed by CCRP in H9c2 cells as well as in LPS-treated mouse serum. In supporting the notion that CCRP repressed the LPS-induced NFκB signaling, pretreatment with pyrrolidinedithiocarbamate, an NFκB signaling inhibitor, or anti-TNF-α antibody before LPS treatment restored heart rates decreased in KO mice after LPS treatment in a dose-dependent manner. Our present study characterized a novel physiological role of CCRP in protecting cardiac functions through the inhibition of NFκB signaling.
  • Marumi Ohno, Masahiko Negishi
    Nuclear Receptor Signaling 15 155076291880107 - 155076291880107 2018年01月 [査読有り][通常論文]
     
    The N-terminal domain (NTD) of nuclear receptor superfamily members has been recently reported to regulate functions of the receptor through the interaction between the NTD and the C-terminal ligand binding domain (LBD), so-called an N/C interaction. Although this N/C interaction has been demonstrated in various nuclear receptors, eg, androgen receptor, this concept has not been observed in glucocorticoid receptor (GR). We hypothesized that GR requires its co-chaperone CCRP (cytoplasmic constitutive active/androstane receptor retention protein) to form a stable N/C interaction. This hypothesis was examined by co-immunoprecipitation assays using GR fragments overexpressing COS-1 cell lysate. Here, we demonstrated that GR undergoes the N/C interaction between the 26VMDFY30 motif in the NTD and the LBD. More importantly, co-chaperone CCRP is now found to induce this interaction. By the fact that a negative charge at Y30 disrupts this interaction, this residue, a potential phosphorylation site, was indicated to regulate the GR N/C interaction critically. Utilizing Y30F and Y30E mutants as N/C interacting and noninteracting forms of GR, respectively, a 2-dimensional blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to examine whether or not the N/C interaction regulated formation of GR complexes. A cDNA microarray analysis was performed with COS-1 cells expressing Y30F or Y30E. We will present experimental data to demonstrate that CCRP is essential for GR to form the N/C interaction and will discuss its implications in GR functions.
  • Yoshihiro NISHIYAMA, Shouta M.M. NAKAYAMA, Kensuke P. WATANABE, Yusuke K. KAWAI, Marumi OHNO, Yoshinori IKENAKA, Mayumi ISHIZUKA
    Journal of Veterinary Medical Science 78 4 675 - 680 2016年 [査読有り][通常論文]
     
    Rat cytochrome P450 (CYP) exhibits inter-strain differences, but their analysis has been scattered across studies under different conditions. To identify these strain differences in CYP more comprehensively, mRNA expression, protein expression and metabolic activity among Wistar (WI), Sprague Dawley (SD), Dark Agouti (DA) and Brown Norway (BN) rats were compared. The mRNA level and enzymatic activity of CYP1A1 were highest in SD rats. The rank order of Cyp3a2 mRNA expression mirrored its protein expression, i.e., DA>BN>SD>WI, and was similar to the CYP3A2-dependent warfarin metabolic activity, i.e., DA>SD>BN>WI. These results suggest that the strain differences in CYP3A2 enzymatic activity are caused by differences in mRNA expression. Cyp2b1 mRNA levels, which were higher in DA rats, did not correlate with its protein expression or enzymatic activity. This suggests that the strain differences in enzymatic activity are not related to Cyp2b1 mRNA expression. In conclusion, WI rats tended to have the lowest CYP1A1, 2B1 and 3A2 mRNA expression, protein expression and enzymatic activity among the strains. In addition, SD rats had the highest CYP1A1 mRNA expression and activity, while DA rats had higher CYP2B1 and CYP3A2 mRNA and protein expression. These inter-strain differences in CYP could influence pharmacokinetic considerations in preclinical toxicological studies.
  • Wageh S. Darwish, Shouta M. M. Nakayama, Yuumi Itotani, Marumi Ohno, Yoshinori Ikenaka, Mayumi Ishizuka
    Journal of Food Science 80 7 T1627 - T1632 2015年07月 [査読有り][通常論文]
     
    Heterocyclic amines get entry into human body mainly through ingestion of pan-fried meats cooked at high temperatures. Exposure of the gastrointestinal tract (GIT) to ingested xenobiotics prior to delivery to the liver may lead to metabolic activation, which may explain the high incidence of GIT carcinogenesis. Therefore, this study investigated the mutagenic activation of 2 heterocyclic amines, 2-aminoanthracene (2-AA) and 3-amino-1-methyl-5H-prydo[4,3-b]indole (Trp-P-2), in the GIT of rats. In addition, the constitutive mRNA expression profiles of xenobiotic-metabolizing enzymes (XMEs) in the GIT of rats were examined. Metabolic activation of 2-AA was detected in all GIT tissues except the duodenum and rectum, and it was detected at high levels in the ileum and cecum. Furthermore, we revealed high metabolic activation of 2-AA and Trp-P-2 in the jejunum. The mRNA expression of phase I and II enzymes in rat GIT corresponded with their mutagenic activation ability. In conclusion, our results suggest that different expression levels of XME among GIT tissues may contribute to the tissue-specific differences in metabolic activation of xenobiotics such as heterocyclic amines in rats. Practical Application: This study declares mutagenic activation of 2 heterocyclic amines namely 2-aminoanthracene (2-AA) and 3-amino-1-methyl-5H-prydo[4,3-b]indole (Trp-P-2), in the gastrointestinal tract (GIT) of rats. In addition, results obtained in this study suggest that GIT tissue-specific expression of xenobiotic metabolizing enzymes may contribute to the tissue-specific mutagenesis/carcinogenesis.
  • Saki Gotoh, Marumi Ohno, Kouichi Yoshinari, Masahiko Negishi, Kaname Kawajiri
    Cytochrome P450: Structure, Mechanism, and Biochemistry, Fourth Edition 787 - 812 2015年01月01日 
    Nuclear receptors such as aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) were first characterized as ligand-bound transcription factors that directly bind their response DNA sequences to regulate cytochrome P450 (CYP) genes. This simple ligand mechanism has evolved into more complex mechanisms in order for us to understand the high specificity and enormous diversity of regulation mediated at various stages by nuclear receptors, including intracellular localization, proteasome-mediated degradation, chromatin-based arrangement, epigenetic modifications, selective recruitment of co-regulators, and cross talk between nuclear receptors. Cell signaling has become a critical regulator that determines various nuclear receptor actions. These nuclear receptors, particularly in CAR, can now be considered as cell signal-regulated nuclear receptors. Through studies of their induction, the cytochrome P450s, once thought to catalyze the oxidation of xenobiotics and therapeutic drugs, are now found to regulate liver functions by metabolizing key endogenous stimuli such as cholesterol, bile acids, and steroid hormones. Recent research on nuclear receptors and CYP induction is creating excitement and is redefining their functions and mechanisms into new frontiers.
  • Marumi Ohno, Tomohiko Kanayama, Rick Moore, Manas Ray, Masahiko Negishi
    PLoS ONE 9 12 e115663 - e115663 2014年12月26日 [査読有り][通常論文]
     
    Cytoplasmic constitutive active/androstane receptor (CAR) retention protein (CCRP and also known as DNAJC7) is a co-chaperone previously characterized to retain nuclear receptor CAR in the cytoplasm of HepG2 cells. Here we have produced CCRP knockout (KO) mice and demonstrated that CCRP regulates CAR at multiple steps in activation of the cytochrome (Cyp) 2b10 gene in liver: nuclear accumulation, RNA polymerase II recruitment and epigenetic modifications. Phenobarbital treatment greatly increased nuclear CAR accumulation in the livers of KO males as compared to those of wild type (WT) males. Despite this accumulation, phenobarbital-induced activation of the Cyp2b10 gene was significantly attenuated. In ChIP assays, a CAR/retinoid X receptor-α (RXRα) heterodimer binding to the Cyp2b10 promoter was already increased before phenobarbital treatment and further pronounced after treatment. However, RNA polymerase II was barely recruited to the promoter even after phenobarbital treatment. Histone H3K27 on the Cyp2b10 promoter was de-methylated only after phenobarbital treatment in WT but was fully de-methylated before treatment in KO males. Thus, CCRP confers phenobarbital-induced de-methylation capability to the promoter as well as the phenobarbital responsiveness of recruiting RNA polymerase II, but is not responsible for the binding between CAR and its cognate sequence, phenobarbital responsive element module. In addition, KO males developed steatotic livers and increased serum levels of total cholesterol and high density lipoprotein in response to fasting. CCRP appears to be involved in various hepatic regulations far beyond CAR-mediated drug metabolism.
  • Marumi Ohno, Wageh Sobhy Darwish, Yoshinori Ikenaka, Wataru Miki, Shoichi Fujita, Mayumi Ishizuka
    Food Chemistry 130 2 356 - 361 2012年01月 [査読有り][通常論文]
     
    The xanthophyll, astaxanthin (Ax), strongly induces cytochrome P450 (CYP) 1A-dependent activity in rats and mice, via increased transcription of Cyp1a1 mRNA through an undetermined mechanism. In this study, we have investigated how aryl hydrocarbon receptor (AhR)-related events are affected by the addition of Ax-rich crude extract from Haematococcus pluvialis in H4IIE rat hepatoma cells. The expression of Cyp1a1 mRNA was significantly increased by exposure to H. pluvialis extract (100 μmol/l, 17.6-fold). The highest level of Cyp1a1 mRNA was observed after a 6-h exposure to H. pluvialis extract (25-fold), but 12- and 24-h exposures resulted in lower levels of induction. Green fluorescence protein-tagged AhR was translocated into the nucleus in COS-7 cells after a 10 min exposure to Ax. Moreover, H. pluvialis extract containing Ax activated the human CYP1A1 enhancer region in a luciferase assay, and a gel shift assay showed that nuclear proteins stimulated by H. pluvialis extract can interact specifically with xenobiotic responsive elements (XREs) in the CYP1A1 promoter. Together these results indicate the possibility that Ax, contained in H. pluvialis extract, caused the induction of Cyp1a1 mRNA through the activation and translocation of an XRE-binding form of AhR. © 2011 Elsevier Ltd. All rights reserved.
  • Marumi Ohno, Yoshinori Ikenaka, Mayumi Ishizuka
    Journal of Biochemical and Molecular Toxicology 26 1 16 - 22 2012年01月 [査読有り][通常論文]
     
    Sudan dyes possess a high affinity to the aryl hydrocarbon receptor (AHR) and potently induce its target genes, such as cytochrome P450 (CYP) 1A1, through unknown mechanisms. We investigated a detailed event occurring in cells after binding of Sudan dye to AHR in HepG2 cells. Treatment with 10 μM Sudan III caused rapid translocation of AHR into the nucleus and increased expression levels of human CYP1A1 mRNA by approximately 20-fold after 16 and 24 h. The transactivation was due to the activation of a region located at -1137 to +59 bp from CYP1A1, in particular, four xenobiotic responsive elements (XREs) existing in the region. AHR and the Ah receptor nuclear translocator interacted with XRE sequences in a gel shift assay using nuclear extract from Sudan III--treated HepG2 cells. Moreover, we suggest that constitutive androstane receptor could modify CYP1A1 transactivation by Sudan III. © 2012 Wiley Periodicals, Inc.
  • Marumi Ohno, Yoshinori Ikenaka, Mayumi Ishizuka
    Biochemical and Biophysical Research Communications 417 1 484 - 489 2012年01月 [査読有り][通常論文]
     
    Aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT) are well-conserved transcription factors among species. However, there are a very limited number of reports on the physiological function of AHR, particularly on the regulation of AHR by endogenous compounds. We hence investigated the effects of all-trans retinoic acid (atRA) on cytochrome P450 (CYP) 1A1 gene transcription as a model of AHR-regulated transcription mechanisms in HepG2 cells, a human hepatoma cell line. Treatment with atRA significantly reduced transactivation and expression of CYP1A1 mRNA to less than half of its control value, and this inhibitory effect was mediated by RARα. The result of chromatin immunoprecipitation assay indicated that treatment with atRA at 1-100. nM drastically inhibited the recruitment of ARNT to DNA regions containing xenobiotic responsive elements. In conclusion, atRA at physiological concentrations could reduce AHR-mediated gene transcription via the inhibition of recruitment of ARNT to relevant DNA regions. © 2011 Elsevier Inc..
  • Marumi Ohno, Wageh S. Darwish, Yoshinori Ikenaka, Wataru Miki, Mayumi Ishizuka
    Food and Chemical Toxicology 49 6 1285 - 1291 2011年06月 [査読有り][通常論文]
     
    Astaxanthin (Ax), a xanthophyll carotenoid, is reported to induce cytochrome P450 (CYP) 1A-dependent activity. CYP1A is one of the most important enzymes participating in phase I metabolism for chemicals, and it can activate various mutagens. To investigate the effect of Ax on the metabolic activation of a typical promutagen, benzo[a]pyrene by CYP1A, we orally administrated Ax-containing oil (100. mg Ax/kg body weight/day for 3. days) to male Wistar rats. In the treated rat liver, expression of CYP1A1 mRNA, protein, and its activity were significantly increased (5.5-, 8.5-, and 2.5-fold, respectively). In contrast, the activities of phase II enzymes (glutathione S-transferase and glucuronosyl-transferase) were not modulated by Ax-containing oil. As a consequence, the mutagenicity of benzo[a]pyrene was more enhanced in Ax-treated rats, compared with controls in the Ames assay. On the other hand, NADPH P450 reductase activity was decreased in liver microsomes from the treated group. This result suggests the possibility that Ax inhibits the electron supply necessary for CYP catalytic activities and decreases CYP1A activity indirectly. In conclusion, Ax-containing oil intake can alter CYP1A-dependent activities through two different mechanisms: (1) induction of CYP1A1 mRNA, protein expression, and activity; and (2) inhibition of the electron supply for the enzyme. © 2011 Elsevier Ltd.
  • Wageh S. Darwish, Yoshinori Ikenaka, Marumi Ohno, Elsaid A. Eldaly, Mayumi Ishizuka
    Food and Chemical Toxicology 48 11 3201 - 3208 2010年11月 [査読有り][通常論文]
     
    Ungulates (deer, cattle and horses) are reported as animal species which show extreme-accelerated metabolism of CYP1A substrates, such as ethoxyresorufin compared to rats. This study was undertaken to investigate whether accumulation of carotenoids is a possible cause for inter-species difference in CYP1A-dependent activity in this group of animals. The relationship between inter-species differences in CYP1A-dependent activity and the accumulated carotenoids and retinoids as candidates of dietary CYP1A inducers in ungulate species was clarified. Interestingly, there were positive correlations between the accumulated carotenoids, such as β-carotene, with both EROD activity and CYP1A protein expression. These correlations were negative with the accumulated retinoids, such as retinol. The β-carotene was major component of carotenoids in ungulates, and known as an inducer of CYP1A. On the other hand, the retinol is reported as the inhibitor of CYP1A. Other factors which affect CYP1A1 expression, such as polycyclic aromatic hydrocarbons, were also analyzed. To cancel the effects of inter-species difference in CYP1A induction signal cascade among these animals, the rat cell line (H4-II-cells) was treated with the extracted carotenoids from the examined animals. In conclusion, carotenoids and retinoids may have direct effects on the inter-species differences in CYP1A-dependent activity and protein expression. © 2010 Elsevier Ltd.
  • Mami Takiguchi, Wageh S. Darwish, Yoshinori Ikenaka, Marumi Ohno, Mayumi Ishizuka
    Toxicological Sciences 116 1 79 - 91 2010年07月 [査読有り][通常論文]
     
    Xenobiotic metabolism in oral tissues, especially in the tongue, has never been reported. In the present study, the metabolic activation/detoxification ability of promutagens in the tongue and the expression levels of related enzymes were investigated. Quantitative PCR analysis of rat tongue demonstrated constitutive messenger RNA (mRNA) expression of numerous drug-metabolizing enzymes. In particular, we detected mRNA, protein expression, and enzymatic activity of cytochrome P450 (CYP)1A1 in the tongue tissue. Metabolic activation of promutagens in the tongue was estimated using benzo[a]pyrene or heterocyclic amines (HCAs), found in cooked meat and tobacco products. Metabolic activation levels ofHCAs in the tongue were comparable to those in the liver. In contrast, the expression levels of glutathione-S-transferase (GST) and uridine diphosphate-glucuronosyltransferase (UGT) in the tongue were considerably lower compared with those in the liver, and as a result, the mutagenic activity in the tongue was not decreased by GST-or UGT-dependent conjugation. Treatment of rats with sudan III, a typical inducer of CYP1A1, resulted in markedly increased CYP1A1 mRNA, protein expressions, and CYP1A-dependent enzymatic and mutagenic activities. In addition, CYP1A1 mRNA expression in carcinoma cells (SAS) was induced by sudan III exposure. In conclusion, mutagenic activation of xenobiotics and an increased risk of cancer in the tongue were observed in this study. Furthermore, ingestion of drug-metabolizing enzyme inducers has the potential to increase the metabolic activation in the tongue tissue and increase the risk of biomolecular attack by promutagens. © The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org.
  • Wageh Sobhy DARWISH, Yoshinori IKENAKA, Elsaid Abozeid ELDALY, Marumi OHNO, Kentaro Q. SAKAMOTO, Shoichi FUJITA, Mayumi ISHIZUKA
    Journal of Veterinary Medical Science 72 5 561 - 566 2010年 [査読有り][通常論文]
     
    The objective of this study was to investigate and characterize the metabolic activities of CYP1A in deer, cattle and horses in comparison to those of rats using ethoxyresorufin O-deethylation (EROD) and methoxyresorufin O-demethylation (MROD) assays. We performed an inhibition study for these activities using anti-rat CYP1A1 antibody and identified that these activities were due to the CYP1A subfamily. Interspecies differences in the CYP1A-dependent activities were highly observed in this study. In particular, we found that the horse had the highest EROD and MROD activities among the examined animal species. In the kinetic analysis, the horses showed the highest Vmax and catalytic efficiency (Vmax/Km), followed by the cattle, deer and rats.
  • Marumi Ohno, Kentaro Q Sakamoto, Mayumi Ishizuka, Shoichi Fujita
    Phytotherapy Research 23 8 1134 - 1139 2009年08月 [査読有り][通常論文]
     
    Polyphenols have been shown to have potent antioxidant activity, and therefore, food containing polyphenols is expected to contribute to the prevention of cancer. However, food contains not only polyphenols but also various other constituents. We used the Ames test to investigate the effects of crude extracts of whole cacao products, which are known to be rich in polyphenols, on the mutagenicity of benzo[a]pyrene (B[a]P) in Salmonella typhimurium strain TA 98 and tert-butyl hydroperoxide (t-BuOOH) in S. typhimurium strain TA 102. B[a]P induces mutagenicity by metabolic activation and t-BuOOH induces it by generation of free radicals. While white chocolate did not modulate the numbers of revertant colonies produced by B[a]P treatment, milk chocolate and cacao powder extracts did. On the other hand, surprisingly, none of the cacao products tested affected the number of revertant colonies when t-BuOOH was used as the mutagen. At maximum concentration (13.25 mg cacao powder/ml), the crude cacao powder extract reduced ethoxyresorufin O-deethylase activity to 17.4% of the control, suggesting that whole cacao products inhibit cytochrome P450 (CYP) 1A activity. In conclusion, inhibition of CYP1A activity by cacao products may prevent DNA damage by reducing metabolic activation of carcinogens. Copyright © 2009 John Wiley & Sons, Ltd.

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