2025/03/01 更新
2025/03/01 更新
１）タンパク質分解系を阻害したストレス誘導性細胞老化のメカニズムを明らかにした（2022年〜）
２）脂肪細胞分化のマスターレギュレーターであるPPARgのマウス新規スプライシングバリアントをクローニングした（2013年〜）
３）海洋プランクトン（カイアシ類）から黄緑色蛍光タンパク質(CpYGFP)と２０種類以上の新規ルシフェラーゼの遺伝子クローニングを行った（2008年〜）
４）加齢にともなうゾウリムシテロメア長の変動を解明し、ゾウリムシテロメラーゼの遺伝子クローニングと新規ゾウリムシ発現ベクターの開発を行った（2001年〜）
博士（農学） （ 筑波大学 ）
ライフサイエンス / 機能生物化学 / 生化学
ライフサイエンス / 応用分子細胞生物学 / 老化
ライフサイエンス / 機能生物化学 / タンパク質
ライフサイエンス / 細胞生物学
ライフサイエンス / 分子生物学
ライフサイエンス / 応用生物化学
ライフサイエンス / 進化生物学
筑波大学 博士課程農学研究科 応用生物化学専攻
1998年4月 - 2002年3月
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日本医科大学 生理学（生体統御学） 講師
2023年10月 - 現在
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国名：日本国
日本医科大学 生理学（生体統御学） 助教
2017年7月 - 2023年9月
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特許庁 審査第一部分析診断
2016年4月 - 2017年6月
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埼玉医科大学 内分泌内科・糖尿病内科 特任研究員
2011年4月 - 2014年12月
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産業技術総合研究所 環境管理技術研究部門 日本産業技術振興協会派遣職員
2010年4月 - 2011年3月
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産業技術総合研究所 健康工学研究センター 日本産業技術振興協会派遣職員
2008年4月 - 2010年3月
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産業技術総合研究所 バイオセラピューティック研究ラボ １号契約職員
2006年10月 - 2008年3月
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産業技術総合研究所 セルダイナミクス研究グループ １号契約職員
2005年10月 - 2006年9月
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インディアナ大学 医学部 ポスドク研究員
2004年8月 - 2005年9月
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独）農業生物資源研究所 蛋白機能研究チーム 開放的融合特別研究員
2002年4月 - 2004年3月
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日本生化学会
2023年6月 - 現在
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日本分子生物学会
2019年8月 - 現在
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日本生理学会
2018年5月 - 現在
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Yasuhiro Takenaka, Yoshihiko Kakinuma, Masaaki Ikeda, Ikuo Inoue
PPAR Research 2024 ( 1 ) Article ID 5518933 2024年6月
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担当区分：筆頭著者, 責任著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Wiley
We have previously reported the identification of a novel splicing variant of the mouse peroxisome proliferator‐activated receptor‐γ (Pparγ), referred to as Pparγ1sv. This variant, encoding the PPARγ1 protein, is abundantly and ubiquitously expressed, playing a crucial role in adipogenesis. Pparγ1sv possesses a unique promoter and 5 ′ untranslated region (5 ′UTR), distinct from those of the canonical mouse Pparγ1 and Pparγ2 mRNAs. We observed a significant increase in DNA methylation at two CpG sites within the proximal promoter region (‐733 to ‐76) of Pparγ1sv during adipocyte differentiation. Concurrently, chromatin immunoprecipitation‐quantitative PCR (ChIP‐qPCR) using antibodies against H3K4me3 and H3K27ac indicated marked elevations in both methylation and acetylation of histone H3, while the repressive histone mark H3K9me2 significantly decreased, at the transcription start sites of both Pparγ1sv and Pparγ2 following differentiation. Knocking down Pparγ1sv using specific siRNA also led to a decrease in Pparγ2 mRNA and PPARγ2 protein levels; conversely, knocking down Pparγ2 resulted in reduced Pparγ1sv mRNA and PPARγ1 protein levels, suggesting synergistic transcriptional regulation of Pparγ1sv and Pparγ2 during adipogenesis. Furthermore, our experiments utilizing the CRISPR‐Cas9 system identified crucial PPARγ‐binding sites within the Pparγ gene locus, underscoring their significance in adipogenesis. Based on these findings, we propose a model of positive feedback regulation for Pparγ1sv and Pparγ2 expression during the adipocyte differentiation process in 3T3‐L1 cells.
DOI： 10.1155/2024/5518933
Transcriptome Analysis Reveals Enhancement of Cardiogenesis-Related Signaling Pathways by S-nitroso-N-pivaloyl-D-penicillamine (SNPiP): Implications for Improved Diastolic Function and Cardiac Performance. 国際誌
Yasuhiro Takenaka, Masataka Hirasaki, Hidemasa Bono, Shigeo Nakamura, Yoshihiko Kakinuma
Journal of cardiovascular pharmacology 2024年2月
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担当区分：筆頭著者, 責任著者 記述言語：英語 掲載種別：研究論文（学術雑誌）
We previously reported a novel compound called S-nitroso-N-pivaloyl-D-penicillamine (SNPiP), which was screened from a group of nitric oxide (NO) donor compounds with a basic chemical structure of S-nitroso-N-acetylpenicillamine (SNAP), to activate the non-neuronal acetylcholine (NNA) system. SNPiP-treated mice exhibited improved cardiac output and enhanced diastolic function, without an increase in heart rate. The NNA-activating effects included increased resilience to ischemia, modulation of energy metabolism preference, and activation of angiogenesis. Here, we performed transcriptome analysis of SNPiP-treated mice ventricles to elucidate how SNPiP exerts beneficial effects on cardiac function. A time-course study (24 and 48 h after SNPiP administration) revealed that SNPiP initially induced Wnt and cGMP-protein kinase G (PKG) signaling pathways, along with upregulation of genes involved in cardiac muscle tissue development and oxytocin signaling pathway. We also observed enrichment of glycolysis-related genes in response to SNPiP treatment, resulting in a metabolic shift from oxidative phosphorylation to glycolysis, which was suggested by reduced cardiac glucose contents while maintaining ATP levels. Additionally, SNPiP significantly upregulated atrial natriuretic peptide (ANP) and sarcolipin (SLN), which play crucial roles in calcium handling and cardiac performance. These findings suggest that SNPiP may have therapeutic potential based on the pleiotropic mechanisms elucidated in this study.
Temporal inhibition of the electron transport chain attenuates stress-induced cellular senescence by prolonged disturbance of proteostasis in human fibroblasts. 査読 国際誌
Yasuhiro Takenaka, Ikuo Inoue, Masataka Hirasaki, Masaaki Ikeda, Yoshihiko Kakinuma
The FEBS journal 290 ( 15 ) 3843 - 3857 2023年8月
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記述言語：英語 掲載種別：研究論文（学術雑誌）
We previously developed a stress-induced premature senescence (SIPS) model in which normal human fibroblast MRC-5 cells were treated with either the proteasome inhibitor MG132 or the vacuolar-type ATPase inhibitor bafilomycin A1 (BAFA1). To clarify the involvement of mitochondrial function in our SIPS model, MRC-5 cells were treated with MG132 or BAFA1 along with an inhibitor targeting either the electron transport chain complex I or complex III, or with a mitochondrial uncoupler. SIPS induced by MG132 or BAFA1 was significantly attenuated by short-term co-treatment with the complex III inhibitor, antimycin A (AA), but not the complex I inhibitor, rotenone or the mitochondrial uncoupler, carbonyl cyanide 3-chlorophenylhydrazone. By co-treatment with AA, mitochondrial and intracellular reactive oxygen species levels, accumulation of protein aggregates and mitochondrial unfolded protein responses (UPRmt ) were remarkably suppressed. Furthermore, AA co-treatment suppressed the hyperpolarization of the mitochondrial membrane and the induction of mitophagy observed in MG132-treated cells and enhanced mitochondrial biogenesis. These findings provide evidence that the temporal inhibition of mitochondrial respiration exerts protective effects against the progression of premature senescence caused by impaired proteostasis.
DOI： 10.1111/febs.16785
Prolonged disturbance of proteostasis induces cellular senescence via temporal mitochondrial dysfunction and subsequent mitochondrial accumulation in human fibroblasts. 査読 国際誌
Yasuhiro Takenaka, Ikuo Inoue, Takanari Nakano, Masaaki Ikeda, Yoshihiko Kakinuma
The FEBS journal 289 ( 6 ) 1650 - 1667 2022年3月
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担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌）
Proteolytic activity declines with age, resulting in the accumulation of aggregated proteins in aged organisms. To investigate how disturbance in proteostasis causes cellular senescence, we developed a stress-induced premature senescence (SIPS) model, in which normal human fibroblast MRC-5 cells were treated with the proteasome inhibitor MG132 or the vacuolar-type ATPase inhibitor bafilomycin A1 (BAFA1) for 5 days. Time-course studies revealed a significant increase in intracellular reactive oxygen species (ROS) and mitochondrial superoxide during and after drug treatment. Mitochondrial membrane potential initially decreased, suggesting temporal mitochondrial dysfunction during drug treatment, but was restored along with mitochondrial accumulation after drug treatment. AMP-activated protein kinase alpha was notably activated during treatment; thereafter, intracellular ATP levels significantly increased. SIPS induction by MG132 or BAFA1 was partially attenuated by co-treatment with vitamin E or rapamycin, in which the levels of ROS, mitochondrial accumulation, and protein aggregates were suppressed, implying the critical involvement of oxidative stress and mitochondrial function in SIPS progression. Rapamycin co-treatment also augmented the expression of HSP70 and activation of AKT, which could recover proteostasis and promote cell survival, respectively. Our study proposes a possible pathway from the disturbed proteostasis to cellular senescence via excess ROS production as well as functional and quantitative changes in mitochondria.
DOI： 10.1111/febs.16249
茂里 康, 中道 夢心, 内藤 玲奈, 山本 玲, 村田 顕也, 竹中 康浩, 村井 稔幸
大学の物理教育 30 ( 3 ) 156 - 158 2024年11月
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記述言語：日本語 出版者・発行元：一般社団法人 日本物理学会
1．はじめに
日本の医学部での医学教育は6年間の一貫教育であり，標準的に一般教養課程 (1～2年次)，基礎医学課程 (2～3年次)，臨床医学課程 (3～4年次)，臨床実習課程 (5～6年次) から構成されて
その他リンク： https://ndlsearch.ndl.go.jp/books/R000000004-I033840372
Yuta Chiba, Yasuhiro Takenaka, Nobuyuki Haga
Microorganisms 12 ( 3 ) 588 - 588 2024年3月
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記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：MDPI AG
The process of sexual reproduction in eukaryotes starts when gametes from two different sexes encounter each other. Paramecium, a unicellular eukaryote, undergoes conjugation and uses a gametic nucleus to enter the sexual reproductive process. The molecules responsible for recognizing mating partners, hypothetically called mating-type substances, are still unclear. We have identified an O3-type mating substance polypeptide and its gene sequence using protein chemistry, molecular genetics, immunofluorescence, RNA interference, and microinjection. The O3-type substance is a polypeptide found in the ciliary membranes, located from the head to the ventral side of cells. The O3-type substance has a kinase-like domain in its N-terminal part located outside the cell and four EF-hand motifs that bind calcium ions in its C-terminal part located inside the cell. RNA interference and immunofluorescence revealed that this polypeptide positively correlated with the expression of mating reactivity. Microinjection of an expression vector incorporating the O3Pc-MSP gene (Oms3) induced additional O3 mating type in the recipient clones of different mating types or syngen. Phylogenetic analysis indicates that this gene is widely present in eukaryotes and exhibits high homology among closely related species. The O3Pc-MSP (Oms3) gene had nine silent mutations compared to the complementary mating type of the E3 homologue gene.
Nobuyuki Haga, Toshinori Usui, Yasuhiro Takenaka, Yuta Chiba, Tomoaki Abe
Microorganisms 11 ( 1 ) 82 - 82 2022年12月
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記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：MDPI AG
Fertilization-initiated development and adult-onset aging are standard features in the life history of eukaryotes. In Paramecium, the number of cell divisions after the birth of a new generation is an essential parameter of sexual phase transition and aging. However, the gene driving this process and its evolutionary origin have not yet been elucidated. Here we report several critical outcomes obtained by molecular genetics, immunofluorescence microscopy, transformation by microinjection, and enzymological analysis. The cloned immaturin gene induces sexual rejuvenation in both mature and senescent cells by microinjection. The immaturin gene originated from proteobacteria’s glutathione-S-transferase (GST) gene. However, immaturin has been shown to lose GST activity and instead acquire nuclease activity. In vitro substrates for immaturin-nuclease are single- and double-stranded DNA, linear and circular DNA, and single-stranded viral genome RNA such as coronavirus. Anti-immaturin antibodies have shown that the subcellular localizations of immaturin are the macronucleus, cytoplasm, cell surface area, and cilia. The phase transition of sexuality is related to a decrease in the intracellular abundance of immaturin. We propose that sexual maturation and rejuvenation is a process programmed by the immaturin gene, and the sexual function of each age is defined by both the abundance and the intracellular localization mode of the immaturin-nuclease.
Yuichi Ikegami, Yasuhiro Takenaka, Daigo Saito, Akira Shimada, Ikuo Inoue
Journal of Clinical Medicine Research 13 ( 10-11 ) 502 - 509 2021年11月
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Effects of Pparγ1 deletion on late-stage murine embryogenesis and cells that undergo endocycle. 査読 国際誌
Takanari Nakano, Hidekazu Aochi, Masataka Hirasaki, Yasuhiro Takenaka, Koji Fujita, Masaru Tamura, Hiroaki Soma, Hajime Kamezawa, Takahiro Koizumi, Hirotoshi Shibuya, Reiko Inomata, Akihiko Okuda, Takayuki Murakoshi, Akira Shimada, Ikuo Inoue
Developmental biology 478 222 - 235 2021年10月
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記述言語：英語 掲載種別：研究論文（学術雑誌）
Peroxisome proliferator-activated receptor (PPAR) γ1, a nuclear receptor, is abundant in the murine placenta during the late stage of pregnancy (E15-E16), although its functional roles remain unclear. PPARγ1 is encoded by two splicing isoforms, namely Pparγ1canonical and Pparγ1sv, and its embryonic loss leads to early (E10) embryonic lethality. Thus, we generated knockout (KO) mice that carried only one of the isoforms to obtain a milder phenotype. Pparγ1sv-KO mice were viable and fertile, whereas Pparγ1canonical-KO mice failed to recover around the weaning age. Pparγ1canonical-KO embryos developed normally up to 15.5 dpc, followed by growth delays after that. The junctional zone of Pparγ1canonical-KO placentas severely infiltrated the labyrinth, and maternal blood sinuses were dilated. In the wild-type, PPARγ1 was highly expressed in sinusoidal trophoblast giant cells (S-TGCs), peaking at 15.5 dpc. Pparγ1canonical-KO abolished PPARγ1 expression in S-TGCs. Notably, the S-TGCs had unusually enlarged nuclei and often occupied maternal vascular spaces, disturbing the organization of the fine labyrinth structure. Gene expression analyses of Pparγ1canonical-KO placentas indicated enhanced S-phase cell cycle signatures. EdU-positive S-TGCs in Pparγ1canonical-KO placentas were greater in number than those in wild-type placentas, suggesting that the cells continued to endoreplicate in the mutant placentas. These results indicate that PPARγ1, a known cell cycle arrest mediator, is involved in the transition of TGCs undergoing endocycling to the terminal differentiation stage in the placentas. Therefore, PPARγ1 deficiency, induced through genetic manipulation, leads to placental insufficiency.
Ezetimibe impairs transcellular lipid trafficking and induces large lipid droplet formation in intestinal absorptive epithelial cells. 査読 国際誌
Takanari Nakano, Ikuo Inoue, Yasuhiro Takenaka, Rina Ito, Norihiro Kotani, Sawako Sato, Yuka Nakano, Masataka Hirasaki, Akira Shimada, Takayuki Murakoshi
Biochimica et biophysica acta. Molecular and cell biology of lipids 1865 ( 12 ) 158808 - 158808 2020年8月
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記述言語：英語 掲載種別：研究論文（学術雑誌）
Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1) protein, which mediates intracellular cholesterol trafficking from the brush border membrane to the endoplasmic reticulum, where chylomicron assembly takes place in enterocytes or in the intestinal absorptive epithelial cells. Cholesterol is a minor lipid constituent of chylomicrons; however, whether or not a shortage of cholesterol attenuates chylomicron assembly is unknown. The aim of this study was to examine the effect of ezetimibe, a potent NPC1L1 inhibitor, on trans-epithelial lipid transport, and chylomicron assembly and secretion in enterocytes. Caco-2 cells, an absorptive epithelial model, grown onto culture inserts were given lipid micelles from the apical side, and chylomicron-like TAG-rich lipoprotein secreted basolaterally were analyzed after a 24-h incubation period in the presence of ezetimibe up to 50 μM. The secretion of lipoprotein and apolipoprotein B48 were reduced by adding ezetimibe (30% and 34%, respectively). Although ezetimibe allowed the cells to take up cholesterol normally, the esterification was abolished. Meanwhile, oleic acid esterification was unaffected. Moreover, ezetimibe activated sterol regulatory element-binding protein 2 by approximately 1.5-fold. These results suggest that ezetimibe limited cellular cholesterol mobilization required for lipoprotein assembly. In such conditions, large lipid droplet formation in Caco-2 cells and the enterocytes in mice were induced, implying that unprocessed TAG was sheltered in these compartments. Although ezetimibe did not reduce the post-prandial lipid surge appreciably in triolein-infused mice, the results of the present study indicated that pharmacological actions of ezetimibe may participate in a novel regulatory mechanism for the efficient chylomicron assembly and secretion.
Redox-dependent PPARγ/Tnpo1 complex formation enhances PPARγ nuclear localization and signaling. 査読 国際誌
Toshiaki Teratani, Kengo Tomita, Sachiko Toma-Fukai, Yutaro Nakamura, Toshimasa Itoh, Hikaru Shimizu, Yasunaga Shiraishi, Nao Sugihara, Masaaki Higashiyama, Takahiko Shimizu, Ikuo Inoue, Yasuhiro Takenaka, Ryota Hokari, Takeshi Adachi, Toshiyuki Shimizu, Soichiro Miura, Takanori Kanai
Free radical biology & medicine 156 45 - 56 2020年8月
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記述言語：英語 掲載種別：研究論文（学術雑誌）
The nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ has been implicated in the pathogenesis of various human diseases including fatty liver. Although nuclear translocation of PPARγ plays an important role in PPARγ signaling, details of the translocation mechanisms have not been elucidated. Here we demonstrate that PPARγ2 translocates to the nucleus and activates signal transduction through H2O2-dependent formation of a PPARγ2 and transportin (Tnpo)1 complex via redox-sensitive disulfide bonds between cysteine (Cys)176 and Cys180 of the former and Cys512 of the latter. Using hepatocyte cultures and mouse models, we show that cytosolic H2O2/Tnpo1-dependent nuclear translocation enhances the amount of DNA-bound PPARγ and downstream signaling, leading to triglyceride accumulation in hepatocytes and liver. These findings expand our understanding of the mechanism underlying the nuclear translocation of PPARγ, and suggest that the PPARγ and Tnpo1 complex and surrounding redox environment are potential therapeutic targets in the treatment of PPARγ-related diseases.
A sudden onset of severe thrombocytopenia while using evolocumab 査読 国際誌
Inoue, I., Takenaka, Y., Kin Y., Yamazaki, M., Ikegami, Y., Saito, D., Shimada, A.
Case Report in Hematology Article ID 3281626 3281626 - 3281626 2020年
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記述言語：英語 掲載種別：研究論文（学術雑誌）
A 72-year-old man with a 10-year history of coronary heart disease started evolocumab treatment once a month after developing excess myalgia due to therapy with a 3-hydroxy-methylglutaryl CoA reductase inhibitor. No side effects such as myalgia symptoms had been reported during the first 14 months of evolocumab treatment; however, he suddenly presented with acute severe thrombocytopenia following the 14th treatment. His platelet count continued to decrease to a nadir of 1,000/μL. His platelet-associated immunoglobulin G level had elevated to 790 ng/107 cells. He started receiving a combination of steroid therapy, high-dose immunoglobulin therapy, and platelet transfusions, but the first-line therapy was ineffective. He was subsequently treated with a thrombopoietin receptor agonist, and his platelet count recovered to 250,000/μL.
DOI： 10.1155/2020/3281626
Shuei Sugama, Takato Takenouchi, Makoto Hashimoto, Hisayuki Ohata, Yasuhiro Takenaka, Yoshihiko Kakinuma
Journal of Neuroinflammation 16 ( 1 ) 2019年12月
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掲載種別：研究論文（学術雑誌） 出版者・発行元：Springer Science and Business Media LLC
DOI： 10.1186/s12974-019-1632-z
その他リンク： http://link.springer.com/article/10.1186/s12974-019-1632-z/fulltext.html
Luminal plant sterol promotes brush border membrane-to-lumen cholesterol efflux in the small intestine. 査読
Nakano T, Inoue I, Takenaka Y, Ikegami Y, Kotani N, Shimada A, Noda M, Murakoshi T
Journal of clinical biochemistry and nutrition 63 ( 2 ) 102 - 105 2018年9月
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Characterization of CoPK02, a Ca2+/calmodulin-dependent protein kinase in mushroom Coprinopsis cinerea. 査読 国際誌
Yamashita M, Sueyoshi N, Yamada H, Katayama S, Senga Y, Takenaka Y, Ishida A, Kameshita I, Shigeri Y
Bioscience, biotechnology, and biochemistry 82 ( 8 ) 1335 - 1343 2018年8月
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Evaluation of Teneligliptin Effects on Transcriptional Activity of PPARγ in Cell-Based Assays. 査読
Takenaka Y, Inoue I, Nakano T, Ikeda M, Kakinuma Y, Ikegami Y, Shimada A, Noda M
Journal of Nippon Medical School = Nippon Ika Daigaku zasshi 85 ( 2 ) 95 - 101 2018年
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A light in the dark: ecology, evolution and molecular basis of copepod bioluminescence 査読
Yasuhiro Takenaka, Atsushi Yamaguchi, Yasushi Shigeri
JOURNAL OF PLANKTON RESEARCH 39 ( 3 ) 369 - 378 2017年5月
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担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：OXFORD UNIV PRESS
Within the calanoid copepods, the bioluminescent species comprise 5-59% of the abundance and 10-15% of the biomass in the world's oceans. Most of the luminous species belong to the superfamily Augaptiloidea. The composition of bioluminescent species within the calanoid copepods shows latitudinal patterns; 5-25% of total calanoid copepods are found in high-latitude oceans, while 34-59% are in low-latitude oceans, reflecting a prey-predator relationship. Bioluminescent species of calanoid copepods are able to produce the light-emitting substrate coelenterazine. It is then transferred to higher predators through the food chain, and might be used for bioluminescence in other luminous organisms. A notable feature of copepod bioluminescence is the secreted-type, and its major function may be as an antipredatory response or a defensive behavior. Identification of more than 20 luciferase genes from calanoid copepods has revealed the highly conserved sequences of those genes. This leads us to the speculation that the genes for luciferase within the group of calanoid copepods have evolved independently of comparable genes outside of this group. We discuss here the ecological and biological functions of copepod bioluminescence, the significant diversity in luminous intensity, which might be evolutionarily relevant to their motility and habitat depth, and the promising future directions of bioluminescence studies.
Ezetimibe Promotes Brush Border Membrane-to-Lumen Cholesterol Efflux in the Small Intestine 査読
Takanari Nakano, Ikuo Inoue, Yasuhiro Takenaka, Hiraku Ono, Shigehiro Katayama, Takuya Awata, Takayuki Murakoshi
PLOS ONE 11 ( 3 ) e0152207 2016年3月
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記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：PUBLIC LIBRARY SCIENCE
Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1), an apical membrane cholesterol transporter of enterocytes, thereby reduces intestinal cholesterol absorption. This treatment also increases extrahepatic reverse cholesterol transport via an undefined mechanism. To explore this, we employed a trans-intestinal cholesterol efflux (TICE) assay, which directly detects circulation-to-intestinal lumen H-3-cholesterol transit in a cannulated jejunal segment, and found an increase of TICE by 45%. To examine whether such increase in efflux occurs at the intestinal brush border membrane(BBM)-level, we performed luminal perfusion assays, similar to TICE but the jejunal wall was labelled with orally-given H-3-cholesterol, and determined elevated BBM-to-lumen cholesterol efflux by 3.5-fold with ezetimibe. Such increased efflux probably promotes circulation-to-lumen cholesterol transit eventually; thus increases TICE. Next, we wondered how inhibition of NPC1L1, an influx transporter, resulted in increased efflux. When we traced orally-given H-3-cholesterol in mice, we found that lumen-to-BBM H-3-cholesterol transit was rapid and less sensitive to ezetimibe treatment. Comparison of the efflux and fractional cholesterol absorption revealed an inverse correlation, indicating the efflux as an opposite-regulatory factor for cholesterol absorption efficiency and counteracting to the naturally-occurring rapid cholesterol influx to the BBM. These suggest that the ezetimibe-stimulated increased efflux is crucial in reducing cholesterol absorption. Ezetimibe-induced increase in cholesterol efflux was approximately 2.5-fold greater in mice having endogenous ATP-binding cassette G5/G8 heterodimer, the major sterol efflux transporter of enterocytes, than the knockout counterparts, suggesting that the heterodimer confers additional rapid BBM-to-lumen cholesterol efflux in response to NPC1L1 inhibition. The observed framework for intestinal cholesterol fluxes may provide ways to modulate the flux to dispose of endogenous cholesterol efficiently for therapeutic purposes.
Molecular Cloning of Secreted Luciferases from Marine Planktonic Copepods. 査読 国際誌
Takenaka Y, Ikeo K, Shigeri Y
Methods in molecular biology (Clifton, N.J.) 1461 33 - 41 2016年
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Feminizing Adrenocortical Carcinoma with Distinct Histopathological Findings 査読
Masako Hatano, Yasuhiro Takenaka, Ikuo Inoue, Keiko Homma, Tomonobu Hasegawa, Hisanobu Sasano, Takuya Awata, Shigehiro Katayama
INTERNAL MEDICINE 55 ( 22 ) 3301 - 3307 2016年
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記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：JAPAN SOC INTERNAL MEDICINE
We herein present a 60-year-old man with adrenocortical carcinoma who had gynecomastia. An endocrinological examination revealed increased levels of serum estradiol and dehydroepiandrosterone-sulfate ( DHEA-S) and reduced levels of free testosterone. Magnetic resonance imaging showed an adrenal tumor with heterogeneous intensity. Iodine-131 adosterol scintigraphy showed an increased uptake at the same site. Because feminizing adrenocortical carcinoma was suspected, right adrenalectomy was performed; the pathological diagnosis was adrenocortical carcinoma. The results of immunostaining indicated a virilizing tumor. Aromatase activity was identified on RT-PCR. As disorganized steroidogenesis is pathologically present in adrenocortical carcinoma, this diagnosis should be made with caution.
Identification of Two Nickel Ion-Induced Genes, NCI16 and PcGST1, in Paramecium caudatum 査読
Yasuhiro Takenaka, Nobuyuki Haga, Ikuo Inoue, Takanari Nakano, Masaaki Ikeda, Shigehiro Katayama, Takuya Awata
EUKARYOTIC CELL 13 ( 9 ) 1181 - 1190 2014年9月
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担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：AMER SOC MICROBIOLOGY
Here, we describe the isolation of two nickel-induced genes in Paramecium caudatum, NCI16 and PcGST1, by subtractive hybridization. NCI16 encoded a predicted four-transmembrane domain protein (similar to 16 kDa) of unknown function, and PcGST1 encoded glutathione S-transferase (GST; similar to 25 kDa) with GST and glutathione peroxidase (GPx) activities. Exposing cells to cobalt chloride also caused the moderate upregulation of NCI16 and PcGST1 mRNAs. Both nickel sulfate and cobalt chloride dose dependently induced NCI16 and PcGST1 mRNAs, but with different profiles. Nickel treatment caused a continuous increase in PcGST1 and NCI16 mRNA levels for up to 3 and 6 days, respectively, and a notable increase in H2O2 concentrations in P. caudatum. NCI16 expression was significantly enhanced by incubating cells with H2O2, implying that NCI16 induction in the presence of nickel ions is caused by reactive oxygen species (ROS). On the other hand, PcGST1 was highly induced by the antioxidant tertbutylhydroquinone (tBHQ) but not by H2O2, suggesting that different mechanisms mediate the induction of NCI16 and PcGST1. We introduced a luciferase reporter vector with an similar to 0.42-kb putative PcGST1 promoter into cells and then exposed the transformants to nickel sulfate. This resulted in significant luciferase upregulation, indicating that the putative PcGST1 promoter contains a nickel-responsive element. Our nickel-inducible system also may be applicable to the efficient expression of proteins that are toxic to host cells or require temporal control.
DOI： 10.1128/EC.00112-14
Computational analysis and functional expression of ancestral copepod luciferase 査読
Yasuhiro Takenaka, Akiko Noda-Ogura, Tadashi Imanishi, Atsushi Yamaguchi, Takashi Gojobori, Yasushi Shigeri
Gene 528 ( 2 ) 201 - 205 2013年10月
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担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：2
We recently reported the cDNA sequences of 11 copepod luciferases from the superfamily Augaptiloidea in the order Calanoida. They were classified into two groups, Metridinidae and Heterorhabdidae/Lucicutiidae families, by phylogenetic analyses. To elucidate the evolutionary processes, we have now further isolated 12 copepod luciferases from Augaptiloidea species ( Metridia asymmetrica, Metridia curticauda, Pleuromamma scutullata, Pleuromamma xiphias, Lucicutia ovaliformis and Heterorhabdus tanneri). Codon-based synonymous/nonsynonymous tests of positive selection for 25 identified copepod luciferases suggested that positive Darwinian selection operated in the evolution of Heterorhabdidae luciferases, whereas two types of Metridinidae luciferases had diversified via neutral mechanism. By in silico analysis of the decoded amino acid sequences of 25 copepod luciferases, we inferred two protein sequences as ancestral copepod luciferases. They were expressed in HEK293 cells where they exhibited notable luciferase activity both in intracellular lysates and cultured media, indicating that the luciferase activity was established before evolutionary diversification of these copepod species. © 2013 Elsevier B.V.
A Novel Splicing Variant of Peroxisome Proliferator-Activated Receptor-γ (Pparγ1sv) Cooperatively Regulates Adipocyte Differentiation with Pparγ2. 査読
Takenaka Y, Inoue I, Nakano T, Shinoda Y, Ikeda M, Awata T, Katayama S
PloS one 8 ( 6 ) e65583 2013年6月
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An active C-terminally truncated form of Ca2+/calmodulin- dependent protein kinase phosphatase-N (CaMKP-N/PPM1E) 査読
Atsuhiko Ishida, Kumiko Tsumura, Megu Oue, Yasuhiro Takenaka, Yasushi Shigeri, Naoki Goshima, Yasuhiro Ishihara, Tetsuo Hirano, Hiromi Baba, Noriyuki Sueyoshi, Isamu Kameshita, Takeshi Yamazaki
BioMed Research International 2013 134813 2013年
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記述言語：英語 掲載種別：研究論文（学術雑誌）
Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) and its nuclear homolog CaMKP-N (PPM1E) are Ser/Thr protein phosphatases that belong to the PPM family. CaMKP-N is expressed in the brain and undergoes proteolytic processing to yield a C-terminally truncated form. The physiological significance of this processing, however, is not fully understood. Using a wheat-embryo cell-free protein expression system, we prepared human CaMKP-N (hCaMKP-N(WT)) and the truncated form, hCaMKP-N(1-559), to compare their enzymatic properties using a phosphopeptide substrate. The hCaMKP-N(1-559) exhibited a much higher V max value than the hCaMKP-N(WT) did, suggesting that the processing may be a regulatory mechanism to generate a more active species. The active form, hCaMKP-N(1-559), showed Mn2+ or Mg2+-dependent phosphatase activity with a strong preference for phospho-Thr residues and was severely inhibited by NaF, but not by okadaic acid, calyculin A, or 1-amino-8-naphthol-2,4-disulfonic acid, a specific inhibitor of CaMKP. It could bind to postsynaptic density and dephosphorylate the autophosphorylated Ca2+/calmodulin-dependent protein kinase II. Furthermore, it was inactivated by H2O2 treatment, and the inactivation was completely reversed by treatment with DTT, implying that this process is reversibly regulated by oxidation/reduction. The truncated CaMKP-N may play an important physiological role in neuronal cells. © 2013 Atsuhiko Ishida et al.
DOI： 10.1155/2013/134813
Evolution of Bioluminescence in Marine Planktonic Copepods 査読
Yasuhiro Takenaka, Atsushi Yamaguchi, Naoki Tsuruoka, Masaki Torimura, Takashi Gojobori, Yasushi Shigeri
MOLECULAR BIOLOGY AND EVOLUTION 29 ( 6 ) 1669 - 1681 2012年6月
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担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：OXFORD UNIV PRESS
Copepods are the dominant taxa in zooplankton communities of the ocean worldwide. Although bioluminescence of certain copepods has been known for more than a 100 years, there is very limited information about the structure and evolutionary history of copepod luciferase genes. Here, we report the cDNA sequences of 11 copepod luciferases isolated from the superfamily Augaptiloidea in the order Calanoida. Highly conserved amino acid residues in two similar repeat sequences were confirmed by the multiple alignment of all known copepod luciferases. Copepod luciferases were classified into two groups of Metridinidae and Heterorhabdidae/Lucicutiidae families based on phylogenetic analyses, with confirmation of the interrelationships within the Calanoida using 18S ribosomal DNA sequences. The large diversity in the specific activity of planktonic homogenates and copepod luciferases that we were able to express in mammalian cultured cells illustrates the importance of bioluminescence as a protective function against predators. We also discuss the relationship between the evolution of copepod bioluminescence and the aspects of their ecological characteristics, such as swimming activity and vertical habitat.
A Bioluminescent Probe for Salivary Cortisol 査読
Sung Bae Kim, Yasuhiro Takenaka, Masaki Torimura
BIOCONJUGATE CHEMISTRY 22 ( 9 ) 1835 - 1841 2011年9月
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記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：AMER CHEMICAL SOC
Cortisol is a classical biomarker for the stress levels of human beings. We fabricated highly sensitive bioluminescent probes for salivary cortisol. The following strategies were contrived in the molecular design. Gaussia princeps luciferase (GLuc) was dissected into two fragments, between which an N-terminal-extended ligand binding domain of glucocorticoid receptor (GR HLBD), named Simgr4, was inserted. First, this unique single-chain probe was then situated downstream of a glucocorticoid response element (GRE) promoter in a reporter-gene system for constructing two ON-OFF switches for cortisol. Second, a circularly permutated (CP) variant of Simgr4 was formulated. The reporter-gene system exerted an improved signal-to-background (S/B) ratio of 8.5 to cortisol. Furthermore, a circularly permutated (CP) variant of Simgr4 exerted a 10 x enhanced detection limit to cortisol and a long dynamic range from 10(-9) to 10(-6) M cortisol, covering all of the normal clinical ranges of serum, urine, and saliva. This optimized probe successfully determined daily fluctuations of salivary cortisol and the correlations with those by ELISA. This study is the first to investigate the contribution of the HLBD of a nuclear receptor and multiple ON-OFF switches for molecular probes and salivary cortisols.
DOI： 10.1021/bc260220k
Structural basis for red-shifted emission of a GFP-like protein from the marine copepod Chiridius poppei 査読
Kyoko Suto, Hiromi Masuda, Yasuhiro Takenaka, Frederick I. Tsuji, Hiroshi Mizuno
GENES TO CELLS 14 ( 6 ) 727 - 737 2009年6月
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記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC
The fluorescence excitation and emission maxima of a GFP-like protein from the marine copepod Chiridius poppei (CpYGFP) show a significant red shift (lambda(ex) = 509 nm, lambda(em) = 517 nm) compared with those of GFP from Aequorea victoria (avGFP) and other GFP-like proteins from marine copepods. We performed crystallographic and biochemical studies to understand why this shift occurs in CpYGFP. The structure of CpYGFP showed that the imidazole side chain of His52 is involved in stacking on the phenol moiety of the chromophore. We investigated the potential role of His52 in causing the red-shifted spectral properties by performing mutational analyses of H52T, H52D and H52F. The emission wavelengths of H52T and H52D were blue-shifted and that of H52F was red-shifted relative to the wild type. Comparison of its structure of another copepod GFP (ppluGFP2) having an emission maximum at 502 nm showed that the imidazole ring of His54 (corresponding to His52 in CpYGFP) is flipped out of the stacking position with the chromophore. These findings suggest that pi-pi stacking interaction between His52 and the phenol moiety of the chromophore is the likely cause of the red-shift in light emission.
Two forms of secreted and thermostable luciferases from the marine copepod crustacean, Metridia pacifica 査読
Yasuhiro Takenaka, Hiromi Masuda, Atsushi Yamaguchi, Satoshi Nishikawa, Yasushi Shigeri, Yasukazu Yoshida, Hiroshi Mizuno
GENE 425 ( 1-2 ) 28 - 35 2008年12月
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担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：ELSEVIER SCIENCE BV
We cloned two forms of the secreted and thermostable luciferase genes, MpLuc1 and MpLuc2, from the marine copepod, Metridia pacifica. The 840-bp MpLuc1 cDNA comprised a 630-bp open reading frame encoding a 210-amino acid polypeptide (22.7 kDa). MpLuc1 had the closest homology with Metridia longa luciferase. The 753-bp MpLuc2 cDNA consisted of a 567-bp open reading frame (20.3 kDa), and it had the closest homology with Gaussia princeps luciferase. Single-specimen genomic PCR confirmed the presence of two luciferase genes in M. pacifica, and single-specimen RT-PCR revealed that both luciferase mRNAs were expressed. Both MpLuc1 and MpLuc2 (MpLucs) specifically reacted with the substrate coelenterazine producing identical bioluminescent spectra (lambda max, 485 nm), but with different kinetics. Adding salt such as MgCl2 and CaCl2 to the reaction mixture significantly enhanced MpLuc1 and MpLuc2 activities. Wild-type MpLucs were remarkably thermostable; MpLuc1 retained about 60% of the original activity even after incubation at 90 degrees C for 30 min. MpLucs expressed in NIH-3T3 and HeLa cells were largely secreted into the culture medium. Continuous monitoring of secreted MpLuc1 driven by the c-fos promoter demonstrated the potential usefulness of MpLuc1 in nondisruptive reporter assays. (c) 2008 Elsevier B.V. All rights reserved.
Random insertion of injected DNA molecules into the macronuclear chromosome of Paramecium caudatum 査読
Takenaka Yasuhiro, Yanagi Akira, Masuda Hiromi, Mitsui Youji, Mizuno Hiroshi, Haga Nobuyuki
原生動物学雑誌 41 ( 2 ) 159 - 168 2008年
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担当区分：筆頭著者 記述言語：英語 出版者・発行元：日本原生生物学会
DOI： 10.18980/jjprotozool.41.2_159
その他リンク： http://id.ndl.go.jp/bib/9796684
Yasuhiro Takenaka, Akira Yanagi, Hiromi Masuda, Youji Mitsui, Hiroshi Mizuno, Nobuyuki Haga
GENE 395 ( 1-2 ) 108 - 115 2007年6月
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担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：ELSEVIER SCIENCE BV
Cytoplasmic exchange between conjugating cells of Paramecium caudatum has been implicated by mating experiments using wild-type and behavioral mutant cells. To observe macromolecular transport between mating cells, we cloned and expressed the P caudatum histone H2B gene as a fusion protein attached to an enhanced yellow fluorescent protein (YFP) named PcVenus. Significant fluorescent signals derived from histone H2B-PeVenus were detected throughout the macro- and micronuclei of transformant cells after microinjection of the expression vector. The normal growth and high mating reactivity of the transformants indicated that H2B-PcVenus functioned normally. Seven hours after a transformant cell expressing histone 1-1213-PeVenus was mated with an untransformed complementary mating-type cell, fluorescence derived from histone H2B-PcVenus was emitted from the macronuclei of the untransformed cell. About 48 h later, the fluorescent signal was detected not only in the macro- and micronuclei of untransformed cells but also in the macronuclear ardagen of both mating cells. This suggests that conjugant cells share parental histones during meiosis and subsequent DNA rearrangement. Single-cell RT-PCR analysis demonstrated the presence of H2B-PcVenus mRNA in untransformed cells 15 and 24 h after conjugation. We concluded that at least the mRNA of histone H2B-PcVenus was transferred from the transformed, to the untransformed cell during conjugation. (c) 2007 Elsevier B.V. All rights reserved.
H Masuda, Y Takenaka, A Yamaguchi, S Nishikawa, H Mizuno
GENE 372 18 - 25 2006年5月
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担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：ELSEVIER SCIENCE BV
A crustacean gene, encoding for a new class of GFP-like protein, has been isolated from a cDNA library of the deep-sea (benthic) copepod crustacean, Chiridius poppei, by expression cloning. The cDNA library was constructed in a pBluescript II vector and screened using a non-UV transilluminator, obtaining a positive clone. The clone consisted of a 781 -bp fragment of cDNA with a 660-bp open reading frame, which encoded for a 219-amino acid polypeptide with a calculated molecular mass of 24.7 kDa. The protein was overexpressed in Escherichia coli, purified to homogeneity by anion-exchange and size-exclusion chromatographies. The protein, CpYGFP, had excitation and emission maxima at 507 and 517 nm, respectively. CpYGFP existed as a dimer in solution and could be expressed either alone or as a fusion protein in HeLa cells. Dual labeling experiments carried out with CpYGFP-actin and DsRed2-Nuc demonstrated the usefulness of CpYGFP as a reporter in the subcellular localization of actin. (c) 2006 Elsevier B.V. All rights reserved.
Accumulation and altered localization of telomere-associated protein TRF2 in immortally transformed and tumor-derived human breast cells. 査読 国際誌
Tarlochan Nijjar, Ekaterina Bassett, James Garbe, Yasuhiro Takenaka, Martha R Stampfer, David Gilley, Paul Yaswen
Oncogene 24 ( 20 ) 3369 - 76 2005年5月
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記述言語：英語 掲載種別：研究論文（学術雑誌）
We have used cultured human mammary epithelial cells (HMEC) and breast tumor-derived lines to gain information on defects that occur during breast cancer progression. HMEC immortalized by a variety of agents (the chemical carcinogen benzo(a)pyrene, oncogenes c-myc and ZNF217, and/or dominant negative p53 genetic suppressor element GSE22) displayed marked upregulation (10-15 fold) of the telomere-binding protein, TRF2. Upregulation of TRF2 protein was apparently due to differences in post-transcriptional regulation, as mRNA levels remained comparable in finite lifespan and immortal HMEC. TRF2 protein was not upregulated by the oncogenic agents alone in the absence of immortalization, nor by expression of exogenously introduced hTERT genes. We found TRF2 levels to be at least twofold higher than in control cells in 11/15 breast tumor cell lines, suggesting that elevated TRF2 levels are a frequent occurrence during the transformation of breast tumor cells in vivo. The dispersed distribution of TRF2 throughout the nuclei in some immortalized and tumor-derived cells indicated that not all the TRF2 was associated with telomeres in these cells. The process responsible for accumulation of TRF2 in immortalized HMEC and breast tumor-derived cell lines may promote tumorigenesis by contributing to the cells' ability to maintain an indefinite lifespan.
Chromatography of isoforms of recombinant apoaequorin and method for the preparation of aequorin 査読
H Masuda, Y Takenaka, Y Shikamoto, M Kagawa, H Mizuno, FI Tsuji
PROTEIN EXPRESSION AND PURIFICATION 31 ( 2 ) 181 - 187 2003年10月
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記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE
Gradient elution chromatography of recombinant apoaequorin carried out in the presence of Ca2+ revealed two isoforms of apoaequorin, reduced and oxidized, whereas in the presence of EDTA 3 isoforms were observed. In a regeneration mixture of apoaequorin, coelenterazine, EDTA, and 2-mercaptoethanol, four isoforms were obtained, of which only one, aequorin, gave light with Ca2+. A method is described for the preparation of highly pure aequorin. The aequorin was stable in solution for approximately 10 days at 4 degreesC and pH 7.6, and then it gradually lost activity with a half-life of about 20 days until it was almost completely inactive on day 30. (C) 2003 Elsevier Science (USA). All rights reserved.
Y Takenaka, N Haga, T Harumoto, T Matsuura, Y Mitsui
GENE 284 ( 1-2 ) 233 - 240 2002年2月
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担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：ELSEVIER SCIENCE BV
We have developed a novel expression vector, pTub-tel3, for transformation in Paramecium caudatum. The vector was constructed by cloning P. caudatum alpha-tubulin 5' and 3' non-coding regions. To examine transformation with the pTub-tel3 construct, we chose the green fluorescent protein (GFP) as a selection marker. When a linearized pTub-tel3 vector containing a GFP open reading frame was injected into the macronucleus, the GFP transcript was expressed in many clones whereas protein expression was detected only after extensive optimization of original GFP codons. GFP-derived fluorescence was distributed throughout the nuclei and cytoplasm except for contractile and food vacuoles. Upon continuous cell division, notable heterogeneity of GFP fluorescence among descendants from the same transformant has emerged. This expression vector can be applied to the analysis of protein trafficking and localization in addition to exogenous gene expression in P. caudatum. (C) 2002 Published by Elsevier Science B,V.
Y Takenaka, T Matsuura, N Haga, Y Mitsui
GENE 264 ( 2 ) 153 - 161 2001年2月
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記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：ELSEVIER SCIENCE BV
Paramecium caudatum has a sexually immature period that lasts for about 60 fissions. To examine the possibility that telomere length is one of the determining factors of the duration of immaturity, we cloned the telomerase reverse transcriptase (TERT) gene from P. candatum, and analyzed its expression levels at mRNA, telomerase activity, and telomere length during the course of clonal division. Paramecium TERT (Pc_TERT) cDNA encodes a basic protein of 107 kDa that harbors conserved RT motifs, T motif, CP motif, and N motif. Pc_TERT mRNA is expressed at very low levels only detectable by RT-PCR, but constitutively, during immature and mature periods, exhibiting abundant telomerase activity. No clear phase shift in Pc_TERT expression, telomerase activity, or telomere length was observed at the point of maturation in P, caudatum. Instead, the telomere elongates successively as cells divide in P. caudatum, although a close species, P. tetraurelia, was reported to keep the length constant. We discuss possible mechanisms for the expression of sexual activity associated with telomere length in P. caudatum. (C) 2001 Elsevier Science B.V. All rights reserved.
Selective induction of apoptosis of renal proximal tubular cells caused by inorganic mercury in vivo 査読
S Homma-Takeda, Y Takenaka, Y Kumagai, N Shimojo
ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY 7 ( 3 ) 179 - 187 1999年7月
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記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：ELSEVIER SCIENCE BV
A recent notion, that a variety of toxicants causing necrosis can lead to apoptosis as well, has been demonstrated with cultured cells, but not with in an vivo system. In the present study, we examined the induction of both apoptosis and necrosis in the kidneys of Wistar rats exposed to mercuric chloride (HgCl(2)). A single injection of HgCl(2), to rats at a dose of 4 mg/kg resulted in an increase in the renal DNA fragmentation evaluated as an occurrence of apoptosis, prior to urinary excretion of alkaline phosphatase (ALP) and renal morphological changes assessed as necrotic phenomena. The mercury-promoted DNA fragmentation was induced in a dose-dependent manner. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and morphological observation of the nuclei revealed that apoptotic cells caused by HgCl(2) were predominantly found in the proximal tubules, but not in the distal tubules, glomeruli or medullary tubules. When we confirmed the proximal tubular-selective apoptosis by inorganic mercury with a combined technique of TUNEL staining with synchrotron radiation X-ray fluorescence (SR-XRF) imaging, it was shown that the apoptotic cells localized in the proximal tubules did contain higher level of mercury. Thus these results indicate that the proximal tubular cells-dominant site-specific distribution of mercury appears to be associated with induction of renal apoptosis and necrosis. (C) 1999 Published by Elsevier Science B.V. All rights reserved.
Various changes in nitric oxide synthase and arginase II in rat kidney caused by inorganic mercury 査読
H. Kanda, Y. Kumagai, H. Nakajima, Y. Takenaka, S. Homma-Takeda, N. Shimojo
Sangyō eiseigaku zasshi = Journal of occupational health 40 ( 5 ) 212 - 213 1998年1月
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Induction of Apoptosis by Mercuric Chloride in Rats
Shino Homma-Takeda, Yoshito Kumagai, Nobuhiro Shimojo, Yasuhiro Takenaka, Yutaka Kugenuma
Japanese Journal of Toxicology and Environmental Health 44 ( 1 ) 1998年
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Hironori Kanda, Yoshito Kumagai, Hiromi Nakajima, Yasuhiro Takenaka, Shino Homma-Takeda, Nobuhiro Shimojo
Japanese Journal of Toxicology and Environmental Health 44 ( 1 ) 1998年
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A dose-response study on inorganic mercury-induced DNA fragmentation in rat kidney 査読
Y. Takenaka, S. Homma-Takeda, Y. Kumagai, N. Shimojo
Sangyō eiseigaku zasshi = Journal of occupational health 39 ( 5 ) 184 - 185 1997年9月
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Exposure of rat to inorganic mercury induces DNA fragmentation responsible for apoptosis in kidney 査読
S Homma-Takeda, M Ishido, Y Kumagai, Y Takenaka, N Shimojo
JOURNAL OF OCCUPATIONAL HEALTH 39 ( 1 ) 70 - 71 1997年1月
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無機水銀投与ラット腎臓におけるアポトーシス誘導の量-反応関係 査読
竹中康浩, 本間志乃, 熊谷嘉人, 下條信弘
産業衛生学雑誌 39 184 - 185 1997年
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M WAKAYAMA, E WATANABE, Y TAKENAKA, Y MIYAMOTO, Y TAU, K SAKAI, M MORIGUCHI
JOURNAL OF FERMENTATION AND BIOENGINEERING 80 ( 4 ) 311 - 317 1995年
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記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN
The gene (termed daa) encoding N-acyl-D-aspartate (D-Asp) amidohydrolase (D-AAase) from the Alcaligenes xylosoxydans subsp. xylosoxydans (Alcaligenes A-6) was cloned in Escherichia coli (E. cell) JM109. The daa gene consists of 1,494 nucleotides and encodes 498 amino acid residues. The molecular weight of D-AAase was calculated to be 53,581. The N-terminal amino acid sequence (NH2-TDRSTLDDAP-) predicted by the nucleotide sequence matched exactly those of the purified D-AAase from both Alcaligenes A-6 and cloned E. coli, with the exception of the removal of the N-terminal methionine processed after translation. A comparison of the amino acid sequence of D-AAase with that of D-aminoacylase from Alcaligenes A-6 showed high overall homology (56%), D-AAase from Alcaligenes A-6 showed 25 similar to 29% homology with Bacillus stearothermophilus, porcine, and human L-aminoacylases. The daa was highly expressed in E. coli, and the recombinant enzyme was purified to homogeneity with 17.8% yield.
Yasuhiro Takenaka, Masataka Hirasaki, Ikuo Inoue, Masaaki Ikeda, Hisayuki Ohata, Yoshihiko Kakinuma
2023年8月
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担当区分：筆頭著者, 責任著者 掲載種別：機関テクニカルレポート，技術報告書，プレプリント等 出版者・発行元：Cold Spring Harbor Laboratory
Adult heart mostly contains long-lived postmitotic cardiomyocytes and non-cardiomyocytes that have proliferative potential. Here, we isolated cardiomyocytes and non-cardiomyocytes from young and aged mouse heart, and performed transcriptome analyses by RNA sequencing to understand the differences of gene expression in postmitotic and proliferative cells. Gene ontology analyses revealed that genes associated with inflammatory response were upregulated in aged cardiac myocytes, whereas genes including two ATP synthases in mitochondrial respiratory complex V (Atp5landAtp5J2) and two NADH dehydrogenases in complex I (Ndufa11andNdufv3) were significantly downregulated. In aged non-cardiomyocytes, genes related to inflammatory responses were also upregulated, while genes involved in cell cycle and DNA replication process were downregulated. We also found that the expression levels of some small nucleolar RNAs (snoRNAs) are decreased cardiomyocytes with aging. snoRNAs are deeply involved in RNA modification such as pseudouridylation stabilizing ribosomal RNA (rRNA) and mRNA splicing. Therefore, the age-related reduction in snoRNA expression may lead to the destabilization of rRNA, splicing dysfunction, and ultimately a decrease in protein synthesis capacity. A comparison with transcriptome results obtained for non-cardiomyocytes suggests that the decline in the expression of mitochondria-related genes and snoRNAs accompanying aging is specific to cardiomyocytes, implying their potential utility as one of novel aging markers in postmitotic cells.
カイアシ類(Metridia pacifica)由来新規耐熱性ルシフェラーゼ
竹中康浩, 増田洋美, 増田洋美, 山口篤, 西川諭, 水野洋
生化学 1P-0326 2007年
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ゾウリムシの分裂時計は？ -テロメアとテロメラーゼの構造と動態-
竹中康浩, 三井洋司
基礎老化研究 25 ( 2 ) 95 - 100 2001年
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植物ステロールは腸管経由コレステロール逆輸送を促進する
中野 貴成, 井上 郁夫, 竹中 康浩, 池上 裕一, 小谷 典弘, 島田 朗, 野田 光彦, 村越 隆之
糖尿病 61 ( Suppl.1 ) S - 408 2018年4月
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植物ステロールは腸管経由コレステロール逆輸送を促進する
中野 貴成, 井上 郁夫, 竹中 康浩, 池上 裕一, 小谷 典弘, 島田 朗, 野田 光彦, 村越 隆之
糖尿病 61 ( Suppl.1 ) S - 408 2018年4月
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カイアシ類（海洋プランクトン）ルシフェラーゼの構造と進化
竹中康浩, 山口篤, 茂里康
生化学 87 ( 1 ) 138 - 143 2015年
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マウスPPARγスプライシングバリアント(PPARγ3)の脂肪細胞分化誘導における発現制御
竹中 康浩, 井上 郁夫, 中野 貴成, 片山 茂裕, 粟田 卓也
The Lipid 24 ( 3 ) 304 - 305 2013年7月
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C末切断型ヒトCaMキナーゼホスファターゼN(CaMKP‐N/PPM1E)の発現と酵素学的性質
津村公美子, 大上恵, 石原康宏, 平野哲男, 竹中康浩, 茂里康, 五島直樹, 馬場裕美, 末吉紀行, 亀下勇, 山崎岳, 石田敦彦
日本生化学会大会(Web) 85th 3P-558 (WEB ONLY) - 558 2012年
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海洋プランクトン(カイアシ類)よりクローニングされた分泌型ルシフェラーゼの分子系統解析
竹中康浩, 山口篤, 鶴岡直樹, 鳥村政基, 五條堀孝, 茂里康
日本分子生物学会年会プログラム・要旨集(Web) 35th WEB ONLY 4P-0092 2012年
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海洋プランクトン（カイアシ類）から単離された分泌型ルシフェラーゼ 耐熱性に優れた２つの酵素遺伝子を単離 高感度アッセイへの応用に期待
竹中康浩, 増田洋美, 茂里康
化学と生物 47 ( 4 ) 231 - 232 2009年
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記述言語：日本語 掲載種別：記事・総説・解説・論説等（学術雑誌） 出版者・発行元：Japan Society for Bioscience, Biotechnology, and Agrochemistry
光るプランクトン —カイアシ類のGFP・ルシフェラーゼ
竹中康浩, 増田洋美, 茂里康
バイオサイエンスとインダストリー 67 ( 3 ) 100 - 101 2009年
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発光プランクトン由来分泌型耐熱性ルシフェラーゼ
竹中康浩, 増田洋美, 茂里康
バイオサイエンスとインダストリー 67 ( 3 ) 107 - 109 2009年
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テロメア末端とDNA損傷・修復
竹中康浩, 三井洋司
基礎老化研究 30 ( 1 ) 3 - 7 2006年
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ゾウリムシの接合過程におけるhistoneマジック
竹中康浩, 柳明, 増田洋美, 芳賀信幸
原生動物学雑誌 39 ( 1 ) 104 - 105 2006年
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最晩年のゾウリムシにおける安定した遺伝子発現
竹中康浩, 芳賀信幸
原生動物学雑誌 36 ( 1 ) 36 - 37 2003年
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動物組織中のアポトーシスに関与する断片化DNAの定量法の確立
竹中康浩, 本間志乃, 熊谷嘉人, 下條信弘
Biomedical Research on Trace Elements 7 ( 3 ) 103 - 104 1996年
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塩化第二水銀投与による肝臓でのアポトーシス誘導
本間志乃, 竹中康浩, 久下沼裕, 熊谷嘉人, 下條信弘
Biomedical Research on Trace Elements 7 ( 3 ) 103 - 104 1996年
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有害金属とアポトーシスの誘導との量—反応関係を解析するためのテクニック
本間志乃, 熊谷嘉人, 竹中康浩, 下條信弘
Biomedical Research on Trace Elements 7 ( 2 ) 43 - 49 1996年
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Analysis of senescence-associated protein aggregates in replicative senescent MRC-5 cells
竹中 康浩, 平崎 正孝, 池田 正明, 井上 郁夫, 柿沼 由彦
第４５回日本基礎老化学会 2022年7月
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タンパク質恒常性機能の阻害により誘導される細胞老化とビタミンE及びラパマイシンの細胞老化抑制作用
竹中 康浩, 井上 郁夫, 池田 正明, 柿沼 由彦
第９９回日本生理学会大会 2022年3月
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海洋プランクトン（カイアシ類）よりクローニングされた分泌型ルシフェラーゼの分子系統解析
竹中康浩, 山口篤, 鶴岡直樹, 鳥村政基, 五條堀孝, 茂里康
第３５回日本分子生物学会年会 2012年
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カイアシ類（Metridia pacifica）由来新規耐熱性ルシフェラーゼ
竹中康浩, 増田洋美, 水野洋, 西川諭, Frederick I Tsuji
第３０回日本分子生物学会年会 2007年
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マウスPPARγスプライシングバリアントとPPARγ2の脂肪細胞分化誘導における協調的発現制御
竹中康浩, 井上郁夫, 中野貴成, 片山茂裕, 粟田卓也
第８５回日本生化学会大会 2012年
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Secreted and Thermostable Luciferases from the Marine Copepod Crustacean, Metridia pacifica.
Takenaka Y, Yamaguchi A, Shigeri Y
International Symposium on Marine Genomics 2009 2009年
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NPC1L1阻害による腸管経由のコレステロール逆輸送機序の解明
中野 貴成, 井上 郁夫, 竹中 康浩, 片山 茂裕, 村越 隆之
ConBio2017 2017年12月
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マウスPPARgスプライシングバリアントのポジティブフィードバック制御機構の解析
竹中 康浩, 井上 郁夫, 中野 貴成, 池田 正明, 栗原 進, 小野 啓, 後藤 誠一, 片山 茂裕, 粟田 卓也
第５７回日本糖尿病学会年次学術集会 2014年
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タンパク質分解系の阻害による細胞老化誘導メカニズムの解析
竹中 康浩, 井上 郁夫, 中野 貴成, 池田 正明, 柿沼 由彦
第42回日本分子生物学会年会 2019年12月
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タンパク質恒常性機能の阻害による細胞老化誘導メカニズムの解析
竹中 康浩, 井上 郁夫, 中野 貴成, 池田 正明, 柿沼 由彦
第17回 RCGMフロンティアシンポジウム 2019年9月
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ゾウリムシニッケル誘導遺伝子NCI16とPcGST1の単離と発現量解析および発現制御に関する研究
竹中 康浩, 芳賀 信幸, 井上 郁夫, 中野 貴成, 池田 正明, 粟田 卓也, 片山 茂裕
第３７回日本分子生物学会年会 2014年
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マウスPPARγスプライシングバリアントのマウス後期胚における局在と機能解析
竹中康浩, 井上郁夫, 中野貴成, 池田正明, 篠田雄一, 片山茂裕, 粟田卓也
第５６回日本糖尿病学会年次学術集会 2013年
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PPARγの転写制御活性にテネリグリプチンが及ぼす効果
竹中 康浩, 井上 郁夫, 中野 貴成, 池田 正明, 栗原 進, 小野 啓, 後藤 誠一, 片山 茂裕, 粟田 卓也
第４６回日本動脈硬化学会総会・学術集会 2014年
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ニッケル誘導遺伝子NCI16とPcGST1の発現誘導に酸化ストレスが及ぼす影響
竹中 康浩, 芳賀 信幸, 井上 郁夫, 粟田 卓也, 片山 茂裕
第４７回日本原生動物学会大会 2014年
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プロテアソーム阻害剤等で誘導した細胞老化におけるミトコンドリア機能と生合成の解析
竹中 康浩, 井上 郁夫, 中野 貴成, 池田 正明, 柿沼 由彦
第４３回日本基礎老化学会大会 2020年5月
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マウス PPARγスプライシングバリアント(Pparγ1sv)の同定と脂肪細胞分化誘導における機能
竹中 康浩, 井上 郁夫
第八回 高血圧と冠動脈疾患研究会 2013年
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新規マウスPPARγスプライシングバリアントの脂肪細胞分化誘導時における発現
竹中康浩, 井上郁夫, 栗原進, 後藤誠一, 篠田雄一, 犬飼浩一, 片山茂裕, 粟田卓也
第45回日本原生動物学会大会 2012年
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ニッケル誘導プロモーターを用いたゾウリムシ誘導発現ベクターの開発
竹中 康浩, 芳賀 信幸, 井上 郁夫
第４６回日本原生動物学会大会 2013年
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核内受容体PPAR_の新規スプライシングバリアント(Pparγ1sv)の転写制御機構の解析
竹中 康浩, 井上 郁夫, 中野 貴成, 池田 正明, 片山 茂裕, 粟田 卓也
第３６回日本分子生物学会年会 2013年
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マウスPPARγスプライシングバリアント（PPARγ3）の脂肪細胞分化誘導における発現制御
竹中康浩, 井上 郁夫, 中野 貴成, 片山 茂裕, 粟田 卓也
第44回日本動脈硬化学会総会・学術集会 2012年
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Expression of novel mouse PPARγ splicing variant during adipocyte differentiation of 3T3-L1 cells
Takenaka Y, Inoue I, Kurihara S, Goto S, Shinoda Y, Katayama S, Awata T
第５５回日本糖尿病学会年次学術集会 2012年
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ゾウリムシニッケル誘導遺伝子の単離と発現解析
竹中康浩, 芳賀信幸, 井上郁夫
第２６回小児脂肪研究会 2012年
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マウス新規PPARγスプライシングバリアント（Pparγ1sv）の脂肪細胞分化誘導における発現と機能解析
竹中 康浩, 井上 郁夫, 中野 貴成, 池田 正明, 栗原 進, 後藤 誠一, 片山 茂裕, 粟田 卓也
第３１回日本肥満症治療学会学術集会 2013年
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マウス17日胚における新規PPARγスプライシングバリアント(Pparγ1sv)の発現
竹中 康浩, 井上 郁夫, 中野 貴成, 池田 正明, 片山 茂裕, 粟田 卓也
第８６回日本生化学会大会 2013年
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ゾウリムシにおける分子生物学的手法の開発と応用
竹中 康浩
石巻専修大ライフサイエンスセミナー（招待講演） 2013年
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脂質タンパク質複合体を含む多検体試料を分析するための電気泳動用スラブ型ポリアクリルアミドゲル及びその方法
竹中 康浩, 井上 郁夫, 柿沼 由彦
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海洋プランクトン由来発光タンパク質
茂里 康, 山口 篤, 竹中 康浩, 今西 規, 小倉 彰子, 五條堀 孝
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出願人：独立行政法人産業技術総合研究所
出願番号：特願2011-126900 出願日：2011年6月
公開番号：特開2012-249619 公開日：2012年12月
蛍光タンパク質の蛍光波長を変える方法
須藤 恭子, 竹中 洋美, 竹中 康浩
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新規な蛍光性タンパク質とそれをコードする遺伝子
辻, フレデリック 一郎, 水野 洋, 高瀬 研二, 門間 充, 藤本 瑞, 若生 俊行, 竹中 康浩, 奈倉 昇, 竹中 洋美
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出願人：NECソフト株式会社, 独立行政法人農業生物資源研究所
出願番号：JP2005006339 出願日：2005年3月
公開番号：WO2005-095599 公開日：2005年10月
老齢マウス心筋細胞における核小体低分子RNA（snoRNA）の機能解析
研究課題/領域番号：24K14708 2024年4月 - 2027年3月
日本学術振興会 科学研究費助成事業 基盤研究(C)
竹中 康浩
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コリン作動系修飾物を介した血液脳関門強化と抗炎症作用による認知機能介入の可能性
研究課題/領域番号：21K11658 2021年4月 - 2025年3月
日本学術振興会 科学研究費助成事業 基盤研究(C)
柿沼 由彦, 竹中 康浩, 洲鎌 秀永
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分裂終了細胞における新規な老化マーカーの探索とその機能の解析
研究課題/領域番号：21K11605 2021年4月 - 2024年3月
日本学術振興会 科学研究費助成事業 基盤研究(C)
竹中 康浩, 平崎 正孝, 柿沼 由彦, 大畠 久幸
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配分額：4030000円 （ 直接経費：3100000円 、 間接経費：930000円 ）
本研究の目的は、老化個体の分裂終了細胞等に普遍的な新規遺伝子マーカーを同定し、分裂終了細胞の老化が個体の老化にどのように寄与しているのかを本老化マーカーの加齢におけるカイネティクス及び機能解析により解明することである。この目的のために本年度は簡便な単離法が確立されている心筋細胞を分裂終了細胞の材料として研究を行った。以下計画書に記載した研究実施計画に沿って当該年度（２０２１年度）に実施した研究の成果について記載する。
１.老齢マウス心筋細胞の網羅的トランスクリプトーム解析
【目的】老齢および若齢マウス心筋細胞の網羅的トランスクリプトーム解析を行う。
【研究成果】分裂終了細胞に特徴的な新規の老化マーカーを同定するために、まず若齢（１４ー１６週齢）および老齢（１年齢）のマウス心臓からコラゲナーゼ還流法により心筋細胞を単離した。同時に分裂終了細胞と分裂細胞との比較を行う目的から分裂性の非心筋細胞についても調製した。次に若齢および老齢心筋細胞からRNA精製を行い、RNAシーケンス解析に供した。その結果老齢の心筋細胞においてRNA発現量が増加している遺伝子を多数同定した。一方、非心筋細胞については後述の理由によりRNAシーケンス解析については保留中である。
２.分裂終了細胞に特徴的な老化マーカーの同定とそのカイネティクス解析
【目的】トランスクリプトーム解析の結果から候補分子を絞り込み、分裂終了細胞に特徴的な新規老化マーカーを同定する。【研究成果】現在心筋細胞のRNAシーケンス解析により得られた結果からいくつかのGOタームに分けて候補老化マーカー遺伝子を絞り込んでいる。
PPARgamma1特異的プロモータのみの欠失による小人症マウス発症因子探索
研究課題/領域番号：26461367 2014年4月 - 2017年3月
日本学術振興会 科学研究費助成事業 基盤研究(C)
井上 郁夫, 中野 貴成, 竹中 康浩
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配分額：4940000円 （ 直接経費：3800000円 、 間接経費：1140000円 ）
我々は、PPARγ1と異なるプロモータであるエクソンCから転写される、新規スプライシングバリアントを発見し、コンストラクトを構築してきた(Fig.1A)(Takahashi S, Inoue I, et al. J Atheroscler Thromb 17:73-83, 2010)。この新規SVが、PPARγ2と同様脂肪細胞分化誘導に関わり(Y Takenaka, I Inoue: PLoS One 8:e65583,2013)、特異的ノックアウト(KO)ホモマウスが胎生致死で(Fig.1C)、一方、エクソンA1特異的KOホモマウスは、受精18.5日まで正常に成長していた(Fig.2)。
腸管コレステロール吸収制御機構の新規パラダイムの提唱と検証
研究課題/領域番号：25504013 2013年4月 - 2016年3月
日本学術振興会 科学研究費助成事業 基盤研究(C)
中野 貴成, 竹中 康浩
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配分額：4420000円 （ 直接経費：3400000円 、 間接経費：1020000円 ）
血中コレステロール(chol)上昇は動脈硬化を介した心疾患の主要リスク因子である。腸管からのchol吸収亢進はそのリスク増加に寄与するが、その量的制御機構は未解明のままである。我々はその吸収量が取り込まれたcholの一部を管腔内へ戻すこと(排出)により調節されているとする新たなモデルを提唱する。マウス腸管において、管腔内のcholが細胞膜に取り込まれた後、chol輸送膜タンパク質Niemann-PickC1-like 1(NPC1L1)により細胞内の膜輸送システムに渡されるか、 もしくはABCG5/G8で管腔側へ排出されることを確認した。
胎生期に高発現するPPARγ3のマウス後期胚における局在と機能解析
研究課題/領域番号：25460299 2013年4月 - 2015年3月
日本学術振興会 科学研究費助成事業 基盤研究(C)
竹中 康浩, 中野 貴成, 井上 郁夫
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配分額：4160000円 （ 直接経費：3200000円 、 間接経費：960000円 ）
前年度の研究ではQuantiGene ViewRNA法を用いてマウス切片におけるPparγ1svの特異的検出を試みたが、Pparγ1svの発現量が低いため検出できなかった。そこで平成２６年度はPparγの共通領域でプローブ配列を設計してPparγの全てのスプライシングバリアントを検出することを試みた。その結果、微弱ではあるがマウス１７日胚の唾液腺と思われる部位にてPparγの発現を確認することができた。また脂肪組織や毛根などでもPparγの発現が認められた。一方抗Pparγ抗体を用いたマウス胚切片の免疫染色の実験では、残念ながらネガティブコントロールと比較して明らかにPparγの発現が認められる組織は確認できなかった。
Pparγ1svの発現制御機構の研究では、Pparγ1svおよびPparγ2の転写がPPARγタンパク質自身によって制御されている可能性を検証した。まず遺伝子発現バンクGEOに登録されているPPARγ抗体を用いたChIP-seqのデータからPPARγ遺伝子およびそのプロモーター領域にPPARγタンパク質が結合している部位が存在するかを解析したところ、３カ所で顕著な結合が確認された。そこで3T3-L1の脂肪細胞分化誘導時にPPARγタンパク質がこれらの部位に結合しているかどうかをChIP-qPCRを用いて確認したところ、分化２日目に３カ所すべてでPPARγタンパク質の有意な結合が認められた。
一方Pparγ1svの胎生期における機能を解明するため、竹中と研究分担者（中野、井上）は、Pparγ1svにユニークなエクソンCのみを欠損したノックアウトマウスの作製を行った。得られたエクソンCのホモノックアウトマウスは野生型と比較して低体重、脂肪組織量の低下などの表現形が確認された。今後さらに詳細な解析を行う予定である。
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