研究者詳細 - 豊國　伸哉

Long-term proliferation in culture and germline transmission of mouse male germline stem cells Open Access

Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, Shinohara T

BIOLOGY OF REPRODUCTION 69 巻 ( 2 ) 頁： 612 - 616 2003年8月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Biology of Reproduction

Spermatogenesis is a complex process that originates in a small population of spermatogonial stem cells. Here we report the in vitro culture of spermatogonial stem cells that proliferate for long periods of time. In the presence of glial cell line-derived neurotrophic factor, epidermal growth factor, basic fibroblast growth factor, and leukemia inhibitory factor, gonocytes isolated from neonatal mouse testis proliferated over a 5-month period (>1014-fold) and restored fertility to congenitally infertile recipient mice following transplantation into seminiferous tubules. Long-term spermatogonial stem cell culture will be useful for studying spermatogenesis mechanism and has important implications for developing new technology in transgenesis or medicine.

DOI： 10.1095/biolreprod.103.017012

Involvement of death receptor Fas in germ cell degeneration in gonads of Kit-deficient Wv/Wv mutant mice. 査読有り国際誌 Open Access

Sakata S, Sakamaki K, Watanabe K, Nakamura N, Toyokuni S, Nishimune Y, Mori C, Yonehara S

Cell death and differentiation 10 巻 ( 6 ) 頁： 676 - 86 2003年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cell Death and Differentiation

Kit and its ligand stem cell factor (SCF) play a fundamental role in hematopoiesis, melanogenesis and gametogenesis. Homozygous Wv mutant mice with a mutation in kit show abnormalities in these cell lineages. Fas is a member of the death receptor family inducing apoptosis. In this study, we generated double-mutant mice (Wv/Wv:Fas-/-) and analyzed histologically their reproductive organs. In testes and ovaries of the double-mutant mice, testicular germ cells and oocytes were detected, respectively, whereas the same-aged Wv/Wv mice contained neither cells. In addition, inhibition of Kit signals by administration of anti-Kit mAb, which induces degeneration of testicular germ cells in vivo in wild-type mice, did not cause degeneration in Fas-deficient mice. In testicular germ cells of Wv/Wv mutant mice, an increase of Fas expression was observed in spermatogonia. Further, in vitro treatment with SCF was shown to downregulate Fas on fibroblasts expressing exogenous Kit through activation of PI3-kinase/Akt. All the results clearly indicate that Fas-mediated apoptosis is involved in germ cell degeneration accompanied by defects in Kit-mediated signals, and Kit signaling negatively regulates Fas-mediated apoptosis in vivo.

DOI： 10.1038/sj.cdd.4401215

Evidence for oxidative stress in NSAID-induced colitis in IL10-/- mice. 国際誌

Seiko Narushima, Douglas R Spitz, Larry W Oberley, Shinya Toyokuni, Toshio Miyata, Carol A Gunnett, Garry R Buettner, Juan Zhang, Hanan Ismail, Richard G Lynch, Daniel J Berg

Free radical biology & medicine 34 巻 ( 9 ) 頁： 1153 - 66 2003年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

The goal of this study was to evaluate for evidence of oxidative stress in colonic inflammation in a novel model of inflammatory bowel disease, nonsteroidal anti-inflammatory drug- (NSAID-) treated interleukin-10-deficient (IL10(-/-)) mice. IL10(-/-) and wild-type (wt) mice were treated with a nonselective NSAID (piroxicam, 200 ppm in the diet) for 2 weeks to induce colitis, and parameters for oxidative stress in the colonic tissues were evaluated. Mean chemiluminescence enhanced with lucigenin in the colons from IL10(-/-) mice treated with piroxicam was more than 5-fold higher than that of the control wt group. Chemiluminescence was inhibited with diphenylethylene iodinium, but not allopurinol, indomethacin, or N-omega-nitro-L-arginine, indicating that flavin-containing enzymes were the source of the reactive oxygen species. Colonic aconitase activity in NSAID-treated IL10(-/-) mice decreased to 50% of the activity of control mice. There was no difference in the total glutathione levels in the colonic mucosa among the groups; however, glutathione disulfide levels were approximately 2-fold greater in the colon of NSAID-treated IL10(-/-) mice as compared with control groups. Immunohistochemistry studies of colons from NSAID-treated IL10(-/-) mice demonstrated intense staining with two antibodies that recognize advanced glycation endproducts formed through glycation and oxidation: anticarboxymethylysine and antipentosidine. The epithelial cells and lamina propria cells in the colons of NSAID-treated IL10(-/-) mice showed immunostaining with antinitrotyrosine, indicating the presence of reactive nitrogen species. Colonic epithelium of IL10(-/-) mice with colitis showed moderate immunostaining for 8-hydroxy-2'-deoxyguanosine in the nuclei. NSAID-treated IL10(-/-) mice treated with diphenylene idodonium chloride (DPI), an irreversible inhibitor of flavoprotein enzymes, experienced significantly reduced inflammation. Taken together, these results strongly indicate the presence of oxidative stress in the inflammatory bowel disease in NSAID-treated IL10(-/-) mice and suggests a role for oxidative stress in the pathophysiology of this model of inflammatory bowel disease.

DOI： 10.1016/S0891-5849(03)00065-0

Evidence for oxidative stress in NSAID-induced colitis in IL10(-/-) mice

Narushima S, Spitz DR, Oberley LW, Toyckuni S, Miyata T, Gunnett CA, Buettner GR, Zhang J, Ismail H, Lynch RG, Berg DJ

FREE RADICAL BIOLOGY AND MEDICINE 34 巻 ( 9 ) 頁： 1153 - 1166 2003年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

The goal of this study was to evaluate for evidence of oxidative stress in colonic inflammation in a novel model of inflammatory bowel disease, nonsteroidal anti-inflammatory drug- (NSAID-) treated interleukin-10-deficient (IL10-/-) mice. IL10-/- and wild-type (wt) mice were treated with a nonselective NSAID (piroxicam, 200 ppm in the diet) for 2 weeks to induce colitis, and parameters for oxidative stress in the colonic tissues were evaluated. Mean chemiluminescence enhanced with lucigenin in the colons from IL10-/- mice treated with piroxicam was more than 5-fold higher than that of the control wt group. Chemiluminescence was inhibited with diphenylethylene iodinium, but not allopurinol, indomethacin, or N-ω-nitro-L-arginine, indicating that flavin-containing enzymes were the source of the reactive oxygen species. Colonic aconitase activity in NSAID-treated IL10-/- mice decreased to 50% of the activity of control mice. There was no difference in the total glutathione levels in the colonic mucosa among the groups; however, glutathione disulfide levels were approximately 2-fold greater in the colon of NSAID-treated IL10-/- mice as compared with control groups. Immunohistochemistry studies of colons from NSAID-treated IL10-/- mice demonstrated intense staining with two antibodies that recognize advanced glycation endproducts formed through glycation and oxidation: anticarboxymethylysine and antipentosidine. The epithelial cells and lamina propria cells in the colons of NSAID-treated IL10-/- mice showed immunostaining with antinitrotyrosine, indicating the presence of reactive nitrogen species. Colonic epithelium of IL10-/- mice with colitis showed moderate immunostaining for 8-hydroxy-2′-deoxyguanosine in the nuclei. NSAID-treated IL10-/- mice treated with diphenylene idodonium chloride (DPI), an irreversible inhibitor of flavoprotein enzymes, experienced significantly reduced inflammation. Taken together, these results strongly indicate the presence of oxidative stress in the inflammatory bowel disease in NSAID-treated IL10-/- mice and suggests a role for oxidative stress in the pathophysiology of this model of inflammatory bowel disease. © 2003 Elsevier Inc.

DOI： 10.1016/S0891-5849(03)00065-0

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis

Functional assessment of self-renewal activity of male germline stem cells following cytotoxic damage and serial transplantation. 査読有り国際誌 Open Access

Kanatsu-Shinohara M, Toyokuni S, Morimoto T, Matsui S, Honjo T, Shinohara T

Biology of reproduction 68 巻 ( 5 ) 頁： 1801 - 7 2003年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biology of Reproduction

Spermatogenesis is dependent on a small population of stem cells. Although stem cells are believed to expand infinitely, there is little functional evidence regarding whether spermatogonial stem cells can increase in their number. Using the spermatogonial transplantation technique, we evaluated the proliferative potential of spermatogonial stem cells in two models of regeneration. After busulfan injection to deplete stem cells, the surviving stem cells were able to expand by at least 15.8-fold within 2 mo. On the other hand, a serial transplantation study indicated that one transplanted stem cell was able to expand by 3.8-and 12-fold within 2 and 4 mo, respectively. These results provide direct functional evidence for the expansion of stem cells and establish the basis for further characterization of the stem cell self-renewal process.

DOI： 10.1095/biolreprod.102.012575

TOYOKUNI Shinya, KAWAGUCHI Waka, AKATSUKA Shinya, HIROYASU Makoto, HIAI Hiroshi

Pathology international 53 巻 ( 4 ) 頁： 259 - 261 2003年4月

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis

TOYOKUNI Shinya, KAWAGUCHI Waka, AKATSUKA Shinya, HIROYASU Makoto, HIAI Hiroshi

Pathology international 53 巻 ( 4 ) 頁： 259 - 261 2003年4月

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis

Toyokuni S, Kawaguchi W, Akatsuka S, Hiroyasu M, Hiai H

PATHOLOGY INTERNATIONAL 53 巻 ( 4 ) 頁： 259 - 261 2003年4月

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis.

Toyokuni S, Kawaguchi W, Akatsuka S, Hiroyasu M, Hiai H

Pathology international 53 巻 ( 4 ) 頁： 259 - 61 2003年4月

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis. 国際誌

Shinya Toyokuni, Waka Kawaguchi, Shinya Akatsuka, Makoto Hiroyasu, Hiroshi Hiai

Pathology international 53 巻 ( 4 ) 頁： 259 - 61 2003年4月

Glycine ameriorates lung reprefusion injury after cold preservation in an ex vivo rat lung model. Open Access

Omasa M, Fukuse T, Toyokuni S, Mizutani Y, Yoshida H, Ikeyama K, Hasegawa S, Wada H

TRANSPLANTATION 75 巻 ( 5 ) 頁： 591 - 598 2003年3月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Transplantation

Background. The role of glycine has not been investigated in lung ischemia-reperfusion injury after cold preservation. Furthermore, the role of apoptosis after reperfusion following cold preservation has not been fully understood. Methods. Lewis rats were divided into three groups (n=6 each). In the GLY(-) and GLY(+) groups, isolated lungs were preserved for 15 hr at 4°C after a pulmonary artery (PA) flush using our previously developed preservation solution (ET-K; extracellular-type trehalose containing Kyoto), with or without the addition of glycine (5 mM). In the Fresh group, isolated lungs were reperfused immediately after a PA flush with ET-K. They were reperfused for 60 min with an ex vivo perfusion model. Pulmonary function, oxidative stress, apoptosis, and tumor necrosis factor (TNF)-α expression were assessed after reperfusion. Results. Shunt fraction and peak inspiratory pressure after reperfusion in the GLY(-) group were significantly higher than those in the GLY(+) and Fresh groups. Oxidative damage and apoptosis in the alveolar epithelial cells of the GLY(-) group, assessed by immunohistochemical staining and quantification of 8-hydroxy-2′-deoxyguanosine and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method, were significantly higher than those of the GLY(+) and Fresh groups. There were correlations among shunt fraction, oxidative damage, and apoptosis. There was no expression of TNF-α messenger RNA in all groups evaluated by the reverse transcription-polymerase chain reaction. Conclusions. Glycine attenuates ischemia/reperfusion injury after cold preservation by reducing oxidative damage and suppressing apoptosis independent of TNF-α in this model. The suppression of apoptosis might ameliorate lung function after reperfusion.

DOI： 10.1097/01.TP.0000053200.98125.14

Glycine ameliorates lung reperfusion injury after cold preservation in an ex vivo rat lung model. 国際誌

Mitsugu Omasa, Tatsuo Fukuse, Shinya Toyokuni, Yoichi Mizutani, Hiroshi Yoshida, Kazuyuki Ikeyama, Seiki Hasegawa, Hiromi Wada

Transplantation 75 巻 ( 5 ) 頁： 591 - 8 2003年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

BACKGROUND: The role of glycine has not been investigated in lung ischemia-reperfusion injury after cold preservation. Furthermore, the role of apoptosis after reperfusion following cold preservation has not been fully understood. METHODS: Lewis rats were divided into three groups (n=6 each). In the GLY(-) and GLY(+) groups, isolated lungs were preserved for 15 hr at 4 degrees C after a pulmonary artery (PA) flush using our previously developed preservation solution (ET-K; extracellular-type trehalose containing Kyoto), with or without the addition of glycine (5 mM). In the Fresh group, isolated lungs were reperfused immediately after a PA flush with ET-K. They were reperfused for 60 min with an ex vivo perfusion model. Pulmonary function, oxidative stress, apoptosis, and tumor necrosis factor (TNF)-alpha expression were assessed after reperfusion. RESULTS: Shunt fraction and peak inspiratory pressure after reperfusion in the GLY(-) group were significantly higher than those in the GLY(+) and Fresh groups. Oxidative damage and apoptosis in the alveolar epithelial cells of the GLY(-) group, assessed by immunohistochemical staining and quantification of 8-hydroxy-2'-deoxyguanosine and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method, were significantly higher than those of the GLY(+) and Fresh groups. There were correlations among shunt fraction, oxidative damage, and apoptosis. There was no expression of TNF-alpha messenger RNA in all groups evaluated by the reverse transcription-polymerase chain reaction. CONCLUSIONS: Glycine attenuates ischemia/reperfusion injury after cold preservation by reducing oxidative damage and suppressing apoptosis independent of TNF-alpha in this model. The suppression of apoptosis might ameliorate lung function after reperfusion.

Regulation of marginal zone B cell development by MINT, a suppressor of Notch/RBP-J signaling pathway.

Tumor initiating activity of <i>Helicobacter pylori</i> water extract on mouse skin carcinogenesis 査読有り国際誌

Ishikawa, T; Yoshida, N; Tokuda, H; Ichiishi, E; Kuchide, M; Kokura, S; Naito, Y; Toyokuni, S; Nishino, H; Yoshikawa, T

CANCER LETTERS 191 巻 ( 1 ) 頁： 41 - 47 2003年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Letters

Helicobacter pylori (H. pylori) infection has been associated with gastric carcinogenesis, but responsible and detail mechanisms are insufficient by the absence of adequate data. To obtain direct evidence regarding the carcinogenicity of H. pylori, we investigated the initiating and promoting activity of H. pylori water extract (HPE) in two-stage mouse skin carcinogenesis model. HPE treatment, as an initiation, significantly enhanced tumor formation compared with control group. Moreover, HPE treatment increased production of 8-hydroxydeoxyguanosine in epidermal cells and HPE-initiated/TPA-promoted papillomas demonstrated a point mutation of the Ha-ras gene. These results suggest an initiating activity of HPE on two-stage mouse skin carcinogenesis. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0304-3835(02)00628-6

Structural basis of protein-bound endogenous aldehydes. Chemical and immunochemical characterizations of configurational isomers of a 4-hydroxy-2-nonenal-histidine adduct. 査読有り国際誌 Open Access

Hashimoto M, Sibata T, Wasada H, Toyokuni S, Uchida K

The Journal of biological chemistry 278 巻 ( 7 ) 頁： 5044 - 51 2003年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Biological Chemistry

4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, reacts with histidine to form a stable HNE-histidine Michael addition-type adduct possessing three chiral centers in the cyclic hemiacetal structure. In the present study, we characterized configurational isomers of a HNE-Nα-acetylhistidine adduct by NMR spectroscopy and by molecular orbital calculations. In addition, we raised monoclonal antibodies against (R)-HNE-histidine and (S)-HNE-histidine adducts, characterized their specificities, and examined in vivo localizations of each adduct under oxidative stress. To facilitate structural characterization of the configurational isomers of an HNE-histidine adduct, we prepared the (R)-HNE-histidine and (S)-HNE-histidine adducts by incubating Nα-acetylhistidine with each HNE enantiomer, both of which provided two peaks (Ra and Rb from (R)-HNE-histidine and Sa and Sb from (S)-HNE-histidine adducts) in reversed-phase high-performance liquid chromatography. The NMR analysis showed that each peak was a mixture of two diastereomers. In addition, the analysis of the nuclear Overhauser effect enabled the determination of configurations of the eight isomers. The relative amounts of these isomers in the NMR analysis correlated with the relative energies calculated by molecular orbital methods. On the other hand, using (R)-HNE-modified and (S)-HNE-modified keyhole limpet hemocyanins as the antigens, we raised the monoclonal antibodies, mAbR310 and mAbS412, which enantioselectively recognized the (R)-HNE-histidine and (S)-HNE-histidine adducts, respectively. Among the mixtures (Ra, Rb, Sa, and Sb) of diastereomers, mAbR310 showed the highest immunoreactivity to Rb (the mixture of 2R,4S,5R and 2S,4S,5R isomers), whereas mAbS412 preferentially recognized Sa (the mixture of 2R,4S,5S and 2S,4S,5S isomers). The presence of (R)-HNE and (S)-HNE epitopes in vivo was immunohistochemically examined in the kidney of rats exposed to the renal carcinogen, ferric nitrilotriacetate, by which nuclear and cytosolic stainings with mAbR310 and mAbS412, respectively, were detected.

DOI： 10.1074/jbc.M210129200

Kuroda K, Han H, Tani S, Tanigaki K, Tun T, Furukawa T, Taniguchi Y, Kurooka H, Hamada Y, Toyokuni S, Honjo T

Immunity 18 巻 ( 2 ) 頁： 301 - 12 2003年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Immunity

We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless.

DOI： 10.1016/s1074-7613(03)00029-3

Regulation of marginal zone B cell development by MINT, a suppressor of Notch/RBP-J signaling pathway

Kuroda K, Han H, Tani S, Tanigaki K, Tun T, Furukawa T, Taniguchi Y, Kurooka H, Hamada Y, Toyokuni S, Honjo T

IMMUNITY 18 巻 ( 2 ) 頁： 301 - 312 2003年2月

Regulation of marginal zone B cell development by MINT, a suppressor of Notch/RBP-J signaling pathway. 国際誌

Kazuki Kuroda, Hua Han, Shoichi Tani, Kenji Tanigaki, Tin Tun, Takahisa Furukawa, Yoshihito Taniguchi, Hisanori Kurooka, Yoshio Hamada, Shinya Toyokuni, Tasuku Honjo

Immunity 18 巻 ( 2 ) 頁： 301 - 12 2003年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

DOI： 10.1016/S1074-7613(03)00029-3

Astaxanthin limits exercise-induced skeletal and cardiac muscle damage in mice. 査読有り国際誌

Aoi W, Naito Y, Sakuma K, Kuchide M, Tokuda H, Maoka T, Toyokuni S, Oka S, Yasuhara M, Yoshikawa T

Antioxidants & redox signaling 5 巻 ( 1 ) 頁： 139 - 44 2003年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Antioxidants and Redox Signaling

Dietary antioxidants may attenuate oxidative damage from strenuous exercise in various tissues. Beneficial effects of the antioxidant astaxanthin have been demonstrated in vitro, but not yet in vivo. We investigated the effect of dietary supplementation with astaxanthin on oxidative damage induced by strenuous exercise in mouse gastrocnemius and heart. C57BL/6 mice (7 weeks old) were divided into groups: rested control, intense exercise, and exercise with astaxanthin supplementation. After 3 weeks of exercise acclimation, both exercise groups ran on a treadmill at 28 m/min until exhaustion. Exercise-increased 4-hydroxy-2-nonenal-modified protein and 8-hydroxy-2′-deoxyguanosine in gastrocnemius and heart were blunted in the astaxanthin group. Increases in plasma creatine kinase activity, and in myeloperoxidase activity in gastrocnemius and heart, also were lessened by astaxanthin. Astaxanthin showed accumulation in gastrocnemius and heart from the 3 week supplementation. Astaxanthin can attenuate exercise-induced damage in mouse skeletal muscle and heart, including an associated neutrophil infiltration that induces further damage.

DOI： 10.1089/152308603321223630