Updated on 2024/02/02

写真a

 
KOBUCHI Hirotsugu
 
Organization
Faculty of Medicine, Dentistry and Pharmaceutical Sciences Lecturer
Position
Lecturer
External link

Degree

  • Ph. D. ( Okayama University )

Research Interests

  • 創薬化学

  • 腫瘍学

  • 生化学

  • 分子細胞生物学

  • Biochemistry

Research Areas

  • Life Science / Tumor biology

  • Life Science / Pharmaceutical chemistry and drug development sciences

  • Life Science / Medical biochemistry

  • Life Science / Molecular biology

  • Life Science / Physiology

Education

  • Okayama University   医学部  

    1991.4 - 1995.8

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    Country: Japan

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  • Okayama University    

    - 1995

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  • Kochi Medical School   医学部  

    1988.4 - 1991.3

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  • Kochi University   農学部   農芸化学科

    1984.4 - 1988.3

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    Country: Japan

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  • Kochi University    

    - 1988

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Research History

  • Okayama University   細胞化学分野   Lecturer

    2021.4

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    Country:Japan

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  • Okayama University   Department of Cell Chemistry   Lecturer   博士

    2021.4

  • Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences   Department of Cell Chemistry   Assistant Professor

    2005.4

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  • - Senior Assistant Professor,Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,Okayama University

    2005

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  • Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences   Department of Cell Chemistry   Assistant Professor

    2001.4 - 2005.3

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  • Senior Assistant Professor,Okayama University

    2001 - 2005

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  • Okayama University   Medical School   Research Assistant

    1998.10 - 2001.3

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  • Research Associate,Medical School,Okayama University

    1998 - 2001

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  • University of California, Berkeley   Department of Molecular biology   Post doctoral fellow

    1994.12 - 1998.9

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  • Researcher,University of California, Berkeley

    1994 - 1998

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  • 倉敷成人病センター   研究員

    1988.4 - 1998.9

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  • Researcher,Kurasiki Medical center

    1988 - 1998

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Professional Memberships

 

Papers

  • Protein N-myristoylation plays a critical role in the mitochondrial localization of human mitochondrial complex I accessory subunit NDUFB7 Reviewed

    Haruna Harada, Koko Moriya, Hirotsugu Kobuchi, Naotada Ishihara, Toshihiko Utsumi

    Scientific Reports   13 ( 1 )   22991 - 22991   2023.12

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media LLC  

    Abstract

    The present study examined human N-myristoylated proteins that specifically localize to mitochondria among the 1,705 human genes listed in MitoProteome, a mitochondrial protein database. We herein employed a strategy utilizing cellular metabolic labeling with a bioorthogonal myristic acid analog in transfected COS-1 cells established in our previous studies. Four proteins, DMAC1, HCCS, NDUFB7, and PLGRKT, were identified as N-myristoylated proteins that specifically localize to mitochondria. Among these proteins, DMAC1 and NDUFB7 play critical roles in the assembly of complex I of the mitochondrial respiratory chain. DMAC1 functions as an assembly factor, and NDUFB7 is an accessory subunit of complex I. An analysis of the intracellular localization of non-myristoylatable G2A mutants revealed that protein N-myristoylation occurring on NDUFB7 was important for the mitochondrial localization of this protein. Furthermore, an analysis of the role of the CHCH domain in NDUFB7 using Cys to Ser mutants revealed that it was essential for the mitochondrial localization of NDUFB7. Therefore, the present results showed that NDUFB7, a vital component of human mitochondrial complex I, was N-myristoylated, and protein N-myrisotylation and the CHCH domain were both indispensable for the specific targeting and localization of NDUFB7 to mitochondria.

    DOI: 10.1038/s41598-023-50390-z

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    Other Link: https://www.nature.com/articles/s41598-023-50390-z

  • A Novel 89Zr-labeled DDS Device Utilizing Human IgG Variant (scFv): “Lactosome” Nanoparticle-Based Theranostics for PET Imaging and Targeted Therapy Reviewed

    Melissa Siaw Han Lim, Takashi Ohtsuki, Fumiaki Takenaka, Kazuko Kobayashi, Masaru Akehi, Hirotaka Uji, Hirotsugu Kobuchi, Takanori Sasaki, Eiichi Ozeki, Eiji Matsuura

    Life   11 ( 2 )   158 - 186   2021.2

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:MDPI AG  

    “Theranostics,” a new concept of medical advances featuring a fusion of therapeutic and diagnostic systems, provides promising prospects in personalized medicine, especially cancer. The theranostics system comprises a novel 89Zr-labeled drug delivery system (DDS), derived from the novel biodegradable polymeric micelle, “Lactosome” nanoparticles conjugated with specific shortened IgG variant, and aims to successfully deliver therapeutically effective molecules, such as the apoptosis-inducing small interfering RNA (siRNA) intracellularly while offering simultaneous tumor visualization via PET imaging. A 27 kDa-human single chain variable fragment (scFv) of IgG to establish clinically applicable PET imaging and theranostics in cancer medicine was fabricated to target mesothelin (MSLN), a 40 kDa-differentiation-related cell surface glycoprotein antigen, which is frequently and highly expressed by malignant tumors. This system coupled with the cell penetrating peptide (CPP)-modified and photosensitizer (e.g., 5, 10, 15, 20-tetrakis (4-aminophenyl) porphyrin (TPP))-loaded Lactosome particles for photochemical internalized (PCI) driven intracellular siRNA delivery and the combination of 5-aminolevulinic acid (ALA) photodynamic therapy (PDT) offers a promising nano-theranostic-based cancer therapy via its targeted apoptosis-inducing feature. This review focuses on the combined advances in nanotechnology and material sciences utilizing the “89Zr-labeled CPP and TPP-loaded Lactosome particles” and future directions based on important milestones and recent developments in this platform.

    DOI: 10.3390/life11020158

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  • Lactosome-Conjugated siRNA Nanoparticles for Photo-Enhanced Gene Silencing in Cancer Cells. Reviewed International journal

    Melissa Siaw Han Lim, Yuki Nishiyama, Takashi Ohtsuki, Kazunori Watanabe, Hirotsugu Kobuchi, Kazuko Kobayashi, Eiji Matsuura

    Journal of pharmaceutical sciences   110 ( 4 )   1788 - 1798   2021.1

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    The A3B-type Lactosome comprised of poly(sarcosine)3-block-poly(l-lactic acid), a biocompatible and biodegradable polymeric nanomicelle, was reported to accumulate in tumors in vivo via the enhanced permeability and retention (EPR) effect. Recently, the cellular uptake of Lactosome particles was enhanced through the incorporation of a cell-penetrating peptide (CPP), L7EB1. However, the ability of Lactosome as a drug delivery carrier has not been established. Herein, we have developed a method to conjugate the A3B-type Lactosome with ATP-binding cassette transporter G2 (ABCG2) siRNA for inducing in vitro apoptosis in the cancer cell lines PANC-1 and NCI-H226. The L7EB1 peptide facilitates the cellular uptake efficiency of Lactosome but does not deliver siRNA into cytosol. To establish the photoinduced cytosolic dispersion of siRNA, a photosensitizer loaded L7EB1-Lactosome was prepared, and the photosensitizer 5,10,15,20-tetra-kis(pentafluorophenyl)porphyrin (TPFPP) showed superiority in photoinduced cytosolic dispersion. We exploited the combined effects of enhanced cellular uptake by L7EB1 and photoinduced endosomal escape by TPFPP to efficiently deliver ABCG2 siRNA into the cytosol for gene silencing. Moreover, the silencing of ABCG2, a protoporphyrin IX (PpIX) transporter, also mediated photoinduced cell death via 5-aminolevulinic acid (ALA)-mediated PpIX accumulated photodynamic therapy (PDT). The synergistic capability of the L7EB1/TPFPP/siRNA-Lactosome complex enabled both gene silencing and PDT.

    DOI: 10.1016/j.xphs.2021.01.026

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  • Identification and characterization of protein N-myristoylation occurring on four human mitochondrial proteins, SAMM50, TOMM40, MIC19, and MIC25. Reviewed

    Utsumi T, Matsuzaki K, Kiwado A, Tanikawa A, Kikkawa Y, Hosokawa T, Otsuka A, Iuchi Y, Kobuchi H, Moriya K

    PloS one   13 ( 11 )   e0206355   2018.11

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    DOI: 10.1371/journal.pone.0206355

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  • 89Zr標識ヒト抗体バリアントと新規DDSキャリアによるTheranostics技術 Reviewed

    竹中文章, 小林和子, 木村俊作, 小関英一, 大槻高史, 小渕浩嗣, 松浦栄次

    Drug Delivery System   33 ( 3 )   214 - 222   2018.7

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:日本DDS学会 ; 2000-  

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    Other Link: https://search.jamas.or.jp/link/ui/2018300955

  • Phytoestrogen Suppresses Efflux of the Diagnostic Marker Protoporphyrin IX in Lung Carcinoma Reviewed

    Hirofumi Fujita, Keisuke Nagakawa, Hirotsugu Kobuchi, Tetsuya Ogino, Yoichi Kondo, Keiji Inoue, Taro Shuin, Toshihiko Utsumi, Kozo Utsumi, Junzo Sasaki, Hideyo Ohuchi

    CANCER RESEARCH   76 ( 7 )   1837 - 1846   2016.4

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER ASSOC CANCER RESEARCH  

    One promising method to visualize cancer cells is based on the detection of the fluorescent photosensitizer protoporphyrin IX (PpIX) synthesized from 5-aminolevulinic acid (ALA), but this method cannot be used in cancers that exhibit poor PpIX accumulation. PpIX appears to be pumped out of cancer cells by the ABC transporter G2 (ABCG2), which is associated with multidrug resistance. Genistein is a phytoestrogen that appears to competitively inhibit ABCG2 activity. Therefore, we investigated whether genistein can promote PpIX accumulation in human lung carcinoma cells. Here we report that treatment of A549 lung carcinoma cells with genistein or a specific ABCG2 inhibitor promoted ALA-mediated accumulation of PpIX by approximately 2-fold. ABCG2 depletion and overexpression studies further revealed that genistein promoted PpIX accumulation via functional repression of ABCG2. After an extended period of genistein treatment, a significant increase in PpIX accumulation was observed in A549 cells (3.7-fold) and in other cell lines. Systemic preconditioning with genistein in a mouse xenograft model of lung carcinoma resulted in a 1.8-fold increase in accumulated PpIX. Long-term genistein treatment stimulated the expression of genes encoding enzymes involved in PpIX synthesis, such as porphobilinogen deaminase, uroporphyrinogen decarboxylase, and protoporphyrinogen oxidase. Accordingly, the rate of PpIX synthesis was also accelerated by genistein pretreatment. Thus, our results suggest that genistein treatment effectively enhances ALA-induced PpIX accumulation by preventing the ABCG2-mediated efflux of PpIX from lung cancer cells and may represent a promising strategy to improve ALA-based diagnostic approaches in a broader set of malignancies.

    DOI: 10.1158/0008-5472.CAN-15-1484

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  • Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses Reviewed

    Emi Takamitsu, Motoaki Otsuka, Tatsuki Haebara, Manami Yano, Kanako Matsuzaki, Hirotsugu Kobuchi, Koko Moriya, Toshihiko Utsumi

    PLOS ONE   10 ( 8 )   e0136360-e0136360   2015.8

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:PUBLIC LIBRARY SCIENCE  

    To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources.

    DOI: 10.1371/journal.pone.0136360

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  • Erythroid and megakaryocytic differentiation of K562 erythroleukemic cells by monochloramine Reviewed

    T. Ogino, H. Kobuchi, H. Fujita, A. Matsukawa, K. Utsumi

    FREE RADICAL RESEARCH   48 ( 3 )   292 - 302   2014.3

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:INFORMA HEALTHCARE  

    The induction of leukemic cell differentiation is a hopeful therapeutic modality. We studied the effects of monochloramine (NH2Cl) on erythroleukemic K562 cell differentiation, and compared the effects observed with those of U0126 and staurosporine, which are known inducers of erythroid and megakaryocytic differentiation, respectively. CD235 (glycophorin) expression, a marker of erythroid differentiation, was significantly increased by NH2Cl and U0126, along with an increase in c mRNA levels. Other erythroid markers such as. gamma-globin and CD71 (transferrin receptor) were also increased by NH2Cl and U0126. In contrast, CD61 (integrin beta 3) and CD42b (GP1b alpha) expression, markers of megakaryocytic differentiation, was increased by staurosporine, but did not change significantly by NH 2 Cl and U0126. NH2Cl retarded cell proliferation without a marked loss of viability. When ERK phosphorylation (T202/ Y204) and CD235 expression were compared using various chemicals, a strong negative correlation was observed (r =-0.76). Paradoxically, NH2Cl and staurosporine, but not U0126, induced large cells with multiple or lobulated nuclei, which was characteristic to megakaryocytes. NH 2 Cl increased the mRNA levels of gata1 and scl, decreased that of gata2, and did not change those of pu.1 and klf1. The changes observed in mRNA expression were different from those of U0126 or staurosporine. These results suggest that NH2Cl induces the bidirectional differentiation of K562. Oxidative stress may be effective in inducing leukemic cell differentiation.

    DOI: 10.3109/10715762.2013.865840

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  • Necrotic and apoptotic cells serve as nuclei for calcification on osteoblastic differentiation of human mesenchymal stem cells in vitro Reviewed

    Hirofumi Fujita, Masanao Yamamoto, Tetsuya Ogino, Hirotsugu Kobuchi, Naoko Ohmoto, Eriko Aoyama, Takashi Oka, Tohru Nakanishi, Keiji Inoue, Junzo Sasaki

    CELL BIOCHEMISTRY AND FUNCTION   32 ( 1 )   77 - 86   2014.1

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    A close relationship between cell death and pathological calcification has recently been reported, such as vascular calcification in atherosclerosis. However, the roles of cell death in calcification by osteoblast lineage have not been elucidated in detail. In this study, we investigated whether cell death is involved in the calcification on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hMSC) under osteogenic culture in vitro. Apoptosis and necrosis occurred in an osteogenic culture of hMSC, and cell death preceded calcification. The generation of intracellular reactive oxygen species, chromatin condensation and fragmentation, and caspase-3 activation increased in this culture. A pan-caspase inhibitor (Z-VAD-FMK) and anti-oxidants (Tiron and n-acetylcysteine) inhibited osteogenic culture-induced cell death and calcification. Furthermore, calcification was significantly promoted by the addition of necrotic dead cells or its membrane fraction. Spontaneously dead cells by osteogenic culture and exogenously added necrotic cells were surrounded by calcium deposits. Induction of localized cell death by photodynamic treatment in the osteogenic culture resulted in co-localized calcification. These findings show that necrotic and apoptotic cell deaths were induced in an osteogenic culture of hMSC and indicated that both necrotic and apoptotic cells of osteoblast lineage served as nuclei for calcification on osteoblastic differentiation of hMSC in vitro. Copyright (c) 2013 John Wiley & Sons, Ltd.

    DOI: 10.1002/cbf.2974

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  • Acetyl-L-carnitine suppresses thyroid hormone-induced and spontaneous anuran tadpole tail shortening Reviewed

    Hideki Hanada, Hirotsugu Kobuchi, Masanao Yamamoto, Keiko Kashiwagi, Kenjiro Katsu, Toshihiko Utsumi, Akihiko Kashiwagi, Junzo Sasaki, Masayasu Inoue, Kozo Utsumi

    HEREDITAS   150 ( 1 )   1 - 9   2013.2

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    Mitochondrial membrane permeability transition (MPT) plays a crucial role in apoptotic tail shortening during anuran metamor phosis. L-carnitine is known to shuttle free fatty acids (FFAs) from the cytosol into mitochondria matrix for -oxidation and energy production, and in a previous study we found that treatment with L-carnitine suppresses 3, 3', 5-triiodothyronine (T3) and FFA-induced MPT by reducing the level of FFAs. In the present study we focus on acetyl-L-carnitine, which is also involved in fatty acid oxidation, to determine its effect on T3-induced tail regression in Rana rugosa tadpoles and spontaneous tail regression in Xenopus laevis tadpoles. The ladder-like DNA profile and increases in caspase-3 and caspase-9 indicative of apoptosis in the tails of T3-treated tadpoles were found to be suppressed by the addition of acetyl-L-carnitine. Likewise, acetyl-L-carnitine was found to inhibit thyroid hormone regulated spontaneous metamorphosis in X. laevis tadpoles, accompanied by decreases in caspase and phospholipase A2 activity, as well as non-ladder-like DNA profiles. These findings support our previous conclusion that elevated levels of FFAs initiate MPT and activate the signaling pathway controlling apoptotic cell death in tadpole tails during anuran metamorphosis.

    DOI: 10.1111/j.1601-5223.2013.02284.x

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  • Mitochondrial Localization of ABC Transporter ABCG2 and Its Function in 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation Reviewed

    Hirotsugu Kobuchi, Koko Moriya, Tetsuya Ogino, Hirofumi Fujita, Keiji Inoue, Taro Shuin, Tatsuji Yasuda, Kozo Utsumi, Toshihiko Utsumi

    PLOS ONE   7 ( 11 )   e50082-e50082   2012.11

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    Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol.

    DOI: 10.1371/journal.pone.0050082

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  • Serum-dependent export of protoporphyrin IX by ATP-binding cassette transporter G2 in T24 cells Reviewed

    Tetsuya Ogino, Hirotsugu Kobuchi, Kazuaki Munetomo, Hirofumi Fujita, Masanao Yamamoto, Toshihiko Utsumi, Keiji Inoue, Taro Shuin, Junzo Sasaki, Masayasu Inoue, Kozo Utsumi

    MOLECULAR AND CELLULAR BIOCHEMISTRY   358 ( 1-2 )   297 - 307   2011.12

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:SPRINGER  

    Accumulation of protoporphyrin IX (PpIX) in cancer cells is a basis of 5-aminolevulinic acid (ALA)-induced photodymanic therapy. We studied factors that affect PpIX accumulation in human urothelial carcinoma cell line T24, with particular emphasis on ATP-binding cassette transporter G2 (ABCG2) and serum in the medium. When the medium had no fetal bovine serum (FBS), ALA induced PpIX accumulation in a time- and ALA concentration-dependent manner. Inhibition of heme-synthesizing enzyme, ferrochelatase, by nitric oxide donor (Noc18) or deferoxamine resulted in a substantial increase in the cellular PpIX accumulation, whereas ABCG2 inhibition by fumitremorgin C or verapamil induced a slight PpIX increase. When the medium was added with FBS, cellular accumulation of PpIX stopped at a lower level with an increase of PpIX in the medium, which suggested PpIX efflux. ABCG2 inhibitors restored the cellular PpIX level to that of FBS(-) samples, whereas ferrochelatase inhibitors had little effects. Bovine serum albumin showed similar effects to FBS. Fluorescence microscopic observation revealed that inhibitors of ABC transporter affected the intracellular distribution of PpIX. These results indicated that ABCG2-mediated PpIX efflux was a major factor that prevented PpIX accumulation in cancer cells in the presence of serum. Inhibition of ABCG2 transporter system could be a new target for the improvement of photodynamic therapy.

    DOI: 10.1007/s11010-011-0980-5

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  • Mechanism of A23187-induced apoptosis in HL-60 cells: Dependency on mitochondrial permeability transition but not on NADPH oxidase Reviewed

    Noriko Kajitani, Hirotsugu Kobuchi, Hirofumi Fujita, Hiromi Yano, Takuzo Fujiwara, Tatsuji Yasuda, Kozo Utsumi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   71 ( 11 )   2701 - 2711   2007.11

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:TAYLOR & FRANCIS LTD  

    Calcium ions (Ca2+) are involved in a number of physiological cellular functions including apoptosis. An elevation in intracellular levels of Ca2+ in A23187-treated HL-60 cells was associated,with the generation of both intracellular and extracellular reactive oxygen species (ROS) and induction of apoptotic cell death. A23187-induced apoptosis was prevented by cyclosporin A, a potent inhibitor of mitochondrial permeability transition (NIPT). The generation of extracellular ROS was suppressed by the NADPH oxidase inhibitor diphenylene iodonium, and by superoxide dismutase, but these agents had no effect on A23187-induced apoptosis. In contrast, the blocking of intracellular ROS by a cell-permeant antioxidant diminished completely the induction of MPT and apoptosis. In isolated mitochondria, the addition of Ca2+ induced a typical MPT concomitant with the generation of ROS, which leads to augmentation of intracellular ROS levels. These results indicate that intracellular not extracellular ROS generated by A23187 is associated with the opening of NIPT pores that leads to apoptotic cell death.

    DOI: 10.1271/bbb.70304

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  • Cell-permeable cAMP analog suppresses 6-hydroxydopamine-induced apoptosis in PC12 cells through the activation of the Akt pathway Reviewed

    Hirofumi Fujita, Tetsuya Ogino, Hirotsugu Kobuchi, Takuzo Fujiwara, Hiromi Yano, Jitsuo Akiyama, Kozo Utsumi, Junzo Sasaki

    BRAIN RESEARCH   1113 ( 1 )   10 - 23   2006.10

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ELSEVIER SCIENCE BV  

    Although cAMP protects neuronal cells from various apoptotic stimulations, its mechanism is not fully elucidated. We report here the molecular mechanism of the 6-hydroxydopamine (6-OHDA)-induced apoptosis of pheochromocytoma PC12 cells and its suppression by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (pCPT-cAMP), which is a membrane permeable cAMP analog. Treatment of PC12 cells with 6-OHDA resulted in the activation of caspases and apoptosis, as detected by chromatin condensation. 6-OHDA also induced superoxide generation, Bid cleavage and mitochondrial membrane depolarization. In addition, Akt phosphorylation that was favorable to cell survival was decreased and p38 MAPK phosphorylation was increased by 6-OHDA. PC12 cell apoptosis was inhibited by pCPT-cAMP, Z-VAD-fmk (a broad-range caspase inhibitor) and tiron (a superoxide scavenger), although PC12 cell apoptosis was not inhibited by cyclosporine A (an inhibitor of mitochondrial membrane permeability transition). Moreover, pCPT-cAMP promoted Akt phosphorylation, but it did not prevent superoxide generation and mitochondrial membrane depolarization. Conversely, LY294002, an inhibitor of Akt upstream molecule PI3-kinase, enhanced 6-OHDA-induced apoptosis. These results indicated that the 6-OHDA-induced apoptosis of PC12 cells was initiated by superoxide generation followed by caspase cascade activation, which was associated with the suppressed Akt phosphorylation and increased p38 phosphorylation. It is likely that pCPT-cAMP prevented the 6-OHDA-induced apoptosis via activation of the PI3-kinase/Akt pathway without any effect on superoxide generation or mitochondrial membrane depolarization. (c) 2006 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.brainres.2006.06.079

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  • Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA Reviewed

    K Yamato, J Fen, H Kobuchi, Y Nasu, T Yamada, T Nishihara, Y Ikeda, M Kizaki, M Yoshinouchi

    CANCER GENE THERAPY   13 ( 3 )   234 - 241   2006.3

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

    Human cervical cancer is caused by high-risk types of human papillomavirus (HPV) such as HPV16 and HPV18, which possess the E6 and E7 oncogenes, whose concurrent expression is a prerequisite for cancer development and maintaining malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether E6, E7, or both should be silenced to obtain most efficient antitumor activity by an HPV small-interfering RNA (siRNA). Herein, we report two types of siRNAs targeting HPV18 E6, that exerted a negative growth effect on HPV18-positive cervical cancer cells (HeLa and SW756), in part, inducing cell death. One siRNA (Ex-18E6), designed to target both E6-E7 mRNA and its splicing variant, E6*I-E7 mRNA, efficiently knocked down both E6 and E7 expression. The other (Sp-18E6), designed to specifically target E6-E7 mRNA but not E6*I-E7 mRNA, suppressed E6 to a similar level as Ex-18E6; however, it less efficiently inhibited E7 as compared to Ex-18E6. Although both siRNAs induced cell death, Sp-18E6 siRNA induced more prominent cell death than Ex-18E6. Our results suggest that E6-specific suppression may induce more potent anticancer activity than simultaneous E6 and E7 suppression, and that E6-specific targeting is a promising strategy for siRNA-based therapy for HPV-positive cervical cancer.

    DOI: 10.1038/sj.cgt.7700891

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  • アポトーシスとミトコンドリアー神経細胞を加味して Reviewed

    藤田洋史, 小渕浩嗣, 内海耕慥

    Clinical Neuroscience   24 ( 6 )   643 - 646   2006

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  • 志賀毒素誘導の腎上皮様Vero細胞のアポトーシスにおけるAktの関与(Involvement of Akt in Shiga toxin-induced apoptosis of renal epithelial cells)

    Kobuchi Hirotsugu, Kaihara Keiko, Utsumi Kozo, Yasuda Tatsuji

    生化学   76 ( 8 )   1089 - 1089   2004.8

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  • Involvement of ceramide in the mechanism of Cr(VI)-induced apoptosis of CHO cells Reviewed

    S Muranaka, T Kanno, H Fujita, H Kobuchi, J Akiyama, T Yasuda, K Utsumi

    FREE RADICAL RESEARCH   38 ( 6 )   613 - 621   2004.6

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    Mitochondria reduce Cr(VI) to Cr(V) with concomitant generation of reactive oxygen species, thereby exhibiting cytotoxic effects leading to apoptosis in various types of cells. To clarify the mechanism by which Cr(VI) induces apoptosis, we examined the effect of Cr(VI) on Chinese hamster ovary (CHO) cells. Cr(VI) increased cellular levels of ceramide by activating acid sphingomyelinase (ASMase) and inhibiting the phosphorylation of pleckstrin homology domain-containing protein kinase B (Akt). Cr(VI) also induced cyclosporin A- and trifluoperazine-sensitive depolarization of mitochondria and activated caspase-3, 8 and 9, thereby causing fragmentation of cellular DNA. The presence of desipramine, an inhibitor of ASMase, and membrane permeable pCPT-cAMP suppressed the Cr(VI)-induced activation of caspases and DNA fragmentation. These results suggested that accumulation of ceramide play an important role in the Cr(VI)-induced apoptosis of CHO cells through activation of mitochondrial membrane permeability transition.

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  • Cholesteryl-hemisuccinate-induced apoptosis of promyelocytic leukemia HL-60 cells through a cyclosporin A-insensitive mechanism Reviewed

    K Yamada, K Arita, H Kobuchi, S Yamamoto, T Yoshioka, H Tamai, K Utsumi

    BIOCHEMICAL PHARMACOLOGY   65 ( 3 )   339 - 348   2003.2

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    We reported previously that alpha-tocopheryl-succinate (VES) induced apoptosis of cultured human promyelocytic leukemia cells (HL-60) (Free Radic Res 2000;33:407-18). We have now studied the effect of cholesteryl-hemisuccinate (CS) on the fate of HL-60 cells to clarify whether CS has an effect similar to that of VES. CS inhibited the growth of HL-60 cells without differentiation to granulocytes and induced DNA fragmentation and ladder formation. CS inhibited the phosphorylation of pleckstrin homology domain-containing protein kinase B (Akt) and initiated the activation of a caspase cascade. CS triggered the reaction leading to the cleavage of Bid and also released cytochrome c (Cyt. c) from mitochondria. In addition, CS induced mitochondrial membrane depolarization and translocation of Bax to mitochondria in HL-60 cells. However, CS did not induce an increase in the concentration of intracellular calcium ions in HL-60 cells. The membrane depolarization, Cyt. c release, and DNA fragmentation were inhibited by z-VAD-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor, but not by cyclosporin A, an inhibitor of membrane permeability transition. These results suggested that CS-induced apoptosis of HL-60 cells might be caused by inhibiting Akt phosphorylation following cleavage of Bid through caspase-8 activation and subsequently via an Apaf complex-caspase cascade pathway. (C) 2002 Elsevier Science Inc. All rights reserved.

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  • Mitochondria and apoptosis. Reviewed

    Kanno T, Utsumi K, Kobuchi H

    Nihon rinsho. Japanese journal of clinical medicine   60 Suppl 4   189 - 193   2002.4

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  • ミトコンドリアと細胞死 Reviewed

    菅野智子, 小渕浩嗣, 内海耕慥

    日本臨床   60 ( 4 )   189 - 193   2002

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  • Mevastatin, an inhibitor of HMG-CoA reductase, induces apoptosis, differentiation and Rap1 expression in HL-60 cells Reviewed

    Tomoko Kanno, Hirotsugu Kobuchi, Noriko Kajitani, Toshihiko Utsumi, Hiromi Yano, Alan A. Horton, Tatsuji Yasuda, Kozo Utsumi

    Physiological Chemistry and Physics and Medical NMR   34 ( 1 )   1 - 15   2002

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    It has been reported that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase suppress cell proliferation and induce apoptosis. One inhibitor which induces apoptosis is mevastatin. However, the molecular mechanism of apoptosis induction is not well understood so the effects of mevastatin on various functions of HL-60 cells were investigated. We confirmed that mevastatin activated caspase-3 by release of cytochrome c (Cyt. c) from mitochondria through a membrane permeability transition mechanism and also induced typical fragmentation and ladder formation of DNA in HL-60 cells. These effects were inhibited by mevalonate, a metabolic intermediate of cholesterol biosynthesis. Mevalonate and geranylgeraniol (GGOH) inhibited DNA fragmentation whereas farnesol (FOH) did not. Mevastatin also induced cell differentiation to monocytic cells via a mevalonate inhibitable mechanism. Furthermore, mevastatin decreased the amount of an isoprenylated membrane bound Rap1 small GTPase concomitant with an increase in cytosolic Rap1 which occurred before apoptosis and differentiation. On the contrary, both mevastatin and geranylgeranylacetone (GGA), which competes with geranylgeranyl pyrophosphate, induced membrane depolarization of isolated mitochondria without swelling and Cyt. c release. These results suggest that mevastatin-induced apoptosis of HL-60 cells might be caused indirectly by activation of the caspase cascade through the modulation of mitochondrial functions and that some relationship between a certain small GTPase molecule, such as Rap1, and mevastatin-induced apoptosis may exist.

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  • Mechanism of apoptosis in HL-60 cells induced by n-3 and n-6 polyunsaturated fatty acids Reviewed

    K Arita, H Kobuchi, T Utsumi, Y Takehara, J Akiyama, AA Horton, K Utsumi

    BIOCHEMICAL PHARMACOLOGY   62 ( 7 )   821 - 828   2001.10

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    The biochemical properties and specificity of n-3 and n-6 polyunsaturated fatty acids (PUFAs) are not well known. Because PUFAs induce apoptosis of different cells, we studied the effect of various PUFAs, such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosapentaenoic acid (DPA), on the fate of cultured human promyelocytic leukemia cells (HL-60) to elucidate the mechanism of apoptosis and the difference in action between n-3 and n-6 PUFAs. Fairly low concentrations of PUFAs inhibited the growth of HL-60 cells and induced their apoptosis by a mechanism that is sensitive to DMSO, an antioxidant, and z-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor. PUFAs stimulated the generation of reactive oxygen species (ROS) and activated various types of caspase-like proteases, such as caspase-3, -6, -8, and -9, but not caspase-1. In addition, PUFAs triggered the reaction leading to the cleavage of Bid, a death agonist member of the Bcl-2 family, and also released cytochrome c from mitochondria into the cytosol. PUFAs also decreased the mitochondrial membrane potential of intact HL-60 cells. All of these actions of n-3 PUFAs were stronger than those of AA, an n-6 PUFA, although the mechanism is not known. PUFAs stimulate swelling and membrane depolarization of isolated mitochondria in a cyclosporin A-sensitive manner. The results indicated that PUFA-induced apoptosis of HL-60 cells may be caused, in part, by direct action on the cells and by activation of the caspase cascade through cytochrome c release coupled with mitochondrial membrane depolarization. (C) 2001 Elsevier Science Inc. All rights reserved.

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  • 志賀毒素による細胞死誘導とその機序 Reviewed

    小渕 浩嗣

    岡山大学アイソトープセンターニュース   11   4 - 7   2001

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  • Mechanism of dibucaine-induced apoptosis in promyelocytic leukemia cells (HL-60) Reviewed

    K Arita, T Utsumi, A Kato, T Kanno, H Kobuchi, B Inoue, J Akiyama, K Utsumi

    BIOCHEMICAL PHARMACOLOGY   60 ( 7 )   905 - 915   2000.10

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    Dibucaine, a local anesthetic, inhibited the growth of promyelocytic leukemia cells (HL-60) without inducing arrest of the cell cycle and differentiation to granulocytes. Typical DNA fragmentation and DNA ladder formation were induced in a concentration- and time-dependent manner. The half-maximal concentration of dibucaine required to induce apoptosis was 100 mu M. These effects were prevented completely by the pan-caspase inhibitor z-Val-Ala-Asp-(OMe)-fluoromethylketone (z-VAD-fmk), thereby implicating the cysteine aspartase (caspase) cascade in the process. Dibucaine activated various caspases, such as caspase-3, -6, -8, and -9 (-like) activities, but nut caspase-1 (-like) activity, and induced mitochondrial membrane depolarization and the release of cytochrome c (Cyt.c) from mitochondria into the cytosol. Processing of pro-caspase-3, -8, and -9 by dibucaine was confirmed by western blot analysis. Bid, a death agonist member of the Bcl-2 family, was processed by caspases following exposure of cells to dibucaine. However, 100 mu M dibucaine scarcely inhibited oxidative phosphorylation, but it induced membrane permeability transition in isolated rat liver mitochondria. Taken together, these data suggest that dibucaine induced apoptosis of HL-60 cells through activation of the caspase cascade in conjunction with Cyt.c release induced by a processed product of Bid and depolarization of the mitochondrial membrane potential. BIOCHEM PHARMACOL 60;7:905-915, 2000. (C) 2000 Elsevier Science Inc.

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  • Mechanism of alpha-tocopheryl succinate-induced apoptosis of promyelocytic leukemia cells Reviewed

    S Yamamoto, H Tamai, R Ishisaka, T Kanno, K Arita, H Kobuchi, K Utsumi

    FREE RADICAL RESEARCH   33 ( 4 )   407 - +   2000

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    Selective induction of apoptosis in tumor cells is important for treating patients with cancer. Because oxidative stress plays an important role in the process of apoptosis, we studied the effect of alpha-tocopheryl succinate (VES) on the fate of cultured human promyelocytic leukemia cells (HL-60). The presence of fairly low concentrations of VES inhibited the growth and DNA synthesis of HL-60 cells, and also induced their apoptosis via a mechanism that was inhibited by z-VAD-fluoromethylketone (z-VAD-fmk), an inhibitor of pan-caspases. VES activated various types of caspases, including caspase-3, 6, 8, and 9, but not caspase-1. VES triggered the reaction leading to the cleavage of Bid, a member of the death agonist Bcl-2 family, and released cytochrome c (Cyt.c) from the mitochondria into the cytosol by a z-VAD-fmk-inhibitable mechanism. VES transiently increased the intracellular calcium level [Ca2+](i) and stimulated the release of Cyt.c in the presence of inorganic phosphate (Pi). However, high concentrations of VES (similar to 100 mu M) hardly induced swelling of isolated mitochondria but depolarized the mitochondrial membrane potential by a cyclosporin A (CsA)-insensitive mechanism. These results indicate that VES-induced apoptosis of HL-60 cells might be caused by activation of the caspase cascade coupled with modulation of mitochondrial membrane function.

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  • Quercetin inhibits inducible ICAM-1 expression in human endothelial cells through the JNK pathway Reviewed

    H Kobuchi, S Roy, CK Sen, HG Nguyen, L Packer

    AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY   277 ( 3 )   C403 - C411   1999.9

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    Quercetin inhibits inducible ICAM-1 expression in human endothelial cells through the JNK pathway. Am. J. Physiol. 277 (Cell Physiol. 46): C403-C411, 1999.-The cell adhesion molecule intercellular adhesion molecule-1 (ICAM-1) plays a pivotal role in inflammatory responses. Quercetin (3,3',4',5,7-pentahydroxyflavone), a naturally occurring dietary flavonol, has potent anti-inflammatory properties. The effect of quercetin on ICAM-1 expression induced by agonists phorbol 12-myristate 13-acetate (PMA) and tumor necrosis factor-alpha (TNF-alpha) in human endothelial cell line ECV304 (ECV) was investigated. Quercetin treatment downregulated both PMA- and TNF-alpha-induced surface expression, as well as the ICAM-1 mRNA levels, in ECV cells in a dose-dependent (10-50 l-IM) manner. Quercetin had no effect on PMA- or TNF-alpha-induced nuclear factor-KB (NF-KB) activation. However, under similar conditions a remarkable dose-dependent downregulation of activator protein-1 (AP-1) activation was observed. This decrease in AP-1 activation was observed to be associated with the inhibitory effects of quercetin on the c-Jun NH2-terminal kinase (JNK) pathway. These results suggest that quercetin downregulates both PMA- and TNF-alpha-induced ICAM-1 expression via inhibiting both AP-1 activation and the JNK pathway.

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  • Assay of inducible form of nitric oxide synthase activity: Effect of flavonoids and plant extracts Reviewed

    H Kobuchi, F Virgili, L Packer

    NITRIC OXIDE, PT C   301   504 - 513   1999

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  • Antioxidant regulation of phorbol ester-induced adhesion of human jurkat T-cells to endothelial cells Reviewed

    S Roy, CK Sen, H Kobuchi, L Packer

    FREE RADICAL BIOLOGY AND MEDICINE   25 ( 2 )   229 - 241   1998.7

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    Regulation of adhesion molecule expression and function by reactive oxygen species via specific redox sensitive mechanisms have been reported. The effects of clinically safe antioxidants in the regulation of adhesion molecule expression in human endothelial cells (ECV), and adherence of human Jurkat T cells to ECV cells were investigated. The thiol antioxidant, alpha-lipoate, at clinically relevant doses down-regulated phorbol 12-myristate 13-acetate (PMA)-induced adhesion molecule expression and cell-cell adhesion. Inhibition of PMA-induced ICAM-1 and VCAM-1 expression as well as PMA-induced adhesion of Jurkat T-cells to ECV cells by alpha-lipoate was dose dependent (50-250 mu M) The effect was significant for ICAM-1 (p <.01) and VCAM-I (p <.01) expression in cells pretreated with 100 mu M alpha-lipoate compared to PMA-activated untreated cells. Inhibition of PMA-induced adhesion molecule expression and cell-cell adhesion was more pronounced when a combination of antioxidants, alpha-lipoate and alpha-tocopherol, were used compared to the use of either of these antioxidant alone. The regulation of adhesion molecule expression and function by low concentration of antioxidants investigated does not appear to be NF-kappa B regulated or transcription dependent because no change in the mRNA response was observed. Protein kinase C (PKC) has been suggested to regulate PMA-induced adhesion molecule expression by post-transcriptional stabilization of adhesion molecule mRNA. alpha-Lipoate pretreatment did not influence the response of PKC activity to PMA. Oxidants are known to be involved in the regulation of cell adhesion processes. Treatment of ECV cells with PMA induced generation of intracellular oxidants. alpha-lipoate (100 or 250 mu M) treatment decreased PMA-induced generation of intracellular oxidants. The inhibitory effect of low concentration of alpha-lipaote alone or in combination with alpha-tocopherol on agonist-induced adhesion processes observed in this study may be of potential therapeutic value. (C) 1998 Elsevier Science Inc.

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  • Antioxidant regulation of phorbol ester-induced adhesion of human jurkat T-cells to endothelial cells Reviewed

    S Roy, CK Sen, H Kobuchi, L Packer

    FREE RADICAL BIOLOGY AND MEDICINE   25 ( 2 )   229 - 241   1998.7

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    Regulation of adhesion molecule expression and function by reactive oxygen species via specific redox sensitive mechanisms have been reported. The effects of clinically safe antioxidants in the regulation of adhesion molecule expression in human endothelial cells (ECV), and adherence of human Jurkat T cells to ECV cells were investigated. The thiol antioxidant, alpha-lipoate, at clinically relevant doses down-regulated phorbol 12-myristate 13-acetate (PMA)-induced adhesion molecule expression and cell-cell adhesion. Inhibition of PMA-induced ICAM-1 and VCAM-1 expression as well as PMA-induced adhesion of Jurkat T-cells to ECV cells by alpha-lipoate was dose dependent (50-250 mu M) The effect was significant for ICAM-1 (p <.01) and VCAM-I (p <.01) expression in cells pretreated with 100 mu M alpha-lipoate compared to PMA-activated untreated cells. Inhibition of PMA-induced adhesion molecule expression and cell-cell adhesion was more pronounced when a combination of antioxidants, alpha-lipoate and alpha-tocopherol, were used compared to the use of either of these antioxidant alone. The regulation of adhesion molecule expression and function by low concentration of antioxidants investigated does not appear to be NF-kappa B regulated or transcription dependent because no change in the mRNA response was observed. Protein kinase C (PKC) has been suggested to regulate PMA-induced adhesion molecule expression by post-transcriptional stabilization of adhesion molecule mRNA. alpha-Lipoate pretreatment did not influence the response of PKC activity to PMA. Oxidants are known to be involved in the regulation of cell adhesion processes. Treatment of ECV cells with PMA induced generation of intracellular oxidants. alpha-lipoate (100 or 250 mu M) treatment decreased PMA-induced generation of intracellular oxidants. The inhibitory effect of low concentration of alpha-lipaote alone or in combination with alpha-tocopherol on agonist-induced adhesion processes observed in this study may be of potential therapeutic value. (C) 1998 Elsevier Science Inc.

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  • Procyanidins extracted from pinus Maritima (Pycnogenol((R))): Scavengers of free radical species and modulators of nitrogen monoxide metabolism in activated murine raw 264.7 macrophages Reviewed

    F Virgili, H Kobuchi, L Packer

    FREE RADICAL BIOLOGY AND MEDICINE   24 ( 7-8 )   1120 - 1129   1998.5

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    Nitrogen monoxide (NO) has diverse physiological roles and also contributes to the immune defense against viruses, bacteria, and other parasites. However, excess production of NO is associated with various diseases such arthritis, diabetes, stroke, septic shock, autoimmune, chronic inflammatory diseases, and atheriosclerosis. Cells respond to activating or depressing stimuli by enhancing or inhibiting the expression of the enzymatic machinery that produce NO. Thus, maintenance of a tight regulation of NO production is important for human health. Phytochemicals have been traditionally utilized in ways to treat a family of pathologies that have in common the disregulation of NO production. Here we report the scavenging activity of Pycnogenol(R) (the polyphenols containing extract of the bark from Pinus maritima) against reactive oxygen and nitrogen species, and its effects on NO metabolism in the murine macrophages cell line RAW 264.7. Macrophages were activated by the bacterial wall components lipopolysaccharide (LPS) and interferon (LFN-gamma), which induces the expression of large amounts of the enzyme nitric oxide synthase (iNOS). Preincubation of cells with physiological concentrations of Pycnogenol(R) significantly decreased NO generation. it was Found that this effect was due to the combination of several different biological activities, i.e., its ROS and NO scavenging activity,inhibition of iNOS activity, and inhibition of iNOS-mRNA expression. These data begin to provide the basis for the conceptual understanding of the biological activity of Pycnogenol(R) and possibly other polyphenolic compounds as therapeutic agents in various human disorders. (C) 1998 Elsevier Science Inc.

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  • Ginkgo biloba extract (EGb 761): antioxidant properties and regulation of gene expression Reviewed

    S Roy, H Kobuchi, CK Sen, K Gohil, MT Droy-Lefaix, L Packer

    GINKGO BILOBA EXTRACT (EGB 761): LESSONS FROM CELL BIOLOGY   7   1 - 17   1998

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    EGb 761 has proven beneficial in pathologies related to cerebral and circulatory disorders. Such effects of EGb, 761 extract have been substantiated with placebo-controlled, double-blind clinical trials. Mechanisms underlying the beneficial effects of EGb 761 are only partially understood. One of the main mechanisms seems to be related to the antioxidant properties of EGb 761, which appear to be due to the synergistic action of its major constituents, such as flavonoids, terpenoids and the organic acids. EGb 761 directly scavenges hydroxyl, superoxide, peroxyl and nitric oxide radicals in vitro. EGb 761 also protects against free radical-mediated damage in biological model systems, including ischemia-reperfusion injury of organs and oxidative modification of low-density lipoprotein. EGb 761 inhibits nitric oxide production in macrophages by inhibiting nitric oxide synthase (NOS) activity and regulating inducible-NOS gene expression. EGb 761 down-regulates agonist-induced cell adhesion molecule expression and lymphocyte-endothelial cell adhesion. Exposure of human endothelial cells to EGb 761 resulted in modulation of several mRNA transcripts identified by differential display of mRNA. Such regulatory effects of EGb 761 on nitric oxide metabolism, cell adhesion processes and gene expression may be implicated in its beneficial effect in brain and circulatory disorders.

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  • Monochloramine inhibits phorbol ester-inducible neutrophil respiratory burst activation and T cell interleukin-2 receptor expression by inhibiting inducible protein kinase C activity Reviewed

    T Ogino, H Kobuchi, CK Sen, S Roy, L Packer, JJ Maguire

    JOURNAL OF BIOLOGICAL CHEMISTRY   272 ( 42 )   26247 - 26252   1997.10

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    Monochloramine derivatives are long lived physiological oxidants produced by neutrophils during the respiratory burst, The effects of chemically prepared monochloramine (NH2Cl) on protein kinase C (PKC) and PKC-mediated cellular responses were studied in elicited rat peritoneal neutrophils and human Jurkat T cells, Neutrophils pretreated with NH2Cl (30-50 mu M) showed a marked decrease in the respiratory burst activity induced by phorbol 12-myristate 13-acetate (PMA), which is a potent PKC activator. These cells, however, were viable and showed a complete respiratory burst upon arachidonic acid stimulation, which induces the respiratory burst by a PKC-independent mechanism, The NH2Cl-treated neutrophils showed a decrease in both PRC activity and PMA-induced phosphorylation of a 47-kDa protein, which corresponds to the cytosolic factor of NADPH oxidase, p47(phox). Jurkat T cells pretreated with NH2Cl (20-70 mu M) showed a decrease in the expression of the interleukin-a receptor a chain following PMA stimulation. This was also accompanied by a decrease in both PKC activity and nuclear transcription factor-kappa B activation, also without loss of cell viability, These results show that NH2Cl inhibits PKC-mediated cellular responses through inhibition of the inducible PKC activity.

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  • Bio-Normalizer modulates interferon-gamma-induced nitric oxide production in the mouse macrophage cell line RAW 264.7 Reviewed

    H Kobuchi, L Packer

    BIOCHEMISTRY AND MOLECULAR BIOLOGY INTERNATIONAL   43 ( 1 )   141 - 152   1997.9

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    Bio-Normalizer, a natural health food supplement prepared from Carica papaya and some other medicinal plants was investigated to determine its effects on cellular nitric oxide (nitrogen monoxide, NO) production and inducible nitric oxide synthase (iNOS) expression. Bio-Normalizer upregulated interferon (IFN)-gamma-induced NO production by macrophages in a dose-dependent manner. Such an effect of Bio-Normalizer on NO production was not due to changes in the activity of iNOS. Reverse transcription-polymerase chain reaction analysis revealed that the levels of iNOS mRNA were augmented by treatment of the cells with Bio-Normalizer and IFN-gamma. The ability of Bio-Normalizer to augment IFN-gamma-induced iNOS mRNA expression was independent of any changes on the mRNA stability. Treatment of cells with Bio-Normalizer alone did not affect NO production by macrophages. Tumor necrosis factor-alpha and interleukin-1 beta are involved in the induction of iNOS gene as well as the immune system. Bio-Normalizer augmented the mRNA expression of these cytokines in the presence of IFN-gamma. This suggests that Bio-Normalizer is not directly involved in the expression of iNOS, but shows synergistic interaction with IFN-gamma to induce NO synthesis.

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  • Ginkgo biloba extract (EGb 761): Inhibitory effect on nitric oxide production in the macrophage cell line RAW 264.7 Reviewed

    H Kobuchi, MT DroyLefaix, Y Christen, L Packer

    BIOCHEMICAL PHARMACOLOGY   53 ( 6 )   897 - 903   1997.3

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    The present study was conducted to evaluate the effect of Ginkgo blloba extract (EGb 761) on the synthesis of nitric oxide (NO) induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN ?I) in the mouse macrophage cell line RAW 264.7. EGb 761 inhibited nitrite and nitrate production, taken as an index for NO, in a concentration-dependent fashion. The IC50 for inhibition of nitrite production by activated macrophages was about 100 mu g/mL EGb 761, The inducible NO synthase (iNOS) enzyme activity of cytosolic preparations from activated RAW 264.7 cells was inhibited by treatment with EGb 761. In addition, reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the expression of iNOS mRNA in activated macrophages was suppressed by high concentrations of EGb 761. However, NF-KB DNA binding activity induced by activation with LPS/IFN-gamma was not inhibited by EGb 761. These findings indicate that not only does EGb 761 directly act as an NO scavenger but also, that it inhibits NO production in LPS/IFN-gamma-activated macrophages by concomitant inhibiyion of induction of iNOS mRNA and the enzyme activity of iNOS. Thus, EGb 761 may act as a potent inhibitor of NO production under tissue-damaging inflammatory conditions. (C) 1997 Elsevier Science Inc.

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  • Lipoic acid increases de novo synthesis of cellular glutathione by improving cystine utilization Reviewed

    D Han, G Handelman, L Marcocci, CK Sen, S Roy, H Kobuchi, HJ Tritschler, L Flohe, L Packer

    BIOFACTORS   6 ( 3 )   321 - 338   1997

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    Lipoic acid (thiotic acid) is being used as a dietary supplement, and as a therapeutic agent, and is reported to have beneficial effects in disorders associated with oxidative stress, but its mechanism of action remains unclear. We present evidence that lipoic acid induces a substantial increase in cellular reduced glutathione in cultured human Jurkat T cells, human erythrocytes, C6 glial cells, NB41A3 neuroblastoma cells, and peripheral blood lymphocytes. The effect depends on metabolic reduction of lipoic acid to dihydrolipoic acid. Dihydrolipoic acid is released into the culture medium where it reduces cystine. Cysteine thus formed is readily taken up by the neutral amino acid transport system and utilized for glutathione synthesis. By this mechanism lipoic acid enables cystine to bypass the x(c)(-) transport system, which is weakly expressed in lymphocytes and inhibited by glutamate. Thereby lipoic acid enables the key enzyme of glutathione synthesis, gamma -glutamylcysteine synthetase, which is regulated by uptake-limited cysteine supply, to work at optimum conditions. Flow cytometric analysis of freshly prepared human peripheral blood lymphocytes, using monobromobimane labeling of cellular thiols, reveals that lipoic acid acts mainly to normalize a subpopulation of cells severely compromised in thiol status rather than to increase thiol content beyond physiological levels. Hence lipoic acid may have clinical relevance in restoration of severely glutathione deficient cells.

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  • Lipoic acid increases de novo synthesis of cellular glutathione by improving cystine utilization Reviewed

    D Han, G Handelman, L Marcocci, CK Sen, S Roy, H Kobuchi, HJ Tritschler, L Flohe, L Packer

    BIOFACTORS   6 ( 3 )   321 - 338   1997

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    Lipoic acid (thiotic acid) is being used as a dietary supplement, and as a therapeutic agent, and is reported to have beneficial effects in disorders associated with oxidative stress, but its mechanism of action remains unclear. We present evidence that lipoic acid induces a substantial increase in cellular reduced glutathione in cultured human Jurkat T cells, human erythrocytes, C6 glial cells, NB41A3 neuroblastoma cells, and peripheral blood lymphocytes. The effect depends on metabolic reduction of lipoic acid to dihydrolipoic acid. Dihydrolipoic acid is released into the culture medium where it reduces cystine. Cysteine thus formed is readily taken up by the neutral amino acid transport system and utilized for glutathione synthesis. By this mechanism lipoic acid enables cystine to bypass the x(c)(-) transport system, which is weakly expressed in lymphocytes and inhibited by glutamate. Thereby lipoic acid enables the key enzyme of glutathione synthesis, gamma -glutamylcysteine synthetase, which is regulated by uptake-limited cysteine supply, to work at optimum conditions. Flow cytometric analysis of freshly prepared human peripheral blood lymphocytes, using monobromobimane labeling of cellular thiols, reveals that lipoic acid acts mainly to normalize a subpopulation of cells severely compromised in thiol status rather than to increase thiol content beyond physiological levels. Hence lipoic acid may have clinical relevance in restoration of severely glutathione deficient cells.

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  • Efficacy of hypochlorous acid scavengers in the prevention of protein carbonyl formation Reviewed

    LJ Yan, MG Traber, H Kobuchi, S Matsugo, HJ Tritschler, L Packer

    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS   327 ( 2 )   330 - 334   1996.3

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    We observed that protein (bovine serum albumin) carbonyl content increases upon hypochlorite oxidation, and this increase is inhibited in a concentration dependent manner in the presence of hypochlorite scavengers. Based on this observation, we tested whether some known hypochlorite scavengers (lipoic acid, cysteine, and glutathione) and some other antioxidants (uric acid, ascorbic acid, alpha-tocopherol, and probucol) could prevent protein carbonyl formation. N-acetylcysteine, dihydrolipoic acid, cysteine, and glutathione (reduced form, GSH), which otherwise could not be tested in a previously reported 5-thio-2-nitro-benzoic acid test system, were successfully evaluated in our assay. The hypochlorite scavenging capacity of different compounds, compared by determining the IC50 (concentration which produces 50% inhibition), showed that the compounds tested have the following potency: dihydrolipoic acid > GSH, N-acetylcysteine > cysteine > S-methyl glutathione > lipoic acid, ascorbic acid > cystine, GSSG, and uric acid. No scavenging ability was observed for either alpha-tocopherol or probucol. (C) 1996 Academic Press, Inc.

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  • INHIBITION OF NEUTROPHIL SUPEROXIDE GENERATION BY HYPERICIN, AN ANTIRETROVIRAL AGENT Reviewed

    T NISHIUCHI, T UTSUMI, T KANNO, Y TAKEHARA, H KOBUCHI, T YOSHIOKA, AA HORTON, T YASUDA, K UTSUMI

    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS   323 ( 2 )   335 - 342   1995.11

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    We previously reported that phorbol 12-myristate 13-acetate (PMA)-induced superoxide (O-2(.-)) generation of neutrophils was inhibited by hypericin, a photosensitizing pigment found in St, Johnswort (herb Hypericin triquetrifolium Turra), via a mechanism involving protein kinase C (PKC). To obtain further insights into the mechanism of inhibition, the effects of hypericin on stimulation-dependent O-2(.-) generation and related enzymes of neutrophils were investigated, Hypericin inhibited O-2(.-) generation of neutrophils induced by PKC-dependent and -independent stimuli in a ligand concentration-dependent manner. Oxygen was required for the light-dependent inhibition by hypericin. NADPH oxidase activity in a cell-free system and TNF-alpha-induced tyrosyl phosphorylation of neutrophil proteins were also inhibited by hypericin in a concentration- and light-dependent manner. However, tyrosine kinase of p60(sre), an enzyme not bound to a membrane, was not inhibited either in the light or in the dark, Oxygen uptake of neutrophils by photosensitization with hypericin resulted in the formation of singlet oxygen (O-1(2)), O-2(.-), and hydroxyl radical (. OH) and enhanced lipid peroxidation. The formation of O-1(2) was inhibited by azide, a quencher of O-1(2), but not by desferrioxamine (DSF), a ferric ion chelator, By contrast, both generation of . OH and lipid peroxidation were inhibited by DSF but not by azide. Furthermore, PMA-induced O-2(.-) generation inhibited by hypericin partially recovered in the presence of azide but not DSF. These results suggested that the light-dependent inhibition of O-2(.-) generation by hypericin might be due to inhibition of tyrosine kinase, PKC, and NADPH oxidase via an oxygen-dependent mechanism, possibly through both Type I and II photosensitization mechanisms. (C) 1995 Academic Press, Inc.

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  • Bio-Catalyzer alpha center dot rho No 11 (Bio-Normalizer) supplementation: Effect on oxidative stress to isolated rat hearts. Reviewed

    N Haramaki, L Marcocci, R DAnna, LJ Yan, H Kobuchi, L Packer

    BIOCHEMISTRY AND MOLECULAR BIOLOGY INTERNATIONAL   36 ( 6 )   1263 - 1268   1995.8

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    Bio-Catalyzer alpha . rho No. 11 (Bio-Normalizer), a natural health food product prepared by yeast fermentation of medicinal plants, has been recently reported to posses antioxidant properties. To better define its antioxidant action, we investigated the effects of orally supplemented Bio-Normalizer on oxidative damage in the rat heart. Hearts were isolated from control or Bio-Normalizer supplemented animals and 1) exposed to ischemia-reperfusion using the Langendorff technique, or 2) homogenized and exposed to peroxyl radicals generated from (2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN). During reperfusion following 40 minutes of ischemia, leakage of lactate dehydrogenase from hearts isolated from Bio-Normalizer supplemented rats was significantly lower than from hearts of control animals. Furthermore, lower levels of AMVN-induced accumulation of thiobarbituric acid reactive substances and of protein carbonyl derivatives were measured in homogenates prepared from hearts isolated from Bio-Normalizer supplemented rats than in samples from control animals. Our findings confirm an antioxidant action of Bio-Normalizer and show that it protects the heart against ischemia-reperfusion induced damage.

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  • PHOTOACTIVATED INHIBITION OF SUPEROXIDE GENERATION AND PROTEIN-KINASE-C ACTIVITY IN NEUTROPHILS BY BLEPHARISMIN, A PROTOZOAN PHOTODYNAMICALLY ACTIVE PIGMENT Reviewed

    Y WATANABE, K EDASHIGE, H KOBUCHI, Y KATO, T MATSUOKA, T UTSUMI, T YOSHIOKA, AA HORTON, K UTSUMI

    BIOCHEMICAL PHARMACOLOGY   49 ( 4 )   529 - 536   1995.2

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    Blepharismin is an endogenous photosensitizing pigment found in the protozoan Blepharisma. This pigment inhibited the generation of superoxide anion (O(2)(r)adical anion) in neutrophils not only via a diacylglycerol-induced protein kinase C (PKC)-dependent reaction but also by an arachidonate-induced PKC-independent reaction. The inhibition was light and concentration dependent for both reactions. Light-activated inhibition was strong at wavelengths between 520 and 570 nm but not above 610 nm. PKC activity in neutrophils and from rat brain was inhibited by blepharismin in a light- and concentration-dependent manner. Moreover, arachidonate-activated NADPH oxidase activity in a cell-free system was also inhibited by the pigment in a light- and concentration-dependent manner. These results suggest that blepharismin inhibits NADPH oxidase activation through the non-specific inhibition of Various membrane-bound enzymes and that this inhibition may also be correlated with that of PKC.

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  • LIGHT-DEPENDENT INHIBITION OF PROTEIN-KINASE-C AND SUPEROXIDE GENERATION OF NEUTROPHILS BY HYPERICIN, AN ANTIRETROVIRAL AGENT Reviewed

    T UTSUMI, M OKUMA, T UTSUMI, T KANNO, T YASUDA, H KOBUCHI, AA HORTON, K UTSUMI

    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS   316 ( 1 )   493 - 497   1995.1

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    Hypericin, a photosensitizing plant pigment, has antiretroviral activity. When exposed to light, the inhibition of Friend leukemia virus (FLV)-induced splenomegaly by hypericin was increased. The ID50 was decreased to less than 2.5 mu g/mouse by exposure to tungsten light (29 X 10(-3) W/cm(2) for 3 min). This pigment inhibited phorbol 12-myristate 13-acetate (PMA)-activated protein kinase C of rat brain in a light- and concentration-dependent manner. The ID50 of hypericin and the light intensity for inhibition of PKC were 0.1 mu M under a constant light of 29 X 10(-3) W/cm(2) for 3 min and 5 X 10(-3) W/cm(2) in the presence of 1 mu M hypericin for 3 min, respectively. The PMA-induced respiratory burst of neutrophils was inhibited in the light but stimulated in the dark by hypericin. The ID50 for inhibition of the respiratory burst was similar to that for inhibition of PKC. These results suggest that hypericin might inhibited PKC-mediated processes of intact cells, including PMA-induced superoxide generation of neutrophils by some light-dependent mechanism, and that this mechanism might underlie its light-dependent inhibition of FLV infection. (C) 1995 Academic Press, Inc.

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  • AAPHによるリポソームの区画性低下に及ぼすトコフェロールの影響 Reviewed

    竹原良記, 小渕浩嗣, 菅野智子, 保田立二, 秋山実男, 吉岡 保, 内海耕慥

    ビタミンE研究の進歩   5   87 - 92   1995

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  • SELECTIVE-INHIBITION OF HUMAN TNF-ALPHA ACTION BY FLECAINIDE ACETATE, AN ANTIARRHYTHMIC DRUG Reviewed

    S NOJIMA, H KOBUCHI, T SAIBARA, T YASUDA, K UTSUMI

    PHYSIOLOGICAL CHEMISTRY AND PHYSICS AND MEDICAL NMR   27 ( 2 )   77 - 89   1995

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    It is now generally accepted that human tumor necrosis factor-alpha(hTNF-alpha) affects not only tumor cells but also normal cells, providing critical tissue damage, hTNF-alpha also enhanced the response of polymorphonuclear neutrophils (PMN) by its priming action and resulted in the increased generation of active oxygen which in turn may be responsible for the tissue injury. Seeking a conventional drug to attenuate the cytolytic activity of tumor necrosis factor (TNF-alpha) and thereby prevent excessive tissue injury, we focused on the cytolytic action of hTNF-alpha against L929 cells, which are sensitive to TNF-alpha, and found that flecainide acetate [N-(2-piperidylmethyl) 1,5-bis-(2,2,2 trifluoroethoxy) benzamide acetate] inhibited specifically the cytolytic action of hTNF-alpha against L929 cells. Flecainide acetate also specifically inhibited the priming action of hTNF-alpha which enhance the formylmethionyl-leucyl-phenylalanine (FMLP)-induced receptor-mediated superoxide (O-2(.)-) generation of human peripheral polymorphonuclear neutrophils (hPMN). The ID50 values for hTNF-alpha induced cytotoxicity in L929 cells and hTNF-alpha-primed FMLP-induced O-2(.)- generation of hPMN were 30 and 50-60 mu M, respectively. However, the drug does not inhibit the FMLP- or phorbol myristate acetate (PMA)-induced O-2(.)- generation of nonprimed hPMN and has a weak cytotoxic effect on L929 cells. From these results, it is concluded that flecainide acetate suppressed specifically the action of hTNF-alpha.

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  • INHIBITION OF STIMULUS-SPECIFIC NEUTROPHIL SUPEROXIDE GENERATION BY ALPHA-TOCOPHEROL Reviewed

    T KANNO, T UTSUMI, H KOBUCHI, Y TAKEHARA, J AKIYAMA, T YOSHIOKA, AA HORTON, K UTSUMI

    FREE RADICAL RESEARCH   22 ( 5 )   431 - 440   1995

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    Alpha-tocopherol but not 2-carboxy-2,5,7,8-tetramethyl-6-chromanol (trolox or CTMC) and 2,2,5,7,8 pentamethyl-6-hydroxy chromane (PMC), derivatives of cx-tocopherol, inhibited the superoxide (O-2(-)) generation of rat peritoneal neutrophils (RPMN) induced by phorbol 12-myristate 13-acetate (PMA). ID50 for neutrophils obtained from the peritoneal cavity of rat and guinea pig was about 1 mu M. This concentration, however, was much lower than that for the inhibition of PMA-activated phospholipid-dependent protein kinase (PKC) (ID50 = 30 mu M). The alpha-tocopherol sensitive O-2(-) generation was also observed in neutrophils induced by dioctanoylglycerol (diC(8)) and calcium ionophore A23187 but not by formylmethionyl-leucyl-phenylalanine (FMLP), opsonized zymosan (OZ) and sodium dodecyl sulfate (SDS). The pattern of inhibition by alpha-tocopherol was quite similar to that of staurosporine, a specific inhibitor of PKC. The alpha-tocopherol content of RPMN was 12 ng/10(6) cells and a linear increase to 200 ng/10(6) cells by addition of alpha-tocopherol to the cell suspension corresponded with an increased inhibition of O-2(-) generation. These results indicate that both the chemical structure and the content of alpha-tocopherol might be important factors in O-2(-) generation by neutrophils.

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  • キサンチン酸化におけるルミノール化学発光の解析 Reviewed

    小渕浩嗣, 秋丸国広, 岡添陽子, 佐藤英介, 枝重圭祐, 吉岡保, 内海耕慥

    過酸化脂質研究   17 ( 2 )   132 - 135   1993

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  • EFFECT OF INDOMETHACIN AND ASPIRIN ON THE TNF-ALPHA-INDUCED PRIMING AND PROTEIN TYROSYL PHOSPHORYLATION OF HUMAN NEUTROPHILS Reviewed

    K MINAKAMI, Y WATANABE, M MIYAHARA, H KOBUCHI, T KURASHIGE, K UTSUMI

    PHYSIOLOGICAL CHEMISTRY AND PHYSICS AND MEDICAL NMR   25 ( 1 )   55 - 67   1993

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    Receptor-mediated superoxide (O2-.)-generation in human peripheral neutrophils (HPPMN) is enhanced by various priming agents, such as granulocyte colony stimulating factor (G-CSF) and tumor necrosis factor (TNF-alpha). We previously reported that this enhancement occurred in parallel with the priming agent-induced increase in protein tyrosyl phosphorylation which is sensitive to tyrosine kinase (TK) inhibitors [Akimaru K. et al. Arch. Biochem. Biophys. 298.703, 1992]. We describe here that nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and aspirin, modulate the priming and tyrosine phosphorylation of HPPMN. The enhancement by TNF-alpha of formylmethionyl-leucyl-phenylalanine (FMLP)-induced O2-. generation was not inhibited by 5 muM azide, whereas that of FMLP-induced luminol chemiluminescence (LCL) was sensitive. Enhancement of FMLP-induced O2-. generation and LCL by priming agents was inhibited by both aspirin and indomethacin in a concentration dependent manner. This inhibition by the NSAIDs was much stronger than that for FMLP-induced O2-. generation without priming. The half-inhibition dose of indomethacin and aspirin were 50 muM and 1.5 mM, respectively. The priming-induced enhancement of tyrosyl phosphorylation of some neutrophil proteins, such as that of 108 and 115 kDa, was also inhibited by aspirin and indomethacin in a dose-dependent manner. However, dose dependent inhibition of the enhanced O2-. generation by the NSAIDs was not completely similar to that of enhanced tyrosyl phosphorylation. However, PKC-mediated O2-. generation, which has little sensitivity to TNF-alpha or G-CSF, was rather stimulated by the NSAIDs. These findings suggest that aspirin and indomethacin inhibit also the early steps of neutrophil activation as reflected by their ability to inhibit priming and the related tyrosyl phosphorylation of neutrophil proteins.

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  • 好中球の活性化とカルシウム動態 Reviewed

    小渕浩嗣, 吉岡 保, 内海耕慥

    Clinical Calcium   3 ( 12 )   31 - 38   1993

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  • INHIBITION OF NEUTROPHIL PRIMING AND TYROSYL PHOSPHORYLATION BY CEPHARANTHINE, A NONSTEROIDAL ANTIINFLAMMATORY DRUG Reviewed

    H KOBUCHI, MJ LI, T MATSUNO, T YASUDA, K UTSUMI

    CELL STRUCTURE AND FUNCTION   17 ( 6 )   385 - 393   1992.12

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    Receptor-mediated superoxide (O2)-generation and tyrosyl phosphorylation of neutrophil proteins, such as 58, 65, 84, 108 and 115 kDa, were enhanced by priming cells with granulocyte colony stimulating factor (G-CSF) [Akimurs, K. et al. Arch. Biochem. Biophys. 298: 703-709, 1992]. To elucidate the possible involvement of tyrosyl phosphorylation of neutrophil proteins in the enhancing mechanism Of O2 generation, the effect of cepharanthine, a biscoclaurine alkaloid that inhibits phorbol 12-myristate 13-acetate (PMA)- and receptor-mediated 02 generation, on the priming of human peripheral neutrophils (HPPMN) was studied. Both enhancement of formyl-methyonyl-leucyl-phenylalanine (FMLP)-mediated 02 generation and tyrosyl phosphorylation of some neutrophil proteins, i.e., 115, 108 and 84 kDa proteins, by HHPMN after treatment with G-CSF were strongly inhibited by cepharanthine in a concentration- and treatment-time-dependent manner. In contrast, inhibition of PMA-mediated 02 generation by cepharanthine was weak and independent of treatment time. These results suggest that cepharanthine might inhibit the priming step of neutrophil activation concomitantly with its inhibition of the tyrosyl phosphorylation of some neutrophil proteins that might underlie the mechanism for priming of neutrophils with G-CSF.

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  • NEUTROPHIL PRIMING BY GRANULOCYTE COLONY STIMULATING FACTOR AND ITS MODULATION BY PROTEIN-KINASE INHIBITORS Reviewed

    M TANIMURA, H KOBUCHI, T UTSUMI, T YOSHIOKA, S KATAOKA, Y FUJITA, K UTSUMI

    BIOCHEMICAL PHARMACOLOGY   44 ( 6 )   1045 - 1052   1992.9

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    Upon stimulation by various ligands, freshly isolated human peripheral neutrophils (PMN) respond in a variety of ways, such as superoxide (O2-) generation, phagocytosis, enzyme release, migration etc. Chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP) and opsonized zymosan activate neutrophils by a receptor-mediated mechanism, while phorbol myristate acetate and dioctanoylglycerol activate the cells by a mechanism involving Ca2+- and phospholipid-dependent protein kinase (PKC). Receptor-mediated but not PKC-mediated O2- generation in PMN was enhanced by the priming of recombinant human granulocyte colony Stimulating factor (G-CSF). FMLP-dependent luminol chemiluminescence was also enhanced by G-CSF. However, no appreciable enhancement was observed in FMLP-induced intracellular calcium ion concentration ([Ca2+]i). Enhancement of FMLP-induced generation of O2- by G-CSF was inhibited by genistein or alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK), and was stimulated by staurosporine and 1-(5-isoquinolinesulfonyl)-3-methyl-piperazine (H-7), inhibitors of PKC. The ED50 values of genistein and ST 638 for the inhibition of the FMLP-induced O2- generation from G-CSF were 0.5 and 5 muM, respectively. In contrast, O2- generation by PKC activation without G-CSF priming was inhibited by stauroporine and H-7, but was stimulated by genisten and ST 638. These results suggested that the enhancing effect of G-CSF on receptor-mediated generation of the O2- might be regulated by protein kinases, such as TK and PKC, and that the TK inhibitor selectively inhibited the G-CSF-primed receptor-mediated O2- generation of neutrophils.

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  • NEUTROPHIL PRIMING BY GRANULOCYTE COLONY STIMULATING FACTOR AND ITS MODULATION BY PROTEIN-KINASE INHIBITORS Reviewed

    M TANIMURA, H KOBUCHI, T UTSUMI, T YOSHIOKA, S KATAOKA, Y FUJITA, K UTSUMI

    BIOCHEMICAL PHARMACOLOGY   44 ( 6 )   1045 - 1052   1992.9

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    Upon stimulation by various ligands, freshly isolated human peripheral neutrophils (PMN) respond in a variety of ways, such as superoxide (O2-) generation, phagocytosis, enzyme release, migration etc. Chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP) and opsonized zymosan activate neutrophils by a receptor-mediated mechanism, while phorbol myristate acetate and dioctanoylglycerol activate the cells by a mechanism involving Ca2+- and phospholipid-dependent protein kinase (PKC). Receptor-mediated but not PKC-mediated O2- generation in PMN was enhanced by the priming of recombinant human granulocyte colony Stimulating factor (G-CSF). FMLP-dependent luminol chemiluminescence was also enhanced by G-CSF. However, no appreciable enhancement was observed in FMLP-induced intracellular calcium ion concentration ([Ca2+]i). Enhancement of FMLP-induced generation of O2- by G-CSF was inhibited by genistein or alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK), and was stimulated by staurosporine and 1-(5-isoquinolinesulfonyl)-3-methyl-piperazine (H-7), inhibitors of PKC. The ED50 values of genistein and ST 638 for the inhibition of the FMLP-induced O2- generation from G-CSF were 0.5 and 5 muM, respectively. In contrast, O2- generation by PKC activation without G-CSF priming was inhibited by stauroporine and H-7, but was stimulated by genisten and ST 638. These results suggested that the enhancing effect of G-CSF on receptor-mediated generation of the O2- might be regulated by protein kinases, such as TK and PKC, and that the TK inhibitor selectively inhibited the G-CSF-primed receptor-mediated O2- generation of neutrophils.

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  • DYNAMIC ASPECTS OF OVARIAN SUPEROXIDE-DISMUTASE ISOZYMES DURING THE OVULATORY PROCESS IN THE RAT Reviewed

    EF SATO, H KOBUCHI, K EDASHIGE, M TAKAHASHI, T YOSHIOKA, K UTSUMI, M INOUE

    FEBS LETTERS   303 ( 2-3 )   121 - 125   1992.6

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    To investigate the role of superoxide dismutase (SOD) in the ovulatory process, SOD isozymes and their mRNAs were determined in the ovary of 22-day-old rat. After treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), ovarian activity of Mn-SOD decreased markedly while Cu/Zn-SOD remained unchanged. However, the ovarian level of mRNA for Mn-SOD markedly increased after hCG-treatment while that for Cu/Zn-SOD decreased only slightly. Ovulation was inhibited by intravenous injection of a long-acting SOD. These results suggested that superoxide radicals in the ovary might play a critical role in the mechanism for hCG-induced ovulation.

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  • Neutrophil Priming by Cytokines : in Relation to the Protein Tyrosyl Phosphorylation of Neutrophils Proteins. Reviewed

    WATANABE Yoshiya, KOBUCHI Hirotsugu, AKIMARU Kunihiro, SATO Eisuke F., MINAKAMI Kayoko, KANBARA Tetsuya, TOMODA Miho, KOBAYASHI Sumio, YOSHIOKA Tamotsu, UTSUMI Kozo

    Research reports of Kochi Medical School Liberal arts   8   45 - 56   1992

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    We have investigated the effect of tumor necrosis factor-α (TNF-α) and granulocyte colony stimulating factor (G-CSF) on human peripheral neutrophils (HPPMN). TNF-α and G -CSF enhanced fonylmethionyl-1eucyl-phenylalanine (FMLP) -stimiulated O^∸_2 generation of HPPMN in concentration and time dependent manners. The enhancement of 0^∸_2 generation was inhibited by tyrosine kinase inhibitors. These cytokins also enhanced the tyrosyl phosphorylation of neutrophil proteins, such as 115,110,98,83,72,70,60,54 kDa and many of low molecular weight proteins. Both tyrosine kinase and phosphotyr-osine phosphatase might regulate the protein tyrosyl phosphorylation since the phosphorylation inhibited by tyrosine kinase inhibitor, genistein. The TNF-α enhanced tyrosyl phosphorylation of 115 and 108 kDa proteins, which distributed in the membrane fraction, was increased in manners of incubation and concentration dependent. The kinetics of TNF-α enhanced FMLP -dependent O^∸_2 generation was quite similar to that of tyrosyl phosphorylation of neutrophil proteins by TNF-α Furthermore, cepharanthine specifically inhibited either TNF-α or G-CSF enhanced FMLP-stimulated 0^∸_2 generation and tyrosyl phophorylation of some neutrophil proteins, 115 kDa. Taken together with this finding, it is concluded that substrate proteins for tyrosin kinases might act as critical effector molecules in the mechanism for priming in neutrophils.

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  • Mechanism of Superoxide Generation in Human Peripheral Neutrophils : Its Priming, Stimulation, and Protein Kinase Specificities Reviewed

    AKIMARU Kunihiro, AKIMARU Yoko, SATO Eisuke F., EDASHIGE Keisuke, TAKAHASHI Masahiko, TANIMURA Masanobu, KOBUCHI Hirotsugu, KOBAYASHI Sumio, YOSHIOKA Tamotsu, UTSUMI Kozo

    Research reports of Kochi Medical School Liberal arts   7   47 - 59   1991

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    Healthy human peripheral neutrophils (HPPMN) are not primed, and have weak responses to stimuli which activate HPPMN through their membrane receptors. Recombinant human tumor necrosis factor-α (rHuTNF) and recombinant granulocyte colony stimulation factor (rG-CSF) primed HPPMN. Superoxide (O^&minusd;_2) generation by formylmethionyl-leucylphenylalanine (FMLP) or opsonized zymosan (OZ) was enhanced by these primers. However, O^&minusd;_2 generation induced by phorbol myristate acetate (PMA) or dioctanoylglycerol (DOG) was not enhanced by the primers. Receptor mediated O^&minusd;_2 generation in rHuTNF primed neutrophils was inhibited by the genistein or alpha-cyan0-3-ethoxy-4-hydroxy-5phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK). But it was enhanced by 1-(5-isoquinoline sulfonyl)-3-methyl-piperazine (H-7) or staurosporine, inhibitors of Ca^<++>_ and phospholipid-dependent protein kinase (PKO, in their concentration dependent manner. On the contrary, O^&minusd;_2 generation of HPPMN at higher concentration of PMA or DOG was rather stimulated by genistein or ST 638 and was inhibited by H-7 or staurosporine. These results suggest that protein kinases participates in the NADPH-oxidase activation of neutrophils and that two pathways exist in the NADPH-oxidase activation : one in PMA- or DOG-stimulated PKC-dependent pathway and the other in FMLP-stimulated TK-dependent pathway. Moreover, it suggests that TK might be involved in the reaction of neutrophil priming with various ligands.

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  • 口腔内多形核白血球の刺激応答性 Reviewed

    佐藤英介, 秋丸国広, 枝重圭祐, 片岡真一, 山本昌澄, 森下誠治, 高橋正彦, 小渕浩嗣, 内海耕慥

    医学のあゆみ   158 ( 11 )   761 - 762   1991

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  • INHIBITION OF ACTIVE OXYGEN GENERATION IN GUINEA-PIG NEUTROPHILS BY BISCOCLAURINE ALKALOIDS Reviewed

    T MATSUNO, K ORITA, K EDASHIGE, H KOBUCHI, EF SATO, B INOUYE, M INOUE, K UTSUMI

    BIOCHEMICAL PHARMACOLOGY   39 ( 7 )   1255 - 1259   1990.4

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  • Levels of sister-chromatid exchanges in hybrids between Bloom syndrome B-lymphoblastoid cells and various cell lines with lymphoid malignancy. Reviewed

    Shiraishi Y, Kobuchi H, Utsumi K, Minowada J

    Mutat. Res.   243 ( 1 )   13 - 20   1990.1

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  • LEVELS OF SISTER-CHROMATID EXCHANGES IN HYBRIDS BETWEEN BLOOM SYNDROME B-LYMPHOBLASTOID CELLS AND VARIOUS CELL-LINES WITH LYMPHOID MALIGNANCY Reviewed

    Y SHIRAISHI, H KOBUCHI, K UTSUMI, J MINOWADA

    MUTATION RESEARCH   243 ( 1 )   13 - 20   1990.1

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  • Levels of sister-chromatid exchanges in hybrids between Bloom syndrome B-lymphoblastoid cells and various cell lines with lymphoid malignancy Reviewed

    Yukimasa Shiraishi, Hirotsugu Kobuchi, Kozo Utsumi, Jun Minowada

    Mutation Research Letters   243 ( 1 )   13 - 20   1990

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    The present study has been undertaken to examine the effect of cell hybridization of Bloom syndrome (BS) B-lymphoblastoid cell lines (LCLs) and various cell lines from lymphoid malignancies in order to clarify the relationship between sister-chromatid exchange (SCE) and malignant conditions. Cell hybridization studies have shown that though BS high-SCE frequencies were completed by fusion with normal cells, fusion with various malignant cell lines did not result in complete normalization of BS SCEs, with 15-30 SCEs remaining per hybrid cell, demonstrating possibly common defects in DNA of BS and malignant cells. These findings strongly support the idea that the characteristic high SCE frequency in BS cells has some connection with the malignant condition, and that at least one step in carcinogenesis is either accompanied by the production of SCEs, or that SCEs themselves cause such a step to occur. © 1990.

    DOI: 10.1016/0165-7992(90)90117-3

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  • EXPRESSION OF THE CDNA-ENCODING LIPOCORTIN-LIKE 39-KDA PROTEIN OF GUINEA-PIG NEUTROPHILS IN YEAST - PURIFICATION AND BIOLOGICAL CHARACTERIZATION Reviewed

    EF SATO, Y TANAKA, K EDASHIGE, H KOBUCHI, S MORISHITA, YM SUGINO, M INOUE, K UTSUMI

    FEBS LETTERS   255 ( 2 )   231 - 236   1989.9

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    DOI: 10.1016/0014-5793(89)81097-X

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  • Distribution of EGF Receptor/Kinase Substrates in Various Organs of Guinea Pig Reviewed

    UTSUMI Kozo, MORISHITA Seiji, SATO Eisuke F., SUGINO Yasuo M., KOBUCHI Hirotsugu

    Research reports of Kochi Medical School Liberal arts   5   39 - 48   1989

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    Distribution of EGF receptor/kinase substrates in cytoplasmic proteins of guinea pig various organs was studied. 35kDa and 38kDa proteins in SDS-PAGE were detected as substrates for the enzyme in lung, spleen, stomach, urinary bladder and skin. 35kDa protein is confermed as lipocortin-like 39K protein of neutrophils by the method of immunoblotting using antibody of 39K protein. The Ca^<2+>-dependent membrane binding activity protein was an important character for the protein phosphorylation by this enzyme. A recombinant 39K protein was also phosphorylated by EGF receptor/kinase and the amount of phosphorylated 38kDa detected on SDS-PAGE was increased by the time of incubation.

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Books

  • 両備てい園記念財団 平成18年度 研究報告書

    2008 

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  • 生物学に関する試験研究論叢 23

    2008 

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  • 浜崎癌研究助成 平成18年度 研究報告書

    2007 

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  • 財団法人ウエスコ学術振興財団 平成17年度(2005〜2006)研究助成報告

    2006 

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  • 財団法人ウエスコ学術振興財団 平成16年度(2004〜2005)研究助成報告

    2005 

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  • 財団法人ウエスコ学術振興財団 平成15年度研究助成報告

    2004 

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  • 財団法人両備てい園記念財団研究助成報告 生物学に関する試験研究論叢

    2004 

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  • 新ミトコンドリア学

    小渕浩嗣( Role: Sole author ,  細胞内および単離ミトコンドリアの膜電位測定)

    共立出版  2001 

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  • Antioxidant Food Supplements in Human Health (L. Packer, and T. Yoshikawa, eds.)

    Virgili F, Kobuchi H, Noda Y, Cossins E, Packer L( Role: Joint author ,  Procyanidins from Pinus marittima Bark: Antioxidant Activity, Effects on the Immune System, and Modulation of Nitrogen Monoxide Metabolism.)

    Academic Press  1999 

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  • Antioxidant food supplements in human health

    Academic Press  1999 

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  • Methods Enzymol.

    Kobuchi H, Virgili F, Packer L( Role: Joint author ,  Assay of inducible form of nitric oxide synthase activity: effect of flavonoids and plant extracts.)

    Elsevier  1999 

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  • Antioxidant Food Supplements in Human Health (L. Packer, M. Hiramatsu, and T. Yoshikawa, eds.)

    Kobuchi H, Haramaki N, Marcocci L, Packer L( Role: Joint author ,  Biological Effects of the Fermentation Product of Carica papaya (Bio-Normalizer).)

    Academic Press  1999 

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  • Flavonoids in Health and Disease (C. A. Rice-Evans, and L. Packer, eds.)

    Virgili F, Kobuchi H, Packer L( Role: Joint author ,  Antioxidant properties and modulation of inducible NO synthase activity in activated macrophages by procyanidins extracted from Pinus maritima (Pycnogenol®).)

    Marcel Dekker, Inc.  1998 

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  • Advances in Ginkgo biloba Extract Research, vol. 7 Ginkgo biloba Extract (EGb 761): Lessons from Cell Biology (L. Packer, and Y. Christen, eds.)

    Roy S, Kobuchi H, Sen C.K, Gohil K, Droy-Lefaix M.T, Packer L( Role: Joint author ,  Ginkgo biloba extract (EGb 761): antioxidant properties and regulation of gene expression.)

    Elsevier  1998 

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  • Molecular mechanisms and health effects (Packer, L. Traber, M. G., and Xin, W. eds.)

    Packer L, Marcocci L, Haramaki N, Kobuchi H, Christen Y, Droy-Lefaix M.T( Role: Joint author ,  Antioxidant properties of Ginkgo biloba extract EGb 761 and clinical implications.)

    AOCS Press  1996 

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MISC

  • ビタミンE及びコレステロールのコハク酸エステルによるヒト前骨髄性白血病細胞(HL-60)のアポトーシス誘導とその比較

    山田 勝彦, 山本 真司, 竹原 良記, 小渕 浩嗣, 吉岡 保, 内海 耕慥, 玉井 浩

    ビタミン   76 ( 3 )   158 - 158   2002

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  • Antioxidants regulate agonists-induced cell adhesion molecule expression and function

    S Roy, CK Sen, H Kobuchi, L Packer

    FASEB JOURNAL   13 ( 7 )   A1581 - A1581   1999.4

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  • French Maritime Pine bark extract: Antioxidant activity and effects on regulation of nitric oxide metabolism and cell adhesion

    L Packer, T Bito, E Cossins, H Kobuchi, H Moini, Y Noda, G Rimbach, S Roy, J Vaya, F Virgili

    FASEB JOURNAL   13 ( 4 )   A546 - A546   1999.3

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  • Effect of polyphenols extracted from pine bark (pycnogenol) on nitric oxide metabolism and their role in the antioxidant network: Development of tools for the management of free-radical associated diseases?

    L Packer, F Virgill, H Kobuchi, R Lee, E Cossins

    FASEB JOURNAL   12 ( 5 )   A814 - A814   1998.3

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  • Regulation of phorbol ester induced adhesion of human jurkat T-cells to endothelial cells by the thiol antioxidant alpha-lipoate

    S Roy, CK Sen, H Kobuchi, L Packer

    FASEB JOURNAL   12 ( 5 )   A772 - A772   1998.3

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  • Antioxidant regulation of ICAM-1 expression

    CK Sen, S Roy, H Kobuchi, L Packer

    FREE RADICAL BIOLOGY AND MEDICINE   25   S52 - S52   1998

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    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:PERGAMON-ELSEVIER SCIENCE LTD  

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  • Pycnogenol (R): Antioxidant mechanism and regulation of no metabolism and cell adhesion

    L Packer, Y Cossins, Y Noda, F Virgili, H Kobuchi, S Roy, T Bito, J Vaya, H Moini

    FREE RADICAL BIOLOGY AND MEDICINE   25   S36 - S36   1998

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  • 7.胎盤組織における過酸化脂質とビタミンE : ビタミンE研究会第2回研究発表要旨

    吉岡 保, 竹原 良記, 藤井 好孝, 小渕 浩嗣

    ビタミン   64 ( 11 )   671 - 672   1990.11

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  • 17.ヒト卵胞液中の過酸化脂質濃度とビタミンE及びステロイドホルモンの関連性(第1回研究発表要旨,ビタミンE研究会)

    竹原 良記, 藤井 好孝, 小渕 浩嗣, 橋本 光月, 本山 洋明, 吉岡 保

    ビタミン   63 ( 11 )   587 - 588   1989.11

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Presentations

  • 効率的なsiRNAの光依存的細胞内送達能を持つ生分解性ナノキャリアの開発

    田中七星, 渡邉和則, 小渕浩嗣, 久保貴紀, 松浦栄次, 大槻高史

    第44回日本バイオマテリアル学会  2022.11.21 

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    Event date: 2022.11.21 - 2022.11.22

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  • N-myristoylation dependent mitochondrial targeting of NDUFB7, an accessory subunit of mitochondrial respiratory complex I

    2021.11.3 

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    Event date: 2021.11.3 - 2021.11.5

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  • N-ミリストイル化依存的にミトコンドリアに特異的に局在するタンパク質NDUFB7の発見

    原田春菜, 岩本奈津子, 西上美里, 七田真由, 守屋康子, 小渕浩嗣, 内海俊彦

    第62回 日本生化学会 中国・四国支部例会  2021.9.11 

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    Event date: 2021.9.10 - 2021.9.11

    Language:Japanese  

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  • Identification of N-myristoylated protein specifically localized to mitochondria by protein N-myristoylation

    2021.3.18 

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    Event date: 2021.3.18 - 2021.3.21

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  • N-ミリストイル化されたミトコンドリアタンパク質の探索

    原田春菜, 岩本奈津子, 西谷美里, 守屋康子, 小渕浩嗣, 内海俊彦

    第61回 日本生化学大会中四国支部例会  2020.7.18 

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    Event date: 2020.7.18 - 2020.7.20

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  • siRNAを用いたABCG2発現抑制と細胞死誘導の解析

    小渕浩嗣, 藤田洋史

    第61回 日本生化学大会中四国支部例会  2020.7.18 

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    Event date: 2020.7.18 - 2020.7.20

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  • ABCG2トランスポーターの発現抑制によるアポトーシス誘導

    小渕浩嗣, 藤田洋史

    第92回 日本生化学大会  2019.9.18 

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    Event date: 2019.9.18 - 2019.9.20

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  • siRNAを腫瘍組織へ送達するための細胞内侵入性ミセル型キャリアの開発

    西山雄基, 小渕浩嗣, 松浦栄次, 小関英一, 渡邉和則, 大槻高史

    日本核酸医薬学会第5回年会  2019.7.10 

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  • ABCG2 siRNA導入によるがん細胞のアポトーシス誘導

    小渕浩嗣, 藤田洋史

    第90回日本生化学会大会  2017.12.6 

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  • Small interfering RNA targeting of the ABCG2 gene induces apoptosis of cancer cells.

    Small interfering RNA targeting of the ABCG2 gene induces apoptosis of cancer cells.  2017 

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  • ヘム代謝関連遺伝子のRNAi効果による光線力学療法の改善

    小渕浩嗣, 藤田洋史

    第89回日本生化学会大会  2016.9.25 

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  • ヘム代謝制御に基づく光感受性物質protoporphyrin IX蓄積機構の研究

    藤田洋史, 永川恵介, 小渕浩嗣, 荻野哲也, 近藤洋一, 井上啓史, 執印太郎, 内海俊彦, 内海耕慥, 佐々木順造, 大内淑代

    第69回 日本酸化ストレス学会学術集会  2016.8.30 

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  • N-ミリストイル化されたミトコンドリアタンパク質SAMM50, TOMM40, CHCHD3, CHCHD6の間に生ずる相互作用の解析

    松崎嘉奈子, 岩永友花, 守屋康子, 小渕浩嗣, 内海俊彦

    第57回 日本生化学会 中国・四国支部例会  2016.5.27 

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  • がんの光線力学的治療の効率化におけるABCG2抑制の有用性

    小渕浩嗣, 藤田洋史

    第68回 日本酸化ストレス学会学術集会  2015.6.11 

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  • アミノレブリン酸を用いた光線力学的作用におけるABCトランスポーターG2阻害の効果

    小渕浩嗣, 藤田洋史

    第4回 ポルフィリンーALA学会年会  2014.4.26 

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  • アセチル-L-カルニチンによる無尾両生類幼生尾部短縮の抑制

    花田秀樹, 小渕浩嗣, 柏木啓子, 内海俊彦, 井上正康, 佐々木順造, 勝賢二郎, 佐藤英介, 内海耕慥, 柏木昭彦

    第84回 日本動物学会  2013.9.26 

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  • マウス骨髄細胞培養系におけるTNF-a による破骨細胞形成と骨髄間様系細胞によるその抑制効果

    藤田洋史, 小渕浩嗣, 内海耕慥, 佐々木順造, 大内淑代

    第86回 日本生化学大会  2013.9.11 

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  • ALA投与によるプロトポルフィリンIX蓄積におけるABCトランスポーターABCG2の役割とミトコンドリア局在

    小渕浩嗣, 守屋康子, 荻野哲也, 藤田洋史, 井上啓史, 執印太郎, 保田立二, 内海耕慥, 内海俊彦

    第3回 ポルフィリンーALA学会年会  2013.4.27 

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  • Acetyl-L-carnitine suppresses thyroid hormone-induced and spontaneous anuran tadpole tail shortening

    2013 

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  • 5−アミノレブリン酸によるプロトポルフィリンIX蓄積におけるABCG2のミトコンドリア局在と機能

    小渕浩嗣, 藤田洋史

    第85回 日本生化学会大会  2012.9 

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  • ABCG2のミトコンドリア局在とプロトポルフィリンIX蓄積の制御

    小渕浩嗣

    第53回 日本生化学会 中国・四国支部例会  2012.5 

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  • Mitochondrial localization of ABC transporter ABCG2 and its function in 5-aminolevulinic acid-mediated protoporphyrin IX accumulation.

    2012 

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  • 骨髄間様系幹細胞のosteogenic cultureによる細胞死仲介性石灰化と活性酸素の関与

    藤田洋史, 小渕浩嗣, 内海耕慥, 佐々木順造, 大内淑代

    第85回 日本生化学会大会  2012 

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  • がん細胞のポルフィリン蓄積におけるABCG2の役割 International conference

    小渕浩嗣

    第84回日本生化学会大会  2011.9 

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  • がん細胞のプロトポルフィリンIX蓄積におけるABCG2の役割

    小渕浩嗣

    第52回日本生化学会中国四国支部例会  2011 

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  • 志賀毒素のアポトーシスにおけるPKC-deltaの役割

    小渕浩嗣

    題51回 日本生化学会 中国四国支部例会  2010 

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  • アポトーシスにおける活性型カスパーゼ3と細胞骨格構造との結合

    藤田洋史

    第82回日本生化学会大会  2009 

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  • Etoposideによるアポトーシスにおける活性型カスパーゼ3の 細胞内局在-特に細胞骨格構造との結合について

    藤田洋史

    第50回日本生化学会 中国・四国支部例会  2009 

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  • 志賀毒素のアポトーシスにおけるカスパーゼを介したミトコンドリア機能障害のフィードバック増幅

    小渕浩嗣

    第82回日本生化学会大会  2009 

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  • 食餌性ポリフェノールによる志賀毒素誘導の腎上皮細胞アポトーシスの抑制

    小渕浩嗣

    Biochemistry and molecular biology 2008  2008 

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  • 志賀毒素による腎上皮細胞のアポトーシスにおけるProtein kinase C-deltaの関与

    小渕浩嗣

    Biochemistry and Molecular Biology 2007  2007 

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  • 志賀毒素誘導のアポトーシスにおけるPKC-deltaの活性化とロットレリンの効果

    小渕浩嗣

    第48回 日本生化学会中国四国支部例会  2007 

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  • Mechanism of A23187-induced apoptosis in HL-60 cells: dependency on mitochondrial permeability transition but not NADPH oxidase International conference

    小渕浩嗣

    第20回 国際生化学・分子生物学会議 (20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress)  2006 

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  • A23187誘導のアポトーシスにおける活性酸素生成とミトコンドリア膜透過性遷移(MPT)の関与

    小渕浩嗣

    第17回中国四国生体ラジカル研究会  2006 

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  • Involvement of Bax translocation in Shiga toxin-induced apoptosis

    小渕浩嗣

    第28回 日本分子生物学会年会  2005 

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  • 志賀毒素はBax活性化によりアポトーシスを誘導する

    小渕浩嗣

    第78回 日本生化学会大会  2005 

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  • 志賀毒素はJNK依存的Bcl-2リン酸化を介してアポトーシスを誘導する

    小渕浩嗣

    第77回 日本細菌学会総会  2004 

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  • 志賀毒素誘導の腎上皮様Vero細胞のアポトーシスにおけるAktの関与

    小渕浩嗣

    第77回 日本生化学会大会  2004 

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  • 志賀毒素のアポトーシス誘導機構におけるJNKの関与 (ワークショップ)

    第76回 日本細菌学会総会  2003 

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  • 志賀毒素誘導アポトーシスにおけるJNK活性化の意義

    小渕浩嗣

    第44回 日本生化学会中四国支部例会  2003 

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  • Requirement for ceramide-mediated JNK pathway in Shiga toxin-induced apoptosis

    小渕浩嗣

    第76回 日本生化学会大会  2003 

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  • 六価クロムによるCHO細胞のアポトーシス誘導:特に初期機構の解析

    小渕浩嗣

    日本過酸化脂質・フリーラジカル学会第26回大会  2002 

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  • 志賀毒素によるJNKを介したアポトーシス誘導

    小渕浩嗣

    第75回 日本生化学会大会  2002 

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  • コハク酸エステルによるHL-60細胞のアポトーシスとその初期反応

    小渕浩嗣

    第75回 日本生化学会大会  2002 

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  • 志賀毒素誘導のアポトーシスにおけるセラミドの関与

    小渕浩嗣

    第75回 日本細菌学会総会  2002 

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  • 志賀毒素のアポトーシス誘導機構におけるセラミドの関与

    小渕浩嗣

    第43回 日本生化学会中四国支部例会  2002 

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  • Involvement of ceramide in Shiga toxin-induced apoptotic cell death International conference

    小渕浩嗣

    Gordon Conference Glycolipid and Sphingolipid Biology  2002 

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  • 高度不飽和脂肪酸によるHL-60細胞のアポトーシスと脂質過酸化の関与

    小渕浩嗣

    第74回 日本生化学会大会  2001 

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  • カテプシンBによるベロ毒素誘導アポトーシスの制御

    小渕浩嗣

    第74回 日本細菌学会総会  2001 

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  • コレステリルサクシネートによるHL-60細胞のアポトーシス誘導機構

    小渕浩嗣

    第74回 日本生化学会大会  2001 

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  • メバスタチンによるHL-60細胞のアポトーシス誘導とその機構

    小渕浩嗣

    第74回 日本生化学会大会  2001 

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  • 志賀毒素誘導のアポトーシスにおけるセラミド産生とJNK活性化の関与

    小渕浩嗣

    第74回 日本生化学会大会  2001 

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    Language:Japanese   Presentation type:Symposium, workshop panel (nominated)  

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  • 高度不飽和脂肪酸によるHL60細胞のアポトーシスとその機構

    小渕浩嗣

    第73回 日本生化学会大会  2000 

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  • ベロ毒素誘導アポトーシスにおけるカテプシンの関与

    小渕浩嗣

    第73回 日本細菌学会総会  2000 

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  • ベロ毒素誘導アポトーシスにおけるカテプシンBの関与

    小渕浩嗣

    第73回 日本生化学会大会  2000 

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  • ミトコンドリアMPTのカルニチンによる抑制とアポトーシスへの関与

    小渕浩嗣

    第73回 日本生化学会大会  2000 

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  • ベロ毒素によるアポトーシス誘導機構の解析

    小渕浩嗣

    第72回 日本生化学会大会  1999 

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  • ベロ毒素誘導アポトーシスの機構解析

    小渕浩嗣

    日本過酸化脂質・フリーラジカル学会第23回大会  1999 

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  • ベロ毒素のアポトーシス誘導機構の解析

    小渕浩嗣

    第72回 日本細菌学会総会  1999 

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Industrial property rights

  • 修飾RNAを含有する分子集合体及びそれを用いたRNA送達システム

    大槻高史, 小関英一, 小渕浩嗣, 松浦栄次

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    Application no:特願PTC/JP2015/081352  Date applied:2016.5.12

    WO2016/072500 A1

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  • ポリ乳酸修飾RNAを有する分子集合体及びそれを用いたRNA送達システム

    大槻高史, 小関英一, 小渕浩嗣, 松浦栄次

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    Application no:特願2014-227811  Date applied:2014.11.8

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Awards

  • 中四国支部奨励賞

    2002.7   日本生化学会   志賀毒素のアポトーシス誘導機構におけるセラミドの関与

    小渕浩嗣

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    Country:Japan

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Research Projects

  • 骨変性部位の目印「dig-hereシグナル」を認識する破骨細胞特異的受容体の探索

    Grant number:22K09305  2022.04 - 2025.03

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    藤田 洋史, 小渕 浩嗣

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    Grant amount:\4160000 ( Direct expense: \3200000 、 Indirect expense:\960000 )

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  • Analysis of cell death induction and antitumor effect by suppression of the expression of ABC transporter

    Grant number:21K07124  2021.04 - 2024.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    小渕 浩嗣, 藤田 洋史

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    Grant amount:\4160000 ( Direct expense: \3200000 、 Indirect expense:\960000 )

    ATP-binding cassette transporter G2 (ABCG2)は、様々な薬剤や生理活性物質を体外に排出する生体バリア機能を担う輸送体である。代表者はABCG2機能阻害の手段として、siRNAによるABCG2遺伝子ノックダウンが、各種のがん細胞で増殖抑制や細胞死が誘導されることを見出し、その細胞死シグナル伝達機構やABCトランスポーターの新たな生理的意義を明らかにすることを目的としている。
    初年度となる令和3年度は、種々のがん細胞株における各種ABCトランスポーターの発現抑制と細胞生存率の解析を中心に行った。この解析の目的は、ABCG2と同様に、siRNAによるタンパク質発現抑制によって細胞死を誘導するABCトランスポーターの存在を明らかにすることである。
    ABCG2遺伝子の他に、ATP-binding cassette subfamily B Member 1(ABCB1,別名:P-Glycoprotein 1)、Multidrug resistance-associated protein 1(ABCC1)など、主として薬剤や生理活性物質を細胞外へ排出するABCトランスポーターについて検討した。各種siRNAの細胞内導入には市販トランスフェクション試薬を使用し、各遺伝子ノックダウン条件の最適化を行い、最も毒性の低い条件でノックダウンを施行した。膵臓がんをはじめ、様々な種類のヒトがん培養細胞株における各遺伝子ノックダウンによる影響は、それらの細胞生存率をMTT法により経時的に測定することで評価した。
    結果として、ABCG2以外でも細胞生存率を有意に低下させるABCトランスポーター遺伝子の存在を認めた。その割合はABCG2遺伝子抑制と比較すると低いものであったが、何らかの共通点が存在する可能性も否定できない。この点については、今後の課題として解析を試みる予定である。

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  • The role of pattern recognition receptors in osteoclast differentiation and function

    Grant number:19K09625  2019.04 - 2022.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    Fujita Hirofumi

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    Authorship:Coinvestigator(s) 

    Grant amount:\4290000 ( Direct expense: \3300000 、 Indirect expense:\990000 )

    How osteoclasts recognize old bone remains to be elucidated. We focused on pattern recognition receptors and found that some pattern recognition receptors are specifically expressed on osteoclasts. In this study, we aimed to identify old bone recognition molecules from these pattern recognition receptors. We then generated some knockout mice of candidate molecules in mice by genome editing. Contrary to expectations, disruption of the pattern recognition receptor A gene strongly promoted osteoclast differentiation. This result showed that gene A might contribute to the recognition of new bone.

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  • 深部・転移がんへのRadio-induced photodynamic (RIPD) - Theranosticsを実現する89Zr標識・抗体担持生分解性キャリアの開発

    2019.04 - 2022.03

    日本医療研究開発機構 次世代がん医療創生研究事業  次世代がん医療創生研究事業

    松浦栄次、大槻高史、小渕浩嗣、宇治広隆

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    Authorship:Coinvestigator(s) 

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  • 深部・転移がんへのRadio-induced photodynamic (RIPD) - Theranosticsを実現する89Zr標識・抗体担持生分解性キャリアの開発

    2016.06 - 2019.03

    日本医療研究開発機構  次世代がん医療創生研究事業 

    松浦栄次、大槻高史、小渕浩嗣、宇治広隆

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    Grant type:Competitive

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  • Development of a novel radiological-photo-therapy essential for Theranostics targeting deep lesions

    Grant number:16K15582  2016.04 - 2018.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Challenging Exploratory Research

    MATSUURA Eiji, KOBUCHI Hirotsugu, TAKENAKA Fumiaki, AKEHI Masaru

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    Grant amount:\3510000 ( Direct expense: \2700000 、 Indirect expense:\810000 )

    Photodynamic therapy (PDT) using a photosensitizer capable of inducing cytotoxicity by light irradiation is effective only for the lesion closed to the body surface, due to low permeability of the near infrared light, and is ineffective for therapy targeting the deep tissues. To solve the problem, we studied using Cherenkov light generated by β+-ray emission of a PET nuclide as a internal light source. We have given the PDT effect by Cherenkov light to the innovative "Theranostics (a treatment and diagnostic enforcement) technology" consisting of the specific antibody/Zr-89 labeled-drug delivery system (DDS) that we have ever established. We were newly able to verify some of the new basic technologies to overcome the problem (limit) of the PDT effect.

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  • 炎症性サイトカインによる破骨細胞分化を制御するグルタチオン応答性タンパク質の同定

    2015.04 - 2017.03

    日本学術振興会  基盤研究(C)基金 

    藤田洋史

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    Grant type:Competitive

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  • 動脈硬化性疾患の早期診断を可能にする分子イメージング・リピドーム解析技術の構築

    2014.04 - 2017.03

    日本学術振興会  基盤研究(A)補助金 

    松浦栄次

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    Grant type:Competitive

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  • 骨髄間葉系幹細胞が分泌する新規破骨細胞分化調節分子の同定

    2012.04 - 2014.03

    日本学術振興会  基盤研究(C)基金 

    藤田洋史

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    Grant type:Competitive

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  • ミトコンドリア新生の促進因子のスクリーニングと細胞機能亢進の解析

    2011.04 - 2013.03

    日本学術振興会  挑戦的萌芽研究 基金 

    小渕浩嗣

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    Authorship:Principal investigator  Grant type:Competitive

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Other research activities

  • GTP結合タンパク質に関する研究

    2018.04

  • 癌の光線動力学的診断・治療法(PDD・PDT)に関する研究

    2018.04

  • 骨髄間葉系幹細胞が分泌する新規破骨細胞分化調節分子の同定

    2018.04

  • ミリストイル化タンパク質に関する研究

    2018.04

 

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