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INOUE Takao

    Department of Medicine　Research associate
Last Updated :2024/04/25

Researcher Information

Degree

  • Ph. D（2002/03 Okayama University）

URL

Research funding number

  • 00441006

J-Global ID

Research Interests

  • metabolic syndrome   高次機能改善   神経細胞死   神経保護作用   酸化ストレス   運動   脳外傷   神経再生促進効果   

Research Areas

  • Life sciences / Experimental pathology
  • Life sciences / Rehabilitation science

Academic & Professional Experience

  • 2011 - 2012  Kindai UniversityFaculty of Medicine助教

Published Papers

  • Fuka Takeuchi; Aki Sugano; Azusa Yoneshige; Man Hagiyama; Takao Inoue; Akihiro Wada; Yutaka Takaoka; Akihiko Ito
    Cells Tissues Organs S. Karger AG 1422-6405 2023/10 
    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first infects the host nasal mucosa, where the viral spike protein binds to angiotensin-converting enzyme 2 (ACE2) on the mucosal cells. This study aimed at searching host cell surface molecules that could contribute to the infection in two views; abundance on host cells and affinity to the spike protein. Since the nasal mucosa is lined by respiratory and olfactory epithelia, and both express an immunoglobulin superfamily member cell adhesion molecule 1 (CADM1), whether CADM1 would participate in the spike protein binding was examined. Immunohistochemistry on the mouse nasal cavity detected CADM1 strongly in the olfactory epithelium at cell–cell contacts and on the apical surface but just faintly in the respiratory epithelium. In contrast, ACE2 was detected in the respiratory, not olfactory, epithelium. When mice were administered intranasally with SARS-CoV-2 S1 spike protein and an anti-CADM1 ectodomain antibody separately, both were detected exclusively on the olfactory, not respiratory, epithelium. Then, the antibody and S1 spike protein were administered intranasally to mice in this order with an interval of 1 hour. After 3 hours, S1 spike protein was detected as a protein aggregate floating in the nasal cavity. Next, S1 spike protein labeled with fluorescein was added to the monolayer cultures of epithelial cells exogenously expressing ACE2 or CADM1. Quantitative detection of fluorescein bound to the cells revealed that S1 spike protein bound to CADM1 with affinity half as high as to ACE2. Consistently, docking simulation analyses revealed that S1 spike protein could bind to CADM1 three quarters as strongly as to ACE2 and that the interface of ACE2 was similar in both binding modes. Collectively, intranasal S1 spike protein appeared to prefer to accumulate on the olfactory epithelium, and CADM1 was suggested to contribute to this preference of S1 spike protein based on the molecular abundance and affinity.
  • 脳卒中易発症高血圧自然発症ラットSHRSPは廃用性萎縮からの回復時にユビキチン化の亢進が遅延する
    井上 敬夫; 金澤 佑治; 水口 信行; 前西 修; 木村 雅友; 峯 嘉宏; 萩山 満; 米重 あづさ; 和田 昭裕; 筑後 孝章; 伊藤 龍生; 佐藤 隆夫; 伊藤 彰彦
    日本筋学会学術集会プログラム・抄録集 (一社)日本筋学会 8回 129 - 129 2433-975X 2022/08
  • Man Hagiyama; Takahiro Mimae; Akihiro Wada; Fuka Takeuchi; Azusa Yoneshige; Takao Inoue; Naoyuki Kotoku; Hironobu Hamada; Yoshitaka Sekido; Morihito Okada; Akihiko Ito
    Frontiers in Cell and Developmental Biology Frontiers Media SA 10 2022/07 
    Malignant pleural mesothelioma (MPM) is a highly aggressive malignant tumor, and the effective therapeutic drugs are limited. Thus, the establishment of novel therapeutic method is desired. Considerable proportion of MPMs are shown to express cell adhesion molecule 1 (CADM1), and to use CADM1 to bind to and proliferate on the pleural mesothelial surface, suggesting that CADM1 is a possible therapeutic target. Here, anti-CADM1 ectodomain chicken monoclonal antibodies, 3E1 and 9D2, were examined for their possible therapeutic utility. The full-length form of CADM1 was expressed in eight out of twelve human MPM cell lines. MPM cell lines were cultured on a confluent monolayer of mesothelial MeT-5A cells in the presence of 9D2, the neutralizing antibody. 9D2 suppressed the cell growth of CADM1-positive MPM cells with the loss and aggregation of CADM1 molecules on the MPM cell membrane, but not of CADM1-negative MPM cells. Co-addition of 3E1, lacking the neutralizing action, enhanced the growth-suppressive effect of 9D2. The two antibodies were tested as drug delivery vectors. 3E1 was converted into a humanized antibody (h3E1) and conjugated with monomethyl auristatin E (MMAE), a tubulin polymerization inhibitor. When the resulting h3E1–MMAE antibody-drug conjugate (ADC) was added to the standard cultures of CADM1-positive MPM cells, it suppressed the cell growth in a dose-dependent manner. Co-addition of 9D2 enhanced the growth-suppressive effect of h3E1–MMAE ADC. Anti-CADM1 ectodomain antibodies were suggested to serve as both antibody drugs and drug vectors in the treatment of MPM.
  • Man Hagiyama; Fuka Takeuchi; Aki Sugano; Azusa Yoneshige; Takao Inoue; Akihiro Wada; Hiroshi Kajiyama; Yutaka Takaoka; Kenroh Sasaki; Akihiko Ito
    Experimental and therapeutic medicine 23 (4) 274 - 274 2022/04 
    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its S1 spike protein to bind to angiotensin-converting enzyme 2 (ACE2) on human cells in the first step of cell entry. Tryptanthrin, extracted from leaves of the indigo plant, Polygonum tinctorium, using d-limonene (17.3 µg/ml), is considered to inhibit ACE2-mediated cell entry of another type of coronavirus, HCoV-NL63. The current study examined whether this extract could inhibit the binding of the SARS-CoV-2 spike protein to ACE2. Binding was quantified as cell-bound fluorescence intensity in live cell cultures in which canine kidney MDCK cells overexpressing ACE2 were incubated with fluorescein-labeled S1 spike protein. When indigo extract, together with S1 protein, was added at 8,650x and 17,300x dilutions, fluorescence intensity decreased in a dose- and S1 extract-dependent manner, without affecting cell viability. When 4.0-nM tryptanthrin was added instead of the indigo extract, fluorescence intensity also decreased, but to a lesser degree than with indigo extract. Docking simulation analyses revealed that tryptanthrin readily bound to the receptor-binding domain of the S1 protein, and identified 2- and 7-amino acid sequences as the preferred binding sites. The indigo extract appeared to inhibit S1-ACE2 binding at high dilutions, and evidently contained other inhibitory elements as well as tryptanthrin. This extract may be useful for the prevention or treatment of SARS-CoV-2 infection.
  • 井上 敬夫; 金澤 佑治; 水口 信行; 峯 嘉宏; 前西 修; 北村 雅友; 萩山 満; 米重 あづさ; 和田 昭裕; 筑後 孝章; 伊藤 龍生
    日本内分泌学会雑誌 (一社)日本内分泌学会 97 (5) 1503 - 1503 0029-0661 2022/03
  • 井上 敬夫; 金澤 佑治; 水口 信行; 峯 嘉宏; 前西 修; 北村 雅友; 萩山 満; 米重 あづさ; 和田 昭裕; 筑後 孝章; 伊藤 龍生
    日本内分泌学会雑誌 (一社)日本内分泌学会 97 (5) 1503 - 1503 0029-0661 2022/03
  • 脳卒中発症ラットSHRSPは廃用性萎縮からの回復時に筋線維の損傷がみられない
    井上 敬夫; 金澤 佑治; 水口 信行; 峯 嘉宏; 前西 修; 木村 雅友; 萩山 満; 米重 あづさ; 和田 昭裕; 筑後 孝章; 伊藤 龍生; 佐藤 隆夫; 伊藤 彰彦
    日本筋学会学術集会プログラム・抄録集 (一社)日本筋学会 7回 125 - 125 2433-975X 2021/11
  • Azusa Yoneshige; Man Hagiyama; Yasutoshi Takashima; Satoru Ueno; Takao Inoue; Ryuichiro Kimura; Yoshiki Koriyama; Akihiko Ito
    Frontiers in cell and developmental biology 9 664327 - 664327 2021 [Refereed]
     
    Elevation of intraocular pressure is a major risk factor for glaucoma development, which causes the loss of retinal ganglion cells (RGCs). Lipocalin 2 (Lcn2) is upregulated in glaucomatous retinae; however, whether Lcn2 is directly involved in glaucoma is debated. In this study, retinal explant cultures were subjected to increased water pressure using a two-chamber culture device, and Lcn2 protein levels were examined by immunoblotting. In situ TdT-mediated dUTP nick and labeling (TUNEL) and glial fibrillary acidic protein (GFAP) immunohistochemical assays were performed to assess apoptosis and gliosis, respectively. The neurotoxicity of Lcn2 in the retinal explant culture was determined with exogenous administration of recombinant Lcn2. The Lcn2 protein levels, percentage of TUNEL-positive cells, and GFAP-positive area were significantly higher in retinae cultured under 50 cm H2O pressure loads compared to those cultured under 20 cm H2O. We found that Lcn2 exhibited neurotoxicity in retinae at dose of 1 μg/ml. The negative effects of increased hydrostatic pressure were attenuated by the iron chelator deferoxamine. This is the first report demonstrating the direct upregulation of Lcn2 by elevating hydrostatic pressure. Modulating Lcn2 and iron levels may be a promising therapeutic approach for retinal degeneration.
  • Man Hagiyama; Ryuichiro Kimura; Azusa Yoneshige; Takao Inoue; Tomoyuki Otani; Akihiko Ito
    International journal of molecular sciences 21 (11) 2020/06 [Refereed]
     
    When epithelial cells in vivo are stimulated to proliferate, they crowd and often grow in height. These processes are likely to implicate dynamic interactions among lateral membranous proteins, such as cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member. Pulmonary epithelial cell lines that express CADM1, named NCI-H441 and RLE-6TN, were grown to become overconfluent in the polarized 2D culture system, and were examined for the expression of CADM1. Western analyses showed that the CADM1 expression levels increased gradually up to 3 times in a cell density-dependent manner. Confocal microscopic observations revealed dense immunostaining for CADM1 on the lateral membrane. In the overconfluent monolayers, CADM1 knockdown was achieved by two methods using CADM1-targeting siRNA and an anti-CADM1 neutralizing antibody. Antibody treatment experiments were also done on 6 other epithelial cell lines expressing CADM1. The CADM1 expression levels were reduced roughly by half, in association with cell height decrease by half in 3 lines. TUNEL assays revealed that the CADM1 knockdown increased the proportion of TUNEL-positive apoptotic cells approximately 10 folds. Increased expression of CADM1 appeared to contribute to cell survival in crowded epithelial monolayers.
  • 肝星細胞の活性化に着目した肝線維化回復機能の検討
    忍海辺 結実; 森島 美幸; 佐藤 隆夫; 井上 敬夫; 水口 信行; 伊藤 龍生
    日本栄養・食糧学会大会講演要旨集 (公社)日本栄養・食糧学会 74回 276 - 276 2020/04
  • 皮膚表皮剥脱による常在菌の侵入は、女性型脱毛症発症のトリガーとなる
    辻 ひかり; 池上 侑希; 森島 真幸; 佐藤 隆夫; 水口 信行; 井上 敬夫; 伊藤 龍生
    日本栄養・食糧学会大会講演要旨集 (公社)日本栄養・食糧学会 74回 183 - 183 2020/04
  • 肝星細胞の活性化に着目した肝線維化回復機能の検討
    忍海辺 結実; 森島 美幸; 佐藤 隆夫; 井上 敬夫; 水口 信行; 伊藤 龍生
    日本栄養・食糧学会大会講演要旨集 (公社)日本栄養・食糧学会 74回 276 - 276 2020/04
  • Takao Inoue; Man Hagiyama; Osamu Maenishi; Masatomo Kimura; Nobuyuki Mizuguchi; Yoshihiro Mine; Ryuichiro Kimura; Takaaki Chikugo; Tatsuki Itoh; Takao Satou; Akihiko Ito
    Life sciences 237 116919 - 116919 0024-3205 2019/11 [Refereed]
     
    AIMS: Stroke-prone spontaneously hypertensive rats (SHRSP) show significantly lower body weight than normotensive Wistar-Kyoto rats (WKY). Our hypotheses are as follows: weight loss of the skeletal muscle is related to hypertension-related diseases, and muscle hypotrophy is useful as a therapeutic target for hypertension and hypertension-related diseases. In this study, we aimed to investigate the pathophysiological characteristics of muscle hypotrophy in SHRSP to determine the therapeutic target molecule(s). MAIN METHODS: The difference in skeletal muscles in the lower leg between WKY and SHRSP was evaluated mainly through weight/tibial length, histological, gene expression, and protein expression analyses. KEY FINDINGS: SHRSP had a significantly lower weight/tibial length in soleus and gastrocnemius, but not in plantaris and tibialis anterior, indicating that muscles consisting of a relatively high amount of slow muscle fiber were affected. This result was confirmed by the histological analysis of soleus, showing that type I fiber mainly decreased the fiber size. Microarray and protein expression analyses showed that the muscle-specific ubiquitin ligase, muscle RING finger 1 (MuRF1), but not atrogin-1, was highly expressed in soleus, but not in plantaris, in SHRSP. TNF-like weak inducer of apoptosis receptor (TWEAKR) was predicted as a MuRF1 up-regulator by Ingenuity Pathway Analysis and immunostained only in type II fiber in WKY but in both type I and II fibers in SHRSP. SIGNIFICANCE: TWEAKR is a type II-specific receptor in the skeletal muscle. Ectopic TWEAKR expression in type I fiber of SHRSP is most likely involved in slow muscle-specific hypotrophy through MuRF1 overexpression.
  • 遅筋特異的発育異常におけるMuRF1の発現上昇と上流因子の解析
    井上 敬夫; 前西 修; 木村 雅友; 萩山 満; 水口 信行; 峯 嘉宏; 筑後 孝章; 伊藤 龍生; 佐藤 隆夫; 伊藤 彰彦
    日本筋学会学術集会プログラム・抄録集 日本筋学会 5回 146 - 146 2433-975X 2019/08
  • Hagiyama M; Nakatani Y; Takashima Y; Kato T; Inoue T; Kimura R; Otani T; Sato Y; Mori H; Arima S; Ito A
    Frontiers in cell and developmental biology Frontiers Media SA 7 111  2019 [Refereed]
  • Ryuichiro Kimura; Azusa Yoneshige; Man Hagiyama; Tomoyuki Otani; Takao Inoue; Naoki Shiraishi; Kazuyoshi Yanagihara; Tomohiko Wakayama; Akihiko Ito
    Life sciences 213 206 - 213 0024-3205 2018/11 [Refereed]
     
    AIMS: To determine cellular distribution of cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member, in the human oxyntic gastric mucosa, and to explore possible involvement in the development and peritoneal dissemination of signet ring cell (SRC) gastric carcinoma, which often develops in the oxyntic mucosa. MAIN METHODS: Immunohistochemistry and double immunofluorescence were conducted on surgical specimens of normal and SRC-bearing stomachs and peritoneal metastatic foci of SRCs. KATO-III (lacking CADM1) and HSC-43 (expressing CADM1) SRC cell lines were cocultured on a Met-5A mesothelial or TIG-1 fibroblastic cell monolayer. KEY FINDINGS: In the oxyntic gland, some neck and nearly all base glandular cells were CADM1-positive, and mucin 5AC-positive cells were CADM1-negative, while some mucin 6-positive neck cells were CADM1-positive. Foveolar-epithelial, parietal, and endocrine cells were CADM1-negative. CADM1 was negative in all SRC carcinomas that were confined within the submucosa (n = 11) and all but one of those invading deeper (n = 15). In contrast, peritoneal metastatic foci of SRCs were CADM1-positive in five out of eleven cases (P < 0.01). In the cocultures, exogenous CADM1 made KATO-III cells adhere more and grow faster on a Met-5A monolayer, not on TIG-1 monolayers. HSC-43 cells adhered more and grew faster on Met-5A than on TIG-1 monolayers, which were partly counteracted by a function-neutralizing anti-CADM1 antibody. SIGNIFICANCE: Nearly all chief cells and a part of mucous neck cells express CADM1. SRC gastric carcinoma appears to emerge as a CADM1-negative tumor, but CADM1 may help SRCs develop peritoneal dissemination through promoting their adhesion and growth in the serosal tissue.
  • Ri A; Hagiyama M; Inoue T; Yoneshige A; Kimura R; Murakami Y; Ito A
    Frontiers in cell and developmental biology 6 52 - 52 2018 [Refereed]
     
    Pulmonary emphysema usually arises in cigarette smokers, and often progresses after smoking cessation and even in ex-smokers. Lung-epithelial cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member, is extracellularly shed to produce a proapoptotic C-terminal fragment (CTF) within the cell and contribute to the development of emphysema. Here, we made an ex-smoker model using C57BL/6 mice; mice (6-week-old; 5 mice per group) were exposed to passive smoke of eight cigarettes twice a day 5 days a week until 18 weeks of age, and were then left untreated until 30 weeks of age. We calculated the mean linear intercept (Lm) and the alveolar septal thickness in the lung histologic sections to estimate the alveolar space dilatation. At 18 weeks of age, Lm was marginally enlarged (P = 0.023) with a marked increase in the septal thickness (P < 0.001) in comparison with age-matched control mice (5 mice per group), while at 30 weeks, the increase in Lm was much more prominent (P = 0.006) and the septal thickness was normalized, suggesting that emphysema progressed with septal remodeling during smoking cessation. Western blot analyses of the lungs were performed for CADM1, a possible CADM1 sheddase ADAM10, an epithelial marker pan-cytokeratin, and a myofibroblastic marker α-smooth muscle actin to estimate the expression levels of CTF and ADAM10 per epithelial cell and the levels of pan-cytokeratin and αSMA per tissue. CADM1 shedding was increased in the treated mice than in control mice at both ages, in association with an increase in the CTF level at 30 weeks (P = 0.021). In total of the treated and control mice of 30 weeks of age, Lm was positively correlated with the CTF and ADAM10 levels, and pan-cytokeratin was negatively correlated with CTF, suggesting an involvement of CADM1 shedding in emphysema progression. Positive correlations were also found between CTF and ADAM10, and between ADAM10 and αSMA, suggesting that increased septal myofibroblasts might be involved in increased CADM1 shedding. Taken together, persisting increase in ectodomain shedding of CADM1 appeared to contribute to the progression of emphysema in ex-smokers, and might be accounted for by alveolar septal remodeling.
  • Man Hagiyama; Norikazu Yabuta; Daisuke Okuzaki; Takao Inoue; Yasutoshi Takashima; Ryuichiro Kimura; Aritoshi Ri; Akihiko Ito
    FRONTIERS IN PHYSIOLOGY FRONTIERS MEDIA SA 8 997  1664-042X 2017/12 [Refereed]
     
    Intraluminal pressure elevation can cause degenerative disorders, such as ileus and hydronephrosis, and the threshold is fairly low and constant, 20-30 cm H2O. We previously devised a novel two-chamber culture system subjecting cells cultured on a semipermeable membrane to increased culture medium height (water pressure up to 60 cm H2O). Here, we sought to determine how a continuous pressure load of similar to 30 cm H2O affects proliferating epithelial cells with special interest in the link with cell morphology. We cultured several different cell lines using the low static pressure-loadable two-chamber system, and examined cell growth, cell cycle, and cell morphology. Madin-Darby canine kidney (MDCK) columnar epithelial cells were growth-suppressed in a manner dependent on static water pressure ranging from 2 to 50 cm H2O, without cell cycle arrest at any specific phase. Two other types of columnar epithelial cells exhibited similar phenotypes. By contrast, spherical epithelial and mesenchymal cells were not growth-suppressed, even at 50 cm H2O. Phalloidin staining revealed that 50 cm H2O pressure load vertically flattened and laterally widened columnar epithelial cells and made actin fiber distribution sparse, without affecting total phalloidin intensity per cell. When the mucosal protectant irsogladine maleate (100 nM) was added to 50-cm-high culture medium, MDCK cells were reduced in volume and their doubling time shortened. Cell proliferation and morphology are known to be regulated by the Hippo signaling pathway. A pressure load of 50 cm H2O enhanced serine-127 phosphorylation and cytoplasmic retention of YAP, the major constituent of this pathway, suggesting that Hippo pathway was involved in the pressure-induced cell growth suppression. RNA sequencing of MDCK cells showed that a 50 cm H2O pressure load upregulated keratin 14, an intermediate filament, 12-fold. This upregulation was confirmed at the protein level by immunofluorescence, suggesting a role in cytoskeletal reinforcement. These results provide evidence that cell morphology and the cytoskeleton are closely linked to cell growth. Pathological intraluminal pressure elevation may cause mucosal degeneration by acting directly on this linkage and the Hippo pathway.
  • Takao Inoue; Kumiko Takemori; Nobuyuki Mizuguchi; Masatomo Kimura; Takaaki Chikugo; Man Hagiyama; Azusa Yoneshige; Tatsufumi Mori; Osamu Maenishi; Takashi Kometani; Tatsuki Itoh; Takao Satou; Akihiko Ito
    Experimental physiology 102 (11) 1435 - 1447 0958-0670 2017/11 [Refereed]
     
    NEW FINDINGS: What is the central question of this study? An inverse correlation between circulating adiponectin and many diseases has been reported, but some studies have found no correlation. To evaluate this controversy, we investigated the relationship between heart-bound adiponectin and hypertension or cardiac hypertrophy, compared with serum adiponectin. What is the main finding and its importance? Using hypertensive and normotensive rats, we found that heart-bound adiponectin was inversely correlated with cardiac hypertrophy, suggesting that heart-bound adiponectin has a more important function in preventing cardiac hypertrophy than circulating adiponectin. Our study provides new insights regarding the role of adiponectin in diseases. The inverse correlation between circulating adiponectin concentration and hypertension or cardiac hypertrophy is still controversial. In addition to circulating adiponectin, adiponectin is also bound to tissues such as the heart and skeletal muscle. In this study, we investigated the relationship of serum adiponectin and heart-bound adiponectin with hypertension and cardiac hypertrophy. Four types of hypertensive rats presenting different blood pressure levels were used at different ages, as follows: normotensive Wistar-Kyoto rats (WKYs); two sub-strains (strains C and B2, having low and high blood pressure, respectively) of spontaneously hypertensive rats (SHRs); and stroke-prone SHRs (SHRSPs). Blood pressure, heart-to-body weight ratio, serum adiponectin and heart-bound adiponectin were determined. Histopathological analysis of the heart was carried out to evaluate the relationship with heart-bound adiponectin. Serum adiponectin concentration was not inversely correlated with blood pressure or heart-to-body weight ratio. In contrast, heart-bound adiponectin levels were significantly lower in SHRSPs than in other strains at respective ages. This resulted from a decrease in T-cadherin expression, which induced adiponectin binding to tissues. No significant difference in heart-bound adiponectin among WKYs and SHRs (C and B2) was detected, indicating that heart-bound adiponectin is not related to hypertension. In addition, differences in heart-bound adiponectin did not affect AMP-activated protein kinase in the traditional adiponectin activation cascade. Histopathological analysis revealed that heart-bound adiponectin was inversely correlated with cardiomyocyte hypertrophy and left ventricular wall thickness and, in part, with cardiac fibrosis. These results suggest that the decreased level of heart-bound adiponectin in SHRSPs is more related to their cardiac hypertrophy than circulating adiponectin.
  • Azusa Yoneshige; Man Hagiyama; Takao Inoue; Tomonori Tanaka; Aritoshi Ri; Akihiko Ito
    MOLECULAR NEUROBIOLOGY HUMANA PRESS INC 54 (8) 6378 - 6390 0893-7648 2017/10 [Refereed]
     
    Internal pressure is often involved in neurode-generation; intraocular and intraventricular pressure elevations over 20-30 cmH(2)O cause glaucoma and hydrocephalus, respectively. Here, we investigated enteric nerve degeneration in colon segments having tumor-induced stenosis and dilation and examined the mechanism of intraluminal pressure involvement. Histological examination revealed that the enteric ganglion neurons and neurites decreased in density in the dilated colons proportionate to the degree of dilation. Western blot analysis for cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member expressed in enteric neurons, revealed that ectodomain shedding of CADM1 increased proportionate to colon dilation, with increased production of its C-terminal fragment alpha CTF, a proapoptotic intracellular molecule. To link these neuro-degenerative events to increased intraluminal pressure, we devised a two-chamber culture system wherein cells cultured on a semipermeable membrane were subjected to increased medium height (water pressure up to 50 cmH(2)O). Mouse dorsal root ganglion (DRG) neurons were examined for expansion of their neurite networks in this system. As the pressure increased to 15, 30, and 45 cmH(2)O, the neurites decreased in density and became thinner. In addition, CADM1 shedding increased with more aCTF production. CADM1 immunofluorescence and Mitotracker mitochondrial labeling revealed that as the pressure increased, neuritic CADM1 distribution changed from uniform to punctate staining patterns, and neuritic mitochondria decreased in number and appeared as course particles. These pressure-induced phenotypes were reproduced by exogenous expression of alpha CTF in standard DRG neuron cultures. Therefore, increases in colonic intraluminal pressure might cause enteric nerve degeneration by inducing CADM1 shedding and alpha CTF production.
  • Yasutoshi Takashima; Teppei Murakami; Takao Inoue; Man Hagiyama; Azusa Yoneshige; Syunji Nishimura; Masao Akagi; Akihiko Ito
    TUMOR BIOLOGY SAGE PUBLICATIONS LTD 39 (6) 1010428317704365  1010-4283 2017/06 [Refereed]
     
    Epithelial carcinomas occasionally have sarcomatous components that consist primarily of spindle and cuboidal cells, which often resemble osteoblasts. Sarcomatoid carcinomas consist of similar cells. Recent studies have characterized these phenomena as a manifestation of epithelial-mesenchymal transition in carcinoma cells, but the mesenchymal phenotypes that manifest in sarcomatous cells of epithelial carcinomas are not well understood. Here, we examined the expression profiles of four osteoblastic differentiation biomarkers in the sarcomatous components of multiple carcinoma types, including five renal clear cell, four breast invasive ductal, two esophageal, one maxillary squamous cell, three larynx, three lung, one liver, and one skin sarcomatoid carcinoma. Expression was analyzed by immunohistochemistry using antibodies against cell adhesion molecule 1, a member of the IgCAM superfamily, osterix transcription factor (Osterix), cluster of differentiation 151, a transmembrane 4 superfamily member, and alkaline phosphatase. Immunostaining intensity was rated in scale 0 (negative), 0.5 (weak), and 1 (strong) for each marker, and the four scale values were summed to calculate osteoblastic scores. In all, 10 cases had a osteoblastic score >= 3, and all of these 10 cases were cell adhesion molecule 1- and Osterix-positive. Eight and five of the nine samples with a osteoblastic score < 3 were negative for cell adhesion molecule 1 (p < 0.0001) and Osterix (p = 0.006), respectively. The other markers showed no statistical significance. These results indicate that osteoblastic differentiation can occur in carcinoma cells and that cell adhesion molecule 1 could be a useful marker for identifying this phenomenon in carcinoma tissues.
  • Inoue Takao; Takemori Kumiko; Muzuguchi Nobuyuki; Kimura Masatomo; Chikugo Takaaki; Hagiyama Man; Yoneshige Azusa; Mori Tatsufumi; Kometani Takashi; Itoh Tatsuki; Satou Takao; Ito Akihiko
    JOURNAL OF HYPERTENSION LIPPINCOTT WILLIAMS & WILKINS 34 E288 - E288 0263-6352 2016/09 [Refereed]
  • Azusa Yoneshige; Man Hagiyama; Takao Inoue; Takahiro Mimae; Takashi Kato; Morihito Okada; Eisuke Enoki; Akihiko Ito
    RESPIRATORY RESEARCH BIOMED CENTRAL LTD 16 1465-993X 2015/08 [Refereed]
     
    Background: Lung alveolar epithelial cell (AEC) apoptosis has attracted attention as an early pathogenic event in the development of idiopathic interstitial pneumonia (IIP); however, the causative mechanism remains unclear. Cell adhesion molecule 1 (CADM1) is an AEC adhesion molecule in the immunoglobulin superfamily. It generates a membrane-associated C-terminal fragment, alpha CTF, through protease-mediated ectodomain shedding, termed a-shedding. Increased CADM1 alpha-shedding contributes to AEC apoptosis in emphysematous lungs. Methods: Formalin-fixed, paraffin-embedded lung lobes (n = 39) from 36 autopsied patients with IIP were classified as acute IIP (n = 10), fibrosing-type nonspecific IIP (f-NSIP, n = 10), cryptogenic organizing IIP (n = 9), and usual IIP (n = 10). CADM1 expression in the lung sections was examined by western blotting and compared with control lungs (n = 10). The rate of CADM1 alpha-shedding was calculated as the relative amount of alpha CTF to full-length CADM1, and the full-length CADM1 level was estimated per epithelial cell by normalization to cytokeratin 7, a lung epithelial marker. Apoptotic AECs were detected by immunohistochemistry for single-stranded DNA (ssDNA). NCI-H441 and A549 human lung epithelial cells were transfected with small interfering RNA (siRNA) to silence CADM1 expression and analyzed by terminal nucleotide nick end labeling assays. Results: The rate of CADM1 alpha-shedding was higher in all IIP subtypes than in the control (P = 0.019), and the full-length CADM1 level was lower in f-NSIP (P = 0.007). The alpha-shedding rate and full-length CADM1 level were correlated with each other (P = 0.015) and with the proportion of ssDNA-positive AECs (P = 0.024). NCI-H441 cells transfected with siRNA exhibited a 61 % lower rate of expression of full-length CADM1 and a 17-fold increased proportion of apoptotic cells. Similar results were obtained with A549 cells. Conclusions: CADM1 alpha-shedding appeared to be increased in all four IIP subtypes and consequently contributed to AEC apoptosis by decreasing the full-length CADM1 level. This mechanism particularly impacted f-NSIP. The molecular mechanism causing AEC apoptosis may be similar between IIP and emphysema.
  • Yoneshige A; Hagiyama M; Inoue T; Mimae T; Kato T; Okada M; Enoki E; Ito A
    Respiratory research 16 90  1465-9921 2015/08 [Refereed]
  • Man Hagiyama; Azusa Yoneshige; Takao Inoue; Yasufumi Sato; Takahiro Mimae; Morihito Okada; Akihiko Ito
    JOURNAL OF BIOMEDICAL SCIENCE BIOMED CENTRAL LTD 22 (1) 67  1021-7770 2015/08 [Refereed]
     
    Background: Pulmonary emphysema is characterized histologically by destruction of alveolar walls and enlargement of air spaces due to lung epithelial cell apoptosis. Cell adhesion molecule 1 (CADM1) is an immunoglobulin superfamily member expressed in lung epithelial cells. CADM1 generates a membrane-associated C-terminal fragment, alpha CTF, through A disintegrin-and metalloprotease-10-mediated ectodomain shedding, subsequently releasing the intracellular domain (ICD) through gamma-secretase-mediated intramembrane shedding of alpha CTF. alpha CTF localizes to mitochondria and induces apoptosis in lung epithelial cells. alpha CTF contributes to the development and progression of emphysema as a consequence of increased CADM1 ectodomain shedding. The purpose of this study was to examine whether the ICD makes a similar contribution. Results: The ICD was synthesized as a 51-amino acid peptide, and its mutant was synthesized by substituting seven amino acids and deleting two amino acids. These peptides were labeled with fluorescein isothiocyanate and were introduced into various cell lines. ICD peptide-derived fluorescence was well visualized in lung epithelial cells at the site of Mitotracker mitochondrial labeling, but was detected in locations other than mitochondria in other cell types. Mutant peptide-derived fluorescence was detected in locations other than mitochondria, even in lung epithelial cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays revealed that transduction of the ICD peptide increased the proportion of apoptotic cells 2- to 5-fold in the lung epithelial cell lines, whereas the mutant peptide did not. Abundance of the ICD was below the Western blot detection limit in emphysematous (n = 4) and control (n = 4) human lungs. However, the ICD was detected only in emphysematous lungs when it was immunoprecipitated with anti-CADM1 antibody (4/4 vs. 0/4, P = 0.029). Conclusions: As the abundance of ICD molecules was sparse but present, increased CADM1 shedding appeared to contribute to the development of emphysema by generating alpha CTF and the ICD in lung epithelial cells.
  • 上皮悪性腫瘍の肉腫様変化は骨芽細胞分化の側面を有する：骨分化マーカーを用いた検討と解析
    村上哲平; 井上敬夫; 西村俊司; 伊藤彰彦; 赤木将男
    近畿大学医学雑誌 40 (1-2) 39 - 46 2015 [Refereed]
  • Yoshioka H; Okuda T; Fujita M; Inoue T; Tasaki T; Izumoto S; Kato A
    Acta Med Kinki Univ Kinki University Medical Association 39 (2) 105 - 113 0386-6092 2014/12 [Refereed]
     
    [Abstract] Current evidence indicates that glioma stem cell-like cells (GSC_S) in humans play critical roles in the pathogenesis of carcinogenesis ofglioblastoma (GBM ). The GSC_S are known to overexpress members of the adenosine triphosphate-binding cassette (ABC) family transporters to exhibit multidrug resistance. Eradication of the GSC compartment is therefore essential to achieve a stable and long-lasting remission of GBM. To elucidate the characteristics of GSC_S in detail, we generated murine GSC lines from Sleeping Beauty transposon-mediated spontaneous GBM . Using these several cell lines, we evaluated the significance of ABC transporters in the GSC kinetics by cell morphology assays, flow cytometry, and quantitativeRT-PCR for mRNA expressions. As a consequence, we show that siRNA-mediated ABCG2 inhibition enhances the sensitivity of GSC_S to temozolomide (TMZ) and in turn reduces their spheroid-forming capability. Furthermore, we show that GSC_S treated with Abcg2-specific siRNA become sensitive to TMZ and reduce their spheroid-forming capability. In conclusion, our data suggest that targeting of drug transporters in GSC_S is a promising strategy to enhance their chemo-sensitivity for achieving a longlasting remission of GBM .
  • Takao Inoue; Man Hagiyama; Azusa Yoneshige; Takashi Kato; Eisuke Enoki; Osamu Maenishi; Takaaki Chikugo; Masatomo Kimura; Takao Satou; Akihiko Ito
    PLOS ONE PUBLIC LIBRARY SCIENCE 9 (6) e100988  1932-6203 2014/06 [Refereed]
     
    Pulmonary emphysema and type 2 diabetes mellitus (T2DM), both caused by lifestyle factors, frequently concur. Respectively, the diseases affect lung alveolar and pancreatic islet cells, which express cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member. Protease-mediated ectodomain shedding of full-length CADM1 produces C-terminal fragments (CTFs) with proapoptotic activity. In emphysematous lungs, the CADM1 shedding rate and thus the level of CTFs in alveolar cells increase. In this study, CADM1 expression in islet cells was examined by western blotting. Protein was extracted from formalin-fixed, paraffin-embedded sections of pancreata isolated from patients with T2DM (n = 12) or from patients without pancreatic disease (n = 8) at autopsy. After adjusting for the number of islet cells present in the adjacent section, we found that full-length CADM1 decreased in T2DM islets, while ectodomain shedding increased. Hemoglobin A1c levels, measured when patients were alive, correlated inversely with full-length CADM1 levels (P = 0.041) and positively with ectodomain shedding rates (P = 0.001). In immunofluorescence images of T2DM islet cells, CADM1 was detected in the cytoplasm, but not on the cell membrane. Consistently, when MIN6-m9 mouse beta cells were treated with phorbol ester and trypsin to induce shedding, CADM1 immunostaining was diffuse in the cytoplasm. When a form of CTFs was exogenously expressed in MIN6-m9 cells, it localized diffusely in the cytoplasm and increased the number of apoptotic cells. These results suggest that increased CADM1 ectodomain shedding contributes to blood glucose dysregulation in T2DM by decreasing full-length CADM1 and producing CTFs that accumulate in the cytoplasm and promote apoptosis of beta cells. Thus, this study has identified a molecular alteration shared by pulmonary emphysema and T2DM.
  • Minami A. Sakurai; Yuki Ozaki; Daisuke Okuzaki; Yoko Naito; Towa Sasakura; Ayumi Okamoto; Hiroe Tabara; Takao Inoue; Man Hagiyama; Akihiko Ito; Norikazu Yabuta; Hiroshi Nojima
    PLOS ONE PUBLIC LIBRARY SCIENCE 9 (6) e100124  1932-6203 2014/06 [Refereed]
     
    Cyclin G-associated kinase (GAK), a key player in clathrin-mediated membrane trafficking, is overexpressed in various cancer cells. Here, we report that GAK expression is positively correlated with the Gleason score in surgical specimens from prostate cancer patients. Embryonic fibroblasts from knockout mice expressing a kinase-dead (KD) form of GAK showed constitutive hyper-phosphorylation of the epidermal growth factor receptor (EGFR). In addition to the well-known EGFR inhibitors gefitinib and erlotinib, the dietary flavonoid luteolin was a potent inhibitor of the Ser/Thr kinase activity of GAK in vitro. Co-administration of luteolin and gefitinib to PC-3 cells had a greater effect on cell viability than administration of either compound alone; this decrease in viability was associated with drastic down-regulation of GAK protein expression. A comprehensive microRNA array analysis revealed increased expression of miR-630 and miR-5703 following treatment of PC-3 cells with luteolin and/or gefitinib, and exogenous overexpression of miR-630 caused growth arrest of these cells. GAK appears to be essential for cell death because co-administration of gefitinib and luteolin to EGFR-deficient U2OS osteosarcoma cells also had a greater effect on cell viability than administration of either compound alone. Taken together, these findings suggest that GAK may be a new therapeutic target for prostate cancer and osteosarcoma.
  • Takahiro Mimae; Man Hagiyama; Takao Inoue; Azusa Yoneshige; Takashi Kato; Morihito Okada; Yoshinori Murakami; Akihiko Ito
    Thorax 69 (3) 223 - 31 0040-6376 2014/03 [Refereed]
     
    RATIONALE: Alveolar epithelial cell apoptosis and protease/antiprotease imbalance based proteolysis play central roles in the pathogenesis of pulmonary emphysema but molecular mechanisms underlying these two events are not yet clearly understood. Cell adhesion molecule 1 (CADM1) is a lung epithelial cell adhesion molecule in the immunoglobulin superfamily. It generates two membrane associated C terminal fragments (CTFs), αCTF and βCTF, through protease mediated ectodomain shedding. OBJECTIVE: To explore the hypothesis that more CADM1-CTFs are generated in emphysematous lungs through enhanced ectodomain shedding, and cause increased apoptosis of alveolar epithelial cells. METHODS AND RESULTS: Western blot analyses revealed that CADM1-CTFs increased in human emphysematous lungs in association with increased ectodomain shedding. Increased apoptosis of alveolar epithelial cells in emphysematous lungs was confirmed by terminal nucleotide nick end labelling (TUNEL) assays. NCI-H441 lung epithelial cells expressing mature CADM1 but not CTFs were induced to express αCTF both endogenously (by shedding inducers phorbol ester and trypsin) and exogenously (by transfection). Cell fractionation, immunofluorescence, mitochondrial membrane potentiometric JC-1 dye labelling and TUNEL assays revealed that CADM1-αCTF was localised to mitochondria where it decreased mitochondrial membrane potential and increased cell apoptosis. A mutation in the intracytoplasmic domain abrogated all three abilities of αCTF. CONCLUSIONS: CADM1 ectodomain shedding appeared to cause alveolar cell apoptosis in emphysematous lungs by producing αCTF that accumulated in mitochondria. These data link proteolysis to apoptosis, which are two landmark events in emphysema.
  • Masaoki Ito; Man Hagiyama; Takahiro Mimae; Takao Inoue; Takashi Kato; Azusa Yoneshige; Jun Nakanishi; Tadashi Kondo; Morihito Okada; Akihiko Ito
    BREAST CANCER RESEARCH AND TREATMENT SPRINGER 144 (1) 59 - 69 0167-6806 2014/02 [Refereed]
     
    Invasive lobular carcinoma (ILC) is more frequently lymph node positive than is invasive ductal carcinoma (IDC), and ILC cell infiltration shows distinctive histological characteristics, suggesting the action of ILC-specific invasion molecules. To identify such a molecule, we used a proteomic approach in the pseudopodia of MDA-MB-231 breast cancer cells. A pseudopodial constituent was identified using excimer laser ablation, two-dimensional difference gel electrophoresis, mass spectroscopy, and immunocytofluorescence. MDA-MB-231 cells were modified to express various levels of this constituent by transient transfection and were examined for pseudopodia formation and migratory abilities using wound healing and two-chamber assays. Immunohistochemical positivity of human breast cancer cells (56 ILCs and 21 IDCs) was compared with clinicopathological variables. An actin-binding adaptor protein, alpha-parvin, was found to localize to pseudopodia and to form focal adhesions in cells not induced to extend pseudopodia. Pseudopodial length and density and migratory abilities correlated with alpha-parvin expression. Twenty-one (37.5 %) ILCs stained positive for alpha-parvin, whereas the results were negative for all 21 IDCs (P < 0.001). alpha-Parvin positivity in ILC was significantly associated with lymphatic invasion (P = 0.038) and lymph node metastasis (P = 0.003) in univariate analyses and to lymph node metastasis (P = 0.020) in multivariate analyses. alpha-Parvin, a pseudopodial constituent, was found to promote migration of breast cancer cells and to be expressed exclusively by ILC, suggesting that alpha-parvin is an ILC-specific invasion molecule that may have clinical utility as a biomarker for aggressive subsets of ILC.
  • Kumiko Takemori; Takao Inoue; Hiroyuki Ito
    LIPIDS IN HEALTH AND DISEASE BIOMED CENTRAL LTD 12 1476-511X 2013/07 
    Background: Hypoadiponectinemia in lipoatrophy may be related to worsening of hypertension in stroke-prone spontaneously hypertensive rats (SHRSP). One of the beneficial effects of candesartan (Angiotensin II Type 1 receptor blocker) for preventing hypertension may be increasing of adiponectin due to improvement of adipocyte dysfunction. In this study, we determined the effects of candesartan or adiponectin on pathophysiologic features and adipocyte dysfunction in SHRSP. Methods: Candesartan was administered to male SHRSP from 16 to 20 weeks of age (2 mg/kg/day). Adiponectin was cloned and intravenously administered to male SHRSP from 16 to 20 weeks of age. We examined biological parameters, as well as the expression and release of adipokines. Results: The SHRSP exhibited severe atrophy of visceral fat and progression of severe hypertension. The expression and release of leptin and adiponectin were impaired at 6 and 20 weeks of age. Candesartan suppressed the development of lipoatrophy and reduced the incidence of stroke at 20 weeks of age. Candesartan also enhanced the expression of adiponectin and leptin by inducing the overexpression of peroxisome proliferator activated receptor.. Circulating level of leptin was significantly higher in candesartan group than in the control group, whereas adiponectin was similar in both groups. Intravenous administration of adiponectin resulted in enhancement of adiponectin expression in adipose tissue, but no remarkable effects were found in pathophysiology in SHRSP. Conclusions: Our results indicate that candesartan protects against hypertension and adipocyte dysfunction in SHRSP. The induction of leptin expression appeared to be important factor in the inhibition of stroke lesions, whereas adiponectin was not a major regulator of blood pressure in SHRSP with genetic hypertension. Further studies are needed to elucidate the role of the renin-angiotensin system in adipose tissue dysfunction in relation to hypertensive end-organ damage.
  • M. Hagiyama; T. Inoue; T. Furuno; T. Iino; S. Itami; M. Nakanishi; H. Asada; Y. Hosokawa; A. Ito
    BRITISH JOURNAL OF DERMATOLOGY WILEY-BLACKWELL 168 (4) 771 - 778 0007-0963 2013/04 [Refereed]
     
    Background Neuroimmunological disorders are involved in the pathogenesis of atopic dermatitis (AD), partly through enhanced sensory nerve-skin mast cell interaction. Cell adhesion molecule 1 (CADM1) is a mast-cell adhesion molecule that mediates the adhesion to, and communication with, sympathetic nerves. Objectives To investigate the role of mast cell CADM1 in the pathogenesis of AD, CADM1 expression levels by comparing between lesional and nonlesional skin mast cells of an AD mouse model, which was developed by repeated application of trinitrochlorobenzene, and to examine, in cocultures, how the alterations in CADM1 detected in lesional mast cells might affect the sensory nerve-mast cell interaction. Methods AD-like lesional and nonlesional skin mast cells were collected separately by laser capture microdissection. CADM1 expression was examined by reverse transcription-polymerase chain reaction and CADM1 immunohistochemistry. In cocultures, adhesion between dorsal root ganglion (DRG) neurites and IC2 mast cells was analysed by loading a femtosecond laser-induced impulsive force on neurite-attendant IC2 cells, while cellular communication was monitored as the IC2 cellular response ([Ca2+](i) increase) after nerve-specific stimulant-induced DRG activation. Results AD-like lesional mast cells expressed three-fold more CADM1 transcripts than nonlesional cells. This was supported at the protein level, shown by immunohistochemistry. In coculture, CADM1 overexpression in IC2 cells strengthened DRG neurite-IC2 cell adhesion and doubled the population of IC2 cells responding to DRG activation. A function-blocking anti-CADM1 antibody abolished these effects in a dose-dependent manner. Conclusions Increased expression of CADM1 in mast cells appeared to be a cause of enhanced sensory nerve-mast cell interaction in a hapten-induced mouse model of AD.
  • Takao Inoue; Man Hagiyama; Eisuke Enoki; Minami A Sakurai; Akihiro Tan; Tomohiko Wakayama; Shoichi Iseki; Yoshinori Murakami; Kanji Fukuda; Chiaki Hamanishi; Akihiko Ito
    Life sciences 1 92 (1) 91 - 9 0024-3205 2013/01 [Refereed]
     
    AIMS: An immunohistochemical screen for mouse embryos showed that cell adhesion molecule 1 (CADM1), which is an immunoglobulin superfamily member, was expressed in developing bones. Here, we determined the cell types expressing CADM1 and examined its usefulness in the differential diagnosis of osteosarcoma. MAIN METHODS: Serial sections of murine developing mandibles were stained with anti-CADM1 antibody, by a coloring substrate reactive to alkaline phosphatase (ALP), a broad osteoblastic marker for preosteoblasts to osteoblasts, and by in situ hybridization for osteopontin (OPN), a marker for mature osteoblasts. CADM1 immunohistochemistry was also performed on human remodeling bones, osteosarcomas and other soft tissue tumors. KEY FINDINGS: CADM1 immunohistochemistry for the mandible revealed that morphologically identifiable osteoblasts expressed CADM1 on their plasma membranes, but neither osteocytes nor bone lining cells did. At the mandibular margin, not only OPN-positive cells but also OPN-negative, ALP-positive cells were CADM1-positive, whereas inside the mandible, OPN-positive cells were often CADM1-negative. Clear membranous staining was detected in the majority of osteosarcomas (46/57), whereas only 13% (6/46) of the other soft tissue tumors were CADM1-positive (P<0.001). SIGNIFICANCE: These results indicated that CADM1 was a novel osteoblastic adhesion molecule that is expressed transiently during osteoblastic maturation, and a useful diagnostic marker for osteosarcoma cells.
  • Kumiko Takemori; Takao Inoue; Hiroyuki Ito
    Lipids in Health and Disease 12 (1) 108  1476-511X 2013 [Refereed]
     
    Background: Hypoadiponectinemia in lipoatrophy may be related to worsening of hypertension in stroke-prone spontaneously hypertensive rats (SHRSP). One of the beneficial effects of candesartan (Angiotensin II Type 1 receptor blocker) for preventing hypertension may be increasing of adiponectin due to improvement of adipocyte dysfunction. In this study, we determined the effects of candesartan or adiponectin on pathophysiologic features and adipocyte dysfunction in SHRSP. Methods. Candesartan was administered to male SHRSP from 16 to 20 weeks of age (2 mg/kg/day). Adiponectin was cloned and intravenously administered to male SHRSP from 16 to 20 weeks of age. We examined biological parameters, as well as the expression and release of adipokines. Results: The SHRSP exhibited severe atrophy of visceral fat and progression of severe hypertension. The expression and release of leptin and adiponectin were impaired at 6 and 20 weeks of age. Candesartan suppressed the development of lipoatrophy and reduced the incidence of stroke at 20 weeks of age. Candesartan also enhanced the expression of adiponectin and leptin by inducing the overexpression of peroxisome proliferator activated receptor γ. Circulating level of leptin was significantly higher in candesartan group than in the control group, whereas adiponectin was similar in both groups. Intravenous administration of adiponectin resulted in enhancement of adiponectin expression in adipose tissue, but no remarkable effects were found in pathophysiology in SHRSP. Conclusions: Our results indicate that candesartan protects against hypertension and adipocyte dysfunction in SHRSP. The induction of leptin expression appeared to be important factor in the inhibition of stroke lesions, whereas adiponectin was not a major regulator of blood pressure in SHRSP with genetic hypertension. Further studies are needed to elucidate the role of the renin-angiotensin system in adipose tissue dysfunction in relation to hypertensive end-organ damage. © 2013 Takemori et al. licensee BioMed Central Ltd.
  • Mast Cells (version 3.0.)
    Oboki K; Hagiyama M; Inoue T; Ito A
    Encyclopedia of Life Sciences 2013 [Refereed]
  • Tatsuo Inoue; Masatoshi Kudo; Mina Komuta; Sosuke Hayaishi; Taisuke Ueda; Masahiro Takita; Satoshi Kitai; Kinuyo Hatanaka; Norihisa Yada; Satoru Hagiwara; Hobyung Chung; Toshiharu Sakurai; Kazuomi Ueshima; Michiie Sakamoto; Osamu Maenishi; Tomoko Hyodo; Masahiro Okada; Seishi Kumano; Takamichi Murakami
    Journal of Gastroenterology 47 (9) 1036 - 1047 0944-1174 2012/09 [Refereed]
     
    Background We aimed to evaluate gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)- enhanced magnetic resonance imaging (MRI) for the detection of hepatocellular carcinomas (HCCs) and dysplastic nodules (DNs) compared with dynamic multi-detector row computed tomography (MDCT), and to discriminate between HCCs and DNs. Methods Eighty-six nodules diagnosed as HCC or DNs were retrospectively investigated. Gd-EOB-DTPA-enhanced MRI and dynamic MDCT were compared with respect to their diagnostic ability for hypervascular HCCs and detection sensitivity for hypovascular tumors. The ability of hepatobiliary images of Gd-EOB-DTPA-enhanced MRI to discriminate between these noduleswas assessed.Wealso calculated the EOB enhancement ratio of the tumors. Results For hypervascular HCCs, the diagnostic ability of Gd-EOB-DTPA-enhanced MRI was significantly higher than that of MDCT for tumors less than 2 cm (p = 0.048). There was no difference in the detection of hypervascular HCCs between hepatobiliary phase images of Gd-EOBDTPA- enhanced MRI (43/45: 96%) and dynamic MDCT (40/45: 89%), whereas the detection sensitivity of hypovascular tumors by Gd-EOB-DTPA-enhanced MRI was significantly higher than that by dynamic MDCT (39/41: 95% vs. 25/41: 61%, p = 0.001). EOB enhancement ratios were decreased in parallel with the degree of differentiation in DNs and HCCs, although there was no difference between DNs and hypovascular well-differentiated HCCs. Conclusion The diagnostic ability of Gd-EOB-DTPAenhanced MRI for hypervascular HCCs less than 2 cm was significantly higher than that of MDCT. For hypovascular tumors, the detection sensitivity of hepatobiliary phase images of Gd-EOB-DTPA-enhanced MRI was significantly higher than that of dynamic Gd-EOB-DTPA-enhanced MRI and dynamic MDCT. It was difficult to distinguish between DNs and hypovascular well-differentiated HCCs based on the EOB enhancement ratio. © 2012 Springer.
  • Takahiro Mimae; Morihito Okada; Man Hagiyama; Yoshihiro Miyata; Yasuhiro Tsutani; Takao Inoue; Yoshinori Murakami; Akihiko Ito
    CLINICAL CANCER RESEARCH AMER ASSOC CANCER RESEARCH 18 (4) 945 - 955 1078-0432 2012/02 [Refereed]
     
    Purpose: Lung adenocarcinoma often manifests as tumors with mainly lepidic growth. The size of invasive foci determines a diagnosis of in situ, minimally invasive adenocarcinoma, or invasive types and suggests that some adenocarcinomas undergo malignant progression in that order. This study investigates how transcriptional aberrations in adenocarcinoma cells at the early stage define the clinical phenotypes of adenocarcinoma tumors at the advanced stage. Experimental Design: We comprehensively searched for differentially expressed genes between preinvasive and invasive cancer cells in one minimally invasive adenocarcinoma using laser capture micro-dissection and DNA microarrays. We screened expression of candidate genes in 11 minimally invasive adenocarcinomas by reverse transcriptase PCR and examined their involvement in preinvasive-to-invasive progression by transfection studies. We then immunohistochemically investigated the presence of candidate molecules in 64 samples of advanced adenocarcinoma and statistically analyzed the findings, together with clinicopathologic variables. Results: The transcription factors Notch2 and Six1 were upregulated in invasive cancer cells in all 11 minimally invasive adenocarcinomas. Exogenous Notch2 transactivated Six1 followed by Smad3, Smad4, and vimentin, and enlarged the nuclei of NCI-H441 lung epithelial cells. Immunochemical staining for the transcription factors was double positive in the invasive, but not in the lepidic growth component of a third of advanced Ads, and the disease-free survival rates were lower in such tumors. Conclusions: Paired upregulation of Notch2 and Six1 is a transcriptional aberration that contributes to preinvasive-to-invasive adenocarcinoma progression by inducing epithelial-mesenchymal transition and nuclear atypia. This aberration persisted in a considerable subset of advanced adenocarcinoma and conferred a more malignant phenotype on the subset. Clin Cancer Res; 18(4); 945-55. (C)2011 AACR.
  • Akihiko Ito; Naoki Ichiyanagi; Yuki Ikeda; Man Hagiyama; Takao Inoue; Keiko B. Kimura; Minami A. Sakurai; Kazuyuki Hamaguchi; Yoshinori Murakami
    ISLETS LANDES BIOSCIENCE 4 (1) 49 - 55 1938-2014 2012/01 
    Cell adhesion molecule-1 (CADM1) is a recently identified adhesion molecule of pancreatic islet alpha-cells that mediates nerve-alpha-cell interactions via trans-homophilic binding and serves anatomical units for the autonomic control of glucagon secretion. CADM1 also mediates attachment between adjacent alpha-cells. Since gap junctional intercellular communication (GJIC) among islet cells is essential for islet hormone secretion, we examined whether CADM1 promotes GJIC among alpha-cells and subsequently participates in glucagon secretion regulation. Dye transfer assays using alpha TC6 mouse alpha-cells, which endogenously express CADM1, supported this possibility; efficient cell-to-cell spread of gap junction-permeable dye was detected in clusters of alpha TC6 cells transfected with nonspecific, but not with CADM1-targeting, siRNA. Immunocytochemical analysis of connexin 36, a major component of the gap junction among alpha TC6 cells, revealed that it was localized exclusively to the cell membrane in CADM1-non-targeted alpha TC6 cells, but diffusely to the cytoplasm in CADM1-targeted cells. Next, we incubated CADM1-targeted and non-targeted alpha TC6 cells in a medium containing 1 mM glucose and 200 mM arginine for 30 min to induce glucagon secretion, and found that the targeted cells secreted three times more glucagon than did the non-targeted. We conducted similar experiments using pancreatic islets that were freshly isolated from wild-type and CADM1-knockout mice, and expressed glucagon secretion as ratios relative to baseline values. The increase in ratio was larger in CADM1-knockout islets than in wild-type islets. These results suggest that CADM1 may serve as a volume limiter of glucagon secretion by sustaining alpha-cell attachment necessary for efficient GJIC.
  • Akihiko Ito; Naoki Ichiyanagi; Yuki Ikeda; Man Hagiyama; Takao Inoue; Keiko B. Kimura; Minami A. Sakurai; Kazuyuki Hamaguchi; Yoshinori Murakami
    ISLETS LANDES BIOSCIENCE 4 (1) 1938-2014 2012/01 [Refereed]
     
    Cell adhesion molecule-1 (CADM1) is a recently identified adhesion molecule of pancreatic islet alpha-cells that mediates nerve-alpha-cell interactions via trans-homophilic binding and serves anatomical units for the autonomic control of glucagon secretion. CADM1 also mediates attachment between adjacent alpha-cells. Since gap junctional intercellular communication (GJIC) among islet cells is essential for islet hormone secretion, we examined whether CADM1 promotes GJIC among alpha-cells and subsequently participates in glucagon secretion regulation. Dye transfer assays using alpha TC6 mouse alpha-cells, which endogenously express CADM1, supported this possibility; efficient cell-to-cell spread of gap junction-permeable dye was detected in clusters of alpha TC6 cells transfected with nonspecific, but not with CADM1-targeting, siRNA. Immunocytochemical analysis of connexin 36, a major component of the gap junction among alpha TC6 cells, revealed that it was localized exclusively to the cell membrane in CADM1-non-targeted alpha TC6 cells, but diffusely to the cytoplasm in CADM1-targeted cells. Next, we incubated CADM1-targeted and non-targeted alpha TC6 cells in a medium containing 1 mM glucose and 200 mM arginine for 30 min to induce glucagon secretion, and found that the targeted cells secreted three times more glucagon than did the non-targeted. We conducted similar experiments using pancreatic islets that were freshly isolated from wild-type and CADM1-knockout mice, and expressed glucagon secretion as ratios relative to baseline values. The increase in ratio was larger in CADM1-knockout islets than in wild-type islets. These results suggest that CADM1 may serve as a volume limiter of glucagon secretion by sustaining alpha-cell attachment necessary for efficient GJIC.
  • Kumiko Takemori; Takao Inoue; Hiroyuki Ito
    BRAIN RESEARCH ELSEVIER SCIENCE BV 1417 137 - 145 0006-8993 2011/10 
    We investigated the mechanisms responsible for cerebral edema in stroke-prone spontaneously hypertensive rats (SHRSP), including leukocyte activation and nitric oxide (NO) generation, both in vivo and in vitro. We also investigated the effects of angiotensin II (AngII) on leukocyte function in relation to NO production. Leukocyte-endothelial cell adhesion in brain microvessels was investigated by electron tomography using ultra-high voltage electron microscopy. Electron tomography clearly showed that leukocytes had well-developed intracellular organelles and abundant microvilli that were tangled with the endothelial cells in brain microvessels. Under confocal microscopic examination, diaminofluorescein-2 (a NO indicator)-positive cells were closely localized to anti-granulocyte-positive cells. The fluorescence intensity was much stronger in SHRSP than in age-matched Wistar-Kyoto (WKY) rats, as a normotensive control. Mac-1 (leukocyte integrin, CD11b/CD18), angiotensin type 1 (AT1) receptor and inducible nitric oxide synthase (iNOS) expression was higher (or tended to be higher) in SHRSP leukocytes than in WKY leukocytes. The plasma NO metabolite content was also higher in SHRSP than in WKY rats. All of these factors were upregulated by AngII stimulation. Furthermore, NO release from leukocytes was enhanced by AngII or lipopolysaccharide through NF-kappa B activation, but was suppressed by an AT1 receptor blocker or s-methyl-isothiourea. The present study revealed that one of the causative factors for cerebral edema in SHRSP rats is the generation of NO radicals by iNOS activated via NF-kappa B in AngII-stimulated leukocytes. Thus, the pathogenesis of cerebral edema in SHRSP is likely to involve inflammatory processes mediated by AngII. (C) 2011 Elsevier B.V. All rights reserved.
  • Kumiko Takemori; Takao Inoue; Hiroyuki Ito
    BRAIN RESEARCH ELSEVIER SCIENCE BV 1417 137 - 145 0006-8993 2011/10 [Refereed]
     
    We investigated the mechanisms responsible for cerebral edema in stroke-prone spontaneously hypertensive rats (SHRSP), including leukocyte activation and nitric oxide (NO) generation, both in vivo and in vitro. We also investigated the effects of angiotensin II (AngII) on leukocyte function in relation to NO production. Leukocyte-endothelial cell adhesion in brain microvessels was investigated by electron tomography using ultra-high voltage electron microscopy. Electron tomography clearly showed that leukocytes had well-developed intracellular organelles and abundant microvilli that were tangled with the endothelial cells in brain microvessels. Under confocal microscopic examination, diaminofluorescein-2 (a NO indicator)-positive cells were closely localized to anti-granulocyte-positive cells. The fluorescence intensity was much stronger in SHRSP than in age-matched Wistar-Kyoto (WKY) rats, as a normotensive control. Mac-1 (leukocyte integrin, CD11b/CD18), angiotensin type 1 (AT1) receptor and inducible nitric oxide synthase (iNOS) expression was higher (or tended to be higher) in SHRSP leukocytes than in WKY leukocytes. The plasma NO metabolite content was also higher in SHRSP than in WKY rats. All of these factors were upregulated by AngII stimulation. Furthermore, NO release from leukocytes was enhanced by AngII or lipopolysaccharide through NF-kappa B activation, but was suppressed by an AT1 receptor blocker or s-methyl-isothiourea. The present study revealed that one of the causative factors for cerebral edema in SHRSP rats is the generation of NO radicals by iNOS activated via NF-kappa B in AngII-stimulated leukocytes. Thus, the pathogenesis of cerebral edema in SHRSP is likely to involve inflammatory processes mediated by AngII. (C) 2011 Elsevier B.V. All rights reserved.
  • Kumiko Takemori; Takashi Kimura; Norifumi Shirasaka; Takao Inoue; Koichi Masuno; Hiroyuki Ito
    LIFE SCIENCES PERGAMON-ELSEVIER SCIENCE LTD 88 (25-26) 1088 - 1094 0024-3205 2011/06 
    Aims: To determine the effects of food restriction (FR) on the expression of Sirt1 and its down-stream factors related to lipid and glucose metabolism in obese and hypertensive rats (SHRSP/IDmcr-fa), as a model of human metabolic syndrome. Main methods: Male, 10-week-old SHRSP/IDmcr-fa rats were treated with 85% FR for 2 weeks. Metabolic parameters, serum adipocytokines and distribution of serum adiponectin multimers were investigated. Sirt1 expression was determined in epididymal adipose tissue, liver and skeletal muscle. We also determined the expression of PPAR alpha, gamma and other adipocyte-related genes in epididymal adipose tissue, and glucose transporters (GLUT2 and GLUT4) in the liver and skeletal muscle. Key findings: FR improved the general conditions as well as blood chemistry of SHRSP/IDmcr-fa rats. In the epididymal adipose tissue of the FR rats, Sirt1 expression was enhanced, as was adiponectin, whereas leptin was downregulation, findings that were paralleled by the serum protein levels. Furthermore, the serum ratio of high to total adiponectin was increased in the FR group. The mRNA expression of Sirt1 was upregulated in the adipose tissue in the FR group. Sirt1 mRNA expression was downregulated, while PPAR alpha and GLUT2 expression was enhanced in the liver. No differences were found in terms of Sirt1, PPAR or GLUM expression in skeletal muscle. Significance: These results indicate that FR corrects adipokine dysfunction by activating PPAR gamma via Sirt1 in adipose tissue. Furthermore, glucose and lipid metabolism are activated by upregulation of GLUT2 via the activation of PPAR alpha in the liver. (C) 2011 Elsevier Inc. All rights reserved.
  • Kumiko Takemori; Takashi Kimura; Norifumi Shirasaka; Takao Inoue; Koichi Masuno; Hiroyuki Ito
    LIFE SCIENCES PERGAMON-ELSEVIER SCIENCE LTD 88 (25-26) 1088 - 1094 0024-3205 2011/06 [Refereed]
     
    Aims: To determine the effects of food restriction (FR) on the expression of Sirt1 and its down-stream factors related to lipid and glucose metabolism in obese and hypertensive rats (SHRSP/IDmcr-fa), as a model of human metabolic syndrome. Main methods: Male, 10-week-old SHRSP/IDmcr-fa rats were treated with 85% FR for 2 weeks. Metabolic parameters, serum adipocytokines and distribution of serum adiponectin multimers were investigated. Sirt1 expression was determined in epididymal adipose tissue, liver and skeletal muscle. We also determined the expression of PPAR alpha, gamma and other adipocyte-related genes in epididymal adipose tissue, and glucose transporters (GLUT2 and GLUT4) in the liver and skeletal muscle. Key findings: FR improved the general conditions as well as blood chemistry of SHRSP/IDmcr-fa rats. In the epididymal adipose tissue of the FR rats, Sirt1 expression was enhanced, as was adiponectin, whereas leptin was downregulation, findings that were paralleled by the serum protein levels. Furthermore, the serum ratio of high to total adiponectin was increased in the FR group. The mRNA expression of Sirt1 was upregulated in the adipose tissue in the FR group. Sirt1 mRNA expression was downregulated, while PPAR alpha and GLUT2 expression was enhanced in the liver. No differences were found in terms of Sirt1, PPAR or GLUM expression in skeletal muscle. Significance: These results indicate that FR corrects adipokine dysfunction by activating PPAR gamma via Sirt1 in adipose tissue. Furthermore, glucose and lipid metabolism are activated by upregulation of GLUT2 via the activation of PPAR alpha in the liver. (C) 2011 Elsevier Inc. All rights reserved.
  • Man Hagiyama; Tadahide Furuno; Yoichiroh Hosokawa; Takanori Iino; Takeshi Ito; Takao Inoue; Mamoru Nakanishi; Yoshinori Murakami; Akihiko Ito
    JOURNAL OF IMMUNOLOGY AMER ASSOC IMMUNOLOGISTS 186 (10) 5983 - 5992 0022-1767 2011/05 
    Close apposition of nerve and mast cells is viewed as a functional unit of neuro-immune mechanisms, and it is sustained by trans-homophilic binding of cell adhesion molecule-1 (CADM1), an Ig superfamily member. Cerebral nerve-mast cell interaction might be developmentally modulated, because the alternative splicing pattern of four (a-d) types of CADM1 transcripts drastically changed during development of the mouse cerebrum: developing cerebrums expressed CADM1b and CADM1c exclusively, while mature cerebrums expressed CADM1d additionally and predominantly. To probe how individual isoforms are involved in nerve-mast cell interaction, Neuro2a neuroblastoma cells that express CADM1c endogenously were modified to express additionally either CADM1b (Neuro2a-CADM1b) or CADM1d (Neuro2a-CADM1d), and they were cocultured with mouse bone marrow-derived mast cells (BMMCs) and BMMC-derived cell line IC-2 cells, both of which expressed CADM1c. BMMCs were found to adhere to Neuro2a-CADM1d neurites more firmly than to Neuro2a-CADM1b neurites when the adhesive strengths were estimated from the femtosecond laser-induced impulsive forces minimally required for detaching BMMCs. GFP-tagging and cross-linking experiments revealed that the firmer adhesion site consisted of an assembly of CADM1d cis-homodimers. When Neuro2a cells were specifically activated by histamine, intracellular Ca(2+) concentration was increased in 63 and 38% of CADM1c-expressing IC-2 cells that attached to the CADM1d assembly site and elsewhere, respectively. These results indicate that CADM1d is a specific neuronal isoform that enhances nerve-mast cell interaction, and they suggest that nerve-mast cell interaction may be reinforced as the brain grows mature because CADM1d becomes predominant. The Journal of Immunology, 2011, 186: 5983-5992.
  • Man Hagiyama; Tadahide Furuno; Yoichiroh Hosokawa; Takanori Iino; Takeshi Ito; Takao Inoue; Mamoru Nakanishi; Yoshinori Murakami; Akihiko Ito
    JOURNAL OF IMMUNOLOGY AMER ASSOC IMMUNOLOGISTS 186 (10) 5983 - 5992 0022-1767 2011/05 [Refereed]
     
    Close apposition of nerve and mast cells is viewed as a functional unit of neuro-immune mechanisms, and it is sustained by trans-homophilic binding of cell adhesion molecule-1 (CADM1), an Ig superfamily member. Cerebral nerve-mast cell interaction might be developmentally modulated, because the alternative splicing pattern of four (a-d) types of CADM1 transcripts drastically changed during development of the mouse cerebrum: developing cerebrums expressed CADM1b and CADM1c exclusively, while mature cerebrums expressed CADM1d additionally and predominantly. To probe how individual isoforms are involved in nerve-mast cell interaction, Neuro2a neuroblastoma cells that express CADM1c endogenously were modified to express additionally either CADM1b (Neuro2a-CADM1b) or CADM1d (Neuro2a-CADM1d), and they were cocultured with mouse bone marrow-derived mast cells (BMMCs) and BMMC-derived cell line IC-2 cells, both of which expressed CADM1c. BMMCs were found to adhere to Neuro2a-CADM1d neurites more firmly than to Neuro2a-CADM1b neurites when the adhesive strengths were estimated from the femtosecond laser-induced impulsive forces minimally required for detaching BMMCs. GFP-tagging and cross-linking experiments revealed that the firmer adhesion site consisted of an assembly of CADM1d cis-homodimers. When Neuro2a cells were specifically activated by histamine, intracellular Ca(2+) concentration was increased in 63 and 38% of CADM1c-expressing IC-2 cells that attached to the CADM1d assembly site and elsewhere, respectively. These results indicate that CADM1d is a specific neuronal isoform that enhances nerve-mast cell interaction, and they suggest that nerve-mast cell interaction may be reinforced as the brain grows mature because CADM1d becomes predominant. The Journal of Immunology, 2011, 186: 5983-5992.
  • Ito Akihiko; Hagiyama Man; Inoue Takao
    Acta Med Kinki Univ Kinki University Medical Association 35 (2) 77 - 85 0386-6092 2010/12 
    There are a plethora of important biological events that are regulated by cellular interactions among heterotypic cell types. Recent biological achievements have identified many molecules that control heterotypic cell-cell interactions. Although our understandings on these events have lately made remarkable advances at molecular levels, physical aspects of cellular adhesion have not been fully examined yet. Cell Adhesion Molecule-1, CADM1, is a member of the immunoglobulin superfamily, and has multiple functions involved in tumor suppression, synaptogenesis, and spermatogenesis. CADM1 plays a key role as not only simple glue among cells, but also a conductor or promoter of heterotypic intercellular communications. Interestingly, it is now being revealed that the efficiency of CADM1-mediated intercellular communication is closely correlated with the kinetic strength of CADM1-mediated intercellular adhesion, by applying the latest laser technique.
  • Masuno Koichi; Takemori Kumiko; Inoue Takao; Yamamoto Kazuo; Ito Hiroyuki
    Acta Med Kinki Univ The Kinki University Medical Association 35 (1) 33 - 40 0386-6092 2010/06 
    SHRSP/IDmcr-fa/fa (SHRSP/fatty) rats are a new animal model that have the potential to develop severe hypertension, obesity, hyperlipidemia, and hyperglycemia, followed by arteriopathy and glomerulopathy in the kidneys. Thus, SHRSP/fatty rats seem to be the most severe animal model of human metabolic syndrome. Using this unique animal model, we investigated how HMG-CoA reductase inhibitor (statin), a well-known drug for hypercholesterolemia, and angiotensin II receptor blocker (ARB), a widely-used anti-hypertensive drug, affected the pathophysiology related to metabolic syndrome. The statin increased the mRNA expression of adiponectin and leptin, decreased the expression of TNF-alpha gene, and increased the secretion of high molecular weight (HMW) adiponectin, without a lipid-lowering effect. ARB increased both total adiponectin and HMW adiponectin, independent of blood pressure lowering. Histologically, the incidence of renal lesions, such as angionecrosis and glomerulosclerosis, was decreased in both treated groups. Except for well-known pharmacological effects of these drugs, the additional medicinal benefit of the statin depended on its anti-inflammatory effect, and that of ARB probably depended on its direct effect on adipocytes. It was considered that the increase of HMW adiponectin was enhanced by both pathways, and this may be a common factor of the protective effects of both drugs on pathophysiological damages in SHRSP/fatty rats.
  • Takao Inoue; Kumiko Takemori; Kazuo Yamamoto; Hiroyuki Ito
    LIFE SCIENCES PERGAMON-ELSEVIER SCIENCE LTD 86 (9-10) 344 - 350 0024-3205 2010/02 [Refereed]
     
    Aims: Wistar-Kyoto rats (HA-WKY) kept in the author's laboratory showed higher levels of serum adiponectin (approximately 4-fold) compared with Wistar-Kyoto/Izm rats (WKY/Izm), a WKY standard strain, at 6 weeks old. In a preliminary study, HA-WKY but not WKY/Izm showed hyperinsulinemia and severe hypercholesterolemia when fed a high-fat diet. This study analyzed the differences between HA-WKY and WKY/Izm to investigate the causes of hyperadiponectinemia. Main methods: Six-week-old male HA-WKY and WKY/Izm were used for an analysis of adiponectin-related factors. Key findings: The main intermediates in the adiponectin signaling pathway, AMP-activated protein kinase and peroxisome proliferator-activated receptor alpha, were activated at similar levels in liver and skeletal muscle between HA-WKY and WKY/Izm, although HA-WKY had not only higher adiponectin concentrations but also extremely high levels of high-molecular weight (HMW, polymer) adiponectin, which is the active form. The main difference between HA-WKY and WKY/Izm was the existence of adiponectin, mainly middle-molecular weight (MMW, hexamer) and HMW adiponectin multimers, in skeletal muscle extracts from WKY/Izm but not HA-WKY. Skeletal muscle in WKY/Izm expressed much higher amounts of T-cadherin, a receptor for MMW and HMW adiponectin multimers, than that in HA-WKY. In contrast, there was no significant difference in the expression level of adiponectin receptor 2 for trimer, MMW, and HMW adiponectin multimers. Significance: The results suggested that the existence of adiponectin in WKY/Izm skeletal muscle was due to the binding of serum adiponectin. The difference in serum adiponectin concentrations between HA-WKY and WKY/Izm could come from the difference in adiponectin binding to skeletal muscle. (C) 2010 Elsevier Inc. All rights reserved.
  • Rika Haniaguichi; Kumiko Takemori; Takao Inoue; Kouichi Masuno; Hiroyuki Ito
    CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY BLACKWELL PUBLISHING 35 (10) 1151 - 1155 0305-1870 2008/10 
    1. The aim of the present study was to investigate the effects of short-term treatment with an AT, receptor blocker (ARB) on amelioration of hypertensive end-organ damage in stroke-prone spontaneously hypertensive rats (SHRSP). 2. Male SHRSP were divided into two groups: (i) an ARB-treated group, and (ii) a control group. Candesartan (1 mg/kg per day),,vas administered orally from 6 to 11 weeks of age. At 20 weeks of age, plasma renin activity (PRA), angiotensin II concentrations, angiotensin-converting enzyme (ACE) activity and hydroperoxide content were measured. Expression of intercellular adhesion molecule (ICAM)-1, renin, AT(1) and AT(2) receptors was investigated by reverse transcription-polymerase chain reaction. 3. Blood pressure in the ARB group was slightly lower at 7, K. 11, 13-15 and 18 weeks of age, but no significant difference in blood pressure was found between the ARB and control groups at 20 weeks of age. All rats in the control group had cerebral oedema, whereas no lesions were found in the ARB group. In the ARB group, PRA, All and hydroperoxide content were lower than in the control group. In the ARB-treated group, lower ICAM-1 expression was found in the cerebral cortex and slightly, albeit not significantly, lower expression of renin was found in the kidney. In contrast, AT, receptor expression in the cerebrum and kidney, was higher in the ARB group compared with the control group. 4. These results indicate that short-term treatment of SHRSP,with ARB at a young age is effective in preventing cerebral oedema after maturation. Such beneficial effects of ARB may be due, in part, to decreased blood pressure and is likely mainly due to inhibition of total circulating and local renin-angiotensin systems.
  • Rika Haniaguichi; Kumiko Takemori; Takao Inoue; Kouichi Masuno; Hiroyuki Ito
    CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY BLACKWELL PUBLISHING 35 (10) 1151 - 1155 0305-1870 2008/10 [Refereed]
     
    1. The aim of the present study was to investigate the effects of short-term treatment with an AT, receptor blocker (ARB) on amelioration of hypertensive end-organ damage in stroke-prone spontaneously hypertensive rats (SHRSP). 2. Male SHRSP were divided into two groups: (i) an ARB-treated group, and (ii) a control group. Candesartan (1 mg/kg per day),,vas administered orally from 6 to 11 weeks of age. At 20 weeks of age, plasma renin activity (PRA), angiotensin II concentrations, angiotensin-converting enzyme (ACE) activity and hydroperoxide content were measured. Expression of intercellular adhesion molecule (ICAM)-1, renin, AT(1) and AT(2) receptors was investigated by reverse transcription-polymerase chain reaction. 3. Blood pressure in the ARB group was slightly lower at 7, K. 11, 13-15 and 18 weeks of age, but no significant difference in blood pressure was found between the ARB and control groups at 20 weeks of age. All rats in the control group had cerebral oedema, whereas no lesions were found in the ARB group. In the ARB group, PRA, All and hydroperoxide content were lower than in the control group. In the ARB-treated group, lower ICAM-1 expression was found in the cerebral cortex and slightly, albeit not significantly, lower expression of renin was found in the kidney. In contrast, AT, receptor expression in the cerebrum and kidney, was higher in the ARB group compared with the control group. 4. These results indicate that short-term treatment of SHRSP,with ARB at a young age is effective in preventing cerebral oedema after maturation. Such beneficial effects of ARB may be due, in part, to decreased blood pressure and is likely mainly due to inhibition of total circulating and local renin-angiotensin systems.
  • ZY Zhang; T Inoue; M Forgac; S Wilkens
    FEBS LETTERS ELSEVIER SCIENCE BV 580 (8) 2006 - 2010 0014-5793 2006/04 [Refereed]
     
    Vacuolar ATPases (VIVO-ATPases) function in proton translocation across lipid membranes of subcellular compartments. We have used antibody labeling and electron microscopy to define the position of subunit C in the vacuolar ATPase from yeast. The data show that subunit C is binding at the interface of the ATPase and proton channel, opposite from another stalk density previously identified as subunit H [Wilkens S., Inoue T., and Forgac M. (2004) Three-dimensional structure of the vacuolar ATPase - Localization of subunit H by difference imaging and chemical cross-linking. J. Biol. Chem. 279, 41942-41949]. A picture of the vacuolar ATPase stalk domain is emerging in which subunits C and H are positioned to play a role in reversible enzyme dissociation and activity silencing. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • YR Wang; T Inoue; M Forgac
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 280 (49) 40481 - 40488 0021-9258 2005/12 [Refereed]
     
    Bafilomycin and concanamycin are potent and highly specific inhibitors of the vacuolar (H+)-ATPases (V-ATPases), typically inhibiting at nanomolar concentrations. Previous studies have shown that subunit c of the integral V-0 domain participates in bafilomycin binding, and that this site resembles the oligomycin binding site of the F-ATPase ( Bowman, B. J., and Bowman, E. J. ( 2002) J. Biol. Chem. 277, 3965-3972). Because mutations in F-ATPase subunit a also confer resistance to oligomycin, we investigated whether the a subunit of the V-ATPase might participate in binding bafilomycin. Twenty-eight subunit a mutations were constructed just N-terminal to the critical Arg(735) residue in transmembrane 7 required for proton transport, a region similar to that shown to participate in oligomycin binding by the F-ATPase. The mutants appeared to assemble normally and all but two showed normal growth at pH 7.5, whereas all but three had at least 25% of wild-type levels of proton transport and ATPase activity. Of the functional mutants, three displayed Ki values for bafilomycin significantly different from wild-type (0.22 +/- 0.03 nM). These included E721K ( Ki 0.38 +/- 0.03 nM), L724A ( 0.40 +/- 0.02 nM), and N725F ( 0.54 +/- 0.06 nM). Only the N725F mutation displayed a Ki for concanamycin ( 0.84 +/- 0.04 nM) that was slightly higher than wild-type ( 0.60 +/- 0.07 nM). These results suggest that subunit a of V-ATPase participates along with subunit c in binding bafilomycin.
  • T Inoue; YR Wang; K Jefferies; J Qi; A Hinton; M Forgac
    JOURNAL OF BIOENERGETICS AND BIOMEMBRANES SPRINGER/PLENUM PUBLISHERS 37 (6) 393 - 398 0145-479X 2005/12 [Refereed]
     
    The V-ATPases are ATP-dependent proton pumps present in both intracellular compartments and the plasma membrane. They function in such processes as membrane traffic, protein degradation. renal acidification, bone resorption and tumor metastasis. The V-ATPases are composed of a peripheral V, domain responsible for ATP hydrolysis and an integral V-0 domain that carries Out proton transport. Our recent work has focused on structural analysis of the V-ATPase complex using both cysteine-mediated cross-linking and electron microscopy. For cross-linking, Studies, unique cysteine residues were introduced into structurally defined sites within the B and C Subunits and used as points of attachment for the photoactivated cross-linking reagent MBP. Disulfide mediated cross-linking has also been used to define helical contact Surfaces between subunits Within the integral V-0 domain. With respect to regulation of V-ATPase activity, we have investigated the role that intracellular environment, luminal pH and a unique domain of the catalytic A Subunit Play in controlling reversible dissociation in vivo.
  • T Inoue; M Forgac
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 280 (30) 27896 - 27903 0021-9258 2005/07 [Refereed]
     
    The vacuolar (H+)- ATPases (V-ATPases) are multisubunit complexes responsible for ATP-dependent proton transport across both intracellular and plasma membranes. The V-ATPases are composed of a peripheral domain (V-1) that hydrolyzes ATP and an integral domain (V-0) that conducts protons. Dissociation of V1 and V0 is an important mechanism of controlling V-ATPase activity in vivo. The crystal structure of subunit C of the V-ATPase reveals two globular domains connected by a flexible linker (Drory, O., Frolow, F., and Nelson, N. ( 2004) EMBO Rep. 5, 1 - 5). Subunit C is unique in being released from both V1 and V0 upon in vivo dissociation. To localize subunit C within the V-ATPase complex, unique cysteine residues were introduced into 25 structurally defined sites within the yeast C subunit and used as sites of attachment of the photoactivated sulfhydryl reagent 4-(N-maleimido) benzophenone (MBP). Analysis of photocross-linked products by Western blot reveals that subunit E ( part of V1) is in close proximity to both the head domain ( residues 166 - 263) and foot domain ( residues 1 - 151 and 287 - 392) of subunit C. By contrast, subunit G ( also part of V1) shows cross-linking to only the head domain whereas subunit a ( part of V0) shows cross-linking to only the foot domain. The localization of subunit C to the interface of the V1 and V0 domains is consistent with a role for this subunit in controlling assembly of the V-ATPase complex.
  • RC Shen; T Inoue; M Forgac; JA Porco
    JOURNAL OF ORGANIC CHEMISTRY AMER CHEMICAL SOC 70 (9) 3686 - 3692 0022-3263 2005/04 [Refereed]
     
    The lobatamides and related salicylate enamide natural products are potent mammalian V-ATPase inhibitors. To probe details of binding of the lobatamides to mammalian V-ATPase, three photoactivatable analogues bearing benzophenone photoaffinity labels have been prepared. The analogues were designed on the basis of a simplified acyclic analogue 2. Late-stage installation of the enamide side chain and tandem deallylation/amidation were employed in synthetic routes to these derivatives. Simplified analogue 2 showed strong inhibition against bovine clathrin-coated vesicle V-ATPase (10 nM). Analogues 3-5 were also evaluated for inhibition of bovine V-ATPase in order to select a suitable candidate for future photoaffinity labeling studies.
  • S Wilkens; T Inoue; M Forgac
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 279 (40) 41942 - 41949 0021-9258 2004/10 [Refereed]
     
    The structure of the proton-pumping vacuolar ATPase (V-ATPase) from bovine brain clathrin coated vesicles was analyzed by electron microscopy and single molecule image analysis. A three-dimensional structural model of the complex was calculated by the angular reconstitution method at a resolution of 27 Angstrom. Overall, the appearance of the V-0 and V-1 domains in the three-dimensional model of the intact bovine V-ATPase resembles the models of the isolated bovine V0 and yeast V1 domains determined previously (Wilkens, S., and Forgac, M. ( 2001) J. Biol. Chem. 276, 44064 - 44068; Zhang, Z., Charsky, C., Kane, P. M., and Wilkens, S. ( 2003) J. Biol. Chem. 278, 47299 - 47306). To determine the binding position of subunit H in the V-ATPase, electron microscopy and cysteine-mediated photochemical cross-linking were used. Difference maps calculated from projection images of intact bovine V-ATPase and a V-ATPase preparation in which the two H subunit isoforms were removed by treatment with cystine revealed less protein density at the bottom of the V-1 in the subunit H-depleted enzyme, suggesting that subunit H isoforms bind at the interface of the V-1 and V-0 domains. A comparison of three-dimensional models calculated for intact and subunit H-depleted enzyme indicated that at least one of the subunit H isoforms, although poorly resolved in the three-dimensional electron density, binds near the putative N-terminal domain of the a subunit of the V-0. For photochemical cross-linking, unique cysteine residues were introduced into the yeast V-ATPase B subunit at sites that were localized based on molecular modeling using the crystal structure of the mitochondrial F-1 domain. Cross-linking was performed using the photoactivatable sulfhydryl reagent 4-(N-maleimido) benzophenone. Cross-linking to subunit H was observed from two sites on subunit B (E494 and T501) predicted to be located on the outer surface of the subunit closest to the membrane. Results from both electron microscopy and cross-linking analysis thus place subunit H near the interface of the V-1 and V-0 domains and suggest a close structural similarity between the V-ATPases of yeast and mammals.
  • YR Wang; T Inoue; M Forgac
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 279 (43) 44628 - 44638 0021-9258 2004/10 [Refereed]
     
    The vacuolar (H+)-ATPase ( or V-ATPase) is an ATP-dependent proton pump which couples the energy released upon ATP hydrolysis to rotational movement of a ring of proteolipid subunits (c, c', and c") relative to the integral subunit a. The proteolipid subunits each contain a single buried acidic residue that is essential for proton transport, with this residue located in TM4 of subunits c and c' and TM2 of subunit c". Subunit c" contains an additional buried acidic residue in TM4 that is not required for proton transport. The buried acidic residues of the proteolipid subunits are believed to interact with an essential arginine residue (Arg(735)) in TM7 of subunit a during proton translocation. We have previously shown that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM4 of subunit c' bordered by Glu(145) and Leu(147) ( Kawasaki-Nishi et al. ( 2003) J. Biol. Chem. 278, 41908 - 41913). We have now analyzed interaction of subunits a and c" using disulfide-mediated cross-linking. The results indicate that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM2 of subunit c" centered on Ile(105), with the essential glutamic acid residue (Glu(108)) located near the opposite border of this face compared with TM4 of subunit c'. By contrast, TM4 of subunit c" does not form strong cross-links with TM7 of subunit a, suggesting that these transmembrane segments are not normally in close proximity. These results are discussed in terms of a model involving rotation of interacting helices in subunit a and the proteolipid subunits relative to each other.
  • T Sugio; T Inoue; Y Kitano; F Takeuchi; K Kamimura
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING SOC BIOSCIENCE BIOENGINEERING JAPAN 98 (2) 85 - 91 1389-1723 2004/08 [Refereed]
     
    A mesophilic, mixotrophic iron-oxidizing bacterium strain OKM-9 uses ferrous iron as a sole source of energy and L-glutamate as a sole source of cellular carbon. Uptake of L-glutamate into OKM-9 cells is absolutely dependent on ferrous iron oxidation. Thus, the Fe2+-dependent L-glutamate uptake system of strain OKM-9 is crucial for the bacterium to grow mixotrophically in iron medium with L-glutamate. The relationship between iron oxidation and L-glutamate transport activities was studied. Iron oxidase containing cytochrome a was purified 9-fold from the plasma membrane of OKM-9. A purified iron oxidase showed one rust-colored band following disc gel electrophoresis after incubation with Fe2+. The Fe2+-dependent L-glutamate transport system was also purified 14.5-fold from the plasma membrane using the same purification steps as for iron oxidase. Fe2+-dependent L-glutamate and L-Cysteine uptake activities of OKM-9 were 0.36 and 0.24 nmol/mg/min, respectively, when a concentration of 18 mM of these amino acids was used as a substrate. Both uptake activities were completely inhibited by potassium cyanide (KCN), suggesting that cytochrome a in the iron oxidase is involved in the transport process. The iron-oxidizing activity of strain OKM-9 was activated 1.7-fold by 80 mM L-glutamate. In contrast, the activity was noncompetitively inhibited by L-cysteine. The Michaelis constant of iron oxidase for Fe2+ was 12.6 mM and the inhibition constant for L-cysteine was 41.6 mM. A marked inhibition of iron oxidase by 50 MM L-cysteine was completely reversed by the addition of 60 MM L-glutamate. The results suggest the possibility that iron oxidase has a binding site for L-cysteine and the cysteine first bound to the iron oxidase was replaced by the added L-glutamate.
  • T Inoue; S Wilkens; M Forgac
    JOURNAL OF BIOENERGETICS AND BIOMEMBRANES KLUWER ACADEMIC/PLENUM PUBL 35 (4) 291 - 299 0145-479X 2003/08 [Refereed]
     
    The vacuolar (H+)-ATPases (or V-ATPases) are ATP-dependent proton pumps that function both to acidify intracellular compartments and to transport protons across the plasma membrane. Acidification of intracellular compartments is important for such processes as receptor-mediated endocytosis, intracellular trafficking, protein processing, and coupled transport. Plasma membrane V-ATPases function in renal acidification, bone resorption, pH homeostasis, and, possibly, tumor metastasis. This review will focus on work from our laboratories on the V-ATPases from mammalian clathrin-coated vesicles and from yeast. The V-ATPases are composed of two domains. The peripheral V-1 domain has a molecular mass of 640 kDa and is composed of eight different subunits (subunits A-H) of molecular mass 70-13 kDa. The integral V-0 domain, which has a molecular mass of 260 kDa, is composed of five different subunits (subunits a, d, c, c', and c") of molecular mass 100-17 kDa. The V-1 domain is responsible for ATP hydrolysis whereas the V-0 domain is responsible for proton transport. Using a variety of techniques, including cysteine-mediated crosslinking and electron microscopy, we have defined both the overall shape of the V-ATPase and the V-0 domain as well as the location of various subunits within the complex. We have employed site-directed and random mutagenesis to identify subunits and residues involved in nucleotide binding and hydrolysis, proton translocation, and the coupling of these two processes. We have also investigated the mechanism of regulation of the V-ATPase by reversible dissociation and the role of different subunits in this process.
  • T Inoue; K Kamimura; T Sugio
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY TAYLOR & FRANCIS LTD 66 (10) 2030 - 2035 0916-8451 2002/10 [Refereed]
     
    Strain OKM-9 is a mesophilic, mixotrophic iron-oxidizing bacterium that absolutely requires ferrous iron as its energy source and L-amino acids (including L-glutamate) as carbon sources for growth. The properties of the L-glutamate transport system were studied with OKM-9 resting cells, plasma membranes, and actively reconstituted proteoliposomes. L-Glutamate uptake into resting cells was totally dependent on ferrous iron that was added to the reaction mixture. Potassium cyanide, an iron oxidase inhibitor, completely inhibited the activity at 1 mm. The optimum pH for Fe2+-dependent uptake activity of L-glutamate was 3.5-4.0. Uptake activity was dependent on the concentration of the L-glutamate. The K-m and V-max for L-glutamate were 0.4 mM and 11.3 nmol . min(-1) . mg(-1), respectively. L-Aspartate, D-aspartate, D-glutamate, and L-cysteine strongly inhibited L-glutamate uptake. L-Aspartate competitively inhibited the activity, and the apparent K-i for this amino acid was 75.9 muM. 2,4-Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, gramicidin D, valinomycin, and monensin did not inhibit Fe2+-dependent L-glutamate uptake. The OKM-9 plasma membranes had approximately 40% of the iron-oxidizing activity of the resting cells and approximately 85% of the Fe2+-dependent uptake activity. The glutamate transport system was solubilized from the membranes with 1% n-octyl-beta-D-glucopyranoside and reconstituted into a lecithin liposome. The L-glutamate transport activity of the reconstituted proteoliposomes was 8-fold than that of the resting cells. The Fe2+-dependent L-glutamate uptake observed here seems to explain the mixotrophic nature of this strain, which absolutely requires Fe2+ oxidation when using amino acids as carbon sources.
  • T Inoue; K Kamimura; T Sugio
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY TAYLOR & FRANCIS LTD 64 (10) 2059 - 2067 0916-8451 2000/10 [Refereed]
     
    An iron-oxidizing bacterium strain, OKM-9, isolated from mud obtained from the bottom of a pond, Minamikata Ohike, in Okayama prefecture, Japan, grew well in an FeSO4. 7H(2)O (3%)-medium (pH 2.5) with 0.03% yeast extract. However, the strain could not grow either in an FeSO4. 7H(2)O (3%)-medium without yeast extract or in a yeast extract (0.03%)medium (pH 2.5) without Fe2+. The strain did not use elemental sulfur as an energy source and did not have the activity to fix carbon dioxide. Strain OKM-9 could grow in an FeSO4. 7H(2)O (3%)-medium with twenty different L-amino acids instead of yeast extract. Incorporation of [U-C-14] glutamic acid into the cells was dependent on the energy produced by the oxidation of Fe2+, Strain OKM-9 did not grow heterotrophically using amino acids and hexoses as a sole energy and carbon source. The results that strain OKM-9 absolutely required ferrous iron (Fe2+) as a sole energy source and yeast extract or L-amino acids as a carbon source for growth strongly suggest that the strain is a mixotrophic iron-oxidizing bacterium. Strain OKM-9 was a Gram-negative and rod-shaped bacterium (0.4-0.6x1.6-2.2 mum) and the mean G + C content of the DNA of the bacterium was 59.6 mol%. The optimum temperature and pH for growth were 30 degreesC and 2.1, respectively. However, the strain could not grow at temperatures above 45 degreesC. Iron-oxidizing activities of strain OKM-9 measured with intact cells and the plasma membrane were 14.3 and 5.7 mul O-2 uptake/mg protein/min, respectively. The pyridine ferrohemochromes prepared from the plasma membrane of this strain showed absorption peaks characteristic of alpha -bands of heme a and b, but not heme c, at 587 and 557 nm, respectively. The results suggest that the cytochromes composing an iron-oxidation system of strain OKM-9 are different from those of the well-known mesophilic iron-oxidizing bacteria Thiobacillus ferrooxidans and LeptospiviElum ferrooxidans.
  • Tada Y; Inoue T
    J Appl Microbiol 88 (1) 154 - 160 2000/01 [Refereed]

Conference Activities & Talks

  • 病態依存的に発育低下がみられる脳卒中発症ラットSHRSPのヒラメ筋に対する組織学的解析及び遺伝子発現解析  [Not invited]
    井上敬夫; 萩山満; 木村竜一朗; 水口信行; 前西修; 筑後孝章; 木村雅友; 伊藤龍生; 佐藤隆夫; 伊藤彰彦
    日本筋学会 第4回学術集会  2018/08
  • プロバイオティクスの整腸作用による潰瘍性大腸炎発症の予防効果  [Not invited]
    倉本 康平; 豊田 啓士; 金田 雅大; 池上 侑希; 佐藤 隆夫; 井上 敬夫; 伊藤 龍生
    第72回 日本栄養･食糧学会  2018/05
  • 正常血圧ラット、高血圧ラット及び脳卒中発症ラットにおける心筋発現遺伝子の比較解析  [Not invited]
    井上敬夫; 竹森久美子; 水口信行; 萩山満; 木村竜一朗; 前西修; 筑後孝章; 木村雅友; 伊藤龍生; 佐藤隆夫; 伊藤彰彦
    第72回 日本栄養･食糧学会  2018/05
  • 脳卒中ラットSHRSPにおいて発育低下がみられる骨格筋の遺伝子発現解析  [Not invited]
    井上敬夫; 萩山満; 水口信行; 木村竜一朗; 前西修; 筑後孝章; 木村雅友; 伊藤龍生; 佐藤隆夫; 伊藤彰彦
    日本農芸化学会 2018年度大会  2018/03
  • 鍼治療としてのフェムト秒レーザー照射  [Not invited]
    大田美香; 細川陽一郎; 波多野直哉; 菅野亜紀; 井上敬夫; 鈴木高史; 伊藤彰彦; 高岡 裕
    2017年度 生命科学系学会合同年次大会  2017/12
  • アディポネクチン高分子多量体の心筋結合量は心筋細胞肥大と負の相関関係にある  [Not invited]
    井上敬夫; 竹森久美子; 水口信行; 木村雅友; 筑後孝章; 萩山満; 米重あづさ; 森樹史; 前西修; 米谷俊; 伊藤龍生; 佐藤隆夫; 伊藤彰彦
    第71回 日本栄養・食糧学会大会  2017/05
  • 心筋結合型アディポネクチンは血圧ではなく、心肥大と相関する  [Not invited]
    井上敬夫竹森久美子; 水口信行; 木村雅友; 筑後孝章; 萩山満; 米重あづさ; 森樹史; 前西修; 米谷俊; 伊藤龍生; 佐藤隆夫; 伊藤彰彦
    日本農芸学会 2017年度大会  2017/03
  • Heart-bound adiponectin, not serum adiponectin, correlates with cardiac hypertrophy in stroke-prone spontaneously hypertensive rats.  [Not invited]
    Inoue, T; Takemori, K; Mizuguchi, N; Kimura, M; Chikugo, T; Hagiyama, M; Yoneshige; A. Mori, T; Kometani, T; Itoh, T; Satou, T; Ito, A
    The 26th Scientific Meeting of the International Society of Hypertension.  2016/09
  • 接着分子cell adhesion molecule 1の細胞内断片による肺胞上皮アポトーシス誘導：肺気腫発症への関与  [Not invited]
    萩山 満; 米重あづさ; 井上敬夫; 伊藤彰彦
    第105回 日本病理学会  2016/05
  • アクチン結合性アダプター蛋白a-parvinは偽足突起に局在し、小葉乳癌のリンパ節転移に関与する  [Not invited]
    萩山満; 井上敬夫; 米重あづさ; 伊藤彰彦
    第104回 日本病理学会総会  2015/05
  • 特発性間質性肺炎における肺上皮接着分子CADM1の発現異常  [Not invited]
    米重あづさ; 井上敬夫; 萩山満; 伊藤彰彦
    第104回 日本病理学会総会  2015/05
  • 2型糖尿病患者の膵島における接着分子CADM1は細胞外ドメインの切断が亢進している  [Not invited]
    井上敬夫; 萩山満; 米重あづさ; 榎木英介; 前西修; 筑後孝章; 木村雅友; 佐藤隆夫; 伊藤彰彦
    第104回 日本病理学会総会  2015/04
  • 骨芽細胞の新規接着分子CADM1は骨肉腫の診断マーカーとして有用である。  [Not invited]
    井上敬夫; 萩山満; 榎木英介; 伊藤彰彦
    第103回 日本病理学会総会  2014/04
  • 重症高血圧における脂肪萎縮と治療による影響  [Not invited]
    竹森 久美子; 井上 敬夫; 伊藤 浩行
    第63回日本体質医学会総会  2013/10  久留米市  第63回日本体質医学会総会
     
    目的: 低adiponectin血症をともなう脂肪萎縮は重症高血圧ラット（SHRSP）の高血圧を増悪させる可能性が考えられる。そこで本研究ではrenin-angiotensin系(RAS)阻害剤（ARB）ならびにadiponectin 投与がSHRSPに及ぼす影響を検討した。方法: 生後16週齢の雄性SHRSPにARB(Candesartan,2 mg/kg/day, po)およびラットadiponectin組換え体(100 μg/day、iv)を投与した。結果: ARBを投与したSHRSPでは血圧低下とともに脂肪萎縮の改善と脳卒中発症の抑制が見られ、脂肪組織におけるPPARγ発現亢進とleptin分泌量の増加が認められた。しかし、adiponectin投与による影響は認められなかった。結論: ARBによる高血圧続発症の抑制機序の一つには、脂肪組織におけるRASを介するleptinの発現亢進が考えられる。
  • 新規骨芽細胞接着分子cell adhesion molecule 1は骨芽細胞分化過程において一過性に発現する  [Not invited]
    井上敬夫; 萩山満; 榎木英介; 伊藤彰彦
    第101回日本病理学会総会  2012/04
  • メタボリック症候発症における骨格筋アディポネクチン受容体発現の重要性  [Not invited]
    井上 敬夫; 竹森 久美子; 伊藤 浩行; 伊藤 彰彦; 東京大学医科学研究所人癌病因遺伝子分野
    第100回日本病理学会総会  2011/04  横浜  第100回日本病理学会総会
  • 新規メタボリックシンドロームモデルラット (SHRSP/fatty) における発育期食餌制限による糖・脂質代謝の改善  [Not invited]
    竹森 久美子; 井上 敬夫; 増野 功一; 伊藤 浩行
    第99回日本病理学会総会  2010/04  東京都  第99回日本病理学会総会
     
    【目的】新規メタボリックシンドロームモデルであるSHRSP/IDmcr-fa（SHRSP/fatty）を用い、食餌制限による糖・脂質代謝への影響と長寿遺伝子Sirt１の発現を検討した。【方法】生後10週齢の雄性SHRSP/fattyに対し85％の食餌制限を2週間行った。血液生化学的検索、血清adipokine（leptin, adiponectin）、精巣上体脂肪組織におけるadipokine関連因子の発現、肝臓・骨格筋におけるGlucose transporter（GLUT）の発現を検索した。さらにこれらの組織におけるSirt1の発現を解析した。【結果】食餌制限を行ったSHRSP/fattyでは、血清アラキドン酸とLeukotrien B4の上昇が観察されるとともに、血清lepitinの減少とadiponectin（特に高分子体）の増加が認められた。精巣上体脂肪組織においてはPPARγとadiponectinの発現亢進、leptinの発現低下が認められ、血清レベルと良く一致した変動を示した。Sitr１の発現は脂肪組織で亢進、肝臓で低下していた。肝臓では脂肪変性の抑制とグリコーゲンの貯蔵異常が改善されているとともに、PPARα
  • 骨格筋における新規アディポネクチン作用機構の解析  [Not invited]
    井上 敬夫; 竹森 久美子; 山本 和夫; 増野功一; 木村孝志; 伊藤 浩行
    日本農芸化学会2010年度大会  2010/03  東京  日本農芸化学会2010年度大会
  • Impaired Binding of Adiponectin to Skeletal Muscle in Hyperadiponectinemia Wistar-Kyoto rats  [Not invited]
    井上 敬夫; 竹森 久美子; 山本 和夫; 木村 孝志; 増野 功一; 伊藤 浩行
    第45回高血圧関連疾患モデル学会学術総会  2009/09  東京  第45回高血圧関連疾患モデル学会学術総会
  • 高adiponectin血症WKY(HA-WKY)における高脂肪食負荷の影響  [Not invited]
    木村 孝志; 竹森 久美子; 井上 敬夫; 山本 和夫; 増野 功一; 伊藤 浩行
    第45回高血圧関連疾患モデル学会学術総会  2009/09  東京  第45回高血圧関連疾患モデル学会学術総会
     
    [目的]これまでの一連の研究から、我々が系統維持しているWKYは、他の系統のラットに比べて、6週齢時において血中adiponectinレベルが高いということが明らかにされてきた(HA-WKY)。Adiponectinは受容体(AdipoR1、AdipoR2)を介して、AMPKを活性化し、骨格筋における糖の取り込みを促進、肝臓における糖新生の抑制、骨格筋および肝臓での脂肪酸燃焼を促進することで、抗動脈硬化や、抗糖尿病作用があることが知られている。そこで、本実験ではHA-WKYに高脂肪食を負荷し、生活習慣病発症耐性に対する影響をWKY/Izmと比較検討した。 [方法]生後6週齢の雄性HA-WKYおよび、WKY/Izmに高脂肪食(A13270)を4週間負荷し、血液生化学的検索を行うとともに、血清adipokine(adiponectin、leptin)量をELISAで測定した。また、脂肪組織におけるadipokine、adiponectin関連因子の発現および骨格筋・肝臓におけるGlucose transporter(GLUT)の発現をrealtime PCRで測定した。 [結果]高脂肪食を負荷することにより、TP・ALP・T-CHO・インスリン値が
  • メタボリックシンドロームの予防・治療に関する基礎的研究―スタチン・ARB併用投与の影響  [Not invited]
    増野 功一; 竹森 久美子; 井上 敬夫; 山本 和夫; 伊藤 浩行
    第45回高血圧関連疾患モデル学会学術総会  2009/09  東京  第45回高血圧関連疾患モデル学会学術総会
     
    【目的】SHRSP/IDmcr-fa/faは加齢に従って高血圧，肥満，高脂血症及び糖尿病を発症することが知られており，最終的には腎機能不全または脳卒中によって状態悪化及び死亡する．以前，我々はメタボリックシンドロームの治療薬であるスタチン又はアンジオテンシン受容体阻害剤（ARB）を投与したところ，いずれの薬剤においても病態の改善が認められた．今回，両剤を併用投与し，処置の影響を検討した． 【方法】13週齢の雄性ラットを2群に分け，対照群，プラバスタチン（100 mg/kg/day）とオルメサルタン（0.1 mg/kg/day）の併用投与群を設定した．投与開始から5週間，薬剤投与および飲水による食塩負荷（1%食塩水）を実施した．血圧・体重の変動を観察するとともに，生後18週齢で安楽殺し，病態の解析を行った． 【結果】体重は対照群で顕著な減少が認められたものの，血圧は対照・投与群間で有意の差は認められなかった．病理組織検査及び血液化学検査の結果，対照群では腎機能
  • 高アディポネクチン血症を示すWistar-Kyotoラット(HA-WKY)におけるアディポネクチン作用機構の検討  [Not invited]
    井上 敬夫; 竹森 久美子; 山本 和夫; 木村 孝志; 増野 功一; 伊藤 浩行
    第59回日本体質医学会総会  2009/07  東京  第59回日本体質医学会総会
  • 肥満進行期のエネルギー制限によるメタボリックシンドローム発症予防効果  [Not invited]
    竹森 久美子; 木村 孝志; 井上 敬夫; 増野 功一; 伊藤 浩行
    第59回日本体質医学会総会  2009/07  東京  第59回日本体質医学会総会
     
    【目的】重症の高血圧・高脂血症・インスリン抵抗性を併せ持つSHRSP/IDmcr-fa/faを用い、若年期の生活習慣の改善がメタボリックシンドロームの発生に及ぼす影響を、脂肪細胞の機能変化を中心に検討した。【方法】生後10週齢の雄性SHRSP/IDmcr-fa/faに85%食餌制限を2週間行った（ER群:n=5）。【結果】ER群の脂肪組織ではcontrolに比べてleptinの発現低下とadiponectinの発現上昇が見られるとともに、血清leptinの低下とadiponectinの増加が認められた。脂肪組織内でのadiponectinの多量体分布に変化は認められなかったものの、血清では総量の増加とともに、高分子adiponectinの比率が増加していた。【結論】以上のことから肥満進行期のエネルギー制限は、adipokineの分泌異常を改善することでメタボリックシンドロームの発生を抑制する効果があることが示唆された。
  • 肥満進行期の食餌制限によるメタボリックシンドロームの発症予防  [Not invited]
    竹森 久美子; 木村 孝志; 井上 敬夫; 増野 功一; 伊藤 浩行
    第63回日本栄養・食糧学会大会  2009/05  長崎市  第63回日本栄養・食糧学会大会
     
    （目的）肥満をともなう高脂血症はメタボリックシンドロームの中心的要因であり、その是正は続発症予防における必須の課題である。本研究では重症の高血圧・高脂血症・インスリン抵抗性を併せ持つSHRSP/IDmcr-fa/faを用い、肥満進行期の生活習慣の改善がメタボリックシンドロームの発生にどのような影響を及ぼすかを、脂肪細胞の機能変化を中心に検討した。 （方法）生後10週齢の雄性SHRSP/IDmcr-fa/faに85%食餌制限を2週間行った（ER群:n=5）。血液生化学的検索を行うととともに、血清adipokineをELISAで測定した。また脂肪組織におけるadipokine関連因子の発現および脳・肝・大腿筋におけるGlucose transporter（GLUT）の発現をrealtime PCRで検索した。さらに血清および脂肪組織内におけるadiponectin 多量体の解析をWestern blotで行った。 （結果）ER群ではcontrolに比べインスリン・AST・TG・BUNが低値を、T-CHO・TPは高値を示しており、肝機能と腎機能は維持されていた。またこれらのラットの肝臓では、
  • メタボリックシンドロームの病態に関する実験的研究 II.モデル動物を用いた治療法の検討  [Not invited]
    竹森 久美子; 増野 功一; 井上 敬夫; 伊藤 浩行
    第98回日本病理学会総会  2009/05  京都市  第98回日本病理学会総会
     
    【目的】高血圧・肥満ラット（SHRSP/IDmcr-fa/fa）を用いて、病態検索を行うとともに、薬剤や食餌制限の影響を検討した。【方法】雄のSHRSP/IDmcr-fa/fa（生後10-13週）を用いて、５週間のAT1 receptor blocker (ARB、0.1mg/kg) 投与、あるいは２週間の85%食餌制限(ER群)を行い、adipokineの発現や凝固能および線溶活性におよぼす影響を検索した。【結果】ARB投与群では、血清leptin、adiponectin濃度に変化は認められなかったが、血漿PAI-1活性の低下、脂肪組織PAI-1 mRNAの発現亢進が認められた。T-CHOとTGの低下が認められたが、血糖値には影響は認められなかった。一方、ＥＲ群では、血中のleptinの減少、adiponectinの上昇が認められ、脂肪組織内の発現変動とよく一致していた。PAI-1や血液凝固能への影響はみられなかったが、線溶活性の亢進が認められた。【結論】以上の結果は、本モデルがメタボリックシンドロームの治療法の開発に有用であることを示すと共に、臓器障害の発生には凝固・線溶系の異常も重要
  • メタボリックシンドロームの病態に関する実験的研究 I. 高血圧・肥満を有する新規モデルラットの有用性  [Not invited]
    増野 功一; 竹森 久美子; 井上 敬夫; 伊藤 浩行
    第98回日本病理学会総会  2009/05  京都市  第98回日本病理学会総会
     
    【目的】高血圧モデルであるSHRSPに肥満・高脂血症モデルであるZucker Fatty ratの原因遺伝子（leptin受容体の変異）を導入した新規モデルラットSHRSP/IDmcr- fa/fa（以下SP/ZF）を用い，原因遺伝子を持たないSHRSP/IDmcr-+/+（以下Lean）との比較において，メタボリックシンドロームモデルとしての有用性を検討した．【方法】13週齢の雄ラットに食塩を負荷し，5週間後，病理検査，生化学検査とともに血中のadipokineの検索を行った．【結果】SP/ZFではクレアチニン，トリグリセリドおよび血糖値が高値であった．adiponectinの総量は高値を示したが，高分子体はほとんど認められなかった．腎臓の糸球体・血管病変が高度にみられ，膵ラ氏島の増大が見られた。一方、Leanは2週目までに半数以上が脳病変を発症して死亡し，高度の脂肪萎縮が認められた。【考察】SP/ZFでは高血圧，肥満，高脂血症および糖尿病といった複数の因子が合併しており，メタボリックシンドロームの病態モデルとして有用と考えら
  • Some poperties of a hyperadiponectinemia model rat  [Not invited]
    井上 敬夫; 竹森 久美子; 木村孝志; 増野功一; 山本 和夫; 伊藤 浩行
    日本農芸化学会2009年度大会  2009/03  福岡  日本農芸化学会2009年度大会
  • Qualitative Abnormality of Adiponectin in Stroke-prone Spontaneously Hypertensive Rats  [Not invited]
    井上 敬夫; 竹森 久美子; 木村孝志; 増野功一; 伊藤 浩行
    第44回高血圧関連疾患モデル学会  2008/11  島根  第44回高血圧関連疾患モデル学会
  • 幼若期の生活改善によるメタボリックシンドロームの発症予防～食餌制限を中心に～  [Not invited]
    木村 孝志; 竹森 久美子; 増野 功一; 井上 敬夫; 伊藤 浩行
    第44回高血圧関連疾患モデル学会  2008/11  島根  第44回高血圧関連疾患モデル学会
     
    生活習慣の悪化は心筋梗塞・脳血管障害・糖尿病発症のリスクを高めることが指摘されている。特に肥満をともなう高脂血症はメタボリックシンドロームの中心的要因であり、その是正は続発症予防における必須の課題である。そこでメタボリックシンドロームのモデル動物としてSHRSP/IDmcr-fa/faを用い、幼若期の生活習慣の改善がメタボリックシンドロームの発生にどのような影響を及ぼすかを、糖・脂質代謝および脂肪細胞の機能を指標として検討した。生後10週齢の雄性SHRSP/IDmcr-fa/faを2群に分け、うち1群（ER群:n=5）に2週間の食餌制限を行った。ER群の体重減少が実験開始時の20％未満となるように設定した。血液生化学的検索を行うととともに、血清adipokine（leptin, adiponectin, PAI-1活性）の測定をELISAで行った。また精巣上体および後腹膜脂肪組織における脂肪細胞関連因子（PPARγ, leptin, adiponectin, PAI-1）の発現および脳・肝・大腿筋におけるGlucose transporter（GLUT）の発現をrealtime PCRで検索し
  • メタボリックシンドロームの予防・治療に関する基礎的研究―スタチン又はARB投与による病態の改善  [Not invited]
    増野 功一; 竹森 久美子; 井上 敬夫; 山本 和夫; 伊藤 浩行
    第44回高血圧関連疾患モデル学会  2008/11  島根  第44回高血圧関連疾患モデル学会
     
    SHRSP/IDmcr-fa/faは加齢に伴って高血圧，肥満，高脂血症及び糖尿病を発症することが知られており，最終的には腎機能不全または脳卒中によって死亡する．そこでメタボリックシンドロームの病態を解明し、かつ治療に対する基礎資料を得るために、メタボリックシンドロームの治療に有効と考えられているスタチン又はアンジオテンシン受容体阻害剤（ARB）を投与し、病態に及ぼす影響を検討した。13週齢の雄性ラットを対照群，プラバスタチン投与群（100 mg/kg/day），オルメサルタン投与群（0.1 mg/kg/day）に分け、5週間，薬剤投与および飲水による食塩負荷（1%食塩水）を実施した。１８週齢で屠殺し、血液生化学検査とともに血中および後腹膜脂肪組織におけるadipokineの比較検討を行った。体重は対照群で顕著な減少が認められたものの，血圧には３群間で有意差が認められなかった。病理組織検査及び血液化学検査の結果，両群で腎機能（糖尿病性・高血圧性腎病変及び血中クレアチニン
  • The composition of adiponectin multimers in stroke-prone spontaneously hypertensive rats  [Not invited]
    井上 敬夫; 竹森 久美子; 増野 功一; 伊藤 浩行
    13th International SHR Symposium  2008/06  Prague, Czech Republic  13th International SHR Symposium
  • 高血圧性臓器障害の進展・増悪に対する脂肪組織中renin-angioteinsin系の関与  [Not invited]
    竹森 久美子; 増野功一; 井上 敬夫; 伊藤 浩行
    第97回日本病理学会総会  2008/05  金沢  第97回日本病理学会総会
     
    （目的）重症高血圧およびその続発症発生に対する内臓脂肪組織のadipokine産生系とrenin-angiotensin系（RAS）の関与を検討した。（方法）6、12、20週齢の雄性WKYおよびSHRSPを用い、精巣上体脂肪細胞の粒子面積、leptin、adiponectin、PPARγの発現、血清leptin、adiponectin濃度、脂肪細胞におけるAT1、AT2 receptorの発現とACE活性を比較した。（結果）重症高血圧を発症したSHRSPでは内臓脂肪の著しい萎縮が認められ、脳卒中発症例では血清leptin、adiponectin濃度と脂肪細胞におけるleptinの発現が低値を示したが、adiponectinやPPARγの発現には差が認められなかった。脂肪細胞のAT1, AT2 receptorの発現は両系統とも6週齢で最も高く、加齢にともない低下し脳卒中発症例で最も低値を示した。また6週齢における脂肪組織内ACE活性はWKYに比べてSHRSPで有意に高値を示した。（結論）以上の結果は、幼弱期における脂肪組織のRASの亢進が成熟後の脂肪萎縮をもたらし、高血圧性臓器障害発生に大きく関与していることを示唆し
  • Multimer formation of adiponectin in stroke-prone spontaneously hypertensive rats  [Not invited]
    井上 敬夫; 竹森 久美子; 濱口梨花; 増野功一; 伊藤 浩行
    日本農芸化学会2008年度大会  2008/03  名古屋  日本農芸化学会2008年度大会
  • Adipokine Secretion Is Regulated By Local RAS In SHRSP  [Not invited]
    竹森 久美子; 増野 功一; 井上 敬夫; 伊藤 浩行
    13th International SHR Symposium  2008  13th International SHR Symposium
     
    高血圧性臓器障害発生に対する脂肪細胞におけるadipokine産生系と局所renin-angiotensin系の関与を検討した。WKYに比べてSHRSPでは著しい内臓脂肪の萎縮が見られた。さらにSHRSPではadipokineレベルが低値であるとともに、幼弱期におけるrenin-angiotensin系(RAS)の亢進が認められた。また発育早期のSHRSPに短期間ARBを投与し、RASを抑制すると脳病変発症と脂肪萎縮が抑制され、leptinの分泌に改善がみとめられた。以上のことから重症高血圧の際のadipokine産生には脂肪細胞内のRASが関係していることが推測される。
  • Possible involvement of the renin-angiotensin system in adipose tissue in the aggravation of end-organ damage in severe hypertension  [Not invited]
    竹森 久美子; 増野 功一; 井上 敬夫; 伊藤 浩行
    Hypertension 2008(22nd Scientific Meeting of the International Society of Hypertension)  2008  ベルリン  Hypertension 2008(22nd Scientific Meeting of the International Society of Hypertension)
     
    重症高血圧およびその続発症発生に対する脂肪組織におけるadipokine産生系とrenin-angiotensin系の関与を検討し、脂肪萎縮は高血圧性臓器障害発生の増悪に関与すること、また、発育早期にrenin-angiotenisn系を阻害することにより、成熟後の脂肪萎縮とともに臓器発生抑制が抑制されることを明らかにした。以上の結果は脂肪組織におけるadipokine産生系とrenin-angiotensin系が密接に関連していることを示している。
  • Possible Involvement of Adipocyte in Development of Hypertensive End-organ Damage  [Not invited]
    竹森 久美子; 増野 功一; 井上 敬夫; 伊藤 浩行
    第４３回高血圧関連疾患モデル学会学術総会  2007/09  大阪市  第４３回高血圧関連疾患モデル学会学術総会
     
    脂肪細胞はメタボリックシンドロームの中心的役割を演じており、高血圧発症にも深く関わっていると考えられている。一方SHRSPでは高血圧の進行とともに脂肪組織の著しい萎縮が生じる。このような変化は血圧上昇がmildなSHRには見られないことから、肥満のみならず脂肪の萎縮もまた血管の機能や病変の発生に関与する可能性が考えられる。本研究では SHRSPにおける重症高血圧およびその続発症発生に対する内臓脂肪組織の関与を検討した。動物には生後6，12，20週齢の雄性WKYとSHRSPを用いた。脂肪細胞の形態学的相違を調べるために後腹膜脂肪細胞を分離・培養し、粒子面積を計測した。またadipokineの産生を比較するために血清adiponectinおよびleptin濃度を測定するとともに精巣上体脂肪組織におけるadiponectin, leptin, PPARγの発現をrealtime PCRで比較した。SHRSPでは高血圧の進行とともに内臓脂肪、特に後腹膜や腸間膜の脂肪組織の著しい萎縮が認められた。脂肪細胞面積を比較したところ、WKY
  • 重症高血圧の発症に及ぼす脂肪萎縮の影響  [Not invited]
    増野 功一; 竹森 久美子; 井上 敬夫; 伊藤 浩行
    第96回日本病理学会総会  2007/03  大阪市  第96回日本病理学会総会
     
    重症の高血圧を示すSHRSPでは内臓脂肪の萎縮が見られるが、よりマイルドな高血圧を示すSHRでは見られないことから、高血圧の重症化に脂肪細胞の機能異常が関与していることが推測される。そこで、SHRSPにおけるadipokineの加齢に伴う変動を正常血圧のWKYと比較すると共に、脂肪除去あるいは脂肪移植の影響を検討した。SHRSPでもWKYでも血中のadiponectin量は加齢と共に減少し、かつ両者の間に有意の差は認められなかった。血中leptin量はWKYでは上昇が認められたが、SHRSPでは大きな変動は見られず、生後20週ではWKYに比べ明らかに低値であった。SHRSPより大網を除去したところ血圧上昇の促進が見られ、leptinに減少傾向が認められたが、adiponectinは不変であった。成熟WKYの内臓脂肪を背部皮下に移植したSHRSPでは血圧への影響は認められず、leptin量も不変であったが、adiponectinには増加傾向が認められた。以上のことからSHRSPにおける高血圧の重症化には脂肪細胞の萎縮に基づくadipokineの分泌異常

MISC

Research Grants & Projects

  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2022/04 -2025/03 
    Author : 前西 修; 井上 敬夫
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2022/04 -2025/03 
    Author : 井上 敬夫; 金澤 佑治
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2021/04 -2025/03 
    Author : 佐藤 隆夫; 井上 敬夫; 伊藤 龍生
     
    本研究の最終的な目標は，緑茶カテキン（エピガロカテキンガレート、EGCG）飲料を用いて脳外傷後に出現する神経幹細胞の神経再生及び修復に関わる因子を網羅的に調べ，高次脳機能障害発症抑制関連因子を明らかにすることである。脳外傷後の神経再生・修復を行う上で神経幹細胞の生存維持は重要である。脳外傷ラットモデルでのEGCG水溶液摂取（EGCG飲水）群と非摂取（非EGCG飲水）群との行動学的な形質の差異を明らかにし，行動学的表現型と神経幹細胞の生存，分化との関係について解析を行う。 本年度は、申請者らはpneumatic control injury deviceを用いて10週齢ラットオスを用いて緑茶カテキン（エピガロカテキンガレート、EGCG）投与群、Contorol群に分け、脳に外傷を与えたラット脳外傷モデルを作製した。Control群とEGCG群での脳外傷部位で1，3，7，30日後の経時的に神経再生及び神経新生を確認するために切片を作製し、nestin抗体及びDCX抗、GFAP抗体を使用し、免疫染色を行った。損傷周囲部分では損傷後1日後からnestin陽性の神経幹細胞と考えられるnestin陽性細胞が増加し、3日後に最大数を示し、7日後まで確認された。EGCG群がどの日数においてもControl群に比較し、有意な増加が認められた。また、新生神経細胞と考えられるDCX陽性の細胞が外傷7日後より増加し30日で最大数であった。さらにnestin陽性細胞の変化と同様にDCX陽性細胞においてもControl群に比較し、EGCG群で有意な増加が見られた。gliosisの指標であるGFAP陽性細胞はEGCG群に比較し、Control群で有意に増加し、EGCG群でgliosisが抑制されていることが示された。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    Date (from‐to) : 2020/04 -2023/03 
    Author : 木村 雅友; 井上 敬夫
     
    超高齢化社会に突入したことにより、「平均寿命」が延びてきたが、介護を必要とせず生活ができる「健康寿命」との間に10年前後の開きがあることが社会的問題となっている。健康寿命の延伸は高齢者自身の生活の質(QOL)の向上だけでなく、介護を担う家族のQOL向上にも繋がる。この問題を解決するための手段の1つは、老化により骨格筋の衰えが生じる「サルコペニア」に対する治療であるが、現在のところ栄養療法と運動療法しか存在しない。骨格筋の衰えは、運動が制限されるだけでなく、骨格筋より分泌されるマイオカインの分泌異常も引き起こし、身体の恒常性を維持できない状態にさせる。しかしながら、運動がままならない状況にあって期待通りの効果を上げることができない場合も多い。本研究ではこの問題に対して、治療薬によるサルコペニアのより積極的な治療法の確立を目指すため、骨格筋の萎縮に関与する標的分子に対する治療法の開発を行う。標的分子がリガンドと結合する細胞外ドメインを認識する抗体を得るために、細胞外ドメイン配列を持つペプチドを免疫したマウスより作製したハイブリドーマを得たため、本研究の目的に合致した抗体をスクリーニングしている。また、標的分子に特異的に作用する化合物の探索をハイスル―プットで行うために、標的分子を発現する筋芽細胞を用いてリガンドが作用することによって動くシグナルカスケード分子の中から、検出に使用できる分子を選び出した。更にハイスル―プットで検出するための方法として免疫染色とELISAを組み合わせた細胞ベースのELISA法を用いるために、各種条件を検討した。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2019/04 -2022/03 
    Author : MAENISHI Osamu
     
    Spontaneously hypertensive rats (SHR) and stroke-prone spontaneously hypertensive rats (SHRSP) show different cardiac hypertrophy compared to normotensive rats, Wistar-Kyoto rats (WKY). Comprehensive gene expression analysis for left ventricular between WKY, SHR, and SHRSP clarified the upregulated or downregulated genes depending on cardiac hypertrophy. Furthermore, the gene which shows cardiac hypertrophy dependency at protein expression level was selected. The selected protein was known to be involved in cardiac hypertrophy. However, the known mechanism for the protein was not able to explain the relationship with cardiac hypertrophy. New interaction protein was identified by immunoprecipitation using the antibody against the selected protein and clarified the possibility of involvement in energy production.
  • 脳卒中発症ラット骨格筋由来の新規遺伝子群を標的とした病態改善への有効性の検討
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2018/04 -2021/03 
    Author : 井上 敬夫
     
    近年、骨格筋は姿勢維持や動作を生み出すといった運動器としての機能だけでなく、マイオカインとよばれる一群のタンパク質を分泌することによって全身の恒常性維持に重要な役割を果たしていることが明らかになってきている。我々は高血圧ラットにおいて病態依存的に骨格筋の発育低下がみられることを確認している。この骨格筋の発育低下がマイオカインの分泌異常を引き起こすことによって全身の恒常性の維持に影響を与え、疾患の増悪に繋がっているものと考えている。本研究では、正常血圧ラットと高血圧ラットの骨格筋を用いて網羅的な遺伝子発現解析を行い、両者を比較解析した結果、見出された新規の遺伝子群に関してその機能や疾患との関わりについて解析を行っている。これらの遺伝子群と疾患との関わりを明らかにすることによって、骨格筋の機能維持や疾患増悪を予防あるいは改善する治療方法の開発への結びつけることを目標とする。H30年度は骨格筋発育低下に関与すると考えられる新規遺伝子群のクローニングを行い、骨格筋細胞に導入を行った。しかしながら、当初は骨格筋細胞への遺伝子導入が困難だったため、他の細胞株（CHO-K1：H31年度使用予定の細胞株）に遺伝子を導入して細胞外への分泌を含めた性質の検討を行っている。現在は骨格筋細胞への導入が可能になったため、最適導入条件を検討しながら、遺伝子の骨格筋細胞の形態や分化に関する機能解析を順次行っている。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2017/04 -2020/03 
    Author : Itoh Tatsuki
     
    From our current study, we propose that the exercise in the early following traumatic brain injury increases the number of nestin-positive cells-including NSCs- with the potential to differentiate into neurons and glia around the area damaged by TBI, and enhanced proliferation activity of neural stem cells (NSCs) around damaged area after traumatic brain injury(TBI). Therefore, the exercise therapy (rehabilitation) in the early phase following TBI is very important for recuperation of induced cerebral dysfunction following TBI.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2015/04 -2018/03 
    Author : SATOU Takao
     
    A major component of green tea, a widely consumed beverage, is EGCG, which has strong antioxidant properties. In this study, we investigated the effects of EGCG on cerebral function and morphology following TBI. 24-month-old rats male Wistar rats that had access to normal drinking water, or water containing 0.1% (w/v) EGCG ad libitum, received TBI with a pneumatic controlled injury device for 4weeks. Immunohistochemistry and lipid peroxidation studies revealed that at 1, 3 and 7 days post-TBI, the number of 8-OHdG-, 4-HNE- and ssDNA-positive cells, and the levels of MDA around the damaged area after TBI, significantly decreased in the EGCG treatment group compared with the water group. In addition, there was a significant increase in the number of surviving neuronal cells and an improvement in cerebral dysfunction after TBI in the EGCG treatment group compared with the water group. In summary, consumption of green tea may be an effective therapy for TBIaged patients.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2015/04 -2018/03 
    Author : INOUE Takao
     
    Researches on adiponectin have been focused on the relationship between adiponectin in blood and diseases. These researches show that adiponectin in blood and diseases have inverse correlation because adiponectin is anti-metabolic syndrome hormone. Recently, we discovered organ-bound adiponectin as a novel adiponectin. We assessed the relationship between heart-bound adiponectin and hypertension or cardiac hypertrophy. The result showed that heart-bound adiponectin, but not serum adiponectin, inversely correlated with cardiac hypertrophy. Further analysis of gene expression in heart showed that there are genes might be involved in hypertension and cardiac hypertrophy via heart-bound adiponectin.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2014/04 -2017/03 
    Author : ITOH Tatsuki
     
    We investigate the effect of exercise on omorphology and cerebral function following traumatic brain injury(TBI) in aged (two years old) rats. Aged wistar rats received TBI by a pneumatic controled injury device and were randomly divided into two groups :1)non-exercise group and 2) exercise group.The exercise groupran on a treadmill.Immunohistochemical and behavioral studied were permormed follwowing TBI. The number of apoptosis cells early after TBI was significantly rediced in the aged exercise group. Furthermore, most apoptosis cells in the non-exercise group indicated neuronal cells. However, in the exercise group, few apoptosis cells shown. In addition, there was a significant inmprovement in cerebral dysfunction after TBI in the exercise group. These resuls indicate that aged person exercise follwoing TBI inhibits neuronal apoptotic neuronal cell death, which results in an improvement of cerebral dysfunction.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2012/04 -2015/03 
    Author : SATOU Takao; ITOH Tatsuki; INOUE Takao
     
    A major component of green tea is (-)-epigallocatechin gallate (EGCG), which has strong antioxidant properties. we investigated the effect of EGCG on neural stem cell (NSC) proliferation following traumatic brain injury (TBI). Male Wistar rats that had access to normal drinking water, or water containing 0.1% (w/v) EGCG, ad libitum. Immunohistochemistry revealed that the number of nestin-positive cells after TBI in the EGCG group increased significantly. The number of single-stranded DNA (ssDNA)-positive cells after TBI significantly decreased in the EGCG group. Almost all ssDNA-positive cells in the water group co-localized with NeuN and nestin-staining. Spheres could only be isolated in the water group at 3 days.In the EGCG group, spheres could be isolated at all days following TBI. Spheres differentiated into neurons and glia. These results indicate that EGCG inhibits degradation of NSCs, which have the potential to differentiate into neurons and glia following TBI.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
    Date (from‐to) : 2012/04 -2014/03 
    Author : INOUE Takao
     
    Pseudopodia (actin-rich protrusion) are considered to be important for cancer metastasis. Tumor cells were cultured on a porous membrane to form pseudopodia in membrane pores. An excimer laser, which was used for LASIK, was employed for removing cell body on a membrane to selectively isolate pseudopodia in membrane pores. The proteins extracted from pseudopodia were analyzed and identified. Of those proteins, alpha-Parvin, which highly expressed in pseudopodia, was characterized. The results showed that the protein was involved in pseudopodial length and density, and cell migration ability.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2011 -2013 
    Author : ITOH Tatsuki; SATOU Takao; INOUE Takao
     
    we investigate the effect of exercise on morphology and cerebral function following TBI in rats. Wistar rats received TBI by a pneumatic controlled injury device and were randomly divided into two groups: 1) non-exercise group and 2) exercise group. The exercise group ran on a treadmill. Immunohistochemical and behavioral studies were performed following TBI. The number of ssDNA-positive cells early after TBI was significantly reduced in the exercise group. Furthermore, most ssDNA-positive cells in the non-exercise group co-localized with neuronal cells. However, in the exercise group, few ssDNA-positive cells co-localized. In addition, there was a significant increase in neuronal cell number and improvement in cerebral dysfunction after TBI in the exercise group. These results indicate that exercise following TBI inhibits neuronal apoptotic cell death, which results in an improvement of cerebral dysfunction.

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