Okayama University Medical School Acta Medica Okayama 0386-300X 69 3 2015 Eosinophil Cationic Protein Shows Survival Effect on H9c2 Cardiac Myoblast Cells with Enhanced Phosphorylation of ERK and Akt/GSK-3β under Oxidative Stress 145 153 EN Hiroko Ishii Shigeshi Kamikawa Satoshi Hirohata Akifumi Mizutani Koji Abe Masaharu Seno Toshitaka Oohashi Yoshifumi Ninomiya Original Article 10.18926/AMO/53521 Eosinophil cationic protein (ECP) is well known as a cationic protein contained in the basic granules of activated eosinophils. Recent studies have reported that ECP exhibits novel activities on various types of cells, including rat neonatal cardiomyocytes. Here we evaluated the effects of ECP on rat cardiac myoblast H9c2 cells. Our results showed that ECP enhanced the survival of the cells, in part by promoting the ERK and Akt/GSK-3β signaling pathways. ECP attenuated the cytotoxic effects of H2O2 on H9c2 cells as well as the production of reactive oxygen species, the number of apoptotic cells and caspase 3/7 activity in the cells. In conclusion, ECP activated the ERK and Akt/GSK-3β pathways, resulting in anti-oxidative effects on H9c2 cells that attenuated apoptosis. No potential conflict of interest relevant to this article was reported.

Wiley-Blackwell Acta Medica Okayama 1347-9032 103 10 2012 Tumor growth inhibitory effect of ADAMTS1 is accompanied by the inhibition of tumor angiogenesis 1889 1897 EN Masanari Obika Hiroko Ogawa Katsuyuki Takahashi Jiayi Li Omer Faruk Hatipoglu Mehmet Zeynel Cilek Toru Miyoshi Junko Inagaki Takashi Ohtsuki Shozo Kusachi Yoshifumi Ninomiya Satoshi Hirohata Angiogenesis plays an important role in tumor progression. Several reports have demonstrated that a disintegrin and metalloproteinase with thrombospondin motifs1 (ADAMTS1) inhibited angiogenesis via multiple mechanisms. The aim of this study was to investigate the effect of ADAMTS1 on endothelial cells in vitro and on tumor growth with regard to angiogenesis in vivo. We examined the effects of the transfection of ADAMTS1 using two constructs, full-length ADAMTS1 (full ADAMTS1) and catalytic domain-deleted ADAMTS1 (delta ADAMTS1). Transfection of both the full ADAMTS1 and delta ADAMTS1 gene constructs demonstrated the secretion of tagged-ADAMTS1 protein into the conditioned medium, so we examined the effects of ADAMTS1-containing conditioned medium on endothelial cells. Both types of conditioned media inhibited endothelial tube formation, and this effect was completely abolished after immunoprecipitation of the secreted protein from the medium. Both types of conditioned media also inhibited endothelial cell migration and proliferation. We then examined the impact of ADAMTS1 on endothelial cell apoptosis. Both conditioned media increased the number of Annexin V-positive endothelial cells and caspase-3 activity and this effect was attenuated when z-vad was added. These results indicated that ADAMTS1 induced endothelial cell apoptosis. We next examined the effects of ADAMTS1 gene transfer into tumor-bearing mice. Both full ADAMTS1 and delta ADAMTS1 significantly inhibited the subcutaneous tumor growth. Collectively, our results demonstrated that ADAMTS1 gene transfer inhibited angiogenesis in vitro and in vivo, likely as a result of the induction of endothelial cell apoptosis by ADAMTS1 that occurs independent of the protease activity. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 124 1 2012 ランビエ絞輪周囲のECMによるdiffusion barrier形成と跳躍伝導における役割 1 4 EN Yoko Bekku Yoshifumi Ninomiya Toshitaka Oohashi No potential conflict of interest relevant to this article was reported.

Elsevier Science BV Acta Medica Okayama 0945-053X 30 4 2011 Drosophila type XV/XVIII collagen, Mp, is involved in Wingless distribution 258 266 EN Ryusuke Momota Ichiro Naito Yoshifumi Ninomiya Aiji Ohtsuka Multiplexin (Mp) is the Drosophila orthologue of vertebrate collagens XV and XVIII. Like them, Mp is widely distributed in the basement membranes of the developing embryos, including those of neuroblasts in the central and peripheral nervous systems, visceral muscles of the gut, and contractile cardioblasts. Here we report the identification of mutant larvae bearing piggyBac transposon insertions that exhibit decrease Mp production associated with abdominal cuticular and wing margin defects, malformation of sensory organs and impaired sensitivity to physical stimuli. Additional findings include the abnormal ultrastructure of fatbody associated with abnormal collagen IV deposition, and reduced Wingless deposition. Collectively, these findings are consistent with the notion that Mp is required for the proper formation and/or maintenance of basement membrane, and that Mp may be involved in establishing the Wingless signaling gradients in the Drosophila embryo. No potential conflict of interest relevant to this article was reported.

Elsevier Inc. Acta Medica Okayama 0009-9120 41 3 2008 Association of elevated plasma B-type natriuretic peptide levels with paroxysmal atrial fibrillation in patients with nonobstructive hypertrophic cardiomyopathy 134 139 EN Hiroko Matsuura Takashi Murakami Kazuyoshi Hina Keizo Yamamoto Hiroshi Kawamura Taiji Sogo Ryoko Shinohata Shinichi Usui Yoshifumi Ninomiya Shozo Kusachi Objectives: To investigate the relationship between the plasma B-type natriuretic peptide (BNP) level and the occurrence of atrial fibrillation (AF) in nonobstructive hypertrophic cardiomyopathy (HCM) patients. Methods: Patients (n=97) were classified into chronic AF (CAF; n=14), paroxysmal AF (PAF; n=18) and normal sinus rhythm (NSR; n=65) groups. The plasma BNP values were analyzed with logarithmic transformation. Results: The PAF group showed significantly higher plasma BNP levels than the NSR group [mean (range; -1 SD and +1 SD); 248.3 (143.5, 429.5) vs. 78.2 (27.9, 218.8 ng/L), p<0.0001]. The CAF group also showed significantly higher plasma BNP levels than the NSR group [291.1 (161.4, 524.8 ng/L), p<0.0001]. Multivariate analysis with other clinical factors selected association of PAF as one of the factors that increased the plasma BNP level. Conclusions: The present study indicated that plasma BNP level is clinically useful for identification of nonobstructive HCM patients who have a risk of PAF. No potential conflict of interest relevant to this article was reported.

The Company of Biologists Acta Medica Okayama 0950-1991 131 7 2004 Collagen IV is essential for basement membrane stability but dispensable for initiation of its assembly during early development 1619 1628 EN Ernst Pöschl Ursula Schlötzer-Schrehardt Bent Brachvogel Kenji Saito Yoshifumi Ninomiya Ulrike Mayer Basement membranes are specialized extracellular matrices consisting of tissue-specific organizations of multiple matrix molecules and serve as structural barriers as well as substrates for cellular interactions. The network of collagen IV is thought to define the scaffold integrating other components such as, laminins, nidogens or perlecan, into highly organized supramolecular architectures. To analyze the functional roles of the major collagen IV isoform α1(IV 2α2(IV) for basement membrane assembly and embryonic development, we generated a null allele of the Col4a1/2 locus in mice, thereby ablating both α-chains. Unexpectedly, embryos developed up to E9.5 at the expected Mendelian ratio and showed a variable degree of growth retardation. Basement membrane proteins were deposited and assembled at expected sites in mutant embryos, indicating that this isoform is dispensable for matrix deposition and assembly during early development. However, lethality occurred between E10.5-E11.5, because of structural deficiencies in the basement membranes and finally by failure of the integrity of Reichert's membrane. These data demonstrate for the first time that collagen IV is fundamental for the maintenance of integrity and function of basement membranes under conditions of increasing mechanical demands, but dispensable for deposition and initial assembly of components. Taken together with other basement membrane protein knockouts, these data suggest that laminin is sufficient for basement membrane-like matrices during early development, but at later stages the specific composition of components including collagen IV defines integrity, stability and functionality. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 111 14 1998 Initiation of skin basement membrane formation at the epidermo-dermal interface involves assembly of laminins through binding to cell membrane receptors 1929 1940 EN Raul Fleischmajer Atsushi Utani E. Douglas MacDonald II Jerome S Perlish Te-Cheng Pan Mon-Li Chu Motoyoshi Nomizu Yoshifumi Ninomiya Yoshihiko Yamada To study the mechanism of basement membrane formation, we determined by immunochemistry temporal and spatial expression of laminin-5 (Ln-5), laminin-1 (Ln-1) and their integrin receptors during early skin morphogenesis. A 3-dimensional skin culture was used that allows the study of the sequential molecular events of basement membrane formation at the epidermodermal interface. During early anchorage of keratinocytes to the extracellular matrix there is expression of Ln-5, BP-230 antigen and α3, β1 integrin subunits. During epidermal stratification and prior to the formation of the lamina densa there is assembly of Ln-5, Ln-1, collagen IV and nidogen accompanied by keratinocyte basal clustering of α2, α3, α6, β1, and β4 integrin subunits. The assembly pattern of Ln-1 and Ln-5 can be disturbed with functional antibodies against the β1 (AIIB2) and α6 (GoH3) integrin subunits. Ln-1 assembly can also be disturbed with antibodies against its E8 domain and by competitive inhibition with a synthetic peptide (AG-73) derived from its G-4 domain. Quantitative RT-PCR showed that the dermis contributes about 80% of the laminin γ1 chain mRNA while 20% is produced by the epidermis which emphasizes its dual tissue origin and the major contribution of the mesenchyma in laminin production. The laminin γ2 chain mRNA, present in Ln-5, was mostly of epidermal origin. This study presents evidence that during the initiation of basement membrane formation, laminins bind to keratinocyte plasma membrane receptors and thus may serve as nucleation sites for further polymerization of these compounds by a self-assembly process. No potential conflict of interest relevant to this article was reported.

American College of Rheumatology Acta Medica Okayama 0004-3591 52 5 2005 ADAMTS-9 is synergistically induced by interleukin-1 and tumor necrosis factor in OUMS-27 chondrosarcoma cells and in human chondrocytes 1451 1460 EN Kadir Demircan Satoshi Hirohata Keiichiro Nishida Omer F. Hatipoglu Toshitaka Oohashi Tomoko Yonezawa Suneel S. Apte Yoshifumi Ninomiya Objective To compare induction of the aggrecanases (ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-8, ADAMTS-9, and ADAMTS-15) by interleukin-1 (IL-1) and tumor necrosis factor (TNF) in chondrocyte-like OUMS-27 cells and human chondrocytes, and to determine the mechanism of induction of the most responsive aggrecanase gene. Methods OUMS-27 cells were stimulated for different periods of time and with various concentrations of IL-1 and/or TNF. Human chondrocytes obtained from osteoarthritic joints and human skin fibroblasts were also stimulated with IL-1 and/or TNF. Total RNA was extracted, reverse transcribed, and analyzed by quantitative real-time polymerase chain reaction and Northern blotting. ADAMTS-9 protein was examined by Western blotting, and the role of the MAPK signaling pathway for ADAMTS9 induction in IL-1-stimulated OUMS-27 cells was investigated. Results<pr> IL-1 increased messenger RNA (mRNA) levels of ADAMTS4, ADAMTS5, and ADAMTS9 but not ADAMTS1 and ADAMTS8. The fold increase for ADAMTS9 mRNA was greater than that for mRNA of the other aggrecanase genes. The increase of ADAMTS9 mRNA by IL-1 stimulation was greater in chondrocytes than in fibroblasts. The combination of IL-1 and TNF had a synergistic effect, resulting in a considerable elevation in the level of ADAMTS9 mRNA. ADAMTS-9 protein was also induced in IL-1-stimulated OUMS-27 cells. The MAPK inhibitors SB203580 and PD98059 decreased ADAMTS9 up-regulation in OUMS-27 cells. Conclusion ADAMTS9 is an IL-1- and TNF-inducible gene that appears to be more responsive to these proinflammatory cytokines than are other aggrecanase genes. Furthermore, these cytokines had a synergistic effect on ADAMTS9. Together with the known ability of ADAMTS-9 to proteolytically degrade aggrecan and its potential to cleave other cartilage molecules, the data suggest that ADAMTS-9 may have a pathologic role in arthritis. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 54 3 2000 Heat shock protein 72 expression in the right ventricle of patients undergoing congenital cardiac surgery. 103 109 EN Koki Nakamura Hiroyuki Irie Emi Fujisawa Hidekatsu Yoshioka Yoshifumi Ninomiya Isao Sakuma Shunji Sano Article 10.18926/AMO/32300 While heat shock protein (HSP) 72 is known as a stress protein, there have been no reports of HSP 72 expression in patients who have undergone surgery for congenital heart disease. Fourteen patients (7 males and 7 females) who had undergone surgery for congenital heart disease were studied. The ages of the patients ranged from 2 months to 43 years old (mean 6.5 +/- 10.8 years old; median 3.0 years old). The diagnoses were Tetralogy of Fallot in seven, pulmonary atresia with ventricular septal defect (VSD) in three, complex anomalies in three, and VSD in one patient. Histological study and HSP analysis using Western blots and immunostaining with anti-HSP 72 monoclonal antibody were performed for right ventricular muscle samples resected during the surgery. The histological findings showed hypertrophic changes of ventricular cardiomyocytes in all samples studied. Western blots detected HSP 72 expression of various degrees in all specimens. Immunostaining using monoclonal antibody against HSP 72 showed that the protein was present in the nuclei and cytoplasm of cardiomyocytes. In conclusion, although it is difficult to determine the cause of the "stress" that triggers HSP 72 expression in cardiomyocytes, low O2 saturation and pressure overload might act as a "stress", and the only common factor that induced HSP 72 in every sample was hypertrophy. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 63 2 2009 The 3'-untranslated region of ADAMTS1 regulates its mRNA stability 79 85 EN Omer Faruk Hatipoglu Satoshi Hirohata Kursat Oguz Yaykasli Mehmet Zeynel Cilek Kadir Demircan Ryoko Shinohata Tomoko Yonezawa Toshitaka Oohashi Shozo Kusachi Yoshifumi Ninomiya Original Article 10.18926/AMO/31831 ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) is an inflammatory-induced gene. We have previously reported that ADAMTS1 was strongly but transiently expressed in the infarcted heart. In this study, we investigated whether a 3'-untranslated region (UTR) affects the mRNA stability of this gene. When stimulated with tissue necrosis factor (TNF)-alpha, the expression level of ADAMTS1 mRNA rapidly increased, but the induction of ADAMTS1 mRNA peaked at 6h after stimulation, after which the expression levels of ADAMTS1 mRNA decreased. The 3'-UTR ADAMTS1 mRNA contains multiple adenine and uridine-rich elements, suggesting that the 3'-UTR may regulate gene stability. The addition of actinomycin D, an RNA synthesis inhibitor, demonstrated the decay of induced ADAMTS1 mRNA by TNF-alpha. Furthermore, a region containing multiple AUUUA motifs within the ADAMTS1 3'-UTR destabilized transfected Enhanced Green Fluorescence Protein (EGFP) mRNA expression. These results demonstrated that the ADAMTS1 3'-UTR may regulate the expression of ADAMTS1 mRNA. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 56 1 2002 Human BRAL1 and BCAN genes that belong to the link-module superfamily are tandemly arranged on chromosome 1q21-23. 25 29 EN Hiroyuki Nomoto Toshitaka Oohashi Satoshi Hirakawa Yasuyoshi Ueki Hiroshi Ohtsuki Yoshifumi Ninomiya Article 10.18926/AMO/31728 We herein determined by fluorescence in situ hybridization the chromosomal localization of 2 human genes, BRAL1 and BCAN, both of which belong to the link-module superfamily, i.e. to the same band of chromosome 1q21-23. Further analysis of the genomic organization of BRAL1 and BCAN revealed that the BRAL1 gene was located 20-kb upstream of the BCAN start site. We isolated a polymorphic dinucleotide (CA) repeat sequence from a genomic clone containing the BCAN gene. High heterozygosity (0.79) makes this polymorphism a useful marker in the study of genetic disorders. Knowledge of the structure of the genes and the marker provides essential information for further analysis of the gene locus at chromosome 1q21-23. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 50 3 1996 Regulation of Interleukin-2 Receptor y Chain mRNA Expression in Human Monocytic Cell Line THP-1 145 150 EN Hiroyuki Yanai Tadashi Yoshino Kiyoshi Takahashi Yoshifumi Ninomiya Tadaatsu Akagi Article 10.18926/AMO/30509 Circulating hepatitis C virus (HCV) particles can be fractionated by means of differential flotation centrifugation. It is reported that in the bottom fraction HCV is in the form immune complexes, whereas in the top, it is free of antibodies. We evaluated the significance of circulating complex and free HCV in chronic hepatitis C, and assessed the relationship in terms of the response to interferon (IFN) therapy. We examined sera before, just after, and 1 year after administering IFN to 18 patients with chronic hepatitis C, 10 of whom responded (group CR), and 8 did not (group NR). The amounts of virus were similar between both groups before therapy. After differential flotation centrifugation with 1.063 g/ml of NaCl, the top and bottom fractions were assayed for HCV RNA. Before therapy, HCV RNA was detected in the top fraction in 1 of 10 in group CR, and in 6 of 8 in group NR (P < 0.05, chi-square test). HCV RNA was positive in the bottom fraction of all samples. In a follow-up study of group NR, HCV RNA was detected in the top fraction in 3 of 8 just after IFN therapy, and in 7 of 8 after 1 year. This study suggests that the presence of HCV in the top fraction can predict a poor response to IFN therapy. No potential conflict of interest relevant to this article was reported.

岡山実験動物研究会 Acta Medica Okayama 14 1997 コラーゲン遺伝子改変と動物の疾患モデル 7 11 EN Yoshifumi Ninomiya No potential conflict of interest relevant to this article was reported.