Wiley Acta Medica Okayama 2045-7634 13 20 2024 New Anti-Angiogenic Therapy for Glioblastoma With the Anti-Depressant Sertraline e70288 EN Nobushige Tsuboi Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Yoshihiro Otani Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Atsuhito Uneda Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Joji Ishida Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Yasuki Suruga Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Yuji Matsumoto Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Atsushi Fujimura Neutron Therapy Research Center, Okayama University Kentaro Fujii Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Hideki Matsui Neutron Therapy Research Center, Okayama University Kazuhiko Kurozumi Department of Neurosurgery, Hamamatsu University School of Medicine Isao Date Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Hiroyuki Michiue Neutron Therapy Research Center, Okayama University Background and Aims: Anti-angiogenic therapies prolong patient survival in some malignancies but not glioblastoma. We focused on the relationship between the differentiation of glioma stem like cells (GSCs) into tumor derived endothelial cells (TDECs) and, anti-angiogenic therapy resistance. Especially we aimed to elucidate the mechanisms of drug resistance of TDECs to anti-angiogenic inhibitors and identify novel anti-angiogenic drugs with clinical applications.<br> Results: The mouse GSCs, 005, were differentiated into TDECs under hypoxic conditions, and TDECs had endothelial cell characteristics independent of the vascular endothelial growth factor (VEGF) pathway. In vivo, inhibition of the VEGF pathway had no anti-tumor effect and increased the percentage of TDECs in the 005 mouse model. Novel anti-angiogenic drugs for glioblastoma were evaluated using a tube formation assay and a drug repositioning strategy with existing blood-brain barrier permeable drugs. Drug screening revealed that the antidepressant sertraline inhibited tube formation of TDECs. Sertraline was administered to differentiated TDECs in vitro and 005 mouse models in vivo to evaluate genetic changes by RNA-Seq and tumor regression effects by immunohistochemistry and MRI. Sertraline reduced Lama4 and Ang2 expressions of TDEC, which play an important role in non-VEGF-mediated angiogenesis in tumors. The combination of a VEGF receptor inhibitor axitinib, and sertraline improved survival and reduced tumor growth in the 005 mouse model.<br> Conclusion: Collectively, our findings showed the diversity of tumor vascular endothelial cells across VEGF and non-VEGF pathways led to anti-angiogenic resistance. The combination of axitinib and sertraline can represent an effective anti-angiogenic therapy for glioblastoma with safe, low cost, and fast availability. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 0168-3659 330 2020 Self-assembling A6K peptide nanotubes as a mercaptoundecahydrododecaborate (BSH) delivery system for boron neutron capture t (BNCT) 788 196 EN Hiroyuki Michiue Neutron Therapy Research Center, Okayama University Mizuki Kitamatsu Department of Applied Chemistry, Kindai University Asami Fukunaga Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nobushige Tsuboi Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Atsushi Fujimura Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroaki Matsushita Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuyo Igawa Neutron Therapy Research Center, Okayama University Tomonari Kasai Neutron Therapy Research Center, Okayama University Natsuko Kondo Institute for Integrated Radiation and Nuclear Science, Kyoto University Hideki Matsui Neutron Therapy Research Center, Okayama University Shuichi Furuya Neutron Therapy Research Center, Okayama University Boron neutron capture therapy (BNCT) is a tumor selective therapy, the effectiveness of which depends on sufficient 10B delivery to and accumulation in tumors. In this study, we used self-assembling A6K peptide nanotubes as boron carriers and prepared new boron agents by simple mixing of A6K and BSH. BSH has been used to treat malignant glioma patients in clinical trials and its drug safety and availability have been confirmed; however, its contribution to BNCT efficacy is low. A6K nanotube delivery improved two major limitations of BSH, including absence of intracellular transduction and non-specific drug delivery to tumor tissue. Varying the A6K peptide and BSH mixture ratio produced materials with different morphologies―determined by electron microscopy―and intracellular transduction efficiencies. We investigated the A6K/BSH 1:10 mixture ratio and found high intracellular boron uptake with no toxicity. Microscopy observation showed intracellular localization of A6K/BSH in the perinuclear region and endosome in human glioma cells. The intracellular boron concentration using A6K/BSH was almost 10 times higher than that of BSH. The systematic administration of A6K/BSH via mouse tail vein showed tumor specific accumulation in a mouse brain tumor model with immunohistochemistry and pharmacokinetic study. Neutron irradiation of glioma cells treated with A6K/BSH showed the inhibition of cell proliferation in a colony formation assay. Boron delivery using A6K peptide provides a unique and simple strategy for next generation BNCT drugs. No potential conflict of interest relevant to this article was reported.

Blackwell Acta Medica Okayama 0013-9580 49 3 2007 Rasmussen encephalitis associated with SCN1A mutation 521 526 EN Iori Ohmori Departments of Cellular Physiology, Graduate School of Medicine,Dentistry and Pharmaceutical Sciences, Okayama University Mamoru Ouchida Departments of Molecular Genetics, Graduate School of Medicine,Dentistry and Pharmaceutical Sciences, Okayama University Katsuhiro Kobayashi Departments of Child Neurology, Graduate School of Medicine,Dentistry and Pharmaceutical Sciences, Okayama University Yoshimi Jitsumori Takushi Inoue Departments of Child Neurology, Graduate School of Medicine,Dentistry and Pharmaceutical Sciences, Okayama University Kenji Shimizu Departments of Molecular Genetics, Graduate School of Medicine,Dentistry and Pharmaceutical Sciences, Okayama University Hideki Matsui Departments of Cellular Physiology, Graduate School of Medicine,Dentistry and Pharmaceutical Sciences, Okayama University Yoko Ohtsuka Departments of Child Neurology, Graduate School of Medicine,Dentistry and Pharmaceutical Sciences, Okayama University Yoshihiro Maegaki Division of Child Neurology, Institute of Neurological Sciences, Faculty of Medicine, Tottori University Mutations in the SCN 1 A gene, encoding the neuronal voltage-gated sodium channel alpha1 subunit, cause SMEI, GEFS+, and related epileptic syndromes. We herein report the R1575C-SCN 1 A mutation identified in a patient with Rasmussen encephalitis. R1575C were constructed in a recombinant human SCN 1 A and then heterologously expressed in HEK293 cells along with the human beta1 and beta2 sodium channel accessory subunits. Whole-cell patch-clamp recording was used to define biophysical properties. The R1575C channels exhibited increased channel availability and an increased persistent sodium current in comparison to the wild-type. These defects of electrophysiological properties can result in neuronal hyperexitability. The seizure susceptibility allele may influence the pathogenesis of Rasmussen encephalitis in this case. No potential conflict of interest relevant to this article was reported.

Elsevier Science Acta Medica Okayama 09201211 105 1-2 2013 Inhalation of 10% carbon dioxide rapidly terminates Scn1a mutation-related hyperthermia-induced seizures 220 224 EN Iori Ohmori Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Keiichiro Hayashi Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Haijiao Wang Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Mamoru Ouchida Department of Molecular Genetics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Naohiro Fujita Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takushi Inoue Department of Child Neurology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hiroyuki Michiue Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teiichi Nishiki Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hideki Matsui Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University The aim of this study was to assess the anticonvulsant effect of carbon dioxide (CO2) on Scn1a mutation-related febrile seizures. We examined physiological changes in the blood gas levels after the induction of hyperthermia-induced seizures (HISs), which were associated with the Scn1a missense mutation. We determined the efficacy of inhalation of 5% or 10% CO2 to treat HISs. HISs were evoked in Scn1a mutant and wild-type (WT) rats by hot water baths. To determine the anticonvulsant effect of CO2 inhalation, rats were placed in a chamber filled with air or mixed gas containing 5% CO2 or 10% CO2 for 3 min, immediately after the induction of HISs. We also analyzed the blood gas levels at the end of inhalation of CO2. Hot water bathing induced a significant reduction in the partial pressure of CO2 (pCO2) and respiratory alkalosis in the WT and Scn1a mutant rats. HISs were evoked in 100% of the Scn1a mutant rats within 5 min, but in none of the WT rats. The Scn1a mutant rats demonstrated a higher HISs susceptibility associated with respiratory alkalosis than the WT rats. Inhalation of 10% CO2 shortened the seizure duration from 62.6±12.1 s to 15.5±1.0 s. Blood gas analysis after the inhalation of 10% CO2 demonstrated an elevated pCO2 level and respiratory acidosis. Inhalation of 10% CO2 demonstrated a potent and fast-acting anticonvulsant effect against HISs. No potential conflict of interest relevant to this article was reported.

Academic Press Acta Medica Okayama 09699961 50 2013 CACNA1A variants may modify the epileptic phenotype of Dravet syndrome 209 217 EN Iori Ohmori Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Mamoru Ouchida Department of Molecular Genetics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Katsuhiro Kobayashi Yoshimi Jitsumori Akiko Mori Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hiroyuki Michiue Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teiichi Nishiki Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yoko Ohtsuka Hideki Matsui Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Dravet syndrome is an intractable epileptic syndrome beginning in the first year of life. De novo mutations of SCN1A, which encode the Na(v)1.1 neuronal voltage-gated sodium channel, are considered the major cause of Dravet syndrome. In this study, we investigated genetic modifiers of this syndrome. We performed a mutational analysis of all coding exons of CACNA1A in 48 subjects with Dravet syndrome. To assess the effects of CACNA1A variants on the epileptic phenotypes of Dravet syndrome, we compared clinical features in two genotype groups: 1) subjects harboring SCN1A mutations but no CACNA1A variants (n=20) and 2) subjects with SCN1A mutations plus CACNA1A variants (n=20). CACNA1A variants detected in patients were studied using heterologous expression of recombinant human Ca(v)2.1 in HEK 293 cells and whole-cell patch-clamp recording. Nine CACNA1A variants, including six novel ones, were detected in 21 of the 48 subjects (43.8%). Based on the incidence of variants in healthy controls, most of the variants seemed to be common polymorphisms. However, the subjects harboring SCN1A mutations and CACNA1A variants had absence seizures more frequently than the patients with only SCN1A mutations (8/20 vs. 0/20, p=0.002). Moreover, the former group of subjects exhibited earlier onset of seizures and more frequent prolonged seizures before one year of age, compared to the latter group of subjects. The electrophysiological properties of four of the five novel Ca(v)2.1 variants exhibited biophysical changes consistent with gain-of-function. We conclude that CACNA1A variants in some persons with Dravet syndrome may modify the epileptic phenotypes. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 0013-9580 52 5 2011 Therapy for hyperthermia-induced seizures in Scn1a mutant rats 1010 1017 EN Keiichiro Hayashi Satoshi Ueshima Mamoru Ouchida Tomoji Mashimo Teiichi Nishiki Toshiaki Sendo Tadao Serikawa Hideki Matsui Iori Ohmori Purpose: Mutations in the SCN1A gene, which encodes the alpha 1 subunit of voltage-gated sodium channels, cause generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI). N1417H-Scn1a mutant rats are considered to be an animal model of human FS+ or GEFS+. To assess the pharmacologic validity of this model, we compared the efficacies of eight different antiepileptic drugs (AEDs) for the treatment of hyperthermia-induced seizures using N1417H-Scn1a mutant rats. Methods: AEDs used in this study included valproate, carbamazepine (CBZ), phenobarbital, gabapentin, acetazolamide, diazepam (DZP), topiramate, and potassium bromide (KBr). The effects of these AEDs were evaluated using the hot water model, which is a model of experimental FS. Five-week-old rats were pretreated with each AED and immersed in water at 45 degrees C to induce hyperthermia-induced seizures. The seizure manifestations and video-electroencephalographic recordings were evaluated. Furthermore, the effects of each AED on motor coordination and balance were assessed using the balance-beam test. Key Findings: KBr significantly reduced seizure durations, and its anticonvulsant effects were comparable to those of DZP. On the other hand, CBZ decreased the seizure threshold. In addition, DZP and not KBr showed significant impairment in motor coordination and balance. Significance: DZP and KBr showed potent inhibitory effects against hyperthermia-induced seizures in the Scn1a mutant rats, whereas CBZ exhibited adverse effects. These responses to hyperthermia-induced seizures were similar to those in patients with GEFS+ and SMEI. N1417H-Scn1a mutant rats may, therefore, be useful for testing the efficacy of new AEDs against FS in GEFS+ and SMEI patients. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1044-7431 49 1 2012 Ca2+-independent syntaxin binding to the C2B effector region of synaptotagmin 1 8 EN Toshio Masumoto Koichiro Suzuki Iori Ohmori Hiroyuki Michiue Kazuhito Tomizawa Atsushi Fujimura Tei-ichi Nishiki Hideki Matsui Although synaptotagmin I, which is a calcium (Ca2+)-binding synaptic vesicle protein, may trigger soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated synaptic vesicle exocytosis, the mechanisms underlying the interaction between these proteins remain controversial, especially with respect to the identity of the protein(s) in the SNARE complex that bind(s) to synaptotagmin and whether Ca2+ is required for their highly effective binding. To address these questions, native proteins were solubilized, immunoprecipitated from rat brain extracts, and analyzed by immunoblotting. SNARE complexes comprising syntaxin 1, 25-kDa synaptosomal-associated protein (SNAP-25), and synaptobrevin 2 were coprecipitzted with synaptotagmin I in the presence of ethylene glycol tetraacetic acid. The amount of cop recipitated proteins was significantly unaltered by the addition of Ca2+ to the brain extract. To identify the component of the SNARE complex that bound to synaptotagmin, SNARE was coexpressed with synaptotagmin in HEK293 cells and immunoprecipitated. Syntaxin, but not SNAP-25 and synaptobrevin, bound to synaptotagmin in a Ca2+-independent manner, and the binding was abolished in the presence of 1 M NaCl. Synaptotagmin contains 2 Ca2+-binding domains (C(2)A, C2B). Mutating the positively charged lysine residues in the putative effector-binding region of the C2B domain, which are critical for transmitter release, markedly inhibited synaptotagmin-syntaxin binding, while similar mutations in the C(2)A domain had no effect on binding. Synaptotagmin-syntaxin binding was reduced by mutating multiple negatively charged glutamate residues in the amino-terminal half of the syntaxin SNARE motif. These results indicate that synaptotagmin I binds to syntaxin 1 electrostatically through its C2B domain effector region in a Ca2+-independent fashion, providing biochemical evidence that synaptotagmin I binds SNARE complexes before Ca2+ influx into presynaptic nerve terminals. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 126 1 2014 第10回日本中性子捕捉療法学会学術大会報告 73 74 EN Hideki Matsui No potential conflict of interest relevant to this article was reported.

Public Library Science Acta Medica Okayama 1932-6203 6 3 2011 Expression of a Constitutively Active Calcineurin Encoded by an Intron-Retaining mRNA in Follicular Keratinocytes EN Atsushi Fujimura Hiroyuki Michiue Tei-ichi Nishiki Iori Ohmori Fanyan Wei Hideki Matsui Kazuhito Tomizawa Hair growth is a highly regulated cyclical process. Immunosuppressive immunophilin ligands such as cyclosporin A (CsA) and FK506 are known as potent hair growth modulatory agents in rodents and humans that induce active hair growth and inhibit hair follicle regression. The immunosuppressive effectiveness of these drugs has been generally attributed to inhibition of T cell activation through well-characterized pathways. Specifically, CsA and FK506 bind to intracellular proteins, principally cyclophilin A and FKBP12, respectively, and thereby inhibit the phosphatase calcineurin (Cn). The calcineurin (Cn)/NFAT pathway has an important, but poorly understood, role in the regulation of hair follicle development. Here we show that a novel-splicing variant of calcineurin A beta CnA beta-FK, which is encoded by an intron-retaining mRNA and is deficient in the autoinhibitory domain, is predominantly expressed in mature follicular keratinocytes but not in the proliferating keratinocytes of rodents. CnA beta-FK was weakly sensitive to Ca(2+) and dephosphorylated NFATc2 under low Ca(2+) levels in keratinocytes. Inhibition of Cn/NFAT induced hair growth in nude mice. Cyclin G2 was identified as a novel target of the Cn/NFATc2 pathway and its expression in follicular keratinocytes was reduced by inhibition of Cn/NFAT. Overexpression of cyclin G2 arrested the cell cycle in follicular keratinocytes in vitro and the Cn inhibitor, cyclosporin A, inhibited nuclear localization of NFATc2, resulting in decreased cyclin G2 expression in follicular keratinocytes of rats in vivo. We therefore suggest that the calcineurin/NFAT pathway has a unique regulatory role in hair follicle development. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 65 1 2011 Regulation of Mitochondrial Dynamics and Neurodegenerative Diseases 1 10 EN Xiao-Jian Han Kazuhito Tomizawa Atsushi Fujimura Iori Ohmori Tei-ichi Nishiki Masayuki Matsushita Hideki Matsui Review 10.18926/AMO/43824 Mitochondria are important cellular organelles in most metabolic processes and have a highly dynamic nature, undergoing frequent fission and fusion. The dynamic balance between fission and fusion plays critical roles in mitochondrial functions. In recent studies, several large GTPases have been identified as key molecular factors in mitochondrial fission and fusion. Moreover, the posttranslational modifications of these large GTPases, including phosphorylation, ubiquitination and SUMOylation, have been shown to be involved in the regulation of mitochondrial dynamics. Neurons are particularly sensitive and vulnerable to any abnormalities in mitochondrial dynamics, due to their large energy demand and long extended processes. Emerging evidences have thus indicated a strong linkage between mitochondria and neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and Huntington's disease. In this review, we will describe the regulation of mitochondrial dynamics and its role in neurodegenerative diseases. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 61 3 2007 Colocalization of oxytocin and phosphorylated form of elongation factor 2 in the rat hypothalamus 161 166 EN Toru Fujisawa Akiyoshi Moriwaki Masayuki Matsushita Kazuhito Tomizawa Hideki Matsui Original Article 10.18926/AMO/32907 Oxytocin (OT) is one of the neuropituitary hormones and is synthesized in the neurons of the paraventricular nucleus (PVN) and supraoptic nucleus (SON). Previous studies have shown that the mRNAs encoding OT are delivered from the soma to both dendrites and axons of the neurons in the PVN and SON. However, it has not been elucidated whether a translational regulation mechanism to enable local synthesis of the hormone exists in the axons of the neurons of PVN and SON. Elongation factor 2 (EF2) is essential for polypeptide synthesis during protein translation. Moreover, phosphorylation of EF2 by EF2 kinase enhances the translation of certain mRNA species. In the present study, in order to shed light on the mechanisms involved in the translational regulation of OT synthesis, we investigated the localization of phosphorylated EF2. Phospho-EF2 was localized in the soma of the neurons in PVN and SON, and in the swellings of the median eminence where axonal tracts of the neurons in the PVN and SON exist. The phosphorylated form was also observed in the rat hypophysis. Moreover, phospho-EF2 and OT were colocalized in a part of the neurons in the PVN and SON. These results suggest that OT may be partially translated in the axons of neurons in the PVN and SON, and then secreted from the pituitary. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 61 3 2007 Calpain-calcineurin signaling in the pathogenesis of calcium-dependent disorder. 123 137 EN Hai-Yan Wu Kazuhito Tomizawa Hideki Matsui Review 10.18926/AMO/32905 Intracellular calcium is a powerful secondary messenger that affects a number of calcium sensors, including calpain, a Ca2+-dependent cysteine protease, and calcineurin, a Ca2+/calmodulin-dependent protein phosphatase. Maintenance of low basal levels of intracellular calcium allows for the tightly regulated physiological activation of these proteins, which is crucial to a wide variety of cellular processes, such as fertilization, proliferation, development, learning, and memory. Deregulation of calpain and calcineurin has been implicated in the pathogenesis of several disorders, including hypertension, heart disease, diabetes, cerebral ischemia, and Alzheimer's disease. Recent studies have demonstrated an interplay between calpain and calcineurin, in which calpain can directly regulate calcineurin activity through proteolysis in glutamate-stimulated neurons in culture and in vivo. The calpain-mediated proteolytic cleavage of calcineurin increases phosphatase activity, which promotes caspase-mediated neuronal cell death. Thus, the activation of the calpain-calcineurin pathway could contribute to calcium-dependent disorders, especially those associated with Alzheimer's disease and myocardial hypertrophy. Here, we focus briefly on recent advances in revealing the structural and functional properties of these 2 calcium-activated proteins, as well as on the interplay between the 2, in an effort to understand how calpain-calcineurin signaling may relate to the pathogenesis of calcium- dependent disorders. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 61 3 2007 Inhibitory effect of polyunsaturated fatty acids on apoptosis induced by etoposide, okadaic acid and AraC in Neuro2a cells 147 152 EN Yumei Wu Mikiro Tada Kyoya Takahata Kazuhito Tomizawa Hideki Matsui Original Article 10.18926/AMO/32903 Neuronal apoptosis is involved in neurodegenerative diseases such as Alzheimer's disease and Parkinson.s disease. An efficient means of preventing it remains to be found. Some n-3 polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA, 22 : 6n-3) and eicosapentaenoic acid (EPA, 20 : 5n-3) have been reported to be protective against the neuronal apoptosis and neuronal degeneration seen after spinal cord injury (SCI) [1]. However, it is unclear which kinds of PUFAs have the most potent ability to inhibit neuronal apoptosis and whether the simultaneous treatment of PUFAs inhibits the apoptosis. In the present study, we compared the abilities of various n-3- and n-6- PUFAs to inhibit the apoptosis induced after the administration of different apoptotic inducers, etoposide, okadaic acid, and AraC, in mouse neuroblastoma cells (Neuro2a). Preincubation with DHA (22 : 6n-3), eicosapentaenoic acid (EPA, 20 : 5n-3), alpha-linolenic acid (alpha-LNA, 18 : 3n-3), linoleic acid (LA, 18 : 2n-6), arachidonic acid (AA, 20 : 4n-3), and gamma-linolenic acid (gamma-LNA, 18 : 3n-6) significantly inhibited caspase-3 activity and LDH leakage but simultaneous treatment with the PUFAs had no effect on the apoptosis of Neuro2a cells. There were no significant differences of the anti-apoptotic eff ect among the PUFAs. These results suggest that PUFAs may not be effective for inhibiting neuronal cell death after acute and chronic neurodegenerative disorders. However, dietary supplementation with PUFAs may be beneficial as a potential means to delay the onset of the diseases and/or their rate of progression. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 37 2 1983 Modulation of optical density by sulfhydryl reagents in microbiuret method: a modified method for protein determination in the presence of sulfhydryl reagents. 125 129 EN Hideki Matsui Tomohiro Kurosaki Masaaki Tokuda Osamu Hatase Article 10.18926/AMO/32414 <p>2-Mercaptoethanol increases the optical density of assay solutions at wavelengths between 280 to 400 nm, and therefore interferes with the measurement of protein concentration by the microbiuret method. Protein concentration can be determined in the presence of 2-mercaptoethanol up to 6 mM by modification of the method as follows: after the precipitation of protein by trichloroacetic acid in the presence of deoxycholate, the precipitate is resolubilized with NaOH solution. Dithiothreitol interfered with the protein determinations could by made in the presence of 4 mM of dithiothreitol with the modified microbiuret method. This modified method is time-saving and more reliable than other methods for protein determination, such as Lowry's method, in the presence of sulfhydryl reagents.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 58 3 2004 Exposure of mouse to high gravitation forces induces long-term potentiation in the hippocampus. 143 149 EN Masamitsu Ishii Kazuhito Tomizawa Masayuki Matsushita Hideki Matsui Article 10.18926/AMO/32113 <p>The central nervous system is highly plastic and has been shown to undergo both transient and chronic adaptive changes in response to environmental influences. The purpose of this study was to investigate the effect of hypergravic field on long-term potentiation (LTP) in the mouse hippocampus. Exposure of mice to 4G fields for 48 h had no effect on input-output coupling during extracellular stimulation of Schaffer collaterals and paired pulse facilitation, suggesting that the hypergravic exposure had no detrimental effect on basal neurotransmission in the hippocampus. However, the exposure to 4G fields for 48 h significantly induced LTP compared with the control mouse hippocampus. In contrast, no significant changes of late-phase LTP (L-LTP) were found in the hippocampi of mice exposed to the hypergravic field. Exposure of mice to 4G fields for 48 h enhanced AMPA receptor phosphorylation but not cyclic AMP-responsive element binding protein (CREB) phosphorylation. These results suggest that exposure to hyperdynamic fields influences the synaptic plasticity in the hippocampus.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 58 4 2004 2-methoxyestradiol enhances p53 protein transduction therapy-associated inhibition of the proliferation of oral cancer cells through the suppression of NFkappaB activity. 181 187 EN Hidehiko Takata Kazuhito Tomizawa Masayuki Matsushita Hideki Matsui Article 10.18926/AMO/32086 <p>Protein transduction therapy using poly-arginine peptide can deliver the biologically active proteins. A previous study showed that 11 poly-arginine fused p53 protein (11R-p53) effectively penetrated across the plasma membrane and inhibited the proliferation of oral cancer cells. However, the intracellular half-life of the delivered protein was less than 36 h. Previous studies also showed that 2-methoxyestradiol (2-ME), an endogenous non-toxic estrogenic metabolite, induces the stabilization of the wild-type p53 protein in human cancer cells posttranscriptionally. In the present study, we examined whether 2-ME induced the stabilization of 11R-p53 and had an inhibitory effect on the proliferation of oral cancer cells. The application of 2-ME significantly enhanced the inhibitory effect of 11R-p53 on the proliferation of oral cancer cells. However, 2-ME had no effect on the intracellular half-life of 11R-p53 in oral cancer cells. Of interest is the finding that 2-ME suppressed the transcriptional activity of NFkappaB, which has an important role in tumorigenesis, but did not affect p53 transcriptional activity. These results suggest that 2-ME synergistically enhances the 11R-p53-induced inhibition of the proliferation of oral cancer cells through the suppression of NFkB transcription.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 33 3 1979 Partial purification and biological activities and properties of chick growth factors. 167 176 EN Fumio Takahashi Makoto Kuramitsu Masaaki Tokuda Hideki Matsui Toshifumi Itano Tetsu-Hide Murakami Osamu Hatase Isamu Nishida Article 10.18926/AMO/32071 <p>Cellular stimulating factors on cell proliferation in the supernatants of chick embryo carcases and adult muscles were studied. There were plural stimulating factors in embryonic and adult muscular supernatants that promoted cell proliferation without any supplement of sera and other materials. Salting-out methods with ammonium sulfate, ethanol fractionation, and isoelectric precipitation were used to isolate the stimulating factors, and these three methods proved the presence of plural stimulants on cell proliferation in the supernatants of chick embryo and adult muscles. The stimulants had altered physico-chemical properties and biological activities due to embryological development. The embryonic stimulants enhanced the synthesis of DNA and protein remarkably, and RNA synthesis in whole cell systems slightly. The muscular stimulants enhanced protein synthesis without any stimulation of DNA and RNA synthesis. Partial purification of the stimulants from the ethanol fractions was performed by DEAE-cellulose chromatography and Sephadex gel chromatography.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 63 1 2009 Protein Transduction Method for Cerebrovascular Disorders 1 7 EN Tomoyuki Ogawa Shigeki Ono Tomotsugu Ichikawa Seiji Arimitsu Keisuke Onoda Koji Tokunaga Kenji Sugiu Kazuhito Tomizawa Hideki Matsui Isao Date Review 10.18926/AMO/31858 <p>Many studies have shown that a motif of 11 consecutive arginines (11R) is one of the most effective protein transduction domains (PTD) for introducing proteins into the cell membrane. By conjugating this &#34;11R&#34;, all sorts of proteins can effectively and harmlessly be transferred into any kind of cell. We therefore examined the transduction efficiency of 11R in cerebral arteries and obtained results showing that 11R fused enhanced green fluorescent protein (11R-EGFP) immediately and effectively penetrated all layers of the rat basilar artery (BA), especially the tunica media. This method provides a revolutionary approach to cerebral arteries and ours is the first study to demonstrate the successful transductionof a PTD fused protein into the cerebral arteries. In this review, we present an outline of our studies and other key studies related to cerebral vasospasm and 11R, problems to be overcome, and predictions regarding future use of the 11R protein transduction method for cerebral vasospasm (CV).</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 63 6 2009 Amphiphysin I and regulation of synaptic vesicle endocytosis 305 323 EN Yumei Wu Hideki Matsui Kazuhito Tomizawa Review 10.18926/AMO/31822 <p>Amphiphysin I, known as a major dynamin-binding partner localized on the collars of nascent vesicles, plays a key role in clathrin-mediated endocytosis (CME) of synaptic vesicles. Amphiphysin I mediates the invagination and fission steps of synaptic vesicles by sensing or facilitating membrane curvature and stimulating the GTPase activity of dynamin. Amphiphysin I may form a homodimer by itself or a heterodimer with amphiphysin II in vivo. Both amphiphysin I and II function as multilinker proteins in the clathrin-coated complex. Under normal physiological conditions, the functions of amphiphysin I and some other endocytic proteins are known to be regulated by phosphorylation and dephosphorylation. During hyperexcited conditions, the most recent data showed that amphiphysin I is truncated by the ca2-dependent protease calpain. Overexpression of the truncated form of amphi-physin I inhibited transferrin uptake and synaptic vesicle endocytosis (SVE). This suggests that amphi-physin I may be an important regulator for SVE when massive amounts of Ca2 flow into presynaptic terminals, a phenomenon observed in neurodegenerative disorders such as ischemia/anoxia, epilepsy, stroke, trauma and Alzheimer's disease. This review describes current knowledge regarding the general properties and functions of amphiphysin I as well as the functional regulations such as phosphorylation and proteolysis in nerve terminals.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 47 3 1993 Exogenous Basic Fibroblast Growth Factor and Nerve Growth Factor Enhance Sprouting of Acetylcholinesterase Positive Fibers in Denervated Rat Hippocampus 139 144 EN Osamu Miyamoto Toshifumi Itano Mutsuo Fujisawa Masaaki Tokuda Hideki Matsui Seigo Nagao Osamu Hatase Article 10.18926/AMO/31596 <p>Basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) were administered into the rat brain following unilateral fimbria-fornix transection. Both bFGF and NGF stimulated the sprouting of acetylcholinesterase (AChE) positive fibers in the hippocampus on the lesioned side. Furthermore, a small number of AChE-positive fibers were regenerated even when only the vehicle was administered. Rats treated with NGF as well as control group had only thin fibers, whereas those treated with bFGF had not only thin fibers but also thick fibers. These results indicate that intrinsic NGF is released and acts on damaged neurons directly, while bFGF acts them on directly and/or indirectly after brain injury.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 62 1 2008 Critical Differences in Magnitude and Duration of N-methyl-D-aspartate (NMDA) Receptor Activation between Long-term Potentiation (LTP) and Long-term Depression (LTD) Induction 21 28 EN Naoki Taniike Yun-Fei Lu Kazuhito Tomizawa Hideki Matsui Original Article 10.18926/AMO/30989 <p>The induction of both long-term potentiation (LTP) and long-term depression (LTD) in the hippocampal CA1 region is triggered by the activation of N-methyl-D-aspartate (NMDA) receptors and the subsequent postsynaptic intracellular Ca2+ increase. However, how NMDA receptor activation differs between LTP and LTD induction is unclear. In the present study, we examined the eff ects of the magnitude and duration of NMDA receptor activation on the induction of LTP and LTD. Partial blockage of NMDA receptors by a low concentration of aminophosphonovaleric acid (APV)(2 &#956;M) prevented the induction of LTP, but not LTD. In contrast, a high concentration of APV(25 &#956;M) blocked both LTP and LTD. Tetanus stimulation-induced LTP was impaired when hippocampal slices were given the tetanus stimulation for more than 5 min. Under partial blockage of NMDA receptors, the prolonged-tetanus stimulation induced LTD but not LTP. This phenomenon was mimicked by the application of glutamate to the slices. Finally, LTD induced by prolonged activation of NMDA receptors was not aff ected by inhibition of the desensitization of &#945;-amino-3-hydroxy-5 methylisoxazole-4-propionic acid (AMPA) receptors. These results suggest that critical diff erences exist between the induction of LTP and that of LTD in terms of both the magnitude and the duration of NMDA receptor activation. The duration of the increase in intracellular Ca2+ concentration may be critical for determining whether LTP or LTD induction occurs.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 51 1 1997 Comparative analysis of CD45RA- and CD45RO-positive CD4⁺T cells in peripheral blood, synovial fluid, and synovial tissue in patients with rheumatoid arthritis and osteoarthritis 25 31 EN Kazuhiko Ezawa Masahiro Yamamura Hideki Matsui Zensuke Ota Hirofumi Makino Article 10.18926/AMO/30810 <p>&#8195;To determine whether the predominant infiltration with memory CD4⁺T cells in joints is specific to the local immune and inflammatory response in rheumatoid arthritis (RA), the proportions of CD45RA⁺ or CD45RO⁺ cells in the CD4⁺T cell populations in three different compartments (i.e., peripheral blood, synovial fluid, and synovial tissue) from patients with RA and osteoarthritis (OA) were compared by two-color flow-cytometric analysis. In the CD4⁺T cell population of peripheral blood, the number of CD45RO⁺ cells was relatively higher than CD45RA⁺ cells in both RA and OA patients, but their percentages did not differ from those found in healthy individuals. However, the great majority of CD4⁺T cells present in synovial fluid and synovial tissue were CD45RO-positive and CD45RA-negative in both patient groups; although CD4⁺T cells infiltrating both the disease compartments were markedly greater in RA joints, their mean percentages of CD45RO⁺ cells were not significantly different from those in OA joints. These data indicate that an accumulation of CD45RO⁺ memory CD4+T cells is a generalized phenomenon during local inflammatory responses in both RA and OA joints, and may be due mainly to the propensity of these cells to preferentially transmigrate into the inflamed joint via adhesion molecules as compared with CD45RA⁺ naive CD4⁺T cells.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 60 2 2006 Immunosuppression for islet transplantation. 71 76 EN Hirofumi Noguchi Shinichi Matsumoto Masayuki Matsushita Naoya Kobayashi Koichi Tanaka Hideki Matsui Noriaki Tanaka Review 10.18926/AMO/30733 The development by the Edmonton group of a sirolimus-based, steroid-free, low-tacrolimus regimen is a significant breakthrough that allows the rate of insulin independence after islet transplantation to increase from 13% to 80% at 1 year ; however, the rate is reduced to 50% at 3 years, attributed to prolonged tacrolimus exposure. Recently, immunosuppression agents such as cyclosporine, mycophenolate mofetil, and the novel agent FTY 720 have been used instead of tacrolimus. Lymphocytedepleting antibodies such as anti-thymocyte globulin, alemtuzumab, and hOKT3gamma 1 (ala, ala) have been launched, and a costimulatory blockade of anti-CD40 monoclonal antibodies and CTLA4-Ig will be attempted in the near future. Moreover, the potential of a novel immunosuppressing peptide could now be realized using new technology called the protein transduction system. In this review, we show some of the most recent contributions to the advancement of knowledge in this field. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 36 1 1982 Factors inhibiting cell proliferation in rat liver cytoplasm. 1 10 EN Makoto Kuramitsu Hideki Matsui Masaaki Tokuda Osamu Hatase Article 10.18926/AMO/30708 <p>Two factors from normal rat liver cytoplasm inhibited the proliferation of cultured L-929 fibroblasts. One was arginase, the other was a small molecular weight inhibitor stable to trypsin and heat treatment. The small molecular weight inhibitor inhibited the protein and DNA synthesis of L-cells. Inhibition of DNA synthesis was thought to be secondary to the inhibition of protein synthesis.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 44 1 1990 In Vivo Analysis of Extracellular Proteins in Rat Brains with a Newly Developed Intracerebral Microdialysis Probe 1 8 EN Mitsuo Nakamura Toshifumi Itano Fuminori Yamaguchi Masayuki Mizobuchi Masaaki Tokuda Hideki Matsui Siji Etoh Kiyoshi Hosokawa Takashi Ohmoto Osamu Hatase Article 10.18926/AMO/30465 <p>Peptides and proteins in the extracellular space in the central nervous system were investigated in vivo using an intracerebral microdialysis probe. The molecular cut-off of the hollow fiber which was used for the probe was approximately 100 kDa. We examined recovery rates of several compounds in vitro. The recovery rates of proteins and peptides were between 7-28%, with the exceptions of substance P and insulin-like growth factor I. The recovery rates of monoamines and their metabolites were 22-40%. In in vivo studies, two major proteins with apparent molecular weights of 62 kDa and 12 kDa, and several minor proteins (28 kDa, 43 kDa, 52 kDa and 70 kDa) were detected by SDS-polyacrylamide gel electrophoresis in the dialysate from a probe implanted in the striatum of anesthetized rats. These results suggest that the newly developed, intracerebral microdialysis probe might be useful for investigating the dynamic changes of peptides and proteins in the central nervous system.</p> No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 121 3 2009 ARTプログラム（先進医学修練）による医学研究者育成―学部・卒後臨床研修をシームレスにつなぐ早期大学院教育― 189 193 EN Hideki Matsui No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 121 3 2009 Dravet 症候群患者に認められたカルシウムチャネル　機能異常を引き起こす CACNB4 遺伝子変異 149 156 EN Iori Ohmori Mamoru Ouchida Nobuyoshi Mimaki Teiichi Nishiki Kazuhito Tomizawa Hideki Matsui No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 91 3-4 1979 Illudin Sの作用機作の解折:Illudin SのL(929)線維芽細胞の表面形態に及ぼす影響について 461 467 EN Makoto Kuramitsu Toshifumi Itano Hideki Matsui Masaaki Tokuda Osamu Hatase Tetsuhide Murakami Isamu Nisida Hideo Hayashi The effects of Illudin S (a toadstool poison) on surface structures of L(929) mouse fibroblasts were studied by scanning electron microscopy. Illudin S in a concentration of 1 ng/ml strongly inhibited cell proliferation, but had no effect on surface structures of L cells through all the cell phases. These results indicate that the biological effects of Illudin S were not manifested by changes in the cytoskeletal system, the cell ability to attach to the substrate, of the surface structures of the cell. It is suggested that the cellular sensitive sites to Illudin S are inside the cell. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 94 3-4 1982 自然形質転換したL細胞(L-N)の増殖制御機構について 341 348 EN Hideki Matsui 1) The L-0 cell absolutely required serum for proliferation, but L-N cell grew rapidly in serum free medium. 2) L-N cell cultured in MEM medium with TPB reached high saturation density as in serum containing medium. 3) The growth rate of L-N cell in serum free medium was dependent on initial density of cell population just before logarithmic growing phase. 4) L-N cell rounded cell cycle from G-1 phase to S phase 4 hours faster than L-0 cell. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 117 2 2005 蛋白導入システムによる薬剤開発と膵島移植への応用 105 108 EN Naoya Kobayashi No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 00301558 118 3 2007 蛋白導入システム“Protein Transduction System”を利用したプロテインセラピーの発展と現状について― 悪性脳腫瘍に対する蛋白導入法の利用を中心に ― 205 208 EN No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 00301558 120 2 2008 11Rを用いた脳血管に対する新しい蛋白質導入法 129 133 EN Tomoyuki Ogawa Shigeki Ono Tomotsugu Ichikawa Seiji Arimitsu Keisuke Onoda Koji Tokunaga Kenji Sugiu Kazuhito Tomizawa Hideki Matsui Isao Date No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 00301558 119 1 2007 Cdk5によるインスリン分泌の制御機構 17 20 EN No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1982 自然形質転換したL細胞(L-N)の増殖制御機構について EN No potential conflict of interest relevant to this article was reported.