MDPI Acta Medica Okayama 2227-9059 12 2 2024 Morphometric Analysis of the Eye by Magnetic Resonance Imaging in MGST2-Gene-Deficient Mice 370 EN Chaomulige Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Toshihiko Matsuo Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kohei Sugimoto Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mary Miyaji Department of Medical Neurobiology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Osamu Hosoya Department of Medical Neurobiology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Masashi Ueda Department of Biofunctional Imaging Analysis, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Ryosuke Kobayashi Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University Takuro Horii Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University Izuho Hatada Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University Strabismus, a neuro-ophthalmological condition characterized by misalignment of the eyes, is a common ophthalmic disorder affecting both children and adults. In our previous study, we identified the microsomal glutathione S-transferase 2 (MGST2) gene as one of the potential candidates for comitant strabismus susceptibility in a Japanese population. The MGST2 gene belongs to the membrane-associated protein involved in the generation of pro-inflammatory mediators, and it is also found in the protection against oxidative stress by decreasing the reactivity of oxidized lipids. To look for the roles of the MGST2 gene in the development, eye alignment, and overall morphology of the eye as the possible background of strabismus, MGST2 gene knockout (KO) mice were generated by CRISPR/Cas9-mediated gene editing with guide RNAs targeting the MGST2 exon 2. The ocular morphology of the KO mice was analyzed through high-resolution images obtained by a magnetic resonance imaging (MRI) machine for small animals. The morphometric analyses showed that the height, width, and volume of the eyeballs in MGST2 KO homozygous mice were significantly greater than those of wild-type mice, indicating that the eyes of MGST2 KO homozygous mice were significantly enlarged. There were no significant differences in the axis length and axis angle. These morphological changes may potentially contribute to the development of a subgroup of strabismus. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 1424-8247 14 7 2021 The Effect of Cyanine Dye NK-4 on Photoreceptor Degeneration in a Rat Model of Early-Stage Retinitis Pigmentosa 694 EN Shihui Liu Department of Ophthalmology, Okayama University Graduate School of Interdisciplinary Science and Engineering in Health Systems Toshihiko Matsuo Department of Ophthalmology, Okayama University Graduate School of Interdisciplinary Science and Engineering in Health Systems Mary Miyaji Department of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Osamu Hosoya Department of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences The present study aimed to evaluate the effects of NK-4 on the apoptosis of photoreceptors in a rat model of retinitis pigmentosa and explore the mechanism underlying anti-apoptosis activity. The Royal College of Surgeons (RCS) rats received an intravitreous injection of NK-4 solution in the left eye and vehicle control in the right eye. Apoptosis was detected by TUNEL method in frozen sections of the eyes. The retinal tissues of the rats were dissected for RNA-seq analysis. Functional and pathway enrichment analyses of differentially expressed genes (DEGs) were performed by using Metascape and DAVID software. The expression levels of DEGs were confirmed by real-time quantitative PCR (RT-qPCR). The number of apoptotic cells decreased in the outer nuclear layer (ONL) and the thickness of the ONL was significantly thicker in the retina of NK-4-injected eyes, compared with control eyes. Five DEGs were identified by RNA-seq analysis, and Hmox1, Mt1, Atf5, Slc7a11, and Bdh2 were confirmed to be up-regulated by RT-qPCR. Functional and pathway enrichment analysis of the up-regulated genes showed that anti-apoptosis effects of NK-4 in the retina of RCS rats may be related to the pathways of metal ion homeostasis, negative regulation of neuron death, response to toxic substance, and pigment metabolic process. We found a potential mechanism of NK-4, providing a new viewpoint for the development of more therapeutic uses of NK-4 in the future. No potential conflict of interest relevant to this article was reported.

Nature Research Acta Medica Okayama 2045-2322 10 1 2020 Topoisomerase II beta targets DNA crossovers formed between distant homologous sites to induce chromatin opening 18550 EN Mary Miyaji Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ryohei Furuta Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Osamu Hosoya Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kuniaki Sano Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Norikazu Hara Department of Molecular Genetics, Bioresource Science Branch, Center for Bioresources, Brain Research Institute, Niigata University Ryozo Kuwano Department of Molecular Genetics, Bioresource Science Branch, Center for Bioresources, Brain Research Institute, Niigata University Jiyoung Kang Graduate School of Life Science, University of Hyogo Masaru Tateno Graduate School of Life Science, University of Hyogo Kimiko M. Tsutsui Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ken Tsutsui Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Type II DNA topoisomerases (topo II) flip the spatial positions of two DNA duplexes, called G- and T- segments, by a cleavage-passage-resealing mechanism. In living cells, these DNA segments can be derived from distant sites on the same chromosome. Due to lack of proper methodology, however, no direct evidence has been described so far. The beta isoform of topo II (topo II beta) is essential for transcriptional regulation of genes expressed in the final stage of neuronal differentiation. Here we devise a genome-wide mapping technique (eTIP-seq) for topo II beta target sites that can measure the genomic distance between G- and T-segments. It revealed that the enzyme operates in two distinctive modes, termed proximal strand passage (PSP) and distal strand passage (DSP). PSP sites are concentrated around transcription start sites, whereas DSP sites are heavily clustered in small number of hotspots. While PSP represent the conventional topo II targets that remove local torsional stresses, DSP sites have not been described previously. Most remarkably, DSP is driven by the pairing between homologous sequences or repeats located in a large distance. A model-building approach suggested that topo II beta acts on crossovers to unknot the intertwined DSP sites, leading to chromatin decondensation. No potential conflict of interest relevant to this article was reported.

Oxford University Press Acta Medica Okayama 0305-1048 42 14 2014 Nuclear dynamics of topoisomerase II beta reflects its catalytic activity that is regulated by binding of RNA to the C-terminal domain 9005 9020 EN Akihisa Onoda Osamu Hosoya Kuniaki Sano Kazuko Kiyama Hiroshi Kimura Shinji Kawano Ryohei Furuta Mary Miyaji Ken Tsutsui Kimiko M. Tsutsui DNA topoisomerase II (topo II) changes DNA topology by cleavage/re-ligation cycle(s) and thus contributes to various nuclear DNA transactions. It is largely unknown how the enzyme is controlled in a nuclear context. Several studies have suggested that its C-terminal domain (CTD), which is dispensable for basal relaxation activity, has some regulatory influence. In this work, we examined the impact of nuclear localization on regulation of activity in nuclei. Specifically, human cells were transfected with wild-type and mutant topo II beta tagged with EGFP. Activity attenuation experiments and nuclear localization data reveal that the endogenous activity of topo II beta is correlated with its subnuclear distribution. The enzyme shuttles between an active form in the nucleoplasm and a quiescent form in the nucleolus in a dynamic equilibrium. Mechanistically, the process involves a tethering event with RNA. Isolated RNA inhibits the catalytic activity of topo II beta in vitro through the interaction with a specific 50-residue region of the CTD (termed the CRD). Taken together, these results suggest that both the subnuclear distribution and activity regulation of topo II beta are mediated by the interplay between cellular RNA and the CRD. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1434-7229 16 3 2013 Behavior tests and immunohistochemical retinal response analyses in RCS rats with subretinal implantation of Okayama-University-type retinal prosthesis 343 351 EN Alamusi Toshihiko Matsuo Osamu Hosoya Kimiko M. Tsutsui Tetsuya Uchida We have developed a photoelectric dye-coupled polyethylene film as a prototype of retinal prosthesis, which we named Okayama University-type retinal prosthesis. The purposes of this study are to conduct behavior tests to assess vision in Royal College of Surgeons (RCS) rats that underwent subretinal implantation of the dye-coupled film and to reveal retinal response to the dye-coupled film by immunohistochemistry. Polyethylene films were made of polyethylene powder at refined purity, and photoelectric dyes were coupled to the film surface at higher density compared with the prototype. Either dye-coupled film or dye-uncoupled plain film used as a control was implanted subretinally from a scleral incision in both eyes of an RCS rat at 6 weeks of the age. Behavior tests 2, 4, 6, and 8 weeks after implantation were conducted by observing head turning or body turning in the direction consistent with clockwise or counterclockwise rotation of a black-and-white-striped drum around a transparent cage housed with the rat. After the behavior tests at 8 weeks, rats' eyes were enucleated to confirm subretinal implantation of the films and processed for immunohistochemistry. In the behavior tests, the number of head turnings consistent with the direction of the drum rotation was significantly larger in RCS rats with dye-coupled- compared with plain-film implantation [P < 0.05, repeated-measure analysis of variance (ANOVA), n = 7]. The number of apoptotic neurons was significantly smaller in eyes with dye-coupled- compared with plain-film implantation (P < 0.05, Mann-Whitney U test, n = 6). In conclusion, subretinal implantation of photoelectric dye-coupled films restored vision in RCS rats and prevented the remaining retinal neurons from apoptosis. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 56 4 2002 Localization of dynamin 2 in rat seminiferous tubules during the spermatogenic cycle. 205 209 EN Hiroki Iguchi Masami Watanabe Akihiro Kamitani Atsushi Nagai Osamu Hosoya Kimiko Tsutsui Hiromi Kumon Article 10.18926/AMO/31681 <p>Dynamin is a protein essential to endocytosis. Dynamin 2, a dynamin isoform, is expressed most intensely in testicular tissue; however, precise localization has never been studied. Therefore, we investigated the expression of dynamin 2 in rat testicular tissue using immunohistochemical methods, and discuss here the physiological function of this protein. Testicular tissues were obtained from Wistar rats at 10, 21 and 63 days of age. Immunohistochemistrical examination and Western blot analysis were conducted using dynamin 2 specific antibody. Western blot analysis showed that expression in 21- and 63-day-old rats was more intense than that in 10-day-old rats. Dynamin 2 expression was observed using immunohistochemical method in the seminiferous tubules of all rats. In the 63-day-old rats, the expression was intense, especially in spermatids in the earlier maturation stages and in spermatocytes, and was observed in Sertoli cells. However, in spermatids, the expression gradually declined as spermatids matured to spermatozoa. In the 21-day-old rats, the expression was evident in spermatocytes and Sertoli cells, but that in the 10-day-old rats was weak. Intense expression of dynamin 2 during spermatogenesis suggests that this protein plays an important role in this process.</p> No potential conflict of interest relevant to this article was reported.