MDPIActa Medica Okayama2227-90591222024Morphometric Analysis of the Eye by Magnetic Resonance Imaging in MGST2-Gene-Deficient Mice370ENChaomulige Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityToshihikoMatsuoGraduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityKoheiSugimotoGraduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityMaryMiyajiDepartment of Medical Neurobiology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityOsamuHosoyaDepartment of Medical Neurobiology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityMasashiUedaDepartment of Biofunctional Imaging Analysis, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityRyosukeKobayashiBiosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma UniversityTakuroHoriiBiosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma UniversityIzuhoHatadaBiosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma UniversityStrabismus, 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.Public Library of ScienceActa Medica Okayama1932-62031882023Selective DNA-binding of SP120 (rat ortholog of human hnRNP U) is mediated by arginine-glycine rich domain and modulated by RNAe0289599ENMaryMiyajiDepartment of Neurogenomics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityShinjiKawanoFaculty of Science, Department of Biochemistry, Okayama University of ScienceRyoheiFurutaDepartment of Neurogenomics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityEmiMurakamiFaculty of Science, Department of Biochemistry, Okayama University of ScienceShogoIkedaFaculty of Science, Department of Biochemistry, Okayama University of ScienceKimiko M.TsutsuiDepartment of Neurogenomics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKenTsutsuiDepartment of Neurogenomics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityA human protein heterogeneous ribonucleoprotein U (hnRNP U) also known as Scaffold attachment factor A (SAF-A) and its orthologous rat protein SP120 are abundant and multifunctional nuclear protein that directly binds to both DNA and RNA. The C-terminal region of hnRNP U enriched with arginine and glycine is essential for the interaction with RNA and the N-terminal region of SAF-A termed SAP domain has been ascribed to the DNA binding. We have reported that rat hnRNP U specifically and cooperatively binds to AT-rich DNA called nuclear scaffold/matrix-associated region (S/MAR) although its detailed mechanism remained unclear. In the present study analysis of hnRNP U deletion mutants revealed for the first time that a C-terminal domain enriched with Arg-Gly (defined here as 'RG domain') is predominantly important for the S/MAR-selective DNA binding activities. RG domain alone directly bound to S/MAR and coexistence with the SAP domain exerted a synergistic effect. The binding was inhibited by netropsin, a minor groove binder with preference to AT pairs that are enriched in S/MAR, suggesting that RG domain interacts with minor groove of S/MAR DNA. Interestingly, excess amounts of RNA attenuated the RG domain-dependent S/MAR-binding of hnRNP U. Taken together, hnRNP U may be the key element for the RNA-regulated recognition of S/MAR DNA and thus contributing to the dynamic structural changes of chromatin compartments.No potential conflict of interest relevant to this article was reported.MDPI AGActa Medica Okayama1424-82471472021The Effect of Cyanine Dye NK-4 on Photoreceptor Degeneration in a Rat Model of Early-Stage Retinitis Pigmentosa694ENShihuiLiuDepartment of Ophthalmology, Okayama University Graduate School of Interdisciplinary Science and Engineering in Health SystemsToshihikoMatsuoDepartment of Ophthalmology, Okayama University Graduate School of Interdisciplinary Science and Engineering in Health SystemsMaryMiyajiDepartment of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesOsamuHosoyaDepartment of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesThe 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 ResearchActa Medica Okayama2045-23221012020Topoisomerase II beta targets DNA crossovers formed between distant homologous sites to induce chromatin opening18550ENMaryMiyajiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityRyoheiFurutaGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityOsamuHosoyaGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKuniakiSanoGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityNorikazuHaraDepartment of Molecular Genetics, Bioresource Science Branch, Center for Bioresources, Brain Research Institute, Niigata UniversityRyozoKuwanoDepartment of Molecular Genetics, Bioresource Science Branch, Center for Bioresources, Brain Research Institute, Niigata UniversityJiyoungKangGraduate School of Life Science, University of HyogoMasaruTatenoGraduate School of Life Science, University of HyogoKimiko M.TsutsuiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKenTsutsuiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityType 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 PressActa Medica Okayama0305-104842142014Nuclear dynamics of topoisomerase II beta reflects its catalytic activity that is regulated by binding of RNA to the C-terminal domain90059020ENAkihisaOnodaOsamuHosoyaKuniakiSanoKazukoKiyamaHiroshiKimuraShinjiKawanoRyoheiFurutaMaryMiyajiKenTsutsuiKimiko M.TsutsuiDNA 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.岡山医学会Acta Medica Okayama0030-155812132009DNAトポイソメラーゼⅡβによる神経関連遺伝子の活性化機構143147ENKuniakiSanoMaryMiyajiM. KimikoTsutsuiKenTsutsuiNo potential conflict of interest relevant to this article was reported.