Language：English   Publisher：Cold Spring Harbor Laboratory  

<title>Abstract</title><sec><title>Background</title>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 placed far from each other on the same chromosome. However, no direct evidence for this to occur has been described so far due to lack of proper methodology.

</sec><sec><title>Results</title>The beta isoform of topo II (topo IIβ) is essential for transcriptional regulation of genes expressed in the final stage of neuronal differentiation. To elucidate the enzyme’s role in the process, here we devise a genome-wide mapping technique for topo IIβ target sites that can measure the genomic distance between G- and T-segments. It became clear 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 the DSP sites are intertwined or knotted and topo IIβ is engaged in unknotting reaction that leads to chromatin decondensation and gene regulation.

</sec><sec><title>Conclusions</title>When combined with categorized gene expression analysis, the model-based prediction of DSP sites reveals that DSP is one of the key factors for topo IIβ-dependency of neuronal gene regulation.

</sec>

DOI： 10.1101/484956

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

Photoelectric dye-coupled polyethylene film, designated Okayama University type-retinal prosthesis or OUReP (TM), generates light-evoked surface electric potentials and stimulates neurons. The dye-coupled films or plain films were implanted subretinally in both eyes of 10 Royal College of Surgeons rats with hereditary retinal dystrophy at the age of 6 weeks. Visual evoked potentials in response to monocular flashing light stimuli were recorded from cranially-fixed electrodes, 4 weeks and 8 weeks after the implantation. After the recording, subretinal film implantation was confirmed histologically in 7 eyes with dye-coupled films and 7 eyes with plain films. The recordings from these 7 eyes in each group were used for statistical analysis. The amplitudes of visual evoked potentials in the consecutive time points from 125 to 250 ms after flash were significantly larger in the 7 eyes with dye-coupled film implantation, compared to the 7 eyes with plain film implantation at 8 weeks after the implantation (P &lt; 0.05, repeated-measure ANOVA). The photoelectric dye-coupled polyethylene film, as retinal prosthesis, gave rise to visual evoked potential in response to flashing light.

DOI： 10.1007/s10047-016-0943-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT, INC  

Purpose: Our previous study demonstrated that photoelectric dye-coupled polyethylene film (Okayama University-type retinal prosthesis), which was implanted in subretinal space of the eyes of Royal College of Surgeons (RCS) rats, prevented retinal neurons from apoptotic death. In this study, we aimed to examine whether photoelectric dye itself would protect retinal neurons from apoptosis in RCS rats.
Methods: RCS rats received intravitreous injection of different concentrations of the dye in the left eye and housed under a 12-h light-dark cycle. Saline injection in the right eye served as control. In addition, RCS rats with dye injection were kept in 24-h daily dark condition. Sections were processed for terminal deoxynucleotidyl transferase-mediated fluorescein-conjugated-dUTP nick-end-labeling (TUNEL) assay and immunohistochemical staining of glial fibrillary acidic protein (GFAP) and protein kinase C alpha (PKC alpha).
Results: The number of TUNEL-positive cells significantly decreased in the retina of dye-injected eyes compared with those in saline-injected eyes (P = 0.0001, 2-factor analysis of variance [ANOVA]), under 12-h light-dark cycle. Significant decrease of TUNEL-positive cells was noted in the retina of rats with dye injection compared with those with saline injection, kept under 24-h dark condition (P = 0.0001, 2-factor ANOVA). Immunoreactive area for GFAP decreased significantly in the retina of dye-injected eyes compared with that in controls (P = 0.0001, 2-factor ANOVA), whereas immunoreactive area for PKCa increased significantly in the retina of dye-injected eyes compared with that in controls (P = 0.01, 2-factor ANOVA).
Conclusions: Photoelectric dye inhibits apoptotic death of photoreceptor cells in RCS rats and downregulates GFAP expression in retinal Muller cells. Photoelectric dye may be a candidate agent for neuroprotection in retinitis pigmentosa and other retinal diseases.

DOI： 10.1089/jop.016.0093

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DOI： 10.1007/s10577-014-9404-1

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Language：Japanese   Publisher：一般社団法人 日本泌尿器科学会  

DOI： 10.5980/jpnjurol.91.372_1

CiNii Article

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Language：Japanese   Publishing type：Research paper, summary (national, other academic conference)  

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Language：English   Publisher：(Cold Harbor Laboratory, CSHL)  

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 placed far from each other on the same chromosome. However, no direct evidence for this to occur has been described so far due to lack of proper methodology.
(Results)The beta isoform of topo II (topo IIβ) is essential for transcriptional regulation of genes expressed in the final stage of neuronal differentiation. To elucidate the enzyme’s role in the process, here we devise a genome-wide mapping technique for topo IIβ target sites that can measure the genomic distance between G- and T-segments. It became clear 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 the DSP sites are intertwined or knotted and topo IIβ is engaged in unknotting reaction that leads to chromatin decondensation and gene regulation.
(Conclusions) When combined with categorized gene expression analysis, the model-based prediction of DSP sites reveals that DSP is one of the key factors for topo IIβ-dependency of neuronal gene regulation.

DOI： 10.1101/484956

Language：English   Publishing type：Research paper, summary (international conference)  

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Language：Japanese   Publishing type：Research paper, summary (national, other academic conference)  

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Language：English   Publishing type：Research paper, summary (international conference)  

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Language：English   Publishing type：Research paper, summary (international conference)  

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Language：English   Publishing type：Research paper, summary (international conference)  

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Language：English   Publisher：SPRINGER JAPAN KK  

Photoelectric dye-coupled polyethylene film, designated Okayama University-type retinal prosthesis or OUReP (TM), generates light-evoked surface electric potentials and stimulates neurons. In this study, the vision was assessed by behavior tests in aged hereditary retinal dystrophic RCS rats with OUReP (TM), retinal apoptosis and electroretinographic responses were measured in dystrophic eyes with OUReP (TM). The dye-coupled films, or plain films as a control, were implanted in subretinal space of RCS rats. On behavior tests, RCS rats with dye-coupled films, implanted at the old age of 14 weeks, showed the larger number of head-turning, consistent with clockwise and anticlockwise rotation of a surrounding black-and-white-striped drum, compared with rats with plain films, under the dim (50 lux) and bright (150 lux) conditions in the observation period until the age of 22 weeks (n = 5, P &lt; 0.05, repeated-measure ANOVA). The number of apoptotic cells in retinal sections at the site of dye-coupled film implantation was significantly smaller, compared with the other retinal sites, neighboring the film, or opposite to the film, 5 months after film implantation at the age of 6 weeks (P = 0.0021, Friedman test). The dystrophic eyes of RCS rats with dye-coupled films showed positive responses to maximal light stimulus at a significantly higher rate, compared with the eyes with no treatment (P &lt; 0.05, Chi-square test). Electroretinograms in normal eyes of Wistar rats with dye-coupled or plain films showed significantly decreased amplitudes (n = 14, P &lt; 0.05, repeated-measure ANOVA). In conclusions, vision was maintained in RCS rats with dye-coupled films implanted at the old age. The dystrophic eyes with dye-coupled films showed electroretinographic responses. Five-month film implantation caused no additional retinal changes.

DOI： 10.1007/s10047-015-0825-1

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ASSOC RESEARCH VISION OPHTHALMOLOGY INC  

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Language：English   Publisher：OXFORD UNIV PRESS  

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.

DOI： 10.1093/nar/gku640

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Language：English   Publisher：SPRINGER JAPAN KK  

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 &lt; 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 &lt; 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.

DOI： 10.1007/s10047-013-0697-1

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Language：English   Publisher：OXFORD UNIV PRESS  

Lens epithelium-derived growth factor (LEDGF) or p75 is a co-activator of general transcription and also involved in insertion of human immunodeficiency virus type I (HIV-1) cDNA into host cell genome, which occurs preferentially to active transcription units. These phenomena may share an underlying molecular mechanism in common. We report here that LEDGF/p75 binds negatively supercoiled DNA selectively over unconstrained DNA. We identified a novel DNA-binding domain in the protein and termed it &apos;supercoiled DNA-recognition domain&apos; (SRD). Recombinant protein fragments containing SRD showed a preferential binding to supercoiled DNA in vitro. SRD harbors a characteristic cluster of lysine and glutamic/aspartic acid residues. A polypeptide mimicking the cluster (K(9)E(9)K(9)) also showed this specificity, suggesting that the cluster is an essential element for the supercoil recognition. eGFP-tagged LEDGF/p75 expressed in the nucleus distributed partially in transcriptionally active regions that were identified by immunostaining of methylated histone H3 (H3K4me3) or incorporation of Br-UTP. This pattern of localization was observed with SRD alone but abolished if the protein lacked SRD. Thus, these results imply that LEDGF/p75 guides its binding partners, including HIV-1 integrase, to the active transcription site through recognition of negative supercoils generated around it.

DOI： 10.1093/nar/gkr088

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Language：English   Publisher：SPRINGER TOKYO  

We have designed a new type of retinal prosthesis using polyethylene films coupled with photoelectric dye molecules that absorb light and convert photon energy to electric potentials. An extruded-blown film of high-density polyethylene was used as the original polyethylene film. Recrystallized film was made by recrystallization from the melting of the original polyethylene film. A photoelectric dye,2-[2-[4-(dibutylamino)phenyl]ethenyl]-3-carboxymethylbenzothiazolium bromide, was coupled to the two types of polyethylene films through amide linkages. Samples of the original dye-coupled film, the dye-coupled recrystallized film, and the dye-uncoupled plain film were implanted in the subretinal space of normal adult rats. Frozen sections were cut from the eyes enucleated at 1 week or 1 month and were either stained with hematoxylin and eosin, stained immunohistochemically for glial fibrillary acidic protein (GFAP), or processed for in situ apoptosis detection. The results revealed that retinal tissue damage was negligible with no inflammatory cells and few apoptotic cells. GFAP was significantly up-regulated in retinal sites with the implantation of all types of polyethylene films at 1 week, compared with the adjacent retinal sites (P &lt; 0.005, analysis of variance). The GFAP up-regulation was also present at 1 month for the plain film and dye-coupled recrystallized film (P &lt; 0.05). Glial cell encirclement around the films increased significantly between 1 week and 1 month (P = 0.023, two-factor analysis of variance) but was not significantly different among the three types of polyethylene films (P = 0.4531). These results showed evidence of glial reactions to the photoelectric dye-coupled polyethylene films implanted into the subretinal space of rat eyes and also proved their basic biological safety.

DOI： 10.1007/s10047-007-0398-8

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：BLACKWELL PUBLISHING  

Mammalian DNA topoisomerase II beta is a type 11 DNA topoisomerase that catalyses topological transformations of genomic DNA by the transport of one DNA double helix through another. The II beta enzyme is highly expressed in cells that have undergone the final cell division and committed to differentiate into neuronal cells. The Ilp enzyme in the differentiating neuronal cells is located in the nucleoplasm and is actively engaged in its catalytic reaction in vivo. When enzyme action is interfered with a specific inhibitor in vitro, transcriptional induction of a subset of genes fails to occur during neuronal differentiation. Detailed analyses of developing rat cerebellum and the cerebrum of mice with disrupted Ilp genes have revealed that DNA topoisomerase II beta is necessary for the developmentally regulated expression of certain genes in cells committed to a neuronal fate after the final division. Herein, we review a dynamic aspect of DNA topoisomerase Ilp in the brain with special emphasis on developing cerebellar neurons.

DOI： 10.1111/j.1447-073x.2006.00146.x

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：BLACKWELL PUBLISHING  

Mammalian DNA topoisomerase II beta is a type 11 DNA topoisomerase that catalyses topological transformations of genomic DNA by the transport of one DNA double helix through another. The II beta enzyme is highly expressed in cells that have undergone the final cell division and committed to differentiate into neuronal cells. The Ilp enzyme in the differentiating neuronal cells is located in the nucleoplasm and is actively engaged in its catalytic reaction in vivo. When enzyme action is interfered with a specific inhibitor in vitro, transcriptional induction of a subset of genes fails to occur during neuronal differentiation. Detailed analyses of developing rat cerebellum and the cerebrum of mice with disrupted Ilp genes have revealed that DNA topoisomerase II beta is necessary for the developmentally regulated expression of certain genes in cells committed to a neuronal fate after the final division. Herein, we review a dynamic aspect of DNA topoisomerase Ilp in the brain with special emphasis on developing cerebellar neurons.

DOI： 10.1111/j.1447-073x.2006.00146.x

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

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Language：English   Publisher：WILEY-BLACKWELL  

In the vertebrate retina, presynaptic terminals of photoreceptors and bipolar cells form ribbon synapses and release neurotransmitter continuously. Endocytic machinery in the ribbon synapse is likely to differ from that in conventional synapses because of the much higher rate of synaptic vesicle recycling. However, protein components of the ribbon synapse identified so far are quite similar to those of the conventional synapses. Recently we identified amphiphysin I splice variants, termed amphiphysin Ir, that are transcribed specifically in retina from the authentic amphiphysin I gene [Y Terada et al. (2002) FEBS Lett., 519, 185-190]. Amphiphysin I is a nerve terminal-enriched protein, and involved in synaptic vesicle endocytosis as heterodimer with amphiphysin II, an isoform of amphiphysin I. We report here that the retina-specific amphiphysin Ir is expressed exclusively in the ribbon synapse and not in conventional synapses. This is the first endocytosis-related, ribbon synapse-specific protein identified in the retina. By immunoprecipitation and double-immunolabelling, amphiphysin Ir was shown to be associated not only with amphiphysin 11, but also with dynamin, clathrin and alpha-adaptin that are involved in synaptic vesicle recycling. The results suggest that endocytosis of the synaptic vesicle membrane in retinal ribbon synapses proceeds through a pathway similar to the one that is used in conventional synapses, although amphiphysin Ir is substituted for amphiphysin I. Amphiphysin Ir may play an essential role in the avid endocytic activity of ribbon synapses by associating with yet unknown protein partner(s) through its large insertional domain, which is absent from the conventional amphiphysin I.

DOI： 10.1111/j.1460-9568.2004.03340.x

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Language：English   Publisher：OKAYAMA UNIV MED SCHOOL  

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.

DOI： 10.18926/AMO/31681

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Language：English   Publisher：ELSEVIER SCIENCE BV  

Using RT-PCR-based cDNA cloning, we identified novel splice variants of amphiphysin I, termed amph Ir, that are expressed specifically in retina. In comparison with the prototype amphiphysin I, amph Ir contained two novel insertions (inserts A and B) and one deletion. Insert A is only 9 bp in length but appears to be a determinant for the retina-specific expression. In contrast, insert B is a large domain of 1740 bp and two shorter transcripts with 3'-truncated insert B were also expressed. All the insert sequences were present as unidentified exons in the amphiphysin I gene on human chromosome 7. Western blot analysis of various rat tissues with anti-insert B antibody confirmed the presence and tissue specificity of the variant proteins. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(02)02763-1

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DOI： 10.1016/S0168-0102(02)00084-6

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Amphiphysin I is a protein concentrated in nerve terminals and involved in the endocytosis of synaptic vesicle membrane. We show here that amphiphysin I is expressed in the rat testis, localized exclusively in the Sertoli cells. In the postnatal testicular development, expression of amphiphysin I was not evident at birth, but became significant at postnatal day 15 (P15), coinciding with the onset of spermatogenesis. The expression level of amphiphysin I increased 10-fold between P15 and P25 to reach the adult level. In adult testes reversibly damaged by ethane dimethane sulphonate administration, expression of amphiphysin I did not change following the damage, whereas the protein was transiently converted into its phosphorylated form. The increase in levels of phosphorylated amphiphysin I was closely associated with the severe histological damage to germ cells. The present findings suggest that amphiphysin I in Sertoli cells is involved in spermatogenesis, probably through endocytic processes. (C) 2001 Academic Press.

DOI： 10.1006/bbrc.2001.5650

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Amphiphysin I is a protein concentrated in nerve terminals and involved in the endocytosis of synaptic vesicle membrane. We show here that amphiphysin I is expressed in the rat testis, localized exclusively in the Sertoli cells. In the postnatal testicular development, expression of amphiphysin I was not evident at birth, but became significant at postnatal day 15 (P15), coinciding with the onset of spermatogenesis. The expression level of amphiphysin I increased 10-fold between P15 and P25 to reach the adult level. In adult testes reversibly damaged by ethane dimethane sulphonate administration, expression of amphiphysin I did not change following the damage, whereas the protein was transiently converted into its phosphorylated form. The increase in levels of phosphorylated amphiphysin I was closely associated with the severe histological damage to germ cells. The present findings suggest that amphiphysin I in Sertoli cells is involved in spermatogenesis, probably through endocytic processes. (C) 2001 Academic Press.

DOI： 10.1006/bbrc.2001.5650

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Language：English   Publisher：WILEY-BLACKWELL  

In mammalian cells, two isoforms of DNA topoisomerase II (topo II alpha and topo II beta) have been identified. Topo II alpha is essential in mitotic cells, whereas the function of topo II beta remains unclear. In the present study, we investigated the developmental control of topo II isoforms in two different neuronal lineages, cerebellar Purkinje cells and granule cells, by immunohistochemical analysis with isoform-specific monoclonal antibodies. As expected, proliferating cells in the neuroepithelium and in the external germinal layer (EGL) were topo II alpha immunopositive. The migrating as well as differentiating Purkinje cells and granule cells showed an enhanced topo II beta immunoreactivity. The postmitotic granule cells in the post natal EGL showed an abrupt transition of expressed topo II isoforms from II alpha to II beta. The transition was clearly coincident with the completion of final cell division and the initiation of terminal differentiation because no increase of the topo II beta immunoreactivity was observed in the spreading EGL cells that are still in the cell division cycle. The topo II beta signal was detected in both nucleoplasm and nucleolus of differentiating cells. However, the nucleoplas mic signal decreased significantly as the cells reached terminal differentiation. The residual topo II beta in nucleoli was shown to occupy an unique location with respect to other nucleolar proteins, nucleolin and DNA topoisomerase I. Our findings indicate that both Purkinje cells and granule cells express the topo II isoforms in a similar timing during the cerebellar development and also suggest that topo II beta localized in nucleoplasm is the functional entity involved in neuronal differentiation. J. Comp. Neurol. 431:228-239, 2001. (C) 2001 Wiley-Liss, Inc.

DOI： 10.1002/1096-9861(20010305)431:2<228::AID-CNE1067>3.0.CO;2-M

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Language：English   Publisher：WILEY-BLACKWELL  

In mammalian cells, two isoforms of DNA topoisomerase II (topo II alpha and topo II beta) have been identified. Topo II alpha is essential in mitotic cells, whereas the function of topo II beta remains unclear. In the present study, we investigated the developmental control of topo II isoforms in two different neuronal lineages, cerebellar Purkinje cells and granule cells, by immunohistochemical analysis with isoform-specific monoclonal antibodies. As expected, proliferating cells in the neuroepithelium and in the external germinal layer (EGL) were topo II alpha immunopositive. The migrating as well as differentiating Purkinje cells and granule cells showed an enhanced topo II beta immunoreactivity. The postmitotic granule cells in the post natal EGL showed an abrupt transition of expressed topo II isoforms from II alpha to II beta. The transition was clearly coincident with the completion of final cell division and the initiation of terminal differentiation because no increase of the topo II beta immunoreactivity was observed in the spreading EGL cells that are still in the cell division cycle. The topo II beta signal was detected in both nucleoplasm and nucleolus of differentiating cells. However, the nucleoplas mic signal decreased significantly as the cells reached terminal differentiation. The residual topo II beta in nucleoli was shown to occupy an unique location with respect to other nucleolar proteins, nucleolin and DNA topoisomerase I. Our findings indicate that both Purkinje cells and granule cells express the topo II isoforms in a similar timing during the cerebellar development and also suggest that topo II beta localized in nucleoplasm is the functional entity involved in neuronal differentiation. J. Comp. Neurol. 431:228-239, 2001. (C) 2001 Wiley-Liss, Inc.

DOI： 10.1002/1096-9861(20010305)431:2<228::AID-CNE1067>3.0.CO;2-M

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

We have previously reported that glia-like folliculo-stellate (FS) cells in the rat anterior pituitary may be involved in heterotopic differentiation of striated muscles which appear during pituitary cell culture. In the present immunocytochemical study, cytological alterations of FS cells in vitro were investigated by using antibodies to marker proteins (i.e., S-100 and vimentin) for FS cells. Expression of skeletal muscle-specific MyoD1 detected by immunocytochemistry, enabled identification of myogenic cells at a stage when they were still unicellular. Elongated myoblasts containing MyoD1-positive nuclei were found as early as the fifth day of monolayer culture of pituitary cells. The double immunostaining technique showed that some of these myoblasts reacted with antiserum to S-100. Although all of the myoblasts were immunoreactive to vimentin, this marker protein was unable to identify FS cells in vitro because rapidly proliferating fibroblasts were also immunoreactive to vimentin. Since the antiserum to S-100 that we used did not react with already differentiated striated muscle fibers, the demonstration of both MyoD1 and S-100-immunoreactive myoblasts in our pituitary cultures suggests that these myogenic cells are derived from FS cells. The present study does not rule out the possibility that fibroblasts, whose origin is presently unknown, are also involved in myogenesis in pituitary cultures.

DOI： 10.2108/zsj.15.247

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Language：English   Publisher：Zoological Society of Japan  

We have previously reported that glia-like folliculo-stellate (FS) cells in the rat anterior pituitary may be involved in heterotopic differentiation of striated muscles which appear during pituitary cell culture. In the present immunocytochemical study, cytological alterations of FS cells in vitro were investigated by using antibodies to marker proteins (i.e., S-100 and vimentin) for FS cells. Expression of skeletal muscle-specific MyoD1 detected by immunocytochemistry, enabled identification of myogenic cells at a stage when they were still unicellular. Elongated myoblasts containing MyoD1-positive nuclei were found as early as the fifth day of monolayer culture of pituitary cells. The double immunostaining technique showed that some of these myoblasts reacted with antiserum to S-100. Although all of the myoblasts were immunoreactive to vimentin, this marker protein was unable to identify FS cells in vitro because rapidly proliferating fibroblasts were also immunoreactive to vimentin. Since the antiserum to S-100 that we used did not react with already differentiated striated muscle fibers, the demonstration of both MyoD1 and S-100-immunoreactive myoblasts in our pituitary cultures suggests that these myogenic cells are derived from FS cells. The present study does not rule out the possibility that fibroblasts, whose origin is presently unknown, are also involved in myogenesis in pituitary cultures.

DOI： 10.2108/zsj.15.247

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Language：English   Publisher：SOC STUDY AMPHIBIANS REPTILES  

Intersexual differences and annual cytological changes in presumptive gonadotropin-producing cells (gonadotropes) were examined immunohistochemically in mid-sagittal sections of the pituitary gland of adult Hynobius nigrescens by the use of a monoclonal antibody against bullfrog luteinizing hormone beta-subunit (LH beta). Total immunoreactive areas for LH beta and profiles of the pars distalis in the mid-sagittal plane of the gland were estimated with an image analyzer. Gonadotropes consistently were distributed in the pars distalis near the median eminence and the pars intermedia in both sexes throughout the year; an intersexual difference was found in their distribution pattern. Gonadotrope cells other than these consistent ones distributed in the peripheral region of the pars distalis frequently were observed in females, but not in males. The proportion of gonadotropes among all pars distalis cells, as well as the mean area and number of gonadotropes, was significantly greater in females than in males throughout the year. In males that had a typical aquatic-phase morph and actively exhibited reproductive behavior, both area and number of gonadotropes, as well as profile estimates of pars distalis size, were the greatest of all monthly values. In females, gonadotrope cells increased in area and number from July-January during the terrestrial phase of the nonbreeding season. This increase was concurrent with the development of ovaries. This consecutive increase and development may explain the absence of a second oogenic phase during early spring.

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Language：English   Publisher：SOC STUDY AMPHIBIANS REPTILES  

Intersexual differences and annual cytological changes in presumptive gonadotropin-producing cells (gonadotropes) were examined immunohistochemically in mid-sagittal sections of the pituitary gland of adult Hynobius nigrescens by the use of a monoclonal antibody against bullfrog luteinizing hormone beta-subunit (LH beta). Total immunoreactive areas for LH beta and profiles of the pars distalis in the mid-sagittal plane of the gland were estimated with an image analyzer. Gonadotropes consistently were distributed in the pars distalis near the median eminence and the pars intermedia in both sexes throughout the year; an intersexual difference was found in their distribution pattern. Gonadotrope cells other than these consistent ones distributed in the peripheral region of the pars distalis frequently were observed in females, but not in males. The proportion of gonadotropes among all pars distalis cells, as well as the mean area and number of gonadotropes, was significantly greater in females than in males throughout the year. In males that had a typical aquatic-phase morph and actively exhibited reproductive behavior, both area and number of gonadotropes, as well as profile estimates of pars distalis size, were the greatest of all monthly values. In females, gonadotrope cells increased in area and number from July-January during the terrestrial phase of the nonbreeding season. This increase was concurrent with the development of ovaries. This consecutive increase and development may explain the absence of a second oogenic phase during early spring.

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

A major objective of the present study was to examine the possibility that non-granular folliculo-stellate (FS) cells in the rat anterior pituitary are involved in the myogenesis that occurs during pituitary cell culture. Enzymatically dissociated anterior pituitary cells were fractionated by use of the Percoll gradient method. The proportion of FS cells was 5.8% on average before cell fractionation. After employing the Percoll gradient procedure, FS cells were enriched to a ratio of 12.2%. Three of five cell fractions were separately cultured, and the incidence of striated muscle fibers was quantitatively investigated. There was a good correlation between the numbers of muscle fibers and the proportions of FS cells in the fractions obtained from the Percoll gradient. These results suggest that FS cells are the cells that transform into striated muscles in pituitary monolayer cultures.

DOI： 10.2108/zsj.14.141

Web of Science

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Language：English   Publisher：Zoological Society of Japan  

A major objective of the present study was to examine the possibility that non-granular folliculostellate (FS) cells in the rat anterior pituitary are involved in the myogenesis that occurs during pituitary cell culture. Enzymatically dissociated anterior pituitary cells were fractionated by use of the Percoll gradient method. The proportion of FS cells was 5.8% on average before cell fractionation. After employing the Percoll gradient procedure, FS cells were enriched to a ratio of 12.2%. Three of five cell fractions were separately cultured, and the incidence of striated muscle fibers was quantitatively investigated. There was a good correlation between the numbers of muscle fibers and the proportions of FS cells in the fractions obtained from the Percoll gradient. These results suggest that FS cells are the cells that transform into striated muscles in pituitary monolayer cultures.

DOI： 10.2108/zsj.14.141

Scopus

PubMed

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Event date： 2019.1.23 - 2019.1.25

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

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Event date： 2018.4.29 - 2018.5.3

Language：English   Presentation type：Poster presentation  

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Language：English  

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Language：English  

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Language：English   Presentation type：Oral presentation (invited, special)  

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