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.

Okayama University Medical School Acta Medica Okayama 0386-300X 46 6 1992 Detection and analyses by gel electrophoresis of cisplatin-mediated DNA damage. 427 434 EN Bo Zhang Shuji Seki Kosuke Akiyama Ken Tsutsui Ting Li Kazutaka Nagao Article 10.18926/AMO/32639 DNA damage induced by cis-diamminedichloroplatinum (II) (cisplatin: cis-DDP), an anticancer drug, was studied in vitro by monitoring the drug-induced conformational change of pUC18 plasmid DNA, the sensitivity to some restriction enzymes of the damaged DNA and the sequence-dependent termination of DNA synthesis caused by cisplatin. Closed circular, superhelical pUC18 DNA was treated at 37 degrees C for 16 h with various concentrations of cisplatin. Cisplatin-dose-dependent conformational change due to unwinding of the treated DNA was detected by agarose gel electrophoresis. To analyze the base-specificity of the cisplatin damage, the measurement for sensitivity of cisplatin-treated DNA to various types of restriction enzyme and sequence gel analysis of the treated DNA were conducted. The results suggested that cisplatin attacked preferentially the sequence of GG > AG > GNG in the order. In the present assay condition, the cisplatin/DNA nucleotide ratios required for the DNA damage detection were roughly 0.025 for the conformational analysis, 0.001 or more for the restriction enzyme analysis, and less than 0.001 for the sequence gel analysis. By using the present method, it was demonstrated that the cisplatin-mediated DNA damage was inhibited by NaCl, KCl, CaCl2 or MgCl2 at their nearly physiological concentrations, and by reducing agents such as thiourea and 2-mercaptoethanol in the reaction mixture. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 37 4 1983 Susceptibility of Rous sarcoma virus-specific sequences integrated into SR-C3H/He mouse ascites sarcoma cell chromatin to DNase I and DNase II. 283 289 EN Kimiko Tstsui Ken Tsutsui Takuzo Oda Article 10.18926/AMO/32389 The susceptibility of Rous sarcoma virus (RSV) genomes integrated in mouse ascites sarcoma cells (SR-C3H/He cells) to DNase I and DNase II was investigated. Approximately half of the viral sequences were sensitive to DNase I and DNase II when 17% and 7.4% of the chromatin DNA was rendered acid soluble, respectively. The results suggest that newly acquired exogenous proviral sequences are integrated into both transcriptionally active and inactive regions of chromatin in cells lacking related endogenous viral sequences. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 45 2 1991 Peplomycin-induced DNA repair synthesis in permeable mouse ascites sarcoma cells. 89 94 EN Bo Zhang Sekiko Watanabe Kosuke Akiyama Ting Li Keisuke Fukushima Ken Tsutsui Shuji Seki Article 10.18926/AMO/32188 DNA repair synthesis induced in permeable mouse ascites sarcoma cells by peplomycin, an antitumor antibiotic, was studied. Mouse ascites sarcoma (SR-C3H/He) cells were permeabilized with a low concentration of Triton X-100 in an isotonic condition. Permeable cells were treated with an appropriate concentration of peplomycin to introduce single-strand breaks in permeable cell DNA. DNA repair synthesis in peplomycin-treated permeable cells was measured by incubating the cells with four deoxynucleoside triphosphates in an appropriate buffer system. The DNA repair synthesis was enhanced by ATP and NaCl at near physiological concentrations. More than 90% of DNA synthesis in the present system depended on the peplomycin-treatment. The repair nature of the DNA synthesis was confirmed by a BrdUMP density shift technique. The repair patches were largely completed and ligated in the presence of ATP. Analyses using selective inhibitors for DNA polymerases showed that both DNA polymerase Beta and aphidicolin-sensitive DNA polymerases (DNA polymerase alpha and/or delta) were involved in the repair DNA synthesis. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 33 2 1979 Partial purification and properties of bovine heart catalase. 103 111 EN Ken Tsutsui Osamu Hatase Takuzo Oda Article 10.18926/AMO/32055 Catalase was partially purified (about 380-fold purification) from the post-mitochondrial supernatant of bovine heart and compared with catalases from bovine erythrocytes and bovine liver. The electrophoretic mobility in polyacrylamide gel (pH 8.0) of heart catalase was the same as that of erythrocyte catalase and was smaller than that of the liver enzyme. The heart catalase was indistinguishable from erythrocyte catalase in regard to the molecular weights of subunit polypeptides, the inhibition patterns produced by several catalase inhibitors, and specific activity. The pH-activity curve of heart catalase consisted of a characteristic biphasic pattern with a peak at pH 7.5 and a shoulder at pH 10. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 59 4 2005 Dynamic view of the nuclear matrix. 113 120 EN Kimiko M. Tsutsui Kuniaki Sano Ken Tsutsui Review 10.18926/AMO/31953 The nuclear matrix is an operationally defined nuclear skeletal structure that is believed to be involved in many nuclear functions including DNA replication, transcription, repair, and prem RNA processing/transport. Until relatively recently, the nuclear matrix was thought to be a rigid and static structure, but it is now thought to be dynamic. This paradigm shift was based in part on the tracking of the intranuclear movement of proteins tagged with fluorochromes. In this review, we attempt to redefine the nuclear matrix in light of recent findings and describe some useful techniques for the dynamic analysis of nuclear function. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 53 6 1999 Genomic structure of the rat major AP endonuclease gene (Apex) with an adjacent putative O-sialoglycoprotease gene (Prsmg1/Gcpl1) and a processed Apex pseudogene (Apexp1). 245 252 EN Ming Yao Kosuke Akiyama Yunshan Tan Altaf Hossain Sarker Shogo Ikeda Shahjalal Shafiul Alam Ken Tsutsui Michihiro C Yoshida Shuji Seki Article 10.18926/AMO/31622 Genomic sequencing and chromosomal assignment of the gene encoding rat APEX nuclease, a multifunctional DNA repair enzyme, were performed. An active Apex gene and a processed pseudogene were isolated from a rat genomic library. The active Apex gene consists of 5 exons and 4 introns spanning 2.1 kb. The putative promoter region of the Apex gene lacks the typical TATA box, but contains CAAT boxes and a CpG island having putative binding sites for several transcription factors, such as Sp1, AP-2, GATA-1 and ATF. A putative O-sialoglycoprotease (a homologue of Pasteurella haemolytica glycoprotease, gcp; abbreviated as Prsmg1/Gcpl1) gene consisting of 11 exons and 10 introns spanning 7.3 kb lies immediately adjacent to the Apex gene in a 5'-to-5' orientation. The Apex gene locus was mapped to rat chromosome 15p12 using in situ hybridization. The processed pseudogene (designated as rat Apexp1) has a nucleotide sequence 87.1% identical to that of the rat Apex cDNA, although several stop codons interrupting the coding sequences and multiple nucleotide deletions were observed. The Apexp1 is located in an inactive LINE sequence. Calculation of nucleotide substitution rates suggests that the immediate, active progenitor of Apexp1 arose 23 million years ago and that the non-functionalization occurred 15 million years ago. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 39 2 1985 Difference in the homology of two nuclear nonhistone protein fractions as compared by polyacrylamide gel electrophoresis. 99 104 EN Ken Tsutsui Kimiko Tsutsui Koji Aoyama Takuzo Oda Article 10.18926/AMO/31512 The extent of homology between two protein fractions was compared by simple electrophoretic analysis. Nuclear proteins of several rodent cells of different origins were fractionated into acid-soluble and acid-insoluble fractions. The two protein fractions were subjected to polyacrylamide gel electrophoresis in separate gel systems, and protein bands with identical mobilities were sought either in all possible combinational pairs of cell types or in all cell types. The paired and overall homology indices calculated from these data and chi-square testing of the results indicated that acid-soluble nuclear nonhistone proteins are more homologous than acid-insoluble nuclear proteins. Several factors which might have affected the results were discussed. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 30 3 1976 The presence of phosphate-binding protein in inner mitochondrial membrane 147 152 EN Osamu Hatase Ken Tsutsui Takuzo Oda Article 10.18926/AMO/31426 Phosphate-binding protein(s) was found in the inner mitochondrial membrane of calf heart by Sephadex G-200 and G-25 gel filtration. The binding activity was inhibited by N-ethylmaleimide and competed by a large amount of cold phosphate. The amount of phosphate bound to the fraction was 29 nmoles per mg of protein. Affinity chromatography with phosphate-bound Sepharose 4B confirmed the presence of phosphate-binding protein(s) in the active fraction of mitochondrial membrane fractionated by gel filtration. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 43 3 1989 Unique features of monoclonal IgG2b in the cleavage reaction with pepsin. 135 141 EN Hiroshi Sumii Ken Tsutsui Masao Hatsushika Hajime Inoue Gozo Tanabe Takuzo oda Article 10.18926/AMO/30889 Preparations of IgG2b purified from several mouse hybridoma clones were highly susceptible, compared to other subclasses, to peptic digestion under conditions usually used to prepare F (ab')2 fragments. Analyses of the digestion products revealed that no F (ab')2 was produced and that the main product was a Fab-like fragment. Demonstration of the hinge disulfides in the Fc portion clearly indicated that in IgG2b the primary peptic cleavage occurs on the NH2-terminal side of the inter-heavy chain disulfide bridge. The resulting Fab failed to bind with antigen, suggesting the importance of the CH1-hinge region in maintaining the native conformation of the antigen-binding site. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 43 2 1989 Rapid purification of squirrel monkey retrovirus-H major gag protein by high performance liquid chromatography. 127 129 EN Shogo Ikeda Ken Tsutsui Masao Hatsushika Sekiko Watanabe Takuzo Oda Article 10.18926/AMO/30869 The major gag protein (p34) of squirrel monkey retrovirus-H was purified in one chromatographic step by anion-exchange high performance liquid chromatography. The virus in a crude fraction was disrupted with Brij 35 in the presence of three kinds of protease inhibitors. The soluble virus lysate was injected into a Polyanion SI column, and p34 was eluted with a linear salt gradient. The recovery of the protein was about 60%. The purified p34 was nearly homogenous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 31 5 1977 Role of hydrophobic interaction in hapten-antibody binding 289 294 EN Ken Tsutsui Noriko Koide Jun Tomoda Hideo Hayashi Osamu Hatase Takuzo Oda Article 10.18926/AMO/30833 The precipitation reaction of bovine serum albumin coupled with p-azophenylleucine with homologous antibody was inhibited by several structurally related haptens. The isobutyl group substituent on alpha-carbon atom of the leucine residue contributed more than -5.8 Kcal/mol to the free energy of binding. This value was consistent with the free energy change expected from the transfer of n-butane from an aqueous environment to liquid n-butane. The observed contribution was explained, in terms of the hydrophobic interaction of the isobutyl group with the antigen binding site of the antibody molecule. These results were also compared with other hapten-antibody systems. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 51 4 1997 cDNA cloning, sequence analysis and expression of a mouse 44-kDa nuclear protein copurified with DNA repair factors for acid-depurinated DNA 195 206 EN Yuko Nakagawa Sekiko Watanabe Kosuke Akiyama Altaf H Sarker Ken Tsutsui Hajime Inoue Shuji Seki Article 10.18926/AMO/30763 We purified a 44-kDa nuclear protein from salt-extract of permeable mouse ascites sarcoma cells in an effort to isolate factors involved in the repair of acid-depurinated DNA. It was copurified with a major AP endonuclease (APEX nuclease) by sequential column chromatography then further purified by sodium dodecyl sulphate-poly-acrylamide gel electrophoresis as a possible DNA repair support factor. Its partial amino acid sequences were determined, and a cDNA clone for the protein was isolated from a mouse T-cell cDNA library using long degenerate oligonucleotide probes deduced from the amino acid sequence. The complete nucleotide sequence of the cDNA (1.7 kilobases) was determined. Northern hybridization using this cDNA detected two transcripts: 1.8kb being the major one and 2.6 kb being the minor one. The complete amino acid sequence for the protein predicted from the nucleotide sequence of the cDNA indicates that the 44-kDa protein consists of 394 amino acids with a calculated molecular weight of 43,698. In tests performed thus far, the recombinant 44-kDa protein expressed in Escherichia coli has not expressed any repair-support activity. It remains to be analyzed whether the protein attains this activity after appropriate posttranslational modifications. Most parts of the 44-kDa protein cDNA and the deduced amino acid sequence were found to be identical to those of the protein p38 -2G4, recently reported as a cell cycle-specifically modulated nuclear protein of 38kDa. The p38-2G4 may be a truncated form of the present 44-kDa protein. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 38 4 1984 Partial purification of Simian virus 40 large T antigen by immunoaffinity chromatography and its detection by enzyme-linked immunosorbent assay. 341 347 EN Shogo Ikeda Masao Hatsushika Tsuguya Shigehara Sekiko Watanabe Sachiko Omura Ken Tsutsui Takuzo Oda Article 10.18926/AMO/30311 Simian virus 40 (SV40) large T antigen was partially purified from small amounts of SV40-infected and SV40-transformed cells by immunoaffinity chromatography with high recovery. T antigen, in both crude and partially purified states, was detected rapidly by a sensitive and quantitative enzyme-linked immunosorbent assay (ELISA). Stability of the partially purified T antigen was found to increase by addition of 0.01% bovine serum albumin (BSA). No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 88 11-12 1976 低分子性ポリアニオンによる核およびクロマチン構造の研究 977 995 EN Ken Tsutsui Synthetic dye, aluminon has been demonstrated to cause swelling of isolated nuclei, decondensation of chromatin fibers, and dissociation of histones from chromatin. These phenomena are similar to those caused by high molecular weight polyanions such as heparin. In this paper, nuclear swelling with aluminon was studied in more detail in order to establish the role of aluminon as a probe for nuclei and chromatin. Studies were also done on the binding of aluminon to nuclei, and on its effect on RNA synthesis in vitro. Nuclear swelling was monitored from changes in turbidity of nuclear suspension. A stoichiometrical relationship was suggested between the amount of aluminon needed for maximal swelling and that of nuclear DNA in the system. The swelling velocity at pH 5.4 was much slower than that at pH 7.9 and the time course of the swelling was shown to be consisted of four distinct successive processes in which the swelling velocity was constant. Centrifugation of treated chromatin in a linear density gradient of sucrose revealed a decreased density of chromatin and dissociation of histones and certain nonhistone proteins from chromatin. Aggregation of arginine rich histones after dissociation was observed. The release of histones from chromation was also deduced from the fact that aluminon removed the restriction of chromatin template when transcribed with homologous RNA polymerases. Above results indicate that aluminon may be a useful tool for studies on structure and function of chromatin. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 121 3 2009 DNA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 143 147 EN Kuniaki Sano Mary Miyaji M. Kimiko Tsutsui Ken Tsutsui No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1976 低分子性ポリアニオンによる核およびクロマチン構造の研究 EN No potential conflict of interest relevant to this article was reported.