Publications by authors named "Koji Nagao"

119 Publications

Protein phosphatase 1 acts as a RIF1 effector to suppress DSB resection prior to Shieldin action.

Cell Rep 2021 Jul;36(2):109383

Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-Cho, Toyonaka, Osaka 560-0043, Japan. Electronic address:

DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining (NHEJ) or homologous recombination (HR). RIF1 negatively regulates resection through the effector Shieldin, which associates with a short 3' single-stranded DNA (ssDNA) overhang by the MRN (MRE11-RAD50-NBS1) complex, to prevent further resection and HR repair. In this study, we show that RIF1, but not Shieldin, inhibits the accumulation of CtIP at DSB sites immediately after damage, suggesting that RIF1 has another effector besides Shieldin. We find that protein phosphatase 1 (PP1), a known RIF1 effector in replication, localizes at damage sites dependent on RIF1, where it suppresses downstream CtIP accumulation and limits the resection by the MRN complex. PP1 therefore acts as a RIF1 effector distinct from Shieldin. Furthermore, PP1 deficiency in the context of Shieldin depletion elevates HR immediately after irradiation. We conclude that PP1 inhibits resection before the action of Shieldin to prevent precocious HR in the early phase of the damage response.
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http://dx.doi.org/10.1016/j.celrep.2021.109383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8293623PMC
July 2021

Mapping replication timing domains genome wide in single mammalian cells with single-cell DNA replication sequencing.

Nat Protoc 2020 12 23;15(12):4058-4100. Epub 2020 Nov 23.

Department of Biochemistry and Proteomics, Graduate School of Medicine, Mie University, Tsu, Japan.

Replication timing (RT) domains are stable units of chromosome structure that are regulated in the context of development and disease. Conventional genome-wide RT mapping methods require many S-phase cells for either the effective enrichment of replicating DNA through bromodeoxyuridine (BrdU) immunoprecipitation or the determination of copy-number differences during S-phase, which precludes their application to non-abundant cell types and single cells. Here, we provide a simple, cost-effective, and robust protocol for single-cell DNA replication sequencing (scRepli-seq). The scRepli-seq methodology relies on whole-genome amplification (WGA) of genomic DNA (gDNA) from single S-phase cells and next-generation sequencing (NGS)-based determination of copy-number differences that arise between replicated and unreplicated DNA. Haplotype-resolved scRepli-seq, which distinguishes pairs of homologous chromosomes within a single cell, is feasible by using single-nucleotide polymorphism (SNP)/indel information. We also provide computational pipelines for quality control, normalization, and binarization of the scRepli-seq data. The experimental portion of this protocol (before sequencing) takes 3 d.
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http://dx.doi.org/10.1038/s41596-020-0378-5DOI Listing
December 2020

Anti-stress effects of rosemary ( L.) leaf extract on intestinal goblet cells and immobility of forced-swimming test in BALB/c mice.

Biosci Biotechnol Biochem 2020 Nov 2;84(11):2385-2389. Epub 2020 Aug 2.

Department of Human Life Sciences Education, Graduate School of Education, Hiroshima University , Hiroshima, Japan.

We investigated the anti-stress effect of rosemary ( L.) leaf extract (RLE) on restraint-stressed mice and found that RLE alleviated decreases in the number of intestinal goblet cells and amount of hepatic triglycerides. It also decreased the immobility time in the forced-swimming test and activation of microglia in the brain, suggesting that RLE has beneficial effects on stress-induced dysfunctions.
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http://dx.doi.org/10.1080/09168451.2020.1800445DOI Listing
November 2020

Eicosapentaenoic acid-containing polar lipids from seaweed Susabinori (Pyropia yezoensis) alleviate hepatic steatosis in obese db/db mice.

Arch Biochem Biophys 2020 09 23;691:108486. Epub 2020 Jul 23.

Department of Biological Resource Science, Saga University, Saga, 840-8502, Japan. Electronic address:

Nonalcoholic fatty liver disease (NAFLD) is emerging as the most common liver disease in industrialized countries. Because hepatic steatosis is an early pathogenesis of NAFLD, the discovery of food components that could ameliorate hepatic steatosis is of interest. Susabinori (Pyropia yezoensis) is recognized as one of the most delicious edible brown algae, and we prepared lipid component of susabinori (SNL), which is rich in eicosapentaenoic acid (EPA)-containing polar lipids. In this study, we tested whether feeding SNL to db/db mice protects them from developing obesity-induced hepatic steatosis. After four weeks of feeding, hepatomegaly, hepatic steatosis, and hepatic injury were markedly alleviated in SNL-fed db/db mice. These effects were partly attributable to the suppression of activities and mRNA expressions of lipogenic enzymes and enhanced levels of adiponectin due to the SNL diet. Additionally, mRNA expression of monocyte chemoattractant protein-1, an inflammatory chemokine, was markedly suppressed, and the mRNA levels of PPARδ, the anti-inflammatory transcription factor, were strongly enhanced in the livers of db/db mice by the SNL diet. We speculate that the development and progression of obesity-induced hepatic steatosis was prevented by the suppression of chronic inflammation due to the combination of bioactivities of EPA, phospholipids, and glycolipids in the SNL diet.
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http://dx.doi.org/10.1016/j.abb.2020.108486DOI Listing
September 2020

Rare variant of the epigenetic regulator SMCHD1 in a patient with pituitary hormone deficiency.

Sci Rep 2020 07 3;10(1):10985. Epub 2020 Jul 3.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan.

Isolated hypogonadotropic hypogonadism (IHH), combined pituitary hormone deficiency (CPHD), and septo-optic dysplasia (SOD) constitute a disease spectrum whose etiology remains largely unknown. This study aimed to clarify whether mutations in SMCHD1, an epigenetic regulator gene, might underlie this disease spectrum. SMCHD1 is a causative gene for Bosma arhinia microphthalmia syndrome characterized by arhinia, microphthalmia and IHH. We performed mutation screening of SMCHD1 in patients with etiology-unknown IHH (n = 31) or CPHD (n = 43, 19 of whom also satisfied the SOD diagnostic criteria). Rare variants were subjected to in silico analyses and classified according to the American College of Medical Genetics and Genomics guidelines. Consequently, a rare likely pathogenic variant, p.Asp398Asn, was identified in one patient. The patient with p.Asp398Asn exhibited CPHD, optic nerve hypoplasia, and a thin retinal nerve fiber layer, and therefore satisfied the criteria of SOD. This patient showed a relatively low DNA methylation level of the 52 SMCHD1-target CpG sites at the D4Z4 locus. Exome sequencing for the patient excluded additional variants in other IHH/CPHD-causative genes. In vitro assays suggested functional impairment of the p.Asp398Asn variant. These results provide the first indication that SMCHD1 mutations represent a rare genetic cause of the HH-related disease spectrum.
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http://dx.doi.org/10.1038/s41598-020-67715-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335161PMC
July 2020

Homozygous nonsense variant in associated with facioscapulohumeral muscular dystrophy.

Neurology 2020 06 28;94(23):e2441-e2447. Epub 2020 May 28.

From the Department of Neuromuscular Research (K.H., S.M., M.O., S.N., I.N.), National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan; Department of Neurology (K.H.), Graduate School of Medicine, Kyoto University, Japan; Department of Human Genetics (D.Š., R.J.L.F.L., R.G., J.B., S.M.M.), Leiden University Medical Center, the Netherlands; Department of Clinical Development (S.M., I.N.), Medical Genome Center, National Center of Neurology and Psychiatry, Tokyo; Department of Neurology (H.M., Y.S., A.S., K.S., S.K.), Graduate School of Medicine, Chiba University; Department of Biological Sciences (K.N., C.O.), Graduate School of Science, Osaka University; and Department of Pathophysiology (Y.K.H.), Tokyo Medical University, Tokyo, Japan.

Objective: Facioscapulohumeral muscular dystrophy (FSHD) is a heterogenetic disorder predominantly characterized by progressive facial and scapular muscle weakness. Patients with FSHD either have a contraction of the D4Z4 repeat on chromosome 4q35 or mutations in D4Z4 chromatin modifiers SMCHD1 and DNMT3B, both causing D4Z4 chromatin relaxation and inappropriate expression of the D4Z4-encoded gene in skeletal muscle. In this study, we tested the hypothesis whether , a known SMCHD1 protein interactor, is a disease gene for idiopathic FSHD2.

Methods: Clinical examination of a patient with idiopathic FSHD2 was combined with pathologic muscle biopsy examination and with genetic, epigenetic, and molecular studies.

Results: A homozygous mutation was identified in a patient with a clinical phenotype consistent with FSHD. This mutation resulted in the absence of the long isoform of LRIF1 protein, D4Z4 chromatin relaxation, and and DUX4 target gene expression in myonuclei, all molecular and epigenetic hallmarks of FSHD. In concordance, LRIF1 was shown to bind to the D4Z4 repeat, and knockdown of the LRIF1 long isoform in muscle cells results in and DUX4 target gene expression.

Conclusion: is a bona fide disease gene for FSHD2. This study further reinforces the unifying genetic mechanism, which postulates that FSHD is caused by D4Z4 chromatin relaxation, resulting in inappropriate expression in skeletal muscle.
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http://dx.doi.org/10.1212/WNL.0000000000009617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7455367PMC
June 2020

Soy β-Conglycinin Peptide Attenuates Obesity and Lipid Abnormalities in Obese Model OLETF Rats.

J Oleo Sci 2020 ;69(5):495-502

Department of Biological Resource Science, Saga University.

We previously reported that soy β-conglycinin (βCG) improves obesity-induced metabolic abnormalities, but not obesity, in obese model Otsuka Long-Evans Tokushima fatty (OLETF) rats. In the present study, we aimed to investigate the effects of βCG-derived peptide consumption on obesity and lipid abnormality in OLETF rats. To this end, wild-type Long-Evans Tokushima Otsuka and OLETF rats were provided a normal diet containing 20% casein for four weeks as a control. In addition, we prepared βCG peptide by enzymatic hydrolysis, and OLETF rats were fed a diet in which half of the casein was replaced by βCG peptide (βCG peptide group). Consequently, rats in the βCG peptide group showed decreased abdominal white adipose tissue weight and lipid content (serum and liver triglycerides, and serum and liver cholesterol) compared to control OLETF rats. Further analysis demonstrated that βCG peptide consumption decreased lipogenic enzyme activity and increased lipolytic enzyme activity in the liver of OLETF rats. In addition, suppressive effects on both synthesis and absorption of cholesterol were observed in βCG peptide-fed OLETF rats. These findings suggest that peptidization of βCG enhanced the anti-obese and hypolipidemic effects of βCG.
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http://dx.doi.org/10.5650/jos.ess20010DOI Listing
September 2020

Asymmetrical localization of Nup107-160 subcomplex components within the nuclear pore complex in fission yeast.

PLoS Genet 2019 06 6;15(6):e1008061. Epub 2019 Jun 6.

Graduate School of Frontier Biosciences, Osaka University, Suita, Japan.

The nuclear pore complex (NPC) forms a gateway for nucleocytoplasmic transport. The outer ring protein complex of the NPC (the Nup107-160 subcomplex in humans) is a key component for building the NPC. Nup107-160 subcomplexes are believed to be symmetrically localized on the nuclear and cytoplasmic sides of the NPC. However, in S. pombe immunoelectron and fluorescence microscopic analyses revealed that the homologous components of the human Nup107-160 subcomplex had an asymmetrical localization: constituent proteins spNup132 and spNup107 were present only on the nuclear side (designated the spNup132 subcomplex), while spNup131, spNup120, spNup85, spNup96, spNup37, spEly5 and spSeh1 were localized only on the cytoplasmic side (designated the spNup120 subcomplex), suggesting the complex was split into two pieces at the interface between spNup96 and spNup107. This contrasts with the symmetrical localization reported in other organisms. Fusion of spNup96 (cytoplasmic localization) with spNup107 (nuclear localization) caused cytoplasmic relocalization of spNup107. In this strain, half of the spNup132 proteins, which interact with spNup107, changed their localization to the cytoplasmic side of the NPC, leading to defects in mitotic and meiotic progression similar to an spNup132 deletion strain. These observations suggest the asymmetrical localization of the outer ring spNup132 and spNup120 subcomplexes of the NPC is necessary for normal cell cycle progression in fission yeast.
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http://dx.doi.org/10.1371/journal.pgen.1008061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6553703PMC
June 2019

Genome-wide stability of the DNA replication program in single mammalian cells.

Nat Genet 2019 03 25;51(3):529-540. Epub 2019 Feb 25.

Laboratory for Developmental Epigenetics, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan.

Here, we report a single-cell DNA replication sequencing method, scRepli-seq, a genome-wide methodology that measures copy number differences between replicated and unreplicated DNA. Using scRepli-seq, we demonstrate that replication-domain organization is conserved among individual mouse embryonic stem cells (mESCs). Differentiated mESCs exhibited distinct profiles, which were also conserved among cells. Haplotype-resolved scRepli-seq revealed similar replication profiles of homologous autosomes, while the inactive X chromosome was clearly replicated later than its active counterpart. However, a small degree of cell-to-cell replication-timing heterogeneity was present, which was smallest at the beginning and the end of S phase. In addition, developmentally regulated domains were found to deviate from others and showed a higher degree of heterogeneity, thus suggesting a link to developmental plasticity. Moreover, allelic expression imbalance was found to strongly associate with replication-timing asynchrony. Our results form a foundation for single-cell-level understanding of DNA replication regulation and provide insights into three-dimensional genome organization.
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http://dx.doi.org/10.1038/s41588-019-0347-5DOI Listing
March 2019

Evaluating the Content and Distribution of Trans Fatty Acid Isomers in Foods Consumed in Japan.

J Oleo Sci 2019 Feb 17;68(2):193-202. Epub 2019 Jan 17.

Department of Applied Biochemistry and Food Science, Saga University.

Trans fatty acids (TFA) are considered risk factors for cardiovascular disease. However, detailed information on total content of TFA and TFA isomers and distribution of trans-octadecenoic acid positional isomers in foods consumed in Japan is not available till date. In this study, 250 foods, 169 processed foods and 81 foods derived from ruminant meat or milk, were analyzed. According to the results, most foods contained less than 1.0 g TFA / 100 g food. However, almost all foods containing butter had more than 1.0 g TFA / 100 g food. TFA isomers in foods were classified into two categories, monoene-rich type and polyenerich type. We hypothesized that these differences were attributed to diverse TFA formation mechanisms. Furthermore, we observed that trans-10-18:1 was also the dominant trans-18:1 positional isomer in foods consumed in Japan. These results are valuable for future analysis of the role of TFA in epidemiological studies in Japan.
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http://dx.doi.org/10.5650/jos.ess18214DOI Listing
February 2019

glycosylceramide commonly contained in Japanese traditional fermented foods alters cholesterol metabolism in obese mice.

Biosci Biotechnol Biochem 2019 Aug 30;83(8):1514-1522. Epub 2018 Dec 30.

a Department of Environmental Science, Faculty of Agriculture , Saga University , Saga city , Japan.

, which is manufactured by proliferating non-pathogenic fungus on steamed rice, is the base for Japanese traditional fermented foods. We have revealed that and related Japanese fermented foods and drinks such as amazake, shio-, unfiltered sake and miso contain abundant glycosylceramide. Here, we report that feeding of glycosylceramide to obese mice alters the cholesterol metabolism . Liver cholesterol was significantly decreased in obese mice fed with glycosylceramide. We hypothesized that their liver cholesterol was decreased because it was converted to bile acids. Consistent with the hypothesis, many bile acids were increased in the cecum and feces of obese mice fed with glycosylceramide. Expressions of CYP7A1 and ABCG8 involved in the metabolism of cholesterol were significantly increased in the liver of mice fed with glycosylceramide. Therefore, it was considered that glycosylceramide affects the cholesterol metabolism in obese mice.
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http://dx.doi.org/10.1080/09168451.2018.1562877DOI Listing
August 2019

Metabolism of Natural Highly Unsaturated Fatty Acid, Tetracosahexaenoic Acid (24:6n-3), in C57BL/KsJ-db/db Mice.

J Oleo Sci 2018 Dec 15;67(12):1597-1607. Epub 2018 Nov 15.

Department of Health and Nutrition Science, Nishikyushu University.

Tetracosahexaenoic acid (THA; 24:6n-3) is a natural, n-3 highly unsaturated fatty acid (n-3HUFA) that exists in fish, including Baltic herring (Clupea harengus) and the flathead flounder (Hippoglossoides dubius). In this study, natural n-3HUFAs, i.d. eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3), and THA were administrated to C57BL/KsJ-db/db mice for 4 weeks and the liver and serum lipid profiles, hepatic enzyme activity, expression of mRNA related to lipid metabolism, and adiponectin serum levels were then analyzed. The results showed that THA had the highest activity in suppressing hepatic triglyceride (TG) accumulation and increase in liver weight among the test groups. Furthermore, THA increased adiponectin levels in serum. These results indicate that THA is an excellent natural n-3HUFA that can suppress the development of metabolic syndromes and circulatory system diseases. The order of the n-3HUFA activity was THA > DHA > EPA in almost all the factors examined here. In a previous study of ours, the order was DHA > DPA > EPA, so the final order was summarized as THA > DHA > DPA > EPA. This order clearly translates to the rule that "the number of double bonds and carbon atoms in the n-3HUFA structure relates to their clinical functions".
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http://dx.doi.org/10.5650/jos.ess18167DOI Listing
December 2018

Publisher Correction: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells.

Nat Commun 2018 10 8;9(1):4109. Epub 2018 Oct 8.

The Cancer Institute, Japanese Foundation for Cancer Research (JFCR), Koto-ku, Tokyo, 135-8550, Japan.

This Article contains errors in Fig. 4. In panel d, the lanes of the western blot should have been labeled '1.05','1.06, '1.09', '1.11' '1.13', '1.16', '1.19', '1.22', '1.24', '1.25'. The correct version of Figure 4 appears in the associated Publisher Correction.
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http://dx.doi.org/10.1038/s41467-018-06613-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6174156PMC
October 2018

Role of SmcHD1 in establishment of epigenetic states required for the maintenance of the X-inactivated state in mice.

Development 2018 09 25;145(18). Epub 2018 Sep 25.

Medical Institute of Bioregulation, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

X inactivation in mammals is regulated by epigenetic modifications. Functional deficiency of SmcHD1 has been shown to cause de-repression of X-inactivated genes in post-implantation female mouse embryos, suggesting a role of SmcHD1 in the maintenance of X inactivation. Here, we show that de-repression of X-inactivated genes accompanied a local reduction in the enrichment of H3K27me3 in mouse embryonic fibroblasts deficient for SmcHD1. Furthermore, many of these genes overlapped with those having a significantly lower enrichment of H3K27me3 at the blastocyst stage in wild type. Intriguingly, however, depletion of SmcHD1 did not compromise the X-inactivated state in immortalized female mouse embryonic fibroblasts, in which X inactivation had been established and maintained. Taking all these findings together, we suggest that SmcHD1 facilitates the incorporation of H3K27me3 and perhaps other epigenetic modifications at gene loci that are silenced even with the lower enrichment of H3K27me3 at the early stage of X inactivation. The epigenetic state at these loci would, however, remain as it is at the blastocyst stage in the absence of SmcHD1 after implantation, which would eventually compromise the maintenance of the X-inactivated state at later stages.
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http://dx.doi.org/10.1242/dev.166462DOI Listing
September 2018

Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1.

Genes Dev 2018 06 13;32(11-12):806-821. Epub 2018 Jun 13.

Faculty of Science, Kyushu University, Nishi-ku, Fukuoka 819-0395, Japan.

Post-replicative correction of replication errors by the mismatch repair (MMR) system is critical for suppression of mutations. Although the MMR system may need to handle nucleosomes at the site of chromatin replication, how MMR occurs in the chromatin environment remains unclear. Here, we show that nucleosomes are excluded from a >1-kb region surrounding a mismatched base pair in egg extracts. The exclusion was dependent on the Msh2-Msh6 mismatch recognition complex but not the Mlh1-containing MutL homologs and counteracts both the HIRA- and CAF-1 (chromatin assembly factor 1)-mediated chromatin assembly pathways. We further found that the Smarcad1 chromatin remodeling ATPase is recruited to mismatch-carrying DNA in an Msh2-dependent but Mlh1-independent manner to assist nucleosome exclusion and that Smarcad1 facilitates the repair of mismatches when nucleosomes are preassembled on DNA. In budding yeast, deletion of , the homolog of Smarcad1, showed a synergistic increase of spontaneous mutations in combination with or deletion but no significant increase with deletion. Genetic analyses also suggested that the function of Fun30 in MMR is to counteract CAF-1. Our study uncovers that the eukaryotic MMR system has an ability to exclude local nucleosomes and identifies Smarcad1/Fun30 as an accessory factor for the MMR reaction.
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http://dx.doi.org/10.1101/gad.310995.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6049510PMC
June 2018

Study of Trans Fatty Acid Formation in Oil by Heating Using Model Compounds.

J Oleo Sci 2018 Mar 19;67(3):273-281. Epub 2018 Feb 19.

Department of Applied Biochemistry and Food Science, Saga University.

The intake of trans fatty acids (TFAs) in foods changes the ratio of low density lipoprotein (LDL) to high density lipoprotein (HDL) cholesterol in blood, which causes cardiovascular disease. TFAs are formed by trans isomerization of unsaturated fatty acids (UFAs). The most recognized formation mechanisms of TFAs are hydrogenation of liquid oil to form partially hydrogenated oil (PHO,) and biohydrogenation of UFAs to form TFA in ruminants. Heating oil also forms TFAs; however, the mechanism of formation, and the TFA isomers formed have not been well investigated. In this study, the trans isomerization mechanism of unsaturated fatty acid formation by heating was examined using the model compounds oleic acid, trioleate, linoleic acid, and trilinoleate for liquid plant oil. The formation of TFAs was found to be suppressed by the addition of an antioxidant and argon gas. Furthermore, the quantity of formed TFAs correlated with the quantity of formed polymer in trioleate heated with air and oxygen. These results suggest that radical reactions form TFAs from UFAs by heating. Furthermore, trans isomerization by heating oleic acid and linoleic acid did not change the original double bond positions. Therefore, the distribution of TFA isomers formed was very simple. In contrast, the mixtures of TFA isomers formed from PHO and ruminant UFAs are complicated because migration of double bonds occurs during hydrogenation and biohydrogenation. These findings suggest that trans isomerization by heating is executed by a completely different mechanism than in hydrogenation and biohydrogenation.
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http://dx.doi.org/10.5650/jos.ess17209DOI Listing
March 2018

Comparison of the Effect of trans Fatty Acid Isomers on Apolipoprotein A1 and B Secretion in HepG2 Cells.

J Oleo Sci 2017 Oct 15;66(10):1175-1181. Epub 2017 Sep 15.

Department of Food Science and Technology, Tokyo University of Marine Science and Technology.

Intake of trans fatty acid (TFA) is believed to change the ratio of low-density lipoprotein (LDL) to high density lipoprotein (HDL) cholesterol in blood, which leads to cardiovascular disease. In this study, thirteen types of TFA including monoene type TFA (trans-octadecenoic fatty acid isomers, t-18:1 isomers), diene type TFA (t9,t12-18:2), and triene type TFA (t-18:3) were added to cultured HepG2 cells to compare the amount of apolipoprotein A1 and B (those relating to levels of HDL and LDL cholesterol in blood, respectively) being secreted. We found that trans-5-18:1 increased the secretion of apolipoprotein B relative to oleic acid (cis-9-18:1, control). Secretion of apolipoprotein B was also increased by t-18:3; however, the amount was not significant compared with that observed in the control. The secretion amount of apolipoprotein B tended to increase with the number of double bonds in TFA among trans-9-18:1, t9,t12-18:2, and t-18:3. The secretion amount of apolipoprotein A1 after TFA treatment was also measured. No significant difference was detected among t-18:1 groups; however, t-18:3 increased the amount significantly compared to that in the control. These results suggest that the effect of TFA isomers on the ratio of LDL to HDL cholesterol in the blood follows a mechanism different from that in cultured cells.
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http://dx.doi.org/10.5650/jos.ess17074DOI Listing
October 2017

Inhibition of RIF1 by SCAI Allows BRCA1-Mediated Repair.

Cell Rep 2017 07;20(2):297-307

Graduate School of Life Science, Hokkaido University, Kita 21 Nishi 11, Sapporo, Hokkaido 001-0021, Japan; Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan. Electronic address:

DNA double-strand breaks (DSBs) are repaired by either the homology-directed repair (HDR) or the non-homologous end-joining (NHEJ) pathway. RIF1 (RAP1-interacting factor homolog) was recently shown to stimulate NHEJ through an interaction with 53BP1 (p53-binding protein 1) phosphorylated at S/TQ sites, but the molecular mechanism underlying pathway choice remains unclear. Here, we show that SCAI (suppressor of cancer cell invasion) binds to 53BP1 phosphorylated at S/TP sites and facilitates HDR. Upon DNA damage, RIF1 immediately accumulates at damage sites and then gradually dissociates from 53BP1 and is subsequently replaced with SCAI. Depletion of SCAI reduces both the accumulation of HDR factors, including BRCA1 (breast cancer susceptibility gene 1), at damage sites and the efficiency of HDR, as detected by a reporter assay system. These data suggest that SCAI inhibits RIF1 function to allow BRCA1-mediated repair, which possibly includes alt-NHEJ and resection-dependent NHEJ in G1, as well as HDR in S/G2.
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http://dx.doi.org/10.1016/j.celrep.2017.06.056DOI Listing
July 2017

Defects in dosage compensation impact global gene regulation in the mouse trophoblast.

Development 2017 08 6;144(15):2784-2797. Epub 2017 Jul 6.

Department of Bioscience, Graduate School of Agriculture, Kindai University, 3327-204, Nakamachi, Nara 631-8505, Japan

RNA, which is responsible for X inactivation, is a key epigenetic player in the embryogenesis of female mammals. Of the several repeats conserved in RNA, the A-repeat has been shown to be essential for its silencing function in differentiating embryonic stem cells. Here, we introduced a new allele into mouse that produces mutated RNA lacking the A-repeat ( ). RNA expressed in the embryo coated the X chromosome but failed to silence it. Although imprinted X inactivation was substantially compromised upon paternal transmission, allele-specific RNA-seq in the trophoblast revealed that RNA still retained some silencing ability. Furthermore, the failure of imprinted X inactivation had more significant impacts than expected on genome-wide gene expression. It is likely that dosage compensation is required not only for equalizing X-linked gene expression between the sexes but also for proper global gene regulation in differentiated female somatic cells.
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http://dx.doi.org/10.1242/dev.149138DOI Listing
August 2017

Exosomes maintain cellular homeostasis by excreting harmful DNA from cells.

Nat Commun 2017 05 16;8:15287. Epub 2017 May 16.

The Cancer Institute, Japanese Foundation for Cancer Research (JFCR), Koto-ku, Tokyo 135-8550, Japan.

Emerging evidence is revealing that exosomes contribute to many aspects of physiology and disease through intercellular communication. However, the biological roles of exosome secretion in exosome-secreting cells have remained largely unexplored. Here we show that exosome secretion plays a crucial role in maintaining cellular homeostasis in exosome-secreting cells. The inhibition of exosome secretion results in the accumulation of nuclear DNA in the cytoplasm, thereby causing the activation of cytoplasmic DNA sensing machinery. This event provokes the innate immune response, leading to reactive oxygen species (ROS)-dependent DNA damage response and thus induce senescence-like cell-cycle arrest or apoptosis in normal human cells. These results, in conjunction with observations that exosomes contain various lengths of chromosomal DNA fragments, indicate that exosome secretion maintains cellular homeostasis by removing harmful cytoplasmic DNA from cells. Together, these findings enhance our understanding of exosome biology, and provide valuable new insights into the control of cellular homeostasis.
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http://dx.doi.org/10.1038/ncomms15287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5440838PMC
May 2017

Compositionally distinct nuclear pore complexes of functionally distinct dimorphic nuclei in the ciliate .

J Cell Sci 2017 05 6;130(10):1822-1834. Epub 2017 Apr 6.

Advanced ICT Research Institute, National Institute of Information and Communications Technology (NICT), Kobe 651-2492, Japan

The nuclear pore complex (NPC), a gateway for nucleocytoplasmic trafficking, is composed of ∼30 different proteins called nucleoporins. It remains unknown whether the NPCs within a species are homogeneous or vary depending on the cell type or physiological condition. Here, we present evidence for compositionally distinct NPCs that form within a single cell in a binucleated ciliate. In , each cell contains both a transcriptionally active macronucleus (MAC) and a germline micronucleus (MIC). By combining analysis, mass spectrometry analysis for immuno-isolated proteins and subcellular localization analysis of GFP-fused proteins, we identified numerous novel components of MAC and MIC NPCs. Core members of the Nup107-Nup160 scaffold complex were enriched in MIC NPCs. Strikingly, two paralogs of Nup214 and of Nup153 localized exclusively to either the MAC or MIC NPCs. Furthermore, the transmembrane components Pom121 and Pom82 localize exclusively to MAC and MIC NPCs, respectively. Our results argue that functional nuclear dimorphism in ciliates is likely to depend on the compositional and structural specificity of NPCs.
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http://dx.doi.org/10.1242/jcs.199398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5450191PMC
May 2017

Pterostilbene, a dimethylated analog of resveratrol, promotes energy metabolism in obese rats.

J Nutr Biochem 2017 05 8;43:151-155. Epub 2017 Mar 8.

Department of Health and Nutrition Sciences, Nishikyushu University, Kanzaki 842-8585, Japan.

Pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene) is a dimethylated analog of resveratrol and has been reported to exert various pharmacological effects. In this study, we evaluated the effect of pterostilbene on the pathogenesis of obesity and energy metabolism in obese rats. Pterostilbene significantly activates silent mating type information regulation 2 homolog-1 and peroxisome proliferator-activated receptor-alpha in vitro. At 4 weeks a 0.5% pterostilbene diet markedly suppressed the abdominal white adipose tissue (WAT) accumulation in obese rats. The oxygen consumption and energy expenditure were significantly higher in the pterostilbene group, and pterostilbene increased the fat metabolism rather than the carbohydrate metabolism in obese rats. The mRNA level of uncoupling protein, a thermogenic regulator, was increased and the mRNA levels of fatty acid synthase and leptin, which are involved in lipogenesis and fat storage, were markedly decreased in WAT after the pterostilbene feeding. These results suggest that pterostilbene prevents WAT accumulation through the enhancement of energy metabolism and partly the suppression of lipogenesis in obese OLETF rats.
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http://dx.doi.org/10.1016/j.jnutbio.2017.02.009DOI Listing
May 2017

Usp7-dependent histone H3 deubiquitylation regulates maintenance of DNA methylation.

Sci Rep 2017 03 3;7(1):55. Epub 2017 Mar 3.

Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1, Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, 467-8601, Japan.

Uhrf1-dependent histone H3 ubiquitylation plays a crucial role in the maintenance of DNA methylation via the recruitment of the DNA methyltransferase Dnmt1 to DNA methylation sites. However, the involvement of deubiquitylating enzymes (DUBs) targeting ubiquitylated histone H3 in the maintenance of DNA methylation is largely unknown. With the use of Xenopus egg extracts, we demonstrate here that Usp7, a ubiquitin carboxyl-terminal hydrolase, forms a stable complex with Dnmt1 and is recruited to DNA methylation sites during DNA replication. Usp7 deubiquitylates ubiquitylated histone H3 in vitro. Inhibition of Usp7 activity or its depletion in egg extracts results in enhanced and extended binding of Dnmt1 to chromatin, suppressing DNA methylation. Depletion of Usp7 in HeLa cells causes enhanced histone H3 ubiquitylation and enlargement of Dnmt1 nuclear foci during DNA replication. Our results thus suggest that Usp7 is a key factor that regulates maintenance of DNA methylation.
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http://dx.doi.org/10.1038/s41598-017-00136-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427934PMC
March 2017

Soy Protein Isolate Suppresses Lipodystrophy-induced Hepatic Lipid Accumulation in Model Mice.

J Oleo Sci 2017 Feb 18;66(2):161-169. Epub 2017 Jan 18.

Department of Applied Biochemistry and Food Science, Saga University.

Lipodystrophies are acquired and genetic disorders characterized by the complete or partial absence of body fat with a line of metabolic disorders, including hepatic steatosis. Because soy protein isolate (SPI) has been reported to reduce cholesterol and triglyceride levels in animals and humans, we explored the effect of SPI on the pathophysiology of hepatic lipid accumutaion in a diet-induced lipodystrophy model mice. Four weeks of the lipodystrophy model diet induced hepatic lipid accumulation concomitant with marked deficiencies of adipose tissue and serum adipocytokines in mice. However, supplementing the lipodystrophy model diet with SPI could alleviate the hepatic lipid acculation without affecting the lipoatrophic effect of the diet. Enhanced lipogenesis is the principal mechanism of hepatic steatosis in this model, but SPI supplementation significantly attenuated the increase in enzyme activity and/or mRNA expression. Additionally, SPI supplementation upregulated the hepatic mRNA expression of an enzyme involved in cholesterol catabolism. In conclusion, our results indicate the possibility of dietary SPI to attenuate lipodystorophy-induced hepatic steatosis through the direct reduction of hepatic lipogenesis without affecting adipocytokine production.
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http://dx.doi.org/10.5650/jos.ess16145DOI Listing
February 2017

CK2 phospho-independent assembly of the Tel2-associated stress-signaling complexes in Schizosaccharomyces pombe.

Genes Cells 2017 Jan 9;22(1):59-70. Epub 2016 Dec 9.

Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

An evolutionarily conserved protein Tel2 regulates a variety of stress signals. In mammals, TEL2 associates with TTI1 and TTI2 to form the Triple T (TTT: TEL2-TTI1-TTI2) complex as well as with all the phosphatidylinositol 3-kinase-like kinases (PIKKs) and the R2TP (Ruvbl1-Ruvbl2-Tah1-Pih1 in budding yeast)/prefoldin-like complex that associates with HSP90. The phosphorylation of TEL2 by casein kinase 2 (CK2) enables direct binding of PIHD1 (mammalian Pih1) to TEL2 and is important for the stability and the functions of PIKKs. However, the regulatory mechanisms of Tel2 in fission yeast Schizosaccharomyces pombe remain largely unknown. Here, we report that S. pombe Tel2 is phosphorylated by CK2 at Ser490 and Thr493. Tel2 forms the TTT complex with Tti1 and Tti2 and also associates with PIKKs, Rvb2, and Hsp90 in vivo; however, the phosphorylation of Tel2 affects neither the stability of the Tel2-associated proteins nor their association with Tel2. Thus, Tel2 stably associates with its binding partners irrespective of its phosphorylation. Furthermore, the Tel2 phosphorylation by CK2 is not required for the various stress responses to which PIKKs are pivotal. Our results suggest that the Tel2-containing protein complexes are conserved among eukaryotes, but the molecular regulation of their formation has been altered during evolution.
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http://dx.doi.org/10.1111/gtc.12454DOI Listing
January 2017

Erratum to: Japanese traditional dietary fungus koji functions as a prebiotic for through glycosylceramide: Japanese dietary fungus koji is a new prebiotic.

Springerplus 2016 25;5(1):1869. Epub 2016 Oct 25.

Department of Environmental Science, Faculty of Agriculture, Saga University, Honjo-cho, Saga City, Saga Japan.

[This corrects the article DOI: 10.1186/s40064-016-2950-6.].
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http://dx.doi.org/10.1186/s40064-016-3539-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5080276PMC
October 2016

Japanese traditional dietary fungus koji Aspergillus oryzae functions as a prebiotic for Blautia coccoides through glycosylceramide: Japanese dietary fungus koji is a new prebiotic.

Springerplus 2016 11;5(1):1321. Epub 2016 Aug 11.

Department of Environmental Science, Faculty of Agriculture, Saga University, Honjo-cho, Saga City, Saga Japan.

Background: The Japanese traditional cuisine, Washoku, considered to be responsible for increased longevity among the Japanese, comprises various foods fermented with the non-pathogenic fungus Aspergillus oryzae (koji). We have recently revealed that koji contains an abundant amount of glycosylceramide. Intestinal microbes have significant effect on health. However, the effects of koji glycosylceramide on intestinal microbes have not been studied.

Materials And Methods: Glycosylceramide was extracted and purified from koji. C57BL/6N mice were fed a diet containing 1 % purified koji glycosylceramide for 1 week. Nutritional parameters and faecal lipid constituents were analyzed. The intestinal microbial flora of mice on this diet was investigated.

Results: Ingested koji glycosylceramide was neither digested by intestinal enzymes nor was it detected in the faeces, suggesting that koji glycosylceramide was digested by the intestinal microbial flora. Intestinal microbial flora that digested koji glycosylceramide had an increased ratio of Blautia coccoides. Stimulation of B. coccoides growth by pure koji glycosylceramide was confirmed in vitro.

Conclusions: Koji functions as a prebiotic for B. coccoides through glycosylceramide. Since there are many reports of the effects of B. coccoides on health, an increase in intestinal B. coccoides by koji glycosylceramide might be the connection between Japanese cuisine, intestinal microbial flora, and longevity.
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http://dx.doi.org/10.1186/s40064-016-2950-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4980852PMC
August 2016

Corrigendum to "Clinical, muscle pathological, and genetic features of Japanese facioscapulohumeral muscular dystrophy 2 (FSHD2) patients with SMCHD1 mutations": [Neuromuscular Disorders 26/4-5 (2016) 300-308].

Neuromuscul Disord 2016 Jul;26(7):472

Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan; Department of Clinical Development, Medical Genome Center, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan.

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http://dx.doi.org/10.1016/j.nmd.2016.05.015DOI Listing
July 2016

Probiotic Lactobacillus gasseri SBT2055 improves glucose tolerance and reduces body weight gain in rats by stimulating energy expenditure.

Br J Nutr 2016 08 8;116(3):451-8. Epub 2016 Jun 8.

1Laboratory of Nutrition Chemistry,Department of Bioscience and Biotechnology, Faculty of Agriculture,Graduate School,Kyushu University,6-10-1 Hakozaki,Higashi-ku,Fukuoka 812-8581,Japan.

Probiotic Lactobacillus gasseri SBT2055 (LG2055) reduces postprandial TAG absorption and exerts anti-obesity effects in rats and humans; however, the underlying mechanisms are not fully understood. In the present study, we addressed the mechanistic insights of the anti-obesity activity of LG2055 by feeding Sprague-Dawley rats diets containing skimmed milk fermented or not by LG2055 for 4 weeks and by analysing energy expenditure, glucose tolerance, the levels of SCFA in the caecum and serum inflammatory markers. Rats fed the LG2055-containing diet demonstrated significantly higher carbohydrate oxidation in the dark cycle (active phase for rats) compared with the control group, which resulted in a significant increase in energy expenditure. LG2055 significantly reduced cumulative blood glucose levels (AUC) compared with the control diet after 3 weeks and increased the molar ratio of butyrate:total SCFA in the caecum after 4 weeks. Furthermore, the LG2055-supplemented diet significantly reduced the levels of serum amyloid P component - an indicator of the inflammatory process. In conclusion, our results demonstrate that, in addition to the inhibition of dietary TAG absorption reported previously, the intake of probiotic LG2055 enhanced energy expenditure via carbohydrate oxidation, improved glucose tolerance and attenuated inflammation, suggesting multiple additive and/or synergistic actions underlying the anti-obesity effects exerted by LG2055.
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http://dx.doi.org/10.1017/S0007114516002245DOI Listing
August 2016

Histone H4 lysine 20 acetylation is associated with gene repression in human cells.

Sci Rep 2016 Apr 11;6:24318. Epub 2016 Apr 11.

Department of Nephrology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

Histone acetylation is generally associated with gene activation and chromatin decondensation. Recent mass spectrometry analysis has revealed that histone H4 lysine 20, a major methylation site, can also be acetylated. To understand the function of H4 lysine 20 acetylation (H4K20ac), we have developed a specific monoclonal antibody and performed ChIP-seq analysis using HeLa-S3 cells. H4K20ac was enriched around the transcription start sites (TSSs) of minimally expressed genes and in the gene body of expressed genes, in contrast to most histone acetylation being enriched around the TSSs of expressed genes. The distribution of H4K20ac showed little correlation with known histone modifications, including histone H3 methylations. A motif search in H4K20ac-enriched sequences, together with transcription factor binding profiles based on ENCODE ChIP-seq data, revealed that most transcription activators are excluded from H4K20ac-enriched genes and a transcription repressor NRSF/REST co-localized with H4K20ac. These results suggest that H4K20ac is a unique acetylation mark associated with gene repression.
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http://dx.doi.org/10.1038/srep24318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4827026PMC
April 2016
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