Chromatin Compaction Protects Genomic DNA from Radiation Damage

Genomic DNA is organized three-dimensionally in the nucleus, and is thought to form compact chromatin domains. Although chromatin compaction is known to be essential for mitosis, whether it confers other advantages, particularly in interphase cells, remains unknown. Here, we report that chromatin compaction protects genomic DNA from radiation damage. Using a newly developed solid-phase system, we found that the frequency of double-strand breaks (DSBs) in compact chromatin after ionizing irradiation was 5–50-fold lower than in decondensed chromatin. Since radical scavengers inhibited DSB induction in decondensed chromatin, condensed chromatin had a lower level of reactive radical generation after ionizing irradiation. We also found that chromatin compaction protects DNA from attack by chemical agents. Our findings suggest that genomic DNA compaction plays an important role in maintaining genomic integrity.     

Melanocyte stem cells express receptors for canonical Wnt-signaling pathway on their surface

It has been reported that melanocytes play important roles in skin and hair pigmentation and are differentiated from melanocyte stem cells (MSCs) residing in the bulge area of hair follicles. Recently, interest has been growing in MSCs because regulation of the upstream of differentiated melanocytes is essential for the determination of skin and hair pigmentation; however, their precise characteristics remain to be elucidated. The aim of this study is to explore cell-surface markers expressed on MSCs in order to understand their characteristics.To explore genes specifically expressed in the bulge region, we classified a hair follicle into four areas, hair bulb, hair bulb to bulge (lower bulge), bulge, and epidermis to bulge (upper bulge), and collected these areas from back skin sections of C57BL/6 mice by laser microdissection. Real-time RT-PCR performed on these areas revealed that Frizzled (Fzd)-4, Fzd7, low density lipoprotein receptor-related protein 5 (Lrp5), and Lrp6, receptors for Wnt molecules, were expressed higher in the bulge area than other areas. Furthermore, FACS analysis showed that populations of Fzd4⁺ cells and Fzd7⁺ cells were different from those of Kit⁺ cells (precursor of melanocytes: melanoblasts). Fzd4⁺ and Fzd7⁺ cells isolated by FACS required a longer culture period to differentiate into mature melanocytes than Kit⁺ cells. Up-regulation of mRNA expressions of melanocyte markers (dopa chrometautomerase: Dct, tyrosinase: Tyr, tyrosinase-related protein 1: Tyrp1) was observed in Fzd4⁺ and Fzd7⁺ cells following Kit⁺ cells during differentiation. These results suggested that Fzd4⁺ and Fzd7⁺ cells were more immature than melanoblasts, therefore raising the possibility that Fzd4⁺ and Fzd7⁺ cells are MSCs.     

Late S phase-specific recruitment of Mre11 complex triggers hierarchical assembly of telomere replication proteins in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, telomere replication occurs in late S phase and is accompanied by dynamic remodeling of its protein components. Here, we show that MRX (Mre11-Rad50-Xrs2), an evolutionarily conserved protein complex involved in DNA double-strand break (DSB) repair, is recruited to the telomeres in late S phase. MRX is required for the late S phase-specific recruitment of ATR-like kinase Mec1 to the telomeres. Mec1, in turn, contributes to the assembly of the telomerase regulators Cdc13 and Est1 at the telomere ends. Our results provide a model for the hierarchical assembly of telomere-replication proteins in late S phase; this involves triggering by the loading of MRX onto the chromosome termini. The recruitment of DNA repair-related proteins to the telomeres at particular times in the cell cycle suggests that the normal terminus of a chromosome is recognized as a DSB during the course of replication.     

Induction of sister chromatid exchanges and chromosome aberrations in cultured mammalian cells treated with aminophenylnorharman formed by norharman with aniline

Aminophenylnorharman (APNH) is a newly identified mutagenic heterocyclic amine formed by coupling of norharman with aniline in the presence of S9 mix. Furthermore, mutagenic amino-3'-methylphenylnorharman (AMPNH) and aminophenylharman (APH) have been identified from a reaction mixture of norharman and o-toluidine and that of harman and aniline, respectively, with S9 mix. Among these three heterocyclic amines, APNH shows most potent mutagenic activity towards Salmonella typhimurium TA98 and YG1024 with S9 mix. In the present study, the induction of sister chromatid exchanges (SCEs) by APNH was examined in Chinese hamster lung (CHL) cells in vitro, comparing it to those of AMPNH and APH. On incubation with rat S9 for 6h, followed by a recovery culture period of 18h, a dose-dependent effect was found at concentrations between 0.00125 and 0.01 microg/ml for APNH and between 0.3125 and 5 microg/ml for AMPNH and APH. The approximate chemical concentrations leading to a three-fold of control SCE levels calculated from slopes of the linear regressions of induced SCEs were 0.005 for APNH, 0.51 for AMPNH and 1.7 microg/ml for APH. Because of the very strong SCE-causing ability of APNH, we further explored its genotoxicity by examining the induction of chromosome aberrations in CHL cells. A dose-dependent effect was found for chromosome aberrations at concentrations between 0.00125 and 0.04 microg/ml of APNH. The aberrations observed were primarily chromatid exchanges (cte) and breaks (ctb). In conclusion, the potency of SCE induction and clastogenic activity induced by APNH is stronger than Actinomycin D, Mitomycin C (MMC) or 1,8-dinitropyrene which are considered to be the potent clastogens in the literature. Further studies are needed for elucidating mechanisms of the genotoxic actions of these compounds and for evaluating their potential hazards to human health.     

HIV-1-derived self-inactivating lentivirus vector induces megakaryocyte lineage-specific gene expression

Pluripotent, self-renewing, hematopoietic stem cells are considered good targets for gene modification to treat a wide variety of disorders. However, as many genes are expressed in a stage-specific manner during the course of hematopoietic development, it is necessary to establish a lineage-specific gene expression system to ensure the proper expression of transduced genes in hematopoietic stem cells. In this study, we constructed a VSV-G-pseudotyped, human immunodeficiency virus type 1-based, self-inactivating lentivirus vector that expressed green fluorescent protein (GFP) under the control of the human CD41 (glycoprotein 2b; GP2b) promoter; this activity is restricted to megakaryocytic lineage cells. The recombinant virus was used to infect human peripheral blood CD34+ (hematopoietic stem/progenitor) cells, and lineage-specific gene expression was monitored with GFP measurements. The analysis by FACS determined that GFP expression driven by the GP2b promoter was restricted to megakaryocytic progenitors and was not present in erythrocytes. Furthermore, in the hematopoietic colony-forming assay, GFP expression was restricted to colony-forming units-megakaryocyte (CFU-Meg) colonies under the control of the GP2b promoter, whereas all myeloid colonies (burst-forming units-erythroid, colony-forming units-granulocyte-macrophage, and CFU-Meg) expressed GFP when the transgene was regulated by the cytomegalovirus promoter. These results demonstrated lineage-specific expression after gene transduction of hematopoietic stem cells. The application of this vector system should provide a useful tool for gene therapy to treat disorders associated with megakaryocyte (platelet) dysfunction.     

Osteopontin is critical to determine symptom severity of influenza through the regulation of NK cell population

Osteopontin (OPN) is involved in exacerbating various inflammatory diseases. A severe pulmonary inflammation is frequently found in lethal influenza A virus (IAV) infection. However, the function of OPN against the infection was poorly understood. Here, we demonstrate an importance of OPN on immune response and disease severity after IAV infection. We found that the expression level of OPN was increased in mice infected with IAV. The OPN knockout (KO) mice exhibited a severe pathological phenotype and the survival rate decreased after the lethal IAV infection, compared to the wild type mice, while the survival rate increased in OPN transgenic (Tg) mice. The population of natural killer (NK) cells significantly decreased in OPN KO mice at day 5 after the infection, whereas, it increased in OPN Tg mice. These results suggest that OPN plays an important role in host defense against IAV infection through the regulation of NK cell population.     

Vialinin A is a ubiquitin-specific peptidase inhibitor

Vialinin A, a small compound isolated from the Chinese mushroom Thelephora vialis, exhibits more effective anti-inflammatory activity than the widely used immunosuppressive drug tacrolimus (FK506). Here, we show that ubiquitin-specific peptidase 5/isopeptidase T (USP5/IsoT) is a target molecule of vialinin A, identified by using a beads-probe method. Vialinin A inhibited the peptidase activity of USP5/IsoT and also inhibited the enzymatic activities of USP4 among deubiquitinating enzymes tested. Although USPs are a member of thiol protease family, vialinin A exhibited no inhibitions for other thiol proteases, such as calpain and cathepsin.     

Low prevalence of Helicobacter pylori-negative gastric cancer among Japanese

Background and Aims: The true prevalence of Helicobacter pylori‐negative gastric cancer (HpNGC) is unknown. We attempt to clarify the prevalence and clinicopathologic features of HpNGC in Japanese. Methods: Helicobacter pylori infection was detected by antibody titer and microscopic observation. In addition, we confirmed the lack of endoscopic atrophy and histologic gastritis. In these cases, we added urea breath test or rapid urease test to confirm the absence of H. pylori. The mucus phenotype of gastric cancer tissue was also evaluated by immunohistochemistry. Results: We screened 3161 gastric cancer cases from 1996 to 2010, and 21 cases were regarded as H. pylori negative. Clinically, patients with HpNGC were younger than patients with H. pylori‐positive gastric cancer (controls), and revealed a lack of male dominancy. Histologically, diffuse type was frequently found. All patients examined were pepsinogen negative. Among HpNGC cases with endoscopic resection, the depressed macroscopic appearance was dominant. The prevalence of HpNGC was calculated as 0.66% (95% confidence interval = 0.41–1.01). The mucus phenotype of HpNGC was similar to that of the controls. Conclusion: The prevalence of HpNGC is very low and its pathological characteristics are different from common gastric cancer.     

In vivo and in vitro effect of p-chlorophenoxyisobutyrate on insulin binding and glucose transport in isolated rat adipocytes

We studied the in vivo and in vitro effect of p-chlorophenoxyisobutyrate (CPIB) on insulin binding and glucose transport in isolated rat adipocytes. In the in vitro study, adipocytes were incubated with 1mM of CPIB for 2 h at 37 degrees C, pH 7.4, and then insulin binding (37 degrees C, 60 min) and 3-0-methylglucose transport (37 degrees C, 2s) were measured. Incubation with CPIB did not affect either insulin binding or glucose transport in the cells. The addition of insulin (10 ng/ml) with CPIB to the incubation media also did not affect the following insulin binding and glucose transport. In the in vivo study, rats were fed a high sucrose-diet containing 0.25% CPIB for 7 days. Serum cholesterol, plasma free fatty acid, and insulin levels were significantly decreased in the CPIB-treated rats. The treated rats demonstrated an almost 2 fold increased maximal binding capacity for insulin (189,000 sites/cell for treated vs 123,000 sites/cell for control cells). Basal glucose transport (glucose transport in the absence of insulin) significantly decreased in the CPIB-treated rats, although insulin-stimulated glucose transport was comparable in treated and control cells. Thus, CPIB might have no direct effect on glucose transport and insulin binding, as determined by the in vitro studies. Furthermore, a relatively short-term in vivo treatment with CPIB, such as 7 days, did not stimulate glucose transport.