Sap1 promotes the association of the replication fork protection complex with chromatin and is involved in the replication checkpoint in Schizosaccharomyces pombe

Sap1 is involved in replication fork pausing at rDNA repeats and functions during mating-type switching in Schizosaccharomyces pombe. These two roles are dependent on the ability of Sap1 to bind specific DNA sequences at the rDNA and mating-type loci, respectively. In S. pombe, Swi1 and Swi3 form the replication fork protection complex (FPC) and play important roles in the activation of the replication checkpoint and the stabilization of stalled replication forks. Here we describe the roles of Sap1 in the replication checkpoint. We show that Sap1 is involved in the activation of the replication checkpoint kinase Cds1 and that sap1 mutant cells accumulate spontaneous DNA damage during the S- and G2-phases, which is indicative of fork damage. We also show that sap1 mutants have a defect in the resumption of DNA replication after fork arrest. Sap1 is localized at the replication origin ori2004 and this localization is required for the association of the FPC with chromatin. We propose that Sap1 is required to recruit the FPC to chromatin, thereby contributing to the activation of the replication checkpoint and the stabilization of replication forks.     

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) interacts with membrane type 1 matrix metalloproteinase and CD13/aminopeptidase N and modulates their endocytic pathways

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is anchored to the cell surface via glycosylphosphatidylinositol. This molecule antagonizes the function of membrane type 1 matrix metalloproteinase (MT1-MMP) to promote proMMP-2 maturation. Here, we attempt to clarify the mechanism underlying RECK functions. First, we found that RECK forms a complex with MT1-MMP and inhibits its proteolytic activity. Notably, RECK increases the amount of MT1-MMP that associates with detergent-resistant membranes during sucrose gradient ultracentrifugation. Furthermore, perturbation of membrane cholesterol significantly affected the function of RECK in suppressing MT1-MMP function. These findings indicate that RECK possibly regulates MT1-MMP function by modulating its behavior on the cell surface as well as by enzymatic action; this prompted us to find another molecule whose behavior in detergent-resistant membranes is influenced by RECK. Subsequently, we found that RECK interacts with CD13/aminopeptidase N. Further, we found that RECK inhibits the proteolytic activity of CD13 in a cholesterol perturbation-sensitive manner. Finally, we examined whether RECK influences the behavior of MT1-MMP and CD13 during their internalization from the cell surface. In the absence of RECK, MT1-MMP and CD13 were internalized along with the markers of clathrin- or caveolae-dependent endocytosis. However, interestingly, in the presence of RECK these molecules were internalized preferentially with an endocytic marker that is neither clathrinnor caveolae-dependent, indicating that RECK modulates endocytic pathways of MT1-MMP and CD13. This modulation was correlated with the accelerated internalization and decay of MT1-MMP and CD13. This study unveils the novel function and target molecules of RECK.     

Triaxial accelerometry for assessment of physical activity in young children

Objective: The purpose of the present study was to derive linear and non‐linear regression equations that estimate energy expenditure (EE) from triaxial accelerometer counts that can be used to quantitate activity in young children. We are unaware of any data regarding the validity of triaxial accelerometry for assessment of physical activity intensity in this age group. Research Methods and Procedures: EE for 27 girls and boys (6.0 ± 0.3 years) was assessed for nine activities (lying down, watching a video while sitting and standing, line drawing for coloring‐in, playing blocks, walking, stair climbing, ball toss, and running) using indirect calorimetry and was then estimated using a triaxial accelerometer (ActivTracer, GMS). Results: Significant correlations were observed between synthetic (synthesized tri‐axes as the vector), vertical, and horizontal accelerometer counts and EE for all activities (0.878 to 0.932 for EE). However, linear and non‐linear regression equations underestimated EE by >30% for stair climbing (up and down) and performing a ball toss. Therefore, linear and non‐linear regression equations were calculated for all activities except these two activities, and then evaluated for all activities. Linear and non‐linear regression equations using combined vertical and horizontal acceleration counts, synthetic counts, and horizontal counts demonstrated a better relationship between accelerometer counts and EE than did regression equations using vertical acceleration counts. Adjustment of the predicted value by the regression equations using the vertical/horizontal counts ratio improved the overestimation of EE for performing a ball toss. Discussion: The results suggest that triaxial accelerometry is a good tool for assessing daily EE in young children.     

High diversity of Ligularia dictyoneura in chemical composition and DNA sequence

The chemical composition of root extracts of the title species collected at 20 different places in the Hengduan Mountains of China was examined. From these samples, a total of 17 eremophilane derivatives were isolated, three of which were new franoeremophilane derivatives: 3beta-acetoxy-6beta-(angeloyloxy)furanoeremophilan-10beta-ol, 1alpha-acetoxyfuranoeremophilan-15,6alpha-olide, and 6beta-[2-(hydroxymethyl)prop-2-enoyloxy]furanoeremophil-1(10)-ene. Based on the chemical composition, the samples could be classified into as many as seven types: one type containing non-furanoeremophilane derivatives and the other six containing furanoeremophilane derivatives with different oxidation levels. Results of DNA sequencing of the atpB-rbcL region and the internal transcribed spacers of the ribosomal RNA gene also indicated a high diversity in the species.     

Insights into the ubiquinol/dioxygen binding and proton relay pathways of the alternative oxidase

The alternative oxidase (AOX) is a monotopic diiron carboxylate protein which catalyzes the four-electron reduction of dioxygen to water by ubiquinol. Although we have recently determined the crystal structure of Trypanosoma brucei AOX (TAO) in the presence and absence of ascofuranone (AF) derivatives (which are potent mixed type inhibitors) the mechanism by which ubiquinol and dioxygen binds to TAO remain inconclusive. In this article, ferulenol was identified as the first competitive inhibitor of AOX which has been used to probe the binding of ubiquinol. Surface plasmon resonance reveals that AF is a quasi-irreversible inhibitor of TAO whilst ferulenol binding is completely reversible. The structure of the TAO-ferulenol complex, determined at 2.7?Å, provided insights into ubiquinol binding and has also identified a potential dioxygen molecule bound in a side-on conformation to the diiron center for the first time.     

Timing of food intake is more potent than habitual voluntary exercise to prevent diet-induced obesity in mice

Inappropriate eating habits such as skipping breakfast and eating late at night are associated with risk for abnormal weight-gain and adiposity. We previously reported that time-imposed feeding during the daytime (inactive phase) induces obesity and metabolic disorders accompanied by physical inactivity in mice. The present study compares metabolic changes induced in mice by time-imposed feeding under voluntary wheel-running (RW) and sedentary (SED) conditions to determine the effects of voluntary wheel-running activity on obesity induced in mice by feeding at inappropriate times. Mice were individually housed in cages with or without running-wheels. We compared food consumption, core body temperature, hormonal and metabolic variables in the blood, lipid accumulation in the liver, circadian expression of clock and metabolic genes in peripheral tissues, and gains in body weight between mice allowed access to food only during the sleep phase (daytime feeding; DF) or only during the active phase (nighttime feeding; NF) under SED or RW conditions. Only a high-fat high-sucrose diet was available to the mice throughout restricted feeding. Nocturnal activity was maintained in both NF and DF mice under RW conditions, but significantly suppressed during the latter half of the dark phase in DF mice. Nocturnal fluctuations in core body temperature were maintained in DF and NF mice under both SED and RW conditions, although DF attenuated the day–night amplitude more under SED, than RW conditions. The degrees of DF-induced increases in body weight gain, food efficiency, adipose tissue mass, lipogenic gene expression in metabolic tissues, and hepatic lipid accumulation were essentially identical between SED and RW conditions. Daytime feeding also induced hyperinsulinemia and hyperleptinemia under both SED and RW conditions, although DF-induced hyperleptinemia was slightly attenuated by wheel-running. The temporal expression of circadian clock genes became synchronized to feeding cycles in the liver but not in the skeletal muscle of mice under both SED and RW conditions. Chronic voluntary exercise on running-wheels minimally affected obesity and adiposity in mice caused by daily feeding at unusual times. The timing of food intake might be more important than physical exercise for preventing metabolic disorders.

Abbreviations: ANOVA: analysis of variance; DF: daytime feeding; FFA: free fatty acid; GLP-1: glucagon-like peptide-1; HOMA-IR: homeostasis model assessment of insulin resistance; NEAT: non-exercise activity thermogenesis; NF: nighttime feeding; RF: restricted feeding; RW: running-wheel; SCN: suprachiasmatic nucleus; SE: standard error of the mean; SED: sedentary; SPA: spontaneous physical activity; T-Cho: total cholesterol; TG: triglyceride; WAT: white adipose tissues     

东俄洛橐吾遗传变异与分化的ISSR分析

应用ISSR标记对东俄洛橐吾（Ligularia tongolensis）的遗传多样性进行了研究。从100个引物中筛选出8个用于正式扩增。在所研究的8个居群共150个个体中检测到148个多态位点。在居群水平上,多态位点百分率（PPB）为50.45%,Nei′s基因多样性指数（H）和Shannon信息指数（I）分别为0.1595和0.2440。在物种水平上,多态位点百分率（PPB）为88.10%,Nei′s基因多样性指数（H）和Shannon信息指数（I）分别为0.2811和0.4279。居群间的遗传分化系数（Gst）达0.4355。研究结果表明东俄洛橐吾的遗传多样性水平很高,居群间遗传分化较大。这与其多样化的生态环境是有必然联系的。因适应其多样化的生态环境而形成了遗传多样性;且因其生态环境的不连续性阻碍了居群间的基因交流而产生了遗传分化,即东俄洛橐吾高水平的遗传多样性和遗传分化是适应其分布区多样化生态环境的结果。     

Carboxy-terminal modulator protein induces Akt phosphorylation and activation, thereby enhancing antiapoptotic, glycogen synthetic, and glucose uptake pathways

Carboxy-terminal modulator protein (CTMP) was identified as binding to the carboxy terminus of Akt and inhibiting the phosphorylation and activation of Akt. In contrast to a previous study, we found CTMP overexpression to significantly enhance Akt phosphorylation at both Thr³⁰⁸ and Ser⁴⁷³ as well as the kinase activity of Akt, while phosphatidylinositol 3-kinase (PI3-kinase) activity was unaffected. Translocation of Akt to the membrane fraction was also markedly increased in response to overexpression of CTMP, with no change in the whole cellular content of Akt. Furthermore, the phosphorylations of GSK-3β and Foxo1, well-known substrates of Akt, were increased by CTMP overexpression. On the other hand, suppression of CTMP with small interfering RNA partially but significantly attenuated this Akt phosphorylation. The cellular activities reportedly mediated by Akt activation were also enhanced by CTMP overexpression. UV-B-induced apoptosis of HeLa cells was significantly reversed not only by overexpression of the active mutant of Akt (myr-Akt) but also by that of CTMP. Increases in glucose transport activity and glycogen synthesis were also induced by overexpression of either myr-Akt or CTMP in 3T3-L1 adipocytes. Taking these results into consideration, it can be concluded that CTMP induces translocation of Akt to the membrane and thereby increases the level of Akt phosphorylation. As a result, CTMP enhances various cellular activities that are principally mediated by the PI3-kinase/Akt pathway. phosphatidylinositol 3-kinase