ATP hydrolysis by ORC catalyzes reiterative Mcm2-7 assembly at a defined origin of replication

The origin recognition complex (ORC) is a six-subunit, ATP-regulated, DNA binding protein that is required for the formation of the prereplicative complex (pre-RC), an essential replication intermediate formed at each origin of DNA replication. In this study, we investigate the mechanism of ORC function during pre-RC formation and how ATP influences this event. We demonstrate that ATP hydrolysis by ORC requires the coordinate function of the Orc1 and Orc4 subunits. Mutations that eliminate ORC ATP hydrolysis do not support cell viability and show defects in pre-RC formation. Pre-RC formation involves reiterative loading of the putative replicative helicase, Mcm2-7, at the origin. Importantly, preventing ORC ATP hydrolysis inhibits this repeated Mcm2-7 loading. Our findings indicate that ORC is part of a helicase-loading molecular machine that repeatedly assembles Mcm2-7 complexes onto origin DNA and suggest that the assembly of multiple Mcm2-7 complexes plays a critical role in origin function.     

Neurotensin elevates hepatic bile acid secretion in chickens by a mechanism requiring an intact enterohepatic circulation

Neurotensin (NT), given intravenously at 10-50 pmol/kg per min to anesthetized female chickens equipped with a bile duct fistula, dose-dependently elevated hepatic bile flow and bile acid output but only when the enterohepatic circulation was maintained by returning the bile to the intestinal lumen. Infusion of NT at 10 and 50 pmol/kg per min increased the average hepatic bile acid output over a 30-min period to 138 +/- 11 and 188 +/- 13% of control, respectively. During infusion of NT, plasma levels of immunoreactive NT (iNT) increased in time from the basal level (14 +/- 1.3 pM) to reach steady state at 30 min. There was a near linear relationship between the dose of NT infused and the increment in plasma iNT. In addition, infusion of NT at 40 pmol/kg min gave a plasma level of iNT (approximately/= 88 pM) which was within the range of those observed during duodenal perfusion with lipid (54-300 pM) and near to that measured in hepatic portal blood from fed animals (52 +/- 5 pM). Perfusion of duodenum with lipid released endogenous NT and increased the rate of hepatic bile flow. When NT antagonist SR48692 was given, bile flow rate decreased to the basal level. These results suggest that intestinal NT, released by lipid, may participate in the regulation of hepatic bile acid output by a mechanism requiring an intact enterohepatic circulation.     

西藏黄绿卷毛菇生境土壤微生物群落组成

牧草外生菌根菌黄绿卷毛菇Floccularia luteovirens具有较高的生态和经济价值,其生境微生物对其菌丝生长、菌根化和子实体形成具有促进作用。本研究利用高通量测序技术对西藏黄绿卷毛菇菌窝土壤及其周围无菇土壤微生物群落组成进行分析,挖掘促进黄绿卷毛菇生长的有益微生物资源。结果显示,西藏黄绿卷毛菇生境土壤细菌隶属于17门22纲116目161科227属,其中酸杆菌门、变形菌门、拟杆菌门和疣微菌门累计比例高达81.75%;生境土壤真菌隶属于5门17纲34目55科61属,子囊菌门和担子菌门累计比例高达80.89%。对比周围无菇土壤的微生物群落,促进黄绿卷毛菇生长的潜在细菌类群为Flavisolibacter、黄杆菌属Flavobacterium、芽单胞菌属Gemmatimonas、Haliangium、赛格特杆菌Segetibacter和鞘氨醇单胞菌属Sphingomonas;促进黄绿卷毛菇生长的潜在真菌类群为斜盖伞属Clitopilus、丝膜菌属Cortinarius、被孢霉属Mortierella和毛霉属Mucor。土壤微生物促进黄绿卷毛菇生长繁殖的机制有待进一步研究。     

RING-box proteins regulate leaf senescence and stomatal closure via repression of ABA transporter gene ABCG40

Plant hormone abscisic acid (ABA) plays an indispensable role in the control of leaf senescence, during which ABA signaling depends on its biosynthesis. Nevertheless, the role of ABA transport in leaf senescence remains unknown. Here, we identified two novel RING-box protein-encoding genes UBIQUITIN LIGASE of SENESCENCE 1 and 2 (ULS1 and ULS2) involved in leaf senescence. Lack of ULS1 and ULS2 accelerates leaf senescence, which is specifically promoted by ABA treatment. Furthermore, the expression of senescence-related genes is significantly affected in mature leaves of uls1/uls2 double mutant (versus wild type (WT)) in an ABA-dependent manner, and the ABA content is substantially increased. ULS1 and ULS2 are mainly expressed in the guard cells and aging leaves, and the expression is induced by ABA. Further RNA-seq and quantitative proteomics of ubiquitination reveal that ABA transporter ABCG40 is highly expressed in uls1/uls2 mutant versus WT, though it is not the direct target of ULS1/2. Finally, we show that the acceleration of leaf senescence, the increase of leaf ABA content, and the promotion of stomatal closure in uls1/usl2 mutant are suppressed by abcg40 loss-of-function mutation. These results indicate that ULS1 and ULS2 function in feedback inhibition of ABCG40-dependent ABA transport during ABA-induced leaf senescence and stomatal closure.     

Immunisation with a major Trypanosoma cruzi antigen promotes pro-inflammatory cytokines, nitric oxide production and increases TLR2 expression

Innate and adaptive immunity collaborate in the protection of intracellular pathogens including Trypanosoma cruzi infection. However, the parasite molecules that regulate the host immune response have not been fully identified. We previously demonstrated that the immunisation of C57BL/6 mice with cruzipain, an immunogenic T. cruzi glycoprotein, induced a strong specific T-cell response. In this study, we demonstrated that active immunisation with cruzipain was able to stimulate nitric oxide (NO) production by splenocytes. Immune cells also showed increased inducible nitric oxide synthase protein and mRNA expression. Spleen adherent cells secreted high levels of IFN-gamma and IL-12. Microbicidal activity in vitro was mainly mediated by reactive nitrogen intermediaries and IFN-gamma, as demonstrated by the inhibitory effects of NO synthase inhibitor or by IFN-gamma neutralisation. Specific T-cells were essential for NO, IFN-gamma and TNF-alpha production. Furthermore, we reported that cruzipain enhanced CD80 and major histocompatibility complex-II molecule surface expression on F4/80+ spleen cells. Interestingly, we also showed that cruzipain up-regulated toll like receptor-2 expression, not only in F4/80+ but also in total spleen cells which may be involved in the effector immune response. Our findings suggest that a single parasite antigen such as cruzipain, through adaptive immune cells and cytokines, can modulate the macrophage response not only as antigen presenting cells, but also as effector cells displaying enhanced microbicidal activity with reactive nitrogen intermediary participation. This may represent a mechanism that contributes to the immunoregulatory process during Chagas disease.     

Selective detection of superoxide anion radicals generated from macrophages by using a novel fluorescent probe

Quantitation of superoxide radical (O (2)(-).) production at the site of radical generation remains challenging. A simple method to detect nanomolar to micromolar levels of superoxide radical in aqueous solution has been developed and optimized. This method is based on the efficient trapping of O(2)(-). using a novel fluorescent probe (2-chloro-1,3-dibenzothiazolinecyclohexene), coupled with a spectra character-signaling increase event. A high-specificity and high-sensitivity fluorescent probe was synthesized in-house and used to image O(2)(-). in living cells. Better selectivity for O(2)(-). over competing cellular reactive oxygen species and some biological compounds illustrates the advantages of our method. Under optimal conditions, the linear calibration range for superoxide anion radicals was 5.03 x 10(-9)-3.33 x 10(-6) M. The detection limit was 1.68 x 10(-9) M. Fluorescence images of probe-stained macrophages stimulated with 4beta-phorbol 12-myristate 13-acetate were obtained successfully using a confocal laser scanning microscope.     

A new cytotoxic lanostane-type triterpene glycoside from the sea cucumber Holothuria impatiens

A new lanostane-type triterpene glycoside, impatienside A (1), was isolated from the sea cucumber Holothuria impatiens, together with a structurally related, known compound, bivittoside D (2). Their structures were elucidated by in-depth spectroscopic and mass-spectrometric methods, including (1)H-, (13)C-, and 2D-NMR, ESI-MS, and HR-ESI-MS experiments, as well as by chemical evidence. Compounds 1 and 2 possess the same hexasaccharide moieties, but differ slightly in their holostane-type triterpene aglycone. The two glycosides were found to exhibit in vitro cytotoxicities similar to or better than those of the potent anticancer drug etoposide (V-16) against seven different human tumor cells, with IC50 values of 0.37-2.75 microg/ml.     

The Pillars of Hercules as a bathymetric barrier to gene flow promoting isolation in a global deep‐sea shark (Centroscymnus coelolepis)

Knowledge of the mechanisms limiting connectivity and gene flow in deep‐sea ecosystems is scarce, especially for deep‐sea sharks. The Portuguese dogfish (Centroscymnus coelolepis) is a globally distributed and near threatened deep‐sea shark. C. coelolepis population structure was studied using 11 nuclear microsatellite markers and a 497‐bp fragment from the mtDNA control region. High levels of genetic homogeneity across the Atlantic (Φ_ST = ?0.0091, F_ST = 0.0024, P > 0.05) were found suggesting one large population unit at this basin. The low levels of genetic divergence between Atlantic and Australia (Φ_ST = 0.0744, P < 0.01; F_ST = 0.0015, P > 0.05) further suggested that this species may be able to maintain some degree of genetic connectivity even across ocean basins. In contrast, sharks from the Mediterranean Sea exhibited marked genetic differentiation from all other localities studied (Φ_ST = 0.3808, F_ST = 0.1149, P < 0.001). This finding suggests that the shallow depth of the Strait of Gibraltar acts as a barrier to dispersal and that isolation and genetic drift may have had an important role shaping the Mediterranean shark population over time. Analyses of life history traits allowed the direct comparison among regions providing a complete characterization of this shark's populations. Sharks from the Mediterranean had markedly smaller adult body size and size at maturity compared to Atlantic and Pacific individuals. Together, these results suggest the existence of an isolated and unique population of C. coelolepis inhabiting the Mediterranean that most likely became separated from the Atlantic in the late Pleistocene.     

响应面法优化紫山药花青苷提取工艺

以紫山药为实验材料,以酸性乙醇为提取溶剂通过Box-Behnken响应面法及Design-Expert 8.0.6分析软件建立二次多项式数学模型优化紫山药花青苷的提取工艺及参数。研究表明,五种单因素对花青苷得率影响大小的顺序为X5(盐酸质量分数)X2(时间)X4(乙醇体积分数)X3(料液比)X1(温度)。并且温度和料液比、温度和乙醇体积分数、提取时间和料液比、提取时间和盐酸体积分数之间存在交互作用。得到紫山药花青苷最佳提取工艺参数为温度:81℃、时间:3.5 h、料液比:1∶25、乙醇体积分数:70%、盐酸质量分数:18‰。在此条件下花青苷平均得率达到4.928 mg/g,相对标准偏差为0.31%,与数学模型理论得率间的相对误差小于1.0%。