The Mini-Chromosome Maintenance (Mcm) Complexes Interact with DNA Polymerase α-Primase and Stimulate Its Ability to Synthesize RNA Primers

The Mini-chromosome maintenance (Mcm) proteins are essential as central components for the DNA unwinding machinery during eukaryotic DNA replication. DNA primase activity is required at the DNA replication fork to synthesize short RNA primers for DNA chain elongation on the lagging strand. Although direct physical and functional interactions between helicase and primase have been known in many prokaryotic and viral systems, potential interactions between helicase and primase have not been explored in eukaryotes. Using purified Mcm and DNA primase complexes, a direct physical interaction is detected in pull-down assays between the Mcm2∼7 complex and the hetero-dimeric DNA primase composed of the p48 and p58 subunits. The Mcm4/6/7 complex co-sediments with the primase and the DNA polymerase α-primase complex in glycerol gradient centrifugation and forms a Mcm4/6/7-primase-DNA ternary complex in gel-shift assays. Both the Mcm4/6/7 and Mcm2∼7 complexes stimulate RNA primer synthesis by DNA primase in vitro. However, primase inhibits the Mcm4/6/7 helicase activity and this inhibition is abolished by the addition of competitor DNA. In contrast, the ATP hydrolysis activity of Mcm4/6/7 complex is not affected by primase. Mcm and primase proteins mutually stimulate their DNA-binding activities. Our findings indicate that a direct physical interaction between primase and Mcm proteins may facilitate priming reaction by the former protein, suggesting that efficient DNA synthesis through helicase-primase interactions may be conserved in eukaryotic chromosomes.     

Phosphorylation of cardiac troponin I by mammalian sterile 20-like kinase 1

Mst1 (mammalian sterile 20-like kinase 1) is a ubiquitously expressed serine/threonine kinase and its activation in the heart causes cardiomyocyte apoptosis and dilated cardiomyopathy. Its myocardial substrates, however, remain unknown. In a yeast two-hybrid screen of a human heart cDNA library with a dominant-negative Mst1 (K59R) mutant used as bait, cTn [cardiac Tn (troponin)] I was identified as an Mst1-interacting protein. The interaction of cTnI with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK-293 cells (human embryonic kidney cells) and native cardiomyocytes, in which cTnI interacted with full-length Mst1, but not with its N-terminal kinase fragment. in vitro phosphorylation assays demonstrated that cTnI is a sensitive substrate for Mst1. In contrast, cTnT was phosphorylated by Mst1 only when it was incorporated into the Tn complex. MS analysis indicated that Mst1 phosphorylates cTnI at Thr(31), Thr(51), Thr(129) and Thr(143). Substitution of Thr(31) with an alanine residue reduced Mst1-mediated cTnI phosphorylation by 90%, whereas replacement of Thr(51), Thr(129) or Thr(143) with alanine residues reduced Mst1-catalysed cTnI phosphorylation by approx. 60%, suggesting that Thr(31) is a preferential phosphorylation site for Mst1. Furthermore, treatment of cardiomyocytes with hydrogen peroxide rapidly induced Mst1-dependent phosphorylation of cTnI at Thr(31). Protein epitope analysis and binding assays showed that Mst1-mediated phosphorylation modulates the molecular conformation of cTnI and its binding affinity to TnT and TnC, thus indicating functional significances. The results of the present study suggest that Mst1 is a novel mediator of cTnI phosphorylation in the heart and may contribute to the modulation of myofilament function under a variety of physiological and pathophysiological conditions.     

低聚果糖的生物学效应及其安全性研究进展

作为一种应用最早和最为广泛的功能性低聚糖,低聚果糖具有调节肠道菌群、提高机体免疫功能、降低血脂和促进某些营养物质吸收的功能,且对人体无任何生理毒性.通过对其生物学效应与安全性等方面近年来的最新研究进展进行综述,以期推动低聚果糖的进一步深入研究开发和在食品上的广泛应用.     

Structure of the F‐spondin domain of mindin,an integrin ligand and pattern recognition molecule

Mindin (spondin‐2) is an extracellular matrix protein of unknown structure that is required for efficient T‐cell priming by dendritic cells. Additionally, mindin functions as a pattern recognition molecule for initiating innate immune responses. These dual functions are mediated by interactions with integrins and microbial pathogens, respectively. Mindin comprises an N‐terminal F‐spondin (FS) domain and C‐terminal thrombospondin type 1 repeat (TSR). We determined the structure of the FS domain at 1.8‐Å resolution. The structure revealed an eight‐stranded antiparallel β‐sandwich motif resembling that of membrane‐targeting C2 domains, including a bound calcium ion. We demonstrated that the FS domain mediates integrin binding and identified the binding site by mutagenesis. The mindin FS domain therefore represents a new integrin ligand. We further showed that mindin recognizes lipopolysaccharide (LPS) through its TSR domain, and obtained evidence that C‐mannosylation of the TSR influences LPS binding. Through these dual interactions, the FS and TSR domains of mindin promote activation of both adaptive and innate immune responses.     

小蜉属小蜉亚属一新种：蜉蝣目：小蜉科

本文记述小蜉属小蜉亚属 subgenus Ephemerella Walsh 一新种,小蜉亚属种类多,全世界已知有40种左右,广布于北美、苏联、日本等国,但中国尚未发现,因此本文也是小蜉亚属在中国的首次报道。     

Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment

Cell replacement therapy is a promising approach for the treatment of cardiac diseases. It is, however, challenged by a limited supply of appropriate cells. Therefore, we have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem cells (hESCs). In this study, we developed an efficient protocol for the generation of functional cardiomyocytes from hESCs by combining hanging drop culture and 5-azacytidine, a well-known demethylating agent, and then evaluated the expression of cardiac-specific markers. hESCs were cultured both in the medium without or with 0.1, 1, or 10 microM of 5-azacytidine under a hanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR, RT-PCR, immunofluorescence, and confocal microscopy. To verify the structural and functional properties of hESC-derived cardiomyocytes, we performed electron microscopy and electrophysiological recording. The efficiency of beating cell generation was significantly improved in the hanging drop culture compared with that in suspension culture. Treatment of hESCs with 0.1 microM of 5-azacytidine for 1-3 days significantly increased the number of beating cells and simultaneously enhanced the expression of cardiac-specific markers. Transmission electron microscopy and electrophysiological recording showed that hESC-derived cardiomyocytes acquired structural and functional properties of cardiomyocytes. In conclusion, these results suggest that differentiation of hESCs into cardiomyocytes can be enhanced by the combination of hanging drop culture and 5-azacytidine treatment. Also the methylation status of genes related to cardiomyocyte development may play an important role in the differentiation of hESCs into cardiomyocytes.     

Dok5 is substrate of TrkB and TrkC receptors and involved in neurotrophin induced MAPK activation

Tropomyosin-related kinase (Trk) family receptors are a group of high affinity receptors for neurotrophin growth factors, which have pivotal functions in many physiological processes of nervous system. Trk receptors can dimerize and autophosphorylate upon neurotrophin stimulation, then recruit multiple adaptor proteins to transduct signal. In this report, we identified Dok5, a member of Dok family, as a new substrate of TrkB/C receptors. In yeast two-hybrid assay, Dok5 can interact with intracellular domain of TrkB and TrkC receptor through its PTB domain, but not with that of TrkA receptor. The interaction was then confirmed by GST pull-down assay and Co-IP experiment. Dok5 co-localized with TrkB and TrkC in differentiated PC12 cells, providing another evidence for their interaction. By using mutational analysis, we characterized that Dok5 PTB domain bound to Trk receptor NPQY motif in a kinase-activity-dependent manner. Furthermore, competition experiment indicated that Dok5 competed with N-shc for binding to the receptors at the same site. Finally, we showed that Dok5 was involved in the activation of MAPK pathway induced by neurotrophin stimulation. Taken together, these results suggest that Dok5 acts as substrate of TrkB/C receptors and is involved in neurotrophin induced MAPK signal pathway activation.     

Production of inulooligosaccharides from inulin by a novel endoinulinase from Xanthomonas sp.

A novel inulinolytic microorganism, Xanthomonas sp. produced an endoinulinase, to be used for inulooligosaccharide (IOS) formation from inulin, at an activity of 11 units ml^–1 (1.2 mg protein ml^–1). The endoinulinase was optimally active at 45°C and pH 6.0. Batchwise production of IOS was carried out by the partially purified endoinulinase with a maximum yield of about 86% on a total sugar basis with 10 g inulin l^–1. The major IOS components were DP (degree of polymerization) 5 and 6 with trace amount of smaller oligosaccharides.     

Oral Microbiota Distinguishes Acute Lymphoblastic Leukemia Pediatric Hosts from Healthy Populations

In leukemia, oral manifestations indicate aberrations in oral microbiota. Microbiota structure is determined by both host and environmental factors. In human hosts, how health status shapes the composition of oral microbiota is largely unknown. Taking advantage of advances in high-throughput sequencing, we compared the composition of supragingival plaque microbiota of acute lymphoblastic leukemia (ALL) pediatric patients with healthy controls. The oral microbiota of leukemia patients had lower richness and less diversity compared to healthy controls. Microbial samples clustered into two major groups, one of ALL patients and another of healthy children, with different structure and composition. Abundance changes of certain taxa including the Phylum Firmicutes, the Class Bacilli, the Order Lactobacillales, the Family Aerococcaceae and Carnobacteriaceae, as well as the Genus Abiotrophia and Granulicatella were associated with leukemia status. ALL patients demonstrated a structural imbalance of the oral microbiota, characterized by reduced diversity and abundance alterations, possibly involved in systemic infections, indicating the importance of immune status in shaping the structure of oral microbiota.     

Effects of nitrogen application on beetle communities in tea plantations

In contrast to grassland and forest ecosystems, little is known about insect response to nitrogen deposition in agricultural ecosystems. This study was carried out to investigate the effects of short-term (1–2 years) nitrogen application (0, 172.5, 345.0, 690.0, and 1035.0 kg · N ha⁻¹· y⁻¹) on beetle communities in tea plantations. A collection of 34 families, 89 species of beetles, was obtained from a tea plantation in Wuyishan, China. Among them, herbivores, predators and detritivores had 52, 29, and eight species, respectively. Species richness, effective diversity and abundance (measured as the number of individuals and insect biomass) of the beetle community were not significantly related to the rate of nitrogen application. However, nitrogen application changed the species distribution and weakly increased the evenness of species distribution, while this did not significantly change the species evenness. Species richness and abundance of herbivores and predators were not significantly related to the rate of nitrogen application. However, there were some variations in trophic responses to nitrogen. Species richness and abundance of detritivores increased with increasing nitrogen application.