Patterns of [PSI+] aggregation allow insights into cellular organization of yeast prion aggregates

The yeast prion phenomenon is very widespread and mounting evidence suggests that it has an impact on cellular regulatory mechanisms related to phenotypic responses to changing environments. Studying the aggregation patterns of prion amyloids during different stages of the prion life cycle is a first key step to understand major principles of how and where cells generate, organize and turn-over prion aggregates. The induction of the [PSI⁺] state involves the actin cytoskeleton and quality control compartments such as the Insoluble Protein Deposit (IPOD). An initially unstable transitional induction state can be visualized by overexpression of the prion determinant and displays characteristic large ring- and ribbon-shaped aggregates consisting of poorly fragmented bundles of very long prion fibrils. In the mature prion state, the aggregation pattern is characterized by highly fragmented, shorter prion fibrils that form aggregates, which can be visualized through tagging with fluorescent proteins. The number of aggregates formed varies, ranging from a single large aggregate at the IPOD to multiple smaller ones, depending on several parameters discussed. Aggregate units below the resolution of light microscopy that are detectable by fluorescence correlation spectroscopy are in equilibrium with larger aggregates in this stage and can mediate faithful inheritance of the prion state. Loss of the prion state is often characterized by reduced fragmentation of prion fibrils and fewer, larger aggregates.     

Serologic and immunochromatographic detection of oxygenated polyenoic acids in Euglena gracilis var. bacillaris

The extent of evolutionary conservation of DNA complimentary to RNA stored in the mature oocyte of the sea urchin S. purpuratus has been assessed. To do this, such DNA was hybridized with total genomic DNA of S. purpuratus and S. franciscanus and the thermal stability of the resultant duplexes was measured by two methods. In the first method, the duplexes were bound to hydroxylapatite and thermally eluted; the difference in thermal stability between homologous and heterologous duplexes averaged 6.9 degrees C in duplicate determinations. In the second experiment, the same hybrids were thermally melted in 2.4M tetraethylammonium chloride, then assayed with S1 nuclease; the difference in thermal stability of homologous and heterologous duplexes was 4.8 degrees C. Either value is significantly lower than the divergence of total single-copy DNA among these species as measured by the same techniques. This demonstrates that DNA sequences complimentary to maternal RNA are conserved during evolution, and thus that a high fraction of them are likely to be physiologically functional.     

Clinical analysis of human umbilical cord mesenchymal stem cell allotransplantation in patients with premature ovarian insufficiency

ObjectivePremature ovarian insufficiency (POI) is a refractory disease that seriously affects female fertility. Growing body of evidence has indicated mesenchymal stem cells (MSCs) as promising resources in regenerative medicine. In this study, we treated POI patients with umbilical cord‐derived MSCs (UCMSCs) and then investigated the restoration of ovarian function and clinical outcomes through follow‐ups.Materials and methodsSixty‐one patients diagnosed with POI participated in this study. UCMSCs were isolated and cultured according to GMP standards, and then transplanted to the patients’ ovary by orthotopic injection under the guidance of vaginal ultrasound. We monitored side effects, vital signs and changes in clinical and collected haematological and imaging parameters during the follow‐ups.ResultsAll patients showed normal clinical behaviour without serious side effects or complications relevant to the treatment. Transplantation of UCMSCs rescued the ovarian function of POI patients, as indicated by increased follicular development and improved egg collection. POI patients who experienced shorter amenorrhoea durations (<1 year) seemed to obtain mature follicles more easily after stem cell therapy, and patients with better ovarian conditions (pre‐operative antral follicles) were more likely to derive the better outcomes by UCMSC injection. Four successful clinical deliveries were obtained from POI patients after UCMSC transplantation, and all of these babies are developed normally.ConclusionsThe clinical trial result sugggests a possible therapy for POI by UCMSC transplantation.     

基于SOE-PCR的两种鱼类hepcidin基因串联及其在毕赤酵母中的表达

Hepcidin抗菌肽是由生物肝脏细胞表达的一类具有抗菌作用和铁代谢调节功能的碱性小分子肽,它们在机体免疫系统中发挥了重要作用,被认为是抗生素的理想替代品。通过重组DNA表达技术制备抗菌肽无疑是一条良好的途径。为扩大hepcidin的抗菌谱和提高其表达量,通过SOE-PCR将斑点叉尾鮰Ictalurus punctatus hepcidin成熟肽的c DNA"m CH"与尼罗罗非鱼Oreochromis niloticus hepcidin成熟肽的c DNA"m TH"进行串联,并在两端分别添加Eco RⅠ和NotⅠ酶切位点,以p PIC9K为真核表达载体,成功构建了"p PIC9K-m CH-m TH"重组表达载体;电转化进毕赤酵母GS115中,经不同浓度G418和选择性培养基筛选以及对酵母基因组DNA的PCR鉴定,得到高拷贝酵母转化子;在30℃、以1%的甲醇诱导表达不同时间后,经Tricine-SDS-PAGE分析,表明发酵培养72 h时目的蛋白的表达量最高,达77 mg/L。发酵上清经SP-Sepharose阳离子交换层析纯化后获得高纯度的目的蛋白。抑菌实验显示含目的蛋白的发酵上清和经纯化后的重组目的蛋白对革兰氏阳性和革兰氏阴性细菌都有良好的抑菌效果。本研究结果为hepcidin抗菌肽的产业化生产奠定了良好的基础。     

Nesting success in Redshank Tringa totanus breeding on coastal meadows and the importance of habitat features used as perches by avian predators

Capsule Nest survival rates could not be explained by distance to habitat edges or other features used by predators.

Aims To investigate if predation on Redshank nests was affected by habitat characteristics at a local scale.

Methods We examined survival rates of Redshank nests on coastal meadows on the Baltic island of Gotland, Sweden, over two breeding seasons. We analysed nest survival rates in relation to several habitat characteristics that may benefit predators searching for nests. We examined existing studies concerning predation rates on wader nests in relation to edges and habitat features potentially used by avian predators.

Results We found no significant effects of distance to habitat edge or to nearest potential lookout for avian predators or to shoreline. Abundance of Lapwings Vanellus vanellus, an aggressive species with active nest-defence, did not have any significant effect on nest survival rate, nor did vegetation concealment of nests. Nest survival rates were significantly different between years and lower later in the season.

Conclusions There is only weak support for general effects on wader nest predation rates of proximity to edges and features used by avian predators. Simple mechanical management actions such as removal of trees and bushes on coastal meadows may not directly, and by itself, result in higher reproductive success of waders. Further understanding is needed of the behaviour of predators and the composition of the predator community in different landscapes in order to increase the efficiency of management actions to remove threats to vulnerable species on coastal meadows.     

猪β防御素2成熟肽在酵母中的表达

【目的】将猪β防御素2成熟肽基因片段正确整合到酵母基因组染色体上,从而得到稳定的猪β防御素2成熟肽的毕赤酵母表达株。实现猪β防御素2成熟肽的表达。【方法】首先参考酵母偏爱密码子,设计3段引物序列,利用PCR技术扩增得到β防御2成熟肽基因,构建了重组质粒pPIC9k-GST-pBD-2和pPIC9k-pBD-2。将线性化的重组质粒电转化到毕赤酵母KM71细胞中。最后筛选得到酵母阳性克隆,通过不断调节表达条件,实现猪β防御素2成熟肽的表达。【结果】将GST-pBD-2基因序列和pBD-2基因序列分别成功整合到酵母KM71基因组中,重组毕赤酵母工程菌构建成功;重组酵母蛋白GST-pBD-2和PBD-2都成功获得了表达;PBD-2成熟肽表达上清对猪霍乱沙门氏菌弱毒株C500有一定的抑制作用。【结论】获得表达pBD-2成熟肽的酵母菌株,本实验是用真核细胞表达pBD-2成熟肽的一次探索,为后续大量表达pBD-2成熟肽方法的研究打下了基础。     

禾谷镰刀菌粗毒素对不同抗性小麦品种成熟胚脱分化与再分化的影响

以5个赤霉病抗性水平不同的小麦品种为材料,研究了禾谷镰刀菌粗毒素对小麦成熟胚脱分化与再分化的影响.结果表明,小麦成熟胚愈伤组织的诱导率和分化率在品种间、毒素浓度间、毒素浓度×品种间差异极显著.低浓度（5 g/L）禾谷镰刀菌粗毒素能够促进抗性较强品种的出愈速度,高浓度禾谷镰刀菌粗毒素（10 g/L和15 g/L）对成熟胚愈伤组织的出愈速度起到抑制作用,随着浓度提高,抑制作用增强.成熟胚愈伤组织的诱导率和分化率随着禾谷镰刀菌粗毒素浓度升高而下降,品种的抗性越差下降幅度越大.10 g/L毒素浓度,5个品种成熟胚愈伤组织的SOD活性均比对照（0 g/L）增加;20 g/L毒素浓度,5个品种成熟胚愈伤组织的SOD活性均有所下降,抗性品种苏麦3号和郑9023的SOD活性仍高于对照,其它3个感性品种低于对照,SOD活性与小麦品种的赤霉病抗性水平存在一定关系.研究结果为小麦赤霉病抗性育种提供了一定的技术支撑.     

Improved measurements of Na+ fluxes in plants using calixarene-based microelectrodes

Ion-selective microelectrodes are a powerful tool in studying adaptive responses of plant cells and tissues to various abiotic stresses. However, application of this technique in Na⁺ flux measurements was limited due to poor selectivity for Na⁺ ions of commercially available Na⁺ cocktails. Often, these cocktails cannot discriminate between Na⁺ and other interfering ions such as K⁺ and Ca²⁺, leading to inaccurate measurements of Na⁺ concentration and, consequently, inaccurate Na⁺ flux calculations. To overcome this problem, three Na⁺-selective cocktail mixtures were prepared using tetramethoxyethyl ester derivative of p-t-butyl calix[4]arene. These cocktail mixtures were compared with commercially available ETH 227-based Na⁺ cocktail for selectivity for Na⁺ ions over other ions (particularly K⁺ and Ca²⁺). Among the three calixarene-based Na⁺ cocktails tested, cocktail 2 [in % w/w: Na⁺ ionophore (4-tert-butylcalix[4]arene-tetra acetic acid tetraethyl ester) 3.5, the plasticizer (2-nitrophenyl octyl ether) 95.9 and lipophilic anion (potassium tetrakis (4-chlorophenyl) borate) 0.6] showed the best selectivity for Na⁺ ions over K⁺ and Ca²⁺ ions and was highly stable over time (up to 10 h). Na⁺ flux measurements under a wide range of NaCl concentrations (25-150 mM) using Na⁺ cocktail 2 established a clear dose-response relationship between severity of salt stress and magnitude of Na⁺ influx at the distal elongation and mature zones of Arabidopsis thaliana roots. Furthermore, Na⁺ cocktail 2 was compared with commercially available ETH 227-based Na⁺ cocktail by measuring Na⁺ fluxes at the two Arabidopsis root zones in response to 100 mM NaCl treatment. With calixarene-based Na⁺ cocktail 2, a large decreasing Na⁺ influx (0-15 min) followed by small Na⁺ influx (15-45 min) was measured, whereas with ETH-based Na⁺ cocktail Na⁺ influx was short-lived (1-3 min) and was followed by Na⁺ efflux (3-45 min) that might have been due to K⁺ and Ca²⁺ efflux measured together with Na⁺ influx. In conclusion, Na⁺-selective calixarene-based microelectrodes have excellent potential to be used in real-time Na⁺ flux measurements in plants.