铜绿假单胞菌pfm基因对三型分泌系统效应蛋白的影响

[目的]检测铜绿假单胞菌基因pfm对三型分泌系统效应蛋白的影响.[方法]构建pfm基因互补菌株pfmC.提取野生株PAO1、敲除株Δpfm和互补株pfmC的RNA,利用Real-time PCR从转录水平检测效应蛋白ExoS、ExoT和ExoY转录水平的变化.以ExoS为代表,检测细胞内和分泌到细胞外效应蛋白的含量.收集铜绿假单胞菌PAO1、Δpfm和pfmC菌体内和分泌到细胞外的总蛋白,利用ExoS多克隆抗体进行Western杂交,特异检测ExoS的蛋白水平.[结果]与野生型相比,Δpfm中exoS、exoT和exoY转录水平明显降低,而pfmC中这3个蛋白的转录水平得到回补.Δpfm菌体内和分泌到细胞外的ExoS量均明显低于野生株PAO1,pfmC细胞内和细胞外分泌的ExoS蛋白量均得到恢复.[结论]铜绿假单胞菌基因pfm会影响三型分泌系统效应蛋白的水平.     

GRASP55 regulates the unconventional secretion and aggregation of mutant huntingtin

Recent studies demonstrated that the Golgi reassembly stacking proteins (GRASPs), especially GRASP55, regulate Golgi-independent unconventional secretion of certain cytosolic and transmembrane cargoes; however, the underlying mechanism remains unknown. Here, we surveyed several neurodegenerative disease–related proteins, including mutant huntingtin (Htt-Q74), superoxide dismutase 1 (SOD1), tau, and TAR DNA–binding protein 43 (TDP-43), for unconventional secretion; our results show that Htt-Q74 is most robustly secreted in a GRASP55-dependent manner. Using Htt-Q74 as a model system, we demonstrate that unconventional secretion of Htt is GRASP55 and autophagy dependent and is enhanced under stress conditions such as starvation and endoplasmic reticulum stress. Mechanistically, we show that GRASP55 facilitates Htt secretion by tethering autophagosomes to lysosomes to promote autophagosome maturation and subsequent lysosome secretion and by stabilizing p23/TMED10, a channel for translocation of cytoplasmic proteins into the lumen of the endoplasmic reticulum–Golgi intermediate compartment. Moreover, we found that GRASP55 levels are upregulated by various stresses to facilitate unconventional secretion, whereas inhibition of Htt-Q74 secretion by GRASP55 KO enhances Htt aggregation and toxicity. Finally, comprehensive secretomic analysis identified novel cytosolic cargoes secreted by the same unconventional pathway, including transgelin (TAGLN), multifunctional protein ADE2 (PAICS), and peroxiredoxin-1 (PRDX1). In conclusion, this study defines the pathway of GRASP55-mediated unconventional protein secretion and provides important insights into the progression of Huntington’s disease.     

调控巨噬细胞迁移抑制因子基因表达的影响因素

巨噬细胞迁移抑制因子(macrophage migration inhibitory factor, MIF)是一种广泛表达的多效性细胞因子,参与多种炎症和免疫疾病的过程并在其中发挥重要作用,是许多疾病的生物标志物或治疗靶点。MIF基因在系统发育中高度保守,在其启动子区有多种不同转录因子的特定结合位点,借此调节MIF的表达。MIF在细胞内外均发挥作用,且MIF是组成型表达。因此,研究调控MIF基因表达和刺激MIF分泌的相关因素具有重要意义。本文通过对MIF基因和MIF启动子上的结合位点的简述,对影响MIF基因表达的相关因素进行总结和归类。根据与MIF基因结合的方式,可分为：(1)与MIF基因启动子特定位点结合,改变转录活性;(2)与MIF CATT5-8微卫星重复序列结合,改变高表达MIF等位基因;(3)非编码RNA调控MIF表达;(4)影响MIF分泌的相关因素。通过对这4类调控MIF基因表达的相关因素的综述,进而认识MIF基因表达的调控机制和影响因素,以期对其治疗相关疾病提供理论基础。     

盐碱池塘细巧华哲水蚤对浮游植物的摄食生态研究

利用常规显微镜直接计数法和叶绿素 a法研究了盐碱池塘细巧华哲水蚤 ( Sinocalanustenellus)对浮游藻类的原位摄食率及其影响因素。测得该水蚤对水柱浮游植物的滤水率为 1 .73～ 1 5 5 .1 6μl/ ( ind.· h) ,摄食率为 92～ 2 5 38cell/( ind.· h)或 0 .7～ 2 7.7ng C/ ( ind.· h)。两种方法测定的滤水率和摄食率基本相符 ( p>0 .5 ,t检验 )。该水蚤可有选择地滤食诸如小色球藻、光甲藻、绿裸藻 ,单生卵囊藻和小环藻等 ,其选择性指数分别为 0 .81、0 .75、0 .72、0 .6 1和 0 .35。不同光处理下细巧华哲水蚤的滤食率多数情况是无光时摄食率高。在本实验的条件下该水蚤在饥饿后的滤水率和摄食率与正常情况相比均显著降低 ( P<0 .0 1 ,t检验 )。     

Calcium in the micropylar secretion and receptivity of explanted Crocus ovules to intra- and interspecific pollen

The micropylar secrete of the ovules of Crocus vernus ssp. vernus was analyzed for the Ca⁺² content by atomic absorption, and its capacity to germinate and attract pollen was tested by pollinating explanted ovules, and incubating in absence of culture medium. The results display a Ca⁺² concentration of 28.9 mM in the micropylar secrete. On this secrete both compatible- and incompatible pollen germinates with a mean percentage of 53.7%, and their pollen tubes enter the micropylar canal with percentages of 32.3% to 21.0%. In situ the ovules fail to attract tubes of incompatible pollen. The results are discussed in relation to the ovule receptivity and the guided growth of pollen tubes, substantiating the model of the tropic growth towards increasing calcium concentrations.     

A high‐throughput method to quantify feeding rates in aquatic organisms: A case study with Daphnia

1. Food ingestion is one of the most basic features of all organisms. However, obtaining precise—and high‐throughput—estimates of feeding rates remains challenging, particularly for small, aquatic herbivores such as zooplankton, snails, and tadpoles. These animals typically consume low volumes of food that are time‐consuming to accurately measure.
2. We extend a standard high‐throughput fluorometry technique, which uses a microplate reader and 96‐well plates, as a practical tool for studies in ecology, evolution, and disease biology. We outline technical and methodological details to optimize quantification of individual feeding rates, improve accuracy, and minimize sampling error.
3. This high‐throughput assay offers several advantages over previous methods, including i) substantially reduced time allotments per sample to facilitate larger, more efficient experiments; ii) technical replicates; and iii) conversion of in vivo measurements to units (mL^‐1 hr^‐1 ind^‐1) which enables broad‐scale comparisons across an array of taxa and studies.
4. To evaluate the accuracy and feasibility of our approach, we use the zooplankton, Daphnia dentifera, as a case study. Our results indicate that this procedure accurately quantifies feeding rates and highlights differences among seven genotypes.
5. The method detailed here has broad applicability to a diverse array of aquatic taxa, their resources, environmental contaminants (e.g., plastics), and infectious agents. We discuss simple extensions to quantify epidemiologically relevant traits, such as pathogen exposure and transmission rates, for infectious agents with oral or trophic transmission.

     

北京典型树种木质组织碳释放速率温度敏感性的时间变化规律和铅锤分异特征

采用红外气体分析法(IRGA)于2014年1—12月原位测定了北京市4个典型树种(国槐Sophora japonica,旱柳Salix matsudana,华北落叶松Larix principis-rupprechtii和侧柏Platycladus orientalis)在不同高度上的木质组织CO_2通量速率(E_(CO_2)),旨在比较不同树种间E_(CO_2)及其温度敏感性(Q_(10))的时间变化规律和铅锤分异特征。研究结果显示:(1)4个树种的E_(CO_2)均表现为单峰型季节变化规律,生长月份内的E_(CO_2)显著高于非生长月份,温度和枝干的径向生长是影响E_(CO_2)季节变化的主要因素;(2)E_(CO_2)对温度的敏感性在夏季月份明显降低,且出现明显的垂直分异:Q_(10)随测量高度的增加而增加,呈现出非连续的阶梯分布;(3)在日间尺度上,阔叶树种E_(CO_2)对温度的感性系数Q_(10)出现昼夜不对称现象,晚上Q_(10)明显升高。准确量化E_(CO_2)的时间变化规律和铅锤分异特征,细化不同时间尺度下E_(CO_2)对温度的响应特征,成为准确估算木质组织碳排放的前提条件。     

Remote Homology between Munc13 MUN Domain and Vesicle Tethering Complexes

Most core components of the neurotransmitter release machinery have homologues in other types of intracellular membrane traffic, likely underlying a universal mechanism of intracellular membrane fusion. However, no clear similarity between Munc13s and protein families generally involved in membrane traffic has been reported, despite the essential nature of Munc13s for neurotransmitter release. This crucial function was ascribed to a minimal Munc13 region called the MUN domain, which likely participates in soluble N-ethylmaleimide sensitive factor attachment protein receptor complex (SNARE) assembly and is also found in Ca²⁺-dependent activator protein for secretion. We have now used comparative sequence and structural analyses to study the structure and evolutionary origin of the MUN domain. We found weak yet significant sequence similarities between the MUN domain and a set of protein subunits from several related vesicle tethering complexes, such as Sec6 from the exocyst complex and Vps53 from the Golgi-associated retrograde protein complex. Such an evolutionary relationship allows structure prediction of the MUN domain and suggests functional similarities between MUN domain-containing proteins and multisubunit tethering complexes such as exocyst, conserved oligomeric Golgi complex, Golgi-associated retrograde protein complex, and Dsl1p. These findings further unify the mechanism of neurotransmitter release with those of other types of intracellular membrane traffic and, in turn, support a role for tethering complexes in soluble N-ethylmaleimide sensitive factor attachment protein receptor complex assembly.     

固沙树种梭梭在不同水分梯度下的光合生理特征

对不同水分梯度下梭梭光合作用的研究发现,地下水位、土壤含水量、灌水量对梭梭的光合具有明显的影响。在1．4m、2．4m、3．4m地下水位下,梭梭的光合速率、呼吸作用、量子效率随水位的降低而降低,光补偿点随水位的降低而增大;在滴灌供水时,梭梭的光合速率较对照明显提高,且灌水量越大,梭梭的光合速率越高;半流动沙丘、丘间低地、粘土沙障固定沙丘退化梭梭的光合作用的强弱同其林地土壤水分的高低基本一致。7月,不同水分梯度下的梭梭光合作用均表现出午休现象,9月只有立地条件较差的梭梭发生光合午休,而且9月光合作用要强于7月,说明9月环境条件更有利于梭梭生长。     

重组人血清白蛋白在Pichia pastoris中分泌表达影响因素的研究

为获得高产稳产重组人血清白蛋白的Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ细胞株用于高密度发酵,构建了多种分泌表达载体,在Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ中作表达,研究表达载体构建等因素对表达量的影响。发现采用酿酒酵母α性成熟因子前导肽比人血清白蛋白天然前导肽作为分泌信号肽的表达量要高１０％左右。而载体整合方式、整合拷贝数、５’非翻译区的改造和甲醇利用表型等对表达量无规律性影响。筛选到的高表达株经高密度发酵,细