Fluorescence resonance energy transfer reports properties of syntaxin1a interaction with Munc18-1 in vivo

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal alpha-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced approximately 70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca(2+)-regulated secretory events.     

Integrated soil and plant phosphorus management for crop and environment in China. A review

In semi-arid grassland ecosystems, soil biogeochemical processes are controlled by seasonal and inter-annual rainfall variation and temperature, which may override the long-term impact of grazers on N availability and N dynamics. In a three-year (2004?C2006) case study of an Inner Mongolian grassland, we analysed time-integrated (ion-exchange resins) and instantaneous (soil mineral N extractions) inorganic N availability at three sites of varying grazing intensities and combined these data with information on soil water content (SWC), aboveground net primary productivity (ANPP) and plant N uptake. Additionally, the effects of rainfall and grazing on N-form availability (NO ₃ ^? -N, NH ₄ ⁺ -N) were considered. Grazing had less impact on N availability compared to seasonal and annual rainfall distribution. One of the three study years (2004) showed a grazing effect with higher resin-N availability at the ungrazed site compared to the heavily grazed site. Inorganic N availability was low in the driest year (2005) and highest in a year of average rainfall amount and favourable distribution (2004). In general, we found a positive relationship between inorganic N availability and both plant productivity and plant N uptake. Rainfall also controlled the plant available NO ₃ ^? -N and NH ₄ ⁺ -N pools; NH ₄ ⁺ -N dominated the available inorganic N-form in times of low SWC, while the available NO ₃ ^? -N increased with SWC. We observed N availability and plant productivity in a temporal synchronized pattern. Increased rainfall variability and land-use practices affecting SWC will likely alter N availability dynamics (and the relation of N-forms) and, therefore, important processes of semi-arid natural grassland carbon and N cycling.     

SpltMNPV日本分离株gp41的克隆表达及gp41和ph的进化分析

从斜纹夜蛾核型多角体病毒(Spodopteralitura multicapsid nucleopolyhedrovirus,SpltMNPV)日本分离株(C3)基因组中克隆了gp41基因.该基因编码区含993bp核苷酸,编码分子量为36.9kDa的多肽.将该基因克隆至原核表达载体pET28a,经IPTG诱导后在大肠杆菌BL21(DE3)中获得了表达.应用CLUSTAL程序分析表明,SpltMNPV日本株(C3)gp41的核苷酸序列和氨基酸序列与SpltMNPV中国G2株相似性最高,均达99.9%.用MEGA分别构建了基于gp41和ph的聚类分析图和分子进化树,发现它们具有相似的拓扑结构.将这两个基因序列结合在一起构建进化树,该树的结构与基于gp41的进化树相似.突变率分析显示gp41的突变率高于ph,这意味着在杆状病毒进化过程中,gp41和ph面临不同的选择压力.     

杜香植物资源开发利用的基础研究——Ⅰ.杜香油化学成分的研究

应用气相色谱-质谱联用技术,对杜香挥发油的化学成分进行分析,共鉴定出桧烯、α-萜品烯、γ-萜品烯、萜品烯-[4]-醇、桃金娘烯醛等18个化合物,并测定了各成分在挥发油中的含量。     

木质素降解菌Bax的筛选及特性研究

为了高效降解造纸污水中木质素类化合物,采用苯胺兰和鞣酸平板法从腐木分离、筛选得到一株具有高降解木质素活性的丝状真菌,经鉴定为绿色木霉,命名为Bax.最适碳源为葡萄糖和蔗糖,最适氮源为蛋白胨和尿紊,最适酸碱度为pH 5.0,最适温度为30℃.通过对木质素氧化酶系分析,主要起作用的是漆酶和木质素酶,为造纸污水的处理奠定了基础.     

Design and comparison of gene-pyramiding schemes in animals

Marker-assisted gene pyramiding provides a promising way to develop new animal breeds or lines, in which genes responsible for certain favorable characters identified in different breeds or lines are incorporated. In consideration of features of animal populations, we proposed five schemes for pyramiding three genes, denoted Scheme A-E, and five schemes for pyramiding four genes, denoted Scheme F-J. These schemes are representative of the possible alternatives. We also provided an algorithm to compute the population sizes needed in each generation. We compared these schemes with respect to the total population size and the number of generations required under different situations. The results show that there is no scheme that is optimal in all cases. Among the schemes for pyramiding three genes from three lines (L1, L2 and L3), Scheme D (a three-way cross between the three lines are first performed, followed by a backcross to L1 and a subsequent intercross to obtain the desired genotype) has a significant advantage over the other schemes when the recombination rate between adjacent genes ranges from 0.1 to 0.4, while Scheme A (a two-way cross between L1 and L2 and a subsequent intercross are performed, followed by a cross with L3 and a subsequent intercross to obtain the desired genotype) is optimal when recombination rate is 0.5. Among schemes for pyramiding four genes from four lines (L1, L2, L3 and L4), Scheme I (seperately, a two-way cross between L1 and L2 (L3 and L4) followed by a backcross to L1 (L3) and a subsequent intercross are performed, then the offspring from the two sides are crossed and followed by a backcross to L1 and a subsequent intercross to obtain the desired genotype) is optimal when the recombination rate ranges from 0.1 to 0.4, while Scheme F (cross and subsequent intercross between the four lines are performed successively) is the optimal when the recombination rate is 0.5. We also disscuss how the animals' reproductive capacity, the probabilities of obtaining the desired genotypes and genetic distance between adjacent genes would affect the design of an optimal scheme.     

离子对高效液相色谱法分析黄瓜花叶病毒侵染烟草的总RNA水平变化

为研究植物病毒在RNA水平上对寄主植物的基因表达产生的影响,采用离子对高效液相色谱法,对黄瓜花叶病毒侵染烟草造成的总RNA的组成成分变化进行研究。离子对液相色谱法,是近几年才应用于对RNA进行分离、纯化和分析的一种试验技术,具有操作简单、重复性好、所需时间短等优点。选择适宜的离子对试剂,并对选定的离子对试剂正己胺/1,1,1,3,3,3-六氟-2-丙醇进行了优化,达到了较好的总RNA分离效果,并观察到健康植株和染病植株分离峰之间的差异,有差异的RNA种类还需进一步试验来验证。为研究植物病毒的致病机制提供一种新的试验方法和途径。     

RU486对早孕猕猴海马GR的影响

目的探讨早孕猕猴给予大剂量RU486 3天后海马糖皮质激素受体(GR)的表达变化.方法 15只早孕猕猴随机分为空白对照组、赋形剂组和RU486组.空白对照组不予任何处理,赋形剂组和RU486组分别鼻饲赋形剂和RU486 3天.应用单克隆抗体链菌素亲生物蛋白过氧化酶(SP)免疫组织化学方法观察海马GR的表达情况,并用电子计算机图象分析技术进行处理.结果 RU486组猕猴妊娠终止,该组的海马GR表达显著下降,与对照组的差异有显著性.空白对照组和赋形剂组猕猴妊娠没有终止,其海马GR表达无差异.结论 RU486可使早孕猕猴海马的GR表达下降,推测这可能是该药终止妊娠的中枢作用机理之一.