Role of Membrane Microdomains in Compartmentation of cAMP Signaling

Spatially restricting cAMP production to discrete subcellular locations permits selective regulation of specific functional responses. But exactly where and how cAMP signaling is confined is not fully understood. Different receptors and adenylyl cyclase isoforms responsible for cAMP production are not uniformly distributed between lipid raft and non-lipid raft domains of the plasma membrane. We sought to determine the role that these membrane domains play in organizing cAMP responses in HEK293 cells. The freely diffusible FRET-based biosensor Epac2-camps was used to measure global cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. Disruption of lipid rafts by cholesterol depletion selectively altered cAMP responses produced by raft-associated receptors. The results indicate that receptors associated with lipid raft as well as non-lipid raft domains can contribute to global cAMP responses. In addition, basal cAMP activity was found to be significantly higher in non-raft domains. This was supported by the fact that pharmacologic inhibition of adenylyl cyclase activity reduced basal cAMP activity detected by Epac2-CAAX but not Epac2-MyrPalm or Epac2-camps. Responses detected by Epac2-CAAX were also more sensitive to direct stimulation of adenylyl cyclase activity, but less sensitive to inhibition of phosphodiesterase activity. Quantitative modeling was used to demonstrate that differences in adenylyl cyclase and phosphodiesterase activities are necessary but not sufficient to explain compartmentation of cAMP associated with different microdomains of the plasma membrane.     

Crystal structure and novel recognition motif of rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis

Clostridium botulinum C3 exoenzyme inactivates the small GTP-binding protein family Rho by ADP-ribosylating asparagine 41, which depolymerizes the actin cytoskeleton. C3 thus represents a major family of the bacterial toxins that transfer the ADP-ribose moiety of NAD to specific amino acids in acceptor proteins to modify key biological activities in eukaryotic cells, including protein synthesis, differentiation, transformation, and intracellular signaling. The 1.7 A resolution C3 exoenzyme structure establishes the conserved features of the core NAD-binding beta-sandwich fold with other ADP-ribosylating toxins despite little sequence conservation. Importantly, the central core of the C3 exoenzyme structure is distinguished by the absence of an active site loop observed in many other ADP-ribosylating toxins. Unlike the ADP-ribosylating toxins that possess the active site loop near the central core, the C3 exoenzyme replaces the active site loop with an alpha-helix, alpha3. Moreover, structural and sequence similarities with the catalytic domain of vegetative insecticidal protein 2 (VIP2), an actin ADP-ribosyltransferase, unexpectedly implicates two adjacent, protruding turns, which join beta5 and beta6 of the toxin core fold, as a novel recognition specificity motif for this newly defined toxin family. Turn 1 evidently positions the solvent-exposed, aromatic side-chain of Phe209 to interact with the hydrophobic region of Rho adjacent to its GTP-binding site. Turn 2 evidently both places the Gln212 side-chain for hydrogen bonding to recognize Rho Asn41 for nucleophilic attack on the anomeric carbon of NAD ribose and holds the key Glu214 catalytic side-chain in the adjacent catalytic pocket. This proposed bipartite ADP-ribosylating toxin turn-turn (ARTT) motif places the VIP2 and C3 toxin classes into a single ARTT family characterized by analogous target protein recognition via turn 1 aromatic and turn 2 hydrogen-bonding side-chain moieties. Turn 2 centrally anchors the catalytic Glu214 within the ARTT motif, and furthermore distinguishes the C3 toxin class by a conserved turn 2 Gln and the VIP2 binary toxin class by a conserved turn 2 Glu for appropriate target side-chain hydrogen-bonding recognition. Taken together, these structural results provide a molecular basis for understanding the coupled activity and recognition specificity for C3 and for the newly defined ARTT toxin family, which acts in the depolymerization of the actin cytoskeleton. This beta5 to beta6 region of the toxin fold represents an experimentally testable and potentially general recognition motif region for other ADP-ribosylating toxins that have a similar beta-structure framework.     

胡桃楸提取液对肿瘤细胞增殖的抑制作用

目的考察胡桃楸提取液对肿瘤细胞Hela、K562的抑制作用和相关机制。方法用MTT方法分析胡桃楸提取液对Hela、K562细胞增殖的影响。采用端粒酶PCR ELISA试剂盒分析胡桃楸提取液对Hela、K562细胞端粒酶的影响。结果 Hela细胞24、48和72 h的LD50分别为406.18μg/mL、319.48μg/mL和112.84μg/mL。K562细胞24 h LD50为154.50μg/mL。HLF细胞LD50为918.69μg/mL。胡桃楸提取液可抑制Hela细胞和K562细胞的端粒酶活性,而对HLF细胞端粒酶活性影响不大。结论胡桃楸提取液对Hela细胞、K562细胞有抑制作用,在低浓度下对HLF细胞杀伤不大。对肿瘤细胞抑制作用可能与抑制端粒酶活性相关。     

一种利用Cre/loxP系统进行标记基因删除与靶基因置换的细胞模型研究

Cre/lox系统可以介导DNA的定点插入和定点删除,可利用其实现转基因动物中"友好位点"的重复利用及标记基因的有效删除.为直观地评估该系统介导的以上两种重组反应的效果,通过标记基因并利用大鼠乳腺癌细胞系SHZ-88进行了模型研究.首先构建了两个载体:a.整合载体pTE-lox2272-DsRed-loxP-GFP-loxP,含有红色荧光标记基因DsRed和绿色荧光标记基因GFP;b.置换载体pT-lox2272-neo-loxP,含有筛选标记基因neo,用以置换DsRed基因.然后,用整合载体转染SHZ-88细胞,并随机挑取了3个同时表达DsRed和GFP的稳定整合细胞克隆.随后用置换载体和Cre表达载体PBS185对以上每个克隆分别进行了3次共转染,通过G418筛选并扩增培养后,总共获得1 070个克隆.通过分析标记基因DsRed和GFP在这些克隆中的表达情况:Cre介导的删除效率为91.1%,定点置换效率为29.3%.最后对部分克隆进行了PCR和DNA印迹鉴定,分子鉴定结果与发光的表型状况一致.这一方法为Cre/lox系统在转基因家畜生产中的进一步应用提供了实验依据.     

Closing the Achievement Gap through Modification of Neurocognitive and Neuroendocrine Function: Results from a Cluster Randomized Controlled Trial of an Innovative Approach to the Education of Children in Kindergarten

Effective early education is essential for academic achievement and positive life outcomes, particularly for children in poverty. Advances in neuroscience suggest that a focus on self-regulation in education can enhance children’s engagement in learning and establish beneficial academic trajectories in the early elementary grades. Here, we experimentally evaluate an innovative approach to the education of children in kindergarten that embeds support for self-regulation, particularly executive functions, into literacy, mathematics, and science learning activities. Results from a cluster randomized controlled trial involving 29 schools, 79 classrooms, and 759 children indicated positive effects on executive functions, reasoning ability, the control of attention, and levels of salivary cortisol and alpha amylase. Results also demonstrated improvements in reading, vocabulary, and mathematics at the end of kindergarten that increased into the first grade. A number of effects were specific to high-poverty schools, suggesting that a focus on executive functions and associated aspects of self-regulation in early elementary education holds promise for closing the achievement gap.     

氨肽酶N的表达及其与结石形成的关系(英文)

 为研究大鼠高胆固醇饮食时 ,肝脏氨肽酶N(APN)在实验结石形成中可能的结石发生作用 ,采用 1.2 %胆固醇饮食 4周 ,诱发新西兰兔胆囊结石形成 .根据兔APN基因cDNA序列设计引物 ,提取肝脏总RNA .利用RT PCR检测肝脏APNmRNA水平的变化 ,用组织化学方法观察肝脏毛细胆管膜上APN的表达 .观察新西兰兔胆囊结石形成过程中肝脏APN的mRNA水平的变化、APN表达及胆汁中APN活性、胆脂、总蛋白含量的变化 ,探讨APN在胆石形成中可能的作用 .经成石饲料饲养后 ,随着胆汁饱和度增加和APN活性加强 ,胆囊结石组肝脏APNmRNA水平较对照组明显增高 ,胆囊结石组胆汁中总胆固醇、CSI、总蛋白浓度及APN活性均明显高于对照组 ,且胆汁中APN活性与肝脏APN的表达及胆汁CSI增高呈正相关 .结果提示 ,当存在胆汁过饱和的情况下 ,APN很可能作为促成核因子在胆结石形成早期发挥重要作用     

Foliar modifications induced by inhibition of polar transport of auxin

The effects of auxin polar transport inhibitors,9-hydroxy-fluorene-9-carboxylic acid (HFCA);2,3,5-triiodobenzoic acid(TIBA) and trans-cinnamic acid (CA) on leaf pattern formation were investigated with shoots formed from cultured leaf explants of tobacco and cultured pedicel explants of Orychophragmus violaceus,and the seedlings of tobacco and Brassica chinensis,Although the effective concentration varies with the inhibitors used,all of the inhibitors induced the formation of trumpet-shaped and/or fused leaves.The frequency of trumpet-shaped leaf formation was related to the concentration of inhibitors in the medium.Histological observation of tobacco seedlings showed that there was only one main vascular bundle and several minor vascular bundles in normal leaves of the control,but there were several vascular bundles of more or less the same size in the trumpet-shaped leaves of treated ones.These results indicated that auxin polar transport played an important role on bilateral symmetry of leaf growth.     

害虫防治决策的复序贯分析方法及抽样技术研究

复序贯抽样决策技术实际应用的受限 ,原因在于截止限序贯抽样模型的缺乏。本文在检验昆虫种群空间格局回归模型的基础上 ,推导出了目前国内常用检验回归模型的截止限序贯抽样模型 ,并将其运用于复序贯分析决策过程中。实例分析表明 ,对于同一种生物种群 ,在一定的精度 (D)和置信水平(tα)要求下 ,复序贯抽样决策技术可以大幅度地节约抽样成本     

不同污染程度下毛白杨叶表面PM2.5颗粒的数量及性质和叶片气孔形态的比较研究

选择了北京市环境PM_(2.5)浓度不同的两个采样点的毛白杨(Populus tomentosa Carr.)作为研究对象,利用环境扫描电镜及X-射线能谱仪对杨树叶片表面滞留的PM_(2.5)颗粒进行了观察、统计和成分分析,并研究了叶片气孔对环境颗粒物污染的适应性变化。结果表明:夏秋两季西直门叶片样品上下表面的PM_(2.5)数量均多于森林公园样品这说明环境PM_(2.5)浓度是影响叶片表面滞留颗粒物数量的主要原因;其中叶片上表面是滞留PM_(2.5)颗粒的主要区域。森林公园样品中PM_(2.5)颗粒性质比较单一,硅铝酸盐颗粒和石英颗粒占很大比例,二者的主要来源均为天然源,如土壤扬尘、矿物颗粒等;而西直门采样点叶片样品滞留的PM_(2.5)颗粒的元素组成更为复杂,其中50%以上的硅铝酸盐颗粒检测出了明显的铜、钾、氯、钠等元素的谱峰其来源主要是工业排放;西直门样品PM_(2.5)的含硫量高于森林公园样品,且夏季明显高于秋季。研究还发现有少数PM_(2.5)颗粒进入了毛白杨叶片的气孔而且不同污染程度下气孔的形态特征存在差异。与森林公园毛白杨叶片的气孔相比,西直门处的毛白杨叶片气孔的长度、宽度、面积和气孔密度均较小,说明较高的PM_(2.5)污染程度对毛白杨叶片的形态发育有一定影响。研究结果可以为揭示植物叶片阻滞、吸收大气颗粒污染物的机制、合理选择和优化城市绿化树种从而改善空气质量提供一定的科学理论依据。     

amiRNA分子设计及其离体合成策略

详细介绍了基于WMD3 (Web MicroRNA Designer 3) 软件平台的amiRNA (artificial microRNA)分子自动设计方法及其离体合成策略。应用网络在线设计时,只需输入目的基因靶序列相关信息后便可获得候选amiRNA。根据选定的最佳amiRNA,可得到四条含有amiRNA以及载体中miRNA 两侧序列的寡聚核苷酸序列。重叠延伸PCR合成策略可以以这四条序列以及根据质粒模板设计的A、B两段序列作为引物,扩增出目标amiRNA。同样尿嘧啶切除的策略也可合成amiRNA,但这些引物序列需做适当调整变动。此外,本文还介绍了基于特异引物退火的amiRNA合成策略,该策略可保证amiRNA分子能够一步PCR合成,合成后的amiRNA表达盒可通过合适的限制性位点被克隆至目的载体中的相应位点。可以预见,这种amiRNA分子设计的科学性与合成策略的精确性将会使amiRNAi技术在生物基因功能分析中发挥更加重要的作用,对生命科学研究产生深远的影响。