双酰胺类杀虫剂对棉铃虫和松毛虫赤眼蜂的选择毒力

为了指导棉铃虫防控中化学杀虫剂和天敌昆虫协调使用,本文采用浸叶法和玻管药膜法测定了4种双酰胺类杀虫剂对棉铃虫3龄幼虫和松毛虫赤眼蜂成蜂的毒力.结果表明,供试双酰胺类杀虫剂中氯虫苯甲酰胺、氟苯虫酰胺和氟氰虫酰胺对棉铃虫幼虫表现出了较高的毒力水平(LC50分别为0.2882、0.3894、0.8609 mg/L),稍低于对照药剂甲氨基阿维菌素苯甲酸盐(简称:甲维盐)(LC50为0.06493 mg/L);而溴氰虫酰胺对棉铃虫的毒力(LC50为3.7638 mg/L)明显低于甲维盐.从对天敌昆虫安全性来看,氯虫苯甲酰胺、氟苯虫酰胺和氟氰虫酰胺对松毛虫赤眼蜂的毒性(LC50分别为1.0060、0.9933、1.1541 mg/L)明显低于甲维盐(其LC50为0.1646 mg/L),表现出了较高的安全性;但是,溴氰虫酰胺对松毛虫赤眼蜂毒性较高(LC50为0.4952 mg/L),安全性较差.本研究将为棉铃虫防控中药剂与天敌协调使用提供依据.     

蛾类昆虫性信息素受体及其作用机理

蛾类昆虫性信息素受体首先从烟芽夜蛾Heliothis virescens和家蚕Bombyx mori中鉴定出来, 到目前为止已经克隆得到了19种蛾类昆虫的几十种性信息素受体基因, 并且这些基因在系统发育树中聚成一个亚群。性信息素受体从蛾类蛹期开始表达, 主要表达在雄性触角的毛形感器中, 少部分受体在雌性触角、 雄性触角其他感器以及身体其他部位中也有表达。大部分蛾类性信息素受体的配体并不是单一的, 而是能够对多种性信息素组分有反应, 部分性信息素受体还能够识别性信息素以外的其他物质, 还有一部分性信息素受体的识别配体目前尚不清楚。另外发现在雌性蛾类触角中也存在一些嗅觉受体能够识别雄性分泌的性信息素。在蛾类性信息素受体与性信息素识别的过程中, 性信息素结合蛋白不仅能够特异性地运送配体到嗅觉神经元树状突上, 还能够提高性信息素与性信息素受体之间的结合效率。另外, OrCo类受体与性信息素受体共表达在嗅觉神经元中, 在蛾类性信息素受体与配体的识别过程中扮演了重要角色。但是蛾类信息素对神经元刺激的终止并非由性信息素受体控制, 而是由细胞中的气味降解酶等其他因子调控。蛾类性信息素受体研究中还有很多疑问需要解答, 其过程可能比我们想象的更为复杂。     

应用差异蛋白质组学方法分析作物化感作用的分子机理

试验旨在分析运用分子标记技术(QTL)和差异蛋白组学技术研究作物化感作用分子机理的差异性。首先运用差异蛋白组学技术探讨在生物胁迫(稗草)下水稻化感作用潜力变化的内在分子机理。分别用稗草和水稻的根系分泌物培养切自一株5叶龄化感水稻P I312777植株并经恢复的2个分蘖。7d后,提取处理和对照相同叶位叶片的全蛋白质并进行双向电泳,每张电泳胶片上获得800多个电泳胶点,其中差异表达的蛋白质点有4个。采用M ALD I-TOF-M S对各差异蛋白质点进行肽质量指纹图谱分析,经过SW ISS-PROT数据库查询,结果表明化感水稻P I312777在稗草胁迫下的特异蛋白分别与苯丙氨酸氨解酶(PAL)、硫还原型蛋白(T rx-m)、3-羟基-3-甲基戊二酰辅酶A还原酶(HM GR)和过氧化物酶(POD)相匹配。根据编码以上4个差异蛋白质的DNA序列,发现编码以上4个差异蛋白的基因分别位于水稻染色体4、7、8和12上的特定克隆位点,这就是与化感作用相关基因。前人也运用QTL方法开展作物化感作用的分子机理研究,但由于所采用的供体材料、受体植物及对表型性状的评价方法等的不同,定位结果存在较大的差异。综合比较两种方法后认为,运用差异蛋白组学技术分析水稻化感作用的分子机理,比QTL技术更加直接和深入。因为比较胁迫处理和对照植物组织的2-DE图谱将能鉴定出由表达候选基因编码的胁迫蛋白质,氨基酸残基序列的测定将揭示那些功能与胁迫性状密切相关的蛋白质,这种编码的基因就是兼具功能与表达的候选基因。     

褐飞虱对吡虫啉的抗性机理和靶标分子毒理学

褐飞虱Nilaparvata lugens是水稻最重要的害虫之一,长期依赖化学防治导致了该害虫对不同类型杀虫剂抗性的产生,对新烟碱类杀虫剂吡虫啉高水平抗性的产生更是造成了巨大的粮食生产损失。近年来在褐飞虱对吡虫啉抗性机理,以及在抗药性机理研究推动下吡虫啉作用靶标褐飞虱神经系统烟碱型乙酰胆碱受体（nicotinic acetylcholine receptors, nAChRs）毒理学等方面取得了许多研究进展。nAChRs是昆虫神经系统中最重要的神经递质受体,是几类重要杀虫剂的作用靶标,其中以新烟碱类杀虫剂为代表。通过对比敏感品系和室内连续筛选获得的高抗吡虫啉品系,在褐飞虱两个nAChRs亚基Nlα1和Nlα3中均发现了抗性相关点突变Y151S,该突变导致了受体与吡虫啉结合亲和力的显著下降,而对内源神经递质乙酰胆碱的亲和力影响很小。Nlα1与褐飞虱另外两个亚基Nlα2和Nlβ1共聚成一个受体,构成吡虫啉低亲和力结合位点;Nlα3与褐飞虱另外两个亚基Nlα8和Nlβ1共聚成一个受体,构成吡虫啉高亲和力结合位点。不仅褐飞虱nAChRs与吡虫啉抗性相关,某些nAChRs附属蛋白也直接影响褐飞虱对吡虫啉的抗性,如Lynx蛋白。关于褐飞虱nAChRs组成、抗药性相关变异、受体附属蛋白对抗药性的影响等方面的研究,均为国内外前沿报道,不仅有助于对新烟碱类杀虫剂抗性机理的理解,对昆虫nAChRs毒理学同样具有很大的推动作用。     

酰胺类蚊虫驱避化合物与引诱物氨分子缔合作用的计算

【目的】为了探究驱避机理,此前选择萜类驱避化合物及与DEET(避蚊胺)具有类似结构的酰胺类驱避化合物,开展了驱避化合物与引诱气味组分(L-乳酸、羧酸等)缔合作用对驱避活性影响的研究。为了扩大驱避化合物的类型,本研究选择另外一组43个酰胺类驱避化合物,计算了它们与蚊虫引诱物氨之间的双分子缔合作用,以及该缔合作用对驱避活性的影响,从而为驱避机理研究提供帮助。【方法】用Gaussian 03软件优化驱避化合物单体和双分子缔合体的三维结构式;通过Ampac和Codessa软件建立结构与驱避活性之间的定量构效关系模型。【结果】驱避化合物与氨分子的缔合距离、角度和缔合能量分别是2.2~3.0,128~180°和14~25 k J/mol;最佳四参数模型中R2为0.8987,其中2个参数来自驱避化合物单体,分别是(1/6)X GAMMA polarizability(DIP)和ESPminimum net atomic charge for an H atom,另外2个参数来自双分子缔合体,分别是ESP-DPSA-2 difference in CPSAs(PPSA2-PNSA2)[Quantum-Chemical PC]和Minimum valency of a C atom。模型检验中训练集和测试集的相关系数平方的平均值分别为0.9013和0.8666。【结论】驱避化合物与氨分子之间存在弱氢键力缔合作用,驱避化合物分子的极化度及其与氨分子之间的极性相互作用、缔合体中分子间键相互作用及其电荷分布均对驱避活性产生显著影响,说明双分子缔合对驱避活性具有显著影响。模型检验表明最佳四参数模型具有良好的稳定性和预测能力。本研究可为寻找新型蚊虫驱避剂和揭示蚊虫驱避剂的作用机理提供参考。     

Angeli盐对二氢硫辛酰胺脱氢酶的可逆性灭活作用(英文)

二氢硫辛酰胺脱氢酶(dihydrolipoamide dehydrogenase,DLDH)是线粒体3个α-酮酸脱氢酶复合物(丙酮酸脱氢酶复合物、α-酮戊二酸脱氢酶复合物、支链氨基酸脱氢酶复合物)的关键成分,属于吡啶依赖性二硫化物氧化还原酶类,对活性氮自由基(reactive nitrogen species,RNS)和活性氧自由基(reactive oxygenspecies,ROS)造成的氧化修饰非常敏感。本研究探索由Angeli盐所产生的RNS对DLDH的修饰作用及机制。将大鼠脑线粒体分离,与不同浓度的Angeli盐作用,应用分光光度计、蓝色胶、基于二维电泳的蛋白质组学等手段,测定DLDH酶活性。结果显示,Angeli盐呈浓度依赖性方式灭活DLDH,过氧亚硝酸盐在同样条件下对DLDH酶活性无抑制作用,说明Angeli盐对DLDH的作用可能是非随机的。由于Angeli盐在生理pH条件下可分解为硝基阴离子(nitroxyl anion,HNO)和一氧化氮(nitric oxide,NO),故进一步分析了Angeli盐对DLDH的灭活作用是否由HNO引起,结果证实确实如此。最后,二维电泳Western blot结果显示,Angeli盐对DLDH的灭活伴随着DLDH蛋白质的S-亚硝基硫醇形成,提示S-亚硝基硫醇化可能是导致DLDH酶失活的原因。综上,本研究为研究Angeli盐灭活DLDH的机制提供了新证据。     

2类多巴胺受体对心肌缺血后处理保护作用的影响及机制

目的：观察2类多巴胺受体（DR2）在心肌缺血后处理保护中的作用,并探讨其机制。方法：复制原代培养乳鼠心肌细胞缺血后处理模型,细胞随机分为正常组（Control）、缺血/再灌注组（I/R）、缺血后处理组（PC）、缺血后处理＋DR2激动剂组（PC＋Bro）、缺血后处理＋DR2抑制剂组（PC＋Hal）、缺血后处理＋DR2抑制剂＋激动剂组（PC＋Hal＋Bro）。比色法检测细胞培养液乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量;透射电镜观察细胞超微结构改变;流式细胞仪检测细胞凋亡变化;Western blot方法检测DR2蛋白表达、p-p38和p-JNK的活性。结果：与正常组相比,I/R能够增加DR2蛋白的表达,升高LDH活性和MDA含量,降低SOD活性,加重细胞损伤和细胞凋亡,上调p-p38和p-JNK的活性;与I/R组比较,PC进一步增加DR2蛋白的表达,降低LDH活性和MDA含量,增加SOD活性,减轻细胞损伤和细胞凋亡,下调p-p38和p-JNK的活性,Bro增强了PC的心肌保护作用,Hal则可取消Bro的这种作用。结论：DR2激活通过下调p-p38和p-JNK的活性参与缺血后处理对心肌缺血/再灌注损伤的保护作用。     

β 西柏三烯二醇对拟南芥的化感效应及其作用机理研究

以拟南芥幼苗为受体植物,研究了不同浓度二萜类化合物β-西柏三烯二醇对拟南芥生长的活性作用以及作用机理,以揭示萜类化感物质的作用机制。结果表明:(1)β-西柏三烯二醇对拟南芥幼苗的鲜重具有显著抑制作用,当处理浓度为50μmol/L时抑制作用达到显著水平,并对拟南芥鲜重的抑制率均大于30%。(2)经过β-西柏三烯二醇处理后,拟南芥叶片中叶绿素a、b含量显著下降,且抑制作用随处理浓度的增大和处理时间的延长而增加。(3)随着β-西柏三烯二醇处理浓度的增大,拟南芥中过氧化物酶(POD)和超氧化物歧化酶(SOD)的活性逐渐显著降低,而过氧化产物MDA含量显著增加。可见,β-西柏三烯二醇通过抑制拟南芥体内抗氧化酶的活性,引起膜脂过氧化,从而抑制拟南芥幼苗的正常生长和生理过程,且这种抑制作用随浓度的升高而增强。     

A calcium conducting channel akin to a calcium pump

Summary Calcium conducting channels were studied in blebs of sarcoplasmic reticulum described by Stein & Palade (1988). The calcium channels had at least three conductance states (70 pS, 50 pS and 37 pS) and were weakly selective for calcium ions, with a permeability ratio Ca²⁺ to K⁺ of about 3.4. The open probability of the channel was strongly voltage dependent, decreasing at positive membrane voltages. 10 m ryanodine and 5 m ruthenium red had no effect on this channel; neither did millimolar concentrations of ATP, Mg²⁺, caffeine, and Ca²⁺, implying that the calcium conducting channels are not ryanodine receptors. Several calcium pump inhibitors—namely, vanadate, AlF ₄ ^– , reactive red 120, and cyclopiazonic acid—had obvious effects on the calcium conducting channels, suggesting that the calcium conducting channel of SR membrane blebs is some form of the SR calcium pump.We thank the National Science Foundation for steadfast support.We thank Drs. F. Cohen, A. Fox, R. Levis and E. Rios for much useful help and criticism and Dr. G. Inesi for sending us his paper while in press.     

Single-Channel Properties of Inositol (1,4,5)-Trisphosphate Receptor Heterologously Expressed in HEK-293 Cells

The inositol (1,4,5)-trisphosphate receptor (InsP₃R) mediates Ca²⁺ release from intracellular stores in response to generation of second messenger InsP₃. InsP₃R was biochemically purified and cloned, and functional properties of native InsP₃-gated Ca²⁺ channels were extensively studied. However, further studies of InsP₃R are obstructed by the lack of a convenient functional assay of expressed InsP₃R activity. To establish a functional assay of recombinant InsP₃R activity, transient heterologous expression of neuronal rat InsP₃R cDNA (InsP₃R-I, SI− SII+ splice variant) in HEK-293 cells was combined with the planar lipid bilayer reconstitution experiments. Recombinant InsP₃R retained specific InsP₃ binding properties (K _d = 60 nM InsP₃) and were specifically recognized by anti–InsP₃R-I rabbit polyclonal antibody. Density of expressed InsP₃R-I was at least 20-fold above endogenous InsP₃R background and only 2–3-fold lower than InsP₃R density in rat cerebellar microsomes. When incorporated into planar lipid bilayers, the recombinant InsP₃R formed a functional InsP₃-gated Ca²⁺ channel with 80 pS conductance using 50 mM Ba²⁺ as a current carrier. Mean open time of recombinant InsP₃-gated channels was 3.0 ms; closed dwell time distribution was double exponential and characterized by short (18 ms) and long (130 ms) time constants. Overall, gating and conductance properties of recombinant neuronal rat InsP₃R-I were very similar to properties of native rat cerebellar InsP₃R recorded in identical experimental conditions. Recombinant InsP₃R also retained bell-shaped dependence on cytosolic Ca²⁺ concentration and allosteric modulation by ATP, similar to native cerebellar InsP₃R. The following conclusions are drawn from these results. (a) Rat neuronal InsP₃R-I cDNA encodes a protein that is either sufficient to produce InsP₃-gated channel with functional properties identical to the properties of native rat cerebellar InsP₃R, or it is able to form a functional InsP₃-gated channel by forming a complex with proteins endogenously expressed in HEK-293 cells. (b) Successful functional expression of InsP₃R in a heterologous expression system provides an opportunity for future detailed structure–function characterization of this vital protein.     

An anionic ryanoid, 10-O-succinoylryanodol,provides insights into the mechanisms governing the interaction of ryanoids and the subsequent altered function of ryanodine-receptor channels

We have investigated the interactions of a novel anionic ryanoid, 10-O-succinoylryanodol, with individual mammalian cardiac muscle ryanodine receptor channels under voltage clamp conditions. As is the case for all ryanoids so far examined, the interaction of 10-O-succinoylryanodol with an individual RyR channel produces profound alterations in both channel gating and rates of ion translocation. In the continued presence of the ryanoid the channel fluctuates between periods of normal and modified gating, indicating a reversible interaction of the ligand with its receptor. Unlike the majority of ryanoids, we observe a range of different fractional conductance states of RyR in the presence of 10-O-succinoylryanodol. We demonstrate that 10-O-succinoylryanodol is a very flexible molecule and propose that each fractional conductance state arises from the interaction of a different conformer of the ryanoid molecule with the RyR channel. The probability of channel modification by 10-O-succinoylryanodol is dependent on the transmembrane holding potential. Comparison of the voltage dependence of channel modification by this novel anionic ryanoid with previous data obtained with cationic and neutral ryanoids reveals that the major influence of transmembrane potential on the probability of RyR channel modification by ryanoids results from an alteration in receptor affinity. These investigations also demonstrate that the charge of the ryanoid has a major influence on the rate of association of the ligand with its receptor indicating that ionic interactions are likely to be involved in this reaction.     

Calmodulin binding to the 3614-3643 region of RyR1 is not essential for excitation-contraction coupling in skeletal myotubes

Calmodulin is a ubiquitous Ca(2+) binding protein that modulates the in vitro activity of the skeletal muscle ryanodine receptor (RyR1). Residues 3614-3643 of RyR1 comprise the CaM binding domain and mutations within this region result in a loss of both high-affinity Ca(2+)-bound calmodulin (CaCaM) and Ca(2+)-free CaM (apoCaM) binding (L3624D) or only CaCaM binding (W3620A). To investigate the functional role of CaM binding to this region of RyR1 in intact skeletal muscle, we compared the ability of RyR1, L3624D, and W3620A to restore excitation-contraction (EC) coupling after expression in RyR1-deficient (dyspedic) myotubes. W3620A-expressing cells responded normally to 10 mM caffeine and 500 microM 4-chloro-m-cresol (4-cmc). Interestingly, L3624D-expressing cells displayed a bimodal response to caffeine, with a large proportion of cells ( approximately 44%) showing a greatly attenuated response to caffeine. However, high and low caffeine-responsive L3624D-expressing myotubes exhibited Ca(2+) transients of similar magnitude after activation by 4-cmc (500 microM) and electrical stimulation. Expression of either L3624D or W3620A in dyspedic myotubes restored both L-type Ca(2+) currents (retrograde coupling) and voltage-gated SR Ca(2+) release (orthograde coupling) to a similar degree as that observed for wild-type RyR1, although L-current density was somewhat larger and activated at more hyperpolarized potentials in W3620A-expressing myotubes. The results indicate that CaM binding to the 3614-3643 region of RyR1 is not essential for voltage sensor activation of RyR1.     

Endoplasmic Reticulum Dysfunction and Ca<Superscript>2<Emphasis Type="Bold">+</Emphasis></Superscript> Deregulation in Isolated CA1 Neurons during Oxygen and Glucose Deprivation

Intracellular calcium ([Ca²⁺]_i) plays a pivotal role in neuronal ischemia. The aim of the present study was to investigate the routes of Ca²⁺ entry during non-excitotoxic oxygen and glucose deprivation (OGD) in acutely dissociated rat CA1 neurons. During OGD the fluo-3/fura red ratio reflecting [Ca²⁺]_i increased rapidly and irreversibly. [Ca²⁺]_i increased to the same degree in Ca²⁺ depleted medium, and also when both the ryanodine receptors (RyR) and the inositol 1,4,5-trisphosphate (IP₃) receptors were blocked. When the endoplasmic reticulum (ER) Ca²⁺ stores were emptied with thapsigargin no increase in [Ca²⁺]_i was observed independent of extracellular Ca²⁺. The OGD induced Ca²⁺ deregulation in isolated CA1 neurons is not prevented by removing Ca²⁺, or by blocking the IP₃– or RyR receptors. However, when SERCA was blocked, no increase in [Ca²⁺]_i was observed suggesting that SERCA dysfunction represents an important mechanism for ischemic Ca²⁺ overload.     

Atomic force microscopy study of the rabbit skeletal muscle ryanodine receptors in different functional states

Atomic force microscope was applied to investigate the effect of extrinsic phospholipid on the structure of rabbit skeletal muscle ryanodine receptor/calcium release channel (RyR1). In addition, in the presence of extrinsic phospholipid, the height and elasticity of the RyR1s in different functional states were also measured. The results indicate: (i) most of the RyR1s showed a normal structure only in the presence of extrinsic phospholipid; (ii) treatment of the RyR1s with AMP and Ca²⁺ together could increase their Young’s Modulus but not change their apparent height; (iii) no detectable change in either height or Young’s Modulus of the RyR1s appeared, if the RyR1s were treated with other activators or inhibitors.