麦穗鱼脑乙酰胆碱酯酶(AChE)的纯化及其比较性研究

用PEG2000双水相萃取、DEAE-Sephadex A-50和Sephadex G-200方法分离纯化麦穗鱼Pseudorasbora parva脑中的乙酰胆碱酯酶(AChE),然后比较分析纯酶液与粗酶液中鱼脑AChE 的动力学特性和抗抑制性,以便更直接地了解底物与酶以及毒剂与酶的反应关系。研究结果表明,经过一系列步骤的纯化,最后所得的AChE是纯度较高的酶液;通过对AChE的动力学研究发现,纯化后的麦穗鱼脑AChE与底物之间的亲和力和其在粗酶状态时没有显著的差别,而且纯化后的鱼脑AChE对底物抑制作用更敏感;抗抑制性研究发现,纯化后的麦穗鱼脑AChE对经溴水氧化的马拉硫磷(malathion)和三唑磷(triazophos)的敏感性显著高于粗酶状态的AChE,酶更易受抑制。     

水稻纹枯病与白背飞虱同田发生密切相关

小麦穗蚜是我市小麦上一种主要害虫,近年来频繁大发生,对小麦产量影响很大。为了探索一种防治小麦穗蚜的新药剂,1991—1992年我们对新近开发的氨基甲酸酯杀虫剂抗蚜威进行了田间药效试验。 试验设50％抗蚜威可湿性粉剂(江阴市中港有机化工厂生产),每亩10、15和20克;40％氧化乐果(江苏海门农药厂生产)每亩     

家蝇（Musca domestica Vicina L.）头部乙酰胆碱酯酶检测农药残留最佳条件研究

系统研究了敏感家蝇Musca domestica Vicina L.羽化后不同天数其头部乙酰胆碱酯酶(AChE)的比活力、敏感度以及酶源蛋白质含量的变化;缓冲液pH值对家蝇头部AChE比活力的影响。结果表明:家蝇初羽化至羽化后第15 d,其头部AChE的比活力逐渐升高 ,而酶源蛋白质的含量逐渐下降(187.022 2±9.021 3~83.022 2±7.111 1 μg/头)。羽化后2~4 d,其头部AChE的敏感度最高;初羽化和羽化后第15 d,敌敌畏的I50分别是羽化后第3 d的1.4倍和2.1倍。在一定的pH值范围内(6.6~8.0),家蝇头部AChE的比活力随缓冲液pH值的升高而增大。本研究为利用家蝇头部AChE检测有机磷残留量探索了最佳条件。     

噻虫嗪及其代谢物噻虫胺在麦叶和麦穗中的痕量检测方法及其应用

建立了超高效液相色谱-串联质谱(UPLC-MS/MS)测定麦叶和麦穗中噻虫嗪及其代谢物噻虫胺残留的痕量、快速检测方法.麦叶或麦穗中噻虫嗪及其代谢物噻虫胺用乙腈提取,NH2/Carb固相萃取柱净化,内标法定量.结果表明:噻虫嗪和噻虫胺在0.001～～0.5 mg/L范围内线性关系良好,相关系数r大于0.99;噻虫嗪和噻虫...     

田间小菜蛾抗药性监测及毒理机制研究

田间监测结果表明，福州地区小菜蛾Plutella xylostella(L.)已对生产上常用的各类杀虫剂产生不同程度的抗性，从9月至次年5年期间，田间小菜蛾表现出较高水平的抗性，而在8月份则表现出明显低的抗性水平，短期高温和长期低温的环境条件可明显抑制小菜蛾幼虫的AChE和CarE活性，但短期低温的环境条件对小菜蛾幼虫的AChE和CarE活性影响不大，饲养温度的变化不会影响小菜蛾幼虫AChE和Ki值，但在较高的酶与杀虫剂的反应温度下，有机磷对小菜蛾、菜青虫和黄曲条跳甲AChE的抑制作用显著增高，此外，探讨了田间影响小菜蛾抗性水平的环境因素及毒理学机制。     

靶标酶在研究杀虫活性构效关系中的应用

运用已建立的乙酰胆碱酯酶(AChE) 活力分析规范化筛选模型, 研究N-甲基取代苯基氨基甲酸酯类化合物和含硅氨基甲酸酯类新系列化合物与AChE 酶抑制中量( I50)构效关系的规律, 并以CA SAR 软件进行Q SAR 分析, 所得结果迅速反馈给化学合成, 为高效低毒新杀虫剂的创制提供有益的信息和科学依据。     

枸杞木虱乙酰胆碱酯酶活力及三唑磷、辛硫磷对其活性的抑制作用

乙酰胆碱酯酶(acetylcholinesterase,AChE)是有机磷和氨基甲酸酯类农药的靶标酶。研究AChE活性及农药对酶的抑制作用,对昆虫抗药性研究和新农药开发具有重要意义。目前,对枸杞木虱Paratri-oza sinicaYang et Li的抗药性及其AChE尚无系统报道。作者测定枸杞木虱AChE最佳反应体系     

不同体色瓜蚜耐药性差异及其生化机制

试验结果表明,在黄瓜上的绿色瓜蚜(Aphis gossypii Glov.)种群对氧化乐果、抗蚜威和氰戊菊酯的耐性明显高于黄色瓜蚜种群。这两种颜色的瓜蚜乙酰胆碱酯酶(ACHE)具有明显的不同,在绿色种群中,有60.7%的个体AChE活性大于0.8(OD_(412)/mg蛋白质,分钟),而在黄色种群中则仅有25.3%的个体AChE活性大于0.8;氧化乐果(终浓度为1.69×10~(-2)mol/L)对单个蚜虫AChE的抑制率,前者仅有19%的个体AChE抑制率在30%以上,而后者则有46%的个体在30%以上。α-乙酸萘酯羧酸酯酶的活性大于25(×10~(-2)μmol/蚜,15分钟)的个体在绿色种群中占56%,而在黄色种群中仅有20%;β-乙酸萘酯羧酸酯酶活性大于25(×10~(-2)μmol/蚜,15分钟)的个体在绿色瓜蚜中占45%,而在黄色种群中仅有7%。说明绿色和黄色瓜蚜耐药性的差异可能是由于AChE敏感性和羧酸酯酶活性的不同造成的。     

农药残留检测用新型碳纳米管固载酶生物传感器的制备及其电学传感性能分析

采用滴涂法、自组装法及化学键合法制备了基于碳纳米管(CNTs)修饰的乙酰胆碱酯酶(AChE)生物传感器,采用循环伏安法(CV)、计时电流法(CA)、交流阻抗法(EIS)和扫描电镜(SEM)对生物传感器的电化学性能和表面形貌进行表征,通过采用该生物传感器对异丙威(氨基甲酸酯类农药)进行分析的结果,考察了其检测性能,研究了各生物传感器的动力学性质及电化学行为,并构建了新型固载酶生物传感器的等效电路模型。结果表明:各生物传感器表观表面积比裸电极显著提高,其电子传递速率遵循以下顺序逐渐降低:乙酰胆碱酯酶(AChE)/壳聚糖(CS)/功能化碳纳米管(F-CNTs/GCE)生物传感器AChE/F-CNTs/GCE生物传感器CS/双醛纤维素固载酶(DAC-AChE)/F-CNTs/GCE生物传感器,各传感器表观电子传递速率常数分别为:k s AChE/CS/F-CNTs/GCE=0.24 s-1,k s AChE/F-CNTs/GCE=0.23 s-1和k s CS/DAC-AChE/F-CNTs/GCE=0.22 s-1。获得生物传感器电学阻抗谱等效电路模型为R1(CPE1(R2(CPE2(R3)))),计算得到等效电路模型中各具体元件参数,证明该有效电路能有效模拟生物传感器检测异丙威的传感机理。该研究结果可为农药残留检测用生物传感器分析机理研究提供有益参考。     

与昆虫抗药性相关的乙酰胆碱酯酶基因突变研究进展

有机磷和氨基甲酸酯类杀虫剂的广泛使用导致许多害虫产生了明显的抗药性。害虫对这些杀虫剂产生抗性的一个重要原因是其乙酰胆碱酯酶 (AChE)基因发生突变,从而导致AChE敏感度下降。简要概述了AChE基因发生突变的昆虫种类,介绍了AChE基因突变对其结构与功能的影响、变构AChE的特性、AChE基因突变对适合度的影响以及AChE突变不同组合对抗性的影响。这些突变可为设计新颖的反抗性化合物开辟新的途径。     

三唑磷对麦穗鱼脑组织中乙酰胆碱酯酶的诱导

为了检测有机磷杀虫剂对鱼脑组织中乙酰胆碱酯酶(AChE, EC 3.1.1.7)的诱导,采用间接非竞争酶联免疫吸附测定法(ELISA)检测了三唑磷胁迫下麦穗鱼Pseudorasbora parva脑组织中乙酰胆碱酯酶含量的变化。当暴露浓度仅为0.1 μg /L时,三唑磷即对AChE产生诱导。由于存在诱导效应,三唑磷对酶活性的抑制在一定程度上被酶蛋白含量的提高所掩盖。研究结果表明,以酶蛋白替代总蛋白来衡量粗酶液中AChE的活性,可以明显提高对有机磷胁迫作用的检测灵敏度。     

锐劲特与马拉硫磷混用对麦穗鱼的影响研究

锐劲特fipronil(化学名(±)-5-氨基-1-(2,6-二氯-α,α,α-三氟-ρ-甲苯基-)-4-三氟甲基-亚硫酰基吡唑-3-碳化腈),是一广谱的新型杀虫剂,其作用靶标是γ-氨基丁酸调节的神经轴突氯离子通道。进入我国市场后能有效防治蔬菜、水稻害虫[1～3],具有较好的推广潜力。有关该农药对家蚕、鱼、天敌的毒性已有研究[1～5]。但该药与其他农药的相互作用,尚未见报道。本文研究了该农药与马拉硫磷混合使用时,对麦穗鱼的影响。1　材料与方法1.1　实验用鱼麦穗鱼(Pseudorasboraparva),体重0.39-1.09g,体长4.1-5.8cm,购自杭州花鸟市场,试验前在实验室驯养一…     

氰氟草酯、杀虫安亚致死剂量对金鱼Carrasius auratus和麦穗鱼Pseudorasbora parva肝脏酯酶及谷胱甘肽-S-转移酶活性的影响

研究了 10 %千金乳油 (有效成分 :氰氟草酯 )和 78%杀虫安可溶性粉剂对金鱼和麦穗鱼肝脏酯酶及谷胱甘肽-S-转移酶 (GST)活性的亚致死剂量效应。发现杀虫安 (0 .2 34mg· L-1)对两种鱼的 GST活性均具诱导作用 ,而氰氟草酯 (1,2 mg· L-1)仅诱导了麦穗鱼的 GST活性 ;当杀虫安与氰氟草酯混合处理时 ,对麦穗鱼 GST活性的诱导效应最为明显。在一定的浓度范围内 ,氰氟草酯 (1,2 mg· L-1)和杀虫安 (0 .117,0 .2 34mg· L-1)均可诱导金鱼肝脏酯酶活性 ;对麦穗鱼肝脏酯酶而言 ,杀虫安为诱导作用 ,氰氟草酯则抑制其活性。研究结果表明 :两种酶的活性直接或间接地受供试药剂的影响 ,两种试鱼对氰氟草酯和杀虫安的生物反应存在差异。     

鱼类肝脏酯酶的同工酶构成及其对磷酸三苯酯和马拉氧磷的敏感性

用聚丙烯酰胺凝胶电泳法分离了麦穗鱼 (Pseudorasbora p arva)、金鱼 (Carassius auratus)、尼罗罗非鱼 (Tilapja nilotica)、食蚊鱼 (Gambusia affinis)、虹鳟 (Salmo gairdneri)等五种鱼的肝脏酯酶的同工酶 ,以乙酸α-萘酯为底物测定了它们的活性。在麦穗鱼、金鱼、尼罗罗非鱼体内发现两条酶带 ,在食蚊鱼和虹鳟体内发现一条酶带。离体抑制率实验表明 ,五种鱼肝脏内均含有对磷酸三苯酶敏感的酶带 ,而对马拉氧磷敏感的酶带只存在于麦穗鱼、金鱼、尼罗罗非鱼体内。酯酶同工酶分布型的意义及与马拉硫磷选择毒性的关系在本文中进行了讨论。     

Measurement of the bioconcentration factors of pesticides by freshwater fish and their correlation with physicochemical properties or acute toxicities

The bioconcentration factors (BCF) for 15 pesticides by a freshwater fish (topmouth gudgeon) were measured under continuous flow conditions in water containing 5 to 20 μg litre^?1 of each pesticide at the equilibrium condition. A significant correlation was found between the BCF by the fish and the water solubility of the pesticide or its partition coefficient (PC) between octan-1-01 and water. Moreover, a significant correlation was also found between the BCF by topmouth gudgeon and the acute toxicities to carp, rainbow trout and water flea. It would therefore appear that the bioconcentration potential of a pesticide by a fish may be predicted from knowledge of its solubility or PC, and that the acute toxicity of a pesticide to a fish may be predicted from knowledge of the BCF.     

溴氰菊酯对麦穗鱼谷胱甘肽S-转移酶(GSTs)的影响

研究了溴氰菊酯不同浓度、不同处理时间对麦穗鱼体内谷胱甘肽S-转移酶(GSTs)活性的影响,同时测定了麦穗鱼不同组织中谷胱甘肽S-转移酶对低浓度溴氰菊酯处理的反应特点。结果表明,处理48 h后,高浓度(0.009 mg/L)组对肝中GSTs活性的抑制率达到43.7％,对鳃中GSTs活性的抑制率达54.3％;而低浓度组(0.001 mg/L)对鳃中GSTs活性有一定的诱导作用。亚致死剂量的溴氰菊酯(3.0×10-4mg/L)处理96 h后,对卵巢的诱导活性最高,其GSTs活性是对照的2.43倍,但对肝、鳃、肾、肠的诱导作用均较低。溴氰菊酯对麦穗鱼不同组织GSTs活性诱导的时间效应和强度不同。     

Activation of an O-ethyl S-n-propyl phosphorothiolate,TIA-230, in the central nerve of Spodoptera larvae

TIA-230, O-[1-(4-chlorophenyl)-4-pyrazolyl] O-ethyl S-propyl phosphorothiolate, showed strong insecticidal activity against Spodoptera larvae, in spite of its weak in vitro anti-AChE activity. Head AChE of Spodoptera was, however, inhibited with the progress of TIA-230 intoxication. When the isolated central nerve cord was incubated with TIA-230, AChE in the tissue was strongly inhibited even by concentration (10^?5M) lower than in vitro I₅₀ against AChE (10^?4M). The frequency of spontaneous firing of the nerve cord was increased by treatment of TIA-230 at low concentrations (10^?6–10^?5M) after a latent time of several minutes. The firing was increased by fenitroxon, but without the latent time. The length of the latent time agreed well with the time necessary for rising the inhibition of nerve cord AChE by TIA-230. AChE inhibition of TIA-230 in the nerve cord was reduced by the treatment of piperonyl butoxide, an inhibitor of mixed-function oxidases. From these results, TIA-230 was regarded as being activated oxidatively in the nerve cord to inhibit AChE. Profenofos was also activated in the nerve cord. It was concluded, therefore, that O-ethyl S-n-propyl phosphorothiolate insecticides were activated in the central nerve of the insect.     

λ-cyhalothrin and cypermethrin induced in vivo alterations in the activity of acetylcholinesterase in a freshwater fish, Channa punctatus (Bloch)

In the present study, the in vivo effects of λ-cyhalothrin and cypermethrin on the activity of acetylcholinesterase (AChE, EC 3.1.1.7) were evaluated for 96 h in brain, muscle and gills of Channa punctatus. Both compounds exhibited tissue specific as well as dose dependent decrease in the activity of AChE. The treated fish showed a significant decrease in the activity of AChE in brain and a lesser inhibition in muscle and gills in response to the increasing concentrations of λ-cyhalothrin as well as cypermethrin. Our results indicated that the brain was the main target organ for both insecticides, followed by muscle and gills, as determined by AChE inhibition study. However, these organs showed variations in the degree of AChE inhibition for separate treatments of both insecticides. The λ-cyhalothrin was a more potent AChE inhibitor as compared to cypermethrin. These findings indicated that apart from the established mechanism of delayed closure of sodium ion channels, these pyrethroids inhibit the activity of AChE in C. punctatus which could further aggravate their neurotoxic effects.     

Neurotoxic effects of subacute exposure of dichlorvos and methyl parathion at sublethal dosages in rats

The present study was designed to understand the effects of sublethal dosages of dichlorvos (DIC) and methyl parathion (MP) on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in various tissues of rats exposed to 5 and 10 ppm of DIC and MP in drinking water ad libitum for 28 days continuously. According to the results, AChE activity was significantly decreased in all the tissues of rats treated with both dosages of DIC and MP except for in the lungs treated with both dosages of DIC. With regard to the BChE, MP caused a significant decrease in the liver, heart and lungs with 5 ppm dosage whereas it did not change the BChE activity in the other tissues with two dosages. Also, DIC caused a significant decrease in BChE activity in the heart tissue treated with both dosages and in the brain of rats treated with 5 ppm. The observations presented led us to conclude that the administrations of MP and DIC at sublethal concentrations inhibited AChE and BChE activities in the rats. These results suggest that inhibition of AChE may be a better biomarker for the assessment of neurotoxic effects in the living.