鲫鱼肝脏AChE的提取及有机磷农药对AChE的抑制效应研究

本研究利用淡水鲫鱼肝脏为主要原材料提取乙酰胆碱酯酶(acetylcholinesterase,AChE),并设计正交试验确定了鲫鱼肝脏中乙酰胆碱酯酶的最佳提取条件:通过酶抑制法衡量敌敌畏、辛硫磷、三唑磷、乐果等四种不同种类有机磷农药对粗酶液的抑制作用;将鱼肝和鱼脑中乙酰胆碱酯酶活性及蛋白含量的进行对比研究。研究结果表明:提取液pH值、提取液种类以及原料与提取液的质量体积比对酶液的活性影响显著;被测四种有机磷农药中,敌敌畏对AChE活性抑制作用最强。鱼脑的AChE比酶活是肝脏的AChE比酶活的3～7倍。

鲫鱼肌肉AChE的提取、纯化及活性研究

以鲫鱼肌肉为原料提取乙酰胆碱酯酶(Acetylcholinesterase,AChE),然后采用改进的Ellman方法按照正交试验设计分别测定不同环境条件(pH值,温度,时间)下粗酶液活性,得到测定AChE活性的最佳条件组合。再将粗酶液依次通过DEAE-Sephadex A-50和Sephadex G-200柱层析纯化。结果表明,3个因素对AChE活性影响的顺序为温度>pH值>时间;最佳酶活性测定条件组合为环境温度35℃,体系pH值8.0,反应时间30 min;经Sephadex A-50柱层析后的纯化倍

鲫鱼脑乙酰胆碱酯酶(AChE)的活性测定及对有机磷农药的敏感性研究

提取不同生长期的鲫鱼脑中乙酰胆碱酯酶(acetylcholinesterase,AChE)并做比较,以Ellman法测定其酶活。并用正交试验方法确定了最佳测定条件。采用体外实验法研究了在最佳条件下鲫鱼脑AChE对三种有机磷农药的敏感性。研究结果表明,以体长12cm左右的鲫鱼体内AChE活性最高,正交试验得出AChE最佳测定条件组合为:酶的剂量为40μl、底物最终浓度为120mmol/L、pH值7.5、温度35℃;时间15min。鲫鱼脑AChE对农药的敏感性为敌敌畏>辛硫磷>乐果。

Acetylcholinesterase amperometric detection system based on a cobalt(II) tetraphenylporphyrin-modified electrode

An acetylcholinesterase (AChE) activity detection system was fabricated based on the electrocatalysis of cobalt(II) tetraphenylporphyrin of the electrooxidation of thiocholine chloride, which is the product of the hydrolysis of acetylthiocholine chloride by AChE. A simple modified method was used to form the base electrode. AChE was cross-linked on the base electrode by glutaraldehyde. The optimum working conditions are discussed and the characteristics of the detection system are evaluated.

Effects of hexachlorobenzene on antioxidant status of liver and brain of common carp (Cyprinus carpio)

Hexachlorobenzene (HCB)-induced oxidative damages have been published in rats while the effects have not yet been reported in fishes. Juvenile common carps (Cyprinus carpio) were exposed to waterborne HCB from 2 to 200 mu g l(-1) for 5, 10 or 20 days. Liver and brain were analyzed for various parameters of oxidative stress. There were no significant changes of glutathione (GSH) content and superoxide dismutase (SOD) activity in liver after 5 or 10 days exposure, whereas obvious drops were observed at higher concentrations after 20 days exposure. Significant decreases of GSH content and SOD activity in brain were found during all the exposure days. In brain, HCB also significantly elevated the contents of reactive oxygen species (ROS), thiobarbituric acid-reactive substances (TBARS, as an indicator of lipid peroxidation products), glutathione disulfide (GSSG), and activities of nitric oxide synthase (NOS), glutathione peroxidase (GPx), and glutathione reductase (GR), and inhibited activities of acetylcholinesterase (AchE) and glutathione S-transferase (GST). The results clearly demonstrated that environmentally possible level of HCB could result in oxidative stress in fish and brain was a sensitive target organ of HCB toxicity. (c) 2006 Elsevier Ltd. All rights reserved.

Comparative researches on effects of sodium dodecylbenzene sulfonate and sodium dodecyl sulfate upon Lateolabrax japonicus biomarker system

Fish Lateolabrax japonicus were exposed to anion surfactant sodium dodecylbenzene sulfonate (SDBS) and sodium dodecyl sulfate (SDS) at 1 mg/l, respectively, for 6, 12 and 18 d, with one control group. Liver antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH) and glutathione S-transferase (GST) were determined; brain acetylcholinesterase (AChE) and liver inducible nitric oxide synthase (NOS) activities were also measured. The results of the study indicated that these parameters made different, sometimes, adverse responses to SDBS and SDS exposure, such as the activity of NOS can be inhibited by SDBS and induced by SDS, the different physico-chemical characteristics of SDBS and SDS should be responsible for their effects on enzyme activities. (c) 2005 Elsevier B.V. All rights reserved.

Biochemical and biological properties of Trimeresurus jerdonii venom and characterization of a platelet aggregation-inhibiting acidic phospholipase A(2)

Several biochemical and biological activities such as phospholipase A(2), arginine esterase, proteolytic, L-amino acid oxidase, 5'nucleotidase, acetylcholinesterase, thrombin-like, anticoagulant, and hemorrhagic activities were determined for whole desiccated venom of Trimeresurus jerdonii. An acidic phospholipase (named TJ-PLA(2)) was purified by anionic exchange chromatography, gel filtration, and reverse phase HPLC. TJ-PLA(2) had a molecular weight of 16,000 and a pI of 4.8. TJ-PLA(2) was non-lethal to mice up to an i.p. dose of 15 mg/kg body weight and lacked neurotoxicity and myotoxicity. It induced edema in the footpads of mice. The purified enzyme inhibited ADP- and collagen-induced human platelet aggregation in a manner which was both dose- and time-dependent.

Purification and cloning of cysteine-rich proteins from Trimeresurus jerdonii and Naja atra venoms

Three 26 kDa proteins, named as TJ-CRVP, NA-CRVP1 and NA-CRVP2, were isolated from the venoms of Trimeresurus jerdonii and Naja atra, respectively. The N-terminal sequences of TJ-CRVP and NA-CRVPs were determined. These components were devoid of the enzymatic activities tested, such as phospholipase A(2), arginine esterase, proteolysis, L-amino acid oxidase, 5' nucleotidase, acetylcholinesterase. Furthermore, these three components did not have the following biological activities: coagulant and anticoagulant activities, lethal activity, myotoxicity, hemorrhagic activity, platelet aggregation and platelet aggregation-inhibiting activities. These proteins are named as cysteine-rich venom protein (CRVP) because their sequences showed high level of similarity with mammalian cysteine-rich secretory protein (CRISP) family. Recently, some CRISP-like proteins were also isolated from several different snake venoms, including Agkistrodon blomhoffi, Trimeresurus flavoviridis, Lanticauda semifascita and king cobra. We presumed that CRVP might be a common component in snake venoms. Of particular interest, phylogenetic analysis and sequence alignment showed that NA-CRVP1 and ophanin, both from elapid snakes, share higher similarity with CRVPs from Viperidae snakes. (C) 2003 Elsevier Ltd. All rights reserved.

Improving effects of huperzine A on spatial working memory in aged monkeys and young adult monkeys with experimental cognitive impairment

Our previous studies demonstrated that huperzine A, a reversible and selective acetylcholinesterase inhibitor, exerts beneficial effects on memory deficits in various rodent models of amnesia. To extend the antiamnesic action of huperzine A to nonhuman primates, huperzine A was evaluated for its ability to reverse the deficits in spatial memory produced by scopolamine in young adult monkeys or those that are naturally occurring in aged monkeys using a delayed-response task. Scopolamine, a muscarinic receptor antagonist, dose dependently impaired performance with the highest dose (0.03 mg/kg, i.m.) producing a significant reduction in choice accuracy in young adult monkeys. The delayed performance changed from an average of 26.8/30 trials correct on saline control to an average of 20.2/30 trials correct after scopolamine administration. Huperzine A (0.01-0.1 mg/kg, i.m.) significantly reversed deficits induced by scopolamine in young adult monkeys on a delayed-response task; performance after an optimal dose (0.1 mg/kg) averaged 25.0/30 correct. In four aged monkeys, huperzine A (0.001-0.01 mg/kg, i.m.) significantly increased choice accuracy from 20.5/30 on saline control to 25.2/30 at the optimal dose (0.001 mg/kg for two monkeys and 0.01 mg/kg for the other two monkeys). The beneficial effects of huperzine A on delayed-response performance were long lasting; monkeys remained improved for about 24 h after a single injection of huperzine A. This study extended the findings that huperzine A improves the mnemonic performance requiring working memory in monkeys, and suggests that huperzine A may be a promising agent for clinical therapy of cognitive impairments in patients with Alzheimer's disease.

Effect of huperzine A on working memory in reserpine- or yohimbine-treated monkeys

The effect of huperzine A, a reversible and selective acetylcholinesterase inhibitor, on reserpine- or yohimbine-induced spatial working memory deficits in monkeys has been examined using the delayed response task that depends on the integrity of prefront

鸡血液中乙酰胆碱酯酶的提取研究

[目的]为了研究硫酸铵盐析法在乙酰胆碱酯酶分离纯化中的效果,从而利用乙酰胆碱酯酶的活性变化反映农药的污染情况。[方法]采用硫酸铵盐析和等电点区分的方法从鸡血中提取乙酰胆碱酯酶,并探讨其适宜的pH值范围。[结果]结果表明:pH值4.5左右大量的杂蛋白能被除去,可获得较高乙酰胆碱酯酶得率;采用饱和度为50%~60%的硫酸铵盐析效果较好,据此得出较佳的提取工艺。[结论]该研究结果为后续鸡血AChE的提取、分离、纯化等工作提供了相应的参考,同时为利用乙酰胆碱酯酶指示环境受有机磷和氨基甲酸酯类农药污染的后续研究和应

鲮鱼冷休克及其死亡的某些生化因素

采用分光光度法和淀粉凝胶电泳法初步研究了鲮鱼冷休克前后脑乙酰胆碱酯酶(AchE)活力和三种酶的同工酶类的动态变化。并根据鲮鱼在冷休克期间(7—6℃)脑AchE活力显著降低和肝脏组织乳酸脱氢酶(LDH)同工酶活性明显升高以及苹果酸脱氢酶(MDH)、酯酶(EST)同工酶类出现酶活性变化的情况,讨论了导致鲮鱼耐寒能力差的某些生化因素。同时提出脑AchE可以作为评定鲮鱼冷休克期间中枢神经系统受害程度的一种生化指标。

Determination of organophosphate and carbamate pesticides based on enzyme inhibition using a pH-sensitive fluorescence probe

A flow injection system for the determination of organophosphate and carbamate pesticides is described. A sensitive fluorescence probe was synthesized and used as the pH indicator to detect the inhibition of the enzyme acetylcholinesterase (ACNE). The percentage inhibition of enzyme activity is correlated to the pesticide concentration. Several parameters influencing the performance of the system are discussed. The detection limits of 3.5, 50, 12 and 25 mug/l for carbofuran, carbaryl, paraoxon and dichlorvos, in pure water, respectively were achieved with an incubation time of 10 min. A complete cycle of analysis, including incubation time, took 14 min. The detection system has been applied to the determination of carbofuran in spiked vegetable juices (Chinese cabbage and cole), achieving recovery values between 93.2 and 107% for Chinese cabbage juice and 108 and 118% for cole juice at the different concentration levels assayed. (C) 2004 Elsevier B.V. All rights reserved.

文冠果果壳乙醇提取物对大鼠学习记忆障碍的改善作用

目的研究文冠果果壳乙醇提取物(EEHXS)对双侧颈总动脉结扎大鼠学习记忆障碍的改善作用及其作用机制。方法用DigBehv动物行为分析系统分析大鼠的自发活动,Y迷宫法及Morris水迷宫法测试大鼠的学习记忆能力,比色法检测大脑组织中超氧化物歧化酶(SOD)活性和丙二醛(MDA)、乙酰胆碱酯酶(AchE)的水平,尼氏染色观察海马组织形态学改变。结果模型组大鼠自发交替反应正确率、空间记忆能力和工作记忆能力显著降低,SOD活性显著下降,MDA和AchE水平显著升高,海马神经元变性、脱落;EEHXS显著改善大鼠的学习记忆能力、提高SOD活性、降低MDA和AchE水平,显著抑制海马神经元的变性及脱落。结论EEHXS对双侧颈总动脉结扎大鼠的学习障碍有显著的改善作用,其作用机制可能与改善胆碱能神经功能及降低自由基水平有关。

文冠果壳苷对侧脑室注射Aβ_(1-42)致小鼠学习记忆障碍的改善作用

目的研究文冠果壳苷对侧脑室注射β肽淀粉样蛋白1-4(2Aβ1-42)小鼠学习记忆障碍的改善作用,并初步探讨了其作用机制。方法通过避暗和水迷宫实验检测了小鼠的学习记忆能力;使用分光光度计检测小鼠大脑组织中乙酰胆碱酯酶(AchE)和胆碱乙酰转移酶(ChAT)活性的变化。结果小鼠侧脑室注射凝聚态Aβ1-42显著降低了小鼠被动回避学习记忆能力及空间学习记忆能力(P<0.01),并且小鼠大脑AchE和ChAT活性显著降低(P均<0.05);而文冠果壳苷能够显著减少避暗错误次数,延长潜伏期(分别P<0.05、P<0.01);剂量依赖性地缩短水迷宫实验中小鼠到达安全台的游泳时间(分别P<0.05、P<0.01),不同程度地抑制Aβ1-42模型小鼠的AchE和ChAT活性的降低(P<0.05和<0.01)。结论文冠果壳苷对侧脑室注射Aβ1-42小鼠学习记忆障碍具有显著的改善作用,其作用机制可能与保护中枢胆碱能神经细胞,改善中枢胆碱能神经系统功能等有关。