神经毒性酯酶的测定方法

有机磷杀虫剂引起动物迟发性神经毒性问题正日益受到重视。在生产上必须对所使用的农药评价它对人及非靶标生物可能发生的危险及应用后的潜在影响。迟发性神经毒性是其中的一个重要指标。Johnson[1～3]用母鸡鸡脑作材料,研究有机磷化合物的迟发性神经毒性问题,发现只有当鸡脑中水解苯基乙酸苯酯或戊酸苯酯的神经毒性酯酶被抑制时,才出现迟发性神经毒性的综合症状。而在急性或慢性中感时,该酶不受抑制。因此可通过测定神经毒性酯酶是否受抑制来作为有机磷化合物近发性神经毒性的部分测定指标。我们基本上按Johnson的方法[2]并加以改进,…     

昆虫神经分泌细胞体的快速染色方法

<正> 昆虫神经分泌细胞(NSC)分布于中枢神经系统的各个部份,要观察它们的结构与种类,必须做许多切片。为了在取材之前明确NSC的正确位置,提高超薄切片的准确性,作者采用了整体快速染色法。它仅使NSC着色,从而能     

松墨天牛幼虫体内神经毒性酯酶和乙酰胆碱酯酶对甲胺磷敏感性的比较

松墨天牛Monochamus alternatus Hope是传播松材线虫Bursaphelenchus xylophilus Nickle的主要媒介昆虫,在其化学防治中经常使用有机磷杀虫剂,而这类杀虫剂有些可通过作用于神经毒性酯酶（neurotoxic esterase, NTE）使敏感的脊椎动物产生迟发性神经毒性。为了深入理解有机磷杀虫剂对害虫毒杀作用机理,增强对昆虫体内NTE活性的了解,我们在松墨天牛中开展了相关的研究。通过差异测定法和经典的乙酰胆碱酯酶（acetylcholinesterase, AChE）活性测定方法,分别测定了松墨天牛幼虫体内NTE和AChE的活性。结果发现,在松墨天牛体内存在NTE活性,其头部的NTE活力为2.80±0.30 nmol·min^-1·mg^-1,比脂肪体高出了近10倍,但仍较鸟类等脊椎动物为低。体内实验显示,松墨天牛体内NTE可被甲胺磷所抑制; 但在体外实验条件下,甲胺磷对来自松墨天牛头部的NTE活性并没有产生明显抑制作用,而此浓度下的甲胺磷对AChE表现出较高水平的抑制作用。表明松墨天牛体内NTE和AChE对甲胺磷的敏感性完全不同。     

氧化胁迫在硝基苯致小鼠神经毒性中的作用

目的:探讨氧化胁迫在硝基苯致小鼠神经毒性中的作用。方法:用不同剂量的硝基苯对小鼠进行灌胃染毒,每天1次,共30d;用碱性羟胺比色法测定小鼠脑乙酰胆碱酯酶活性,用试剂盒检测脑抗氧化功能。结果:试验组小鼠的中毒症状随着染毒剂量的增加和时间的延长而越来越明显;乙酰胆碱酯酶活性降低;与对照组相比,脑各项抗氧化指标均表现出显著或极显著差异。结论:氧化胁迫在硝基苯发挥神经毒性过程中起重要作用。     

三甲基苯基磷酸酯对神经毒性酯酶的抑制及其酶促动力学分析

以可使人和敏感动物产生迟发性神经毒性的有机磷化合物三甲基苯基磷酸酯（ＴＯＣＰ）为测试药物,研究其在体外对成年产卵来航母鸡不同神经组织神经毒性酯酶（ＮＴＥ）活性抑制敏感性及其抑制的动力学,结果表明,外周神经ＮＴＥ对于ＴＯＣＰ的抑制比中枢神经ＮＴＥ敏感得多,ＴＯＣＰ对鸡脑、脊髓和坐骨神经中ＮＴＥ抑制的Ｉ５０值分别为：１．９３２３、２．３９５０和０．００３５ｍｍｏｌ／Ｌ、ＮＴＥ酶促动力学研究显示,鸡脑Ｎ     

甲基苯丙胺神经毒性及防治的研究进展

甲基苯丙胺近年来在国际上迅速泛滥,作为一类精神刺激药物,世界范围内对其神经毒性的研究一直是热点问题.随着分子生物学及功能影像学等各种技术的快速发展,甲基苯丙胺的神经毒性机制研究日益深入,但是其防治仍然有待进一步研究.本文参考了近年来发表的国内外文献27篇,对其神经毒性及防治作一综述.     

纳米银神经毒性研究进展

纳米银(sliver nanoparticles,AgNPs)性能优异,在肿瘤的早期诊断和神经系统疾病的诊治中应用广泛.然而纳米颗粒可经多种途径进入中枢神经系统,并可能在神经组织中蓄积,导致神经细胞功能紊乱和神经退行性病变.该综述阐述了纳米银的脑累积效应以及进入中枢神经系统的途径,主要包括嗅神经和血脑屏障途径;阐述了纳...     

绿僵菌素对四种昆虫细胞的毒性

【目的】绿僵菌素(Destruxins)是绿僵菌产生的具有杀虫活性的次生代谢产物,本研究以家蚕Bombyx mori Bm12细胞、亚洲玉米螟Ostrinia furnacalis血细胞(Ofh)、果蝇S2细胞和草地贪夜蛾Sf9细胞为对象,探索绿僵菌素对不同昆虫细胞的毒性差异。【方法】采用MTT法和显微观察法比较绿僵菌素A、B(DA、DB)以及两者等量混合物(DABM)对上述4种昆虫细胞的影响,比较其IC50值和形态学变化。【结果】绿僵菌素处理24 h后,在较低处理剂量(<25μg/m L)下,Ofh细胞比Bm12、S2、Sf9细胞对DA、DB和DABM更为敏感,DA、DB和DABM对Ofh细胞IC50值分别219.19、112.29和34.86μg/m L,而对Bm12、S2、Sf9细胞的IC50值均大于250μg/m L;DA和DB对Ofh细胞具有协同增效作用,对Sf9细胞具拮抗作用,对Bm12和S2细胞具相加作用。显微观察发现,绿僵菌素处理后,6.25μg/m L的低剂量下,即可发现细胞的形态变化,剂量越高,变化越显著。Bm12细胞出现瘤状突起、细胞破碎、聚集、扩展及胞内空泡等现象,且细胞数量减少;Ofh细胞扩展,似乎回归到浆血细胞及类绛色细胞的形态,发生凝集现象,少数出现瘤状突起和细胞破碎;S2细胞出现明显的胞内空泡,少数发生扩展、破碎和聚集现象;Sf9细胞细胞膜收缩、细胞空泡、破碎,细胞数量减少等变化。【结论】玉米螟的血细胞Ofh对绿僵菌素最为敏感,而来自非寄主昆虫果蝇的S2细胞最不敏感。绿僵菌素对4种细胞的致死剂量较高,但引起细胞形态改变的剂量却非常低。     

三甲基苯基磷酸酯对神经毒性酯酶的抑制及其酶促动力学分析

以可使人和敏感动物产生迟发性神经毒性的有机磷化合物三甲基苯基磷酸酯（TOCP）为测试药物,研究其在体外对成年产卵来航母鸡不同神经组织神经毒性酯酶（NTE）活性抑制的敏感性及其抑制的动力学．结果表明,外周神经NTE对于TOCP的抑制比中枢神经NTE敏感得多．TOCP对鸡脑、脊髓和坐骨神经中NTE抑制的I50值．分别为：1．9323、2．3950和0．0035mmol／L．NTE酶促动力学研究显示,鸡脑NTE催化分解底物戊酸苯酯（PV）的Vmax为62．10nmol·min-1·mg-1,Km为0．92mmol／L．TOCP对鸡脑NTE的抑制属竞争性抑制类型,并有\"底物抑制\"现象．     

Inhibition of neuropathy target esterase expressing by antisense RNA does not affect neural differentiation in human neuroblastoma (SK-N-SH) cell line

Neuropathy target esterase (NTE) is phosphorylated and aged by oraganophosphorus compounds (OP) that induce delayed neuropathy in human and some animals. NTE has been proposed to play a role in neurite outgrowth and process elongation during neural differentiation. However, to date, there is no direct evidence of the relevance of NTE in neural differentiation under physiological conditions. In this study we have investigated a possible role for NTE in the all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells by antisense RNA. A NTE antisense RNA construct was generated and then transfected into human neuroblastoma SK-N-SH cells. A positive cell clone that can stably express NTE antisense RNA was obtained by G418 selection and then identified by western blotting. NTE activity was depressed in the transfected cells with only about 50% activity of the enzyme in the control cells. ATRA-induced differentiation of the neuroblastoma cells with lowered NTE activity revealed that inhibition of NTE expression does not affect neural differentiation in SK-N-SH cells. The result suggested that organophosphates may inhibit neural differentiation by initially acting on other targets other than NTE.     

Purification and characterization of neuropathy target esterase (NTE) from rat brain

Neuropathy target esterase (NTE) is an integral membrane protein in vertebrate neurons and a member of a novel family of putative serine hydrolases. Neuropathic organophosphates react covalently with the active site serine residue of NTE, causing degeneration of long axons in spinal cord and peripheral nerves which becomes clinically evident 1-3 weeks after exposure to OPs, hence termed as organophosphate induced delayed neuropathy. The present study reports the isolation and characterization of NTE protein from rat brain. Rat brain microsomes were solubilized with phospholipase A2 and they were fractionated by gel filtration chromatography in S-300 column. The sample was eluted in buffer containing polyoxyethylene W1 detergent, which yielded an active fraction of 200 kDa. The most enriched NTE active fraction was further purified by 3-9'-mercaptononylthio-1,1,1-trifluoropropan-2-one bound to sepharose CL4B. The SDS-PAGE confirmed the 155-kDa protein as the most likely candidate for NTE. Database searching of rat N-terminal protein revealed homology with variety of polypeptides from different organisms and suggested that NTE protein has function beyond the nervous system and mediates a biochemical reaction highly conserved through evolution.     

Recovery of neuropathy target esterase activity after inhibition with mipafox and O-hexyl O-2,5-dichlorophenyl phosphoramidate in bovine chromaffin cell cultures

Neuropathy target esterase (NTE) is a membrane protein present in various tissues whose physiological function has been recently suggested to be the maintenance of phosphatidylcholine homeostasis. Inhibition and further modification of NTE by certain organophosphorus compounds (OPs) were related to the induction of the "organophosphorus induced delayed neuropathy". Bovine chromaffin cells were cultured at 75,000cells/well in 96-well plates and exposed to 25microM mipafox or 3microM O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) for 60min. Inhibitors were removed by washing cells three times with Krebs solution. Then NTE activity was assayed at 0, 24, 48 and 120h after exposure using the Biomek 1000 workstation. Immediately after mipafox treatment NTE activity represented 3% of the control (6.7+/-1.9mU/10(6) cells). At 24, 48 and 120h after removing inhibitor, recorded activities were 33%, 42% and 111% of their respective controls (5.7+/-3.1; 5.7+/-1.9; 5.4+/-0.0mU/10(6) cells, respectively). Treatment with HDCP also displayed a time-dependent pattern of NTE recovery. As NTE inhibited by phosphoramidates is not reactivated in homogenized tissues, these results confirm a time-dependent regeneration of NTE after inhibition by neuropathic OPs.     

Influence of lysophospholipid hydrolysis by the catalytic domain of neuropathy target esterase on the fluidity of bilayer lipid membranes

Neuropathy target esterase (NTE) is an integral membrane protein localized in the endoplasmic reticulum in neurons. Irreversible inhibition of NTE by certain organophosphorus compounds produces a paralysis known as organophosphorus compound-induced delayed neuropathy. In vitro, NTE has phospholipase/lysophospholipase activity that hydrolyses exogenously added single-chain lysophospholipids in preference to dual-chain phospholipids, and NTE mutations have been associated with motor neuron disease. NTE's physiological role is not well understood, although recent studies suggest that it may control the cytotoxic accumulation of lysophospholipids in membranes. We used the NTE catalytic domain (NEST) to hydrolyze palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (p-lysoPC) to palmitic acid in bilayer membranes comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and the fluorophore 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (NBD-PC). Translational diffusion coefficients (D_L) in supported bilayer membranes were measured by fluorescence recovery after pattern photobleaching (FRAPP). The average D_L for DOPC/p-lysoPC membranes without NEST was 2.44 µm²s^-1 ± 0.09; the D_L for DOPC/p-lysoPC membranes containing NEST and diisopropylphosphorofluoridate, an inhibitor, was nearly identical at 2.45 ± 0.08. By contrast, the D_L for membranes comprising NEST, DOPC, and p-lysoPC was 2.28 ± 0.07, significantly different from the system with inhibited NEST, due to NEST hydrolysis. Likewise, a system without NEST containing the amount of palmitic acid that would have been produced by NEST hydrolysis of p-lysoPC was identical at 2.26 ± 0.06. These results indicate that NTE's catalytic activity can alter membrane fluidity.     

A rapid spectrophotometric method for the determination of esterase activity

We have developed a spectrophotometric assay method which continuously records esterase activity at 510 nm by monitoring absorbance changes due to the formation of a diazo dye complex. In our method, α-naphthyl ester substrates are hydrolyzed by enzymatic action to α-naphthol which couples to Fast Blue RR salt (a diazonium salt) forming a diazo dye complex. Our method is unique in directly monitoring the formation of the diazo dye complex without extracting the color of the complex as in other methods that use naphthyl esters and diazo coupling of reaction products. The method appears to be limited to α-naphthyl ester substrates, however, since β-naphthyl esters did not give a linear change in absorbance in the enzymatic reactions tested. With this assay method, one can use a single substrate both to determine esterase units quantitatively in solution and to detect esterase staining activity on gel electrophoresis.     

High-performance liquid chromatographic–fluorimetric assay of chymotrypsin-like esterase activity

A sensitive and reproducible assay for the determination of chymotrypsin-like esterase activity is reported. This method is based on fluorimetric detection of a dansylated amino acid, 5-dimethylaminonaphthalene-1-sulfonyl- -phenylalanine, enzymatically formed from the substrate 5-dimethylaminonaphthalene-1-sulfonyl- -phenylalanine ethyl ester, after separation by high-performance liquid chromatography using a C₁₈ reversed-phase column and isocratic elution. This method is sensitive enough to measure 5-dimethylaminonaphthalene-1-sulfonyl- -phenylalanine at concentrations as low as 40 pmol/ml, yields highly reproducible results and requires less than 9.5 min per sample for quantitation. The optimum pH for chymotrypsin-like esterase activity was 7.7–8.3. The K_m and V_max values were, respectively 25 μM and 0.241 pmol/μg protein/h with the use of enzyme extract obtained from mouse kidney. The approximate molecular mass of this enzyme was estimated to be 67 000 by gel filtration. Chymotrypsin-like esterase activity was strongly inhibited by N-tosyl- -phenylalaline chloromethyl ketone. Among the mouse organs examined, the highest specific activity of the enzyme was found in lung. This new method would be useful for clarification of the physiological role of this enzyme.     

Administration of Lactobacillus fermentum CRL1446 increases intestinal feruloyl esterase activity in mice

Aims: To evaluate the effect of oral administration of Lactobacillus fermentum CRL1446 on the intestinal feruloyl esterase (FE) activity and oxidative status of mice. Methods and Results: Adult Swiss albino mice received Lact. fermentum CRL1446 at the doses 10⁷ and 10⁹ cells per day per mouse for 2, 5, 7 and 10 days. Intestinal FE activity, intestinal microbiota counts, plasmatic thiobarbituric acid‐reactive substances (TBARS) percentage and glutathione reductase (GR) activity were determined. Mice that received Lact. fermentum CRL1446 at the dose 10⁷ cells per day for 7 days showed a twofold increase in total intestinal FE activity, compared to the nontreated group. In large intestine content, FE activity increased up to 6·4 times. No major quantitative changes in colonic microbiota were observed in treated animals. Administration of this strain produced an approx. 30–40% decrease in the basal levels of plasmatic TBARS and an approx. twofold increase in GR activity from day 5 of feeding with both doses. Conclusions: Oral administration of Lact. fermentum CRL1446 to mice increases total intestinal FE activity, decreases the basal percentage of plasmatic lipoperoxides and increases GR activity. Significance and Impact of the Study: Lactobacillus fermentum CRL1446 could be orally administered as a dietary supplement or functional food for increasing the intestinal FE activity to enhance the bioavailability of ferulic acid, thus improving oxidative status.     

Development of a model for prediction of target insect mortality following field application of a Bacillus thuringiensis formulation

A model for predicting mortality of Schizura concinna larvae on Cercis occidentalis trees following application of Bacillus thuringiensis formulations is based on an estimation of the effective dose. This is calculated from the initial application level and field persistence characteristics of the pathogen and from the feeding rate of the target insects. Using the estimated effective dose and the time the pathogen was on the host plant, the probability of larval mortality is calculated by means of a multiple logistic dose-response model. Predicted mortalities for B. thuringiensis applications at concentrations 0.00025, 0.0025, 0.025, and 0.25%, $\text{w}\text{v}$, were 7.7, 23.3, 46.2, and 87.1%, respectively. Corresponding, observed mortalities adjusted for larval loss due to forces unrelated to the B. thuringiensis application were 6, 54, 66, and 100%.