高温对田间小菜蛾DNA损伤及4种抗药性相关酶系活性的抑制

研究了高温对福州上街菜田小菜蛾成虫4种抗药性相关酶系活性的影响。与饲养在25℃下的小菜蛾相比,33．5~C或40℃处理72h导致小菜蛾基因组DNA出现DNA凋亡特征梯度化条带。33~C饲养小菜蛾4、8、12或24h对小菜蛾成虫乙酰胆碱酯酶（AChE）和羧酸酯酶（CarE）活性无显著影响,但33℃饲养小菜蛾12或24h导致小菜蛾成虫谷胱甘肽s转移酶（GSTs）酶活性和细胞色素P450含量显著下降。36℃、24h可导致AChE活性显著下降,36℃、12h和24h可导致CarE活性显著下降,36℃、4h,8h,12h和24h可导致GST活性和细胞色素P450含量显著下降。总体上,高温对CarE、GSTs和细胞色素P450的抑制作用大于对AChE的影响,此外,3CC对AChE、CarE、GSTs酶活性和细胞色素P450含量的抑制作用大于33℃的抑制作用。     

脱氧鬼臼毒素对美洲大蠊的毒力及几种酶系的影响

采用药膜法测试了脱氧鬼臼毒素对美洲大蠊Periplaneta americana初孵若虫的触杀活性,并测定了其对成虫中枢神经系统乙酰胆碱酯酶（AChE）和腺苷三磷酸酶(ATPase)离体活性的影响。结果表明：脱氧鬼臼毒素对美洲大蠊初孵若虫具有较强的毒杀活性,在接触时间24、48、72和96 h 的LC₅₀分别为26.26、4.68、1.51和0.62 μg/cm²;其对AChE没有明显影响; 对Na⁺ -K⁺ -ATPase有明显抑制作用,并存在浓度 效应关系,IC₅₀为44.9 μmol/L; 对Ca²⁺-Mg²⁺-ATPase表现出低剂量激活,高剂量抑制的现象。结果提示美洲大蠊的AChE不是脱氧鬼臼毒素靶标,而ATPase可能是脱氧鬼臼毒素的重要靶标之一。     

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。     

二化螟体内乙酰胆碱酯酶的分布及纯化方法

研究了二化螟Chilo suppressalis乙酰胆碱酯酶（AChE）的体躯和亚细胞分布,并用凝胶过滤层析和2种亲和层析方法从二化螟幼虫体内分离、纯化乙酰胆碱酯酶。结果表明：二化螟幼虫乙酰胆碱酯酶的活性主要集中于头部和胸部,而成虫胸部乙酰胆碱酯酶的活性最低,显著低于头部和腹部。成虫体内AChE的活性明显高于幼虫。在亚细胞的分布上,乙酰胆碱酯酶主要位于膜上（86%）,近46%的活性存在微粒体中。在3种纯化乙酰胆碱酯酶的方法中,以3-羧基苯基-乙基二甲基铵作配体的亲和层析法纯化效果最佳,乙酰胆碱酯酶的最高纯化倍数为536.05倍,产率30.46%。     

苦豆子生物碱对萝卜蚜的毒力及其对几种酯酶的影响

苦豆子Sophoraalopecuroids（L．）的次生代谢物质为喹诺里西定生物碱类。本研究明确了该生物碱中的野靛碱对萝卜蚜（Lipaphiserysimi）有很高的毒杀作用,对其无翅成蚜的致死中浓度（LC50,浸渍法）为（4325±2．12）mg／L,优于著名的杀蚜生物碱毒黎碱和烟碱,两者对该试虫的LC50分别为（684．70±2．28）mg／L和（1090．65±2．01）mg／L。用小菜蛾（Plutellaxylostella）幼虫作试虫,得知苦豆子7种主要生物碱对昆虫的乙酰胆碱酯酶（AChE）有抑制作用,其抑制程度排序为：总碱＞野靛碱＞槐胺碱＞槐定碱＞槐果碱＞氧化苦参碱＞苦参碱＞苦豆碱。野靛碱和苦豆碱对α-乙酸萘酯酶、α-乙酸萘酯羧酸酯酶及酯酶同功酶的活性亦表现不同程度的抑制作用。     

昆虫乙酰胆碱酯酶基因研究进展

对昆虫乙酰胆碱酯酶（acetylcholinesterase, AChE,EC 3.1.1.7）的基因结构和表达等方面的研究进展进行了综述。分析了昆虫乙酰胆碱酯酶基因的结构,包括10个外显子的特征。对已经报道的昆虫AChE基因进行了系统归纳,并基于已知全序列的昆虫AChE基因,进行了昆虫AChE基因的分子进化分析。对昆虫AChE基因的结构特点及其功能,以及昆虫AChE基因的活性位点、AChE的变构与昆虫抗药性的关系进行了探讨。最后对昆虫AChE基因研究中存在的问题和前景进行了分析和展望。     

甲酸乙酯对松材线虫的熏蒸效果

【背景】在溴甲烷面临禁用的情形下,探讨新熏蒸剂甲酸乙酯对松材线虫的处理效果,可以科学地评估甲酸乙酯的使用前景。【方法】采用松材线虫分离液和带疫松木段,设置甲酸乙酯5个剂量梯度、5个处理温度和5个处理时间,测定其对松材线虫的毒力及CT值。【结果】在25℃下处理3、6、12、24、48h,甲酸乙酯对松材线虫的Lc50分别为2．63、1．60、0．99、0．41、0．20mg·L-1。温度对毒力有显著影响,在10～29℃,随温度升高,甲酸乙酯对松材线虫的毒力降低,19和29℃下,1．85mg·L-1甲酸乙酯处理松材线虫的死亡率分别为63％和100％。甲酸乙酯熏蒸12h内,能完全杀灭木段中的松材线虫。在23℃下处理6和12h,松材线虫死亡率达到99％时,甲酸乙酯的cr值分别为453．94和424．14mg·h-1·L-1。【结论与意义】甲酸乙酯可用于松材线虫的检疫处理。     

玉米花粉形成过程不同阶段特异蛋白变化规律及酯酶同工酶研究

利用薄层胶电泳技术对玉米花粉形成过程不同阶段的酯酶同工酶及可溶性蛋白种类和含量变化进行了研究。结果表明在玉米花粉形成过程中的不同阶段酯酶同工酶的种类和活性均有不同。四分体时期出现两种小分子量酯酶同工酶到小孢子阶段即很快消失,早期小孢子阶段酯酶同工活性及种类达到高峰,以后则逐步递减。玉米花粉可溶性蛋白电泳显示玉米花粉形成过程中不同发育时期蛋白类和含量有很大变化。四分体时期存在有特异性蛋白（ＴＰ）,它     

菜蛾绒茧蜂和小菜蛾对杀虫剂的敏感性及酶学特性的比较研究

分别采用药膜法和浸叶法测定了菜蛾绒茧蜂Apanteles plutellae和小菜蛾Plutella xylostella对杀虫剂的敏感度。结果显示： 有机磷、氨基甲酸酯、拟除虫菊酯类杀虫剂、阿维菌素和锐劲特对菜蛾绒茧蜂高毒,而抑太保和Bt为低毒,然而,短时间(1 h)接触常规防治剂量的锐劲特、氰戊菊酯、氯氰菊酯和乙酰甲胺磷对菜蛾绒茧蜂低毒。增效剂胡椒基丁醚（PB）、磷酸三苯酯（TPP）和马来酸二乙酯（DEM）对菜蛾绒茧蜂的甲胺磷、克百威、氰戊菊酯、氯氰菊酯、阿维菌素和锐劲特敏感性增效显著,但对抑太保无增效作用。PB的增效作用显著高于TPP 和DEM。PB和TPP对菜蛾绒茧蜂羧酸酯酶(CarE),以及DEM对谷胱甘肽S转移酶(GST)具显著的活体抑制作用,但PB,TPP和 DEM对菜蛾绒茧蜂乙酰胆碱酯酶（AChE）无抑制作用。菜蛾绒茧蜂AChE的米氏常数(K_m)、最大反应速度(V_max)、CarE和GST活性分别为小菜蛾的0.22、2.08、4.60和0.45倍,甲胺磷、敌敌畏和克百威对菜蛾绒茧蜂AChE的双分子速度常数(Ki)分别为对小菜蛾的14.7、10.5 和26.0倍。酶与抑制剂反应温度增高将导致酶抑制率增高,尤其对菜蛾绒茧蜂AChE的抑制作用更为显著。上述结果表明,菜蛾绒茧蜂对有机磷和氨基甲酸酯类杀虫剂的高敏感性与其显著高的AChE敏感性有关,氧化代谢的解毒作用对菜蛾绒茧蜂耐药性的影响大于水解作用。此外,对小菜蛾和菜蛾绒茧蜂杀虫剂敏感性差异的毒理学原因进行了讨论。     

细辛醚对粘虫幼虫的毒力及几种重要酶系的影响

采用点滴法测定了细辛醚对粘虫Mythimna separate Walker 5龄幼虫的触杀毒力,观察了细辛醚对粘虫的致毒症状,并测定了对不同中毒阶段其头部或中肠乙酰胆碱酯酶（AChE）和腺苷三磷酸酶(ATPase)离体活性以及乙酰胆碱（ACh）含量的影响。结果表明：细辛醚对粘虫5龄幼虫12 h的触杀LD₅₀值为106.93 μg/头;中毒粘虫依次表现出兴奋、痉挛、麻痹和死亡等症状,类似于神经毒剂的致毒症状,但试虫从中毒到死亡历期较长,且中毒试虫肠内有气泡产生,此现象异于典型的神经毒剂;对Na+,K+-ATPase有较强的抑制作用,在兴奋期、痉挛期和麻痹期,酶抑制率分别为33.29%、44.45%和23.98%;对AChE活力在不同阶段的抑制率分别是21.64%、29.75%和35.22%;ACh含量有所增加。     

Cytochrome c oxidase inhibition in the rice weevil Sitophilus oryzae (L.) by formate, the toxic metabolite of volatile alkyl formates

Volatile alkyl formates are potential replacements for the ozone-depleting fumigant, methyl bromide, as postharvest insecticides and here we have investigated their mode of insecticidal action. Firstly, a range of alkyl esters, ethanol and formic acid were tested in mortality bioassays with adults of the rice weevil, Sitophilus oryzae (L.) and the grain borer, Rhyzopertha dominica (F.) to determine whether the intact ester or one of its components was the toxic moiety. Volatile alkyl formates and formic acid caused similar levels of mortality (LC(50) 131-165 micromol l(-1)) to S. oryzae and were more potent than non-formate containing alkyl esters and ethanol (LC(50)>275 micromol l(-1)). The order of potency was the same in R. dominica. Ethyl formate was rapidly metabolised in vitro to formic acid when incubated with insect homogenates, presumably through the action of esterases. S. oryzae and R. dominica fumigated with a lethal dose of ethyl formate had eight and 17-fold higher concentrations of formic acid, respectively, in their bodies than untreated controls. When tested against isolated mitochondria from S. oryzae, alkyl esters, alcohols, acetate and propionate salts were not inhibitory towards cytochrome c oxidase (EC 1.9.3.1), but sodium cyanide and sodium formate were inhibitory with IC(50) values of 0.0015 mM and 59 mM, respectively. Volatile formate esters were more toxic than other alkyl esters, and this was found to be due, at least in part, to their hydrolysis to formic acid and its inhibition of cytochrome c oxidase.     

Interactions of carbaryl with susceptible and multiresistant house flies (Diptera: Muscidae).

The in vivo and in vitro fate of [14C]carbaryl was compared in adult male and female house flies from an insecticide-susceptible (S) strain and a resistant (R) strain with multiple resistance to different classes of insecticides. Cuticular penetration of topically applied carbaryl (0.01 microgram/insect) was very rapid and rates were essentially the same among males and females of both strains. Rates of penetration were dramatically reduced as the concentration of applied carbaryl was increased over a range of 0.01-5.0 micrograms/insect. In vivo and in vitro tests demonstrated that the R strain had an enhanced capability for the metabolic degradation of carbaryl. In evaluations of topical toxicity and in vitro metabolic degradation, coadministration of the metabolic synergists piperonyl butoxide (a microsomal oxidase inhibitor) and S,S,S-tributyl phosphorothioate (DEF, an esterase inhibitor) with carbaryl provided conclusive evidence that microsomal oxidases were the major factor in enhanced metabolism and that hydrolytic enzymes had only a minor effect. Studies of the in vitro inhibition of acetylcholinesterase (AChE) activity by carbaryl demonstrated that there was no difference between males and females of a given strain and that the R strain AChE was considerably less sensitive to inhibition. These tests also indicated that homogenates of brains from the R strain contained more than one form of AChE with different sensitivities to the inhibitor. This information and results of toxicity tests with other insecticides suggest that the R strain is not homozygous in its resistance to carbaryl.     

Methanol Inhibition in Continuous Culture of Hansenula polymorpha

Growth inhibition of Hansenula polymorpha DL-1 by methanol, formaldehyde, formate, and formic acid was examined to determine the causes of unstable behavior observed during continuous cultures on methanol. The much greater inhibition of growth by formic acid than by formate and the effect of formic acid excretion and assimilation on pH helped to explain culture dynamics observed after transitory oxygen limitations. Oxygen limitation caused by temporary reduction of agitation in a continuous fermentation caused methanol to accumulate to inhibitory concentrations. Immediately after resumption of agitation, formic acid was produced and caused culture inhibition. To ensure the stability of H. polymorpha in continuous culture, it was therefore necessary to prevent transient methanol accumulation.     

16S acetylcholinesterase of the extracellular matrix is assembled within mouse muscle cells in culture.

The present paper examines where the extracellular-matrix (ECM) 16S acetylcholinesterase (AChE, EC 3.1.1.7) is assembled in muscle cells in culture. The existence of an internal pool of 16S AChE was detected by using AChE inhibitors of differing membrane permeability. After irreversible inhibition of all cellular esterase, the newly synthesized 16S form appears in an intracellular compartment and is only later detected on the cell surface. Results show that the ECM 16S AChE is assembled within muscle cells.     

The aryl acylamidase activity is much more sensitive to Alzheimer drugs than the esterase activity of acetylcholinesterase in chicken embryonic brain

The appearance of cholinergic trait often precedes synaptogenesis, indicating the involvement of cholinesterase proteins in nervous system development, particularly so acetylcholinesterase (AChE). In addition to AChE's acclaimed esterase activity, its lesser known non-cholinergic functions have gained much attention, because of AChE protein expression in areas other than cholinergic innervations; one such function could be exerted by its associated aryl acylamidase (AAA) activity. In this study, an attempt has been made in profiling esterase and AAA activities of AChE at different developmental stages of the chick embryo, e.g. at embryonic day 6 (E6), E9, E12, E15 and E18. AAA activity showed a correlated expression with esterase activity at all stages, but the relative ratios of AAA to esterase activity were higher at younger stages. The inhibition of AAA activity was shown to be more sensitive towards Huperzine, Donepezil whereas inhibition of esterase activity was sensitive to Tacrine and DFP. Remarkably, the major Alzheimer drugs- Huperzine and Donepezil, much more strongly inhibited AAA activity of AChE at younger developmental stages whose IC50 values are 0.01 μM and 0.1 μM respectively. In the case of BW284c51, inhibition was more pronounced at older stages and IC50 value was 0.1 μM. Since in Alzheimer's disease (AD), embryonic forms of AChE have been reported to reappear, a possible role of AAA activity in the pathogenesis of AD should be considered.     

MetR-mediated repression of the glyA gene in Escherichia coli

Abstract Both lactic and acetic acids cause mixed inhibition of acid production in mutans streptococci. This inhibition is partly irreversible due to cell death, an important factor when considering acidogenicity and aciduricity of these organisms, and their role in the caries process. Other monocarboxylic end-products may be present and may also be important inhibitors of acid production in dental plaque. This study considered the effects of varying concentrations of the end-product formic acid on acid production rates in Streptococcus mutans R9, measured using the pH-stat. Undissociated formic acid caused mixed inhibition with constants of K _iu (uncompetitive) of 6.07 ± 1.27 mmol 1⁻¹ and K _ic (competitive) of 0.2 ± 10.11 mmol I ⁻¹. Inhibition was found to be fully reversible, with no loss of cell viability. It is concluded that at those concentrations found in vivo, formate is not a significant inhibitor of acid production by S. mutans in dental plaque at any time, and is not important in determining the acidogenicity or aciduricity of this organism.     

A comparative study of inhibition of acetylcholinesterase,trypsin, neuropathy target esterase,and spleen cell activation by structurally related organophosphorus compounds

Organophosphorus (OP) compounds can bind to and inactivate several target molecules other than acetylcholinesterase (AChE). In the present study, five sets of structurally related organophosphorus compounds were used to evaluate the relationships between organophosphorus binding sites of AChE, neuropathy target esterase (NTE), trypsin, and the target molecule(s) involved in inhibition of splenocyte activation by OP compounds. The concentration of each OP compound required to inhibit enzyme activity or splenocyte activation by concanavalin A by 50% was determined. The pattern of IC₅₀ values indicated that AChE, trypsin, NTE, and the molecule(s) involved in inhibition of splenocyte activation are distinct with regard to patterns of inhibition by OP compounds. However, there was a striking similarity in the patterns of inhibition for trypsin and NTE with substantial differences for only 2 of 20 compounds. This pattern suggests similarity in the active sites of these molecules. There were also similarities in the IC₅₀ patterns for lymphocyte activation and trypsin or NTE activity. However, the correlation was not as strong as between NTE and trypsin, and the data suggested the possibility of multiple target molecules for inhibition of splenocyte activation by OP compounds. More importantly, there was essentially no correlation between the pattern of IC₅₀ values for AChE and splenocyte activation. This strongly suggests that acetylcholine and AChE of the type found in the brain are not important in the regulation of splenocyte activation by concanavalin A.     

Effect of carbamate esters on neurite outgrowth in differentiating human SK-N-SH neuroblastoma cells

Carbamate esters are widely used as pesticides and can cause neurotoxicity in humans and animals; the exact mechanism is still unclear. In the present investigation, the effects of carbamates at sublethal concentration on neurite outgrowth and cytoskeleton as well as activities of acetylcholinesterase (AChE) and neuropathy target esterase (NTE) in differentiating human SK-N-SH neuroblastoma cells were studied. The results showed that 50 microM of either aldicarb or carbaryl significantly decreased neurite length in the retinoic acid-induced differentiation of the neuroblastoma cells, compared to cells treated with vehicle. Western blot analyses revealed that neither carbamate had significant effects on the levels of actin, or total neurofilament high molecular proteins (NF-H). However, increased NF-H phosphorylation was observed following carbamate treatment. These changes may represent a useful in vitro marker of carbamate neurotoxicity within a simple model of neuronal cell differentiation. Furthermore, activity of AChE, but not NTE, was significantly inhibited by aldicarb and carbaryl in differentiating cells, which suggested that cytoskeletal protein changes induced by carbamate esters in differentiating cells was associated with inhibition of AChE but not NTE.     

Analysis of methanol or formic acid in body fluids by headspace solid-phase microextraction and capillary gas chromatography

Methanol and its metabolite formic acid have been found extractable from human whole blood and urine by headspace solid-phase microextraction (SPME) with a Carboxen/polydimethylsiloxane fiber. The headspace SPME for formic acid was carried out after derivatization to methyl formate under acidic conditions. The determinations of both compounds were made by using acetonitrile as internal standard (IS) and capillary gas chromatography (GC) with flame ionization detection. The headspace SPME–GC gave sharp peaks for methanol, methyl formate and I.S.; and low background noises for whole blood and urine samples. Extraction efficiencies were 0.25–1.05% of methanol and 0.38–0.84% formic acid for whole blood and urine. The calibration curves for methanol and formic acid showed excellent linearity in the range of 1.56 to 800 and 1.56 to 500 μg/0.5 ml of whole blood or urine, respectively. The detection limits were 0.1–0.5 μg/0.5 ml for methanol and 0.6 μg/0.5 ml for formic acid for both body fluids. The within-day relative standard deviations in terms of extraction efficiency for both compounds in whole blood and urine samples were not greater than 9.8%. By using the established SPME method, methanol and formic acid were successfully separated and determined in rat blood after oral administration of methanol.     

(S)-Thiirancarboxylic acid as a reactive building block for a new class of cysteine protease inhibitors

For (S)-thiirancarboxylic acid a second-order rate constant of k2nd = 222 M(-1) min(-1) for the irreversible inhibition of papain was determined. The ethyl and methyl ester do not inhibit the enzyme time-dependently. An improved synthesis of enantiomerically pure thiirancarboxylic acid is described. It is shown that thiirancarboxylates can be substrates for serine proteases (alpha-chymotrypsin) and esterases (pig liver esterase) and even for metallo proteases (thermolysin).