The essential oil of Eucalyptus tereticornis, and its constituents α- and β-pinene,potentiate acetylcholine-induced contractions in isolated rat trachea

The effects of the essential oil of Eucalyptus tereticornis (EOET), especially the effects of its constituents α- and β-pinene, were studied on rat trachea in vitro. In tracheal rings, EOET, α- or β-pinene potentiated the contractions induced by acetylcholine (ACh). Contractions induced by K⁺ (60 mM) were also potentiated by α- and β-pinene, but were reduced by EOET. Our findings show that EOET has myorelaxant effects on rat airways, but potentiates ACh-induced contractions. Monoterpenes α- and β-pinene are involved in its potentiating actions, but are not responsible for its myorelaxant effects. A putative inhibition of the acetylcholinesterase enzyme is involved.     

朱砂叶螨乙酰胆碱酯酶的表达纯化及其酶活测定条件优化

乙酰胆碱酯酶(Acetylcholinesterase,AChE)是生物体内保证神经信号正常传递的关键酶,是有机磷和氨基甲酸酯类农药的作用靶标。现研究拟采用发酵罐培养含pET-30a/ace的表达菌株E.coli BL21(DE3),大量表达纯化螨AChE,利用单因素试验和正交试验优化AChE活性的测定条件。结果表明:发酵培养的重组菌株大量表达约为68kD的朱砂叶螨AChE蛋白。底物浓度、温度、pH和酶浓度等各测定因素对朱砂叶螨乙酰胆碱酯酶活性存在显著影响。确定朱砂叶螨乙酰胆碱酯酶活性测定的最佳反应条件为:底物浓度1mmol/L,pH值7,温度35℃,酶浓度0.5mg/mL。通过体外发酵培养纯化获得了大量的朱砂叶螨AChE重组蛋白,确定了螨AChE活性测定的最佳条件,为进一步研究其酶学性质以及AChE抑制剂的体外筛选奠定基础。     

Frequencies and evolution of organophosphate insensitive acetylcholinesterase alleles in laboratory and field populations of the house fly, Musca domestica L.

Resistance to organophosphate (OP) and/or carbamate insecticides can be due to mutations in the acetylcholinesterase gene (Ace). Genotypes of house fly, Musca domestica L., Ace were determined in twelve laboratory maintained strains (originally from North America, Europe and Asia) and two field collected populations from New York and Florida. There were 15 Ace alleles found and 11 of the alleles coded for a susceptible form of the enzyme (i.e., V260, A316, G342 and F407). Three of the four resistance alleles were previously described, while one is new. Phylogenetic analysis of the alleles suggests multiple origins of the F407Y mutation and multiple origins of the G342A mutation that confer OP resistance. Genotyping of field collected house flies from New York and Florida populations revealed the presence of only one resistance allele, Acev10 (containing the non-synonymous mutations for A342 and Y407). All other alleles detected from the field-collected flies coded for a susceptible AChE. Thus, we were able to categorize individual flies as having homozygous susceptible (Ace^S/Ace^S), homozygous insensitive (Ace^I/Ace^I or Acev10/Acev10) or heterozygous AChE. The frequencies of Ace^S and Ace^I were not different between the NY2002 and FL2002 populations. Both populations were out of Castle-Hardy-Weinberg equilibrium, having an excess of Ace^S/Ace^I individuals and very few Ace^S/Ace^S individuals. Comparison of Ace, Vssc and CYP6D1 genotypes indicates individual house flies commonly have resistance alleles at multiple loci. Comparison of genotype data with bioassays, as well as the use of genotype data in resistance studies is discussed.     

A new lupene triterpenetriol and anticholinesterase activity of Salvia sclareoides

A new lupene triterpenetriol was isolated from the acetone extract of the aerial parts of Salvia sclareoides and characterised as (1beta,3beta)-lup-20(29)-ene-1,3,30-triol (1). In addition, nepetidin (2), nepeticin (3), lupendiol (4), (1beta,11alpha)-dihydroxy-lup-20(29)-en-3-one (5), ursolic acid (6), sumaresinolic acid (7) and hederagenin (8), were identified in this Salvia sp. To the best of our knowledge, the compounds 2 and 7 are new constituents in Salvia spp. The acetone, ethanol, butanol and water extracts of the plant were screened for the in vitro inhibitory activity of acetylcholinesterase (AChE) and butyrilcholinesterase (BChE), enzymes which play a role in the Alzheimer disease. All extracts inhibited acetylcholinesterase activity at 10 microg/ml, a remarkable activity since the standard drug rivastigmine does not inhibit acetylcholinesterase at the same concentration. Regarding the butyrilcholinesterase, the acetone extract at 1000 microg/ml was able to inhibit completely the enzyme activity and the butanol and ethanol extracts, at this concentration, produced a potent inhibition of BchE.     

小菜蛾对辛硫磷的抗性选育及其乙酰胆碱酯酶和羧酸酯酶活力变化

在室内模拟田间药剂的选择压力,用辛硫磷对小菜蛾（Plutella xylostella）逐代处理,以选育其抗性种群,并测定乙酰胆碱酯酶（acetylcholinesterase,AChE）和羧酸酯酶（carboxylesterase,CarE）的活性变化。选育至12代,对辛硫磷抗性增长到4.524倍。生化分析表明,抗性品系AChE和CarE活性均显著高于敏感品系。在抗性选育过程中,小菜蛾AChE酶活性增长迅速,其比活力由F0的0.035μmol/L·mg·min上升为F12的0.209μmol/L·mg·min;小菜蛾不同选育世代CarE酶活性差异显著,F12比活力增长到F0的12.944倍。     

丙硫磷对乙酰胆碱酯酶的抑制能力及其毒力与药效研究*

以两种对称型有机磷杀虫剂倍硫磷和敌敌畏为对照，利用Ｅｌｌｍａｎ法测定了不对称有机磷杀虫剂丙硫磷（Ｐｒｏｔｈｉｏｆｏｓ）对体外乙酰胆碱酯酶（ＡｃｈＥ）的抑制能力，并对褐刺蛾（Ｓｅｔｏｒａｐｏｓｔｏｒ－ｎａｔａＨａｍｐｓｏｎ）进行了３种杀虫剂的触杀毒力测定和野外药效试验。结果表明，丙硫磷为弱的ＡｃｈＥ体外抑制剂，酶抑制能力与毒力和药效间无相关性，抑制能力最差的丙硫磷对褐刺蛾幼虫毒力最强，野外药效也最好。说明丙硫磷的三元不对称结构和特殊的毒效基团与其高效低毒的特点之间可能有着密切的联系。     

Potent and selective butyrylcholinesterase inhibitors from Ficus foveolata

Four stilbenes (1-4), one inseparable mixture of two alkyl diferulates (5a, 5b), one alkyl ferulate (6) and four flavonoids (7-10) were isolated from Ficus foveolata Wall. Except for quercetin (10), the other ten constituents were isolated from F. foveolata for the first time. In addition, one of the two components in mixture 5 is a new compound identified as (1E,22E)-1,22-docosanediol diferulate (5a). All nine isolated compounds, plus the mixture of 5a and 5b known as 5, exhibited a low or no activity against acetylcholinesterase. However, and interestingly, the stilbenes 1-4 showed a high inhibition towards butyrylcholinesterase. Gnetol (4) had the lowest IC(50) value of 1.3μM towards butyrylcholinesterase and showed a reversible and competitive inhibition in the kinetic study.     

2,4-D and MCPA and their derivatives: Effect on the activity of membrane erythrocytes acetylcholinesterase (in vitro)

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA), and their derivatives: phenol, 2,4-dichlorophenol (2,4-DCP), 2,4-dimethylphenol (2,4-DMP), and catechol on the activity of acetylcholinesterase (AChE, EC 3.1.1.7) in human erythrocytes was studied. Phenol, MCPA, and 2,4-DMP did not significantly change AChE activity in human erythrocytes (in vitro). Decrease of AChE activity was observed under the highest applied dose of 2,4-D—500 and 1000 ppm. Decrease of AChE activity exposed to 2,4-DCP and catechol was noted and depended on the doses of applied compounds. The relationship between activities and substrate concentrations (curves) was analyzed for reactions of acetylcholinesterase. Catalytic constants K_m and V_max were calculated from the Michaelis curve. Statistically significant decrease of V_max and K_m was observed in the activity of AChE incubated with 2,4-DCP and catechol, revealing mixed inhibition type of AChE inhibition (this compound may affect not only on enzyme but also on complex ES as well). 2,4-D decreases V_max but do not change K_m value, what reveals non-competitive type of AChE inhibition by this compounds. Non-competitive inhibition does not depend on the substrate concentrations but only on the inhibitor concentration and its K_i value, characterizes the affinity of inhibitors towards enzyme. In conclusion, changes of AChE activity upon 2,4-D, 2,4-DCP, and catechol are the consequences of direct interactions between compounds and the enzyme and indirect via membrane modification and increase of Reactive Oxygen Species.     

Detection of resistance-associated point mutations of organophosphate-insensitive acetylcholinesterase in the olive fruit fly, Bactrocera oleae (Gmelin)

We have recently identified two resistance-associated point mutations of organophosphate (OP)-insensitive acetylcholinesterase in the olive fruit fly Bactrocera oleae, the most important olive orchard pest world-wide. We have developed simple PCR-restriction fragment length polymorphism assays for each mutation, utilising an AccI restriction site created by Ile214Val, and a BssHII restriction site destroyed by a neutral change always accompanying the second mutation Gly488Ser. Samples from Greece homozygous for both mutations proved the most insensitive to dimethoate. The frequencies of these mutations in field-collected samples from several countries were investigated. Ninety-three percent of samples from Greece and Albania, where OPs have been extensively used in B. oleae control, were homozygous for both mutations. Resistance-associated alleles were detected at lower frequencies, but still with both mutations in conjunction in the majority of cases, in western Mediterranean countries with limited use of OPs. Samples from South Africa, however, did not have either of the resistance-associated mutations. The double mutation haplotype clearly confers a strong selective advantage in field populations of B. oleae exposed to OPs.     

Susceptibilities to methamidophos and enzymatic characteristics in 18 species of pest insects and their natural enemies in crucifer vegetable crops

The susceptibilities to methamidophos as well as the kinetic and inhibitory parameter of acetylcholinesterases (AChE) and the activities of carboxyestsrases (CarE) and glutathione-S-transferases (GST) were studied in 18 species field populations of insects collected in Fuzhou, China during April and May 2000 and 2001. The insect species included five hymenopteran endoparasitoids, one hymenopteran exoparasitoid, one hymenopteran hyperparasitoid, one dipteran predator, four coleopteran predator ladybirds, six herbivorous pest insects of lepidoptera, diptera, homoptera, and coleoptera, respectively. There existed significant correlations between the susceptibility to methamidophos and the k_i values of AChE to methamidophos, dichlorvos, and carbofuran and between the k_i and V_max values of AChE among 18 species of insects. The six herbivorous pests and four ladybirds showed significantly low k_i and V_max values of AChE compared to the seven parasitoids and predator Epistrophe balteate. It was difficult to correlate the susceptibility to methamidophos or the k_i values with the K_m values of AChE, or with the activity of CarE and GST. The activities of CarE and GST varied depending on the different insect species. Significant synergisms of piperonyl butoxide (PB), triphenyl phosphate (TPP), and diethyl maleate (DEM) with methamidophos were observed in 14 pest insects and their natural enemies. Synergisms of PB were found to be the greatest. Reduced k_i values suggested that insensitive AChE might play a critical role in the tolerance to methamidophos in the 18 insect species. The detoxification enzymes, mixed-function oxidase (MFO), CarE, and GST, were believed to be involved in the tolerance to methamidophos. MFO might play the most important role, and CarE or GST might be important in the tolerance in some insect species. Different models of tolerance to methamidophos and enzymatic potential were existed in parasitoids, predators, and herbivores based on the different selection of insecticide pressure (either directly by exposing to the spray in the field, or indirectly by the insecticides penetrated into the body of host insects) as well as different ecological and biological habitats.