Fournier D


Full name : Fournier Didier

First name : Didier

Mail : Lab. d'Entomologie, Universite Paul Sabatier 118 route de Narbonne, 310162 Toulouse, France

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Country : France

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References (103)

Title : Biochemical Characterization of the Esterases Al and Bl Associated with Organophosphate Resistance in the Culex pipiens L. - Fournier_1987_Pestic.Biochem.Physiol_27_211
Author(s) : Fournier D , Bride JM , Mouches C , Raymond M , Magnin M , Berge JB , Pasteur N , Georghiou GP
Ref : Pestic Biochem Physiol , 27 :211 , 2015
Abstract : Two esterases, A1 and B1, displaying a high activity in organophosphate (OP) resistant Culex pipiens L. from southern France and in C. quinquefasciatus Say from California, respectively, have been analyzed. Both enzymes are shown to be soluble and to constitute a large proportion of the proteins (1-3% for esterase A1 and 6-12% for esterase B1). The size of native esterase A1 was estimated between 118 and 134 kDa, that of esterase B1 67 kDa. Upon SDS denaturation, esterase B1 leads a single polypeptide of 67 kDa which suggests that it is a monomeric protein; esterase A1 leads also a single polypeptide of 60 kDa suggesting a homodimeric structure of the protein. These observations are discussed with regards to esterase E4 of Myzus persicae Sultz.
ESTHER : Fournier_1987_Pestic.Biochem.Physiol_27_211
PubMedSearch : Fournier_1987_Pestic.Biochem.Physiol_27_211

Title : Correction: Evidence for inhibition of cholinesterases in insect and mammalian nervous systems by the insect repellent deet - Corbel_2012_BMC.Biol_10_86
Author(s) : Corbel V , Stankiewicz M , Pennetier C , Fournier D , Stojan J , Girard E , Dimitrov M , Molgo J , Hougard JM , Lapied B
Ref : BMC Biol , 10 :86 , 2012
Abstract : ABSTRACT After the publication of this work Corbel et al BMC Biol 2009 7:47 it was brought to our attention that there were insufficient details given on the final composition of the experimental and control samples used in some experiments in this study We would like to clarify that the statement made in the methods Final dilutions in physiological saline contained at most 0.1 DMSO and absolute ethanol applies to the electrophysiological experiments done on insect preparations but not to those on mammalian preparations Note that 0.1 DMSO has no effect on insect synaptic transmission We supply all previously missing details in this correction.
ESTHER : Corbel_2012_BMC.Biol_10_86
PubMedSearch : Corbel_2012_BMC.Biol_10_86
PubMedID: 23114027

Title : Accumulation of tetrahedral intermediates in cholinesterase catalysis: a secondary isotope effect study - Tormos_2010_J.Am.Chem.Soc_132_17751
Author(s) : Tormos JR , Wiley KL , Wang Y , Fournier D , Masson P , Nachon F , Quinn DM
Ref : Journal of the American Chemical Society , 132 :17751 , 2010
Abstract : In a previous communication, kinetic beta-deuterium secondary isotope effects were reported that support a mechanism for substrate-activated turnover of acetylthiocholine by human butyrylcholinesterase (BuChE) wherein the accumulating reactant state is a tetrahedral intermediate ( Tormos , J. R. ; et al. J. Am. Chem. Soc. 2005 , 127 , 14538 - 14539 ). In this contribution additional isotope effect experiments are described with acetyl-labeled acetylthiocholines (CL(3)COSCH(2)CH(2)N(+)Me(3); L = H or D) that also support accumulation of the tetrahedral intermediate in Drosophila melanogaster acetylcholinesterase (DmAChE) catalysis. In contrast to the aforementioned BuChE-catalyzed reaction, for this reaction the dependence of initial rates on substrate concentration is marked by pronounced substrate inhibition at high substrate concentrations. Moreover, kinetic beta-deuterium secondary isotope effects for turnover of acetylthiocholine depended on substrate concentration, and gave the following: (D3)k(cat)/K(m) = 0.95 +/- 0.03, (D3)k(cat) = 1.12 +/- 0.02 and (D3)betak(cat) = 0.97 +/- 0.04. The inverse isotope effect on k(cat)/K(m) is consistent with conversion of the sp(2)-hybridized substrate carbonyl in the E + A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis, whereas the markedly normal isotope effect on k(cat) is consistent with hybridization change from sp(3) toward sp(2) as the reactant state for deacylation is converted into the subsequent transition state. Transition states for Drosophila melanogaster AChE-catalyzed hydrolysis of acetylthiocholine were further characterized by measuring solvent isotope effects and determining proton inventories. These experiments indicated that the transition state for rate-determining decomposition of the tetrahedral intermediate is stabilized by multiple protonic interactions. Finally, a simple model is proposed for the contribution that tetrahedral intermediate stabilization provides to the catalytic power of acetylcholinesterase.
ESTHER : Tormos_2010_J.Am.Chem.Soc_132_17751
PubMedSearch : Tormos_2010_J.Am.Chem.Soc_132_17751
PubMedID: 21105647

Title : Biochemical determination of acetylcholinesterase genotypes conferring resistance to the organophosphate insecticide chlorpyriphos in field populations of Bemisia tabaci from Benin, West Africa - Houndete_2010_Pestic.Biochem.Physiol_98_115
Author(s) : Houndete TA , Fournier D , Ketoh GK , Glitho IA , Nauen R , Martin T
Ref : Pesticide Biochemistry and Physiology , 98 :115 , 2010
Abstract : Resistance to chlorpyriphos insecticide in Bemisia tabaci from a field population collected in Benin, West Africa was suggested with bioassay showing the presence of two sub-populations. Patterns of acetylcholinesterase (AChE) inhibition by the organophosphate chlorpyriphos-oxon were analyzed to estimate the number of possible genotypes with different sensitivity expected in three B. tabaci field populations collected in Benin. The analysis of inhibition patterns in these populations compared with four laboratory strains of B. tabaci using chlorpyriphos-oxon allowed the differentiation of three possible genotypes. In the reference strain SUD-S we detected two different acetylcholinesterases with different sensitivity to chlorpyriphos oxon suggesting the presence of two genes ace 1 and ace 2. The proportion of the insensitive enzyme (ace 2) was estimated to be 31%. In field populations we can detect two alleles at the same gene locus ace 1: one susceptible ace1S and one resistant ace1R. Both strains called Arizona University and Mexico-S2 have lost sensitive ace1S but the field populations from Benin clearly contained at least three genotypes confirming heterogeneous populations not completely resistant..
ESTHER : Houndete_2010_Pestic.Biochem.Physiol_98_115
PubMedSearch : Houndete_2010_Pestic.Biochem.Physiol_98_115

Title : Evidence for inhibition of cholinesterases in insect and mammalian nervous systems by the insect repellent deet - Corbel_2009_BMC.Biol_7_47
Author(s) : Corbel V , Stankiewicz M , Pennetier C , Fournier D , Stojan J , Girard E , Dimitrov M , Molgo J , Hougard JM , Lapied B
Ref : BMC Biol , 7 :47 , 2009
Abstract : BACKGROUND: N,N-Diethyl-3-methylbenzamide (deet) remains the gold standard for insect repellents. About 200 million people use it every year and over 8 billion doses have been applied over the past 50 years. Despite the widespread and increased interest in the use of deet in public health programmes, controversies remain concerning both the identification of its target sites at the olfactory system and its mechanism of toxicity in insects, mammals and humans. Here, we investigated the molecular target site for deet and the consequences of its interactions with carbamate insecticides on the cholinergic system. RESULTS: By using toxicological, biochemical and electrophysiological techniques, we show that deet is not simply a behaviour-modifying chemical but that it also inhibits cholinesterase activity, in both insect and mammalian neuronal preparations. Deet is commonly used in combination with insecticides and we show that deet has the capacity to strengthen the toxicity of carbamates, a class of insecticides known to block acetylcholinesterase. CONCLUSION: These findings question the safety of deet, particularly in combination with other chemicals, and they highlight the importance of a multidisciplinary approach to the development of safer insect repellents for use in public health.
ESTHER : Corbel_2009_BMC.Biol_7_47
PubMedSearch : Corbel_2009_BMC.Biol_7_47
PubMedID: 19656357

Title : Kinetic insight into the mechanism of cholinesterasterase inhibition by aflatoxin B1 to develop biosensors - Hansmann_2009_Biosens.Bioelectron_24_2119
Author(s) : Hansmann T , Sanson B , Stojan J , Weik M , Marty JL , Fournier D
Ref : Biosensors & Bioelectronics , 24 :2119 , 2009
Abstract : In this paper, the inhibition effect of aflatoxin B1 on different species of cholinesterases was investigated to unravel action mechanism. The inhibition curves of several cholinesterase mutants (obtained by spectrophotometric measurements of enzyme activity, pS curves) were analyzed. They showed that this toxin reversibly inhibits cholinesterases by binding to a peripheral site located at the entrance of the active site gorge without entering inside the site. Electric eel enzyme revealed the highest inhibition extent with a binding constant estimated to 0.35 microM. This binding prevents the entrance of substrate en route to the catalytic site and also decreases chemical steps of the reaction at the catalytic site: acetylation is reduced to the half and deacetylation is reduced to the third. Electric eel acetylcholinesterase was used to settle an amperometric biosensor. The best detection was obtained by using 0.3 mU enzyme on the electrode and 0.5mM ATCh in the solution. The limit of detection was 3 microM corresponding to 20% inhibition.
ESTHER : Hansmann_2009_Biosens.Bioelectron_24_2119
PubMedSearch : Hansmann_2009_Biosens.Bioelectron_24_2119
PubMedID: 19109006

Title : Automated resolution of dichlorvos and methylparaoxon pesticide mixtures employing a Flow Injection system with an inhibition electronic tongue - Valdes-Ramirez_2009_Biosens.Bioelectron_24_1103
Author(s) : Valdes-Ramirez G , Gutierrez M , Del Valle M , Ramirez-Silva MT , Fournier D , Marty JL
Ref : Biosensors & Bioelectronics , 24 :1103 , 2009
Abstract : An amperometric biosensor array has been developed to resolve pesticide mixtures of dichlorvos and methylparaoxon. The biosensor array has been used in a Flow Injection system, in order to operate automatically the inhibition procedure. The sensors used were three screen-printed amperometric biosensors that incorporated three different acetylcholinesterase enzymes: the wild type from Electric eel and two different genetically modified enzymes, B1 and B394 mutants, from Drosophila melanogaster. The inhibition response triplet was modelled using an Artificial Neural Network which was trained with mixture solutions that contain dichlorvos from 10(-4) to 0.1 microM and methylparaoxon from 0.001 to 2.5 microM. This system can be considered an inhibition electronic tongue.
ESTHER : Valdes-Ramirez_2009_Biosens.Bioelectron_24_1103
PubMedSearch : Valdes-Ramirez_2009_Biosens.Bioelectron_24_1103
PubMedID: 18644713

Title : Phosphotriesterase: a complementary tool for the selective detection of two organophosphate insecticides: chlorpyrifos and chlorfenvinfos - Istamboulie_2009_Talanta_77_1627
Author(s) : Istamboulie G , Fournier D , Marty JL , Noguer T
Ref : Talanta , 77 :1627 , 2009
Abstract : This work shows the possibility of combining the high sensitivity of genetically-modified Drosophila melanogaster acetylcholinesterase (B394) with the ability of phosphotriesterase (PTE) to hydrolyse organophosphate compounds, in the aim of developing a biosensor selective to two insecticides of interest: chlorpyrifos and chlorfenvinfos. The studies clearly demonstrate that chlorfenvinfos is a substrate that acts as competitive inhibitor of PTE, therefore preventing the efficient hydrolysis of other pesticides, including chlorpyrifos. A bi-enzymatic sensor was designed by immobilizing both B394 and PTE in a polyvinylalcohol matrix. The sensor was shown to be able to discriminate between chlorpyrifos and chlorfenvinfos inhibitions.
ESTHER : Istamboulie_2009_Talanta_77_1627
PubMedSearch : Istamboulie_2009_Talanta_77_1627
PubMedID: 19159775

Title : Evidence for subdomain flexibility in Drosophila melanogaster acetylcholinesterase - Stojan_2008_Biochemistry_47_5599
Author(s) : Stojan J , Ladurantie C , Siadat OR , Paquereau L , Fournier D
Ref : Biochemistry , 47 :5599 , 2008
Abstract : The catalytic domain of the acetylcholinesterases is composed of a single polypeptide chain, the folding of which determines two subdomains. We have linked these two subdomains by mutating two residues, I327 and D375, to cysteines, to form a disulfide bridge. As a consequence, the hydrodynamic radius of the protein was reduced, suggesting that there is some flexibility in the subdomain connection. In addition to the smaller size, the mutated protein is more stable than the wild-type protein. Therefore, the flexibility between the two domains is a weak point in terms of protein stability. As expected from the location of the disulfide bond at the rim of the active site, the kinetic studies show that it affects interactions with peripheral ligands and the entrance of some of the bulkier substrates, like o-nitrophenyl acetate. In addition, the mutations affect the catalytic step for o-nitrophenyl acetate and phosphorylation by organophosphates, suggesting that this movement between the two subdomains is connected with the cooperativity between the peripheral and catalytic sites.
ESTHER : Stojan_2008_Biochemistry_47_5599
PubMedSearch : Stojan_2008_Biochemistry_47_5599
PubMedID: 18439026

Title : Insights into substrate and product traffic in the Drosophila melanogaster acetylcholinesterase active site gorge by enlarging a back channel - Nachon_2008_FEBS.J_275_2659
Author(s) : Nachon F , Stojan J , Fournier D
Ref : Febs J , 275 :2659 , 2008
Abstract : To test a product exit differing from the substrate entrance in the active site of acetylcholinesterase (EC, we enlarged a channel located at the bottom of the active site gorge in the Drosophila enzyme. Mutation of Trp83 to Ala or Glu widens the channel from 5 A to 9 A. The kinetics of substrate hydrolysis and the effect of ligands that close the main entrance suggest that the mutations facilitate both product exit and substrate entrance. Thus, in the wild-type, the channel is so narrow that the 'back door' is used by at most 5% of the traffic, with the majority of traffic passing through the main entrance. In mutants Trp83Ala and Trp83Glu, ligands that close the main entrance do not inhibit substrate hydrolysis because the traffic can pass via an alternative route, presumably the enlarged back channel.
ESTHER : Nachon_2008_FEBS.J_275_2659
PubMedSearch : Nachon_2008_FEBS.J_275_2659
PubMedID: 18422651

Title : Hysteresis of insect acetylcholinesterase - Badiou_2008_Chem.Biol.Interact_175_410
Author(s) : Badiou A , Froment MT , Fournier D , Masson P , Belzunces LP
Ref : Chemico-Biological Interactions , 175 :410 , 2008
Abstract : Pre-steady-state catalytic properties of insect acetylcholinesterase (AChE, EC were studied with the neutral substrate N-methylindoxylacetate. Kinetics of soluble Apis mellifera and Drosophila melanogaster AChE forms showed lags (v(i)=0) before reaching the steady-state. Results were interpreted in terms of slow equilibrium between two conformational states E and E' of insect AChE. Hysteresis of insect AChE has been pointed out for the first time. The hysteretic behaviour was found to depend on the NMIA concentration and the nature of the enzyme. The maximum induction times (tau(max)) to reach the steady-state were 800 and 1000s with soluble AChE from A. mellifera and D.melanogaster, respectively. The orders of magnitude of the tau(max) were high and similar to human AChE and BuChE.
ESTHER : Badiou_2008_Chem.Biol.Interact_175_410
PubMedSearch : Badiou_2008_Chem.Biol.Interact_175_410
PubMedID: 18602377

Title : Sensitive amperometric biosensor for dichlorovos quantification: Application to detection of residues on apple skin - Valdes-Ramirez_2008_Talanta_74_741
Author(s) : Valdes-Ramirez G , Fournier D , Ramirez-Silva MT , Marty JL
Ref : Talanta , 74 :741 , 2008
Abstract : This paper presents the construction of an amperometric biosensor for the highly sensitive detection of the organophosphorus insecticide dichlorvos, based on the inhibition of acetylcholinesterase (AChE). The sensitivity of three AChEs from different sources were tested and compared: AChEs from Electric eel (Ee) and genetically engineered (B394) and wild type (B1) from Drosophila melanogaster (Dm). The enzymes were immobilized by entrapment in a photocrosslinkable PVA-SbQ polymer on a screen printed graphite electrode. The enzyme activity was estimated amperometrically at 100mV versus Ag/AgCl by measuring the thiocholine produced by the enzymatic hydrolysis of the acetylthiocholine substrate using cobalt phthalocyanine as electron mediator. The pesticide was measured in the presence of 5% acetonitrile without loss of enzyme activity. The best sensitivity was achieved with the Dm mutant B394 with a detection limit of 7x10(-11)M as compared to 1x10(-8)M with the B1 Dm and 6x10(-7)M with the Ee. The B394 biosensor was used to quantify dichlorvos in a sample of skin apple after extraction with acetonitrile.
ESTHER : Valdes-Ramirez_2008_Talanta_74_741
PubMedSearch : Valdes-Ramirez_2008_Talanta_74_741
PubMedID: 18371703

Title : Shoot-and-Trap: use of specific x-ray damage to study structural protein dynamics by temperature-controlled cryo-crystallography - Colletier_2008_Proc.Natl.Acad.Sci.U.S.A_105_11742
Author(s) : Colletier JP , Bourgeois D , Sanson B , Fournier D , Sussman JL , Silman I , Weik M
Ref : Proc Natl Acad Sci U S A , 105 :11742 , 2008
Abstract : Although x-ray crystallography is the most widely used method for macromolecular structure determination, it does not provide dynamical information, and either experimental tricks or complementary experiments must be used to overcome the inherently static nature of crystallographic structures. Here we used specific x-ray damage during temperature-controlled crystallographic experiments at a third-generation synchrotron source to trigger and monitor (Shoot-and-Trap) structural changes putatively involved in an enzymatic reaction. In particular, a nonhydrolyzable substrate analogue of acetylcholinesterase, the "off-switch" at cholinergic synapses, was radiocleaved within the buried enzymatic active site. Subsequent product clearance, observed at 150 K but not at 100 K, indicated exit from the active site possibly via a "backdoor." The simple strategy described here is, in principle, applicable to any enzyme whose structure in complex with a substrate analogue is available and, therefore, could serve as a standard procedure in kinetic crystallography studies.
ESTHER : Colletier_2008_Proc.Natl.Acad.Sci.U.S.A_105_11742
PubMedSearch : Colletier_2008_Proc.Natl.Acad.Sci.U.S.A_105_11742
PubMedID: 18701720
Gene_locus related to this paper: torca-ACHE

Title : Does mercury interact with the inhibitory effect of dichlorvos on Palaemon serratus (Crustacea: Decapoda) cholinesterase? - Frasco_2008_Sci.Total.Environ_404_88
Author(s) : Frasco MF , Fournier D , Carvalho F , Guilhermino L
Ref : Sci Total Environ , 404 :88 , 2008
Abstract : Mercury is one of the most hazardous metals that may contaminate estuarine ecosystems and induce toxic effects on wildlife organisms. It has been suggested that impairment of cholinesterase (ChE) activity may be involved in the resulting mercury toxicity. Following Palaemon serratus exposure to mercury chloride (HgCl2), no effect on ChE activity was observed whatever the concentration used (to 37.5 microM) or the time of exposure (to 7 days). By contrast, following 24 h exposure to dichlorvos, an organophosphate insecticide with a well-characterised anti-ChE action, decrease of ChE activity was observed until 30 to 40% basal activity, which seems to be the minimum activity required for prawn survival. In addition, HgCl2 does not affect dichlorvos toxicity and treatments with a mixture of both compounds can be interpreted as the sum of the two independent toxicities. Therefore, mercury and insecticide toxicities are independent and ChE activity from P. serratus eyes seems to be a reliable and sensitive biomarker for organophosphate insecticides even when organisms are simultaneously exposed to mercury.
ESTHER : Frasco_2008_Sci.Total.Environ_404_88
PubMedSearch : Frasco_2008_Sci.Total.Environ_404_88
PubMedID: 18639316

Title : Probing gorge dimensions of cholinesterases by freeze-frame click chemistry - Radic_2008_Chem.Biol.Interact_175_161
Author(s) : Radic Z , Manetsch R , Fournier D , Sharpless KB , Taylor P
Ref : Chemico-Biological Interactions , 175 :161 , 2008
Abstract : Freeze-frame click chemistry is a proven approach for design in situ of high affinity ligands from bioorthogonal, reactive building blocks and macromolecular template targets. We recently described in situ design of femtomolar reversible inhibitors of fish and mammalian acetylcholinesterases (EC; AChEs) using several different libraries of acetylene and azide building blocks. Active center gorge geometries of those AChEs are rather similar and identical triazole inhibitors were detected in situ when incubating the same building block libraries in different AChEs. Drosophila melanogaster AChE crystal structure and other insect AChE homology models differ more in their overall 3D structure than other members of the cholinesterase family. The portion of the gorge proximal to the catalytic triad and choline binding site has a approximately 50% reduction in volume, and the gorge entrance at the peripheral anionic site (PAS) is more constricted than in the fish and mammalian AChEs. In this communication we describe rationale for using purified recombinant Drosophila AChE as a template for in situ reaction of tacrine and propidium based libraries of acetylene and azide building blocks. The structures of resulting triazole inhibitors synthesized in situ are expected to differ appreciably from the fish and mammalian AChEs. While the latter AChEs exclusively promote synthesis of syn-substituted triazoles, the best Drosophila AChE triazole inhibitors were always anti-substituted. The anti-regioisomer triazoles were by about one order of magnitude better inhibitors of Drosophila than mammalian and fish AChEs. Moreover, the preferred site of acetylene+azide reaction in insect AChE and the resulting triazole ring formation shifts from near the base of the gorge to closer to its rim due to substantial differences of the gorge geometry in Drosophila AChE. Thus, in addition to synthesizing high affinity, lead inhibitors in situ, freeze-frame, click chemistry has capacity to generate species-specific AChE ligands that conform to the determinants in the gorge.
ESTHER : Radic_2008_Chem.Biol.Interact_175_161
PubMedSearch : Radic_2008_Chem.Biol.Interact_175_161
PubMedID: 18555981

Title : Mechanisms of cholinesterase inhibition by inorganic mercury - Frasco_2007_FEBS.J_274_1849
Author(s) : Frasco MF , Colletier JP , Weik M , Carvalho F , Guilhermino L , Stojan J , Fournier D
Ref : Febs J , 274 :1849 , 2007
Abstract : The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl2) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl2 was investigated by kinetic studies, X-ray crystallography, and dynamic light scattering. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo-first-order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electricus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury-induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment.
ESTHER : Frasco_2007_FEBS.J_274_1849
PubMedSearch : Frasco_2007_FEBS.J_274_1849
PubMedID: 17355286
Gene_locus related to this paper: human-BCHE

Title : [Liposomes: support for the formation of stable capsules made of reticulated polyelectrolytes or silicum] - Germain_2007_Ann.Pharm.Fr_65_134
Author(s) : Germain M , Paquereau L , Winterhalter M , Hochepied JF , Fournier D
Ref : Ann Pharm Fr , 65 :134 , 2007
Abstract : Uses of enzymes for therapeutic purpose or for biosensing require a well-controlled nanoenvironnement to avoid degradation by proteolytic agents, pH variations or dilution effects. A solution is encapsulation under undenaturating conditions into a nanometer sized and stable capsule. The nanometer scall decreases recognition by the reticulo-endothelial system recognition and subsequent immune reaction. Liposomes are the method of choice since they allow protein encapsulation under mild conditions. However they lack in stability. In contrast, other type of capsules exhibit strong stability but with conditions required for formation that are incompatible with enzyme integrity. Here we combine different capsule formation techniques and use liposomes as templates for further stabilization. Here we demonstrate two types of multicomposite capsules. The first type is to coat the liposome surface with polyelectrolytes followed by secondary covalent crosslinking of the polyelectrolytes multilayer. In the second type of capsules we used silica to build an inorganic shell around liposome. Both techniques allow the formation of detergent stable nanocapsules which exhibits properties protective against acetylcholinesterase protein degradation, an enzyme of much interest for pesticide detection.
ESTHER : Germain_2007_Ann.Pharm.Fr_65_134
PubMedSearch : Germain_2007_Ann.Pharm.Fr_65_134
PubMedID: 17404547

Title : Inhibition and protection of cholinesterases by methanol and ethanol - Fekonja_2007_J.Enzyme.Inhib.Med.Chem_22_407
Author(s) : Fekonja O , Zorec-Karlovsek M , El Kharbili M , Fournier D , Stojan J
Ref : J Enzyme Inhib Med Chem , 22 :407 , 2007
Abstract : The cholinesterases have been investigated in terms of the effects of methanol and ethanol on substrate and carbamate turnover, and on their phosphorylation. It was found: 1) that at low substrate concentrations the two alcohols inhibit all three tested cholinesterases and that the optimum activities are shifted towards higher substrate concentrations, but with a weak effect on horse butyrylcholinesterase; 2) that methanol slows down carbamoylation by eserine and does not influence decarbamoylation of vertebrate and insect acetylcholinesterase and 3) that ethanol decreases the rate of phosphorylation of vertebrate acetylcholinesterase by DFP. Our results are in line with the so-called 'approach-and-exit' hypothesis. By hindering the approach of substrate and the exit of products, methanol and ethanol decrease cholinesterase activity at low substrate concentrations and allow for the substrate inhibition only at higher substrate concentrations. Both effects appears to be a consequence of the lower ability of substrate to substitute alcohol rather than water. It also seems that during substrate turnover in the presence of alcohol the transacetylation is negligible.
ESTHER : Fekonja_2007_J.Enzyme.Inhib.Med.Chem_22_407
PubMedSearch : Fekonja_2007_J.Enzyme.Inhib.Med.Chem_22_407
PubMedID: 17847706

Title : The effect of engineered disulfide bonds on the stability of Drosophila melanogaster acetylcholinesterase - Siadat_2006_BMC.Biochem_7_12
Author(s) : Siadat OR , Lougarre A , Lamouroux L , Ladurantie C , Fournier D
Ref : BMC Biochem , 7 :12 , 2006
Abstract : BACKGROUND: Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use in biosensors for detection of these insecticides. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, its stability has to be improved for extensive utilization.
RESULTS: To create a disulfide bond that could increase the stability of the Drosophila melanogaster acetylcholinesterase, we selected seven positions taking into account first the distance between Cbeta of two residues, in which newly introduced cysteines will form the new disulfide bond and second the conservation of the residues in the cholinesterase family. Most disulfide bonds tested did not increase and even decreased the stability of the protein. However, one engineered disulfide bridge, I327C/D375C showed significant stability increase toward denaturation by temperature (170 fold at 50 degrees C), urea, organic solvent and provided resistance to protease degradation. The new disulfide bridge links the N-terminal domain (first 356 aa) to the C-terminal domain. The quantities produced by this mutant were the same as in wild-type flies. CONCLUSION: Addition of a disulfide bridge may either stabilize or unstabilize proteins. One bond out of the 7 tested provided significant stabilisation.
ESTHER : Siadat_2006_BMC.Biochem_7_12
PubMedSearch : Siadat_2006_BMC.Biochem_7_12
PubMedID: 16686937

Title : Cholinesterase from the common prawn (Palaemon serratus) eyes: catalytic properties and sensitivity to organophosphate and carbamate compounds - Frasco_2006_Aquat.Toxicol_77_412
Author(s) : Frasco MF , Fournier D , Carvalho F , Guilhermino L
Ref : Aquat Toxicol , 77 :412 , 2006
Abstract : The main purpose of this study was to describe the kinetic properties of the cholinesterase (ChE) enzyme present in the eyes of the prawn Palaemon serratus, an abundant, ecological and commercially relevant species of European coastal environments. The obtained results suggest that the studied enzyme is a ChE and not a non-specific esterase, due to its apparent affinity for choline esters and the high sensitivity to eserine sulphate. This ChE displays a distinct preference for the substrate acetylthiocholine, showing a triphasic behaviour, with activation at low concentrations and inhibition by excess of substrate. Moreover, irreversible ChE inhibition by several organophosphate and carbamate compounds was characterized. All the irreversible inhibitions were homogeneous following a second-order rate reaction. The bimolecular rate constant (k(i)) values of ChE inhibition by the tested pesticides were also estimated and compared with available data from other invertebrate and vertebrate species. In conclusion, the results of the present study showed that prawn eyes possess only one ChE with typical properties of acetylcholinesterase, which is highly sensitive to the tested anti-cholinesterase compounds.
ESTHER : Frasco_2006_Aquat.Toxicol_77_412
PubMedSearch : Frasco_2006_Aquat.Toxicol_77_412
PubMedID: 16497396

Title : Structural insights into substrate traffic and inhibition in acetylcholinesterase - Colletier_2006_EMBO.J_25_2746
Author(s) : Colletier JP , Fournier D , Greenblatt HM , Stojan J , Sussman JL , Zaccai G , Silman I , Weik M
Ref : EMBO Journal , 25 :2746 , 2006
Abstract : Acetylcholinesterase (AChE) terminates nerve-impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active-site gorge.
ESTHER : Colletier_2006_EMBO.J_25_2746
PubMedSearch : Colletier_2006_EMBO.J_25_2746
PubMedID: 16763558
Gene_locus related to this paper: torca-ACHE

Title : An electrochemical bioassay for dichlorvos analysis in durum wheat samples - Del Carlo_2006_J.Food.Prot_69_1406
Author(s) : Del Carlo M , Pepe A , De Gregorio M , Mascini M , Marty JL , Fournier D , Visconti A , Compagnone D
Ref : J Food Prot , 69 :1406 , 2006
Abstract : The use of an acetylcholinesterase inhibition assay for the detection of dichlorvos in durum wheat samples by a simplified extraction procedure is reported. After an incubation step, the residual activity was determined with an amperometric biosensor using a portable potentiostat. The use of electric eel and recombinant acetylcholinesterase was compared. The effect of the matrix extract was evaluated by using various sample:solvent ratios, 1:2.5, 1:5, 1:10, and 1:20. The optimal extraction ratio, considering the electrochemical interferences and the effect on enzyme activity and bioavailability of the pesticide, was 1:10. Calibrations were performed in buffer and durum wheat extract. The calculated detection limits in buffer solution were 10 ng/ ml and 0.045 ng/ml for electric eel and recombinant acetylcholinesterase, respectively, whereas operating in the matrix extract they increased up to 45 ng/ml and 0.07 ng/ml, corresponding to 0.45 mg/kg (extraction ratio 1:10) and 0.07 mg/kg in samples. These characteristics allowed the detection of contaminated samples at the maximum residue limit, which is 2 mg/kg and well below. Fortified samples of durum wheat were obtained with both dichlorvos and the commercial product Didivane, which contains dichlorvos as an active molecule. At all the tested levels, the occurrence of contaminant was detected with an average recovery of 75%. The total assay time, including the extraction step, was 30 min. Because several extractions as well as most of the assay steps can be run simultaneously, the throughput for one operator is 12 determinations per hour.
ESTHER : Del Carlo_2006_J.Food.Prot_69_1406
PubMedSearch : Del Carlo_2006_J.Food.Prot_69_1406
PubMedID: 16786864

Title : Genetically engineered acetylcholinesterase-based biosensor for attomolar detection of dichlorvos - Sotiropoulou_2005_Biosens.Bioelectron_20_2347
Author(s) : Sotiropoulou S , Fournier D , Chaniotakis NA
Ref : Biosensors & Bioelectronics , 20 :2347 , 2005
Abstract : The design of a biosensor for the detection of dichlorvos at attomolar levels is described based on a highly sensitive double mutant (E69Y Y71D) of the Drosophila melanogaster acetylcholinesterase (Dm. AChE). This enzyme has a k(i) for dichlorvos equal to 487 microM(-1)min(-1), which is 300 and 20,000 times higher than that of the wild type Dm. AChE and the Electrophorus electricus AChE (E.el. AChE), respectively. The enzyme is immobilized into microporous-activated conductive carbon, and is used as such for the development of an inhibitor electrochemical biosensor. This E69Y Y71D mutant enables the decrease in the detection limit of the biosensor down to 10(-17) M, which is five orders of magnitude lower compared to the Electropharus electricus-based biosensor and eight orders of magnitude lower than the biosensors described so far.
ESTHER : Sotiropoulou_2005_Biosens.Bioelectron_20_2347
PubMedSearch : Sotiropoulou_2005_Biosens.Bioelectron_20_2347
PubMedID: 15797338

Title : Mutations of acetylcholinesterase which confer insecticide resistance in insect populations - Fournier_2005_Chem.Biol.Interact_157-158_257
Author(s) : Fournier D
Ref : Chemico-Biological Interactions , 157-158 :257 , 2005
Abstract : Resistance-modified acetylcholinesterases have been described in many insect species and sequencing of their genes has allowed several point mutations to be described. Most mutations line the active site gorge. Each mutation provides a specific resistance pattern: it confers resistance to one insecticide but may increase sensitivity to another. Most mutations alter hydrolysis of the substrate by decreasing the rate of enzyme deacetylation and by diminishing the stability of the enzyme. Mutations are often found in combination in the same protein. This has several consequences: it increases the level of resistance, it enlarges the spectrum of resistance and it may restore the catalytic efficiency of the enzyme. Natural populations are heterogeneous, composed of a mixture of different alleles.
ESTHER : Fournier_2005_Chem.Biol.Interact_157-158_257
PubMedSearch : Fournier_2005_Chem.Biol.Interact_157-158_257
PubMedID: 16274684

Title : Do metals inhibit acetylcholinesterase (AChE)? Implementation of assay conditions for the use of AChE activity as a biomarker of metal toxicity - Frasco_2005_Biomarkers_10_360
Author(s) : Frasco MF , Fournier D , Carvalho F , Guilhermino L
Ref : Biomarkers , 10 :360 , 2005
Abstract : The enzymatic activity of acetylcholinesterase (AChE) has been shown to be altered by environmental contaminants such as metals. However, the available literature illustrates a background of contradictory results regarding these effects. Therefore, the main purpose of this study was to investigate the potential of five metal ions (nickel, copper, zinc, cadmium and mercury) to inhibit AChE activity in vitro. First, to accomplish this objective, the possible interference of metals as test toxicants in the Ellman's assay, which is widely used to assess AChE activity, was studied. The potential influence of two different reaction buffers (phosphate and Tris) was also determined. The results suggest that the selected metals react with the products of this photometric technique. It is impossible to ascertain the artefactual contribution of the interaction of the metals with the technique when measuring AChE inhibition. This constitutes a major obstacle in obtaining accurate data. The presence of phosphate ions also makes enzymatic inhibition difficult to analyse. Attending to this evidence, an assay using the substrate o-nitrophenyl acetate and Tris buffer was used to investigate the effects of metals on AChE activity. O-nitrophenyl acetate is also a substrate for esterases other than cholinesterases. It is therefore only possible to use it for the measurement of cholinesterase activity with purified enzymes or after a previous verification of the absence of other esterases in the sample tissue. Under these conditions, the results indicate that with the exception of nickel, all tested metals significantly inhibit AChE activity.
ESTHER : Frasco_2005_Biomarkers_10_360
PubMedSearch : Frasco_2005_Biomarkers_10_360
PubMedID: 16243721

Title : Effects of soman inhibition and of structural differences on cholinesterase molecular dynamics: a neutron scattering study - Gabel_2005_Biophys.J_89_3303
Author(s) : Gabel F , Weik M , Masson P , Renault F , Fournier D , Brochier L , Doctor BP , Saxena A , Silman I , Zaccai G
Ref : Biophysical Journal , 89 :3303 , 2005
Abstract : Incoherent elastic neutron scattering experiments on members of the cholinesterase family were carried out to investigate how molecular dynamics is affected by covalent inhibitor binding and by differences in primary and quaternary structure. Tetrameric native and soman-inhibited human butyrylcholinesterase (HuBChE) as well as native dimeric Drosophila melanogaster acetylcholinesterase (DmAChE) hydrated protein powders were examined. Atomic mean-square displacements (MSDs) were found to be identical for native HuBChE and for DmAChE in the whole temperature range examined, leading to the conclusion that differences in activity and substrate specificity are not reflected by a global modification of subnanosecond molecular dynamics. MSDs of native and soman-inhibited HuBChE were identical below the thermal denaturation temperature of the native enzyme, indicating a common mean free-energy surface. Denaturation of the native enzyme is reflected by a relative increase of MSDs consistent with entropic stabilization of the unfolded state. The results suggest that the stabilization of HuBChE phosphorylated by soman is due to an increase in free energy of the unfolded state due to a decrease in entropy.
ESTHER : Gabel_2005_Biophys.J_89_3303
PubMedSearch : Gabel_2005_Biophys.J_89_3303
PubMedID: 16100272

Title : Fluorescence detection of enzymatic activity within a liposome based nano-biosensor - Vamvakaki_2005_Biosens.Bioelectron_21_384
Author(s) : Vamvakaki V , Fournier D , Chaniotakis NA
Ref : Biosensors & Bioelectronics , 21 :384 , 2005
Abstract : The encapsulation of enzymes in microenvironments and especially in liposomes, has proven to greatly improve enzyme stabilization against unfolding, denaturation and dilution effects. Combining this stabilization effect, with the fact that liposomes are optically translucent, we have designed nano-sized spherical biosensors. In this work liposome-based biosensors are prepared by encapsulating the enzyme acetylcholinesterase (AChE) in L-a phosphatidylcholine liposomes resulting in spherical optical biosensors with an average diameter of 300+/-4 nm. Porins are embedded into the lipid membrane, allowing for the free substrate transport, but not that of the enzyme due to size limitations. The enzyme activity within the liposome is monitored using pyranine, a fluorescent pH indicator. The response of the liposome biosensor to the substrate acetylthiocholine chloride is relatively fast and reproducible, while the system is stable as has been shown by immobilization within sol-gel.
ESTHER : Vamvakaki_2005_Biosens.Bioelectron_21_384
PubMedSearch : Vamvakaki_2005_Biosens.Bioelectron_21_384
PubMedID: 16023967

Title : Mutations of acetylcholinesterase which confer insecticide resistance in Drosophila melanogaster populations - Menozzi_2004_BMC.Evol.Biol_4_4
Author(s) : Menozzi P , Shi MA , Lougarre A , Tang ZH , Fournier D
Ref : BMC Evol Biol , 4 :4 , 2004
Abstract : BACKGROUND: Organophosphate and carbamate insecticides irreversibly inhibit acetylcholinesterase causing death of insects. Resistance-modified acetylcholinesterases(AChEs) have been described in many insect species and sequencing of their genes allowed several point mutations to be described. However, their relative frequency and their cartography had not yet been addressed.
RESULTS: To analyze the most frequent mutations providing insecticide resistance in Drosophila melanogaster acetylcholinesterase, the Ace gene was cloned and sequenced in several strains harvested from different parts of the world. Sequence comparison revealed four widespread mutations, I161V, G265A, F330Y and G368A. We confirm here that mutations are found either isolated or in combination in the same protein and we show that most natural populations are heterogeneous, composed of a mixture of different alleles. In vitro expression of mutated proteins showed that combining mutations in the same protein has two consequences: it increases resistance level and provides a wide spectrum of resistance. CONCLUSION: The presence of several alleles in natural populations, offering various resistance to carbamate and organophosphate compounds will complicate the establishment of resistance management programs.
ESTHER : Menozzi_2004_BMC.Evol.Biol_4_4
PubMedSearch : Menozzi_2004_BMC.Evol.Biol_4_4
PubMedID: 15018651

Title : Encapsulation of enzymes in liposomes: high encapsulation efficiency and control of substrate permeability - Chaize_2004_Artif.Cells.Blood.Substit.Immobil.Biotechnol_32_67
Author(s) : Chaize B , Colletier JP , Winterhalter M , Fournier D
Ref : Artif Cells Blood Substit Immobil Biotechnol , 32 :67 , 2004
Abstract : Enzyme encapsulation into liposomes is a promising technique to stabilize and prevent them from denaturation and proteolysis. We demonstrate this using acetylcholinesterase which is the main target for pesticides. In order to achieve a reasonable encapsulation yield, we analyzed the parameters involved in each step of various encapsulation procedures. The only encapsulation method which did not denature the protein was the lipid film hydration technique, however the encapsulation efficiency was usually low. The efficiency could be increased up to more than 40% by induction of a specific interaction between the enzyme and the lipid surface. Once encapsulated, the enzyme encountered another problem: the permeability barrier of the lipid membrane drastically diminished the activity of the enzyme entrapped in the liposome by reducing the entrance rate of the substrate molecules and then reducing the substrate concentration inside the liposome. To solve this problem, we controlled the permeability of the liposome wall by reconstituting a porin from Escherichia coli. We succeeded to recover the full functionality of the enzyme, while retaining the protection against denaturation and proteolytic enzymes.
ESTHER : Chaize_2004_Artif.Cells.Blood.Substit.Immobil.Biotechnol_32_67
PubMedSearch : Chaize_2004_Artif.Cells.Blood.Substit.Immobil.Biotechnol_32_67
PubMedID: 15027802

Title : Acetylcholinesterase alterations reveal the fitness cost of mutations conferring insecticide resistance - Shi_2004_BMC.Evol.Biol_4_5
Author(s) : Shi MA , Lougarre A , Alies C , Fremaux I , Tang ZH , Stojan J , Fournier D
Ref : BMC Evol Biol , 4 :5 , 2004
Abstract : BACKGROUND: Insecticide resistance is now common in insects due to the frequent use of chemicals to control them, which provides a useful tool to study the adaptation of eukaryotic genome to new environments. Although numerous potential mutations may provide high level of resistance, only few alleles are found in insect natural populations. Then, we hypothesized that only alleles linked to the highest fitness in the absence of insecticide are selected.
RESULTS: To obtain information on the origin of the fitness of resistant alleles, we studied Drosophila melanogaster acetylcholinesterase, the target of organophosphate and carbamate insecticides. We produced in vitro 15 possible proteins resulting from the combination of the four most frequent mutations and we tested their catalytic activity and enzymatic stability. Mutations affected deacetylation of the enzyme, decreasing or increasing its catalytic efficiency and all mutations diminished the stability of the enzyme. Combination of mutations result to an additive alteration. CONCLUSION: Our findings suggest that the alteration of activity and stability of acetylcholinesterase are at the origin of the fitness cost associated with mutations providing resistance. Magnitude of the alterations was related to the allelic frequency in Drosophila populations suggesting that the fitness cost is the main driving force for the maintenance of resistant alleles in insecticide free conditions.
ESTHER : Shi_2004_BMC.Evol.Biol_4_5
PubMedSearch : Shi_2004_BMC.Evol.Biol_4_5
PubMedID: 15018650

Title : Molecular recognition of cocaine by acetylcholinesterases for affinity purification and bio-sensing - Knosche_2004_Biosens.Bioelectron_20_153
Author(s) : Knosche K , Halamek J , Makower A , Fournier D , Scheller FW
Ref : Biosensors & Bioelectronics , 20 :153 , 2004
Abstract : Cholinesterases can be used as sensitive biorecognition elements for widely used agricultural pesticides. This requires highly purified and inhibitor-free enzyme preparations. In the present work the cocaine derivative benzoylecgonine was for the first time used as the molecular recognition element for the purification of acetylcholinesterase from Electrophorus electricus by affinity chromatography. The preparation of enriched enzyme without the contamination by an inhibitor, which is traditionally used for eluting the "affinity" bound protein, was achieved. The specific activity was 2.2-fold increased to 3100 Umg(-1). The same cocaine derivative was immobilized on the surface of a piezoelectric crystal in order to analyze the binding of acetylcholinesterases from two different species, E. electricus and Drosophila melanogaster, to the immobilized inhibitor. Evaluation of the binding curves allowed the analysis of the binding kinetics. These experiments are fundamental for the development of a (competitive) biosensor for inhibitors of cholinesterase.
ESTHER : Knosche_2004_Biosens.Bioelectron_20_153
PubMedSearch : Knosche_2004_Biosens.Bioelectron_20_153
PubMedID: 15308216

Title : Poster (74) Structural studies on torpedo californica acetylcholinesterase in complex with a substrate analogue -
Author(s) : Colletier JP , Fournier D , Greenblatt HM , Sussman JL , Zaccai G , Silman I , Weik M
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :359 , 2004

Title : Inhibition of Drosophila melanogaster acetylcholinesterase by high concentrations of substrate - Stojan_2004_Eur.J.Biochem_271_1364
Author(s) : Stojan J , Brochier L , Alies C , Colletier JP , Fournier D
Ref : European Journal of Biochemistry , 271 :1364 , 2004
Abstract : Acetylcholine hydrolysis by acetylcholinesterase is inhibited at high substrate concentrations. To determine the residues involved in this phenomenon, we have mutated most of the residues lining the active-site gorge but mutating these did not completely eliminate hydrolysis. Thus, we analyzed the effect of a nonhydrolysable substrate analogue on substrate hydrolysis and on reactivation of an analogue of the acetylenzyme. Analyses of various models led us to propose the following sequence of events: the substrate initially binds at the rim of the active-site gorge and then slides down to the bottom of the gorge where it is hydrolyzed. Another substrate molecule can bind to the peripheral site: (a) when the choline is still inside the gorge - it will thereby hinder its exit; (b) after choline has dissociated but before deacetylation occurs - binding at the peripheral site increases deacetylation rate but (c) if a substrate molecule bound to the peripheral site slides down to the bottom of the active-site before the catalytic serine is deacetylated, its new position will prevent the approach of water, thus blocking deacetylation.
ESTHER : Stojan_2004_Eur.J.Biochem_271_1364
PubMedSearch : Stojan_2004_Eur.J.Biochem_271_1364
PubMedID: 15030487

Title : Sorting out molecules reacting with acetylcholinesterase by enzyme encapsulation in liposome - Chaize_2004_Biosens.Bioelectron_20_628
Author(s) : Chaize B , Fournier D
Ref : Biosensors & Bioelectronics , 20 :628 , 2004
Abstract : Enzymes are considered as providential molecules for biosensor design because of their sensitivity and the high specificity of the reactions they catalyse. However, their active sites often display low selectivity, a lot of molecules may enter and interfere with catalysis. These molecules may be either competitive inhibitors, activators or molecules which change the physico-chemical environment of the enzyme (pH, ionic strength). They produce the "matrix effect" that lowers the reliability of biosensors. We show here that encapsulation of enzymes in liposomes inserts a barrier between the enzyme and the external environment and protects the enzyme in a stable nano-environment for an optimal activity. This barrier sorts out the molecules that could react with the enzyme according to their hydrophobicity. Acetylcholinesterase is used to detect organophosphorous and carbamate insecticide residues but several molecules (reversible inhibitors, pH and ionic strength modifiers) generate matrix effects in free conditions. These perturbations were completely ineffective following enzyme encapsulation.
ESTHER : Chaize_2004_Biosens.Bioelectron_20_628
PubMedSearch : Chaize_2004_Biosens.Bioelectron_20_628
PubMedID: 15494248

Title : Rational polynomial equation as an unbiased approach for the kinetic studies of Drosophila melanogaster acetylcholinesterase reaction mechanism - Stojan_2004_Biochim.Biophys.Acta_1703_53
Author(s) : Stojan J , Golicnik M , Fournier D
Ref : Biochimica & Biophysica Acta , 1703 :53 , 2004
Abstract : The hydrolysis of substrates by cholinesterases does not follow the Michaelis-Menten reaction mechanism. The well-known inhibition by excess substrate is often accompanied by an unexpectedly high activity at low substrate concentrations. It appears that these peculiarities are the consequence of an unusual architecture of the active site, which conducts the substrate molecule over many stages before it is cleaved and released. Structural and kinetic data also suggest that two substrate molecules can attach at the same time to the free, as well as to the acetylated, enzyme. We present a procedure which provides an unbiased framework for mathematical modelling of such complex reaction mechanisms. It is based on regression analysis of a rational polynomial using classical initial rate data. The determination of polynomial degree reveals the number of independent parameters that can be evaluated from the available information. Once determined, these parameters can substantially facilitate the construction and evaluation of a kinetic model reflecting the expected molecular events in an enzymic reaction. We also present practical suggestions for testing the postulated kinetic model, using an original thermodynamic approach and an isolated effect in a specifically mutated enzyme.
ESTHER : Stojan_2004_Biochim.Biophys.Acta_1703_53
PubMedSearch : Stojan_2004_Biochim.Biophys.Acta_1703_53
PubMedID: 15588702

Title : The influence of solvent composition on global dynamics of human butyrylcholinesterase powders: a neutron-scattering study - Gabel_2004_Biophys.J_86_3152
Author(s) : Gabel F , Weik M , Doctor BP , Saxena A , Fournier D , Brochier L , Renault F , Masson P , Silman I , Zaccai G
Ref : Biophysical Journal , 86 :3152 , 2004
Abstract : A major result of incoherent elastic neutron-scattering experiments on protein powders is the strong dependence of the intramolecular dynamics on the sample environment. We performed a series of incoherent elastic neutron-scattering experiments on lyophilized human butyrylcholinesterase (HuBChE) powders under different conditions (solvent composition and hydration degree) in the temperature range from 20 to 285 K to elucidate the effect of the environment on the enzyme atomic mean-square displacements. Comparing D(2)O- with H(2)O-hydrated samples, we were able to investigate protein as well as hydration water molecular dynamics. HuBChE lyophilized from three distinct buffers showed completely different atomic mean-square displacements at temperatures above approximately 200 K: a salt-free sample and a sample containing Tris-HCl showed identical small-amplitude motions. A third sample, containing sodium phosphate, displayed highly reduced mean-square displacements at ambient temperature with respect to the other two samples. Below 200 K, all samples displayed similar mean-square displacements. We draw the conclusion that the reduction of intramolecular protein mean-square displacements on an Angstrom-nanosecond scale by the solvent depends not only on the presence of salt ions but also on their type.
ESTHER : Gabel_2004_Biophys.J_86_3152
PubMedSearch : Gabel_2004_Biophys.J_86_3152
PubMedID: 15111428

Title : Mutation of exposed hydrophobic amino acids to arginine to increase protein stability - Strub_2004_BMC.Biochem_5_9
Author(s) : Strub C , Alies C , Lougarre A , Ladurantie C , Czaplicki J , Fournier D
Ref : BMC Biochem , 5 :9 , 2004
Abstract : BACKGROUND: One strategy to increase the stability of proteins is to reduce the area of water-accessible hydrophobic surface.
RESULTS: In order to test it, we replaced 14 solvent-exposed hydrophobic residues of acetylcholinesterase by arginine. The stabilities of the resulting proteins were tested using denaturation by high temperature, organic solvents, urea and by proteolytic digestion. CONCLUSION: Although the mutational effects were rather small, this strategy proved to be successful since half of the mutants showed an increased stability. This stability may originate from the suppression of unfavorable interactions of nonpolar residues with water or from addition of new hydrogen bonds with the solvent. Other mechanisms may also contribute to the increased stability observed with some mutants. For example, introduction of a charge at the surface of the protein may provide a new coulombic interaction on the protein surface.
ESTHER : Strub_2004_BMC.Biochem_5_9
PubMedSearch : Strub_2004_BMC.Biochem_5_9
PubMedID: 15251041

Title : New biosensors for improved detection of environmental and food contamination by anticholinesterase pesticides -
Author(s) : Toutant JP , Massoulie J , Fournier D , Marty JL , Schmid RD , Pfeiffer D , Selkirk ME , Sussman JL , Silman I , Talesa V , Wodak SJ , Stojan J , Magearu V
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :233 , 2004

Title : Poster (24) New biosensors for improved detection of environmental contamination by anticholinesterase pesticides -
Author(s) : Toutant JP , Massoulie J , Fournier D , Schmid RD , Pfeiffer D , Selkirk ME , Sussman JL , Silman I , Talesa V , Wodak SJ
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :333 , 2004

Title : Engineering of acetylcholinesterase for insecticide detection: example of Aldicarbe -
Author(s) : Shi MA , Lougarre A , Alies C , Ladurantie C , Fournier D
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :237 , 2004

Title : Poster (25) Acetylcholinesterase engineering for detection of insecticide residues. -
Author(s) : Fournier D
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :334 , 2004

Title : Poster (26) Strategy for the development of biosensors based on bio-engineered acetylcholinesterases. -
Author(s) : Marty JL , Andreescu S , Bonnet C , Fournier D
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :334 , 2004

Title : Incidence of insecticide resistance alleles in sexually-reproducing populations of the peach-potato aphid Myzus persicae (Hemiptera: Aphididae) from southern France - Guillemaud_2003_Bull.Entomol.Res_93_289
Author(s) : Guillemaud T , Brun A , Anthony N , Sauge MH , Boll R , Delorme R , Fournier D , Lapchin L , Vanlerberghe-Masutti F
Ref : Bull Entomol Res , 93 :289 , 2003
Abstract : Intensive chemical treatments have led to the development of a number of insecticide resistance mechanisms in the peach-potato aphid Myzus persicae (Sulzer). Some of these mechanisms are known to be associated with negative pleiotropic effects (resistance costs). Molecular and biochemical methods were used to determine the genotypes or phenotypes associated with four insecticide resistance mechanisms in single aphids from sexually-reproducing populations in southern France. The mechanisms considered were E4 and FE4 carboxylesterase overproduction, modified acetycholinesterase, and kdr and rdl resistance-associated mutations. A new method for determining individual kdr genotypes is presented. Almost all resistant individuals overproduced FE4 carboxylesterase, whereas modified acetylcholinesterase was rare. Both the kdr and rdl resistance mutations were present at high frequencies in French sexually-reproducing populations. The frequencies of insecticide resistance genes were compared before and after sexual reproduction in one peach orchard at Avignon to evaluate the potential impact of selection on the persistence of resistance alleles in the over-wintering phase. The frequencies of the kdr and rdl mutations varied significantly between autumn and spring sampling periods. The frequency of the kdr mutation increased, probably due to pyrethroid treatments at the end of the winter. Conversely, the frequency of the rdl mutation decreased significantly during winter, probably because of a fitness cost associated with this mutation.
ESTHER : Guillemaud_2003_Bull.Entomol.Res_93_289
PubMedSearch : Guillemaud_2003_Bull.Entomol.Res_93_289
PubMedID: 12908914

Title : Encapsulation of acetylcholinesterase in preformed liposomes -
Author(s) : Chaize B , Winterhalter M , Fournier D
Ref : Biotechniques , 34 :1158 , 2003
PubMedID: 12813881

Title : Protein encapsulation in liposomes: efficiency depends on interactions between protein and phospholipid bilayer - Colletier_2002_BMC.Biotechnol_2_9
Author(s) : Colletier JP , Chaize B , Winterhalter M , Fournier D
Ref : BMC Biotechnol , 2 :9 , 2002
Abstract : BACKGROUND: We investigated the encapsulation mechanism of enzymes into liposomes. The existing protocols to achieve high encapsulation efficiencies are basically optimized for chemically stable molecules. Enzymes, however, are fragile and encapsulation requires in addition the preservation of their functionality. Using acetylcholinesterase as a model, we found that most protocols lead to a rapid denaturation of the enzyme with loss in the functionality and therefore inappropriate for such an application. The most appropriate method is based on lipid film hydration but had a very low efficiency.
RESULTS: To improve it and to propose a standard procedure for enzyme encapsulation, we separate each step and we studied the effect of each parameter on encapsulation: lipid and buffer composition and effect of the different physical treatment as freeze-thaw cycle or liposomes extrusion. We found that by increasing the lipid concentration, increasing the number of freeze-thaw cycles and enhancing the interactions of the enzyme with the liposome lipid surface more than 40% of the initial total activity can be encapsulated. CONCLUSION: We propose here an optimized procedure to encapsulate fragile enzymes into liposomes. Optimal encapsulation is achieved by induction of a specific interaction between the enzyme and the lipid surface.
ESTHER : Colletier_2002_BMC.Biotechnol_2_9
PubMedSearch : Colletier_2002_BMC.Biotechnol_2_9
PubMedID: 12003642

Title : Acetylcholinesterase engineering for detection of insecticide residues - Boublik_2002_Protein.Eng_15_43
Author(s) : Boublik Y , Saint-Aguet P , Lougarre A , Arnaud M , Villatte F , Estrada-Mondaca S , Fournier D
Ref : Protein Engineering , 15 :43 , 2002
Abstract : To detect traces of insecticides in the environment using biosensors, we engineered Drosophila acetylcholinesterase (AChE) to increase its sensitivity and its rate of phosphorylation or carbamoylation by organophosphates or carbamates. The mutants made by site-directed mutagenesis were expressed in baculovirus. Different strategies were used to obtain these mutants: (i) substitution of amino acids at positions found mutated in AChE from insects resistant to insecticide, (ii) mutations of amino acids at positions suggested by 3-D structural analysis of the active site, (iii) Ala-scan analysis of amino acids lining the active site gorge, (iv) mutagenesis at positions detected as important for sensitivity in the Ala-scan analysis and (v) combination of mutations which independently enhance sensitivity. The results highlighted the difficulty of predicting the effect of mutations; this may be due to the structure of the site, a deep gorge with the active serine at the bottom and to allosteric effects between the top and the bottom of the gorge. Nevertheless, the use of these different strategies allowed us to obtain sensitive enzymes. The greatest improvement was for the sensitivity to dichlorvos for which a mutant was 300-fold more sensitive than the Drosophila wild-type enzyme and 288 000-fold more sensitive than the electric eel enzyme, the enzyme commonly used to detect organophosphate and carbamate.
ESTHER : Boublik_2002_Protein.Eng_15_43
PubMedSearch : Boublik_2002_Protein.Eng_15_43
PubMedID: 11842237

Title : Improvement of Drosophila acetylcholinesterase stability by elimination of a free cysteine - Fremaux_2002_BMC.Biochem_3_21
Author(s) : Fremaux I , Mazeres S , Brisson-Lougarre A , Arnaud M , Ladurantie C , Fournier D
Ref : BMC Biochem , 3 :21 , 2002
Abstract : BACKGROUND: Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use for residue detection with biosensors. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, it is not sufficiently stable for extensive utilization. It is a homodimer in which both subunits contain 8 cysteine residues. Six are involved in conserved intramolecular disulfide bridges and one is involved in an interchain disulfide bridge. The 8th cysteine is not conserved and is present at position 290 as a free thiol pointing toward the center of the protein.
RESULTS: The free cysteine has been mutated to valine and the resulting protein has been assayed for stability using various denaturing agents: temperature, urea, acetonitrile, freezing, proteases and spontaneous-denaturation at room temperature. It was found that the C290V mutation rendered the protein 1.1 to 2.7 fold more stable depending on the denaturing agent. CONCLUSION: It seems that stabilization resulting from the cysteine to valine mutation originates from a decrease of thiol-disulfide interchanges and from an increase in the hydrophobicity of the buried side chain.
ESTHER : Fremaux_2002_BMC.Biochem_3_21
PubMedSearch : Fremaux_2002_BMC.Biochem_3_21
PubMedID: 12149129

Title : Pyrethroid resistance mechanisms in the cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) from West Africa - Martin_2002_Pestic.Biochem.Physiol_74_17
Author(s) : Martin T , Chandre F , Ochou OG , Vaissayre M , Fournier D
Ref : Pesticide Biochemistry and Physiology , 74 :17 , 2002
Abstract : In West Africa, the cotton bollworm Helicoverpa armigera has recently developed resistance to deltamethrin and cypermethrin. Resistance mechanisms of the strain BK99R9 collected in Bouak, Ivory Coast in 1999 and selected with deltamethrin were investigated by comparison with a susceptible strain BK77 collected in the same area in 1977. Several approaches were performed: evaluation of the cross-resistance spectrum to various pyrethroids and DDT, effect of a synergist, and by determination of the biochemical characteristics of three enzyme systems (esterases, glutathione-S-transferases, and mixed function oxidases). Deltamethrin resistance in BK99R9 was correlated to an increase of mixed function oxidase. Enhanced monooxygenase levels were then confirmed in several H. armigera field strains collected in cotton areas of West Africa from 1999 to 2001.
ESTHER : Martin_2002_Pestic.Biochem.Physiol_74_17
PubMedSearch : Martin_2002_Pestic.Biochem.Physiol_74_17

Title : Acceleration of Drosophila melanogaster acetylcholinesterase methanesulfonylation: peripheral ligand D-tubocurarine enhances the affinity for small methanesulfonylfluoride - Golicnik_2002_Chem.Biol.Interact_139_145
Author(s) : Golicnik M , Fournier D , Stojan J
Ref : Chemico-Biological Interactions , 139 :145 , 2002
Abstract : D-Tubocurarine, a reversible peripheral inhibitor of cholinesterases accelerates methanesulfonylation of Drosophila melanogaster wild type and W359L mutant. The kinetic evaluation of the process was performed in a step-by-step analysis. The second order overall sulfonylation rate constants, determined from classical residual activity measurements, were used in the subsequent analysis of progress curves. The latter were obtained by measuring the hydrolysis of acetylthiocholine in a complex reaction system of enzyme, substrate, irreversible and reversible inhibitor. The underlying kinetic mechanisms, from such a complex data, could only be untangled by targeted inspection and successive incorporation of reaction steps for which experimental evidence existed. The study showed that the peripheral ligand D-tubocurarine, by binding at the entrance into the active site of the two investigated enzymes (Golicnik et al., Biochemistry 40 (2001) 1214), enhances the affinity for small methanesulfonylfluoride, rather to speeding up the formation of a stable covalent enzyme-inhibitor complex. The specific arrangements at the rim of the active site of each individual enzyme dictate the actual events which can be detected by kinetic means.
ESTHER : Golicnik_2002_Chem.Biol.Interact_139_145
PubMedSearch : Golicnik_2002_Chem.Biol.Interact_139_145
PubMedID: 11823003

Title : Detection of anatoxin-a(s) in environmental samples of cyanobacteria by using a biosensor with engineered acetylcholinesterases - Devic_2002_Appl.Environ.Microbiol_68_4102
Author(s) : Devic E , Li D , Dauta A , Henriksen P , Codd GA , Marty JL , Fournier D
Ref : Applied Environmental Microbiology , 68 :4102 , 2002
Abstract : Bioassays are little used to detect individual toxins in the environment because, compared to analytical methods, these assays are still limited by several problems, such as the sensitivity and specificity of detection. We tentatively solved these two drawbacks for detection of anatoxin-a(s) by engineering an acetylcholinesterase to increase its sensitivity and by using a combination of mutants to obtain increased analyte specificity. Anatoxin-a(s), a neurotoxin produced by some freshwater cyanobacteria, was detected by measuring the inhibition of acetylcholinesterase activity. By using mutated enzyme, the sensitivity of detection was brought to below the nanomole-per-liter level. However, anatoxin-a(s) is an organophosphorous compound, as are several synthetic molecules which are widely used as insecticides. The mode of action of these compounds is via inhibition of acetylcholinesterase, which makes the biotest nonspecific. The use of a four-mutant set of acetylcholinesterase variants, two mutants that are sensitive to anatoxin-a(s) and two mutants that are sensitive to the insecticides, allows specific detection of the cyanobacterial neurotoxin.
ESTHER : Devic_2002_Appl.Environ.Microbiol_68_4102
PubMedSearch : Devic_2002_Appl.Environ.Microbiol_68_4102
PubMedID: 12147513

Title : Levels of Total Acetylcholinesterase in Drosophila melanogaster in Relation to Insecticide Resistance - Charpentier_2001_Pestic.Biochem.Physiol_70_100
Author(s) : Charpentier A , Fournier D
Ref : Pesticide Biochemistry and Physiology , 70 :100 , 2001
Abstract : Titration of acetylcholinesterase active sites was used to estimate the level of acetylcholinesterase in 82 Drosophila melanogaster strains originating from several countries. The amount of enzyme varied by up to a factor of twofold and was directly correlated with insecticide resistance. This suggests that increased acetylcholinesterase production contributes to resistance. This mechanism adds to the qualitative modifications of acetylcholinesterase, which render the enzyme less sensitive to inhibition by insecticides. As these qualitative modifications of the active site of the enzyme usually result in a decrease in the rate of neurotransmitter hydrolysis, they may affect the fitness of the flies. Overproduction of a modified acetylcholinesterase appears to be a potential modifier since it compensates for the decrease in neurotransmitter hydrolysis..
ESTHER : Charpentier_2001_Pestic.Biochem.Physiol_70_100
PubMedSearch : Charpentier_2001_Pestic.Biochem.Physiol_70_100

Title : Involvement of deacylation in activation of substrate hydrolysis by Drosophila acetylcholinesterase - Brochier_2001_J.Biol.Chem_276_18296
Author(s) : Brochier L , Pontie Y , Willson M , Estrada-Mondaca S , Czaplick J , Klaebe A , Fournier D
Ref : Journal of Biological Chemistry , 276 :18296 , 2001
Abstract : Insect acetylcholinesterase (AChE), an enzyme whose catalytic site is located at the bottom of a gorge-like structure, hydrolyzes its substrate over a wide range of concentrations (from 2 microm to 300 mm). AChE is activated at low substrate concentrations and inhibited at high substrate concentrations. Several rival kinetic models have been developed to try to describe and explain this behavior. One of these models assumes that activation at low substrate concentrations partly results from an acceleration of deacetylation of the acetylated enzyme. To test this hypothesis, we used a monomethylcarbamoylated enzyme, which is considered equivalent to the acylated form of the enzyme and a non-hydrolyzable substrate analog, 4-oxo-N,N,N-trimethylpentanaminium iodide. It appears that this substrate analog increases the decarbamoylation rate by a factor of 2.2, suggesting that the substrate molecule bound at the activation site (K(d) = 130 +/- 47 microm) accelerates deacetylation. These two kinetic parameters are consistent with our analysis of the hydrolysis of the substrate. The location of the active site was investigated by in vitro mutagenesis. We found that this site is located at the rim of the active site gorge. Thus, substrate positioning at the rim of the gorge slows down the entrance of another substrate molecule into the active site gorge (Marcel, V., Estrada-Mondaca, S., Magn, F., Stojan, J., Klab, A., and Fournier, D. (2000) J. Biol. Chem. 275, 11603-11609) and also increases the deacylation step. This results in an acceleration of enzyme turnover.
ESTHER : Brochier_2001_J.Biol.Chem_276_18296
PubMedSearch : Brochier_2001_J.Biol.Chem_276_18296
PubMedID: 11278288

Title : Interaction of Drosophila acetylcholinesterases with D-tubocurarine: an explanation of the activation by an inhibitor - Golicnik_2001_Biochemistry_40_1214
Author(s) : Golicnik M , Fournier D , Stojan J
Ref : Biochemistry , 40 :1214 , 2001
Abstract : Homotropic cooperativity in Drosophila melanogaster acetylcholinesterase seems to be a consequence of an initial substrate binding to a high-affinity peripheral substrate binding site situated around the negative charge of D413 (G335, Torpedo numbering). An appropriate mutation which turns the peripheral binding site to a low-affinity spot abolishes apparent activation but improves the overall enzyme effectiveness. This contradiction can be explained as less effective inhibition due to a shorter occupation of such a peripheral site. A similar effect can be achieved by an appropriate peripheral inhibitor such as TC, which can in special cases, when less effective heterotropic inhibition prevails over homotropic, acts as an activator. At the highest substrate concentrations, however, these enzymes are always inhibited, although steric components may influence the strength of inhibition like in the F368G mutant (F290, Torpedo numbering). Cooperative effects thus may include a steric component, but covering of the entrance must affect influx and efflux to different extents.
ESTHER : Golicnik_2001_Biochemistry_40_1214
PubMedSearch : Golicnik_2001_Biochemistry_40_1214
PubMedID: 11170446

Title : Exploration of the Drosophila acetylcholinesterase substrate activation site using a reversible inhibitor (Triton X-100) and mutated enzymes - Marcel_2000_J.Biol.Chem_275_11603
Author(s) : Marcel V , Estrada-Mondaca S , Magne F , Stojan J , Klaebe A , Fournier D
Ref : Journal of Biological Chemistry , 275 :11603 , 2000
Abstract : Cholinesterases are activated at low substrate concentration, and this is followed by inhibition as the level of substrate increases. However, one of these two components is sometimes lacking. In Drosophila acetylcholinesterase, the two phases are present, allowing both phenomena to be studied. Several kinetic schemes can explain this complex kinetic behavior. Among them, one model assumes that activation results from the binding of a substrate molecule to a non-productive site affecting the entrance of a substrate molecule into the active site. To test this hypothesis, we looked for an inhibitor competitive for activation and we found Triton X-100. Using organophosphates or carbamates as hemisubstrates, we showed that Triton X-100 inhibits or increases phosphorylation or carbamoylation of the enzyme. In vitro mutagenesis of the residues lining the active site gorge allowed us to locate the Triton X-100 binding site at the rim of the gorge with glutamate 107 playing the major role. These results led to the hypothesis that substrate binding at this site affects the entrance of another substrate molecule into the active site cleft.
ESTHER : Marcel_2000_J.Biol.Chem_275_11603
PubMedSearch : Marcel_2000_J.Biol.Chem_275_11603
PubMedID: 10766776

Title : A High Number of Mutations in Insect Acetylcholinesterase May Provide Insecticide Resistance - Villatte_2000_Pestic.Biochem.Physiol_67_95
Author(s) : Villatte F , Ziliani P , Marcel V , Menozzi P , Fournier D
Ref : Pesticide Biochemistry and Physiology , 67 :95 , 2000
Abstract : Many insect pest species have developed insecticide resistance through modifications of acetylcholinesterase. Seven mutations, issued from one nucleotide change, have been associated with resistance in natural populations of Drosophila and housefly (A. Mutero, M. Pralavorio, J. M. Bride, and D. Fournier, Resistance-associated point mutations in insecticide insensitive acetylcholinesterase, Proc. Natl. Acad Sci. USA91, 5922 (1994); A. L. Devonshire, F. J Byrne, G. D. Moores, and M. S. Williamson, Biochemical and molecular characterisation of insecticide-insensitive acetylcholinesterases in resistant insects, in -YStructure and function of cholinesterases and related proteins (B. P. Doctor, P. Taylor, D. M. Quinn, R. L. Rotundo, and M. K. Gentry, Eds.), pp. 491-496, Plenum Press, New York, 1998). In order to study the number of mutations which can lead to resistance, we first analyzed the effects of a set of amino acid replacements in the Drosophila acetylcholinesterase on inhibition by several carbamate and organophosphate insecticides. It appeared that most of the mutations led to a reduced sensitivity to insecticides. Second, we investigated the effect of mutations on substrate hydrolysis. We found that most of the variants retained sufficient levels of substrate hydrolysis. These data suggest that more mutations in acetylcholinesterase may be involved in organophosphate and carbamate resistance in addition to the previously known seven mutations.
ESTHER : Villatte_2000_Pestic.Biochem.Physiol_67_95
PubMedSearch : Villatte_2000_Pestic.Biochem.Physiol_67_95

Title : Improved multianalyte detection of organophosphates and carbamates with disposable multielectrode biosensors using recombinant mutants of Drosophila acetylcholinesterase and artificial neural networks - Bachmann_2000_Biosens.Bioelectron_15_193
Author(s) : Bachmann TT , Leca B , Vilatte F , Marty JL , Fournier D , Schmid RD
Ref : Biosensors & Bioelectronics , 15 :193 , 2000
Abstract : Engineered variants of Drosophila melanogaster acetylcholinesterase (AChE) were used as biological receptors of AChE-multisensors for the simultaneous detection and discrimination of binary mixtures of cholinesterase-inhibiting insecticides. The system was based on a combination of amperometric multielectrode biosensors with chemometric data analysis of sensor outputs using artificial neural networks (ANN). The multisensors were fully manufactured by screen-printing, including enzyme immobilisation. Two types of multisensors were produced that consisted of four AChE variants each. The AChE mutants were selected in order to obtain high resolution, enhanced sensitivity and minimal assay time. This task was successfully achieved using multisensor I equipped with wild-type Drosophila AChE and mutants Y408F, F368L, and F368H. Each of the AChE variants was selected on the basis of displaying an individual sensitivity pattern towards the target analytes. For multisensor II, the inclusion of F368W, which had an extremely diminished paraoxon sensitivity, increased the sensor's capacity even further. Multisensors I and II were both used for inhibition analysis of binary paraoxon and carbofuran mixtures in a concentration range 0-5 microg/l, followed by data analysis using feed-forward ANN. The two analytes were determined with prediction errors of 0.4 microg/l for paraoxon and 0.5 microg/l for carbofuran. A complete biosensor assay and subsequent ANN evaluation was completed within 40 min. In addition, multisensor II was also investigated for analyte discrimination in real water samples. Finally, the properties of the multisensors were confirmed by simultaneous detection of binary organophosphate mixtures. Malaoxon and paraoxon in composite solutions of 0-5 microg/l were discriminated with predication errors of 0.9 and 1.6 microg/l, respectively.
ESTHER : Bachmann_2000_Biosens.Bioelectron_15_193
PubMedSearch : Bachmann_2000_Biosens.Bioelectron_15_193
PubMedID: 11286337

Title : Is acetyl\/butyrylcholine specificity a marker for insecticide-resistance mutations in insect acetylcholinesterase? - Villatte_2000_Pest.Manag.Sci_56_1023
Author(s) : Villatte F , Ziliani P , Estrada-Mondaca S , Menozzi P , Fournier D
Ref : Pest Manag Sci , 56 :1023 , 2000
Abstract : Substrate specificity has been widely studied in vertebrate cholinesterases and it has been shown that two phenylalanines in the acyl pocket of acetylcholinesterase govern the acceptance of the acetyl/butyryl moiety of the choline esters. As an insecticide-resistance mutation has been evidenced in the acyl pocket of Drosophila melanogaster and Musca domestica acetylcholinesterase we investigated the possibility of linking changes in acetyl/butyrylthiocholine specificity with mutations in insect acetylcholinesterase. We thus analyzed the effect of 28 mutations in Drosophila enzyme on acetyl/butyrylthiocholine, N-methyl/N-propyl-carbamates and ethyl/methyl-paraoxon preference. It appeared that the highest changes on acetyl/butyrylthiocholine and N-propyl/N-methyl-carbamates preference were due to mutations in the acyl pocket. Nevertheless, other insecticide-resistance mutations, not located in the acyl pocket, also modified these substrate preferences. Moreover, the effect of mutations in the acyl pocket was hidden when some other insecticide-resistance mutations were combined in the enzyme. Consequently, acetyl/butyrylthiocholine preference alteration cannot be used as a marker to localize a mutation in the insect AChE.
ESTHER : Villatte_2000_Pest.Manag.Sci_56_1023
PubMedSearch : Villatte_2000_Pest.Manag.Sci_56_1023

Title : A method to estimate acetylcholinesterase-active sites and turnover in insects - Charpentier_2000_Anal.Biochem_285_76
Author(s) : Charpentier A , Menozzi P , Marcel V , Villatte F , Fournier D
Ref : Analytical Biochemistry , 285 :76 , 2000
Abstract : Acetylcholinesterase is the primary target of organophosphorous and carbamate insecticides. Quantitative changes in acetylcholinesterase are suspected to confer resistance to these insecticides, but a method to estimate the amount in insect is not available. A method using irreversible inhibitors has been developed. Among the irreversible inhibitors tested, 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide, chlorpyrifos-ethyl-oxon, and coumaphos-oxon were found to be sufficiently potent and specific.
ESTHER : Charpentier_2000_Anal.Biochem_285_76
PubMedSearch : Charpentier_2000_Anal.Biochem_285_76
PubMedID: 10998265

Title : Inhibition of Drosophila acetylcholinesterase by 7-(methylethoxyphosphinyloxy)1-methyl-quinolinium iodide - Stojan_1999_Chem.Biol.Interact_119-120_147
Author(s) : Stojan J , Marcel V , Fournier D
Ref : Chemico-Biological Interactions , 119-120 :147 , 1999
Abstract : The kinetic behaviour of Drosophila melanogaster acetylcholinesterase toward its substrate shows, in comparison with classic Michaelis-Menten kinetics, an apparent homotropic cooperative double activation-inhibition pattern. In order to construct an appropriate kinetic model and obtain further information on the mechanism of the catalytic action of this enzyme, the hydrolysis of acetylthiocholine in the absence and presence of different concentrations of synthetic quaternary methylphosphonate, 7-(methylethoxyphosphinyloxy)1-methyl-quinolinium iodide (MEPQ), was followed on a stopped-flow apparatus. The reaction at low substrate concentrations was followed until the change of the absorbance became negligible and at high concentrations only initial parts were recorded. A simultaneous analysis of the progress curves using numerical integration showed that the powerful organophosphonate inhibitor binds and compete with the substrate for the same binding sites. The results are also in accordance with the hypothesis that virtually every substrate or quasi-substrate molecule that enters into the gorge of active site is hydrolysed.
ESTHER : Stojan_1999_Chem.Biol.Interact_119-120_147
PubMedSearch : Stojan_1999_Chem.Biol.Interact_119-120_147
PubMedID: 10421448

Title : Cholinesterases from the marine mussels Mytilus galloprovincialis Lmk. and M. edulis L. from the freshwater bivalve Corbicula fluminea Muller - Mora_1999_Comp.Biochem.Physiol.C.Pharmacol.Toxicol.Endocrinol_122_353
Author(s) : Mora P , Fournier D , Narbonne JF
Ref : Comparative Biochemistry & Physiology C Pharmacology Toxicology & Endocrinology , 122 :353 , 1999
Abstract : In order to improve the molecular basis for the use of bivalve cholinesterases as a reliable biomarker for aquatic pollution, the polymorphism and characterization of these enzymes in Mytilus edulis, Mytilus galloprovincialis and Corbicula fluminea were investigated. All results are consistent with the presence of only one pharmacological form of cholinesterase in each species. The molecular masses were 180 kDa for the two marine mussels and 240 kDa for C. fluminea. The cholinesterases are anchored to the membrane by a glycosyl inositol phosphate like the Ga form (type I) described in vertebrates. Surprisingly, these cholinesterases were poorly inhibited by organophosphorous compounds compared to enzymes from other sources. This suggests that these bivalves could be used as a biomarker for acute rather than chronic contaminations by anticholinesterase insecticides.
ESTHER : Mora_1999_Comp.Biochem.Physiol.C.Pharmacol.Toxicol.Endocrinol_122_353
PubMedSearch : Mora_1999_Comp.Biochem.Physiol.C.Pharmacol.Toxicol.Endocrinol_122_353
PubMedID: 10336096

Title : Negative Cross-Insensitivity in Insecticide-Resistant Cotton Aphid Aphis gossypii Glover, - Villatte_1999_Pestic.Biochem.Physiol_65_55
Author(s) : Villatte F , Auge D , Touton P , Delorme R , Fournier D
Ref : Pesticide Biochemistry and Physiology , 65 :55 , 1999
Abstract : The cotton aphid, Aphis gossypii G., is a widespread agricultural pest and many populations around the world display resistance to pirimicarb. This resistance originates mainly from acetylcholinesterase modification. Hydrolysis of 14 carbamate and organophosphate insecticides by altered acetylcholinesterase of a resistant strain from southern France has been analyzed. It appeared that the modified enzyme provides a specific resistance to pirimicarb but also a greater sensitivity to bendiocarb. Bioassays showed that the resistant strain was slightly more sensitive to bendiocarb. Utilization of this antiresistance property was tested in the laboratory: a mixed population containing 50% pirimicarb-resistant and 50% pirimicarb-susceptible aphids was established and repetitively treated with bendiocarb. Frequency of resistant individuals in this population was monitored and compared to that of a nontreated mixed population. Treatments with bendiocarb delayed the increase of resistant individuals but as the resistant strain displayed a higher fitness than the sensitive strain and as antiresistance was weak, this selection assay did not allow the population to revert. These data showed that application of this strategy in fields requires compounds displaying high antiresistance properties.
ESTHER : Villatte_1999_Pestic.Biochem.Physiol_65_55
PubMedSearch : Villatte_1999_Pestic.Biochem.Physiol_65_55

Title : Interaction of 3-alkylpyridinium polymers from the sea sponge Reniera sarai with insect acetylcholinesterase - Sepcic_1999_J.Protein.Chem_18_251
Author(s) : Sepcic K , Poklar N , Vesnaver G , Fournier D , Turk T , Macek P
Ref : J Protein Chem , 18 :251 , 1999
Abstract : 3-Alkylpyridinium polymers (poly-APS), composed of 29 or 99 N-butyl-3-butyl pyridinium units, were isolated from the marine sponge Reniera sarai. They act as potent cholinesterase inhibitors. The inhibition kinetics pattern reveals several successive phases ending in irreversible inhibition of the enzyme. To provide more information on mechanism of inhibition, interaction of poly-APS and N-butyl-3-butyl pyridinium iodide (NBPI) with soluble dimeric and monomeric insect acetylcholinesterase (AChE) was studied by using enzyme intrinsic fluorescence and light scattering, conformational probes ANS and trypsin, and SDS-PAGE. Poly-APS quenched tryptophan fluorescence emission of AChE more extensively than NBPI. Both inhibitors exhibited a pseudo-Lehrer type of quenching. Interaction of poly-APS with dimeric AChE did not induce significant changes of the enzyme conformation as assayed by using the hydrophobic probe ANS and trypsin digestion. In contrast to NBPI, titration of both monomeric and dimeric AChE with poly-APS resulted in the appearance of large complexes detected by measuring light scattering. An excess of poly-APS produced AChE precipitation as proved on SDS-PAGE. None of the effects were observed with trypsin as a control. It was concluded that AChE aggregation and precipitation rather than the enzyme conformational changes accounted for the observed irreversible component of poly-APS inhibition.
ESTHER : Sepcic_1999_J.Protein.Chem_18_251
PubMedSearch : Sepcic_1999_J.Protein.Chem_18_251
PubMedID: 10395443

Title : Effect of tetramethylammonium, choline and edrophonium on insect acetylcholinesterase: test of a kinetic model - Stojan_1999_Chem.Biol.Interact_119-120_137
Author(s) : Stojan J , Marcel V , Fournier D
Ref : Chemico-Biological Interactions , 119-120 :137 , 1999
Abstract : Cholinesterases display a non-Michaelian behaviour with respect to substrate concentration. With the insect enzyme, there is an activation at low substrate concentrations and an inhibition at high concentrations. Previous studies allow us to propose a kinetic model involving a secondary non-productive binding site for the substrate. Unexpectedly, this secondary site has a very high affinity for the substrate when the enzyme is free. On the contrary, when the catalytic site of the enzyme is occupied a strong decrease of this affinity was observed. Moreover, a substrate molecule bound to the peripheral site results in a global decrease of the acylation and/or the deacylation step. Kinetic studies with three reversible inhibitors, tetramethylammonium, edrophonium and choline supported the kinetic model and enable its further refinement.
ESTHER : Stojan_1999_Chem.Biol.Interact_119-120_137
PubMedSearch : Stojan_1999_Chem.Biol.Interact_119-120_137
PubMedID: 10421447

Title : Two invertebrate acetylcholinesterases show activation followed by inhibition with substrate concentration - Marcel_1998_Biochem.J_329_329
Author(s) : Marcel V , Palacios LG , Pertuy C , Masson P , Fournier D
Ref : Biochemical Journal , 329 :329 , 1998
Abstract : In vertebrates there are two cholinesterases, with differences in catalytic behaviour with respect to substrate concentration: butyrylcholinesterase displays an increased activity at low substrate concentrations, whereas acetylcholinesterase displays inhibition by excess substrate. In two invertebrates, Drosophila melanogaster and Caenorhabditis elegans, we found cholinesterases that showed both kinetic complexities: substrate activation at low substrate concentrations followed by inhibition at higher concentrations. These triphasic kinetics can be explained by the presence of two enzymes with different kinetic behaviours or more probably by the existence of a single enzyme regulated by the substrate concentration.
ESTHER : Marcel_1998_Biochem.J_329_329
PubMedSearch : Marcel_1998_Biochem.J_329_329
PubMedID: 9425116

Title : A new attempt to assess the effect of learning processes on the cholinergic system: studies on fruitflies and honeybees - Fresquet_1998_Comp.Biochem.Physiol.B.Biochem.Mol.Biol_119_349
Author(s) : Fresquet N , Fournier D , Gauthier M
Ref : Comp Biochem Physiol B Biochem Mol Biol , 119 :349 , 1998
Abstract : The effect of training on the functioning of the cholinergic system was investigated in fruitflies and in honeybees. Drosophila were submitted to a passive avoidance conditioning of the proboscis extension response (PER). Flies had to learn to suppress the sugar-induced PER to avoid an aversive quinine reinforcement. In a yoked control group, the punishment was administered with no relation to the response displayed. Honeybees underwent a five-trial olfactory conditioning of the PER elicited by an antennal gustatory stimulation. In the control group, olfactory and gustatory stimulations were unpaired to prevent a learning process from developing. Immediately at the end of the learning session, acetylcholinesterase (AChE) activity was individually measured on the whole animal for Drosophila and on the head for the honeybee in experimental and in control groups. In fruitflies and honeybees, the AChE rate did not differ between the experimental group and its respective control group. Moreover, no significant correlation could be found individually between the learning performance and the AChE rate in either Drosophila or in honeybees. This experiment did not reveal any modulatory effect of the learning acquisition level on the AChE activity in insects as was previously reported in honeybees.
ESTHER : Fresquet_1998_Comp.Biochem.Physiol.B.Biochem.Mol.Biol_119_349
PubMedSearch : Fresquet_1998_Comp.Biochem.Physiol.B.Biochem.Mol.Biol_119_349
PubMedID: 9629668

Title : A putative kinetic model for substrate metabolisation by Drosophila acetylcholinesterase - Stojan_1998_FEBS.Lett_440_85
Author(s) : Stojan J , Marcel V , Estrada-Mondaca S , Klaebe A , Masson P , Fournier D
Ref : FEBS Letters , 440 :85 , 1998
Abstract : Insect acetylcholinesterase, an enzyme whose catalytic site is located at the bottom of a gorge, can metabolise its substrate in a wide range of concentrations (from 1 microM to 200 mM) since it is activated at low substrate concentrations. It also presents inhibition at high substrate concentrations. Among the various rival kinetic models tested to analyse the kinetic behaviour of the enzyme, the simplest able to explain all the experimental data suggests that there are two sites for substrate molecules on the protein. Binding on the catalytic site located at the bottom of the gorge seems to be irreversible, suggesting that each molecule of substrate which enters the active site gorge is metabolised. Reversible binding at the peripheral site of the free enzyme has high affinity (2 microM), suggesting that this binding increases the probability of the substrate entering the active site gorge. Peripheral site occupation decreases the entrance rate constant of the second substrate molecule to the catalytic site and strongly affects the catalytic activity of the enzyme. On the other hand, catalytic site occupation lowers the affinity of the peripheral site for the substrate (34 mM). These effects between the two sites result both in apparent activation at low substrate concentration and in general inhibition at high substrate concentration.
ESTHER : Stojan_1998_FEBS.Lett_440_85
PubMedSearch : Stojan_1998_FEBS.Lett_440_85
PubMedID: 9862431

Title : Stabilization of recombinant Drosophila acetylcholinesterase - Estrada-Mondaca_1998_Protein.Expr.Purif_12_166
Author(s) : Estrada-Mondaca S , Fournier D
Ref : Protein Expr Purif , 12 :166 , 1998
Abstract : The uses of pure and stable acetylcholinesterase can range from simple basic research to applications in environment quality assessment. In order to satisfy some of these needs its recombinant expression is routinely performed. Affinity-purified recombinant Drosophila melanogaster acetylcholinesterase proved to be instable; an apparent cause of this seemed to be the presence of contaminants with protease activity as evidenced by SDS-PAGE. The elimination of these accompanying products was achieved by anion-exchange, hydrophobic interaction, and cibacron blue affinity chromatography applied downstream from procainamide affinity chromatography. The utilization of a parallel affinity acting via an engineered histidine tail permitted the elimination of the copurified proteases as well. Despite the elimination of the contaminants, the apparently pure extracts were still unstable. It is shown that such instability can be counterbalanced by provoking protein-protein interactions, either between enzyme molecules or with other molecules such as bovine serum albumin. Another way to reduce instability is the addition of a reversible inhibitor or polyethylene glycol 3350.
ESTHER : Estrada-Mondaca_1998_Protein.Expr.Purif_12_166
PubMedSearch : Estrada-Mondaca_1998_Protein.Expr.Purif_12_166
PubMedID: 9518457

Title : Stabilization of Recombinant Drosophila Acetylcholinesterase -
Author(s) : Estrada-Mondaca S , Fournier D
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :151 , 1998

Title : Acetylcholinesterase and Insecticide Resistance in the Mosquito Culex Pipiens -
Author(s) : Bourguet D , Fournier D , Toutant JP , Arpagaus M , Raymond M
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :483 , 1998

Title : Acetylcholinesterase Increase in Drosophila as a Mechanism of Resistance to Insecticide -
Author(s) : Charpentier A , Villatte F , Fournier D
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :503 , 1998

Title : Engineering sensitive acetylcholinesterase for detection of organophosphate and carbamate insecticides - Villatte_1998_Biosens.Bioelectron_13_157
Author(s) : Villatte F , Marcel V , Estrada-Mondaca S , Fournier D
Ref : Biosensors & Bioelectronics , 13 :157 , 1998
Abstract : High quantities of various acetylcholinesterases can now be produced following in vitro expression and it is possible to use them as biosensors to detect organophosphates and carbamates insecticides. In order to check the potentialities of acetylcholinesterase from various sources, we have studied enzyme from bovine erythrocyte, Electrophorus electricus, Drosophila melanogaster, Torpedo californica and Caenorhabditis elegans. It appears that insect acetylcholinesterase is more susceptible to a broad range of organophosphates and carbamates insecticides than the other tested enzymes. D. melanogaster is 8-fold more sensitive than E. electricus enzyme and this sensitivity has been increased to 12-fold by introducing a mutation at position 408.
ESTHER : Villatte_1998_Biosens.Bioelectron_13_157
PubMedSearch : Villatte_1998_Biosens.Bioelectron_13_157
PubMedID: 9597732

Title : Engineering Sensitive Acetylcholinesterase for Detection of Organophosphate and Carbamate Insecticides -
Author(s) : Villatte F , Marcel V , Estrada-Mondaca S , Fournier D
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :553 , 1998

Title : Inhibition of acetylcholinesterase by an alkylpyridinium polymer from the marine sponge, reniera sarai - Sepcic_1998_Biochim.Biophys.Acta_1387_217
Author(s) : Sepcic K , Marcel V , Klaebe A , Turk T , Suput D , Fournier D
Ref : Biochimica & Biophysica Acta , 1387 :217 , 1998
Abstract : Large polymeric 3-alkylpyridinium salts have been isolated from the marine sponge Reniera sarai. They are composed of N-butyl(3-butylpyridinium) repeating subunits, polymerized head-to-tail, and exist as a mixture of two main polymers with molecular weights without counterion of about 5520 and 18900. The monomer analogue of the inhibitor, N-butyl-3-butylpyridinium iodide has been synthesized. This molecule shows mixed reversible inhibition of acetylcholinesterase. The polymers also act as acetylcholinesterase inhibitors and show an unusual inhibition pattern. We tentatively describe it as quick initial reversible binding, followed by slow binding or irreversible inhibition of the enzyme. This kinetics suggests that there are several affinity binding sites on the acetylcholinesterase molecule where the polymer can bind. The first binding favors binding to other sites which leads to an apparently irreversibly linked enzyme-inhibitor complex.
ESTHER : Sepcic_1998_Biochim.Biophys.Acta_1387_217
PubMedSearch : Sepcic_1998_Biochim.Biophys.Acta_1387_217
PubMedID: 9748587

Title : Variation of dominance of newly arisen adaptive genes - Bourguet_1997_Genetics_147_1225
Author(s) : Bourguet D , Lenormand T , Guillemaud T , Marcel V , Fournier D , Raymond M
Ref : Genetics , 147 :1225 , 1997
Abstract : Newly arisen adaptive alleles such as insecticide resistance genes represent a good opportunity to investigate the theories put forth to explain the molecular basis of dominance and its possible evolution. Dominance levels of insecticide resistance conferred by insensitive alleles of the acetylcholinesterase gene were analyzed in five resistant strains of the mosquito Culex pipiens. Dominance levels were found to differ between strains, varying from partial recessivity to complete dominance. This variation was not explained by differences in catalytic properties of the enzyme, since four of the five resistant strains had identical inhibition properties for the insensitive acetylcholinesterase. Among these four laboratory strains and in individuals collected from natural populations, we found a correlation between increased acetylcholinesterase activities and higher dominance levels. We propose a molecular explanation for how variation in acetylcholinesterase activity may result in variation of dominance level. We also conjecture that the four resistant strains did not differ in their amino acid sequence in the catalytically active regions of acetylcholinesterase, but that the expression of the gene was regulated by either neighboring or distant sites, thereby modifying the dominance level. Under this interpretation, dominance levels may evolve in this system, since heritable variation in acetylcholinesterase activity was found.
ESTHER : Bourguet_1997_Genetics_147_1225
PubMedSearch : Bourguet_1997_Genetics_147_1225
PubMedID: 9383065

Title : Transposition-mediated transcriptional overexpression as a mechanism of insecticide resistance - Berrada_1997_Mol.Gen.Genet_256_348
Author(s) : Berrada S , Fournier D
Ref : Molecular & General Genetics , 256 :348 , 1997
Abstract : It has been proposed that amplification of genes for esterase that provide resistance to insecticides may originate from transposition events. To test this hypothesis, we have constructed a minigene coding for a soluble acetylcholinesterase under the control of a nontissue-specific promoter (hsp70). When introduced into Drosophila, the gene is expressed in all tissues and the extra acetylcholinesterase produced confers a low level of insecticide resistance (twofold). The minigene was mobilized by crossing the initial transformant with a strain providing a source of P-element transposase. After 34 generations of exposure to the organophosphate parathion, we obtained a strain with a higher resistance (fivefold). This strain had only one extra Ace gene, which overexpressed acetylcholinesterase. Thus, following transposition, resistance resulted from the overexpression of a single copy of the gene and not from gene amplification.
ESTHER : Berrada_1997_Mol.Gen.Genet_256_348
PubMedSearch : Berrada_1997_Mol.Gen.Genet_256_348
PubMedID: 9393432

Title : Cholinesterases from the common oyster (Crassostrea gigas). Evidence for the presence of a soluble acetylcholinesterase insensitive to organophosphate and carbamate inhibitors - Bocquene_1997_FEBS.Lett_407_261
Author(s) : Bocquene G , Roig A , Fournier D
Ref : FEBS Letters , 407 :261 , 1997
Abstract : Marine bivalves such as oysters and mussels are widely used as bioindicators of contamination in the monitoring of pollutant effects. As filter feeders, these species are known to be good general indicators of chemical contamination. However, the efficient use of decreased acetylcholinesterase activity in the oyster as a biomarker of exposure to neurotoxic compounds requires a definition of the different types of cholinesterases coexisting in this mollusk. This study reports the partial purification, separation and characterization of two cholinesterases extracted from the oyster Crassostrea gigas. Differences in apparent molecular weight, type of glycosylation and hydrophobicity, and sensitivity to inhibitors suggest that they are encoded by two different genes. 'A' cholinesterase (apparent molecular weight 200 kDa) is anchored to the membrane via a glycolipid, is not glycosylated but sensitive to organophosphate and carbamate inhibitors. 'B' cholinesterase (molecular weight 330 kDa) is hydrophilic, glycosylated and highly resistant to organophosphate and carbamate inhibitors. The kinetic properties of these two cholinesterases were compared with those of other invertebrate cholinesterases. The presence of a cholinesterase insensitive to insecticides suggests that a significant improvement in the use of oyster cholinesterases as biomarkers of pollutant effects could be achieved by simple separation of the two forms.
ESTHER : Bocquene_1997_FEBS.Lett_407_261
PubMedSearch : Bocquene_1997_FEBS.Lett_407_261
PubMedID: 9175864

Title : Existence of two acetylcholinesterases in the mosquito Culex pipiens (Diptera:Culicidae) - Bourguet_1996_J.Neurochem_67_2115
Author(s) : Bourguet D , Raymond M , Fournier D , Malcolm CA , Toutant JP , Arpagaus M
Ref : Journal of Neurochemistry , 67 :2115 , 1996
Abstract : Two acetylcholinesterases (AChEs), AChE1 and AChE2, differing in substrate specificity and in some aspects of inhibitor sensitivity, have been characterized in the mosquito Culex pipiens. The results of ultracentrifugation in sucrose gradients and nondenaturing gel electrophoresis of AChE activity peak fractions show that each AChE is present as two molecular forms: one amphiphilic dimer possessing a glycolipid anchor and one hydrophilic dimer that does not interact with nondenaturing detergents. Treatment by phosphatidylinositol-specific phospholipase C converts each type of amphiphilic dimer into the corresponding hydrophilic dimer. Molecular forms of AChE1 have a lower electrophoretic mobility than those of AChE2. However, amphiphilic dimers and hydrophilic dimers have similar sedimentation coefficients (5.5S and 6.5S, respectively). AChE1 and AChE2 dimers, amphiphilic or hydrophilic, resist dithiothreitol reduction under conditions that allow reduction of Drosophila AChE dimers. In the insecticide-susceptible strain S-LAB, AChE1 is inhibited by 5 x 10(-4) M propoxur (a carbamate insecticide), whereas AChE2 is resistant. All animals are killed by this concentration of propoxur, indicating that only AChE1 fulfills the physiological function of neurotransmitter hydrolysis at synapses. In the insecticide-resistant strain, MSE, there is no mortality after exposure to 5 x 10(-4) M propoxur: AChE2 sensitivity to propoxur is unchanged, whereas AChE1 is now resistant to 5 x 10(-4) M propoxur. The possibility that AChE1 and AChE2 are products of tissue-specific posttranslational modifications of a single gene is discussed, but we suggest, based on recent results obtained at the molecular level in mosquitoes, that they are encoded by two different genes.
ESTHER : Bourguet_1996_J.Neurochem_67_2115
PubMedSearch : Bourguet_1996_J.Neurochem_67_2115
PubMedID: 8863521

Title : Oxidative Degradation of Diazinon by Drosophila: Metabolic Changes Associated with Insecticide Resistance and Induction - Pisani-Borg_1996_Pestic.Biochem.Physiol_54_56
Author(s) : Pisani-Borg E , Cuany A , Brun A , Amichot M , Fournier D , Berge JB
Ref : Pesticide Biochemistry and Physiology , 54 :56 , 1996
Abstract : Using [14C]diazinon, it was found that four molecules accounted for most of the metabolites afterin vitroorin vivoincubation withDrosophila melanogaster.RalDDTR, an insecticide-resistant strain ofDrosophila,produced higher amounts of each metabolite than CantonS, a susceptible strain. However, the degradative metabolic pathway giving hydroxydiazinon and pyrimidine was twofold faster than the activating metabolic pathway giving hydroxydiazoxon and diazoxon. Clofibrate and phenobarbital increased the metabolism of diazinon in induced, susceptible flies. Phenobarbital was more potent than clofibrate in stimulating the hydroxylation of diazinon. Kinetic analysis ofin vivoinhibition of brain acetylcholinesterase by diazinon or hydroxydiazinon demonstrated that degradation of diazinon, especially in RalDDTR, was delayed compared to the degradation of hydroxydiazinon. Varying levels of diazinon tolerance amongDrosophilastrains may be explained by differential metabolic pathways. The most conclusive result was a strong interaction between tolerance and the rate of formation of hydroxylated derivatives.
ESTHER : Pisani-Borg_1996_Pestic.Biochem.Physiol_54_56
PubMedSearch : Pisani-Borg_1996_Pestic.Biochem.Physiol_54_56

Title : Properties of Class a Acetylcholinesterase, the Enzyme Encoded by ACE-1 in Caenorhabditis elegans -
Author(s) : Arpagaus M , Schirru N , Culetto E , Talesa V , Cousin X , Chatonnet A , Fedon Y , Berge JB , Fournier D , Toutant JP
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :7 , 1995

Title : Insect Acetylcholinesterase and Resistance to Insecticides -
Author(s) : Fournier D , Maturano M , Gagnoux L , Ziliani P , Pertuy C , Pralavorio M , Bride JM , Elmarbouh L , Klaebe A , Masson P
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :149 , 1995

Title : Identification of Resistance Mechanisms in a Selected Laboratory Strain of Cacopsylla pyri (Homoptera: Psyllidae): Altered Acetylcholinesterase and Detoxifying Oxidases - Berrada_1994_Pestic.Biochem.Physiol_48_41
Author(s) : Berrada S , Fournier D , Cuany A , Nguyen TX
Ref : Pesticide Biochemistry and Physiology , 48 :41 , 1994
Abstract : A resistant strain (TL) of pear psylla, Cacopsylla pyri (L.), was obtained by laboratory selection with monocrotophos. In comparison with a sensitive strain (LS), resistance mechanisms in TL were investigated on the basis of both the effects of two synergists (piperonyl butoxide and S,S,S-tributyl phosphorotrithioate) and the in vitro analysis of the activity of four enzymes known to be often involved in organophosphate resistance (acetylcholinesterase, mixed-function oxidases, esterases, and glutathione S-transferases). The presence of a modified acetylcholinesterase, less sensitive to the inhibition by the insecticide (70-fold) combined with a slight increase in the oxidative metabolism (5-fold) was found to be responsible for the high resistance level developed in TL (150-fold).
ESTHER : Berrada_1994_Pestic.Biochem.Physiol_48_41
PubMedSearch : Berrada_1994_Pestic.Biochem.Physiol_48_41

Title : Drosophila melanogaster acetylcholinesterase: identification and expression of two mutations responsible for cold- and heat-sensitive phenotypes - Mutero_1994_Mol.Gen.Genet_243_699
Author(s) : Mutero A , Bride JM , Pralavorio M , Fournier D
Ref : Molecular & General Genetics , 243 :699 , 1994
Abstract : AceIJ29 and AceIJ40 are cold- and heat-sensitive variants of the gene coding for acetylcholinesterase in Drosophila melanogaster. In the homozygous condition, these mutations are lethal when animals are raised at restrictive temperatures, i.e., below 23 degrees C for AceIJ29 or above 25 degrees C for AceIJ40. The coding regions of the gene in these mutants were sequenced and mutations changing Ser374 to Phe in AceIJ29 and Pro75 to Leu in AceIJ40 were found. Acetylcholinesterases bearing these mutations were expressed in Xenopus oocytes and we found that these mutations decrease the secretion rate of the protein most probably by affecting its folding. This phenomenon is exacerbated at restrictive temperatures decreasing the amount of secreted acetylcholinesterase below the lethality threshold. In parallel, the substitution of the conserved Asp248 by an Asn residue completely inhibits the activity of the enzyme and its secretion, preventing the correct folding of the protein in a non-conditional manner.
ESTHER : Mutero_1994_Mol.Gen.Genet_243_699
PubMedSearch : Mutero_1994_Mol.Gen.Genet_243_699
PubMedID: 8028587

Title : Biochemical characterization of Drosophila melanogaster acetylcholinesterase expressed by recombinant baculoviruses - Chaabihi_1994_Biochem.Biophys.Res.Commun_203_734
Author(s) : Chaabihi H , Fournier D , Fedon Y , Bossy JP , Ravallec M , Devauchelle G , Cerutti M
Ref : Biochemical & Biophysical Research Communications , 203 :734 , 1994
Abstract : Recombinant baculoviruses expressing full length and 3' truncated forms of c-DNA encoding the Drosophila melanogaster acetylcholinesterase (AChE) were constructed. Biochemical analyses showed that full length recombinant protein was enzymatically active and anchored to the cell membrane via a glycolipidic residue. DTT treatment dissociated the native form into monomers migrating as did the corresponding form of AChE extracted from drosophila heads. Finally, DFP labelling demonstrated that the specific proteolytic cleavage leading to the formation of 55 and 16 kDa subunits occurred in Sf9 cells. In contrast with the full-length enzyme, C-terminal-truncated forms were highly secreted, confirming the prominent role of the C-terminal hydrophobic peptide for the addition of the glycolipidic residue. Accumulation of inactive precursor was observed when recombinant proteins were overproduced using an improved baculovirus, suggesting a saturation of insect cell machineries.
ESTHER : Chaabihi_1994_Biochem.Biophys.Res.Commun_203_734
PubMedSearch : Chaabihi_1994_Biochem.Biophys.Res.Commun_203_734
PubMedID: 94354885

Title : cDNA sequence, gene structure, and in vitro expression of ace-1, the gene encoding acetylcholinesterase of class A in the nematode Caenorhabditis elegans - Arpagaus_1994_J.Biol.Chem_269_9957
Author(s) : Arpagaus M , Fedon Y , Cousin X , Chatonnet A , Berge JB , Fournier D , Toutant JP
Ref : Journal of Biological Chemistry , 269 :9957 , 1994
Abstract : Three genes, ace-1, ace-2, and ace-3, encode three acetylcholinesterase classes (A, B, and C) in the nematode Caenorhabditis elegans. A fragment of genomic DNA was amplified by a polymerase chain reaction (PCR) using degenerate oligonucleotides based on sequences conserved in the cholinesterase family. This fragment mapped to chromosome X at a position that perfectly matched the location of ace-1 previously determined by genetic methods. Comparison of genomic and cDNA sequences showed that the open reading frame was interrupted by eight introns. The product of ace-1 (ACE-1, 620 amino acids) presented 42% identity with Torpedo and human acetylcholinesterases, 41% with human butyrylcholinesterase, and 35% with Drosophila acetylcholinesterase. The overall structure of cholinesterases was conserved in ACE-1 as indicated by the conserved sequence positions of Ser-216, His-468, and Glu-346 (S200, H440, E327 in Torpedo (AChE) as components of the catalytic triad, of the six cysteines which form three intrachain disulfide bonds, and of Trp-99(84), a critical side chain in the choline binding site. Spodoptera Sf9 cells were infected by a recombinant baculovirus containing ace-1 cDNA. The secreted enzyme was active and existed as hydrophilic 5 and 11.5 S molecular forms. It hydrolyzed both acetylthiocholine and butyrylthiocholine and was inhibited by acetylthiocholine above 10 mM.
ESTHER : Arpagaus_1994_J.Biol.Chem_269_9957
PubMedSearch : Arpagaus_1994_J.Biol.Chem_269_9957
PubMedID: 8144590
Gene_locus related to this paper: caeel-ACHE1

Title : Resistance-associated point mutations in insecticide-insensitive acetylcholinesterase - Mutero_1994_Proc.Natl.Acad.Sci.U.S.A_91_5922
Author(s) : Mutero A , Pralavorio M , Bride JM , Fournier D
Ref : Proceedings of the National Academy of Sciences of the United States of America , 91 :5922 , 1994
Abstract : Extensive utilization of pesticides against insects provides us with a good model for studying the adaptation of a eukaryotic genome to a strong selective pressure. One mechanism of resistance is the alteration of acetylcholinesterase (EC, the molecular target for organophosphates and carbamates. Here, we report the sequence analysis of the Ace gene in several resistant field strains of Drosophila melanogaster. This analysis resulted in the identification of five point mutations associated with reduced sensitivities to insecticides. In some cases, several of these mutations were found to be combined in the same protein, leading to different resistance patterns. Our results suggest that recombination between resistant alleles preexisting in natural populations is a mechanism by which insects rapidly adapt to new selective pressures.
ESTHER : Mutero_1994_Proc.Natl.Acad.Sci.U.S.A_91_5922
PubMedSearch : Mutero_1994_Proc.Natl.Acad.Sci.U.S.A_91_5922
PubMedID: 8016090

Title : Action of Esterase B1 on Chlorpyrifos in Organophosphate-Resistant Culex Mosquitos - Cuany_1993_Pestic.Biochem.Physiol_45_1
Author(s) : Cuany A , Handani J , Berge J , Fournier D , Raymond M , Georghiou GP , Pasteur N
Ref : Pesticide Biochemistry and Physiology , 45 :1 , 1993
Abstract : Esterase B1 activity in Culex pipiens mosquitoes was strongly inhibited by oxidized organophosphates (OP), but not by nonoxidized forms or by carbamates. Inhibition by chlorpyrifos oxon and paraoxon remained total during the 2 hr following the removal of free insecticide molecules, indicating that hydrolysis by esterase B1 is either very slow or absent. This hypothesis was confirmed by comparing the fate of [14C]chlorpyrifos in larvae of strains TEM-R (with the over-produced esterase B1) and MSE (lacking an overproduced esterase). As expected, large quantities of chlorpyrifos oxon were observed in the two strains, but no other metabolite was found in TEM-R. It is concluded that esterase B1 confers resistance at least to diethyl OPs through sequestering rather than metabolism, as is also the case with the overproduced esterase E4 of Myzus persicae.
ESTHER : Cuany_1993_Pestic.Biochem.Physiol_45_1
PubMedSearch : Cuany_1993_Pestic.Biochem.Physiol_45_1

Title : Drosophila acetylcholinesterase: mechanisms of resistance to organophosphates - Fournier_1993_Chem.Biol.Interact_87_233
Author(s) : Fournier D , Mutero A , Pralavorio M , Bride JM
Ref : Chemico-Biological Interactions , 87 :233 , 1993
Abstract : Quantitative and qualitative changes of acetylcholinesterase can affect the sensitivity of insects to insecticides. First, the amount of acetylcholinesterase in the central nervous system is important in Drosophila melanogaster, flies which overexpress the enzyme are more resistant than wild-type flies. On the contrary, flies which express low levels of acetylcholinesterase are more susceptible. An overproduction of acetylcholinesterase outside the central nervous system also protects against organophosphate poisoning, that is, flies producing a soluble acetylcholinesterase, secreted in the haemolymph, are resistant to organophosphates. Second, resistance can also result from a qualitative modification of acetylcholinesterase. Four mutations have been identified in resistant strains: Phe115 to Ser, Ileu199 to Val, Gly303 to Ala and Phe368 to Tyr. Each of these mutations led to a different pattern of resistance and combinations between these mutations led to highly resistant enzymes.
ESTHER : Fournier_1993_Chem.Biol.Interact_87_233
PubMedSearch : Fournier_1993_Chem.Biol.Interact_87_233
PubMedID: 8343979

Title : Minigene rescues acetylcholinesterase lethal mutations in Drosophila melanogaster - Hoffmann_1992_J.Mol.Biol_223_17
Author(s) : Hoffmann F , Fournier D , Spierer P
Ref : Journal of Molecular Biology , 223 :17 , 1992
Abstract : The gene encoding acetylcholinesterase in Drosophila melanogaster is over 34,000 base-pairs long. We have constructed a 5800 base-pair minigene containing 1500 base-pairs of genomic sequence upstream from the transcription start spliced to the coding sequence, but lacking the nine introns. After germline genetic transformation, this minigene rescues acetylcholinesterase lethal mutants. Tissue-specific distribution appears normal. This allows us to test site-directed mutations of acetylcholinesterase. In a first effort, deletion of most of the unusual 1000 bases leader and its intriguing short open reading frames showed no effect on gene expression. The way is open to study in vivo the structure-function relationships of acetylcholinesterase and insecticide resistance.
ESTHER : Hoffmann_1992_J.Mol.Biol_223_17
PubMedSearch : Hoffmann_1992_J.Mol.Biol_223_17
PubMedID: 1731068

Title : Drosophila acetylcholinesterase. Expression of a functional precursor in Xenopus oocytes - Fournier_1992_Eur.J.Biochem_203_513
Author(s) : Fournier D , Mutero A , Rungger D
Ref : European Journal of Biochemistry , 203 :513 , 1992
Abstract : In insects, acetylcholinesterase is mainly found in the central nervous system. It is expressed in the synapse where it hydrolyzes the neurotransmitter acetylcholine. Maturation of this protein involves several post-translational modifications. The precursor polypeptide is cut at three sites; the N-terminal signal peptide is removed, the C-terminal hydrophobic polypeptide is clipped off and replaced by a glycolipid anchor and the resulting peptide is cut into two polypeptides, corresponding to active subunits. Two of these active subunits are associated to form the final active glycosylated protein. We have expressed the protein via microinjection of an expression vector into Xenopus oocyte nuclei. When the complete cDNA is injected, the acetylcholinesterase formed is biochemically similar to the Drosophila-head acetylcholinesterase. However, the hydrophobic C-terminal peptide is not replaced by a glycolipid anchor. As a consequence, the enzyme is no longer externalized, the proteolytic cutting of the main peptide does not occur and a new polymerization form occurs. Although incompletely processed, this protein is enzymatically active. When a cDNA lacking the coding region of the C-terminal hydrophobic peptide is injected, the resulting acetylcholinesterase is hydrophilic, cleaved into two subunits and secreted into the incubation medium free of contaminants.
ESTHER : Fournier_1992_Eur.J.Biochem_203_513
PubMedSearch : Fournier_1992_Eur.J.Biochem_203_513
PubMedID: 1310468

Title : Catalytic properties of cholinesterases: importance of tyrosine 109 in Drosophila protein - Mutero_1992_Neuroreport_3_39
Author(s) : Mutero A , Pralavorio M , Simeon-Rudolf V , Fournier D
Ref : Neuroreport , 3 :39 , 1992
Abstract : Tyrosine 109 in the acetylcholinesterase sequence of Drosophila melanogaster corresponds to an aspartate in vertebrate cholinesterases. Mutation of this amino acid to a glycine in the human butyrylcholinesterase gives rise to the 'atypic' phenotype characterized by a reduced activity for charged compounds. We investigated the importance of tyrosine 109 in the Drosophila sequence by in vitro mutagenesis and its expression in the Xenopus oocyte. We show here that tyrosine 109 contributes to the conformation of the active site and the charge of the residue at position 109 is important for catalytic properties. Sensitivity of the enzyme to organophosphorus and carbamate compounds is modified depending on residues present in position 109, therefore this amino acid is a potential site of resistance for insects to insecticides.
ESTHER : Mutero_1992_Neuroreport_3_39
PubMedSearch : Mutero_1992_Neuroreport_3_39
PubMedID: 1611033

Title : Acetylcholinesterase. Two types of modifications confer resistance to insecticide - Fournier_1992_J.Biol.Chem_267_14270
Author(s) : Fournier D , Bride JM , Hoffmann F , Karch F
Ref : Journal of Biological Chemistry , 267 :14270 , 1992
Abstract : Quantitative and qualitative changes in acetylcholinesterase confer resistance to insecticides. We have constructed several Drosophila melanogaster strains producing various amounts of enzyme by P-mediated transformation. Toxicological analysis of these strains demonstrates that resistance to organophosphorus insecticides is correlated with the amount of acetylcholinesterase in the central nervous system. Resistance may also be qualitatively determined. Comparison of the Drosophila acetylcholinesterase gene between a resistant strain caught in the wild and a wild type susceptible strain only revealed one nucleotide transition resulting in the replacement of a phenylalanine by a tyrosine. Flies mutant for acetylcholinesterase and rescued with a minigene mutagenized for this same transition produced an altered enzyme which renders flies resistant to pesticides.
ESTHER : Fournier_1992_J.Biol.Chem_267_14270
PubMedSearch : Fournier_1992_J.Biol.Chem_267_14270
PubMedID: 1629220

Title : Drosophila acetylcholinesterase: characterization of different mutants resistant to insecticides - Pralavorio_1992_Biochem.Genet_30_77
Author(s) : Pralavorio M , Fournier D
Ref : Biochemical Genetics , 30 :77 , 1992
Abstract : Selection of field populations originating from several countries allowed us to isolate 13 strains of Drosophila melanogaster resistant to parathion. In vitro studies of acetylcholinesterase inhibition by paraoxon have been carried out on purified enzymes: most of the resistant strains harbor an altered acetylcholinesterase. Enzymes with higher resistance levels have been characterized with respect to their cross-resistance toward several insecticides. The patterns obtained have permitted us to group them and to delineate four categories. The existence of four distinct types of protein suggests that several mutations of acetylcholinesterase are responsible for insecticide resistance in Drosophila.
ESTHER : Pralavorio_1992_Biochem.Genet_30_77
PubMedSearch : Pralavorio_1992_Biochem.Genet_30_77
PubMedID: 1520255

Title : Post-translational modifications of Drosophila acetylcholinesterase. In vitro mutagenesis and expression in Xenopus oocytes - Mutero_1992_J.Biol.Chem_267_1695
Author(s) : Mutero A , Fournier D
Ref : Journal of Biological Chemistry , 267 :1695 , 1992
Abstract : Drosophila acetylcholinesterase (EC is a 150-kDa glycoprotein anchored in plasmic membranes via a glycolipid. It is composed of two active subunits which are themselves made of two noncovalently linked polypeptides of 18 and 55 kDa resulting from the proteolysis of a single precursor of 75 kDa. Active Drosophila acetylcholinesterase can be expressed in Xenopus oocytes as an excreted protein. We have identified some of the amino acids essential in post-translational modifications of the protein by site-directed mutagenesis and expression of mutants in this system. The intersubunit disulfide bond involves cysteine at position 615. Cleavage of the 75-kDa precursor, as observed in Drosophila, originates from a hydrophilic peptide (in position 148 to 180) which does not exist in cholinesterase sequences from vertebrates. This cleavage is associated with excretion out of the cell. Drosophila acetylcholinesterase exhibits four effective sites of asparagine-linked glycosylation in positions 126, 174, 331, and 531. We show that glycosylations and dimerization protect the protein against proteolytic digestion. In contrast, none of these post-translational modifications significantly affects the activity of acetylcholinesterase or affinity for its substrate.
ESTHER : Mutero_1992_J.Biol.Chem_267_1695
PubMedSearch : Mutero_1992_J.Biol.Chem_267_1695
PubMedID: 1730712

Title : Drosophila Acetylcholinesterase: Analysis of Structure and Sensitivity to Insecticides by In Vitro Mutagenesis and Expression -
Author(s) : Fournier D , Mutero A
Ref : In Multidisciplinary approaches to cholinesterase functions - Proceedings of Fourth International Meeting on Cholinesterases , (Shafferman, A. and Velan, B., Eds) Plenum Press, New York :75 , 1992

Title : Drosophila Acetylcholinesterase Structure -
Author(s) : Mutero A , Fournier D
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :141 , 1991

Title : Drosophila melanogaster acetylcholinesterase gene. Structure, evolution and mutations - Fournier_1989_J.Mol.Biol_210_15
Author(s) : Fournier D , Karch F , Bride JM , Hall LM , Berge JB , Spierer P
Ref : Journal of Molecular Biology , 210 :15 , 1989
Abstract : Acetylcholinesterase is a key component of cholinergic neurotransmission. In Drosophila melanogaster, acetylcholinesterase is encoded by the Ace locus. We have determined the complete organization of the locus. The transcription unit is 34 kb (1 kb = 10(3) bases) long and encompasses ten exons. We have mapped the 5' end of the transcript, sequenced all the intron/exon boundaries, as well as the 3' end of the transcript. The deduced mature transcript is 4291 nucleotides long without poly(A). Sequencing of the promoter region reveals a potential TATA box and (GA)n motives. The Drosophila coding sequence is more split than its vertebrate counterparts, but the splicing sites of the two last exons are precisely conserved among Drosophila and vertebrate cholinesterases, and intriguingly also with the bovine thyroglobulin gene. Finally, a number of the mutations isolated in earlier genetic work are precisely placed on our molecular map in introns, exons and promoter regions. Among them, for example, a short deletion known to affect acetylcholinesterase level and tissue distribution removes promoter regions and the first non-coding exon.
ESTHER : Fournier_1989_J.Mol.Biol_210_15
PubMedSearch : Fournier_1989_J.Mol.Biol_210_15
PubMedID: 2511327

Title : Acetylcholinesterase from Drosophila melanogaster. Identification of two subunits encoded by the same gene - Fournier_1988_FEBS.Lett_238_333
Author(s) : Fournier D , Bride JM , Karch F , Berge JB
Ref : FEBS Letters , 238 :333 , 1988
Abstract : Purified acetylcholinesterase from Drosophila melanogaster is composed of a 55 kDa and a 16 kDa noncovalently associated subunit. Cleavage of disulfide bonds reveals that two 55 kDa polypeptides are linked together in native dimeric AChE. Western blots with two antibodies directed against the N- and C-termini of the predicted AChE primary sequence show that the 55 and 16 kDa polypeptides originate from proteolysis of the same precursor encoded by the Ace locus.
ESTHER : Fournier_1988_FEBS.Lett_238_333
PubMedSearch : Fournier_1988_FEBS.Lett_238_333
PubMedID: 3139459

Title : Native molecular forms of head acetylcholinesterase from adult Drosophila melanogaster: quaternary structure and hydrophobic character - Toutant_1988_J.Neurochem_50_209
Author(s) : Toutant JP , Arpagaus M , Fournier D
Ref : Journal of Neurochemistry , 50 :209 , 1988
Abstract : The native molecular forms of acetylcholinesterase (AChE) present in adult Drosophila heads were characterized by sedimentation analysis in sucrose gradients and by nondenaturing electrophoresis. The hydrophobic properties of AChE forms were studied by comparing their migration in the presence of Triton X100, 10-oleyl ether, or sodium deoxycholate, or in the absence of detergent. We examined the polymeric structure of AChE forms by disulfide bridge reduction. We found that the major native molecular form is an amphiphilic dimer which is converted into hydrophilic dimer and monomer on autolysis of the extracts, or into a catalytically active amphiphilic monomer by partial reduction. The latter component exists only as trace amounts in the native enzyme. Two additional minor native forms were identified as hydrophilic dimer and monomer. Although a significant proportion of AChE was only solubilized in high salt, following extractions in low salt, this high salt-soluble fraction contained the same molecular forms as the low salt-soluble fractions: thus, we did not detect any molecular form resembling the asymmetric forms of vertebrate cholinesterases.
ESTHER : Toutant_1988_J.Neurochem_50_209
PubMedSearch : Toutant_1988_J.Neurochem_50_209
PubMedID: 3121787

Title : Acetylcholinesterases from Musca domestica and Drosophila melanogaster brain are linked to membranes by a glycophospholipid anchor sensitive to an endogenous phospholipase - Fournier_1988_J.Neurochem_50_1158
Author(s) : Fournier D , Berge JB , Cardoso de Almeida ML , Bordier C
Ref : Journal of Neurochemistry , 50 :1158 , 1988
Abstract : The sensitivity of acetylcholinesterases (AChEs) from Musca domestica and from Drosophila melanogaster to the phosphatidylinositol-specific phospholipase C from Bacillus cereus and to the glycosylphosphatidylinositol-specific phospholipase C from Trypanosoma brucei was investigated. B. cereus phospholipase C solubilizes membrane-bound AChE, and both phospholipases convert amphiphilic AChEs into hydrophilic forms of the enzyme. The lipases uncover an immunological determinant that is found on other glycosylphosphatidylinositol-anchored membrane proteins after the same treatment. This immunological determinant is also present on the native hydrophilic form of AChE. The polypeptide bearing the active site of the membrane-bound enzyme migrates faster during sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the same polypeptide from the soluble enzyme. We conclude that AChE from insect brain is attached to membranes via a glycophospholipid anchor. This anchor is covalently linked to the polypeptide bearing the active esterase site of the enzyme and can be cleaved by an endogenous lipase.
ESTHER : Fournier_1988_J.Neurochem_50_1158
PubMedSearch : Fournier_1988_J.Neurochem_50_1158
PubMedID: 2831298

Title : Analysis of methidathion resistance mechanisms in Phytoseiulus persimilis A.H. - Fournier_1987_Pestic.Biochem.Physiol_28_271
Author(s) : Fournier D , Cuany A , Pralavorio M , Bride JM , Berge JB
Ref : Pesticide Biochemistry and Physiology , 28 :271 , 1987
Abstract : A resistant strain of Phytoseiulus persimilis selected by methidathion pressure for several years metabolizes the [14C]methidathion faster than does the corresponding susceptible strain. The metabolism is for the main part glutathione dependent and gives the methidathion conjugate on glutathione as a first metabolite: S[5-methoxy-2-oxo-1,3,4-thiadiazol-3(2H)-yl]-l-glutathione. In addition, glutathione transferase with chlorodinitrobenzene as a substrate has a threefold lower Km in R strain than in S strain. Furthermore, this reaction is competitively inhibited by methidathion with a Ki which is threefold lower in R than in S strain. These results indicated that in this strain of P. persimilis resistance is due to an elevated detoxication of methidathion by a glutathione transferase. Other parameters known to be able to induce resistance in arthropods have been compared in resistant and sensitive strains. Esterase and monooxygenase activity measured with chromogenic substrates are the same in the two strains as is the level of acetylcholinesterase and its inhibition by methidathion oxon. No difference between the two strains has been found in the penetration kinetics measured with [14C]methidathion. These results indicated that glutathione transferase is the only mechanism which has been selected in P. persimilis, although other mechanisms are known to be involved in resistance to other insecticides in phytoseiid mites.
ESTHER : Fournier_1987_Pestic.Biochem.Physiol_28_271
PubMedSearch : Fournier_1987_Pestic.Biochem.Physiol_28_271

Title : Molecular polymorphism of head acetylcholinesterase from adult houseflies (Musca domestica L.) - Fournier_1987_J.Neurochem_49_1455
Author(s) : Fournier D , Cuany A , Bride JM , Berge JB
Ref : Journal of Neurochemistry , 49 :1455 , 1987
Abstract : Acetylcholinesterase (AChE) from housefly heads was purified by affinity chromatography. Three different native forms were separated by electrophoresis on polyacrylamide gradient gels. Two hydrophilic forms presented apparent molecular weights of 75,000 (AChE1) and 150,000 (AChE2). A third component (AChE3) had a migration that depended on the nature and concentration of detergents. In the presence of sodium deoxycholate in the gel, AChE3 showed an apparent molecular weight very close to that of AChE2. Among the three forms, AChE3 was the only one found in purified membranes. The relationships among the various forms were investigated using reduction with 2-mercaptoethanol or proteolytic treatments. Such digestion converted purified AChE3 into AChE2 and AChE1, and reduction of AChE3 and AChE2 by 2-mercaptoethanol gave AChE1, in both cases with a significant loss of activity. These data indicate that the three forms of purified AChE may be classified as an active hydrophilic monomeric unit (G1) plus hydrophilic and amphiphilic dimers. These two components were termed G2s and G2m, where "s" refers to soluble and "m" to membrane bound.
ESTHER : Fournier_1987_J.Neurochem_49_1455
PubMedSearch : Fournier_1987_J.Neurochem_49_1455
PubMedID: 3668532

Title : Identification of resistance mechanisms in Culex pipiens (Diptera: Culicidae) from southern France: insensitive acetylcholinesterase and detoxifying oxidases -
Author(s) : Raymond M , Fournier D , Bride JM , Cuany A , Berge JB , Magnin M , Pasteur N
Ref : J Econ Entomol , 79 :1452 , 1986
PubMedID: 3805481

Title : Single-mosquito test to determine genotypes with an acetylcholinesterase insensitive to inhibition to propoxur insecticide - Raymond_1985_J.Am.Mosquito.Control.Assoc_1_425
Author(s) : Raymond M , Fournier D , Berge JB , Cuany A , Bride JM , Pasteur N
Ref : Journal of the American Mosquito Control Association , 1 :425 , 1985
Abstract : A sensitive technique allowing to identify the three genotypes (AceSS, AceRR and AceRS) of the Ace gene existing in natural populations of Culex pipiens in southern France is described. The technique is based on the comparison of AChE (acetylcholinesterase) activity in 3 equal aliquots taken from the homogenate of a single mosquito (a) in absence of inhibitor (RA), (b) in presence of eserine that inhibits the AChE encoded by AceS and AceR alleles (RI) and (c) in presence of a concentration of propoxur inhibiting the AChE coded by the AceS allele but not by the AceR allele (RG). The mosquito tested is AceSS when RG = RI, AceRR when RG = RA and AceRS when RI less than RG less than RA.
ESTHER : Raymond_1985_J.Am.Mosquito.Control.Assoc_1_425
PubMedSearch : Raymond_1985_J.Am.Mosquito.Control.Assoc_1_425
PubMedID: 3880259