Grassi J


Full name : Grassi Jacques

First name : Jacques

Mail : Service de Pharmacologie et d'Immunologie LERI\/SPI\/DRM Batiment 136, CEA Saclay

Zip Code : 91191

City : Saclay

Country : France

Email :

Phone :

Fax :

Website :

Directory :

References (48)

Title : Molecular Characterization of Monoclonal Antibodies that Inhibit Acetylcholinesterase by Targeting the Peripheral Site and Backdoor Region - Bourne_2013_PLoS.One_8_e77226
Author(s) : Bourne Y , Renault L , Essono S , Mondielli G , Lamourette P , Boquet D , Grassi J , Marchot P
Ref : PLoS ONE , 8 :e77226 , 2013
Abstract : The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9A-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis.
ESTHER : Bourne_2013_PLoS.One_8_e77226
PubMedSearch : Bourne_2013_PLoS.One_8_e77226
PubMedID: 24146971

Title : Quantitative immunoassay to measure plasma and intracellular atazanavir levels: analysis of drug accumulation in cultured T cells - Roucairol_2007_Antimicrob.Agents.Chemother_51_405
Author(s) : Roucairol C , Azoulay S , Nevers MC , Creminon C , Lavrut T , Garraffo R , Grassi J , Burger A , Duval D
Ref : Antimicrobial Agents & Chemotherapy , 51 :405 , 2007
Abstract : We have developed an enzyme immunoassay to measure atazanavir (ATV) levels in plasma and cells. Anti-ATV polyclonal antibodies were raised in rabbits by using a synthetic ATV derivative coupled to bovine serum albumin as the immunogen, and the enzyme tracer was prepared by chemically coupling the ATV derivative with acetylcholinesterase. These reagents were used to develop a sensitive competitive enzyme immunoassay performed in microtitration plates, and the lowest limit of quantification was 150 pg/ml, which is about 10 times more sensitive than previously published techniques. The plasma assay was performed, after a simple methanol extraction, with a minimum of 30 microl of plasma. This assay showed good precision and efficiency, since the rates of recovery from human plasma and cell extracts spiked with ATV ranged form 93 to 113%, with coefficients of variation of less than 10%. ATV concentrations were measured in peripheral blood mononuclear cells incubated with various ATV concentrations and in CEM cells in the absence or presence of antiretroviral drugs and drug transporter inhibitors. The results indicated a dose-dependent uptake (intracellular concentration/extracellular concentration ratio range, 0.04 to 19). A significant increase in the accumulation of ATV was noticed in the presence of P-glycoprotein and MRP1 inhibitors (dipyridamole, inter alia). Interestingly, efavirenz significantly increased the baseline accumulation of ATV, whereas nevirapine induced a marked reduction. This new enzyme immunoassay for measuring plasma and intracellular ATV levels was fully validated and provides an inexpensive and useful tool for routine therapeutic drug monitoring. Moreover, in vitro results suggested the implication of drug transporters and interactions with other antiviral drugs that should be further explored in human immunodeficiency virus-infected patients.
ESTHER : Roucairol_2007_Antimicrob.Agents.Chemother_51_405
PubMedSearch : Roucairol_2007_Antimicrob.Agents.Chemother_51_405
PubMedID: 17116661

Title : An enzyme immunoassay for the quantification of plasma and intracellular lopinavir in HIV-infected patients - Azoulay_2004_J.Immunol.Methods_295_37
Author(s) : Azoulay S , Nevers MC , Creminon C , Heripret L , Garraffo R , Durant J , Dellamonica P , Grassi J , Guedj R , Duval D
Ref : Journal of Immunological Methodsods , 295 :37 , 2004
Abstract : The protease inhibitor lopinavir (LPV; [1S-[1R*(R*), 3R*,4R*]]-N-[4-[[(2,6-dimethylphenoxy)-acetyl]amino]-3-hydroxy-5-phenyl-1-(phenyl methyl) pentyl] tetrahydro-alpha-(1-methylethyl)-2-oxo-1(2H)-pyrimide acetamide) is widely used in anti-human immunodeficiency virus (HIV) therapy. Knowledge of the plasma and intracellular concentrations of the drug would be useful for a better understanding of LPV action and for therapeutic monitoring. The aim of this study was to develop a sensitive and specific immunoassay for LPV in plasma and cells. Anti-LPV polyclonal antibodies were raised in rabbits using a synthetic LPV derivative coupled to keyhole limpet hemocyanin (KLH) as immunogen. The enzyme tracer was prepared by chemically coupling the LPV derivative with acetylcholinesterase. These reagents were used to develop a competitive enzyme immunoassay (EIA) performed in microtitration plates. The assay was performed on a minimum of 50 microl of plasma or 2 x 10(6) cells. It showed good precision and efficiency in as much as recovery from human plasma and cell extracts spiked with LPV ranged between 87% and 104%, with coefficients of variation of less than 10%. The limit of detection (LOD) was 100 pg/ml, i.e., a value at least 10 times lower than those currently achieved using previously described techniques. Cross-validation with high-performance liquid chromatography (HPLC) revealed a good correlation between methods (r2=0.96). Intracellular concentrations of LPV were measured in cultured human T lymphoblastoid cells (CEM). A pharmacokinetic analysis of plasma and intracellular LPV was performed on a healthy volunteer, and measurements were done in patients infected with HIV. The results obtained indicated a high intracellular/extracellular concentration ratio in cultured cells (19.3) but not in cells from HIV patients (1.3). In contrast, in peripheral blood mononuclear cells (PBMC) the accumulation of ritonavir (RTV) was six times higher than LPV. To date, this is the first reported immunoassay for LPV, and this method is sensitive enough for monitoring plasma and intracellular LPV levels in HIV-infected patients and for intracellular studies.
ESTHER : Azoulay_2004_J.Immunol.Methods_295_37
PubMedSearch : Azoulay_2004_J.Immunol.Methods_295_37
PubMedID: 15627609

Title : Sensitive enzyme immunoassay for measuring plasma and intracellular nevirapine levels in human immunodeficiency virus-infected patients - Azoulay_2004_Antimicrob.Agents.Chemother_48_104
Author(s) : Azoulay S , Nevers MC , Creminon C , Heripret L , Durant J , Dellamonica P , Grassi J , Guedj R , Duval D
Ref : Antimicrobial Agents & Chemotherapy , 48 :104 , 2004
Abstract : We have developed an enzyme immunoassay to measure nevirapine (NVP) in plasma and peripheral blood mononuclear cells. Anti-NVP polyclonal antibodies were raised in rabbits by using a synthetic NVP derivative coupled to keyhole limpet hemocyanin as the immunogen, and the enzyme tracer was prepared by chemically coupling the NVP derivative with acetylcholinesterase. These reagents were used to develop a sensitive competitive enzyme immunoassay performed in microtitration plates with a 100-pg ml(-1) limit of detection and thus approximately 100 times more sensitive than previously published techniques. The plasma assay was performed directly without extraction (in this case, a 500-pg ml(-1) limit of detection was observed) on a minimum of 30 micro l of plasma. This assay shows good precision and efficiency, since recovery from human plasma and cell extracts spiked with NVP ranged between 87 and 104%, with coefficients of variation of <10%. A pharmacokinetic analysis of plasma NVP was performed for seven patients infected with human immunodeficiency virus (HIV), and it gave results similar to published findings. Intracellular concentrations of NVP were measured in cultured human T-lymphoblastoid cells and peripheral blood mononuclear cells from HIV-infected patients. The results indicated a very low intracellular/extracellular concentration ratio (0.134), thus demonstrating the absence of intracellular drug accumulation. This is the first intracellular assay of a nonnucleoside reverse-transcriptase inhibitor, and this method could be useful in monitoring plasma and intracellular NVP levels in HIV-infected patients.
ESTHER : Azoulay_2004_Antimicrob.Agents.Chemother_48_104
PubMedSearch : Azoulay_2004_Antimicrob.Agents.Chemother_48_104
PubMedID: 14693526

Title : Design and synthesis of chemiluminescent probes for the detection of cholinesterase activity - Sabelle_2002_J.Am.Chem.Soc_124_4874
Author(s) : Sabelle S , Renard PY , Pecorella K , de Suzzoni-Dezard S , Creminon C , Grassi J , Mioskowski C
Ref : Journal of the American Chemical Society , 124 :4874 , 2002
Abstract : Acetylcholinesterase is one of the most widely used and studied enzymes. Not only does this enzyme regulate neurotransmission (and thus play a key role in neurodegenerative processes) but it is also a prime target for pest control agents and warfare agents. Above all, due to its particularly high turnover rate, acetylcholinesterase is among the most efficient reporter enzymes yet described (for use as enzymatic tracer in immunoassays, for instance). However, its activity is detected through a colorimetric reagent, the Ellman reagent, which displays low detection limits and is often subject to background perturbations. In the course of our search for a more sensitive detection assay, we describe here a first-generation 1,2-dioxetane chemiluminescent probe, based on chemically induced electron exchange luminescence, which has an approximately 10 times lower detection limit than the Ellman colorimetric assay (2.5 x 10(-19) mol for Electrophorus electricus AChE in its tetrameric form).
ESTHER : Sabelle_2002_J.Am.Chem.Soc_124_4874
PubMedSearch : Sabelle_2002_J.Am.Chem.Soc_124_4874
PubMedID: 11971738

Title : Use of free radical chemistry in an immunometric assay for 17 beta-estradiol - Buscarlet_2001_Clin.Chem_47_102
Author(s) : Buscarlet L , Volland H , Dupret-Carruel J , Jolivet M , Grassi J , Creminon C , Taran F , Pradelles P
Ref : Clinical Chemistry , 47 :102 , 2001
Abstract : BACKGROUND: We wished to develop an enzyme immunometric assay for 17 beta-estradiol (E2) in human serum using solid-phase immobilized epitope immunoassay (SPIE-IA) technology and free radical chemistry.
METHODS: We used an anti-estradiol monoclonal antibody as capture antibody and Fenton-like reagents to cross-link it to E2. The same antibody, labeled with acetylcholinesterase, was used for detection. Serum was diluted 10-fold before assay.
RESULTS: After correction by the dilution factor, the detection limit was 5 ng/L for human serum and intra- and interassay CVs were <7% and 15%, respectively, at concentrations of 169-2845 ng/L. No cross-reactivity was seen with other natural steroids. In comparison with a competitive commercial RIA performed on 88 undiluted human sera, the slope (SD) of the regression line was 1.05 (+/- 0.02) and the intercept was 47 (+/-27) ng/L (S(y/x) = 186 ng/L) at concentrations of 20-5000 ng/L (r(2) = 0.97).
CONCLUSIONS: The use of Fenton-like chemistry in SPIE-IA technology allows a sensitive measurement of E2 in human serum and could be a new approach for the development of sensitive immunoassays.
ESTHER : Buscarlet_2001_Clin.Chem_47_102
PubMedSearch : Buscarlet_2001_Clin.Chem_47_102
PubMedID: 11148184

Title : Toward antibody-catalyzed hydrolysis of organophosphorus poisons - Vayron_2000_Proc.Natl.Acad.Sci.U.S.A_97_7058
Author(s) : Vayron P , Renard PY , Taran F , Creminon C , Frobert Y , Grassi J , Mioskowski C
Ref : Proc Natl Acad Sci U S A , 97 :7058 , 2000
Abstract : We report here our preliminary results on the use of catalytic antibodies as an approach to neutralizing organophosphorus chemical weapons. A first-generation hapten, methyl-alpha-hydroxyphosphinate Ha, was designed to mimic the approach of an incoming water molecule for the hydrolysis of exceedingly toxic methylphosphonothioate VX (1a). A moderate protective activity was first observed on polyclonal antibodies raised against Ha. The results were further confirmed by using a mAb PAR 15 raised against phenyl-alpha-hydroxyphosphinate Hb, which catalyzes the hydrolysis of PhX (1b), a less toxic phenylphosphonothioate analog of VX with a rate constant of 0.36 M(-1) x min(-1) at pH 7.4 and 25 degrees C, which corresponds to a catalytic proficiency of 14,400 M(-1) toward the rate constant for the uncatalyzed hydrolysis of 1b. This is a demonstration on the organophosphorus poisons themselves that mAbs can catalytically hydrolyze nerve agents, and a significant step toward the production of therapeutically active abzymes to treat poisoning by warfare agents.
ESTHER : Vayron_2000_Proc.Natl.Acad.Sci.U.S.A_97_7058
PubMedSearch : Vayron_2000_Proc.Natl.Acad.Sci.U.S.A_97_7058
PubMedID: 10860971

Title : Evaluation of a high IgE-responder mouse model of allergy to bovine beta-lactoglobulin (BLG): development of sandwich immunoassays for total and allergen-specific IgE, IgG1 and IgG2a in BLG-sensitized mice - Adel-Patient_2000_J.Immunol.Methods_235_21
Author(s) : Adel-Patient K , Creminon C , Bernard H , Clement G , Negroni L , Frobert Y , Grassi J , Wal JM , Chatel JM
Ref : Journal of Immunological Methods , 235 :21 , 2000
Abstract : An animal model of food allergy represents an important tool for studying the mechanisms of induction and repression of an allergic reaction, as well as for the development of an immunotherapy to prevent or minimize such an adverse reaction. IgE and IgG1 (Th2 response) vs. IgG2a (Th1 response) are good markers for the induction of an allergic response in mice. Nevertheless, while the total serum concentrations of these isotypes are easy to measure using classical sandwich immunoassays, this is not the case for allergen-specific isotypes. To develop an animal model of allergy to bovine beta-lactoglobulin (BLG), we set up quantitative assays for total and for allergen-specific IgE, IgG1 and IgG2a. Microtiter plates coated either with anti-isotype antibodies (Abs) or with allergen were used for Ab capture, while anti-isotype Fab' fragments coupled to acetylcholinesterase were used for visualization. These assays of anti-BLG specific Abs are original in two ways. First, assay calibration is performed using anti-BLG specific mAbs, thus allowing good quantification of the different isotypes and subclasses of serum antibodies. Second, the detection of all anti-BLG specific Abs, i.e., those recognizing both the native and denatured forms of the protein, is achieved through indirect coating of BLG using biotin-streptavidin binding. The present assays are quantitative, specific to the isotype (cross-reactivity <0.5%), very sensitive (detection limit in the 10 pg/ml range), and reproducible (coefficient of variation less than 10%). Applied to the humoral response in mice sensitized with BLG adsorbed on alum, these assays proved to be a very useful tool for monitoring high IgE-responder mice following BLG immunization, and for an immunotherapy directed at polarizing the immune response.
ESTHER : Adel-Patient_2000_J.Immunol.Methods_235_21
PubMedSearch : Adel-Patient_2000_J.Immunol.Methods_235_21
PubMedID: 10675754

Title : Ultrastructural evidence for dendritic release of acetylcholinesterase in the rat substantia nigra - Anglade_1999_Folia.Histochem.Cytobiol_37_243
Author(s) : Anglade P , Grassi J , Motelica-Heino I , Hashikawa T , Tsuji S
Ref : Folia Histochemica et Cytobiologica , 37 :243 , 1999
Abstract : Morphological evidence for dendritic secretion of acetylcholinesterase (AChE) in rat substantia nigra--a physiologically known phenomenon--was searched by means of a modified cytochemical method devised for fine localization of AChE activity at the electron microscopic level. DAB precipitate was observed in cluster of small vesicles in contact with the plasma membrane and in the extracellular space in the vicinity of the vesicles. Single coated or uncoated large vesicles filled with stained material were found in the cytoplasm of the dendrites at distance from or in contact with the plasma membrane. Immunoperoxidase staining with specific anti-serum against rat AChE gave similar localization of AChE. These results suggest that AChE is released from the dendrites of the nigral neurons by a process of vesicular exocytosis and captured by endocytosis. The relation of this process to a putative release from the smooth endoplasmic reticulum remains to be elucidated.
ESTHER : Anglade_1999_Folia.Histochem.Cytobiol_37_243
PubMedSearch : Anglade_1999_Folia.Histochem.Cytobiol_37_243
PubMedID: 10598325

Title : The binding sites of inhibitory monoclonal antibodies on acetylcholinesterase. Identification of a novel regulatory site at the putative back door - Simon_1999_J.Biol.Chem_274_27740
Author(s) : Simon S , Le Goff A , Frobert Y , Grassi J , Massoulie J
Ref : Journal of Biological Chemistry , 274 :27740 , 1999
Abstract : We investigated the target sites of three inhibitory monoclonal antibodies on Electrophorus acetylcholinesterase (AChE). Previous studies showed that Elec-403 and Elec-410 are directed to overlapping but distinct epitopes in the peripheral site, at the entrance of the catalytic gorge, whereas Elec-408 binds to a different region. Using Electrophorus/rat AChE chimeras, we identified surface residues that differed between sensitive and insensitive AChEs: the replacement of a single Electrophorus residue by its rat homolog was able to abolish binding and inhibition, for each antibody. Reciprocally, binding and inhibition by Elec-403 and by Elec-410 could be conferred to rat AChE by the reverse mutation. Elec-410 appears to bind to one side of the active gorge, whereas Elec-403 covers its opening, explaining why the AChE-Elec-410 complex reacts faster than the AChE-Elec-403 or AChE-fasciculin complexes with two active site inhibitors, m-(N,N, N-trimethyltammonio)trifluoro-acetophenone and echothiophate. Elec-408 binds to the region of the putative "back door," distant from the peripheral site, and does not interfere with the access of inhibitors to the active site. The binding of an antibody to this novel regulatory site may inhibit the enzyme by blocking the back door or by inducing a conformational distortion within the active site.
ESTHER : Simon_1999_J.Biol.Chem_274_27740
PubMedSearch : Simon_1999_J.Biol.Chem_274_27740
PubMedID: 10488117

Title : Conformational flexibility of the acetylcholinesterase tetramer suggested by x-ray crystallography - Bourne_1999_J.Biol.Chem_274_30370
Author(s) : Bourne Y , Grassi J , Bougis PE , Marchot P
Ref : Journal of Biological Chemistry , 274 :30370 , 1999
Abstract : Acetylcholinesterase, a polymorphic enzyme, appears to form amphiphilic and nonamphiphilic tetramers from a single splice variant; this suggests discrete tetrameric arrangements where the amphipathic carboxyl-terminal sequences can be either buried or exposed. Two distinct, but related crystal structures of the soluble, trypsin-released tetramer of acetylcholinesterase from Electrophorus electricus were solved at 4.5 and 4.2 A resolution by molecular replacement. Resolution at these levels is sufficient to provide substantial information on the relative orientations of the subunits within the tetramer. The two structures, which show canonical homodimers of subunits assembled through four-helix bundles, reveal discrete geometries in the assembly of the dimers to form: (a) a loose, pseudo-square planar tetramer with antiparallel alignment of the two four-helix bundles and a large space in the center where the carboxyl-terminal sequences may be buried or (b) a compact, square nonplanar tetramer that may expose all four sequences on a single side. Comparison of these two structures points to significant conformational flexibility of the tetramer about the four-helix bundle axis and along the dimer-dimer interface. Hence, in solution, several conformational states of a flexible tetrameric arrangement of acetylcholinesterase catalytic subunits may exist to accommodate discrete carboxyl-terminal sequences of variable dimensions and amphipathicity.
ESTHER : Bourne_1999_J.Biol.Chem_274_30370
PubMedSearch : Bourne_1999_J.Biol.Chem_274_30370
PubMedID: 10521413
Gene_locus related to this paper: eleel-ACHE

Title : A sensitive sandwich enzyme immunoassay for calcitonin gene-related peptide (CGRP): characterization and application - Frobert_1999_Peptides_20_275
Author(s) : Frobert Y , Nevers MC , Amadesi S , Volland H , Brune P , Geppetti P , Grassi J , Creminon C
Ref : Peptides , 20 :275 , 1999
Abstract : Thirty mouse monoclonal antibodies (mAbs) directed against rat calcitonin gene-related peptide-alpha (CGRP-alpha) have been obtained. These mAbs are classified in 2 groups, one recognizing the peptide N-terminus and the other binding the C-terminus. A two-site immunometric assay was developed using mAb CGRP-83 as capture antibody, whereas mAb CGRP-72 acts as tracer, covalently labeled with enzyme acetylcholinesterase. This assay appeared sensitive (limit of detection: 2 pg/ml) and precise, allowing quantitative measurement of all human and murine CGRP isoforms. The assay was used to determine specific concentrations of CGRP in different rat, mice and guinea pig samples. The validity of the test was demonstrated by HPLC fractionation experiments.
ESTHER : Frobert_1999_Peptides_20_275
PubMedSearch : Frobert_1999_Peptides_20_275
PubMedID: 10422884

Title : A new specific enzyme immunoassay allowing an efficient pharmacokinetic evaluation of gamma-cyclodextrin after intravenous administration to rats - Creminon_1999_Pharm.Res_16_1407
Author(s) : Creminon C , Djedaini-Pilard F , Vienet R , Pean C , Grognet JM , Grassi J , Perly B , Pradelles P
Ref : Pharm Res , 16 :1407 , 1999
Abstract : PURPOSE Because of its ability to form complexes with drugs, gamma-cyclodextrin is of great potential value in pharmaceutical formulations. The biological fate of y-cyclodextrin must therefore be considered in safety evaluation, using sensitive and specific methods applicable to biological fluids. METHODS: Antibodies were raised against gamma-cyclodextrin, allowing the development of a new enzyme immunoassay. The analytical characteristics of this assay were evaluated. Rats were given a single intravenous 25 mg/kg dose of gamma-cyclodextrin. Plasma and urine samples were collected and assayed. RESULTS: This new enzyme immunoassay was sensitive (limit of detection close to 94 pg/mL) and suitable for quantification of gamma-cyclodextrin in urine and plasma after methanol extraction. The use of different linear and cyclic compounds demonstrated the high specificity of the assay. After i.v. administration, the concentration of gamma-cyclodextrin rapidly decreased in the plasma while the molecule was probably distributed into the tissues. Although urinary elimination predominates, only 50% of the injected gamma-cyclodextrin was recovered in urine, suggesting enzymatic degradation and/or tissular storage.
CONCLUSIONS: This assay may provide important information on the fate of gamma-cyclodextrin inclusion complexes dedicated to drug-delivery using various modes of administration (oral, parenteral, transmucosal or dermal).
ESTHER : Creminon_1999_Pharm.Res_16_1407
PubMedSearch : Creminon_1999_Pharm.Res_16_1407
PubMedID: 10496657

Title : A solid-phase immobilized epitope immunoassay (SPIE-IA) permitting very sensitive and specific measurement of angiotensin II in plasma - Volland_1999_J.Immunol.Methods_228_37
Author(s) : Volland H , Pradelles P , Ronco P , Azizi M , Simon D , Creminon C , Grassi J
Ref : Journal of Immunological Methods , 228 :37 , 1999
Abstract : We have developed a new enzyme immunometric assay for angiotensin II (AII) based on SPIE-IA technology (solid-phase immobilized epitope-immunoassay). A monoclonal antibody with optimal properties (mAb3 131) was selected from a series of 19 anti-AII mAbs. The mAb had to be purified from ascitic fluid in a specific manner in order to remove endogenous AII from the antibody-binding sites. We established a sensitive (minimum detectable concentration 0.5 pg/ml) and precise (CV below 15% in the 2-100 pg/ml range) SPIE-IA. Using different AII-related peptides, we observed that this new assay has a specificity profile that compares favourably with the corresponding competitive immunoassay. We have used the assay to measure AII in 42 plasma samples, and demonstrated a good correlation with values obtained using a commercial radioimmunoassay. Assay specificity was supported by HPLC fractionation experiments, confirming the absence of interference induced by endogenous AII-related products.
ESTHER : Volland_1999_J.Immunol.Methods_228_37
PubMedSearch : Volland_1999_J.Immunol.Methods_228_37
PubMedID: 10556541

Title : Acetylcholinesterase in Elapid Snakes -
Author(s) : Cousin X , Bon S , Grassi J , Massoulie J , Bon C
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. :99 , 1998

Title : Inhibition of Electrophorus acetylcholinesterase by monoclonal antibodies -
Author(s) : Simon S , Le Goff A , Frobert Y , Grassi J , Massoulie J
Ref : Journal de Physiologie (Paris) , 92 :492 , 1998

Title : Monitoring of intracellular levels of 5'-monophosphate-AZT using an enzyme immunoassay - Goujon_1998_J.Immunol.Methods_218_19
Author(s) : Goujon L , Brossette T , Dereudre-Bosquet N , Creminon C , Clayette P , Dormont D , Mioskowski C , Lebeau L , Grassi J
Ref : Journal of Immunological Methods , 218 :19 , 1998
Abstract : We have developed a competitive enzyme immunoassay suitable for routine monitoring of intracellular levels of 5'-monophosphate-AZT (AZT-MP). This assay is performed in 96-well microtiter plates coated with anti-rabbit immunoglobulin antibodies and is based on the use of rabbit polyclonal antibodies raised against an AZT-MP analog and of an AZT-MP/acetylcholinesterase conjugate as tracer. It is very sensitive, with a detection limit close to 0.1 ng/ml (0.2 pmol/ml), and precise (CV < 20% from 20 to 0.3 ng/ml). Very low cross-reactivities were observed with AZT and the corresponding di- and triphosphate derivatives as well as with other related nucleotides and nucleosides. The validity of the assay was demonstrated by measuring intracellular concentrations of AZT-MP in peripheral blood mononuclear cells (PBMCs) and in monocyte-derived macrophages (MDMs) cultured in the presence of various concentrations of AZT (from 0.01 microM to 10 microM). We observed very high levels of AZT-MP in stimulated (PHA + IL2) PBMCs (> 100 pmol/10(6) cells) while, as expected, much lower concentrations were measured in resting PBMCs or MDMs (0.1 to 2 pmol/10(6) cells). The assay constitutes a very convenient tool permitting easy, precise studies of the first step of the intracellular metabolism of AZT leading to the formation of AZT-TP in cultured cells.
ESTHER : Goujon_1998_J.Immunol.Methods_218_19
PubMedSearch : Goujon_1998_J.Immunol.Methods_218_19
PubMedID: 9819120

Title : Screening of monoclonal antibodies using antigens labeled with acetylcholinesterase -
Author(s) : Frobert Y , Grassi J
Ref : Methods in Molecular Biology , 80 :57 , 1998
PubMedID: 9664363

Title : A competitive enzyme hybridization assay for plasma determination of phosphodiester and phosphorothioate antisense oligonucleotides - Deverre_1997_Nucleic.Acids.Res_25_3584
Author(s) : Deverre JR , Boutet V , Boquet D , Ezan E , Grassi J , Grognet JM
Ref : Nucleic Acids Research , 25 :3584 , 1997
Abstract : An enzyme competitive hybridization assay was developed and validated for determination of mouse plasma concentrations of a 15mer antisense phosphodiester oligodeoxyribonucleotide and of two phosphorothioate analogs. Assays were performed in 96-well microtiter plates. The phosphodiester sense sequence was covalently bound to the microwells. The 5'-biotinylated antisense sequence was used as tracer. The principle of the assay involves competitive hybridization of tracer and antisense nucleotide to the solid phase-immobilized sense oligonucleotide. Solid phase- bound tracer oligonucleotide was assayed after reaction with a streptavidin-acetylcholinesterase conjugate, using the colorimetric method of Ellman. As in competitive enzyme immunoassays, coloration was inversely related to the amount of analyte initially present in the sample. The limit of quantification was 900 pM for phosphodiester antisense oligonucleotide using a 100 microl volume of plasma without extraction. Cross-reactivity was negligible after a four base deletion in either the 3'or 5'position. The assay was simple and sensitive, suitable for in vitro screening of oligonucleotide hybridization potency in biological fluids and for measuring the plasma pharmacokinetics of phosphorothioate and phosphodiester sequences.
ESTHER : Deverre_1997_Nucleic.Acids.Res_25_3584
PubMedSearch : Deverre_1997_Nucleic.Acids.Res_25_3584
PubMedID: 9278477

Title : Acetylcholinesterases from Elapidae snake venoms: biochemical, immunological and enzymatic characterization - Frobert_1997_Biochim.Biophys.Acta_1339_253
Author(s) : Frobert Y , Creminon C , Cousin X , Remy MH , Chatel JM , Bon S , Bon C , Grassi J
Ref : Biochimica & Biophysica Acta , 1339 :253 , 1997
Abstract : We analyzed 45 batches of venom from 20 different species belonging to 11 genera from the 3 main families of venomous snakes (Elapidae, Viperidae and Crotalidae). We found high acetylcholinesterase (AChE) activity in all venoms from Elapidae, except in those from the Dendroaspis genus. AChE was particularly abundant in Bungarus venoms which contain up to 8 mg of enzyme per gram of dried venom. We could not detect acetylcholinesterase activity in any batch of venom from Viperidae or Crotalidae. Titration of active sites with an organophosphorous agent (MPT) revealed that the AChE of all venoms have similar turnovers (6000 to 8000 s(-1)) which are clearly higher than those of Torpedo and mammalian enzymes but lower than that of Electrophorus. AChEs from the venom of elapid snakes of the Bungarus, Naja, Ophiophagus and Haemacatus genera were purified by affinity chromatography. SDS-PAGE analysis and sucrose gradient centrifugation demonstrated that AChE is exclusively present as a nonamphiphilic monomer. These enzymes are true AChEs, hydrolyzing acetylthiocholine faster than propionylthiocholine and butyrylthiocholine and exhibiting excess substrate inhibition. Twenty-seven different monoclonal antibodies directed against AChE from Bungarus fasciatus venom were raised in mice. Half of them recognized exclusively the Bungarus enzyme while the others cross-reacted with AChEs from other venoms. Polyspecific mAbs were used to demonstrate that venoms from Dendroaspis, which contain the AChE inhibitor fasciculin but lack AChE activity, were also devoid of immunoreactive AChE protein. AChE inhibitors acting at the active site (edrophonium, tacrine) and at the peripheral site (propidium, fasciculin), as well as bis-quaternary ligands (BW284C51, decamethonium), were tested against the venom AChEs from 11 different species. All enzymes had a very similar pattern of reactivity with regard to the different inhibitors, with the exception of fasciculin. AChEs from Naja and Haemacatus venoms were relatively insensitive to fasciculin inhibition (IC50 >> 10(-6) M), while Bungarus (IC50 approximately 10(-8) M) and especially Ophiophagus (IC50 < 10(-10) M) AChEs were inhibited very efficiently. Ophiophagus and Bungarus AChEs were also efficiently inhibited by a monoclonal antibody (Elec-410) previously described as a specific ligand for the Electrophorus electricus peripheral site. Taken together, these results show that the venoms of most Elapidae snakes contain large amounts of a highly active non-amphiphilic monomeric AChE. All snake venom AChEs show strong immunological similarities and possess very similar enzymatic properties. However, they present quite different sensitivity to peripheral site inhibitors, fasciculin and the monoclonal antibody Elec-410.
ESTHER : Frobert_1997_Biochim.Biophys.Acta_1339_253
PubMedSearch : Frobert_1997_Biochim.Biophys.Acta_1339_253
PubMedID: 9187246

Title : Competitive enzyme immunoassay with monoclonal antibody for homovanillic acid measurement in human urine samples - Taran_1997_Clin.Chem_43_363
Author(s) : Taran F , Frobert Y , Creminon C , Grassi J , Olichon D , Mioskowski C , Pradelles P
Ref : Clinical Chemistry , 43 :363 , 1997
Abstract : A fast competitive enzyme immunoassay (EIA) for measuring homovanillic acid in human urine samples was developed with a monoclonal antibody and acetylcholinesterase as enzyme label. Enzyme detection was performed by an easy colorimetric assay. Monoclonal antibodies were screened on the basis of sensitivity, specificity, and correlation studies. EIA has a detection limit of 0.5 micromol/L, a CV <10% in the 1.25-10 micromol/L range, and intra- and interassay CVs of <10%. Cross-reactivity with vanillylmandelic acid was 0.5% and <8% for other structurally related catecholamine metabolites. Parallelism of the EIA was shown in dilution studies and the correlation with routine HPLC assay in 62 normal and pathologic samples was EIA = 1.492 (HPLC) - 3.46, S(y/x), = 47.52, range = 4-1800 micromol/L, r2 = 0.977. Additional data concerning the validity of this assay were provided by HPLC analysis of urinary immunoreactive material.
ESTHER : Taran_1997_Clin.Chem_43_363
PubMedSearch : Taran_1997_Clin.Chem_43_363
PubMedID: 9023141

Title : Acetylcholinesterase from Bungarus venom: a monomeric species - Cousin_1996_FEBS.Lett_387_196
Author(s) : Cousin X , Creminon C , Grassi J , Meflah K , Cornu G , Saliou B , Bon S , Massoulie J , Bon C
Ref : FEBS Letters , 387 :196 , 1996
Abstract : The venom of Bungarus fasciatus, an Elapidae snake, contains a high level of AChE activity. Partial peptide sequences show that it is closely homologous to other AChEs. Bungarus venom AChE is a non-amphiphilic monomeric species, a molecular form of AChE which has not been previously found in significant levels in other tissues. The composition of carbohydrates suggests the presence of N-glycans of the 'complex' and 'hybrid' types. Ion exchange chromatography, isoelectric focusing and electrophoresis in non-denaturing and denaturing conditions reveal a complex microheterogeneity of this enzyme, which is partly related to its glycosylation.
ESTHER : Cousin_1996_FEBS.Lett_387_196
PubMedSearch : Cousin_1996_FEBS.Lett_387_196
PubMedID: 8674549

Title : Extracellular and asymmetric forms of acetylcholinesterase are expressed on cholinergic and noncholinergic terminal neuropil of the developing chick retina - Reiss_1996_Cell.Tiss.Res_286_13
Author(s) : Reiss Y , Kroger S , Grassi J , Tsim KWK , Willbold E , Layer PG
Ref : Cell & Tissue Research , 286 :13 , 1996
Abstract : Only two out of four major acetylcholinesterase (AChE) subbands in the inner plexiform layer (IPL) of vertebrate retinae correspond to sites of cholinergic synaptic transmission, as has been shown by the co-distribution of AChE and choline acetyltransferase (ChAT) staining. The function and molecular identity of AChE in non-cholinergic subbands is unknown. We have used immunocytochemical methods to compare the development of asymmetric or extracellularly localized AChE with that of total AChE and ChAT in embryonic and adult chicken retinae. After injection of the AChE-specific monoclonal antibody 3D10 into the vitreous body of live embryos, a method that labels only extracellular AChE, five subbands in the IPL were labelled, whereas cell somata or their radial processes remained unstained. In contrast, the entire cell including processes was immunoreactive, when the 3D10 antibody was applied to permeabilized cryosections, suggesting that in cell bodies the enzyme is exclusively localized intracellularly. Compared with total AChE, detection of asymmetric AChE with the monoclonal antibody 6B6 was delayed, first being seen in cells of the inner nuclear layer and finally appearing on all subbands, reflecting more closely the course of synaptogenesis. Thus, extracellular and asymmetric forms of AChE are predominantly found on the terminal arbor neuropil of both cholinergic and non-cholinergic IPL subbands. These data show a differential distribution of extra- and intracellular AChE and suggest novel roles for the AChE in non-cholinergic IPL subbands.
ESTHER : Reiss_1996_Cell.Tiss.Res_286_13
PubMedSearch : Reiss_1996_Cell.Tiss.Res_286_13
PubMedID: 8781208

Title : Characterization of monoclonal antibodies that strongly inhibit Electrophorus electricus acetylcholinesterase - Remy_1995_Eur.J.Biochem_231_651
Author(s) : Remy MH , Frobert Y , Grassi J
Ref : European Journal of Biochemistry , 231 :651 , 1995
Abstract : In this study, we describe three different monoclonal antibodies (mAbs Elec-403, Elec-408, and Elec-410) directed against Electrophorus electricus acetylcholinesterase (AChE) which were selected as inhibitors for this enzyme. Two of these antibodies (Elec-403 and Elec-410), recognized overlapping but different epitopes, competed with snake venom toxin fasciculin for binding to the enzyme, and thus apparently recognized the peripheral site of AChE. In addition, the binding of Elec-403 was antagonized by 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284C51) and propidium, indicating that the corresponding epitope encompassed the anionic site involved in the binding of these low-molecular-mass inhibitors. The third mAb (Elec-408), was clearly bound to another site on the AChE molecule, and its inhibitory effect was cumulative with those of Elec-403, Elec-410, and fasciculin. All mAbs bound AChE with high affinity and were as strong inhibitors with an apparent Ki values less than 0.1 nM. Elec-403 was particularly efficient with an inhibitory activity similar to that of fasciculin. Inhibition was observed with both charged (acetylthiocholine) and neutral substrates (o-nitrophenyl acetate) and had the characteristics of a non-competitive process. Elec-403 and Elec-410 probably exert their effect by triggering allosteric transitions from the peripheral site to the active site. The epitope recognized by mAb Elec-408 has not been localized, but it may correspond to a new regulatory site on AChE.
ESTHER : Remy_1995_Eur.J.Biochem_231_651
PubMedSearch : Remy_1995_Eur.J.Biochem_231_651
PubMedID: 7649165

Title : Two-site immunoradiometric assay of chicken acetylcholinesterase: active and inactive molecular forms in brain and muscle - Chatel_1994_J.Neurochem_63_1111
Author(s) : Chatel JM , Eichler J , Vallette FM , Bon S , Massoulie J , Grassi J
Ref : Journal of Neurochemistry , 63 :1111 , 1994
Abstract : Several monoclonal antibodies were raised against chicken acetylcholinesterase (AChE; EC Some of these antibodies react with quail AChE but not with AChEs from nonavian vertebrates or invertebrates and not with butyrylcholinesterase. They may be classified in several mutually compatible groups, i.e., that can bind simultaneously to the monomeric form of AChE. Most antibodies recognize a peptidic domain that does not exist in mammalian AChE and that may be digested by trypsin without loss of activity or dissociation of quaternary structure. The only exception is the antibody C-131, which is conformation dependent and preferentially recognizes active AChE. We have set up two-site immunoradiometric assays, using an immobilized capture antibody, C-6 or C-131, and a radiolabeled antibody, 125I-C-54. The C-6/C-54 assay quantifies the totality of inactive and active AChE subunits: It detects 10(-3) Ellman unit (approximately 40 pg of protein) and yields a linear response up to at least 25 10(-3) Ellman units. An analysis of gradient fractions, using C-6/C-54 and C-131/C-54 assays as well as activity determination, shows that the A12 and G4 forms are exclusively composed of active subunits, whereas inactive molecules cosediment with the active G2 and G1 forms. Both active and inactive G2 and G1 forms are amphiphilic, as indicated by the influence of detergents on their sedimentation coefficients and Stokes radii. In brain, the proportion of inactive forms decreases from 40% at embryonic day 11 (E11) to 20% at birth [day 1 (D1)]. In muscle, we observed no inactive AChE at E11 and a small proportion of inactive G1 at D1.
ESTHER : Chatel_1994_J.Neurochem_63_1111
PubMedSearch : Chatel_1994_J.Neurochem_63_1111
PubMedID: 8051552

Title : Enzyme immunometric assay for leukotriene C4 - Volland_1994_J.Immunol.Meth_175_97
Author(s) : Volland H , Vulliez Le Normand B , Mamas S , Grassi J , Creminon C , Ezan E , Pradelles P
Ref : Journal of Immunological Methods , 175 :97 , 1994
Abstract : An enzyme immunometric assay of LTC4 named SPIE-IA is described. The assay involves different sequential steps: (1) immunocapture of LTC4 by monoclonal anti-LTC4 antibodies coated on 96-well microtiter plates; (2) cross-linking of LTC4 via its amino group to the wells using glutaraldehyde; (3) treatment with HCl; (4) measurement of linked LTC4 using the same monoclonal anti-LTC4 antibodies labeled with acetylcholinesterase. A minimal detectable concentration of 2 pg/ml after 60 min of enzymatic reaction was obtained. Cross-reactivity was less than 15% with LTD4 or LTE4. The coefficient of variation was less than 6% in the 20-1000 pg/ml range. Good correlation was observed between SPIE-IA and a competitive enzyme immunoassay for biological samples. The different sequential steps of the assay are investigated.
ESTHER : Volland_1994_J.Immunol.Meth_175_97
PubMedSearch : Volland_1994_J.Immunol.Meth_175_97
PubMedID: 7930643

Title : Subpicogram determination of oxytocin by an enzyme immunoassay using acetylcholinesterase as label - Marnet_1994_J.Immunoassay_15_35
Author(s) : Marnet PG , Volland H , Pradelles P , Grassi J , Beaufils M
Ref : Journal of Immunoassay , 15 :35 , 1994
Abstract : The pure tetrameric form of Acetylcholinesterase (EC- from the electric eel electrophorus electricus has been covalently coupled to oxytocin. This conjugate has been used as tracer in a heterologous competitive immunoassay. Microtiter plates coated with a mouse monoclonal anti-rabbit immunoglobulin antibody were used to separate bound and free moieties of the tracer. Acetylcholinesterase activity bound to the solid phase was measured by a colorimetric assay. The minimum detectable concentration was 0.075 pg/well (ie 1.5 pg/ml) and precision was less than 8% at concentration above 0.15 pg/well. An extraction step improved sensitivity up to 10 times with good recoveries. To assess the validity of this assay, basal levels of oxytocin were measured during the oestrous cycle of a cow.
ESTHER : Marnet_1994_J.Immunoassay_15_35
PubMedSearch : Marnet_1994_J.Immunoassay_15_35
PubMedID: 8150985

Title : Cholinesterases display genuine arylacylamidase activity but are totally devoid of intrinsic peptidase activities - Checler_1994_J.Neurochem_62_756
Author(s) : Checler F , Grassi J , Vincent JP
Ref : Journal of Neurochemistry , 62 :756 , 1994
Abstract : The purpose of this article was to evaluate the intrinsic character of arylacylamidase and peptidase activities that are often detected along with cholinesterase activities. Various pools of commercial or affinity-purified acetylcholinesterases (AChEs) were examined. Affinity-purified AChE displays esterase- and amidase-specific activities that are similarly enriched when compared with commercial AChE. By contrast, commercial AChE exhibits much higher tryptic-like and carboxypeptidase-specific activities than the affinity-purified enzyme. The parallel enrichment in esterase and arylacylamidase suggests that these two activities are copurified, whereas peptidases do not seem to behave similarly. We show that trypsinolysis or spontaneous degradation of affinity-purified AChE leads to the conversion of the 75-kDa monomer protein into two fragments of 50 and 25 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. However, these modifications are without effect on the esterase, arylacylamidase, and peptidase activities. This clearly shows that AChE does not behave as a zymogen of peptidases that would have been activated on autolysis of AChE. Immunoprecipitation of AChEs with a purified monoclonal antibody directed toward electric eel AChE totally separated the esterase and arylacylamidase activities (pellet) from peptidase activities (supernatant). The immunoprecipitated AChEs could be dissociated from the interaction with IgGs. These resolubilized AChE preparations have kept the same percentage of initial esterase and arylacylamidase activities but were totally devoid of peptidase activities.
ESTHER : Checler_1994_J.Neurochem_62_756
PubMedSearch : Checler_1994_J.Neurochem_62_756
PubMedID: 8294938

Title : A conformation-dependent monoclonal antibody against active chicken acetylcholinesterase - Chatel_1993_FEBS.Lett_319_12
Author(s) : Chatel JM , Vallette FM , Massoulie J , Grassi J
Ref : FEBS Letters , 319 :12 , 1993
Abstract : We show that the C-131 monoclonal antibody, directed against chicken AChE, recognizes active chicken AChE, but not the SDS-denatured or heat-inactivated protein. Previous results indicated that C-131 only binds to the active enzyme, and not to inactive molecules which also occur in the embryonic chicken brain. In contrast with C-131, other monoclonal antibodies obtained in the same series, such as C-6 and C-54, also recognize denatured or inactive AChE. It is noteworthy that these antibodies all seem to react with a trypsin-sensitive peptide which is present in chicken but not in mammalian or Torpedo AChE, whereas the C-131 antibody binds trypsin-modified as well as intact molecules. These results show that C-131 is highly conformation-dependent, specific for active AChE. They confirm our previous conclusion that active and inactive molecules arise from different folding processes.
ESTHER : Chatel_1993_FEBS.Lett_319_12
PubMedSearch : Chatel_1993_FEBS.Lett_319_12
PubMedID: 8454042

Title : Existence of an inactive pool of acetylcholinesterase in chicken brain - Chatel_1993_Proc.Natl.Acad.Sci.U.S.A_90_2476
Author(s) : Chatel JM , Grassi J , Frobert Y , Massoulie J , Vallette FM
Ref : Proceedings of the National Academy of Sciences of the United States of America , 90 :2476 , 1993
Abstract : We analyzed acetylcholinesterase (AcChoEase; EC activity and AcChoEase immunoreactive protein in chicken brain by using five monoclonal antibodies raised against chicken AcChoEase. Four of them specifically recognized AcChoEase catalytic subunits in Western blots and one, C-131, recognized only enzymatically active AcChoEase. We observed considerable differences in the ratio of immunoreactive protein to catalytic activity in various fractions, indicating the existence of inactive AcChoEase protein. This inactive AcChoEase component was more abundant in a low-salt-soluble extract than in a subsequent detergent-soluble extract. On the basis of the ratio between activity and immunoreactivity, we calculated that the inactive component represents about 30% of the total AcChoEase subunits in chicken brain. The immunoreactive AcChoEase protein sedimented in sucrose gradients like the active molecular forms; the G1 and G2 peaks contained inactive molecules, whereas the G4 peak appeared to contain only active AcChoEase. The bulk of inactive AcChoEase reacted with the organophosphate cholinesterase inhibitor O-ethyl S-[2-(diisopropylamino)ethyl]methylphosphonothioate (MTP) but was found to bind the active site affinity ligand N-methylacridinium poorly and was not recognized by the active-form-specific monoclonal antibody, C-131. In addition, most of this fraction is sensitive to endoglycosidase H and binds the lectin wheat germ agglutinin poorly, suggesting that it was not processed in the Golgi apparatus. From these observations, we propose that the active and inactive AcChoEase components are differently folded.
ESTHER : Chatel_1993_Proc.Natl.Acad.Sci.U.S.A_90_2476
PubMedSearch : Chatel_1993_Proc.Natl.Acad.Sci.U.S.A_90_2476
PubMedID: 8460161

Title : Molecular architecture of acetylcholinesterase collagen-tailed forms\; construction of a glycolipid-tailed tetramer - Duval_1992_EMBO.J_11_3255
Author(s) : Duval N , Krejci E , Grassi J , Coussen F , Massoulie J , Bon S
Ref : EMBO Journal , 11 :3255 , 1992
Abstract : Asymmetric forms of Torpedo acetylcholinesterase (AChE) are produced in COS cells by the simultaneous expression of collagenic subunits (Q) and catalytic T subunits (AChET). Truncated AChET delta subunits, from which most of the C-terminal peptide (TC) had been deleted by mutagenesis, did not associate with Q subunits. The TC peptide is therefore necessary for the association of the AChET and Q subunits. In order to determine the orientation of the Q subunit in the collagen-tailed forms, we have developed an antiserum against its non-collagenic C-terminal domain, expressed as a fusion protein in Escherichia coli. This antiserum, which recognized the Q subunit in Western blots, was found to react with intact asymmetric forms, but not with collagenase-treated forms, from which the distal part of the tail had been cleaved, suggesting that the N-terminal non-collogenic domain (QN) is responsible for the interaction with the AChET subunits. This was confirmed by creating a chimeric subunit (QN/HC), in which QN was linked to the C-terminal peptide of the H subunit of Torpedo AChE, which contains the glycophosphatidylinositol (GPI) cleavage/attachment signal: co-expression of AChET and QN/NC produced GPI-anchored tetramers, which were sensitive to PI-PLC and largely exposed to the external surface of the cells. We thus demonstrate that: (i) the HC peptide is sufficient to determine the addition of a glycolipid anchor and (ii) the QN domain is sufficient to bind a catalytic AChET tetramer by interacting with the TC peptide.
ESTHER : Duval_1992_EMBO.J_11_3255
PubMedSearch : Duval_1992_EMBO.J_11_3255
PubMedID: 1380451

Title : The Use of Acetylcholinesterase as a Universal Marker in Enzyme-Immunoassays -
Author(s) : Grassi J , Pradelles P
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 :221 , 1991

Title : The Structure and Significance of Multiple Cholinesterase Forms -
Author(s) : Massoulie J , Bon S , Krejci E , Coussen F , Duval N , Chatel JM , Anselmet A , Legay C , Vallette FM , Grassi J
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 :2 , 1991

Title : Monoclonal antibodies allow precipitation of esterasic but not peptidasic activities associated with butyrylcholinesterase - Checler_1990_J.Neurochem_55_750
Author(s) : Checler F , Grassi J , Masson P , Vincent JP
Ref : Journal of Neurochemistry , 55 :750 , 1990
Abstract : Commercially available and affinity-purified butyrylcholinesterases isolated from human serum were examined for their esterasic activity and their ability to hydrolyze various neuropeptides, including neurotensin, substance P, and leucine-enkephalin. The three pools that displayed the lowest esterasic activities were shown to hydrolyze neurotensin with the same HPLC degradative pattern. By contrast, noticeable qualitative and quantitative discrepancies were observed when hydrolyses of substance P and leucine-enkephalin by these three butyrylcholinesterase pools were studied. The pool that exhibited the highest esterasic activity appeared to be homogeneously constituted by 90- and 180-kDa protein bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and was totally unable to hydrolyze these three neuropeptides. This suggested that the three other butyrylcholinesterase preparations could be contaminated by exogenous peptidases. This was confirmed by means of three distinct monoclonal antibodies directed toward human serum butyrylcholinesterase. The three IgG-purified fractions precipitated the esterasic activity, whereas they failed to precipitate the neuropeptide-hydrolyzing activities whatever the substrate examined. Altogether, these results demonstrate that peptidases associated with butyrylcholinesterase are contaminating enzymes that cannot be considered as intrinsic activities of this enzyme.
ESTHER : Checler_1990_J.Neurochem_55_750
PubMedSearch : Checler_1990_J.Neurochem_55_750
PubMedID: 1696618

Title : NGF treatment promotes development of basal forebrain tissue grafts in the anterior chamber of the eye - Eriksdotter-Nilsson_1989_Exp.Brain.Res_74_89
Author(s) : Eriksdotter-Nilsson M , Skirboll S , Ebendal T , Hersh L , Grassi J , Massoulie J , Olson L
Ref : Experimental Brain Research , 74 :89 , 1989
Abstract : The effects of nerve growth factor (NGF) on developing central cholinergic neurons were studied using intraocular grafts of rat fetal (E17) basal forebrain tissue. Prior to grafting, grafts were incubated in NGF or saline. Transplants were allowed to mature for six weeks, receiving weekly intraocular injections of NGF or saline. Measurements of NGF levels in oculo after one single injection showed that NGF slowly decreases in the anterior chamber fluid, and after one week, low but significant levels were still present in the eye. Following pretreatment with diisopropylfluorophosphate (DFP), the cholinergic neurons in the grafts were analyzed using three morphological markers: antibodies to cholineacetyltransferase (ChAT), antibodies to acetylcholinesterase (AChE Ab) and acetylcholinesterase histochemistry (AChE). The transplants grew well and became vascularized within the first week. The growth of the NGF-treated basal forebrain grafts was significantly enhanced as compared to the growth of the saline-treated grafts evaluated with repeated stereomicroscopical observations directly through the cornea of the ether-anaesthetized hosts. The NGF-treated grafts contained almost twice as many cholinergic neurons seen with all the cholinergic markers used, as the saline-treated grafts. However, there was no difference in cholinergic cell density between the two groups. The morphology and size of an individual cholinergic neuron was similar in the two groups. The fiber density as evaluated with AChE-immunohistochemistry did not change after NGF-treatment. The DFP-treatment did not seem to affect the AChE-immunoreactivity since an extensive fiber network was found, whereas almost no fibers were seen using conventional AChE histochemistry. We have demonstrated that in oculo transplantation of basal forebrain is a useful model for examining in vivo effects of NGF on central cholinergic function. The marked volume increase of NGF-treated grafts and the unchanged density of cholinergic cells and terminals suggests, that NGF increases the survival of not only developing cholinergic neurons, but possibly other non-cholinergic neurons and non-neuronal cells as well. These results support the notion that NGF acts as a neurotrophic factor on cholinergic and possibly non-cholinergic cells in the central nervous system.
ESTHER : Eriksdotter-Nilsson_1989_Exp.Brain.Res_74_89
PubMedSearch : Eriksdotter-Nilsson_1989_Exp.Brain.Res_74_89
PubMedID: 2924843

Title : Monoclonal antibodies against acetylcholinesterase from electric organs of Electrophorus and Torpedo - Musset_1987_Biochimie_69_147
Author(s) : Musset F , Frobert Y , Grassi J , Vigny M , Boulla G , Bon S , Massoulie J
Ref : Biochimie , 69 :147 , 1987
Abstract : We studied the reactivity of monoclonal antibodies (mAbs) raised against acetylcholinesterase (AChE) purified from Electrophorus and Torpedo electric organs. We obtained IgG antibodies (Elec-21, Elec-106, Tor-3E5, Tor-ME8, Tor-1A5), all of them directed against the catalytic subunit of the corresponding species, with no significant cross-reactivity. These antibodies do not inhibit the enzyme and recognize all molecular forms, globular (G) and asymmetric (A). Tor-ME8 reacts specifically with the denatured A and G subunits of Torpedo AChE, in immunoblots. Several hybridomas raised against Electrophorus AChE produced IgM antibodies (Elec-39, Elec-118, Elec-121). These antibodies react with the A forms of Electrophorus electric organs and also with a subset of dimers (G2) from Torpedo electric organ. In addition, they react with a number of non-AChE components, in immunoblots. In contrast, they do not recognize AChE from other Electrophorus tissues or A forms from Torpedo electric organs.
ESTHER : Musset_1987_Biochimie_69_147
PubMedSearch : Musset_1987_Biochimie_69_147
PubMedID: 3105603

Title : An immunoglobulin M monoclonal antibody, recognizing a subset of acetylcholinesterase molecules from electric organs of Electrophorus and Torpedo, belongs to the HNK-1 anti-carbohydrate family - Bon_1987_J.Neurochem_49_1720
Author(s) : Bon S , Meflah K , Musset F , Grassi J , Massoulie J
Ref : Journal of Neurochemistry , 49 :1720 , 1987
Abstract : An immunoglobulin M (IgM) monoclonal antibody (mAb Elec-39), obtained against asymmetric acetylcholinesterase (AChE) from Electrophorus electric organs, also reacts with a fraction of globular AChE (amphiphilic G2 form) from Torpedo electric organs. This antibody does not react with asymmetric AChE from Torpedo electric organs or with the enzyme from other tissues of Electrophorus or Torpedo. The corresponding epitope is removed by endoglycosidase F, showing that it is a carbohydrate. The subsets of Torpedo G2 that react or do not react with Elec-39 (Elec-39+ and Elec-39-) differ in their electrophoretic mobility under nondenaturing conditions; the Elec-39+ component also binds the lectins from Pisum sativum and Lens culinaris. Whereas the Elec-39- component is present at the earliest developmental stages examined, an Elec-39+ component becomes distinguishable only around the 70-mm stage. Its proportion increases progressively, but later than the rapid accumulation of the total G2 form. In immunoblots, mAb Elec-39 recognizes a number of proteins other than AChE from various tissues of several species. The specificity of Elec-39 resembles that of a family of anti-carbohydrate antibodies that includes HNK-1, L2, NC-1, NSP-4, as well as IgMs that occur in human neuropathies. Although some human neuropathy IgMs that recognize the myelin-associated glycoprotein did not react with Elec-39+ AChE, mAbs HNK-1, NC-1, and NSP-4 showed the same selectivity as Elec-39 for Torpedo G2 AChE, but differed in the formation of immune complexes.
ESTHER : Bon_1987_J.Neurochem_49_1720
PubMedSearch : Bon_1987_J.Neurochem_49_1720
PubMedID: 2445915

Title : Presynaptic or postsynaptic origin of acetylcholinesterase at neuromuscular junctions? An immunological study in heterologous nerve- muscle cultures - De La Porte_1986_Dev.Biol_116_69
Author(s) : De La Porte S , Vallette FM , Grassi J , Vigny M , Koenig J
Ref : Developmental Biology , 116 :69 , 1986
Abstract : Numerous studies have shown that the acetylcholine receptor (AChR) is inserted in the plasma membrane of the muscle fiber, and that it is focalized at the site of neuromuscular junctions, as an effect of neural influence. In contrast, acetylcholinesterase (AChE) may be presynaptic or anchored in the basal lamina, as well as postsynaptic at neuromuscular junctions. We investigated the origin of the junctional enzyme, particularly the collagen-tailed asymmetric A12 forms, by studying the AChE contents of heterologous rat and chicken neuromuscular cocultures by immunohistochemical and biochemical methods. We found that the overall content of AChE, in the neuromuscular cocultures, including the A12 form, was essentially identical to the sum of the contents of separate myotube and motoneuron cultures. The sedimentation coefficients of the rat and chicken asymmetric forms are sufficiently different to clearly differentiate these enzymes in sucrose gradients: 16 S for rat, 20 S for chicken A12 AChE. Sedimentation analyses of AChE in cocultures thus showed that the A12 form was of muscular origin. In the case of aneural cultures of myotubes, histochemical staining of AChE activity or immunohistochemical staining with specific antibodies showed only very scarce, faint concentrations of enzyme. Some patches of acetylcholine receptor (AChR) were, however, visible in these cultures. Neuromuscular contacts are readily established in cocultures of myotubes with embryonic motoneurons from spinal cords. In the presence of motoneurons, the myotubes presented a larger number of AChR patches. The most remarkable feature of neuromuscular cocultures was the presence of numerous intense AChE patches which always coincided with AChR clusters. By specifically staining nerve terminals with tetanus toxin, we could show an excellent correlation between neuromuscular contacts and the presence of AChE-AChR patches. We found that the AChE patches in heterologous cocultures could be stained exclusively by the anti-myotube AChE antiserum. The focalized enzyme is therefore exclusively, or very predominantly, provided by the myotube.
ESTHER : De La Porte_1986_Dev.Biol_116_69
PubMedSearch : De La Porte_1986_Dev.Biol_116_69
PubMedID: 3525279

Title : Enzyme immunoassays of eicosanoids using acetylcholine esterase as label: an alternative to radioimmunoassay -
Author(s) : Pradelles P , Grassi J , Maclouf J
Ref : Analytical Chemistry , 57 :1170 , 1985
PubMedID: 3898913

Title : Isolation of a cDNA clone for a catalytic subunit of Torpedo marmorata acetylcholinesterase - Sikorav_1985_FEBS.Lett_193_159
Author(s) : Sikorav JL , Vallette FM , Grassi J , Massoulie J
Ref : FEBS Letters , 193 :159 , 1985
Abstract : We have constructed a cDNA library from Torpedo marmorata electric organ poly(A+) RNA in the lambda phage expression vector lambda gt11. This library has been screened with polyclonal anti-acetylcholinesterase antibodies. One clone, lambda AChE1, produced a fusion protein which was recognized by the antibodies and which prevented the binding of native acetylcholinesterase in an enzymatic immune assay. These results indicate that lambda AChE1 contains a cDNA insert coding for a part of a catalytic subunit of Torpedo acetylcholinesterase. The 200-base-pair cDNA insert hybridized to three mRNAs (14.5, 10.5 and 5.5 kb) from Torpedo electric organs. These mRNAs were also detected in Torpedo electric lobes.
ESTHER : Sikorav_1985_FEBS.Lett_193_159
PubMedSearch : Sikorav_1985_FEBS.Lett_193_159
PubMedID: 2415396

Title : The polymorphism of cholinesterases: classification of molecular forms\; Interactions and solubilization characteristics metabolic relationships and regulations -
Author(s) : Massoulie J , Bon S , Lazar M , Grassi J , Marsh D , Meflah K , Toutant JP , Vallette FM , Vigny M
Ref : In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases , (Brzin M, Barnard EA, Sket D, Eds) De Gruyter :73 , 1984

Title : Synthesis in vitro of precursors of the catalytic subunits of acetylcholinesterase from Torpedo marmorata and Electrophorus electricus - Sikorav_1984_Eur.J.Biochem_145_519
Author(s) : Sikorav JL , Grassi J , Bon S
Ref : European Journal of Biochemistry , 145 :519 , 1984
Abstract : We translated poly(A-rich messenger RNA prepared from the electric organs of Electrophorus electricus and Torpedo marmorata in a reticulocyte lysate system. In the case of Electrophorus, which appears to contain only one type of acetylcholinesterase catalytic subunit, an anti-(Electrophorus acetylcholinesterase) antiserum precipitated a single 65-kDa polypeptide from the products translation obtained in vitro. In the case of Torpedo, where a number of distinct catalytic subunits corresponding to different fractions of the enzyme have been described, an anti-(Torpedo acetylcholinesterase) antiserum precipitated two main polypeptides, 61 kDa and 65 kDa, both of which could be displaced by unlabelled purified Torpedo acetylcholinesterase. Synthesis in vitro thus appears to produce a single type of precursor of the acetylcholinesterase catalytic subunit for Electrophorus, and at least two distinct precursors for Torpedo, suggesting that several mRNAs code for the catalytic subunits in the latter species.
ESTHER : Sikorav_1984_Eur.J.Biochem_145_519
PubMedSearch : Sikorav_1984_Eur.J.Biochem_145_519
PubMedID: 6150849

Title : Poster 15. An Immunological study of mammalian acetylcholinesterase using anti-rat serum -
Author(s) : Grassi J , Marsh D , Vigny M , Massoulie J
Ref : In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases , (Brzin M, Barnard EA, Sket D, Eds) De Gruyter , 1984

Title : Poster 24 Relationship of collagen-tailed acetylcholinesterase with basal lamina components: Absence of binding with laminin, fibronectin and collagens type IV and V and lack of reactivity with different anti-collagen antisera -
Author(s) : Grassi J , Massoulie J , Timpl R
Ref : In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases , (Brzin M, Barnard EA, Sket D, Eds) De Gruyter , 1984

Title : Poster 41. Acetylcholinesterase focalized at neuromuscular contacts: Neuronal or muscular origin? -
Author(s) : De La Porte S , Marsh D , Grassi J , Vallette FM , Vigny M , Koenig J
Ref : In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases , (Brzin M, Barnard EA, Sket D, Eds) De Gruyter , 1984

Title : An immunological study of rat acetylcholinesterase: comparison with acetylcholinesterases from other vertebrates - Marsh_1984_J.Neurochem_43_204
Author(s) : Marsh D , Grassi J , Vigny M , Massoulie J
Ref : Journal of Neurochemistry , 43 :204 , 1984
Abstract : We have examined the immunoreactivity of acetylcholinesterase from different vertebrate species with a rabbit antiserum raised against the purified rat brain hydrophobic enzyme (G4 form). We found no significant interaction with enzymes from Electrophorus, Torpedo, chicken, and rabbit. The antiserum reacted with acetylcholinesterases from the brains of the other mammalian species studied, with titers decreasing in the following order: rat = mouse greater than human greater than bovine. The serum was inhibitory with murine and human acetylcholinesterases, but not with the bovine enzyme. The inhibition was partially depressed in the presence of salt (e.g., 1 M NaCl). In those species whose acetylcholinesterase was recognized by the antiserum, both soluble and detergent-soluble fractions behaved in essentially the same manner, interacting with the same antibodies. The apparent immunoprecipitation titer was decreased in the presence of salt, and it did not make any difference whether NaCl was included in the solubilization procedure or added to the extracts. Both G1 and G4 forms of acetylcholinesterase in the soluble and detergent-soluble fractions were recognized by the antiserum, and in the case of the human enzyme, by monoclonal antibodies produced against human erythrocyte acetylcholinesterase. However, the monomer G1 showed a clear tendency to form smaller complexes and precipitate less readily than the tetramer G4. Although we cannot exclude the existence of significant differences between the various molecular forms of acetylcholinesterase, our results are consistent with the hypothesis that they all derive from the same gene or set of genes by posttranslational modifications.
ESTHER : Marsh_1984_J.Neurochem_43_204
PubMedSearch : Marsh_1984_J.Neurochem_43_204
PubMedID: 6374038

Title : Relationship of collagen-tailed acetylcholinesterase with basal lamina components. Absence of binding with laminin, fibronectin, and collagen types IV and V and lack of reactivity with different anti-collagen sera - Grassi_1983_Eur.J.Biochem_133_31
Author(s) : Grassi J , Massoulie J , Timpl R
Ref : European Journal of Biochemistry , 133 :31 , 1983
Abstract : In view of their supposed localization in extracellular structures, such as basal lamina, we have investigated the possible interactions of collagen-tailed forms of acetylcholinesterase from Electrophorus and bovine superior cervical ganglion with matrix proteins: laminin, fibronectin and types IV and V collagens. Using binding and sedimentation assays, with iodinated or non-radioactive matrix proteins, we have not observed any significant interaction, in conditions of high or low ionic strength. We also examined whether the collagen tail of acetylcholinesterase asymmetric forms possessed an immunological relationship with known collagen types (I, III, IV, V) from mammalian sources. We found no specific immunoreactivity with any of the 32 sera studied, either with the iodinated Electrophorus or with the native bovine enzyme. We conclude from these negative results that the collagen-like tail of acetylcholinesterase is clearly distinct from the classical types of collagen and that asymmetric forms of the enzyme do not interact specifically with the matrix proteins studied. This does not exclude the possibility of specific interactions with other components, remaining to be identified.
ESTHER : Grassi_1983_Eur.J.Biochem_133_31
PubMedSearch : Grassi_1983_Eur.J.Biochem_133_31
PubMedID: 6852032

Title : Molecular forms of acetylcholinesterase in bovine caudate nucleus and superior cervical ganglion: solubility properties and hydrophobic character -
Author(s) : Grassi J , Vigny M , Massoulie J
Ref : Journal of Neurochemistry , 38 :457 , 1982
PubMedID: 7108551