The Experts below are selected from a list of 41220 Experts worldwide ranked by ideXlab platform
Jordi Molgó - One of the best experts on this subject based on the ideXlab platform.
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Cyclic imine toxins survey in coastal european shellfish samples: Bioaccumulation and mode of action of 28-O-palmitoyl ester of pinnatoxin-G. first report of portimine-A bioaccumulation
Harmful Algae, 2020Co-Authors: Rómulo Aráoz, Armen Zakarian, Jordi Molgó, Paul Barnes, Véronique Séchet, Muriel Delepierre, Sophie Zinn-justin, Philipp Hess, Denis ServentAbstract:Cyclic imine toxins exhibit fast acting neurotoxicity and lethality by respiratory arrest in mice explained by their potent antagonistic activity against muscular nicotinic acetylcholine Receptors. We performed a survey of gymnodimine-A, 13-desmethyl spirolide-C, 13,19-didesmethyl spirolide-C, 20-methyl spirolide-G, pinnatoxin-A, pinnatoxin-G, portimine-A and 28-O-palmitoyl ester of pinnatoxin-G in 36 shellfish samples collected in coastal areas of 8 European countries using a microplate Receptor Binding Assay and UPLC-MS/MS for toxin identification and quantification. The major toxins found in these samples were pinnatoxin-G, 20-methyl spirolide-G, 13-desmethyl spirolide-C, gymnodimine-A and portimine-A. Traces of 13,19-didesmethyl spirolide-C, pinnatoxin-A and 28-O-palmitoyl ester of pinnatoxin-G were also detected. The rapid death of mice was correlated with higher pinnatoxin-G concentrations in mussel digestive gland extracts injected intraperitoneally. Our survey included nontoxic control samples that were found to contain moderate to trace amounts of several cyclic imine toxins. Shellfish may bioaccumulate not only cyclic imine toxins but also a large number of acyl derivatives as a product of metabolic transformation of these neurotoxins. This is the first report in which portimine-A and 28-O-palmitoyl ester of pinnatoxin-G were detected in shellfish extracts from digestive glands of mussels collected in Ingril lagoon. The bioaccumulation of portimine-A is particularly of concern because it is cytotoxic and is able to induce apotosis. The mode of action of 28-O-palmitoyl ester of pinnatoxin-G was studied by Receptor Binding-Assay and by two-electrode voltage clamp electrophysiology. The antagonistic behavior of the acylated pinnatoxin-G towards nicotinic acetylcholine Receptor of muscle type is shown here for the first time. Since cyclic imine toxins are not regulated further monitoring of these emerging toxins is needed to improve evidence gathering of their occurrence in shellfish commercialized for human consumption in Europe given their potent antagonism against muscle and neuronal nicotinic acetylcholine Receptors.
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Colorimetric microtiter plate Receptor-Binding Assay for the detection of freshwater and marine neurotoxins targeting the nicotinic acetylcholine Receptors.
Toxicon, 2014Co-Authors: Fernando Rubio, Jordi Molgó, Lisa Kamp, Justin Carpino, Erin Faltin, Keith Loftin, Rómulo AráozAbstract:Anatoxin-a and homoanatoxin-a, produced by cyanobacteria, are agonists of nicotinic acetylcholine Receptors (nAChRs). Pinnatoxins, spirolides, and gymnodimines, produced by dinoflagellates, are antagonists of nAChRs. In this study we describe the development and validation of a competitive colorimetric, high throughput functional Assay based on the mechanism of action of freshwater and marine toxins against nAChRs. Torpedo electrocyte membranes (rich in muscle-type nAChR) were immobilized and stabilized on the surface of 96-well microtiter plates. Biotinylated α-bungarotoxin (the tracer) and streptavidin-horseradish peroxidase (the detector) enabled the detection and quantitation of anatoxin-a in surface waters and cyclic imine toxins in shellfish extracts that were obtained from different locations across the US. The method compares favorably to LC/MS/MS and provides accurate results for anatoxin-a and cyclic imine toxins monitoring. Study of common constituents at the concentrations normally found in drinking and environmental waters, as well as the tolerance to pH, salt, solvents, organic and inorganic compounds did not significantly affect toxin detection. The Assay allowed the simultaneous analysis of up to 25 samples within 3.5 h and it is well suited for on-site or laboratory monitoring of low levels of toxins in drinking, surface, and ground water as well as in shellfish extracts.
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Coupling the torpedo microplate-Receptor Binding Assay with mass spectrometry to detect cyclic imine neurotoxins.
Analytical Chemistry, 2012Co-Authors: Rómulo Aráoz, Suzanne Ramos, Franck Pelissier, Vincent Guérineau, Evelyne Benoit, Natalia Vilariño, Luis M Botana, Armen Zakarian, Jordi MolgóAbstract:Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine Receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine Receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine Receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin Binding to Torpedo-nicotinic acetylcholine Receptors in a concentration-dependent manner. The microplate-Receptor Binding Assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine Receptors as a class, the Receptor Binding Assay cannot identify a given analyte. To address the low selectivity of the microplate-Receptor Binding Assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic Receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine Receptors directly in shellfish matrixes with high sensitivity and reproducibility.
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coupling the torpedo microplate Receptor Binding Assay with mass spectrometry to detect cyclic imine neurotoxins
Analytical Chemistry, 2012Co-Authors: Rómulo Aráoz, Suzanne Ramos, Franck Pelissier, Vincent Guérineau, Evelyne Benoit, Natalia Vilariño, Luis M Botana, Armen Zakarian, Jordi MolgóAbstract:Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine Receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine Receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine Receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin Binding to Torpedo-nicotinic acetylcholine Receptors in a concentration-dependent manner. The microplate-Receptor Binding Assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine recepto...
Rómulo Aráoz - One of the best experts on this subject based on the ideXlab platform.
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Cyclic imine toxins survey in coastal european shellfish samples: Bioaccumulation and mode of action of 28-O-palmitoyl ester of pinnatoxin-G. first report of portimine-A bioaccumulation
Harmful Algae, 2020Co-Authors: Rómulo Aráoz, Armen Zakarian, Jordi Molgó, Paul Barnes, Véronique Séchet, Muriel Delepierre, Sophie Zinn-justin, Philipp Hess, Denis ServentAbstract:Cyclic imine toxins exhibit fast acting neurotoxicity and lethality by respiratory arrest in mice explained by their potent antagonistic activity against muscular nicotinic acetylcholine Receptors. We performed a survey of gymnodimine-A, 13-desmethyl spirolide-C, 13,19-didesmethyl spirolide-C, 20-methyl spirolide-G, pinnatoxin-A, pinnatoxin-G, portimine-A and 28-O-palmitoyl ester of pinnatoxin-G in 36 shellfish samples collected in coastal areas of 8 European countries using a microplate Receptor Binding Assay and UPLC-MS/MS for toxin identification and quantification. The major toxins found in these samples were pinnatoxin-G, 20-methyl spirolide-G, 13-desmethyl spirolide-C, gymnodimine-A and portimine-A. Traces of 13,19-didesmethyl spirolide-C, pinnatoxin-A and 28-O-palmitoyl ester of pinnatoxin-G were also detected. The rapid death of mice was correlated with higher pinnatoxin-G concentrations in mussel digestive gland extracts injected intraperitoneally. Our survey included nontoxic control samples that were found to contain moderate to trace amounts of several cyclic imine toxins. Shellfish may bioaccumulate not only cyclic imine toxins but also a large number of acyl derivatives as a product of metabolic transformation of these neurotoxins. This is the first report in which portimine-A and 28-O-palmitoyl ester of pinnatoxin-G were detected in shellfish extracts from digestive glands of mussels collected in Ingril lagoon. The bioaccumulation of portimine-A is particularly of concern because it is cytotoxic and is able to induce apotosis. The mode of action of 28-O-palmitoyl ester of pinnatoxin-G was studied by Receptor Binding-Assay and by two-electrode voltage clamp electrophysiology. The antagonistic behavior of the acylated pinnatoxin-G towards nicotinic acetylcholine Receptor of muscle type is shown here for the first time. Since cyclic imine toxins are not regulated further monitoring of these emerging toxins is needed to improve evidence gathering of their occurrence in shellfish commercialized for human consumption in Europe given their potent antagonism against muscle and neuronal nicotinic acetylcholine Receptors.
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Colorimetric microtiter plate Receptor-Binding Assay for the detection of freshwater and marine neurotoxins targeting the nicotinic acetylcholine Receptors.
Toxicon, 2014Co-Authors: Fernando Rubio, Jordi Molgó, Lisa Kamp, Justin Carpino, Erin Faltin, Keith Loftin, Rómulo AráozAbstract:Anatoxin-a and homoanatoxin-a, produced by cyanobacteria, are agonists of nicotinic acetylcholine Receptors (nAChRs). Pinnatoxins, spirolides, and gymnodimines, produced by dinoflagellates, are antagonists of nAChRs. In this study we describe the development and validation of a competitive colorimetric, high throughput functional Assay based on the mechanism of action of freshwater and marine toxins against nAChRs. Torpedo electrocyte membranes (rich in muscle-type nAChR) were immobilized and stabilized on the surface of 96-well microtiter plates. Biotinylated α-bungarotoxin (the tracer) and streptavidin-horseradish peroxidase (the detector) enabled the detection and quantitation of anatoxin-a in surface waters and cyclic imine toxins in shellfish extracts that were obtained from different locations across the US. The method compares favorably to LC/MS/MS and provides accurate results for anatoxin-a and cyclic imine toxins monitoring. Study of common constituents at the concentrations normally found in drinking and environmental waters, as well as the tolerance to pH, salt, solvents, organic and inorganic compounds did not significantly affect toxin detection. The Assay allowed the simultaneous analysis of up to 25 samples within 3.5 h and it is well suited for on-site or laboratory monitoring of low levels of toxins in drinking, surface, and ground water as well as in shellfish extracts.
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Coupling the torpedo microplate-Receptor Binding Assay with mass spectrometry to detect cyclic imine neurotoxins.
Analytical Chemistry, 2012Co-Authors: Rómulo Aráoz, Suzanne Ramos, Franck Pelissier, Vincent Guérineau, Evelyne Benoit, Natalia Vilariño, Luis M Botana, Armen Zakarian, Jordi MolgóAbstract:Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine Receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine Receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine Receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin Binding to Torpedo-nicotinic acetylcholine Receptors in a concentration-dependent manner. The microplate-Receptor Binding Assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine Receptors as a class, the Receptor Binding Assay cannot identify a given analyte. To address the low selectivity of the microplate-Receptor Binding Assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic Receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine Receptors directly in shellfish matrixes with high sensitivity and reproducibility.
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coupling the torpedo microplate Receptor Binding Assay with mass spectrometry to detect cyclic imine neurotoxins
Analytical Chemistry, 2012Co-Authors: Rómulo Aráoz, Suzanne Ramos, Franck Pelissier, Vincent Guérineau, Evelyne Benoit, Natalia Vilariño, Luis M Botana, Armen Zakarian, Jordi MolgóAbstract:Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine Receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine Receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine Receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin Binding to Torpedo-nicotinic acetylcholine Receptors in a concentration-dependent manner. The microplate-Receptor Binding Assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine recepto...
Zhan-yun Guo - One of the best experts on this subject based on the ideXlab platform.
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application of the novel bioluminescent ligand Receptor Binding Assay to relaxin rxfp1 system for interaction studies
Amino Acids, 2016Co-Authors: Lei Zhang, Ya-li Liu, Xiao-xia Shao, Jiahui Wang, Yu Gao, Zhan-yun GuoAbstract:Relaxin is a prototype of the relaxin family peptide hormones and plays important biological functions by Binding and activating the G protein-coupled Receptor RXFP1. To study their interactions, in the present work, we applied the newly developed bioluminescent ligand-Receptor Binding Assay to the relaxin-RXFP1 system. First, a fully active easily labeled relaxin, in which three Lys residues of human relaxin-2 were replaced by Arg, was prepared through overexpression of a single-chain precursor in Pichia pastoris and in vitro enzymatic maturation. Thereafter, the B-chain N-terminus of the easily labeled relaxin was chemically cross-linked with a C-terminal cysteine residue of an engineered NanoLuc through a disulfide linkage. Receptor-Binding Assays demonstrated that the NanoLuc-conjugated relaxin retained high Binding affinity with the Receptor RXFP1 (K d = 1.11 ± 0.08 nM, n = 3) and was able to sensitively monitor Binding of a variety of ligands with RXFP1. Using the novel bioluminescent Binding Assay, we demonstrated that three highly conserved B-chain Arg residues of relaxin-3 had distinct contributions to Binding of the Receptor RXFP1. In summary, our present work provides a novel bioluminescent ligand-Receptor Binding Assay for the relaxin-RXFP1 system to facilitate their interaction studies, such as characterization of relaxin analogues or screening novel agonists or antagonists of RXFP1.
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Novel bioluminescent Receptor-Binding Assays for peptide hormones: using ghrelin as a model.
Amino acids, 2015Co-Authors: Yu Liu, Ya-li Liu, Xiao-xia Shao, Lei Zhang, Ge Song, Zhan-yun GuoAbstract:Peptide hormones perform important biological functions by Binding specific cell membrane Receptors. For hormone–Receptor interaction studies, Receptor-Binding Assays are widely used. However, conventional Receptor-Binding Assays rely on radioactive tracers that have drawbacks. In recent studies, we established novel non-radioactive Receptor-Binding Assays for some recombinant protein hormones based on the ultrasensitive bioluminescence of a newly developed nanoluciferase (NanoLuc) reporter. In the present work, we extended the novel bioluminescent Receptor-Binding Assay to peptide hormones that have small size and can be conveniently prepared by chemical synthesis. Human ghrelin, a 28-amino acid peptide hormone carrying a special O-fatty acid modification, was used as a model. To prepare a bioluminescent ghrelin tracer, a chemically synthesized ghrelin analog with a unique cysteine residue at the C-terminus was site-specifically conjugated with an engineered NanoLuc with a unique exposed cysteine residue at the C-terminus via a reversible disulfide linkage. The NanoLuc-conjugated ghrelin retained high Binding affinity with the ghrelin Receptor GHSR1a (Kd = 1.14 ± 0.13 nM, n = 3) and was able to sensitively monitor the Receptor-Binding of various GHSR1a ligands. The novel bioluminescent Receptor-Binding Assay will facilitate the interaction studies of ghrelin with its Receptor. We also proposed general procedures for convenient conjugation of other peptide hormones with NanoLuc for novel bioluminescent Receptor-Binding Assays.
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a novel ultrasensitive bioluminescent Receptor Binding Assay of insl3 through chemical conjugation with nanoluciferase
Biochimie, 2013Co-Authors: Lei Zhang, Ya-li Liu, Xiao-xia Shao, Ge Song, Zhan-yun GuoAbstract:Abstract Insulin-like peptide 3 (INSL3) is a reproduction-related peptide hormone belonging to the insulin/relaxin superfamily, which mediates testicular descent in the male fetus, suppresses male germ cell apoptosis and promotes oocyte maturation in adults by activating the relaxin family peptide Receptor 2 (RXFP2). To establish an ultrasensitive Receptor-Binding Assay for INSL3−RXFP2 interaction studies, in the present work we labeled a recombinant INSL3 peptide with a newly developed nanoluciferase (NanoLuc) reporter through a convenient chemical conjugation approach, including the introduction of an active disulfide bond to INSL3 by chemical modification and engineering of a 6× His-Cys-NanoLuc carrying a unique exposed cysteine at the N-terminus. The bioluminescent NanoLuc-conjugated INSL3 retained high Binding affinity with the target Receptor RXFP2 (Kd = 2.0 ± 0.1 nM, n = 3) and was able to sensitively monitor the Receptor-Binding of a variety of ligands, representing a novel ultrasensitive tracer for non-radioactive Receptor-Binding Assays. Our present chemical conjugation approach could readily be adapted for conjugation of NanoLuc with other proteins, even other macrobiomolecules, for various highly sensitive bioluminescent Assays.
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design recombinant preparation and europium labeling of a fully active easily labeled insl3 analog for Receptor Binding Assays
Process Biochemistry, 2012Co-Authors: Weijie Zhang, Ya-li Liu, Xiao-xia Shao, Xuejuan Gao, Zhan-yun GuoAbstract:Insulin-like peptide 3 (INSL3) is a peptide hormone belonging to the insulin/relaxin superfamily, which mediates testes descent in the male fetus, and suppresses male germ cell apoptosis and promotes oocyte maturation in adults by activating the leucine-rich repeat-containing G-protein coupled Receptor RXFP2. In a previous work, we prepared mature two-chain INSL3 by recombinant expression of a designed single-chain precursor in Escherichia coli and subsequent in vitro maturation. To establish a convenient high throughput Receptor-Binding Assay for screening novel RXFP2 agonists or antagonists, in the present study we designed and recombinantly prepared a fully active easily-labeled INSL3 analog. Due to presence of a single primary amine moiety, the easily-labeled analog was conveniently mono-labeled by a DTPA/Eu3+-moiety at the A-chain N-terminus through reacting with excess modification reagent in a simple one-step procedure. The DTPA/Eu3+-labeled INSL3 analog bound Receptor RXFP2 with high affinity and low non-specific Binding. Using this non-radioactive tracer, we established a high throughput cell-based Receptor-Binding Assay for screening of novel RXFP2 agonists or antagonists in future studies.
Luis M Botana - One of the best experts on this subject based on the ideXlab platform.
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determination of gonyautoxin 4 in echinoderms and gastropod matrices by conversion to neosaxitoxin using 2 mercaptoethanol and post column oxidation liquid chromatography with fluorescence detection
Toxins, 2015Co-Authors: Marisa Silva, Veronica Rey, Ana M Botana, Vitor Vasconcelos, Luis M BotanaAbstract:Paralytic Shellfish Toxin blooms are common worldwide, which makes their monitoring crucial in the prevention of poisoning incidents. These toxins can be monitored by a variety of techniques, including mouse bioAssay, Receptor Binding Assay, and liquid chromatography with either mass spectrometric or pre- or post-column fluorescence detection. The post-column oxidation liquid chromatography with fluorescence detection method, used routinely in our laboratory, has been shown to be a reliable method for monitoring paralytic shellfish toxins in mussel, scallop, oyster and clam species. However, due to its high sensitivity to naturally fluorescent matrix interferences, when working with unconventional matrices, there may be problems in identifying toxins because of naturally fluorescent interferences that co-elute with the toxin peaks. This can lead to erroneous identification. In this study, in order to overcome this challenge in echinoderm and gastropod matrices, we optimized the conversion of Gonyautoxins 1 and 4 to Neosaxitoxin with 2-mercaptoethanol. We present a new and less time-consuming method with a good recovery (82.2%, RSD 1.1%, n = 3), requiring only a single reaction step.
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Coupling the torpedo microplate-Receptor Binding Assay with mass spectrometry to detect cyclic imine neurotoxins.
Analytical Chemistry, 2012Co-Authors: Rómulo Aráoz, Suzanne Ramos, Franck Pelissier, Vincent Guérineau, Evelyne Benoit, Natalia Vilariño, Luis M Botana, Armen Zakarian, Jordi MolgóAbstract:Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine Receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine Receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine Receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin Binding to Torpedo-nicotinic acetylcholine Receptors in a concentration-dependent manner. The microplate-Receptor Binding Assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine Receptors as a class, the Receptor Binding Assay cannot identify a given analyte. To address the low selectivity of the microplate-Receptor Binding Assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic Receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine Receptors directly in shellfish matrixes with high sensitivity and reproducibility.
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coupling the torpedo microplate Receptor Binding Assay with mass spectrometry to detect cyclic imine neurotoxins
Analytical Chemistry, 2012Co-Authors: Rómulo Aráoz, Suzanne Ramos, Franck Pelissier, Vincent Guérineau, Evelyne Benoit, Natalia Vilariño, Luis M Botana, Armen Zakarian, Jordi MolgóAbstract:Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine Receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine Receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine Receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin Binding to Torpedo-nicotinic acetylcholine Receptors in a concentration-dependent manner. The microplate-Receptor Binding Assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine recepto...
Armen Zakarian - One of the best experts on this subject based on the ideXlab platform.
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Cyclic imine toxins survey in coastal european shellfish samples: Bioaccumulation and mode of action of 28-O-palmitoyl ester of pinnatoxin-G. first report of portimine-A bioaccumulation
Harmful Algae, 2020Co-Authors: Rómulo Aráoz, Armen Zakarian, Jordi Molgó, Paul Barnes, Véronique Séchet, Muriel Delepierre, Sophie Zinn-justin, Philipp Hess, Denis ServentAbstract:Cyclic imine toxins exhibit fast acting neurotoxicity and lethality by respiratory arrest in mice explained by their potent antagonistic activity against muscular nicotinic acetylcholine Receptors. We performed a survey of gymnodimine-A, 13-desmethyl spirolide-C, 13,19-didesmethyl spirolide-C, 20-methyl spirolide-G, pinnatoxin-A, pinnatoxin-G, portimine-A and 28-O-palmitoyl ester of pinnatoxin-G in 36 shellfish samples collected in coastal areas of 8 European countries using a microplate Receptor Binding Assay and UPLC-MS/MS for toxin identification and quantification. The major toxins found in these samples were pinnatoxin-G, 20-methyl spirolide-G, 13-desmethyl spirolide-C, gymnodimine-A and portimine-A. Traces of 13,19-didesmethyl spirolide-C, pinnatoxin-A and 28-O-palmitoyl ester of pinnatoxin-G were also detected. The rapid death of mice was correlated with higher pinnatoxin-G concentrations in mussel digestive gland extracts injected intraperitoneally. Our survey included nontoxic control samples that were found to contain moderate to trace amounts of several cyclic imine toxins. Shellfish may bioaccumulate not only cyclic imine toxins but also a large number of acyl derivatives as a product of metabolic transformation of these neurotoxins. This is the first report in which portimine-A and 28-O-palmitoyl ester of pinnatoxin-G were detected in shellfish extracts from digestive glands of mussels collected in Ingril lagoon. The bioaccumulation of portimine-A is particularly of concern because it is cytotoxic and is able to induce apotosis. The mode of action of 28-O-palmitoyl ester of pinnatoxin-G was studied by Receptor Binding-Assay and by two-electrode voltage clamp electrophysiology. The antagonistic behavior of the acylated pinnatoxin-G towards nicotinic acetylcholine Receptor of muscle type is shown here for the first time. Since cyclic imine toxins are not regulated further monitoring of these emerging toxins is needed to improve evidence gathering of their occurrence in shellfish commercialized for human consumption in Europe given their potent antagonism against muscle and neuronal nicotinic acetylcholine Receptors.
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Coupling the torpedo microplate-Receptor Binding Assay with mass spectrometry to detect cyclic imine neurotoxins.
Analytical Chemistry, 2012Co-Authors: Rómulo Aráoz, Suzanne Ramos, Franck Pelissier, Vincent Guérineau, Evelyne Benoit, Natalia Vilariño, Luis M Botana, Armen Zakarian, Jordi MolgóAbstract:Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine Receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine Receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine Receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin Binding to Torpedo-nicotinic acetylcholine Receptors in a concentration-dependent manner. The microplate-Receptor Binding Assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine Receptors as a class, the Receptor Binding Assay cannot identify a given analyte. To address the low selectivity of the microplate-Receptor Binding Assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic Receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine Receptors directly in shellfish matrixes with high sensitivity and reproducibility.
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coupling the torpedo microplate Receptor Binding Assay with mass spectrometry to detect cyclic imine neurotoxins
Analytical Chemistry, 2012Co-Authors: Rómulo Aráoz, Suzanne Ramos, Franck Pelissier, Vincent Guérineau, Evelyne Benoit, Natalia Vilariño, Luis M Botana, Armen Zakarian, Jordi MolgóAbstract:Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine Receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine Receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine Receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin Binding to Torpedo-nicotinic acetylcholine Receptors in a concentration-dependent manner. The microplate-Receptor Binding Assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine recepto...
