Jordi Molgó
Publications
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8.98Impact points
Preferential Entry of Botulinum Neurotoxin A Hc Domain through Intestinal Crypt Cells and Targeting to Cholinergic Neurons of the Mouse Intestine.
PLoS pathogens. 03/2012; 8(3):e1002583.
Botulism, characterized by flaccid paralysis, commonly results from botulinum neurotoxin (BoNT) absorption across the epithelial barrier from the digestive tract and then dissemination through the blood circulation to target autonomic and motor nerve terminals. The trafficking pathway of BoNT/A pass... [more] Botulism, characterized by flaccid paralysis, commonly results from botulinum neurotoxin (BoNT) absorption across the epithelial barrier from the digestive tract and then dissemination through the blood circulation to target autonomic and motor nerve terminals. The trafficking pathway of BoNT/A passage through the intestinal barrier is not yet fully understood. We report that intralumenal administration of purified BoNT/A into mouse ileum segment impaired spontaneous muscle contractions and abolished the smooth muscle contractions evoked by electric field stimulation. Entry of BoNT/A into the mouse upper small intestine was monitored with fluorescent HcA (half C-terminal domain of heavy chain) which interacts with cell surface receptor(s). We show that HcA preferentially recognizes a subset of neuroendocrine intestinal crypt cells, which probably represent the entry site of the toxin through the intestinal barrier, then targets specific neurons in the submucosa and later (90-120 min) in the musculosa. HcA mainly binds to certain cholinergic neurons of both submucosal and myenteric plexuses, but also recognizes, although to a lower extent, other neuronal cells including glutamatergic and serotoninergic neurons in the submucosa. Intestinal cholinergic neuron targeting by HcA could account for the inhibition of intestinal peristaltism and secretion observed in botulism, but the consequences of the targeting to non-cholinergic neurons remains to be determined.
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5.20Impact points
Pharmacological characterization of a novel μ-conopeptide, CnIIIC, indicates potent and preferential inhibition of sodium channel subtypes (Na(V) 1.2/1.4) and reveals unusual activity on neuronal nicotinic acetylcholine receptors.
British journal of pharmacology. 01/2012;
BACKGROUND AND PURPOSE The µ-conopeptide family is defined by its ability to block voltage-gated sodium channels (VGSCs), a property that can be used for the development of myorelaxants and analgesics. We performed a pharmacological characterisation of a new µ-conopeptide (μ-CnIIIC) on multiple pre... [more] BACKGROUND AND PURPOSE The µ-conopeptide family is defined by its ability to block voltage-gated sodium channels (VGSCs), a property that can be used for the development of myorelaxants and analgesics. We performed a pharmacological characterisation of a new µ-conopeptide (μ-CnIIIC) on multiple preparations and molecular targets to gauge its potential as a myorelaxant. EXPERIMENTAL APPROACH The µ-CnIIIC was sequenced, synthesized, and characterized by its ability to block directly-elicited twitch tension in mouse skeletal muscle and action potentials in mouse sciatic and pike olfactory nerves. µ-CnIIIC was also studied on HEK-293 cells expressing various rodent VGSCs. Pharmacological investigations were extended to voltage-gated potassium channels and nAChRs to assess cross-interactions. Nuclear magnetic resonance (NMR) experiments were carried out for structural data. KEY RESULTS Synthetic μ-CnIIIC potently decreased twitch tension in mouse hemidiaphragms (IC(50) = 150 nM), and displayed a higher blocking effect in mouse extensor digitorum longus muscles (IC = 46 nM), as compared to µ-SIIIA, µ-SmIIIA and µ-PIIIA. μ-CnIIIC blocked Na(V) 1.4 (IC(50) = 1.3 nM) and Na(V) 1.2 in a long-lasting manner. Cardiac Na(V) 1.5 and DRG-specific Na(V) 1.8 were not blocked at 1 μM. An activity was unveiled on the α3β2 nAChR subtype (IC(50) = 450 nM) and, to a lesser extent, on the α7 and α4β2 subtypes. Structure determination of µ-CnIIIC revealed some similarities to α-conotoxins acting on nAChRs. CONCLUSION AND IMPLICATIONS μ-CnIIIC potently blocks VGSCs in skeletal muscle and nerve, and hence is applicable to myorelaxation. Its new atypical pharmacological profile suggests some common structural features between VGSCs and nAChR channels.
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3.76Impact points
6,6-Spiroimine analogs of (-)-gymnodimine A: synthesis and biological evaluation on nicotinic acetylcholine receptors.
Organic & biomolecular chemistry. 12/2011; 9(23):8112-8.
Simple models of the spiroimine core of (-)-gymnodimine A have been synthesized in racemic and optically active forms. The quaternary carbon of the racemic spiroimines was created by Michael addition of a β-ketoester to acrolein, whereas the asymmetric allylic alkylation of the same β-ketoester was ... [more] Simple models of the spiroimine core of (-)-gymnodimine A have been synthesized in racemic and optically active forms. The quaternary carbon of the racemic spiroimines was created by Michael addition of a β-ketoester to acrolein, whereas the asymmetric allylic alkylation of the same β-ketoester was used to access the spiroimines in an enantioselective fashion. Both racemic and enantio-enriched mixtures were tested for their biological activities on Xenopus oocytes either expressing (human α4β2) or having incorporated (Torpedoα1(2)βγδ) nicotinic acetylcholine receptors (nAChRs). These spiroimine analogs of (-)-gymnodimine A inhibited acetylcholine-evoked nicotinic currents, but were less active than the phycotoxin. Our results reveal that the 6,6-spiroimine moiety is important for the blockade of nAChRs and support the hypothesis that it is one of the pharmacophores of this group of toxins.
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4.31Impact points
First direct fluorescence polarization assay for the detection and quantification of spirolides in mussel samples.
Analytica chimica acta. 09/2011; 701(2):200-8.
In 2009, we achieve the first inhibition FP assay to detect imine cyclic toxins. In the present paper we propose a new FP assay for direct quantify spirolides. This new method has resulted in significant improvement of sensitivity, rapidity and accessibility. In the method design, nicotinic acetylch... [more] In 2009, we achieve the first inhibition FP assay to detect imine cyclic toxins. In the present paper we propose a new FP assay for direct quantify spirolides. This new method has resulted in significant improvement of sensitivity, rapidity and accessibility. In the method design, nicotinic acetylcholine receptor from Torpedo marmorata membranes labelled with a derivative of fluorescein was used. Spirolides, 13-desmethyl spirolide C (13-desMeC) and 13,19-didesmethyl spirolide C (13,19-didesMeC) were extracted and purified from cultures of the Alexandrium ostenfeldii dinoflagellate. Data showed the decrease of FP when toxin concentration was increased. Thus, a relationship between the FP units and the spirolides amount present in a sample was obtained. This direct assay is a reproducible, simple and very sensitive method with a detection limit about 25 nM for 13-desMeC and 150 nM for 13,19-didesMeC. The procedure was used to measure spirolides in mussel samples using an extraction and clean up protocol suitable for the FP assay. Results obtained show that this method is able to quantify 13-desMeC in the range of 50-350 μg kg(-1) meat. Other liposoluble toxins did not interfere with the assay, proving a specific method. Moreover, the matrix do not affect in the range of toxin concentrations that involving risk of spirolides intoxication.
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5.21Impact points
Solid-phase receptor-based assay for the detection of cyclic imines by chemiluminescence, fluorescence, or colorimetry.
Analytical chemistry. 06/2011; 83(15):5857-63.
The spirolides and gymnodimines are marine phycotoxins included in the group of cyclic imines. The toxicity of these compounds to humans is still unknown, although their toxicity by intraperitoneal injection in rodents is very high. A receptor-based method was developed using the competition of the ... [more] The spirolides and gymnodimines are marine phycotoxins included in the group of cyclic imines. The toxicity of these compounds to humans is still unknown, although their toxicity by intraperitoneal injection in rodents is very high. A receptor-based method was developed using the competition of the 13-desmethyl spirolide C with biotin-labeled α-bungarotoxin for binding to nicotinic acetylcholine receptors and the immobilization of the α-bungarotoxin-receptor complex on streptavidin-coated surfaces. The quantification of the immobilized receptor can be achieved using a specific antibody. Finally, after the addition of a secondary antibody labeled with horseradish peroxidase, three alternative substrates of this enzyme generate a chemiluminescent, fluorescent, or colorimetric signal. The assay performs well in shellfish extracts and the detection range is 5-150 nM of 13-desmethyl spirolide C in shellfish extracts, which is at least 5 times more sensitive than the existing fluorescence polarization assay. This assay can also detect gymnodimine, although with 10 times lower sensitivity than the spirolide. The detection of cyclic imines with microplate assays would be useful for screening purposes in order to reduce the number of samples to be processed by bioassays or analytical methods.
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8.58Impact points
Total synthesis of pinnatoxins A and G and revision of the mode of action of pinnatoxin A.
Journal of the American Chemical Society. 06/2011; 133(27):10499-511.
Pinnatoxins belong to an emerging class of potent marine toxins of the cyclic imine group. Detailed studies of their biological effects have been impeded by unavailability of the complex natural product from natural sources. This work describes the development of a robust, scalable synthetic sequenc... [more] Pinnatoxins belong to an emerging class of potent marine toxins of the cyclic imine group. Detailed studies of their biological effects have been impeded by unavailability of the complex natural product from natural sources. This work describes the development of a robust, scalable synthetic sequence relying on a convergent strategy that delivered a sufficient amount of the toxin for detailed biological studies and its commercialization for use by other research groups and regulatory agencies. A central transformation in the synthesis is the highly diastereoselective Ireland-Claisen rearrangement of a complex α,α-disubstituted allylic ester based on a unique mode for stereoselective enolization through a chirality match between the substrate and the lithium amide base. With synthetic pinnatoxin A, a detailed study has been performed that provides conclusive evidence for its mode of action as a potent inhibitor of nicotinic acetylcholine receptors selective for the human neuronal α7 subtype. The comprehensive electrophysiological, biochemical, and computational studies support the view that the spiroimine subunit of pinnatoxins is critical for blocking nicotinic acetylcholine receptor subtypes, as evidenced by analyzing the effect of a synthetic analogue of pinnatoxin A containing an open form of the imine ring. Our studies have paved the way for the production of certified standards to be used for mass-spectrometric determination of these toxins in marine matrices and for the development of tests to detect these toxins in contaminated shellfish.
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3.76Impact points
Synthesis of the gymnodimine tetrahydrofuran core through a Ueno-Stork radical cyclization.
Organic & biomolecular chemistry. 04/2011; 9(10):3726-32.
A straightforward access to the C10-C20 skeleton of gymnodimine, incorporating a tetrahydrofuran fragment, is described. The elaboration of the THF moiety is based on a stereocontrolled Ueno-Stork cyclization. A Lewis-acid mediated allylation of the resulting acetal at C13 and a Horner-Wadsworth-Emm... [more] A straightforward access to the C10-C20 skeleton of gymnodimine, incorporating a tetrahydrofuran fragment, is described. The elaboration of the THF moiety is based on a stereocontrolled Ueno-Stork cyclization. A Lewis-acid mediated allylation of the resulting acetal at C13 and a Horner-Wadsworth-Emmons olefination on the ketone at C17 complete the synthesis.
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2.13Impact points
The marine polyether gambierol enhances muscle contraction and blocks a transient K(+) current in skeletal muscle cells.
Toxicon : official journal of the International Society on Toxinology. 10/2010; 56(5):785-91.
Gambierol is a complex marine toxin first isolated with ciguatoxins from cell cultures of the toxic dinoflagellate Gambierdiscus toxicus. Despite the chemical complexity of the polycyclic ether toxin, the total successful synthesis of gambierol has been achieved by different chemical strategies. In ... [more] Gambierol is a complex marine toxin first isolated with ciguatoxins from cell cultures of the toxic dinoflagellate Gambierdiscus toxicus. Despite the chemical complexity of the polycyclic ether toxin, the total successful synthesis of gambierol has been achieved by different chemical strategies. In the present work the effects of synthetic gambierol on mouse and frog skeletal neuromuscular preparations and Xenopus skeletal myocytes have been studied. Gambierol (0.1-5 muM) significantly increased isometric twitch tension in neuromuscular preparations stimulated through the motor nerve. Less twitch augmentation was observed in directly stimulated muscles when comparing twitch tension-time integrals obtained by nerve stimulation. Also, gambierol induced small spontaneous muscle contraction originating from presynaptic activity that was completely inhibited by d-tubocurarine. Gambierol slowed the rate of muscle action potential repolarization, triggered spontaneous and/or repetitive action potentials, and neither affected action potential amplitude nor overshoot in skeletal muscle fibers. These results suggest that gambierol through an action on voltage-gated K(+) channels prolongs the duration of action potentials, enhances the extent and time course of Ca(2+) release from the sarcoplasmic reticulum, and increases twitch tension generation. Further evidence is provided that gambierol at sub-micromolar concentrations blocks a fast inactivating outward K(+) current that is responsible for action potential prolongation in Xenopus skeletal myocytes.
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4.26Impact points
IP(3)-dependent, post-tetanic calcium transients induced by electrostimulation of adult skeletal muscle fibers.
The Journal of general physiology. 10/2010; 136(4):455-67.
Tetanic electrical stimulation induces two separate calcium signals in rat skeletal myotubes, a fast one, dependent on Cav 1.1 or dihydropyridine receptors (DHPRs) and ryanodine receptors and related to contraction, and a slow signal, dependent on DHPR and inositol trisphosphate receptors (IP(3)Rs) ... [more] Tetanic electrical stimulation induces two separate calcium signals in rat skeletal myotubes, a fast one, dependent on Cav 1.1 or dihydropyridine receptors (DHPRs) and ryanodine receptors and related to contraction, and a slow signal, dependent on DHPR and inositol trisphosphate receptors (IP(3)Rs) and related to transcriptional events. We searched for slow calcium signals in adult muscle fibers using isolated adult flexor digitorum brevis fibers from 5-7-wk-old mice, loaded with fluo-3. When stimulated with trains of 0.3-ms pulses at various frequencies, cells responded with a fast calcium signal associated with muscle contraction, followed by a slower signal similar to one previously described in cultured myotubes. Nifedipine inhibited the slow signal more effectively than the fast one, suggesting a role for DHPR in its onset. The IP(3)R inhibitors Xestospongin B or C (5 µM) also inhibited it. The amplitude of post-tetanic calcium transients depends on both tetanus frequency and duration, having a maximum at 10-20 Hz. At this stimulation frequency, an increase of the slow isoform of troponin I mRNA was detected, while the fast isoform of this gene was inhibited. All three IP(3)R isoforms were present in adult muscle. IP(3)R-1 was differentially expressed in different types of muscle fibers, being higher in a subset of fast-type fibers. Interestingly, isolated fibers from the slow soleus muscle did not reveal the slow calcium signal induced by electrical stimulus. These results support the idea that IP(3)R-dependent slow calcium signals may be characteristic of distinct types of muscle fibers and may participate in the activation of specific transcriptional programs of slow and fast phenotype.
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6.40Impact points
Abnormal distribution of inositol 1,4,5-trisphosphate receptors in human muscle can be related to altered calcium signals and gene expression in Duchenne dystrophy-derived cells.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 09/2010; 24(9):3210-21.
Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) drive calcium signals involved in skeletal muscle excitation-transcription coupling and plasticity; IP(3)R subtype distribution and downstream events evoked by their activation have not been studied in human muscle nor has their possible alter... [more] Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) drive calcium signals involved in skeletal muscle excitation-transcription coupling and plasticity; IP(3)R subtype distribution and downstream events evoked by their activation have not been studied in human muscle nor has their possible alteration in Duchenne muscular dystrophy (DMD). We studied the expression and localization of IP(3)R subtypes in normal and DMD human muscle and in normal (RCMH) and dystrophic (RCDMD) human muscle cell lines. In normal muscle, both type 1 IP(3)Rs (IP(3)R1) and type 2 IP(3)Rs (IP(3)R2) show a higher expression in type II fibers, whereas type 3 IP(3)Rs (IP(3)R3) show uniform distribution. In DMD biopsies, all fibers display a homogeneous IP(3)R2 label, whereas 24 +/- 7% of type II fibers have lost the IP(3)R1 label. RCDMD cells show 5-fold overexpression of IP(3)R2 and down-regulation of IP(3)R3 compared with RCMH cells. A tetanic stimulus induces IP(3)-dependent slow Ca(2+) transients significantly larger and faster in RCDMD cells than in RCMH cells as well as significant ERK1/2 phosphorylation in normal but not in dystrophic cells. Excitation-driven gene expression was different among cell lines; 44 common genes were repressed in RCMH cells and expressed in RCDMD cells or vice versa. IP(3)-dependent Ca(2+) release may play a significant role in DMD pathophysiology.
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31.15Impact points
Essential regulation of cell bioenergetics by constitutive InsP3 receptor Ca2+ transfer to mitochondria.
Cell. 07/2010; 142(2):270-83.
Mechanisms that regulate cellular metabolism are a fundamental requirement of all cells. Most eukaryotic cells rely on aerobic mitochondrial metabolism to generate ATP. Nevertheless, regulation of mitochondrial activity is incompletely understood. Here we identified an unexpected and essential role ... [more] Mechanisms that regulate cellular metabolism are a fundamental requirement of all cells. Most eukaryotic cells rely on aerobic mitochondrial metabolism to generate ATP. Nevertheless, regulation of mitochondrial activity is incompletely understood. Here we identified an unexpected and essential role for constitutive InsP(3)R-mediated Ca(2+) release in maintaining cellular bioenergetics. Macroautophagy provides eukaryotes with an adaptive response to nutrient deprivation that prolongs survival. Constitutive InsP(3)R Ca(2+) signaling is required for macroautophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to diminished mitochondrial Ca(2+) uptake. Mitochondrial uptake of InsP(3)R-released Ca(2+) is fundamentally required to provide optimal bioenergetics by providing sufficient reducing equivalents to support oxidative phosphorylation. Absence of this Ca(2+) transfer results in enhanced phosphorylation of pyruvate dehydrogenase and activation of AMPK, which activates prosurvival macroautophagy. Thus, constitutive InsP(3)R Ca(2+) release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration and maintenance of normal cell bioenergetics.
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5.21Impact points
First toxin profile of ciguateric fish in Madeira Arquipelago (Europe).
Analytical chemistry. 07/2010; 82(14):6032-9.
Ciguatera fish poisoning (CFP) is a human foodborne intoxication caused by ingestion of tropical fishes contaminated with the potent polyether toxins known as ciguatoxins (CTXs). These toxins are issued from Gambierdiscus species of dinoflagellates. Herbivorous fish accumulate these toxins in their ... [more] Ciguatera fish poisoning (CFP) is a human foodborne intoxication caused by ingestion of tropical fishes contaminated with the potent polyether toxins known as ciguatoxins (CTXs). These toxins are issued from Gambierdiscus species of dinoflagellates. Herbivorous fish accumulate these toxins in their musculature and viscera after ingesting dinoflagellates. Epidemiological studies showed that CFP has been present in areas between 35 degrees North and 35 degrees South latitude, mainly, Indo-pacific and Caribbean areas, but not in waters closed to European and African continent. In the present paper, a specimen of Seriola dumerili weighing 70 kg and a smaller Seriola fasciata specimen, captured in waters belonging to Selvagens Islands (Madeira Arquipelago), were analyzed. Fishes from this genus were implicated in previous suspected ciguatera poisoning outbreaks in the Portuguese Madeira Arquipelago in the North Atlantic Ocean. Analysis was performed by two approaches, a functional method using cerebellar granule cells and by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) method. The study was carried out in one portion of the tail muscle of Seriola fasciata and five parts of the body of Seriola dumerili (tail muscle, head, ventral muscle, mid muscle, and liver). The functional method consisted in the modification of the inward sodium current in cerebellar granule cells and the chemical method was a high resolution chromatography, which allowed elucidating the toxin profile in the samples. In addition, UPLC-MS technique was optimized and used for detecting and quantifying CTXs for the first time. After fish extraction and clean up, the chromatograms revealed the presence of CTX-1B at 1111.6 m/z, CTX-3C at 1023.5 m/z, a CTX analogue at 1040.6 m/z, and a CTX from the Caribbean or Indic waters at 1141.6 m/z. Therefore, the results obtained in the present paper for both methods confirm, for the first time, the presence of CTX in fish from Madeira Arquipelago.
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3.29Impact points
Detection of 13,19-didesmethyl C spirolide by fluorescence polarization using Torpedo electrocyte membranes.
Analytical biochemistry. 04/2010; 403(1-2):102-7.
Fluorescence polarization (FP) is a powerful tool for studying molecular interactions by monitoring changes in the apparent size of fluorescent molecules. In this paper, a previously described fluorescence polarization assay was used to detect 13,19-didesmethyl C spirolide. The assay is based on the... [more] Fluorescence polarization (FP) is a powerful tool for studying molecular interactions by monitoring changes in the apparent size of fluorescent molecules. In this paper, a previously described fluorescence polarization assay was used to detect 13,19-didesmethyl C spirolide. The assay is based on the competition of cyclic imine marine biotoxins with alpha-bungarotoxin for binding to nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata. The 13,19-didesmethyl C spirolide was detected in buffer and mussel matrix. The sensitivity of the assay for the 13,19-didesmethyl C spirolide and the 13-desmethyl C spirolide was similar. After an acetone/chloroform extraction of spiked mussel meat, the average recovery rate of 13,19-didesmethyl C spirolide was 77.7 +/- 1.9%. The quantification range for this toxin in mussel was 40-200 microg/kg of shellfish meat. This assay can be used to detect the spirolides 13,19-didesmethyl C spirolide and 13-desmethyl C spirolide, in shellfish as a screening assay.
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6.14Impact points
Sustained synaptic-vesicle recycling by bulk endocytosis contributes to the maintenance of high-rate neurotransmitter release stimulated by glycerotoxin.
Journal of cell science. 03/2010; 123(Pt 7):1131-40.
Glycerotoxin (GLTx), a large neurotoxin isolated from the venom of the sea worm Glycera convoluta, promotes a long-lasting increase in spontaneous neurotransmitter release at the peripheral and central synapses by selective activation of Ca(v)2.2 channels. We found that GLTx stimulates the very high... [more] Glycerotoxin (GLTx), a large neurotoxin isolated from the venom of the sea worm Glycera convoluta, promotes a long-lasting increase in spontaneous neurotransmitter release at the peripheral and central synapses by selective activation of Ca(v)2.2 channels. We found that GLTx stimulates the very high frequency, long-lasting (more than 10 hours) spontaneous release of acetylcholine by promoting nerve terminal Ca(2+) oscillations sensitive to the inhibitor omega-conotoxin GVIA at the amphibian neuromuscular junction. Although an estimate of the number of synaptic vesicles undergoing exocytosis largely exceeds the number of vesicles present in the motor nerve terminal, ultrastructural examination of GLTx-treated synapses revealed no significant change in the number of synaptic vesicles. However, we did detect the appearance of large pre-synaptic cisternae suggestive of bulk endocytosis. Using a combination of styryl dyes, photoconversion and horseradish peroxidase (HRP)-labeling electron microscopy, we demonstrate that GLTx upregulates presynaptic-vesicle recycling, which is likely to emanate from the limiting membrane of these large cisternae. Similar synaptic-vesicle recycling through bulk endocytosis also occurs from nerve terminals stimulated by high potassium. Our results suggest that this process might therefore contribute significantly to synaptic recycling under sustained levels of synaptic stimulation.
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9.43Impact points
Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism.
Proceedings of the National Academy of Sciences of the United States of America. 03/2010; 107(13):6076-81.
Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Analysis of 13-desmethyl spirolide C and gymnodimine A by binding and voltage-clamp recordings on muscle-type alpha1(2)betagammadelta and... [more] Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Analysis of 13-desmethyl spirolide C and gymnodimine A by binding and voltage-clamp recordings on muscle-type alpha1(2)betagammadelta and neuronal alpha3beta2 and alpha4beta2 nicotinic acetylcholine receptors reveals subnanomolar affinities, potent antagonism, and limited subtype selectivity. Their binding to acetylcholine-binding proteins (AChBP), as soluble receptor surrogates, exhibits picomolar affinities governed by diffusion-limited association and slow dissociation, accounting for apparent irreversibility. Crystal structures of the phycotoxins bound to Aplysia-AChBP ( approximately 2.4A) show toxins neatly imbedded within the nest of ar-omatic side chains contributed by loops C and F on opposing faces of the subunit interface, and which in physiological conditions accommodates acetylcholine. The structures also point to three major features: (i) the sequence-conserved loop C envelops the bound toxins to maximize surface complementarity; (ii) hydrogen bonding of the protonated imine nitrogen in the toxins with the carbonyl oxygen of loop C Trp147 tethers the toxin core centered within the pocket; and (iii) the spirolide bis-spiroacetal or gymnodimine tetrahydrofuran and their common cyclohexene-butyrolactone further anchor the toxins in apical and membrane directions, along the subunit interface. In contrast, the se-quence-variable loop F only sparingly contributes contact points to preserve the broad receptor subtype recognition unique to phycotoxins compared with other nicotinic antagonists. These data offer unique means for detecting spiroimine toxins in shellfish and identify distinctive ligands, functional determinants and binding regions for the design of new drugs able to target several receptor subtypes with high affinity.
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3.84Impact points
Ligand-binding assays for cyanobacterial neurotoxins targeting cholinergic receptors.
Analytical and bioanalytical chemistry. 03/2010; 397(5):1695-704.
Toxic cyanobacterial blooms are a threat to public health because of the capacity of some cyanobacterial species to produce potent hepatotoxins and neurotoxins. Cyanobacterial neurotoxins are involved in the rapid death of wild and domestic animals by targeting voltage gated sodium channels and chol... [more] Toxic cyanobacterial blooms are a threat to public health because of the capacity of some cyanobacterial species to produce potent hepatotoxins and neurotoxins. Cyanobacterial neurotoxins are involved in the rapid death of wild and domestic animals by targeting voltage gated sodium channels and cholinergic synapses, including the neuromuscular junction. Anatoxin-a and its methylene homologue homoanatoxin-a are potent agonists of nicotinic acetylcholine receptors. Since the structural determination of anatoxin-a, several mass spectrometry-based methods have been developed for detection of anatoxin-a and, later, homoanatoxin-a. Mass spectrometry-based techniques provide accuracy, precision, selectivity, sensitivity, reproducibility, adequate limit of detection, and structural and quantitative information for analyses of cyanobacterial anatoxins from cultured and environmental cyanobacterial samples. However, these physicochemical techniques will only detect known toxins for which toxin standards are commercially available, and they require highly specialized laboratory personnel and expensive equipment. Receptor-based assays are functional methods that are based on the mechanism of action of a class of toxins and are thus, suitable tools for survey of freshwater reservoirs for cyanobacterial anatoxins. The competition between cyanobacterial anatoxins and a labelled ligand for binding to nicotinic acetylcholine receptors is measured radioactively or non-radioactively providing high-throughput screening formats for routine detection of this class of neurotoxins. The mouse bioassay is the method of choice for marine toxin monitoring, but has to be replaced by fully validated functional methods. In this paper we review the ligand-binding assays developed for detection of cyanobacterial and algal neurotoxins targeting the nicotinic acetylcholine receptors and for high-throughput screening of novel nicotinic agents.
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4.31Impact points
Feasibility of gymnodimine and 13-desmethyl C spirolide detection by fluorescence polarization using a receptor-based assay in shellfish matrixes.
Analytica chimica acta. 01/2010; 657(1):75-82.
The detection of toxins in shellfish through reliable methods is essential for human health preservation and prevention of economic losses in the aquaculture industry. Although no human intoxication has been unequivocally linked to gymnodimines or spirolides, these phycotoxins are highly toxic by in... [more] The detection of toxins in shellfish through reliable methods is essential for human health preservation and prevention of economic losses in the aquaculture industry. Although no human intoxication has been unequivocally linked to gymnodimines or spirolides, these phycotoxins are highly toxic by intraperitoneal injection causing false positives in lipophilic toxin detection by the mouse bioassay. Based on the detection of molecular interactions by fluorescence polarization an inhibition assay was developed using fluorescent alpha-bungarotoxin and nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata to detect gymnodimine and 13-desmethyl C spirolide. Both toxins, classified into the cyclic imine group, inhibit the interaction of alpha-bungarotoxin with Torpedo nicotinic acetylcholine receptors in the nM range. In this study we analyze the matrix effect of four shellfish species on the fluorescence polarization assay. Mussels, clams, cockles and scallops were extracted with acetone and sequentially partitioned with n-hexane and chloroform. The interference of these shellfish extracts with the alpha-bungarotoxin fluorescence or its binding to the nicotinic acetylcholine receptor was lower than 11%. The average recovery rates of gymnodimine and 13-desmethyl C spirolide using these solvents were 90.6+/-7.8% and 89.6+/-3.2%, respectively with variations among species. The quantification range of this fluorescence polarization assay for gymnodimine and 13-desmethyl C spirolide in all tested species was 80-2000 microg kg(-1) and 85-700 microg kg(-1) of shellfish meat, respectively. This assay format can be used to detect gymnodimine and 13-desmethyl C spirolide in shellfish as a screening assay.
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2.59Impact points
Quantitative Determination of Gymnodimine-A by High Performance Liquid Chromatography in Contaminated Clams from Tunisia Coastline.
Marine biotechnology (New York, N.Y.). 12/2009;
Quantitative determination by high performance liquid chromatography (HPLC) was performed for gymnodimine-A (GYM-A), a phycotoxin responsible for the contamination of Tunisian clams. This study demonstrates a rapid and reproducible HPLC-ultraviolet (UV) method for extraction, detection and quantific... [more] Quantitative determination by high performance liquid chromatography (HPLC) was performed for gymnodimine-A (GYM-A), a phycotoxin responsible for the contamination of Tunisian clams. This study demonstrates a rapid and reproducible HPLC-ultraviolet (UV) method for extraction, detection and quantification of GYM-A in toxic clams. The extraction of GYM-A from the digestive gland of clams in acetone, subsequent clean-up with diethyl ether and extraction with dichloromethane is the more valid protocol. Chromatography analyses were performed using a gradient of acetonitrile-water (10:90 to 90:10), containing trifluoroacetic acid (0.1%) for 20 min at 1 mL/min rate with a C18 column. Recovery rates exceeded 96%, and limits of detection and quantification were 5 ng/mL and 8 ng/g digestive gland, respectively. Repeatability and reproducibility were tested for various samples containing different levels of GYM-A. A significant correlation was observed between toxicity level of samples and the determined amount of GYM-A. Also, the persistence of GYM-A in contaminated clams from Boughrara lagoon was demonstrated. The kinetics discharge study of GYM-A in controlled medium, during 1 month, showed that the process of depuration was biphasic with an exponential discharge of 75% of the total amount of sequestered GYM-A during the first 12 days followed by a slow discharge (>10%) for the subsequent days up to the seventeenth day. This is the first time that a quantitative study of GYM-A in clams from Tunisian coasts is performed through the development of a new method for detection and quantify of this phycotoxin. We found HPLC-UV a reliable and suitable alternative to the mouse bioassay.
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2.13Impact points
Neurotoxic cyanobacterial toxins.
Toxicon : official journal of the International Society on Toxinology. 09/2009;
Worldwide development of cyanobacterial blooms has significantly increased in marine and continental waters in the last century due to water eutrophication. This phenomenon is favoured by the ability of planktonic cyanobacteria to synthesize gas vesicles that allow them to float in the water column.... [more] Worldwide development of cyanobacterial blooms has significantly increased in marine and continental waters in the last century due to water eutrophication. This phenomenon is favoured by the ability of planktonic cyanobacteria to synthesize gas vesicles that allow them to float in the water column. Besides, benthic cyanobacteria that proliferate at the bottom of lakes, rivers and costal waters form dense mats near the shore. Cyanobacterial massive proliferation is of public concern regarding the capacity of certain cyanobacterial strains to produce hepatotoxic and neurotoxic compounds that can affect public health, human activities and wild and stock animals. The cholinergic synapses and voltage-gated sodium channels constitute the targets of choice of cyanobacterial neurotoxins. Anatoxin-a and homoanatoxin-a are agonists of nicotinic acetylcholine receptors. Anatoxin-a(s) is an irreversible inhibitor of acetylcholinesterase. Saxitoxin, kalkitoxin and jamaicamide are blockers of voltage-gated sodium channels, whereas antillatoxin is an activator of such channels. Moreover the neurotoxic amino acid L-beta-N-methylamino-L-alanine was shown to be produced by diverse cyanobacterial taxa. Although controversial, increasing in vivo and in vitro evidence suggest a link between the ingestion of L-beta-N-methylamino-L-alanine and the development of amyotrophic lateral sclerosis/parkinsonism-dementia complex, neurodegenerative diseases. This paper reviews the occurrence of cyanobacterial neurotoxins, their chemical properties, mode of action and biosynthetic pathways.
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Ciguatoxin-induced catecholamine secretion in bovine chromaffin cells: mechanism of action and reversible inhibition by brevenal.
Toxicon : official journal of the International Society on Toxinology. 09/2009;
Ciguatoxin (P-CTX-1B) from the dinoflagellate Gambierdiscus toxicus, belongs to the family of polyether neurotoxins responsible for the neurological poisoning disorder ciguatera. Although it is the most widespread marine-borne disease affecting humans, there is no current FDA-approved treatment avai... [more] Ciguatoxin (P-CTX-1B) from the dinoflagellate Gambierdiscus toxicus, belongs to the family of polyether neurotoxins responsible for the neurological poisoning disorder ciguatera. Although it is the most widespread marine-borne disease affecting humans, there is no current FDA-approved treatment available except for symptomatic therapies. In this paper, we report that P-CTX-1B produces large catecholamine secretion from bovine chromaffin cells that is insensitive to concomitant activation of capacitative Ca(2+) entry. Moreover, we confirm that brevenal, a polyether from the dinoflagellate Karenia brevis, blocks P-CTX-1B-induced catecholamine secretion. This effect is partially reversible. Our results raised the prospect of finding functional antagonists for P-CTX-1B that could be useful for the treatment of ciguatera.
Following (22)
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Giampietro Schiavo
Cancer Research UK -
Frederic A Meunier
University of Queensland -
Tam Hong Nguyen
University of Illinois at Urbana Champaign -
Dany Spencer Adams
Tufts University -
Veronique Bernard
Institut national de la santé et de la recherche médicale
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