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Domoic acid neurotoxicity: Electrophysiological and behavioral investigations
1084
Reports
and Abstracts
Solution structure andpharmacological activity of scorpion toxins active on KCa channels. E. Blanc’, V. Fremont?, and H. Darbon’ (‘Laboratoire de Cristallographie et Cristallisation des Macromolecules Biologiques, CNRS URA 1296, IFRl, Chemin Joseph-Aiguier, 13008 Marseille; ‘Laboratoire d’lngtnierie des Proteines, CNRS URA 1455, IFR Jean Roche, Fact&e de Medecine-Nord, 13916 Marseille cedex 20. France). P05, POl, and Leiurotoxin I (Lei) have been purified from scorpion venoms. They bind on calcium activated-potassium channels apamin sensitive (SK or small conductance). We synthesized them as well as two analogs: [M7R]-Leiurotoxin (R7Lei) et [L6R, L7R]-PO5 (L6L7PO5) (Sabatier Y( al., 1994, It-n. J. Peptide Protein Res. 43, 486495) and determined their solution structure by ‘H-2D NMR, demonstrating that they all shared the same overall conformation, i.e. 2.5 turns of cc-helix connected by a tight turn to a two antiparallel strands of P-sheet, with the exception of PO1 in which the cc-helix is shortened to two turns with a proline in the first turn. The three disuhides bonds are topologically homologous and are in equilibrium between two conformations. The structure of these molecules is present in all scorpion toxins whatever their activity is. The pharmacological activity of these molecules, as tested in competition experiment against “‘I-apamin on SK channels is unconstant: PO5 = 2 x 10 ” M, R7Lei = 2 x IO-” M. Lei = 3 x IO-” M, L6L7PO5 = lO_‘M, PO1 = 10 -’ M. This activity is related to the electropositivity of the solvent exposed surface of the a-helix. However, analysis of the charges beared by the surface in interaction with the receptor cannot explain by itself the detected activities. One has to take into account the repartition of charges all over the molecule.
Capillary electrophoresis: a highly sensitive techniqur,for the determination of’ marine phycotoxins. N. Bouai’cha’. M. C. Hennion’, and P. Sandra’ (‘CEMATMA, Laboratoire de Chimie Analytique, E.S.P.C.I., IO rue Vauquelin, 75231 Paris, France; Universite de Gand, Laboratoire de Chimie Organique Krijgslaan 281 (S4), 9000 Gand, Belgique). Marine phycotoxins are among the most virulent nonproteineous known toxins. They usually elicit very specific physiological responses and often possess complex chemical structures. They present a major public health problem due to their ability to contaminate seafoods. Okadaic acid (OA), a toxin from the dinoflagellate. Prorocentrum lima, with a polyether structure, has been shown to be a powerful tumor promotor. Thus considerable effort has been undertaken to find sensitive and easy detection techniques for the determination of phycotoxins from various matrices (microalgae, mussels, fish, etc.). Different biological and chemical methods have been developed to identify these toxins in shellfish and fish. The mouse bioassay method has been widely utilized in the detection of toxins such as maitotoxin and ciguatoxin, that are not easily determined by chemical procedures. In this report we describe a capillary electrophoretic method that enables the detection of okadaic acid and maitotoxin in picogram range.
Immunochemical and electrophysiological properties of a novel recombinant scorpion ulpha insect toxin. B. Bouhaouala-Zahar’, R. Ben Khalifa’,‘, I. Zanouaki’, L. Borchani’, F. Ducancel’. M. Pelhate’, J. C. Boulain’, A. Menez’, M. El Ayeb ’ and H. Karoui’ (‘Laboratory of Venoms and Toxins, Institut Pasteur de Tunis, BP 74, 1002 Tunis-Belvedere, Tunisia; ‘Laboratory of Neurophysiology, CNRS ERS 108, University of Angers, 49045 Angers Cedex, France: ‘Department of Protein Studies and Engineering, C.E.A. Saclay, Gif sur Yvette, France). A cDNA library was constructed from the venom glands of the scorpion Buthus occitanus tunetanus and a DNA sequence, encoding a previously unknown a-toxin, was cloned. This clone was efficiently expressed in Ekherichia coli as a fusion protein with two synthetic domains (ZZ) of the protein A from Staplzylococcus aureus. Following CNBr treatment and HPLC purification, a recombinant cc-toxin. called BotXIV r., was obtained. This latest displays no toxicity towards mammals, but is active on insects as shown by contractive paralysis effects it induces on the cockroach Blattellu germanica. Interestingly the fused protein is highly immunogenic in mice and induces production of sera capable of recognizing and neutralizing specifically highly toxic components previously injected to mice. Thus, it could constitute a potentially well suited material to develop new antisera against scorpion envenomation. Finally, electrophysiological studies on the cockroach Periplaneta americuna isolated giant axons, showed an evident slowing of the sodium current inactivation mechanism, a typical property of an cc-insect toxin.
Domoic acid neurotoxicity: electrophysiological and behavioral investigations. P. Breton. X. Manciaux, I. de la Manche, and J. Buee (Departement de Biologic, Centre d’Etudes du Bouchet, B.P. Vert-le-Petit, France).
J. C. Bizot, n 3-91710
Domoic acid is a phytoplankter neurotoxin extracted from Nitzschia pungens alga Implicated in a severe collective intoxication which occurred in Canada in 1987, this molecule is one of the most potent glutamate agonists presently known. This neurotoxicological study, related to limbic seizure, correlated data of symptomatology. electrophysiology, behavioral and histology. Fifty-nine Wistar rats received a 13 mm long
Reports
and Abstracts
10x5
stainless steel guide cannula for intracerebroventricular injection (coordinates were A: 8,2; L: 1.3; P: 3.6 from bregma skull surface according to Paxinos). In addition. 29 received a subcortical ipsilateral bipolar electrode within the hippocampus (CA1 field or dentate gyrus region: CD) and two or three cortical electrodes. Animals received a single dose of toxin (or solvant = phosphate buffer 0.01 M). For infusion into lateral ventricule, the injection cannula was made from a 30 gauge needle. The cannula was filled prior to placement into the guide and was connected to a glass microsyringe (Hamilton 5 ~1) by a thin catheter. Four doses of toxin were tested: 15, 20, 25, 50 ng. A toxic threshold dose was evaluated at 25 ng. Under this dose, epileptic induced process were not significant (15% of the cases for 20 ng) without any organic sequel. They appeared in numerous short repetitive episodes and was greatly attenuated after 1 hr. Theta rhythm, the most characteristic component of the hippocampal EEG in rodents, was not significantly modified. For highest doses tested (25-50 ng) sustained seizure or repetitive epileptic episodes were recorded at least during the first 2 hr. These activities were nolt observed at day 3. Theta rhythm remained alterated mainly near GD. Neuronal induced damages observed at day 7 were located in hippocampus (CA3-CA4 fields and. in the case of severe disease. in CAI held) and entorhinal cortex. Nevertheless it was difficult to establish any correlation between EEG modifications and the spreading of lesions. MK 801 (I mg/kg S.C. 1 hr before the toxin: 50 ng ICV) was able to prevent the maintenance of seizure (but not appearance) and neuronal damages. Effects of domoic acid on spatial memory were studied with the Moris water mage test. Rats received domoic acid (25 or 50 ng ICV; II = 17) or phosphate buffer (control group n = 13). Learning test was undertaken 7 and 8 days later. Learning was disrupted in rats previously injected with domoic acid. This effect reached a statistically significant level only for animals that exhibited dramatic symptoms (long-lasting and pronounced status epilepticus) following injection. These animals exhibited significant lesions in hippocampus (CA3-CA4 and CA1 fields) at day 14. The results indicate that lasting seizure activities seem a prerequisite to neuronal damages and their spreading. These neurotoxic effects may be partly dependent on NMDA receptors activation and induce mnemonic impairments.
Synthesi.s qf’cJ~&peptidic
CNRS
46%UniversitP
and cyclodepsipeptidic
Montpellier
to.\-ins: tet1to.xin md de.smr.vins. F. Cavelier
II, 34095 Montpellier
and J. Verducci
(URA
cedex 05-France).
We describe original chemical approaches for the total synthesis of two kinds of fungal toxins. Tentoxin (isolated from Al/ernnrin rem&) which possesses herbicidal properties and a tentoxin analogue. with N-methyl serine in place of N-methyl alanine, are prepared in col~abordtion with the CEA( I). In a second part. we present the synthesis of some analogues of Destruxin E (isolated from Me/harhi;ium anisopliae) which are bioinsecticides. We replaced hydroxy acid by various N-methyl amino acids or modified side chain of the hydroxy acid, studying implications on bioactivity(2).
rNMe
Ala-Leu-NMe
APhe-Gly
1
Tentoxin ?
-Pro-Ile-NMe
Val-NMe
Ala-p
Ala-X-CH-$0
Detruxin analogues (X=O,NH or N-Me) (1) F. Andre. G. Girault, F. Haraux, E. Pinet and C. Sigalat. CEA, Dkpartement de Biologic Cellulaire et Moltculaire, Section de biotnergCtique, Centre d’Etudes de Saclay. (2) Contract CEE, DG XII, Agriculture and Agroindustry. including Fisheries: ‘Fungal Cyclic Peptides ah new Biopesticides‘ N AIR3 -CT93-1253.
Cloning Y~N(.(‘f?./(,l7f~lineStCruSefrom Bungdrus fasciatus r~nom. X. Cousin’ ‘. C. Bon’ (‘Unit& des Venins. Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France; ‘Laboratoire de Neurobiologie. Ecole Normale Supbrieure. 26 rue d’Ulm. 75005 Paris, France).
The enzyme
acetylcholinesterase (AChE) is present in the venom of snakes of the Ekupidaz family, except in the genus. In addition with the active site, AChE possesses a peripheral anionic site which has been revealed by the inhibitory effect of some compounds such as propidium. The Dendroaspis venom toxin fasciculin also bound to this peripheral site. The AChE from Bungcrrus venom is much more resistant to fasciculin and Dmdronspis
Reports
and Abstracts
Solution structure andpharmacological activity of scorpion toxins active on KCa channels. E. Blanc’, V. Fremont?, and H. Darbon’ (‘Laboratoire de Cristallographie et Cristallisation des Macromolecules Biologiques, CNRS URA 1296, IFRl, Chemin Joseph-Aiguier, 13008 Marseille; ‘Laboratoire d’lngtnierie des Proteines, CNRS URA 1455, IFR Jean Roche, Fact&e de Medecine-Nord, 13916 Marseille cedex 20. France). P05, POl, and Leiurotoxin I (Lei) have been purified from scorpion venoms. They bind on calcium activated-potassium channels apamin sensitive (SK or small conductance). We synthesized them as well as two analogs: [M7R]-Leiurotoxin (R7Lei) et [L6R, L7R]-PO5 (L6L7PO5) (Sabatier Y( al., 1994, It-n. J. Peptide Protein Res. 43, 486495) and determined their solution structure by ‘H-2D NMR, demonstrating that they all shared the same overall conformation, i.e. 2.5 turns of cc-helix connected by a tight turn to a two antiparallel strands of P-sheet, with the exception of PO1 in which the cc-helix is shortened to two turns with a proline in the first turn. The three disuhides bonds are topologically homologous and are in equilibrium between two conformations. The structure of these molecules is present in all scorpion toxins whatever their activity is. The pharmacological activity of these molecules, as tested in competition experiment against “‘I-apamin on SK channels is unconstant: PO5 = 2 x 10 ” M, R7Lei = 2 x IO-” M. Lei = 3 x IO-” M, L6L7PO5 = lO_‘M, PO1 = 10 -’ M. This activity is related to the electropositivity of the solvent exposed surface of the a-helix. However, analysis of the charges beared by the surface in interaction with the receptor cannot explain by itself the detected activities. One has to take into account the repartition of charges all over the molecule.
Capillary electrophoresis: a highly sensitive techniqur,for the determination of’ marine phycotoxins. N. Bouai’cha’. M. C. Hennion’, and P. Sandra’ (‘CEMATMA, Laboratoire de Chimie Analytique, E.S.P.C.I., IO rue Vauquelin, 75231 Paris, France; Universite de Gand, Laboratoire de Chimie Organique Krijgslaan 281 (S4), 9000 Gand, Belgique). Marine phycotoxins are among the most virulent nonproteineous known toxins. They usually elicit very specific physiological responses and often possess complex chemical structures. They present a major public health problem due to their ability to contaminate seafoods. Okadaic acid (OA), a toxin from the dinoflagellate. Prorocentrum lima, with a polyether structure, has been shown to be a powerful tumor promotor. Thus considerable effort has been undertaken to find sensitive and easy detection techniques for the determination of phycotoxins from various matrices (microalgae, mussels, fish, etc.). Different biological and chemical methods have been developed to identify these toxins in shellfish and fish. The mouse bioassay method has been widely utilized in the detection of toxins such as maitotoxin and ciguatoxin, that are not easily determined by chemical procedures. In this report we describe a capillary electrophoretic method that enables the detection of okadaic acid and maitotoxin in picogram range.
Immunochemical and electrophysiological properties of a novel recombinant scorpion ulpha insect toxin. B. Bouhaouala-Zahar’, R. Ben Khalifa’,‘, I. Zanouaki’, L. Borchani’, F. Ducancel’. M. Pelhate’, J. C. Boulain’, A. Menez’, M. El Ayeb ’ and H. Karoui’ (‘Laboratory of Venoms and Toxins, Institut Pasteur de Tunis, BP 74, 1002 Tunis-Belvedere, Tunisia; ‘Laboratory of Neurophysiology, CNRS ERS 108, University of Angers, 49045 Angers Cedex, France: ‘Department of Protein Studies and Engineering, C.E.A. Saclay, Gif sur Yvette, France). A cDNA library was constructed from the venom glands of the scorpion Buthus occitanus tunetanus and a DNA sequence, encoding a previously unknown a-toxin, was cloned. This clone was efficiently expressed in Ekherichia coli as a fusion protein with two synthetic domains (ZZ) of the protein A from Staplzylococcus aureus. Following CNBr treatment and HPLC purification, a recombinant cc-toxin. called BotXIV r., was obtained. This latest displays no toxicity towards mammals, but is active on insects as shown by contractive paralysis effects it induces on the cockroach Blattellu germanica. Interestingly the fused protein is highly immunogenic in mice and induces production of sera capable of recognizing and neutralizing specifically highly toxic components previously injected to mice. Thus, it could constitute a potentially well suited material to develop new antisera against scorpion envenomation. Finally, electrophysiological studies on the cockroach Periplaneta americuna isolated giant axons, showed an evident slowing of the sodium current inactivation mechanism, a typical property of an cc-insect toxin.
Domoic acid neurotoxicity: electrophysiological and behavioral investigations. P. Breton. X. Manciaux, I. de la Manche, and J. Buee (Departement de Biologic, Centre d’Etudes du Bouchet, B.P. Vert-le-Petit, France).
J. C. Bizot, n 3-91710
Domoic acid is a phytoplankter neurotoxin extracted from Nitzschia pungens alga Implicated in a severe collective intoxication which occurred in Canada in 1987, this molecule is one of the most potent glutamate agonists presently known. This neurotoxicological study, related to limbic seizure, correlated data of symptomatology. electrophysiology, behavioral and histology. Fifty-nine Wistar rats received a 13 mm long
Reports
and Abstracts
10x5
stainless steel guide cannula for intracerebroventricular injection (coordinates were A: 8,2; L: 1.3; P: 3.6 from bregma skull surface according to Paxinos). In addition. 29 received a subcortical ipsilateral bipolar electrode within the hippocampus (CA1 field or dentate gyrus region: CD) and two or three cortical electrodes. Animals received a single dose of toxin (or solvant = phosphate buffer 0.01 M). For infusion into lateral ventricule, the injection cannula was made from a 30 gauge needle. The cannula was filled prior to placement into the guide and was connected to a glass microsyringe (Hamilton 5 ~1) by a thin catheter. Four doses of toxin were tested: 15, 20, 25, 50 ng. A toxic threshold dose was evaluated at 25 ng. Under this dose, epileptic induced process were not significant (15% of the cases for 20 ng) without any organic sequel. They appeared in numerous short repetitive episodes and was greatly attenuated after 1 hr. Theta rhythm, the most characteristic component of the hippocampal EEG in rodents, was not significantly modified. For highest doses tested (25-50 ng) sustained seizure or repetitive epileptic episodes were recorded at least during the first 2 hr. These activities were nolt observed at day 3. Theta rhythm remained alterated mainly near GD. Neuronal induced damages observed at day 7 were located in hippocampus (CA3-CA4 fields and. in the case of severe disease. in CAI held) and entorhinal cortex. Nevertheless it was difficult to establish any correlation between EEG modifications and the spreading of lesions. MK 801 (I mg/kg S.C. 1 hr before the toxin: 50 ng ICV) was able to prevent the maintenance of seizure (but not appearance) and neuronal damages. Effects of domoic acid on spatial memory were studied with the Moris water mage test. Rats received domoic acid (25 or 50 ng ICV; II = 17) or phosphate buffer (control group n = 13). Learning test was undertaken 7 and 8 days later. Learning was disrupted in rats previously injected with domoic acid. This effect reached a statistically significant level only for animals that exhibited dramatic symptoms (long-lasting and pronounced status epilepticus) following injection. These animals exhibited significant lesions in hippocampus (CA3-CA4 and CA1 fields) at day 14. The results indicate that lasting seizure activities seem a prerequisite to neuronal damages and their spreading. These neurotoxic effects may be partly dependent on NMDA receptors activation and induce mnemonic impairments.
Synthesi.s qf’cJ~&peptidic
CNRS
46%UniversitP
and cyclodepsipeptidic
Montpellier
to.\-ins: tet1to.xin md de.smr.vins. F. Cavelier
II, 34095 Montpellier
and J. Verducci
(URA
cedex 05-France).
We describe original chemical approaches for the total synthesis of two kinds of fungal toxins. Tentoxin (isolated from Al/ernnrin rem&) which possesses herbicidal properties and a tentoxin analogue. with N-methyl serine in place of N-methyl alanine, are prepared in col~abordtion with the CEA( I). In a second part. we present the synthesis of some analogues of Destruxin E (isolated from Me/harhi;ium anisopliae) which are bioinsecticides. We replaced hydroxy acid by various N-methyl amino acids or modified side chain of the hydroxy acid, studying implications on bioactivity(2).
rNMe
Ala-Leu-NMe
APhe-Gly
1
Tentoxin ?
-Pro-Ile-NMe
Val-NMe
Ala-p
Ala-X-CH-$0
Detruxin analogues (X=O,NH or N-Me) (1) F. Andre. G. Girault, F. Haraux, E. Pinet and C. Sigalat. CEA, Dkpartement de Biologic Cellulaire et Moltculaire, Section de biotnergCtique, Centre d’Etudes de Saclay. (2) Contract CEE, DG XII, Agriculture and Agroindustry. including Fisheries: ‘Fungal Cyclic Peptides ah new Biopesticides‘ N AIR3 -CT93-1253.
Cloning Y~N(.(‘f?./(,l7f~lineStCruSefrom Bungdrus fasciatus r~nom. X. Cousin’ ‘. C. Bon’ (‘Unit& des Venins. Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France; ‘Laboratoire de Neurobiologie. Ecole Normale Supbrieure. 26 rue d’Ulm. 75005 Paris, France).
The enzyme
acetylcholinesterase (AChE) is present in the venom of snakes of the Ekupidaz family, except in the genus. In addition with the active site, AChE possesses a peripheral anionic site which has been revealed by the inhibitory effect of some compounds such as propidium. The Dendroaspis venom toxin fasciculin also bound to this peripheral site. The AChE from Bungcrrus venom is much more resistant to fasciculin and Dmdronspis