Abel Aneiros’s research while affiliated with Instituto de Oceanología and other places

CgNa (Condylactis gigantea neurotoxin) is a 47-amino-acid- residue toxin from the giant Caribbean sea anemone Condylactis gigantea. The structure of CgNa, which was solved by 1H-NMR spectroscopy, is somewhat atypical and displays significant homology with both type I and II anemone toxins. CgNa also displays a considerable number of exceptions to the canonical structural elements that are thought to be essential for the activity of this group of toxins. Furthermore, unique residues in CgNa define a characteristic structure with strong negatively charged surface patches. These patches disrupt a surface-exposed cluster of hydrophobic residues present in all anemone-derived toxins described to date. A thorough characterization by patch-clamp analysis using rat DRG (dorsal root ganglion) neurons indicated that CgNa preferentially binds to TTX-S (tetrodotoxin-sensitive) voltage-gated sodium channels in the resting state. This association increased the inactivation time constant and the rate of recovery from inactivation, inducing a significant shift in the steady state of inactivation curve to the left. The specific structural features of CgNa may explain its weaker inhibitory capacity when compared with the other type I and II anemone toxins.

We have characterized the actions of ApC, a sea anemone polypeptide toxin isolated from Anthopleura elegantissima, on neuronal sodium currents (I(Na)) using current and voltage-clamp techniques. Neurons of the dorsal root ganglia of Wistar rats (P5-9) in primary culture were used for this study. These cells express tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) I(Na). In current-clamp experiments, application of ApC increased the average duration of the action potential. Under voltage-clamp conditions, the main effect of ApC was a concentration-dependent increase in the TTX-S I(Na) inactivation time course. No significant effects were observed on the activation time course or on the current peak-amplitude. ApC also produced a hyperpolarizing shift in the voltage at which 50% of the channels are inactivated and caused a significant decrease in the voltage dependence of Na+ channel inactivation. No effects were observed on TTX-R I(Na). Our results suggest that ApC slows the conformational changes required for fast inactivation of the mammalian Na+ channels in a form similar to other site-3 toxins, although with a greater potency than ATX-II, a highly homologous anemone toxin.

A new peptide toxin exhibiting a molecular weight of 5043Da (av.) and comprising 47 amino acid residues was isolated from the sea anemone Condylactis gigantea. Purification of the peptide was achieved by a multistep chromatographic procedure monitoring its strong paralytic activity on crustacea (LD(50) approx. 1microg/kg). Complete sequence analysis of the toxic peptide revealed the isolation of a new member of type I sea anemone sodium channel toxins containing the typical pattern of the six cysteine residues. From 11kg of wet starting material, approximately 1g of the peptide toxin was isolated. The physiological action of the new toxin from C. gigantea CgNa was investigated on sodium currents of rat dorsal root ganglion neurons in culture using whole-cell patch clamp technique (n=60). Under current clamp condition (CgNa) increased action potential duration. This effect is due to slowing down of the TTX-S sodium current inactivation, without modifying the activation process. CgNa prolonged the cardiac action potential duration and enhanced contractile force albeit at 100-fold higher concentrations than the Anemonia sulcata toxin ATXII. The action on sodium channel inactivation and on cardiac excitation-contraction coupling resemble previous results with compounds obtained from this and other sea anemones [Shapiro, B.I., 1968. Purification of a toxin from tentacles of the anemone C. gigantea. Toxicon 5, 253-259; Pelhate, M., Zlotkin, E., 1982. Actions of insect toxin and other toxins derived from the venom of scorpion Androtonus australis on isolated giant axons of the cockroach Periplaneta americana. J. Exp. Biol. 97, 67-77; Salgado, V., Kem, W., 1992. Actions of three structurally distinct sea anemone toxins on crustacean and insect sodium channels. Toxicon 30, 1365-1381; Bruhn, T., Schaller, C., Schulze, C., Sanchez-Rodriquez, J., Dannmeier, C., Ravens, U., Heubach, J.F., Eckhardt, K., Schmidtmayer, J., Schmidt, H., Aneiros, A., Wachter, E., Béress, L., 2001. Isolation and characterization of 5 neurotoxic and cardiotoxic polypeptides from the sea anemone Anthopleura elegantissima. Toxicon, 39, 693-702]. Comprehensive analysis of the purified active fractions suggests that CgNa may represent the main peptide toxin of this sea anemone species.

Marine organisms represent a valuable source of new compounds. The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited resource of new active substances in the field of the development of bioactive products. In this paper, the molecular diversity of different marine peptides is described as well as information about their biological properties and mechanisms of action is provided. Moreover, a short review about isolation procedures of selected bioactive marine peptides is offered. Novel peptides from sponges, ascidians, mollusks, sea anemones and seaweeds are presented in association with their pharmacological properties and obtainment methods.

Toxin research in Cuba has made some important contributions to the field in recent decades. Most of the work carried out on marine toxins has been devoted to the isolation, purification, and characterization of polypeptide substances. The purification, molecular, and functional characterization as well as the pharmacological properties of these toxins are revised. The toxin battery described includes new biomolecules: sticholysins I and II, cytolysins from Stichodactyla helianthus; BgK and ShK, two K + channel blockers purified from Bunodosoma granulifera and S. helianthus, respectively; and BgII and III, two Na + channeltoxinsfrom B. granulifera.

Two sodium channel toxins, BgII and BgIII, isolated from the sea anemone Bunodosoma granulifera, have been subjected to an elaborate electrophysiological and pharmacological comparison between five different cloned sodium channels expressed in Xenopus laevis oocytes in order to determine their efficacy, potency and selectivity. Our results reveal large differences in toxin-induced effect between the different sodium channels. These toxins possess the highest efficacy for the insect sodium channel (para). Our data also show that BgII, generally known as a neurotoxin, is especially potent on the insect sodium channel with an EC(50) value of 5.5+/-0.5 nM. Therefore, this toxin can be used as a template for further development of new insecticides. Based on our findings, an evolutionary relationship between crustaceans and insects is also discussed.

Two sodium channel toxins, BgII and BgIII, have been isolated and purified from the sea anemone Bunodosoma granulifera. Combining different techniques, we have investigated the electrophysiological properties of these toxins. We examined the effect of BgII and BgIII on rat ventricular strips. These toxins prolong action potentials with EC50 values of 60 and 660 nM and modify the resting potentials. The effect on Na+ currents in rat cardiomyocytes was studied using the patch-clamp technique. BgII and BgIII slow the rapid inactivation process and increase the current density with EC50 values of 58 and 78 nM, respectively. On the cloned hH1 cardiac Na+ channel expressed in Xenopus laevis oocytes, BgII and BgIII slow the inactivation process of Na+ currents (respective EC50 values of 0.38 and 7.8 μM), shift the steady-state activation and inactivation parameters to more positive potentials and the reversal potential to more negative potentials. The amino acid sequences of these toxins are almost identical except for an asparagine at position 16 in BgII which is replaced by an aspartic acid in BgIII. In all experiments, BgII was more potent than BgIII suggesting that this conservative residue is important for the toxicity of sea anemone toxins. We conclude that BgII and BgIII, generally known as neurotoxins, are also cardiotoxic and combine the classical effects of sea anemone Na+ channels toxins (slowing of inactivation kinetics, shift of steady-state activation and inactivation parameters) with a striking decrease on the ionic selectivity of Na+ channels. British Journal of Pharmacology (2001) 134, 1195–1206; doi:10.1038/sj.bjp.0704361

Five toxins (APE 1 to APE 5) of the sea anemone species Anthopleura elegantissima (Brandt) have been isolated from a toxic by-product fraction of its concentrated crude watery-methanolic extract, prepared previously for the isolation of a neuropeptide (the head-activator) by Schaller and Bodenmüller (Proc. Natl. Acad. Sci. USA 78 (1981) 7000) from 200kg sea anemones. Toxin purification was performed by desalting of the starting material by dialysis (MWCO 3500) against distilled water, anion exchange chromatography on QAE-Sephadex A25 at pH 8, twice gel filtration on Sephadex G50 m, repeated chromatography on QAE-Sephadex at pH 10 and chromatography on the cation exchanger Fractogel EMD SO(3)(-)-650 M.Final purification of the toxins was achieved by HPLC on MN SP 250/10 Nucleosil 500-5 C(18) PPN and MN SP 250/21 Nucleosil 300-7 C(18). Each toxin was composed of at least two isotoxins of which APE 1-1, APE 1-2, APE 2-1, APE 2-2 and APE 5-3 were isolated in preparative scale. With exception of APE 5-3 the sequences of the isotoxins have been elucidated. They resemble the 47 residue type-I long polypeptide toxins native to Anemonia sulcata (Pennant). All isotoxins paralyse the shore crab (Carcinus maenas) by tetanic contractions after i.m. application. The toxins modify current passing through the fast Na(+) channel in neuroblastoma cells, leading to delayed and incomplete inactivation. APE 1-1, APE 2-1 and APE 5-3 produce a positive inotropic effect in mammalian heart muscle, although they differ in potency. The order of potency is APE 2-1>APE 1-1>APE 5-3 (i.e. threshold concentrations are 1, 10 and 300nM, respectively). In addition, they enhance the spontaneous beating frequency in isolated right atria (guinea pig). The most potent cardiotoxic isotoxin is APE 2-1, its sequence is identical with that of AP-C, a toxin isolated and characterised previously by Norton et al. (Drugs and Foods from the Sea, 1978, University of Oklahoma Press, p. 37-50).LD50 APE 2-1:1 micro g/kg b.w. C. maenas (i.m.). LD50 APE 1-1:10 microg/kg b.w. C. maenas (i. m.). LD50 APE 5-3:50 microg/kg b.w. C. maenas (i.m.).

We studied the effects of BgK toxin on outward K+ currents in isolated neurons of the snail Helix aspersa, using the whole cell patch clamp technique. BgK partially and reversibly blocked K+ currents in the 1 pM to 100 nM concentration range (n=53). The dose–response curve for BgK current inhibition had a maximum blocking effect at 100 nM. Our results indicate that BgK is a potent, apparently non-selective, K+ channel blocker in molluscan neurons.

The effects were studied of a toxin (Bainh) isolated from the secretion of the Caribbean sea anemone Bunodosoma granulifera on electrical and mechanical activities of rat ventricular muscle. The effects on the ionic currents of single rat and dog ventricular cardiomyocytes were studied using the whole-cell recording patch-clamp technique. In the concentration range from 1 to 10 mg/ml, Bainh increased the force of contraction and induced an increase in action potential duration of ventricular multicellular preparations. In single cardiomyocytes, at concentrations up to 10 mg/ml Bainh showed no significant effects on the sodium current. However, at 0.5-1 mg/ml it increased the L-type Ca current (ICaL) by 25-50%. This increase in ICaL was not voltage dependent and was reversible after washout. The transient outward current was not significantly affected by Bainh (1-10 mg/ml). In this concentration range, Bainh markedly (approximately 75%) increased the inward-going rectifier current, IKI. This effect that was not voltage dependent and was fully reversible upon returning to control solution. It is suggested that these effects on ionic currents could explain the positive inotropic action of Bainh on cardiac multicellular preparations.