Tom Turk

Primary and secondary structure of a pore-forming toxin from the sea anemone, Actinia equina L., and its association with lipid vesicles

The complete amino acid sequence of equinatoxin II, a potent pore-forming toxin with hemolytic, cytotoxic and cardiotoxic activity from the venom of the sea anemone, Actinia equina L., is reported. In addition, circular dicroism was used to estimate the secondary structure of this toxin either in the water-soluble or in the membrane-anchored form. Equinatoxin II when in water was found to contain about 29-33% of alpha-helical structure, 53-58% of beta-strand+beta-turn and 10-16% of random structure. Upon association with phospholipids, in particular with sphingomyelin, a rearrangement of the secondary structure occurs resulting in an increase of the alpha-helix content. An amphiphilic alpha-helical segment is predicted at the N-terminus, which shares structural homology with membrane active peptides like melittin and viral fusion peptides. In analogy to the behaviour of these peptides we propose that at least part of the alpha-helix content increase of equinatoxin II is due to the insertion of its N-terminus into the lipid bilayer. As in the case of melittin, association of 3-4 equinatoxin molecules is necessary to induce membrane permeabilisation.

Pleurotus and Agrocybe hemolysins, new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg(2+) but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.

Cytolytic Toxins from Sea Anemones

AbstractA number of cytolytic toxins are commonly found in sea anemones. They represent a unique class of animal toxins considering their biochemical characteristics and biological activity. Sea anemone cytolysins are lethal polypeptides which act directly at the membrane level and have potent cardiotoxic activity. Cytotoxic, cytocidal and cytostatic activity were also detected. In general they are basic proteins which usually lack Cys or Met residues. Sea anemone cytolysins are slightly hydrophilic molecules which, however, possess large hydrophobic domains which could insert and span across cell membranes and thus form an ion channel. Cytolytic activity of all except one cytolysin can be prevented by sphingomyelin. Purification, biochemical characterization of sea anemone cytolysins and their mechanisms of action are discussed in the following review article.

The phylum Cnidaria and investigations of its toxins and venoms until 1990.

Cnidarians are the largest phylum of generally toxic animals, yet their toxins and venoms have not received as much scientific attention as those of many terrestrial (snakes, scorpions, spiders, etc.) and even some marine animals (i.e. cone snails). Approximately 13,000 living cnidarian species have been described by systematists. A major rationale for their study in the past, besides scientific curiosity, was to better treat victims of their envenomation. While that goal remains a high priority, it is now appreciated that the toxins of these mostly marine animals can be very useful molecular probes for the analysis of ion channels involved in electrical signaling, immune responses and other signal transduction processes of biomedical interest. For instance, anaphylaxis was discovered by Richet (1905) during experiments with sea anemone and hydrozoan tentacular extracts. Similarly, it has recently been shown that a toxin from another sea anemone is able to potently inhibit T-lymphocyte proliferation in models of certain autoimmune diseases. Thus, these natural substances continue to be of relevance for understanding and treating human diseases. In addition to introducing phylum Cnidaria (Coelenterata), we provide a short history of early (until about 1990) research on cnidarian toxins and venoms, to provide a perspective for appreciating the scientific advances of the past two decades that are summarized in the ensuing 19 papers in this special Toxicon issue.

Inhibition of acetylcholinesterase by an alkylpyridinium polymer from the marine sponge, Reniera sarai

Large polymeric 3-alkylpyridinium salts have been isolated from the marine sponge Reniera sarai. They are composed of N-butyl(3-butylpyridinium) repeating subunits, polymerized head-to-tail, and exist as a mixture of two main polymers with molecular weights without counterion of about 5520 and 18900. The monomer analogue of the inhibitor, N-butyl-3-butylpyridinium iodide has been synthesized. This molecule shows mixed reversible inhibition of acetylcholinesterase. The polymers also act as acetylcholinesterase inhibitors and show an unusual inhibition pattern. We tentatively describe it as quick initial reversible binding, followed by slow binding or irreversible inhibition of the enzyme. This kinetics suggests that there are several affinity binding sites on the acetylcholinesterase molecule where the polymer can bind. The first binding favors binding to other sites which leads to an apparently irreversibly linked enzyme-inhibitor complex.

Non-toxic antifouling activity of polymeric 3-alkylpyridinium salts from the Mediterranean sponge Reniera sarai (Pulitzer-Finali).

The antifouling activity and toxicity of polymeric 3-alkylpyridinium salts (poly-APS) isolated from the Mediterranean sponge Reniera sarai were studied. The activity of these natural products was compared to that of zinc and copper complexes of pyrithione, two non-persistent booster biocides successfully used in current antifouling coatings. Larvae of Balanus amphitrite (cyprids and nauplii) were used to monitor settlement inhibition and the extent to which inhibition was due to toxicity. The microalga Tetraselmis suecica and larvae of the mussel Mytilus galloprovincialis were used in toxicity bioassays. Compared to the booster biocides, poly-APS were less effective at inhibiting cyprid settlement, but their effects were non toxic and reversible, with very low toxicity against the organisms used in the toxicity bioassays. Although encouraging, these results are not enough to warrant the use of poly-APS as a potential commercial antifoulant. They however justify possible future efforts to chemically synthesize poly-APS analogues for further tests.

Synthesis and bioactivity of linear oligomers related to polymeric alkylpyridinium metabolites from the Mediterranean sponge Reniera sarai

Dimers and tetramers of linear 3-alkylpyridinium salts have been synthesized by an efficient synthetic pathway, which is also applicable to the preparation of higher oligomers. Mono-, di- and tetrameric compounds have been tested for antibacterial and hemolytic activities and for the inhibition of acetylcholinesterase and protein phosphatase 2A. Their activities were compared to those of the natural poly-3-octylpyridinium alkaloids isolated from the Mediterranean sponge Reniera sarai. Relatively high antibacterial and anti-acetylcholinesterase activities were observed that increase with higher degrees of oligomerization.

Biological Activities of Aqueous Extracts from Marine Sponges and Cytotoxic Effects of 3-Alkylpyridinium Polymers from Reniera sarai

We screened the biological activity of 21 marine sponges collected in the northern Adriatic sea. Hemolytic, hemagglutinating, antimicrobial, cytotoxic, and anti-acetilcholinesterase activities of the extracts were monitored. We found that hemolytic activity was generally weak; only extracts from three sponge species possess considerable activity. Hemagglutinating activity was present in almost half of extracts but with little specificity against human erythrocytes of different blood groups. Detectable antimicrobial activity was present in only two extracts, while most of them possessed cytotoxic activity. Strong anti-cholinesterase activity was present only in one sample. 3-alkypyridinium polymers isolated from Reniera sarai were hemolytic and strongly cytotoxic against different cell lines with slightly expressed specificity against transformed cells.

Biological Activities of Aqueous and Organic Extracts from Tropical Marine Sponges

We report on screening tests of 66 extracts obtained from 35 marine sponge species from the Caribbean Sea (Curaçao) and from eight species from the Great Barrier Reef (Lizard Island). Extracts were prepared in aqueous and organic solvents and were tested for hemolytic, hemagglutinating, antibacterial and anti-acetylcholinesterase (AChE) activities, as well as their ability to inhibit or activate cell protein phosphatase 1 (PP1). The most interesting activities were obtained from extracts of Ircinia felix, Pandaros acanthifolium, Topsentia ophiraphidites, Verongula rigida and Neofibularia nolitangere. Aqueous and organic extracts of I. felix and V. rigida showed strong antibacterial activity. Topsentia aqueous and some organic extracts were strongly hemolytic, as were all organic extracts from I. felix. The strongest hemolytic activity was observed in aqueous extracts from P. acanthifolium. Organic extracts of N. nolitangere and I. felix inhibited PP1. The aqueous extract from Myrmekioderma styx possessed the strongest hemagglutinating activity, whilst AChE inhibiting activity was found only in a few sponges and was generally weak, except in the methanolic extract of T. ophiraphidites.

Mechanisms of toxicity of 3-alkylpyridinium polymers from marine sponge Reniera sarai.

Polymeric 3-alkylpyridinium salts (poly-APS) present in the marine sponge Reniera sarai show a broad spectrum of biological activities. They are lytic to erythrocytes and various other mammalian cells, enabling the transfection of the latter with alien DNA. Furthermore, they show inhibitory effects to marine bacteria and can inhibit fouling of micro- and macroorganisms to submerged surfaces. Finally, poly-APS act as potent cholinesterase inhibitors. The kinetics of acetylcholinesterase inhibition by poly-APS in vitro is complex and comprises several successive phases ending in irreversible inhibition of the enzyme. The latter is accounted for by aggregation and precipitation of the enzyme-inhibitor complexes. Poly-APS are lethal to rats in concentrations above 2.7 mg/kg. Monitoring of the basic vital functions and histopathological analysis showed that the effects directly ascribable to acetylcholinesterase inhibition are only observed after application of lower concentrations of poly-APS. At higher concentrations, such effects were masked by other, more pronounced and faster developing lethal effects of the toxin, such as haemolysis and platelet aggregation.