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The heteronemertine Parborlasia corrugatus contains a cytolytic protein, parborlysin, which after extensive purification was found by Edman sequencing to be a mixture of several homologues. To investigate this microheterogeneity and enable the analysis of single toxins, we have obtained seven parborlysin isoform genes from P. corrugatus collected in Antarctica. Total RNA was isolated from the homogenized head region and parborlysin genes were identified from a cDNA library using degenerate primers. The translated sequences reveal that the isoforms are ∼ 10 kDa basic (pI ∼ 10) proteins of which all but one harbour six cysteine residues. We generated a model of the three dimensional structure of parborlysins, which suggests that they are composed of five alpha-helical segments that include large, exposed hydrophobic surfaces. Finally, we constructed plasmids and inserted them into Escherichia coli to obtain overexpressed amino- or carboxy-terminal polyhistidine-tagged parborlysin isoforms fused to the third domain of the E. coli periplasmic-protein TolA to facilitate toxin isolation. One of the isoforms adversely affected growth in the E. coli expressing it. Although we succeeded in isolating one of the recombinant parborlysin constructs, it lacked haemolytic activity.

Sponges (Porifera) currently represent one of the richest sources of natural products and account for almost half of the pharmacologically active compounds of marine origin. However, to date very little is known about the pharmacological potential of the sponges from polar regions. In this work we report on screening of ethanolic extracts from 24 Antarctic marine sponges for different biological activities. The extracts were tested for cytotoxic effects against normal and transformed cell lines, red blood cells, and algae, for modulation of the activities of selected physiologically important enzymes (acetylcholinesterase, butyrylcholinesterase, and α-amylase), and for inhibition of growth of pathogenic and ecologically relevant bacteria and fungi. An extract from Tedania (Tedaniopsis) oxeata was selectively cytotoxic against the cancer cell lines and showed growth inhibition of all of the tested ecologically relevant and potentially pathogenic fungal isolates. The sponge extracts from Isodictya erinacea and Kirkpatrickia variolosa inhibited the activities of the cholinesterase enzymes, while the sponge extracts from Isodictya lankesteri and Inflatella belli reduced the activity of α-amylase. Several sponge extracts inhibited the growth of multiresistant pathogenic bacterial isolates of different origins, including extended-spectrum beta-lactamase and carbapenem-resistant strains, while sponge extracts from K. variolosa and Myxilla (Myxilla) mollis were active against a human methicillin-resistant Staphylococcus aureus strain. We conclude that Antarctic marine sponges represent a valuable source of biologically active compounds with pharmacological potential.

Polymeric alkylpyridinium salts (poly-APS) isolated from the Mediterranean marine sponge, Haliclona (Rhizoniera) sarai, effectively inhibit barnacle larva settlement and natural marine biofilm formation through a non-toxic and reversible mechanism. Potential use of poly-APS-like compounds as antifouling agents led to the chemical synthesis of monomeric and oligomeric 3-alkylpyridinium analogues. However, these are less efficient in settlement assays and have greater toxicity than the natural polymers. Recently, a new chemical synthesis method enabled the production of poly-APS analogues with antibacterial, antifungal and anti-acetylcholinesterase activities. The present study examines the antifouling properties and toxicity of six of these synthetic poly-APS using the barnacle (Amphibalanus amphitrite) as a model (cyprids and II stage nauplii larvae) in settlement, acute and sub-acute toxicity assays. Two compounds, APS8 and APS12-3, show antifouling effects very similar to natural poly-APS, with an anti-settlement effective concentration that inhibits 50% of the cyprid population settlement (EC$_{50}$) after 24 h of 0.32 mg/L and 0.89 mg/L, respectively. The toxicity of APS8 is negligible, while APS12-3 is three-fold more toxic (24-h LC$_{50}$: nauplii, 11.60 mg/L cyprids, 61.13 mg/L) than natural poly-APS. This toxicity of APS12-3 towards nauplii is, however, 60-fold and 1200-fold lower than that of the common co-biocides, Zn- and Cu-pyrithione, respectively. Additionally, exposure to APS12-3 for 24 and 48 h inhibits the naupliar swimming ability with respective IC$_{50}$ of 4.83 and 1.86 mg/L.

We report on the screening of ethanolic extracts from 33 deep-sea Antarctic marine sponges for different biological activities. We monitored hemolysis, inhibition of acetylcholinesterase, cytotoxicity towards normal and transformed cells and growth inhibition of laboratory, commensal and clinically and ecologically relevant bacteria. The most prominent activities were associated with the extracts from sponges belonging to the genus Latrunculia, which show all of these activities. While most of these activities are associated to already known secondary metabolites, the extremely strong acetylcholinesterase inhibitory potential appears to be related to a compound unknown to date. Extracts from Tetilla leptoderma, Bathydorus cf. spinosus, Xestospongia sp., Rossella sp., Rossella cf. racovitzae and Halichondria osculum were hemolytic, with the last two also showing moderate cytotoxic potential. The antibacterial tests showed significantly greater activities of the extracts of these Antarctic sponges towards ecologically relevant bacteria from sea water and from Arctic ice. This indicates their ecological relevance for inhibition of bacterial microfouling.

Analogs of marine sponge-derived 3-alkylpyridinium compounds (3-APS) were synthesized and screened for possible antibacterial and antifungal activities. They were found to exhibit moderate antibacterial activity. Antifungal potential was tested on pathogenic fungus Candida albicans, baker's yeast Saccharomyces cerevisiae and hypersaline species Wallemia sebi. S. cerevisiae was the most susceptible to the action of selected 3-APS. Inhibitory effects on fungal growth were also studied on two wood-rotting fungi, brown-rot fungus Gloeophyllum trabeum and a white-rot fungus Trametes versicolor. The former showed a higher susceptibility to the action of 3-APS. The highest antifungal potential was observed with the poly-1,3-dodecyl pyridinium chloride (APS12-3, 7), while a complete loss of activity was noticed with the poly-1,3-butyl pyridinium chloride (APS3, 1), suggesting that this activity may closely correlate to the length of their alkyl chains. Based on our results, synthetic APS12-3 is a good candidate to be used as biocide or wood preservative against wood-rotting fungi.

Six new steroidal saponins, pandarosides K-M (1-3) and their methyl esters (4-6), were isolated as minor components, after a careful chemical reinvestigation of the Caribbean sponge Pandaros acanthifolium. Their structures were established on the basis of spectroscopic analyses and comparison with the data obtained from previous metabolites of this family. All new compounds showed moderate to weak activity against four parasitic protozoa. Additionally, these compounds and previously reported pandarosides and acanthifoliosides were tested on three human tumour cell lines, and their haemolytic and liposome permeabilizing activity were assessed. Two pandarosides exhibited moderate to strong cytotoxic effect, while three acanthifoliosides showed strong haemolytic activity.

The structural peculiarity of saraines A, B and C, wherein a zwitterionic-like form is present due to the coordination of a nitrogen atom to the C-2 aldehyde by a strong proximity effect, has been further extended by experimental and theoretical evidences. They include the detection of [2M + H] + clusters in electrospray ionization mass spectrometry in the positive ion mode of a water/acetonitrile solution, whereas only signals corresponding to [M + H] + ions are detectable after addition of trifluoroacetic acid to the same solution. The zwitterionic form can be trapped to give O-methyl derivatives only by reaction with a strong alkylating agent such as

Eggs of the common barbel (Barbus barbus) cause intoxication in humans after ingestion. In this work, the chemical composition of the haemolytically active fraction from methanolic barbel roe extract was analyzed. Compounds showing haemolytic activity and cytotoxicity towards normal and transformed cell lines were isolated and identified as polyunsaturated fatty acids, using online liquid chromatography-electrospray ionization mass spectrometry through tandem fragmentation experiments (HPLC-MS/MS). Arachidonic acid (C20:4), docosahexaenoic acid (C22:6) and eicosapentaenoic acid (C20:5) proved to be the three most abundant members of a complex series of free fatty acids ranging from C14:0 to C24:5.

Disulfide pairings and active site residues are highly conserved in secretory phospholipases A2 (PLA2s). However, secretory PLA2s of marine invertebrates display some distinctive structural features. In this study, we report the isolation and characterization of a PLA2 from the northern Pacific sea anemone, Urticina crassicornis (UcPLA2), containing a C27N substitution and a truncated C-terminal sequence. This novel cnidarian PLA2 shares about 60% identity and almost 70% homology with two putative PLA2s identified in the starlet sea anemone (Nematostella vectensis) genome project. UcPLA2 lacks hemolytic and neurotoxic activities. A search of available sequences revealed that Asn27-‘type’ PLA2s are present in a few other marine animal species, including some vertebrates. The possibility that the C27N replacement represents a structural adaptation for PLA2 digestion/activity in the marine environment was not supported by experiments testing the influence of ionic strength on UcPLA2 enzymatic activity. Because of the highly divergent sequences among invertebrate group I PLA2s, it is currently not possible to identify orthologous relationships. As the Asn27-containing PLA2s are scattered among the other invertebrate group I PLA2s, they do not constitute a new, monophyletic PLA2 clade.

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.

Polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai act as antifouling and anticholinesterase agents. They also show moderate haemolytic and cytotoxic activities against different cell lines. The haemolytic activity of poly-APS is due to their detergent-like structure and behaviour in aqueous solutions. In this work, the lytic activity of poly-APS against freshwater and marine algae, and inhibitory effects on wood decay fungi, were investigated. The results show that poly-APS inhibit the proliferation and movements of susceptible algae. Effects of poly-APS were time- and concentration-dependent and differed between various algal species. No growth inhibition effects were observed towards the examined wood fungi.

The extent of pollution with organotin compounds and Cd, Pb, Ni, Cu, Zn, Cr, Mn, V, Co and Al was investigated in sediments and mussels (Mytilus galloprovincialis) from the Slovenian costal area of the Northern Adriatic Sea. Sampling was performed in Marina Portoroz, Dockyard Izola, non exposed area of the Bay of Mesecev zaliv and in Mariculture Secovlje. Mussels were taken in the summer and winter time, while sediments were collected during the winter sampling. Organotin compounds were determined by gas chromatography-mass spectrometry and metals by flame or electrothermal atomic absorption spectrometry. The accuracies of the analytical procedures were checked by the analysis of standard reference materials CRM 477 mussel tissue and PACS 2 marine sediment (organotin compounds) and SRM 2976 mussel tissue and CRM 320 river sediment (metals). Good agreements between certified and determined values were obtained. Normalization procedure to Al was applied to estimate the anthropogenic inputs of metals in sediments. The analyses of sediments demonstrated moderate pollution with organotin compounds in Marina Portoroz and in Dockyard Izola. Concentrations of tributyltin species were higher than those of dibutyltin and monobutyltin. In mussels substantial contamination with tributyltin was observed in Marina Porotroz and Dockyard Izola. The extent of pollution was higher in the winter time. The analysis of metals in sediments exhibited elevated concentrations in Marina Portoroz and Dockyard Izola. Data from the normalization procedure indicated the anthropogenic inputs of Cu, Zn and Cr in Marina Portoroz and Mn in Bay of Mesecev zaliv and Dockyard Izola. Mussels, as accumulators of pollutants, in general contained higher metal concentrations during winter time in Dockyard Izola.

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.

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 Hg2+ 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.

A synthetic linear tetrazacyclopent(f)azulene compound, parazoanthoxanthin A (m.w. 214.2), strongly fluorescent pigment occurring in zoanthids, was characterized and assayed for anticholinesterase activity. The pigment, emitting fluorescence at lambda(em) 420 nm, was found to be a pure competitive inhibitor of cholinesterases. At pH 8.0, a Ki value of 19 and 26 microM was determined with insect recombinant, and electric eel acetylcholinesterase. Horse serum butyrylcholinesterase was less sensitive with a Ki of 70 microM.

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.

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.

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 curiousity, 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.

A 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.

A new pore-forming cytolytic protein was isolated from the Northern red sea anemone, Urticina crassicornis. Its biochemical properties were characterized and partial N-terminal amino acid sequence was determined. The cytolysin, named UcI, has a molecular mass of around 30 kDa and lacks phospholipase A2 activity. UcI lyses bovine erythrocytes at nanomolar concentrations. Hemolysis is a result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores and can be prevented by osmotic protectants of size >600 Da. The functional radius of an average pore was estimated to be about 0.66 nm. A more detailed study of the cytolytic activity of UcI was performed with lipid vesicles and monolayers. The toxin binds to monolayers and efficiently permeabilizes small lipid vesicles composed of sphingomyelin and cholesterol. However, the cytolytic activity is not prevented by preincubation with either pure cholesterol or sphingomyelin dispersions. We conclude that the presence of both sphingomyelin and cholesterol, key components of lipid rafts, greatly enhances toxin binding to membranes and probably facilitates pore formation. Alignment of the toxin partial amino acid sequence with sequences of cytolysins belonging to the actinoporin family reveals no sequence homology. We conclude that partial sequence of UcI resembles only the N-terminal part of UpI, a cytolytic protein isolated from a related sea anemone species, Urticina piscivora. The two proteins most probably belong to a separate family of sea anemone cytolysins that are worthy of further characterization.

Polymeric 3-alkylpyridinium salts (poly-APS), surface-active compounds from the marine sponge Reniera sarai, have been shown to stimulate the fruit body formation from Pleurotus ostreatus mycelium. In nutrient media supplemented with poly-APS (>or= 0.01 microg ml(-1)), the formation of primordia and development of fruit bodies were detected approximately 10d earlier than in the absence of poly-APS, and also led to a considerably larger quantity of young mushrooms. This effect appears to be specific, as other surface-active compounds, lysophospholipids and fatty acids, showed no induction of fruiting.

During the last 30 years, a number of simple 3-alkylpyridine and 3-alkylpyridinium (3-AP) compounds have been isolated from marine sponges belonging to the order Haplosclerida, suggesting these compounds as chemical markers for systematic determination of haplosclerid sponges. They were isolated from haplosclerid sponges either as (i) monomers differing in the length, saturation, branching and termination of the alkyl chains, (ii) cyclic or linear oligomers, or (iii) a mixture of high-molecular weight polymers. What the biosynthetic pathways are in marine sponges, and how these compounds can be made by organic synthesis is also an interesting question addressed in our review. In this regard we focus particularly on organic syntheses by which selective polymerization of 3-alkylpyridinium polymers may be achieved. Structural investigation of alkylpyridinium compounds and the role played by mass spectrometry has also been reviewed. In spite of their relatively simple chemical structure, all these compounds exert a broad spectrum of biological activities. More than 40 monomeric 3-AP compounds have been isolated from marine sponges and the majority bear a nitrogenous functionality at the end of the alkyl chain. Almost all reported 3-AP monomers exert moderate cytotoxicity, in the concentration range of a few micrograms per millilitre, against certain transformed cell lines. An example of 3-alkylpyridines is niphatyne A, from the marine sponge of the genus Niphates which exhibits an IC 50 of 0.5 μg/mL against several transformed cell lines. The majority of 3-AP dimers and trimers were isolated from haplosclerid sponges in cyclic forms. Haliclamines are a typical representative of this group. They inhibit the division of fertilized sea urchin eggs as well as the growth of several transformed cell lines. On the other hand cyclostellettamines are typical example of cyclic alkylpyridinium dimers that modulate muscarinic receptors. Recently an increasing number of polymeric alkylpyridinium compounds have been isolated from haplosclerid sponges. Halitoxins, amphitoxins and alkylpyridinium salts from Mediterranean sponge Reniera sarai are the most studied. The latter exhibit a plethora of interesting biological activities. For instance, they are able to make pores in membranes through which DNA can be transfected into the cell, show selective cytotoxic activity against certain human cancer cells and possess non-toxic inhibitory antifouling properties against larvae of several important fouling organisms. The biological activities and potential use of alkylpyridinium polymers are discussed.

Fruiting initiation in mushrooms can be triggered by a variety of environmental and biochemical stimuli, including substances of natural or synthetic origin. In this work ostreolysin, a cytolytic protein specifically expressed during the formation of primordia and fruit bodies of Pleurotus ostreatus, was applied to nutrient media inoculated with mycelium of P. ostreatus, and its effects on mycelial growth and fructification of the mushroom studied. The addition of ostreolysin slightly inhibited the growth of mycelium, but strongly induced the formation of primordia, which appeared 10 d earlier than in control plates supplemented with bovine serum albumin or with the dissolving buffer alone. Moreover, ostreolysin stimulated the subsequent development of primordia into fruit bodies. However, direct involvement of this protein in the sporulation of the mushroom is unlikely, as it was also detected in large amounts in the non-sporulating strain of P. ostreatus.

To date, around thirty bioactive 3-alkylpyridinium compounds, either in monomeric or oligomeric forms, have been identified in marine sponges belonging to the order Haplosclerida In this work, we have reviewed their biological activities, which include mainly cytotoxicity, ichthyotoxicity, inhibition of bacterial growth, and enzyme inhibition. Most of these activities increase with the increasing degree of oligomerization of the corresponding 3-alkylpyridinium compound. It was shown recently that 3-alkylpyridines also exhibit promising antifouling activities. Linear 3-octylpyridinium polymers (Poly-APS), isolated from the Mediterranean sponge Reniera sarai, showed a non-toxic reversible mechanism of settlement inhibition of Balanus amphitrite cypris larvae with an EC50 of 0.27 microg/mL. At the same time, their toxicity towards the organisms used in the toxicity bioassays (B. amphitrite nauplii, microalga Tetraselmis suecica and larvae of Mytilus galloprovincialis) was almost negligible in comparison to commercially available and currently used booster biocides based on copper and zinc complexes with pyrithione. Poly-APS and some other natural 3-alkylpyridines were also found to be very effective in preventing microbial biofilm formation. Preliminary tests have confirmed that some monomeric and oligomeric synthetic analogues of poly-APS also exert antifouling activity, which makes these compounds promising candidates as new environmentally-friendly ingredients in the new generation of antifouling coatings.

Metabolites from marine sponges are considered a promising alternative to heavy metals in antifouling coatings. Water-soluble polymeric 3-alkylpyridinium salts and 14 related synthetic analogues showed considerable antibacterial activity against marine biofilm bacteria and may represent good candidates as natural biocides for marine technology applications.

In the mushrooms Pleurotus ostreatus and Agrocybe aegerita , expression of the hemolytic proteins ostreolysin and aegerolysin, which belong to the aegerolysin family, has been shown to be initiated specifically during formation of primordia and fruiting bodies. We used rabbit anti-ostreolysin and fluorescent rhodamine-labelled secondary goat antibodies for monitoring ostreolysin and aegerolysin in situ during the mushrooms' development. In parallel, the protein level in developing tissues was monitored with SDS-PAGE and hemolytic assay. Immunolocalization of ostreolysin, visualized by epifluorescence, together with biochemical tests, confirmed specific expression of ostreolysin and aegerolysin in the primordia and fruiting bodies but not in the vegetative mycelia. In the primordia the proteins were disposed diffusely. In growing and mature fruit bodies they persisted in the lower part of the pileus, in particular in basidia and basidiospores, while in other parts only a few focal remains were observed. Confocal microscopy of immunolabelled sections showed that intracellular ostreolysin was located specifically along the inner edges of hyphae. Since both proteins were found preferentially in the rapidly growing primordia, and in the basidia and basidiospores of maturing fruit bodies, it is suggested that they might play a role in the processes of fructification and/or sporulation.

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.

From freeze dried mucus of the Antarctic nemertine Parborlasia corrugatus we have isolated 10.3 kDa basic (pI>9.0) cytolytic protein, referred to as parborlysin. Although the purified protein sample was homogeneous by reversed phase HPLC chromatography profiles and several gel electrophoretic techniques, N-terminal sequence and mass spectrometric analyses revealed that it consisted of few very similar isotoxins. The N-terminal sequence of the parborlysin sample shows a high degree of homology with the sequence of cytolysin A-III from the heteronemertine Cerebratulus lacteus. Parborlysin in micromolar concentration range disrupts mammalian erythrocytes with an apparent detergent mode of action. Hemolytic activity was inhibited by preincubation of parborlysin with pure phosphatidic acid or with rather high concentrations of small unilamellar vesicles composed of phosphatidylcholine (PC)/phosphatidyglycerol, PC/phosphatidylinositol, and PC/phosphatidylserine. Osmotic protectants as large as 3000 Da failed to protect red cells from lysis induced by parborlysin. Further structural and pharmacological analysis of the heteronemertine cytolysins may provide new insights regarding the mechanisms by which some water soluble proteins are able to penetrate into lipid membranes and form pores or, acting as detergents, disrupt their normal structure and function.

University ofLjubljana, Slovenia Ostreolysin is a 16-kDa cytolytic protein specifically expressed in primordia and fruiting bodies of the edible mushroom Pleurotus ostreatus. To understand its interaction with lipid membranes,we compared its effects on mammalian cells,on vesicles prepared with either pure lipids or total lipid extracts,and on dispersions of lysophospholipids or fatty acids. At nanomolar concentrations,the protein lysed human,bovine and sheep erythrocytes by a colloid-osmotic mechanism,compatible with the formation of pores of 4 nm diameter,and was cytotoxic to mammalian tumor cells. A search for lipid inhibitors of hemolysis revealed a strong effect of lysophospholipids and fatty acids,occurring below their critical micellar concentration. This effect was distinct from the capacity of ostreolysin to bind to and permeabilize lipid membranes. In fact,permeabilization of vesicles occurred only when they were prepared with lipids extracted from erythrocytes,and not with lipids extracted from P. ostreatus or pure lipid mixtures,even if lysophospholipids or fatty acids were included. Interaction with lipid vesicles, and their permeabilization,correlated with an increase in the intrinsic fluorescence and a-helical content of the protein, and with aggregation,which were not detected with lysophospholipids. It appears that either an unknown lipid acceptor or a specific lipid complex is required for binding, aggregation and pore formation. The inhibitory effect of lysophospholipids may reflect a regulatory role for these components on the physiological action of ostreolysin and related proteins during fruiting.

Polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai act as potent anticholinesterase agents; in addition they show moderate hemolytic and cytotoxic activities. The hemolytic activity of poly-APS is due to their detergent-like structure and behavior in aqueous solutions. In this work, the hemolytic activity of poly-APS is analyzed and compared to that of structurally-related monomeric cationic surfactants. The influence of different divalent cations and lipids on poly-APS induced hemolysis is discussed. The dimensions of lesions caused by poly-APS in erythrocyte membranes are determined by the use of osmotic protectants. Finally, the possible role of poly-APS in their natural environment is proposed.

An ethanolic extract from a zoanthid crust coral Parazoanthus axinellae was lethal to mice and crabs and exhibited anticholinesterase activity. The isolation of several AChE inhibitors with the aid of RP-HPLC is described. The most abundant of the inhibitors present in the P. axinellae extract was identified as pseudozoanthoxanthin or an almost identical compound which belongs to the chemically well-characterized series of tetrazacyclopentazulene natural pigments from the genera Parazoanthus, Epizoanthus, Zoanthus and Palythoa. The inhibitor has a mol. wt of 242 and acts as a competitive inhibitor with a Ki of 4 μM. The inhibitor exhibited a strong blue fluorescence. In vivo action of crude extract and the isolated inhibitor showed a typical picture of systemic acetylcholinesterase inhibition. Atropinization of experimental animals prior to injection of the inhibitor almost entirely neutralized its activity.

Summary The stoichiometric relationship between thiol oxidized and NO reduced was studied for the reaction catalyzed by nitric oxide reductase from Paracoccus denitrificans . The reaction systems consisted of dithiothreitol, ascorbate, phenazine methosulfate, enzyme and NO, or that system minus ascorbate. The mole ratio of thiol groups oxidized to NO reduced was observed to be 2.3 to 1.5 over a range of NO from 0.09 to 0.35 μ mol. A ratio of 1.0 was expected for the simple reduction of NO by 1-electron to N 2 O. The oxidation of additional thiol is attributed to the trapping of nitrosyl hydride (nitroxyl, NO − /NOH) by thiol.

Equinatoxin II, a pore forming polypeptide from the sea anemone Actinia equina L. was subjected to chemical modifications with group specific reagents. Lysine residues were modified with pyridoxal-5′-phosphate, histidine residues with diethyl pyrocarbonate and carboxyl groups with the use of a water soluble carbodiimide. Modification of charged residues had no significant influence on the toxin interaction with serum lipoproteins. Lysine 5′-phosphopyridoxylated and histidine carbethoxylated derivatives of the toxin retained lethal and hemolytic activities, but the pH profile of hemolytic activity of 5′-phospho-pyridoxylequinatoxin II was markedly altered. Modification of the toxin carboxyl groups impaired both hemolytic and lethal activities, the latter, however, to the greater extent.

Acetylcholinesterase is an enzyme responsible for the timely termination of acetylcholine action at muscarinic and nicotinic receptors, so it is essential for the normal function of cholinergic synapses. Naturally occurring acetylcholinesterase inhibitors are relatively rare, but several synthetic acetylcholinesterase inhibitors have been developed as potent remedies, pesticides, and neurotoxins. Here we describe the inhibition of soluble and membrane-bound cholinesterases with a recently isolated and purified acetylcholinesterase inhibitor isolated from the crust coral Parazoanthus axinellae Adriaticus. This substance with a novel chemical structure might serve as a new prototype of cholinesterase inhibitors.

The role of arginine and tyrosine in cytolytic properties of equinatoxin II, isolated from the sea anemone Actinia equina L., was studied by means of chemical modifications. The toxin was modified with 2,3 butanedione and tetranitromethane, respectively. The extent of modification and physico-chemical properties of the modified proteins were checked with amino acid analysis, isoelectric focusing and circular dichroic spectra. Extensive treatment of the toxin with 2,3 butanedione modified seven arginines and also two tyrosines, with resulting loss of hemolytic activity. Modification of two out of nine arginine residues resulted in a 25% loss of hemolytic activity, whereas nitration of three out of ten tyrosines decreased hemolytic activity by 95%. The nitrated toxin had at least a 30-fold higher i.v. ld 50 than the native toxin. None of the modifications significantly affected the secondary structure of the toxin as revealed by the CD spectra. It is concluded that tyrosine residues are involved in both lethal and cytolytic activity, while the role of arginine residues is not evident because of the non-specific alteration of tyrosine residues with 2,3 butanedione.