A review on the effects of environmental conditions on growth and toxin production of Ostreopsis ovata.

Since the end of the 1990s the occurrence of blooms of the benthic dinoflagellates Ostreopsis spp. is spreading in many tropical and temperate regions worldwide, sometimes causing benthonic biocenosis suffering and occasional human distress. Ostreopsis ovata has been found to produce palytoxin-like compounds, a class of highly potent toxins. As general, the highest abundances of Ostreopsis spp. are recorded during warmer periods characterized by high temperature, salinity, and water column stability. Moreover, as these cells are easily resuspended in the water column, the role of hydrodynamism in the blooms development and decline has been highlighted. The environmental conditions appear, therefore, to be one of the main factors determining the proliferation of these species as testified by several field surveys. Laboratory studies on the effect of environmental parameters on growth and toxicity of O. ovata are rather scarce. With regard to the effects of temperature, culture results indicate that different strains blooming along Italian coasts displayed different optima, in accordance to blooming periods, and that higher toxin levels correlated with best growth conditions. Additionally, in relation to an Adriatic strain, cell growth positively correlated with the increase in salinity, while toxicity was lowest at the highest salinity value (i.e. 40). For the same strain, both nitrogen and phosphorus limitation determined a decrease in cell toxicity showing different behaviour with respect to many other toxic dinoflagellates.

Old and new targets for innovative antimalarial compounds: the different strategies of the Italian Malaria Network.

Clinical treatment-failures to affordable drugs encouraged new investigation for discovery and development of new prophylactic and therapeutic interventions against malaria. The Drug Discovery Cluster (DDcl) of the Italian Malaria Network gathers several highly integrated and complementary laboratories from different Italian Institutions to identify, synthesise, screen in vitro and in vivo new antimalarial molecules directed against the intraerythrocytic stage of P. falciparum parasites and/or with transmission blocking activity to select lead compounds for further development. Complementary research activities, both in vitro and in the clinics, aim at investigating the pathogenetic mechanisms of severe malaria anaemia and the different manifestations of the disease in malaria-HIV co-infected patients to identify new therapies and improve survival.

Bivalve molluscs as vectors of marine biotoxins involved in seafood poisoning.

Molluscs of many sorts, which are high in protein and trace minerals, have always been a substantial portion of the human diet. A great variety of mollusc species are therefore of commercial importance throughout the world. Episodes of poisoning occasionally happen to the consumers of molluscs, the main hazard being represented by bivalve molluscs. These organisms are filter-feeders, feeding mainly on a wide range of phytoplankton species. Among the thousands of species of microscopic algae at the base of the marine food chain, there are a few dozen which produce potent toxins. One major category of impact occurs when toxic phytoplankton are filtered from the water as food by shellfish, which then accumulate the algal toxins to levels which can be lethal to humans. Incidences of poisoning related to marine algal toxins come under the main categories of paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), diarrhetic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP), depending upon the toxins and the symptoms that they cause. Since the beginning of the 1990s, a research program has been initiated to examine the toxin profiles in mussels from the Adriatic Sea. Since then, a number of polyether toxins have been isolated and characterized, some of which represent new additions to the DSP class of biotoxins. During this investigation, new types of toxins have also been isolated. The recent application of LC-MS methods for the detection of Adriatic marine biotoxins made it possible to speed up the analysis of toxic samples.

Sustainable production of bioactive compounds from sponges: primmorphs as bioreactors.

Sponges [phylum Porifera] are a rich source for the isolation of biologically active and pharmacologically valuable compounds with a high potential to become effective drugs for therapeutic use. However, until now, only one compound has been introduced into clinics because of the limited amounts of starting material available for extraction. To overcome this serious problem in line with the rules for a sustainable use of marine resources, the following routes can be pursued; first, chemical synthesis, second, cultivation of sponges in the sea (mariculture), third, growth of sponge specimens in a bioreactor, and fourth, cultivation of sponge cells in vitro in a bioreactor. The main efforts to follow the latter strategy have been undertaken with the marine sponge Suberites domuncula. This species produces compounds that affect neuronal cells, such as quinolinic acid, a well-known neurotoxin, and phospholipids. A sponge cell culture was established after finding that single sponge cells require cell-cell contact in order to retain their telomerase activity, one prerequisite for continuous cell proliferation. The sponge cell culture system, the primmorphs, comprises proliferating cells that have the potency to differentiate. While improving the medium it was found that, besides growth factors, certain ions (e.g. silicate and iron) are essential. In the presence of silicate several genes required for the formation of the extracellular matrix are expressed (silicatein, collagen and myotrophin). Fe3+ is essential for the synthesis of the spicules, and causes an increased expression of the ferritin-, septin- and scavenger receptor genes. Furthermore, high water current is required for growth and canal formation in the primmorphs. The primmorph system has already been successfully used for the production of pharmacologically useful, bioactive compounds, such as avarol or (2'-5')oligoadenylates. Future strategies to improve the sponge cell culture are discussed; these include the elucidation of those genes which control the proliferation phase and the morphogenesis phase, two developmental phases which the cells in primmorphs undergo. In addition, immortalization of sponge cells by transfection with genomic DNA appears to be a promising way, since recent studies underscore the applicability of this technique for sponges.

Caspase activation and death induced by yessotoxin in HeLa cells.

We have studied the death response induced by yessotoxin (YTX) in cultured HeLa cells, and have compared it to that triggered by okadaic acid (OA) in the same experimental system. Sub-nanomolar concentrations of YTX were found to induce HeLa cell death after a 48-96-h incubation. YTX caused loss of intact poly(ADP-ribose)-polymerase (PARP) in HeLa cells, and detection of the 85kDa fragment, which is indicative of proteolytic attack by caspases. Measurements of caspase activities using extracts prepared from YTX-treated cells and substrates of the caspase-3/7 and caspase-2 isoforms, showed that the relative proteolysis of caspase-3/7 substrate was about eight-fold higher than that of caspase-2, the levels of which were about twice those measured with extracts from control cells. These findings were matched by Western blot analyses of caspase-2, -3 and -7 in HeLa cell extracts, which showed that the levels of pro-caspase-2 were not greatly affected by YTX treatment, whereas pro-caspase-3 and -7 were activated in YTX-treated cells. Taken together, these data complement others previously obtained with OA, and support the notion that caspase isoforms involved in cell death induced by OA and YTX are cell- and toxin-specific.

Turbinamide, a new selective cytotoxic agent from the Mediterranean tunicate Sidnyum turbinatum.

A unique cytotoxic metabolite, turbinamide (1), has been isolated from the marine tunicate Sidnyum turbinatum through a bioassay-guided approach. Its structure has been elucidated by an extensive spectroscopic analysis. Turbinamide demonstrated a strong and selective cytotoxic effect against neuronal cells rather than immune system cells. Structure: see text.

Structural elucidation of a new cytotoxin isolated from mussels of the Adriatic sea.

A detailed analysis of the toxic composition in the hepatopancreas of mussels from northern Adriatic sea has been performed. Along with some polyether toxins of DSP (diarrhetic shellfish poisoning) type, such as yessotoxin and its analogues, which are responsible for a variety of human seafood poisonings throughout the world, we have now isolated a new type of toxin, the chlorosulfolipid 1, which is completely different in structure from the polyether DSP-toxins isolated so far. The structural determination of the new toxin, including its absolute stereochemistry, has been performed by extensive NMR analysis and molecular mechanics and dynamics calculations.

New bioactive sulfated metabolites from the Mediterranean tunicate Sidnyum turbinatum.

In addition to the known sodium 3,7,11,15-tetramethylhexadeca-1,19-diyl sulfate (4), the BuOH extract of the Mediterranean tunicate Sidnyum turbinatum was shown to contain four new metabolites: 1-heptadecanyl sulfate (1), 1-octadecanyl sulfate (2), sodium (2S)-2,6,10,14-tetramethylpentadeca-1,18-diyl sulfate (3), and 1-hexyl sulfate (5). Their structures were determined by spectroscopic and chemical methods. Compounds 1-5 exhibited in vitro antiproliferative activity estimated on the WEHI 164 cell line.

The flavonoids of leek, Allium porrum.

A phytochemical investigation of the extracts obtained from bulbs of leek. Allium porrum L. has led to the isolation of five flavonoid glycosides based on the kaempferol aglycone. Two of them are new compounds and have been identified as kaempferol 3-O-[2-O-(trans-3-methoxy-4-hydroxycinnamoyl)-beta-D-galactopyranosyl]-(1-->4)-O-beta-D-glucopyranoside, and kaempferol 3-O-[2-O-(trans-3-methoxy-4-hydroxycinnamoyl)-beta-D-glucopyranosyl]-(1-->6)-O-beta-D-glucopyranoside, on the basis of spectroscopic methods, including 2D NMR. The isolated compounds have been evaluated for their human platelet anti-aggregation activity.

42,43,44,45,46,47,55-Heptanor-41-oxohomoyessotoxin, a new biotoxin from mussels of the northern Adriatic sea.

The diarrhetic shellfish toxin composition in the digestive glands of mussels from the northern Adriatic sea was investigated. Along with known yessotoxins, identified by comparison of their chromatographic and spectral properties with those reported in the literature, we isolated a new analogue of yessotoxin, 42,43,44,45,46,47,55-heptanor-41-oxohomoyessotoxin, 1. Its structure was determined by (1)H NMR spectroscopy and mass spectrometry.

Novel bioactive sulfated alkene and alkanes from the mediterranean ascidian Halocynthia papillosa.

Three sulfated alkene and alkanes-(R)-2,6-dimethylheptyl sulfate (1), 6-methylheptyl sulfate (2a), and (E)-5-octenyl sulfate (3a)-with cytotoxic activity in vitro, have been isolated from the Mediterranean ascidian Halocynthia papillosa. The structures of the new compounds 2a and 3a have been elucidated by spectroscopic analysis.

Structure determination of carboxyhomoyessotoxin, a new yessotoxin analogue isolated from adriatic mussels.

The contamination of shellfish with marine biotoxins derived from microalgae represents a serious problem for shellfish industries and public health. This study investigated the composition of diarrhetic shellfish toxins in the digestive glands of mussels from the Northern Adriatic Sea. Along with known yessotoxins, identified by comparison of their chromatographic and spectral properties with those reported in the literature, we isolated a new analogue of yessotoxin, carboxyhomoyessotoxin, whose structure was determined by mass spectrometry and (1)H NMR spectroscopy.

Cytotoxic saponins from bulbs of Allium porrum L.

An extensive phytochemical analysis of the saponin content has been undertaken on leek, Allium porrum L., sown and collected at different seasons. As a result of this investigation, eight saponins (1-8) have been isolated, four of them (5-8) being novel compounds. Compounds 5 and 6, possessing the same tetrasaccharide moiety of compounds 1 and 3, display very unusual spirostane aglycones, 12-ketoporrigenin and 2,12-diketoporrigenin (named porrigenin C), respectively, recently isolated for the first time as free sapogenin in the same plant. Compounds 7 and 8 are rare cholestane bidesmosides possessing a di- and trisaccharide residues linked to a polyhydroxycholesterol aglycone, respectively. The structures of the isolated compounds have been determined by nondegradative spectroscopic analysis, mainly based on NMR. All the eight saponins isolated from leek were tested for their cytotoxic activity against two different cell lines in vitro, and compounds 1, 2, and 6 resulted particularly active.

Saxitoxin and neosaxitoxin as toxic principles of Alexandrium andersoni (Dinophyceae) from the Gulf of Naples, Italy.

A clonal culture of Alexandrium andersoni, obtained from germination of a resting cyst, collected from the Gulf of Naples, was found positive for PSP toxicity by mouse bioassay. The toxicity profile of this dinoflagellate consists mainly of toxins belonging to the saxitoxin class, in particular of Saxitoxin (STX) and Neosaxitoxin (NEO), as determined by a wide MS and (1)H NMR analysis. This represents the first report of the presence of A. andersoni in the Mediterranean Sea, as well as of its toxicity.

Sulcatin, a novel antiproliferative N-methylpyridinium alkaloid from the ascidian Microcosmus vulgaris.

A new N-methylpyridinium alkaloid, with an interesting antiproliferative activity in vitro, has been isolated from the Mediterranean tunicate Microcosmus vulgaris. Its structure has been elucidated by spectroscopic analysis, including extensive 2D NMR experiments.

Ecdysteroids from the caribbean sponge Iotrochota birotulata.

The sterol composition of the Caribbean sponge Iotrochota birotulata was investigated. Structure of a new ecdysteroid 2beta,3beta,14alpha, 20beta-tetrahydroxy-22alpha-(2-hydroxyacetiloxy)-5b eta-colest-7-en-6- one (1) was assigned on the basis of spectroscopic and chemical evidence and molecular mechanics calculations. Isolation of the widespread ecdysteroids 2-5 is also reported.

Chemistry of verongida sponges. 10. Secondary metabolite composition of the caribbean sponge Verongula gigantea.

A detailed analysis of the secondary metabolites of the Caribbean sponge Verongula gigantea has been performed. A number of bromotyrosine derivatives, 1, 2, and 6-17, were identified, one of which (17) is a novel compound. Its structure was determined on the basis of spectroscopic evidence. Additionally, aureol (18) and 5, 6-dibromo-N,N-dimethyltryptamine (19) were isolated from one of the five analyzed specimens.

Spirostanol saponins of Allium porrum L.

An investigation of the extracts from bulbs of Allium porrum L. has led to the isolation of four spirostanol saponins. Two of them are new compounds and have been identified as: (25R)-5 alpha-spirostan-3 beta, 6 beta-diol 3-O-{O-beta-D-glucopyranosyl-(1-->2)-O-[beta-D-xylopyranosyl-(1-->3)]-O- beta -D-glucopyranosyl-(1-->4)-beta-D-galactopyranoside} (3) and (25R)-5 alpha-spirostan-3 beta,6 beta-diol 3-O-{O-beta-D-glucopyranosyl-(1-->3)-O-beta-D-glucopyranosyl-(1-->2)-O- [beta-D-xylopyranosyl-(1-->3)]-O-beta-D-glucopyranosyl-(1-->4)-beta-D- galactopyranoside} (4). The isolated compounds were evaluated for their antifungal activity.

Chemistry of verongida sponges. 9.1 secondary metabolite composition of the caribbean sponge aplysina cauliformis

A detailed analysis of the secondary metabolites of the sponge Aplysina cauliformis has been performed. Eight compounds were identified, two of which (13 and 14) are new bromotyrosine derivatives whose structures were determinated from spectroscopic evidence, including 2D NMR. The new compounds were analyzed for cytotoxic activity, and compound 14 was shown to inhibit mammalian protein synthesis and cell proliferation.