PRELIMINARY FINDINGS ON GENE EXPRESSION STUDY OF SXTA1 IN ALEXANDRIUM MINUTUM

The genus Alexandrium is known for the production of potent neurotoxins affecting health of marine organisms, as well as of human seafood consumers via paralytic shellfish poisoning (PSP). Recently, Alexandrium spp. population genetic and gene expression studies have received considerable attention, in the attempt to identify genes involved in PSP toxin production. The aim of this work was to investigate the relationship between toxin content and expression level of gene sxtA1 in order to provide a new tool for predicting toxins??? presence in environmental samples. Indeed, this gene encodes for a poliketide synthase, the enzyme responsible for initiating the synthesis of saxitoxins. In our study, chemical analyses on PSP toxin composition and content in different Mediterranean A. minutum isolates confirmed that A. minutum was able to synthesize only the saxitoxin derivatives GTX1 and GTX4. Average cellular toxin content in standard condition varied considerably among different strains, ranging from 3.4 fmol/cell to traces per cell. The quantification of intracellular sxtA1 mRNA was very variable with significant differences between the phases of growth, culture conditions or strains. Moreover, the sxtA1 expression decreased during stationary phases and significant differences in strains that were grown in limitation of phosphorus or nitrogen were also observed compared to the standard culture conditions. Unexpectedly, the amount of sxtA1 messengers were not correlated with the amount of intracellular toxins in the strains analyzed suggesting that the production of toxins may be regulated probably by post-transcriptional mechanisms and/or by the concerted actions of alternative genes belonging to PSP toxin biosynthesis gene cluster

Cell division cycle control in embryonal and alveolar rhabdomyosarcomas

In this study, we investigated the mRNA level of several genes involved in cell cycle regulation in alveolar (ARMS) and embryonal rhabdomyosarcomas (ERMS). p21(Cip1), Cyclin D1, Cyclin D2, Cyclin D3, CDK2, and CDK4 were evaluated by RT-PCR. All (13 out of 13) ERMS expressed the p21(Cip1) gene compared with only 40% (4 out of 10) of the ARMS. Moreover, the amount of p21(Cip1) mRNA was noticeably higher in the ERMS samples than in the positive ARMS specimens. p27(Kip1) protein were analysed by immunohistochemical and immunoblotting. A noticeable difference was observed, in that ERMS had higher amounts of the cell cycle inhibitor compared with the ARMS. Finally, treatment of two rhabdomyosarcoma cell lines, RH-30 and RD, with butyrate, resulted in complete growth inhibition and in the upregulation of the p21(Cip1) and p27(Kip1) levels. Our results demonstrate that ERMS have a much higher level of p27(Kip1) and p21(Cip1) than the alveolar types, explaining, at least in part, the distinct features and outcomes (i.e. a poor prognosis of the alveolar type) of the two forms of this childhood solid cancer. Moreover, the data on butyrate-treated cell lines suggest that the two genes are potential novel therapeutic targets for the treatment of rhabdomyosarcomas

Full relative stereochemistry assignment and conformational analysis of 13,19-didesmethyl spirolide C via NMR- and Molecular Modeling-based techniques. A step towards the comprehension of spirolide mechanism of action.

The spirolide content of massive cultures of Alexandrium ostenfeldii1 collected along the North-western Adriatic coasts of Italy in November 2003 was determined by Liquid Chromatography- Mass Spectrometry and 1D- and 2D-NMR techniques. Among the detected spirolides, three resulted major compounds and were unambiguously identified as 13-desmethyl spirolide C2, 13,19-didesmethyl spirolide C3 and 27-hydroxy-13,19-didesmethyl spirolide C4. During our ongoing studies on Adriatic A. ostenfeldii, we have recently succeeded in elucidating the full relative stereochemistry of 13,19-didesmethyl spirolide C through NMR- and Molecular Modeling-based techniques. Besides this, our studies have also contributed to shed some light upon 13,19-didesmethyl spirolide C conformational behavior in solution. This could pave the way towards a more in-depth comprehension of spirolide mechanism of action. In fact, so far pharmacological studies have identified spirolides as fast-acting toxins5, as in the mouse bioassay they induce rapid onset of symptoms akin to those reported for the acute toxicity of paralytic shellfish poisoning (PSP) toxins, followed by death within minutes from the intraperitoneal injection. It has been shown that spirolides affect Ca2+ channels and hypothesized that their pharmacophore is represented by the uncommon cyclic imine moiety. Beyond this information, though, spirolide toxicity is far from being totally and unambiguously defined. 1) Ciminiello, P., Dell'Aversano, C., Fattorusso, E., Magno, S., Tartaglione, L., Cangini, M., Pompei, M., Guerrini, F., Boni, L., Pistocchi, R. (2006). Toxicon 47, 597-604. 2) T. Hu, I. W. Burton, A. D. Cembella, J. M. Curtis, M. A. Quilliam, J. A. Walter and J. L. C. Wright, J. Nat. Prod., 2001, 64, 308. 3) S. L. MacKinnon, J. A. Walter, M. A. Quilliam, A. D. Cembella, P. LeBlanc, I. W. Burton, W. R. Hardstaff and N. I. Lewis, J. Nat. Prod., 2006, 69, 983. 4) P. Ciminiello, C. Dell'Aversano, E. Fattorusso, M. Forino, L. Grauso, L. Tartaglione, F. Guerrini, and R. Pistocchi, J. Nat. Prod., 2007, 70, 1878. 5) Richard, D., Arsenault, E., Cembella, A., Quilliam, M. A. (2000) In Intergovernmental Oceanographic Commission of UNESCO: Harmful Algal Blooms 2000. (Hallegraeff, G. M., Blackburn, S. I., Bolch, C. J., Lewis, R. J., Eds.) pp. 383-386

Investigation of toxin profile of Mediterranean and Atlantic strains of Ostreopsis cf. siamensis (Dinophyceae) by Liquid Chromatography-High Resolution Mass Spectrometry

Blooms of Ostreopsis spp. once confined to tropical and subtropical areas have recently spread to more temperate regions such as the Mediterranean and the Southern-Atlantic coasts of Europe. However, while O. confronta (cf.) ovata has caused several toxic outbreaks, the presence of O. cf. siamensis has been reported rather occasionally and in very few regions; as a consequence, O. cf. ovata toxin profile has been in-depth studied while poor information exists on toxicity of the Mediterranean and Atlantic O. cf. siamensis. In the present study toxin profile of Mediterranean and Atlantic O. cf. siamensis isolates also phylogenetically related has been studied through liquid chromatography–high resolution mass spectrometry (LC–HRMS) versus a palytoxin standard, a crude extract of O. cf. ovata containing all the ovatoxins so far known (ovatoxin-a to -f), and a Japanese O. siamensis extract which contained ostreocin- d and ostreocin-b. The Mediterranean and Atlantic O. cf. siamensis strains were shown not to produce either ostreocins, which are produced by the Japanese O. siamensis strain, or ovatoxins, which are produced by the Mediterranean O. cf. ovata. Only sub-fg levels of palytoxin on a per cell basis were detected in the Mediterranean strain. This study demonstrates that the Mediterranean and the Atlantic O. cf. siamensis strains are devoid of any appreciable toxicity. Thus, at least in the European area, O. cf. siamensis seems to present a much lower risk to human health than O. cf. ovata