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An exclusive club : The first member of an unprecedented class of polyunsaturated and polyhydroxylated macrocycle, belizeanolide (see structure), has been isolated from a marine dinoflagellate. The corresponding open form, belizeanolic acid, was also found. Their structures were determined primarily by analysis of NMR spectroscopic data. Both compounds showed significant antiproliferative activities.

Belizentrin (1), a novel 25-membered polyketide-derived macrocycle, was isolated from cultures of the marine dinoflagellate Prorocentrum belizeanum. This metabolite is the first member of an unprecedented class of polyunsaturated and polyhydroxylated macrolactams. The structure of 1 was primarily determined by NMR and computational methods. Pharmacological assays with cerebellar cells showed that 1 produces important changes in neuronal network integrity at nanomolar concentrations.

Benthic marine dioflagellate microalgae belonging to the genus Prorocentrum are a major source of okadaic acid (OA), OA analogues and polyketides. However, dinoflagellates produce these valuable toxins and bioactives in tiny quantities, and they grow slowly compared to other commercially used microalgae. This hinders evaluation in possible large-scale applications. The careful selection of producer species is therefore crucial for success in a hypothetical scale-up of culture, as are appropriate environmental conditions for optimal growth. A clone of the marine toxic dinoflagellate P. belizeanum was studied in vitro to evaluate its capacities to grow and produce OA as an indicator of general polyketide toxin production under the simultaneous influence of temperature (T) and irradiance (I0). Three temperatures and four irradiance levels were tested (18, 25 and 28 °C; 20, 40, 80 and 120 µE·(m-2)·s(-1)), and the response variables measured were concentration of cells, maximum photochemical yield of photosystem II (PSII), pigments and OA. Experiments were conducted in T-flasks, since their parallelepipedal geometry proved ideal to ensure optically thin cultures, which are essential for reliable modeling of growth-irradiance curves. The net maximum specific growth rate (µ(m)) was 0.204 day(-1) at 25 °C and 40 µE·(m-2)·s(-1). Photo-inhibition was observed at I040 μEm(-2)s(-1), leading to culture death at 120 µE·m(-2)·s(-1) and 28 °C. Cells at I0 ≥ 80 µE·m(-2)·s(-1) were photoinhibited irrespective of the temperature assayed. A mechanistic model for µ(m)-I0 curves and another empirical model for relating µ(m)-T satisfactorily interpreted the growth kinetics obtained. ANOVA for responses of PSII maximum photochemical yield and pigment profile has demonstrated that P. belizeanum is extremely light sensitive. The pool of photoprotective pigments (diadinoxanthin and dinoxanthin) and peridinin was not able to regulate the excessive light-absorption at high I0-T. OA synthesis in cells was decoupled from optimal growth conditions, as OA overproduction was observed at high temperatures and when both temperature and irradiance were low. T-flask culture observations were consistent with preliminary assays outdoors.

Three new benthic dinoflagellate species, Prorocentrum belizeanum, Prorocentrum elegans, and Prorocentrum caribbaeum, from mangrove floating detritus are described from scanning electron micrographs. Species were identified based on shape, size, surface micromorphology, ornamentation of thecal plates, and architecture of the periflagellar area and intercalary band. Cells of P. belizeanum are round to slightly oval with a cell size of 55–60 μm long and 50–55 μm wide. Areolae are round and numerous (853–1024 per valve) and range from 0.66 to 0.83 μm in size. The periflagellar area of P. belizeanum is a broad V-shaped depression; it accommodates a flagellar and an auxiliary pore and a flared, curved apical collar. The intercalary band of P. belizeanum is horizontally striated. Prorocentrum elegans is a small species 15–20 μm long and 10–14 μm wide, with an ovate cell shape. The thecal surface is smooth. Two sizes of valve pores were recognized: large, round pores (20–22 per valve) arranged in a distinct pattern and smaller pores situated in an array along the intercalary band. The periflagellar area is V-shaped; it accommodates an uneven sized flagellar pore, an auxiliary pore, and an angled protuberant flagellar plate. The intercalary band is transversely striated. It is a bloom-forming species. Prorocentrum caribbaeum cells are heart-shaped with a rounded anterior end and a pointed posterior end. Cells range from 40 to 45 μm long and 30 to 35 μm wide. Thecal surface has two different-sized pores: large, round pores (145–203 per valve) arranged perpendicularly from the posterior margins, and small, round pores unevenly distributed on the thecal surface. The periflagellar area is ornate. It is V-shaped with a curved apical collar located next to the auxiliary pore; a smaller protuberant apical plate is adjacent to the flagellar pore. The intercalary band is transversely striated and sinuous. Cells are active swimmers.

Okadaic acid (1) (OA) and its congeners are mainly responsible for diarrhetic shellfish poisoning (DSP) syndrome. The presence of several OA derivatives have already been confirmed in Prorocentrum and Dinophysis spp. In this paper, we report on the detection and identification of a new DSP toxin, the OA isomer 19-epi-okadaic acid (2) (19-epi-OA), isolated from cultures of Prorocentrum belizeanum, by determining its retention time (RT) and fragmentation pattern using liquid chromatography coupled with mass spectrometry (LC–MS/MS).

En el marco de nuestro programa de búsqueda de nuevas moléculas bioactivas de origen marino, hemos estudiado el dinoflagelado Prorocentrum belizeanum, a partir del cual se encontró el ácido belizeánico. Este es un nuevo derivado del ácido okadaico, del que puede considerarse un precursor de interés. Utilizando fundamentalmente técnicas de resonancia magnética nuclear se determinó su estructura y mediante la utilización de diferentes técnicas de modelización molecular se ha estudiado su conformación en disolución. Es interesante el hecho de que este metabolito también presenta una potente actividad inhibidora de la proteína fosfatasa 2A pese a las diferencias estructurales que presenta con el ácido okadaico.

7 páginas, 3 figuras., Okadaic acid (1) (OA) and its congeners are mainly responsible for diarrhetic shellfish poisoning (DSP) syndrome. The presence of several OA derivatives have already been confirmed in Prorocentrum and Dinophysis spp. In this paper, we report on the detection and identification of a new DSP toxin, the OA isomer 19-epi-okadaic acid (2) (19-epi-OA), isolated from cultures of Prorocentrum belizeanum, by determining its retention time (RT) and fragmentation pattern using liquid chromatography coupled with mass spectrometry (LC–MS/MS)., The authors acknowledge financial support from the Spanish MEC (AGL2005-07924-C04-01 and 02/ALI).

[ESP] En el presente estudio, las principales toxinas producidas por el dinoflagelado Prorocentrum belizeanum (Ácido okadaico, DTX-5c y 7- hidroximetil-2-metilen-octa-4,7-dienil okadaato) fueron analizadas en un sistema de cromatografía líquida acoplado a espectrometría de masas (LC- MS), lo que permitió determinar los tiempos de retención (tR) respectivos y los patrones de fragmentación asociados. Como una muestra de la aplicabilidad de la metodología empleada, diversas muestras provenientes de cultivos in vitro de P. belizeanum fueron analizadas. Los autores agradecen el financiamiento del MEC (AGL2005- 07924-C04-01 y 02/ALI), VALBIOMAR, ICIC (PGC) y CajaCanarias (JGN). La cepa de P. belizeanum fue facilitada por el Dr. S. Fraga, del CCVIEO (Vigo, España).

9 páginas, 5 figuras, The main toxins produced by the dinoflagellate Prorocentrum belizeanum, DTX5c and 7-hydroxymethyl-2methyleneocta- 4,7 dienyl okadaate, were studied by liquid chromatography-coupled with ion trap mass spectrometry (LC–MS/MS). Their retention times (RTs) and fragmentation patterns were established, in particular those of DTX5c, as there is a lack of data about these water soluble OA sulphated derivatives. As an application of the LC–MS methodology, a sample of the toxic P. belizeanum culture was analysed for these metabolites. Both metabolites were detected in cells as majority compounds, whereas levels of these compounds in the culture media were undetectable., The authors acknowledge financial support from the Spanish MEC (AGL2005-07924-C04-01 and 02/ ALI), VALBIOMAR, ICIC (PGC) and CajaCanarias (JGN); Strain of P. belizeanum was obtained from the CCVIEO (Vigo) courtesy of S. Fraga.