20 research outputs found
Identification of PalytoxinâCa<sup>2+</sup> Complex by NMR and Molecular Modeling Techniques
More
than 40 years after its isolation, the understanding of how
palytoxin interacts with biological systems has yet to be fully determined.
The Na<sup>+</sup>,K<sup>+</sup>-ATPase pump constitutes a molecular
receptor for palytoxin that is able to convert the pump into an open
channel, with consequent loss of cellular K<sup>+</sup> and remarkable
rise of cytosolic Na<sup>+</sup> levels. In addition, a slight permeability
to Ca<sup>2+</sup> is detected when palytoxin binds to the pump. It
has been demonstrated that the increase of cytosolic free Ca<sup>2+</sup> concentration gives rise to downstream events ultimately leading
to cell death. The widely accepted recognition of the dependence of
important cellular events on calcium ion concentration propelled us
to investigate the occurrence of palytoxinâCa<sup>2+</sup> complex
in aqueous solution by NMR- and molecular modeling-based approach.
We identified two specific regions of palytoxin where Ca<sup>2+</sup> is preferentially coordinated. This study constitutes the first
characterization of a calcium complex with palytoxin and, as such,
is expected to support the investigation of the toxin molecular bioactivity
Variability in Toxin Profiles of the Mediterranean <i>Ostreopsis</i> cf. <i>ovata</i> and in Structural Features of the Produced Ovatoxins
Fifty-five
strains of <i>Ostreopsis</i> were collected
in the Mediterranean Sea and analyzed to characterize their toxin
profiles. All the strains were grown in culture under the same experimental
conditions and identified by molecular PCR assay based on the ITS-5.8S
rDNA. A liquid chromatography-high resolution multiple stage mass
spectrometry (LC-HRMS<i><sup>n</sup></i>) approach was used
to analyze toxin profiles and to structurally characterize the detected
toxins. Despite morphological and molecular characterization being
consistent within the species <i>O.</i> cf. <i>ovata</i>, a certain degree of toxin variability was observed. All the strains
produced ovatoxins (OVTXs), with the exception of only one strain.
Toxin profiles were quite different from both qualitative and quantitative
standpoints: 67% of the strains contained OVTX-a to -e, OVTX-g, and
isobaric PLTX, in 25% of them only OVTX-a, -d, -e and isobaric PLTX
were present, while 4% produced only OVTX-b and -c. None of the strains
showed a previously identified profile, featuring OVTX-f as dominant
toxin, whereas OVTX-f was a minor component of very few strains. Toxin
content was mostly in the range 4â70 pg/cell with higher levels
(up to 238 pg/cell) being found in strains from the Ligurian and South
Adriatic Sea. Structural insights into OVTX-b, -c, -d, and -e were
gained, and the new OVTX-l was detected in 36 strains
The <i>sxt</i> Gene and Paralytic Shellfish Poisoning Toxins as Markers for the Monitoring of Toxic <i>Alexandrium</i> Species Blooms
Paralytic shellfish
poisoning (PSP) is a serious human illness
caused by the ingestion of seafood contaminated with saxitoxin and
its derivatives (STXs). These toxins are produced by some species
of marine dinoflagellates within the genus <i>Alexandrium</i>. In the Mediterranean Sea, toxic <i>Alexandrium</i> spp.
blooms, especially of <i>A. minutum</i>, are frequent and
intense with negative impact to coastal ecosystem, aquaculture practices
and other economic activities. We conducted a large scale study on
the <i>sxt</i> gene and toxin distribution and content in
toxic dinoflagellate <i>A. minutum</i> of the Mediterranean
Sea using both quantitative PCR (qPCR) and HILIC-HRMS techniques.
We developed a new qPCR assay for the estimation of the <i>sxtA1</i> gene copy number in seawater samples during a bloom event in Syracuse
Bay (Mediterranean Sea) with an analytical sensitivity of 2.0 Ă
10° <i>sxtA1</i> gene copy number per reaction. The
linear correlation between <i>sxtA1</i> gene copy number
and microalgal abundance and between the <i>sxtA1</i> gene
and STX content allowed us to rapidly determine the STX-producing
cell concentrations of two <i>Alexandrium</i> species in
environmental samples. In these samples, the amount of <i>sxtA1</i> gene was in the range of 1.38 Ă 10<sup>5</sup> â 2.55
Ă 10<sup>8</sup> copies/L and the STX concentrations ranged from
41â201 nmol/L. This study described a potential PSP scenario
in the Mediterranean Sea
Stereoisomers of 42-Hydroxy Palytoxin from Hawaiian <i>Palythoa toxica</i> and <i>P. tuberculosa</i>: Stereostructure Elucidation, Detection, and Biological Activities
Palytoxin ranks among the most potent
marine biotoxins. Its lethality
was well known to native Hawaiians that used to smear a âmossâ
containing the toxin on their spears to cause instant death to their
victims. Human intoxications due to exposure to palytoxin and to its
many congeners have been reported worldwide. Currently, palytoxins
constitute the main threat to public health across the Mediterranean
Sea. In the present work we report on the isolation and stereostructural
determination of a new palytoxin analogue from a Hawaiian <i>Palythoa tuberculosa</i> sample. This new toxin is a stereoisomer
of 42-hydroxypalytoxin isolated from <i>Palythoa toxica</i>. The whole absolute configuration of this latter toxin is also reported
in the paper. Interestingly, the two 42-hydroxypalytoxins do not share
the same biological activity. The stereoisomer from <i>P. tuberculosa</i> showed cytotoxicity toward skin HaCaT keratinocytes approximately
1 order of magnitude lower than that of 42-hydroxypalytoxin from <i>P. toxica</i> and about 2 orders of magnitude lower than that
of palytoxin itself. This finding holds the prospect of interesting
structureâactivity relationship evaluations in the future
Ovatoxin-a, A Palytoxin Analogue Isolated from <i>Ostreopsis</i> cf. <i>ovata</i> Fukuyo: Cytotoxic Activity and ELISA Detection
This
study provides the first evaluation of the cytotoxic effects
of the recently identified palytoxin (PLTX) analog, ovatoxin-a (OVTX-a),
the major toxin produced by <i>Ostreopsis</i> cf. <i>ovata</i> in the Mediterranean Sea. Its increasing detection
during <i>Ostreopsis</i> blooms and in seafood highlights
the need to characterize its toxic effects and to set up appropriate
detection methods. OVTX-a is about 100 fold less potent than PLTX
in reducing HaCaT cells viability (EC<sub>50</sub> = 1.1 Ă 10<sup>â9</sup> M vs 1.8 Ă 10<sup>â11</sup> M, MTT test)
in agreement with a reduced binding affinity (<i>K</i><sub>d</sub> = 1.2 Ă 10<sup>â9</sup> vs 2.7 Ă 10<sup>â11</sup> M, saturation experiments on intact cells). Similarly,
OVTX-a hemolytic effect is lower than that of the reference PLTX compound.
Ost-D shows the lowest cytotoxicity toward HaCaT keratinocytes, suggesting
the lack of a hydroxyl group at C44 as a critical feature for PLTXs
cytotoxic effects. A sandwich ELISA developed for PLTX detects also
OVTX-a in a sensitive (LOD = 4.2 and LOQ = 5.6 ng/mL) and accurate
manner (Bias = 0.3%), also in <i>O.</i> cf. <i>ovata</i> extracts and contaminated mussels. Although in vitro OVTX-a appears
less toxic than PLTX, its cytotoxicity at nanomolar concentrations
after short exposure time rises some concern for human health. The
sandwich ELISA can be a viable screening method for OVTXs detection
in monitoring program
<i>Ostreopsis</i> cf. <i>ovata</i> trichocysts: Scanning and transmission electron microscopy.
<p>(A) Scanning electron microscopy. Cell ventral end: numerous trichocysts extruded through thecal pores converge at the ventral end of the cell where they coalesce together forming a single thicker filament (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057291#pone-0057291-g002" target="_blank">Figs 2D, 2F</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057291#pone-0057291-g003" target="_blank">3D</a>). Scale bar 3 ”m. (B) Scanning electron microscopy. Trichocysts emerging through thecal pores distributed over the whole cell surface. Most trichocysts are directed towards the ventral end of the cell. Scale bar 2 ”m. (C) Scanning electron microscopy. Bundle of trichocysts forming a filament (see also Fig. 2D). Scale bar 1 ”m. (D) Transmission electron microscopy: negative staining Trichocyst shafts: they show a banded appearance with major bands period of 68 nm. Scale bar 100 nm. (E) Transmission electron microscopy. Peripheral cytoplasm section showing a trichocyst (t) with crystalline core and terminal fibres perperdicular to the amphiesma. Fixation 1. Scale bar 500 nm. (F) Transmission electron microscopy. Peripheral cytoplasm section showing a thecal pore in connection with a trichocyst (t) (only terminal fibres are visible), two mucocysts (m) with granular content and an the empty sac of an extruded trichocyst. Fixation 1. Scale bar 500 nm.</p
Raman spectrum of palytoxin.
<p>400â1800 cm-1 region of the Raman spectrum of palytoxin (solid); bands are labeled with the corresponding Raman shifts and with tentative vibrational modes assignments. Excitation wavelength is 785 nm.</p
New Insights on Cytological and Metabolic Features of <em>Ostreopsis</em> cf. <em>ovata</em> Fukuyo (Dinophyceae): A Multidisciplinary Approach
<div><p>The harmful dinoflagellate <i>Ostreopsis</i> cf. <i>ovata</i> has been causing toxic events along the Mediterranean coasts and other temperate and tropical areas, with increasing frequency during the last decade. Despite many studies, important biological features of this species are still poorly known. An integrated study, using different microscopy and molecular techniques, Raman microspectroscopy and high resolution liquid chromatography-mass spectrometry (HR LC-MS), was undertaken to elucidate cytological aspects, and identify main metabolites including toxins. The species was genetically identified as <i>O.</i> cf. <i>ovata</i>, Atlantic-Mediterranean clade. The ultrastructural results show unique features of the mucilage network abundantly produced by this species to colonize benthic substrates, with a new role of trichocysts, never described before. The amorphous polysaccharidic component of mucilage appears to derive from pusule fibrous material and mucocysts. In all stages of growth, the cells show an abundant production of lipids. Different developmental stages of chloroplasts are found in the peripheral cytoplasm and in the centre of cell. <i>In vivo</i> Raman microspectroscopy confirms the presence of the carotenoid peridinin in <i>O</i>. cf. <i>ovata</i>, and detects in several specimen the abundant presence of unsaturated lipids structurally related to docosahexaenoic acid. The HR LC-MS analysis reveals that ovatoxin-a is the predominant toxin, together with decreasing amounts of ovatoxin-b, -d/e, -c and putative palytoxin. Toxins concentration on a per cell basis increases from exponential to senescent phase. The results suggest that benthic blooms of this species are probably related to features such as the ability to create a unique mucilaginous sheath covering the sea bottom, associated with the production of potent toxins as palytoxin-like compounds. In this way, <i>O.</i> cf. <i>ovata</i> may be able to rapidly colonize benthic substrates outcompeting other species.</p> </div
Raman spectra of peridinin and living <i>O.</i> cf. <i>ovata</i> cells.
<p>400â1800 cm-1 region of (a) the Raman spectrum of peridinin (solid); (b) average normalized Raman spectrum (in black) plus and minus the intensity S.D. (in grey) of a set of 24 spectra collected from 24 different <i>O.</i> cf. <i>ovata</i> cells; (c) S.D. of the Raman intensity for the same set of 24 cells, plotted with a magnified intensity scale. In all spectra, bands are labelled with their Raman shift. In (b) and (c) bands assigned to chlorophyll are labelled as chl, whereas in (c) bands assigned to carotenoids are labelled as car. Excitation wavelength 785 nm.</p
Bright field transmission micrographs and Raman maps of fixed cells at different growth phases.
<p>(aâc) bright field transmission micrograph of fixed <i>O.</i>cf. <i>ovata</i> cells in the (a) exponential, (b) stationary and (c) senescence phase, and (aââcâ) the corresponding Raman map depicting carotenoids concentration based on the un-normalized intensity of the Îœ<sub>1</sub> carotenoid band at 1527 cm<sup>â1</sup>.</p