16 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
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
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
Isolation and Structure Elucidation of Ovatoxin-a, the Major Toxin Produced by Ostreopsis ovata
Since 2005, the benthic dinoflagellate Ostreopsis cf. ovata has bloomed across the
Mediterranean basin, provoking serious toxic outbreaks. LC/MS studies
have identified a number of palytoxin-like compounds, termed ovatoxins,
along with trace amounts of putative palytoxin as the causative agents
of the O. cf. ovata-related human sufferings. So far, any risk assessment for ovatoxins
as well as establishment of their allowance levels in seafood has
been prevented by the lack of pure toxins. The present paper reports
on the isolation, NMR-based structural determination, and preliminary
mouse lethality evaluation of ovatoxin-a, the major toxic compound
contained in O. cf. ovata extracts. Availability of pure ovatoxin-a will
open the double prospect of fully evaluating its toxicity and preparing
reference standards to be employed in LC/MS quantitative analyses.
Elucidation of ovatoxin-aâs complex structure will ultimately
herald the understanding of the molecular bases of ovatoxins bioactivity
MAPKs activation and toxin-induced COX-2, TNF-α, IL-8 and IÎșBα mRNA expression upon p38 MAPK and JNK inhibition.
<p>(<b>A</b>) Western blot analysis of MAPK phosphorylation in macrophages incubated 4 h with the <i>O</i>. cf. <i>ovata</i> toxin extract (final OSTRTX concentration 2 ng/ml and 20 ng/ml) (lane 2, 3), 2 ng/ml PLTX (lane 4) or the vehicle (lane 1). 20 ”g of whole cell extracts were separated by SDS-PAGE on 8% gel and submitted to western immunoblot with phospho-specific antibodies against ERK1/2, JNK and p38 MAPK. Actin was stained as a loading control. (<b>B</b>) Total RNA was extracted from macrophages pre-incubated 1 h with 1 ”M p38 inhibitor (ip38) or 1 ”M and 5 ”M JNK inhibitor (iJNK), and then exposed 4 h to 2 ng/ml palytoxin. In parallel, macrophages were left untreated and then exposed to PLTX or to the vehicle alone. RNA was amplified with gene-specific primers. Expression data, normalized to the housekeeping B2M gene, were analyzed by the 2<sup>âÎÎCT</sup> method and referred to the value obtained in control cells (CTR). The boxplots show the results of 3 independent experiments, run in duplicate. Asterisks indicate statistical significance versus cells receiving only PLTX (*<i>p</i><0.05; **<i>p</i><0.01).</p
IÎșBα and p65 protein levels in cells treated with the vehicle, the OSTRTX extract and PLTX.
<p>(<b>A</b>) IÎșBα protein levels were determined in whole cell lysates obtained from macrophages incubated 4 h with the vehicle (lane 1), the OSTRTX extract (lanes 2â3) or PLTX (lane 4). Protein extracts (15 ”g) were resolved by SDS-PAGE on 8% gel and then submitted to Western immunoblotting. Blots were probed with an anti-IÎșBα (upper panel) and an anti-p65 (RelA) antibody to check for protein loading (lower panel). (<b>B</b>) intracellular content and subcellular distribution of p65 (RelA) in PLTX- versus vehicle-treated cells was assessed by immunoblotting analysis of whole (15 ”g, lanes 1â2), cytosolic (15 ”g, lanes 3â4) and nuclear (10 ”g, lane 5â6) extracts. Whole cell extracts were obtained by lysing cells in SDS buffer. In parallel, cells were sub-fractionated by extraction in Buffer A (BUFF A, cytosolic proteins) followed by Buffer B (nuclear proteins, BUFF B), the residual material, containing insoluble proteins, including those associated with cytoskeletal structures, was solubilized in SDS-PAGE sample buffer and run in parallel (lanes 7â8). (<b>C</b>) Approaches to inhibit p65 <i>in vitro</i> degradation during extraction in native conditions. Cell pellets, containing an equivalent number of macrophages, were lysed in SDS buffer (SDS BUFF, lane 1), Buffer A (BUFF A, lane 2) or Buffer A supplemented with palytoxin (BUFF A+PLTX, lane 3). An equal volume of SDS sample buffer was added to all tubes and comparable volumes of the resulting protein extracts were resolved by electrophoresis and immunoblotted with an anti p65 antibody.(<b>D</b>) cell pellets as in C were lysed in SDS buffer (lane 1), Buffer A (lane 2) and Buffer A further supplemented with lysososmal protease inhibitors (BUFF A PLUS, lane 3) and submitted to SDS-PAGE and immunoblotting with an anti p65 antibody.</p
IÎșB-α mRNA levels in human macrophages exposed to PLTX and to the OSTRTX extract.
<p>Evaluation of IÎșB-α mRNA levels by quantitative Real-Time PCR assay. Total RNA, extracted from primary human macrophages exposed 4 h to PLTX, to the <i>O</i>. cf. <i>ovata</i> toxin extract (final OSTRTX concentration 2 ng/ml and 20 ng/ml) or to the vehicle (i.e. 0.05% MetOH), was amplified with IÎșB-α gene-specific primers. Expression data, normalized to the housekeeping B2M gene, were analysed by the 2<sup>âÎÎCT</sup> method and referred to the value obtained in untreated cells (CTR). The boxplots show the results of 8 and 5 independent experiments performed with PLTX and the OSTRTX extract, respectively. Asterisks indicate statistical significance versus cells receiving the vehicle alone (*<i>p</i><0.05; **<i>p</i><0.01).</p
COX-2, IL-8 and TNF-α mRNA levels in human macrophages exposed to PLTX and to the OSTRTX extract.
<p>Evaluation of COX-2, IL-8 and TNF-α mRNA levels by quantitative Real-Time PCR assay. Total RNA, extracted from primary human macrophages exposed 4 h to PLTX (2 ng/ml), to the <i>O</i>. cf. <i>ovata</i> toxin extract (final OSTRTX concentration 2 ng/ml and 20 ng/ml) or the vehicle (i.e. 0.05% MetOH), was amplified with gene-specific primers. Expression data, normalized to the housekeeping B2M gene, were analyzed by the 2<sup>âÎÎCT</sup> method and referred to the value obtained in untreated cells (CTR). The boxplots show the results of 8 and 5 independent experiments performed with PLTX and the OSTRTX extract, respectively. Asterisks indicate statistical significance versus cells receiving the vehicle alone (*<i>p</i><0.05; **<i>p</i><0.01).</p