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1.
Mar Drugs ; 15(12)2017 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-29258236

RESUMO

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the detection and quantitation of karlotoxins in the selected reaction monitoring (SRM) mode. This novel method was based upon the analysis of purified karlotoxins (KcTx-1, KmTx-2, 44-oxo-KmTx-2, KmTx-5), one amphidinol (AM-18), and unpurified extracts of bulk cultures of the marine dinoflagellate Karlodinium veneficum strain CCMP2936 from Delaware (Eastern USA), which produces KmTx-1 and KmTx-3. The limit of detection of the SRM method for KmTx-2 was determined as 2.5 ng on-column. Collision induced dissociation (CID) spectra of all putative karlotoxins were recorded to present fragmentation patterns of each compound for their unambiguous identification. Bulk cultures of K. veneficum strain K10 isolated from an embayment of the Ebro Delta, NW Mediterranean, yielded five previously unreported putative karlotoxins with molecular masses 1280, 1298, 1332, 1356, and 1400 Da, and similar fragments to KmTx-5. Analysis of several isolates of K. veneficum from the Ebro Delta revealed small-scale diversity in the karlotoxin spectrum in that one isolate from Fangar Bay produced KmTx-5, whereas the five putative novel karlotoxins were found among several isolates from nearby, but hydrographically distinct Alfacs Bay. Application of this LC-MS/MS method represents an incremental advance in the determination of putative karlotoxins, particularly in the absence of a complete spectrum of purified analytical standards of known specific potency.


Assuntos
Organismos Aquáticos/química , Dinoflagelados/química , Toxinas Marinhas/química , Cromatografia Líquida/métodos , Dinoflagelados/isolamento & purificação , Mar Mediterrâneo , Polienos/química , Piranos/química , Espectrometria de Massas em Tandem/métodos
2.
Mar Drugs ; 15(10)2017 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-29027912

RESUMO

Guanidinium toxins, such as saxitoxin (STX), tetrodotoxin (TTX) and their analogs, are naturally occurring alkaloids with divergent evolutionary origins and biogeographical distribution, but which share the common chemical feature of guanidinium moieties. These guanidinium groups confer high biological activity with high affinity and ion flux blockage capacity for voltage-gated sodium channels (NaV). Members of the STX group, known collectively as paralytic shellfish toxins (PSTs), are produced among three genera of marine dinoflagellates and about a dozen genera of primarily freshwater or brackish water cyanobacteria. In contrast, toxins of the TTX group occur mainly in macrozoa, particularly among puffer fish, several species of marine invertebrates and a few terrestrial amphibians. In the case of TTX and analogs, most evidence suggests that symbiotic bacteria are the origin of the toxins, although endogenous biosynthesis independent from bacteria has not been excluded. The evolutionary origin of the biosynthetic genes for STX and analogs in dinoflagellates and cyanobacteria remains elusive. These highly potent molecules have been the subject of intensive research since the latter half of the past century; first to study the mode of action of their toxigenicity, and later as tools to characterize the role and structure of NaV channels, and finally as therapeutics. Their pharmacological activities have provided encouragement for their use as therapeutants for ion channel-related pathologies, such as pain control. The functional role in aquatic and terrestrial ecosystems for both groups of toxins is unproven, although plausible mechanisms of ion channel regulation and chemical defense are often invoked. Molecular approaches and the development of improved detection methods will yield deeper understanding of their physiological and ecological roles. This knowledge will facilitate their further biotechnological exploitation and point the way towards development of pharmaceuticals and therapeutic applications.


Assuntos
Guanidina/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio Disparados por Voltagem/efeitos dos fármacos , Animais , Cianobactérias/metabolismo , Dinoflagelados/metabolismo , Guanidina/química , Humanos , Saxitoxina/química , Saxitoxina/farmacologia , Bloqueadores dos Canais de Sódio/química , Tetrodotoxina/química , Tetrodotoxina/farmacologia , Toxinas Biológicas/química , Toxinas Biológicas/farmacologia , Canais de Sódio Disparados por Voltagem/metabolismo
3.
Toxins (Basel) ; 8(5)2016 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-27164145

RESUMO

Saxitoxin (STX) and its analogs are paralytic alkaloid neurotoxins that block the voltage-gated sodium channel pore (Nav), impeding passage of Na⁺ ions into the intracellular space, and thereby preventing the action potential in the peripheral nervous system and skeletal muscle. The marine dinoflagellate Gymnodinium catenatum produces an array of such toxins, including the recently discovered benzoyl analogs, for which the mammalian toxicities are essentially unknown. We subjected STX and its analogs to a theoretical docking simulation based upon two alternative tri-dimensional models of the Nav1.4 to find a relationship between the binding properties and the known mammalian toxicity of selected STX analogs. We inferred hypothetical toxicities for the benzoyl analogs from the modeled values. We demonstrate that these toxins exhibit different binding modes with similar free binding energies and that these alternative binding modes are equally probable. We propose that the principal binding that governs ligand recognition is mediated by electrostatic interactions. Our simulation constitutes the first in silico modeling study on benzoyl-type paralytic toxins and provides an approach towards a better understanding of the mode of action of STX and its analogs.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Saxitoxina/análogos & derivados , Saxitoxina/metabolismo , Dinoflagelados/metabolismo , Simulação de Acoplamento Molecular , Canal de Sódio Disparado por Voltagem NAV1.4/química , Saxitoxina/química
4.
Harmful Algae ; 55: 191-201, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-28073532

RESUMO

Harmful Algal Bloom (HAB) surveillance is complicated by high diversity of species and associated phycotoxins. Such species-level information on taxonomic affiliations and on cell abundance and toxin content is, however, crucial for effective monitoring, especially of aquaculture and fisheries areas. The aim addressed in this study was to determine putative HAB taxa and related phycotoxins in plankton from aquaculture sites in the Ebro Delta, NW Mediterranean. The comparative geographical distribution of potentially harmful plankton taxa was established by weekly field sampling throughout the water column during late spring-early summer over two years at key stations in Alfacs and Fangar embayments within the Ebro Delta. Core results included not only confirmed identification of HAB taxa that are common for the time period and geographical area, but also provided evidence of potentially new taxa. At least 25 HAB taxa were identified to species level, and an additional six genera were confirmed, by morphological criteria under light microscopy and/or by molecular genetics approaches involving qPCR and next generation DNA pyrosequencing. In particular, new insights were gained by the inclusion of molecular techniques, which focused attention on the HAB genera Alexandrium, Karlodinium, and Pseudo-nitzschia. Noteworthy is the discovery of Azadinium sp., a potentially new HAB species for this area, and Gymnodinium catenatum or Gymnodinium impudicum by means of light microscopy. In addition, significant amounts of the neurotoxin domoic acid (DA) were found for the first time in phytoplankton samples in the Ebro Delta. While the presence of the known DA-producing diatom genus Pseudo-nitzschia was confirmed in corresponding samples, the maximal toxin concentration did not coincide with highest cell abundances of the genus and the responsible species could not be identified. Combined findings of microscopic and molecular detection approaches underline the need for a synoptic strategy for HAB monitoring, which integrates the respective advantages and compensates for limitations of individual methods.


Assuntos
Dinoflagelados/química , Dinoflagelados/fisiologia , Monitoramento Ambiental , Toxinas Marinhas/análise , Diatomáceas/química , Diatomáceas/fisiologia , Dinoflagelados/classificação , Eutrofização , Proliferação Nociva de Algas , Mar Mediterrâneo , Fitoplâncton/química , Fitoplâncton/classificação , Fitoplâncton/fisiologia
5.
Ann Rev Mar Sci ; 4: 143-76, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22457972

RESUMO

The public health, tourism, fisheries, and ecosystem impacts from harmful algal blooms (HABs) have all increased over the past few decades. This has led to heightened scientific and regulatory attention, and the development of many new technologies and approaches for research and management. This, in turn, is leading to significant paradigm shifts with regard to, e.g., our interpretation of the phytoplankton species concept (strain variation), the dogma of their apparent cosmopolitanism, the role of bacteria and zooplankton grazing in HABs, and our approaches to investigating the ecological and genetic basis for the production of toxins and allelochemicals. Increasingly, eutrophication and climate change are viewed and managed as multifactorial environmental stressors that will further challenge managers of coastal resources and those responsible for protecting human health. Here we review HAB science with an eye toward new concepts and approaches, emphasizing, where possible, the unexpected yet promising new directions that research has taken in this diverse field.


Assuntos
Mudança Climática , Monitoramento Ambiental/métodos , Proliferação Nociva de Algas , Projetos de Pesquisa , Animais , Humanos , Oceanos e Mares
6.
Harmful Algae ; 14: 10-35, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22308102

RESUMO

The dinoflagellate genus Alexandrium is one of the major harmful algal bloom (HAB) genera with respect to the diversity, magnitude and consequences of blooms. The ability of Alexandrium to colonize multiple habitats and to persist over large regions through time is testimony to the adaptability and resilience of this group of species. Three different families of toxins, as well as an as yet incompletely characterized suite of allelochemicals are produced among Alexandrium species. Nutritional strategies are equally diverse, including the ability to utilize a range of inorganic and organic nutrient sources, and feeding by ingestion of other organisms. Many Alexandrium species have complex life histories that include sexuality and often, but not always, cyst formation, which is characteristic of a meroplanktonic life strategy and offers considerable ecological advantages. Due to the public health and ecosystem impacts of Alexandrium blooms, the genus has been extensively studied, and there exists a broad knowledge base that ranges from taxonomy and phylogeny through genomics and toxin biosynthesis to bloom dynamics and modeling. Here we present a review of the genus Alexandrium, focusing on the major toxic and otherwise harmful species.

7.
Mar Drugs ; 9(9): 1625-48, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22131962

RESUMO

Marine chemical ecology comprises the study of the production and interaction of bioactive molecules affecting organism behavior and function. Here we focus on bioactive compounds and interactions associated with phytoplankton, particularly bloom-forming diatoms, prymnesiophytes and dinoflagellates. Planktonic bioactive metabolites are structurally and functionally diverse and some may have multiple simultaneous functions including roles in chemical defense (antipredator, allelopathic and antibacterial compounds), and/or cell-to-cell signaling (e.g., polyunsaturated aldehydes (PUAs) of diatoms). Among inducible chemical defenses in response to grazing, there is high species-specific variability in the effects on grazers, ranging from severe physical incapacitation and/or death to no apparent physiological response, depending on predator susceptibility and detoxification capability. Most bioactive compounds are present in very low concentrations, in both the producing organism and the surrounding aqueous medium. Furthermore, bioactivity may be subject to synergistic interactions with other natural and anthropogenic environmental toxicants. Most, if not all phycotoxins are classic secondary metabolites, but many other bioactive metabolites are simple molecules derived from primary metabolism (e.g., PUAs in diatoms, dimethylsulfoniopropionate (DMSP) in prymnesiophytes). Producing cells do not seem to suffer physiological impact due to their synthesis. Functional genome sequence data and gene expression analysis will provide insights into regulatory and metabolic pathways in producer organisms, as well as identification of mechanisms of action in target organisms. Understanding chemical ecological responses to environmental triggers and chemically-mediated species interactions will help define crucial chemical and molecular processes that help maintain biodiversity and ecosystem functionality.


Assuntos
Ecossistema , Plâncton/química , Animais , Fitoplâncton/química , Fitoplâncton/fisiologia , Plâncton/fisiologia , Zooplâncton/química , Zooplâncton/fisiologia
8.
BMC Genomics ; 11: 248, 2010 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-20403159

RESUMO

BACKGROUND: The dinoflagellate Alexandrium minutum typically produces paralytic shellfish poisoning (PSP) toxins, which are known only from cyanobacteria and dinoflagellates. While a PSP toxin gene cluster has recently been characterized in cyanobacteria, the genetic background of PSP toxin production in dinoflagellates remains elusive. RESULTS: We constructed and analysed an expressed sequence tag (EST) library of A. minutum, which contained 15,703 read sequences yielding a total of 4,320 unique expressed clusters. Of these clusters, 72% combined the forward-and reverse reads of at least one bacterial clone. This sequence resource was then used to construct an oligonucleotide microarray. We analysed the expression of all clusters in three different strains. While the cyanobacterial PSP toxin genes were not found among the A. minutum sequences, 192 genes were differentially expressed between toxic and non-toxic strains. CONCLUSIONS: Based on this study and on the lack of identified PSP synthesis genes in the two existent Alexandrium tamarense EST libraries, we propose that the PSP toxin genes in dinoflagellates might be more different from their cyanobacterial counterparts than would be expected in the case of a recent gene transfer. As a starting point to identify possible PSP toxin-associated genes in dinoflagellates without relying on a priori sequence information, the sequences only present in mRNA pools of the toxic strain can be seen as putative candidates involved in toxin synthesis and regulation, or acclimation to intracellular PSP toxins.


Assuntos
Dinoflagelados/genética , Perfilação da Expressão Gênica , Toxinas Marinhas/genética , Dinoflagelados/metabolismo , Etiquetas de Sequências Expressas , Análise de Sequência com Séries de Oligonucleotídeos , Intoxicação por Frutos do Mar/microbiologia
9.
Mol Ecol ; 18(10): 2122-33, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19389181

RESUMO

Genotypic or phenotypic markers for characterization of natural populations of marine microalgae have typically addressed questions regarding differentiation among populations, usually with reference to a single or few clonal isolates. Based upon a large number of contemporaneous isolates from the same geographical population of the toxigenic species Alexandrium tamarense from the North Sea, we uncovered significant genetic substructure and low but significant multilocus linkage disequilibrium (LD) within the planktonic population. Between the alternative molecular genotyping approaches, only amplified fragment length polymorphism (AFLP) revealed cryptic genetic population substructure by Bayesian clustering, whereas microsatellite markers failed to yield concordant patterns. Both markers, however, gave evidence for genetic differentiation of population subgroups as defined by AFLP. A considerable portion of multilocus LD could be attributed to population subdivision. The remaining LD within population subgroups is interpreted as an indicator of frequency shifts of clonal lineages during vegetative growth of planktonic populations. Phenotypic characters such as cellular content and composition of neurotoxins associated with paralytic shellfish poisoning (PSP) and allelochemical properties may contribute to intra- or inter-annual differentiation of planktonic populations, if clonal lineages that express these characters are selectively favoured. Nevertheless, significant phenotypic differentiation for these characters among the genetically differentiated subgroups was only detected for PSP toxin content in two of the four population subgroups. By integrating the analysis of phenotypic and genotypic characteristics, we developed a conceptual population genetic model to explain the importance of life-cycle dynamics and transitions in the evolutionary ecology of these dinoflagellates.


Assuntos
Dinoflagelados/genética , Genética Populacional , Desequilíbrio de Ligação , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Animais , Marcadores Genéticos , Genótipo , Toxinas Marinhas/genética , Repetições de Microssatélites , Fenótipo , Reprodução , Análise de Sequência de DNA
10.
Toxicon ; 52(8): 927-35, 2008 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-18938193

RESUMO

Lipophilic phycotoxins in size-fractionated plankton net tows (20 mum mesh-size) were measured on-board during a month-long oceanographic cruise in North Sea coastal waters. Tandem mass spectrometry coupled to liquid chromatography (LC-MS/MS) was employed for the detection and quantification of a broad spectrum of known and putative phycotoxins. For pectenotoxins (PTXs) the following ion masses ([M + NH(4)](+)) were monitored: m/z 876 for PTX-2, m/z 892 for PTX-11 and PTX-13, and m/z 874 for PTX-12 and PTX-14. The PTX levels in net plankton were highest along the Danish north coast, but levels over 50 ng per net tow were also detected on the southern Scottish East coast and in the northern Skagerrak. Abundance of PTXs was highly correlated with the occurrence of the marine dinoflagellate Dinophysis spp. Whereas in the eastern North Sea PTX-2 was the most abundant PTX, in the western North Sea PTX-1 was the major component, but it was also present in lower proportions in the Norwegian and Danish waters than in the western North Sea. Isobaric PTX-11 was absent or only detected at trace levels throughout the entire cruise, and PTX-13 and PTX-14 were not detected at all. The identity of PTX-1 was confirmed by comparison of retention time and mass spectrum of the North Sea phytoplankton sample to PTX-1 previously isolated from shellfish. Statistical analysis showed the best correlation between the occurrence of PTX-1 and Dinophysis acuminata cell concentration. Nevertheless, we could not rule out the possibility of metabolic transformations of PTXs by organisms that have grazed upon Dinophysis. Such biotransformations could conceivably occur in heterotrophic dinoflagellates or ciliates, or even via oxidation in copepod fecal pellets. In any case, this study confirmed the presence of PTX-1 in the plankton and is the first definitive report of this toxin in the North Sea.


Assuntos
Dinoflagelados/química , Toxinas Marinhas/análise , Plâncton/química , Piranos/análise , Animais , Cromatografia Líquida , Toxinas Marinhas/química , Toxinas Marinhas/isolamento & purificação , Mar do Norte , Piranos/química , Piranos/isolamento & purificação , Espectrometria de Massas em Tandem
11.
J Org Chem ; 71(23): 8724-31, 2006 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-17080999

RESUMO

Biosynthetic origins of the cyclic imine toxin 13-desmethyl spirolide C were determined by supplementing cultures of the toxigenic dinoflagellate Alexandrium ostenfeldii with stable isotope-labeled precursors [1,2-13C2]acetate, [1-13C]acetate, [2-13CD3]acetate, and [1,2-13C2,15N]glycine and measuring the incorporation patterns by 13C NMR spectroscopy. Despite partial scrambling of the acetate labels, the results show that most carbons of the macrocycle are polyketide-derived and that glycine is incorporated as an intact unit into the cyclic imine moiety. This work represents the first conclusive evidence that such cyclic imine toxins are polyketides and provides support for biosynthetic pathways previously defined for other polyether dinoflagellate toxins.


Assuntos
Dinoflagelados/metabolismo , Compostos Macrocíclicos/metabolismo , Toxinas Marinhas/metabolismo , Compostos de Espiro/metabolismo , Animais , Isótopos de Carbono , Dinoflagelados/química , Compostos Macrocíclicos/química , Compostos Macrocíclicos/isolamento & purificação , Espectroscopia de Ressonância Magnética/métodos , Toxinas Marinhas/química , Toxinas Marinhas/isolamento & purificação , Estrutura Molecular , Sensibilidade e Especificidade , Compostos de Espiro/química , Compostos de Espiro/isolamento & purificação , Estereoisomerismo
12.
J Nat Prod ; 69(7): 983-7, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16872129

RESUMO

Using LC/MS methodology, spirolides were detected in two clonal isolates of Alexandrium ostenfeldii isolated from Limfjorden, Denmark. Examination of the LC/MS profiles of extracts from these Danish cultures revealed the presence of two dominant peaks representing two previously unidentified spirolide components and one minor peak identified as the previously reported desmethyl spirolide C (1). Culturing of these clonal strains, LF 37 and LF 38, of A. ostenfeldii resulted in the accumulation of sufficient cell biomass to allow for the isolation and structure elucidation of two new spirolides, 13,19-didesmethylspirolide C (2) and spirolide G (3). While 2 was found to differ from 1 only in that it contained one less methyl group, 3 was the first spirolide to be isolated that contained a 5:6:6-trispiroketal ring system. The effect of this new feature on the toxicity of 3 relative to other spirolides is presently being pursued.


Assuntos
Dinoflagelados/química , Iminas , Toxinas Marinhas , Compostos de Espiro , Animais , Dinamarca , Iminas/química , Iminas/isolamento & purificação , Iminas/farmacologia , Toxinas Marinhas/química , Toxinas Marinhas/isolamento & purificação , Toxinas Marinhas/farmacologia , Estrutura Molecular , Compostos de Espiro/química , Compostos de Espiro/isolamento & purificação , Compostos de Espiro/farmacologia
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