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1.
Mar Drugs ; 18(2)2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32033403

RESUMO

Saxitoxin is an alkaloid neurotoxin originally isolated from the clam Saxidomus giganteus in 1957. This group of neurotoxins is produced by several species of freshwater cyanobacteria and marine dinoflagellates. The saxitoxin biosynthesis pathway was described for the first time in the 1980s and, since then, it was studied in more than seven cyanobacterial genera, comprising 26 genes that form a cluster ranging from 25.7 kb to 35 kb in sequence length. Due to the complexity of the genomic landscape, saxitoxin biosynthesis in dinoflagellates remains unknown. In order to reveal and understand the dynamics of the activity in such impressive unicellular organisms with a complex genome, a strategy that can carefully engage them in a systems view is necessary. Advances in omics technology (the collective tools of biological sciences) facilitated high-throughput studies of the genome, transcriptome, proteome, and metabolome of dinoflagellates. The omics approach was utilized to address saxitoxin-producing dinoflagellates in response to environmental stresses to improve understanding of dinoflagellates gene-environment interactions. Therefore, in this review, the progress in understanding dinoflagellate saxitoxin biosynthesis using an omics approach is emphasized. Further potential applications of metabolomics and genomics to unravel novel insights into saxitoxin biosynthesis in dinoflagellates are also reviewed.


Assuntos
Dinoflagelados/genética , Dinoflagelados/metabolismo , Saxitoxina/biossíntese , Saxitoxina/química , Vias Biossintéticas , Cianobactérias/metabolismo , Genômica , Metabolômica , Neurotoxinas/metabolismo , Biossíntese de Proteínas , Proteômica , Saxitoxina/metabolismo , Transcriptoma
2.
Chemistry ; 26(9): 2025-2033, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-31769085

RESUMO

A novel series of C12-keto-type saxitoxin (STX) derivatives bearing an unusual nonhydrated form of the ketone at C12 has been synthesized, and their NaV -inhibitory activity has been evaluated in a cell-based assay as well as whole-cell patch-clamp recording. Among these compounds, 11-benzylidene STX (3 a) showed potent inhibitory activity against neuroblastoma Neuro 2A in both cell-based and electrophysiological analyses, with EC50 and IC50 values of 8.5 and 30.7 nm, respectively. Interestingly, the compound showed potent inhibitory activity against tetrodotoxin-resistant subtype of NaV 1.5, with an IC50 value of 94.1 nm. Derivatives 3 a-d and 3 f showed low recovery rates from NaV 1.2 subtype (ca 45-79 %) compared to natural dcSTX (2), strongly suggesting an irreversible mode of interaction. We propose an interaction model for the C12-keto derivatives with NaV in which the enone moiety in the STX derivatives 3 works as Michael acceptor for the carboxylate of Asp1717 .


Assuntos
Saxitoxina/química , Bloqueadores dos Canais de Sódio/síntese química , Canais de Sódio Disparados por Voltagem/metabolismo , Potenciais de Ação/efeitos dos fármacos , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Técnicas de Patch-Clamp , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Teoria Quântica , Saxitoxina/metabolismo , Saxitoxina/farmacologia , Bloqueadores dos Canais de Sódio/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/química , Tetrodotoxina/metabolismo , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/genética
3.
PLoS One ; 14(11): e0222468, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31697710

RESUMO

Optical tweezers have enabled the exploration of picoNewton forces and dynamics in single-molecule systems such as DNA and molecular motors. In this work, we used optical tweezers to study the folding/unfolding dynamics of the APTSTX1-aptamer, a single-stranded DNA molecule with high affinity for saxitoxin (STX), a lethal neurotoxin. By measuring the transition force during (un)folding processes, we were able to characterize and distinguish the conformational changes of this aptamer in the presence of magnesium ions and toxin. This work was supported by molecular dynamics (MD) simulations to propose an unfolding mechanism of the aptamer-Mg+2 complex. Our results are a step towards the development of new aptamer-based STX sensors that are potentially cheaper and more sensitive than current alternatives.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Saxitoxina/química , Simulação de Dinâmica Molecular , Nanotecnologia/métodos , Neurotoxinas/química , Pinças Ópticas
4.
Toxicon ; 167: 76-81, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31170406

RESUMO

To understand phycotoxin contamination in shellfish in the sub-Arctic and Arctic areas, scanning for the presence of 13 hydrophilic and lipophilic toxin components each was by liquid chromatography tandem quadrupole mass spectrometry analysis in shellfish samples collected from the Northern Bering Sea and the Chukchi Sea in 2014. The results showed that shellfish collected in both areas werecontaminated to different extents. Saxitoxin (STX), decarbamoylsaxitoxin (dcSTX) and decarbamoylneosaxitoxin (dcNEO) were the most frequently detected hydrophilic components, with maximum concentrations of 90.1 µg/kg, 112.25 µg/kg and 23.09 µg/kg, respectively. Although gonyautoxins (GTXs) were only detected in 3 samples, they were the main contributors to overall toxicity of high-latitude samples, especially GTX1. For lipophilic toxins, spirolide-1 (SPX1) and yessotoxin (YTX) were present in all samples at low levels (< 7 µg/kg and < 50 µg/kg, respectively). Only 5 samples showed evidence of okadaic acid (OA) and dinophysistoxin-2 (DTX-2) at low concentrations, ranging from 0.42 µg/kg to 7.23 µg/kg and 3.03 µg/kg to 30.59 µg/kg, respectively. Notably, a high level of pectenotoxin-1 (PTX-1) at 467.40 µg/kg was found in the shellfish collected at the northernmost station, exceeding the safety regulation standard by nearly 3 times. For both lipophilic and hydrophilic toxins, contamination in shellfish in the sub-Arctic and the Arctic area may be much more widespread and severe than was previously thought. This study highlighted the need to monitor toxins in a wider variety of shellfish, especially economic or commercial species, and across a wider range of sub-Arctic and Arctic waters, as well as the potential sources of these toxins.


Assuntos
Contaminação de Alimentos/análise , Saxitoxina/análise , Frutos do Mar , Regiões Árticas , Cromatografia Líquida , Saxitoxina/análogos & derivados , Saxitoxina/química , Espectrometria de Massas em Tandem
5.
Science ; 363(6433): 1303-1308, 2019 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-30765606

RESUMO

Voltage-gated sodium channel Nav1.7 represents a promising target for pain relief. Here we report the cryo-electron microscopy structures of the human Nav1.7-ß1-ß2 complex bound to two combinations of pore blockers and gating modifier toxins (GMTs), tetrodotoxin with protoxin-II and saxitoxin with huwentoxin-IV, both determined at overall resolutions of 3.2 angstroms. The two structures are nearly identical except for minor shifts of voltage-sensing domain II (VSDII), whose S3-S4 linker accommodates the two GMTs in a similar manner. One additional protoxin-II sits on top of the S3-S4 linker in VSDIV The structures may represent an inactivated state with all four VSDs "up" and the intracellular gate closed. The structures illuminate the path toward mechanistic understanding of the function and disease of Nav1.7 and establish the foundation for structure-aided development of analgesics.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/química , Peptídeos/química , Saxitoxina/química , Venenos de Aranha/química , Tetrodotoxina/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/química , Subunidade beta-2 do Canal de Sódio Disparado por Voltagem/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Células HEK293 , Humanos , Conformação Proteica
6.
Biosens Bioelectron ; 128: 45-51, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30620920

RESUMO

Saxitoxin (STX) has high toxicity, and is water soluble, acid stable and thermostable. Therefore, STX in seawater can be accumulated by marine organism to form bioaccumulation. To ensure the safety of seafood for consumption, it is crucial to accurately determine trace STX in seawater and seafood. We herein developed a novel magnetic electrochemical immunosensor for ultra-sensitive detection of STX in seawater and seafood by using non-competitive strategy. The immunosensor employs STX-specific antibody-functionalized magnetic beads (MBs) for STX recognition, palladium-doped graphitic carbon nitride (g-C3N4-PdNPs) peroxidase mimetic for catalyzing H2O2-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to generate signal. The immunosensor combines the merits of g-C3N4-PdNPs peroxidase mimetic, non-competitive strategy, MBs-based antibody recognition and magnetic gold electrode, and thus has excellent stability, lower cost, no risk of false positive result, high sensitivity and strong ability resist to matrix interference. The proposed immunosensor has been successfully used to detect trace STX in seawater and shellfish samples with a detection limit of 1.2 pg/mL (4.0 × 10-12 M), a recovery of 93-107% and a relative standard deviation (RSD, n = 5) < 5%. The success of this study provided a promising approach for the rapid and on-site detection of trace STX in seawater and seafood.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Saxitoxina/isolamento & purificação , Alimentos Marinhos/análise , Ouro/química , Humanos , Limite de Detecção , Saxitoxina/química , Frutos do Mar
7.
Mar Drugs ; 18(1)2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31888062

RESUMO

Voltage-gated sodium channels (NaVs) are membrane proteins that are involved in the generation and propagation of action potentials in neurons. Recently, the structure of a complex made of a tetrodotoxin-sensitive (TTX-s) NaV subtype with saxitoxin (STX), a shellfish toxin, was determined. STX potently inhibits TTX-s NaV, and is used as a biological tool to investigate the function of NaVs. More than 50 analogs of STX have been isolated from nature. Among them, zetekitoxin AB (ZTX) has a distinctive chemical structure, and is the most potent inhibitor of NaVs, including tetrodotoxin-resistant (TTX-r) NaV. Despite intensive synthetic studies, total synthesis of ZTX has not yet been achieved. Here, we review recent efforts directed toward the total synthesis of ZTX, including syntheses of 11-saxitoxinethanoic acid (SEA), which is considered a useful synthetic model for ZTX, since it contains a key carbon-carbon bond at the C11 position.


Assuntos
Saxitoxina/análogos & derivados , Bloqueadores do Canal de Sódio Disparado por Voltagem/síntese química , Animais , Saxitoxina/síntese química , Saxitoxina/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/química
8.
Harmful Algae ; 80: 158-170, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30502809

RESUMO

Strains of the freshwater filamentous, benthic cyanobacterium Scytonema crispum Agardh isolated from six sites in subtropical south-east Queensland were characterised using a combination of phenotypic and genetic traits. Morphologically, the strains were consistent with the description of Scytonemataceae sensu stricto, and the description of Scytonema crispum. However, phylogenetic analysis of the 16S rRNA gene, the 16S-23S rRNA operon, and the nifH gene revealed that these strains and three others from outside Australia formed a monophyletic clade distinct from Scytonema and other species in the Scytonemataceae. Collectively, this data suggests this group is sufficiently evolutionarily distinct to be placed in a new family, Heteroscytonemataceae fam. nov. Accordingly, the taxon previously known as S. crispum has been transferred to a new genus Heteroscytonema gen nov., as H. crispum. Some strains of H. crispum exhibited facultative production of paralytic shellfish toxins (PSTs). The concentration of PSTs produced by individual strains varied widely, from 2.7 µg g-1 to 171.3 µg g-1, and included C toxins, decarbamoyl saxitoxin (dcSTX), gonyautoxins (GTX2, GTX3 and GTX5), saxitoxin (STX) and uncharacterised PSTs. The majority of the Australian strains produced dcSTX as the dominant saxitoxin analogue, a significant finding given that dcSTX has approximately half the relative toxicity of STX. The PST profile varied within and between Australian strains of H. crispum and in strains collected from New Zealand and the United States. The sxtA gene, one of the determinants for the production of PSTs, was present in all strains in which PSTs were detected. The discovery of PST-producing H. crispum in the headwaters of a major drinking water reservoir presents a serious risk for potential human and animal exposure to these neurotoxic compounds and further highlights the importance of monitoring benthic cyanobacteria populations for potentially toxigenic species.


Assuntos
Toxinas Bacterianas/metabolismo , Cianobactérias/classificação , Toxinas Marinhas/metabolismo , Microcistinas/metabolismo , Filogenia , Biodiversidade , Cianobactérias/metabolismo , Queensland , RNA Ribossômico 16S/química , Saxitoxina/análise , Saxitoxina/química , Análise de Sequência de DNA
9.
ACS Chem Biol ; 13(11): 3107-3114, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30296060

RESUMO

The neurotoxin saxitoxin and related paralytic shellfish toxins are produced by multiple species of cyanobacteria and dinoflagellates. This study investigates the two saxitoxin-producing strains of Scytonema crispum, CAWBG524 and CAWBG72, isolated in New Zealand. Each strain was previously reported to have a distinct paralytic shellfish toxin profile, a rare observation between strains within the same species. Sequencing of the saxitoxin biosynthetic clusters ( sxt) from S. crispum CAWBG524 and S. crispum CAWBG72 revealed the largest sxt gene clusters described to date. The distinct toxin profiles of each strain were correlated to genetic differences in sxt tailoring enzymes, specifically the open-reading frame disruption of the N-21 sulfotransferase sxtN, adenylylsulfate kinase sxtO, and the C-11 dioxygenase sxtDIOX within S. crispum CAWBG524 via genetic insertions. Heterologous overexpression of SxtN allowed for the proposal of saxitoxin and 3'-phosphoadenosine 5'-phosphosulfate as substrate and cofactor, respectively, using florescence binding assays. Further, catalytic activity of SxtN was confirmed by the in vitro conversion of saxitoxin to the N-21 sulfonated analog gonyautoxin 5, making this the first known report to biochemically confirm the function of a sxt tailoring enzyme. Further, SxtN could not convert neosaxitoxin to its N-21 sulfonated analog gonyautoxin 6, indicating paralytic shellfish toxin biosynthesis most likely occurs along a predefined route. In this study, we identified key steps toward the biosynthetic conversation of saxitoxin to other paralytic shellfish toxins.


Assuntos
Família Multigênica , Neurotoxinas/classificação , Neurotoxinas/genética , Saxitoxina/classificação , Saxitoxina/genética , Cianobactérias/genética , Dioxigenases/genética , Genes Bacterianos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Neurotoxinas/química , Fosfoadenosina Fosfossulfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Filogenia , Ligação Proteica , Saxitoxina/análogos & derivados , Saxitoxina/síntese química , Saxitoxina/química , Sulfotransferases/química , Sulfotransferases/genética , Sulfotransferases/metabolismo , Transposases/genética
10.
J Agric Food Chem ; 66(37): 9801-9809, 2018 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-30153406

RESUMO

In this study, a novel magnetic separation-based multiple systematic evolution of ligands by exponential enrichment (SELEX) was applied to select aptamers simultaneously against three kinds of marine biotoxins, including domoic acid (DA), saxitoxin (STX), and tetrodotoxin (TTX). Magnetic reduced graphene oxide (MRGO) was prepared to adsorb unbound ssDNAs and simplify the separation step. In the multiple SELEX, after the initial twelve rounds of selection against mixed targets and the subsequent four respective rounds of selection against each single target, the three resulting ssDNA pools were cloned, sequenced, and analyzed. Several aptamer candidates were selected and subjected to the binding affinity and specificity test. Finally, DA-06 ( Kd = 62.07 ± 19.97 nM), TTX-07 ( Kd = 44.12 ± 15.38 nM), and STX-41 ( Kd = 61.44 ± 23.18 nM) showed high affinity and good specificity for DA, TTX, and STX, respectively. They were also applied to detect and quantify DA, TTX, and STX successfully. The other two multitarget aptamers, DA-01 and TTX-27, were also obtained, which can bind with either DA or TTX. These aptamers provide alternative recognition molecules to antibodies for biosensor applications.


Assuntos
Ácido Caínico/análogos & derivados , Magnetismo/métodos , Toxinas Marinhas/isolamento & purificação , Técnica de Seleção de Aptâmeros/métodos , Saxitoxina/isolamento & purificação , Tetrodotoxina/isolamento & purificação , Aptâmeros de Nucleotídeos/química , Grafite/química , Ácido Caínico/química , Ácido Caínico/isolamento & purificação , Cinética , Magnetismo/instrumentação , Toxinas Marinhas/química , Óxidos/química , Técnica de Seleção de Aptâmeros/instrumentação , Saxitoxina/química , Tetrodotoxina/química
11.
Science ; 362(6412)2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30049784

RESUMO

Animal toxins that modulate the activity of voltage-gated sodium (Nav) channels are broadly divided into two categories-pore blockers and gating modifiers. The pore blockers tetrodotoxin (TTX) and saxitoxin (STX) are responsible for puffer fish and shellfish poisoning in humans, respectively. Here, we present structures of the insect Nav channel NavPaS bound to a gating modifier toxin Dc1a at 2.8 angstrom-resolution and in the presence of TTX or STX at 2.6-Å and 3.2-Å resolution, respectively. Dc1a inserts into the cleft between VSDII and the pore of NavPaS, making key contacts with both domains. The structures with bound TTX or STX reveal the molecular details for the specific blockade of Na+ access to the selectivity filter from the extracellular side by these guanidinium toxins. The structures shed light on structure-based development of Nav channel drugs.


Assuntos
Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/química , Venenos de Aranha/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Canais de Sódio Disparados por Voltagem/química , Sequência de Aminoácidos , Animais , Microscopia Crioeletrônica , Proteínas de Insetos/ultraestrutura , Ativação do Canal Iônico/efeitos dos fármacos , Periplaneta , Domínios Proteicos , Saxitoxina/química , Tetrodotoxina/química , Canais de Sódio Disparados por Voltagem/ultraestrutura
12.
Spectrochim Acta A Mol Biomol Spectrosc ; 204: 180-187, 2018 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-29933153

RESUMO

Aptamers could be used to construct simple and effective biosensor because the conformational switch of aptamer upon target binding is easy to be transferred to optical or electrochemical signals. Nevertheless, we found that the binding between saxitoxin (STX) and aptamer (M-30f) is not accompanied with conformational switch. Here, the circular dichroism spectra, fluorophore and quencher labeled aptamer, and crystal violet-based assays were used to identify the binding way between STX and aptamer. The results show that the conformation of aptamer is stabilized in PBS buffer (10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, pH 7.4) and this conformation may provide an exactly suitable cave for STX binding. Through the analysis of UV-melting curves and circular dichroism-melting curves, it is found that different concentrations of STX produce different unfolding extents of the aptamer under high temperature. Then, a simple temperature-assisted "turn-on" fluorescent aptasensor was developed to detect STX and the application in real sample detection demonstrates its feasibility. The proposed method provides not only an alternative for STX detection but also a strategy for simple aptasensor design using aptamers that do not switch conformation upon targets binding.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/metabolismo , Saxitoxina/análise , Saxitoxina/metabolismo , Aptâmeros de Nucleotídeos/química , Corantes Fluorescentes/química , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Saxitoxina/química
13.
J Am Chem Soc ; 140(7): 2430-2433, 2018 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-29390180

RESUMO

Like many complex natural products, the intricate architecture of saxitoxin (STX) has hindered full exploration of this scaffold's utility as a tool for studying voltage-gated sodium ion channels and as a pharmaceutical agent. Established chemical strategies can provide access to the natural product; however, a chemoenzymatic route to saxitoxin that could provide expedited access to related compounds has not been devised. The first step toward realizing a chemoenzymatic approach toward this class of molecules is the elucidation of the saxitoxin biosynthetic pathway. To date, a biochemical link between STX and its putative biosynthetic enzymes has not been demonstrated. Herein, we report the first biochemical characterization of any enzyme involved in STX biosynthesis. Specifically, the chemical functions of a polyketide-like synthase, SxtA, from the cyanobacteria Cylindrospermopsis raciborskii T3 are elucidated. This unique megasynthase is comprised of four domains: methyltransferase (MT), GCN5-related N-acetyltransferase (GNAT), acyl carrier protein (ACP), and the first example of an 8-amino-7-oxononanoate synthase (AONS) associated with a multidomain synthase. We have established that this single polypeptide carries out the formation of two carbon-carbon bonds, two decarboxylation events and a stereospecific protonation to afford the linear biosynthetic precursor to STX (4). The synthetic utility of the SxtA AONS is demonstrated by the synthesis of a suite of α-amino ketones from the corresponding α-amino acid in a single step.


Assuntos
Cylindrospermopsis/enzimologia , Policetídeo Sintases/metabolismo , Saxitoxina/biossíntese , Estrutura Molecular , Policetídeo Sintases/química , Saxitoxina/química
14.
Talanta ; 181: 380-384, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29426529

RESUMO

Potentiometric chemical sensors for the detection of paralytic shellfish toxins have been developed. Four toxins typically encountered in Portuguese waters, namely saxitoxin, decarbamoyl saxitoxin, gonyautoxin GTX5 and C1&C2, were selected for the study. A series of miniaturized sensors with solid inner contact and plasticized polyvinylchloride membranes containing ionophores, nine compositions in total, were prepared and their characteristics evaluated. Sensors displayed cross-sensitivity to four studied toxins, i.e. response to several toxins together with low selectivity. High selectivity towards paralytic shellfish toxins was observed in the presence of inorganic cations with selectivity coefficients ranging from 0.04 to 0.001 for Na+ and K+ and 3.6*10-4 to 3.4*10-5 for Ca2+. Detection limits were in the range from 0.25 to 0.9 µmolL-1 for saxitoxin and decarbamoyl saxitoxin, and from 0.08 to 1.8 µmolL-1 for GTX5 and C1&C2, which allows toxin detection at the concentration levels corresponding to the legal limits. Characteristics of the developed sensors allow their use in the electronic tongue multisensor system for simultaneous quantification of paralytic shellfish toxins.


Assuntos
Técnicas Biossensoriais/métodos , Potenciometria/métodos , Saxitoxina/análogos & derivados , Saxitoxina/análise , Animais , Portugal , Reprodutibilidade dos Testes , Saxitoxina/química , Frutos do Mar/análise
15.
Bioanalysis ; 10(4): 229-239, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29333869

RESUMO

AIM: An immunomagnetic capture protocol for use with LC-MS was developed for the quantitation of saxitoxin (STX) in human urine. MATERIALS & METHODS: This method uses monoclonal antibodies coupled to magnetic beads. STX was certified reference material grade from National Research Council, Canada. Analysis was carried out using LC-MS. RESULTS: With an extraction efficiency of 80%, accuracy and precision of 93.0-100.2% and 5.3-12.6%, respectively, and a dynamic range of 1.00-100 ng/ml, the method is well suited to quantify STX exposures based on previously reported cases. CONCLUSION: Compared with our previously published protocols, this method has improved selectivity, a fivefold increase in sensitivity and uses only a third of the sample volume. This method can diagnose future toxin exposures and may complement the shellfish monitoring programs worldwide.


Assuntos
Cromatografia Líquida/métodos , Testes Imunológicos , Saxitoxina/urina , Intoxicação por Frutos do Mar/urina , Espectrometria de Massas em Tandem/métodos , Anticorpos Monoclonais/imunologia , Calibragem , Humanos , Imãs , Microesferas , Padrões de Referência , Saxitoxina/química , Saxitoxina/normas , Intoxicação por Frutos do Mar/diagnóstico , Fatores de Tempo
16.
Artigo em Inglês | MEDLINE | ID: mdl-29195146

RESUMO

Saxitoxin (STX) and neosaxitoxin (NEO) are water-soluble toxins and their cleanup in bio-matrix is a hot topic but difficult problem. A fast and quantitative determination method for STX and NEO in urine was developed using ultra performance liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) based on the cleanup of solid phase extraction (SPE) with hydrophilic interaction (HILIC) mechanism. Acetonitrile/methanol/water mixture was used to extract the toxins in urine. Polyamide (PA) was used as HILIC SPE material to clean the toxins in sample matrix. The limits of detection were 0.2ngmL-1 for STX and 1ngmL-1 for NEO in urine. The linear ranges were 0.5ngmL-1-99.2ngmL-1 with the correlation coefficient of r=0.9992 for STX and 2.1ngmL-1-207ngmL-1 with r=0.997 for NEO in urine matrix. The recoveries at three spiking levels were 81.5%-117% with the relative standard deviations (RSDs) of 5.4%-8.5% for STX and 89.0%-118% with the RSDs of 6.7%-9.1% for NEO. STX was found in all the 6 patients' urines while NEO was only found in one sample from an intoxication case.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Saxitoxina/análogos & derivados , Saxitoxina/urina , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Saxitoxina/química , Saxitoxina/isolamento & purificação
17.
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
18.
J Agric Food Chem ; 65(27): 5494-5502, 2017 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-28616979

RESUMO

A seafood poisoning event occurred in Qinhuangdao, China, in April 2016. Subsequently, the causative mussels (Mytilus galloprovincialis) were harvested and analyzed to reveal a high concentration [∼10 758 µg of saxitoxin (STX) equiv kg-1] of paralytic shellfish toxins (PSTs), including gonyautoxin (GTX)1/4 and GTX2/3, as well as new metabolites 11-hydroxy-STX (M2), 11,11-dihydroxy-STX (M4), open-ring 11,11-dihydroxy-STX (M6), 11-hydroxy-neosaxitoxin (NEO) (M8), and 11,11-dihydroxy-NEO (M10). To understand the origin and biotransformation pathways of these new metabolites, uncontaminated mussels (M. galloprovincialis) were fed with either of two Alexandrium tamarense strains (ATHK and TIO108) under laboratory conditions. Similar PST metabolites were also detected in mussels from both feeding experiments. Results supposed that 11-hydroxy-C2 toxin (M1) and 11,11-dihydroxy-C2 (M3) are transformed from C2, while 11-hydroxy-C4 toxin (M7) and 11,11-dihydroxy-C4 (M9) are converted from C4. In addition, the metabolites M2, M4, and M6 appear to be products of GTX2/3, and the metabolites M8 and M10 are likely derived from GTX1/4.


Assuntos
Bivalves/química , Toxinas Marinhas/metabolismo , Intoxicação por Frutos do Mar/metabolismo , Frutos do Mar/análise , Animais , Biotransformação , Bivalves/metabolismo , Dinoflagelados/metabolismo , Toxinas Marinhas/química , Toxinas Marinhas/toxicidade , Estrutura Molecular , Saxitoxina/análogos & derivados , Saxitoxina/química , Saxitoxina/metabolismo , Frutos do Mar/toxicidade
19.
Angew Chem Int Ed Engl ; 56(19): 5327-5331, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28370934

RESUMO

Saxitoxin (STX) and its analogues are potent voltage-gated sodium channel blockers biosynthesized by freshwater cyanobacteria and marine dinoflagellates. We previously identified genetically predicted biosynthetic intermediates of STX at early stages, Int-A' and Int-C'2, in these microorganisms. However, the mechanism to form the tricyclic skeleton of STX was unknown. To solve this problem, we screened for unidentified intermediates by analyzing the results from previous incorporation experiments with 15 N-labeled Int-C'2. The presence of monohydroxy-Int-C'2 and possibly Int-E' was suggested, and 11-hydroxy-Int-C'2 and Int-E' were identified from synthesized standards and LC-MS. Furthermore, we observed that the hydroxy group at C11 of 11-hydroxy-Int-C'2 was slowly replaced by CD3 O in CD3 OD. Based on this characteristic reactivity, we propose a possible mechanism to form the tricyclic skeleton of STX via a bicyclic intermediate from 11-hydroxy-Int-C'2.


Assuntos
Cianobactérias/metabolismo , Dinoflagelados/metabolismo , Saxitoxina/biossíntese , Bloqueadores do Canal de Sódio Disparado por Voltagem/metabolismo , Conformação Molecular , Saxitoxina/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/química
20.
Toxicon ; 130: 47-55, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28235579

RESUMO

Last decades, cyanobacterial blooms have been commonly reported in Russia. Among the boom-forming species, potential toxin producers have been identified. The aim of this paper was to study the presence of neurotoxic compounds - saxitoxins and anatoxin-a - in water bodies from different regions of Russia. We also made attempts to identify the neurotoxin-producing genera. The good convergence of the results obtained by light microscopy, PCR and LC-MS/MS analyses indicated the presence of active neurotoxin producing species in all investigated water bodies. Saxitoxin was detected in phytoplankton from 4 water bodies in Central European Russia and West Siberia, including lake and reservoirs used as a source for potable water. The water bodies differed with the respect of saxitoxin producers which belonged to Aphanizomenon and/or Dolichospermum genera. For the first time, we obtained quantitative data on the intracellular saxitoxin concentration in Russian freshwaters using LC-MS/MS. Anatoxin-a was detected only in lakes of Northwestern Russia. In the eutrophic shallow Lower Suzdal Lake, Aphanizomenon was the stated anatoxin-a-producing genus. In the large shallow artificial hypertrophic Sestroretskij Razliv Lake, it was very likely that both dominant species - Aphanizomenon flos-aquae and Dolichospermum planctonicum - were anatoxin-a producers.


Assuntos
Aphanizomenon/metabolismo , Cianobactérias/metabolismo , Água Doce/química , Neurotoxinas/metabolismo , Aphanizomenon/genética , Aphanizomenon/isolamento & purificação , Cromatografia Líquida , Cianobactérias/genética , Cianobactérias/isolamento & purificação , Monitoramento Ambiental , Água Doce/microbiologia , Espectrometria de Massas , Neurotoxinas/química , Neurotoxinas/isolamento & purificação , Federação Russa , Saxitoxina/química , Saxitoxina/isolamento & purificação , Saxitoxina/metabolismo , Tropanos/química , Tropanos/isolamento & purificação , Tropanos/metabolismo
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