Method performance verification for the combined detection and quantitation of the marine neurotoxins cyclic imines and brevetoxin shellfish metabolites in mussels (Mytilus edulis) and oysters (Crassostrea gigas) by UHPLC-MS/MS.

A single laboratory method performance verification is reported for a rapid sensitive UHPLC-MS/MS method for the quantification of eight cyclic imine and two brevetoxin analogues in two bivalve shellfish matrices: mussel (Mytilus edulis) and Pacific oyster (Crassostrea gigas). Targeted cyclic imine analogues were from the spirolide, gymnodimine and pinnatoxin groups, namely 20-Me-SPX-C, 13-desMe-SPX-C, 13,19-didesMe-SPX-C, GYM-A, 12-Me-GYM, PnTx-E, PnTx-F and PnTx-G. Brevetoxin analogues consisted of the shellfish metabolites BTX-B5 and S-desoxy-BTX-B2. A rapid dispersive extraction was used as well as a fast six-minute UHPLC-MS/MS analysis. Mobile phase prepared using ammonium fluoride and methanol was optimised for both chromatographic separation and MS/MS response to suit all analytes. Method performance verification checks for both matrices were carried out. Matrix influence was acceptable for the majority of analogues with the MS response for all analogues being linear across an appropriate range of concentrations. In terms of limits of detection and quantitation the method was shown to be highly sensitive when compared with other methods. Acceptable recoveries were found with most analogues, with laboratory precision in terms of intra- and inter-batch precision deemed appropriate. The method was applied to environmental shellfish samples with results showing low concentrations of cyclic imines to be present. The method is fast and highly sensitive for the detection and quantification of all targeted analogues, in both mussel and oyster matrices. Consequently, the method has been shown to provide a useful tool for simultaneous monitoring for the presence or future emergence of these two toxin groups in shellfish.

Synthesis-Driven Stereochemical Assignment of Marine Polycyclic Ether Natural Products.

Marine polycyclic ether natural products have gained significant interest from the chemical community due to their impressively huge molecular architecture and diverse biological functions. The structure assignment of this class of extraordinarily complex natural products has mainly relied on NMR spectroscopic analysis. However, NMR spectroscopic analysis has its own limitations, including configurational assignment of stereogenic centers within conformationally flexible systems. Chemical shift deviation analysis of synthetic model compounds is a reliable means to assign the relative configuration of "difficult" stereogenic centers. The complete configurational assignment must be ultimately established through total synthesis. The aim of this review is to summarize the indispensable role of organic synthesis in stereochemical assignment of marine polycyclic ethers.

Two new epimers of C15-acetogenin, 4-epi-isolaurallene and 4-epi-itomanallene A as diastereomeric model.

Two new C15-acetogenins, 4-epi-isolaurallene (1) and 4-epi-itomanallene A (2) were isolated from a population of marine red alga Laurencia nangii Masuda from Carrington Reef. The structures of these compounds were determined intensively by NMR and HRESIMS data. Their configurations were elucidated by detailed comparison of chemical shifts, germinal protons splitting and NOE correlations with known and synthesized analogues. In addition, antibacterial activities of these compounds were evaluated. These compounds would serve as diastereomeric models for future reference. Since the isolaurallene, neolaurallene, 9-acetoxy-1,10,12-tribromo-4,7:6,13-bisepoxypentadeca-1,2-diene, itomanallene A and laurendecumallene A were isolated, compounds 1 and 2 were the sixth example of C15-acetogenin with dioxabicyclo[7.3.0]dodecene skeleton.

Toxicology of Gambierdiscus spp. (Dinophyceae) from Tropical and Temperate Australian Waters.

Ciguatera Fish Poisoning (CFP) is a human illness caused by the consumption of marine fish contaminated with ciguatoxins (CTX) and possibly maitotoxins (MTX), produced by species from the benthic dinoflagellate genus Gambierdiscus. Here, we describe the identity and toxicology of Gambierdiscus spp. isolated from the tropical and temperate waters of eastern Australia. Based on newly cultured strains, we found that four Gambierdiscus species were present at the tropical location, including G. carpenteri, G. lapillus and two others which were not genetically identical to other currently described species within the genus, and may represent new species. Only G. carpenteri was identified from the temperate location. Using LC-MS/MS analysis we did not find any characterized microalgal CTXs (P-CTX-3B, P-CTX-3C, P-CTX-4A and P-CTX-4B) or MTX-1; however, putative maitotoxin-3 (MTX-3) was detected in all species except for the temperate population of G. carpenteri. Using the Ca2+ influx SH-SY5Y cell Fluorescent Imaging Plate Reader (FLIPR) bioassay we found CTX-like activity in extracts of the unidentified Gambierdiscus strains and trace level activity in strains of G. lapillus. While no detectable CTX-like activity was observed in tropical or temperate strains of G. carpenteri, all species showed strong maitotoxin-like activity. This study, which represents the most comprehensive analyses of the toxicology of Gambierdiscus strains isolated from Australia to date, suggests that CFP in this region may be caused by currently undescribed ciguatoxins and maitotoxins.

Ciguatoxins and Maitotoxins in Extracts of Sixteen Gambierdiscus Isolates and One Fukuyoa Isolate from the South Pacific and Their Toxicity to Mice by Intraperitoneal and Oral Administration.

Ciguatoxins (CTXs), and possibly maitotoxins (MTXs), are responsible for Ciguatera Fish Poisoning, an important health problem for consumers of reef fish (such as inhabitants of islands in the South Pacific Ocean). The habitational range of the Gambierdiscus species is expanding, and new species are being discovered. In order to provide information on the potential health risk of the Gambierdiscus species, and one Fukuyoa species (found in the Cook Islands, the Kermadec Islands, mainland New Zealand, and New South Wales, Australia), 17 microalgae isolates were collected from these areas. Unialgal cultures were grown and extracts of the culture isolates were analysed for CTXs and MTXs by liquid chromatography tandem mass spectrometry (LC-MS/MS), and their toxicity to mice was determined by intraperitoneal and oral administration. An isolate of G. carpenteri contained neither CTXs nor MTXs, while 15 other isolates (including G. australes, G. cheloniae, G. pacificus, G.honu, and F. paulensis) contained only MTX-1 and/or MTX-3. An isolate of G. polynesiensis contained both CTXs and MTX-3. All the extracts were toxic to mice by intraperitoneal injection, but those containing only MTX-1 and/or -3 were much less toxic by oral administration. The extract of G. polynesiensis was highly toxic by both routes of administration.

Rapid Extraction and Identification of Maitotoxin and Ciguatoxin-Like Toxins from Caribbean and Pacific Gambierdiscus Using a New Functional Bioassay.

BACKGROUND: Ciguatera is a circumtropical disease produced by polyether sodium channel toxins (ciguatoxins) that enter the marine food chain and accumulate in otherwise edible fish. Ciguatoxins, as well as potent water-soluble polyethers known as maitotoxins, are produced by certain dinoflagellate species in the genus Gambierdiscus and Fukuyoa spp. in the Pacific but little is known of the potential of related Caribbean species to produce these toxins. METHODS: We established a simplified procedure for extracting polyether toxins from Gambierdiscus and Fukuyoa spp. based on the ciguatoxin rapid extraction method (CREM). Fractionated extracts from identified Pacific and Caribbean isolates were analysed using a functional bioassay that recorded intracellular calcium changes (Ca2+) in response to sample addition in SH-SY5Y cells. Maitotoxin directly elevated Ca2+i, while low levels of ciguatoxin-like toxins were detected using veratridine to enhance responses. RESULTS: We identified significant maitotoxin production in 11 of 12 isolates analysed, with 6 of 12 producing at least two forms of maitotoxin. In contrast, only 2 Caribbean isolates produced detectable levels of ciguatoxin-like activity despite a detection limit of >30 pM. Significant strain-dependent differences in the levels and types of ciguatoxins and maitotoxins produced by the same Gambierdiscus spp. were also identified. CONCLUSIONS: The ability to rapidly identify polyether toxins produced by Gambierdiscus spp. in culture has the potential to distinguish ciguatoxin-producing species prior to large-scale culture and in naturally occurring blooms of Gambierdiscus and Fukuyoa spp. Our results have implications for the evaluation of ciguatera risk associated with Gambierdiscus and related species.

Yessotoxin, a Promising Therapeutic Tool.

Yessotoxin (YTX) is a polyether compound produced by dinoflagellates and accumulated in filter feeding shellfish. No records about human intoxications induced by this compound have been published, however it is considered a toxin. Modifications in second messenger levels, protein levels, immune cells, cytoskeleton or activation of different cellular death types have been published as consequence of YTX exposure. This review summarizes the main intracellular pathways modulated by YTX and their pharmacological and therapeutic implications.

Fenton reaction-based colorimetric immunoassay for sensitive detection of brevetoxin B.

We designed a new colorimetric immunoassay for sensitive monitoring of brevetoxin B (BTB) using enzyme-controlled Fenton reaction with a high-resolution 3,3',5,5'-tetramethylbenzidine (TMB)-based visual colored system. Upon addition of hydrogen peroxide (H2O2), the equivalent iron(II) could be first converted into iron(III) and free hydroxyl radical (•OH) via the classical Fenton reaction. Then the as-produced iron(III) and •OH could cause a perceptible change from colorless to blue with the increasing H2O2 concentration in the presence of TMB. Based on Fenton reaction-triggered visual colored system, a novel competitive-type colorimetric enzyme immunoassay was developed for the quantitative screening of target BTB on the bovine serum albumin-BTB-modified magnetic bead using glucose oxidase/anti-BTB antibody-labeled gold nanoparticle as the signal-transduction tag. Upon target BTB introduction, the analyte competed with the conjugated BTB on the magnetic bead for anti-BTB antibody on gold nanoparticle. The carried glucose oxidase with the gold nanoparticle could implement the oxidation of glucose to produce H2O2, and the generated H2O2 promoted the above-mentioned Fenton reaction for color development. Under the optimal conditions, the absorbance decreased with the increasing target BTB in the range from 0.1 to 150 ng kg(-1) with a low detection limit (LOD) of 0.076 ng kg(-1). The LOD was 500-fold lower than that of commercialized Abraxis BTB ELISA kit. Non-specific adsorption was not observed. The precision, reproducibility and specificity were acceptable. Finally, the method accuracy was also validated for monitoring spiked seafood samples, giving results well matched with the referenced brevetoxin ELISA kit.

Characterization of a Strain of Fukuyoa paulensis (Dinophyceae) from the Western Mediterranean Sea.

A single cell of the dinoflagellate genus Fukuyoa was isolated from the island of Formentera (Balearic Islands, west Mediterranean Sea), cultured, and characterized by morphological and molecular methods and toxin analyses. This is the first report of the Gambierdiscus lineage (genera Fukuyoa and Gambierdiscus) from the western Mediterranean Sea, which is cooler than its eastern basin. Molecular analyses revealed that the Mediterranean strain belongs to F. paulensis and that it bears LSU rDNA sequences identical to New Zealand, Australian, and Brazilian strains. It also shared an identical sequence of the more variable ITS-rDNA with the Brazilian strain. Toxin analyses showed the presence of maitotoxin, 54-deoxyCTX1B, and gambieric acid A. This is the first observation of the two latter compounds in a Fukuyoa strain. Therefore, both Gambierdiscus and Fukuyoa should be considered when as contributing to ciguatera fish poisoning. Different strains of Fukuyoa form a complex of morphologically cryptic lineages where F. paulensis stands as the most distantly related nominal species. The comparison of the ITS2 secondary structures revealed the absence of CBCs among strains. The study of the morphological and molecular traits depicted an unresolved taxonomic scenario impacted by the low strains sampling.

Antimicrobial activity and synergy of antibiotics with two biphenyl compounds, protosappanins A and B from Sappan Lignum against methicillin-resistant Staphylococcus aureus strains.

OBJECTIVES: This study aims to investigate antimicrobial ingredients from Sappan Lignum and to evaluate their synergy on methicillin-resistant Staphylococcus aureus strains with antibiotics. METHODS: Bioactivity-guided phytochemical procedures were used to screen the active compounds. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were assayed by broth microdilution. The synergy was evaluated through checkerboard microdilution and loss of viability assays. KEY FINDINGS: Protosappanins A (PsA) and B (PsB) were identified from Sappan Lignum extracts. They showed active against both S. aureus and MRSA with MIC or MIC50 at 64 (PsA) and 128 (PsB) mg/L alone. When they were used in combination with antibiotics, they showed best synergy with amikacin and gentamicin with MIC50 (mg/L) of amikacin reduced more significantly from 32 to four (with PsA) and eight (with PsB), and the fractional inhibitory concentration index (FICI) ranged between 0.078 and 0.500 (FICI50 = 0.375). Moreover, the resistance of MRSA towards amikacin and gentamicin could be reversed by the Clinical and Laboratory Standards Institute criteria. The combined bactericidal mode could as well be synergy. PsA and PsB showed very low cytotoxicity in comparison with their promising activity against MRSA. CONCLUSIONS: Protosappanins A and B showed both alone activities and resistance reversal effects of amikacin and gentamicin against MRSA, which warrant further investigations for potential combinatory therapy of MRSA infection.

New method for the analysis of lipophilic marine biotoxins in fresh and canned bivalves by liquid chromatography coupled to high resolution mass spectrometry: a quick, easy, cheap, efficient, rugged, safe approach.

A new method for the analysis of lipophilic marine biotoxins (okadaic acid, dinophysistoxins, azaspiracids, pectenotoxins, yessotoxins, spirolids) in fresh and canned bivalves has been developed. A QuEChERS methodology is applied; i.e. the analytes are extracted with acetonitrile and clean-up of the extracts is performed by dispersive solid phase extraction with C18. The extracts are analyzed by ultra-high performance liquid chromatography coupled to a hybrid quadrupole-Orbitrap mass spectrometer, operating in tandem mass spectrometry mode, with resolution set at 70,000 (m/z 200, FWHM). Separation of the analytes, which takes about 10min, is carried out in gradient elution mode with a BEH C18 column and mobile phases based on 6.7mM ammonia aqueous solution and acetonitrile mixtures. For each analyte the molecular ion and 1 or 2 product ions are acquired, with a mass accuracy better than 5ppm. The quantification is performed using surrogate matrix matched standards, with eprinomectin as internal standard. The high-throughput method, which has been successfully validated, fulfills the requirements of European Union legislation, and has been implemented as a routine method in a public health laboratory.

Brevisulcatic acids, marine ladder-frame polyethers from the red tide dinoflagellate Karenia brevisulcata in New Zealand.

The isolation and structural determination of new marine ladder-frame polyethers, brevisulcatic acids-1 (1) and -4 (2) are reported. Brevisulcatic acids were isolated from the dinoflagellate Karenia brevisulcata, which was identified as the causative species of a major red tide event in New Zealand in 1998. The ether ring composition and a ß-hydroxy, γ-methylene valeric acid side chain of 1 and 2 are common, but 2 has a γ-lactone as the 5-membered A-ring while 1 is the seco acid analogue. Compound 2 has structural and bioactivity similarities to brevetoxin A.

Further insights into brevetoxin metabolism by de novo radiolabeling.

The toxic dinoflagellate Karenia brevis, responsible for early harmful algal blooms in the Gulf of Mexico, produces many secondary metabolites, including potent neurotoxins called brevetoxins (PbTx). These compounds have been identified as toxic agents for humans, and they are also responsible for the deaths of several marine organisms. The overall biosynthesis of these highly complex metabolites has not been fully ascertained, even if there is little doubt on a polyketide origin. In addition to gaining some insights into the metabolic events involved in the biosynthesis of these compounds, feeding studies with labeled precursors helps to discriminate between the de novo biosynthesis of toxins and conversion of stored intermediates into final toxic products in the response to environmental stresses. In this context, the use of radiolabeled precursors is well suited as it allows working with the highest sensitive techniques and consequently with a minor amount of cultured dinoflagellates. We were then able to incorporate [U-¹4C]-acetate, the renowned precursor of the polyketide pathway, in several PbTx produced by K. brevis. The specific activities of PbTx-1, -2, -3, and -7, identified by High-Resolution Electrospray Ionization Mass Spectrometer (HRESIMS), were assessed by HPLC-UV and highly sensitive Radio-TLC counting. We demonstrated that working at close to natural concentrations of acetate is a requirement for biosynthetic studies, highlighting the importance of highly sensitive radiolabeling feeding experiments. Quantification of the specific activity of the four, targeted toxins led us to propose that PbTx-1 and PbTx-2 aldehydes originate from oxidation of the primary alcohols of PbTx-7 and PbTx-3, respectively. This approach will open the way for a better comprehension of the metabolic pathways leading to PbTx but also to a better understanding of their regulation by environmental factors.

Graphene based pipette tip solid phase extraction of marine toxins in shellfish muscle followed by UPLC-MS/MS analysis.

Graphene is a novel carbonic material with great potentials for the use as sorbent due to its ultrahigh surface area. Herein, we report the use of graphene as sorbent in solid-phase extraction (SPE) using pipette tip as cartridge namely GPT-SPE, together with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), for the analysis of lipophilic marine toxins (LMTs), including yessotoxins (YTX), okadaic acid (OA), dinophysistoxin-1 (DTX1), gymnodimine (GYM), spirolides-1 (SPX1), pectenotoxin-2 (PTX2) and azaspiracid-1 (AZA1) in shellfish. The GPT-SPE procedure was optimized and the performance of graphene was fully validated. Results with high-sensitivity and good reproducibility was obtained and compared with that of other sorbents like C18 silica, multi-walled carbon nanotubes (MWCNTs), commercial Oasis HLB, and Strata-X for the extraction of LMTs, which showed superiority and advantages of graphene, such as good recoveries, stability and compatibility with various solvents. In order to exhibit the potentials of graphene as an excellent sorbent material, 67 mussel samples from six coastal cities of China were analyzed. OA was found to be the dominant contaminant, while YTX was also detected with low level.

First detection and seasonal variation of lipophilic toxins okadaic acid, dinophysistoxin-1, and yessotoxin in Korean gastropods.

Okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2, and yessotoxin (YTX) are classes of lipophilic toxins found in marine animals. OA and DTX1 accumulation causes diarrhetic shellfish poisoning, a worldwide public health problem. Diarrhetic shellfish poisoning has not previously been reported in gastropods, which are widely consumed in Korea. Seasonal variation in marine lipophilic toxins in gastropods was investigated using liquid chromatography-tandem mass spectrometry. Eighty specimens of Neptunea cumingii, 65 specimens of Rapana venosa, and 95 specimens of Batillus cornutus were collected at the Tongyeong fish market on the southern coast of Korea between May 2009 and December 2010. OA, DTX1, and YTX were detected in meat and digestive glands in all gastropod species studied. Pectenotoxin-2 was not found in any sample tested. Lipophilic toxins were detected in the digestive glands of gastropods; no lipophilic toxin was detected in the salivary glands of the carnivorous gastropods, N. cumingii and R. venosa. The highest concentrations of OA (21.5 ng/g) and DTX1 (8.4 ng/g) were detected in the digestive glands of R. venosa, and the maximum concentration of YTX (13.7 ng/g) was found in the digestive glands of N. cumingii. The maximum toxicities in gastropod tissues were lower than the European standard for acceptable levels. The concentrations of lipophilic toxins in carnivorous gastropods showed a high degree of seasonal variation; lipophilic toxins in carnivorous gastropods were found predominantly in spring and summer. This is the first report of the occurrence of lipophilic toxins in Korean gastropods.

Simultaneous multiplexed stripping voltammetric monitoring of marine toxins in seafood based on distinguishable metal nanocluster-labeled molecular tags.

Marine toxins from microscopic algae can accumulate through the food chain and cause various neurological and gastrointestinal illnesses for human health. Herein, we designed a new ultrasensitive multiplexed immunoassay protocol for simultaneous electrochemical determination of brevetoxin B (BTX-2) and dinophysistoxin-1 (DTX-1) in seafood using distinguishable metal nanocluster-labeled molecular tags as traces on bifunctionalized magnetic capture probes. To construct such a bifunctionalized probe, monoclonal mouse anti-BTX-2 (mAb(1)) and anti-DTX-1 (mAb(2)) antibodies were co-immobilized on a magnetic bead (MB-mAb(1,2)). The distinguishable metal nanoclusters including cadmium nanoclusters (CdNC) and copper nanoclusters (CuNC) were synthesized using the artificial peptides with amino acid sequence CCCYYY, which were used as distinguishable signal tags for the label of the corresponding bovine serum albumin-BTX-2 and bovine serum albumin-DTX-1 conjugates. A competitive-type immunoassay format was adopted for the online simultaneous monitoring of BTX-2 and DTX-1 on a homemade flow-through magnetic detection cell. The assay was based on the stripping voltammetric behaviors of the labeled CdNC and CuNC at the various peak potentials in pH 2.5 HCl containing 0.01 M KCl using square wave anodic stripping voltammetry (SWASV). Under optimal conditions, the multiplexed immunoassays enabled simultaneous detection of BTX-2 and DTX-1 in a single run with wide working ranges of 0.005-5 ng mL(-1) for two marine toxins. The limit of detection (LOD) and limit of quantification (LOQ) were 1.8 and 6.0 pg mL(-1) for BTX-2, while those for DTX-1 were 2.2 and 7.3 pg mL(-1), respectively. No non-specific adsorption and electrochemical cross-talk between neighboring sites were observed during a series of procedures to detect target analytes. The covalent conjugation of biomolecules onto the nanoclusters and magnetic beads resulted in good repeatability and intermediate precision down to 9.5%. The method featured unbiased identification of negative (blank) and positive samples. No significant differences at the 0.05 significance level were encountered in the analysis of 12 spiked samples, including Sinonovacula constricta , Musculista senhousia , and Tegillarca granosa , between the multiplexed immunoassay and commercially available enzyme-linked immunosorbent assay (ELISA) for analysis of BTX-2 and DTX-1.

Artificial ladder-shaped polyethers that inhibit maitotoxin-induced Ca2+ influx in rat glioma C6 cells.

Maitotoxin (MTX) is a ladder-shaped polyether produced by the epiphytic dinoflagellate Gambierdiscus toxicus. It is known to elicit potent toxicity against mammals and induce influx of Ca(2+) into cells. An artificial ladder-shaped polyether possessing a 6/7/6/6/7/6/6 heptacyclic ring system, which was designed for elucidating interactions with transmembrane proteins, was found to be the most potent inhibitor against MTX-induced Ca(2+) influx that has ever been reported.

First report on the detection of pectenotoxin groups in Chinese shellfish by LC-MS/MS.

Chinese shellfish samples were harvested from different locations along the Chinese coast. These shellfish were analyzed by liquid chromatography in combination with mass spectrometry to detect the following toxins: okadaic acid (OA), dinophysistoxins (DTXs), petenotoxins (PTXs), azaspiracids (AZAs), yessotoxins (YTXs), spirlides (SPXs) and gymnodimines (GYM). The results revealed the lipophilic toxin profiles varied with shellfish sampling locations. In addition to OA, GYM and YTX derivatives, PTX-2 and its derivatives were found for the first time in the following Chinese shellfish: Crassostrea gigas, Mactra chinensis and Mytilus galloprovincialis. The presence of GYM, YTXs, OA and PTXs in Chinese shellfish collected from regions where no previous record of DSP-neutral toxic compounds was reported. Serious efforts should therefore be made to conduct a phycotoxin monitoring program to detect the presence of lipophilic toxins in biological materials of marine origin, which may ensure that Chinese seafood products do not present a health risk. With respect to suspected carcinogenicity, further research on the distribution and concentrations of toxic compounds are needed, in order to carry out long-term risk assessments, particularly sub-acute and chronic toxicity tests associated with of lower doses.

The extract of the jellyfish Phyllorhiza punctata promotes neurotoxic effects.

Phyllorhiza punctata (P. punctata) is a jellyfish native to the southwestern Pacific. Herewith we present the biochemical and pharmacological characterization of an extract of the tentacles of P. punctata. The tentacles were subjected to three freeze-thaw cycles, homogenized, ultrafiltered, precipitated, centrifuged and lyophilized to obtain a crude extract (PHY-N). Paralytic shellfish poisoning compounds such as saxitoxin, gonyautoxin-4, tetrodotoxin and brevetoxin-2, as well as several secretory phospholipase A(2) were identified. PHY-N was tested on autonomic and somatic neuromuscular preparations. In mouse vas deferens, PHY-N induced phasic contractions that reached a peak of 234 ± 34.7% of control twitch height, which were blocked with either 100 µ m of phentolamine or 1 m m of lidocaine. In mouse corpora cavernosa, PHY-N evoked a relaxation response, which was blocked with either L-N(G) -Nitroarginine methyl ester (0.5 m m) or 1 m m of lidocaine. PHY-N (1, 3 and 10 µg ml(-1) ) induced an increase in tonus of the biventer-cervicis neuromuscular preparation that was blocked with pre-treatment of galamine (10 µ m). Administration of 6 mg kg(-1) PHY-N intramuscularly produced death in broilers by spastic paralysis. In conclusion, PHY-N induces nerve depolarization and nonspecifically increases neurotransmitter release.

Characterization of tamulamides A and B, polyethers isolated from the marine dinoflagellate Karenia brevis.

Florida red tides occur as the result of blooms of the marine dinoflagellate Karenia brevis. K. brevis is known to produce several families of fused polyether ladder compounds. The most notable compounds are the brevetoxins, potent neurotoxins that activate mammalian sodium channels. Additional fused polyether ladder compounds produced by K. brevis include brevenal, brevisin, and hemibrevetoxin B, all with varying affinities for the same binding site on voltage-sensitive sodium channels. The structure elucidation and biological activity of two additional fused polyether ladder compounds containing seven fused ether rings will be described in this paper. Tamulamide A (MW = 638.30) and tamulamide B (MW = 624.29) were isolated from K. brevis cultures, and their structures elucidated using a combination of NMR spectroscopy and high-resolution mass spectrometry. Tamulamides A and B were both found to compete with tritiated brevetoxin-3 ([(3)H]-PbTx-3) for its binding site on rat brain synaptosomes. However, unlike the brevetoxins, tamulamides A and B showed no toxicity to fish at doses up to 200 nM and did not cause significant bronchoconstriction in sheep pulmonary assays.