Sequencing and phylogenetic analysis of neurotoxin gene from an environmental isolate of Clostridium sp.: comparison with other clostridial neurotoxins

A Clostridium sp. isolated from intestine of decaying fish exhibited 99% sequence identity with C. tetani at 16S rRNA level. It produced a neurotoxin that was neutralized by botulinum antitoxin (A+B+E) as well as tetanus antitoxin. The gene fragments for light chain, C-terminal and N-terminal regions of the heavy chain of the toxin were amplified using three reported primer sets for tetanus neurotoxin (TeNT). The neurotoxin gene fragments were cloned in Escherichia coli and sequenced. The sequences obtained exhibited approximately 98, 99 and 98% sequence identity with reported gene sequences of TeNT/LC, TeNT/HC and TeNT/HN, respectively. The phylogenetic interrelationship between the neurotoxin gene of Clostridium sp. with previously reported gene sequences of Clostridium botulinum A to G and C. tetani was examined by analysis of differences in the nucleotide sequences. Six amino acids were substituted at four different positions in the light chain of neurotoxin from the isolate when compared with the reported closest sequence of TeNT. Of these, four were located in the β15 motif at a solvent inaccessible, buried region of the protein molecule. One of these substitutions were on the solvent accessible surface residue of α1 motif, previously shown to have strong sequence conservation. A substitution of two amino acids observed in N-terminal region of heavy chain were buried residues, located in the β21 and β37 motifs showing variability in other related sequences. The C-terminal region responsible for binding to receptor was conserved, showing no changes in the amino acid sequence.Nucleotide sequence data reported are available in the EMBL database under the accession numbers CAI79619, AM076940, and CAI79618.’     

Reductive cleavage of tetanus toxin and botulinum neurotoxin A by the thioredoxin system from brain

Summary Inhibition of neurotransmitter release by tetanus toxin and botulinum neurotoxin A can be mimicked by intracellular application of the corresponding toxin light chains. The aim of this study was to determine whether the two-chain toxins are reduced by brain preparations to yield free light chains which would represent the ultimate toxins.The interchain disulfide of two-chain tetanus toxin was cleaved by rat cortex homogenate fortified with NADPH. Reduction was promoted further by addition of thioredoxin. Thioredoxin reductase was demonstrated in and purified from porcine brain cortex. The thioredoxin system which consisted of purified enzyme, thioredoxin and NADPH reduced both toxins. The resulting light chains appeared homogeneous in SDS gel electrophoresis. The complementary heavy chain of tetanus but not of botulinum toxin migrated in two bands, the faster one with the velocity of heavy chain obtained by chemical reduction. The major, slower form was converted into the faster by chemical but not by enzymatic reduction. Tetanus toxin, whether in its single-chain or two-chain version also occurred in two forms which differed by their electrophoretic mobility. The two forms of single-chain toxin were interconverted by chemical reduction or oxidation but not by the thioredoxin system.It is concluded that a) a thioredoxin system in brain tissue reduces the interchain disulfide of two-chain tetanus toxin and botulinum neurotoxin A, b) tetanus toxin but not botulinum neurotoxin A consists of two electrophoretically distinct forms which differ by the thiol-disulfide status of their heavy chains, c) the disulfide loop within the heavy chain of tetanus toxin is resistant to the thioredoxin system. Send offprint requests to E. Habermann at the above address     

An in vitro assay for detection of tetanus neurotoxin activity: Using antibodies for recognizing the proteolytically generated cleavage product

Tetanus neurotoxin (TeNT¹) is a bacterial protease which specifically cleaves the vesicle protein synaptobrevin-2 (vesicle associated membrane protein-2, VAMP-2). This proteolytic feature of the toxin has been used to develop a sensitive endopeptidase assay for the detection of TeNT activity as an alternative to the in vivo assay for TeNT toxicity. Recombinant synaptobrevin-2 (rSyb2) is immobilized onto a microtiter plate, and the cleavage of immobilized rSyb2 by TeNT is detected with a polyclonal antibody directed against the newly generated C-terminus of the cleavage product. This antibody is shown to be a highly specific tool for detecting rSyb2 proteolysis by TeNT. The method reaches a detection limit of less than 1 pg TeNT/ml. To our knowledge, this is the most sensitive in vitro assay for the detection of TeNT activity, and it is easy to perform. Besides, the assay can also detect the activity of botulinum neurotoxin type B (BoNT/B). The method can be applied to examine the toxicity of TeNT or BoNT/B preparations as well as the influence of chemicals on TeNT and BoNT/B activity. In the future, the assay may also serve as a basis for the replacement of the in vivo safety control of tetanus vaccines.     

Effects of purification on the bioavailability of botulinum neurotoxin type A

Botulinum neurotoxins (BoNTs) are among the most potent biological toxins for humans. They are primarily produced by the gram-positive, anaerobic spore-forming bacterium, Clostridium botulinum. In bacterial cultures, secreted BoNTs are associated with non-toxic accessory proteins forming large complexes. Neurotoxin-associated proteins have been shown to play an important role in the oral toxicity of BoNTs by protecting them from degradation and digestion by gastric acid and enzymes. Most toxicity studies using BoNTs have been performed using highly purified toxin. In this study, the toxicities of purified and crude BoNT/A toxin preparations were compared. Protein components secreted into culture supernatants along with BoNT/A were identified by mass spectrometry and the contribution of extra proteins found in the soluble crude toxin extracts to the toxicity of BoNTs was determined in mouse models of oral and parenteral botulinum intoxication. Analysis of crude toxin composition permitted assessment of the impact of accessory proteins on the oral bioavailability of BoNT/A toxin in food matrices.     

Isolation and characterization of a neutralizing antibody specific to internalization domain of Clostridium botulinum neurotoxin type B.

Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. In this study, a neutralizing mouse monoclonal antibody against botulinum neurotoxin serotype B (BoNT/B), named BTBH-N1, was developed from mice immunized with BoNT/B toxoid without non-toxic components, which are generally associated with the toxin. Western blot analysis, using recombinant toxin fragments containing light (L), N-terminal half of heavy (HN) and C-terminal half of heavy chains, indicated that BTBH-N1 recognizes linear epitopes located on the HN domain. An in vivo neutralization assay with mice, was conducted to characterize the neutralization capacity of the BTBH-N1. Only 10 microg of BTBH-N1 completely neutralized 20 units (1 unit = one 50% lethal dose) of BoNT/B. Even though the Mab (up to 100 microg) failed to protect mice challenged with 100 units, it significantly prolonged the time to death in a dose dependent manner. BTBH-N1, the first neutralizing antibody against BoNT/B, could be further developed as effective biological therapeutics for preventing and treating botulism, as well as other diseases caused by BoNT/B.     

Expression, purification, and efficacy of the type A botulinum neurotoxin catalytic domain fused to two translocation domain variants.

Clostridial neurotoxins are potent inhibitors of synaptic function, with the zinc-dependent proteolytic light chain (LC) portion of the toxin cleaving one of three neural SNARE proteins. In nature, the LC is expressed as a part of a much larger toxin and hemagglutinin complex, protecting it from environmental degradation and preserving its catalytic activity. We developed forms of the LC of type A botulinum neurotoxin (BoNT-A) with parts of the larger toxin gene, for use as reagents in high-throughput assays to screen for potential LC antagonists, to further elucidate the toxin's mechanism of action, and to study immunological responses to the toxin. Three BoNT-A constructs were engineered and expressed: the LC, LC with translocation region (LC+H(n)), and the LC with the belt portion of the translocation region (LC+Belt). Purification was optimized to a two-step process, with relatively high yields of all three constructs obtained. Activity assays showed all three constructs to be active, with the LC being the most active. Immunogenic protection against native BoNT-A toxin challenge was observed for all three constructs, with the best protection observed with the LC+H(n) and LC+Belt proteins.     

Comparison of oral toxicological properties of botulinum neurotoxin serotypes A and B

Botulinum neurotoxins (BoNTs) are among the most potent biological toxins for humans. Of the seven known serotypes (A-G) of BoNT, serotypes A, B and E cause most of the foodborne intoxications in humans. BoNTs in nature are associated with non-toxic accessory proteins known as neurotoxin-associated proteins (NAPs), forming large complexes that have been shown to play important roles in oral toxicity. Using mouse intraperitoneal and oral models of botulism, we determined the dose response to both BoNT/B holotoxin and complex toxins, and compared the toxicities of BoNT/B and BoNT/A complexes. Although serotype A and B complexes have similar NAP composition, BoNT/B formed larger-sized complexes, and was approximately 90 times more lethal in mouse oral intoxications than BoNT/A complexes. When normalized by mean lethal dose, mice orally treated with high doses of BoNT/B complex showed a delayed time-to-death when compared with mice treated with BoNT/A complex. Furthermore, we determined the effect of various food matrices on oral toxicity of BoNT/A and BoNT/B complexes. BoNT/B complexes showed lower oral bioavailability in liquid egg matrices when compared to BoNT/A complexes. In summary, our studies revealed several factors that can either enhance or reduce the toxicity and oral bioavailability of BoNTs. Dissecting the complexities of the different BoNT serotypes and their roles in foodborne botulism will lead to a better understanding of toxin biology and aid future food risk assessments.     

Separation of the components of type A botulinum neurotoxin complex by electrophoresis.

Clostridium botulinum neurotoxins (BoNTs) are the most toxic substances known. They exert potent neuroparalysis on vertebrates. C. botulinum produces seven serotypes of neurotoxin (A-G). BoNT/A, found in bacterial cultures of C. botulinum type A, is produced as a complex with a group of neurotoxin associated proteins (NAPs). Botulinum neurotoxin complex is the only known example of a protein complex where a group of proteins (NAPs) protect another protein (BoNT) against the acidity and proteases of the stomach. Here, we used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for separation and identification of the constituent proteins of BoNT/A complex. A range of homogenous and gradient SDS-PAGE gels was used to resolve the BoNT/A complex. These gels were run under constant voltage and constant current conditions. The molecular weight and relative amount of each protein band were determined. On a 12.5% homogenous SDS-PAGE under reducing conditions, seven protein bands were identified with average molecular weights of 118, 106, 90, 56, 36, 23 and 17 kDa. The relative amounts of these seven proteins were determined densitometrically as 10, 6, 13, 27, 22, 13 and 8%, respectively. The separation and identification of BoNT/A complex will help in understanding the molecular structure and function of BoNT/A NAPs and their interaction with the toxin, in the toxico-infection process of the botulism diseased state. In particular, the stoichiometry of the individual components is established for a typical preparation of BoNT/A complex. Furthermore, the studies reported here identify the most favorable conditions for the baseline resolution of BoNT/A NAPs proteins for other workers in this field.     

Inhibition in vivo of the activity of botulinum neurotoxin A by small molecules selected by virtual screening

To search for small molecular size inhibitors of botulinum neurotoxin A (BoNT/A) endopeptidase activity, we have screened the NCI library containing about 1 million structures against the substrate binding pocket of BoNT/A. Virtual screening (VS) was performed with the software Glide (Grid-based ligand docking energetics) and the findings were confirmed by AutoDock. Ten compounds were found that had favorable energetic and glide criteria and 5 of these compounds were selected for their ability to protect mice in vivo against a lethal dose of BoNT/A. Each compound was incubated at different molar excesses with a lethal dose of the toxin and then the mixture injected intravenously into mice. At 4690 M excess, compounds NSC94520 and NSC99639 protected all (100%) the mice from lethal toxicity. Compounds NSC48461 and NSC627733 gave 75% protection. Compound NSC348884 showed the least inhibition of toxicity allowing only a fraction (25%) of the mice to survive challenge with a lethal dose; and in the case of the mice that did not survive there was a considerable delay of mortality. At 2400 M excess compounds NSC94520 remained fully protective while and NSC99639 afforded 75% protection and at 1200 M excess each of these two compounds gave 50% protection. The two compounds gave no protection at 600 or less molar excess. When each compound was administered intravenously at 4690 M excess at different times (from 1 h to 6 h) after the intravenous injection of the active toxin, none of the compounds was able to protect the animals from toxicity. The findings show the value of VS in identifying potential inhibitors of the toxin for further development and improvement.     

Complete amino acid sequence and phylogenetic analysis of a long-chain neurotoxin from the venom of the African banded water cobra, Boulengerina annulata.

The sequence of a long-chain neurotoxin (71 amino acid residues, 10 half-cystines) from the venom of the African banded water cobra (Boulengerina annulata) was determined by Edman degradation. It exhibits high sequence similarity with long-chain neurotoxins from the venoms of four species of African cobras (genus Naja), which are collectively more similar to the Boulengerina toxin than to those of Asian Naja species. These results are discussed in the light of phylogenetic hypotheses on the relationships of African cobras.     

Detection of the neurotoxin BMAA within cyanobacteria isolated from freshwater in China

The cyanobacterial neurotoxin, β-N-methylamino-l-alanine (BMAA), has been suggested as an important environmental factor for neurodegenerative disease such as amyotrophic lateral sclerosis- Parkinsonism dementia complex (ALS/PDC) in Guam. BMAA was detected within the majority of cyanobacterial isolates surveyed in both free and symbiotic cyanobacteria, living in freshwater as well as marine environments. In this study, we report two methods using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) each coupled with a different type of hydrophilic interaction liquid chromatography (HILIC) column to detect BMAA. A third method using AQC-derivatized BMAA was also used for comparison. Axenic cultures of Microcystis aeruginosa and Nostoc sp. isolated from Chinese freshwater were analyzed for both free and protein-bound BMAA at the exponential growth stage. Cultures of two strains of M. aeruginosa collected at four growth stages were also analyzed for the presence of BMAA. BMAA was detected in the Nostoc sp. at very low concentrations (<0.07 pmoles on column) only when precolumn AQC derivatization was used. No BMAA was detected in the Chinese derived axenic cultures of Microcystis; detection limits for the LC-ESI-MS and LC-ESI-MS/MS without precolumn derivatization were 10 ng and 2 pg BMAA on column, respectively. We suggest that cyanobacteria grown under some culture conditions may be relatively free of BMAA.     

Analysis of the substrate recognition domain determinants of botulinum type B toxin using phage display.

The botulinum neurotoxin endopeptidases appear to recognise their intracellular protein substrates via two distinct sites: the cleavage site sequence and a 'recognition site' motif. In the present study phage display has been employed to generate a library of vesicle-associated membrane protein (VAMP2) variants in which the toxin recognition motif (part of the SNARE motif ELDDRADA) has been modified. VAMP (1-94) was displayed on the surface of M13 bacteriophage and this fragment was recognised and cleaved by botulinum neurotoxin type B (BoNT/B). A phage-displayed library was constructed in which six residues of the recognition domain (VAMP residues 63-68; wild-type sequence LDDRAD) were randomised, and a selection method established for identifying cleaved VAMP variants. Sequence analysis of 24 clones revealed that 5 contained two acidic residues although none corresponded to the native sequence. Cleavage was reduced compared to wild-type VAMP, and cleavage of mutants containing no acidic residues was also observed. The data are discussed in relation to the substrate recognition mechanism of BoNT/B.     

Presence of presynaptic neurotoxin complexes in the venoms of Australo-Papuan death adders (Acanthophis spp.)

Australo-papuan death adders (Acanthophis spp.) are a cause of serious envenomations in Papua New Guinea and northern Australia often resulting in neurotoxic paralysis. Furthermore, victims occasionally present with delayed-onset neurotoxicity that sometimes responds poorly to antivenom or anticholinesterase treatment. This clinical outcome could be explained by the presence of potent snake presynaptic phospholipase A₂ neurotoxin (SPAN) complexes and monomers, in addition to long- and short-chain postsynaptic α-neurotoxins, that bind irreversibly, block neurotransmitter release and result in degeneration of the nerve terminal. The present study therefore aimed to determine within-genus variations in expression of high molecular mass SPAN complexes in the venoms of six major species of Acanthophis, four geographic variants of Acanthophis antarcticus. Venoms were separated by size-exclusion liquid chromatography under non-denaturing conditions and fractions corresponding to proteins in the range of 22 to >60 kDa were subjected to pharmacological characterization using the isolated chick biventer cervicis nerve-muscle (CBCNM) preparation. All venoms, except Acanthophis wellsi and Acanthophis pyrrhus, contained high mass fractions with phospholipase A₂ activity that inhibited twitch contractions of the CBCNM preparation. This inhibition was of slow onset, and responses to exogenous nicotinic agonists were not blocked, consistent with the presence of SPAN complexes. The results of the present study indicate that clinicians may need to be aware of possible prejunctional neurotoxicity following envenomations from A. antarcticus (all geographic variants except perhaps South Australia), Acanthophis praelongus, Acanthophis rugosus and Acanthophis. laevis species, and that early antivenom intervention is important in preventing further development of toxicity.     

Evidence for a link between specific proteolysis and inhibition of [3H]-noradrenaline release by the light chain of tetanus toxin

Summary The light chain of tetanus toxin is known to inhibit the Ca²⁺-evoked release of [³H]-noradrenaline from digitonin-permeabilized bovine adrenomedullary cells in culture but does not change the basal outflow or the total cellular radioactivity. Evidence for the involvement of proteolysis in this effect was obtained by three approaches.First, the permeabilized cells were exposed to a series of enzymes. The endoproteinase Glu-C mimicked the inhibition produced by the light chain. Second, protease inhibitors of different specificities were assessed for blockade of the action of light chain on [3H]-noradrenaline release from permeabilized cells. Blockade was complete with EDTA (2.5 mmol/1) or 1,10-o-phenanthroline (1 mmol/1), and absent with the highest concentrations tested of diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, pepstatin, leupeptin, bestatin, phosphoramidon, thiorphan or trans-epoxysuccinic acid (E64) which is regarded as an inhibitor of thiol proteases. This inhibitor spectrum suggested that light chain might be a metalloprotease. Finally a sequence-His-Glu-Leu-x-Hisoccurring in the light chains of tetanus toxin and of the botulinum neurotoxins A, C, D, E was also found in many endoproteinases and an aminopeptidase. The motif is known to constitute their active site and to bind Zn²⁺. In fact Zn²⁺. (0.6–0.9 mol/mol) was found in thoroughly dialysed two-chain tetanus toxin.The three approaches jointly support the hypothesis that the light chain of tetanus toxin, and probably of all clostridial neurotoxins, inhibits [³H]-noradrenaline release from adrenomedullary cells by degradation of (a) specific, still unknown protein(s) involved in exocytosis. Correspondence to E. Habermann at the above address     

过表达HMGR催化结构域以优化酵母工程菌原人参二醇代谢途径的研究

目的 过表达3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMGR)催化结构域提高酵母工程菌中原人参二醇的产量.方法 分别克隆酿酒酵母中编码3-羟基-3-甲基戊二酸单酰辅酶A还原酶催化结构域的基因tHMG1、人参中达玛烯二醇-Ⅱ合酶基因ds和原人参二醇合酶基因cyp1、拟南芥中CYP450还原酶基因cyp-re,构建相应表达质粒,转化酿酒酵母INVSc1,并检测重组菌中原人参二醇产量.结果 重组菌INVSc1-DS-GFP/CYP1／CYP-Re和INVSc 1-DS-GFP/CYP1/CYP-Re/tHMG1中原人参二醇产量分别为3.04 mg·L-1和26.5 mg·L-1.结论 导入基因tHMG1,使原人参二醇产量提高了8.7倍,该结果为优化酵母工程菌中人参皂苷代谢途径奠定了基础.     

A powder X-ray diffraction method for detection of polyprenylated benzophenones in plant extracts associated with HPLC for quantitative analysis

A robust, direct, rapid and non-destructive X-ray diffraction crystallography method to detect the polyprenylated benzophenones 7-epi-clusianone (1) and guttiferone A (2) in extracts from Garcinia brasiliensis is presented. Powder samples of benzophenones 1 and 2, dried hexane extracts from G. brasiliensis seeds and fruit's pericarp, and the dried ethanolic extract from G. brasiliensis seeds were unambiguously characterized by powder X-ray diffractometry. The calculated X-ray diffraction peaks from crystal structures of analytes 1 and 2, previously determined by single-crystal X-ray diffraction technique, were overlaid to those of the experimental powder diffractograms, providing a practical identification of these compounds in the analyzed material and confirming the pure contents of the powder samples. Using the X-ray diffraction crystallography method, the studied polyprenylated benzophenones were selectively and simultaneously detected in the extracts which were mounted directly on sample holder. In addition, reference materials of the analytes were not required for analyses since the crystal structures of the compounds are known. High performance liquid chromatography analyses also were comparatively carried out to quantify the analytes in the same plant extracts showing to be in agreement with X-ray diffraction crystallography method.     

Structural determinants of efficacy at A3 adenosine receptors: modification of the ribose moiety

We have found previously that structural features of adenosine derivatives, particularly at the N6- and 2-positions of adenine, determine the intrinsic efficacy as A3 adenosine receptor (AR) agonists. Here, we have probed this phenomenon with respect to the ribose moiety using a series of ribose-modified adenosine derivatives, examining binding affinity and activation of the human A3 AR expressed in CHO cells. Both 2'- and 3'-hydroxyl groups in the ribose moiety contribute to A3 AR binding and activation, with 2'-OH being more essential. Thus, the 2'-fluoro substitution eliminated both binding and activation, while a 3'-fluoro substitution led to only a partial reduction of potency and efficacy at the A3 AR. A 5'-uronamide group, known to restore full efficacy in other derivatives, failed to fully overcome the diminished efficacy of 3'-fluoro derivatives. The 4'-thio substitution, which generally enhanced A3 AR potency and selectivity, resulted in 5'-CH2OH analogues (10 and 12) which were partial agonists of the A3 AR. Interestingly, the shifting of the N6-(3-iodobenzyl)adenine moiety from the 1'- to 4'-position had a minor influence on A3 AR selectivity, but transformed 15 into a potent antagonist (16) (Ki = 4.3 nM). Compound 16 antagonized human A3 AR agonist-induced inhibition of cyclic AMP with a K(B) value of 3.0 nM. A novel apio analogue (20) of neplanocin A, was a full A3 AR agonist. The affinities of selected, novel analogues at rat ARs were examined, revealing species differences. In summary, critical structural determinants for human A3 AR activation have been identified, which should prove useful for further understanding the mechanism of receptor activation and development of more potent and selective full agonists, partial agonists and antagonists for A3 ARs.     

Structural basis for the interaction of [E160A-E189A]-trichosanthin with adenine.

Trichosanthin is a ribosome-inactivating protein that cleaves specifically the N-glycosidic bond of A-4324 of 28S rRNA. Trichosanthin and its variant [E160A-E189A]-trichosanthin were found to bind an adenine base with a K(d) value of approximately 0.2mM. To determine how this doubly mutated variant of trichosanthin interacts with adenine, the co-crystal structure of [E160A-E189A]-trichosanthin and adenine was resolved to 0.193nm which revealed that the active site conformation of the doubly mutated variant is isomorphous to wild-type trichosanthin. Water molecules were found at locations corresponding to the eliminated side chain of Glu-160 and Glu-189. On the other hand, the adenine base interacted with [E160A-E189A]-trichosanthin in a manner similar to that in wild-type trichosanthin. Our structural analysis illustrates that Glu-160 and Glu-189 in trichosanthin do not play an important role in maintaining the active site conformation and binding adenine, an essential step for substrate-enzyme interaction. On the other hand, removal of two glutamate residues changed a large patch of negatively charged surface to a positive charge, which may account for the destabilization of the oxocarbenium-like transition-state and the significant decrease in ribosome-inactivating activity in [E160A-E189A]-trichosanthin.     

Accumulation and depuration profiles of PSP toxins in the short-necked clam Tapes japonica fed with the toxic dinoflagellate Alexandrium catenella.

A toxic dinoflagellate responsible for paralytic shellfish poisoning (PSP), Alexandrium catenella (Ac) was fed to the short-necked clam Tapes japonica, and the accumulation and depuration profiles of PSP toxins were investigated by means of high-performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD). The short-necked clams ingested more than 99% of the Ac cells (4 x 10(7)cells) supplied once at the beginning of experiment, and accumulated a maximal amount of toxin (185 nmol/10 clams) after 12h. The rate of toxin accumulation at that time was 23%, which rapidly decreased thereafter. Composition of the PSP toxin accumulated in the clams obviously different from that of Ac even 0.5h after the cell supply, the proportion of C1+2 being much higher than in Ac, although the reason remains to be elucidated. In contrast, a higher ratio of gonyautoxin (GTX)1+4 than in Ac was detected in the toxin profiles of clam excrements. The variation in toxin composition derived presumably from the transformation of toxin analogues in clams was observed from 0.5h, such as reversal of the ratio of C1 to C2, and appearance of carbamate (saxitoxin (STX), neoSTX and GTX2, 3) and decarbamoyl (dc) derivatives (dcSTX and dcGTX2, 3), which were undetectable in Ac cells. The total amount of toxin distributed over Ac cells, clams and their excrements gradually declined, and only 1% of supplied toxin was detected at the end of experiment.     

Identification and quantification of tetrodotoxin in the marine gastropod Nassarius by LC-MS.

Tetrodotoxin (TTX) has been usually analyzed in marine and other sources by HPLC separation, followed by alkaline treatment and fluorescence assay. However, the applications of such methods were limited because of their tedious extraction and derivation procedures. A simpler, rapid LC-MS method, using selected ion-monitoring, has now been developed, and used to identify and quantitate TTX in a marine gastropod of the genus Nassarius, collected from Fijian province of China. The toxin was confirmed comparable to authentic TTX by MS and MSn. In addition, a moderate amount of TTX was detected in the Nassarius by this novel approach, suggesting that Nassarius would be a potential food poison source.