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PubMed2001

Microbial reclamation of shellfish wastes for the production of chitinases.

Shrimp and crab shell powder (SCSP), prepared by treating shellfish processing waste with boiling and crushing, was used as a substrate for isolating chitinolytic microorganisms. Three potential strains (E1, J1, and J1-1) were isolated and identified as Bacillus cereus, B. alvei, and B. sphaericus, respectively. Three extracellular chitinases (FB1, FB2, and FB3) were purified from the culture supernatants of Bacillus cereus E1, B. alvei J1, and B. sphaericus J1-1, respectively. The molecular weights of FB1, FB2, and FB3 were 71,000, 71,000, and 65,000, respectively, by SDS-PAGE. The pIs for FB1, FB2, and FB3 were 7.1, 7.2, and 7.4, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of FB1 were pH 9, 50 degrees C, pH 7 to 10, and 70 degrees C; those of FB2 were pH 9, 60 degrees C, pH 5 to 9, and 70 degrees C; and those of FB3 were pH 7, 50 degrees C, pH 5 to 9, and 60 degrees C. The activities of all enzymes were strongly inhibited by Hg(2+) and completely inhibited by glutathione, dithiothreitol, and 2-mercaptoethanol.

Journal Article↗
PubMed2001

The purine degradation pathway: possible role in paralytic shellfish toxin metabolism in the cyanobacterium Planktothrix sp. FP1.

The paralytic shellfish toxins (PSTs) are potent neurotoxic alkaloids and their major biological effect is due to the blockage of voltage-gated sodium channels in excitable cells. They have been recognised as an important health risk for humans, animals, and ecosystems worldwide. The metabolic pathways that lead to the production and the degradation of these toxic metabolites are still unknown. In this study, we investigated the possible link between PST accumulation and the activation of the metabolism that leads to purine degradation in the filamentous freshwater cyanobacterium Planktothrix sp. FP1. The purine catabolic pathway is related to the nitrogen microcycle in water environments, in which cyanobacteria use traces of purines and ureides as a nitrogen source for growth. Thus, the activity of allantoicase, a key inducible enzyme of this metabolism, was used as tool for assaying the activation of the purine degradation pathway. The enzyme and the pathway were induced by allantoic acid, the direct substrate of allantoicase, as well as by adenine and, to a lower degree, by urea, one of the main products of purine catabolism. Crude cell extract of Escherichia coli was also employed and showed the best induction of allantoicase activity. In culture, Planktothrix sp. FP1 showed a differential accumulation of PST in consequence of the induction with different substrates. The cyanobacterial culture induced with allantoic acid accumulated 61.7% more toxins in comparison with the control. On the other hand, the cultures induced with adenine, urea, and the E. coli extract showed low PST accumulation, respectively, 1%, 38%, and 5% of the total toxins content detected in the noninduced culture. A degradation pathway for the PSTs can be hypothesised: as suggested for purine alkaloids in higher plants, saxitoxin (STX) and derivatives may also be converted into xanthine, urea, and further to CO2 and NH4+ or recycled in the primary metabolism through the purine degradation pathway.

Cyanobacteria↗
PubMed2003

Saccharification of chitin using solid-state culture of Aspergillus sp. S1-13 with shellfish waste as a substrate.

Saccharification of chitin was performed in a suspension (mash) of a solid-state culture of chitinase-producing Aspergillus sp. Sl-13 with acid-treated shellfish waste as a substrate. The conditions for the saccharifying reaction and the solid-state cultivation were examined from the viewpoint of saccharification in the mash. Optimum cultivation conditions were defined: a solid-state medium consisting of 5 g of 10% lactic acid-treated crab shells (0.50-2.36 mm in size) and 3 ml of a basal medium (0.028% KH2PO4 0.007% CaCl2.2H2O, and 0.025% MgSO4.7H2O) supplemented with 0.3% peptone was inoculated with 4 ml of spore suspension (1 x 10(7) spores/ml), and the water content of the medium was adjusted to 75%; static cultivation at 37 degrees C for 7 d. When a culture obtained under the optimum conditions was suspended in 70 ml of 50 mM sodium phosphate-citrate buffer (pH 4.0) and incubated at 45 degrees C for 11-13 d, 55 mM N-acetylglucosamine (GlcNAc) was formed in the solid-state culture mash, indicating that at least 33% of the initial chitin in the solid material was hydrolyzed. Through the experiments, the amounts of G1cNAc formed in the solid-state culture mash varied in a way similar to that of the water-extractable pnitrophenyl beta-D-N-acetylglucosaminide-hydrolyzing enzyme in the culture, but not to that of the colloidal chitin-hydrolyzing enzyme. G1cNAc-assimilating lactic acid bacteria, which were inoculated into the mash after or at the start of the saccharification, formed lactic acid with decreasing GlcNAc.

Journal Article↗
PubMed2003

[Microphytobenthos assemblage mapping by spatial visible-infrared remote sensing in a shellfish ecosystem].

The aim of this work is to assess the use of (SPOT) multispectral visible infrared remote sensing to study microphytobentos assemblages in a shellfish ecosystem (Bay of Bourgneuf, France). SPOT satellite images (acquired at low tide in spring or autumn between 1986 and 1998) were calibrated using in situ radiometric data, and the normalised vegetation index (NDVI) obtained from these images showed microphytobenthos on bay mudflats. Proliferation was mainly along a north-south strip, essentially localised around the +2 m isobath and covering a surface area of 19 to 25% of the total mudflat area studied (420 to 550 ha). Three factors seem to be responsible for the spatial structure of the assemblages: bathymetry, nutrient input from the Falleron River and its channel, and the location of oyster-farming areas. Although spatial and spectral resolutions of multispectral remote sensing data have certain limitations, this approach opens up a new field of application for hyperspectral remote sensing, particularly for synoptic mapping of biomass distribution.

Animals↗
PubMed2002

Structure and stereochemistry of a new cytotoxic polychlorinated sulfolipid from Adriatic shellfish.

A detailed analysis of the causative toxins contained in the hepatopancreas of toxic mussels from the northern Adriatic sea has been carried out. Along with some DSP (diarrhetic shellfish poisoning) type toxins, such as okadaic acid, yessotoxin, and their derivatives, which are involved in a number of human intoxications throughout the world, we have now isolated a new cytotoxin, a polychlorinated sulfolipid 1, whose gross structure has been elucidated by spectral analysis, including various 2D NMR techniques. The relative stereochemistry of 1 was elucidated by successful application of the J-based configuration analysis developed for acyclic compounds using carbon-proton spin-coupling constants ((2,3)J(C,H)) and proton-proton spin-coupling constants ((3)J(H,H)); its absolute stereochemistry was established by the Mosher method. Compound 1 possesses in vitro cytotoxicity against WEHI 164 and RAW 264.7 cells.

Animals↗
PubMed2002

The detection and identification of 42,43,44,45,46,47,55-heptanor-41-oxoyessotoxin, a new marine toxin from adriatic shellfish, by liquid chromatography-mass spectrometry.

The diarrhetic shellfish toxin composition in the digestive glands of mussels collected in June 2001 from the Northern Adriatic sea was investigated by high-performance liquid chromatography coupled with electrospray ion trap mass spectrometry. Along with known yessotoxins (1, 3-6), identified by comparison of their retention times and mass spectra with those of appropriate standards, a new marine toxin, 42,43,44,45,46,47,55-heptanor-41-oxoyessotoxin, 7, was detected. MS/MS experiments were used to gain structural information. 7 represents a new addition to the class of yessotoxins.

Animals↗
PubMed2004

A novel pectenotoxin, PTX-12, in Dinophysis spp. and shellfish from Norway.

Two novel pectenotoxins (PTXs) were detected by LC-MS in solid phase extracts of net hauls taken at Flødevigen, Norway, in June 2002 that were dominated by Dinophysis acuminata and Dinophysis norvegica. The new compounds were isolated as minor components from a large collection of a Dinophysis acuta-dominated bloom obtained from Skjer, Sognefjorden, Norway, in October 2002. LC-MS and NMR analyses revealed that the new components, 36S-PTX-12 and 36R-PTX-12, occurred as a pair of equilibrating diastereoisomers differing from PTX-2 in that they contained an exocylic olefinic methylene rather than a methyl group at C-38. Analyses of shellfish extracts revealed that PTX-12 accumulated in Norwegian blue mussels (Mytilus edulis) and cockles (Cerastoderma edule), along with PTX-12 seco acids occurring as a complex mixture of diastereoisomers. LC-MS analysis of algal cells picked from the net haul from Flødevigen revealed that PTX-12 predominated in D. acuta and D. norvegica, whereas PTX-2 was the predominant pectenotoxin in D. acuminata. Preliminary observations indicate that the relative contents of PTX-2 and PTX-12 vary between sites and years in Norway, even within a single species of Dinophysis. Our data also suggest that heterotrophic dinoflagellates may accumulate toxins from their prey.

Animals↗
PubMed1992

Neural and behavioural effects of domoic acid, an amnesic shellfish toxin, in the rat.

To examine the neurotoxic effects of domoic acid, an amnesic shellfish toxin, electroencephalographic and behavioural experiments were conducted on 38 rats. Injection of domoic acid (0.5-1.0 mg/kg intravenously, or 0.04-0.08 microgram intraventricularly) caused seizure discharges in the hippocampus, tonic-clonic convulsions, and death within a few days. Convulsions and ensuing death were prevented by diazepam. Animals pretreated with diazepam (5 mg/kg, ip) tolerated intraventricular dose of domoic acid 0.4 microgram, but showed a loss of pyramidal neurons mainly in the CA3, CA4, and a part of CA1 areas of the dorsal hippocampus. Learning of a radial maze task was severely impaired in naive rats after intraventricular injection of domoic acid (and diazepam, ip). In the animals previously trained on the maze task, domoic acid interfered with relearning of the same task. These effects appear similar to those of kainic acid and are analogous to the symptoms observed in humans who ingested mussels tainted with domoic acid.

Animals↗
PubMed1997

Bacterial influence on the production of paralytic shellfish toxins by dinoflagellated algae.

This study investigated the role of intracellular and extracellular bacteria in the production of paralytic shellfish toxins by dinoflagellated algal cells. Three strains of the toxic dinoflagellate species, Alexandrium tamarense, were purified by external bacteria using penicillin G (Pen. G) at levels of 500 and 1000 p.p.m. Levels of toxicity of the resulting purified dinoflagellate cultures were similar to those of the original strains contaminated with external bacteria, indicating that the external bacteria had no influence on toxicity. No bacterial colony forming units (cfu) arose from disruption of algal cells derived from penicillin-treated cultures, indicating that intracellular bacteria were not responsible for the toxicity of cultures.

Animals↗
PubMed1996

Inhibition of protein synthesis in a cell-free system and Vero cells by okadaic acid, a diarrhetic shellfish toxin.

Okadaic acid, a diarrhetic shellfish toxin, is a potent promoter of tumors in mouse skin and a specific inhibitor of protein phosphatases 1 and 2A. In the present study, we investigated its effects on protein synthesis in Vero cells and rabbit reticulocyte lysate. Protein synthesis was inhibited by okadaic acid in Vero cells in a concentration-dependent manner (IC50 = 3.3 x 10(8) M-1). DNA synthesis was also inhibited by okadaic acid in Vero cells in a concentration-dependent manner (IC50 = 5.3 x 10(8) M-1). DNA synthesis inhibition in Vero cells occurred only after 8 h or longer. RNA synthesis was inhibited with an IC50 of 8.2 x 10(8) M-1. The time lag before DNA and RNA synthesis inhibition occurred was longer than the time lag before protein synthesis inhibition occurred in the same cells (4 h), indicating that protein synthesis is probably the main target and the first of okadaic acid's cytotoxic effects. Moreover, the inhibition of DNA and RNA synthesis is probably a consequence of the inhibition of protein synthesis. Since okadaic acid does not impair the uptake of the precursor of protein synthesis, it is assumed that the inhibition is due to a direct effect on one of the components of the protein synthesis machinery. We then used a cell-free system of rabbit reticulocyte lysate in which specific mRNA is translated into globin to ensure that protein synthesis is a direct target of okadaic acid in vitro. In rabbit reticulocyte lysate, okadaic acid inhibited protein synthesis in a concentration-dependent manner (IC50 = 6.3 x 10(12) M-1) with a correlation coefficient for percent inhibition values of r = .918. The molecular target of okadaic acid inside the cell whereby protein synthesis is inhibited remains to be discovered.

Animals↗
PubMed1999

Use of immunoaffinity columns for clean-up of diarrhetic toxins (okadaic acid and dinophysistoxins) extracts from shellfish prior to their analysis by HPLC/fluorimetry.

Diarrhetic Shellfish Poisoning (DSP) is a severe gastro-intestinal disease caused by consumption of seafood contaminated by microalgal toxins, mainly okadaic acid (OA) and structurally related toxins, dinophysistoxins (DTXs). Regulatory monitoring is generally based on rodent bioassays which, however, present some technical and ethical disadvantages. The most promising technique of analysis of these toxins involves an HPLC separation with spectrofluorimetric detection after derivatization of the toxins with a fluorescent reagent. The lack of specificity of the extraction procedure (liquid-liquid partition), and the presence of interfering compounds in the matrix, does not allow the determination and the quantification of low amounts of toxins in seafood. In this paper, the authors report the development and the characterization of immunoaffinity columns (IAC), which were elaborated using anti-okadaic acid monoclonal antibodies, for a specific retention of the OA group of toxins. The coupling yield and the stability of these columns were investigated as well as their capacity to remove interfering compounds. Cross-reactivity was observed between the antibodies and the DTX-1 and the DTX-2, allowing the detection of the different toxins in a single analysis. Different spiked (1 microgram OA/g) or naturally-contaminated (mussel digestive gland: 2 micrograms OA/g; algae: 165 micrograms OA/g) matrices were tested. The recovery for OA varied from 55 to 95% according to the matrices. The IAC purification was then included as a step of a global [IAC/HPLC/spectrofluorimetric detection] method and the performance of the method was evaluated. Estimations of the linearity and the accuracy (percentages of the presumptive response for OA in the range +101% to +114%) were satisfactory in accordance with the method validation criteria.

Animals↗
PubMed2001

Fish and shellfish consumption in relation to death from myocardial infarction among men in Shanghai, China.

Between 1986 and 1989, 18,244 men aged 45-64 years in Shanghai, China, participated in a prospective study of diet and cancer. All participants completed an in-person, structured interview and provided blood and urine samples. As of September 1, 1998, 113 deaths from acute myocardial infarction were identified. After analyses were adjusted for age, total energy intake, and known cardiovascular disease risk factors, men who consumed >or=200 g of fish/shellfish per week had a relative risk of 0.41 (95% confidence interval: 0.22, 0.78) for fatal acute myocardial infarction compared with men consuming <50 g per week. Similarly, dietary intake of n-3 fatty acids derived from seafood also was significantly associated with reduced mortality from myocardial infarction. Neither dietary seafood nor n-3 fatty acid intake was associated with a reduced risk of death from stroke or ischemic heart disease other than acute myocardial infarction. However, approximately a 20% reduction in total mortality associated with weekly fish/shellfish intake was observed in the study population (relative risk = 0.79, 95% confidence interval: 0.69, 0.91). These prospective data suggest that eating fish and shellfish weekly reduces the risk of fatal myocardial infarction in middle-aged and older men in Shanghai, China.

Analysis of Variance↗
PubMed1982

Purification and characterization of a lectin from the shellfish, Saxidomus purpuratus.

A lectin was purified from a shellfish, Saxidomus purpuratus, using ion-exchange chromatography on DEAE-cellulose and affinity chromatography on N-acetylglucosamine-Sepharose. The lectin purified by affinity chromatography showed seven protein bands in polyacrylamide gel electrophoresis. The two major lectins (SPA-I and SPA-III) were purified by a second DEAE-cellulose column chromatography. The molecular weights of the lectins were almost the same and were estimated to be around 40,000 by gel filtration on a Sepharose 6B column. On SDS-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol, the lectins showed molecular weights of 14,000. The isoelectric points of SPA-I and -III were estimated to be 4.4 and 4.1, respectively. The two lectins (SPA-I and -III) differed slightly in amino acid composition and were glycoproteins containing 2.1 and 3.8 mol of GlcNAc per 40,000 g of the lectin, respectively. The binding constant of SPA-I or SPA-III for methyl N-acetyl-a-D-glucosamide, the strongest inhibitor of hemagglutination in this experiment, was estimated to be 1.3 X 10(3) or 4.2 X 10(4) M-1, respectively, by the UV difference spectroscopy method.

Amino Acids↗
PubMed2006

Marinobacter algicola sp. nov., isolated from laboratory cultures of paralytic shellfish toxin-producing dinoflagellates.

Phylogenetic and phenotypic analysis of cultivable marine bacteria isolated from laboratory cultures of two paralytic shellfish toxin-producing dinoflagellates, Gymnodinium catenatum and Alexandrium tamarense, showed the presence of a novel group of Gram-negative, aerobic, moderately halophilic and hydrocarbon-degrading bacteria, related to the genus Marinobacter. The strains, designated DG893T, DG1136 and ATAM407-13, grew optimally in media with 3-6 % NaCl and at 25-30 degrees C, and all could utilize n-hexadecane and n-tetradecane as the sole carbon source. The strains had a 16S rRNA gene sequence similarity of 94.2-94.3 % to Marinobacter hydrocarbonoclasticus ATCC 27132, and a similarity of 97.5-97.8 % to the closest phylogenetically related type strain, Marinobacter flavimaris DSM 16070T. DNA-DNA hybridization levels to M. flavimaris and other Marinobacter type strains were < or = 42 %, while DNA-DNA reassociation values among DG893T, DG1136 and ATAM407-13 were > or = 83 %. The DNA G + C content was 54-55 mol% and the major isoprenoid quinone was ubiquinone-9. On the basis of phenotypic, chemotaxonomic, DNA-DNA hybridization and phylogenetic analysis, it is proposed that these three strains represent a novel species, Marinobacter algicola sp. nov. The type strain is DG893T (= DSM 16394T = NCIMB 14009T).

Alteromonadaceae↗
PubMed1988

Diarrhetic shellfish toxin, dinophysistoxin-1, is a potent tumor promoter on mouse skin.

Dinophysistoxin-1, 35-methylokadaic acid, is a causative agent of diarrhetic shellfish poisoning. The biological activities and tumor-promoting activity of dinophysistoxin-1 were studied together with those of okadaic acid and 7-O-palmitoyl okadaic acid. Dinophysistoxin-1 is a skin irritant and induces ornithine decarboxylase in mouse skin with the same potency as okadaic acid. 7-O-Palmitoyl okadaic acid induced a lower activity than the other compounds. Dinophysistoxin-1 inhibited the specific [3H]okadaic acid binding to a particulate fraction of mouse epidermis. The binding affinities of dinophysistoxin-1 and okadaic acid to a particulate fraction were almost the same. Dinophysistoxin-1 showed a tumor-promoting activity as strong as that of okadaic acid in a two-stage carcinogenesis experiment on mouse skin. The percentages of tumor-bearing mice in the groups treated with 100 micrograms of 7,12-dimethylbenz[a]anthracene (DMBA) followed by 5 micrograms of dinophysistoxin-1, twice a week, and with DMBA followed by 5 micrograms of okadaic acid twice a week were 86.7% and 80.0% in week 30, respectively. The average number of tumors per mouse was 4.6 in the former group and 3.9 in the latter. Dinophysistoxin-1 and okadaic acid act on cells through different pathways from the 12-O-tetradecanoylphorbol-13-acetate-type tumor promoters.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1969

Differences between the effects of saxitoxin (paralytic shellfish poison) and tetrodotoxin on the frog neuromuscular junction.

1. End-plate potentials (e.p.p.) have been recorded from the neuromuscular junctions of frog sartorius and extensor longus dig. IV muscles, using intracellular micropipettes. Either curare or MgCl(2) were present in the Ringer solution, to keep the e.p.p. amplitude below the threshold for a muscle action potential and contraction.2. It has been shown that saxitoxin (paralytic shellfish poison) usually caused a progressive reduction in the amplitude of the e.p.p. Occasionally, when it was applied in the presence of MgCl(2), the e.p.p. disappeared abruptly.3. Tetrodotoxin usually caused the e.p.p. to disappear abruptly. Occasionally, when applied in the presence of curare, the e.p.p. declined progressively for a short time before disappearing abruptly.4. It is concluded that at the frog neuromuscular junction the preferential site of action of saxitoxin is at the nerve terminals, but tetrodotoxin preferentially blocks nerve conduction at a site proximal to the junction.5. It is suggested that this preparation would be a convenient and reliable test object for distinguishing saxitoxin from tetrodotoxin.

Animals↗
PubMed2001

Comparison of paralytic shellfish toxin (PST) production by the dinoflagellates Alexandrium lusitanicum NEPCC 253 and Alexandrium tamarense NEPCC 407 in the presence and absence of bacteria.

The ability of two Alexandrium species to produce paralytic shellfish toxins (PST) in laboratory culture following the generation of bacteria-free cultures was investigated. The dinoflagellates Alexandrium lusitanicum NEPCC 253 and Alexandrium tamarense NEPCC 407 were cultured in the presence of antibiotics and tested for residual bacteria. After treatment with a cocktail of streptomycin, ciprofloxacin, gentamicin and penicillin G, bacteria could not be detected in either of the treated Alexandrium cultures using 17 different solid and broth bacterial growth media, by epifluorescence microscopy with the dye Sybr green 1, or polymerase chain reaction amplification using universal eubacterial primers designed to target the 16S rRNA gene. Subsequent analysis of A. lusitanicum for PST using high performance liquid chromatography demonstrated that the growth rate and toxin profile remained similar in both bacteria-free and control cultures, although the quantity of toxins produced differed with the bacteria-free culture producing generally more of each compound and also having a greater toxin content in terms of saxitoxin equivalents. A. tamarense also retained similarities between the bacteria-free and control cultures in terms of growth rates and toxin profile, although in this instance, depending on the growth stage and the toxin, the control culture produced more of some toxins than the bacteria-free culture. The control culture was also more toxic in terms of saxitoxin equivalents than the axenic culture. These results suggest that bacteria can influence toxin production in laboratory cultures of Alexandrium species although the mechanisms remain unknown.

Journal Article↗