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Structural studies on glycolipid of shellfish. II. Occurrence of 3-O-methylgalactosamine in oyster glycolipid.

3-O-Methyl hexosamine was found for the first time and characterized as one of the sugar components of oyster glycolipid. The alditol acetate of this sugar was identified as 3-O-methyl-N-acetylgalactosaminitol acetate by comparing its retention time on gas chromatography and mass spectrum with those of the authentic sample synthesized in this laboratory. The new sugar, 3-O-methylgalactosamine, occupied the non-reducing terminal position of the carbohydrate moiety of the lipid and was linked to the penultimate hexose by a (1 leads to 3) bond.

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Structural studies on glycolipids of shellfish. V. Gala-6 series glycosphingolipids of the marine snail, Chlorostoma argyrostoma turbinatum.

A series of glycosphingolipids and phosphonoglycosphingolipid containing only galactose as the sugar component were isolated from the marine snail, Chlorostoma argyrostoma turbinatum. The structures of these lipids were studied by methylation analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy and fast atom bombardment mass spectrometry, and characterized as follows: the glycosphingolipids galactosyl beta(1-1)ceramide, galactosyl beta(1-6)galactosyl beta(1-1)ceramide, galactosyl beta(1-6)galactosyl beta(1-6)galactosyl beta(1-1)ceramide and galactosyl beta(1-6)galactosyl beta(1-6)galactosyl beta(1-6)galactosyl beta(1-1)ceramide, and phosphonoglycosphingolipid N-methylaminoethylphosphonyl galactosyl(1-1)ceramide. The main molecular species of the ceramide moiety were hexadecanoyl-octadecasphingenine and hydroxyhexadecanoyl-octadecasphingadienine in all of these sphingolipids.

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Potent neurotoxic action of the shellfish biotoxin yessotoxin on cultured cerebellar neurons.

Yessotoxin (YTX) and its analogues are disulphated polyether compounds of increasing occurrence in seafood. The biological effects of these algal toxins on mammals and the risk associated to their ingestion have not been clearly established. We have used primary cultures of rat cerebellar neurons to investigate whether YTX affected survival and functioning of central nervous system neurons. Exposure to YTX (> or =25 nM) caused first (approximately 8 h) weakening, granulation, and fragmentation of neuronal network, and later (approximately 48 h) complete disintegration of neurites and extensive neuronal death, with a significant decrease in the amount of filamentous actin. The concentration of YTX that reduced by 50% the maximum neuronal survival (EC50(48)) was approximately 20 nM. Lower toxin concentrations (approximately 15 nM) also caused visible signs of toxicity affecting neuronal network primarily. Removal of YTX after 5 h exposure delayed the onset of neurotoxicity but did not prevent neuronal degeneration and death. YTX induced a two-fold increase in cytosolic calcium that was prevented by the voltage-sensitive calcium channel antagonists nifedipine and verapamil. These antagonists were, however, completely ineffective in reducing neurotoxicity. Voltage-sensitive sodium channel antagonists saxitoxin and nefopam, and the NMDA receptor antagonist MK-801 also failed to prevent YTX neurotoxicity. Neuronal death by YTX involved typical hallmarks of apoptosis and required the synthesis of new proteins. Our data suggest neuronal tissue to be a vulnerable biological target for YTX. The potent neurotoxicity of YTX we report raises reasonable concern about the potential risk that exposure to YTX may represent for neuronal survival in vivo.

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Neuroexcitatory and neurotoxic actions of the amnesic shellfish poison, domoic acid.

We have investigated the action of domoic acid in the mouse brain following systemic exposure. Domoic acid increased c-fos mRNA within 15 min and its translational product (c-Fos) within 1 h. c-Fos immunoreactivity was most prominent in the hippocampal formation, lateral septal nucleus, olfactory bulb, area postrema and the nucleus of the solitary tract. We next examined irreversible toxic effects of domoic acid. Domoic acid caused extensive degeneration in CA1-2 of the hippocampus, lateral septal nucleus and olfactory bulb. No degeneration was evident in the dentate gyrus or brain stem. These studies demonstrate that domoic acid has only neuroexcitatory effects on brain stem regions associated with visceral function whereas it has permanent neurotoxic effects on brain regions associated with memory formation.

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Congenital endophthalmitis following maternal shellfish ingestion.

PURPOSE: To highlight an unusual organism causing a unilateral endophthalmitis by transplacental spread. METHOD: We report a case of Plesiomonas shigelloides endophthalmitis, presenting in a newborn, with co-existing septicaemia and meningitis. There was a significant maternal history of diarrhoea associated with the ingestion of oysters 2 weeks prior to delivery. RESULT: The endophthalmitis was treated with parenteral antibiotics and topical mydriatics with complete resolution, although subsequent assessment of the affected eye suggests a poor visual outcome. CONCLUSION: Endophthalmitis in the newborn is an unusual clinical finding and usually presents with other manifestations of bacteraemia. Plesiomonas shigelloides is fortunately an infrequent cause of neonatal infection, but is associated with a high degree of morbidity and mortality. We postulate that this neonate acquired P. shigelloides via the transplacental route, and suggest that this organism be included in the list of 'other' causes of transplacental infection that has been abbreviated to 'O' in the acronym 'TORCH'.

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Australia antigen: detection and transmission in shellfish.

Australia antigen was found in clams contaminated by drainage of untreated sewage from a coastal hospital. In closed-system aquariums, the antigen was ingested by clams and transmitted to previously uninfected clams. In opensystem aquariums, the titer of Australia antigen decreased with time, suggesting viral concentration rather than replication.

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Vibrio anguillarum and larval mortality in a California coastal shellfish hatchery.

Vibrio anguillarum was isolated as a pathogen in the commercial culture of oyster spat at Pigeon Point, Calif. A water-soluble, heat-stable exotoxin extracted from cultures of the vibrio inhibited larval swimming and contributed to larval mortality. Although the vibrio was insensitive to penicillin in standard plate testing, this antibiotic proved useful in preventing mass larval mortalities in the hatchery.

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Bacteria associated with false-positive most-probable-number coliform test results for shellfish and estuaries.

Aerobic and facultatively anaerobic bacteria isolated from false-positive, presumptive, total coliform, most-probable-number tests of Chesapeake Bay oyster, water, and sediment samples were characterized and then classified by numerical taxonomy. A total of 538 bacterial strains clustered into 17 phena, the predominant groups of which were Enterobacteriaceae (including Escherichia coli), Aeromonas spp., and Bacillus spp. Bacillus spp. were recovered most frequently from sediment samples. Gas-producing strains which were not members of the Enterobacteriaceae were not isolated during this study. However, disproportionately large numbers of atypical and anaerogenic lactose-fermenting strains were encountered. We concluded that no single, specific bacterial group can be identified as being responsible for the false-positive reaction in the presumptive coliform test. Instead, the false-positive reaction is a result of complex interactions among various genera, representing predominantly bacteria other than coliforms.

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Effects of temperature and salinity on the survival of Vibrio vulnificus in seawater and shellfish.

Sterilized seawater was used to assess the effects of temperature and salinity on the survival of Vibrio vulnificus. In the temperature range of 13 to 22 degrees C, numbers of V. vulnificus increased during the 6-day incubation. Temperatures outside this range reduced the time of V. vulnificus survival in sterile 10-ppt seawater. At these restrictive temperatures, V. vulnificus numbers were reduced by 90% after 6 days of incubation. Incubation between 0.5 and 10.5 degrees C demonstrated that V. vulnificus survives poorly below 8.5 degrees C. At salinities between 5 and 25 ppt and at 14 degrees C, V. vulnificus numbers actually increased or remained unchanged after 6 days of incubation. At salinities of 30, 35, and 38 ppt, numbers of V. vulnificus decreased 58, 88, and 83%, respectively. V. vulnificus could not be recovered from deionized water, indicating lysis. When a rifampin-resistant strain of V. vulnificus was used to inoculate sterilized and unsterilized seawater (20 ppt, 20 degrees C), numbers increased in sterile seawater but decreased to undetectable levels in 14 days in the unsterilized seawater, indicating that biological factors may play a role in the survival of V. vulnificus in the environment. Since our studies demonstrated sensitivity to low temperatures, the survival of V. vulnificus in naturally contaminated oysters at temperatures of 0, 2, and 4 degrees C was also determined. Numbers of endogenous V. vulnificus in oyster shellstock increased by more than 100-fold in shellstock stored at 30 degrees C but were reduced approximately 10- and 100-fold after 14 days at 2 to 4 degrees C and 0 degrees C, respectively. We conclude that both biological and physicochemical factors are important to the survival of V. vulnificus in the environment and that temperature is critical to controlling its growth in oyster shellstock.

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An estuarine agar medium for enumeration of aerobic heterotrophic bacteria associated with water, sediment, and shellfish.

A plate count agar was formulated for use in bacteriological analysis of estuarine samples and was tested together with standard plate count agar and an estuarine salts yeast extract agar for growth of aerobic, heterotrophic bacteria in water, sediment, and oysters. The estuarine agar was found to be efficient for enumerating aerobic, heterotrophic bacterial populations of water, sediment, and oysters, and is recommended for plate counts of estuarine samples.

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Specificity of mouse monoclonal anti-okadaic acid antibodies to okadaic acid and its analogs among diarrhetic shellfish toxins.

The specificity of five mouse monoclonal antibodies to okadaic acid was studied for use in an enzyme-linked immunosorbent assay of okadaic acid and its analogs. OA8-2 and OA22-22 antibodies (IgG2a-kappa), which bind more strongly to dinophysistoxin-1 and 7-O-palmitoyl-dinophysistoxin-1 than to okaic acid or 7-O-palmitoyl-okadaic acid in 50% aqueous methanol, were useful in the detection of dinophysistoxins-1 and -3. OA10-8 (IgG1-kappa), which binds more strongly to 7-O-palmitoyl-okadaic acid and 7-O-palmitoyl-dinophysistoxin-1 than to okadaic or dinophysistoxin-1 in 50% aqueous methanol, was useful in the detection of dinophysistoxin-3. OA423-3 (IgG1-kappa), which binds weakly to dinophysistoxin-1 and 7-O-palmitoyl-dinophysistoxin-1 in 20% aqueous methanol, was useful in the selective detection of okadaic acid. OA958-2 (IgG1-kappa), which binds with equal strength to each of the four toxins in methanol, was useful in the detection of all okadaic acid analogs, and the minimum detectable concentration was 30 ng/ml. OA423-3 and OA958-2 retained their binding ability in 50% acetone, ethyl ether, or benzene in methanol.

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