Kanehisa Hashimoto

Vibrio alginolyticus, a tetrodotoxin-producing bacterium, in the intestines of the fish Fugu vermicularis vermicularis

To clarify the mechanism of toxification in animals contaminated with tetrodotoxin, the intestinal contents of the puffer Fugu vermicularis vermicularis were examined for bacterial flora in 1985. Twenty-six out of 33 strains belonged to the genus Vibrio. These bacteria were classified into Groups I to VII, based on biological and biochemical characters. High performance liquid chromatography and gas chromatography-mass spectrometry, together with mouse bioassay for toxicity, clearly demonstrated that Group I produced tetrodotoxin and anhydrotetrodotoxin under cultivation with a medium composed of Phytone peptone (BBL) and NaCl. Some other groups also produced this toxin and/or related substances to some extent. Strains of Group I were all identified as Vibrio alginolyticus. Two strains among four produced a detectable amount of tetrodotoxin and/or anhydrotetrodotoxin, as measured by all instrumental analyses applied. Our findings suggest that some strains of V. alginolyticus are closely related to the toxification of the puffer, and probably of other species.

Mycalolides A – C, hybrid macrolides of ulapualides and halichondramide, from a sponge of the genus Mycale

Three cytotoxic macrolides, mycalolides A – C have been isolated from a sponge Mycale sp., and their structures elucidated to be hybrids of ulapualides and halichondramide mainly by analyses of 2D NMR spectra as well as by comparison of spectral data.

Tetrodotoxin-producing bacteria from the blue-ringed octopus Octopus maculosus

Several live specimens of the blue-ringed octopus Octopus maculosus were collected from the Philippines in November 1985, and from Japan in February 1986, and the distribution of toxicity, along with toxin composition, in the posterior salivary gland and other soft parts were examined. Tetrodotoxin (TTX: 1400 mouse units g-1) was detected in the posterior salivary gland of a Japanese specimen, while not only the salivary gland but other soft parts were toxic in the Philippine specimens. The Philippine specimens contained TTX and anhydrotetrodotoxin, the Japanese specimen TTX, 4-epitetrodotoxin, and an unknown toxin. The posterior salivary gland, intestine and other parts were excised from the Philippine specimens and examined for bacterial flora. Twenty-two dominant strains were isolated and cultured in a 2xORI medium (Ocean Research Institute, Simidu and Tsukamoto 1985) at 20°C for 20 to 48 h. Cells were harvested by centrifugation, and disrupted by ultrasonication. The toxins were partially purified from the cell lyzate by ultrafiltration and Bio-Gel P-2 column-chromatography. Instrumental analyses disclosed that 16 of the 22 strains produced TTX and/or related substances. Six strains which clearly exhibited TTX productivity were identified as Alteromonas (2 strains), Bacillus (2), Pseudomonas (1) and Vibrio (1), based on biochemical and biological characteristics. Of these, one strain each of Bacillus and Pseudomonas produced TTX at a level detectable by the mouse assay.

Changes in carp myosin ATPase induced by temperature acclimation

SummaryMyosins were isolated from dorsal ordinary muscles of carp acclimated to 10°C and 30°C for a minimum of 5 weeks and examined for their ATPase activities. Ca2+-ATPase activity was different between myosins from cold-and warm-acclimated carp, especially at KCl concentrations ranging from 0.1 to 0.2 M, when measured at pH 7.0. The highest activity was 0.32 μmol Pi·min-1·mg-1 at 0.2 M KCl for cold-acclimated carp and 0.47 μmol Pi·min-1·mg-1 at 0.1 M KCl for warm-acclimated fish. The pH-dependency of Ca2+-ATPase activity at 0.5 M KCl for both carp was, however, similar exhibiting two maxima around 0.3 μmol Pi·min-1·mg-1 at pH 6 and 0.4 μmol Pi·min-1·mg-1 at pH 9. K+(EDTA)-ATPase activity at pH 7.0 neither exhibited differences between both myosins. It increased with increasing KCl concentration showing the highest value of about 0.4 μmol Pi·min-1·mg-1 at 0.6–0.7 M KCl. Actin-activated myosin Mg2+-ATPase activity was markedly different between cold-and warm-acclimated carp. The maximum initial velocity was 0.53 μmol Pi·min-1·mg-1 myosin at pH 7.0 and 0.05 M KCl for cold-acclimated carp, which was 1.6 times as high as that for warm-acclimated carp. These differences were in good agreement with those obtained with myofibrillar Mg2+-ATPase activity between both carp. No differences were, however, observed in myosin affinity to actin. Differences in myosin properties between cold- and warm-acclimated carp were further evidenced by its thermal stability. The inactivation rate constant of myosin Ca2+-ATPase was 25·10-4·s-1 at 30°C and pH 7.0 for cold-acclimated carp, which was about 4 times as high as that for warm-acclimated carp. Light chain composition did not differ between both carp myosins. The differences in a primary structure of the heavy chain subunit was, however, clearly demonstrated between both myosins by peptide mapping.

(+)-Curcuphenol and dehydrocurcuphenol, novel sesquiterpenes which inhibit H,K-ATPase, from a marine sponge Epipolasis sp.

Two H, K-ATPase inhibitors and an inactive related compound have been isolated from a marine spongeEpipolasis sp. They are aromatic sesquiterpene α-curcumenes

Occurrence of tetrodotoxin in the flatworm Planocera multitentaculata

The paralytic toxin which occurs in a flatworm Planocera multitentaculata was partially purified by column chromatography using Bio-Gel P-2 and Bio-Rex 70 (H+ form). Thin-layer chromatographic, electrophoretic and gas chromatography-mass spectrometric analyses demonstrated that the flatworm toxin is tetrodotoxin.

Occurrence of saxitoxin and other toxins in the liver of the pufferfish Takifugu pardalis

Highly toxic livers of the pufferfish Takifugu pardalis were extracted with acidic ethanol. The toxins extracted were partially purified by chromatography on Bio-Gel P-2 and then Bio-Rex 70, resulting in separation into three fractions I, II and III. ratios of total mouse units per fraction were approximately 0.1:100:0.01, respectively, with tetrodotoxin (TTX) as standard. By TLC, electrophoresis and a TTX analyzer, Fr. II was identified as TTX and, unexpectedly, Fr. III as saxitoxin, while Fr. I remains unidentified.

Poison arrowworms: a tetrodotoxin venom in the marine phylum Chaetognatha

Abstract We have conclusively detected a neurotoxin which blocks the sodium channel of cell membranes in extracts of the heads of six diverse chaetognath species through the use of a new bioassay method utilizing mouse neuroblastoma tissue culture. Analysis by gas chromatography—mass spectrometry has revealed that the major bioactive component in chaetognath venom is tetrodotoxin or a tetrodotoxin analogue. The existence of a venom which contains tetrodotoxin is previously known from only one other animal and is produced by symbiotic bacteria. Although the source of tetrodotoxin in chaetognaths remains unknown, similarities in the vestibular morphology between different chaetognath species further suggest that the majority of the phylum Chaetognatha may be capable of subduing prey with a tetrodotoxin venom.

Bioactive marine metabolites XX. Petrosynol and petrosynone, antimicrobial C30 polyacetylenes from the marine sponge Petrosia sp.: Determination of the absolute configuration☆

Abstract An antimicobial C30 polyacetylene alcohol and its tetraketo analog have been isolated from the marine sponge Petrosia sp. and their structures including absolute configuration determined by spectral and chemical methods.

Hypnins, low-molecular weight peptidic agglutinins isolated from a marine red alga, Hypnea japonica

Abstract Four electrophoretically homogeneous agglutinins have been isolated from the aqueous ethanolic extract of the marine red alga Hypnea japonica by precipitation with organic solvents, gel filtration, and reversed-phase and gel permeation high-performance liquid chromatography. Since these agglutinins were found to be new peptides, they were designated hypnins A, B, C and D after the generic name of the seaweed. The major agglutinin, hypnin A, was a monomeric peptide with a molecular weight of 4200. It had an isoelectric point of 4.3, and contained large amounts of serine and glycine. The N- and C-terminal amino acids were identified as tyrosine and serine, respectively. On the other hand, hypnins B and C were suggested to be dimeric or trimeric forms of hypnin A or the related peptide, while hypnin D was quite different from the others. Hypnin A agglutinated not only animal erythrocytes other than human, but also mouse FM3A tumour cells. The hemagglutinating activity was inhibited only by glycoproteins with N -glycosidic sugar chains of a complex type. The hemagglutinating activity of hypnin A was not affected by heating for 30 min at 100°C or by addition of divalent cations. Hypnins B, C and D were similar to hypnin A in hemagglutination pattern and sugar-binding specificity.