Sachio Nishio

Ostreopsis sp., a possible origin of palytoxin (PTX) in parrotfish Scarus ovifrons

A clone of toxic dinoflagellate Ostreopsis sp. and six specimens of a parrotfish Scarus ovifrons were collected in October 1997 at Tokushima Prefecture, Japan. Ostreopsis sp. was cultured in ESM medium for 16 days, and after rearing the cell pellet (about 4.0x10(5) cells) was extracted with 50% methanol, partitioned between an aqueous layer and 1-butanol layer, and biochemically tested. Similarly, the crude toxin from S. ovifrons was extracted, and tested. The mice injected with each 1-butanol layer from Ostreopsis sp. and S. ovifrons showed the common symptoms of convulsion, drowsiness and collapse, and died within 48 h. The lethal potency of Ostreopsis sp. was calculated to be 1.0x10(-4) MU/cell. All specimens of S. ovifrons were found to be toxic, where the highest potency was determined as 2 MU/g in muscle of one specimen. After being injected with toxins, the serum creatine phosphokinase levels of mice were found to be elevated. Toxins from Ostreopsis sp. and S. ovifrons showed delayed haemolytic activity with mouse and human erythrocytes, which was inhibited by an anti-palytoxin (PTX) antibody antibody and ouabain. Toxins from Ostreopsis sp. and S. ovifrons thus resembled each other, and strongly suggested to be PTX or its akin substance. Additionally, a considerable number of adherent Ostreopsis sp. was found in the gut contents of S. ovifrons during the heavy occurrence of Ostreopsis sp. in October 1997 at Tokushima Prefecture. From the above results, it can be strongly postulated that the dinoflagellate Ostreopsis sp. is the origin of PTX which is sequestered by the parrotfish S. ovifrons through food chain.

Two new isomers of domoic acid from a red alga, Chondria armata

Isodomoic acids G and H, two new isomers of the neurotoxin domoic acid, along with isodomoic acids A, B, E and F, were isolated from a red alga, Chondria armata, collected at the southern tip of Kyushu Island. The structures of two of these were deduced to be (E, E) and (Z, E) isomers of 2-carboxy-4-(5-carboxy-l-methyl-2-hexenylidene)-3-pyrro- lidineacetic acid, based on electrospray ionization mass and [1H]nuclear magnetic resonance spectral analyses including [1H-1H]correlation spectroscopy and nuclear Overhauser effect correlation spectroscopy.

Genetic diversity of Gambierdiscus spp. (Gonyaulacales, Dinophyceae) in Japanese coastal areas

The genetic diversity of the ciguatera fish poisoning‐related dinoflagellate distributed in Japanese coastal areas was investigated. The entire sequence of the 5.8S rRNA gene and two internal transcribed (ITS) regions were determined, which included putative pseudogenes, from 19 strains of dinoflagellates assigned to the genus Gambierdiscus Adachi et Fukuyo collected from Japanese subtropical and temperate coastal areas. The sequences obtained from the 19 strains were divided into two types based on sequence similarity. Here we designate the two types as type 1 and type 2. For the relationship between the genotypes and origins of the strains used, the strains collected from subtropical areas possessed the type 1 sequence; whereas those from temperate areas possessed the type 2. This observation led us to question former reputations that Gambierdiscus cells observed in Japanese temperate areas are immigrants from Japanese subtropical areas. Subsequently, we sequenced a part of the 18S rRNA gene from two strains from subtropical areas and two from temperate areas. Unfortunately, phylogenetic analysis including the sequences obtained from various gonyaulacales dinoflagellates failed to determine the species phylogenetically closely related to and possible origin(s) of the Gambierdiscus sp. from the Japanese coastal areas.

A new saxitoxin analogue from a xanthid crab Atergatis floridus

A novel paralytic toxin was isolated from toxic specimens of a xanthid crab Atergatis floridus inhabiting the Pacific coast of Shikoku Island. Its structure was deduced to be 11-saxitoxinethanoic acid (SEA) based on high-performance liquid chromatography, electrospray ionization mass spectrometry, and 1H and 13C-NMR spectrometries. This acid was assumed to exist as an equilibrium mixture of three tautomers, the main tautomer being the hydrate form 11 beta-epimer. SEA showed a specific toxicity of 830 mouse units per mumole on i.p. injection into mice.

Detection of tetrodotoxin by thin-layer chromatography/fast atom bombardment mass spectrometry.

A new method for detection of tetrodotoxin (TTX) by thin-layer chromatography/fast atom bombardment (FAB) mass spectrometry was developed. TTX and/or related substances were separated by TLC on LHP-K high-performance precoated plates, with a solvent system of pyridine:ethyl acetate:acetic acid:water (15:5:3:4). The plates were subjected to positive FAB mass spectrometry, under scanning within a mass range from m/z 100 to 500. TTX was identified by selected ion-monitored chromatograms at m/z 320 (M + H)+ and 302 (M + H - H2O)+, along with full scan positive ion FAB mass spectrometry. The limit of detection for TTX was about 0.1 micrograms. TTX was also detected by cellulose acetate membrane electrophoresis/FAB mass spectrometry.

Subcellular distribution of tetrodotoxin in puffer fish liver

The liver homogenate of puffer fish was fractionated into blood cell, nuclear, mitochondrial, microsomal and cytosol fractions by the differential centrifugation method. All the five fractions were toxic to mice, although the toxin amount was significantly high in the cytosol fraction. Analyses by HPLC and LC-FABMS demonstrated that tetrodotoxin is the major toxic principle in each fraction. These results reveal that tetrodotoxin is widely distributed in organelles in liver cells, though predominantly in the cytosol fraction.

Tetrodonic acid-like substance; A possible precursor of tetrodotoxin

A tetrodonic acid-like substance which was hardly distinguishable from authentic tetrodonic acid in thin-layer chromatography, high performance liquid chromatography, etc., was successfully purified from the ribbon worm and flatworm by a method consisting mainly of Bio-Gel P-2 column chromatography. The tetrodonic acid-like substance showed a specific toxicity of approximately 700 mouse units/mg as tetrodotoxin, unlike tetrodonic acid which is a completely non-toxic substance. Instrumental analyses including gas chromatography-mass spectrometry, secondary ion mass spectrometry and thin-layer chromatography/fast atom bombardment mass spectrometry disclosed that the tetrodonic acid-like substance had a structure similar to, but not the same as, that of tetrodotoxin. This, along with its high convertibility into tetrodotoxin during storage and other experimental operations, suggested that the tetrodonic acid-like substance is a precursor of tetrodotoxin.

Occurrence of the polyamines caldopentamine and homocaldopentamine in axenic cultures of the red tide flagellates Chattonella antiqua and Heterosigma akashiwo (Raphidophyceae)

The polyamines caldopentamine and homocaldopentamine were detected in axenic strains of Chattonella antiqua and Heterosigma akashiwo (Raphidophyceae), respectively, as well as spermidine, the most abundant polyamine in both phytoplankton species. Trace amounts of putrescine, diaminopropane and norspermine were also detected in both species. Spermine was detected only from C. antiqua. These long linear polyamines are characteristic components of thermophilic bacteria. The detection from two species of Raphidophyceae indicates that the occurrence of long linear polyamines is not restricted to thermophilic microorganisms.

Occurrence of PSP-producing dinoflagellate Alexandrium tamiyavanichii in Bingo-Nada, the central coastal water of the Seto Inland Sea, Hiroshima Prefecture, Japan

During surveillance of the distribution of the paralytic shellfish poison (PSP)-producing dinoflagellate in 2003, 2004 and 2005 along the coastlines of the Seto Inland Sea, Hiroshima Prefecture, Japan, some species of toxic phytoplankton were isolated from the eastern coasts, Bingo-Nada, the central regions of the Seto Inland Sea. It was rather unexpectedly revealed from the basis of the morphological characteristics that they were unambiguously identified as Alexandrium tamiyavanichii and Alexandrium catenella. Two strains (ATY041106, ATY051018) of A. tamiyavanichii showed a specific toxicity of 38.7 x 10(-6) and 111.5 x 10(-6)MU/cell, respectively. These values seemed to be several times or much higher than that of A. catenella (AC030816, AC040614), having a specific toxicity of 4.5 x 10(-6) and 4.1 x 10(-6)MU/cell, respectively, isolated in the same area. From the results of HPLC-furuorometric analysis, it revealed that the toxins in ATY041106 exist almost exclusively as beta-epimers (C2, GTX3, GTX4), which accounted for 72.7 mol%. The toxin profiles of this strain are featured by the presence of a large amount of GTX3 (59.1 mol%) and a small amount (20.6%) of C1 and 2 in comparison with the PSP compositions of A. tamarense, which is isolated as the main responsible species in Hiroshima Bay, a western part of coastal sea in Hiroshima Prefecture. On the other hand, it revealed that the toxin profiles of two strains (AC030816, AC040614) of A. catenella exist almost exclusively as beta-epimers (C2, GTX3, GTX4), which accounted for 81.8 and 56.5 mol%, as the same manner. The toxin profiles of these two strains are featured by the presence of a large amount of C2 (80.5 and 46.3 mol%) in comparison with the PSP compositions of A. tamiyavanichii. To our knowledge, this is the first record to show the distribution and harmful influence of A. tamiyavanichii and A. catenella in Bingo-Nada in Hiroshima Prefecture. Though contamination of bivalves with these PSP-producing planktons in this area has not occurred yet so far, attention should be paid to this species as well as the other causative dinoflagellate from the stand point of public health and food hygiene.

Free amino acid contents of roots of the lotus cultivars "Bicchu" and "Lotus" grown in Tokushima prefecture

(平成28年6月17日受付,平成28年10月8日受理) Free amino acid contents of roots of the lotus cultivars “Bicchu” and “Lotus” grown in Tokushima prefecture Kozue Edo , Tamiko Hashimoto , Sachio Nishio , Eiji Sawada , Hideyuki Nakagawa * Junior College Division, Department of Science for Human Health, Shikoku University, Furukawa, Ojin-cho, Tokushima-shi, Tokushima, 771-1192 Department of Food Science and Nutrition, Mukogawa Women’s University, 6-46, Ikebiraki-cho, Nishinomiya-shi, Hyogo, 633-8558 Tokushima Agriculture, Forestry, and Fisheries Technology Support Center, 1660, Ishii, Ishii-cho, Myohzai-gun, Tokushima, 779-3233 Department of Nursing, Shikoku University, Furukawa, Ojin-cho, Tokushima-shi, Tokushima, 771-1192