Hideaki Yamanaka

Isolation and characterization of a lethal hemolysin in the sea anemone Parasicyonis actinostoloides

Four species of sea anemones in the coastal waters of Japan were surveyed for hemolysins. Powerful hemolysins were detected in Parasicyonis actinostoloides and Anthopleura japonica, whereas extracts of A. fuscoviridis showed much weaker hemolytic activity and those of of Haliplanella luciae no activity. Among the animal erythrocytes tested sheep were most sensitive to the hemolysins of the three positive species. The major hemolysin (parasitoxin) in whole bodies of P. actinostoloides was isolated by ion exchange chromatography, gel filtration and chromatofocusing. In addition to hemolytic activity (119900 HU/mg) it exhibited lethal activity in mice (LD50 65 micrograms/kg, i.v.) and fish Oryzias latipes (approximate minimum lethal concentration 1.5 micrograms/ml). Parasitoxin was slightly basic (pI 7.9) in nature and its amino acid composition was characterized by the absence of half-cystine. The molecular weight was 19,000 by SDS-polyacrylamide gel electrophoresis or 17,000 by sedimentation equilibrium, indicating that parasitoxin has no subunit structure.

Venoms from six species of marine fish: lethal and hemolytic activities and their neutralization by commercial stonefish antivenom

A specimen of the stonefish Synanceja verrucosa was captured in Okinawa in March 1988. Live specimens of the scorpionfish Inimicus japonicus were purchased from the Tokyo Central Wholesale Market in November 1988 and those of four species of zebrafish Pterois lumulata, P. volitans, P. antennata and Dendrochirus zebra from an aquarium in December 1988. Crude venoms were extracted from dorsal spines of the six species. All venoms exhibited lethal activity against mice and hemolytic activity specific for rabbit erythrocytes. The lethal activity (or hemolytic activity) of each venom was very unstable to freezing, lyophilization and heating. Both lethal and hemolytic activities of S. verrucosa venom were remarkably neutralized by the commerical stonefish (Synanceja trachynis) antivenom. This antivenom was also effective, to some extent, in counteracting the other venom activities. The neutralizing capacities of the antivenom were calculated to be 7310 LD50 ml-1 for S. verrucosa venom and 1 220 to 2 990 LD50 ml-1 for the other venoms. Results of neutralization tests suggest that venoms from the six species were comparable in terms of antigenecity.

Purification and characterization of a lethal factor in venom from the crown-of-thorns starfish (Acanthaster planci)

A lethal factor in venom of the crown-of-thorns starfish (Acanthaster planci) was obtained in an electrophoretically pure state by chromatography on CM-cellulose and Sephadex G-100. The purified lethal factor is a basic (pI 10.6) glycoprotein (carbohydrate content 3.5%). The mol. wt was estimated to be 20,000 by gel filtration or 25,000 by SDS-disc electrophoresis, suggesting that the lethal factor has no subunit structure. Despite its basicity, the lethal factor was richer in acidic amino acids than in basic amino acids. The lethal factor had an LD50 of 0.43 mg/kg (i.p. injection into mice). Hemolytic, edema-forming and capillary permeability-increasing activities, though very weak, were also exhibited by the lethal factor, while hemorrhagic and phospholipase A activities were not present.

Toxins in the skin secretion of the oriental catfish (Plotosus lineatus): immunological properties and immunocytochemical identification of producing cells.

Antiserum against toxin I, one of the lethal factors in the skin secretion from the oriental catfish (Plotosus lineatus), was used to examine immunological properties of the toxic factors and identify toxin-producing cells by an immunocytochemical technique. In immunodiffusion tests, the antiserum formed a precipitin line with toxin I while it formed no precipitin line either with another lethal factor (toxin II) or with a hemolysin. Lethal and edema-forming activities of toxin I were neutralized by the antiserum but lethal activity of toxin II and lytic activity of the hemolysin were not. These results suggested that toxin I can be antigenically distinguished from both toxin II and hemolysin. In immunocytochemical tests using the antiserum, club cells in the epidermis were positively stained, indicating that toxin I is produced in the club cells. Interestingly, venom glandular cells surrounding the dorsal and pectoral spines were also stained. The venom glandular cells appear to produce toxin I or a toxin with the same antigen determinants as toxin I.

Liver damage by the crown-of-thorns starfish (Acanthaster planci) lethal factor

Upon autopsy of mice injected with the crown-of-thorns starfish (Acanthaster planci) lethal factor, a change in color of the liver, swelling of the gall bladder and jaundice were observed. After administration of the lethal factor into mice, activities of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), lactate dehydrogenase (LDH), acid phosphatase (ACP) and alkaline phosphatase (ALP) were significantly elevated in serum. Hepatic ALP also increased markedly but hepatic GOT, GPT and ACP showed a tendency to decrease. Activity of hepatic LDH first decreased and then returned to control level. Histopathological studies showed that severe degenerative changes such as enlargement and necrosis of hepatocytes were caused in the mouse liver by the lethal factor. These results strongly suggested that the A. planci lethal factor is a potent hepatotoxin.

Evidence for the presence of arsenobetaine as a major arsenic compound in the shrimpSergestes lucens

The major arsenic compound in the shrimpSergestes lucens was isolated and identified as arsenobetaine (CH3)3A S + CH2COO−. Arsenobetaine accounted for 80% of the total arsenic in the shrimp.

Purification and characterization of a galactose-binding lectin from the skin mucus of the conger eel Conger myriaster.

1. A galactose-binding lectin was purified from the skin mucus of the conger eel Conger myriaster by affinity chromatography and HPLC. 2. The lectin was a simple protein having the same two subunits with a mol. wt of 12,500 and a N-terminal amino acid of phenylalanine. 3. Electrofocusing suggested that the purified lectin was composed of several isolectins. 4. From the ultraviolet difference spectra attributable to tryptophanyl residues in the binding site, the binding constant of the lectin for D-galactose was estimated to be 5.3 x 10(3)/M.

Hemolytic, lethal and edema-forming activities of the skin secretion from the oriental catfish (Plotosus lineatus)

The crude extract from the skin secretion of the oriental catfish, Plotosus lineatus, contained at least one hemolysin, two lethal factors and two edema-forming factors; the lethal and edema-forming factors seem to be identical. The molecular weights of the hemolysin and the lethal factors (edema-forming factors) were estimated to be 180,000 and 12,000, respectively. Peculiar secretory cells, which resembled the venom glandular cells of the dorsal and pectoral stings, were observed in the epidermis.

Development of an octopine biosensor and its application to the estimation of scallop freshness

Abstract An octopine sensor was developed which was based on the immobilized enzymes pyruvate oxidase and octopine dehydrogenase. The sensor consisted of a reactor, oxygen electrode, flow cell, peristaltic pump, recorder, and a buffer tank. The optimum conditions for the sensor were as follows: pH 7.4; temperature, 26°C; flow rate, 0.77 ml min−1; sample volume, 50 μl; and 0.05 m phosphate buffer transfer solution. A good correlation was obtained between the octopine contents in scallop adductor muscle determined by the sensor and those determined by high performance liquid chromatography (HPLC). These results show that scallop freshness can be estimated by the determination of octopine using the proposed sensor system.

Purification and comparison of water-soluble arsenic compounds in a flatfish Limanda herzensteini, sea squirt Halocynthia roretzi, and sea cucumber Stichopus japonicus

Abstract 1. The water-soluble arsenic compounds were purified frorn a flatfish Limanda herzensteini , sea squirt Halocynthia roretzi , and sea cucumber Stichopus japonicus . 2. In the cases of flatfish and sea cucumber the major arsenic compound was judged to be arsenobetaine. 3. The sea squirt contained one acidic and two basic arsenic compounds differing from arsenobetaine.