Rina Goto

Purification and properties of agglutinins from conger eel, Conger myriaster (Brevoort), skin mucus

The skin mucus of the conger eel, Conger myriaster, contains galactose-specific agglutinins. Hemagglutinating activity is independent of divalent cations and is destroyed by heating at 50 degrees C for 15 min. The mucus agglutinins, named congerins, are a mixture of proteins with different electrical charges. Three of these molecules were isolated by affinity chromatography on acid-treated Sepharose 4B and by ion-exchange chromatography. They are simple proteins with the same molecular weight of 30,000 and consisting of two subunits (each 13,000 daltons). The agglutinins inhibited the normal embryonic development of the starfish Asterina pectinifera, and lysed the fertilized eggs at a concentration of 25 micrograms protein/ml. They also agglutinated but did not inhibit the growth of a marine bacterium, Vibrio anguillarum.

Purification and characterization of an antibacterial and antineoplastic protein secretion of a sea hare, Aplysia juliana

The fetid secretion of a sea hare, Aplysia juliana, was lethal to crabs and also inhibited the growth of bacteria. When the secretion was partitioned between water and n-hexane, only the n-hexane layer, which had a nauseating odor, was lethal to crabs. The water-soluble fraction showed strong antibacterial activity and inhibited the growth of both Gram-positive and Gram-negative bacteria. Antibacterial activity of the water-soluble fraction was destroyed by heating at 50 degrees C for 15 min, but was resistant to treatment with proteolytic enzymes. The active principle, named julianin-S, was purified by gel filtration and ion exchange chromatography. The purified specimen gave a single protein showing a mol. wt of approximately 67,000, as determined by gel filtration. Julianin-S inhibited the growth of Bacillus subtilis by 50% at a concentration of 70 ng protein/ml. It was also cytotoxic to murine tumor cells and inhibited in vitro growth of L1210 cells by 50% at a concentration of 8 ng protein/ml.

Fatty acids as antifoulants in a marine sponge

The antifouling factor against the blue mussel Mytilus edulis found in the organic extract of the marine sponge Phyllospongia papyracea collected in Kagoshima was purified by solvent partition, silica gel column chromatography and HPLC on Toyopearl HW‐40 and on ODS. The active substance was analyzed by gas chromatography after methylesterification and identified as a mixture of several free fatty acids containing C16: 0 (59.9%), C16: l (11.8%) and C18: l (13.8%) as the major components. Among the various authentic free fatty acids tested, C10: 0, C14: l, C16: 0, C16.1, C18: 2, C20: 4 and fatty acids from porcine liver (FAPL) showed antifouling activity against M. edulis. The stronger activity of fatty acid mixtures compared with the same amount of each used singly suggested a synergistic effect of fatty acids on antifouling activity. FAPL inhibited larval settlement in the barnacle Balanus amphitrite without killing the larvae.

Lectins in the Rock Lobster Jasus Novaehollandiae Hemolymph

Hcmolymph of three different species of rock lobsters was examined for its agglutinating activity using mammalian erythrocytes and bacteria. The hemolymph of the red rock lobster Jasus novaehollandiae agglutinated human ABO erythrocytes and also a marine bacterium Pseudomonas aeruginosa. The hemagglutinating activity was, however, inconsistent, and varied from a titer of 4 to 1024 for human erythrocytes. J. novaehollandiae lectins were heat-labile and the hemagglutinating activity was dependent on the presence of Ca2+. The major lectin named JN-2 was a glycoprotein having a molecular weight of 400,000 and dissociated into subunits of different molecular sizes (85, 81, and 63kD). Porcine stomach mucin (PSM), asialo-PSM, and fetuin were effective inhibitors to J. novaehollandiae lectins. N-acetylneuraminic acid did not inhibit the hemagglutinating activity. Simple sugars such as D-ribose, D-arabinose, and D-galactose also inhibited the hemagglutinating activity to some extent. Another species of the red rock lobster J. edwardsii was found to possess lectins similar to those of J. novaehollandiae. On the contrary, the humoral lectin(s) of the green rock lobster J. verreauxi was sialic acid-specific, and the lectin activity was independent of the presence of Ca2+.

Purification and characterization of an agglutinin of the soft coral Sinularia species

A D-galactose-specific agglutinin, named sinularian, has been isolated from the soft coral Sinularia sp. by affinity chromatography on acid-treated Sepharose 4B and by gel filtration on HPLC. Sinularian was a glycoprotein containing 11% sugar. It gave a single band corresponding to 78 kDa in SDS-PAGE, irrespective of a treatment with 2-mercaptoethanol. Sinularian agglutinated rabbit erythrocytes and murine leukemia cells but not sheep or human ABO erythrocytes. Its hemagglutinating activity was Ca(++)-independent. Sinularian promoted binding of macrophages to tumor cells.

Isolation and characterization of agglutinins from the hemolymph of an acorn barnacle, Megabalanusvolcano

Abstract Two agglutinins. MVA-1 and MVA-2, were isolated from the hemolymph of the acorn barnacle, Megabalanus volcano . They agglutinated human erythrocytes irrespective of the ABO blood group and also rabbit and sheep blood cells. Lactose and fetuin strongly inhibited the hemagglutinating activity. D-galactose, D-arabinose and N-acetylneuraminic acid were also moderate inhibitors. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both MVA-1 and MVA-2 gave a single band corresponding to 38,000 daltons. It split into one major band with a molecular weight of 23,000 in the presence of 2-mercaptoethanol. The two agglutinins showed the same apparent molecular weight of 116,000 by gel filtration. In isoelectric focusing MVA-1 showed one band at pH 4.8. whereas MVA-2 gave a main band at pH 4.4 with few faint ones in the range between pH 4.0 and 4.8. The agglutinins were glycoproteins containing D-mannose and L-fucose as carbohydrate components. No precipitation reaction was observed in Ouchterlony immuno-diffusion tests using rabbit antisera against the agglutinins from the phylogenetically related Megabalanus rosa .

Purification of the major allergen of red soft coral (Dendronephthya nipponica).

Red soft coral (RSC; Dendronephthya nipponica, a marine coelenterate) causes spiny lobster fishermen living along the Pacific coast of Miyazaki Prefecture in Japan to develop occupational allergies, such as conjunctivitis, rhinitis, dermatitis and bronchial asthma. The aim of this study was to purify and to characterize RSC allergen, which causes occupational asthma in spiny lobster fishermen. The allergic responsiveness of spiny lobster fishermen to RSC was examined. The examinations included specific IgE production, skin test responses, lymphocyte stimulation tests and specific IgG production. We found that RSC has a strong sensitizing activity in humans at a molecular weight of 10 kD or more, while it has no IgE-producing activity at a molecular weight of less than 10 kD. Neither the nonatopic controls nor the atopic non-coral-allergic controls exhibited any RAST-binding activity to any fraction. For the purification and the identification of this new allergen component, repeated gel filtration of the RSC extract was performed on a Sephacryl S-200 column, followed by gel filtration on a Superose-6 column. The purified major allergen component Den n 1, which is separated on a Mono-Q column, showed intradermal responses, lymphocyte stimulating activity and specific IgG-producing activity in RSC-induced bronchial asthma patients. The 53-kD component was electroblotted on a polyvinylidene difluoride membrane. The N-terminal amino acid sequence of this new allergen component (Den n 1) was determined as Asp-Asp-Ile-Asn-Arg-Tyr-Ala-Phe-Asp-Asn-Lys-Ile-Asn- Asp-Lys-Leu-Phe-Asp-His-Trp-Gln-Ser.