Characterization of peptides in sea anemone venom collected by a novel procedure.

Peptide neurotoxins were isolated from the venom obtained by electrical stimulation of the sea anemone Bunodosoma caissarum. This technique allows almost pure venom to be collected, and the animals to survive. Three neurotoxins (assayed on crustacean nerves) were isolated by gel filtration and reversed-phase high performance liquid chromatography. Hemolysins were also detected in the venom. The amino acid sequence of a major neurotoxin BcIII was determined. BcIII has 48 amino acid residues with six half-cystine residues. This sequence has homology with the type 1 long sea anemone neurotoxins. Two minor toxins (BcI and II) have similar amino acid composition and amino-terminal sequences to BcIII.

A case of envenoming by Portuguese man-of-war from the Brazilian coast.

This is an individual case report on envenoming caused by the cnidarian Portuguese man-of-war. The reported local reactions and the clinical symptoms are similar to those already known for Portuguese man-of-war envenoming. As far as we know it is the first clinical case documented for the Brazilian shores.

Occurrence of toxins, other than paralysing type, in the skin of Tetraodontiformes fish.

Puffer fish (Tetraodontidae and Diodontidae) possess paralysing toxins (tetrodotoxin and analogues) that are secreted upon stimulation. In a previous work it was demonstrated that mucous secretion from the puffer fish Sphoeroides spengleri, when mixed in sea water passing through the orobranchial cavity of groupers, induced cardiorespiratory alterations. In the present study, skin secretions from Ciclichthys spinosus, S. spengleri and Diodon hystrix were tested on crustacean nerves, sea urchin eggs and mouse erythrocyte suspensions to verify neurotoxic and cytotoxic activities. Ciclichthys spinosus and D. hystrix secretions induced transient depolarizations with 0.16 mg and blocked crustacean nerve conduction after prolonged exposure. Both secretions had cytotoxic effects; when applied to sea urchin eggs they caused cleavage inhibition and anomalies in a dose-dependent manner (ED50 +/- S. E. M. = 2.59 +/- 0.08 mg/ml for C. spinosus and 1.23 +/- 0.07 mg/ml for D. hystrix); moreover, hemolysis occurred with an ED50 = 0.76 mg/ml of 0.5% mouse erythrocyte suspensions to C. spinosus and 0.59 mg/ml to D. hystrix. These secretions were not lethal in acute toxicity tests, even at 335 mg/ml. The neurotoxic components were thermolabile while the hemolytic activity was resistant to boiling. Tests with the secretion from S. spengleri did not show cytotoxic effects but promptly blocked action potentials of crustacean nerves and were lethal for mice in acute toxicity rests. When applied to groupers, the C. spinosus secretion caused cardiorespiratory alterations. These results suggest the presence of neurotoxins (other than tetrodotoxin) and cytotoxins in skin of diodontid puffer fish.

Hemolytic activity in extracts of the diatom Nitzschia.

The few reports about diatom toxins are related to central nervous system toxicity, induced by domoic acid. In the present work Nitzschia sp. (Bacillariophyceae) was studied. The cells were cultured in f/2 medium, under 4000 lux and 14/10 hr light/dark cycle. After massive growth (5 x 10(6) cells/ml) the diatom cells were filtered, and an extract was prepared and partitioned in two fractions (polar and apolar). After cell harvesting by filtration, the diatom cells were shaken in artificial sea water to extract the water-soluble extracellular matrix (mucilage). An extract was prepared with the washed cells (free of mucilage), and polar and apolar fractions were obtained. Hemolytic assays were performed using 4.0 and 0.5% erythrocyte suspensions. Both the diatom polar and apolar fractions showed hemolytic activity. The membrane phospholipid sphingomyelin was tested as an acceptor for the hemolysins in the polar and apolar fractions. The mucilage did not exhibit hemolytic activity.

Hemolytic activity of the nematocyst venom from the sea anemone Bunodosoma caissarum.

The nematocyst venom of the sea anemone Bunodosoma caissarum obtained by electric stimulation of the animals has hemolytic activity on fish, toad, snake, mice and rat erythrocytes. The hemolytic action was dose-dependent and the ED50 varied between 2.9 and 7.6 micrograms venom/ml erythrocyte suspension (0.5%, v/v). Toad erythrocytes were the most sensitive while rat erythrocytes were the most resistant to the sea anemone venom. The hemolytic activity of venom in mice was partially inhibited by preincubation of the venom with sphingomyelin for 1 h. The ED50 was increased 3.8-fold when 10.0 micrograms sphingomyelin per ml erythrocyte suspension was used. Venom concentrations above 33.6 micrograms/ml erythrocyte suspension produced approximately 95% hemolysis and were inhibited only by 40.0 micrograms sphingomyelin. The hemolysin activity was inhibited by heating to 90 degrees C but not to 70 degrees C.

Antimitotic effect of an extract of the sea anemone Bunodosoma caissarum on sea urchin egg development.

A methanolic extract of the sea anemone Bunodosoma caissarum has an antimitotic effect on sea urchin egg development. The extract produces a dose-dependent inhibition of cell cleavage. When the extract is added together with sperm to unfertilized sea urchin eggs, the ED50 is 0.60 +/- 0.03 mg/ml (mean +/- SEM). When added shortly after fertilization, the extract produces the same kind of progressive inhibition but with an ED50 of 0.98 +/- 0.16 mg/ml. In the first case, detachment of the vitelline layer is inhibited whereas in the second case the extract inhibits cleavage even when the membrane is present.

Cytotoxic activity of a methanol extract of Phallusia nigra (Tunicata, Ascidiacea).

Tunicates have been reported to be a rich source of biologically active compounds. In this study, we demonstrate the presence of cytotoxic substances in Phallusia nigra, a common tunicate from Brazilian coastal waters. An extract of tunicate tissue was obtained by homogenizing the visceral organs from 50 specimens in methanol, followed by filtration and concentration in a rotary vacuum evaporator. Finally, the concentrate was partitioned with chloroform to remove lipids. The resulting extract possessed antimitotic and hemolytic activity. The former was demonstrated as a delay in the development of sea urchin eggs by partially inhibiting the process of cleavage (first cleavage, EC50 +/- SEM = 3.44 +/- 0.84 mg/ml). The < 500 molecular fraction of the extract obtained by ultrafiltration also inhibited cell proliferation (the number of viable cells was decreased by 68% with 500 micrograms/ml) and DNA synthesis of T47D cells derived from human breast carcinoma as measured by [3H]-thymidine incorporation (66% of the control value after 24-h incubation with 100 micrograms/ml). Dose-dependent hemolysis obtained with P. nigra extract on mouse erythrocytes had an EC50 +/- SEM = 1.12 +/- 0.02 mg/ml for a 0.5% erythrocyte suspension. Hemolysis could be reduced by pre-incubating the cells with choline-containing phospholipid. Sphingomyelin (40 micrograms/ml) increased the EC50 by two-fold to 2.86 +/- 0.04 mg/ml, but phosphatidylcholine (80 micrograms/ml) did not modify hemolysis.

Antimitotic action of extracts of Petiveria alliacea on sea urchin egg development.

The hydroethanol extract of the roots of Petiveria alliacea L. (Phytolaccaceae) has been investigated previously as an antitumor agent against mouse Ehrlich ascites. The extract and its methanol, butanol and ether fractions exhibited an antimitotic effect on sea urchin egg development. The aqueous fraction did not produce inhibition of cell cleavage. At the first cleavage the inhibition, at the lowest concentration (10 micrograms/ml), produced by the ether fraction was 42%, whereas the inhibition produced by the total extract and by the other fractions was only 5 to 10% showing that the ether fraction was the most active. Even at higher concentrations the butanol and methanol fractions inhibit the cleavage about 30%. At the first cleavage, the ED50 of the hydroethanol extract and of the ether fraction were 45.02 and 12.40 micrograms/ml, respectively. Furthermore, in the second cleavage, the hydroethanol extract was about twice as potent as the methanol or butanol fractions (ED50 of 22.40, 44.80 and 54.10 micrograms/ml, respectively).

This histamine-like effects of Phallusia nigra extract: evidences for direct activity at H1 receptors.

The methanol extract (mol. wt lower than 3,000 Da) of the sea squirt Phallusia nigra has stimulatory activity on guinea-pig ileum preparations. This effect was inhibited by cyproheptadine and mepyramine, but not be atropine. Mepyramine antagonized competitively the extract activity with a pA2 of 10.09 +/- 1.12, suggesting a direct activity on H1 histamine receptors. The extract was also assayed on guinea-pig right atria, however, only a mild increase in spontaneous contractions was observed compared to histamine, showing that the extract was a rather poor activator of cardiac H2 receptors. Histamine was not detected upon TLC analysis of the extract by comparison with an authentic standard.

Chemical and pharmacological characterization of halitoxin from Amphimedon viridis (Porifera) from the southeastern Brazilian coast.

The halitoxin complex from the marine sponge Amphimedon viridis, collected in the São Sebastião channel (southeastern Brazilian coast), was isolated by gel-filtration chromatography on Sephadex LH-20. Spectroscopic data (1H, 13C and 2D-NMR) of halitoxin from A. viridis indicated that it has the same two alkylpyridine monomers composition of the previously isolated halitoxin from Amphimedon compressa (as Haliclona rubens). Nevertheless, analysis by High Performance Gel Permeation Chromatography indicated that the halitoxin complex of A. viridis has a lower molecular weight (500, 2000 and 5000 Da; the major component corresponding to the fraction of 2000 Da MW) when compared to the previously isolated halitoxin complex from A. compressa. Some pharmacological properties of the halitoxin complex from A. viridis were evaluated in terms of lethality, antimitosis, hemolysis and neurotoxicity. The possible chemotaxonomic value of alkyl pyridine alkaloids is discussed.

1,3-Dimethylisoguanine, a new purine from the marine sponge Amphimedon viridis.

A new purine, 1,3-dimethylisoguanine (1), has been isolated from the marine sponge Amphimedon viridis and identified by analysis of spectroscopic data. Compound 1 increased the contractions obtained by transmural electrical stimulation in the guinea pig longitudinal muscle/myenteric plexus in a dose-dependent manner.

Antimitotic effect of an extract of the sea anemone bunodosoma caissarum on sea urchin EGG development

A methanolic extract of the sea anemone Bunodosoma caissarum has an antimiototic effect on sea urchin egg development. The extract produces a dose-dependent inhibition of cell cleavage. When the extract is alded together with sperm to unfertilized sea urchin eggs, the ED50 is 0.60 ñ 0.03 mg/ml (x ñ SEM). When added shortly after fertilization, the extract produces the same kind of progressive inhibition but with an ED50 of 0.98 ñ 0.16 mg/ml. In the first case, detachment of the vitelline layer is inhibited whereas in the second case the extract inhibits cleavage even when the membrane is present

Hemolytic activity of the nematocyst venom from the sea anemone Bundodosoma Caissarum

The nematocyst venom of the sea anemone Bunodosoma caisarum obtained by electric stimulation of the animals has hemolytic activity on fish, toad, snake, mice and rat erythrocytes. The hemolytic action was dose-dependent and the ED50 varied between 2.9 and 7.6*g venom/ml erythrocyte suspension (0.5%, v/v). Toad erythrocytes were the most sensitive while rat erythrocytes were the most resistant to the sea anemone venom. The hemolytic activity of venom in mice was partially inhibited by preincubation of the venom with sphingomyelin for 1h. The ED50 was increased 3.8-fold when 10.0* sphingomyelin per ml erythrocyte suspension produced approximately 95% hemolysis and were inhibited only by 40.0*g sphingomyelin. The hemolysin activity was inhibited by heating to 90-C but not to 70-C

Antimitotic action of extracts of Petiveria alliacea on sea urchin egg development

The hydroethanol extract of the roots of Petiveria alliacea L. (Phytolaccaceae) has been investigated previously as an antitumor agent against mouse Ehrlich ascites. The extract and its methanol, butanol and ether fractions exhibited an antimitotic effect on sea urchin egg development. The aqueous fraction did not produce inhibition of cell cleavage. At the first cleavage the inhibition, at the lowest concentration (10 micrograms/ml), produced by the ether fraction was 42 per cent , whereas the inhibition produced by the total extract and by the other fractions was only 5 to 10 per cent showing that the ether fraction was the most active. Even at higher concentrations the butanol and methanol fractions inhibit the cleavage about 30 per cent . At the first cleavage, the ED50 of the hydroethanol extract and of the ether fraction were 45.02 and 12.40 micrograms/ml, respectively. Furthermore, in the second cleavage, the hydroethanol extract was about twice as potent as the methanol or butanol fractions (ED50 of 22.40, 44.80 and 54.10 micrograms/ml, respectively)

Cytotoxic activity of a methanol extract of Phallusia nigra (Tunicata, Ascidiacea)

Tunicates have been reported to be a rich source of biologically active compounds. In this study, we demonstrate the presence of cytotoxic substances in Phallusia nigra, a common tunicate from Brazilian coastal waters. An extract of tunicate tissue was obtained by homogenizing the visceral organs from 50 specimens in methanol, followed by filtration and concentration in a rotary vacuum evaporator. Finally, the concentrate was partitioned with chloroform to remove lipids. The resulting extract possessed antimitotic and hemolytic activity. The former was demonstrated as a delay in the development of sea urchin eggs by partially inhibiting the process of cleavage (first cleavage, EC50 ñ SEM = 3.44 ñ 0.84 mg/ml). The <500 molecular fraction of the extract obtained by ultrafiltration also inhibited cell proliferation (the number of viable cells was decreased by 68 per cent with 500 mug/ml) and DNA synthesis of T47D cells derived from human breast carcinoma as measured by [3H]-thymidine incorporation (66 per cent of the control value after 24-h incubation with 100 mug/ml). Dose-dependent hemolysis obtained with P. nigra extract on mouse erythrocytes had an EC50 ñ SEM = 1.12 ñ 0.02 mglml for a 0.5 per cent erythrocyte suspension. Hemolysis could be reduced by pre-incubating the cells with choline-containing phospholipid. Sphingomyelin (40 mug/ml) increased the EC50 by twofold to 2.86 ñ 0.04 mg/ml, but phosphatidylcholine (80 mug/ml) did not modify hemolysis.