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.

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.

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.

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.

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.

The sea anemone purine, caissarone: adenosine receptor antagonism.

Caissarone, a sea anemone iminopurine, produced an increase in the twitch response of the electrically stimulated guinea-pig ileum-myenteric plexus. In the same assay, caissarone reduced the inhibitory response to the endogenous neuromodulator, adenosine, the A1 adenosine receptor agonist, R-phenylisopropyladenosine (R-PIA), and the A2 agonist, 5'-(N-cyclopropyl)-carboxamidoadenosine (CPCA) in a dose-dependent manner. Schild plot analysis of antagonism by caissarone yielded slopes of near unity, indicating that caissarone acts as a simple competitive antagonist at the adenosine receptor. The dissociation constants (KB) for caissarone ranged from 0.53 mM to 0.78 mM. In functional nicotinic receptor assays in two human cell lines, caissarone failed either to potentiate or to reduce carbamylcholine-mediated 86Rb+ efflux. Thus, the enhancing activity of caissarone on the gut could not be attributed to activity at the ganglionic nicotinic receptor. Based on structure and pharmacological activity, caissarone appears to be the first marine product described as an adenosine receptor antagonist.

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.

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.

Pharmacological evidence for the presence of a beta-adrenoceptor-like agonist in the amphinoid polychaete Eurythoe complanata.

1. Methanolic extracts from the body wall of Eurythoe complanata (ExEc) were tested for biological activity on the isolated rat ileum. 2. ExEc produced either relaxation or relaxation followed by contraction of the rat ileum in a concentration-dependent manner. 3. The predominant relaxation response to ExEc was completely blocked by the beta-adrenoceptor antagonist propranolol and was unaffected by the alpha-adrenoceptor antagonist phenoxybenzamine. 4. The results indicate that the relaxation induced by ExEc is mediated by beta-adrenoceptors. The presence of a myorelaxing substance in E. complanata that selectively activates the beta-adrenoceptors is suggested.

Neurobehavioral study of the effect of beta-myrcene on rodents.

Tea prepared from lemongrass (Cymbopogon citratus) is used for its supposed anxiolytic, hypnotic and analgesic properties in Brazilian folk medicine. beta-Myrcene, a major constituent of lemongrass, produces analgesia in rodents but there is some controversy about whether this action is central or peripheral or both. Rats and mice received beta-myrcene, 1 g/kg po in corn oil, or corn oil alone 1 h before being evaluated for a series of responses which included exploratory and emotional behavior, anxiolytic activity in a plus maze and inhibition of conditioned avoidance. No evidence was demonstrable for an effect of beta-myrcene on any of these behaviors. Similarly, beta-myrcene had no protective effect on pentylenetetrazol (PTZ)-induced seizures in mice. These data suggest that beta-myrcene has no benzodiazepine-like anxiolytic activity and that an activity on the central nervous system (antidepressive or antipsychotic) is unlikely. Despite the negative results of this study, folk use of lemongrass tea may still be justified by its analgesic properties.

Virulence factors of avian Escherichia coli.

A total of 45 strains of Escherichia coli isolates from chickens with colisepticemia were examined for virulence factors commonly found in pathogenic groups of E. coli. These strains were studied for the following: pathogenicity in 1-day-old chicks; toxin, hemolysin, and colicin production; cell invasiveness and adherence; hemagglutination for fimbriae detection; serum resistance; aerobactin production in iron-limited conditions; and plasmid content. The characteristics exhibited by virulent strains were invasion for HeLa and chicken fibroblast cells, serum resistance, colicin V, and aerobactin production. None of the isolates were toxigenic or positive in hemagglutination tests. The molecular genetic studies of the virulence factors by agarose electrophoresis showed that the plasmids of these strains are of high molecular weight.

Increase of mammalian intestinal motility by the iminopurine caissarone isolated from the sea anemone Bunodosoma caissarum.

The effects of caissarone (C8H11N5O) on guinea-pig ileum and rat duodenum preparations are reported. Caissarone evoked, at first, a small response, as compared with that of applied acetylcholine. This effect was blocked by atropine, hexamethonium and slightly reduced by tetrodotoxin. The main caissarone effect seems to be on the myenteric plexus as it increased spontaneous activity, tone and peristalsis; these effects being counteracted by tetrodotoxin, hexamethonium and atropine. Extracellular electrophysiological recording of the action potentials of myenteric plexus neurons showed an increase in frequency in the presence of caissarone. Caissarone potentiated the longitudinal muscle tension responses elicited by transmural electric stimulation in both control conditions, and when tension was reduced by pretreatment with tetrodotoxin or adenosine triphosphate. Caissarone also potentiated the longitudinal muscle tension responses to applied acetylcholine and this effect was reduced, but not abolished, by tetrodotoxin. Caissarone may have an indirect excitatory action on nicotinic receptors at the ganglia or may act as a reversible antagonist of purine transmitters released by neurons of the myenteric plexus involved in the physiological inhibition of intestinal motility.

Electrophysiology of the swimming system of hydromedusae and the effects of atropine-induced crumpling.

1. In order to evaluate how crumpling behavior influences the swimming system in Liriope tetraphylla, extracellular recordings of muscle action potentials related to both behaviors were performed. 2. The swimming pattern of Liriope consists of irregular bursts of muscle potentials. A correlation was obtained between number of muscle potentials and burst length and between burst and interburst periods. The swimming pattern could be predicted from the burst length since a long burst was followed by a short period of quiescence and by a burst as long as the preceding one. 3. The addition of atropine to induce radial muscle contraction first caused an increase in swimming activity and then an irreversible reduction. The enhancement of swimming activity was due to the increase of burst length and of the frequency of swimming bursts (due to the decrease of the interburst period), and the reduction of swimming activity, to the enhancement of the interburst period and to the reduction of the burst length. 4. It is proposed that, when facing a noxious stimulus, Liriope will first "escape" from it by enhancing its swimming activity and, if the stimulus persists, blockade of the pacemaker system becomes accentuated as the radial muscle contractions become more sustained.

Anomalies in sea-urchin egg development induced by a novel purine isolated from the sea-anemone Bunodosoma caissarum.

Caissarone (mol. wt 229.5; melting point 285-290 degrees C) is a novel purine isolated and purified from the sea-anemone Bunodosoma caissarum. The purine inhibits the detachment of the vitelline layer from the sea-urchin egg plasma membrane after fertilization and this effect leads to polyspermy. Various abnormalities were detected at various embryonic stages, from multipolar egg division through unequal cleavages and exogastrulation up to teratogenic effects on the sea-urchin larvae (echinopluteus).

Interactions of immunoglobulin G, fibrinogen and fibronectin with Staphylococcus hyicus and Staphylococcus intermedius.

Binding of immunoglobulin G, fibrinogen and fibronectin to 112 cultures of coagulase-positive staphylococci together with 7 of coagulase-negative S. hyicus subsp. chromogenes were investigated. Of the coagulase-positive staphylococcal cultures 45 were S. hyicus subsp. hyicus, 51 S. intermedius and 16 S. aureus. All 45 S. hyicus subsp. hyicus cultures coagulated plasma preparations from pigs and not always those from sheep, rabbits and dogs. Labelled IgG was bound by all cultures of S. hyicus subsp. hyicus and S. aureus, but only by 6 of 51 S. intermedius cultures. Fibrinogen interacted with 28 of the 45 S. hyicus subsp. hyicus cultures, with 17 of the 51 S. intermedius cultures and with S. aureus throughout. Fibronectin reacted with 19 cultures of S. hyicus subsp. hyicus, 11 of S. intermedius and all S. aureus. The binding activities for labelled IgG were more pronounced than those for fibrinogen and fibronectin. None of the 7 cultures of S. hyicus subsp. chromogenes bound any of these plasma proteins. Bindings of fibrinogen and fibronectin to S. hyicus subsp. hyicus and S. intermedius elicited only in part distinct clumping reactions of the staphylococci in the respective plasma proteins.

In vitro inactivation of the neurotoxic action of beta-bungarotoxin by the marine natural product, manoalide.

The irreversible neurotoxic action of beta-bungarotoxin (beta-BuTx) can be prevented by preincubation of the toxin with manoalide, a non-steroidal anti-inflammatory agent. Manoalide was also found to inactivate purified phospholipase A2 and thus prevent hydrolysis of phosphatidylcholine. PLA2 is a component found in several neurotoxic venoms and is also a rate limiting enzyme important in phospholipid metabolism and prostaglandin synthesis in man.

The responses of isolated preparations of Bunodosoma caissarum (Correa, 1964) (Cnidaria, Anthozoa) to drugs.

Body wall strips and oral rings of Bunodosoma caissarum respond to the application of at least four categories of drugs with sustained contractions. Propionylcholine, butyrylcholine and nicotine never failed to evoke a response; acetylcholine eventually was inactive. Occasionally, a correlation between dose and response was possible. Adrenaline and noradrenaline also elicited responses, although, occasionally, both were ineffective. Tryptamine and serotonin (less) were also effective. L-Glutamate was particularly effective and its action could be depressed by GABA. Histamine, betaine, taurine and aspartate were ineffective. The results are discussed in terms of depolarization due to specific membrane receptors or to unspecific causes.