Identification, isolation and characterization of tetracosapolyenoic acids in lipids of marine coelenterates.

Several tetracosapolyenoic acids (TPA) were detected in lipids of different marine coelenterates. Two of these acids were isolated and their structures were confirmed by chemical and spectral methods as all-cis-6,9,12,15,18-tetracosapentaenoic and all-cis-6,9,12,15,18,21-tetracosahexaenoic acid. Their distribution among lipids of a number of species of different classes of coelenterates from the northern and tropical seas, among neutral and polar lipids of these organisms was investigated. Significant quantities of TPA were found in all of the Octacorallia species studied. In some cases the sum of TPA reaches the level of 20% of total lipid fatty acids. The fatty acid composition of different coelenterates is also discussed.

Biochemical and pharmacological studies of the mechanism of action of tenebrosin-C, a cardiac stimulatory and haemolytic protein from the sea anemone, Actinia tenebrosa.

Tenebrosin-C is a protein of mol. wt 19,500 that displays potent cardiac stimulatory and haemolytic activities. Its haemolytic activity is inhibited by sphingomyelin but not phosphatidylcholine, and is not affected by Ca2+. The positive inotropic effect of tenebrosin-C on isolated guinea pig right atria is inhibited by the cyclooxygenase blockers indomethacin and aspirin, the lipoxygenase blocker and leukotriene antagonist RG5901, and the phospholipase A2 inhibitor mepacrine. This activity of tenebrosin-C therefore appears to be due to stimulation of the release of arachidonic acid and subsequent formation of prostaglandins and leukotrienes. Phospholipase A2-like activity was found with some tenebrosin-C preparations, but did not correlate with their positive inotropic or haemolytic activities and was too weak to account for either of these effects. Treatment of tenebrosin-C with various proteases in order to obtain active fragments showed that the protein is remarkably resistant to proteolysis.

Paciforgine: a new macrolide antifungicidal isolated from Paciforgia sp-2.

A new macrolide, paciforgine, has been isolated from Paciforgia sp.-2. The structure of the new macrolide was determined by spectrometric methods. Paciforgine is active in vitro against Trichophyton verrucosum and Candida albicans.

[Determination of calcium carbonate, trace elements and amino acids in Os Sepiae].

This paper reports the determination methods for calcium carbonate, trace elements and amino acids in the traditional Chinese medicine Os Sepiae. The data provide a reference for the quality control of the drug.

New antitumor promoters: (-)-epigallocatechin gallate and sarcophytols A and B.

EGCG, the main constituent of green tea, and sarcophytols A and B, isolated from a soft coral, inhibited tumor promotion by teleocidin in a two-stage carcinogenesis experiment on mouse skin. EGCG and sarcophytols showed inhibition of tumor development by chemical carcinogenesis. A possibility of developing these compounds as cancer chemopreventives for human beings is discussed.

A new spermatostatic glycoside from the soft coral Sinularia crispa.

The steroid glycoside 1 has been isolated from the Sri Lankan soft coral Sinularia crispa. The structure has been determined by spectroscopic (1H and 13C nmr) techniques. Glycoside 1 showed spermatostatic activity on rat cauda epididymal spermatozoa.

New phospholipid fatty acids from the Caribbean sponge Ectyoplasia ferox.

The phospholipid fatty acids from the Caribbean sponge Ectyoplasia ferox were studied. The novel fatty acids 25-methyl-5,9-heptacosadienoic (1) and 26-methyl-5,9-heptacosadienoic (2) were identified in 3.4 and 2.0% abundance, respectively, representing the longest set of delta 5,9 iso and anteiso acids yet isolated from a marine sponge. The new acid 10,13-dimethyltetradecanoic (3), the unusual acid 15-methyl-11-hexadecenoic (4) and the also novel acid 9-methyl-11-hexadecenoic (5) were also identified in E. ferox. The principal sterols isolated from E. ferox were 24-ethylcholest-5-en-3 beta-ol (46%) and 24(R)-methylcholesta-5,22-dien-3 beta-ol (14%).

[Anti-tumor natural products isolated from marine organisms].

Marine organisms comprise over half a million species. Due to their unusual living environment as compared with terrestrial organisms, marine organisms produce a variety of metabolic substances which often have various unprecedented chemical structures. In recent years, an increasing number of marine natural products have been reported to exhibit various biological activities such as antimicrobial, physiological, and pharmacological ones. Some metabolites have also been noted by their significant toxicities. The effort to find out anti-tumor substances from marine organisms has been also undertaken in recent years, and several novel compounds (e.g. peptide, polyether, alkaloid, prostanoid etc.), with anti-tumor activities, have been isolated from marine sponges, octocorals, marine algae, tunicates, nudibranchs, bryozoans, and so on. In this article, those chemically clarified anti-tumor compounds isolated from marine organisms were reviewed.

Isolation of less than Glu-Gly-Leu-Arg-Trp-NH2 (Antho-RWamide II), a novel neuropeptide from sea anemones.

Using a radioimmunoassay for the peptide sequence Arg-Phe-NH2 (RFamide), a novel peptide has been purified from acetic acid extracts of the sea anemone Anthopleura elegantissima. This peptide has the structure less than Glu-Gly-Leu-Arg-Trp-NH2, and was named Antho-RWamide II. Antho-RWamide II is a neuropeptide. Its structure is closely related to an earlier characterized neuropeptide from Anthopleura less than Glu-Ser-Leu-Arg-Trp-NH2 (Antho-RWamide I).

Morphological, cytochemical and neuropharmacological evidence for the presence of catecholamines in hydrozoan planulae.

Planula larvae of Halocordyle disticha were examined for the presence of catecholamines using a multipronged approach. Transmission electron micrographs of planular sensory cells and ganglionic cells demonstrated dense-cored vesicles and electron-dense droplets in both cell types. These vesicles and droplets were similar in morphology to catecholamine-containing granules of vertebrates. Planulae processed with the SPG histofluorescence technique, specific only for catecholamines, exhibited blue-green fluorophores which were most prominent in the anterior ectoderm. Such fluorescence was associated with sensory cells, ganglionic cells and the neural plexus. Pretreatment of planulae with neuropharmacological agents which prevent reuptake (reserpine) or cause release (nicotine, ephedrine) of catecholamines caused a diminution of the fluorophores. Pretreatment of animals with 6-hydroxydopamine, which causes destruction of catecholamine-containing cells, prevented any fluorescent response. Ultrastructural examination of reserpine-treated planulae revealed a dramatic reduction in the populations of dense-cored vesicles and electron-dense droplets. Furthermore, many of the vesicles and droplets remaining in reserpinized animals appeared washed out, i.e. stained faintly. Exposure of planulae to exogenous norepinephrine caused premature, rapid metamorphosis and produced polyps with slightly stunted tentacles and pitted, irregular hypostomes. Exposure of planulae to nicotine caused similar effects. Rearing planulae in sea water containing alpha blockers, phentolamine and tolazoline, had no discernible effect on behaviour (motility, phototactic response) or gross morphology. However, planulae raised in sea water containing propranolol, a beta blocker, ceased all movement, became tack-shaped and died within 72 h. These results meet multiple criteria for the identification of catecholamines in hydrozoan planulae and suggest that such catecholamines may function as neurotransmitters, neurohormones or neuromodulators during larval development.

Isolation and characterization of three lethal and hemolytic toxins from the sea anemone Actinia equina L.

Lethal and hemolytic toxins were purified by acetone precipitation, Sephadex G-50, CM-cellulose and CM-Sephadex column chromatography from the tentacles and bodies of the sea anemone Actinia equina. The isolated toxins, with a mol. wt of 19,000 determined by SDS-polyacrylamide gel electrophoresis, differed only slightly in amino acid composition and had a high tryptophan content. The isoelectric points were estimated to be 9.8 for equinatoxin I and 10.5 for equinatoxins II and III. The pure toxins exhibited high lethal potency; the acute i.v. LD50 in mice of equinatoxins I, II and III were 23, 35 and 83 micrograms/kg, respectively. The sigmoidal time course of hemolysis is characteristic of toxins.

Phylogenetic study of the oxytocin-like immunoreactive system in invertebrates.

1. A phylogenetic study of oxytocin (OXT)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Oncidium verrucosum, Limax marginatus and Meretrix lamarckii of the Mollusca; and Baratha brassica of the Arthropoda. 3. No immunoreactive cells were found in Bipalium sp. of the Platyhelminthes; Pomacea canaliculata, Aplysia kurodai, Bradybaena similaris and Achatina fulica of the Mollusca; and Gnorimosphaeroma rayi, Procambarus clarkii, Hemigrapsus sanguineus, Helice tridens and Gryllus bimaculatus of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 4. These results demonstrate that an OXT-immunoreactive substance is widely present not only in vertebrates but also in invertebrates. 5. OXT seems to have been introduced into these invertebrates at an early stage of their phylogenetic history.

Phylogenetic study of the arginine-vasotocin/arginine-vasopressin-like immunoreactive system in invertebrates.

1. A phylogenetic study of arg-vasotocin (AVT)/arg-vasopressin (AVP)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Pomacea canaliculata, Aplysia kurodai, Oncidium verrucosum, Bradybaena similaris, Achatina fulica, Limax marginatus and Meretrix lamarckii of the Mollusca; Gnorimosphaeroma rayi, Hemigrapsus sanguineus, Gryllus bimaculatus and Baratha brassicae of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 3. No immunoreactivity was detected in Bipalium sp. of the Platyhelminthes, or in Procambarus clarkii and Helice tridens of the Arthropoda. 4. From these results, it appears that AVT/AVP is a phylogenetically ancient peptide which is present in a wide variety of invertebrates. 5. The actions of AVT/AVP and its presence in invertebrates are discussed.

A D-mannose-specific lectin from Gerardia savaglia that inhibits nucleocytoplasmic transport of mRNA.

A new lectin has been isolated from the coral Gerardia savaglia by affinity chromatography, using locust gum as an absorbent, and D-mannose as eluant. Final purification was achieved by Bio-Gel P300 gel filtration. The agglutinin is a protein composed of two polypeptide chains with a Mr of 14800; the two subunits are not linked by disulfide bond(s). The isoelectric point is 4.8, the amino acid composition is rich in the acidic amino acids aspartic acid and glutamic acid. The absorption maximum for the protein was at 276 nm; with a molar absorption coefficient of 1.27 X 10(5) M-1 cm-1. The lectin precipitated erythrocytes from humans (A, B and O), sheep, rabbit and carp with a titer between 2(5) and 10(10); the affinity constant for lectin binding to sheep red blood cells was 2.8 X 10(8) M-1 and the number of binding sites, 3.2 X 10(5)/cell. Ca2+ ions are required for full activity; the pH optimum lies in the range between 6 and 11. Inhibition experiments revealed that the lectin is specific for D-mannose. The lectin is mitogenic only for those spleen lymphocytes from mice which had been activated by lipopolysaccharide. An interesting feature of this lectin is its ability to bind to glycoproteins present in nuclei from CV-1 monkey kidney cells. The fluorescein-isothiocyanate-labelled lectin reacted with six polypeptides in the nuclear envelope from rat liver (Mr 190,000, 115,000, 80,000, 62,000, 56,000 and 42,000) and with two polypeptides in the nuclear matrix or pore complex lamina fraction (Mr 190,000 and 62,000). The lectin inhibited the nuclear envelope mRNA translocation system in vitro. It is suggested that this effect is due to an interaction of the lectin with the nuclear glycoproteins gp190 and/or gp62.

Tenebrosin-A, a new cardiostimulant protein from the Australian sea anemone Actinia tenebrosa.

A new cardiac stimulatory protein, tenebrosin-A, has been isolated from the Australian sea anemone Actinia tenebrosa by gel filtration and cation-exchange chromatography, followed by cation-exchange HPLC. Its purity is established by analytical reversed-phase HPLC and N-terminal sequence analysis. According to SDS-PAGE, its apparent Mr is 20,000 daltons. Amino acid analysis indicates that it contains 186 residues, and is devoid of cysteine or cystine. Tenebrosin-A exerts a strong positive inotropic effect on isolated guinea pig atria at a concentration of 1.4 nM, with little chronotropic activity.

2,3-Dihydrolinderazulene, a new bioactive azulene pigment from the gorgonian Acalycigorgia sp.

A new azulene pigment, 2,3-dihydrolinderazulene, has been isolated along with guaiazulene and linderazulene as bioactive metabolites from the gorgonian Acalycigorgia sp.

The amino acid sequence of toxin RpIII from the sea anemone, Radianthus paumotensis.

The amino acid sequence of the sodium channel toxin RpIII from the sea anemone Radianthus paumotensis has been determined. The protein is homologous with five analogous toxins from three anemone species, and is most similar to a less toxic protein, RpII, from the same organism. Twelve residues are conserved in all six toxins, one of which is an arginine residue thought to be essential for toxicity. The others (Cys, Gly, Pro and Trp) tend to be conserved in other sets of homologous proteins to maintain functional folds. Comparisons of the sequences suggest the existence of two separate but related classes of toxins cumon the three species of anemone.

Separation of two myotoxins from nematocysts of the box jellyfish (Chironex fleckeri).

Two myotoxins, both lethal to mice by i.v. injection, were obtained by chromatography on Sephadex G-200 of material released from isolated microbasic mastigophores of C. fleckeri. Both toxins elicit contractures of skeletal (diaphragm) musculature of the rat and of smooth (ileum and vas deferens) and atrial musculature of the guinea-pig, although consistent differences in the parameters of the contractures of each muscle type elicited by the two toxins are shown. Moreover, one toxin, with a molecular weight of approximately 150,000, also elicits activity on crustacean (barnacle) musculature, whilst the other toxin, with a molecular weight of approximately 600,000, elicits no activity in barnacle musculature at the concentrations tested. The toxins are labile when released from nematocysts and they lose all myotoxic activity within 3 days at 5 degrees C. They can also be isolated chromatographically from crude extracts of the contents of mixed nematocysts of C. fleckeri. They are considered to be the principal toxins injected by C. fleckeri during nematocyst discharge and appear to be different from the C. fleckeri toxins described by other workers.