Cardiovascular effects of lepadiformine, an alkaloid isolated from the ascidians Clavelina lepadiformis (Müller) and C. moluccensis (Sluiter).

The effects of lepadiformine, a natural marine alkaloid isolated from the ascidians Clavelina lepadiformis (Müller) and C. moluccensis (Sluiter), were studied in vivo by arterial blood pressure (aBP) recordings and electrocardiograms (ECG) in anaesthetised rats and in situ by peripheral vascular pressure recordings on perfused rabbit ear. Transmembrane resting (RP) and action (AP) potentials were also recorded by intracellular microelectrodes on electrically stimulated left ventricular papillary muscle and spontaneously beating atrium isolated from rat and frog hearts, respectively. Intravenous injection of lepadiformine (6mg/kg) produced marked bradycardia and a lengthening of ECG intervals as well as a transient decrease of aBP, which rapidly returned to normal. The decrease of aBP may have been related to a vasoconstrictor effect observed in the perfused ear experiment. Lepadiformine did not alter RP, but significantly lengthened the repolarising phase of AP in rat papillary muscle and frog atrium. Lepadiformine also mimicked the effect of Ba(2+) (0.2mM) on the rat AP repolarising phase. Moreover, the lengthening of the AP in frog atrium induced by lepadiformine still developed after the delayed outward K(+) current (I(K)) was blocked by tetraethylammonium (10mM). These observations suggest that lepadiformine-induced lengthening of AP duration was not due to a decrease of I(K), but may reasonably be attributed to a reduction of the inward rectifying K(+) current (I(K1)). This blockade of I(K1) could account for the cardiovascular effects of lepadiformine in vivo and in vitro and suggests that lepadiformine has antiarrhythmic properties.

Toxigenic saprophytic fungi in marine shellfish farming areas.

Toxigenic saprophytic fungi were isolated from samples of shellfish, sediment and seawater obtained from marine shellfish farming areas. The 456 strains identified included 12 different genera, with a clear predominance (68%) of Penicillium, Aspergillus, Trichoderma and Cladosporium. To assess the risk of poisoning due to the presence of these fungi in shellfish farming areas, the strains were cultured in liquid medium, filtered, and tested on larvae of Artemia salina, a small crustacean highly sensitive to mycotoxins. Thirty-five point five percent of the strains proved active with this test. This study confirms the existence of fungi in shellfish farming areas, as suggested by our earlier work showing that filter-feeding shellfish accumulate toxic metabolites of fungal origin. The presence of fungi in the marine environment represents a real risk of poisoning through the consumption of contaminated shellfish.

Toxicity of French strains of the dinoflagellate Prorocentrum minimum experimental and natural contaminations of mussels.

Mediterranean strains of Prorocentrum minimum do not appear to have the same toxic component as Japanese strains since they showed no cytotoxicity for hepatocytes in culture. However, their toxic components, which appear to block calcium channels, were detectable by the immobilisation test on Diptera larvae. A bio-accumulation experiment in the laboratory showed that the toxins could accumulate in nearly equivalent amounts in the hepatopancreas and meat of cultured mussels. The same toxicity was found in natural samples collected in a period of bloom of P. minimum. These results suggest that P. minimum could be responsible for shellfish toxicity in the natural environment and thus present a risk for human health.

Antiproliferative effects of a product isolated from the gorgonian Rumphella aggregata.

A fraction isolated from the gorgonian Rumphella aggregata (Plexauridae) was studied vitro on asynchronous cells of a human non-small-cell-bronchopulmonary-carcinoma line (NSCLC-N6). Cell growth appeared to be inhibited in the Gl phase of the cell cycle, and kinetic studies in pretreated cells showed that this growth arrest was irreversible. These events seem to show a terminal maturation induced by this new product.

Antifungal metabolites from the marine sponge Pachastrissa sp.: new bengamide and bengazole derivatives.

This paper reports the studies of components of an undescribed sponge in the genus Pachastrissa sp., collected along the Djibouti coast. The extract showed activity against Candida albicans. Six new bengazoles (1-6) and a new bengamide, named bengamide L (16), in addition to the known bengazoles (7-11), bengamides A (12), B (13), E (14), and F (15), and a lactone (17) are described in this paper. All structures were determined on the basis of spectroscopic studies.

Bioaccumulation of mycotoxins by shellfish: contamination of mussels by metabolites of a Trichoderma koningii strain isolated in the marine environment.

To determine whether toxic metabolites produced by fungi could cause shellfish toxicities, mussels were contaminated in laboratory conditions by sterile filtrates of a liquid culture of a strain of the fungus Trichoderma koningii previously isolated from a shellfish, the cockle (Cerastoderma edule). Mussels were kept in aerated natural seawater and fed with a culture of the microalga Isochrysis galbana, to which a filtrate of liquid fungal culture was added. Mussels were exposed to contamination for 7 days at 16 or 20 degrees C and extractions were then performed and their activity tested on blowfly larvae. The same toxicity was found in the fungal filtrate and the shellfish, indicating bioaccumulation. The digestive gland was the most toxic part of the mussel, confirming contamination by filtration. Treated mussels produced a mucus which appeared to be a means of eliminating toxic metabolites.

Combined use of analytical high-performance liquid chromatography and cell morphology transformation assay to detect new protein phosphatase inhibitors of okadaic acid type.

The search for new protein phosphatase inhibitors in shellfish contaminated by toxin-producing dinoflagellates generally relies on preliminary separation techniques followed by biological tests. To detect such substances without purifying them initially, we developed an approach based on a correlation of the results of two different analytical techniques applied to toxic extracts: high-performance liquid chromatography after derivation of the toxins and the cell morphology transformation assay on KB cells. Application of this protocol to stored frozen mussels showed a decrease in okadaic acid concentration during storage, with formation of degradation derivatives, some of which possessed notable protein phosphatase inhibition activity.

Specificity of the test based on modification of cell morphology for detection of lipophilic inhibitors of protein phosphatases.

The test based on morphological changes in KB cells was assayed with different toxins. Only lipophilic inhibitors of protein phosphatases, such as okadaic acid or calyculin A, induced visible changes in cell morphology. The activity of contaminated mussel extracts on KB cells was evaluated comparatively by direct interpretation of morphological changes and by a colorimetric method estimating the number of viable cells after staining. The latter technique revealed interferences (not detected by the former) with mussel cytotoxins. These results show that the technique, based on determination of the minimal active concentration of toxic extracts inducing morphological changes, is more specific, faster and preferable to the determination of IC50 for the detection of protein phosphatase inhibitors in shellfish.

Antiinflammatory mechanism of cordiachromene A action.

The mechanism of antiinflammatory action of cordiachromene A, isolated from the chloromethylenic extract of the ascidian Aplidium antillense or chemically synthesized, was studied using different in vivo and in vitro inhibition tests on enzymes of the cyclooxygenase cascade. Cordiachromene A inhibits prostacyclin synthesis and arachidonic acid metabolism but not phospholipase A2 and peroxidase. The mechanism of action, already known to be stereospecific, operates by cyclooxygenase inhibition.

Pachymatisma johnstonii extract opens ATP-sensitive potassium channels in cardiac myocytes of the frog heart.

The effects of a proteic extract (PachyTX), isolated from the sponge Pachymatisma johnstonii, were studied respectively with intracellular microelectrode and the "whole cell" patch clamp techniques on fibers and myocytes isolated from the sinoatrial region of frog heart. PachyTX significantly and reversibly shortened the action potential duration and decreased the stimulated peak contraction. Under voltage clamp, PachyTX increased, in a dose-dependent manner, a tetraethylammonium-insensitive but glibenclamide-inhibited outward current. This current failed to develop when adenosine 5'-triphosphate (ATP) was intracellularly applied to the cell by diffusion through the patch electrode. PachyTX mimics the effect of the ATP-sensitive K channel opener SR 44866 on frog atrial action potential and peak contraction. From this evidence, it can be concluded that PachyTX is a new natural potent opener of ATP-sensitive K channels in frog atrial heart muscle and that the opening of these channels is associated with the inhibition of the contraction of cardiac muscle.

Bistramides A, B, C, D, and K: a new class of bioactive cyclic polyethers from Lissoclinum bistratum.

The isolation and characterization is described of four novel cyclic polyethers, bistramides B [2], C [3], D [4], and K [5], which are closely related to the previously reported bistramide A [1] from the New Caledonian urochordata Lissoclinum bistratum. The structures of these metabolites were defined by spectroscopic methods. The four compounds exhibited in vitro cytotoxicity toward six tumor cell lines, including the human non-small cell lung carcinoma (NSCLC-N6) line. Cytofluorimetric analysis with bistramide K showed a complete block of NSCLC-N6 cells in the G1 phase. Bistramide D and particularly bistramide K are less toxic than bistramides A, B, and C and are thereby effective in vivo against NSCLC-N6.

Influence of Bistramide A on the twitch tension in rat atrial heart muscle.

Bistramide A, a new toxin isolated from the Urochordate Lissoclinum bistratum Sluiter, was applied to rat auricular heart muscle bundles. At a stimulation frequency of 0.2 Hz, the toxin induces a dose-dependent reduction of the stimulated twitch tension force; it decreases Vmax and shortens the duration of the plateau and the slow repolarizing phase of the action potential. In the control solution, switching from a stimulation frequency of 0.2 Hz to 1 Hz decreases the force with which a positive potentiation develops either at a maintained high frequency or after switching from 1 Hz to 0.2 Hz. Bistramide A reduces both the force evoked at 1 Hz and the potentiation. The data suggest that Bistramide A blocks Na+ conductance; inhibits Ca++ channels in a time- and frequency-dependent manner; reduces Na(+)-Ca++ exchange activity; but does not modify the ability of the sarcoplasmic reticulum to be refilled although the rate of Ca++ accumulation is decreased.

Chemotherapeutic inhibition of erb-B2 oncogene expression on a non-small-cell cancer line (NSCLC-N6) by marine substances.

The inhibitory effect of natural substances of marine origin on the erb-B2 oncogene of a human NSCLC-N6 line was demonstrated in vitro by simultaneous study of the expression of the gene and its product, using respectively an erb-B2 specific probe and an anti-c-erb-B2 polyclonal antibody. Preliminary results indicate inhibition ranging from 17-77% of oncogene expression and from 77-90% of product expression. The fact that substances of this type, with different chemical structures, have the common ability to induce terminal differentiation in an experimental model after irreversible blockade in G1 phase suggests a relationship between the inhibition of certain oncogenes and terminal differentiation.

Cytotoxic and antibacterial labdane-type diterpenes from the aerial parts of Cistus incanus subsp. creticus.

Seven labdane-type diterpenoids were isolated from the leaves of Cistus incanus subsp. creticus; their structures were established by spectroscopic means. All compounds were tested in vitro for their cytotoxicity against three cell line systems: KB, P-388; and NSCLC-N6. Their antibacterial and antifungal activities were tested against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosae, Enterobacter cloacae, Escherichia coli, Klebsiella pneumonia, Torulopsis glabrata, Saccharomyces cerevisiae, and Candida albicans as well.

Comparative study of the antitumor activity of bistramides A, D and K against a non-small cell broncho-pulmonary carcinoma.

Bistramides A, D and K are substances extracted from the marine ascidian Lissoclinum bistratum Sluiter that are capable of inducing in vitro terminal differentiation (G1DT) of cells from a non-small cell broncho-pulmonary carcinoma (NSCLCN6), but present different in vitro toxicities. This study shows that only the least toxic bistramides D and K possess an antitumor activity. These two substances could be administered as a continuous treatment which would induce terminal differentiation of stem cells at their entry into the cell cycle, thereby causing their destruction.

Alteration of the voltage-dependence of the twitch tension in frog skeletal muscle fibres by a polyether, Bistramide A.

Bistramide A, a toxin isolated from Lissoclinum bistratum Sluiter, was applied to frog skeletal muscle fibres. Micromolar concentrations of toxin decreased the amplitude of the twitch tension. Bistramide A shifted the activation and the steady-state inactivation characteristics of the tension on the voltage axis towards more positive and negative membrane potentials respectively. The data suggest that Bistramide A binds to the activated and inactivated state of the dihydropyridine-receptor which senses the T-tubule membrane potential.

Synthesis and antitumor activity of some new 2-chloroethylnitrosoureas.

The synthesis of a series of N-(2-chloroethyl)-N'-(9H-xanthen-9-yl)-N-nitrosoureas and N-(2-chloroethyl)-N'-(9H-thioxanthen-9-yl)-N-nitrosoureas is described. The title compounds were evaluated against NSCLCN6 L16 bronchial epidermoid carcinoma in vitro and some of them were found to be active. N-(2-chloroethyl)-N'-(2-methoxy-9H-xanthen-9-yl)-N-nitrosourea (8e) was active against leukemia P388 tumor system in mice.

Bioactive Diterpenoids Isolated from Dilophus ligulatus.

Nine diterpenoids, acetyldictyolal ( 1), epoxyoxodolabelladiene ( 2), dictyotalide B ( 3), neodictyolactono ( 4), pachylactone ( 5), the acetals 6A and 6B, isoacetoxycrenulatin ( 7), and dictyolactone ( 8) were isolated from the brown alga DILOPHUS LIGULATUS (Kütz.) Feldm. Their structures have been elucidated by comparison of mass, IR, (1)H- and (13)C-NMR spectra with reported literature data. Compounds 1, 2, 4, 7, and 8 exhibited antifungal activity. Cytotoxic activity against several types of mammalian cells (KB, P-388, P-388/DOX, and NSCLCN6-L16) in culture was examined for the 8 diterpenoids. Some of them showed stronger cyto-toxicity than mercaptopurine which was used as a positive control in this study.