Antifouling activity of synthetic polymer gels against cyprids of the barnacle (Balanus amphitrite) in vitro.

Barnacle (Balanus amphitrite) settlement on synthetic hydrogels with various chemical structures was tested in laboratory assays. The results demonstrated that cyprids settle less or not at all on hydrogels and PDMS elastomer compared with the polystyrene control. The low settlement on gels is most likely due to the 'easy release' of initially attached cyprids from the gel surfaces. This low adhesion of cyprids is independent of surface hydrophilicity or hydrophobicity, and of surface charge. The results also revealed that hydrogels can be categorized into two groups. One group showed an extremely strong antifouling (AF) performance that was independent of the elasticity (E) or swelling degree (q) of the gels. The second group showed relatively less strong AF performance that was E- or q-dependent. In the latter case, E, rather than the q, may be the more important factor for cyprid settlement.

Sex pheromones of the hair crab Erimacrus isenbeckii. II. Synthesis of ceramides.

To confirm their structures and to assess the pheromonal activity, novel ceramides, possible sex pheromones of the hair crab Erimacrus isenbeckii, were synthesized from D-galactose. The synthetic ceramides were identical with the natural ceramides.

Two new antimicrobial lysoplasmanylinositols from the marine sponge Theonella swinhoei.

Two new antimicrobial lysoplasmanylinositols have been isolated from the marine sponge Theonella swinhoei. Their structures were elucidated on the basis of spectroscopic and chemical analysis.

Dogger Bank Itch revisited: isolation of (2-hydroxyethyl) dimethylsulfoxonium chloride as a cytotoxic constituent from the marine sponge Theonella aff. mirabilis.

(2-Hydroxyethyl) dimethylsulfoxonium chloride (1), the well-known causative agent of Dogger Bank Itch, has been isolated as a cytotoxic constituent from the marine sponge Theonella aff. mirabilis. The structure of 1 was determined by spectral and X-ray crystallographic analyses.

Membrane permeabilization induced by discodermin A, a novel marine bioactive peptide.

The effects of discodermin A (DC-A), a novel marine bioactive peptide extracted from sea sponge Discodermia kiiensis, on the vascular smooth muscle cells and tissues were examined. Analysis with a confocal laser microscope showed that DC-A (0.1-30 microM) permeabilized the plasma membrane of A10 cells to the non-permeable fluorescent agents, ethidium homodimer-1 (MW = 857) and calcein (MW = 623), in a concentration-dependent manner. In the vascular tissue treated with 30 microM DC-A, addition of a micromolar concentration of Ca(2+) evoked a sustained contraction in the presence of ATP, suggesting that DC-A increased the permeability of the membrane to Ca(2+) and ATP. DC-A at higher concentrations (30 microM) significantly increased the leakage of lactate dehydrogenase (140 kD) from the vascular tissue. These results suggest that DC-A has a permeabilizing effect on the plasma membrane possibly by interacting with plasma membrane phospholipids with its six successive hydrophobic amino acid residues at N-terminal.

Four new bioactive pyrrole-derived alkaloids from the marine sponge Axinella brevistyla.

Four new alkaloids (1-4) were isolated from the marine sponge Axinella brevistyla, and their structures were determined on the basis of spectroscopic analysis. The alkaloids 1-4 were antifungal against the yeast Saccharomyces cerevisiae at <1.0, <1.0, 30, and 100 microg/disk, respectively. Compounds 1-3 also exhibited cytotoxicity against L1210 cells with IC(50) values of 1.1, 0.66, and 2.5 microg/mL, respectively.

Effect of calyculin A on the surface structure of unfertilized sea urchin eggs.

Calyculin A, a potent inhibitor of type 1 and type 2A protein phosphatases, induces contractile ring formation when applied to unfertilized sea urchin eggs [Tosuji et al., 1992: Proc. Natl. Acad. Sci. USA 89:10613-10617]. We report here the elongation of microvilli in the unfertilized eggs exposed to calyculin A. The elongated microvilli and associated sperm-egg binding sites (egg receptor for sperm) then became concentrated into a constriction site corresponding to the cleavage furrow. The egg receptor for sperm was also in close connection to the microfilaments. Okadaic acid is another known inhibitor of protein phosphatase type-1 and type-2A. Its effect, however, is about a hundredfold feebler for type-1 phosphatase than type-2A. Even after treatment with okadaic acid, no change was observed, suggesting that these morphological changes were induced by calyculin A solely though its inhibitory effect on the type-1 protein phosphatase.

Hachijodines A-G: seven new cytotoxic 3-alkylpyridine alkaloids from two marine sponges of the genera Xestospongia and amphimedon.

Seven cytotoxic 3-alkylpyridine alkaloids, hachijodines A-G, have been isolated from two marine sponges of the genera Xestospongia and Amphimedon. Their structures were determined on the basis of spectral data. These alkaloids are moderately cytotoxic against P388 murine leukemia cells with IC(50) values of 1.0-2.3 microg/mL.

An antimicrobial C(14) acetylenic acid from a marine sponge Oceanapia species.

A C(14) acetylenic acid has been isolated as an antimicrobial principle from a marine sponge Oceanapia sp. Its structure was determined on the basis of spectral data.

Species specificity of barnacle settlement-inducing proteins.

We previously isolated a larval settlement-inducing protein complex (SIPC) from adult extracts of the barnacle, Balanus amphitrite using a nitrocellulose membrane settlement assay. In the present study, we found that the extracts of other adult barnacles, Megabalanus rosa and Balanus eburneus, also induced the settlement of B. amphitrite cyprids although the inductive activity was slightly lower than that of conspecific extracts. Furthermore, we examined reactivity to anti-SIPC antibody in adult extracts from six species of Japanese barnacles other than B. amphitrite, brine shrimp and eight marine sessile organisms besides barnacles. The results showed that all barnacles examined contained SIPC-like proteins with slightly different molecular weight, while the other animals did not react to the antibody by immunoblot analysis. These findings suggest that species specificity in settlement-inducing proteins of barnacles is not so strict, but these proteins are characteristic to barnacle species.

Interaction of Cytotoxic Bicyclic Peptides, Theonellamides A and F, with Glutamate Dehydrogenase and 17beta-Hydroxysteroid Dehydrogenase IV.

Theonellamide A, a bicyclic peptide isolated from a Theonella sponge, was fixed on hydrazide-containing gel beads and screened for its binding proteins from rabbit liver tissues. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that two major proteins of 80 kDa and 55 kDa interacted with theonellamide A. The interaction between theonellamide A and two proteins was confirmed by competition experiments in which these two proteins failed to bind to theonellamide A-conjugated gel beads in the presence of theonellamide A or F. Amino-terminal amino acid sequence analysis of peptide fragments derived from the binding proteins by lysylendopeptidase digestion demonstrated that the 80-kDa and 55-kDa proteins were 17beta-hydroxysteroid dehydrogenase IV and glutamate dehydrogenase, respectively. In an in vitro assay system, amination of alpha-ketoglutarate by glutamate dehydrogenase was activated with theonellamide F, although this effect was weaker than that with adenosine diphosphate, a well-known activator.

Assessment of the marine toxins by monitoring the integrity of human intestinal Caco-2 cell monolayers.

The effects of marine substances with various cytotoxic mechanisms on the integrity of the human intestinal Caco-2 cell monolayer were examined by measuring the transepithelial electrical resistance (TEER). TEER was rapidly decreased by apical exposure of the monolayers to discodermin A, a membrane pore-forming substance. The decrease in TEER occurred in an earlier stage of incubation than the release of intracellular lactate dehydrogenase (LDH) which is commonly used as a parameter of cell damage or death. Mycalolide B (an actin-depolymerizing substance), calyculin A and okadaic acid (protein phosphatase inhibitors) also rapidly decreased the TEER value, although no cell membrane damage or resultant LDH release by these toxicants were detected. The TEER decrease caused by the toxicants was associated with the increased transepithelial permeability of the cell monolayer. Treatment with these toxicants, except calyculin A, caused morphological changes in the intracellular actin filament, suggesting that these toxicants altered the cytoskeletal structure, by which the tight junction was opened. Calyculin A was likely to loosen the cellular junctions rapidly and induce cell detachment from the monolayer. Although onnamide A, a protein synthesis inhibitor, did not cause any decrease in TEER, at least during a 90-min incubation, TEER sensitively reflects the cytotoxic effects of various types of toxicants with acute toxicity.

New isomalabaricane triterpenes from the marine sponge Stelletta globostellata that induce morphological changes in rat fibroblasts.

Three new isomalabaricane triterpenes, 29-hydroxystelliferin D (2), 3-epi-29-hydroxystelliferin E (3), and 3-epi-29-hydroxystelliferin A (4), were isolated from the marine sponge Stelletta globostellata. Their structures, including absolute stereochemistry, were determined on the basis of spectral data and chemical methods. Rat fibroblasts treated with 0.2 microM of 2-4 exhibited unusual morphological characteristics, followed by death in 5 days.

Four new steroids from two octocorals

In search of analogues of isogosterones A-D (1-4), a group of antifouling 13,17-seco-steroids found in octocorals of the order Alcyonacea, we have isolated four new steroids possessing aromatic, enone, or dienone A-rings from two octocorals, Alcyonium gracillimum and Dendronephthya sp. These compounds, 3-methoxy-19-norpregna-1,3, 5(10),20-tetraene (5), 3-(4-O-acetyl-6-deoxy-beta-galactopyranosyloxy)-19-norpregna-1,3, 5(10),20-tetraene (6), 22,23-dihydroxycholesta-1,24-dien-3-one (7), and methyl 3-oxochola-4,22-dien-24-oate (8), showed no antifouling activity against barnacle (Balanus amphitrite) larvae, but lethality to barnacle larvae at a concentration of 100 &mgr;g/mL (LD(100)).

Lumichrome. A larval metamorphosis-inducing substance in the ascidian Hhalocynthia roretzi.

It has long been known that metamorphosis of ascidian larvae is induced by exposure to adult tunic extract or larval-conditioned seawater. However, such a natural 'inducer' has not been identified, probably due to its very low concentration in organisms. Here we have succeeded in isolating the same metamorphosis-inducing substance from the larvae, the larval-conditioned seawater, and the adult tunic of the ascidian Halocynthia roretzi. Structural analysis revealed that this substance was identical to lumichrome. Lumichrome was active toward H. roretzi larvae, but inactive toward another ascidian larvae, suggesting that lumichrome is species-specific. Riboflavin (vitamin B2), from which lumichrome might be derived from, was found to be inactive in induction of larval metamorphosis. In addition, it was demonstrated that lumichrome is localized predominantly in the basal region of the adhesive organ and the posterior part of the larval trunk. Thus, we propose that lumichrome functions as a natural inducer for larval metamorphosis in H. roretzi. This is the first natural metamorphosis-inducing substance to be identified in ascidians.

Theonellamide F, a Bicyclic Peptide Marine Toxin, Induces Formation of Vacuoles in 3Y1 Rat Embryonic Fibroblast.

: The effect of a marine bicyclic peptide, theonellamide F, on vacuole formation in exponentially growing 3Y1 rat embryonic fibroblasts was studied in comparison with the effect of monensin. Many abnormally large vacuoles appeared around the nucleus in the cells treated with 6 µM theonellamide F within 24 hours. Following prolonged treatment with this agent, the number of enlarged vacuoles increased. Such vacuoles accumulated many granules that showed Brownian movement. The effect of theonellamide F on the cells was more drastic in an amino-acid-deficient medium, in which all cells died within 1 hour at a 3-µM concentration of the agent. Theonellamide F probably affects cellular autophagy, inhibiting the degradation of the organelles and turnover of proteins. Monensin, a well-known Na(+) ionophore that disintegrates the Golgi apparatus, induced similar types of vacuole formation, although the vacuoles were localized in a region slightly distant from the nucleus. Monensin readily affected cell morphology, resulting in cell death. We propose that theonellamide F, like monensin, is a useful agent for investigating membrane structures in cells.

On topography and functionality in the B-D rings of cephalostatin cytotoxins.

Analogues 12'beta-hydroxycephalostatin 1 (9), 7'-deoxyritterazine G (10), and 14-epi-7'-deoxyritterazine B (11) were prepared via our protocol for unsymmetrical pyrazine synthesis. Cytotoxicity against human tumors was also determined for the first time for ritterazines, with femtomolar potency and a high correlation to cephalostatins observed. The SAR of these and related compounds provide insight into the importance of topography and certain chemical functionality in the B-D and B'-D' rings of cephalostatin type antineoplastics.

Three new antibacterial alkaloids from a marine sponge Stelletta species.

From a marine sponge of the genus Stelletta we isolated three alkaloids, stellettazole B (3), stellettazole C (4), and stellettamide C (5), which were antibacterial against Escherichia coli. Their structures were determined on the basis of spectral and chemical methods.

Aurantosides D, E, and F: new antifungal tetramic acid glycosides from the marine sponge Siliquariaspongia japonica.

Aurantosides D-F (4-6), new polyene tetramic acids comprising an N-trisaccharide unit, have been isolated from the marine sponge Siliquariaspongia japonica. Their structures were determined by spectral and chemical methods. A reinvestigation of NMR data of the previously isolated aurantosides A and B led to revision of the geometry of the terminal double bond. Aurantosides exhibit potent antifungal activity against Aspergillus fumigatus and Candida albicans.

The solution structures of calyculin A and dephosphonocalyculin A by NMR.

The NMR solution structure of calyculin A (1) in chloroform exhibits intramolecular interactions, resembling the original crystal structure. In methanol, calyculin A has the hydrogen bonding moieties solvent exposed. Dephosphonocalyculin A in chloroform resembles calyculin A in chloroform and the crystal structure of calyculin A. Dephosphonocalyculin A in methanol resembles calyculin A in methanol.