Biomedical subjects
Publications and source records attributed to N Fusetani.
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.
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)).
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.
Immunological investigation has revealed that a settlement-inducing protein complex (SIPC), which induces cypris settlement of the barnacle Balanus amphitrite, is synthesized during larval development and accumulates in the cypris larva. We previously purified the SIPC from adult B. amphitrite, which was active when bound to a substratum. The SIPC is a glycoprotein of high molecular mass, consisting of three major subunits of 76, 88 and 98 kDa with lentil lectin (LCA)-binding sugar chains. In the present study, we prepared antiserum against each LCA-binding subunit of SIPC, and performed immunoblot analyses. Immunoblotting of adult extracts showed that anti-76-kDa antibody reacted only with the 76-kDa protein, whereas anti-88-kDa and anti-98-kDa antibodies reacted with both the 88-kDa and the 98-kDa proteins. Immunoblotting of larval extracts indicated that reactivity of the 76-kDa protein to anti-76-kDa antiserum increased during larval development and cyprid extracts reacted strongly. Moreover, by using immunostaining we found that the SIPC was contained in 'footprints' of cyprids, which have been shown to act as a settlement-inducing pheromone, and is secreted onto the antennular attachment discs. The results suggest that the SIPC (or SIPC-like proteins) is involved in both adult-larva and larva-larva interactions during settlement of the barnacle B. amphitrite.
The effects of extended swimming on short-lived lecithotrophic larvae of the marine bryozoan Bugula neritina (L.) were examined. Larvae were forced to swim for 2 or 24 h by bath application of serotonin. Settlement and metamorphosis success were significantly reduced, larval dimensions were unaffected and ancestrulae were smaller after 24 h of swimming. Larvae settled predominantly on seawater-conditioned glass after 2 h, but became less discriminative after 24 h. Lipid content in intact larvae and dissociated surface ciliated and interior cell fractions was analysed by thin-layer chromatography. Hydrophilic lipids were unaffected by swimming regime. The hydrophobic fraction contained triglyceride, confirmed by proton nuclear magnetic resonance spectroscopy (1H-NMR) analysis and correlation spectroscopy (1H-1H COSY) patterns, which was significantly depleted after 24 h, and diacylglycerol, which was not. NMR spectra suggested no differences in fatty acid chain compositions between larvae swimming for 2 and 24 h. Triglyceride depletion was limited to the ciliated cell fraction. We propose that the functional partitioning of lipid reserves has evolved in association with the costs and benefits linked with larval dispersal.
During our study on signal transduction systems involved in larval settlement and metamorphosis of the barnacle Balanus amphitrite, we could detect calmodulin, a major intracellular calcium-binding protein, in both cyprids and adult barnacles using immunoblot technique. In order to examine roles of calmodulin in larval settlement and metamorphosis, we examined the effects of calmodulin inhibitors involved in phosphodiesterase (PDE), myosin light chain kinase (MLCK), calmodulin kinase II (CaM-II) on cyprids. Calcium-calmodulin-dependent PDE inhibitors inhibited larval attachment and metamorphosis in a dose-dependent manner. In contrast, cyclic AMP (cAMP)-dependent PDE inhibitors promoted larval attachment and metamorphosis, while cyclic GMP (cGMP)-dependent PDE inhibitors did not show such effect. Thus, PDE activity may depend on cAMP rather than calmodulin or cGMP in promotion of larval attachment and metamorphosis. Moreover, calmodium inhibitors involved in MLCK or CaM-II inhibited larval behaviors for attachment and metamorphosis. These results suggest that calmodulin, which is present in both adult barnacles and cyprids, may be involved in enzyme reactions such as MLCK or CaM-II rather than PDE in B. amphitrite cyprids.
Two new cytotoxic trisoxazole macrolides, thiomycalolides A (2) and B (3), have been isolated from a marine sponge Mycale sp. The structures were determined to be glutathione adducts of mycalolides A and B by interpretation of spectral data and chemical transformation. Thiomycalolides A and B are highly cytotoxic against P388 murine leukemia cells.
1-Carboxymethylnicotinic acid (1) has been isolated from a marine sponge Anthosigmella cf. raromicrosclera as a cysteine protease inhibitor. The structure was elucidated by spectral data and chemical synthesis.
Two new potent serine protease inhibitors, cyclotheonamides E2 (3) and E3 (4), have been isolated from a marine sponge of the genus Theonella. Their structures were determined by interpretation of spectral data and chemical degradation studies. They are closely related to the previously reported cyclotheonamide E, from which they differ in the N-acyl group of the alanyl side chain. Cyclotheonamides E, E2, and E3 were more active against thrombin than against trypsin.
Three new macrolides, 30-hydroxymycalolide A (4), 32-hydroxymycalolide A (5), and 38-hydroxymycalolide B (6), were isolated from the marine sponge Mycale magellanica. Their structures were assigned on the basis of spectroscopic data. They were cytotoxic against L1210 cells with IC50 values of 0.019, 0.013, and 0.015 microg/mL, respectively. Chemical interconversion of the known mycalolides A-C (1-3) with 4-6 established their stereochemical relationships.
Ten new steroidal sulfates, acanthosterol sulfates A-J (1-10), have been isolated from a marine sponge, Acanthodendrilla sp., collected in western Japan. Acanthosterol sulfates I and J (9 and 10) showed antifungal activity against the yeast Saccharomyces cerevisiae A364A and its mutants at 0.1 mg/disk.
We compared the effects of dimeric marine toxins, bistheonellide A, and swinholide A, on actin polymerization. Bistheonellide A and swinholide A possess two identical side chains with similar structures to those of other marine toxins, mycalolide B, and aplyronine A. By monitoring changes in fluorescent intensity of pyrenyl-actin, bistheonellide A was found to inhibit polymerization of G-actin and to depolymerize F-actin in a concentration-dependent manner. The relationship between the concentration of bistheonellide A and its inhibitory activity on actin polymerization suggested that one molecule of bistheonellide A binds two molecules of G-actin. We demonstrated by SDS-PAGE that the complex of G-actin with bistheonellide A, swinholide A, or mycalolide B could not interact with myosin. No evidence was found that bistheonellide A severs F-actin at the concentrations examined (molar ratio to actin; 0. 025-2.5), while swinholide A showed severing activity, although it was weaker than that of mycalolide B. We also demonstrated that the depolymerizing effect of bistheonellide A or mycalolide B is irreversible. Bistheonellide A increased, while swinholide A decreased, the rate of nucleotide exchange in G-actin, suggesting that binding of these toxins induces different conformational changes in the actin molecule. These results suggest that bistheonellide A intervenes between two actin molecules, forms a tertiary complex with each of its side chains bound to G-actin, and inhibits polymerization by sequestering G-actin from incorporation into F-actin. A difference in structure at the end of the side chain between dimeric macrolides and mycalolide B may account for the weak severing activity of the former.
An actin-depolymerizing marine natural product, mycalolide B, and a related compound, kabiramide D, were labeled with biocytin, a biotin derivative, and used to specify target molecules in cultured rat 3Y1 fibroblasts. Mycalolide B exhibited the ability to bind to various intracellular proteins, probably through the Michael addition of a sulfhydryl group to C5 of mycalolide B. However, no intracellular proteins other than actin apparently reacted with biocytinylated kabiramide D, demonstrating that the binding of kabiramide D to actin was highly specific. Cells treated with biocytinylated kabiramide D followed by staining with fluorescein isothiocyanate-conjugated avidin showed that biocytinylated kabiramide D bound to stress fibers composed of F-actin, although the staining intensity was weaker than the fluorescent phalloidin staining. The assay for the binding of kabiramide D to actin, which had previously been treated with other actin-depolymerizing agents, showed that the actin-binding site for kabiramide D was the same as that for bistheonellide A, but not those for latrunculin A and cytochalasin D.
Saponins in bulbs of a mutant of elephant garlic were investigated, and three new steroidal saponins named yayoisaponins A-C were obtained together with the known dioscin and aginoside. Their structures, including the relative stereochemistry, were elucidated by spectral data interpretation, while the absolute stereochemistry of the sugar moieties was assigned on the basis of a chiral gas chromatographic analysis of the acid hydrolysates. Yayoisaponins A-C and aginoside exhibited not only in vitro cytotoxicity against P388 cells at 2.1 micrograms/ml, but also antifungal activity against Mortierella ramanniana at 10 micrograms/disk.
In vitro effects of mycalolide-B (MB), isolated from marine sponge, were investigated with regard to the activation of rat platelets. Collagen-induced platelet aggregation in platelet-rich plasma (PRP) was slightly but significantly potentiated by lower concentrations of MB (0.3 and 1 microM) but was inhibited by higher concentrations (3 and 10 microM). ADP-induced platelet aggregation in PRP was also significantly prevented by MB (1-10 microM). Potentiation of ADP-induced aggregation by MB (0.3 microM) was hardly observed. G-actin contents, determined by DNase I inhibition assay, were increased in resting washed platelets incubated with MB (3 microM). In contrast, cytochalasin-D (CD) at 3 microM slightly reduced G-actin contents in resting platelets. After platelet aggregation with collagen (3 microg/ml) or ADP (10 microM), G-actin contents in platelets were reduced, indicating de novo actin polymerization. MB (3 microM) and CD (3 microM) abolished both ADP (10 microM)- and collagen (3 microg/ml)-induced platelet aggregation and actin polymerization in washed platelets. MB (1-10 microM) had no effects on intracellular Ca2+ concentrations in ADP (10 microM)-stimulated platelets. [125I]-fibrinogen binding to activated platelets with ADP (10 microM)(was inhibited by MB (0.3-3 microM) in a concentration-dependent manner. Thrombin-induced platelet-fibrin clot retraction was inhibited by MB (1 and 10 microM). These results suggest that MB inhibits platelet activation by interfering with actin polymerization through a different mechanism of action from CD. MB may be a useful tool for studying the role of actin polymerization in various cells.
1. Stellettamide A (ST-A), a novel marine toxin isolated from a marine sponge, inhibited high K+(72.7 mM)-induced contraction in the smooth muscle of guinea-pig taenia coli with an IC50 of 88 microM. 2. In the taenia permeabilized with Triton X-100, ST-A inhibited Ca2+ (3 and 10 microM)-induced contractions with an IC50 of 46 microM for 3 microM Ca2+ and 105 microM for 10 microM Ca2+. In the permeabilized taenia, calyculin-A (300 nM), a potent inhibitor of type-1 and type-2A phosphatases, induced sustained contraction in the absence of Ca2+. ST-A had no effect on this contraction. 3. ST-A inhibited Mg2+-ATPase activity in native actomyosin prepared from chicken gizzard with an IC50 of 25 microM. 4. In a reconstituted smooth muscle contractile system containing calmodulin, myosin light chain (MLC) and MLC kinase, ST-A inhibited MLC phosphorylation with an IC50 of 152 microM. The inhibitory effect of ST-A was antagonized by increasing the concentration of calmodulin. 5. ST-A inhibited calmodulin activity, assessed by Ca2+/calmodulin-dependent enzymes, (Ca2+-Mg2+)-ATPase of erythrocyte membrane, with an IC50 of 100 microM and phosphodiesterase prepared from bovine cardiac muscle with an IC50 of 52 microM. The inhibitory effect on phosphodiesterase activity was antagonized by increasing the calmodulin concentration. 6. Interaction between ST-A and calmodulin was demonstrated by instantaneous quenching of the intrinsic tyrosine fluorescence of calmodulin by ST-A (3-300 microM). Similar results were obtained in the presence or absence of Ca2+ suggesting that ST-A binds to calmodulin and that Ca2+ is not essential for the binding of ST-A to calmodulin. 7. These results suggest that ST-A, isolated from marine metabolites, is a novel inhibitor of calmodulin.
A highly cytotoxic extract from marine sponge, polytheonamide B, is a linear 48-residue peptide. Alternative D- and L-forms of unusual amino acids suggest formation of beta-helix that is stable in membrane and serves for ion conducting pore. The NMR study indicated that polytheonamide B forms beta-helix in methanol/chloroform solution. Channel activity of polytheonamide B was examined using planar lipid bilayers. Ionic current appeared from pM concentration. Measurements of the reversal potentials revealed that the channel showed cation selectivity. Single channel current was recorded in symmetrical 1 M solutions. The selectivity sequence was: H+ > Cs+ > Rb+ > K+ > Na+. Single-channel I-V curve exhibited slight inward rectification. Voltage-dependent transitions between brief openings and long closures were observed. Orientation of the peptide in the membrane was fixed when the peptide was added to one side of the chamber. The asymmetric behaviors, such as single channel rectification, voltage-dependent gating and oriented incorporation into the membrane, must be correlated to the molecular structure of polytheonamide B.
The effects of discodermin A, an antimicrobial peptide extracted from sea sponge Discodermia kiiensis, on cell membranes were investigated using vascular smooth muscle cells and erythrocytes. At lower concentrations (0.1-3 microM), discodermin A increased muscle tension with an increase in intracellular free Ca2+ concentration ([Ca2+]i) indicated by the fluorescence of Ca2+ indicator, fura-PE3, in rat aortic smooth muscle. On the other hand, the higher concentration of discodermin A (10-30 microM) accelerated the leakage of loaded fura-PE3 from cells. In rabbit mesenteric artery treaded with discodermin A, addition of micromolar concentration of Ca2+ evoked contraction in the presence of ATP, suggesting that permeability of the membrane to Ca2+ and ATP is increased by discodermin A. Confocal fluorescence microscopy showed that discodermin A permeabilized the plasma membrane of A10 cells to fluorescent agents EthD-1 and the intracellular esterase coupled with another fluorescent agent calcein. Discodermin A also showed a hemolytic effect on rabbit erythrocytes, suggesting that discodermin A permitted transmembrane passage of hemoglobin. These results suggest that discodermin A form pores of different sizes on the cytoplasm membrane in concentration- and time-dependent manners. Discodermin A may be a saponin-like bioactive peptide.