Biomedical subjects
Publications and source records attributed to N Fusetani.
Three new cytotoxic sesterterpenes, 1-3, have been isolated from the marine sponge Hyrtios cf. erectus. Their structures have been determined by interpretation of NMR data. They exhibited cytotoxicity against P-388 murine leukemia cells with IC50 values of 0.4-2.1 micrograms/mL.
Four new cytotoxic triterpenes, globostellatic acids A-D (1-4), have been isolated from the marine sponge Stelletta globostellata. Their structures have been determined by interpretation of spectral data. They exhibited cytotoxicity against P-388 murine leukemia cells with IC50 values of 0.1-0.46 microgram/mL.
Two new polyacetylene carboxylic acids, montiporic acids A (1) and B (2), have been isolated from the eggs of the scleractinian coral Montipora digitata and their structures elucidated on the basis of spectroscopic data. They exhibited antimicrobial activity against Escherichia coli and cytotoxicity against P-388 murine leukemia cells.
Halicylindramides A-C (1-3) have been isolated from the Japanese marine sponge Halichondria cylindrata. They are tetradecapeptides with the N-terminus blocked by a formyl group and the C-terminus lactonized with a threonine residue. Their total structures including absolute stereochemistry were determined by a combination of spectral and chemical methods. Halicylindramides A-C were antifungal against Mortierella ramanniana and cytotoxic against P388 murine leukemia cells.
Three new thrombin and trypsin inhibitors, cyclotheonamides C (3), D (4), and E (8) were isolated from the marine sponge Theonella swinhoei. Their structures were determined by spectral and chemical methods.
We investigated the effects of a novel marine toxin, mycalolide B, on actin polymerization and actin-activated myosin Mg(2+)-ATPase activity using purified actin and myosin from rabbit skeletal muscle. The results were compared with cytochalasin D which inhibits actin polymerization by binding to the barbed end of F-actin. By monitoring fluorescent intensity of pyrenyl-actin, mycalolide B did not accelerate actin polymerization but quickly depolymerized F-actin, whereas cytochalasin D accelerated actin nucleation and depolymerized F-actin at slower rate. The kinetics of depolymerization suggest that mycalolide B severs F-actin. The relationship between the concentration of total actin and F-actin at different concentration of mycalolide B suggests that mycalolide B forms 1:1 complex with G-actin. Viscometry and electron microscopic observation further suggest that actin filament was depolymerized by mycalolide B. Unlike cytochalasin D, furthermore, mycalolide B suppressed actin-activated myosin Mg(2+)-ATPase activity. We concluded that mycalolide B severs F-actin and sequesters G-actin and may serve as a novel pharmacological tool for analyzing actin-mediated cell functions.
Three new acetylenic acids, corticatic acids A-C [1-3] have been isolated from the marine sponge, Petrosia corticata. Their structures were determined by spectroscopic methods, and 1-3 exhibited antifungal activity against Mortieralla ramanniana.
Four new antibacterial and antifungal sulfates, 2,6-dimethylheptyl sulfate [1], (4Z,7Z)-4,7-decadienyl sulfate [2], (4Z,7E)-4,7-decadienyl sulfate [3], and (3Z,6Z)-3,6,9-decatrienyl sulfate [4], have been isolated from the hepatopancreas of the ascidian Halocynthia roretzi. The structures were determined by spectral analysis.
A toxin isolated from marine sponge, mycalolide-B, inhibited smooth muscle contractions without changing cytosolic Ca2+ levels. It also inhibited Ca(2+)-induced contraction in permeabilized smooth muscles. In native actomyosin prepared from chicken gizzard, mycalolide-B inhibited superprecipitation and Mg(2+)-ATPase activity stimulated by Ca2+ without changing myosin light chain phosphorylation. In the permeabilized muscle and native actomyosin preparation thiophosphorylated with ATP gamma S, mycalolide-B inhibited ATP-induced contraction and Mg(2+)-ATPase activity, respectively, in the absence of Ca2+. Mycalolide-B also inhibited Mg(2+)-ATPase activity of skeletal muscle native actomyosin. Mycalolide-B had no effect on calmodulin-stimulated (Ca(2+)-Mg2+)-ATPase activity of erythrocyte membranes. These results suggest that mycalolide-B selectively inhibits actin-myosin interaction.
Cyclotheonamide A (CA), a cyclic peptide isolated from the marine sponge of the genus Theonella was shown to be a slow-binding inhibitor of several trypsin-like serine proteinases. Values of 4.6 x 10(4), 4.8 x 10(4), 9.3 x 10(3), 2.1 x 10(3) and 2.7 x 10(2) M-1 s-1 were determined for the second-order rate constants for formation of CA complexes with thrombin, trypsin, plasmin, 2-chain t-PA and factor Xa, respectively. The equilibrium constant (Ki) was measured for dissociation of CA from the CA complex with human thrombin (Ki = 1.0 nM), bovine trypsin (Ki = 0.2 nM), human plasmin (Ki = 12 nM), human factor Xa (Ki = 50 nM) and human 2-chain tissue plasminogen activator (t-PA) (Ki = 40 nM). CA produces dose dependent increases in clotting time assays. The clotting time in the thrombin time, activated partial thromboplastin time and prothrombin time assays, were doubled by 1.5, 0.9 and 48 microM CA, respectively. A model for the binding of CA to the active site of thrombin is proposed.
Calyculin A, a protein phosphatase inhibitor, induced cleavage-like morphological change in unfertilized sea urchin eggs. A contractile ring-like apparatus containing both filamentous actin and myosin was formed in the cleavage furrow. Wheat germ agglutinin receptors were also found in the same region. The eggs did not develop further after constriction of the ring. No aster-like microtubular structure was found in the calyculin A-treated eggs. The cleavage was not inhibited by the antimicrotubule drug griseofulvin. Calyculin A also increased histone H1 kinase activity and induced chromosome condensation. These changes also occurred in the presence of emetine (an inhibitor of protein synthesis) and aphidicolin (an inhibitor of DNA synthesis). It is suggested that calyculin A induced these changes in the sea urchin eggs by inhibiting the activity of protein phosphatase 1.
Addition of the protein phosphatase inhibitor, calyculin-A, to 3T3 fibroblasts causes a marked change in cell morphology. Initially the cells become rounded, develop surface blebs and then detach from the substratum. In the detached cells an unusual ball-like structure is observed. This study focuses on the cytoskeleton during these calyculin-A-induced morphological changes. Stress fibres disappear as the cells begin to round and aggregates of actin are formed towards the apical surface of the cell. These aggregates condense, in the detached cells, to form the ball structure of approximately 3 microns diameter. Between the ball and the nucleus are cables of intermediate filaments that appear to be attached to the surface of the ball and to the nuclear lamina. Using a procedure designed for the isolation of nuclei the nucleus-ball complex can be obtained. Analysis of the nucleus-ball preparation by immunofluorescence and electron microscopy demonstrate that the ball contains actin and that intermediate filaments are located between the ball and the nucleus. In this preparation, the intermediate filaments also appear to attach to the surfaces of the ball and the nucleus. Electrophoretic analysis of the nucleus-ball preparation indicates that, in addition to actin, a major component of the ball is myosin. It is suggested that the formation of the ball is caused by an actin-myosin-based contractile process, initiated by the phosphorylation of myosin. The aggregation of the actomyosin draws together the intermediate filaments into the area between the ball and nucleus. This hypothesis requires that vimentin binds both to the nucleus and to some component of the ball.
This review article deals with significant effects of marine natural products in carcinogenesis, namely as chemical probes to understand the process of carcinogenesis and as possible cancer preventive agents in humans.
The effects of a potent phosphatase inhibitor, calyculin A (CL-A), on inward currents in guinea pig taenia coli smooth muscle cells were examined. CL-A increased the inward current, and this effect of CL-A was inhibited by a protein kinase C inhibitor, H-7, and by nifedipine. Phorbol 12,13-dibutyrate, an activator of protein kinase C, also increased the inward current and this effect was antagonized by H-7. These results suggest that in guinea pig taenia coli smooth muscle cells CL-A may facilitate the opening of the L-type Ca2+ channels through the protein kinase C-dependent phosphorylation system.
Calyculin-A, an inhibitor of type 1 and 2A phosphatases, was applied extracellularly to 3T3 fibroblasts. At 0.1 microM, calyculin-A caused a marked increase in protein phosphorylation in both the cytosolic and insoluble cellular fractions. This effect was independent of external Ca2+. An immunoprecipitate, formed with an antibody to myosin, contained several cytoskeletal components. Increased phosphorylation following treatment with calyculin-A was observed in vimentin, the 20-kD myosin light chain, and an unidentified 440-kD component. An enhanced level of vimentin phosphorylation was found in intermediate filament preparations from treated cells. Calyculin-A also caused marked shape changes of 3T3 cells. Within minutes after addition of calyculin-A (0.1 microM) cells became rounded and lost attachment to the substratum. Stress fibers, intermediate filaments, and microtubules, prominent in the attached control cells, were not evident in the rounded cells. Shape changes were reversible and after removal of calyculin-A the rounded cells attached to the substratum, resumed a flattened shape, and were active mitotically. In the cells treated with calyculin-A an unusual "ball-like" structure was observed with transmission electron microscopy. This unique structure was 2-3 microM in diameter and was located close to the nucleus. The use of calyculin-A adds further support to the idea that cell shape is controlled, at least in part, by concerted actions of a kinase-phosphatase couple.
1. Relationships between cytosolic Ca2+ concentration ([Ca2+]cyt), myosin light chain (MLC) phosphorylation and muscle tension were examined in circular smooth muscle of canine gastric antrum. 2. Electrical slow waves induced a transient increase in [Ca2+]cyt and muscle tension. [Ca2+]cyt increased before the initiation of contraction and reached a maximum before the peak of the phasic contractions. Following the first Ca2+ transient, a second rise in [Ca2+]cyt was often observed. The second Ca2+ transient was of similar magnitude to the first, but only in some cases was this increase in [Ca2+]cyt associated with a second phase of contraction. Relaxation occurred more rapidly than the restoration of resting levels of [Ca2+]cyt. 3. Acetylcholine (ACh; 3 x 10(-7) M) increased the amplitude of Ca2+ transients, caused MLC phosphorylation and increased the force of contraction. The decay of contraction and MLC dephosphorylation preceded that of [Ca2+]cyt. 4. Increasing external K+ (to 25-40 mM) caused a sustained increase in [Ca2+]cyt, but little change in resting tension. This suggests that the Ca2+ sensitivity decreased as [Ca2+]cyt increased. Increasing K+ to 59.5 mM further increased the level of [Ca2+]cyt, induced MLC phosphorylation and caused a transient contraction. When normal levels of K+ were restored, the rates of MLC dephosphorylation and relaxation exceeded the rate of decay in [Ca2+]cyt. 5. Removal of external Ca2+ in depolarized muscles decreased [Ca2+]cyt below the resting level without affecting resting tension. Readmission of Ca2+ to depolarized muscles caused force to develop at [Ca2+]cyt levels below the original resting level, suggesting that Ca2+ sensitivity was increased when the resting level of [Ca2+]cyt was decreased. 6. The phosphatase inhibitor, calyculin-A (10(-6) M), induced tonic contraction and MLC phosphorylation without an increase in [Ca2+]cyt. During these contractures, electrical activity caused transient increases in [Ca2+]cyt and phasic contractions which were superimposed upon the Ca(2+)-independent contracture. In the presence of calyculin-A, relaxation occurred in two phases. The initial, rapid phase of relaxation was not significantly affected by calyculin-A, but the slow phase was significantly decreased. 7. These results suggest that the relationship between [Ca2+]cyt, MLC phosphorylation and contraction changes as a function of [Ca2+]cyt in canine antral muscles. This may be due to a Ca(2+)-and time-dependent phosphatase that regulates the level of myosin phosphorylation.
The effects of calyculin A, a phosphatase inhibitor isolated from the marine sponge Discodermia calyx, on the electrical activity of colonic and gastric muscles were studied. Calyculin A reduced the amplitude and duration of slow waves, primarily by inhibiting the plateau component. Okadaic acid, another phosphatase inhibitor, also reduced the amplitude and duration of gastric slow waves. The mechanism of action of calyculin A was investigated by studying its effects on inward currents of isolated gastric and colonic myocytes. Calyculin A reduced the amplitude of the peak and the sustained components of the inward current. Okadaic acid had similar effects. These data suggest that phosphorylation of Ca2+ channels of gastrointestinal smooth muscles may inhibit Ca2+ currents. This mechanism may provide an important means of regulating the currents responsible for excitation-contraction coupling in these muscles.
Many proteins including ion channels are regulated by phosphorylation. We tested the effect of 10 U/ml catalytic subunit protein kinase A on 260-pS Ca(2+)-activated K+ channels in excised inside-out membrane patches from freshly dispersed smooth muscle cells of the canine proximal colon. At +50 mV with 10(-7) M Ca2+ and -50 mV with 10(-6) M Ca2+, open probability of the channels was increased to 270 +/- 48% of control (n = 12). This increase was due to a shift in voltage-dependent activation by 13.9 +/- 3.2 mV (n = 3) to more negative potentials. Protein kinase A in the absence of ATP had no effect on channel activity (n = 3). Regulation by phosphorylation must be accompanied by dephosphorylation. We tested the effect of two potent inhibitors of protein phosphatases, calyculin A and okadaic acid. Application of 10(-9) to 10(-6) M of each inhibitor in the presence of protein kinase A further increased open probability by up to 250%. Calyculin A appeared to be less effective in increasing open probability than okadaic acid, suggesting that the phosphatase involved is neither type 1, 2A, nor 2B. Calyculin A in the absence of protein kinase A was ineffective. These data suggest that endogenous phosphatases are found in excised membrane patches and that a balance between phosphorylation and dephosphorylation may provide an important control of colonic motility.