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Persistence of yessotoxin under light and dark conditions.

The change in concentration of the disulfated polyether yessotoxin (YTX) produced by a culture of the marine dinoflagellate Protoceratium reticulatum was measured in laboratory experiments under light and dark conditions. Experimental cultures were inoculated and grew at a growth rate of 0.14 d(-1) until stationary phase was reached, after approximately 21 days. Cultures were maintained in the stationary phase until 31 days after inoculation. Cells of P. reticulatum contained a concentration of approximately 10-15 pg YTX cell(-1) during stationary phase but this was considerably lower (<5 pg cell-1) during the growth phase. Low amounts of 45-hydroxy-YTX were also detected. At day 32, P. reticulatum was killed by cooling to 1 degrees C (confirmed microscopically) and YTX concentrations were measured periodically under light and dark conditions. YTX concentrations decreased rapidly to approximately 10% of the initial concentration within the first 3 days and depleted to near zero within a week in the light treatment. In the dark environment, YTX persisted longer with approximately 10% of the initial YTX concentration still remaining after 18 days.

Animals↗

Okadaic acid: an additional non-phorbol-12-tetradecanoate-13-acetate-type tumor promoter.

Okadaic acid is a polyether compound of a C38 fatty acid, isolated from a black sponge, Halichondria okadai. Previous studies showed that okadaic acid is a skin irritant and induces ornithine decarboxylase (OrnDCase; 3-hydroxyl-L-glutamate 1-carboxy-lyase, EC 4.1.1.17) in mouse skin 4 hr after its application to the skin. This induction was strongly inhibited by pretreatment of the skin with 13-cis-retinoic acid. A two-stage carcinogenesis experiment in mouse skin initiated by a single application of 100 micrograms of 7,12-dimethylbenz[a]anthracene (DMBA) and followed by application of 10 micrograms of okadaic acid twice a week revealed that okadaic acid is a potent additional tumor promoter: tumors developed in 93% of the mice treated with DMBA and okadaic acid by week 16. In contrast, tumors were found in only one mouse each in the groups treated with DMBA alone or okadaic acid alone. An average of 2.6 tumors per mouse was found in week 30 in the group treated with DMBA and okadaic acid. Unlike phorbol 12-tetradecanoate 13-acetate (TPA), teleocidin, and aplysiatoxin, okadaic acid did not inhibit the specific binding of [3H]TPA to a mouse skin particulate fraction when added up to 100 microM or activate calcium-activated, phospholipid-dependent protein kinase (protein kinase C) in vitro when added up to 1.2 microM. Therefore, the actions of okadaic acid and phorbol ester may be mediated in different ways. These results show that okadaic acid is a non-TPA-type tumor promoter in mouse skin carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗

Goniodomin A induces modulation of actomyosin ATPase activity mediated through conformational change of actin.

Goniodomin A, one of the polyether macrolides, isolated from the dinoflagellate Goniodoma pseudogoniaulax stimulated skeletal actomyosin ATPase activity 2-3 times, with a peak at 3 x 10(-8) to 3 x 10(-7) M. The effect of goniodomin A was dependent on the concentration of actin, but not of myosin. The actomyosin ATPase activity was increased by pretreatment of actin (but not of myosin) with goniodomin A. Goniodomin A induced a sustained and concentration-dependent increase in the fluorescence intensity (excitation wavelength, 277 nm; emission wavelength, 329 nm) of actin. The maximum response was obtained with concentrations of goniodomin A in the 10(-5) to 10(-4) M range in the presence of 5 microM F-actin. However, the ATPase activity and fluorescence intensity of myosin were not changed by goniodomin A at concentrations from 10(-8) to 10(-5) M. Interestingly, goniodomin A induced a remarkable but transient increase in the fluorescence intensity of actomyosin in a concentration-dependent manner, with a peak at 3 x 10(-7) M. This profile was quite similar to that found in the stimulation of the actomyosin ATPase activity induced by goniodomin A. To investigate further the effect of goniodomin A, actin was labeled with N-(1-pyrenyl)iodoacetamide. Goniodomin A at 10(-6) M had no effect on the fluorescence intensity of pyrenyl-actin (excitation wavelength, 365 nm; emission wavelength, 407 nm), but increasing concentrations of goniodomin A to 3 x 10(-6) M remarkably decreased its intensity. This effect was potentiated by heavy meromyosin. Actin molecules treated with goniodomin A were completely sedimented by mild centrifugation (for 15 min at 12,000 x g). Electron microscopic observations suggest that actin filaments associate with each other to form a gel in the presence of 3 x 10(-6) M goniodomin A. The conformational change of actin molecules, resulting from stoichiometric binding of goniodomin A to actin monomers in filaments, may modify the interaction between actin and myosin.

Actins↗

Cytostatic and cytotoxic properties of the marine product bistratene A and analysis of the role of protein kinase C in its mode of action.

Bistratene A is a polyether which was isolated from the marine ascidian Lissoclinum bistratum Sluiter. The hypothesis has been tested that the cytostatic effect of bistratene A is mediated by modulation of protein kinase C (PKC). Human-derived A549 lung and MCF-7 breast adenocarcinoma cells are extremely sensitive to growth inhibition induced by activators of PKC. Therefore, the effect of bistratene A on these cell lines was compared with that of the known PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA). The ability of bistratene A to modulate PKC activity in cellular cytosol was assessed to determine the involvement of PKC in the induction of cytostasis. Bistratene A inhibited the growth of both cell lines and initial seeding density determined its cytostatic potency. IC50 values were between 1.0 and 2.9 nM. Bistratene A also had a profound effect on the colony forming ability of A549 cells, preventing clonal growth at 5 nM. Using the incorporation of [3H]thymidine into cells to assess DNA synthetic activity and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay to define cytotoxicity, the compound was found to have both cytostatic and cytotoxic properties. Bistratene A decomposed by 50% after only 2.8 hr in cell culture medium. TPA induced rapid motility and the formation of a network of branched colonies in both cell lines grown on Matrigel, whereas bistratene A did not cause the same effect. Cell cytosol was analysed for phorbol ester binding sites after treatment with bistratene A or TPA. Incubation with TPA (10 nM) caused a reduction in binding sites to 57% of binding in control cells after 30 min and to 35% after 24 hr. Bistratene A did not cause a significant change in binding sites. Assays of PKC activity in cellular cytosol revealed that bistratene A was unable to activate or inhibit the enzyme at concentrations of up to 10 microM. The results suggest that bistratene A is an exquisitely potent cytostatic agent in the two cell lines studied, but modulation of PKC is not involved in the mode of action by which it elicits this effect.

Acetamides↗