The cytotoxic and cytolytic activity of equinatoxin II from the sea anemone Actinia equina

The cytotoxic and cytolytic effects of equinatoxin II (EqT II) from the sea anemone Actinia equina L. were studied on exponentially growing and synchronized V-79-379 A cell line in culture. The cell viability test and the determination of the cytolytic effect by cell counting confirmed both cytotoxic and cytolytic activity of EqT II. Additionally, cytocidal and cytostatic effects depending on the toxin concentration were observed. The presence of fetal calf serum in the cell culture medium reduced both cytocidal and cytostatic effects by two magnitudes and prevented cytolysis. Combining EqT II and serum resulted in an insoluble complex which was cytostatic even when isolated and resuspended in the culture medium, while the supernatant retained both cytocidal and cytostatic activity. No significant difference in sensitivity between synchronized and exponentially growing cells could be detected after EqT II treatment.     

Programmed cell death and cell necrosis activity during hyperthermic stress-induced bleaching of the symbiotic sea anemone Aiptasia sp.

Different cell death pathways were investigated during bleaching in the sea anemone Aiptasia sp. in response to hyperthermic treatment. Using a suite of techniques, (haematoxylin and eosin staining of paraffin wax-embedded tissue sections, in-situ end labelling (ISEL) of fragmented DNA, agarose gel electrophoresis electron microscopy) both necrotic and programmed cell death (PCD) activity were indicated. After a treatment period of 4 days, the host endoderm tissues underwent necrotic cell death. This was indicated by widespread cellular degradation, dilation of cell cytoplasm and organelles, cell swelling and rupture, irregular pyknotic condensation of nuclear chromatin, and abundant cell debris. Host cell necrosis was associated with the release of zooxanthellae with a normal, healthy appearance into the coelenteron. Longer periods of hyperthermic treatment (7 days) were correlated with further animal cell degradation and the in-situ degradation of zooxanthellae remaining within the degraded endoderm. Within the same degraded endoderm tissue, the degradation of zooxanthellae resulted from two forms of cell death occurring simultaneously, which were identified as programmed cell death and cell necrosis. Programmed cell death of zooxanthellae was characterised by condensation of the cytoplasm and organelles, cell shrinkage, formation of accumulation bodies at the periphery of the cell wall, and DNA fragmentation. Cell necrosis of zooxanthellae was characterised by dilation of the cytoplasm and organelles, cell swelling and lysis, dispersion of cell component debris, and DNA fragmentation. The existence of a programmed cell death pathway within zooxanthellae is important to the understanding of coral bleaching events, raising interesting questions regarding the evolution of this process and the activation of the cellular trigger mechanisms involved.     

Structure-function relationships of sea anemone toxin II from Anemonia sulcata

Chemical modifications of sea anemone toxin II from Anemonia sulcata have been used to study the residues involved in its toxic action on crabs and mice and in its binding properties to the Na+ channel of rat brain synaptosomes. Guanidination of th epsilon-amino groups of lysines 35, 36, and 46 with O-methylisourea hydrogen sulfate did not change the net charge of the toxin molecule and had no effect upon its toxic and binding properties. Either acetylation or fluorescamine treatment of the toxin that destroyed the positive charges of the three epsilon-amino groups and of the alpha-amino function of Gly produced an almost complete loss of toxicity and a considerable decrease in the binding activity. Iodination of the toxin on His induced practically no loss of toxic or binding properties. Carbethoxylation of both histidines 32 and 37 with diethyl pyrocarbonate provoked an important decrease of both the toxicity and the binding activity. Modifications of the guanidine side chain of Arg with 1,2-cyclohexanedione fully destroyed both toxicity and binding of the toxin to the Na+ channel. Modification of the carboxylate functions of Asp, Asp, and of the COOH-terminal Gln with glycine ethyl ester in the presence of a soluble carbodiimide completely abolished the toxicity but left the affinity for the sea anemone toxin receptor unchanged. The antagonist character of this carboxylate-modified derivative was further confirmed by electrophysiological and Na+ flux experiments. The theoretical and practical significance of these results are discussed.     

Binding of the sea anemone polypeptide BDS II to the voltage-gated sodium channel.

BDS II, a 43-residue polypeptide from the sea anemone Anemonia sulcata, is reported to have both antihypertensive and antiviral activity. This polypeptide possesses a number of sequence and structural similarities to a class of cardiotonic proteins which bind to receptor site 3 of the voltage-gated sodium channel. In contrast to these cardiostimulant proteins, which produce positive inotropic effects at concentrations of 2-15 nM, BDS II produced a weak negative inotropic effect upon isolated guinea-pig atria, with doses of 90 and 180 nM depressing contractile strength by 15 and 28%, respectively. BDS II also competed with a 125-iodine labelled derivative of AP-A (a representative of the cardiostimulant proteins) bound to sodium channels in rat brain synaptosomes. The IC50 for BDS II versus AP-A was 5.2 microM. BDS II may therefore be considered an antagonist for receptor site 3 of the voltage-gated sodium channel. Structural differences between BDS II and the agonist AP-A which may give rise to their different effects on the sodium channel are considered.     

Binding of sea anemone pore-forming toxins sticholysins I and II to interfaces--modulation of conformation and activity,and lipid-protein interaction

Sticholysins I and II (St I and St II) are water-soluble toxins produced by the sea anemone Stichodactyla helianthus. St I and St II bind to biological and model membranes containing sphingomyelin (SM), forming oligomeric pores that lead to leakage of internal contents. Here we describe functional and structural studies of the toxins aiming at the understanding at a molecular level of their mechanism of binding, as well as their effects on membrane permeabilization. St I and St II caused potassium leakage from red blood cells and temperature-dependent hemolysis, the activation energy of the process being lower for the latter toxin. Protein intrinsic fluorescence measurements provided evidence for toxin binding to model membranes composed of 1:1 (mol:mol) egg phosphatidyl choline (ePC):SM. The fluorescence intensity increased and the maximum emission wavelength decreased as a result of binding. The changes were quantitatively different for both toxins. Circular dichroism spectra showed that both St I and St II exhibit a high content of beta-sheet structure and that binding to model membranes did not alter the toxin's conformation to a large extent. Changing the lipid composition by adding 5 mol% of negatively charged phosphatidic acid (PA) or phosphatidyl glycerol (PG) had small, but detectable, effects on protein conformation. The influence of lipid composition on toxin-induced membrane permeabilization was assessed by means of fluorescence measurements of calcein leakage. The effect was larger for ePC:SM bilayers containing 5 mol% of negative curvature-inducing lipids. Electron paramagnetic resonance (EPR) spectra of intercalated fatty acid spin probes carrying the nitroxide moiety at different carbons (5, 7, 12, and 16) evidenced the occurrence of lipid-protein interaction. Upon addition of the toxins, two-component spectra were observed for the probe labeled at C-12. The broader component, corresponding to a population of strongly immobilized spin probes, was ascribed to boundary lipid. The contribution of this component to the total spectrum was larger for St II than for St I. Moreover, it was clearly detectable for the C-12-labeled probe, but it was absent when the label was at C-16, indicating a lack of lipid-protein interaction close to the lipid terminal methyl group. This effect could be either due to the fact that the toxins do not span the whole bilayer thickness or to the formation of a toroidal pore leading to the preferential interaction with acyl chain carbons closer to the phospholipids head groups.     

Chromatin-associated proteins of the developing sea urchin embryo. II. Acid-soluble proteins

Of the five well-characterized histories, only the slightly lysine-rich histories F2a and F2b are present in sea urchin embryos before the 16-cell stage. At the 16-cell stage, the arginine-rich (F3) and lysine-rich (Fla) histones appear and all the major histones are then present in the same relative proportions until the pluteus stage except for a second lysine-rich protein, Flb, which is first detected at 12 to 16 hours of development and increases to the pluteus stage. From 16 cells to pluteus at 70 hours, all the histones are labeled by a 30-minute incubation with radioactive lysine, with the exception of the lysine-rich histone Fla which does not incorporate label after 20 to 30 hours of development and Fib which is labeled only after 20 to 30 hours. Fla is conserved, however, to the pluteus stage.The total acid-soluble protein content of chromatin remains constant to 22 hours of development. During the period of 22 to 45 hours, there is a slight loss of protein followed by a rapid loss from 45 to 70 hours such that at 70 hours only 20% remains.     

NMR analysis and sequence of toxin II from the sea anemone Radianthus paumotensis

Toxin II from Radianthus paumotensis (RpII) has been investigated by high-resolution NMR and chemical sequencing methods. Resonance assignments have been obtained for this protein by the sequential approach. NMR assignments could not be made consistent with the previously reported primary sequence for this protein, and chemical methods have been used to determine a sequence with which the NMR data are consistent. Analysis of the 2D NOE spectra shows that the protein secondary structure is comprised of two sequences of beta-sheet, probably joined into a distorted continuous sheet, connected by turns and extended loops, without any regular alpha-helical segments. The residues previously implicated in activity in this class of proteins, D8 and R13, occur in a loop region.     

Spontaneous contractions and nerve net activity in the sea anemone calliactis parasitica

Suction electrodes attached to tentacles of the sea anemone Calliactis parasitica record regular bursts of activity associated with the through‐conducting nerve net. Most bursts consist of 10–15 pulses at a frequency of 1 every 4 sec to 1 every 10 sec. The interval between bursts is usually 10–20 min. Regularity in pulse number and frequency in successive bursts suggests that the activity originates from a pacemaker. Bursts are always followed by slow contraction of endodermal longitudinal (parietal) muscles after a short delay, and endo‐dermal circular muscles after a long delay. A simple model for nervous pacemaker control of rhythmic contractions cannot be proposed as slow contractions can also occur in the absence of recorded nerve net activity.     

Amino acid sequence of neurotoxin II from the sea anemone Radianthus macrodactylus

Amino acid sequence of neurotoxin II isolated from the sea anemone Radianthus macrodactylus was determined by analysis of peptides obtained after its digestion with trypsin and staphylococcal proteinase. It is shown that the polypeptide chain of the toxin consists of 48 amino acid residues, including six cysteines.     

Genetics and asexual reproduction of the sea anemone Metridium senile.

1. Metridium senile was studied for phosphohexose-isomerase variation at three locations on Cape Cod, Massachusetts: Woods Hole, Cape Cod Canal, and Barnstable Town Boat Harbor. 2. All three locations exhibited significant polymorphism for PHI. 3. Mapping of individual polyps was performed at Barnstable to analyze spatial distributions of clones and genotypes. 4. In Barnstable, PHI does not depart significantly from Hardy-Weinberg expectations at the time of establishment of new polyps, and establishment of larvae is spatially random with respect to PHI genotype. 5. Asexual reproduction was uses as a meausre of the relative success of different PHI genotypes. There are indications that not all genotypes are equally likely to produce large clones. 6. There is significant heterogeneity among the three locations with respect to PHI genotype frequencies, suggesting that there may be geographical differentiation of the populations. 7. Sessile, asexual organisms provide powerful tools for examining the dynamic aspects of genetic structure in natural populations.     

Proline inhibition of a sea anemone alarm pheromone response.

1. L-proline, by itself or in animal tissue extracts, inhibits the response of the sea anemone Anthopleura elegantissima to the alarm phermone, anthopeurine. 2. The effect of proline is mediated by a receptor that is specific for the structure and configuration of the part of the L-proline molecule containing the carboxyl and imino groups. 3. Proline inhibition is competitive, in the sense that the effects of a given proline concentration can be overridden by an increase in anthopeurine concentration. 4. The magnitude of proline inhibition increases with proline concentration and decreases as the duration of exposure to proline increases. 5. Neither the final conducting system mediating the alarm response nor the responding muscles are inhbited by proline. Inhibition presumably occurs at or soon after the level of anthopleurine receptors. 6. Proline inhibition may resolve the potential conflict between Anthopleura's mutually exclusive feeding and alarm pheromone responses.     

Characterisation of nitric oxide synthase activity in the tropical sea anemone Aiptasia pallida

The presence of nitric oxide synthase (EC 1.14.23 NOS) activity is demonstrated in the tropical marine cnidarian Aiptasia pallida (Verrill). Enzyme activity was assayed by measuring the conversion of [³H]arginine to [³H]citrulline. Optimal NOS activity was found to require NADPH. Activity was inhibited by the competitive NOS inhibitor N^G-methyl- -arginine ( -NMA), but not the arginase inhibitors -valine and -ornithine. NOS activity was predominantly cytosolic, and was characterised by a K_m for arginine of 19.05 μM and a V_max of 2.96 pmol/min per μg protein. Histochemical localisation of NOS activity using NADPH diaphorase staining showed the enzyme to be predominantly present in the epidermal cells and at the extremities of the mesoglea. These results provide a preliminary biochemical characterisation and histochemical localisation of NOS activity in A. pallida, an ecologically important sentinel species in tropical marine ecosystems.     

Effect of membrane-partitioned n-alcohols and fatty acids on pore-forming activity of a sea anemone toxin

Equinatoxin II, a 19.8 kDa pore-forming toxin from the sea anemone Actinia equina, was examined for hemolytic activity and permeabilization of small unilamellar lipid vesicles (SUV) in the presence of increasing amounts of n-alcohols (methanol to n-octanol) and fatty acids (palmitic and palmitoleic acid). We observed an enhancement of toxin activity which was dependent on the concentration of the membrane partitioning additive. An exception was palmitic acid which exerted a bimodal role. While at low bulk concentrations it increased toxin-induced hemolysis, above 3 μM bulk concentration it was inhibitory; in neither case was it efficient in promoting release of the fluorescent marker calcein from SUV. The increased permeabilization activity was correlated with an increase in the amount of toxin bound as indicated by changes in the intrinsic toxin fluorescence. In the case of n-alcohols, at least, these effects appeared to depend on the actual amount of alcohol present inside the membrane rather than on its specific chemical nature. This suggests that the observed effects could be due to changes of the biophysical properties of the lipid bilayer, such as thickness, lipid acyl-chain ordering, and dielectric constant induced by the partitioned additives. Received: 27 March 1996 / Accepted: 10 October 1996     

Biological activity of paramyxovirus fusion proteins: factors influencing formation of syncytia.

The fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of the paramyxovirus simian virus 5 (SV5) were expressed individually or coexpressed in CV-1 cells by using SV40-based vectors and recombinant vaccinia viruses. The extent of detectable fusion in a syncytium formation assay was found to be affected by the expression system used. In addition, when HN was coexpressed with F, it was found that the expression vector system influenced the contribution of HN in forming syncytia. The abilities of the SV5, human parainfluenza virus type 3, and Newcastle disease virus F glycoproteins to cause fusion, when expressed alone or coexpressed with HN, were directly compared by using the SV40-based vector system in CV-1 cells. The F proteins exhibited various degrees of fusion activity independent of HN expression, but the formation of syncytia could be enhanced to different extents by the coexpression of the homotypic HN protein.     

Multiple conformations of the sea anemone polypeptide anthopleurin-A in solution.

Anthopleurin-A (AP-A) is a member of a family of sea anemone-derived polypeptides that interact with sodium channels in a voltage-dependent manner, producing a positive inotropic effect on the mammalian heart. There has been considerable interest in this molecule as a lead compound for the development of novel therapeutic agents. Earlier attempts to define the 3-dimensional structure of AP-A were complicated by the fact that it was found to exist in 2 conformations in solution. Using 1H- and 13C-NMR spectroscopy, we have now shown that this conformational heterogeneity arises from cis-trans isomerization about the Gly 40-Pro 41 peptide bond and that in the major form of the protein this peptide bond adopts a cis conformation. Furthermore, the increased sensitivity afforded by higher-field NMR has allowed identification of additional minor conformations of AP-A, the origin of which is presently unknown. We believe there will be many more examples of the detection by high-field NMR of previously unobserved minor conformations of proteins in solution.     

Biological activity and pharmacological prospects of lupane terpenoids: II. Semisynthetic lupane derivatives

The discussion of lupane triterpenoids as prospective medicinal preparations is continued, and semisynthetic triterpenoids are being discussed. Acyl derivatives that mainly exhibit high anti-HIV, antitumor, and organo-protective activities are described. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.     

Biological activity and pharmacological prospects of lupane terpenoids: II. Semisynthetic lupane derivatives

The discussion of lupane triterpenoids as prospective medicinal preparations is continued, and semisynthetic triterpenoids are being discussed. Acyl derivatives that mainly exhibit high anti-HIV, antitumor, and organoprotective activities are described.