Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes

Summary The interaction ofActinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.     

Lysine 77 is a Key Residue in Aggregation of Equinatoxin II, a Pore-forming Toxin from Sea Anemone Actinia equina

Among eighteen point mutants of equinatoxin II produced in E. coli, containing a single cystein substitution at variable position, EqtIIK77C was chosen for its peculiar properties. It was almost 100 times less hemolytic than the wild-type, but its hemolytic activity could be restored by chemical modification of the thiol group, provided that a positive charge was reintroduced. This indicates that a positive charge at this position is necessary for toxin activity. The mutant formed larger pores as compared to the wild type, but displayed the same cation selectivity. The pores reverted to normal size upon reintroduction of the positive charge. The conformation of EqtIIK77C and its binding to lipid membranes, either vesicles or red blood cells, was almost normal. However the kinetics of calcein release from lipid vesicles was substantially slower than that of the wild-type. Taken together with the different size of the pore formed, this is an indication that mutation of Lys77 → Cys influences the normal development of the aggregate which is required for assembling the functional pore. Received: 18 May 1999/Revised: 18 September 1999     

Gametogenic cycles and reproduction in the beadlet sea anemone Actinia equina (Cnidaria: Anthozoa)

Sexual Actinia equina have been maintained in the laboratory. Monthly biopsies show that these animals have annual gametogenic cycles. Regular samples from several natural populations suggest that animals in the wild show similar gametogenic periodicity. In the laboratory, males release sperm, but females brood young, at least for a few weeks. Members of these broods are released sporadically, usually after a water change. Males and non-sexual adults kept in isolation for over a year release a few juveniles during and at the end of that time. These young are likely to be asexually reproduced. There appears to be geographical variation of anemone size and in the frequency of sexual individuals in populations round the British Coast, so that modes of reproduction may vary.     

Effects of feeding regime on growth rate in the Mediterranean Sea anemone Actinia equina (Linnaeus)

Polyps of Actinia equina are the most common sea anemones in the rocky intertidal zone of the Mediterranean coast of Israel, where they occur in one of the southernmost populations of this species in the northern hemisphere. We examined effects of feeding rate on polyp growth at ambient sea temperature for this population. Under laboratory conditions, polyps were left unfed, or were fed with brine shrimp (Artemia) once every 2 weeks, once a week, or twice a week. Of the four experimental treatments, only feeding twice a week resulted in polyp growth; under all other regimes, the sea anemones lost body mass. We conclude that a high rate of feeding is required at sea temperatures in the eastern Mediterranean, where these sea anemones may have high metabolic rates relative to more northern populations.     

Kunitz-type protease inhibitors from acrorhagi of three species of sea anemones

Sea anemones are rich in biologically active polypeptides such as toxins and protease inhibitors. These polypeptides have so far been isolated from whole bodies, tentacles or secreted mucus. Recently, two novel peptide toxins with crab lethality have been isolated from acrorhagi (specialized aggressive organs elaborated by only certain species of sea anemones belonging to the family Actiniidae) of Actinia equina. This prompted us to survey biologically active polypeptides in the acrorhagi of two species of sea anemones, Anthopleura aff. xanthogrammica and Anthopleura fuscoviridis. No potent crab lethality was displayed by the acrorhagial extracts of both species. However, significantly high protease inhibitory activity was instead detected in the acrorhagial extracts of the two species and also in that of A. equina. From the acrorhagi of A. equina, A. aff. xanthogrammica and A. fuscoviridis, one (AEAPI), one (AXAPI) and two (AFAPI-I and AFAPI-III) protease inhibitors were isolated, respectively. The complete amino acid sequences of the four inhibitors were elucidated by N-terminal sequencing and sequencing of the C-terminal peptide fragment produced upon asparaginylendopeptidase digestion. The determined amino acid sequences revealed that all the four inhibitors are new members of the Kunitz-type protease inhibitor family.     

Experiments on food preference and chemical sense in Pycnogonida

Food choice experiments were carried out with three species of pycnogonids: Nymphon rubrum, N. gracile , and Endeis spinosa.
Running sea-water that had circulated over different possible food-sources (hydroids, actiniarians, octocorals, synascidians) was offered to the pycnogonids at one side of a basin, pure sea-water or sea-water that had flowed over a different source of food at the other.
The experiments, though few in number, seemed to indicate that:
(1) Pycnogonids are capable of detecting the presence or absence of various coelenterate species.
(2) They are able to discriminate between different food-sources, i.e. the nature of the chemical substances produced by the food determines the degree of attraction.
(3) The preferential sequence for the three pycnogonids tested is: Nymphon rubrum prefers Dynamena over Tubularia and Laomedea ; these are preferred over Actinia and Metridium ; there is no attraction to Alcyonium. Nymphon gracile prefers Laomedea over Dynamena. Endeis spinosa prefers Laomedea over Dynamena ; these are preferred over Actinia ; there is no attraction to Morchellium.
(4) Experimental amputation of appendages indicates a high probability that the chemo-receptors of the pycnogonids are not located on the chelifores, palps, or ovigers. They may be located (a) all over the body, possibly in innervated epidermal bristles; and/or (b) in the distal part of the proboscis, bearing sensory setae; and/or (c) on the walking legs.     

An ultrastructural investigation of the early stages of oocyte differentiation in Actinia fragacea (Cnidaria; Anthozoa)

Individuals from a population of the intertidal sea anemone Actinia fragacea (Tugwell) were collected at approximately monthly intervals over an 18 month period. Samples of gonad were removed from each anemone and examined by light and electron microscopy. During late spring and early summer, large numbers of small cells were seen in the endoderm of the female gonads, lying close to the mesoglea. For convenience, these cells were classified into three types. Type I cells are 6–9 μm in diameter, with relatively very large nuclei, which may contain synaptinemal complexes, and scant cytoplasm containing few organelles. Type II cells are larger, reaching 15 μ m in diameter, with more abundant cytoplasm containing more organelles and inclusions. The nucleus is more dense, but may also contain synaptinemal complexes. Type III cells are less common. They are similar in size to Type II cells, but their nuclei contain irregular dense chromatin masses, and the nuclear envelope is incomplete or absent. The possible significance of the various cell types is discussed. It is suggested that Type I cells are oocytes at a very early stage of differentiation and that Type II cells are rather later oocytes. The status of the Type III cells is uncertain.     

海葵毒素多肽的分离和初步表征

经冻融法提取,丙酮沉淀,SP Sephadex C 25强离子交换和HPLC ODS C₁₈反相色谱层析,从海葵Actinia cari中提纯到两个新的毒素多肽,命名为RSAPⅠ和RSAPⅡ,经SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）鉴定RSAPⅠ和RSAPⅡ均为单一成分,相对分子质量分别为5 008和4 992.初步钠通道药理学实验表明,RSAPⅠ和RSAPⅡ对豚鼠心室肌细胞钠通道表现出活性,RSAPⅠ使钠离子内流增加,起开放作用;RSAPⅡ使钠离子内流减少,起阻滞作用.二者的圆二色谱均表现为100% β转角.     

Preliminary ultrastructural and autoradiographic evidence that the trophonema of the sea anemone Actinia fragacea has a nutritive function

The developing oocyte of the sea anemone Actinia fragacea is associated with a distinct group of gonad epithelial cells which constitute the trophonema. Electron microscopy has shown that the cells of the trophonema extend through a pore in the mesoglea which surrounds the oocyte, and make intimate contact with the oocyte surface. The ooplasm beneath this region of contact differs from the rest of the oocyte in containing numerous small vesicles, but few yolk granules or other organelles. Light microscope autoradiography has shown that oocytes within gonads can take up and incorporate tritiated glucose and leucine from solution. The cells of the trophonema appear more active in precursor incorporation than other gonad epithelial cells. The evidence therefore suggests that these cells have a nutritive function during oogenesis.