Diversity and antimicrobial activity of culturable fungi associated with sea anemone Anthopleura xanthogrammica

Background: The main objective of this study was to isolate fungi associated with Anthopleura xanthogrammica and measure their antimicrobial and enzymatic activities. A total of 93 fungal strains associated with A. xanthogrammica were isolated in this study, of which 32 isolates were identified using both morphological characteristics and internal transcribed spacer (ITS) sequence analysis. The antibacterial activities of 32 fungal isolates were tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Edwardsiella tarda, Vibrio harveyi, Fusarium oxysporum, and Pyricularia oryzae by agar diffusion assay. Extracellular hydrolytic enzyme activities of the fungal isolates were determined by agar diffusion assays. Enzyme activities were detected from clear halo size. Results: The isolated fungi belonged to 18 genera within 7 taxonomic orders of 1 phylum. The genera Aspergillaceae were the most diverse and common. The antimicrobial activities of 32 isolates were evaluated, and 19 (59.4%) of fungi isolate displayed unique antimicrobial activities. All fungal strains displayed at least one enzyme activity. The most common enzyme activities in the fungi isolates were amylase and protease, while the least common were pectinase and xylanase. Conclusions: This is first report on the sea anemone-derived fungi with antimicrobial and enzyme activities. Results indicated that sea anemone is a hot spot of fungal diversity and a rich resource of bioactive natural products.

Identification of a pore-forming protein from sea anemone Anthopleura dowii Verrill (1869) venom by mass spectrometry

Background: Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Anthopleura dowii Verrill (1869). 17. Methods: Cytolytic activity of A. dowii Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18. Results: The amount of protein at which the venom produced 50% hemolysis (HU50) was determined in hemolysis assays using erythrocytes from sheep (HU50 = 10.7 ± 0.2 µg), goat (HU50 = 13.2 ± 0.3 µg), rabbit (HU50 = 34.7 ± 0.5 µg), and human (HU50 = 25.6 ± 0.6 µg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC50) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 µg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction containing the pore-forming protein was osmotically protected by PEG-3350 Da molecular mass, which corroborated that the loss of integrity in the plasma membrane was produced via pore formation. 19. Conclusion: A. dowii Verrill (1869) venom contains a pore-forming protein suitable for designing new drugs for cancer therapy.(AU)

Estudio bioquímico del veneno de la anémona de mar Phymactis papillosa (Actiniidae)

En este trabajo se ha estudiado bioquímicamente el veneno de Phymactis papillosa, colectadas en la bahía de Ancón. El veneno fue obtenido mediante shock hipotónico y luego se liofilizó. El análisis electroforético del veneno soluble mostró la presencia de 5 bandas proteicas con pesos moleculares entre 5 y 25.1 kDa. El veneno soluble fue fraccionado por cromatografía de filtración en una columna de Sephadex G-50, obteniéndose cuatro picos de proteína (I, II, III y IV). Tanto en el veneno soluble como en las fracciones colectadas se midió actividad de proteasa, fosfolipasa, hialuronidasa, fosfatasa ácida y fosfatasa alcalina; así como, actividad hemolítica y neurotóxica. Se encontró actividad proteolítica sobre caseína, en el veneno soluble y en los picos I y III. No se detectó actividad de fosfolipasa, hialuronidasa, fosfatasa ácida y fosfatasa alcalina. La actividad hemolítica, ensayada sobre eritrocitos humanos, se encontró en el veneno soluble y en el pico II. Finalmente, tanto el veneno soluble como el pico III mostraron ser neurotóxicos al ser inyectados en ratones albinos vía intraperitoneal. Se concluye que el veneno soluble de P. papillosa tiene actividad proteolítica, hemolítica y neurotóxica

In this work, the poison of Phymactis papillosa collected in Ancón bay has been studied biochemically. The venom was obtained by hypotonic shock and then lyophilized. Electrophoretic analysis of the soluble poison showed the presence of 5 protein bands with molecular weights between 5 and 25.1 kDa. The soluble venom was fractionated by filtration chromatography on a Sephadex G-50 column, yielding four protein peaks (I, II, III and IV). In the soluble venom and collected fractions was measured protease activity, phospholipase, hyaluronidase, acid phosphatase and alkaline phosphatase; as well as hemolytic and neurotoxic activity. Proteolytic activity on casein was found in the soluble venom and peaks I and III. Was not detected phospholipase activity, hyaluronidase, acid phosphatase and alkaline phosphatase. Hemolytic activity on human red cells tested, was found in the soluble venom and peak II. Finally, the soluble venom as the peak III showed be neurotoxic when injected into white mice intraperitoneally. It is concluded that the soluble venom of P. papillosa has proteolytic, hemolytic and neurotoxic activity

Antimicrobial properties of sea anemone Anthopleura nigrescens from Pacific coast of Costa Rica

Objective: To evaluate the antimicrobial and antifungal activities of the aqueous and partitioned extract of sea anemone Anthopleura nigrescens (A. nigrescens). Methods: The sea anemone A. nigrescens was collected, minced, homogenized, lyophilized and then further partitioned with diethyl ether, acetone, ethanol and water. These fractions were evaluated for antimicrobial activity against bacterial and fungal pathogens. Results: Acetone extract was found to produce a pronounced inhibition of 7.0 mm against Proteus vulgaris and diethyl ether extract inhibited Pseudomonas aeruginosa with an inhibition zone of 6.5 mm. In antifungal activity, ethanol extract showed good activity against Botrytis cinerea, Trichoderma harzianum and Rhizopus oryzae compared with other strains. Acetone and ethanol extract of A. nigrescens showed activity against all of pathogens tested. Slight activity was observed in the water extract with inhibition zone of 1.5 mm. Conclusions: The present study revealed that sea anemone A. nigrescens may also contain some biologically active agents which have potential activity against pathogenic microorganisms.

Antimicrobial properties of sea anemone Anthopleura nigrescens from Pacific coast of Costa Rica

Objective: To evaluate the antimicrobial and antifungal activities of the aqueous and partitioned extract of sea anemone Anthopleura nigrescens (A. nigrescens). Methods: The sea anemone A. nigrescens was collected, minced, homogenized, lyophilized and then further partitioned with diethyl ether, acetone, ethanol and water. These fractions were evaluated for antimicrobial activity against bacterial and fungal pathogens. Results: Acetone extract was found to produce a pronounced inhibition of 7.0 mm against Proteus vulgaris and diethyl ether extract inhibited Pseudomonas aeruginosa with an inhibition zone of 6.5 mm. In antifungal activity, ethanol extract showed good activity against Botrytis cinerea, Trichoderma harzianum and Rhizopus oryzae compared with other strains. Acetone and ethanol extract of A. nigrescens showed activity against all of pathogens tested. Slight activity was observed in the water extract with inhibition zone of 1.5 mm. Conclusions: The present study revealed that sea anemone A. nigrescens may also contain some biologically active agents which have potential activity against pathogenic microorganisms.

Ocurrence of the sea anemone Telmatactis panamensis (Verrill, 1869) (Cnidaria: Anthozoa: Actiniaria) at Isla del Coco National Park, Costa Rica / Ocurrencia de la anémona de mar Telmatactis panamensis (Verrill, 1869) (Cnidaria: Anthozoa: Actiniaria) en el Parque Nacional Isla del Coco, Costa Rica

The sea anemone fauna of Isla del Coco National Park (also known as Cocos Island Nacional Park), Pacific Costa Rica is poorly known. In the present work we report the first occurrence of the species Telmatactis panamensis. Individuals of this sea anemone (n=24) were collected at Chatham Bay intertidal and at 15m depth in Punta Ulloa, in both cases attached to rocks; during the expedition UCR-UNA-COCO-I in April 2010. We provide photographs of live individuals, external anatomy and an inventory of cnidae of the studied specimens. Possibly this species is extended to greater depth as observed by other authors in the Galápagos Islands.

La fauna de anémonas de mar es prácticamente desconocida para el Parque Nacional Isla del Coco (Costa Rica). En el presente trabajo se reporta por primera vez la presencia de la especie Telmatactis panamensis. Individuos de esta anémona de mar fueron colectados en el intermareal de Bahía Chatham y a 15m de profundidad en Punta Ulloa, en ambos casos adheridas a rocas; durante la expedición UCR-UNA-COCO-I en Abril de 2010. Se proveen fotografías de ejemplares vivos, datos de su anatomía externa y un inventario del cnidae de los especímenes estudiados. Posiblemente esta especie se extienda a mayor profundidad, tal como fue observado por otros autores para ejemplares de las Islas Galápagos.

Differential susceptibilities of human lung, breast and skin cancer cell lines to killing by five sea anemone venoms

Although sea anemones are well known for being rich sources of toxins, including cytolysins and neurotoxins, their venoms and toxins have been poorly studied. In the present study, the venoms from five sea anemones (Heteractis crispa, Heteractis magnifica, Heteractis malu, Cryptodendrum adhaesivum and Entacmaea quadricolor) were obtained by the milking technique, and the potential of these venoms to kill cancer cells was tested on three cell lines (A549 lung cancer, T47D breast cancer and A431 skin cancer). The total protein level in the crude extract was determined by the bicinchoninic acid (BCA) protein assay. The cytotoxicity on different cell lines was assayed using the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay which measures survival based on the detection of mitochondrial activity and by the crystal violet assay, which measures survival based on the ability of cells to remain adherent to microplates. The results indicate that the sea anemone venom is cytotoxic to human cancer cells. The A549 cell line was the most sensitive of the cell lines tested with a significant reduction in viability observed at 40 µg/mL. H. malu, C. adhaesivum and E. quadricolor had a significant inhibitory effect on A431 cells. Furthermore, H. malu and C. adhaesivum had a significant inhibitory effect on T47D cell line at 40 µg/mL. In conclusion, the sea anemone venoms tested have the potential to be developed as anticancer agents.(AU)

Biological activity of sea anemone proteins: II. Cytolysis and cell line toxicity.

Potent cytolytic activity was exhibited by proteins extracted from three sea anemones viz. Heteractis magnifica, Stichodactyla haddoni and Paracodylactis sinensis by affecting the red blood corpuscles (RBC) and the mouse fibroblast cell line (L929) and leukemia cell line (P388). Crude toxin of all the three anemone species induced spontaneous hemolysis of chicken, goat and human erythrocytes. The crude toxin of H. magnifica (0.98 mg/ml) elicited hemolysis at levels of 4096, 512 and 4096 HU (hemolytic unit) in chicken, goat and human erythrocytes respectively. Subsequently, the crude toxin of S.haddoni (0.82 mg/ml) exhibited a hemolytic activity of 256, 128 and 512 HU and that of P. sinensis (0.60 mg/ml) had a hemolytic activity of 128, 4096 and 512 HU. Most of the partially purified proteins of these anemones also exhibited the activity against the three different erythrocytes. The viability of L929 and P388 was adversely affected on adding the crude toxins. The symptoms of toxicity shown by the cells were rounding, lysis and detachment from the substratum. These effects were the least in S. haddoni, as compared to those the crude toxins of the other two species. Inhibition of growth of L929 exhibited by the toxin of the three species ranged between 61.08 and 93.38%. Similarly, inhibition of the growth of P388 ranged between 51.32 and 86.16%. The present investigation reveal the cytotoxic nature of anemone toxins.

Biological activity of sea anemone proteins: I. Toxicity and histopathology.

The crude as well as partially purified protein fractions from anemone species viz. Heteractis magnifica, Stichodactyla haddoni and Paracodylactis sinensis, collected from the Gulf of Mannar, south east coast of India were found to be toxic at different levels to mice. The mice showed behavioral changes such as loss of balance, opaque eyes, tonic convulsions, paralysis, micturiction, flexing of muscles, prodding (insensitive to stimulii), foaming from mouth and exophthalmia. The toxic proteins upon envenomation produced several chronic and lethal histopathological changes like formation of pycnotic nuclii and glial nodules in the brain; heamolysis, thrombosis and myocardial haemorrhage in the heart; granulomatous lesions, and damage to the hepatic cells in the liver and haemorrhage throughout the kidney parenchyma and shrinkage of glomerular tufts in the kidney. The toxins proved to be neurotoxic, cardiotoxic, nephrotoxic and hepatotoxic by their action on internal organ systems. The toxins were also thermostable till 60oC and had considerable shelf life.

Effects of sea anemone toxin anthopleurin-Q on potassium currents in rats and guinea pig ventricular myocytes / 老年心脏病学杂志（英文版）

To investigate the effect of sea anemone toxin anthopleurin-Q (AP-Q) on potassium currents in isolated rats and guinea pig ventricular myocytes.Methods The ventricular cells of guinea pigs and SD rats were obtained by enzymatic dissociation method.Whole cell patch clamp technique was used to record potassium currents (Ito,IK,and IK1).Results AP-Q 3-100 nmol/L increased Ito in a concentration-dependent manner,with an EC50 value of 12.7 nmol/L.At a potential of +50mV,AP-Q 10nmol/L increased Ito from (13.3±3.4) pA pF-1 to (19.46±4.3) pA pF-1.AP-Q 0.1-100 nmol/L increased IK and IK tail in a concentration-dependent manner with EC50 values of 4.7 nmol/L and 5.0 nmol/L,respectively.AP-Q 1 pmol/L-100 nmol/L increased IK1 in dose-dependent manner,with an EC50 of 0.2 nmol/L.Conclusions The effect of AP-Q on Ito,IK and IK1 may partly explain its mechanism in shortening APD and increasing RP.(J Geriatr Cardiol 2008;5:243-247)

A Case of Sea Anemone Dermatitis / 대한피부과학회지

Sea anemone dermatitis, caused by the toxin of sea anemones (member of phylum Cnidaria), is characterized by erythematous papules, wheals, or vesicles, accompanied by pain and itching. A 25-year old woman presented with multiple erythematous papules on her right forearm, after contact with a sea anemone while scuba diving. She took both courses of an acute toxic reaction and a delayed allergic reaction.

A STUDY OF SAG ARTIA ROSEA IN BOHAI SEA (A RED SEA ANEMONE) ON BIOMEDICINE / 中国海洋药物

On the basis of the preliminary study of the prevention and treatment of Bohai Sea divers' dermatitis from 1983 to 1987, the morphological characteristic of 5. rosea (see section I ), the pathogenic organism of the divers' dermatitis, was studied in the field of systematic zoology, some aspect concerning medical pathogenic characteristic of the nematocysts from the red sea anemone (section I ) was studied from the point of view of pathophysiology,a preliminary study on active proteins from 5. rosea (section I ) was studied according to toxicology. The article further stated the pathogenic mechanism of the sea anemones' dermatitis, in order to go further into the new ways and methods for preventing and treating the divers' dermatitis.