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...(AU)

Hemolytic, anticancer and antigiardial activity of Palythoa caribaeorum venom

Background Cnidarian venoms and extracts have shown a broad variety of biological activities including cytotoxic, antibacterial and antitumoral effects. Most of these studied extracts were obtained from sea anemones or jellyfish. The present study aimed to determine the toxic activity and assess the antitumor and antiparasitic potential of Palythoa caribaeorum venom by evaluating its in vitro toxicity on several models including human tumor cell lines and against the parasite Giardia intestinalis. Methods The presence of cytolysins and vasoconstrictor activity of P. caribaeorum venom were determined by hemolysis, PLA2 and isolated rat aortic ring assays, respectively. The cytotoxic effect was tested on HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma), K562 (human chronic myelogenous leukemia), U251 (human glyoblastoma), PC-3 (human prostatic adenocarcinoma) and SKLU-1 (human lung adenocarcinoma). An in vivo toxicity assay was performed with crickets and the antiparasitic assay was performed against G. intestinalis at 24 h of incubation. Results P. caribaeorum venom produced hemolytic and PLA2 activity and showed specific cytotoxicity against U251 and SKLU-1 cell lines, with approximately 50% growing inhibition. The venom was toxic to insects and showed activity against G. intestinalis in a dose-dependent manner by possibly altering its membrane osmotic equilibrium. Conclusion These results suggest that P. caribaeorum venom contains compounds with potential therapeutic value against microorganisms and cancer.(AU)

Hemolytic, anticancer and antigiardial activity of Palythoa caribaeorum venom

Background Cnidarian venoms and extracts have shown a broad variety of biological activities including cytotoxic, antibacterial and antitumoral effects. Most of these studied extracts were obtained from sea anemones or jellyfish. The present study aimed to determine the toxic activity and assess the antitumor and antiparasitic potential of Palythoa caribaeorum venom by evaluating its in vitro toxicity on several models including human tumor cell lines and against the parasite Giardia intestinalis. Methods The presence of cytolysins and vasoconstrictor activity of P. caribaeorum venom were determined by hemolysis, PLA2 and isolated rat aortic ring assays, respectively. The cytotoxic effect was tested on HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma), K562 (human chronic myelogenous leukemia), U251 (human glyoblastoma), PC-3 (human prostatic adenocarcinoma) and SKLU-1 (human lung adenocarcinoma). An in vivo toxicity assay was performed with crickets and the antiparasitic assay was performed against G. intestinalis at 24 h of incubation. Results P. caribaeorum venom produced hemolytic and PLA2 activity and showed specific cytotoxicity against U251 and SKLU-1 cell lines, with approximately 50% growing inhibition. The venom was toxic to insects and showed activity against G. intestinalis in a dose-dependent manner by possibly altering its membrane osmotic equilibrium. Conclusion These results suggest that P. caribaeorum venom contains compounds with potential therapeutic value against microorganisms and cancer.

Pharmacological characterization of cnidarian extracts from the Caribbean Sea: evaluation of anti-snake venom and antitumor properties

Background: Cnidarians produce toxins, which are composed of different polypeptides that induce pharmacological effects of biotechnological interest, such as antitumor, antiophidic and anti-clotting activities. This study aimed to evaluate toxicological activities and potential as antitumor and antiophidic agents contained in total extracts from five cnidarians: Millepora alcicornis, Stichodactyla helianthus, Plexaura homomalla, Bartholomea annulata and Condylactis gigantea (total and body wall). Methods: The cnidarian extracts were evaluated by electrophoresis and for their phospholipase, proteolytic, hemorrhagic, coagulant, fibrinogenolytic, neuromuscular blocking, muscle-damaging, edema-inducing and cytotoxic activities. Results: All cnidarian extracts showed indirect hemolytic activity, but only S. helianthus induced direct hemolysis and neurotoxic effect. However, the hydrolysis of NBD-PC, a PLA2 substrate, was presented only by the C gigantea (body wall) and S. helianthus. The extracts from P. homomalla and S. helianthus induced edema, while only C gigantea and S. helianthus showed intensified myotoxic activity. The proteolytic activity upon casein and fibrinogen was presented mainly by B. annulata extract and all were unable to induce hemorrhage or fibrinogen coagulation. Cnidarian extracts were able to neutralize clotting induced by Bothrops jararacussu snake venom, except M. alcicornis. All cnidarian extracts were able to inhibit hemorrhagic activity induced by Bothrops moojeni venom. Only the C. gigantea (body wall) inhibited thrombin-induced coagulation. All cnidarian extracts showed antitumor effect against Jurkat cells, of which C. gigantea (body wall) and S. helianthus were the most active; however, only C. gigantea (body wall) and M. alcicornis were active against B16F10 cells...(AU)

Pharmacological characterization of cnidarian extracts from the Caribbean Sea: evaluation of anti-snake venom and antitumor properties

Background: Cnidarians produce toxins, which are composed of different polypeptides that induce pharmacological effects of biotechnological interest, such as antitumor, antiophidic and anti-clotting activities. This study aimed to evaluate toxicological activities and potential as antitumor and antiophidic agents contained in total extracts from five cnidarians: Millepora alcicornis, Stichodactyla helianthus, Plexaura homomalla, Bartholomea annulata and Condylactis gigantea (total and body wall). Methods: The cnidarian extracts were evaluated by electrophoresis and for their phospholipase, proteolytic, hemorrhagic, coagulant, fibrinogenolytic, neuromuscular blocking, muscle-damaging, edema-inducing and cytotoxic activities. Results: All cnidarian extracts showed indirect hemolytic activity, but only S. helianthus induced direct hemolysis and neurotoxic effect. However, the hydrolysis of NBD-PC, a PLA2 substrate, was presented only by the C gigantea (body wall) and S. helianthus. The extracts from P. homomalla and S. helianthus induced edema, while only C gigantea and S. helianthus showed intensified myotoxic activity. The proteolytic activity upon casein and fibrinogen was presented mainly by B. annulata extract and all were unable to induce hemorrhage or fibrinogen coagulation. Cnidarian extracts were able to neutralize clotting induced by Bothrops jararacussu snake venom, except M. alcicornis. All cnidarian extracts were able to inhibit hemorrhagic activity induced by Bothrops moojeni venom. Only the C. gigantea (body wall) inhibited thrombin-induced coagulation. All cnidarian extracts showed antitumor effect against Jurkat cells, of which C. gigantea (body wall) and S. helianthus were the most active; however, only C. gigantea (body wall) and M. alcicornis were active against B16F10 cells...

In vitro characterization of jellyfish venom fibrin(ogen)olytic enzymes from Nemopilema nomurai

Background: Because jellyfish are capable of provoking envenomation in humans, they are considered hazardous organisms. Although the effects of their toxins are a matter of concern, information on the venom components, biological activity and pathological mechanisms are still scarce. Therefore, the aim of the present study was to investigate a serine protease component of Nemopilema nomurai jellyfish venom (NnV) and unveil its characteristics. Methods: To determine the relationship between fibrinolytic activity of NnV and the serine protease, fibrin zymography was performed using metalloprotease and serine protease inhibitors. The biochemical characterization of serine proteases of NnV were determined by the amidolytic assay. Fractions with fibrinolytic activity were obtained by DEAE cation exchange column. Results: NnV displayed fibrinolytic activities with molecular masses of approximately 70, 35, 30, and 28 kDa. The fibrinolytic activity of NnV was completely obliterated by phenylmethylsulfonyl fluoride, a prototype serine protease inhibitor. Based on amidolytic assays using chromogenic substrates specific for various kinds of serine proteases, NnV predominantly manifested a chymotrypsin-like feature. Its activity was completely eliminated at low pH (< 6) and high temperatures (> 37 °C). Some metal ions (Co2+, Cu2+, Zn2+ and Ni2+) strongly suppressed its fibrinolytic activity, while others (Ca2+ and Mg2+) failed to do so. Isolation of a serine protease with fibrionolytic activity from NnV revealed that only p3 showed the fibrinolytic activity, which was completely inhibited by PMSF. Conclusion: The present study showed that N. nomurai jellyfish venom has a chymotrypsin-like serine protease with fibrinolytic activity. Such information might be useful for developing clinical management of jellyfish envenomation and pharmacological agents with therapeutic potential for thrombotic diseases in the future.(AU)

In vitro characterization of jellyfish venom fibrin(ogen)olytic enzymes from Nemopilema nomurai

Background: Because jellyfish are capable of provoking envenomation in humans, they are considered hazardous organisms. Although the effects of their toxins are a matter of concern, information on the venom components, biological activity and pathological mechanisms are still scarce. Therefore, the aim of the present study was to investigate a serine protease component of Nemopilema nomurai jellyfish venom (NnV) and unveil its characteristics. Methods: To determine the relationship between fibrinolytic activity of NnV and the serine protease, fibrin zymography was performed using metalloprotease and serine protease inhibitors. The biochemical characterization of serine proteases of NnV were determined by the amidolytic assay. Fractions with fibrinolytic activity were obtained by DEAE cation exchange column. Results: NnV displayed fibrinolytic activities with molecular masses of approximately 70, 35, 30, and 28 kDa. The fibrinolytic activity of NnV was completely obliterated by phenylmethylsulfonyl fluoride, a prototype serine protease inhibitor. Based on amidolytic assays using chromogenic substrates specific for various kinds of serine proteases, NnV predominantly manifested a chymotrypsin-like feature. Its activity was completely eliminated at low pH (< 6) and high temperatures (> 37 °C). Some metal ions (Co2+, Cu2+, Zn2+ and Ni2+) strongly suppressed its fibrinolytic activity, while others (Ca2+ and Mg2+) failed to do so. Isolation of a serine protease with fibrionolytic activity from NnV revealed that only p3 showed the fibrinolytic activity, which was completely inhibited by PMSF. Conclusion: The present study showed that N. nomurai jellyfish venom has a chymotrypsin-like serine protease with fibrinolytic activity. Such information might be useful for developing clinical management of jellyfish envenomation and pharmacological agents with therapeutic potential for thrombotic diseases in the future.(AU)

Characteristics of hemolytic activity induced by the aqueous extract of the Mexican fire coral Millepora complanata

Background Millepora complanata is a plate-like fire coral common throughout the Caribbean. Contact with this species usually provokes burning pain, erythema and urticariform lesions. Our previous study suggested that the aqueous extract of M. complanata contains non-protein hemolysins that are soluble in water and ethanol. In general, the local damage induced by cnidarian venoms has been associated with hemolysins. The characterization of the effects of these components is important for the understanding of the defense mechanisms of fire corals. In addition, this information could lead to better care for victims of envenomation accidents.Methods An ethanolic extract from the lyophilized aqueous extract was prepared and its hemolytic activity was compared with the hemolysis induced by the denatured aqueous extract. Based on the finding that ethanol failed to induce nematocyst discharge, ethanolic extracts were prepared from artificially bleached and normal M. complanata fragments and their hemolytic activity was tested in order to obtain information about the source of the heat-stable hemolysins.Results Rodent erythrocytes were more susceptible to the aqueous extract than chicken and human erythrocytes. Hemolytic activity started at ten minutes of incubation and was relatively stable within the range of 28-50°C. When the aqueous extract was preincubated at temperatures over 60°C, hemolytic activity was significantly reduced. The denatured extract induced a slow hemolytic activity (HU50= 1,050.00 ± 45.85 μg/mL), detectable four hours after incubation, which was similar to that induced by the ethanolic extract prepared from the aqueous extract (HU50= 1,167.00 ± 54.95 μg/mL). No significant differences were observed between hemolysis induced by ethanolic extracts from bleached and normal fragments, although both activities were more potent than hemolysis induced by the denatured extract.Conclusions The results showed that the aqueous extract of M. complanata possesses one or more powerful heat-labile hemolytic proteins that are slightly more resistant to temperature than jellyfish venoms. This extract also contains slow thermostable hemolysins highly soluble in ethanol that are probably derived from the body tissues of the hydrozoan.(AU)

Identification of two novel cytolysins from the hydrozoan Olindias sambaquiensis (Cnidaria)

Although the hydrozoan Olindias sambaquiensis is the most common jellyfish associated with human envenomation in southeastern and southern Brazil, information about the composition of its venom is rare. Thus, the present study aimed to analyze pharmacological aspects of O. sambaquiensis venom as well as clinical manifestations observed in affected patients. Crude protein extracts were prepared from the tentacles of animals; peptides and proteins were sequenced and submitted to circular dichroism spectroscopy. Creatine kinase, cytotoxicity and hemolytic activity were evaluated by specific methods.(AU)

Identification of two novel cytolysins from the hydrozoan Olindias sambaquiensis (Cnidaria)

Although the hydrozoan Olindias sambaquiensis is the most common jellyfish associated with human envenomation in southeastern and southern Brazil, information about the composition of its venom is rare. Thus, the present study aimed to analyze pharmacological aspects of O. sambaquiensis venom as well as clinical manifestations observed in affected patients. Crude protein extracts were prepared from the tentacles of animals; peptides and proteins were sequenced and submitted to circular dichroism spectroscopy. Creatine kinase, cytotoxicity and hemolytic activity were evaluated by specific methods.

Characteristics of hemolytic activity induced by the aqueous extract of the Mexican fire coral Millepora complanata

Background Millepora complanata is a plate-like fire coral common throughout the Caribbean. Contact with this species usually provokes burning pain, erythema and urticariform lesions. Our previous study suggested that the aqueous extract of M. complanata contains non-protein hemolysins that are soluble in water and ethanol. In general, the local damage induced by cnidarian venoms has been associated with hemolysins. The characterization of the effects of these components is important for the understanding of the defense mechanisms of fire corals. In addition, this information could lead to better care for victims of envenomation accidents.Methods An ethanolic extract from the lyophilized aqueous extract was prepared and its hemolytic activity was compared with the hemolysis induced by the denatured aqueous extract. Based on the finding that ethanol failed to induce nematocyst discharge, ethanolic extracts were prepared from artificially bleached and normal M. complanata fragments and their hemolytic activity was tested in order to obtain information about the source of the heat-stable hemolysins.Results Rodent erythrocytes were more susceptible to the aqueous extract than chicken and human erythrocytes. Hemolytic activity started at ten minutes of incubation and was relatively stable within the range of 28-50°C. When the aqueous extract was preincubated at temperatures over 60°C, hemolytic activity was significantly reduced. The denatured extract induced a slow hemolytic activity (HU50= 1,050.00 ± 45.85 g/mL), detectable four hours after incubation, which was similar to that induced by the ethanolic extract prepared from the aqueous extract (HU50= 1,167.00 ± 54.95 g/mL). No significant differences were observed between hemolysis induced by ethanolic extracts from bleached and normal fragments, although both activities were more potent than hemolysis induced by the denatured extract...(AU)

Characteristics of hemolytic activity induced by the aqueous extract of the Mexican fire coral Millepora complanata

Background Millepora complanata is a plate-like fire coral common throughout the Caribbean. Contact with this species usually provokes burning pain, erythema and urticariform lesions. Our previous study suggested that the aqueous extract of M. complanata contains non-protein hemolysins that are soluble in water and ethanol. In general, the local damage induced by cnidarian venoms has been associated with hemolysins. The characterization of the effects of these components is important for the understanding of the defense mechanisms of fire corals. In addition, this information could lead to better care for victims of envenomation accidents.Methods An ethanolic extract from the lyophilized aqueous extract was prepared and its hemolytic activity was compared with the hemolysis induced by the denatured aqueous extract. Based on the finding that ethanol failed to induce nematocyst discharge, ethanolic extracts were prepared from artificially bleached and normal M. complanata fragments and their hemolytic activity was tested in order to obtain information about the source of the heat-stable hemolysins.Results Rodent erythrocytes were more susceptible to the aqueous extract than chicken and human erythrocytes. Hemolytic activity started at ten minutes of incubation and was relatively stable within the range of 28-50°C. When the aqueous extract was preincubated at temperatures over 60°C, hemolytic activity was significantly reduced. The denatured extract induced a slow hemolytic activity (HU50= 1,050.00 ± 45.85 g/mL), detectable four hours after incubation, which was similar to that induced by the ethanolic extract prepared from the aqueous extract (HU50= 1,167.00 ± 54.95 g/mL). No significant differences were observed between hemolysis induced by ethanolic extracts from bleached and normal fragments, although both activities were more potent than hemolysis induced by the denatured extract...

Dose and time dependence of box jellyfish antivenom

Background The effectiveness of the currently available box jellyfish (Chironex fleckeri) antivenom has been subject of debate for many years. To assess whether the box jellyfish antivenom has the ability to attenuate venom-induced damage at cellular level, the present study analyzed the dose and time dependence of the antivenom in a cell-based assay.Methods Different doses of antivenom were added to venom and subsequently administered to cells and the cell index was measured using xCelligence Technology (ACEA Biosciences). Similarly, antivenom and venom were incubated over different time periods and cell survival measured as stated above. For both experiments, the cell index was plotted as a measure of cell survival against the dose or incubation time and significance was determined with the use of a one-way ANOVA with a LSD post hoctest.Results Increasing concentrations of antivenom significantly augmented cell survival, with a concentration of approximately five times the currently recommended dose for human envenomation, causing the first significant increase in cell survival compared venom alone. Further, cell survival improved with increasing incubation time of venom and antivenom prior to addition to the cells, indicating that box jellyfish antivenom requires approximately 70 minutes to neutralize C. fleckeri venom.Conclusion The presented results suggest that the currently recommended dose of antivenom requires adjustment, and more importantly, a human trial to test the effects of higher concentrations is also necessary. Further, antivenom has delayed neutralizing effects (i.e. after 70 minutes) which underlines the eminence of immediate and prolonged cardiopulmonary resuscitation in victims suffering from a C. fleckerivenom-induced cardiovascular collapse.(AU)

Dose and time dependence of box jellyfish antivenom

Background The effectiveness of the currently available box jellyfish (Chironex fleckeri) antivenom has been subject of debate for many years. To assess whether the box jellyfish antivenom has the ability to attenuate venom-induced damage at cellular level, the present study analyzed the dose and time dependence of the antivenom in a cell-based assay.Methods Different doses of antivenom were added to venom and subsequently administered to cells and the cell index was measured using xCelligence Technology (ACEA Biosciences). Similarly, antivenom and venom were incubated over different time periods and cell survival measured as stated above. For both experiments, the cell index was plotted as a measure of cell survival against the dose or incubation time and significance was determined with the use of a one-way ANOVA with a LSD post hoctest.Results Increasing concentrations of antivenom significantly augmented cell survival, with a concentration of approximately five times the currently recommended dose for human envenomation, causing the first significant increase in cell survival compared venom alone. Further, cell survival improved with increasing incubation time of venom and antivenom prior to addition to the cells, indicating that box jellyfish antivenom requires approximately 70 minutes to neutralize C. fleckeri venom.Conclusion The presented results suggest that the currently recommended dose of antivenom requires adjustment, and more importantly, a human trial to test the effects of higher concentrations is also necessary. Further, antivenom has delayed neutralizing effects (i.e. after 70 minutes) which underlines the eminence of immediate and prolonged cardiopulmonary resuscitation in victims suffering from a C. fleckerivenom-induced cardiovascular collapse.

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)

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)

Assessment of biomedical and pharmacological activities of sea anemones Stichodactyla mertensii and Stichodactyla gigantea from Gulf of Mannar Biosphere Reserve, southeast coast of India

Cnidarians comprise an old and diverse animal phylum, and possess a wide variety of biologically active substances. Sea anemones contain a diversity of interesting biologically active compounds including some potent toxins. In the present work, the sea anemones Stichodactyla mertensii and Stichodactyla gigantea, collected from the Mandapam coast, are characterized biomedically and pharmacologically. The crude protein was obtained by using methanol and aqueous extracts. The respective protein contents of S. mertensii and S. gigantea were found to be 2.10 µg/mL and 1.87 µg/mL. The methanol and aqueous extracts of S. mertensii and S. gigantea yielded six and nine bands by SDS-PAGE on 12 percent gel. In the hemolytic assay, both extracts exhibited hemolytic effect on chicken, goat, cow and human erythrocytes ('A', 'B' and 'O'). The neurotoxic effects of these crude extracts were determined in vivo using the sea shore crab Ocypode macrocera and mortality was observed. The mouse bioassay for lethality was performed on male albino mice. The crude extract of S. mertensii showed higher lethality (58 seconds at 1 mL-dose) than that of S. gigantea (2 minutes and 10 seconds at 0.75 mL-dose). The analgesic activity test was also carried out on albino mice by Eddy's hot plate and tail-flick methods. The extracts showed moderate analgesic effect by both hot-plate and tail-flick methods. These characteristics emphasize the need for the isolation and molecular characterization of new active toxins in S. mertensii and S. gigantea.(AU)

Structure of nematocysts isolated from the fire corals Millepora alcicornis and Millepora complanata (Cnidaria: Hydrozoa)

Structural characteristics of discharged and undischarged nematocysts from the hydrozoans Millepora alcicornis and Millepora complanata, two fire corals collected in the Mexican Caribbean, were examined using transmission electron, scanning and light microscopy. In this study, we report for the first time images of the nematocysts found in these Mexican Caribbean venomous species. Two types of nematocysts were observed in both species, the more abundant identified as macrobasic mastigophore and the other a stenotele type. Macrobasic mastigophores were present in medium and large size classes while stenoteles appeared in only one size.(AU)

Hemolytic toxin from the soft coral Sarcophyton trocheliophorum: isolation and physiological characterization

The unifying characteristic of cnidarians is the production of protein and polypeptide toxins. The present study describes the identification of a hemolytic toxin from the soft coral Sarcophyton trocheliophorum. The crude extract was highly cytotoxic (EC50 = 50 ng/mL) against human erythrocytes. It was also tested for hemolytic activity by the blood agar plate method, resulting in a hemolytic halo of 12 mm with 50 µg of protein. The stability of the venom under different physiological conditions was analyzed. The venom hemolytic activity was augmented by alkaline and neutral pH whereas it was reduced in acidic pH. The activity was stable up to 60º C. The hemolytic activity was completely abolished by the addition of serum and reduced significantly during frequent freezing-thawing cycles. Toxin purification was performed by ammonium sulfate precipitation and subsequently desalted by dialysis against 10 mM sodium phosphate buffer (pH 7.2), followed by anion exchange chromatography on DEAE cellulose column and gel filtration chromatography using Sephadex G-50 matrix. The purified active fractions possessed a prominent protein of approximately 45 kDa, as revealed by SDS-PAGE.(AU)

Hemolytic toxin from the soft coral Sarcophyton trocheliophorum: isolation and physiological characterization

The unifying characteristic of cnidarians is the production of protein and polypeptide toxins. The present study describes the identification of a hemolytic toxin from the soft coral Sarcophyton trocheliophorum. The crude extract was highly cytotoxic (EC50 = 50 ng/mL) against human erythrocytes. It was also tested for hemolytic activity by the blood agar plate method, resulting in a hemolytic halo of 12 mm with 50 µg of protein. The stability of the venom under different physiological conditions was analyzed. The venom hemolytic activity was augmented by alkaline and neutral pH whereas it was reduced in acidic pH. The activity was stable up to 60º C. The hemolytic activity was completely abolished by the addition of serum and reduced significantly during frequent freezing-thawing cycles. Toxin purification was performed by ammonium sulfate precipitation and subsequently desalted by dialysis against 10 mM sodium phosphate buffer (pH 7.2), followed by anion exchange chromatography on DEAE cellulose column and gel filtration chromatography using Sephadex G-50 matrix. The purified active fractions possessed a prominent protein of approximately 45 kDa, as revealed by SDS-PAGE.(AU)