Biochemical and physiological analyses of a hemolytic toxin isolated from a sea anemone Actineria villosa.

A species of venomous sea anemone Actineria villosa was recently found inhabiting the coastal areas of Okinawa, Japan. This marine animal produces various proteinous toxins, so that a local health organization was called for medical treatment for those who had accidental contact with this animal. In this study we analyzed the biochemical and physiological properties of hemolytic protein from A. villosa. The toxin purified from the tentacles of the animals was found to be a protein with a molecular weight of approximately 19 kDa. We named this newly found hemolytic toxin of A. villosa, Avt-I. Incubation of the toxin with sphingomyelin inhibited hemolytic activity by up to 85%, showing that Avt-I may target sphingomyelin on the erythrocyte membrane. The hemolytic activity was stably maintained at temperatures below 45 degrees C, however, a sharp linear decrease in heat stability was observed within the range of 45-55 degrees C. Our results provide the first evidence that A. villosa produces a toxin with strong hemolytic activity similar in biochemical and physiological properties to other members of actinoporin family previously isolated from related species of sea anemones.     

Characterization of PsTX-60B, a new membrane-attack complex/perforin (MACPF) family toxin, from the venomous sea anemone Phyllodiscus semoni

The sea anemone Phyllodiscus semoni has been known as one of the most venomous sea anemones. Previously, we reported the isolation of the major lethal protein toxins, PsTX-60A and PsTX-60B, from P. semoni and the primary structure of PsTX-60A. In this paper, we report the complete sequence of cDNA (1600 base pairs) encoding PsTX-60B and the deduced primary structure (488 amino acids) of PsTX-60B. The amino acid sequence of PsTX-60B showed the homology with those of PsTX-60A and Actineria villosa toxin, AvTX-60A. The results showed that PsTX-60B is a new member of the membrane-attack complex/perforin (MACPF) family toxin.     

Purification and partial characterization of the phospholipase A2 and co-lytic factor from sea anemone (Aiptasia pallida) nematocyst venom.

Functional nematocysts of one specific morphological class, the penetrant microbasic mastigophores, were isolated from the sea anemone, Aiptasia pallida. These nematocysts contain a multicomponent venom composed of several proteins, including those with neurotoxic, hemolytic, and lethal activities. Hemolytic activity is produced by at least three synergistic venom proteins. One of these proteins is identified as a phospholipase A₂ (EC 3.1.1.4) which exists in two isozymic forms, and β, with molecular weights of 45,000 and 43,000, respectively. The β isozyme has been purified to homogeneity. It is a single-chained glycoprotein with an isoelectric point (pI) of 8.8 and represents 70% of the phospholipase activity of the venom. The activity of the β isozyme is relatively labile and is inactivated by 3.5 M urea or by heating at 45°C. It is most stable at pH 4.0 and loses 50% of its activity at pH values below 3.5 and above 8.0. A second venom protein has also been purified. It is essential for the hemolytic activity of the venom and is termed co-lytic factor (CLF). It is a monomeric glycoprotein having a pI of 4.5. CLF has a molecular weight of approximately 98,000, a sedimentation coefficient of 4.8 S, and is prolate in shape, having a frictional ratio of about 1.6. CLF constitutes about 1.25% of the total venom protein and is assayed by reversing fatty acid inhibition of the venom hemolysis activity.     

Purification and partial characterization of the phospholipase A2 and co-lytic factor from sea anemone (Aiptasia pallida) nematocyst venom

Functional nematocysts of one specific morphological class, the penetrant microbasic mastigophores, were isolated from the sea anemone, Aiptasia pallida. These nematocysts contain a multicomponent venom composed of several proteins, including those with neurotoxic, hemolytic, and lethal activities. Hemolytic activity is produced by at least three synergistic venom proteins. One of these proteins is identified as a phospholipase A₂ (EC 3.1.1.4) which exists in two isozymic forms, α and β, with molecular weights of 45,000 and 43,000, respectively. The β isozyme has been purified to homogeneity. It is a single-chained glycoprotein with an isoelectric point (pI) of 8.8 and represents 70% of the phospholipase activity of the venom. The activity of the β isozyme is relatively labile and is inactivated by 3.5 M urea or by heating at 45°C. It is most stable at pH 4.0 and loses 50% of its activity at pH values below 3.5 and above 8.0. A second venom protein has also been purified. It is essential for the hemolytic activity of the venom and is termed co-lytic factor (CLF). It is a monomeric glycoprotein having a pI of 4.5. CLF has a molecular weight of approximately 98,000, a sedimentation coefficient of 4.8 S, and is prolate in shape, having a frictional ratio of about 1.6. CLF constitutes about 1.25% of the total venom protein and is assayed by reversing fatty acid inhibition of the venom hemolysis activity.     

Molecular characterization on the genome structure of hemolysin toxin isoforms isolated from sea anemone Actineria villosa and Phyllodiscus semoni

We recently identified the existence of new isoforms of Avt-I (from sea anemone Actineria villosa) and Pstx20 (from sea anemone Phyllodiscus semoni) hemolytic toxins, and named them Avt-II and Pst-I. Avt-II and Pst-I differ in length by 14 and 7 bp, respectively, as compared to their corresponding isoform genes. Both newly found isoform genes have the coding regions with the identical length of 1033 bp. The restriction fragment length polymorphism analysis with endonuclease HphI was able to clearly distinguish between the two Avt isoforms, but not Pstx isoforms, and based on the densitometric analysis of DNA bands, it indicated that relative expression levels of Avt-I and Avt-II genes were 18.3% and 81.7%, respectively. PCR amplification of the two Avt isoform genes using the genomic DNA as template indicated the existence of two introns within each toxin isoform gene. The first intron with the identical 242 bp in length for both Avt isoform was found within the 5′-untranslated region, and the second intron with lengths of 654 bp and 661 bp in Avt-I and Avt-II isoforms, respectively, was found within the signal sequence coding region. This is for the first time to identify the existence of introns within hemolysin genes of sea anemone. Having several unique characteristics that have identified only for a new member of actinoporin family of A. villosa and P. semoni, e.g., strong toxicity and genes with introns, it is plausible to speculate that these toxins have a unique genetic evolutionary linage differed from that for other sea anemone hemolytic toxins.     

A new toxin from the sea anemone Condylactis gigantea with effect on sodium channel inactivation.

A new peptide toxin exhibiting a molecular weight of 5043Da (av.) and comprising 47 amino acid residues was isolated from the sea anemone Condylactis gigantea. Purification of the peptide was achieved by a multistep chromatographic procedure monitoring its strong paralytic activity on crustacea (LD(50) approx. 1microg/kg). Complete sequence analysis of the toxic peptide revealed the isolation of a new member of type I sea anemone sodium channel toxins containing the typical pattern of the six cysteine residues. From 11kg of wet starting material, approximately 1g of the peptide toxin was isolated. The physiological action of the new toxin from C. gigantea CgNa was investigated on sodium currents of rat dorsal root ganglion neurons in culture using whole-cell patch clamp technique (n=60). Under current clamp condition (CgNa) increased action potential duration. This effect is due to slowing down of the TTX-S sodium current inactivation, without modifying the activation process. CgNa prolonged the cardiac action potential duration and enhanced contractile force albeit at 100-fold higher concentrations than the Anemonia sulcata toxin ATXII. The action on sodium channel inactivation and on cardiac excitation-contraction coupling resemble previous results with compounds obtained from this and other sea anemones [Shapiro, B.I., 1968. Purification of a toxin from tentacles of the anemone C. gigantea. Toxicon 5, 253-259; Pelhate, M., Zlotkin, E., 1982. Actions of insect toxin and other toxins derived from the venom of scorpion Androtonus australis on isolated giant axons of the cockroach Periplaneta americana. J. Exp. Biol. 97, 67-77; Salgado, V., Kem, W., 1992. Actions of three structurally distinct sea anemone toxins on crustacean and insect sodium channels. Toxicon 30, 1365-1381; Bruhn, T., Schaller, C., Schulze, C., Sanchez-Rodriquez, J., Dannmeier, C., Ravens, U., Heubach, J.F., Eckhardt, K., Schmidtmayer, J., Schmidt, H., Aneiros, A., Wachter, E., Béress, L., 2001. Isolation and characterization of 5 neurotoxic and cardiotoxic polypeptides from the sea anemone Anthopleura elegantissima. Toxicon, 39, 693-702]. Comprehensive analysis of the purified active fractions suggests that CgNa may represent the main peptide toxin of this sea anemone species.     

Isolation and cDNA cloning of a potassium channel peptide toxin from the sea anemone Anemonia erythraea.

A potassium channel peptide toxin (AETX K) was isolated from the sea anemone Anemonia erythraea by gel filtration on Sephadex G-50, reverse-phase HPLC on TSKgel ODS-120T and anion-exchange HPLC on Mono Q. AETX K inhibited the binding of (125)I-alpha-dendrotoxin to rat synaptosomal membranes, although much less potently than alpha-dendrotoxin. Based on the determined N-terminal amino acid sequence, the nucleotide sequence of the full-length cDNA (609bp) encoding AETX K was elucidated by a combination of degenerate RT-PCR, 3'RACE and 5'RACE. The precursor protein of AETX K is composed of a signal peptide (22 residues), a propart (27 residues) ended with a pair of basic residues (Lys-Arg) and a mature peptide (34 residues). AETX K is the sixth member of the type 1 potassium channel toxins from sea anemones, showing especially high sequence identities with HmK from Heteractis magnifica and ShK from Stichodactyla helianthus. It has six Cys residues at the same position as the known type 1 toxins. In addition, the dyad comprising Lys and Tyr, which is considered to be essential for the binding of the known type 1 toxins to potassium channels, is also conserved in AETX K.     

Revisiting cangitoxin, a sea anemone peptide: purification and characterization of cangitoxins II and III from the venom of Bunodosoma cangicum.

Sodium channel toxins from sea anemones are employed as tools for dissecting the biophysical properties of inactivation in voltage-gated sodium channels. Cangitoxin (CGTX) is a peptide containing 48 amino acid residues and was formerly purified from Bunodosoma cangicum. Nevertheless, previous works reporting the isolation procedures for such peptide from B. cangicum secretions are controversial and may lead to incorrect information. In this paper, we report a simple and rapid procedure, consisting of two chromatographic steps, in order to obtain a CGTX analog directly from sea anemone venom. We also report a substitution of N16D in this peptide sample and the co-elution of an inseparable minor isoform presenting the R14H substitution. Peptides are named as CGTX-II and CGTX-III, and their effects over Nav1.1 channels in patch clamp experiments are demonstrated.     

Novel peptide toxins from acrorhagi, aggressive organs of the sea anemone Actinia equina.

Two peptide toxins, acrorhagin I (50 residues) and II (44 residues), were isolated from special aggressive organs (acrorhagi) of the sea anemone Actinia equina by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T. The LD50 against crabs of acrorhagin I and II were estimated to be 520 and 80 microg/kg, respectively. 3'- and 5'-RACE established the amino acid sequences of the acrorhagin precursors. The precursor of acrorhagin I is composed of both signal and mature peptides and that of acrorhagin II has an additional sequence (propart) between signal and mature peptides. Acrorhagin I has no sequence homologies with any toxins, while acrorhagin II is somewhat similar to spider neurotoxins (hainantoxin-I from Selenocosmia hainana and Tx 3-2 from Phoneutria nigriventer) and cone snail neurotoxin (omega-conotoxin MVIIB from Conus magus). In addition, analogous peptides (acrorhagin Ia and IIa) were also cloned during RT-PCR experiments performed to confirm the nucleotide sequences of acrorhagins. This is the first to demonstrate the existence of novel peptide toxins in the sea anemone acrorhagi.     

An epidermal growth factor-like toxin and two sodium channel toxins from the sea anemone Stichodactyla gigantea.

Three peptide toxins (gigantoxins I-III) with crab toxicity were isolated from the sea anemone Stichodactyla gigantea by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T and their complete amino acid sequences were determined. Gigantoxins II (44 residues) and III (48 residues) have LD(50) (against crabs) of 70 and 120 microg/kg, respectively, and are analogous to the known type 1 and 2 sea anemone sodium channel toxins, respectively. On the other hand, gigantoxin I (48 residues) is potently paralytic to crabs (ED(50) 215 microg/kg), although its lethality is very weak (LD(50)>1000 microg/kg). Interestingly, gigantoxin I has 31-33% homologies with mammalian epidermal growth factors (EGFs), with the same location of six cysteine residues. In accordance with the sequence similarity, gigantoxin I exhibits EGF activity as evidenced by rounding of A431 cells and tyrosine phosphorylation of the EGF receptor in the cells, although much less potently than human EGF. Gigantoxin I is the first example of EGF-like toxins of natural origin.     

Isolation and molecular cloning of novel peptide toxins from the sea anemone Antheopsis maculata.

Three peptide toxins (Am I-III) with crab toxicity were isolated from the sea anemone Anthopleura maculata by gel filtration and reverse-phase HPLC. Am I was weakly lethal to crabs (LD50 830 microg/kg) and Am III was potently lethal (LD50 70 microg/kg), while Am II was only paralytic (ED50 420 microg/kg). The complete amino acid sequences of the three toxins were determined by cDNA cloning based on 3'-Race and 5'-Race. Although Am III (47 residues) is an analogue of the well-known type 1 sea anemone sodium channel toxins, both Am I (27 residues) and II (46 residues) are structurally novel peptide toxins. Am I is a new toxin having no sequence homologies with any toxins. Am II shares 28-39% identity with the recently characterized sea anemone toxins inhibiting specialized ion channels, BDS-I and II from Anemonia sulcata and APETx1 and 2 from Anthopleura elegantissima. The precursor proteins of the three toxins are commonly composed of a signal peptide, a propart with a pair of basic residues (Lys-Arg) at the end and the remaining portion. Very interestingly, the Am I precursor protein contains as many as six copies of Am I.     

Purification and characterization of Heteroscorpine-1 (HS-1) toxin from Heterometrus laoticus scorpion venom.

Crude venom from Thai giant scorpion, Heterometrus laoticus, most commonly found in the northeastern area of Thailand, was evaluated for PD50 (paralytic dose 50) activities from abdominal injection to cricket (Gryllus sp.) and activities against various kinds of microorganisms. It exhibited good results in disc diffusion assay for Bacillus subtilis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. After purification, toxin showed acceptable results for PD50 determination in entrapped cricket as well as inhibitory activity against B. subtilis, K. pneumoniae, and P. aeruginosa with activities over 300 times higher than that of the crude venom. The purified fraction was showed to contain a single band in SDS-PAGE. MALDI-TOF-MS/MS analysis showed one peak of major protein with 8293Da. Partial amino acid sequence show high homology to Scorpine-a polypeptide toxin family with potassium channel blocking and defensin activity. The novel toxin was named "Heteroscorpine-1" (HS-1) as the first Scorpine from genus Heterometrus. After full length determination by PCR, HS-1 gene was found to be composed of two exons, separated by an intron. Deduction revealed 95 amino acid residues with 19 residues as the leading sequence. It showed about 80% similarity to Panscorpine and Opiscorpine.     

A lethal cardiotoxic-cytotoxic protein from the Indian monocellate cobra (Naja kaouthia) venom

A lethal cardiotoxic-cytotoxic protein (mol. wt. 6.76 kDa) has been purified from the Indian monocellate cobra (Naja kaouthia) venom by ion-exchange chromatography and HPLC. CD spectra indicated the presence of 23% α helix, 19% β sheets and 35% coil. Complete amino acid sequence was determined by MALDI, which showed similar homology with cardiotoxins/cytotoxins isolated from venom of other Naja species. Intraperitoneal LD₅₀ was 2.5 mg kg⁻¹ in BalbC male mice. In vitro cardiotoxicity studies on isolated guinea pig auricle showed that the molecule produced auricular blockade that was abolished after trypsin treatment. Cytotoxicity studies on human leukemic U937 and K562 cells showed that it significantly inhibited cell proliferation in a dose and time dependent manner, as observed by trypan blue exclusion method and tetrazolium bromide reduction assay. IC₅₀ on U937 and K562 cells were 3.5 μg/ml and 1.1 μg/ml respectively. Morphometry and cell sorting studies indicated apoptosis induction in toxin treated leukemic cells. Apoptosis was caspase 3 and 9 dependent and the treated leukemic cells were arrested in sub-G1 stage. There was an increase in Bax-Bcl2 ratio, decrease in HSP (Heat shock protein) 70 and HSP90 and induction of PARP cleavage after NK-CT1 treatment. The toxin showed low cytotoxic effect on normal human leukocytes as compared with imatinib mesylate. Further detailed cytotoxic and cardiotoxic effects at the molecular level are in progress.     

Cardiovascular effects and lethality of venom from nematocysts of the box-jellyfish Chiropsalmus quadrigatus (Habu-kurage) in anaesthetized rats.

Haemodynamic effects of saline-extracted venom from nematocysts isolated from Chiropsalmus quadrigatus (Habu-kurage) were studied in anaesthetized rats. Intravenous administration of venom (0.2-5 microg protein/kg) produced immediately dose-dependent hypertension and bradycardia. Femoral blood flow transiently increased but calculated femoral vascular conductance decreased. Changes caused by 1 microg/kg of venom were reproducible, and were not affected by prazosin, atropine or BQ123 (ET(A) receptor antagonist) but were significantly attenuated by nicardipine. At doses over 2 microg/kg, hypotension and a decrease in pulse pressure were observed subsequent to transient hypertension. In 5 of 8 rats received 5 microg/kg venom and 6 of 6 rats at 10 microg/kg, death due to irreversible cardiac arrest occurred within 30 min after intravenous injection. However, during nicardipine infusion, venom (10 microg/kg) exerted only modest effects and the rats survived. Heating venom (50 degrees C for 10 min) before injection practically abolished the haemodynamic effects of 10 microg/kg venom, indicating its thermolability. Data show that C. quadrigatus venom has both vasoconstrictor and cardiodepressive effects in rats, and suggest that a calcium channel blocker can protect against the cardiovascular and lethal effects of the venom.     

The crude extract from the sea anemone, Bunodosoma caissarum elicits convulsions in mice: possible involvement of the glutamatergic system

The crude extract from the sea anemone, Bunodosoma caissarum caused dose-dependent convulsions by i.c.v. route in mice. The involvement of the glutamatergic system in the convulsions was investigated. MK-801 and ketamine, non-competitive NMDA receptor antagonists, prolonged the latencies for convulsion onset. AP-5, a competitive NMDA receptor antagonist, reduced the number of animals convulsing and also increased the latency for convulsion onset. 7-Chlorokynurenic acid, an antagonist of the glycine site on the NMDA receptor, reduced the incidence of convulsions. GMP, a nucleotide known to antagonize some NMDA actions, reduced the incidence and the severity of convulsions and prolonged the latency for their onset. Riluzole, a neuroprotective and anticonvulsant agent, blocked the appearance of convulsions. In vitro, the crude extract inhibited [³H]glutamate binding to cerebral cortical membranes and enhanced [³H]glutamate release from cortical synaptosomes. Heating the crude extract to 100 °C for 30 min or preincubating it with sphingomyelin, abolished its effect on glutamate release, but did not alter its ability to induce convulsions and to inhibit glutamate binding. However, the convulsant action was inhibited when the crude extract was submitted to trypsin treatment. Our data suggest that the convulsions elicited by the crude extract are not due to the presence of cytolysin and are not related to an increase in glutamate release, but seem to be dependent on the interaction between a peptide component of the extract and NMDA receptors.     

Paralytic shellfish toxin profiles and toxin variability of the genus Alexandrium (Dinophyceae) isolated from the Southeast China Sea.

Paralytic shellfish toxin (PST) profiles of 16 Alexandrium isolates from the Southeast China Sea were analyzed by high-pressure liquid chromatography. Toxin content and composition of three A. tamarense isolates, ATDH01, ATGX02 and ATMJ02, were also investigated at different growth phases and under various culture conditions. Our results showed that six strains of A. affine were non-toxic, while 10 strains of A. tamarense and A. catenella were toxic. These toxic isolates grown in the same culture conditions consistently produced an unusually high proportion of the N-sulfocarbamoyl toxin C1/2 (around 60-80% of total toxins) and medium amounts of gonyautoxin GTX5 (around 15-30% of total) with only trace quantities (<5% of total) of other saxitoxin derivatives (i.e. GTX1, GTX3, GTX4 and neoSTX). The toxin composition of three A. tamarense isolates did not vary with the growth phases, although higher toxin contents (Qt, fmolcell(-1)) were found in the exponential phase. Variations in temperature, salinity and nutrient levels affected toxin content of three A. tamarense isolates but they did not have pronounced effects on the toxin composition (mole %). These results indicate that toxin composition remained relatively constant under various culture conditions, suggesting that toxin composition could be used as a stable biomarker for the Alexandrium species in this region. However, comparison of toxin profiles between isolates from different localities require special caution since isolates even from the same region can have distinct toxin profiles.     

Toxicity of Bothrops neuwiedi complex (“yarará chica”) venom from different regions of Argentina (Serpentes, Viperidae)

We report a study of toxic and enzymatic activities of Bothrops neuwiedi complex venoms collected from specimens of different regions of Argentina and a pool of these same venoms. Were determined lethal, hemorrhagic and pro-coagulant (plasma and fibrinogen) doses and the neutralization of these activities by a bivalent antivenom. The electrophoretic pattern of different regions venom was studied by SDS-PAGE. All samples exhibited lethal potencies, hemorrhagic and coagulant (plasma and fibrinogen) activities with potencies concordant with previous studies. The only conspicuous difference in the toxicological pattern of Bothrops diporus venoms was the low-thrombin-like activity found in one sample. The antivenom used in this study could neutralize all the toxic activities tested and the neutralizing potency of the antivenom was comparable for all samples. Despite the wide distribution of B. neuwiedi complex throughout Argentina and the evident morphological variation between B. diporus (B. neuwiedi complex), this study establishes a remarkably similar toxicity profile throughout its range. This is the first systematic study on the regional variation of enzymatic and toxic activities of venom from species belonging to the B. neuwiedi complex, one of the snakes of highest sanitary importance in South America and their neutralization by the type of antivenom most commonly used in the South of South America.     

Comparative study on extracts from the tissue covering the stingers of freshwater (Potamotrygon falkneri) and marine (Dasyatis guttata) stingrays.

Stingrays are elasmobranchs found along the seacoast and in some rivers of Brazil. Pain is the most conspicuous symptom observed in patients wounded by the bilaterally retroserrate stingers located in the tail, which are covered by glandular and integument tissues. In addition, cutaneous necrosis is commonly observed in injuries caused by freshwater stingrays. The aim of this work was to characterize and compare certain properties of tissue extracts obtained from the glandular tissues covering the stinger apparatus of Potamotrygon falkneri and Dasyatis guttata stingrays. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), tissue extracts have similar bands above 80 kDa, but most differences were observed below this molecular mass. Lethal, dermonecrotic and myotoxic activities were detected only in P. falkneri tissue extract. Edematogenic activity was similar and dose dependent in both tissue extracts. Nociceptive activity was verified in both tissue extracts, but P. falkneri presented a two-fold higher activity than D. guttata tissue extract. No direct hemolysis, phospholipase A2 and coagulant activities were observed in both tissue extracts. Antigenic cross-reactivity was noticed by ELISA and Western blotting, using antisera raised in rabbits. Species-specific sera reacted with several components of both tissue extracts, noticeably above 22kDa. Both tissue extracts presented gelatinolytic, caseinolytic and fibrinogenolytic activities, which were not caused by the action of metalloproteinases. Hyaluronidase activity was detected only in P. falkneri tissue extract. Our experimental observations suggest that P. falkneri tissue extract is more toxic than D. guttata tissue extract. These results may explain why injuries caused by freshwater stingrays are more severe in human accidents.     

Primary structure, behavioral and electroencephalographic effects of an epileptogenic peptide from the sea anemone Bunodosoma cangicum.

The primary structure of cangitoxin (CGX), a 4958 Da peptide from the sea anemone Bunodosoma cangicum, was determined: GVACRCDSDGPTVRGNSLSGTLWLTGGCPSGWHNCRGSGPFIGYCCKK. CGX contains all the 11 residues that are conserved and the 5 that are conservatively substituted within or between the type 1 and type 2 sequences of sea anemone peptides with specific action on voltage-sensitive sodium channels. Furthermore, it also has 6 identities (Asp9, Arg14, Asn16, Leu18, Trp33 and Lys48) and 1 homology (Arg36) in the 8 residues of the pharmacophore of the sea anemone ApB which are essential for interaction with mammalian sodium channels. The intrahippocampal injection of CGX induces several sequential behavioral alterations--episodes of akinesia alternating with facial automatisms and head tremor, salivation, rearing, jumping, barrel-rolling, wet dog shakes and forelimb clonic movements--and the electroencephalography analysis shows that they were followed by important seizure periods that gradually evolved to status epilepticus that lasted 8-12 h, similar to that observed in the acute phase of the pilocarpine model of epilepsy. These results suggest that CGX may be an important tool to develop a new experimental model of status epilepticus which may contribute to understanding the etiology of epilepsy and to test the effects of new antiepileptic drugs.     

Purification and characterization of the biological effects of phospholipase A2 from sea anemone Bunodosoma caissarum

Sea anemones contain a variety of biologically active substances. Bunodosoma caissarum is a sea anemone from the Cnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of PLA₂ isolated from the sea anemone B. caissarum on the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion. Specimens of B. caissarum were collected from the São Vicente Channel on the southern coast of the State of São Paulo, Brazil. Reverse phase HPLC analysis of the crude extract of B. caissarum detected three PLA₂ proteins (named BcPLA₂1, BcPLA₂2 and BcPLA₂3) found to be active in B. caissarum extracts. MALDI-TOF mass spectrometry of BcPLA₂1 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of BcPLA₂1 showed high amino acid sequence identity with PLA₂ group III protein isolated from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA₂ and 1POC_A). In addition, BcPLA₂1 also showed significant overall homology to bee PLA₂. The enzymatic activity induced by native BcPLA₂1 (20 μg/well) was reduced by chemical treatment with p-bromophenacyl bromide (p-BPB) and with morin. BcPLA₂1 strongly induced insulin secretion in presence of high glucose concentration. In isolated kidney, the PLA₂ from B. caissarum increased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA₂1, however, did not increase the perfusion pressure on the mesenteric vascular bed. In conclusion, PLA₂, a group III phospholipase isolated from the sea anemone B. caissarum, exerted effects on renal function and induced insulin secretion in conditions of high glucose concentration.