Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin: endothelial cell membrane phospholipids as targets for toxicity

Brown spider dermonecrotic toxins (phospholipases-D) are the most well-characterized biochemical constituents of Loxosceles spp. venom. Recombinant forms are capable of reproducing most cutaneous and systemic manifestations such as dermonecrotic lesions, hematological disorders, and renal failure. There is currently no direct confirmation for a relationship between dermonecrosis and inflammation induced by dermonecrotic toxins and their enzymatic activity. We modified a toxin isoform by site-directed mutagenesis to determine if phospholipase-D activity is directly related to these biological effects. The mutated toxin contains an alanine substitution for a histidine residue at position 12 (in the conserved catalytic domain of Loxosceles intermedia Recombinant Dermonecrotic Toxin - LiRecDT1). LiRecDT1H12A sphingomyelinase activity was drastically reduced, despite the fact that circular dichroism analysis demonstrated similar spectra for both toxin isoforms, confirming that the mutation did not change general secondary structures of the molecule or its stability. Antisera against whole venom and LiRecDT1 showed cross-reactivity to both recombinant toxins by ELISA and immunoblotting. Dermonecrosis was abolished by the mutation, and rabbit skin revealed a decreased inflammatory response to LiRecDT1H12A compared to LiRecDT1. Residual phospholipase activity was observed with increasing concentrations of LiRecDT1H12A by dermonecrosis and fluorometric measurement in vitro. Lipid arrays showed that the mutated toxin has an affinity for the same lipids LiRecDT1, and both toxins were detected on RAEC cell surfaces. Data from in vitro choline release and HPTLC analyses of LiRecDT1-treated purified phospholipids and RAEC membrane detergent-extracts corroborate with the morphological changes. These data suggest a phospholipase-D dependent mechanism of toxicity, which has no substrate specificity and thus utilizes a broad range of bioactive lipids.     

Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea anemone Bunodosoma caissarum: purification and biological characterization

Two cationic proteins, C1 and C3, were purified to homogeneity from the hemolytic fraction of the venom of Bunodosoma caissarum sea anemone. The purification processes employed gel filtration followed by ion exchange chromatography, being the purity and molecular mass confirmed by SDS-PAGE and mass spectrometry. Protein C1 represented the second major peak of the hemolytic fraction and was previously believed to be a cytolysin belonging to a new class of hemolysins. The C1 protein has a molecular mass of 15495 Da and was assayed for hemolysis, PLA2 activity and acute toxicity in crabs and mice, showing no activity in these assays. It has an amino terminal with no similarity to all known hemolysins and, therefore, should not be considered a toxin, being its function completely unknown. The protein C3 (19757 Da), that also lacks PLA2 activity, was recognized by antiserum against Eqt II and presented high hemolytic activity to human erythrocytes (ED50 of 0.270 microg/ml), being named Caissarolysin I (Bcs I). Its activity was inhibited by pre-incubation with sphingomyelin (SM) and also when in presence of erythrocytes pre-treated with the SMase P2, a phospholipase D from the brown spider Loxosceles intermedia, indicating that SM is the main target of Bcs I. Caissarolysin I is the first hemolysin purified from a sea anemone belonging to the genus Bunodosoma and belongs to the Actinoporin family of sea anemone hemolysins.     

Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea anemone Bunodosoma caissarum: Purification and biological characterization

Two cationic proteins, C1 and C3, were purified to homogeneity from the hemolytic fraction of the venom of Bunodosoma caissarum sea anemone. The purification processes employed gel filtration followed by ion exchange chromatography, being the purity and molecular mass confirmed by SDS-PAGE and mass spectrometry. Protein C1 represented the second major peak of the hemolytic fraction and was previously believed to be a cytolysin belonging to a new class of hemolysins. The C1 protein has a molecular mass of 15495 Da and was assayed for hemolysis, PLA₂ activity and acute toxicity in crabs and mice, showing no activity in these assays. It has an amino terminal with no similarity to all known hemolysins and, therefore, should not be considered a toxin, being its function completely unknown. The protein C3 (19757 Da), that also lacks PLA₂ activity, was recognized by antiserum against Eqt II and presented high hemolytic activity to human erythrocytes (ED₅₀ of 0.270 μg/ml), being named Caissarolysin I (Bcs I). Its activity was inhibited by pre-incubation with sphingomyelin (SM) and also when in presence of erythrocytes pre-treated with the SMase P2, a phospholipase D from the brown spider Loxosceles intermedia, indicating that SM is the main target of Bcs I. Caissarolysin I is the first hemolysin purified from a sea anemone belonging to the genus Bunodosoma and belongs to the Actinoporin family of sea anemone hemolysins.     

Two novel dermonecrotic toxins LiRecDT4 and LiRecDT5 from brown spider (Loxosceles intermedia) venom: from cloning to functional characterization

Loxoscelism (the condition produced by the bite of brown spiders) has been reported worldwide, but especially in warmer regions. Clinical manifestations include skin necrosis with gravitational spreading while systemic loxoscelism may include renal failure, hemolysis and thrombocytopenia. The venom contains several toxins, of which the best biochemically and biologically studied is the dermonecrotic toxin, a phospholipase-D. Purified toxin induces cutaneous and systemic loxoscelism, especially necrotic lesions, hematological disturbances and renal failure. Herein, we describe cloning, heterologous expression and purification of two novel dermonecrotic toxins: LiRecDT4 and LiRecDT5. The recombinant proteins stably expressed in Escherichia coli cells were purified from culture supernatants in a single step using Ni(2+)-chelating chromatography producing soluble proteins of 34 kDa (LiRecDT4) and 37 kDa (LiRecDT5). Circular dichroism analysis evidenced correctly folding for toxins but differences in secondary structures. Both proteins were recognized by whole venom serum antibodies and by a specific antibody to dermonecrotic toxin. Also, recombinant toxins with phospholipase activity induced experimental skin lesions and caused a massive inflammatory response in rabbit skin dermis. Nevertheless, toxins displayed different effects upon platelet aggregation, increase in vascular permeability and not caused death in mice. These characteristics in combination with functional studies illustrates that a family of dermonecrotic toxins exists, and includes two novel members that are useful for future structural and functional studies. They will also be useful in biotechnological ends, for example, as inflammatory and platelet aggregating studies, as antigens for serum therapy source and for lipids biochemical research.     

Omega-agatoxins differentially block calcium channels in locust, chick and rat synaptosomes.

Three toxins (omega-Agatoxins IA, IIA and IIIA) isolated from the venom of the funnel web spider, Agelenopsis aperta, differentially block depolarization-induced calcium influx in chick, rat and locust synaptosomes. In chick, this block of calcium influx is observed with omega-Agatoxins IIA and IIIA but not with omega-Agatoxin IA. Block by omega-Agatoxin IIA and IIIA is maximal at 70 and 82% respectively of the total depolarization-induced calcium influx; maximal suppression of calcium influx by omega-Conotoxin GVIA (omega-CgTx) is 100%. The IC50 for block with omega-Agatoxin IIA is ca 3 nM as compared with an IC50 of 38 nM for omega-CgTx. Incomplete block of calcium influx at saturating concentrations of omega-Agatoxins IIA and IIIA (above 100 nM) suggests that both omega-Agatoxin-sensitive and -insensitive calcium channels occur in chick brain synaptosomes. In rat cerebrocortical synaptosomes, omega-Agatoxins IA and IIA are only partially effective at blocking depolarization-induced calcium influx, as is omega-CgTx, whilst IIIA blocks 47% of this effective at blocking depolarization-induced calcium influx, as is omega-CgTx, whilst IIIA blocks 47% of this influx. In synaptosomes prepared from the CNS of adult locusts, omega-Agatoxins IA and IIA are most effective at blocking depolarization-induced calcium influx; omega-CgTx and omega-Agatoxin IIIA are ineffective. Block of depolarization-induced calcium influx in chick brain synaptosomes by omega-Agatoxins IIA, IIIA and omega-CgTx suggests that the spider toxin interacts directly with the voltage-dependent calcium channel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hemolytic effect of phospholipase A2 and orientotoxin from venom of the great hornet, Vespa orientalis

The effect of toxic phospholipase A2 and orientotoxin from the venom of the giant hornet Vespa orientalis on human erythrocytes was studied. It was shown that these venom components are potent hemolytic agents, the efficiency of the latter being by about two orders of magnitude as high as that of phospholipase A2. The hemolytic function of the both components is enhanced in the presence of low concentrations of Ca2+, whereas high concentrations of this cation exert an inhibiting action. Polyvalent cations, in particular, ruthenium red, peptide HR-1, mellitin and cytotoxins Us-1 and Us-5 synergetically increase the hemolytic effect of phospholipase A2. During erythrocyte hemolysis the synergistic effect is manifested upon a combined action of phospholipase A2 and orientotoxin. The combination of these toxins increases the total hemolytic activity and produces a far greater effect than in could be expected in the case of each of these compounds taken separately.     

Experimental evidence for a direct cytotoxicity of Loxosceles intermedia (brown spider) venom in renal tissue.

Brown spider (Loxosceles genus) venom causes necrotic lesions often accompanied by fever, hemolysis, thrombocytopenia, and acute renal failure. Using mice exposed to Loxosceles intermedia venom, we aimed to show whether the venom directly induces renal damage. The experimental groups were composed of 50 mice as controls and 50 mice that received the venom. Light microscopic analysis of renal biopsy specimens showed alterations including hyalinization of proximal and distal tubules, erythrocytes in Bowman's space, glomerular collapse, tubule epithelial cell blebs and vacuoles, interstitial edema, and deposition of eosinophilic material in the tubule lumen. Electron microscopic findings indicated changes including glomerular epithelial and endothelial cell cytotoxicity as well as disorders of the basement membrane. Tubule alterations include epithelial cell cytotoxicity with cytoplasmic membrane blebs, mitochondrial changes, increase in smooth endoplasmic reticulum, presence of autophagosomes, and deposits of amorphous material in the tubules. We also found that the venom caused azotemia with elevation of blood urea levels but did not decrease C3 complement concentration or cause hemolysis in vivo. Confocal microscopy with antibodies against venom proteins showed direct binding of toxins to renal structures, confirmed by competition assays. Double-staining immunofluorescence reactions with antibodies against type IV collagen or laminin, antibodies to venom toxins, and fluorescent cytochemistry with DAPI revealed deposition of toxins in glomerular and tubule epithelial cells and in renal basement membranes. Two-dimensional electrophoresis showed venom rich in low molecular mass and cationic toxins. By immunoblotting with antibodies to venom toxins on renal extracts from venom-treated mice, we detected a renal binding toxin at 30 kD. The data provide experimental evidence that L. intermedia venom is directly involved in nephrotoxicity.     

Red blood cell lysis induced by the venom of the brown recluse spider: the role of sphingomyelinase D.

Red blood cell lysis induced by the venom of Loxosceles reclusa, the brown recluse spider, may be related to the hemolytic anemia observed in several cases of spider envenomation. These investigations demonstrate that the venom of the brown recluse spider contains a calcium-dependent, heat-labile hemolysin of molecular weight approximately 19,000. The pH optimum for the hemolytic reaction was 7.1, and the optimum calcium concentration for venom-induced lysis was observed within the range of 6 to 10 mm. Sheep red blood cells were more susceptible to the spider hemolysin than human red blood cells, although both types exhibited appreciable lysis. Digestion of sheep red blood cell membranes with partially purified venom lysin resulted in degradation of the sphingomyelin component. However, reaction of the membranes with the venom lysin produced no release of water-soluble phosphate, and no free fatty acids were generated. These results indicate that the sphingomyelin-degrading activity of the venom is not a phospholipase C- or a phospholipase A₂-type activity. Sphingomyelin was employed as substrate for the venom hemolysin, and the organic and aqueous fractions of the reaction mixtures were analyzed by thin-layer chromatography. Analysis of the organic fraction revealed a phosphate-containing product with the solubility and chromatographic characteristics of N-acylsphingosine phosphate (ceramide phosphate), and analysis of the aqueous fraction demonstrated the presence of choline. The isolation and identification of these products indicate that the sphingomyelin of the red cell membrane is hydrolyzed by a sphingomyelinase D-type activity expressed by the partially purified venom hemolysin. A close correspondence between the hemolytic and sphingomyelinase D activities was observed when the partially purified hemolysin was further characterized in polyacrylamide gel electrophoresis at pH 8.3 and pH 4.9. The hemolytic and sphingomyelinase activities were coincident within the electrophoretic pattern at both pHs. The results presented demonstrate conclusively a direct lytic action of brown recluse venom upon red blood cells and report for the first time the presence of sphingomyelinase D in spider venom.     

Molecular cloning and functional characterization of two isoforms of dermonecrotic toxin from Loxosceles intermedia (brown spider) venom gland

Brown spider (Genus Loxosceles) bites are normally associated with necrotic skin degeneration, gravitational spreading, massive inflammatory response at injured region, platelet aggregation causing thrombocytopenia and renal disturbances. Brown spider venom has a complex composition containing many different toxins, of which a well-studied component is the dermonecrotic toxin. This toxin alone may produce necrotic lesions, inflammatory response and platelet aggregation. Biochemically, dermonecrotic toxin belongs to a family of toxins with 30-35 kDa characterized as sphingomyelinase-D. Here, employing a cDNA library of Loxosceles intermedia venom gland, we cloned and expressed two recombinant isoforms of the dermonecrotic toxin LiRecDT2 (1062 bp cDNA) and LiRecDT3 (1007 bp cDNA) that encode for signal peptides and complete mature proteins. Phylogenetic tree analysis revealed a structural relationship for these toxins compared to other members of family. Recombinant molecules were expressed as N-terminal His-tag fusion proteins in Escherichia coli and were purified to homogeneity from cell lysates by Ni(2+) chelating chromatography, resulting in proteins of 33.8 kDa for LiRecDT2 and 34.0 kDa for LiRecDT3. Additional evidence for related toxins containing sequence/epitopes identity comes from antigenic cross-reactivity using antibodies against crude venom toxins and antibodies raised with a purified dermonecrotic toxin. Recombinant toxins showed differential functionality in rabbits: LiRecDT2 caused a macroscopic lesion with gravitational spreading upon intradermal injection, while LiRecDT3 evoked transient swelling and erythema upon injection site. Light microscopic analysis of skin biopsies revealed edema, a collection of inflammatory cells in and around blood vessels and a proteinaceous network at the dermis. Moreover, differential functionality for recombinant toxins was also demonstrated by a high sphingomyelinase activity for LiRecDT2 and low activity for LiRecDT3 as well as greater in vitro platelet aggregation and blood vessel permeability induced by LiRecDT2 and residual activity for LiRecDT3. Cloning and expression of two recombinant dermonecrotic toxins demonstrate an intraspecific family of homologous toxins that act in synergism for deleterious activities of the venom and open possibilities for biotechnological applications for recombinant toxins as research tools for understanding the inflammatory response, vascular integrity and platelet aggregation modulators.     

ImKTx1, a new Kv1.3 channel blocker with a unique primary structure

Toxins from the venoms of scorpion, snake, and spider are valuable tools to probe the structure-function relationship of ion channels. In this investigation, a new toxin gene encoding the peptide ImKTx1 was isolated from the venom gland of the scorpion Isometrus maculates by constructing cDNA library method, and the recombinant ImKTx1 peptide was characterized physiologically. The mature peptide of ImKTx1 has 39 amino acid residues including six cross-linked cysteines. The electrophysiological experiments showed that the recombinant ImKTx1 peptide had a pharmacological profile where it inhibited Kv1.3 channel currents with IC(50) of 1.70 n± 1.35 μM, whereas 10 μM rImKTx1 peptide inhibited about 40% Kv1.1 and 42% Kv1.2 channel currents, respectively. In addition, 10 μM rImKTx1 had no effect on the Nav1.2 and Nav1.4 channel currents. Multiple sequence alignments showed that ImKTx1 had no homologous toxin peptide, but it was similar with Ca(2+) channel toxins from scorpion and spider in the arrangement of cysteine residues. These results indicate that ImKTx1 is a new Kv1.3 channel blocker with a unique primary structure. Our results indicate the diversity of K(+) channel toxins from scorpion venoms and also provide a new molecular template targeting Kv1.3 channel.     

Clostridium perfringens alpha-toxin-induced hemolysis of horse erythrocytes is dependent on Ca2+ uptake

Clostridium perfringens alpha-toxin is able to lyse various erythrocytes. Exposure of horse erythrocytes to alpha-toxin simultaneously induced hot-cold hemolysis and stimulated production of diacylglycerol and phosphorylcholine. When A23187-treated erythrocytes were treated with the toxin, these events were dependent on the concentration of extracellular Ca2+ . Incubation with the toxin of BAPTA-AM-treated horse erythrocytes caused no hemolysis or production of phosphorylcholine, but that of the BAPTA-treated erythrocytes did. When Quin 2-AM-treated erythrocytes were incubated with the toxin in the presence of 45Ca2+, the cells accumulated 45Ca2+ in a dose- and a time-dependent manner. These results suggest that the toxin-induced hemolysis and hydrolysis of phosphatidylcholine are closely related to the presence of Ca2+ in the cells. Flunarizine, a T-type Ca2+ channel blocker, and tetrandrine, an L- and T-type Ca2+ channel blocker, inhibited the toxin-induced hemolysis and Ca2+ uptake. However, L-type Ca2+ channel blockers, nifedipine, verpamil and diltiazem, an N-type blocker, omega-conotoxin SVIB, P-type blockers, omega-agatoxin TK and omega-agatoxin IVA, and a Q-type blocker, omega-conotoxin MVII C, had no such inhibitory effect. The observation suggests that Ca2+ taken up through T-type Ca2+ channels activated by the toxin plays an important role in hemolysis induced by the toxin.     

Identification of a direct hemolytic effect dependent on the catalytic activity induced by phospholipase‐D (dermonecrotic toxin) from brown spider venom

Brown spiders have world‐wide distribution and are the cause of health problems known as loxoscelism. Necrotic cutaneous lesions surrounding the bites and less intense systemic signs like renal failure, DIC, and hemolysis were observed. We studied molecular mechanism by which recombinant toxin, biochemically characterized as phospholipase‐D , causes direct hemolysis (complement independent). Human erythrocytes treated with toxin showed direct hemolysis in a dose‐dependent and time‐dependent manner, as well as morphological changes in cell size and shape. Erythrocytes from human, rabbit, and sheep were more susceptible than those from horse. Hemolysis was not dependent on ABO group or Rhesus system. Confocal and FACS analyses using antibodies or GFP‐phospholipase‐D protein showed direct toxin binding to erythrocytes membrane. Moreover, toxin‐treated erythrocytes reacted with annexin‐V and showed alterations in their lipid raft profile. Divalent ion chelators significantly inhibited hemolysis evoked by phospholipase‐D , which has magnesium at the catalytic domain. Chelators were more effective than PMSF (serine‐protease inhibitor) that had no effect on hemolysis. By site‐directed mutation at catalytic domain (histidine 12 by alanine), hemolysis and morphologic changes of erythrocytes (but not the toxin's ability of membrane binding) were inhibited, supporting that catalytic activity is involved in hemolysis and cellular alterations but not toxin cell binding. The results provide evidence that L. intermedia venom phospholipase‐D triggers direct human blood cell hemolysis in a catalytic‐dependent manner. J. Cell. Biochem. 107: 655–666, 2009. © 2009 Wiley‐Liss, Inc.     

Characterization of Ca2+ influx induced by dimethylphytosphingosine and lysophosphatidylcholine in U937 monocytes

Calcium is a ubiquitous second messenger controlling a broad range of cellular functions. We previously observed that N,N-dimethyl-D-ribo-phytosphingosine (DMPH) and lysophosphatidylcholine (LPC) induced Ca2+ influx across the plasma membrane in U937 monocytes. In this study, we characterized the Ca2+ influx induced by DMPH and LPC. L-type voltage-gated Ca2+ channel blockers, verapamil and nifedipine, significantly reduced LPC-induced Ca2+ influx, but not DMPH-induced one. On the other hand, non-specific Ca2+ channel blockers, Ga3+ and La3+, considerably reduced DMPH- and LPC-induced Ca2+ influx. Preincubation of the cells with forskolin enhanced DMPH-induced Ca2+ influx, however, LPC-induced Ca2+ influx was not affected by the treatment. The enhancement by forskolin was blocked by KT5720, a PKA inhibitor. We also confirmed the presence of TRPM7 and absence of TRPM3 in U937 cells. Therefore, our characterization of Ca2+ influx in U937 human monocytes shows the presence of two different types of Ca2+ channels modulated by lysolipid molecules, DMPH and LPC. LPC may induce Ca2+ influx via L-type Ca2+ channels and DMPH seems to induce Ca2+ influx through TRPM7 in U937 human monocytes.     

Translocation-specific conformation of adenylate cyclase toxin from Bordetella pertussis inhibits toxin-mediated hemolysis

Bordetella pertussis adenylate cyclase (AC) toxin belongs to the RTX family of toxins but is the only member with a known catalytic domain. The principal pathophysiologic function of AC toxin appears to be rapid production of intracellular cyclic AMP (cAMP) by insertion of its catalytic domain into target cells (referred to as intoxication). Relative to other RTX toxins, AC toxin is weakly hemolytic via a process thought to involve oligomerization of toxin molecules. Monoclonal antibody (MAb) 3D1, which binds to an epitope (amino acids 373 to 399) at the distal end of the catalytic domain of AC toxin, does not affect the enzymatic activity of the toxin (conversion of ATP into cAMP in a cell-free system) but does prevent delivery of the catalytic domain to the cytosol of target erythrocytes. Under these conditions, however, the ability of AC toxin to cause hemolysis is increased three- to fourfold. To determine the mechanism by which the hemolytic potency of AC toxin is altered, we used a series of deletion mutants. A mutant toxin, DeltaAC, missing amino acids 1 to 373 of the catalytic domain, has hemolytic activity comparable to that of wild-type toxin. However, binding of MAb 3D1 to DeltaAC enhances its hemolytic activity three- to fourfold similar to the enhancement of hemolysis observed with 3D1 addition to wild-type toxin. Two additional mutants, DeltaN489 (missing amino acids 6 to 489) and DeltaN518 (missing amino acids 6 to 518), exhibit more rapid hemolysis with quicker onset than wild-type toxin does, while DeltaN549 (missing amino acids 6 to 549) has reduced hemolytic activity compared to wild-type AC toxin. These data suggest that prevention of delivery of the catalytic domain or deletion of the catalytic domain, along with additional amino acids distal to it, elicits a conformation of the toxin molecule that is more favorable for hemolysis.     

Expression and characterization of LTx2, a neurotoxin from Lasiodora sp. effecting on calcium channels

Here, we described the expression and characterization of the recombinant toxin LTx2, which was previously isolated from the venomous cDNA library of a Brazilian spider, Lasiodora sp. (Mygalomorphae, Theraphosidae). The recombinant toxin found in the soluble and insoluble fractions was purified by reverse phase high-performance liquid chromatography (HPLC). Ca2+ imaging analysis revealed that the recombinant LTx2 acts on calcium channels of BC3H1 cells, blocking L-type calcium channels.     

The hemolytic activity of six arachnid cationic peptides is affected by the phosphatidylcholine-to-sphingomyelin ratio in lipid bilayers

The hemolytic activity of six cationic amphipathic peptides (Oxki1, Oxki2, Pin1, Pin2, IsCT1 and IsCT2) from arachnids strongly depends on the source of red blood cells. The hemolytic activity of the amphipathic peptides was correlated to the phosphocholine-to-sphingomyelin ratio (PC/SM) content, the potency order of which on mammal erythrocytes ranked as follows Guinea pig>pig>sheep. The spider peptides, Oxki1 and Oxki2, prefer small unilamellar vesicles (SUV) composed of PC, but they could not disrupt SUVs made of SM only. Moreover, the membrane-disrupting activity of the scorpion peptide Pin1 was affected by increasing concentrations of SM. Only the scorpion hemolytic peptide Pin2 was able to disrupt SUVs composed merely of SM at high concentrations. Finally, the short scorpion peptides IsCT1 and IsCT2 seem to tolerate high concentrations of SM in the presence of PC for disruption of SUVs; however, the disrupting activities of IsCT1 and IsCT2 are much lower than that of the other four hemolytic peptides. The hemolytic activity caused by all six cationic peptides in mammalian erythrocytes was positively correlated to increases in temperature and increases in the concentration of benzyl alcohol, a membrane fluidizing agent. It was concluded that the hemolytic activity of the cationic peptides strongly depends on the PC/SM content of mammalian erythrocytes, in which cell membranes with a low PC/SM ratio (i.e., of low fluidity) were less disturbed than membranes with a high PC/SM ratio (i.e., of high fluidity).     

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.     

Identification,cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.     

Characteristics of lysophosphatidylcholine-induced Ca2+ response in human neuroblastoma SH-SY5Y cells

Lysophosphatidylcholine (LPC) is an important bioactive lipid. In the nervous system, elevated levels of LPC have been shown to produce demyelination. In the present study, we examined the effect of exogenous LPC on intracellular Ca2+ mobilization in human neuroblastoma SH-SY5Y cells. In Ca2+-containing medium, introduction of LPC induced a steady rise in cytosolic Ca2+ levels ([Ca2+]i) in a dose-dependent manner, and this rise was provoked by LPC itself, not by its hydrolysis product produced by lysophospholipase. The increase in [Ca2+]i was reduced by 36% by removal of extracellular Ca2+, while preincubation of the cells with verapamil, an L-type Ca2+ channel blocker, inhibited the response by 23%, part of the Ca2+ influx. Conversely, Ni2+, which inhibits the Na+-Ca2+ exchanger, or Na+-deprivation did not affect LPC-induced Ca2+ influx. In Ca2+-free medium, depletion of Ca2+ stores in the endoplasmic reticulum (ER) by thapsigargin, an ER Ca2+-ATPase inhibitor, abolished the Ca2+ increase. Moreover, LPC-induced [Ca2+]i increase was fully blocked by ruthenium red and procaine, inhibitors of ryanodine receptor (RyR), but was not affected by 2-aminoethoxydiphenyl borate, an inhibitor of inositol triphosphate receptor, or by pertussis toxin, a G(i/o) protein inhibitor. Combined treatment with verapamil plus thapsigargin markedly inhibited but did not abolish the LPC-induced Ca2+ response. These findings indicate that LPC-induced [Ca2+]i increase depends on both external Ca2+ influx and Ca2+ release from ER Ca2+ stores, in which L-type Ca2+ channels and RyRs may be involved. However, in digitonin-permeabilized SH-SY5Y cells, LPC could not induce any [Ca2+]i increase in Ca2+-free medium, suggesting that LPC may act indirectly on RyRs of ER.     

Phospholipase A2 as a probe of phospholipid distribution in erythrocyte membranes. Factors influencing the apparent specificity of the reaction.

The action of snake venom phospholipases A2 in intact human erythrocytes was investigated in detail. The basis phospholipase from Agkistrodon halys blomhifii was found to induce both hydrolysis of membrane phospholipids and total cell hemolysis under certain experimental conditions. The hydrolytic action of the basic enzyme was found to consist of two sequential events: (a) hydrolysis of 70% of the total cell ph osphatidylcholine without any evident hemolysis; and (b) complete hydrolysis of the remaining phosphatidylcholine, followed closely by extensive phosphatidylethanolamine hydrolysis and finally with onset of hemolysis, attack on the phosphatidylserine. At pH 7.4 and 10 mM Ca2+ only stage (a) occurred. However, a slight elevation of the pH of incubation to pH 8.0 and/or inclusion of 40 mM Ca2+ in the reaction mixture caused both events (a) and (b) to occur. The addition of glucose limited the action of the enzyme to stage (a) under any reaction conditions. An investigation showed that enzymically induced hemolysis occurred under conditions where the intracellular ATP levels were lowered. Data are presented which suggest that stage (b) is mediated by in influx of Ca2+ into the cell when the levels of ATP are low. Interestingly the phosphllipase from Naja naja venom (Pakistan) yielded results similar to those observed with the basic enzyme from Agkistrodon venom. However, the enzyme from Crotalus adamanteus and the acidic enzyme also present in the Agkistrodon venom produced only slight hydrolysis or hemolysis under any of the conditions studied. Other species of erythrocytes, e.g., guinea pig, monkey, pig, and rat, were tested but only those from guinea pig behaved similarly to the human cells. Pig, monkey, and rat erythrocytes underwent very limited hydrolysis and hemolysis. It is evident that the use of these phospholipases to probe the localization of phospholipds in erythrocyte membranes must be approached with caution. Certain facets of this problem are discussed.