**3. Biotechnological and pharmacological applications of scorpion venom toxins**

Scorpions are venomous arthropods, members of Arachnida class and order Scorpiones. These animals are found in all continents except Antarctica, and are known to cause prob‐ lems in tropical and subtropical regions. Actually these animals are represented by 16 fami‐ lies and approximately 1500 different species and subspecies which conserved their morphology almost unaltered [92-93]. The scorpion species that present medically impor‐ tance belonging to the family Buthidae are represented by the genera *Androctonus, Buthus, Mesobuthus, Buthotus, Parabuthus*, and *Leirus* located in North Africa, Asia, the Middle East, and India. *Centruroides* spp. are located in Southwest of United States, Mexico, and Central America, while *Tityus* spp. are found in Central and South America and Caribbean. In these different regions of the world the scorpionism is considered a public health problem, with frequent statements that scorpion stings are dangerous [8]. It is generally known that scor‐ pion venom is a complex mixture composed of a wide array of substances. It contains muco‐ polysaccharides, hyaluronidase, phopholipase, low relative molecular mass molecules like serotonin and histamine, protease inhibitors, histamine releasers and polypeptidyl com‐ pounds. Scorpion venoms are a particularly rich source of small, mainly neurotoxic proteins or peptides interacting specifically with various ionic channels in excitable membranes [94].

### **3.1. Toxins acting on cardiovascular system**

**2.6. Toxins with anticancer and cytotoxic activities**

ins from snake venoms with anticancer potential.

**toxins**

Applications

28

Anticancer therapy is an important area for the application of proteins and peptides from venomous animals. Integrins play multiple important roles in cancer pathology including tumor cell proliferation, angiogenesis, invasion and metastasis [72]. Inhibition of angiogene‐ sis is one of the heavily explored treatment options for cancer, and in this scenario snake venom disintegrins represent a library of molecules with different structure, potency and specificity [1]. RGD-containing disintegrins was identified in several snake venoms, inhibit‐ ing tumor angiogenesis and metastasis, such as accutin (from *Agkistrodon acutus*) [73], sal‐ mosin (from *Agkistrodon halys brevicaudus*) [74], contortrostatin (from *Agkistrodon contortrix*) [75], jerdonin (from *Trimeresurus jerdonii*) [76], crotatroxin (from *Crotalus atrox*) [77], rhodos‐ tomin (from *Calloselasma rhodostoma*) [78] and a novel desintegrin from *Naja naja* [79]. The cytostatic effect of L-amino acid oxidases (LAAOs) have been demonstrated using various models of human and animal tumors. Studies show that LAAOs induces apoptosis in vascu‐ lar endothelial cells and inhibits angiogenesis [80]. Examples of LAAOs isolated from snake venoms with anticancer potential are a LAAO isolated from *Ophiophagus hannah* [81], ACTX-6 from *A. acutus* [82], OHAP-1 from *Trimeresurus flavoviridis* [83] and Bl-LAAO from *Bothrops leucurus* [84]. Secretory phospholipases A2 (sPLA2) also figures the snake toxins with anticancer potential [1]. sPLA2 with cytotoxic activity to tumor cells was described in *Bothrops neuwiedii* [85], *Bothrops brazili* [86], *Naja naja naja* [87], among others. Crotoxin, the main polypeptide isolated from *C. d. terrificus* has shown potent antitumor activity as well the whole venom, highlighting thereby the potential of venom as a source of pharmaceutical templates for cancer therapy [88]. BJcuL, a lectin purified from *Bothrops jararacussu* venom [89] and a metalloproteinase [90] and a lectin from *B. leucurus* [91] are other examples of tox‐

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

**3. Biotechnological and pharmacological applications of scorpion venom**

Scorpions are venomous arthropods, members of Arachnida class and order Scorpiones. These animals are found in all continents except Antarctica, and are known to cause prob‐ lems in tropical and subtropical regions. Actually these animals are represented by 16 fami‐ lies and approximately 1500 different species and subspecies which conserved their morphology almost unaltered [92-93]. The scorpion species that present medically impor‐ tance belonging to the family Buthidae are represented by the genera *Androctonus, Buthus, Mesobuthus, Buthotus, Parabuthus*, and *Leirus* located in North Africa, Asia, the Middle East, and India. *Centruroides* spp. are located in Southwest of United States, Mexico, and Central America, while *Tityus* spp. are found in Central and South America and Caribbean. In these different regions of the world the scorpionism is considered a public health problem, with frequent statements that scorpion stings are dangerous [8]. It is generally known that scor‐ pion venom is a complex mixture composed of a wide array of substances. It contains muco‐ polysaccharides, hyaluronidase, phopholipase, low relative molecular mass molecules like

The first peptide from scorpion endowed effects of bradykinin and on arterial blood pres‐ sure was isolated from the Brazilian scorpion *Tityus serrulatus* [95]. These peptides named *Tityus serrulatus* Hypotensins have molecular masses ranging approximately from 1190 to 2700 Da [96]. Other scorpion bradykinin-potentiating peptides (BPPs) were reported to be found in the venom of the scorpions *Buthus martensii* Karsch [97] and *Leiurus quinquestriatus* [98]. These molecules can display potential as new drugs and could be of interest for bio‐ technological purposes.

#### **3.2. Toxins with antibiotic activity**

In order to defend themselves against the hostile environment, scorpions have developed potent defensive mechanisms that are part of innate and adaptive immunity [99]. Cysteinefree antimicrobial peptides have been identified and characterized from the venom of six scorpion species [100]. Antimicrobial peptides isolated from scorpion venom are important in the discovery of novel antibiotic molecules [101]. The first antimicrobial peptide isolated from scorpions were of the defensin type from *Leiurus quinquestriatus hebraeus* [102]. Later cytolitic and/or antibacterial peptides were isolated from scorpions belonging to the Buthi‐ dae, Scorpionidae, Ischnuridae, and Iuridae superfamilies hemo-lymph and venom [103-108]. The discovery of these peptides in venoms from Eurasian scorpions, Africa and the Americas, confirmed their widespread occurrence and significant biological function. Scorpine, a peptide from *Pandinus imperator* with 75 amino acids, three disulfide bridges, and molecular mass of 8350 Da has anti-bacterial and anti-malaria effects [104]. A cationic amphipatic peptide consisting of 45 amino acids has been purified from the venom of the southern African scorpion, *Parabuthus schlechteri*. At higher concentrations it forms non-se‐ lective pores into membranes causing depolarization of the cells [109]. Opistoporin1 and 2 (OP 1 and 2) was isolated from the venom of *Opistophthalmus carinatus*. These are amphi‐ pathic, cationic peptides which differ only in one amino acid residue. OP1 and PP were ac‐ tive against Gram-negative bacteria and both had hemolytic activity and antifungal activity. These effects are related to membrane permeabilization [106]. A new antimicrobial peptide, hadrurin, was isolated from *Hadrurus aztecus*. It is a basic peptide composed of 41 aminoacid residues with a molecular mass of 4436 Da, and contains no cysteines. It is a unique peptide among all known antimicrobial peptides described, only partially similar to the Nterminal segment of gaegurin 4 and brevinin 2e, isolated from frog skin. It would certainly be a model molecule for studying new antibiotic activities and peptide-lipid interactions [110]. Pandinin 1 and 2 are antimicrobial peptides have been identified and characterized from venom of the African scorpion *Pandinus imperator* [101]. Recently six novel peptides, named bactridines, were isolated from *Tityus discrepans* scorpion venom by mass spectrome‐ try. The antimicrobial effects on membrane Na+ permeability induced by bactridines were

observed on *Yersinia enterocolitica* [111]. The profile of gene in the venom glands of *Tityus stigmurus* scorpions was studied by transcriptome. Data revealed that 41 % of ESTs belong to recognized toxin-coding sequences, with transcripts encoding antimicrobial toxins (AMPlike) being the most abundant, followed by alfa KTx-like, beta KTx-like, beta NaTx-like and alfa NaTx-like. Parallel, 34% of the transcripts encode "other possible venom molecules", which correspond to anionic peptides, hypothetical secreted peptides, metalloproteinases, cystein-rich peptides and lectins [7].

tide inhibitor of shaker-type (K(v)1) K+ channels have been purified to homogeneity from venom of the scorpion *Centruroides limbatus* [123]. Noxiustoxin, component II-11 from the venom of scorpion *Centruroides noxius* Hoffmann, was obtained in pure form after fractiona‐ tion by Sephadex G-50 chromatography followed by ion exchange separation on carboxymethylcellulose columns. This peptide is the first short toxin directed against mammals and the first K+ channel blocking polypeptide-toxin found in scorpion venoms [124]. Pi1 is a peptide purified and characterized from the venom of the scorpion *Pandinus imperato,* show‐ ing ability to block the shaker K+ channel [125]. All of these peptides obtained from scor‐ pions venoms are potential toxins acting on immunological system as immunosuppressant

Toxins from Venomous Animals: Gene Cloning, Protein Expression and Biotechnological Applications

http://dx.doi.org/10.5772/52380

31

One of the most notable active principles found in scorpion venom is chlorotoxin (Cltx), a peptide isolated from the species *Leiurus quinquestriatus*. Cltx has 36 amino acids with four disulfide bonds, and inhibits chloride influx in the membrane of glioma cells [126]. This pep‐ tide binds only to glioma cells, displaying little or no activity at all in normal cells. The toxin appears to bind matrix metalloproteinase II [117]. A synthetic version of this peptide (TM601) is being produced by the pharmaceutical industry coupled to iodine 131 (131I-TM601), to carry radiation to tumor cells [127]. A recent study shows that TM601 inhibited angiogenesis stimulated by pro-angiogenic factors in cancer cells, and when TM601 was coadministered with bevacizumab, the combination was significantly more potent than a tenfold increase in bevacizumab dose [128]. A chlorotoxin-like peptide has also been isolated, cloned and sequenced from the venom of another scorpion species, *Buthus martensii* Karsch [129]. In reference [130] was expressed the recombinant chlorotoxin like peptide from *Leiu‐ rus quinquestriatus* and named rBmK CTa. Two novel peptides named neopladine 1 and neo‐ pladine 2 were purified from *Tityus discrepans* scorpion venom and found to be active on human breast carcinoma SKBR3 cells. Inmunohistochemistry assays revealed that neopla‐ dines bind to SKBR3 cell surface inducing FasL and BcL-2 expression [131]. Results indicate the venom from this scorpion represents a great candidate for the development of new clini‐

Evidence for the potential application of scorpions toxins as insecticides has emerged in re‐ cent years. The precise action mechanism of several of these molecules remains unknown; many have their effects via interactions with specific ion channels and receptors of neuro‐ muscular systems of insects and mammals. These highly potent and specific interactions make venom constituents attractive candidates for the development of novel therapeutics,

Toxin Lqhα IT from the scorpion *Leiurus quinquestriatus hebraeus* venom is the best represen‐ tative of anti-insect alpha toxins [133-134]. A similar effect was observed after applying the insect-selective toxin Bot IT1 from *Buthus occitanus tunetanus* venom [135]. Selective inhibi‐ tion of the inactivation process of the insect para/tipNav expressed in *Xenopus oocyteswas*

for autoimmune diseases.

cal treatments against tumors.

**3.6. Toxins with insecticides applications**

pesticides and as molecular probes of target molecules [132].

**3.5. Toxins with anticancer and cytotoxic activities**

### **3.3. Toxins acting on acting on inflammatory and nociceptive response**

The use of toxins as novel molecular probes to study the structure-function relationship of ion-channels and receptors as well as potential therapeutics in the treatment of wide variety of diseases is well documented. The high specificity and selectivity of these toxins have at‐ tracted a great deal of interest as candidates for drug development [8]. At least five peptides have been identified from *Buthus martensii* (Chinese scorpion) venom that have anti-inflam‐ matory and antinociceptive properties [61]. One peptide, J123, blocks potassium channels that activate memory T-cells [112]. The venom also contains a 61-amino acid peptide that has demonstrated antiseizure properties in an animal model [113] as well as other constitu‐ ents that act as analgesics in mice, rats, and rabbits [114]. The polypeptide BmK IT2 from scorpion *Buthus martensi* Karsh stops rats from reacting to experimentally-induced pain [115]. A protein from the Indian black scorpion, *Heterometrus bengalensis*, bengalin caused human leukemic cells to undergo apoptosis *in vitro* [116]. The peptide chlorotoxin, found in the venom of the scorpion *Leiurus quinquestriatus*, retarded the activity of human glioma cells *in vitro* [117]. An investigation about the role of kinins, prostaglandins and nitric oxide in mechanical hypernociception, spontaneous nociception and paw oedema after intraplan‐ tar have been done with *Tityus serrulatus* venom in male wistar rats, proving the potential of use of the venom to alleviate pain and oedema formation [118].

### **3.4. Toxins acting on acting on immunological system**

OSK1 (alpha-KTx3.7) is a 38-residue toxin cross-linked by three disulphide bridges initial‐ ly purified from the venom of the central Asian scorpion *Orthochirus scrobiculosus* [119]. OSK1 and several structural analogues were produced by solid-phase chemical synthesis, and were tested for lethality in mice and for their efficacy in blocking a series of 14 voltage-gated and Ca2+ activated K+ channels *in vitro*. The literature report that OSK1 could serve as leads for the design and production of new immunosuppressive drugs [119]. Margatoxin, a peptid‐ yl inhibitor of K+ channels has been purified to homogeneity from venom of the new world scorpion *Centruroides margaritatus* showed that could be used as immunosuppressive agent [120]. Kaliotoxin, a peptidyl inhibitor of the high conductance Ca2+-activated K+ channels (KCa) has been purified to homogeneity from the venom of the scorpion *Androctonus maur‐ etanicus mauretanicus*. This peptide appears to be a useful tool for elucidating the molecu‐ lar pharmacology of the high conductance Ca2+-activated K+ channel [121]. Agitoxin 1, 2, and 3, from the venom of the scorpion *Leiurus quinquestriatus* var. hebraeus have been identi‐ fied on the basis of their ability to block the shaker K+ channel [122]. Hongotoxin, a pep‐ tide inhibitor of shaker-type (K(v)1) K+ channels have been purified to homogeneity from venom of the scorpion *Centruroides limbatus* [123]. Noxiustoxin, component II-11 from the venom of scorpion *Centruroides noxius* Hoffmann, was obtained in pure form after fractiona‐ tion by Sephadex G-50 chromatography followed by ion exchange separation on carboxymethylcellulose columns. This peptide is the first short toxin directed against mammals and the first K+ channel blocking polypeptide-toxin found in scorpion venoms [124]. Pi1 is a peptide purified and characterized from the venom of the scorpion *Pandinus imperato,* show‐ ing ability to block the shaker K+ channel [125]. All of these peptides obtained from scor‐ pions venoms are potential toxins acting on immunological system as immunosuppressant for autoimmune diseases.

### **3.5. Toxins with anticancer and cytotoxic activities**

observed on *Yersinia enterocolitica* [111]. The profile of gene in the venom glands of *Tityus stigmurus* scorpions was studied by transcriptome. Data revealed that 41 % of ESTs belong to recognized toxin-coding sequences, with transcripts encoding antimicrobial toxins (AMPlike) being the most abundant, followed by alfa KTx-like, beta KTx-like, beta NaTx-like and alfa NaTx-like. Parallel, 34% of the transcripts encode "other possible venom molecules", which correspond to anionic peptides, hypothetical secreted peptides, metalloproteinases,

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

The use of toxins as novel molecular probes to study the structure-function relationship of ion-channels and receptors as well as potential therapeutics in the treatment of wide variety of diseases is well documented. The high specificity and selectivity of these toxins have at‐ tracted a great deal of interest as candidates for drug development [8]. At least five peptides have been identified from *Buthus martensii* (Chinese scorpion) venom that have anti-inflam‐ matory and antinociceptive properties [61]. One peptide, J123, blocks potassium channels that activate memory T-cells [112]. The venom also contains a 61-amino acid peptide that has demonstrated antiseizure properties in an animal model [113] as well as other constitu‐ ents that act as analgesics in mice, rats, and rabbits [114]. The polypeptide BmK IT2 from scorpion *Buthus martensi* Karsh stops rats from reacting to experimentally-induced pain [115]. A protein from the Indian black scorpion, *Heterometrus bengalensis*, bengalin caused human leukemic cells to undergo apoptosis *in vitro* [116]. The peptide chlorotoxin, found in the venom of the scorpion *Leiurus quinquestriatus*, retarded the activity of human glioma cells *in vitro* [117]. An investigation about the role of kinins, prostaglandins and nitric oxide in mechanical hypernociception, spontaneous nociception and paw oedema after intraplan‐ tar have been done with *Tityus serrulatus* venom in male wistar rats, proving the potential of

OSK1 (alpha-KTx3.7) is a 38-residue toxin cross-linked by three disulphide bridges initial‐ ly purified from the venom of the central Asian scorpion *Orthochirus scrobiculosus* [119]. OSK1 and several structural analogues were produced by solid-phase chemical synthesis, and were tested for lethality in mice and for their efficacy in blocking a series of 14 voltage-gated and Ca2+ activated K+ channels *in vitro*. The literature report that OSK1 could serve as leads for the design and production of new immunosuppressive drugs [119]. Margatoxin, a peptid‐

scorpion *Centruroides margaritatus* showed that could be used as immunosuppressive agent [120]. Kaliotoxin, a peptidyl inhibitor of the high conductance Ca2+-activated K+ channels (KCa) has been purified to homogeneity from the venom of the scorpion *Androctonus maur‐ etanicus mauretanicus*. This peptide appears to be a useful tool for elucidating the molecu‐

3, from the venom of the scorpion *Leiurus quinquestriatus* var. hebraeus have been identi‐ fied on the basis of their ability to block the shaker K+ channel [122]. Hongotoxin, a pep‐

channels has been purified to homogeneity from venom of the new world

channel [121]. Agitoxin 1, 2, and

**3.3. Toxins acting on acting on inflammatory and nociceptive response**

use of the venom to alleviate pain and oedema formation [118].

**3.4. Toxins acting on acting on immunological system**

lar pharmacology of the high conductance Ca2+-activated K+

yl inhibitor of K+

cystein-rich peptides and lectins [7].

Applications

30

One of the most notable active principles found in scorpion venom is chlorotoxin (Cltx), a peptide isolated from the species *Leiurus quinquestriatus*. Cltx has 36 amino acids with four disulfide bonds, and inhibits chloride influx in the membrane of glioma cells [126]. This pep‐ tide binds only to glioma cells, displaying little or no activity at all in normal cells. The toxin appears to bind matrix metalloproteinase II [117]. A synthetic version of this peptide (TM601) is being produced by the pharmaceutical industry coupled to iodine 131 (131I-TM601), to carry radiation to tumor cells [127]. A recent study shows that TM601 inhibited angiogenesis stimulated by pro-angiogenic factors in cancer cells, and when TM601 was coadministered with bevacizumab, the combination was significantly more potent than a tenfold increase in bevacizumab dose [128]. A chlorotoxin-like peptide has also been isolated, cloned and sequenced from the venom of another scorpion species, *Buthus martensii* Karsch [129]. In reference [130] was expressed the recombinant chlorotoxin like peptide from *Leiu‐ rus quinquestriatus* and named rBmK CTa. Two novel peptides named neopladine 1 and neo‐ pladine 2 were purified from *Tityus discrepans* scorpion venom and found to be active on human breast carcinoma SKBR3 cells. Inmunohistochemistry assays revealed that neopla‐ dines bind to SKBR3 cell surface inducing FasL and BcL-2 expression [131]. Results indicate the venom from this scorpion represents a great candidate for the development of new clini‐ cal treatments against tumors.

### **3.6. Toxins with insecticides applications**

Evidence for the potential application of scorpions toxins as insecticides has emerged in re‐ cent years. The precise action mechanism of several of these molecules remains unknown; many have their effects via interactions with specific ion channels and receptors of neuro‐ muscular systems of insects and mammals. These highly potent and specific interactions make venom constituents attractive candidates for the development of novel therapeutics, pesticides and as molecular probes of target molecules [132].

Toxin Lqhα IT from the scorpion *Leiurus quinquestriatus hebraeus* venom is the best represen‐ tative of anti-insect alpha toxins [133-134]. A similar effect was observed after applying the insect-selective toxin Bot IT1 from *Buthus occitanus tunetanus* venom [135]. Selective inhibi‐ tion of the inactivation process of the insect para/tipNav expressed in *Xenopus oocyteswas*

was observed in the presence of Bjα IT [136] and OD1 [137], which are toxins from *Buthotus judaicus* and *Odonthobuthus doriae* scorpion venom, respectively. A second group of scorpion toxins slowing insect sodium channel inactivation was called alpha-like toxins. The first pre‐ cisely described toxins from this group were the Lqh III/Lqh3 (from *L. q. hebraeus*), Bom III/ Bom 3 and Bom IV/ Bom 4 (from *B. o. mardochei*). They were all tested on cockroach axonal preparation [138-139]. BmKM1 toxin from *B. martensi* Karsch was the first alpha-like toxin available in recombinant form that was tested also on cockroach axonal preparation [140]. Toxins Lqh6 and Lqh7 from *L. q. hebraeus* scorpion venom show high structural similarity with Lqh3 toxin. Their toxicity to cockroach is in the range found for other alpha-like toxins [141]. Alpha-like toxins from scorpion venoms show lower efficiency when applied to in‐ sects, as compared to α anti-insect toxins. Therefore they seem to be less interesting from the point of view of future insecticide development [132]. Scorpion contractive and depressant toxins are highly selective for insect sodium channels. Several of these toxins were tested on cockroach axonal preparations; toxin AaH IT1 from the *A. australis* scorpion venom was the first one [142-143]. All other contractive toxins tested on cockroach axon produced very sim‐ ilar effects, as for example Lqq IT1 from *L. q. quinquestriatus* [133]; Bj IT1 from *B. judaicus* [143], Bm 32-1 and Bm 33-1 from *B. martensi* [144].

**4.3. Toxins with antibiotic activity**

portant medical implications [150].

**4.4. Toxins acting on inflammatory and nociceptive response**

veal new targets for the treatment of envenomation [10].

venom for anti-venom production and therapy [155].

**4.6. Toxins with anticancer and cytotoxic activities**

Psalmotoxin 1 was evaluated on inhibited Na+

**4.5. Toxins acting on immunological system**

Two peptide toxins with antimicrobial activity, lycotoxins I and II, were identified from ven‐ om of the wolf spider *Lycosa carolinensis* (Araneae: Lycosidae). The lycotoxins may play a dual role in spider-prey interaction, functioning both in the prey capture strategy as well as to protect the spider from potentially infectious organisms arising from prey ingestion. Spi‐ der venoms may represent a potentially new source of novel antimicrobial agents with im‐

Toxins from Venomous Animals: Gene Cloning, Protein Expression and Biotechnological Applications

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33

Psalmotoxin 1, a peptide extracted from the South American tarantula *Psalmopoeus cambridg‐ ei*, has very potent analgesic properties against thermal, mechanical, chemical, inflammatory and neuropathic pain in rodents. It exerts its action by blocking acid-sensing ion channel 1a, and this blockade results in an activation of the endogenous enkephalin pathway [151]. Phospholipases from both *Loxosceles laeta* and *Loxosceles reclusa* cleaved LPC (lysophosphati‐ dylcholine) to LPA (lysophosphatidic acid) and choline. LPA receptors are potential targets for *Loxosceles* sp. envenomation treatment [152]. The possibilities for biotechnological appli‐ cations in this area are enormous. Recombinant dermonecrotic toxins could be used as re‐ agents to establish a new model to study the inflammatory response, as positive inducers of the inflammatory response and edema [9, 153-154]. The phospholipase-D from *Loxosceles* venom could be used in phospholipid studies, specially studies on cell membrane constitu‐ ents with emphasis upon sphingophospholipids, lysophospholipids, lysophosphatidic acid and ceramide-1-phosphate, as models for elucidating lipid product receptors, signaling pathways and biological activities; this new wide field of *Loxosceles* research could also re‐

The antiserum most commonly used for treatment of loxoscelism in Brazil is anti-arachnidic serum. This serum is produced by the Instituto Butantan (São Paulo, Brazil) by hyperimmu‐ nization of horses with venoms of the spiders *Loxosceles gaucho* and *Phoneutria nigriventer* and the scorpion *Tityus serrulatus*. Several studies have indicated that sphingomyelinase D (SMase D) in venom of *Loxosceles* sp. spiders is the main component responsible for local and systemic effects observed in loxoscelism [153, 155]. Neutralization tests showed that an‐ ti-SMase D serum has a higher activity against toxic effects of *L. intermedia* and *L. laeta* ven‐ oms and similar or slightly weaker activity against toxic biological effects of *L. gaucho* than that of Arachnidic serum. These results demonstrate that recombinant SMase D can replace

cells (glioblastoma multiforme, or GBM). These observations suggest this toxin may prove useful in determining whether GBM cells express a specific ASIC-containing ion channel

currents in high-grade human astrocytoma
