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J. Biol. Chem., Vol. 277, Issue 26, 23627-23637, June 28, 2002

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Oxyopinins, Large Amphipathic Peptides Isolated from the Venom of the Wolf Spider Oxyopes kitabensis with Cytolytic Properties and Positive Insecticidal Cooperativity with Spider Neurotoxins^*

Gerardo Corzo^§, Elba Villegas, Froylan Gómez-Lagunas^¶, Lourival D. Possani, Olga S. Belokoneva, and Terumi Nakajima

From the  Suntory Institute for Bioorganic Research, Mishima-Gun, Shimamoto-Cho, Wakayamadai 1-1-1, Osaka 618-8503, Japan, the ^¶ Department of Physiology, School of Medicine, National Autonomous University of Mexico (UNAM), Cd. Universitaria, Mexico City 04510, Mexico, and the  Department of Molecular Recognition and Structural Biology, Institute of Biotechnology, Av. Universidad 2001, Cuernavaca, Morelos 62210, Mexico

Received for publication, January 17, 2002, and in revised form, April 25, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Five amphipathic peptides with antimicrobial, hemolytic, and insecticidal activity were isolated from the crude venom of the wolf spider Oxyopes kitabensis. The peptides, named oxyopinins, are the largest linear cationic amphipathic peptides from the venom of a spider that have been chemically characterized at present. According to their primary structure Oxyopinin 1 is composed of 48 amino acid residues showing extended sequence similarity to the ant insecticidal peptide ponericinL2 and to the frog antimicrobial peptide dermaseptin. Oxyopinins 2a, 2b, 2c, and 2d have highly similar sequences. At least 27 out of 37 amino acid residues are conserved. They also show a segment of sequence similar to ponericinL2. Circular dichroism analyses showed that the secondary structure of the five peptides is essentially -helical. Oxyopinins showed disrupting activities toward both biological membranes and artificial vesicles, particularly to those rich in phosphatidylcholine. Electrophysiological recordings performed on insect cells (Sf9) showed that the oxyopinins produce a drastic reduction of cell membrane resistance by opening non-selective ion channels. Additionally, a new paralytic neurotoxin named Oxytoxin 1 was purified from the same spider venom. It contains 69 amino acid residue cross-linked by five disulfide bridges. Application of mixtures containing oxyopinins and Oxytoxin 1 to insect larvae showed a potentiation phenomenon, by which an increase lethality effect is observed. These results suggest that the linear amphipathic peptides in spider venoms and neuropeptides cooperate to capture insects efficiently.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Amphipathic and cationic -helical peptides are broadly found in animals as a part of their biological defense system (1). Since the discovery of melittins (2) and cecropins (3) a large number of amphiphilic and cationic peptides have been characterized in invertebrates, especially from the phyla Arthropoda and in vertebrates from the class Amphibia. Linear cationic peptides with -helical conformation share some common characteristics such as antimicrobial activities at low micromolar concentrations and -helix formation in hydrophobic environments. However, other features distinguish them such as their disruptive activity toward eukaryotic cells, particularly red blood cells, and their hydrophilic/hydrophobic amino acid distribution along the structure. In arthropods, based on the source of the antimicrobial peptides, these molecules could be roughly classified in two types. The first type is cecropin-like peptides, which are antimicrobial with low hemolytic activity acting as a part of the insect immune defense system against the invasion of pathogenic microorganisms. This type of molecule is mainly found in the hemolymph of various arthropods such as sarcotoxin A in house fly (4), cecropins in lepidoptera (3), and spinigerin in termites (5). The second type is melittin-like peptides, which are antimicrobial with hemolytic activity higher than that of the first type acting mainly as weapons for capturing prey or as self-defense against other animals. This type of peptide is mostly found in the venom glands of bees (6), wasps (7), spiders (8), ants (9), and scorpions (10, 11). Amphipathic -helical peptides cause mainly the disruption of cell membranes by molecular mechanisms depending principally on the charge distribution of the peptide and on the chemical composition of the cell membranes. In the order Arachnida, the biological function of the linear amphipathic peptides may play an important role in capturing insects by dissipating ion and voltage gradients across the membrane of excitable cells (8) and as a defense mechanism against external pathogens (10) or possible microbial infection after digestion caused by decomposition of the prey (8). In this work, we report the isolation and chemical characterization of five amphiphilic peptides (oxyopinins) and one neurotoxin (Oxytoxin 1) from the venom of the spider Oxyopes kitabensis. Oxyopinins are the largest linear cationic amphipathic peptides from the venom of spiders that have been chemically characterized thus far. We have analyzed their membrane lytic activities in biological and in artificial phospholipid vesicles and their mode of action in insect cells.

For comparative purposes we have included here a discussion of the effects of the pandinins, extracted from the venom of the scorpion Pandinus imperator. Additionally, we have demonstrated a positive cooperativity effect of oxyopinins with that of neurotoxic peptides found in spider venom.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Biological Materials-- O. kitabensis crude venom was purchased from SpiderPharm (Yarnell, AZ). Endoproteinase Glu-C (EC 3.4.21.19) was from Sigma Chemical Co. (St. Louis, MO). L--Phosphatidylcholine (PC)¹ and L--phosphatidic acid (PA) from egg yolk were purchased from Nacalai Tesque (Osaka, Japan). Phosphatidylethanolamine (PE) from Escherichia coli was from Doosan Serdary Research Laboratories (Toronto, Canada). All phospholipids were of reagent grade with purity higher than 98%. The microbial strains E. coli (ATCC 11775) and Bacillus subtilis (ATCC 1175) were obtained from the American Type Culture Collection. Staphylococcus aureus (IAM 1098, from the Institute of Applied Microbiology Culture Collection, University of Tokyo) was obtained from the Suntory Institute for Fundamental Research. Pandinins and -paluIT1 were obtained according to Corzo et al. (11, 12).

Isolation of Oxyopinins and Oxytoxin-- O. kitabensis crude venom (70 µl) was resuspended in 0.1% aqueous trifluoroacetic acid containing 10% acetonitrile (CH₃CN), and the insoluble material was removed by centrifugation at 14,000 × g for 5 min. The supernatant was used directly for HPLC separation. The diluted venom was fractionated using a reverse-phase semipreparative C₁₈ column (5C₁₈MS, 10 × 250 mm, Nacalai Tesque, Japan) equilibrated in 0.1% trifluoroacetic acid and eluted with a linear gradient of acetonitrile from 0 to 60% in 0.1% trifluoroacetic acid, run for 60 min at a flow rate of 2 ml/min. Effluent absorbance was monitored at 215 nm. Fractions with antimicrobial (oxyopinins) or insecticidal (oxytoxin) activity were further fractionated by cation exchange HPLC on a TSK-gel sulfopropyl column (Tosoh SP-5PW, 7.5 × 75 mm, Japan). The column was equilibrated with 5 mM sodium phosphate buffer, pH 6.5, containing 30% CH₃CN. A linear gradient from 10 mM to 1.0 M sodium chloride was applied using the same buffer and run at 1 ml/min. The peptides were finally purified by a C₄ reverse-phase column (4.6 × 250 mm, Nacalai Tesque, Japan) using the same gradient as that for HPLC described above but with HFBA as an ion pairing instead of trifluoroacetic acid, run at a flow rate of 1 ml/min.

Sequence Analysis-- Oxyopinins (Oxki1-5) and oxytoxin (OxyTx1) were reduced with tributylphosphine (Nacalai Tesque, Japan) and were alkylated with 4-vinyl-pyridine (Wako, Japan), in 0.5 M NaHCO₃ buffer (pH 8.3) for 2 h at 37 °C in the dark prior to sequencing. The molecular masses of oxyopinins did not change after reduction and alkylation. However, the mass of the pyridylethylated peptide OxyTx1, 9,109.4 Da, exceeded that of the native peptide by 1,050 Da, indicating the presence of five disulfide bonds. All peptides were directly sequenced on a Shimadzu PPSQ-10 automated gas-phase sequencer. The spider peptides were dissolved in 30 µl of a 37% CH₃CN solution and applied to trifluoroacetic acid-treated glass fiber membranes, precycled with Polybrene (Aldrich, Milwaukee, WI). Data were recorded on a Shimadzu CR-7A integrator.

Enzymatic Digestions-- The peptides were subjected to enzymatic hydrolysis. Hydrolysis with type XVII-B endoproteinase Glu-C from Staphylococcus aureus V8 was carried out in 0.1 M sodium bicarbonate buffer (pH 7.6), at 37 °C for 3 h, using a 1:20 (w/w) enzyme to substrate ratio. The endoproteinase digest was fractionated by reverse-phase HPLC using a C₄ column (250 × 4.6 mm, Nacalai Tesque, Japan) and a linear gradient of acetonitrile in 0.1% aqueous trifluoroacetic acid. The endoproteinase fractions were analyzed by MALDI-TOF MS, and chemically sequenced.

Mass Spectrometry-- MALDI mass spectra were obtained on a PerSeptive Voyager Elite time-of-flight (TOF) spectrometer (PerSeptive Biosystems Inc., League City, TX) equipped with a model VSL-337ND nitrogen laser (Laser Science, Cambridge, MA). The accelerating voltage in the ion source was set to 20 kV, and data were acquired in the positive linear mode of operation. Time-to-mass conversion was achieved by external and/or internal calibration using standards of bradykinin (m/z 1061.2), bovine pancreatic beta insulin (m/z 3496.6), bovine pancreatic insulin (m/z 5734.5), and ubiquitin (m/z 8565.8) (Sigma). All experiments were performed using -cyano-4-hydroxycinnamic acid (Aldrich) as the matrix.

Peptide Synthesis of Oxyopinins-- Oxki1 was chemically synthesized by a solid-phase method using the Fmoc methodology on an Applied Biosystems 433 A peptide synthesizer. Fmoc-Gln(tBu)-PEG resin (Watanabe Ltd., Hiroshima, Japan) was used to provide a free carboxyl at the C terminus of synthetics Oxki1-OH and Oxki2b-OH. Chemical synthesis, cleavage, and deprotection of peptide resins were performed according to the instructions from the 1999 Novabiochem peptide synthesis handbook. The crude synthetic peptide was dissolved in a 30% aqueous acetonitrile solution and separated by reverse-phase HPLC on a semipreparative C₁₈ column (10 × 250 mm, Nacalai Tesque, Japan). Cation exchange chromatography and C₄ reverse-phase HPLC, as described above, were further used to purify the synthetic peptides. The structural identity of the synthetic and natural peptides was verified by co-elution experiments using capillary zone electrophoresis and cation exchange HPLC. The mass identity between synthetic and natural peptides was verified by MALDI-TOF mass spectrometry.

Circular Dichroism (CD) Measurements-- CD spectra were obtained on a Jasco J-725 spectropolarimeter (Jasco, Japan). The spectra were measured from 260 to 180 nm in 10 mM potassium phosphate buffer, pH 7.2, and in 60% TFE, pH 7.1, at room temperature, with a 1-mm path-length cell. Data were collected at 0.1 nm with a scan rate of 100 nm/min and a time constant of 0.5 s. The concentration of the peptides was 40 µg/ml. Data were the average of 10 separate recordings and were analyzed by the method of Bohm et al. (13).

Antimicrobial and Hemolytic Assays-- Detection and isolation of oxyopinins was done following plate growth inhibition of E. coli and B. subtilis by vacuum-dried HPLC fractions that were resuspended in 20 µl of distilled water according to Corzo et al. (11). Growth inhibition curves and minimal inhibitory concentrations (MIC) were obtained using pure peptides at concentrations of 1.6, 3.1, 6.2, 12.5, 25, and 50 µM. Briefly, the inoculum was prepared from fresh bacteria cultures. Serial dilutions of peptides were arranged, and an aliquot of cell suspension was added to each vial. The final volume in each vial was 100 µl, and the cell count was 1.2 × 10⁶ CFU/ml for S. aureus and 1.6 × 10⁶ colony-forming units/ml for E. coli. The final peptide concentration ranged from 1.6 to 50 µM. After 16-18 h of incubation at 37 °C, the optical density (OD) of each vial was measured at 630 nm in an enzyme-linked immunosorbent assay reader (Bio-Rad, model 450, Hercules, CA). The positive control contained only the bacterial suspension, and the negative control contained only sterile culture medium. The MIC values were defined as the lowest concentration of peptide at which 100% growth inhibition was observed. Hemolytic activity was determined by incubating suspensions of sheep, pig, or guinea pig red blood cells with serial dilutions of each selected peptide. Red blood cells (10% v/v) were rinsed several times in PBS by centrifugation for 3 min at 3000 × g until the OD of the supernatant reached the OD of the control (PBS only). Red blood cells were counted by an hematocytometer and adjusted to approximately 7.7 × 10⁶ ± 0.3 × 10⁶ cells/ml. Red blood cells were then incubated at room temperature for 1 h in 10% Triton X-100 (positive control), in PBS (blank), or with amphipathic peptides at concentrations of 1.6, 3.1, 6.2, 12.5, 25, and 50 µM. The samples were then centrifuged at 10,000 × g for 5 min; the supernatant was separated from the pellet, and its absorbance was measured at 570 nm. The relative optical density compared with that of the suspension treated with 10% Triton X-100 defined the percentage of hemolysis.

Artificial Vesicles-- Small unilamellar vesicles (SUVs) containing calcein were prepared according to Wieprecht et al. (14). Calcein leakage from vesicles was monitored fluorometrically using a Hitachi fluorescence spectrophotometer (F-4500, Tokyo, Japan) by measuring the time-dependent increase in fluorescence of calcein release. As calcein leaks from the vesicles by the disrupting activity of oxyopinins it becomes diluted and therefore dequenched, and an increase in fluorescence is recorded (excitation = 490 nm, emission = 520 nm). A final volume of 0.95 ml of SUVs was placed in a stirred cuvette at room temperature. An aliquot of peptide solution (50 µl) was added to the cuvette. The percentage of calcein released by the addition of peptides was evaluated by the equation: 100 × (F  F_o)/(F_t  F_o), where F is the fluorescence intensity achieved by the peptides. F_o is the fluorescence intensity observed without the peptides, and F_t is the fluorescence intensity corresponding to 100% calcein release determined by the addition of 50 µl of 10% Triton X-100. The phosphorus content in phospholipid vesicles was estimated by spectrophotometric analysis (15).

Hemocytes-- The hemolymph of Spodoptera litura larvae was collected by an aseptic puncture in the prolegs in 1.5-ml polypropylene vials containing 0.5 ml of an anticoagulant solution (16). The diluted hemolymph was centrifuged at 720 × g for 5 min. The supernatant was discarded, and the pellet containing the hemocytes was washed twice with the anticoagulant solution. Hemocytes were resuspended in Grace's insect media (Invitrogen). Eighty microliters of Grace's medium containing hemocytes was added to each vial containing 20 µl of the cultured medium with serial dilutions of peptides. The final suspension (100 µl) contained ~2 × 10⁶ hemocytes/ml (total hemocytes count). Viability of hemocytes was detected after 60 min of incubation at room temperature by a lactic dehydrogenase-based assay kit (Sigma). The positive control (100% hemolysis) was determined by applying a lytic solution (supplied with the kit) to disrupt hemocyte cells. The hemocytes in Grace's medium were used as a negative control.

Sf9 Insect Cell Culture and Electrophysiological Recordings-- Insect Sf9 cells (Spodoptera frugiperda cells) were kept in culture in Grace's media at 27 °C as previously reported (17). The membrane resistance was monitored by recording the ionic current elicited by test pulses applied every second from a holding potential of 0 mV under whole-cell patch clamp, using an Axopatch-1D amplifier (Axon Instruments, Burlingame, CA). Currents were filtered at 5 KHz and sampled at 100 µs per point, with a TL1 interface (Axon Instruments). Electrodes were pulled from borosilicate glass (KIMAX 51) to a final resistance of 2 M. The bath solution contained (millimolar): 115 NaCl, 10 CaCl₂, 10 HEPES-NaOH buffer, pH 7.2. The internal solution contained (millimolar): 90 KF, 30 KCl, 10 EGTA, 10 HEPES-KOH buffer, pH 7.2. Oxyopinins were diluted in the bath solution before being added to the recording chamber.

Microinjection Assay-- Insect paralytic activity was evaluated with a previously described microinjection assay using S. litura (tobacco cutworm) early third-instar larvae (2-3 mg) (18). Larvae were injected in the pronotum with glass capillary pipettes and up to 300 nl of neurotoxic and/or cytolytic peptide resuspended in water and placed in 55 mm-diameter plastic Petri dishes with an artificial diet. Paralytic and lethal effects were observed at different time intervals up to 24 h. The lethal dose corresponding to 50% of mortality (LD₅₀) was calculated by probit analysis (19).

Statistical Analysis-- The last significant difference method was used to determine whether statistically significance differences occurred among the mean values obtained.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification and Sequence Analysis of Oxyopinins and Oxytoxin-- The crude venom of O. kitabensis was fractionated using reverse-phase HPLC (Fig. 1). Sixty fractions were manually collected and vacuum-dried. Dried HPLC fractions were resuspended in distilled water and assayed for antimicrobial activity toward E. coli and B. subtilis. Fractions 40, 41, and 45 showed antimicrobial activity toward both E. coli and B. subtilis, and they were further fractionated by cation-exchange chromatography (CE). Fraction 45 yielded a pure peptide under CE, and it was named oxyopinin1 (Oxki1). Fractions 40 and 41 produced two antimicrobial peptides each by CE, and they were named oxyopinins 2, 3, 4, and 5 (Oxki2-5). All five antimicrobial peptides (Oxki1-5) were further resolved under reverse-phase HPLC conditions using HFBA for ion pairing. Oxyopinins were obtained in homogenous form as analyzed by capillary zone electrophoresis (data not shown). Direct Edman degradation sequencing of Oxki1 allowed unequivocal identification of the first 42 amino acid residues. The complete amino acid sequence of Oxki1 was obtained after its enzymatic digestion with endoproteinase Glu-C. The endoproteinase digestion of Oxki1 yielded two fragments that permitted the full identification of all amino acid residues in Oxki1 (Fig. 2A). Oxyopinin1 is composed of 48 amino acid residues, and its molecular mass determined by mass spectrometry was 5221.2 Da, in good agreement with the theoretically expected molecular weight of 5221.3. Direct sequencing of Oxki2 to Oxki5 permitted the identification of their 37 amino acid residues and their theoretical molecular weights were 4126.9, 4146.9, 4064.8, and 4156.9, which also agree with their masses found by mass spectrometry of 4127.1, 4146.9, 4064.7, and 4156.8 Da. Because the oxyopinins 2, 3, 4, and 5 had similar number of residues and they were identical in at least 27 of 37 amino acid residues (see Fig. 2A), they were named oxyopinins 2a, 2b, 2c, and 2d (Oxki 2a-2d). All four Oxki2 analogs were digested with Glu-C, and, as expected from their primary structure, only Oxki2b and Oxki2c produced peptide fragments, which is a confirmatory result of the primary structure obtained. Analysis of known antimicrobial peptide sequences using the data base (bbcm1.univ.trieste.it/~tossi/pag1.htm) showed that at least 240 -linear peptides from animals and plants have been reported thus far. From the entire repertoire of peptides, we have chosen some antimicrobial and insecticidal -helical peptides from arthropods and frogs to compare their structural similarities. The primary structure of Oxyopinin 1 is 29% identical to that of the frog antimicrobial peptide dermaseptin and to that of the ant insecticidal peptide ponericinL2. Similarly, the Oxyopinin 2 analogs are about 20% identical to that of the amphipathic peptide ponericinL2 (Fig. 2A).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   RP-HPLC chromatogram of crude O. kitabensis venom. The numbers 40, 41, and 45 represent HPLC fractions that gave positive results in antimicrobial bioassays. The asterisk represents the major insecticidal neurotoxin OxyTx1 from the crude venom.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Amino acid sequences of oxyopinins and oxytoxin (OxyTx1). A, alignment of oxyopinins with known amphipathic antimicrobial peptides. The sequences compared are: esculentin 1 from Rana esculenta (24); cecropin A from Hyalophora cecropia (3); sarcotoxin IA from Sarcohaga peregrina (4); dermaseptin from Phyllomedusa sauvagii (23), pandinins from P. imperator (11); lycotoxins from Lycosa carolinensis (8); hadrurin from Hadrurus aztecus (10); ponericins from Pachycondyla goeldii (9). Percentage of identity is shown based in either oxyopinin 1 or oxyopinin 2a. B, alignment of oxytoxin with known insecticidal neurotoxins. The sequences compared are; Tx2-1, Tx2-5, Tx2-6, and Tx4(6-1) from P. nigriventer (20, 21). The percent identity is shown based on OxyTx1. The sequence alignment was obtained from www.ch.embnet.org/software/ClustalW.html. Gaps (hyphens) have been introduced to enhance similarities.

A new neurotoxic peptide named Oxytoxin 1 (OxyTx1) was also isolated from the same spider venom. OxyTx1 is the major paralytic neurotoxin of O. kitabensis, which elutes from HPLC at the position indicated with an asterisk in Fig. 1. It contains 69 amino acid residues with a molecular mass of 8059.2 Da closely packed by five disulfide bridges (Fig. 2B). OxyTx1 is a basic peptide (calculated pI 9.28) with a carboxylated free C-terminal residue as suggested by mass spectrometry data. OxyTx1 has some sequence similarity to insecticidal neuropeptides isolated from the crude venom of the Brazilian spider Phoneutria nigriventer (20) (Fig. 2B). Tx4(6-1), from P. nigriventer, is an insecticidal toxin that is non-toxic to mice (21) and slows down the inactivation of the sodium channel in insect CNS via binding to receptor site 3 (22). Similarly to Tx4(6-1), OxyTx1 is an insecticidal toxin that is non-toxic to mice up to 1 µg/20-g mouse, but it is surmised to be specific for sodium channels of insects (complete characterization to be described elsewhere). The use of mixtures of OxyTx1 with oxyopinins increased the lethality effect of individual components, as will be briefly described below.

Circular Dichroism and Peptide Synthesis of Oxyopinins-- The secondary structures of Oxki1 and Oxki2 analogs were analyzed by circular dichroism spectrometry in phosphate buffer and in 60% solution of TFE. Aqueous solutions of TFE promote hydrogen bonding, and it is considered to mimic a cell membrane environment (25). Oxyopinins adopted unordered structures in potassium phosphate buffer with negative ellipticities around 195-200 nm (data not shown). Oxyopinins adopted -helix structures with negative ellipticities at 208 and 222 nm in 60% TFE (Fig. 3). Similar CD spectra of oxyopinins in hydrophobic environments have been previously observed with other amphiphilic peptides containing -helix structures (11, 23, 26, 27).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Circular dichroism spectra of native oxyopinins in 60% TFE. The secondary structures of Oxki1 and Oxki2 analogs were analyzed by circular dichroism spectrometry in phosphate buffer and in 60% solution of TFE. The concentration of the peptides was 40 µg/ml. Data are the average of 10 separate recordings.

To conduct biological experiments and to confirm the primary and secondary structures of oxyopinins, Oxki1 and Oxki2b were chemically synthesized. Despite the fact that Oxki1 and Oxki2b are peptides of 48 and 37 amino acid residues long, the final yield of their chemical synthesis was 44 and 56%. In our experience, these good yields are probably a consequence of their linear nature, which would favor amino acid assembly throughout the automatic synthesis. Shortly after their chemical synthesis and purification, co-elution analysis of the synthetic peptides with their respective native forms was verified. Mass spectrometry determinations confirmed the identity of the synthetic and native oxyopinins (data not shown). Further biological experiments were carried out using the synthetic oxyopinins with the prevailing names of Oxki1 and Oxki2.

Antimicrobial Assays-- The antibacterial and hemolytic activities of oxyopinins were compared with those of pandinins (Pin1 and Pin2), which are -linear antimicrobial peptides isolated from the venom of the scorpion P. imperator. Pandinin 1 is a low hemolytic and antimicrobial peptide. Pandinin 2 is a highly hemolytic and antimicrobial peptide with biological and structural characteristics similar to melittin (11). The minimal inhibitory concentrations against S. aureus were 6.2, 6.2, 25, and 1.6 µM for Oxki1, Oxki2, Pin1, and Pin2, respectively (Fig. 4A). The minimal inhibitory concentrations against E. coli were 1.6, 12.5, 50, and 6.2 µM for Oxki1, Oxki2, Pin1, and Pin2, respectively (Fig. 4B). Oxki1 showed strong antimicrobial activity toward both E. coli and S. aureus. Oxki2 and the comparative controls Pin1 and Pin2 were more lytic to S. aureus (Gram-positive) than to E. coli. The high antimicrobial activity of Oxki1 toward E. coli could be of a great significance as a potential antibiotic, because most of the antimicrobial peptides already discovered usually had low activity toward Gram-negative bacteria.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Antimicrobial activity of oxyopinins and pandinins. A, S. aureus; B, E. coli. Data are the average of three independent experiments. Error bars represent the standard deviation.

Hemolytic Assays and Calcein Leakage from Phospholipid Vesicles-- The content of PC in mammalian red blood cells varies depending on the animal source. The percentages of PC of the total membrane phospholipids in guinea pig, pig, and sheep red blood cells are 41, 23, and 1%, respectively (28, 29). The hemolytic activities of Oxki1, Oxki2, and Pin1 in guinea pig, pig, and sheep erythrocytes were lower compared with that of the hemolytic activities of Pin2 (Fig. 5, A-C). With pig and sheep blood the hemolytic activity of Oxki1 was slightly higher than that of Oxki2, whereas with all blood cells the hemolytic activity of Oxki2 was higher than that of the peptide Pin1. Pig and guinea pig erythrocytes whose cell membranes are rich in PC were more susceptible to the hemolysis induced by all four arachnic peptides. Sheep erythrocytes were less susceptible to the hemolysis induced by all four amphipathic peptides. Moreover, at higher concentrations of sheep erythrocytes (approximately 6.6 × 10⁷ cells/ml) non-significant hemolytic activity of Oxki1, Oxki2, and Pin1 was observed using peptide concentrations up to 50 µM, except for Pin2.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Hemolytic activity and percent release of calcein from artificial vesicles following the addition of oxyopinins and pandinins. A, guinea pig; B, pig; C, sheep; D, phosphatidic acid; E, phosphatidylethanolamine; and F, phosphatidylcholine. Data are the average of three independent experiments for the pig and sheep red blood cells and the average of two independent experiments for guinea pig red blood cells and the artificial vesicles. Error bars represent the standard deviation.

A comparison of pandinins and oxyopinins in their ability to release calcein from SUVs with different lipid composition is shown in Fig. 5 (D-F). Oxki1 was more effective in releasing calcein from zwitterionic and anionic vesicles when compared with Oxki2 and pandinins. All four amphipathic peptides were more lytic toward PC vesicles than toward PE and PA vesicles. Although pandinin 2 is shorter than Oxki2, it was more effective than Oxki2 in releasing calcein from artificial vesicles. The disrupting activities of Oxki1 and Oxki2 to the three different phospholipid vesicles demonstrated that the mode of action of these peptides might be phospholipid-mediated. Moreover, oxyopinins are able to bind to the phospholipid bilayers with different specificities and eventually can produce leakage of their content. Overall, oxyopinin 1 was more effective in disrupting both biological and artificial vesicles than oxyopinin 2.

Hemocytes and Whole-cell Patch Clamp of Sf9 Insect Cells-- Because oxyopinins were obtained from the venom gland of a predator specialized in insect prey capture; we looked for a possible significant biological effect in insect cells. Therefore, the membrane integrity of insect hemocytes from S. litura larvae in the presence of oxyopinins and pandinins was measured. However, no cytolytic or membrane disruption effect using up to 200 µM amphipathic peptides was observed when compared with both positive and negative controls under the assay conditions. The positive control gave a clear reaction produced by the release of cytoplasmic lactate dehydrogenase from insect hemocytes. Although the cell surface of insect hemocytes could be a target of amphipathic peptides, because they are negatively charged (30), hemocytes respond to foreign molecules by adhesion mechanisms that could subdue the activity of pore-forming peptides. Moreover we specifically tested the effect of oxyopinins on the membrane resistance of S. frugiperda pupal ovary cells (Sf9). After the whole-cell configuration of the patch clamp was established, the stability of the membrane was monitored with the delivery of 10 mV/3.5-ms test voltage pulses applied every second from the holding potential (HP) of 0 mV. Fig. 6A shows the last three superimposed currents elicited by the test pulses, after compensation of the stray capacitance. The traces show the typical resistor-capacitor passive electrical behavior of biological membranes (31). The large capacitive current transients mark the beginning and the end of the test pulses. Once the stability of the membrane was verified, Oxki1 was added to the bath solution at a final concentration of 1 µM. Fig. 6B shows that the addition of Oxki1 produced a sudden fall of the membrane resistance, which is seen by the increase in both the holding current (labeled I₁) and the current during the pulse (labeled I₂). Interestingly, it was observed that (a) the holding current transiently changes its sign (further more detailed studies are needed to determine the reason of this change) and (b) after its initial increase, both the holding and the pulse currents relaxed to a smaller value (I₁ dropped from 0.6 to 0.01 nA) where they remained stable for some minutes (variable from cell to cell) until the cell was finally lost (not shown). The latter is best seen in Fig. 6C, which shows the membrane resistance during each pulse. The arrow points to the addition of Oxki1.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   Effect of Oxki1 on Sf9 cells. A, control currents elicited by three test pulses of 10 mV/3.5 ms. B, currents after the addition of 1 µM Oxki1 to the external solution, rate of pulsing was 1 Hz. C, membrane resistance versus time of the whole traces in B. D, current elicited by stepping up the membrane potential from 50 to +30 mV (labeled I3) after the initial 10-mV test pulse. The horizontal scale bar indicates 2.3 ms in panels A, B, and D. The vertical scale bar indicates 1.1 nA in A, 0.93 nA in B, and 1.7 nA in D. E, current versus voltage relationship of the traces in D (circles) and the traces recorded 3 min later (not shown in D, triangles). HP = 0 mV, Cm = 8 picofarads. The dotted line in the traces indicates the zero current level.

Finally, to explore the properties of the conductance pathway open by Oxki1, a current versus voltage (I-V) relationship was constructed (Fig. 6D) by stepping up the membrane potential from 50 to +30 mV in 10-mV increments after a former 10-mV test pulse (applied to verify the constancy of the membrane resistance during the I-V). Fig. 6E shows the I-V relationship of both the traces in D (circles) and the traces (triangles) recorded 3 min later. Fig. 6E illustrates that Oxki1 drops the membrane resistance by opening a non-selective (reversal potential = 0 mV) ohmic (approximately linear) pathway in the membrane.

It is important to mention that qualitatively similar results were also observed with all other four native oxyopinins 2a-d tested, including the initial drop in membrane resistance followed by the subsequent partial relaxation shown in Fig. 6C. It is also important to mention that appropriate controls were conducted to show that the effects registered were due neither to buffer components nor to mechanical artifacts.

Insecticidal Cooperativity between Oxyopinins and Neurotoxic Peptides-- It is well known that the primary function of spider venom is to paralyze their prey (32). Therefore, a possible toxic effect of oxyopinins in insects was tested. Oxki1, Oxki2, and Pin2 were injected into S. litura larvae. Upon peptide injection the larvae presented necrotic spots after 60-90 min post-injection at the site of needle insertion. Furthermore, depending on the concentration of peptide injected, the size of such a necrotic spot would increase with time. The LD₅₀ values of Oxki1, Oxki2, and Pin2 were 166.3 ± 13.4 (n = 3), 500.4 ± 26.3 (n = 3), and 553.3 ± 90.4 (n = 3) nmol/g of larvae, respectively (Fig. 7). The insecticidal activities of oxyopinins were compared with those of the spider neurotoxins OxyTx1 and -paluIT1 (12). OxyTx1 is insecticidal to the lepidopteran larvae S. litura with an LD₅₀ of 5.1 ± 0.5 (n = 2) nmol/g of larvae (Fig. 7). Also, the neurotoxin -paluIT1 is lethal to the same insect with a LD₅₀ of 1.8 ± 0.2 (n = 2) nmol/g of larvae (Fig. 7). Comparative studies of the insecticidal activity of oxyopinins to those of the spider neurotoxins showed that the latter were from 30 to 90 times more potent than the insecticidal activity of the oxyopinin 1. Because the paralysis inflicted by the venom on insects is considered to be a synergistic effect caused by several factors, such as neurotransmitters, neuropeptides, and enzymes, the insecticidal activity of the neurotoxins OxyTx1 and -paluIT1 were tested in the presence of the individual oxyopinins. The relative concentrations of the pure peptides Oxki1, Oxki2, and OxyTx1 in the soluble venom, as estimated by the chromatogram areas obtained from Fig. 1, were 7.2, 12.0, and 4.1%, respectively. Based on these data, the area ratio of amphipathic peptides to OxyTx1 is roughly 4.6. Therefore, a molar mixture 1:5 (neurotoxin:Oxki1) was prepared for use for injection into S. litura larvae. Although the concentration of Oxki1 was lower than the total of Oxki2a-d peptides in the crude venom, individually, Oxki1 was the most potent among all five amphipathic peptides. The LD₅₀ values of the OxyTx1/Oxki1 and -paluIT1/Oxki1 mixtures containing molar ratios 1:5 were 0.4 ± 0.1 (n = 2) and 0.1 ± 0.01 (n = 2), respectively. They were significant lower (p < 0.05, n = 2) than the LD₅₀ values of the individual neurotoxins, suggesting the existence of a cooperative lethality effect when mixtures of both neurotoxins and Oxki1 were used for bioassay. Additionally, mixtures at molar ratio 1:1 (neurotoxin:Oxki1) were also tested (see the legend of Fig. 7). In these conditions, the LD₅₀ values for the pair OxyTx1/Oxki1 and -paluIT1/Oxki1 were 5.8 ± 1.6 (n = 2) and 1.6 ± 0.1 (n = 2), respectively. They were not significant different (p > 0.05, n = 2) from those of the insecticidal activities of pure neurotoxins OxyTx1 and -paluIT1. Furthermore, the time required for the larvae to become paralyzed or eventually to die, under the effect of mixtures (ratio 1:5) of neurotoxin/amphipathic peptides, were greatly reduced (Fig. 8). After the injection of the mixtures the S. litura larvae showed a faster paralytic effect. Both neurotoxins OxyTx1 and -paluIT1 act gradually over several hours depending on the concentration injected into the insects (Fig. 8, A and B). Therefore, co-injection of neurotoxins and amphipathic peptides enhanced the paralytic and lethal activity of neurotoxins in a time- and dose-dependent manner.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Insecticidal activity of oxyopinins and neurotoxins toward insect paralysis. Dose response of the individual and the mixtures of amphipathic and neurotoxic peptides. The concentration on the abscissa scale corresponds to nanomoles of individual peptide per gram of larvae for the peptides Pin2, Oxki1, Oxki2, -paluIT1, and OxyTx1. In the case of the peptide mixtures OxyTx1/Oxki1 and -paluIT1/Oxki1, the scale corresponds to the values based on the concentration of the neurotoxins. The lethal dose corresponding to 50% of mortality (LD50) was calculated by probit analysis. The LD50 values are Oxki1, 166.3 ± 13.4 (n = 3); Oxki2, 500.4 ± 26.3 (n = 3); Pin2, 553.3 ± 90.4 (n = 3); -PaluIT1, 1.8 ± 0.2 (n = 2); OxyTx1, 5.1 ± 0.5 (n = 2); -PaluIT1/Oxki1(1:5), 0.1 ± 0.01 (n = 2); OxyTx1/Oxki1(1:5), 0.4 ± 0.1 (n = 2); -PaluIT1/Oxki1(1:1), 1.6 ± 0.1 (n = 2); and OxyTx1/Oxki1(1:1), 5.8 ± 1.6 (n = 2). Each point represents the average of two to three independent experiments using at least ten S. litura larvae. Error bars represent the standard deviation.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Insecticidal cooperativity of neurotoxins and oxyopinins. A, insecticidal effect over time of the individual neurotoxin OxyTx1 and the peptide mixtures OxyTx1/Oxki1, OxyTx1/Oxki2, and OxyTx1/Pin2. (Concentration used: 0.5 nmol of OxyTx1 plus 2.5 nmol of amphipathic peptide per gram of larvae.) B, insecticidal effect over time of individual neurotoxin -paluIT1 and the peptide mixtures -paluIT1/Oxki1, -paluIT1/Oxki2, and -paluIT1/Pin2. (Concentration used: 0.5 nmol of -paluIT1 plus 2.5 nmol of amphipathic peptide per gram of larvae.) A control sample of 2.5 nmol of amphipathic peptide/g of larvae did not produce paralysis of insects (data not shown). Each point represents the average of two independent experiments using at least ten S. litura larvae. Error bars represent the standard deviation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Analysis of the primary structure of all five oxyopinins shows the absence of the amino acid residues tryptophan and tyrosine. The only aromatic residue present in these amphipathic peptides was phenylalanine, which has the lowest molar extinction of all aromatic amino acids. Consequently, oxyopinins were detected at low UV wavelengths. Oxyopinin 1 and the analogs of oxyopinin 2 were eluted at a high percentage of acetonitrile from the reverse-phase HPLC column (Fig. 1) indicating their hydrophobic character. Oxyopinin 1 is more hydrophobic than the oxyopinin 2 analogs, and this characteristic could reflect possible differences in their biological activity. The degree of hydrophobicity and the distribution of cationic charges in oxyopinins certainly play a crucial role in determining the cytotoxic and the antimicrobial effects. These characteristics also should define their ability to interact with membrane phospholipids of different compositions and chemical structures. Oxyopinins belong to a large group of -helical antimicrobial peptides that may contain two different amphipathic -helices separated by flexible hinge regions such as Val²²-Leu²³-Pro²⁴ and Gly¹⁹-Val²⁰-Gly²¹, respectively. Separated segments of -helices have been determined in the antimicrobial peptides cecropin A (33) and sarcotoxin A (34). For the first peptide, Pro²³ was described as the kink-forming point of the structure, whereas for the second a segment of four residues in positions Gly²⁴-Ile²⁷ were the ones disrupting the continuity of the of -helices. The length occupied by these 37- to 48-amino acid peptides was estimated to be 55.5-72 Å (1.5 Å per residue), which is therefore enough to span at least once through the 30-Å-thick phospholipid bilayer of the biological membranes, facilitating the formation of a pore-like structure. Although an extended shape of oxyopinins would be large enough to span twice through the lipid bilayer, the apparent absence of a secondary structure, such as disulfide bridges, -turns, or -sheets would preclude such physical occurrence. The possibility that oxyopinins form helicoidal structures in a lipidic environment seems to be plausible, as shown by the results of Fig. 3, obtained in the presence of TFE. Natural (35-37) and artificial linear (38, 39) amphipathic peptides larger than 24 amino acid residues can form well-defined membrane ion channels. Thus, oxyopinins are more likely pore-forming peptides. This view is partially supported by the experiments performed with the Sf9 ovarian cell membranes (Fig. 6), but it was not directly proven here. Moreover, it has been recently observed that the pore-forming peptide magainin2 and PGLa form a heterodimer, and this heterodimer exhibited membrane permeabilization activity an order of magnitude higher compared with the monomeric species (40). Therefore, there is an additional possibility of synergistic interaction between Oxki1 and Oxki2, either for increasing the cytolytic activity or for enhancement of the neurotoxic effects of the venom.

For amphipathic peptides several mechanisms of membrane permeabilization have been proposed. Cecropin and dermaseptin are suggested to disrupt membranes by the so-called "carpet-like" mechanism (41). In the case of alamethicin, the "barrel-stave" model of pore formation was proposed (42), and in the case of magainin, a peptide-lipid toroidal pore (43, 44). The oxyopinins 1 and 2 act on bacterial cells and also lyse red blood cell membranes (Figs. 4 and 5). The results in vivo correspond well with in vitro studies, which demonstrated that peptides bind and permeate both negatively charged and zwitterionic vesicles. However, the mode of action could differ between acidic and zwitterionic phospholipids. The dose dependence curves obtained with these peptides assayed on PC vesicles are sigmoidal, and peptides permeabilize vesicles at a low peptide-to-lipid ratio, which is consistent with the pore-forming model. On the other hand, the curves for PA and PE vesicles required a 5 to 10 times higher peptide-to-lipid ratio for vesicle permeabilization. The latter is consistent with a non-pore-forming process whereby cationic peptides first adsorbed onto the negatively charged membrane surface to form a carpet-like layer and then (when a threshold concentration of peptides bound to membrane has been reached) form transient holes, capable of producing complete lysis. It is thought that the density or size of the lipid headgroup present in the bilayers plays an important function in this behavior. The headgroups of PE or PA are substantially smaller than that of PC (45, 46). Therefore, PC-rich membranes would be less dense or less compact permitting the insertion of oxyopinins into the bilayers. Interestingly, the addition of PE to PC artificial vesicles has resulted in a decrement of the disruptive activity of some natural antimicrobial peptides such as alamethicin (46) and PGLa (47). Moreover, protegrins have a deleterious effect toward human erythrocytes that are rich in PC lipids but non-toxic to those of pigs that are less rich in PC lipids (29, 48). Oxyopinins have a higher propensity to lyse PC vesicles rather than PE and PA vesicles. This concept correlates well with the increasing hemolytic activity observed in red blood cells containing higher amounts of PC (Fig. 5, A-C). The phospholipid composition of cell membranes may influence parameters of the pore structure, for example, the increase of membrane fluidity shortens the pore lifetime (44). Therefore, the different lytic potency of oxyopinins against erythrocytes of different lipid composition also suggest the pore-forming mechanism.

Wolf spiders are vagabonds that lie in ambush or freely hunt their prey (49). Although the antimicrobial activity of oxyopinins from O. kitabensis could be just fortuitous, it is unlikely that bacteria were the exclusive targets of these amphipathic molecules from a venom gland specialized in prey capture. Despite the fact that several antimicrobial peptides are produced by organisms throughout the phylogenetic tree (50), the biological function of antimicrobial -linear peptides makes sense when they are produced and excreted on the skin of amphibians or produced inside cells specialized in host immunity. In the case of oxyopinins, because of their origin, it seems that the main targets are insect cells rather than microorganisms. The insecticidal activity of amphipathic molecules has been previously shown by Orivel et al. (9). They found that some amphipathic molecules from the ant Pachycondyla goeldii are insecticidal to house crickets. Yan and Adams (8) proposed that the biological function of linear amphipathic peptides from spiders might play an important role in capturing insect prey by dissipating ion and voltage gradients across the membranes of excitable cells. The whole-cell patch clamp experiments in Sf9 cells showed that oxyopinins can effectively disturb the membrane potential of insect cells leading to its complete lysis at higher concentrations (Fig. 6). This effect was also observed with the enlargement of necrotic spots in larvae after post-injection of oxyopinins. However, it seems that not all the insect cell lines are susceptible to oxyopinins as demonstrated here with the hemocytes, which were not susceptible to lysis in the presence of high concentrations of these peptides. Therefore, the detrimental effect of oxyopinins could be more pronounced in insect cell lines containing a substantial amount of PC lipids in their membranes. This hypothesis is supported by the enhanced hemolytic activity of red blood cells with a higher content of PC.

The insecticidal cooperativity between neurotoxins and oxyopinins is not a particular characteristic of these amphipathic molecules from spider venoms. Insecticidal cooperativity was observed in the mixtures OxyTx1/Pin2 and -paluIT1/Pin2 (Fig. 8), where Pin2 is an amphipathic molecule from the scorpion P. imperator venom. Additionally, positive cooperativity in venoms of spiders has been observed between polyamines and neuropeptides (51) as well as between adenosine triphosphate and venom toxins (52). Therefore, other arachnid venoms may contain amphipathic molecules used for potentiation of their neurotoxic activities, as demonstrated for the case of scorpions (53). Recently Elazar et al. (54) have shown that lower levels of neurotoxin AaIT (35-fold lower) are needed to kill or paralyze lepidoptera larvae when the neurotoxin AaIT is expressed internally by baculovirus infection than that when AaIT is injected directly to the hemolymph of the larvae. This phenomenon is called toxin potentiation. Usually upon prey injection, the venom components have to overcome accessibility barriers and internal processes of degradation before reaching their target (55). The larger components and the hydrophilic molecules from spider venom would find it more difficult to diffuse toward their receptor sites. Therefore, neurotoxin diffusion toward their target molecules, inside the prey, might be facilitated by the presence of amphipathic peptides acting as co-factors of insect toxicity. Oxyopinins as well as other linear amphipathic peptides could function as spreading factors, by clearing the way and helping the larger neurotoxins to reach their receptor sites. Spreading factors have been often observed in the venom of spiders or poisonous animals such as snakes and lizards. However, the best known spreading factors are enzymes like hyaluronidase and other proteases that degrade the extracellular matrix molecules of tissues and disrupt cell membranes spreading the deleterious effect over the injured tissue (56). The hypothesis of oxyopinins as spreading factors or co-factors could explain the positive insecticidal cooperativity of oxyopinins and the neurotoxic peptides toward lepidoptera larvae. Oxyopinins, therefore, are an additional ingredient in the lethal mixture of gland venom components that are used by the spiders to achieve a quick and effective mode of capturing their prey.

The antimicrobial properties of oxyopinins are certainly a model for the design and development of alternative drugs to be use in the therapeutic treatment of infection by pathogenic bacteria. Unfortunately, their cytolytic effect should still be addressed in this context. In addition, the oxyopinins could be used to enhance the insecticidal activity of genetically modified insect-specific baculoviruses in the field of biopesticides.

	ACKNOWLEDGEMENTS

We are grateful to T. Maeda for insect rearing and to Dr. Pierre Escoubas for helping with mass spectrometry analysis.

	Addendum

During the original submission of this work, Kuhn-Nentwig et al. (57) reported the antimicrobial peptide structures of four cupiennins from the spider Cupiennius salei (Ctenidae). Although these spiders are of a different genus and from different geographic zones, it seems that they are evolutionarily related.

	FOOTNOTES

^* This work was supported by a grant from the Research for the Future Program from the Japanese Society for the Promotion of Science and by Grant z-005 from the National Council of Science and Technology (to F. G. L. and L. D. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The amino acid sequences reported in this paper has been submitted to the Swiss Protein Database under Swiss-Prot accession no. P83247 for Oxyopinin 1 (Oxki1), P83248 for Oxyopinin 2a (Oxki2a), P83249 for Oxyopinin 2b (Oxki2b), P83250 for Oxyopinin 2c (Oxki2c), P83251 for Oxyopinin 2d (Oxki2d), and P83288 for Oxytoxin 1 (OxyTx1).

^§ To whom correspondence should be addressed. Tel.: 81-75-962-8792; Fax: 81-75-962-2115; E-mail: corzo@sunbor.or.jp.

Published, JBC Papers in Press, April 25, 2002, DOI 10.1074/jbc.M200511200

	ABBREVIATIONS

The abbreviations used are: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PA, phosphatidic acid; CD, circular dichroism; CZE, capillary zone electrophoresis; MALDI-TOF MS, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry; OD, optical density; Oxki1, oxyopinin 1; Oxki2a-d, oxyopinins 2a, 2b, 2c, and 2d; Pin1, pandinin 1; Pin 2, pandinin 2; PBS, phosphate-buffered saline; TFE, trifluoroethanol; HFBA, hepta-fluorobutyric acid; MIC, minimum inhibitory concentration; SUV, small unilamellar vesicles; Fmoc, N-(9-fluorenyl)methoxycarbonyl; LD₅₀, lethal dose that kills 50% of the animals; -paluIT1, insecticidal toxin from the spider Paracoelotes luctuosus; OxyTx1, insecticidal toxin from the spider O. kitabensis; HPLC, high performance liquid chromatography; CD, circular dichroism; CE, cation-exchange chromatography; HP, holding potential; AaIT, insect specific neurotoxin from the venom of the scorpion Androctonus australis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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