A prokaryote system optimization for rMEPLox expression: A promising non-toxic antigen for Loxosceles antivenom production.

Loxoscelism is the most dangerous araneism form in Brazil and antivenom therapy is the recommended treatment. Antivenom is produced by horse immunization with Loxosceles spider venom, which is toxic for the producer animal. Moreover, due to the high amount of venom required for horse hyperimmunization, new strategies for antigens obtention have been proposed. In this sense, our research group has previously produced a non-toxic recombinant multiepitopic protein derived from Loxosceles toxins (rMEPLox). rMEPLox was a successful immunogen, being able to induce the production of neutralizing antibodies, which could be used in the Loxoscelism treatment. However, rMEPLox obtention procedure requires optimization, as its production needs to be scaled up to suit antivenom manufacture. Therefore, an effective protocol development for rMEPlox production would be advantageous. To achieve this objective, we evaluated the influence of different cultivation conditions for rMEPLox optimum expression. The optimum conditions to obtain large amounts of rMEPlox were defined as the use of C43(DE3)pLysS as a host strain, 2xTY medium, 0.6 mM IPTG, biomass pre induction of OD600nm = 0.4 and incubation at 30 °C for 16 h. Following the optimized protocol, 39.84 mg/L of soluble rMEPLox was obtained and tested as immunogen. The results show that the obtained rMEPLox preserved the previously described immunogenicity, and it was able to generate antibodies that recognize different epitopes of the main Loxosceles venom toxins, which makes it a promising candidate for the antivenom production for loxoscelism treatment.

GiTx1(ß/κ-theraphotoxin-Gi1a), a novel toxin from the venom of Brazilian tarantula Grammostola iheringi (Mygalomorphae, Theraphosidae): Isolation, structural assessments and activity on voltage-gated ion channels.

Spider venoms, despite their toxicity, represent rich sources of pharmacologically active compounds with biotechnological potential. However, in view of the large diversity of the spider species, the full potential of their venom molecules is still far from being known. In this work, we report the purification and structural and functional characterization of GiTx1 (ß/κ-TRTX-Gi1a), the first toxin purified from the venom of the Brazilian tarantula spider Grammostola iheringi. GiTx1 was purified by chromatography, completely sequenced through automated Edman degradation and tandem mass spectrometry and its structure was predicted by molecular modeling. GiTx1 has a MW of 3.585 Da, with the following amino acid sequence: SCQKWMWTCDQKRPCCEDMVCKLWCKIIK. Pharmacological activity of GiTx1 was characterized by electrophysiology using whole-cell patch clamp on dorsal root ganglia neurons (DRG) and two-electrode voltage-clamp on voltage-gated sodium and potassium channels subtypes expressed in Xenopus laevis oocytes. GiTx1, at 2 µM, caused a partial block of inward (∼40%) and outward (∼20%) currents in DRG cells, blocked rNav1.2, rNav1.4 and mNav1.6 and had a significant effect on VdNav, an arachnid sodium channel isoform. IC50 values of 156.39 ± 14.90 nM for Nav1.6 and 124.05 ± 12.99 nM for VdNav, were obtained. In addition, this toxin was active on rKv4.3 and hERG potassium channels, but not Shaker IR or rKv2.1 potassium channels. In summary, GiTx1 is a promiscuous toxin with multiple effects on different types of ion channels.

Jingzhaotoxin-X, a gating modifier of Kv4.2 and Kv4.3 potassium channels purified from the venom of the Chinese tarantula Chilobrachys jingzhao

Background:The tarantula Chilobrachys jingzhao is one of the largest venomous spiders in China. In previous studies, we purified and characterized at least eight peptides from C. jingzhao venom. In this report, we describe the purification and characterization of Jingzhaotoxin-X (JZTX-X), which selectively blocks Kv4.2 and Kv4.3 potassium channels.Methods:JZTX-X was purified using a combination of cation-exchange HPLC and reverse-phase HPLC. The amino-acid sequence was determined by automated Edman degradation and confirmed by mass spectrometry (MS). Voltage-gated ion channel currents were recorded in HEK293t cells transiently transfected with a variety of ion channel constructs. In addition, the hyperalgesic activity of JZTX-X and the toxin´s effect on motor function were assessed in mice.Results:JZTX-X contained 31 amino acids, with six cysteine residues that formed three disulfide bonds within an inhibitory cysteine knot (ICK) topology. In whole-cell voltage-clamp experiments, JZTX-X inhibited Kv4.2 and Kv4.3 potassium channels in a concentration- and voltage-dependent manner, without affecting other ion channels (Kv1.1, 1.2, 1.3, 2.1, delayed rectifier potassium channels, high- and low-voltage-activated Ca2+ channels, and voltage-gated sodium channels Nav1.5 and 1.7). JZTX-X also shifted the voltage-dependent channel activation to more depolarized potentials, whereas extreme depolarization caused reversible toxin binding to Kv4.2 channels. JZTX-X shifted the Kv4.2 and Kv4.3 activities towards a resting state, since at the resting potential the toxin...(AU)

Gene sequence analysis of toxins from the spider Phoneutria nigriventer revealed an intronless feature

Background: Phoneutria nigriventer spider venom contains several cysteine-rich peptide toxins that act on different ion channels. Despite extensive studies on its venom and description of cDNA sequences of several of its toxin precursors, the gene structure of these toxins remains unknown. Methods: Genomic regions encoding the precursors of three previously characterized P. nigriventer toxins - PnTx1, PnTx2-5 and PnTx4(5-5) - were amplified by PCR using specific primers. PCR fragments were cloned and sequenced. Obtained sequences were compared with their corresponding cDNA sequences. Results: The size of PCR fragments obtained and sequences corresponding to genomic regions encoding for the toxin precursors matched their cDNA sequences. Conclusions: Despite a few nucleotide substitutions in the genomic regions encoding for the toxin precursors when compared with cDNA sequences, the results of the present work indicate that P. nigriventer toxins do not contain introns in their genes sequences.(AU)

Drosophila bioassays are very sensitive methods to assess tarantula species venoms.

INTRODUCTION: Assaying venom toxicity in a suitable model system is often tricky, since normally the amount of venom is in short supply, and the assay subjects, i.e., typically mice, require large amounts. There is also no guarantee that the effects observed in the bioassay reflect the true nature of the venom's intended effects, as the animals used for assessment might not be the prey items to which the venom has evolved. METHODS: We harvested tarantula venoms from the Indian Poecilotheria regalis and the Mexican Bonnetina papalutlensis using light anesthesia and electrical stimulation. We follow the definition of venom as stated in (Nelsen et al., 2014). The recovered venom was lyophilized and reconstituted in sterile saline solution for injections. Drosophila melanogaster third instar larvae and adult flies were injected with 4.6 nanoliters of different concentrations of the venoms into the sixth abdominal segment, and scored for survival and development to adulthood. RESULTS: The injected venoms were very effective in provoking lethality of injected larvae and adults, with an LD50 of 1-5 nanomoles protein /gram wet weight. Comparison with other toxicity bioassays, i.e., mice and crickets -using the same P. regalis venom- renders the Drosophila bioassays three orders of magnitude more sensitive. The P. regalis and B. papalutlensis venoms have similar LD50. DISCUSSION: These bioassays use a small amount of venom compared to other bioassays, allowing characterization with far fewer starting material. As it uses insects, phylogenetically close to the intended prey victims, it also points to the efficiency of the tarantula venom for its preferred prey items, and thus, links as well to the tarantulas' ecology.

Age-Related Modulations of AQP4 and Caveolin-1 in the Hippocampus Predispose the Toxic Effect of Phoneutria nigriventer Spider Venom.

We have previously demonstrated that Phoneutria nigriventer venom (PNV) causes blood-brain barrier (BBB) breakdown, swelling of astrocytes end-feet and fluid permeation into brain interstitium in rats. Caveolae and water channels respond to BBB alterations by co-participation in shear stress response and edema formation/resolution. Herein, we showed post-natal developmental-related changes of two BBB-associated transporter proteins: the endothelial caveolin-1 (Cav-1), the major scaffolding protein from caveolae frame, and the astroglial aquaporin-4 (AQP4), the main water channel protein expressed in astrocytic peri-vascular end-feet processes, in the hippocampus of rats intraperitoneally-administered PNV. Western blotting protein levels; immunohistochemistry (IHC) protein distribution in CA1, CA2, and CA3 subfields; and gene expression by Real Time-Polymerase Chain Reaction (qPCR) were assessed in post-natal Day 14 (P14) and 8-10-week-old rats over critical periods of envenomation. The intensity and duration of the toxic manifestations indicate P14 neonate rats more vulnerable to PNV than adults. Histologically, the capillaries of P14 and 8-10-week-old rats treated with PNV showed perivascular edema, while controls did not. The intensity of the toxic manifestations in P14 decreases temporally (2 > 5 > 24 h), while inversely the expression of AQP4 and Cav-1 peaked at 24 h when clinically PNV-treated animals do not differ from saline controls. IHC of AQP4 revealed that hippocampal CA1 showed the least expression at 2 h when toxic manifestation was maximal. Subfield IHC quantification revealed that in P14 rats Cav-1 peaked at 24 h when toxic manifestations were absent, whereas in 8-10-week-old rats Cav-1 peaked at 2 h when toxic signs were highest, and progressively attenuated such increases until 24 h, remaining though significantly above baseline. Considering astrocyte-endothelial physical and functional interactions, we hypothesize that age-related modulations of AQP4 and Cav-1 might be linked both to changes in functional properties of astrocytes during post-natal development and in the BBB breakdown induced by the venom of P. nigriventer.

Spinal blockage of P/Q- or N-type voltage-gated calcium channels modulates functional and symptomatic changes related to haemorrhagic cystitis in mice.

BACKGROUND AND PURPOSE: Spinal voltage-gated calcium channels (VGCCs) are pivotal regulators of painful and inflammatory alterations, representing attractive therapeutic targets. We examined the effects of epidural administration of the P/Q- and N-type VGCC blockers Tx3-3 and Phα1ß, respectively, isolated from the spider Phoneutria nigriventer, on symptomatic, inflammatory and functional changes allied to mouse cyclophosphamide (CPA)-induced haemorrhagic cystitis (HC). The effects of P. nigriventer-derived toxins were compared with those displayed by MVIIC and MVIIA, extracted from the cone snail Conus magus. EXPERIMENTAL APPROACH: HC was induced by a single i.p. injection of CPA (300 mg·kg(-1) ). Dose- and time-related effects of spinally administered P/Q and N-type VGCC blockers were assessed on nociceptive behaviour and macroscopic inflammation elicited by CPA. The effects of toxins were also evaluated on cell migration, cytokine production, oxidative stress, functional cystometry alterations and TRPV1, TRPA1 and NK1 receptor mRNA expression. KEY RESULTS: The spinal blockage of P/Q-type VGCC by Tx3-3 and MVIIC or N-type VGCC by Phα1ß attenuated nociceptive and inflammatory events associated with HC, including bladder oxidative stress and cytokine production. CPA produced a slight increase in bladder TRPV1 and TRPA1 mRNA expression, which was reversed by all the toxins tested. Noteworthy, Phα1ß strongly prevented bladder neutrophil migration, besides HC-related functional alterations, and its effects were potentiated by co-injecting the selective NK1 receptor antagonist CP-96345. CONCLUSIONS AND IMPLICATIONS: Our results shed new light on the role of spinal P/Q and N-type VGCC in bladder dysfunctions, pointing out Phα1ß as a promising alternative for treating complications associated with CPA-induced HC.

Extraction and partial characterization of venom from the Colombian spider Pamphobeteus aff. nigricolor (Aranae:Theraphosidae).

We report the first studies of characterization and extraction of the Pamphobeteus aff. nigricolor (Pocock, 1901) (Aranae:Theraphosidae) venom done in Colombia using the electro-stimulation technique previous anesthesia with isofluorane. After each extraction process, a low viscosity, colorless venom was obtained. This venom showed a 1.01 mg/µl density and a pH of 5. The humidity percentage did not show a significance difference between males and females (P > 0.05) with a general media of 77.49 ± 1.74%. In all cases the venom yielded was variable between males and females, with a media of 22.45 ± 5.17 mg (wet weight) and 4.58 ± 0.94 mg (dry weigh), obtaining larger amounts in females, 28.34 ± 7.49 mg and 5.69 ± 1.36 (wet and dry weight respectively). Venom showed a hemolytic activity dependent of enzymatic active phospholipase and neither coagulant nor proteolytic activities were observed. Electrophoretic profile showed a main protein content with a molecular mass below 14 kDa. RP-HPLC venom profile revealed a difference among male and female venom's content where 17 and 21 main fractions were obtained respectively. Three peptides, Theraphotoxin-Pn1a, Theraphotoxin-Pn1b and Theraphotoxin-Pn2a, were identified using HPLC-nESI-MS/MS. These peptides showed a high identity with other peptides found on Theraphosides which are proved to affect voltage-gated calcium channels.

Characterization of a novel peptide toxin from Acanthoscurria paulensis spider venom: a distinct cysteine assignment to the HWTX-II family.

Spider venom toxins have raised interest in prospecting new drugs and pesticides. Nevertheless, few studies are conducted with tarantula toxins, especially with species found in Brazil. This study aims to characterize chemically and biologically the first toxin isolated from Acanthoscurria paulensis venom. Ap1a consists of 48 amino acid residues and has a molecular mass of 5457.79 Da. The cloned gene encodes a putative sequence of 23 amino acid residues for the signal peptide and 27 for the pro-peptide. The sequence of the mature peptide is 60-84% identical with those of toxins of the HWTX-II family. Different from the structural pattern proposed for these toxins, the disulfide pairing of Ap1a is of the ICK type motif, which is also shared by the U1-TRTX-Bs1a toxin. Ap1a induced a dose-dependent and reversible paralytic effect in Spodoptera frugiperda caterpillars, with an ED50 of 13.0 ± 4.2 µg/g 8 h after injections. In the Drosophila melanogaster Giant Fiber circuit, Ap1a (1.14-22.82 µg/g) reduces both the amplitude and frequency of responses from GF-TTM and GF-DLM pathways, suggesting an action at the neuromuscular junction, which is mediated by glutamatergic receptors. It is also lethal to mice (1.67 µg/g, intracranial route), inducing effects similar to those reported with intracerebroventricular administration of NMDA. Ap1a (1 µM) does not alter the response induced by acetylcholine on the rhabdomyosarcoma cell preparation and shows no significant effects on hNav1.2, hNav1.4, hNav1.5, and hNav1.6 channels. Because of its unique sequence and cysteine assignment to the HWTX-II family, Ap1a is a significant contribution to the structure-function study of this family of toxins.

An evaluation of the antinociceptive effects of Phα1ß, a neurotoxin from the spider Phoneutria nigriventer, and ω-conotoxin MVIIA, a cone snail Conus magus toxin, in rat model of inflammatory and neuropathic pain.

Voltage-sensitive calcium channels (VSCCs) underlie cell excitability and are involved in the mechanisms that generate and maintain neuropathic and inflammatory pain. We evaluated in rats the effects of two VSCC blockers, ω-conotoxin MVIIA and Phα1ß, in models of inflammatory and neuropathic pain induced with complete Freund's adjuvant (CFA) and chronic constrictive injury (CCI), respectively. We also evaluated the effects of the toxins on capsaicin-induced Ca(2+) influx in dorsal root ganglion (DRG) neurons obtained from rats exposed to both models of pain. A single intrathecal injection of Phα1ß reversibly inhibits CFA and CCI-induced mechanical hyperalgesia longer than a single injection of ω-conotoxin MVIIA. Phα1ß and MVIIA also inhibited capsaicin-induced Ca(2+) influx in DRG neurons. The inhibitory effect of Phα1ß on capsaicin-induced calcium transients in DRG neurons was greater in the CFA model of pain, while the inhibitory effect of ω-conotoxin MVIIA was greater in the CCI model. The management of chronic inflammatory and neuropathic pain is still a major challenge for clinicians. Phα1ß, a reversible inhibitor of VSCCs with a preference for N-type Ca(2+) channels, has potential as a novel therapeutic agent for inflammatory and neuropathic pain. Clinical studies are necessary to establish the role of Phα1ß in the treatment of chronic pain.

Peptídeos antimicrobianos do veneno de Avicularia juruensis (Mygalomorphae, Theraphosidae) / Antimicrobial peptides venom Avicularia juruensis (Mygalomorphae, Theraphosidae)

Peptídeos Antimicrobianos (PAMs) são os principais elementos da imunidade inata contra bactérias e fungos em ambos os reinos: animal e vegetal. PAMs são um grupo extremamente diversificado de pequenas proteínas e sua função é essencial para a resposta imunológica dos organismos. Venenos animais são boas fontes de substâncias antimicrobianas, pois contêm um grande número de diferentes componentes biologicamente ativos de diferentes estruturas químicas, tais como proteínas, polipeptídeos e aminas. Toxinas que apresentam o nó cistina (CKTs) em venenos de aranhas representam uma rica fonte de novos ligantes para canais iônicos, e estão entre os constituintes mais estudados de venenos de aranha. Eles são moléculas pequenas, compactas ligadas por três a cinco pontes de dissulfeto, assegurando uma maior estabilidade na conformação da molécula que a contém, e, portanto, oferece um elevado potencial para aplicações na engenharia de proteínas. O nó d cistina ocorre numa grande variedade de peptídeos e proteínas e é relativamente comum em pequenas toxinas ricas em cisteína e pequenos peptídeos, com massas moleculares variando de 3,5 a 7 kDa. As toxinas que contêm o nó de cistina possuem uma alta gama de atividades biológicas, como atividade antimicrobiana, anti-HIV e bloqueio de canais iônicos. Este estudo teve como principal objetivo a caracterização do veneno e a identificação e caracterização de moléculas bioativas presentes no veneno da aranha caranguejeira Avicularia juruensis (Mygalomorphae, Theraphosidae), em especial peptídeos antimicrobianos. O veneno foi obtido por estimulação elétrica, diluído em água acidificada (TFA – ácido trifluoracético 0,05%) e centrifugado. A parte solúvel foi submetida à cromatografia líquida de alta eficiência (CLAE) em uma coluna semi preparativa Júpiter C18 de fase reversa. A presença de atividade antimicrobiana foi determinada por ensaio líquido de inibição de crescimento...

Nigriventrine: a low molecular mass neuroactive compound from the venom of the spider Phoneutria nigriventer.

Nigriventrine was isolated from the "armed" spider Phoneutria nigriventer, in which it constitutes about 0.4% of the total venom content. Its structure was determined to be [1,1'-(1-hydroxyhydrazine-1,2-diyl)bis(oxy)bis(4-hydroxy-2,6-dioxopiperidine-4 carboxylic acid)] by NMR, HR-ES/IMS and MS/MS methods. The intracerebroventricular application of nigriventrine in rat brain, followed by the detection of c-Fos protein expression, indicated that the compound was neuroactive in the motor cortex, sensory cortex, piriform cortex, median preoptic nucleus, dorsal endopiriform nucleus, lateral septal nucleus and hippocampus of rat brain. Nigriventrine causes convulsions in rats, even when peripherally applied.

Cardiotoxic effects of Loxosceles intermedia spider venom and the recombinant venom toxin rLiD1.

Loxosceles spider bites cause many human injuries worldwide. Injections in mice of whole Loxosceles (L.) intermedia venom or a recombinant toxin (rLiD1) produce systemic symptoms similar to those detected in envenomed humans. This animal model was used to characterize the effects of Loxosceles intermedia venom in cardiac tissues. L. intermedia antigens were detected by ELISA in kidney, heart, lung and liver of experimentally envenomed mice. In addition, rLiD1 binding to cardiomyocytes was demonstrated by immunofluorescence and confocal microscopy. Furthermore, isolated perfused heart preparations and ventricular cardiomyocytes from envenomed mice showed heart function impairment, and a significant increase of I(Ca,L) density and intracellular Ca(2+) transients, respectively. Thus, L. intermedia spider venom, as shown through the use of the recombinant toxin rLiD1, causes cardiotoxic effects and a protein from the sphingomyelinase D family plays a key role in heart dysfunction. Thus, L. intermedia spider venom and the Loxtox rLiD1 play a key role in heart dysfunction.

A novel expression profile of the Loxosceles intermedia spider venomous gland revealed by transcriptome analysis.

Spiders of the Loxosceles genus are cosmopolitan, and their venom components possess remarkable biological properties associated with their ability to act upon different molecules and receptors. Accidents with Loxosceles intermedia specimens are recognized as a public health problem in the south of Brazil. To describe the transcriptional profile of the L. intermedia venom gland, we generated a wide cDNA library, and its transcripts were functionally and structurally analyzed. After initial analyses, 1843 expressed sequence tags (ESTs) produced readable sequences that were grouped into 538 clusters, 281 of which were singletons. 985 reads (53% of total ESTs) matched to known proteins. Similarity searches showed that toxin-encoding transcripts account for 43% of the total library and comprise a great number of ESTs. The most frequent toxins were from the LiTx family, which are known for their insecticidal activity. Both phospholipase D and astacin-like metalloproteases toxins account for approximately 9% of total transcripts. Toxins components such as serine proteases, hyaluronidases and venom allergens were also found but with minor representation. Almost 10% of the ESTs encode for proteins involved in cellular processes. These data provide an important overview of the L. intermedia venom gland expression scenario and revealed significant differences from profiles of other spiders from the Loxosceles genus. Furthermore, our results also confirm that this venom constitutes an amazing source of novel compounds with potential agrochemical, industrial and pharmacological applications.

Functional expression of a recombinant toxin - rPnTx2-6 - active in erectile function in rat.

In the current study, the putative cDNA for PnTx2-6 toxin of the Phoneutria nigriventer spider venom was cloned and expressed as tioredoxin fusion protein in the cytoplasm of Escherichia coli. The fusion protein was purified from the bacterial extracts by combination of immobilized Ni-ion affinity and gel filtration chromatographies. Then, it was cleaved by enterokinase and the generated recombinant PnTx2-6 (rPnTx2-6) was further purified by reverse-phase HPLC. Likewise the native toxin purified from the spider venom, rPnTx2-6 potentiates the erectile function when injected in rats. This result indicates that the production of functional recombinant PnTx2-6 might be an alternative to provide this basic and valuable tool for study, as well as for further understanding such complex physiological system, including its correlation with the central nervous system and local tissue factors.

Inflammatory events induced by brown spider venom and its recombinant dermonecrotic toxin: a pharmacological investigation.

Accidents involving Brown spider (Loxosceles sp.) venom produce a massive inflammatory response in injured region. This venom has a complex mixture of different toxins, and the dermonecrotic toxin is the major contributor to toxic effects. The ability of Loxosceles intermedia venom and a recombinant isoform of dermonecrotic toxin to induce edema and increase in vascular permeability was investigated. These toxins were injected into hind paws and caused a marked dose and time-dependent edema and increase in vascular permeability in mice. Furthermore, the enzymatic activity of venom toxins may be primal for these effects. A mutated recombinant isoform of dermonecrotic toxin, that has only residual enzymatic activity, was not able to induce these inflammatory events. Besides the previous heating of toxins markedly reduced the paw edema and vascular permeability showing that thermolabile constituents can trigger these effects. In addition, the ability of these venom toxins to evoke inflammatory events was partially reduced in compound 48/80-pretreated animals, suggesting that mast cells may be involved in these responses. Pretreating mice with histamine (prometazine and cetirizine) and serotonin (methysergide) receptor antagonists significantly attenuated toxins induced edema and vascular permeability. Moreover, HPLC analysis of whole venom showed the presence of histamine sufficient to induce inflammatory responses. In conclusion, these inflammatory events may result from the activation of mast cells, which in turn release bioamines and may be related to intrinsic histamine content of venom.

A new family of small (4kDa) neurotoxins from the venoms of spiders of the genus Phoneutria.

A family of 4kDa neurotoxic peptides was purified from venoms of Phoneutria spiders. All have six cysteine residues, and low similarity with other neurotoxins. Three toxins caused moderate inhibition of L-type Ca(2+) channels. The structure of toxin PRTx27C3 was modeled and compared with toxin ADO1. The importance of four residues is suggested.

Toxicity of two North American Loxosceles (brown recluse spiders) venoms and their neutralization by antivenoms.

The toxic, biochemical, and immunological characteristics of L. boneti and L. reclusa venoms and its neutralization by anti-L. boneti and anti-L. reclusa antivenoms were studied. The electrophoretic profile showed very similar patterns and the toxic activities were very close. Immunological studies showed cross-reactivity among L. boneti and L. reclusa venoms, with L. boneti and L. reclusa experimental antivenoms, and anti-L. gaucho and anti-L. laeta antivenoms. The venom of L. laeta showed low immunological reactivity with the North American Loxosceles antivenoms. Experimental anti-North American Loxosceles antivenoms protected mice of the systemic toxicity and were able to prevent necrosis in rabbit skin after the injection of the venom. Both antivenoms displayed cross neutralization. The results showed that both Loxosceles venoms have very close toxic, biochemical, and immunological characteristics, and that either monospecific antivenoms or an antivenom raised with L. boneti and L. reclusa venoms as immunogens could be useful for treating bites by North American Loxosceles spiders.

Leftward shift in the voltage-dependence for Ca2+ currents activation induced by a new toxin from Phoneutria reidyi (Aranae, Ctenidae) venom.

Various neurotoxins have been described from the venom of the Brazilian spider Phoneutria nigriventer, but little is known about the venoms of the other species of this genus. In the present work, we describe the purification and some structural and pharmacological features of a new toxin (PRTx3-7) from Phoneutria reidyi that causes flaccid paralysis in mice. The observed molecular mass (4627.26 Da) was in accordance with the calculated mass for the amidated form of the amino acid sequence (4627.08 Da). The presence of an alpha-amidated C-terminus was confirmed by MS/MS analysis of the C-terminal peptide, isolated after enzymatic digestion of the native protein with Glu-C endoproteinase. The purified protein was injected (intracerebro-ventricular) into mice at dose levels of 5 microg/mouse causing immediate agitation and clockwise gyration, followed by the gradual development of general flaccid paralysis. PRTx3-7 at 1 microM inhibited by 20% the KCl-induced increase on [Ca2+]i in rat brain synaptosomes. The HEK cells permanently expressing L, N, P/Q and R HVA Ca2+ channels were also used to better characterize the pharmacological features of PRTx3-7. To our surprise, PRTx3-7 shifted the voltage-dependence for activation towards hyperpolarized membrane potentials for L (-4 mV), P/Q (-8 mV) and R (-5 mV) type Ca2+ currents. In addition, the new toxin also affected the steady state of inactivation of L-, N- and P/Q-type Ca2+ currents.

Comparison of the partial proteomes of the venoms of Brazilian spiders of the genus Phoneutria.

The proteomes of the venoms of the Brazilian wandering "armed" spiders Phoneutria nigriventer, Phoneutria reidyi, and Phoneutria keyserlingi, were compared using two-dimensional gel electrophoresis. The venom components were also fractionated using a combination of preparative reverse phase HPLC on Vydac C4, analytical RP-HPLC on Vydac C8 and C18 and cation exchange FPLC on Resource S at pH 6.1 and 4.7, or anion exchange HPLC on Synchropak AX-300 at pH 8.6. The amino acid sequences of the native and S-pyridyl-ethylated proteins and peptides derived from them by enzymatic digestion and chemical cleavages were determined using a Shimadzu PPSQ-21(A) automated protein sequencer, and by MS/MS collision induced dissociations. To date nearly 400 peptides and proteins (1.2-27 kDa) have been isolated in a pure state and, of these, more than 100 have had their complete or partial amino acid sequences determined. These sequences demonstrate, as might be expected, that the venoms of P. reidyi and P. keyserlingi (Family: Ctenidae) both contain a similar range of isoforms of the neurotoxins as those previously isolated from P. nigriventer which are active on neuronal ion (Ca(2+), Na(+) and K(+)) channels and NMDA-type glutamate receptors. In addition two new families of small (3-4 kDa) toxins, some larger protein (>10 kDa) components, and two serine proteinases of the venom of P. nigriventer are described. These enzymes may be responsible for some of the post-translational modification observed in some of the venom components.