V.R.D. Eickstedt

Purification and amino acid sequences of six Tx3 type neurotoxins from the venom of the Brazilian ‘armed’ spider Phoneutria Nigriventer (keys.)

Six neurotoxic peptides (Tx3-1 to Tx3-6) were purified from the venom of the spider Phoneutria nigriventer by a combination of gel filtration, reverse phase FPLC on PEP-RPC and PRO-RPC columns, reverse phase HPLC on Vydac C18, and ion exchange HPLC on cationic and anionic columns. These toxins caused different neurological symptoms in mice after intracerebroventricular injection. At dose levels of 5 micrograms/mouse, Tx3-3 and Tx3-4 caused rapid general flaccid paralysis followed by death in 10-30 min; Tx3-2 induced immediate clockwise gyration and flaccid paralysis after 6 hr; Tx3-1, Tx3-5 and Tx3-6 produced paralysis only in the posterior limbs and gradual decreases in movement and aggression during 24 hr. The mol. wt of these cystine-rich peptides were found to be in the range of 3500-8500 by mass spectroscopy and SDS-PAGE. The complete amino acid sequences of the neurotoxins Tx3-1 (40 residues), Tx3-2 (34 residues) and Tx3-6 (55 residues), and the N-terminal sequences of Tx3-3 (34 res.), Tx3-4 (40 res.) and Tx3-5 (36 res.) were established by direct automated Edman degradation, and manual DABITC/PITC microsequence analyses of peptides obtained from digests with various proteases. The structures of these Tx3 neurotoxins from Phoneutria nigriventer exhibited sequence similarities to one another and to the neurotoxins from the venoms of the spiders Hololena curta and Agelenopsis aperta, which were most evident in the location of the Cys residues.

The purification and amino acid sequences of four Tx2 neurotoxins from the venom of the Brazilian ‘armed’ spider Phoneutria nigriventer (Keys)

Four neurotoxic polypeptides (Tx2.1, Txt2‐5, Tx2‐6 and Tx2‐9) were purified from the venom of the South American ‘armed’ spider Phoneutria nigriventer (Keys) by gel filtration and reverse phase FPLC and HPLC. These cysteine‐rich polypeptides exhibited different levels of neurotoxicity in mice after intracerebroventricular injection. Tx2‐1, Tx2‐5 and Tx2‐6 caused spastic paralysis and death, but the less toxic Tx2‐9 produced only tail erection and scratching. The molecular weights of the polypeptides as determined by desorption mass spectroscoopy were 5838.8 for Tx2‐1, 5116.6 (Tx2‐5), 5291.3 (Tx2‐6) and 3742.1 (Tx2‐9). The complete amino acid sequences of the neurotoxins were determined by automated Edman degradation and by manual DABITC‐PITC microseqeunce analysis of peptides obtained after digestions with various proteases. The amino acid sequences of Tx2‐1 (53 residues), Tx2‐5 (48 residues) and Tx2‐6 (48 residues) were homologous, but had only limited similarities to the less toxic Tx2‐9 (32 residues). All four polypeptides had varying sequence identities with other neurotoxins from different spider species and biologically active peptides from scorpions, a sea snail and seeds of Mirabilis jalapa.

Dermonecrotic and lethal components of Loxosceles gaucho spider venom

Loxosceles gaucho spider venom causes a typical dermonecrotic lesion in bitten patients and rarely causes lethal systemic effects. Gel filtration on Sephadex G 100 of L. gaucho spider venom resulted in three fractions: fraction A, containing the higher mol. wt components (approximately 35,000); fraction B, containing lower mol. wt components (approximately 15,000); and fraction C, containing very low mol. wt components (probably small peptides). The dermonecrotic and lethal activities were detected exclusively in fraction A. The venom and fraction A produced large dermonecrotic lesions in rabbits with necrosis spreading by gravity to the skin of the lateral body wall. Analysis by SDS-PAGE showed that the proteins contained in fraction A are approximately 35,000 and 33,000 mol. wt. Immunoblotting analysis showed that the proteins responsible for the dermonecrotic and lethal activity are very immunogenic and the first to be detected by antibodies during the course of immunization.

Antigenic cross-reactivity among the venoms from several species of Brazilian scorpions

The venoms of seven species of scorpions living in different regions of Brazil were analysed with regard to their lethality, antigenic cross-reactivity and ability to induce antibody production. In mice, the tested scorpion venoms can be grouped as: (a) highly toxic: Tityus stigmurus Thorell (LD50 = 0.773 mg/kg), Tityus bahiensis (Perty) (LD50 = 1.062 mg/kg), Tityus serrulatus Lutz and Mello (LD50 = 1.160 mg/kg), and Tityus costatus (Karsch) (LD50 = 1.590 mg/kg); (b) moderately toxic: Tityus cambridgei Pocock (LD50 = 12.136 mg/kg); and (c) practically nontoxic: Rhopalurus agamemnon (Koch) (LD50 = 36.363 mg/kg), and Brotheas amazonicus Lourenço (LD50 = 90.909 mg/kg). On electrophoresis the venoms showed many protein bands displayed along the chromatogram, most of them cross-reacting in immunoelectrophoresis and immunoblotting using horse anti-T. serrulatus, anti-T. bahiensis or anti-T. serrulatus+T. bahiensis sera as probes. The antibodies present in these antivenoms combine with venom components as measured in vitro by the ELISA assay, and neutralize their lethal effects in vivo. These results indicate that horse anti-venoms against a mixture of T. serrulatus and T. bahiensis venoms or only against T. serrulatus venom yield an antibody population able to neutralize the toxic effects found in all venoms studied.

Compared chemical properties of dermonecrotic and lethal toxins from spiders of the genus Loxosceles (Araneae)

Loxosceles spider venom usually causes a typical dermonecrotic lesion in bitten patients, but it may also cause systemic effects that may be lethal. Gel filtration on Sephadex G-100 ofLoxosceles gaucho, L. laeta, orL. intermedia spider venoms resulted in three fractions (A, containing higher molecular mass components, B containing intermediate molecular mass components, and C with lower molecular mass components). The dermonecrotic and lethal activities were detected exclusively in fraction A of all three species. Analysis by SDS-PAGE showed that the major protein contained in fraction A has molecular weight approximately 35 kDa inL. gaucho andL. intermedia, but 32 kDa inL. laeta venom. These toxins were isolated from venoms ofL. gaucho, L. laeta, andL. intermedia by SDS-PAGE followed by blotting to PVDF membrane and sequencing. A database search showed a high level of identity between each toxin and a fragment of theL. reclusa (North American spider) toxin. A multiple sequence alignment of theLoxosceles toxins showed many common identical residues in their N-terminal sequences. Identities ranged from 50.0% (L. gaucho andL. reclusa) to 61.1% (L. intermedia andL. reclusa). The purified toxins were also submitted to capillary electrophoresis peptide mapping afterin situ partial hydrolysis of the blotted samples. The results obtained suggest thatL. intermedia protein is more similar toL. laeta toxin thanL. gaucho toxin and revealed a smaller homology betweenL. intermedia andL gaucho. Altogether these findings suggest that the toxins responsible for most important activities of venoms ofLoxosceles species have a molecular mass of 32–35 kDa and are probably homologous proteins.

Antigenic cross-reactivity of venoms from medically important Loxosceles (Araneae) species in Brazil

Antigenic cross-reactivity between the components of venoms from three spiders of the genus Loxosceles, L. gaucho, L. laeta and L. intermedia, was studied. Species-specific antisera were prepared by immunization of rabbits with each venom. Anti-L. gaucho horse hyperimmune serum provided by the Butantan Institute for treatment of accidents with these spiders was also used. Separation by SDS-PAGE showed the existence of many common components in the three antigens. No individual antigen was observed. Analysis of the antisera by ELISA and Western blotting showed cross-reactivity as well as several common bands between the three venoms. The horse anti-L. gaucho venom serum recognized many common proteins when antigens of the other two species were used. Antigens in the range of 33,000-35,000 mol. wt showed most cross-reactivity. Both horse and rabbit anti-venom sera contained antibodies able to neutralize the lethal and dermonecrotic activities of the venom of the three species studied.

IgG antibodies to Loxosceles sp. spider venom in human envenoming

The presence and specificity of IgG antibodies produced by patients with loxoscelism were studied. The loxoscelism diagnosis was supported mainly by clinical parameters. A search for IgG antibodies anti-Loxosceles gaucho venom in patients with loxoscelism submitted to serumtherapy showed antibodies in four out of 20 patients. The IgG antibodies were detected as early as 9 days and as late as 120 days after bite. The highest IgG antibody titer was 1:640 and the lowest was 1:80. Immunoblotting tests showed that human anti-L. gaucho IgG antibodies recognize preferentially the components responsible for the dermonecrotic and lethal activities of the venom. A comparison of the clinical picture, the level of serum IgG antibodies and the dose of antivenom administered suggest that there is no relationship between these parameters.

Adjuvant effect of Loxosceles gaucho (South American brown spider) venom

Injection of L. gaucho venom and antigens (ovalbumin, ovomucoid and bovine gamma globulin) into rabbit skin induced an intense local inflammatory lesion and resulted in a significant increase in the level of IgG antibodies to the antigen in both the primary and secondary humoral immune response. The adjuvant activity of the venom was associated with its high mol. wt components, which are responsible for the inflammatory lesion. Rabbits rendered unresponsive to the venom and injected with venom plus antigen presented a very mild local inflammatory reaction and no increase in antibody formation. When venom and antigen were injected simultaneously but at different skin sites no adjuvant effect was induced. However, when antigen was injected 4 hr after venom injection but at the same skin site a significant adjuvant effect was produced. Furthermore, when venom plus antigen was injected intradermally into mice, a species in which the venom does not cause an inflammatory skin lesion, no adjuvant effect was detected. It is suggested that the adjuvant effect of L. gaucho venom in rabbits is probably due to its ability to cause a local severe inflammatory reaction.

Ocorrência de Cupiennius Simon na América do Sul e redescricão de Cupiennius celerrimus Simon (Araneae, Ctenidae)

The ocurrence of Cupiennius Simon. 1891 restricted to Central America, Colombia, Jamaica, Haiti and Cuba, is now confirmed to South America and the geographical distribution of C. celerrimus is extended to Venezuela and north and northeaster regions of Brazil. A redescription of C. celerrimus is given based on specimens from the type locality and adjacent localities.

Sinopse das espécies de Tityus do Nordeste do Brasil, com a redescrição da T. neglectus Mello-Leitão (Scorpiones, Burthidae)

Tityus neglectus Mello-Leitao, 1932, originally described from a female specimen collected in the state of Rio Grande do Norte (Brazil) is redescribed. The type-specimen, deposited in the Instituto Oswaldo Cruz (Rio de Janeiro) is lost; a male of the Instituto Butantan (Sao Paulo) scorpion collection is proposed as the neotype. A synopsis of the known species of Tityus from the northeastern part of Brazil is added, as well as a key for the identification of those species.