Inhibition of crotoxin binding to synaptosomes by a receptor-like protein from Crotalus durissus terrificus (the South American rattlesnake).

Crotoxin (Ctx) is a potent neurotoxin of the venom of Crotalus durissus terrificus (the South American rattlesnake). Ctx is a heterodimer composed of CB, a toxic PLA(2) subunit, and CA, a non-toxic and non-enzymatic subunit, that potentiates the neurotoxicity of CB in vivo. The deleterious action of Ctx upon C. d. terrificus snakes themselves is known to be prevented by a PLA(2) inhibitor (CNF) present in their blood serum. CNF acts by replacing CA in Ctx, thus forming a new stable complex CNF-CB. This complex no longer interacts with the target receptor (TR) to deliver CB to cause its lethal effect. Furthermore, CNF-CB seems to be reminiscent of the interaction Ctx-TR at the pre-synaptic site. In the present work, the binding competition between rat brain synaptosomes (TR) and CNF for Ctx was investigated. Radiolabeled Ctx, made of CA and one isoform of CB (CA-(125)ICB(2)), was used as ligand. The competition by unlabeled Ctx was taken as a reference. The potency of CNF as a competitor was evaluated under different incubation conditions with varying time scale addition of reagents (CA-(125)ICB(2), synaptosomes and CA-CB(2) or CNF). CNF was able to inhibit the binding of the toxin to synaptosomes as well as to partially displace the toxin already bound to its membrane target. The mechanisms of competition involved were discussed and a previous schematic model of interactions between Ctx, TR and CNF was updated.

ß/δ-PrIT1, a highly insecticidal toxin from the venom of the Brazilian spider Phoneutria reidyi (F.O. Pickard-Cambridge, 1897).

A potent insecticidal toxin, ß/δ-PrIT1, molecular mass of 5598.86 [M+H](+), was characterized from Phoneutria reidyi spider venom. Its partial amino acid sequence showed high similarity with insecticidal spider toxins from the genus Phoneutria. ß/δ-PrIT1 was very toxic (LD50 = 4 nmol/g) to flies (Musca domestica), but not to mice (Mus musculus). Kinetic studies showed that (125)I-ß/δ-PrIT1 binds to two distinct sites in insect sodium channels, with close affinity (Kd1 = 34.7 pM and Kd2 = 35.1 pM). Its association is rather fast (t1/2(1) = 1.4 min, t1/2(2) = 8.5 min) and its dissociation is a slower process (t1/2(1) = 5.4 min, t1/2(2) = 32.8 min). On rat brain synaptosomes ß/δ-PrIT1 partially competed (∼30%) with the beta-toxin (125)I-CssIV, but did not compete with the alpha-toxin of reference (125)I-AaII, nor with the beta-toxin (125)I-TsVII. On cockroach nerve cord synaptosomes, ß/δ-PrIT1 did not compete with the anti-insect toxin (125)I-LqqIT1, but it competed (IC50 = 80 pM) with the "alpha-like" toxin (125)I-BomIV. In cockroach neurons, ß/δ-PrIT1 inhibited the inactivation of Nav-channels and it shifted the sodium channel activation to hyperpolarizing potentials. These results indicate two different binding sites for ß/δ-PrIT1, leading to two different pharmacological responses. ß/δ-PrIT1 is one of the most toxic spider toxins to insects without apparent toxicity to mammals, and provide new model for the development of insecticides.

PnTx4-3, a new insect toxin from Phoneutria nigriventer venom elicits the glutamate uptake inhibition exhibited by PhTx4 toxic fraction.

Several pools of neurotoxic peptides obtained from fractionated Phoneutria nigriventer venom induce different toxicological effects. One of them, PhTx4, is highly toxic towards insects and displays only a slight toxicity when injected in mice. Also, this fraction contains a class of peptides that are able to inhibit glutamate uptake in preparations of mammalian central nervous systems (CNS). In this work a new toxin called PnTx4-3 was isolated from the PhTx4 fraction by reverse phase and anion exchange steps using high performance liquid chromatography (HPLC). Edman sequencing of PnTx4-3 revealed that it was a polypeptide of 48 amino acid residues, containing 10 cysteines cross-linked by five disulfide bridges. The molecular mass measured by ES-Q-TOF mass spectrometry was 5199.49+/-0.64 Da, which is very close to the calculated mass from amino acid sequence (5199.99 Da). This toxin induces immediate excitatory effects when injected intrathoracically in house flies and cockroaches. Intracerebroventricular injections of 30 microg of PnTx4-3 in mice resulted in no apparent signs of intoxication. In order to make an orthologous comparison, pharmacological characterisation were carried out in rat brain synaptosomes by using [3H]-L-glutamate, showed that the whole PhTx4 fraction as well as the pure toxins PnTx4-3, Tx4(6-1) and Tx4(5-5) obtained of this fraction, were able to inhibit the glutamate uptake in the micromolar concentration range. PnTx4-3 inhibits the glutamate uptake in a dose dependent manner, with an IC50 of approximately 1 microM. PnTx4-3 is highly homologous to the Tx4(6-1) and Tx4(5-5) toxins previously described from the same fraction.

Prevalência de Tracoma em crianças em idade escolar no município de Turmalina, MG / Trachoma prevalence among schoolchildren in the municipality of Turmalina, Minas Gerais state

RESUMO O tracoma, tido equivocadamente como erradicado em nosso meio, encontra-se na lista de doenças negligenciadas. Trata-se da maior causa de cegueira evitável do mundo, sendo encontrado predominantemente nos países subdesenvolvidos. Diversos trabalhos têm demonstrado que esta doença atualmente se faz presente em todas as regiões do Brasil, o que evidencia que tanto o governo (nas esferas federal, estadual e municipal), quanto a academia devem continuar a considerar o tracoma entre as causas de cegueira em nosso meio. Neste trabalho, procurou-se levantar a prevalência de tracoma entre as crianças de sete a quinze anos matriculadas nas escolas da rede pública do município de Turmalina, MG. A pesquisa foi realizada neste município, situado no Vale do Jequitinhonha, considerando que existem nele regiões com populações em situação de alta vulnerabilidade social, portanto com características propícias para o surgimento da doença. Participaram da pesquisa seis médicos e onze enfermeiros com atuação na atenção primária, capacitados e padronizados para tal. Os profissionais de saúde realizaram o exame de 635 estudantes entre 7 a 15 anos nas escolas públicas de Turmalina, MG. Os estudantes com diagnóstico clínico de tracoma foram submetidos à raspagem de conjuntiva com swab e o material enviado para análise laboratorial. Todos os estudantes diagnosticados com tracoma foram tratados no Sistema Único de Saúde (SUS). Os dados coletados foram analisados com auxílio do software Statistical Package for Social Sciences, IBM Inc., USA - SPSS, versão 22.0. Entre os estudantes foi encontrada uma prevalência do tracoma de 4,7%, com predomínio nas áreas rurais.

ABSTRACT Trachoma has been mistakenly assumed as having been eradicated, but is on the list of neglected diseases. It is the leading cause of preventable blindness in the world, being found predominantly in developing countries. Several studies have shown that this disease is currently present in all regions of Brazil, which shows that both the government (at the federal, state and municipal levels) and academia must continue to treat trachoma as one of the causes of blindness. In this study, we sought to identify the prevalence of trachoma among schoolchildren aged between seven and 15 years from public schools in the municipality of Turmalina, Minas Gerais state. The survey was conducted in this municipality, located in the Jequitinhonha Valley, a region that contains populations who live in situations of high social vulnerability, and thus display the characteristics that are conducive to the emergence of the disease. The participants were six doctors and 11 nurses working in primary healthcare, trained and standardized to do so. Health professionals examined 635 students aged 7-15 years in the public schools of Turmalina. Students with a clinical diagnosis of trachoma underwent conjunctiva scraping with a swab and the material was sent for laboratory analysis. All school children diagnosed with trachoma were treated at the Unified Health System (SUS). The collected data were analyzed using the Statistical Package for Social Sciences software, IBM Inc., USA - SPSS, version 22.0. Among the students, a 4.7% prevalence rate of trachoma was found, predominantly in rural areas.

Endothelium-dependent relaxation of rat mesenteric arterial rings by a Phoneutria nigriventer venom fraction.

Phoneutria nigriventer spider venom has been described as acting on several cardiovascular sites. In the present paper, a semi-purified fraction of this spider venom was studied to observe any contractile or relaxing effect in rat mesenteric arterial rings (MAR). Spider venom was first fractionated by gel filtration and subsequently by gradual isocratic steps in 0.1% trifluoroacetic acid. The first fraction of this last fractionation step is studied in the present paper and due to its main effect, it was named NORF (nitric oxide releasing fraction). No direct contractile effect was induced by NORF in relaxed MAR, suggesting no NORF-induced neurotransmitter release in this preparation. No significant influence of NORF was observed on concentration-response curves to phenylephrine on endothelium-denuded MAR, but a significant inhibitory shift of concentration-respense curves was observed on endothelium-preserved MAR (EC50 = 0.39 +/- 0.07 microM for control and EC50 = 0.68 +/- 0.14 microM with NORF). NORF induced concentration-dependent relaxation in endothelium-preserved phenylephrine pre-contracted MAR but not in endothelium-denuded MAR. NORF-induced relaxation was inhibited by the nitric oxide synthase inhibitor L-NAME (N(omega)-nitro-arginine methyl ester). Indomethacin or HOE-140 (D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-bradykinin) had no significant effect on NORF-induced relaxation. Acetylcholine- and NORF-induced relaxation of pre-contracted MAR were differently inhibited by atropine. The pA2 value for atropine-acetylcholine was 9.78 +/- 0.06 and that for atropine-NORF was 8.53 +/- 0.30 (P<0.01). These observations suggest that NORF induces concentration-dependent liberation of nitric oxide from MAR endothelium and that a non-muscarinic mechanism might be involved in this effect. Our data suggest no involvement of prostanoids or bradykinin in the relaxing mechanism.