Alphastatin-C a new inhibitor of endothelial cell activation is a pro-arteriogenic agent in vivo and retards B16-F10 melanoma growth in a preclinical model.

Most characterized angiogenic modulators are proteolytic fragments of structural plasma and/or matrix components. Herein, we have identified a novel anti-angiogenic peptide generated by the in vitro hydrolysis of the C-terminal moiety of the fibrinogen alpha chain, produced by the snake venom metalloprotease bothropasin (SVMP), a hemorrhagic proteinase in Bothrops jararaca venom. The 14-amino acids peptide (alphastatin-C) is a potent antagonist of basic fibroblast growth factor, induced endothelial cell (HUVEC-CS) proliferation, migration and capillary tube formation in matrigel. It also inhibits cell adhesion to fibronectin. The basis of the antagonism between bFGF and alphastatin-C is elucidated by the inhibition of various bFGF induced signaling pathways and their molecular components modification, whenever the combination of the stimuli is provided, in comparison to the treatment with bFGF only. To corroborate to the potential therapeutic use of alphastatin-C, we have chosen to perform in vivo assays in two distinct angiogenic settings. In chick model, alphastatin-C inhibits chorioallantoic membrane angiogenesis. In mouse, it efficiently reduces tumor number and volume in a melanoma model, due to the impairment of tumor neovascularization in treated mice. In contrast, we show that the alphastatin-C peptide induces arteriogenesis, increasing pial collateral density in neonate mice. alphastatin-C is an efficient new antiangiogenic FGF-associated agent in vitro, it is an inhibitor of embryonic and tumor vascularization in vivo while, it is an arteriogenic agent. The results also suggest that SVMPs can be used as in vitro biochemical tools to process plasma and/or matrix macromolecular components unraveling new angiostatic peptides.

Intrahippocampal injection of TsTX-I increases the levels of INF-γ in the cerebral tissue but not the levels of glutamate.

TsTX-I, isolated from Tityus serrulatus scorpion venom, causes epileptic-like discharges when injected into the central nervous system. The involvement of excitatory amino acids and cytokines in this activity was investigated. Our results have demonstrated that TsTX-I increases the release of IFN-γ but does not alter the intracerebral concentration of the excitatory amino acids in rats. Thus, this cytokine seems to be more important in the convulsive process than glutamate.

Enzymatic properties of venoms from Brazilian scorpions of Tityus genus and the neutralisation potential of therapeutical antivenoms.

Tityus scorpion stings are an important public health problem in Brazil, where the incidence of such stings exceeds the incidence of the health problems caused by other venomous animals, including snakes. In this study, we have analysed specific enzymatic activities of the venom from the Brazilian scorpions of Tityus genus, i.e., Tityus serrulatus, Tityus bahiensis and Tityus stigmurus. The data presented here revealed that Tityus spp. venoms exhibited significant hyaluronidase activity but no phospholipase activity. All the venom samples exhibited the ability to hydrolyse Abz-FLRRV-EDDnp and dynorphin 1-13 substrates. These activities were inhibited by 1,10-phenanthroline but not by PMSF, indicating the presence of metalloproteinases in the Tityus spp. venoms. The venom peptidase activity on Abz-FLRRV-EDDnp and on dynorphin 1-13 was partially inhibited by therapeutic Brazilian anti-scorpion and anti-arachnidic antivenoms. Dynorphin 1-13 (YGGFLRRIRPKLK) contains two scissile bonds between the residues Leu-Arg and Arg-Arg that are susceptible to cleavage by the Tityus venom metallopeptidase(s). Their cleavage releases leu-enkephalin, an important bioactive peptide. The detection of metalloproteinase(s) with specificity for both dynorphin 1-13 degradation and leu-enkephalin releasing can be important for the mechanistic understanding of hypotension and bradycardia induction in cases of scorpion stings, whereas hyaluronidases might contribute to the diffusion of the toxins present in these venoms. Furthermore, the limited inhibition of the toxic enzymatic activities by commercial antivenoms illustrates the necessity of improvements in current antivenom preparation.

Insights into scorpion venom peptides: alternative processing of ß-KTx propeptide from Tityus serrulatus venom results in a new naturally occurring thimet oligopeptidase inhibitor.

Most functions attributed to Tityus serrulatus venom (TsV) are related to active molecules on ion-channels; however, here we describe a new pentapeptide that was discovered through enzymatic assay selection using EP24.15. The primary structure analysis revealed the sequence KEXXG (X means Ile or Leu), similar to the sequence present in the ß-KTX propeptide described from the venom of Tityus spp. We confirmed through HPLC analysis that KEILG is the peptide present in TsV, but that KELLG also inhibits EP24.15 although through different mechanisms.

Análise dos componentes proteolíticos e petídicos do veneno do escorpião Tityus serrulatus / Analysis of proteolytic and peptidic compounds from the venom of the scorpion Tityus serrulatus

Acidentes causados por escorpiões representam um grave problema de saúde pública no Brasil, sendo a principal espécie o Tityus serrulatus, devido a sua ecologia, sua estratégia reprodutiva e a potência do envenenamento. Na composição deste veneno, diversos polipeptídiosneurotóxicos que afetam canais de Na+ e K+ podem ser encontrados, sendo esse o maior foco das pesquisas relacionadas ao assunto. Em contrapartida, pouca informação sobre componentes proteolíticos e peptídicos no veneno do escorpião Tityus serrulatus (VTs) está disponível naliteratura. Deste modo, este trabalho visou estudar novos peptídeos presentes no VTs capazes deinteragir com oligopeptidases, além de caracterizar a atividade proteolítica do veneno do escorpião amarelo, relacionando substratos biológicos de importância para o envenenamento,tendo em vista que até o presente momento não existe tal assimilação. Para o estudo de atividade proteolítica do VTs, foram utilizados substratos de fluorescência apagada, sendo queAbz-GGFLRRV-EDDnp foi selecionado como substrato ideal, além da família enzimática ser determinada pelo uso de inibidores específicos. O ponto de clivagem deste substrato foiestimado ser entre leucina e arginina. Por meio de ensaios de fluorescência, padrões bioquímicos de atividade ótima do veneno total, como pH, temperatura e influência de sais,foram estabelecidos. A partir disso, foram realizados experimentos para determinação substratospeptídicos com atividade biológica, analisados em sistema de HPLC, sendo que somente foi observada hidrólise para a dinorfina 1-13, onde os fragmentos foram analisados porespectrometria de massas para a determinação dos pontos de clivagem. Foi evidenciada a liberação do peptídeo opióide leu-encefalina, podendo estar atuando sobre canais de K+, comoindicam dados da literatura, o que estaria relacionado com os efeitos neurotóxicos do veneno.Na tentativa de verificarmos se apenas uma peptidase estava sendo responsável...