RESUMO
BACKGROUND: Alpha-scorpion toxins with long-chain peptide and four disulfide bonds represent diverse pharmacological profiles for various subtypes of voltage-gated sodium channels. Obtaining the natural toxins are difficult and time-consuming process, which represents the major difficulty to interpreting analysis of their structural and functional properties. METHODS AND RESULTS: This study describes the toxin peptide and plasmid construct containing the gene coding for mammalian toxin AnCra1 from the scorpion Androctonus crassicauda venom. We have established genetic construction of fusion protein in pET32a + vector containing thioredoxin (Trx-tag), enterokinase cleavage site and 6xhistidine-tag for efficient expression in Escherichia coli strain RG2 (DE3). The soluble expressed peptide, then purified by Ni-NTA resin affinity chromatography and its purity was confirmed by reverse-phase HPLC and mass spectrometry (7433.54 Da.). The electrophysiological data showed that recombinant AnCra1 selectively inhibits the fast inactivation of hNav1.7 channel (EC50 = 136.7 ± 6.6 nM). CONCLUSIONS: Our findings demonstrate that the AnCra1 is structurally and functionally analogous to alpha excitatory toxins; furthermore, expression and purification of bioactive scorpion toxins in bacterial cells can be a practicable and efficient way to obtain a novel source of toxin peptides as tools to study the function and physiological responses of ion channels.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Venenos de Escorpião/isolamento & purificação , Venenos de Escorpião/farmacologia , Escorpiões/genética , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Dose Letal Mediana , Espectrometria de Massas/métodos , Camundongos , Peptídeos/química , Peptídeos/genética , Plasmídeos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Venenos de Escorpião/química , Venenos de Escorpião/genéticaRESUMO
The present study was aimed to investigate the pre-treatment effect of Centella asiatica (CeA) extract on long-term potentiation (LTP) in a rat model of Alzheimer's disease (AD). A total of 32 male Wistar rats weighing 380 ± 30 g were randomly divided into four groups (n = 8). Group 1 (C: Control): the control group. Group 2 (L: Lesion): The nucleus basalis of Meynert (NBM) of rats' brain was bilaterally destroyed by injection of Ibotenic acid. Group 3 (CeA): Animals in this group received the CeA leaf extract for only a period of six weeks. Group 4 (CeA + L): The NBM of rats was destroyed by Ibotenic acid after six weeks of a diet containing the CeA leaf extract. In all groups, LTP was recorded using the electrophysiological technique and fEPSP after high frequency stimulation (HFS). The results showed that the slope and amplitude of PS as well as the sub-curve level significantly increased in the CeA + L group compared with the L and CeA groups. The CeA extract improved and strengthened the slope, amplitude and sub-curve surface of cumulative waves in animals with NBM lesion. The results showed that administration CeA extract for six weeks before induction of NBM lesion and induction of Alzheimer could enhance memory. In other words, the CeA extract had a preventive or protective role. The present study showed that CeA had a protective role for neurons among rats with NBM lesion.