RESUMO
Selected channel-lining cysteine mutants from the M2 segment of rat alpha1 gamma-aminobutyric acid (GABA) type A receptor subunit, at positions 257, 261, 264, and 272 were co-expressed with beta1 and gamma2 subunits in Xenopus oocytes. They generated functional receptors displaying conductance and response to both GABA and picrotoxinin similar to the wild type alpha1beta1gamma2 receptor. Three chemically reactive affinity probes derived from non-competitive blockers were synthesized to react with the engineered cysteines: 1) dithiane bis-sulfone derivative modified by an isothiocyanate function (probe A); 2) fiprole derivatives modified by an alpha-chloroketone (probe B) and alpha-bromoketone (probe C) moiety. These probes blocked the GABA-induced currents on all receptors. This blockade could be fully reversed by a washing procedure on the wild type, the alpha1T261Cbeta1gamma2 and alpha1L264Cbeta1gamma2 mutant receptors. In contrast, an irreversible effect was observed for all three probes on both alpha1V257Cbeta1gamma2 and alpha1S272Cbeta1gamma2 mutant receptors. This effect was probe concentration-dependent and could be abolished by picrotoxinin and/or t-butyl bicyclophosphorothionate. These data indicate a major interaction of non-competitive blockers at position 257 of the presumed M2 segment of rat alpha1 subunit but also suggest an interaction at the more extracellular position 272.
Assuntos
Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/metabolismo , Antagonistas de Receptores de GABA-A , Peptídeos/farmacologia , Receptores de GABA-A/metabolismo , Sulfonas/farmacologia , Animais , Canais de Cloreto/genética , Cisteína/genética , Ligantes , Mutagênese Sítio-Dirigida , Peptídeos/química , Mutação Puntual , Ratos , Receptores de GABA-A/genética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfonas/química , XenopusRESUMO
Lepidoptera have been reported to produce several antibacterial peptides in response to septic injury. However, in marked contrast to other insect groups, no inducible antifungal molecules had been described so far in this insect order. Surprisingly, also cysteine-rich antimicrobial peptides, which predominate in the antimicrobial defense of other insects, had not been discovered in Lepidoptera. Here we report the isolation from the hemolymph of immune induced larvae of the lepidopteran Heliothis virescens of a cysteine-rich molecule with exclusive antifungal activity. We have fully characterized this antifungal molecule, which has significant homology with the insect defensins, a large family of antibacterial peptides directed against Gram-positive strains. Interestingly, the novel peptide shows also similarities with the antifungal peptide drosomycin from Drosophila. Thus, Lepidoptera appear to have built their humoral immune response against bacteria on cecropins and attacins. In addition, we report that Lepidoptera have conferred antifungal properties to the well conserved structure of antibacterial insect defensins through amino acid replacements.
Assuntos
Antifúngicos/farmacologia , Proteínas de Drosophila , Lepidópteros/imunologia , Proteínas/isolamento & purificação , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Defensinas , Drosophila , Eletroforese Capilar , Escherichia coli/imunologia , Hemolinfa/química , Proteínas de Insetos/química , Larva/imunologia , Lepidópteros/química , Micrococcus luteus/imunologia , Dados de Sequência Molecular , Proteínas/química , Proteínas/genética , Homologia de Sequência de AminoácidosRESUMO
A simple, rapid, direct method for the HPLC analysis of 1-aminocyclopropane-1-carboxylic acid (ACC) as its o-phthaldialdehyde derivative is described. The method is sensitive to about 1 pmol and can be used on plant tissue extracts with no cleanup. It will prove valuable in plant extracts where the chemical conversion of ACC in the tissue extracts to ethylene is variable, or when analyzing the specific radioactivity of ACC produced from radiolabeled precursors.