RESUMO
Calcium signalling is little understood in filamentous fungi largely because easy and routine methods for calcium measurement in living hyphae have previously been unavailable. We have developed the recombinant aequorin method for this purpose. High levels of aequorin expression were obtained in Neurospora crassa, Aspergillus niger and Aspergillus awamori by codon optimization of the aequorin gene. Three external stimuli (mechanical perturbation, hypo-osmotic shock and high external calcium) were found transiently to increase [Ca(2+)](c). Each of the calcium signatures associated with these physico-chemical treatments was unique, suggesting the involvement of three distinct calcium-mediated signal transduction pathways. The fungal calcium channel blocker KP4 inhibited the [Ca(2+)](c) responses to hypo-osmotic shock and high external calcium, but not to mechanical perturbation. The divalent cation chelator BAPTA inhibited [Ca(2+)](c) responses to mechanical perturbation and hypo-osmotic shock. The calcium agonists A23187 and cyclopiazonic acid increased [Ca(2+)](c) levels.
Assuntos
Equorina/genética , Equorina/metabolismo , Aspergillus/metabolismo , Cálcio/metabolismo , Códon , Ácido Egtázico/análogos & derivados , Neurospora crassa/metabolismo , Aspergillus/citologia , Aspergillus/genética , Sequência de Bases , Calcimicina/metabolismo , Bloqueadores dos Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Quelantes/metabolismo , Ácido Egtázico/metabolismo , Inibidores Enzimáticos/metabolismo , Indóis/metabolismo , Ionóforos/metabolismo , Dados de Sequência Molecular , Neurospora crassa/citologia , Neurospora crassa/genética , Pressão Osmótica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Estresse MecânicoRESUMO
Following the discovery of RPR200765, a series of pyrimidine analogues have been prepared as backups. Amongst them, RPR203494 was identified with a better in vitro profile than RPR200765A.
