Conformational changes on ligand binding in wild-type and mutants from Spodoptera frugiperda midgut trehalase.

Trehalase specifically hydrolyses trehalose into two glucose units and is most important in insects and fungi. Previous evidence suggested that Spodoptera frugiperda midgut trehalase (wild type, WT) has substantial conformational changes on binding different substances. Our goal is to understand this mobility. For this, two deletion mutants were produced, lacking regions supposed to be the cause of mobility [(102 residues from the N-terminus (NT) and this portion plus 31 residues from the C-terminus (NCT)]. Circular dichroism spectra before and after denaturation of the enzymes support the assertion that they are appropriately folded. The overall results show that the removal of 102 or 133 amino acids does not greatly change the interaction with the substrate and competitive inhibitors, but leads to a considerable decrease in kcat/Km values from WT 74,500 M-1 s-1 to NT 647 M-1 s-1 and NCT 1,044 M-1 s-1. Diethyl pyrocarbonate His modification only occurs in wild and truncated trehalases in the presence of some ligands. Looking for changes in folding WT, NT, and NCT were incubated with different compounds in the presence of Sypro Orange, that binds to hydrophobic regions increasing its fluorescence. The dye fluorescence is affected by 2 compounds when WT is present, and at least by 5 compounds when NT or NCT are present, suggesting that conformational changes caused by ligand binding occur only in the vicinity of the active site. These data provide physical evidence in favor of a change in folding around the active site caused by ligand binding, in agreement to prior chemical modification and other kinetic data and challenging the hypothesis that N- and C-terminal are the mobile regions.

Midgut proteins released by microapocrine secretion in Spodoptera frugiperda.

Microapocrine vesicles bud from the lepidopteran midgut microvilli as double membrane vesicles. To identify the proteins secreted by this process, antibodies raised against isolated microapocrine vesicles from Spodoptera frugiperda were used for screening a midgut cDNA expression library. Positive clones were sequenced, assembled and N blasted against S. frugiperda sequences obtained by pyrosequencing midgut mRNA. This procedure led to the extension of microapocrine sequences that were annotated. A similar procedure was used to identify midgut microvillar proteins that necessarily are part of the microapocrine vesicle. Forty-eight proteins were associated with microvillar membranes. They pertain to 8 functional groups: digestive enzymes, peritrophic membrane, protection, transporters, receptors, secretory machinery, cytoskeleton and signaling, and unknown. Twenty-eight proteins are putatively secreted by microapocrine secretion. Most of them are digestive enzymes, but the list also includes proteins involved in protection and in peritrophic membrane formation. Among the identified digestive enzymes, aminopeptidases are typically microvillar and group into the classes 1, 2, 3, 5, and 6. There are two amylases secreted by microapocrine secretion: one is a digestive enzyme and the other is a transporter-like amylase with no clear function. One lipase has a predicted transmembrane loop, whereas the others are supposed to be secreted by microapocrine secretion and be digestive. Trypsin is membrane bound and is delivered by microapocrine secretion, but has no predicted features to bind membranes. It may remain bound through the signal peptide till be delivered into the midgut lumen. Proteins supposed to be involved in the microapocrine secretory machinery were: calmodulin, annexin, myosin 7a, and gelsolin 1. Their putative roles are discussed, but more research is necessary to settle this subject.

Effects of croton urucurana extracts and crude resin on Anagasta kuehniella (Lepidoptera: Pyralidae)

Hundreds of plant species have been studied in order to find out the active ingredient responsible for their insecticidal activity against the pests of economic importance. To verify the insecticidal activity in the husk of stem of Croton urucurana Baillon 1864 (Euphorbiaceae) against Anagasta kuehniella Zeller 1879 (Lepidoptera: Pyralidae), the methanolic (EMeOH) extract, dichloromethane fraction (FDM), ethyl acetate fraction (FAE) and crude resin, incorporated into an artificial diet were evaluated. EMeOH (0.5, 1.0 and 2.0 percent) and crude resin (2.0 percent) interfered with neither the weight nor the survival of fourth instar larvae and other analyzed parameters. FDM (2.0 percent) fraction caused mortality of 65 percent, and the artificial diet containing 2.0, 1.0 and 0.5 percent FAE caused 100, 55 and 68 percent mortality respectively when compared with the control, confirming the least efficiency rates of food conversion for FDM(2.0 percent) and FAE(1.0 percent). The tryptic analysis performed with the midgut fluid of fourth-instar larvae demonstrated that tryptic and chymiotryptic activities for the larvae fed artificial diet containing EMeOH and crude resin were not different.

Atualmente centenas de plantas são investigadas para se conhecer os princípios ativos responsáveis pela atividade inseticida contra as diversas pragas de importância econômica. Com o objetivo de verificar a atividade inseticida das cascas do caule de Croton urucurana Baillon 1864 (Euphorbiaceae) em relação a Anagasta kuehniella Zeller 1879 (Lepidoptera: Pyralidae), avaliou-se o extrato metanólico (EMeOH), fração diclorometano (FDM), a fração acetato de etila (FAE) e a resina in natura, os quais foram adicionados à dieta artificial. O EMeOH (0,5, 1,0 e 20, por cento) e a resina in natura (2,0 por cento), não interferiram no peso, sobrevivência das larvas de 4ª ínstar, bem como nos demais parâmetros analisados. A fração FDM (2,0 por cento) causou mortalidade de 65 por cento, e a dieta artificial contendo 2,0, 1,0, e 0,5 por cento de FAE causou 100, 55 e 68 por cento de mortalidade respectivamente quando comparado com o controle, constatando-se as menores eficiências de conversão do alimento para a FDM (2,0 por cento) e FAE (1,0 por cento). Através do ensaio tríptico realizado com o fluido do intestino médio das larvas, foi possível verificar que a atividade tríptica e quimiotríptica para as larvas alimentadas com dieta artificial contendo o EMeOH e a resina in natura não apresentaram diferenças significativas.

Insecticidal and antifungal activity of a protein from Pouteria torta seeds with lectin-like properties.

This paper describes the purification and characterization of a novel protein from the seeds of Pouteria torta (family Sapotaceae). The protein was purified by a combination of gel filtration, ion-exchange, and reverse phase chromatographies. SDS-PAGE of the purified protein resulted in a single protein band of 14 kDa in the presence and absence of DTT. The lectin-like activity of pouterin was best inhibited by glycoproteins such as fetuin, asialofetuin, heparin, orosomucoid, and ovoalbumin. Pouterin inhibited the growth of the fungi Fusarium oxysporum and Colletotrichum musae and of the yeast Saccharomyces cerevisiae. The incorporation of pouterin into an artificial diet (final concentration = 0.12%, w/w) caused 50% mortality in larvae of the insect Callosobruchus maculatus, whereas 0.08% pouterin produced an ED50.