RESUMEN
Few chronic food protein models have described the relationship between allergenicity and the molecular structure of food protein after physical processing. The effect of γ-radiation on the structure of food protein was measured by fluorescence, circular dichroism and microcalorimetry. BALB/c mice were intraperitoneally sensitized and then given non-irradiated and irradiated Con-A by daily gavage for 28days. The tendency to form insoluble amorphous aggregates and partially unfolded species was observed after irradiation. The administration of non-irradiated and irradiated samples at low-dose significantly increased weight loss as well as plasma levels of eotaxin in animals repeatedly exposed to Con-A. Significant lymphocytic infiltrate filling completely the stroma of microvilli and tubular glands was observed in the small intestinal of the group given Con-A irradiated at a low dose. This phenotype was not observed in animals treated with Con-A irradiated at a high dose.
Asunto(s)
Concanavalina A/química , Concanavalina A/inmunología , Concanavalina A/efectos de la radiación , Hipersensibilidad a los Alimentos/etiología , Administración Oral , Animales , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Concanavalina A/administración & dosificación , Modelos Animales de Enfermedad , Relación Dosis-Respuesta en la Radiación , Femenino , Hipersensibilidad a los Alimentos/patología , Rayos gamma , Intestino Delgado/inmunología , Intestino Delgado/patología , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Microvellosidades/inmunología , Microvellosidades/patología , Conformación Proteica , Pérdida de PesoRESUMEN
Bacillus thuringiensis Cry toxins are used worldwide as insecticides in agriculture, in forestry, and in the control of disease transmission vectors. In the lepidopteran Manduca sexta, cadherin (Bt-R(1)) and aminopeptidase-N (APN) function as Cry1A toxin receptors. The interaction with Bt-R(1) promotes cleavage of the amino-terminal end, including helix alpha-1 and formation of prepore oligomer that binds to APN, leading to membrane insertion and pore formation. Loops of domain II of Cry1Ab toxin are involved in receptor interaction. Here we show that Cry1Ab mutants located in domain II loop 3 are affected in binding to both receptors and toxicity against Manduca sexta larvae. Interaction with both receptors depends on the oligomeric state of the toxin. Monomers of loop 3 mutants were affected in binding to APN and to a cadherin fragment corresponding to cadherin repeat 12 but not with a fragment comprising cadherin repeats 7-12. In contrast, the oligomers of loop 3 mutants were affected in binding to both Bt-R(1) fragments but not to APN. Toxicity assays showed that either monomeric or oligomeric structures of Cry1Ab loop 3 mutations were severely affected in insecticidal activity. These data suggest that loop 3 is differentially involved in the binding with both receptor molecules, depending on the oligomeric state of the toxin and also that possibly a "ping pong" binding mechanism with both receptors is involved in toxin action.
Asunto(s)
Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/química , Antígenos CD13/química , Cadherinas/química , Endotoxinas/química , Proteínas Hemolisinas/química , Manduca/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Dicroismo Circular , Larva/metabolismo , Larva/microbiología , Microvellosidades/inmunología , Mutagénesis Sitio-Dirigida , Mutación , Plásmidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The objective of this study was to identify midgut microvillar proteins in insects appearing earlier (Coleoptera) and later (Lepidoptera) in evolution. For this, cytoskeleton-free midgut microvillar membrane from Spodoptera frugiperda (Lepidoptera) and Tenebrio molitor (Coleoptera) were used to raise antibodies. These were used for screening midgut cDNA expression libraries. Positive clones were sequenced, assembled and searched for similarities with gene/protein databases. The predicted midgut microvillar proteins from T. molitor were: cockroach allergens (unknown function), peritrophins (peritrophic membrane proteins), digestive enzymes (aminopeptidase, alpha-mannosidase) and unknown proteins. Predicted S. frugiperda midgut proteins may be grouped into six classes: (a) proteins involved in protection of midgut (thioredoxin peroxidase, aldehyde dehydrogenase, serpin and juvenile hormone epoxide hydrolase); (b) digestive enzymes (astacin, transporter-like amylase, aminopeptidase, and carboxypeptidase); (c) peritrophins; (d) proteins associated with microapocrine secretion (gelsolin, annexin); (e) membrane-tightly bound-cytoskeleton proteins (fimbrin, calmodulin) and (f) unidentified proteins. The novel approach is compared with others and microvillar function is discussed in the light of the predicted proteins.
Asunto(s)
Tracto Gastrointestinal/metabolismo , Biblioteca de Genes , Proteínas de Insectos/metabolismo , Microvellosidades/metabolismo , Spodoptera/metabolismo , Tenebrio/metabolismo , Animales , Anticuerpos/inmunología , Evolución Biológica , Tracto Gastrointestinal/inmunología , Perfilación de la Expresión Génica , Proteínas de Insectos/genética , Proteínas de Insectos/inmunología , Microvellosidades/inmunología , Spodoptera/genética , Spodoptera/inmunología , Tenebrio/genética , Tenebrio/inmunologíaRESUMEN
Antiserum raised against Rhodnius prolixus perimicrovillar membranes (PMM) and midgut tissue interfered with the midgut structural organization and reduced the development of Trypanosoma cruzi in the R. prolixus insect vector. SDS-PAGE and Western blot analyses confirmed the specific recognition of midgut proteins by the antibody. Feeding, mortality, molt, and oviposition of the insects were unaffected by feeding with the antiserum. However, the eclosion of the eggs were reduced from R. prolixus females treated with antiserum. Additionally, in vivo evaluation showed that after oral treatment with the antiserum, the intensity of infection with the Dm-28c clone of T. cruzi decreased in the digestive tract of fifth-instar nymphs and in the excretions of R. prolixus adults. These results suggest that the changes observed in the PMM organization in the posterior midgut of R. prolixus may not be important for triatomine survival but the antiserum acts as a transmission-reduction vaccine able to induce significant decreases in T. cruzi infection in the vector.