RESUMEN
BACKGROUND: Lipid transfer protein (LTP) is a major fruit allergen. It has, however, recently been revealed that the systemic reaction in peach-allergic patients is related not only to LTP (Pru p 3) but also to gibberellin-regulated protein (Pru p 7). We investigated recombinant Pru p 7 (rPru p 7) for its potential use in worldwide standardization for the diagnosis of peach allergy. METHODS: Natural Pru p 7 (nPru p 7) was purified from peach crude extract using a monoclonal antibody affinity column. Complementary DNA for Pru p 7 was cloned and expressed in Escherichia coli and Pichia pastoris. Serum immunoglobulin (Ig) E in peach-allergic patients was examined by enzyme-linked immunosorbent assay (ELISA) using nPru p 7 and rPru p 7 (E. coli product: erPru p 7 and P. pastoris product: prPru p 7). RESULTS: Peach-allergic patients (n=27) were diagnosed and categorized into oral reaction (n=10) or systemic reaction (n=17). The nPru p 7 positivity based on serum IgE levels was 52% in the systemic-reaction group and 0% in the oral-reaction group (P<0.05). In the systemic-reaction group, there was no significant difference in reactivity between nPru p 7 and prPru p 7, but the reactivity of erPru p 7 was significantly lower than those of nPru p 7 and prPru p 7 (P<0.05). CONCLUSIONS: We found that prPru p 7 exhibited reactivity in ELISA comparable to that of nPru p 7 for the diagnosis of peach allergy with systemic reaction.
Asunto(s)
Antígenos de Plantas/inmunología , Hipersensibilidad a los Alimentos/diagnóstico , Inmunoglobulina E/sangre , Prunus persica/efectos adversos , Adolescente , Adulto , Antígenos de Plantas/administración & dosificación , Antígenos de Plantas/efectos adversos , Proteínas Portadoras/administración & dosificación , Proteínas Portadoras/efectos adversos , Proteínas Portadoras/inmunología , Niño , Ensayo de Inmunoadsorción Enzimática , Femenino , Hipersensibilidad a los Alimentos/sangre , Hipersensibilidad a los Alimentos/inmunología , Giberelinas/administración & dosificación , Giberelinas/efectos adversos , Giberelinas/inmunología , Humanos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/inmunología , Adulto JovenRESUMEN
Caveolae have been postulated as a center for signal transduction, because many signaling molecules are concentrated in caveolin-rich fractions. We took Gi2alpha as an example and examined whether it is constitutively concentrated in caveolae. First, the behavior of caveolin and Gi2alpha in density-equilibrium ultracentrifugation was reexamined. By collecting fractions efficiently, caveolin and Gi2alpha were found to distribute differently. Secondly, by novel immunocytochemical methods it was found that the labeling density of Gi2alpha was 2.29 times higher in caveolae than in the non-caveolar plasma membrane. The results indicate that the concentration of Gi2alpha in caveolae is lower than deduced from most biochemical studies.
Asunto(s)
Caveolinas , Membrana Celular/química , Proteínas de Unión al GTP/análisis , Proteínas Proto-Oncogénicas/análisis , Caveolina 1 , Fraccionamiento Celular , Línea Celular , Membrana Celular/ultraestructura , Centrifugación por Gradiente de Densidad , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Técnica de Fractura por Congelación , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Immunoblotting , Inmunohistoquímica , Proteínas de la Membrana/análisis , Microscopía Inmunoelectrónica , Octoxinol , Transducción de SeñalRESUMEN
BACKGROUND: Lipid transfer protein (LTP) is a major fruit allergen. It has, however, recently been revealed that the systemic reaction in peach-allergic patients is related not only to LTP (Pru p 3) but also to gibberellin-regulated protein (Pru p 7). We investigated recombinant Pru p 7 (rPru p 7) for its potential use in worldwide standardization for the diagnosis of peach allergy. METHODS: Natural Pru p 7 (nPru p 7) was purified from peach crude extract using a monoclonal antibody affinity column. Complementary DNA for Pru p 7 was cloned and expressed in Escherichia coli and Pichia pastoris. Serum immunoglobulin (Ig) E in peach-allergic patients was examined by enzyme-linked immunosorbent assay (ELISA) using nPru p 7 and rPru p 7 (E. coli product: erPru p 7 and P. pastoris product: prPru p 7). RESULTS: Peach-allergic patients (n = 27) were diagnosed and categorized into oral reaction (n=10) or systemic reaction (n = 17). The nPru p 7 positivity based on serum IgE levels was 52% in the systemic-reaction group and 0% in the oral-reaction group (P<0.05). In the systemic-reaction group, there was no significant difference in reactivity between nPru p 7 and prPru p 7, but the reactivity of erPru p 7 was significantly lower than those of nPru p 7 and prPru p 7 (P < 0.05). CONCLUSIONS: We found that prPru p 7 exhibited reactivity in ELISA comparable to that of nPru p 7 for the diagnosis of peach allergy with systemic reaction
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