RESUMEN
BACKGROUND: Actinidin has previously been reported as the major allergen in kiwifruit. Objectives To investigate the relevance of actinidin in a well-characterized population of UK patients with kiwifruit allergy. METHODS: To identify the allergens in kiwifruit, using Western blots, we examined the IgE-binding patterns of 76 patients with a history of kiwifruit allergy, 23 of who had had a positive double-blind, placebo-controlled food challenge. In addition, IgE binding to purified native actinidin was studied in 30 patients, and to acidic and basic isoforms of recombinant actinidin in five patients. Inhibition of IgE binding to kiwifruit protein extract by purified native actinidin was investigated by both inhibition immunoblots and inhibition ELISAs using pooled sera. RESULTS: Twelve protein bands in kiwifruit protein extract were bound by IgE. A protein band with a molecular weight of 38 kDa was the major allergen recognized by 59% of the population. IgE did not bind to actinidin in the kiwifruit protein extract, or to purified native or recombinant forms of actinidin during Western blotting. Pooled sera bound to kiwifruit protein extract but not purified actinidin on ELISA, and pre-incubating sera with actinidin did not inhibit IgE binding to kiwifruit protein extract on immunoblot or ELISA. CONCLUSION: A novel 38 kDa protein, not actinidin, is the major allergen in this large study population. Identification of major allergens in one patient group is therefore not necessarily reproducible in another; therefore, major allergens should not be defined until there is a sufficient body of data from diverse geographical and cultural populations.
Asunto(s)
Actinidia/inmunología , Antígenos de Plantas/clasificación , Antígenos de Plantas/inmunología , Cisteína Endopeptidasas/inmunología , Hipersensibilidad/diagnóstico , Adolescente , Adulto , Western Blotting , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Femenino , Frutas/inmunología , Humanos , Hipersensibilidad/inmunología , Inmunoglobulina E/sangre , Masculino , Persona de Mediana Edad , Pruebas Cutáneas , Reino Unido/epidemiologíaRESUMEN
In kiwifruit, much of the softening process occurs prior to the respiratory climacteric and production of ethylene. This fruit therefore represents an excellent model system for dissecting the process of softening in the absence of endogenous ethylene production. We have characterized the expression of three polygalacturonase (PG) cDNA clones (CkPGA, B and C) isolated from fruit of Actinidia chinensis. Expression of CkPGA and B was detected by northern analysis only in fruit producing endogenous ethylene, and by RT-PCR in other tissues including flower buds, petals at anthesis, and senescent petals. CkPGA promoter fragments of 1296, 860 and 467 bp fused to the beta-glucuronidase (uidA) reporter gene directed fruit-specific gene expression during the climacteric in transgenic tomato. CkPGC gene expression was observed in softening fruit, and reached maximum levels (50-fold higher than for CkPGA and B) as fruit passed through the climacteric. However, expression of this gene was also readily detected during fruit development and in fruit harvested prior to the onset of softening. Using RT-PCR, expression of CkPGC was also detected at low levels in root tips and in senescent petals. These results suggest that PG expression is required not only during periods of cell wall degeneration, but also during periods of cell wall turnover and expansion.
Asunto(s)
Frutas/genética , Poligalacturonasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN de Plantas/genética , Etilenos/biosíntesis , Frutas/enzimología , Frutas/crecimiento & desarrollo , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glucuronidasa/genética , Glucuronidasa/metabolismo , Solanum lycopersicum/genética , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente/genética , Regiones Promotoras Genéticas/genética , ARN de Planta/genética , ARN de Planta/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Distribución TisularRESUMEN
A beta-galactosidase was purified from cortical tissue of ripe apples (Malus domestica Borkh. cv Granny Smith) using a procedure involving affinity chromatography on lactosyl-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that two polypeptides of 44 and 32 kD were present in the fraction that showed activity against the synthetic substrate p-nitrophenol-beta-D-galactopyranoside. The enzyme preparation was incubated with polysaccharide extracts from apple cell walls containing beta-(1-->4)-linked galactans, and products of digestion were analyzed by gas chromatography. Small amounts of monomeric galactose were released during incubation, showing that the enzyme was active against native substrates. Amino acid sequence information was obtained from the purified protein, and this showed high homology with the anticipated polypeptide coded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldborough, W.R. Woodson [1991] Plant Mol Biol 17: 61-71) and a harvest-related pTIP31 cDNA from asparagus (G. King, personal communication). Using the asparagus cDNA clone as a probe, an apple homolog (pABG1) was isolated. This clone contains a 2637-bp insert, including an open reading frame that codes for a polypeptide of 731 amino acids. Cleavage of an N-terminal signal sequence would leave a predicted polypeptide of 78.5 kD. Genomic DNA analysis and the isolation of other homologous apple clones suggest that pABG1 represents one member of an apple beta-galactosidase gene family. Northern analysis during fruit development and ripening showed accumulation of pABG1-homologous RNA during fruit ripening. Enzyme activity as measured in crude extracts increased during fruit development to a level that was maintained during ripening.
Asunto(s)
Frutas/enzimología , Polisacáridos/metabolismo , beta-Galactosidasa/aislamiento & purificación , beta-Galactosidasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Pared Celular/metabolismo , Cromatografía de Afinidad , Clonación Molecular , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Frutas/genética , Frutas/crecimiento & desarrollo , Expresión Génica , Cinética , Datos de Secuencia Molecular , Peso Molecular , Plantas/enzimología , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Verduras/enzimología , beta-Galactosidasa/genéticaRESUMEN
Methods for the estimation of hydrogen peroxide in acetone extracts using titanium(IV) are likely to overestimate hydrogen peroxide when applied to plant leaves. Pigments appear to co-precipitate with the titanium complex and cannot be removed by washing with solvents. Fluoride, which specifically removes the color of the titanium-peroxide complex, removes only some of the color from the reactions with plant extracts. This problem has been avoided by extracting tissues with trichloroacetic acid, and measuring peroxide against catalase-treated blanks by its reaction with the complex of titanium(IV) with 4-(2-pyridylazo) resorcinol. Levels of hydrogen peroxide in leaves of a variety of species were found to range from about 0.1 to 0.6 mumol X g-1.