RESUMO
BACKGROUND: Anti-gliadin IgE are expressed in patients with food allergy associated to skin immediate hypersensitivity to hydrolyzed wheat proteins (IHHWP). It is not known if they react with omega5-gliadins, the major allergens in wheat dependant exercise-induced food anaphylaxis (WDEIA), encoded on wheat chromosomes 1B. METHODS: Unmodified gliadins from 14 wheat varieties expressing most of the 1B omega-gliadin alleles, were immunoprobed after SDS-PAGE and blotting, with four sera from patients with IHHWP, and two with WDEIA. Gliadins reacting with IgE were visualized using chemiluminescence and identified according to their mobility and typical SDS-PAGE pattern. The resulting signal was also measured to compare their IgE reactivity. RESULTS: IHHWP and WDEIA sera exhibited distinct patterns of reactivity. IgE of patients with IHHWP reacted mainly with all omega-gliadins alleles and one gamma-gliadin encoded respectively on chromosomes 1D and 1B, but not with any omega5-gliadins alleles as for WDEIA. A few other reactive alleles of omega-gliadins were encoded on chromosomes 1A. Unassigned additional bands of the whole gliadin pattern were also reactive. The four patients with IHHWP exhibited almost the same pattern of reactivity. Main differences concerned band reactivity which modulated the overall reactivity of each wheat variety. CONCLUSIONS: The IgE epitopes involved in IHHWP and WDEIA are different. This suggests that the protein state and the route of exposure to very similar gluten structures, probably orientate the pattern of epitope reactivity and the wheat food allergy manifestations.
Assuntos
Gliadina/genética , Gliadina/imunologia , Hipersensibilidade Imediata/genética , Hipersensibilidade a Trigo/genética , Alelos , Alérgenos/efeitos adversos , Alérgenos/genética , Alérgenos/imunologia , Anafilaxia/genética , Anafilaxia/imunologia , Dermatite Atópica/etiologia , Dermatite Atópica/genética , Dermatite Atópica/imunologia , Eletroforese em Gel de Poliacrilamida/métodos , Exercício Físico , Humanos , Hipersensibilidade Imediata/sangue , Hipersensibilidade Imediata/imunologia , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Imunoglobulina E/sangue , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Medições Luminescentes/métodos , Triticum/efeitos adversos , Triticum/genética , Triticum/imunologia , Hipersensibilidade a Trigo/sangue , Hipersensibilidade a Trigo/imunologiaRESUMO
1. Interactions between the fungicide prochloraz and the hepatic cytochrome P-450 of rainbow trout were studied by determination of enzymic activities in vitro using the microsomal fraction, and by kinetic studies. 2. Prochloraz inhibited 7-ethoxyresorufin-O-deethylase (EROD) in vitro. This inhibition was partially non-competitive: the enzyme-substrate-inhibitor (ESI) complex was catalytically active (68% of the activity of the enzyme-substrate complex). 3. Prochloraz in vitro inhibited aldrin epoxidase (AE) by a linear mixed-type mechanism. This inhibition might be high because of high affinity for prochloraz and lack of catalytic activity of the ESI complex. 4. Effects of prochloraz in vivo were studied as a function of time after intraperitoneal injection. Total cytochrome P-450 increased for more than 21 days. 5. EROD increased slightly at day 4 and then returned to control level. Kinetics showed an increase in apparent Km and Vmax. AE was strongly inhibited at day 4 (large increase in apparent Km) and then returned to control level in 21 days. 6. Spectral interactions with aniline showed strong inhibition and recovery to an activated level at day 21.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Fungicidas Industriais/farmacologia , Glucuronosiltransferase/metabolismo , Imidazóis/farmacologia , Microssomos Hepáticos/enzimologia , Oxigenases de Função Mista/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Salmonidae/metabolismo , Truta/metabolismo , Animais , Citocromo P-450 CYP1A1 , Cinética , Microssomos Hepáticos/efeitos dos fármacosRESUMO
1. Rainbow trout were dosed with prochloraz by i.p. injection of sprayed food pellets. Cytochrome P-450, two P-450-dependent activities, and two conjugase activities were measured in vitro in microsomal or cytosolic fractions. 2. Prochloraz increased cytochrome P-450 in liver, intestine, and pyloric caeca: maximum response occurred at 30-100 mg/kg i.p. In cold conditions, this increase persisted for more than 8 days after injection. 3. Hepatic 7-ethoxycoumarin-O-dealkylase (ECOD) and 7-ethoxyresorufin-O-dealkylase (EROD) were inhibited by prochloraz except in one assay in warm water where they increased. In intestine and pyloric caeca, ECOD and EROD were not detected, even when cytochrome P-450 was increased. 4. UDP-glucuronosyltransferase (1-naphthol as substrate) was unchanged or inhibited after prochloraz dosing. 5. Glutathione-S-transferase (o-dinitrobenzene as substrate), was unchanged or inhibited by prochloraz. 6. The measured level of enzymic activities was the result of induction and inhibition by prochloraz residues. Variations in basal activities and perhaps in prochloraz interactions were due to temperature acclimatization.
Assuntos
Fungicidas Industriais/farmacologia , Imidazóis/farmacologia , Salmonidae/metabolismo , Truta/metabolismo , Xenobióticos/metabolismo , Administração Oral , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Digestório/efeitos dos fármacos , Sistema Digestório/enzimologia , Relação Dose-Resposta a Droga , Indução Enzimática/efeitos dos fármacos , Injeções Intraperitoneais , Fígado/efeitos dos fármacos , Fígado/enzimologia , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologiaRESUMO
A method for analysing UDP-glucuronosyltransferase activity in rainbow trout hepatic microsomes is described, using 1-naphthol as a substrate and fluorometric determination of glucuronide. Kinetic constants are computed with a classical plot in a weighted regression. The computer uses the least-squares method for each value of a variable which is set and incremented. To get confidence intervals, the computer generates random values around experimental data (in a confidence interval they determine), and then computes again. With each simulation, a weighted regression with classical secondary plots gives simulated kinetic parameters. From each population of simulated values, an interval containing a given percentage of the population is determined.
