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1.
Anal Bioanal Chem ; 408(22): 5973-84, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26973237

RESUMO

Enzyme-linked immunosorbent assay (ELISA) is commonly used to determine food allergens in food products. However, a significant number of ELISAs give an erroneous result, especially when applied to highly processed food. Accordingly, an improved ELISA, which utilizes an extraction solution comprising the surfactant sodium lauryl sulfate (SDS) and reductant 2-mercaptoethanol (2-ME), has been specially developed to analyze food allergens in highly processed food by enhancing analyte protein extraction. Recently, however, the use of 2-ME has become undesirable. In the present study, a new extraction solution containing a human- and eco-friendly reductant, which is convenient to use at the food manufacturing site, has been established. Among three chemicals with different reducing properties, sodium sulfite, tris(3-hydroxypropyl)phosphine, and mercaptoethylamine sodium sulfite was selected as a 2-ME substitute. The protein extraction ability of SDS/0.1 M sodium sulfite solution was comparable to that of SDS/2-ME solution. Next, the ELISA performance for egg, milk, wheat, peanut, and buckwheat was evaluated by using model-processed foods and commercially available food products. The data showed that the SDS/0.1 M sulfite ELISA significantly correlated with the SDS/2-ME ELISA for all food allergens examined (p < 0.01), thereby establishing the validity of the SDS/0.1 M sulfite ELISA performance. Furthermore, the new SDS/0.1 M sulfite solution was investigated for its applicability to the lateral-flow (LF) test. The result demonstrated the successful analysis of food allergens in processed food, showing consistency with the SDS/0.1 M sulfite ELISA results. Accordingly, a harmonized analysis system for processed food comprising a screening LF test and a quantitative ELISA with identical extraction solution has been established. The ELISA based on the SDS/0.1 M sulfite extraction solution has now been authorized as the revised official method for food allergen analysis in Japan.


Assuntos
Alérgenos/isolamento & purificação , Fracionamento Químico/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Análise de Alimentos/métodos , Substâncias Redutoras/química , Alérgenos/análise , Animais , Arachis/química , Ovos/análise , Hipersensibilidade Alimentar/diagnóstico , Humanos , Mercaptoetilaminas/química , Leite/química , Fosfinas/química , Sulfitos/química , Triticum/química
2.
Biochem Biophys Res Commun ; 336(2): 617-24, 2005 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-16140268

RESUMO

Signal transducer and activator of transcription 3 (STAT3), which mediates biological actions in many physiological processes, is activated by cytokines and growth factors via specific tyrosine phosphorylation, dimerization, and nuclear translocation. However, the mechanism involved in its nuclear translocation remains unclear. A previous study demonstrated that STAT3 with Arg-214/215 mutations in the coiled-coil domain (R214A/R215A; STAT3 RA) failed to undergo nuclear translocation. Here, we re-examined the nature of the STAT3 RA mutant and found that it showed higher and more extensive tyrosine-phosphorylation as well as much higher STAT3 transcriptional activity in response to stimuli. Furthermore, STAT3 RA showed nuclear translocation and faster nuclear export than wild-type STAT3 after stimulation. Moreover, nuclear retention of STAT3 RA by a chromosomal region maintenance 1 (CRM1) inhibitor, leptomycin B, decreased the enhanced STAT3 activation by stimuli. These data demonstrate that Arg-214/215 are involved in CRM1-mediated STAT3 nuclear export and the regulation of STAT3 activity.


Assuntos
Transporte Ativo do Núcleo Celular , Carcinoma Hepatocelular/metabolismo , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transativadores/química , Transativadores/metabolismo , Substituição de Aminoácidos , Carcinoma Hepatocelular/ultraestrutura , Linhagem Celular , Humanos , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Fator de Transcrição STAT3 , Relação Estrutura-Atividade , Proteína Exportina 1
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