RESUMO
The increasing prevalence of endocrine-disrupting chemicals (EDCs) in our environment is a growing concern, with numerous studies highlighting their adverse effects on the human endocrine system. Among the EDCs, estrogenic endocrine-disrupting chemicals (eEDCs) are exogenous compounds that perturb estrogenic hormone function by interfering with estrogen receptor (ER) homo (α/α, ß/ß) or hetero (α/ß) dimerization. To date, a comprehensive screening approach for eEDCs affecting all ER dimer forms in live cells is lacking. Here, we developed ER dimerization-detecting biosensors (ERDDBs), based on bioluminescence resonance energy transfer, for dimerization detection and rapid eEDC identification. To enhance the performance of these biosensors, we determined optimal donor and acceptor locations using computational analysis. Additionally, employing HaloTag as the acceptor and incorporating the P2A peptide as a linker yielded the highest sensitivity among the prototypes. We also established stable cell lines to screen potential ER dimerization inducers among estrogen analogs (EAs). The EAs were categorized through cross-comparison of ER dimer responses, utilizing EC values derived from a standard curve established with 17ß-estradiol. We successfully classified 26 of 72 EAs, identifying which ER dimerization types they induce. Overall, our study underscores the effectiveness of the optimized ERDDB for detecting ER dimerization and its applicability in screening and identifying eEDCs.
RESUMO
BACKGROUND: Honey is a natural product used as food, medicine, or cosmetics for very long time and is made by bees. Honey contains various components such as sugar, protein, minerals, and vitamins. Honey is made by Apis cerana or Apis mellifera, which commonly has major royal jelly proteins (MRJPs) as a major protein. To discriminate between natural honey (NH) and artificial honey (AH), many researchers tried method of physicochemical analysis. However, the analysis results were ambiguous and not stable. RESULTS: We have produced a monoclonal antibody that recognizes MRJPs of honeys in common. Monoclonal antibody has advantage such as accuracy, sensitivity, and stability as the standard. The specificity and affinity of produced antibody were measured by western blotting and enzyme-linked immunosorbent assay. As a result, this monoclonal antibody specifically recognized MRJPs of NH and did not recognize AH which has not including MRJPs. CONCLUSION: Natural honey could be able to distinguish from AH accurately by using this monoclonal antibody. Also, this method could be commercially applicable.
RESUMO
Mast cells play crucial roles in the initiation of allergic inflammatory responses by releasing various mediators such as histamines, cytokines, and leukotrienes. In addition, signaling cascade pathways, such as the mitogen-activated protein kinase (MAPK) pathway, contribute to the regulation of mast cell degranulation. Accordingly, different research strategies have been pursued to develop anti-inflammatory and anti-allergic drugs by regulating these signaling pathways. The development of new drugs that inhibit mast cell degranulation may help in the treatment of allergies. In this study, we investigated the effects of coumarin derivatives on mast cell degranulation. The effect of coumarin derivatives on degranulation in rat basophilic leukemia (RBL)-2H3 cells was determined by a ß-hexosaminidase assay and histamine assay. A coumarin derivative 1 (C1), 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate, inhibited degranulation in a dose-dependent manner and demonstrated maximum therapeutic effect when used at 25µM. Additionally, these compounds inhibited the phosphorylation of the extracellular signal-regulated kinase (ERK) pathway. Taken together, these results indicate that 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate inhibits mast cell degranulation by suppressing the activation of the ERK pathway and this inhibitory effect suggests potential therapeutic strategies towards the prevention of allergic disorders.