New Screening Methods for Chemicals with Hormonal Activities Using Interaction of Nuclear Hormone Receptor with Coactivator

Evaluation of wastewater reclamation technologies based on in vitro and in vivo bioassays

“…pGBT9-NRs and pGAD424-TIF2 were integrated into Saccharomyces cerevisiae Y190, which was provided by Osaka University. The assay procedure was the same as described in a previous paper (Nishikawa et al, 1999).…”

Tools to evaluate estrogenic potency of dietary phytoestrogens:A consensus paper from the EU Thematic Network “Phytohealth” (QLKI-2002-2453)

“…One of the most utilized is the "GST (glutathione S-transferase) pulldown assay" (Nishikawa et al, 1999), based on a fusion protein of GST and the ER of interest (GST-ER). The GST-ER fusion protein is incubated with a radio-labeled co-activator in the presence of an ER ligand.…”

“…The interaction between the co-activator and the ER is associated to the characteristics of the ligand and proportional to its response. These associations allow the determination of the potency of a compound in triggering the binding of ERα and ERβ with a coactivator (Onate et al, 1995;Nishikawa et al, 1999;Kraichely et al, 2000;Routledge et al, 2000).…”

“…However, the transformation of a suitable mammalian cell type with a third expression plasmid carrying the yeast β-galactosidase as a reporter gene, makes possible to perform a "two-hybrid" assay in the proper cell context, overcoming any possible interference with different posttranslational modifications (Nishikawa et al, 1999). McDonnell et al, 1995;Shelby et al, 1996;McDonnell, 1999).…”

Estrogenic and Antiestrogenic Activities of Phloridzin

“…Yeast Two-Hybrid Assay The yeast two-hybrid assay was carried out according to the method of Nishikawa et al 19) And yeast cells expressing estrogen receptor (ERs) were allowed to grow overnight at 30°C with shaking in synthetic defined (SD) medium lacking tryptophan and leucine. Yeast cells were treated with a test compound for 4 h at 30°C, and b-galactosidase activity was determined as follows.…”