Detection of juvenile hormone agonists by a new reporter gene assay using yeast expressing Drosophila methoprene-tolerant.

Juvenile hormones (JHs) are sesquiterpenoids that play important roles in the regulation of growth, metamorphosis, and reproduction in insects. Synthetic JH agonists (JHAs) have been used as insecticides and are categorized as a class of insect growth regulators (IGRs). Natural JHs and synthetic JHAs bind to the JH receptor methoprene-tolerant (Met), which forms a functional JH-receptor complex with steroid receptor coactivators, such as Drosophila melanogaster Taiman (Tai). The ligand-bound Met-Tai complex induces the transcription of JH response genes by binding to specific DNA elements referred to as JH response elements (JHREs). In the present study, we established a reporter gene assay (RGA) for detecting natural JHs and synthetic JHAs in a yeast strain expressing D. melanogaster Met and Tai. The yeast RGA system detected various juvenoid ligands in a dose-dependent manner. The rank order of the ligand potencies of the juvenoids examined in the yeast RGA linearly correlated with those of RGAs for Met-Tai established in mammalian and insect cells. Our new yeast RGA is rapid, easy to handle, cost-effective, and valuable for screening novel JHAs.

Facile fabrication of PEG-coated PLGA microspheres via SPG membrane emulsification for the treatment of scleroderma by ECM degrading enzymes.

We developed a facile fabrication method for preparing poly(ethylene glycol)(PEG)-coated poly (lactic-co-glycolic acid) (PLGA) microspheres with homogeneous size distribution via a combination of mPEG-b-PLGA and Shirasu Porous Glass membrane emulsification. Subsequently, extracellular matrix (ECM) degrading enzymes, collagenase (COLase) or hyaluronidase (HAse) were loaded into the microspheres. The obtained microspheres exhibited a sustained release of COLase or HAse over 10 days. The degradation of ECM polymers by the released COLase and HAse was confirmed in vitro. Reversal of established dermal fibrosis via degradation of over-deposited ECM is a promising treatment for scleroderma. The therapeutic effects of COLase- and HAse-loaded PLGA microspheres on scleroderma were evaluated in vivo following their intradermal administration to a bleomycin-induced mice model of scleroderma. COLase- and HAse-loaded PLGA microspheres decreased scleroderma dermal thickness without altering the mechanical properties of skin, whereas the administration of free COLase and HAse solution induced overdecomposition of skin ECM and α-SMA expression. The facile one-pot synthesis of PEG-coated PLGA microspheres with high colloidal stability and narrow size distribution could be employed as a drug carrier for various diseases in future.

New reporter gene assays for detecting natural and synthetic molting hormone agonists using yeasts expressing ecdysone receptors of various insects.

Synthetic nonsteroidal ecdysone agonists, a class of insect growth regulators (IGRs), target the ecdysone receptor (EcR), which forms a heterodimer with ultraspiracle (USP) to transactivate ecdysone response genes. These compounds have high binding affinities to the EcR-USP complexes of certain insects and their toxicity is selective for certain taxonomic orders. In the present study, we developed reporter gene assay (RGA) systems to detect molting hormone (ecdysone) activity by introducing EcR-USP cDNA and a bacterial lacZ reporter gene into yeast. EcR and USP were derived from the insect species of three different taxonomic orders: Drosophila melanogaster (Diptera), Chilo suppressalis (Lepidoptera), and Leptinotarsa decemlineata (Coleoptera). Transcriptional coactivator taiman (Tai) cDNA cloned from D. melanogaster was also used in this RGA system. This yeast RGA system responded to various EcR ligands in a dose-dependent and ecdysteroid-specific manner. Furthermore, the insect order-selective ligand activities of synthetic nonsteroidal ecdysone agonists were linearly related to their binding activities, which were measured against in vitro translated EcR-USP complexes. Our newly established yeast RGA is useful for screening new molting hormone agonists that work selectively on target insects.