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The phytohormone ethylene is widely involved in many developmental processes and is a crucial regulator of defense responses against biotic and abiotic stresses in plants. Ethylene-responsive element binding protein, a member of the APETALA2/ethylene response factor (AP2/ERF) superfamily, is a transcription factor that regulates stress-responsive genes by recognizing a specific cis-acting element of target DNA. A previous study showed only the NMR structure of the AP2/ERF domain of AtERF100 in complex with a GCC box DNA motif. In this report, we determined the crystal structure of AtERF96 in complex with a GCC box at atomic resolution. We analyzed the binding residues of the conserved AP2/ERF domain in the DNA recognition sequence. In addition to the AP2/ERF domain, an N-terminal α-helix of AtERF96 participates in DNA interaction in the flanking region. We also demonstrated the structure of AtERF96 EDLL motif, a unique conserved motif in the group IX of AP2/ERF family, might involve in the transactivation of defense-related genes. Our study establishes the structural basis of the AtERF96 transcription factor in complex with the GCC box, as well as the DNA binding mechanisms of the N-terminal α-helix and AP2/ERF domain. [ABSTRACT FROM AUTHOR]

We report on a fluorescence polarization assay for the detection of the target analyte ATP by making use of an aptasensor and of mass-amplifying CdTe-CdS quantum dots. The ATP aptamer was modified with digoxin antigen and hybridized with its complementary DNA that was modified with the CdTe-CdS quantum dots. Following the addition of digoxin antibody, the mass-amplifying aptasensor probe is formed as a result of the immuno reaction. In the presence of ATP, the polarization of fluorescence decreases because the digoxin antibody becomes dissociated due to the recognition of the ATP by the ATP aptamer. Under optimized conditions, the method has a linear response to ATP in the 10 to 350 μM concentration range, and the limit of detection is 3.7 μM. The method combines the specific recognition capability of aptamers with the sensitivity of an immunoreaction. It has good selectivity and sensitivity, and can be used to detect ATP in serum samples. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Aim: To develop and optimize a competitive fluorescence polarization (FP) method, and use it as a high-throughput screening (HTS) assay for drug discovery. Methods: Human lectin-like oxidized low-density lipoprotein receptor-1 (hLOX-1) and oxidized low-density lipoprotein (oxLDL) were used to establish a high-throughput fluorescence polarization assay to screen ligands of human LOX-1. A 96-well plate assay was performed with a fast plate reader. Three fluorescein isothiocyanate-labeled hLOX-1 concentrations (100, 200, and 400 nmol/L) were selected to be titrated by oxLDL (from 0.05 nmol/L to 100 μmol/L) in order to obtain optimal reactive concentrations. The concentration of Me2SO used (0%, 1%, 3%, 5%) and incubation time (15 min, 30 min, 1h, 2h) were optimized. The Z' factor was calculated to estimate the quality of FP-based HTS. Results: Concentrations of 200 nmol/L for human LOX-1 and 50 μmol/L for oxLDL were used in the actual assay. Concentrations of 0% to 5% Me2SO and different reaction times did not affect the FP-based HTS. The Z' value was 0.66. By using this detection and screening system, 12 700 compounds were screened and 3 ligands with an IC50 of less than 4.5 μmol/L were found. Conclusion: The established competitive FP-based assay is sensitive, stable, highly reproducible and robust, and suitable for HTS for ligands of the hLOX-1 receptor. [ABSTRACT FROM AUTHOR]