A quantitative affinity-based technique for the identification of potential lead compounds.

Here, we describe the development and validation of a quantitative analytical method for rapid evaluation of protein-compound interactions. The method uses size-exclusion chromatography in a 96-well format with liquid chromatography/mass spectrometry (qSEC-LC/MS) by which the amount of a compound that was originally in complex with a target protein is determined as an indicator of the binding affinity. Proof of concept of this new analytical approach was performed using a thrombin-inhibitor model.

A quantitative analysis to unveil specific binding proteins for bioactive compounds.

The mechanisms of the action of bioactive compounds discovered via 'black box' assays using phenotypic indicators generally remain unknown, with the major challenges being the identification of target proteins. In this study, we aimed to develop an efficient methodology to unveil target proteins that are rarely characterised. Proof-of-concept experiments were performed using N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a well-known calmodulin (CaM) antagonist, as a bait compound.

A fluorescence polarization-based assay for the identification and evaluation of calmodulin antagonists.

A fluorescence polarization (FP) assay was developed to identify calmodulin (CaM) antagonists. A fluorescent tracer was newly designed by covalently labeling N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which is a well-known CaM antagonist, with the Cy5 dye. In the FP assay, the tracer (Cy5-W-7) was bound to CaM with a dissociation constant (K(d)) of 6.

Novel membrane protein containing glycerophosphodiester phosphodiesterase motif is transiently expressed during osteoblast differentiation.

Osteoblast maturation is a multistep series of events characterized by an integrated cascade of gene expression that are accompanied by specific phenotypic alterations. To find new osteoblast-related genes we cloned differentially expressed cDNAs characteristic of specific differentiation stages in the mouse osteoblast-like MC3T3-E1 cells by a differential display method. We identified a novel cDNA encoding a putative glycerophosphodiester phosphodiesterase, GDE3, which specifically was expressed at the stage of matrix maturation.

The Serratia marcescens bioH gene encodes an esterase.

The 3.9 kb chromosomal DNA was cloned from Serratia marcescens Sr41, which confers on Escherichia coli cells a phenotype of clear halo formation on tributyrin agar plates. Three complete open reading frames (ORFs) were identified in the inserted DNA, and one ORF was demonstrated to encode a 28 kDa protein of 255 amino acids related to esterase activity.

Receptor activator of NF-kappaB ligand (RANKL) activates TAK1 mitogen-activated protein kinase kinase kinase through a signaling complex containing RANK, TAB2, and TRAF6.

The receptor activator of NF-kappaB (RANK) and its ligand RANKL are key molecules for differentiation and activation of osteoclasts. RANKL stimulates transcription factors AP-1 through mitogen-activated protein kinase (MAPK) activation, and NF-kappaB through IkappaB kinase (IKK) activation. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is essential for activation of these kinases.