No abstract

Transport assays using P-gp-expressing cell lines are commonly used to identify P-gp substrates and inhibitors in drug discovery. The P-gp cell-based assay is performed manually in 12- or 24-well plates and requires improvement for high-throughput screening. In this study, we established an efficient semiautomated 96-well transport assay using LLC-PK1 cells transfected with human P-gp. The protocol was optimized with a microplate washer for exchanging media and buffer to enhance throughput. P-gp substrates and inhibitors, and the paracellular marker Dextran Texas Red® were used to validate the 96-well transport assay. Cell monolayer integrity after washing by a microplate washer was confirmed by measuring paracellular permeability of Dextran Texas Red. Permeability and net flux ratio of the P-gp substrates and the inhibitory potency of the P-gp inhibitors were comparable in 24- and 96-well plates. The regression value of net flux ratio of P-gp substrates was high between the two formats (r²=0.99). The optimized 96-well transport assay using the microplate washer was found to be an efficient high-throughput screening tool that provided the same quality data as the 24-well plate for the identification of P-gp substrates and inhibitors in drug discovery.

Two-component signal transduction is the primary signaling mechanism used for global regulation of cell response to changes in the environment. DNA microarray analysis identified genes up-regulated by acid stress in cyanobacteria Synechocystis sp. PCC 6803. Several of these altered genes are thought to be response regulators that are directly involved in this type of stress. Deletion mutants of response regulator genes were constructed and survivability was compared between the cells transfected with mutant and wild-type genes in a low-pH medium. Among these, deletion of slr0081 affected the growth rate under conditions of acid stress (pH 6.0). We examined the genome-wide expression of genes in ǻslr0081 mutant cells by using DNA microarray in an attempt to determine whether slr0081 is involved in the regulation of other acid stress responsive genes. Our findings by quantitative real-time RT PCR revealed that down regulation of acid responsive genes slr0967 and sll0939 occurs by deletion of slr0081.