Chandrasekaran Vasudevan

PubMed2005

Improving high-content-screening assay performance by using division-arrested cells.

As cell-based assays are used more commonly in robotic high-throughput compound screening, cells themselves have become critical reagents. Thus, it has become essential to produce cell reagents with high consistency and quality. We experimented with cells division-arrested with low-level mitomycin C treatment and demonstrate that they perform with better consistency than non-division-arrested counterparts in high-content screening imaging assays. We propose that for cell-based screening, it is possible to uncouple the cell production process from the screening process. Cells can be produced en masse, treated to become irreversibly division-arrested, and cryopreserved. These "ready-to-use" reagents can be thawed, plated, and used in screening with improved consistency and convenience.

Antibiotics, Antineoplastic↗

PubMed2003

The guanine nucleotide exchange factor ARNO mediates the activation of ARF and phospholipase D by insulin.

BACKGROUND: Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin. RESULTS: Wild type ARNO transiently transfected in HIRcB cells was translocated to the plasma membrane in an insulin-dependent manner and promoted the translocation of ARF to the membranes. ARNO mutants: DeltaCC-ARNO and CC-ARNO were partially translocated to the membranes while DeltaPH-ARNO and PH-ARNO could not be translocated to the membranes. Sec7 domain mutants of ARNO did not facilitate the ARF translocation. Overexpression of wild type ARNO significantly increased insulin-stimulated PLD activity, and mutations in the Sec7 and PH domains, or deletion of the PH or CC domains inhibited the effects of insulin. CONCLUSIONS: Small ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by the insulin receptor.

ADP-Ribosylation Factors↗