Several cationic amphiphilic drugs cause local or systemic phospholipidosis (PLD) after chronic exposure in preclinical species. PLD is characterized by the accumulation of drug, phospholipid, and concentric lamellar bodies in cellular lysosomes. We have developed a fluorescence-based in vitro screen that is predictive of PLD using the Cellomics ArrayScan high-content screening platform, which captures and analyzes images from 96-well cell culture microtiter plates using multichannel fluorescence microscopy. I-13.35 adherent mouse spleen macrophage cells were cultured with drug and a fluorescently tagged phospholipid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). Drug concentrations were used in a range from 1 to 100 micro mol/L. After 24 h incubations, the cells were fixed with formalin. NBD-PE uptake was quantified in controls and treated cells. Nuclei were identified by Hoechst 33258 staining and dead cells were identified using ethidium homodimer-2 incorporation. Thus, confounding accumulation of NBD-PE due to cytotoxicity that produces false-positive results at high concentrations was eliminated from quantitation by ethidium staining and employing cell gating (dead cell rejection). The assay was found to be both sensitive and selective in that 26 of 28 positive, phospholipidogenic controls and 8 of 8 negative, non-phospholipidogenic controls were correctly called.

No abstract

No abstract

Translation elongation factor EF-1 alpha became stably associated with potato tuber polysomes at the onset of hypoxia, coincident with a sharp rise in lactate and decrease in tissue pH. This aberrant association of EF-1 alpha with polysomes also occurred when aerobic tuber extracts were acidified in vitro. Upon resumption of protein synthesis, an increase in the steady-state levels of EF-1 alpha, and expression of an EF-1 alpha/GUS transgene was observed. These results indicate that translational arrest results from to the failure of EF-1 alpha to dissociate from ribosomes during the elongation cycle, and that restoration of protein synthesis is coordinated with expression of EF-1 alpha.