Quantitative determination of cardiac glycosides in Digitalis lanata leaves by reversed-phase thin-layer chromatography

We have recently chosen to undertake a comprehensive evaluation of the modulation of gene expression by oxidative stress, using mRNA as a marker. Our model system is HA-1 hamster fibroblasts, using conditions under which we observe an adaptive response. Under these conditions, the HA-1 cells respond to a minimally toxic pretreatment dose of hydrogen peroxide by synthesizing RNAs and proteins that protect them against subsequent exposure to a highly cytotoxic concentration of hydrogen peroxide. Using the differential display technique to screen for modulated RNAs, we have recently reported an RNA species, adapt15/gadd7, whose steady-state level is significantly induced by a pretreatment dose of hydrogen peroxide (D. R. Crawford, G. P. Schools, S. L. Salmon, and K. J. A. Davies (1996) Arch. Biochem. Biophys. 325, 256-264). Here we report a second induced mRNA, designated adapt33. Two homologous adapt33 mRNAs were revealed by Northern blot hybridization. Both of these species were inducible by hydrogen peroxide, and they were sized at 1.46 and 0.99 kb. These inductions appeared to be dependent upon calcium, occurred as early as 90 min, and were maximal at 5 h. Cell fractionation revealed that a significant proportion of adapt33 RNA is associated with active translation. adapt33 is a novel sequence, as determined by cloning, sequencing, and GenBank analysis. adapt33 represents a new oxidant-inducible RNA and marker of cellular oxidative stress and a potential aid in the study, detection, and possible therapy of oxidant-related disorders.