ABSTRACT
Nitric oxide (NO) has numerous physiological roles in the cell. One of the actions of NO is gene regulation through protein modification and signal transduction. In neurons, NO can be produced from neuronal NO synthase, which is activated by calcium following N-methyl-D-aspartate (NMDA) receptor activation. Differential analysis of cDNA library expression (DAzLE) was used to identify differentially expressed genes by NO. Fundamentally, this technique combines differential hybridization to isolate genes whose expression is differentially regulated with microarray to analyze the expression of the isolated genes. The expression of genes identified by the DAzLE method is verified further by quantitative real-time polymerase chain reaction (RT-PCR) and/or Northern blot analysis. The high selectivity and sensitivity of this technique for detecting differentially expressed gene transcripts enable the investigation and identification of a panel of genes that are regulated by NO.
Subject(s)
DNA, Complementary/genetics , Gene Expression Regulation/physiology , Nitric Oxide/physiology , Animals , Blotting, Northern , Cells, Cultured , Mice , Mice, Inbred C57BL , NADPH Dehydrogenase/metabolism , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/physiology , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The mammalian BAD protein belongs to the BH3-only subgroup of the BCL-2 family. In contrast to its known pro-apoptotic function, we found that endogenous and overexpressed BAD(L) can inhibit cell death in neurons and other cell types. Several mechanisms regulate the conversion of BAD from an anti-death to a pro-death factor, including alternative splicing that produces the N-terminally truncated BAD(S). In addition, caspases convert BAD(L) into a pro-death fragment that resembles the short splice variant. The caspase site that is selectively cleaved during cell death following growth factor (interleukin-3) withdrawal is conserved between human and murine BAD. A second cleavage site that is required for murine BAD to promote death following Sindbis virus infection, gamma-irradiation, and staurosporine treatment is not conserved in human BAD, consistent with the inability of human BAD to promote death with these stimuli. However, loss of the BAD N terminus by any mechanism is not always sufficient to activate its pro-death activity, suggesting that the N terminus is a regulatory domain rather than an anti-death domain. These findings suggest that BAD is more than an inert death factor in healthy cells; it is also a pro-survival factor, prior to its role in promoting cell death.
