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Expression of Escherichia coli purine nucleoside phosphorylase (PNP) activates prodrugs and kills entire populations of mammalian cells, even when as few as 1% of the cells express this gene. This phenomenon of bystander killing has been previously investigated for herpes simplex virus-thymidine kinase (HSV-TK) and has been shown to require cell to cell contact. Using silicon rings to separate E. coli PNP expressing cells from nonexpressing cells sharing the same medium, we demonstrate that bystander cell killing by E. coli PNP does not require cell-cell contact. Initially, cells expressing E. coli PNP convert the non-toxic prodrug, 6-methylpurine-2-deoxyriboside (MeP-dR) to the highly toxic membrane permeable toxin, 6-methylpurine (MeP). As the expressing cells die, E. coli PNP is released into the culture medium, retains activity, and continues precursor conversion extracellularly (as determined by reverse phase high performance liquid chromatography of both prodrug and toxin). Bystander killing can also be observed in the absence of extracellular E. coli PNP by removing the MeP-dR prior to death of the expressing cells. In this case, 100% of cultured cells die when as few as 3% of the cells of a population express E. coli PNP. Blocking nucleoside transport with nitrobenzylthioinosine reduces MeP-dR mediated cell killing but not MeP cell killing. These mechanisms differ fundamentally from those previously reported for the HSV-TK gene.

The amygdala is critical for associating predictive cues with primary rewarding and aversive outcomes. This is particularly evident in tasks in which information about expected outcomes is required for normal responding. Here we used a pavlovian overexpectation task to test whether outcome signaling by amygdala might also be necessary for changing those representations in the face of unexpected outcomes. Rats were trained to associate several different cues with a food reward. After learning, two of the cues were presented together, in compound, followed by the same reward. Before each compound training session, rats received infusions of 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide or saline into either the basolateral (ABL) or central nucleus (CeN) of amygdala. We found that infusions into CeN abolished the normal decline in responding to the compounded cue in a later probe test, whereas infusions into ABL had no effect. These results are inconsistent with the proposal that signaling of information about expected outcomes by ABL contributes to learning, at least in this setting, and instead implicate the CeN in this process, perhaps attributable to the hypothesized involvement of this area in attention and variations in stimulus processing.

more listened to my ramblings about my research or, most importantly, subtly reminded me of other, much more important things in life and I offer my thanks to all.

No abstract