Nucleoside transporter expression profiles in human cardiac tissue show striking individual variability with overall predominance of hENT1.

Nucleoside transporters (NTs) are integral membrane transport proteins that modulate the flux of nucleosides such as adenosine across cell membranes. Two families of NTs exist, the concentrative NTs (CNTs, SLC28) and the equilibrative NTs (ENTs, SLC29). CNTs and ENTs transport anti-cancer and anti-viral nucleoside analog drugs and ENTs are also targets of drugs used to treat cardiac pathologies. Levels of some NT profiles have been shown to relate to clinical outcomes in the use of nucleoside analog drugs. However, currently, patient NT profile is not assessed prior to pharmacological administration of analog drugs. Here we describe a reliable method to determine a complete individual NT expression profile from human tissue using quantitative real-time PCR. We developed this assay on tissue (right atrial appendage, left internal mammary, aorta) from individuals undergoing cardiac surgery and compared these findings to the NT expression profiles in pooled whole heart tissue (normal and diseased). Data show that hENT1 is the most abundantly expressed NT, with highest expression levels in the aorta. However, NT expression profiles are highly variable among individuals and changes in NT expression between normal and diseased tissues were observed. These data are the first to describe the RNA expression patterns of all seven NT isoforms in the human heart. The methodology described here may be useful for quantitatively characterizing complete NT expression profiles in any human target tissue.

Genetic mapping of activity determinants within cellular prion proteins: N-terminal modules in PrPC offset pro-apoptotic activity of the Doppel helix B/B' region.

The PrP-like Doppel (Dpl) protein causes apoptotic death of cerebellar neurons in transgenic mice, a process prevented by expression of the wild type (wt) cellular prion protein, PrP(C). Internally deleted forms of PrP(C) resembling Dpl such as PrPDelta32-121 produce a similar PrP(C)-sensitive pro-apoptotic phenotype in transgenic mice. Here we demonstrate that these phenotypic attributes of wt Dpl, wt PrP(C), and PrPDelta132-121 can be accurately recapitulated by transfected mouse cerebellar granule cell cultures. This system was then explored by mutagenesis of the co-expressed prion proteins to reveal functional determinants. By this means, neuroprotective activity of wt PrP(C) was shown to be nullified by a deletion of the N-terminal charged region implicated in endocytosis and retrograde axonal transport (PrPDelta23-28), by deletion of all five octarepeats (PrPDelta51-90), or by glycine replacement of four octarepeat histidine residues required for selective binding of copper ions (Prnp"H/G"). In the case of Dpl, overlapping deletions defined a requirement for the gene interval encoding helices B and B' (DplDelta101-125). These data suggest contributions of copper binding and neuronal trafficking to wt PrP(C) function in vivo and place constraints upon current hypotheses to explain Dpl/PrP(C) antagonism by competitive ligand binding. Further implementation of this assay should provide a fuller understanding of the attributes and subcellular localizations required for activity of these enigmatic proteins.