Drug inhibition of Gly-Sar uptake and hPepT1 localization using hPepT1-GFP fusion protein

An hPepT1-GFP fusion construct was made to study drug inhibition of dipeptide uptake and apical, basolateral, or subcellular hPepT1 localization. The hPepT1 stop codon was mutated by polymerase chain reaction and was subsequently cloned into the pEGFP-N1 vector. The hPepT1-GFP fusion construct was then transfected into Caco-2 and HeLa cells, and drug inhibition was studied by inhibiting 3H-Gly-Sar uptake. Western blot analysis was used to determine hPepT1-GFP expression levels and confocal microscopy was used to examine the localization. Both anti-hPepT1 antibody and anti-GFP antibody recognized a 120kd hPepT1-GFP fusion protein in the transfected cells. The 3H-Gly-Sar uptake in transfected HeLa cells was enhanced more than 20 times compared with the control. Valacyclovir (5 mmol/L) was able to completely inhibit 3H-Gly-Sar uptake in these transfected cells. Confocal microscopy showed that the hPepT1-GFP mainly localized in the Caco-2 cell apical membrane, but was present throughout the entire HeLa cell membranes. The hPepT1-GFP fusion protein was not found in either early endosome or lysosome of Caco-2 cells under normal conditions; however, it was detected in some subsets of lysosomes and early endosomes in phorbol 12-myristate 13-acetate (PMA)-treated Caco-2 cells

The processing of antigens delivered as DNA vaccines

The ability of DNA vaccines to provide effective immunological protection against infection and tumors depends on their ability to generate good CD4+ and CD8+ T-cell responses. Priming of these responses is a property of dendritic cells (DCs), and so the efficacy of DNA-encoded vaccines is likely to depend on the way in which the antigens they encode are processed by DCs. This processing could either be via the synthesis of the vaccine-encoded antigen by the DCs themselves or via its uptake by DCs following its synthesis in bystander cells that are unable to prime T cells. These different sources of antigen are likely to engage different antigen-processing pathways, which are the subject of this review. Understanding how to access different processing pathways in DCs may ultimately aid the rational development of plasmid-based vaccines to pathogens and to cancer