Characterization of Ku702–NLS as Bipartite Nuclear Localization Sequence for Non-Viral Gene Delivery

Several barriers have to be overcome in order to achieve gene expression in target cells, e.g. cellular uptake, endosomal release and translocation to the nucleus. Nuclear localization sequences (NLS) enhance gene delivery by increasing the uptake of plasmid DNA (pDNA) to the nucleus. So far, only monopartite NLS were analysed for non-viral gene delivery. In this study, we examined the characteristics of a novel bipartite NLS like construct, namely NLS Ku70. We synthesized a dimeric structure of a modified NLS from the Ku70 protein (Ku702-NLS), a nuclear transport active mutant of Ku702-NLS (s1Ku702-NLS) and a nuclear transport deficient mutant of Ku702-NLS (s2Ku702). We examined the transfection efficiency of binary Ku702-NLS/DNA and ternary Ku702-NLS/PEI/DNA gene vector complexes in vitro by using standard transfection protocols as well as the magnetofection method. The application of Ku702-NLS and s1Ku702-NLS increased gene transfer efficiency in vitro and in vivo. This study shows for the first time that the use of bipartite NLS compounds alone or in combination with cationic polymers is a promising strategy to enhance the efficiency of non-viral gene transfer

Dominance of biologically produced nitrate in upland waters of Great Britain indicated by stable isotopes

Atmospheric deposition of nitrogen (N) compounds is the major source of anthropogenic N to most upland ecosystems, where leaching of nitrate (NO3−) into surface waters contributes to eutrophication and acidification as well as indicating an excess of N in the terrestrial catchment ecosystems. Natural abundance stable isotopes ratios, 15N/14N and 18O/16O (the “dual isotope” technique) have previously been used in biogeochemical studies of alpine and forested ecosystems to demonstrate that most of the NO3− in upland surface waters has been microbially produced. Here we present an application of the technique to four moorland catchments in the British uplands including a comparison of lakes and their stream inflows at two sites. The NO3− concentrations of bulk deposition and surface waters at three sites are very similar. While noting the constraints imposed by uncertainty in the precise δ18O value for microbial NO3−, however, we estimate that 79–98% of the annual mean NO3− has been microbially produced. Direct leaching of atmospheric NO3− is a minor component of catchment NO3− export, although greater than in many similar studies in forested watersheds. A greater proportion of atmospheric NO3− is seen in the two lake sites relative to their inflow streams, demonstrating the importance of direct NO3− deposition to lake surfaces in catchments where terrestrial ecosystems intercept a large proportion of deposited N. The dominance of microbial sources of NO3− in upland waters suggests that reduced and oxidised N deposition may have similar implications in terms of contributing to NO3− leaching