Untersuchungen kombinierter DNA-Protein-Transduktion zur Optimierung nicht-viralen Gentransfers

Zur Etablierung eines rekombinanten phiC31-TAT-Systems für den nicht-viralen Gentransfer wurden verschiedene phiC31-Integrase-Konstrukte in E. coli BL21 Bakterien rekombinant exprimiert, chromatographisch aufgereinigt und sowohl im Reagenzglas als auch in renalen, alveolären und T-Zelllinien auf Funktionalität getestet. Zur Transduktion dieser Integrase-Plasmid-Komplexe in unterschiedliche Zelltypen wurden verschiedene Transduktionsmethoden verwendet. Stellvertretend für Lunge, Niere und Lymphozyten wurden A549-, 293- und Jurkat-T-Zellen mit Komplexen aus rekombinanter Integrase und einem Luziferase-attB-Plasmid behandelt und über einen Zeitraum von einem Monat auf Luziferaseexpression getestet. Der kombinierte phiC31-Integraseprotein-Plasmid-Transfer ist eine vielversprechende Anwendung für den nicht-viralen Gentransfer, was insbesondere durch die Vermittlung stabiler Langzeittransgenexpression in verschiedenen Zelltypen gezeigt werden konnte

Corporate Governance, Opaque Bank Activities, and Risk/Return Efficiency: Pre- and Post-Crisis Evidence from Turkey

Does better corporate governance unambiguously improve the risk/return efficiency of banks? Or does either a re-orientation of banks' revenue mix towards more opaque products, an economic downturn, or tighter supervision create off-setting or reinforcing effects? The authors relate bank efficiency to shortfalls from a stochastic risk/return frontier. They analyze how internal governance mechanisms (CEO duality, board experience, political connections, and education profile) and external governance mechanisms (discipline exerted by shareholders, depositors, or skilled employees) determine efficiency in a sample of Turkish banks. The 2000 financial crisis was a wakeup call for bank efficiency and corporate governance. As a result, better corporate governance mechanisms have been able to improve risk/return efficiency when the economic, regulatory, and supervisory environments are more stable and bank products are more complex.corporate governance;bank risk;noninterest income;crisis;frontier

Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus.

Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes

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

Cell type differences in activity of the Streptomyces bacteriophage ϕC31 integrase

Genomic integration by the Streptomyces bacteriophage ϕC31 integrase is a promising tool for non-viral gene therapy of various genetic disorders. We investigated the ϕC31 integrase recombination activity in T cell derived cell lines, primary T lymphocytes and CD34+ haematopoietic stem cells in comparison to mesenchymal stem cells and cell lines derived from lung-, liver- and cervix-tissue. In T cell lines, enhanced long-term expression above control was observed only with high amounts of integrase mRNA. Transfections of ϕC31 integrase plasmids were not capable of mediating enhanced long-term transgene expression in T cell lines. In contrast, moderate to high efficiency could be detected in human mesenchymal stem cells, human lung, liver and cervix carcinoma cell lines. Up to 100-fold higher levels of recombination product was found in ϕC31 integrase transfected A549 lung than Jurkat T cells. When the ϕC31 integrase activity was normalized to the intracellular integrase mRNA levels, a 16-fold difference was found. As one possible inhibitor of the ϕC31 integrase, we found 3- to 5-fold higher DAXX levels in Jurkat than in A549 cells, which could in addition to other yet unknown factors explain the observed discrepancy of ϕC31 integrase activity

The chromatin remodelling enzymes SNF2H and SNF2L position nucleosomes adjacent to CTCF and other transcription

Within the genomes of metazoans, nucleosomes are highly organised adjacent to the binding sites for a subset of transcription factors. Here we have sought to investigate which chromatin remodelling enzymes are responsible for this. We find that the ATP-dependent chromatin remodelling enzyme SNF2H plays a major role organising arrays of nucleosomes adjacent to the binding sites for the architectural transcription factor CTCF sites and acts to promote CTCF binding. At many other factor binding sites SNF2H and the related enzyme SNF2L contribute to nucleosome organisation. The action of SNF2H at CTCF sites is functionally important as depletion of CTCF or SNF2H affects transcription of a common group of genes. This suggests that chromatin remodelling ATPase's most closely related to the Drosophila ISWI protein contribute to the function of many human gene regulatory elements

The Drosophila speciation factor HMR localizes to genomic insulator sites

Hybrid incompatibility between Drosophila melanogaster and D. simulans is caused by a lethal interaction of the proteins encoded by the Hmr and Lhr genes. In D. melanogaster the loss of HMR results in mitotic defects, an increase in transcription of transposable elements and a deregulation of heterochromatic genes. To better understand the molecular mechanisms that mediate HMR's function, we measured genome-wide localization of HMR in D. melanogaster tissue culture cells by chromatin immunoprecipitation. Interestingly, we find HMR localizing to genomic insulator sites that can be classified into two groups. One group belongs to gypsy insulators and another one borders HP1a bound regions at active genes. The transcription of the latter group genes is strongly affected in larvae and ovaries of Hmr mutant flies. Our data suggest a novel link between HMR and insulator proteins, a finding that implicates a potential role for genome organization in the formation of species

CARM1 regulates alveolar epithelial senescence and elastase-induced emphysema susceptibility.

Chronic obstructive pulmonary disease (COPD) is characterized by an irreversible loss of lung function and is one of the most prevalent and severe diseases world-wide. A major feature of COPD is emphysema -the progressive loss of alveolar tissue. Coactivator-associated arginine methyltransferase-1 (CARM1) regulates histone-methylation and the transcription of genes involved in senescence, proliferation and differentiation. Complete loss of CARM1 leads to disrupted differentiation and maturation of alveolar epithelial type-II cells (ATII). We thus hypothesized that CARM1 regulates the development and progression of emphysema. To address this, we investigated the contribution of CARM1 to alveolar rarefication using the mouse model of elastase-induced emphysema in vivo and siRNA-mediated knockdown in ATII-like LA4 cells in vitro. We demonstrate that emphysema progression in vivo is associated with a time-dependent down-regulation of CARM1. Importantly, elastase-treated CARM1 haploinsufficient mice show significantly increased airspace enlargement (52.5±9.6 µm vs. 38.8±5.5 µm, p<0.01) and lung compliance (2.8±0.32 µl/cmH20 vs. 2.4±0.4 µl/cmH20, p<0.04) compared with controls. The knockdown of CARM1 in LA4 cells led to decreased SIRT1 expression (0.034±0.003 vs. 0.022±0.001, p<0.05), but increased expression of p16 (0.27±0.013 vs. 0.31±0.010, p<0.5), p21 (0.81±0.088 vs. 1.28± 0.063, p<0.01) and higher beta-galactosidase-positive senescent cells (50.57%±7.36 vs. 2.21%±0.34, p<0.001), compared with scrambled siRNA. We further demonstrated that CARM1 haploinsufficiency impairs trans-differentiation and wound healing (32.18%±0.9512 vs. 8.769%±1.967, p<0.001) of alveolar epithelial cells. Overall, these results reveal a novel function of CARM1 in regulating emphysema development and premature lung aging via alveolar senescence, as well as impaired regeneration, repair and differentiation of ATII cells