References of "Maass, Alexander H."
     in
Bookmark and Share    
Full Text
Peer Reviewed
See detailOptimization of gene transfer into neonatal rat cardiomyocytes and unmasking of cytomegalovirus promoter silencing.
Bauer, Sebastian; Maier, Sebastian K. G.; Neyses, Ludwig UL et al

in DNA and cell biology (2005), 24(6), 381-7

Cardiomyocytes are notoriously difficult to transfect using standard techniques unless viral vectors such as recombinant adenoviruses are used. Generation of recombinant adenoviruses is, however, a ... [more ▼]

Cardiomyocytes are notoriously difficult to transfect using standard techniques unless viral vectors such as recombinant adenoviruses are used. Generation of recombinant adenoviruses is, however, a complex and time-consuming procedure and not possible for every DNA construct. We therefore optimized DNA/polylysine/adenovirus complexing for efficient gene transfer in neonatal rat cardiomyocytes determining the critical parameters for this method. Importantly, not only the concentration of the various components but also the method used for plasmid purification is critical for this transfection technique. Cesium-chloride-purified DNA is inferior to anion-exchange methods for this purpose possibly because of altered ionic properties. In the second part of this study, we could demonstrate silent gene transfer into cardiomyocytes applying this optimized technique to plasmids encoding luciferase or beta-galactosidase cDNAs under the control of the cytomegalovirus immediate-early promoter. Phorbol myristate acetate and/or forskolin increased the amount of beta-galactosidase positive cells up to fivefold. Luciferase activity could even be increased as much as ninefold. These results demonstrate that the cytomegalovirus promoter is not maximally active in neonatal rat cardiomyocytes under basal conditions. In fact, a large proportion of cells is silently transfected and seems to express (an) inhibitor(s) of transcription from the CMV promoter that can be overcome by stimulation of cAMP- or protein kinase C-dependent pathways. [less ▲]

Detailed reference viewed: 178 (0 UL)
Full Text
Peer Reviewed
See detailThe sarcolemmal calcium pump inhibits the calcineurin/nuclear factor of activated T-cell pathway via interaction with the calcineurin A catalytic subunit.
Buch, Mamta H.; Pickard, Adam; Rodriguez, Antonio et al

in The Journal of biological chemistry (2005), 280(33), 29479-87

The calcineurin/nuclear factor of activated T-cell (NFAT) pathway represents a crucial transducer of cellular function. There is increasing evidence placing the sarcolemmal calcium pump, or plasma ... [more ▼]

The calcineurin/nuclear factor of activated T-cell (NFAT) pathway represents a crucial transducer of cellular function. There is increasing evidence placing the sarcolemmal calcium pump, or plasma membrane calcium/calmodulin ATPase pump (PMCA), as a potential modulator of signal transduction pathways. We demonstrate a novel interaction between PMCA and the calcium/calmodulin-dependent phosphatase, calcineurin, in mammalian cells. The interaction domains were located to the catalytic domain of PMCA4b and the catalytic domain of the calcineurin A subunit. Endogenous calcineurin activity, assessed by measuring the transcriptional activity of its best characterized substrate, NFAT, was significantly inhibited by 60% in the presence of ectopic PMCA4b. This inhibition was notably reversed by the co-expression of the PMCA4b interaction domain, demonstrating the functional significance of this interaction. PMCA4b was, however, unable to confer its inhibitory effect in the presence of a calcium/calmodulin-independent constitutively active mutant calcineurin A suggesting a calcium/calmodulin-dependent mechanism. The modulatory function of PMCA4b is further supported by the observation that endogenous calcineurin moves from the cytoplasm to the plasma membrane when PMCA4b is overexpressed. We suggest recruitment by PMCA4b of calcineurin to a low calcium environment as a possible explanation for these findings. In summary, our results offer strong evidence for a novel functional interaction between PMCA and calcineurin, suggesting a role for PMCA as a negative modulator of calcineurin-mediated signaling pathways in mammalian cells. This study reinforces the emerging role of PMCA as a molecular organizer and regulator of signaling transduction pathways. [less ▲]

Detailed reference viewed: 149 (0 UL)