Restoration of mutant cytochrome P450 reductase activity by external flavin

Cytochrome P450 oxidoreductase (POR) supplies electrons from NADPH to steroid and drug metabolizing reactions catalyzed by the cytochrome P450s located in endoplasmic reticulum. Mutations in human POR cause a wide spectrum of disease ranging from disordered steroidogenesis to sexual differentiation. Previously we and others have shown that POR mutations can lead to reduced activities of steroidogenic P450s CYP17A1, CYP19A1 and CYP21A1. Here we are reporting that mutations in the FMN binding domain of POR may reduce CYP3A4 activity, potentially influencing drug and steroid metabolism; and the loss of CYP3A4 activity may be correlated to the reduction of cytochrome b(5) by POR. Computational molecular docking experiments with a FMN free structural model of POR revealed that an external FMN could be docked in close proximity to the FAD moiety and receive electrons donated by NADPH. Using FMN supplemented assays we have demonstrated restoration of the defective POR activity in vitro.

Restoration of Akt activity by the bisperoxovanadium compound bpV(pic) attenuates hippocampal apoptosis in experimental neonatal pneumococcal meningitis

Pneumococcal meningitis causes apoptosis of developing neurons in the dentate gyrus of the hippocampus. The death of these cells is accompanied with long-term learning and memory deficits in meningitis survivors. Here, we studied the role of the PI3K/Akt (protein kinase B) survival pathway in hippocampal apoptosis in a well-characterized infant rat model of pneumococcal meningitis. Meningitis was accompanied by a significant decrease of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and of phosphorylated (i.e., activated) Akt in the hippocampus. At the cellular level, phosphorylated Akt was decreased in both the granular layer and the subgranular zone of the dentate gyrus, the region where the developing neurons undergo apoptosis. Protein levels and activity of PTEN, the major antagonist of PI3K, were unaltered by infection, suggesting that the observed decrease in PIP(3) and Akt phosphorylation is a result of decreased PI3K signaling. Treatment with the PTEN inhibitor bpV(pic) restored Akt activity and significantly attenuated hippocampal apoptosis. Co-treatment with the specific PI3K inhibitor LY294002 reversed the restoration of Akt activity and attenuation of hippocampal apoptosis, while it had no significant effect on these parameters on its own. These results indicate that the inhibitory effect of bpV(pic) on apoptosis was mediated by PI3K-dependent activation of Akt, strongly suggesting that bpV(pic) acted on PTEN. Treatment with bpV(pic) also partially inhibited the concentration of bacteria and cytokines in the CSF, but this effect was not reversed by LY294002, indicating that the effect of bpV(pic) on apoptosis was independent of its effect on CSF bacterial burden and cytokine levels. These results indicate that the PI3K/Akt pathway plays an important role in the death and survival of developing hippocampal neurons during the acute phase of pneumococcal meningitis.

Laser scanning microscopy combined with image restoration to analyse a 3D model of the human epithelial airway barrier

A laser scanning microscope collects information from a thin, focal plane and ignores out of focus information. During the past few years it has become the standard imaging method to characterise cellular morphology and structures in static as well as in living samples. Laser scanning microscopy combined with digital image restoration is an excellent tool for analysing the cellular cytoarchitecture, expression of specific proteins and interactions of various cell types, thus defining valid criteria for the optimisation of cell culture models. We have used this tool to establish and evaluate a three dimensional model of the human epithelial airway wall.

In vivo evaluation of the first dedicated combined flow-restoration and mechanical thrombectomy device in a swine model of acute vessel occlusion

The use of self-expanding retrievable stents is an emerging and promising treatment strategy for acute stroke treatment. The concept combines the advantages of stent deployment with immediate flow-restoration and of mechanical thrombectomy with definitive thrombus removal. The present study was performed to gain more knowledge about the principle of combined flow restoration and thrombectomy in an established animal model using radiopaque thrombi evaluating efficiency, thrombus-device interaction and possible complications of the first dedicated flow-restoration and mechanical thrombectomy device.

Summer drought reduces total and litter-derived soil CO2 effluxes in temperate grassland - clues from a 13C litter addition experiment

Current climate change models predict significant changes in rainfall patterns across Europe. To explore the effect of drought on soil CO2 efflux (FSoil) and on the contribution of litter to FSoil we used rain shelters to simulate a summer drought (May to July 2007) in an intensively managed grassland in Switzerland by reducing annual precipitation by around 30% similar to the hot and dry year 2003 in Central Europe. We added 13C-depleted as well as unlabelled grass/clover litter to quantify the litter-derived CO2 efflux (FLitter). Soil CO2 efflux and the 13C/12C isotope ratio (δ13C) of the respired CO2 after litter addition were measured during the growing season 2007. Drought significantly decreased FSoil in our litter addition experiment by 59% and FLitter by 81% during the drought period itself (May to July), indicating that drought had a stronger effect on the CO2 release from litter than on the belowground-derived CO2 efflux (FBG, i.e. soil organic matter (SOM) and root respiration). Despite large bursts in respired CO2 induced by the rewetting after prolonged drought, drought also reduced FSoil and FLitter during the entire 13C measurement period (April to October) by 26% and 37%, respectively. Overall, our findings show that drought decreased FSoil and altered its seasonality and its sources. Thus, the C balance of temperate grassland soils respond sensitively to changes in precipitation, a factor that needs to be considered in regional models predicting the impact of climate change on ecosystems C balance.