Pivotal Role of Reduced Glutathione in Oxygen-induced Regulation of the Na + /K + Pump in Mouse Erythrocyte Membranes

This study addresses the mechanisms of oxygen-induced regulation of ion transport pathways in mouse erythrocyte, specifically focusing on the role of cellular redox state and ATP levels. Mouse erythrocytes possess Na+/K+ pump, K+-Cl− and Na+-K+-2Cl− cotransporters that have been shown to be potential targets of oxygen. The activity of neither cotransporter changed in response to hypoxia-reoxygenation. In contrast, the Na+/K+ pump responded to hypoxic treatment with reversible inhibition. Hypoxia-induced inhibition was abolished in Na+-loaded cells, revealing no effect of O2 on the maximal operation rate of the pump. Notably, the inhibitory effect of hypoxia was not followed by changes in cellular ATP levels. Hypoxic exposure did, however, lead to a rapid increase in cellular glutathione (GSH) levels. Decreasing GSH to normoxic levels under hypoxic conditions abolished hypoxia-induced inhibition of the pump. Furthermore, GSH added to the incubation medium was able to mimic hypoxia-induced inhibition. Taken together these data suggest a pivotal role of intracellular GSH in oxygen-induced modulation of the Na+/K+ pump activit

Non-Markoffian effects of a simple nonlinear bath

We analyze a model of a nonlinear bath consisting of a single two-level system coupled to a linear bath (a classical noise force in the limit considered here). This allows us to study the effects of a nonlinear, non-Markoffian bath in a particularly simple situation. We analyze the effects of this bath onto the dynamics of a spin by calculating the decay of the equilibrium correlator of the spin's z-component. The exact results are compared with those obtained using three commonly used approximations: a Markoffian master equation for the spin dynamics, a weak-coupling approximation, and the substitution of a linear bath for the original nonlinear bath.Comment: 7 pages, 6 figure

Temporal Rac1 – HIF-1 crosstalk modulates hypoxic survival of aged neurons

Neurodegenerative diseases are frequently associated with hypoxic conditions. During hypoxia the neuronal cytoskeleton is rapidly reorganized and such abnormalities are directly linked to adverse outcomes. Besides their roles as master regulators of the cytoskeleton, the Rho GTPases are also involved in cellular processes stimulated by hypoxic stress. We investigated the contribution of Rac1-mediated signaling to hypoxic responses of mature neurons using primary cortical cells cultured for 17 days in vitro. We show Rac1 is both upregulated and activated during hypoxia. Pharmacological inhibition of Rac1, but not RhoA, completely abrogated hypoxic HIF-1α stabilization and expression of the HIF-1 targets VEGF and GLUT1. Furthermore activity of JNK and GSK3β were also highly dependent on Rac1 activity and biphasic effects were observed after 6 and 24 h of exposure. Notably, inhibition of either pathway suppressed HIF-1α accumulation. Although inhibition of Rac1 did not affect neuronal viability during acute exposure cell death was strongly induced after 24 h revealing a time-dependent effect of Rac1 signaling on survival. Thus hypoxia-activated Rac1 is critical for neuronal HIF-1α stabilization and survival during oxygen deprivation via integration of complex signaling cascades

NF-κB contributes to transcription of placenta growth factor and interacts with metal responsive transcription factor-1 in hypoxic human cells

Placenta growth factor (PlGF) is a member of the vascular endothelial growth factor family of cytokines that control vascular and lymphatic endothelium development. It has been implicated in promoting angiogenesis in pathological conditions via signaling to vascular endothelial growth factor receptor-1. PlGF expression is induced by hypoxia and proinflammatory stimuli. Metal responsive transcription factor 1 (MTF-1) was shown to take part in the hypoxic induction of PlGF in Ras-transformed mouse embryonic fibroblasts. Here we report that PlGF expression is also controlled by NF-κB. We identified several putative binding sites for NF-κB in the PlGF promoter/enhancer region by sequence analyses, and show binding and transcriptional activity of NF-κB p65 at these sites. Expression of NF-κB p65 from a plasmid vector in HEK293 cells caused a substantial increase of PlGF transcript levels. Furthermore, we found that hypoxic conditions induce nuclear translocation and interaction of MTF-1 and NF-κB p65 proteins, suggesting a role for this complex in hypoxia-induced transcription of PlG

Low cost microfluidic device for partial cell separation: micromilling approach

Several studies have already demonstrated that it is possible to perform blood flow studies in microfluidic systems fabricated by using low-cost techniques. However, most of these techniques do not produce microchannels smaller than 100 microns and as a result they have several limitations related to blood cell separation. Recently, manufacturers have been able to produce milling tools smaller than 100 microns, which consequently have promoted the ability of micromilling machines to fabricate microfluidic devices able to perform separation of red blood cells (RBCs) from plasma. In this work, we show the ability of a micromilling machine to manufacture microchannels with dimensions down to 30 microns. Additionally, we show for the first time the ability of the proposed microfluidic device to enhance the cell-free layer close to the walls, leading to perform partial separation of RBCs from plasma.The authors acknowledge the financial support provided by PTDC/SAU-ENB/116929/2010 and EXPL/EMSSIS/2215/2013 from FCT (Science and Technology Foundation), COMPETE, QREN and European Union (FEDER). RR and DP acknowledge, respectively, the PhD scholarships SFRH/BD/97658/2013 and SFRH/BD/89077/2012 attributed by FCT.info:eu-repo/semantics/publishedVersio

Spectroscopic evidence of anthropogenic compounds extraction from polymers by fluorescent dissolved organic matter in natural water

FDOM is one of the most important carriers of anthropogenic compounds in natural waters. It can combine with environmental contaminants and polymers to form diverse chemical structures. To this end, here a microfluidic chip was designed for the analysis of these changes in fluorescent dissolved organic matter (FDOM) fingerprints due to thermal treatment and varying time intervals of exposure. Excitation Emission Matrix Spectroscopy (EEMS) approach was utilized to detect and identify the inherent compounds in sampled FDOM. Strong direct correlations were founded, Spearman rank correlation values (ρ = 0.85 at α = 0.1, n = 4) and linear correlation R² = 0.8359 were noted between thermal treatment pattern 2 and fluorescence intensity of samples. Materials, acrylic based glue and cyclic olefin copolymer (COC) polymer, used to design the microfluidic sensor were determined to possess unique spectral features in the ultraviolet to green spectrum using EEMS. The study therefore provides an insight on methods to identify contaminants in natural waters. This underlines the potential of optical sensors providing measurements at fast intervals, enabling environmental monitoring

Furin inhibition prevents hypoxic and TGFβ-mediated blood-brain barrier disruption

Hypoxic blood-brain barrier (BBB) dysfunction is a common feature of CNS diseases however mechanisms underlying barrier disturbance are still largely unknown. This study investigated the role of transforming growth factor β (TGFβ), a cytokine known to induce expression of the proprotein convertase Furin, in hypoxia-mediated barrier compromise.We show that exposure of brain endothelial cells (ECs) to hypoxia (1% O2) rapidly stimulates their migration. Additional exogenous TGFβ (0.4nM) exposure potentiated this effect and increased Furin expression in a TGFβ type I receptor activin-like kinase 5 (ALK5) - dependent manner (prevented by 10μM SB431542). Furin inhibition prevented hypoxia-induced EC migration and blocked TGFβ-induced potentiation suggesting existence of a feedback loop. TGFβ and Furin were also critical for hypoxia-induced BBB dysfunction. TGFβ treatment aggravated hypoxia-induced BBB permeability but ALK5 or Furin blockade reversed injury-induced permeability changes. Thus during insult Furin compromises endothelial integrity by mediating the effects of TGFβ. Targeting the Furin or ALK5 pathway may offer novel therapeutic strategies for improving BBB stability and CNS function during disease