Plasma membrane calcium ATPase 4b inhibits nitric oxide generation through calcium-induced dynamic interaction with neuronal nitric oxide synthase.

The activation and deactivation of Ca(2+)- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved Förster resonance energy transfer (FRET), we determined the occurrence of Ca(2+)-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca(2+) concentrations ([Ca(2+)]i), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca(2+)]i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation.

Stable photoluminescent ZnO@Cd(OH)2 core-shell nanoparticles synthesized via ultrasonication-assisted sol-gel method.

Core-shell structured ZnO@Cd(OH)(2) nanoparticles with stable and improved luminescence have been prepared successfully via a facile ultrasonication-assisted sol-gel method. Their composition and structure have been confirmed by high resolution transmission electron microscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and infrared spectra. The size of the nanoparticles decreases gradually along with the increase in the shell thickness, indicating that Cd(OH)(2) shells can hider ZnO cores growth and aggregation effectively. The as-prepared core-shell nanoparticles can be stored at room temperature for several weeks without luminescence efficiency reduction, and they are quite stable at elevated temperatures or in moderate alkaline solutions due to the protection of the Cd(OH)(2) shell.

ESeroS-GS modulates lipopolysaccharide-induced macrophage activation by impairing the assembly of TLR-4 complexes in lipid rafts.

The binding of lipopolysaccharides (LPS) to macrophages results in inflammatory responses. In extreme cases it can lead to endotoxic shock, often resulting in death. A broad range of antioxidants, including tocopherols, can reduce LPS activity in vitro and in vivo. To elucidate the underlying mechanisms of their action, we investigated the effect of the sodium salt of γ-L-glutamyl-S-[2-[[[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl]oxy]carbonyl]-3-[[2-(1H-indol-3-yl)ethyl]amino]-3-oxopropyl]-L-cysteinylglycine (ESeroS-GS), a novel α-tocopherol derivative, on LPS-induced inflammation in vitro and in vivo. ESeroS-GS reduced the transcription of TNF-α, IL-1ß, IL-6 and iNOS genes in a dose-dependent manner in RAW264.7 macrophages, and inhibited the release of these inflammatory factors. In addition, ESeroS-GS inhibited LPS-induced mortality in a mouse sepsis model. Electrophoretic mobility shift assays (EMSA) and reporter gene assays revealed that ESeroS-GS down-regulated the transcriptional activity of NF-κB. By analyzing the partitioning of CD14 and Toll-like receptor 4 (TLR-4) in cell membrane microdomains, we found that ESeroS-GS attenuates the binding of LPS to RAW264.7 cells via interfering with the relocation of CD14 and TLR-4 to lipid rafts, blocking the activation of interleukin-1 receptor-associated kinase 1 (IRAK-1), and inhibiting the consequent phosphorylation of TAK1 and IKKα/ß, which together account for the suppression of NF-κB activation. Taken together, our data suggest that ESeroS-GS can modulate LPS signaling in macrophages by impairing TLR-4 complex assembly via a lipid raft dependent mechanism. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Induction of inducible nitric oxide synthase increases the production of reactive oxygen species in RAW264.7 macrophages.

Macrophages produce a large volume of ROS (reactive oxygen species) through respiratory burst. However, the influence of iNOS [inducible NOS (nitric oxide synthase)] activation on ROS production remains unclear. In the present study, the kinetic generation of ROS in RAW264.7 murine macrophages was monitored by chemiluminescence. PMA induces a robust chemiluminescence in RAW264.7 cells, suggesting PKC (protein kinase C)-related assembly and activation of NOX (NADPH oxidase). The effects of iNOS induction on ROS production were examined. Induction of iNOS expression in RAW264.7 cells with LPS (lipopolysaccharide; 1 microg/ml) causes a significant increase in PMA-induced chemiluminescence, which could be enhanced by the NOS substrate, L-arginine, and could be abolished by the NOS inhibitor, L-NNA (NG-nitro-L-arginine). Further experiments reveal that induction of iNOS expression enhances the PMA-stimulated phosphorylation of the p47phox subunit of NOX, and promotes the relocalization of cytosolic p47phox and p67phox subunits to the membrane. Inhibition of PKCzeta by its myristoylated pseudosubstrate significantly decreased the PMA-stimulated phosphorylation of the p47phox in LPS-pretreated cells, suggesting that PKCzeta is involved in the iNOS-dependent assembly and activation of NOX. Taken together, the present study suggests that the induction of iNOS upregulates the PMA-induced assembly of NOX and leads to the enhanced production of ROS via a PKCzeta-dependent mechanism.

Melatonin impairs NADPH oxidase assembly and decreases superoxide anion production in microglia exposed to amyloid-beta1-42.

Melatonin shows significant protective effects in Alzheimer's disease (AD) models in vitro and in vivo; these effects are related to its function as an antioxidant. The source of reactive oxygen species (ROS) generation in the AD brain is primarily the amyloid-beta (Abeta)- activated microglial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. However, the effects of melatonin on the activation of NADPH oxidase remain unclear. In the present study, the cultures of microglia were incubated in the presence of fibrillar Abeta(1-42), which induces the assembly and the activation of NADPH oxidase, and triggers the production of superoxide anion-derived ROS. Pretreatment of microglia with melatonin dose-dependently prevents the activation of NADPH oxidase and decreases the production of ROS. Melatonin inhibits the phosphorylation of the p47(phox) subunit of NADPH oxidase via a PI3K/Akt-dependent signalling pathway, blocks the translocation of p47(phox) and p67(phox) subunit to the membrane, down-regulates the binding of p47(phox) to gp91(phox), and impairs the assembly of NADPH oxidase. Our data offer new insights into the mechanism of inhibiting ROS generation by melatonin in Abeta-activated microglia. Inhibition of ROS production indirectly might be the underlying mechanism for the neuroprotection by melatonin in the AD brain.

Two-photon fluorescence measurements of reversible photodegradation in a dye-doped polymer.

We report on the dynamics of photodegradation and subsequent recovery of two-photon fluorescence in a dye-doped polymer. The energy dependence suggests that photodegradation is a linear process, while recovery is entropic. Such recovery could be useful to high-intensity devices such as two-photon absorbers, which can be used in many applications.

Pushing the hyperpolarizability to the limit.

We use numerical optimization to find a one-dimensional potential energy function that yields the largest hyperpolarizability, which we find is within 30% of the fundamental limit. Our results reveal insights into the character of the potential energy functions and wave functions that lead to the largest hyperpolarizability. We suggest that donor-acceptor molecules with a conjugated bridge with many sites of reduced conjugation to impart conjugation modulation may be the best paradigm for making materials with huge hyperpolarizabilities that approach the fundamental limit.