Construction of a Near-Infrared-Activatable Enzyme Platform To Remotely Trigger Intracellular Signal Transduction Using an Upconversion Nanoparticle.

Photoactivatable (caged) bioeffectors provide a way to remotely trigger or disable biochemical pathways in living organisms at a desired time and location with a pulse of light (uncaging), but the phototoxicity of ultraviolet (UV) often limits its application. In this study, we have demonstrated the near-infrared (NIR) photoactivatable enzyme platform using protein kinase A (PKA), an important enzyme in cell biology. We successfully photoactivated PKA using NIR to phosphorylate its substrate, and this induced a downstream cellular response in living cells with high spatiotemporal resolution. In addition, this system allows NIR to selectively activate the caged enzyme immobilized on the nanoparticle surface without activating other caged proteins in the cytosol. This NIR-responsive enzyme-nanoparticle system provides an innovative approach to remote-control proteins and enzymes, which can be used by researchers who need to avoid direct UV irradiation or use UV as a secondary channel to turn on a bioeffector.

Influence of extremely low frequency magnetic fields on Ca2+ signaling and NMDA receptor functions in rat hippocampus.

Extremely low frequency (ELF<300Hz) electromagnetic fields affect several neuronal activities including memory. Because ELF magnetic fields cause altered Ca(2+) homeostasis in neural tissues, we examined their influence on Ca(2+) signaling enzymes in hippocampus and related them with NMDA receptor functions. Hippocampal regions were obtained from brains of 21-day-old rats that were exposed for 90 days to 50Hz magnetic fields at 50 and 100 microT intensities. In comparison to controls, ELF exposure caused increased intracellular Ca(2+) levels concomitant with increased activities of Ca(2+)-dependent protein kinase C (PKC), cAMP-dependent protein kinase and calcineurin as well as decreased activity of Ca(2+)-calmodulin-dependent protein kinase in hippocampal regions. Simultaneous ligand-binding studies revealed decreased binding to N-methyl-D-aspartic acid (NMDA) receptors. The combined results suggest that perturbed neuronal functions caused by ELF exposure may involve altered Ca(2+) signaling events contributing to aberrant NMDA receptor activities.

Molecular dynamics simulation of laser desorption of a fragment of protein kinase A from two MALDI matrices.

We have carried out molecular dynamics simulations to study the desorption of a dephosphorylated fragment of protein kinase A from two matrices, sinapic acid (SA) and 2,5-dihydroxybenzoic acid (DHB), after laser excitation. We have examined the results as a function of the laser fluence and of the burial depth of the guest peptide in the matrices. In most cases, we found that the energy transferred from the matrix to the guest peptide was not sufficiently large to fragment the peptide. Exceptions occurred when the peptide was more buried. This finding suggested that protein analytes might be less likely to break into smaller fragments if they were placed closer to the surface of the matrix. We have also examined how likely the guest peptide could form small clusters with the matrix molecules and found that the results depended on the degree of burial of the peptide, on the laser fluence, and on which matrix was used. Generally, stable clusters were more likely to be formed for guest peptides that were more buried, at a lower laser fluence, and in the SA rather than the DHB matrix. In addition, we found that the DHB matrix was broken down more easily by the laser than the SA matrix.

[Effect of x-irradiation on the properties of cyclic AMP-dependent protein kinases from rat spleen lymphocytes]. / Vliianie rentgenovskogo oblucheniia na svoistva tsAMF-zavisimykh proteinkinaz iz limfotsitov selezenki krys.

It was shown, that in conditions of acute radiation affection in doze 0.5 and 1 Gy, there was a decrease of 32 and 55% in activity of cyclic AMP-dependent protein kinases, isolated from cytosol of lymphocytes. The analysis of cAMP-dependent protein kinases properties has shown, that disturbance in the interaction of the enzyme with protein substrate of phosphotransferase reaction and main modulater enzymes activity--cAMP proceeds of the ionising radiation effect.

[Autophosphorylation in regulation of activity of cAMP-dependent protein kinases from spleen lymphocytes under ionizing radiation]. / Rol' avtofosforilirovaniia vreguliatsii aktivnosti cAMP-zavisimykh proteinkinaz iz limfotsitov selezenki krys v usloviiakh vzaimodelstviia ioniziruiushchel radiatsii.

It was found that the irradiation in doses 0.5 and 1 Gy does not make any essential effect on the process of the autophosphorylation of cAMP-dependent protein kinases, extracted from the spleen lymphocytes 12 hours after exposure to the radiation. However, the decrease in the affinity of the holoenzyme to the cyclic nucleotide was found. The increase of the dose leads to the intensity of the effect (0.5 Gy--1.5 times decrease, 1 Gy--3 times decrease).