Phosphatidylinositol 3‐kinase (PI 3‐kinase) has a regulatory 85 kDa adaptor subunit whose SH2 domains bind phosphotyrosine in specific recognition motifs, and a catalytic 110 kDa subunit. Mutagenesis of the p110 subunit, within a sequence motif common to both protein and lipid kinases, demonstrates a novel intrinsic protein kinase activity which phosphorylates the p85 subunit on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This protein‐serine kinase activity is detectable only upon high affinity binding of the p110 subunit with its unique substrate, the p85 subunit. Tryptic phosphopeptide mapping revealed that the same major peptide was phosphorylated in p85 alpha both in vivo in cultured cells and in the purified recombinant enzyme. N‐terminal sequence and mass analyses were used to identify Ser608 as the major phosphorylation site on p85 alpha. Phosphorylation of the p85 subunit at this serine causes an 80% decrease in PI 3‐kinase activity, which can subsequently be reversed upon treatment with protein phosphatase 2A. These results have implications for the role of inter‐subunit serine phosphorylation in the regulation of the PI 3‐kinase in vivo.
In order to identify aqueous species formed in Plasma activated media (PAM), quantitative investigations of reactive oxygen and nitrogen species (ROS, RNS) were performed and compared to Milli-Q water and culture media without and with Fetal Calf Serum. Electron paramagnetic resonance, fluorometric and colorimetric analysis were used to identify and quantify free radicals generated by helium plasma jet in these liquids. Results clearly show the formation of ROS such as hydroxyl radical, superoxide anion radical and singlet oxygen in order of the micromolar range of concentrations. Nitric oxide, hydrogen peroxide and nitrite-nitrate anions (in range of several hundred micromolars) are the major species observed in PAM. The composition of the medium has a major impact on the pH of the solution during plasma treatment, on the stability of the different RONS that are produced and on their reactivity with biomolecules. To emphasize the interactions of plasma with a complex medium, amino acid degradation by means of mass spectrometry was also investigated using methionine, tyrosine, tryptophan and arginine. All of these components such as long lifetime RONS and oxidized biological compounds may contribute to the cytotoxic effect of PAM. This study provides mechanistic insights into the mechanisms involved in cell death after treatment with PAM.
By means of Delta-SCF and time-dependent density functional theory (DFT) calculations on [Ru(LL)3]2+ (LL = bpy = 2,2'-bipyridyl or bpz = 2,2' -bipyrazyl) complexes, we have found that emission of these two complexes could originate from two metal-to-ligand charge-transfer triplet states (3MLCT) that are quasi-degenerate and whose symmetries are D3 and C2. These two states are true minima. Calculated absorption and emission energies are in good agreement with experiment; the largest error is 0.14 eV, which is about the expected accuracy of the DFT calculations. For the first time, an optimized geometry for the metal-centered (MC) state is proposed for both of these complexes, and their energies are found to be almost degenerate with their corresponding 3MLCT states. These [RuII(LL)(eta1-LL)2]2+ MC states have two vacant coordination sites on the metal, so they may react readily with their environment. If these MC states are able to de-excite by luminescence, the associated transition (ca. 1 eV) is found to be quite different from those of the 3MLCT states (ca. 2 eV).
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