Redox regulation is emerging as an important post-translational modification in cell signaling and pathogenesis. Cysteine (Cys) is the most redox active of the commonly coded amino acids and is thus an important target for redox-based modifications. Reactions that oxidize the Cys sulfur atom to low oxidation states (e.g., disulfide) are reversible, while further reactions to higher oxidation states (e.g., sulfonic acid) may be irreversible under biological conditions. Reversible modifications are particularly interesting as they mediate redox signaling and regulation of proteins under physiological conditions and during adaptation to oxidant stress. An enrichment method that relied on rapid and specific alkylation of free Cys, followed by thiol-based reduction and resin capture by thiol-disulfide exchange chemistry was applied to isolate reversibly modified Cys-containing peptides. Chromatographic conditions were optimized to provide increased specificity by removal of noncovalent interactions. The technique was highly efficient, based on near equimolar reactions with the resin, reproducible and linear for peptide elution, as quantified by label-free mass spectrometry. The method was applied to a complex protein lysate generated from rat myocardial tissue and 6559 unique Cys-containing peptides from 2694 proteins were identified. Comparison with the rat database and previous studies showed effective enrichment of proteins modified by S-nitrosylation, disulfide formation, and Cys-sulfenic acid. Analysis of amino acid sequence features indicated a preference for acidic residues and increased hydrophilicity in the regions immediately up- or downstream of the reactive Cys. This technique is ideally suited for the enrichment and profiling of reversible Cys modifications on a proteome-wide scale.

Arousal is largely controlled by the ascending arousal system of the hypothalamus and brainstem, which projects to the corticothalamic system responsible for electroencephalographic (EEG) signatures of sleep. Quantitative physiologically based modelling of brainstem dynamics theory is described here, using realistic parameters, and links to EEG are outlined. Verification against a wide range of experimental data is described, including arousal dynamics under normal conditions, sleep deprivation, stimuli, stimulants and jetlag, plus key features of wake and sleep EEGs.

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We examined the response of Na(+),K(+)-ATPase (NKA) to monensin, a Na(+) ionophore, with and without ouabain, an NKA inhibitor, in suspensions of human erythrocytes (red blood cells). A combination of (13)C and (23)Na NMR methods allowed the recording of intra- and extracellular Na(+), and (13)C-labeled glucose time courses. The net influx of Na(+) and the consumption of glucose were measured with and without NKA inhibited by ouabain. A Bayesian analysis was used to determine probability distributions of the parameter values of a minimalist mathematical model of the kinetics involved, and then used to infer the rates of Na(+) transported and glucose consumed. It was estimated that the numerical relationship between the number of Na(+) ions transported by NKA per molecule of glucose consumed by a red blood cell was close to the ratio 6.0:1.0, agreeing with theoretical prediction.