Membrane flickering of the human erythrocyte: constrained random walk used with Bayesian analysis.

The involvement of adenosine triphosphate (ATP) in erythrocyte (red blood cell; RBC) membrane flickering is of particular interest, because ATP turnover in the cell as a whole is not yet fully accounted for. We sought the origins of flickering by deriving a mathematical model of it, on the basis of the idea of thermally driven collisions of small molecules with the membrane, which responds like an over-damped spring. The model gave simulated responses that were similar to a constrained random walk and had the same frequency-spectral characteristics of membrane displacement as those recorded from RBCs by use of differential interference contrast light microscopy. Bayesian analysis was used as the basis for determination, from experimental results, of the values of the parameters in the model. The analysis was used in the accompanying article in which we investigated the response of membrane flickering to different effector molecules and physicochemical conditions. The results implied ATP was involved only indirectly in membrane flickering.

Membrane flickering of the human erythrocyte: physical and chemical effectors.

Recent studies suggest a link between adenosine triphosphate (ATP) concentration and the amplitude of cell membrane flickering (CMF) in the human erythrocyte (red blood cell; RBC). Potentially, the origin of this phenomenon and the unique discocyte shape could be active processes that account for some of the ATP turnover in the RBC. Active flickering could depend on several factors, including pH, osmolality, enzymatic rates and metabolic fluxes. In the present work, we applied the data analysis described in the previous article to study time courses of flickering RBCs acquired using differential interference contrast light microscopy in the presence of selected effectors. We also recorded images of air bubbles in aqueous detergent solutions and oil droplets in water, both of which showed rapid fluctuations in image intensity, the former showing the same type of spectral envelope (relative frequency composition) to RBCs. We conclude that CMF is not directly an active process, but that ATP affects the elastic properties of the membrane that flickers in response to molecular bombardment in a manner that is described mathematically by a constrained random walk.

Transmembrane exchange of hyperpolarized 13C-urea in human erythrocytes: subminute timescale kinetic analysis.

The rate of exchange of urea across the membranes of human erythrocytes (red blood cells) was quantified on the 1-s to 2-min timescale. (13)C-urea was hyperpolarized and subjected to rapid dissolution and the previously reported (partial) resolution of (13)C NMR resonances from the molecules inside and outside red blood cells in suspensions was observed. This enabled a stopped-flow type of experiment to measure the (initially) zero-trans transport of urea with sequential single-pulse (13)C NMR spectra, every second for up to ~2 min. Data were analyzed using Bayesian reasoning and a Markov chain Monte Carlo method with a set of simultaneous nonlinear differential equations that described nuclear magnetic relaxation combined with transmembrane exchange. Our results contribute to quantitative understanding of urea-exchange kinetics in the whole body; and the methodological approach is likely to be applicable to other cellular systems and tissues in vivo.

Stoichiometric relationship between Na(+) ions transported and glucose consumed in human erythrocytes: Bayesian analysis of (23)Na and (13)C NMR time course data.

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.

Large-scale capture of peptides containing reversibly oxidized cysteines by thiol-disulfide exchange applied to the myocardial redox proteome.

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.

7Li+ NMR quadrupolar splitting in stretched hydrogels: developments in relaxation time estimation from z-spectra.

(7)Li NMR z-spectra were recorded from the cation constituted in gelatin gels that were held stretched. The system has been studied previously, but we revisited the disparity that was noted between estimates of some of the relaxation times of spin-states of various ranks and orders made using a global data-fitting strategy and estimates made from data acquired by using multiple-quantum-filter pulse sequences. The global data fitting was performed with a probability approach along with the Markov chain Monte Carlo (MCMC) method applied to z-spectra from (7)Li(+) dissolved in (1)H(2)O using more refined experimental methods than hitherto. We also present a more extensive explanation of the MCMC method as it applies in the present NMR context. We achieved much closer agreement between the estimates of relaxation times made by using the two methods of analysis and attribute the previous discrepancies to spectral drift and z-spectrum asymmetry.

A Global Profile of Reversible and Irreversible Cysteine Redox Post-Translational Modifications During Myocardial Ischemia/Reperfusion Injury and Antioxidant Intervention.

Aims: Cysteine (Cys) is a major target for redox post-translational modifications (PTMs) that occur in response to changes in the cellular redox environment. We describe multiplexed, peptide-based enrichment and quantitative mass spectrometry (MS) applied to globally profile reversible redox Cys PTM in rat hearts during ischemia/reperfusion (I/R) in the presence or absence of an aminothiol antioxidant, N-2-mercaptopropionylglycine (MPG). Parallel fractionation also allowed identification of irreversibly oxidized Cys peptides (Cys-SO2H/SO3H). Results: We identified 4505 reversibly oxidized Cys peptides of which 1372 were significantly regulated by ischemia and/or I/R. An additional 219 peptides (247 sites) contained Cys-SO2H/Cys-SO3H modifications, and these were predominantly identified from hearts subjected to I/R (n = 168 peptides). Parallel reaction monitoring MS (PRM-MS) enabled relative quantitation of 34 irreversibly oxidized Cys peptides. MPG attenuated a large cluster of I/R-associated reversibly oxidized Cys peptides and irreversible Cys oxidation to less than nonischemic controls (n = 24 and 34 peptides, respectively). PRM-MS showed that Cys sites oxidized during ischemia and/or I/R and "protected" by MPG were largely mitochondrial, and were associated with antioxidant functions (peroxiredoxins 5 and 6) and metabolic processes, including glycolysis. Metabolomics revealed I/R induced changes in glycolytic intermediates that were reversed in the presence of MPG, which were consistent with irreversible PTM of triose phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), altered GAPDH enzyme activity, and reduced I/R glycolytic payoff as evidenced by adenosine triphosphate and NADH levels. Innovation: Novel enrichment and PRM-MS approaches developed here enabled large-scale relative quantitation of Cys redox sites modified by reversible and irreversible PTM during I/R and antioxidant remediation. Conclusions: Cys sites identified here are targets of reactive oxygen species that can contribute to protein dysfunction and the pathogenesis of I/R.

Quantum confinement effects in gallium nitride nanostructures: ab initio investigations.

We present ab initio density functional investigations of the atomic and electronic structure of gallium nitride nanodots and nanowires. With increasing diameter, the average Ga-N bond length in the nanostructures increases, as does the relative stability (heat of formation), approaching the values for bulk GaN. As the diameter decreases, the band gap increases, with the variation for the nanodots greater than that for the nanowires, in qualitative accordance with expectations based on simple geometrical quantum confinement considerations. Interestingly, in contrast to nanowires, the lowest unoccupied states of the nanodots exhibit an extended delocalized (Ga-derived) character, weighted in the centre of the nanodot.

NMR of (133)Cs(+) in stretched hydrogels: One-dimensional, z- and NOESY spectra, and probing the ion's environment in erythrocytes.

(133)Cs nuclear magnetic resonance (NMR) spectroscopy was conducted on (133)Cs(+) in gelatin hydrogels that were either relaxed or stretched. Stretching generated a septet from this spin-7/2 nucleus, and its nuclear magnetic relaxation was studied via z-spectra, and two-dimensional nuclear Overhauser (NOESY) spectroscopy. Various spectral features were well simulated by using Mathematica and the software package SpinDynamica. Spectra of CsCl in suspensions of human erythrocytes embedded in gelatin gel showed separation of the resonances from the cation inside and outside the cells. Upon stretching the sample, the extracellular (133)Cs(+) signal split into a septet, while the intracellular peak was unchanged, revealing different alignment/ordering properties of the environment inside and around the cells. Differential interference contrast light microscopy confirmed that the cells were stretched when the overall sample was elongated. Analysis of the various spectral features of (133)Cs(+) reported here opens up applications of this K(+) congener for studies of cation-handling by metabolically-active cells and tissues in aligned states.

Simultaneous estimation of T1 and the flip angle in hyperpolarized NMR experiments using acquisition at non-regular time intervals.

In NMR spectroscopy of the liquid state T(1) is typically measured using an inversion recovery pulse sequence; but with hyperpolarized spins use is made of a sequence of multiple small radiofrequency (RF) induced nutations, α. Depending on the values of α and τ, the time interval between the pulses, the estimate of T(1) can be artifactually smaller than the real value; so without knowing the value of α the estimate of T(1) can be incorrect. Thus, we propose a method that involves a series of pulses with timing governed by a geometric sequence (or in general, any mathematically specified non-uniformly spaced sequence). This approach enables the simultaneous estimation of both the intrinsic T(1) value and α. The method was successfully applied to obtain T(1)=(44.9 ± 0.3)s and α=(4.0 ± 0.2)° (n=3) for a sample of hyperpolarized (13)C-urea in solution, matching with the inversion recovery pulse sequence estimate of T(1)=44 ± 2s using non-hyperpolarized (13)C-urea in solution.

Relaxation times of spin states of all ranks and orders of quadrupolar nuclei estimated from NMR z-spectra: Markov chain Monte Carlo analysis applied to 7Li+ and 23Na+ in stretched hydrogels.

The NMR z-spectra of 7Li+ and 23Na+ in stretched hydrogels contain five minima, or critical values, with a sharp "dagger" on the central dip. The mathematical representation of such z-spectra from spin-3/2 nuclei contains nine distinct (the total is 15 but there is redundancy of the ±order-numbers) relaxation rate constants that are unique for each of the spin states, up to rank 3, order 3. We present an approach to multiple-parameter-value estimation that exploits the high level of separability of the effects of each of the relaxation rate constants on the features of the z-spectrum. The Markov chain Monte Carlo (MCMC) method is computationally demanding but it yielded statistically robust estimates (low coefficients of variation) of the parameter values. We describe the implementation of the MCMC analysis (in the present context) and posit that it can obviate the need for using multiple-quantum filtered RF-pulse sequences to estimate all relaxation rate constants/times under experimentally favorable, but readily achievable, circumstances.