Proteomic analysis of arginine adducts on glyoxal-modified ribonuclease

Accumulation of advanced glycation end-products (AGEs) on proteins is associated with the development of diabetic complications. Although the overall extent of modification of protein by AGEs is limited, localization of these modifications at a few critical sites might have a significant effect on protein structure and function. In the present study, we describe the sites of modification of RNase by glyoxal under physiological conditions. Arg(39) and Arg(85), which are closest to the active site of the enzyme, were identified as the primary sites of formation of the glyoxal-derived dihydroxyimidazolidine and hydroimidazolone adducts. Lower amounts of modification were detected at Arg(10), while Arg(33) appeared to be unmodified. We conclude that dihydroxyimidazolidine adducts are the primary products of modification of protein by glyoxal, that Arg(39) and Arg(85) are the primary sites of modification of RNase by glyoxal, and that modification of arginine residues during Maillard reactions of proteins is a highly selective process.

Mass spectrometric analysis of glucose-modified ribonuclease

RNase A (1 mM) was incubated with glucose (0.4 M) at 37°C for up to 14 days in phosphate buffer (0.2 M, pH 7.4), digested with trypsin and analysed by LC-MS. The major sites of fructoselysine formation were Lys1, Lys7, Lys37 and Lys41. Three of these sites (Lys7, Lys37 and Lys41) were also the major sites of Ne-(carboxymethyl)lysine formation.

Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease

Proteomic analysis using electrospray liquid chromatography-mass spectrometry (ESI-LC-MS) has been used to compare the sites of glycation (Amadori adduct formation) and carboxymethylation of RNase and to assess the role of the Amadori adduct in the formation of the advanced glycation end-product (AGE), N-is an element of-(carboxymethyl)lysine (CIVIL). RNase (13.7 mg/mL, 1 mM) was incubated with glucose (0.4 M) at 37 degreesC for 14 days in phosphate buffer (0.2 M, pH 7.4) under air. On the basis of ESI-LC-MS of tryptic peptides, the major sites of glycation of RNase were, in order, K41, K7, K1, and K37. Three of these, in order, K41, K7, and K37 were also the major sites of CIVIL formation. In other experiments, RNase was incubated under anaerobic conditions (1 mM DTPA, N-2 purged) to form Amadori-modified protein, which was then incubated under aerobic conditions to allow AGE formation. Again, the major sites of glycation were, in order, K41, K7, K1, and K37 and the major sites of carboxymethylation were K41, K7, and K37. RNase was also incubated with 1-5 mM glyoxal, substantially more than is formed by autoxidation of glucose under experimental conditions, but there was only trace modification of lysine residues, primarily at K41. We conclude the following: (1) that the primary route to formation of CIVIL is by autoxidation of Amadori adducts on protein, rather than by glyoxal generated on autoxidation of glucose; and (2) that carboxymethylation, like glycation, is a site-specific modification of protein affected by neighboring amino acids and bound ligands, such as phosphate or phosphorylated compounds. Even when the overall extent of protein modification is low, localization of a high proportion of the modifications at a few reactive sites might have important implications for understanding losses in protein functionality in aging and diabetes and also for the design of AGE inhibitors.

Decadal predictability of regional-scale peak winds over Europe using the Earth System Model of the Max-Planck-Institute for Meteorology

The predictability of high impact weather events on multiple time scales is a crucial issue both in scientific and socio-economic terms. In this study, a statistical-dynamical downscaling (SDD) approach is applied to an ensemble of decadal hindcasts obtained with the Max-Planck-Institute Earth System Model (MPI-ESM) to estimate the decadal predictability of peak wind speeds (as a proxy for gusts) over Europe. Yearly initialized decadal ensemble simulations with ten members are investigated for the period 1979–2005. The SDD approach is trained with COSMO-CLM regional climate model simulations and ERA-Interim reanalysis data and applied to the MPI-ESM hindcasts. The simulations for the period 1990–1993, which was characterized by several windstorm clusters, are analyzed in detail. The anomalies of the 95 % peak wind quantile of the MPI-ESM hindcasts are in line with the positive anomalies in reanalysis data for this period. To evaluate both the skill of the decadal predictability system and the added value of the downscaling approach, quantile verification skill scores are calculated for both the MPI-ESM large-scale wind speeds and the SDD simulated regional peak winds. Skill scores are predominantly positive for the decadal predictability system, with the highest values for short lead times and for (peak) wind speeds equal or above the 75 % quantile. This provides evidence that the analyzed hindcasts and the downscaling technique are suitable for estimating wind and peak wind speeds over Central Europe on decadal time scales. The skill scores for SDD simulated peak winds are slightly lower than those for large-scale wind speeds. This behavior can be largely attributed to the fact that peak winds are a proxy for gusts, and thus have a higher variability than wind speeds. The introduced cost-efficient downscaling technique has the advantage of estimating not only wind speeds but also estimates peak winds (a proxy for gusts) and can be easily applied to large ensemble datasets like operational decadal prediction systems.

A sodar for profiling in a spatially inhomogeneous urban environment

The urban boundary layer, above the canopy, is still poorly understood. One of the challenges is obtaining data by sampling more than a few meters above the rooftops, given the spatial and temporal inhomogeneities in both horizontal and vertical. Sodars are generally useful tools for ground-based remote sensing of winds and turbulence, but rely on horizontal homogeneity (as do lidars) in building up 3-component wind vectors from sampling three or more spatially separated volumes. The time taken for sound to travel to a typical range of 200 m and back is also a limitation. A sodar of radically different design is investigated, aimed at addressing these problems. It has a single vertical transmitted sound pulse. Doppler shifted signals are received from a number of volumes around the periphery of the transmitted beam with microphones which each having tight angular sensitivity at zenith angles slightly off-vertical. The spatial spread of sampled volumes is therefore smaller. By having more receiver microphones than a conventional sodar, the effect of smaller zenith angle is offset. More rapid profiling is also possible with a single vertical transmitted beam, instead of the usual multiple beams.A prototype design is described, together with initial field measurements. It is found that the beam forming using a single dish antenna and the drift of the sound pulse downwind both give rise to reduced performance compared with expectations. It is concluded that, while the new sodar works in principle, the compromises arising in the design mean that the expected advantages have not been realized