Analysis of carbon and nitrogen dynamics in riparian soils: model development.

The quality of riparian soils and their ability to buffer contaminant releases to aquifers and streams are connected intimately to moisture content and nutrient dynamics, in particular of carbon (C) and nitrogen (N). A multi-compartment model-named the Riparian Soil Model (RSM)-was developed to help investigate the influence and importance of environmental parameters, climatic factors and management practices on soil ecosystem functioning in riparian areas. The model improves existing tools, in particular regarding its capability to simulate a wide range of temporal scales, from days to centuries, along with its ability to predict the concentration and vertical distribution of dissolved organic matter (DOM). It was found that DOM concentration controls the amount of soil organic matter (SOM) stored in the soil as well as the respiration rate. The moisture content was computed using a detailed water budget approach, assuming that within each time step all the water above field capacity drains to the layer underneath, until it becomes fully saturated. A mass balance approach was also used for nutrient transport, whereas the biogeochemical reaction network was developed as an extension of an existing C and N turnover model. Temperature changes across the soil profile were simulated analytically, assuming periodic temperature changes in the topsoil. To verify the consistency of model predictions and to illustrate its capabilities, a synthetic but realistic soil profile in a deciduous forest was simulated. Model parameters were taken from the literature, and model predictions were consistent with experimental observations for a similar scenario. Modelling results stressed the importance of environmental conditions on SOM cycling in soils. The mineral and organic C and N stocks fluctuate at different time scales in response to oscillations in climatic conditions and vegetation inputs/uptake.

Analysis of carbon and nitrogen dynamics in riparian soils: model validation and sensitivity to environmental controls.

The Riparian Soil Model (RSM) of Brovelli et al. (2012) was applied to study soil nutrient turnover in a revitalized section of the Thur River, North-East Switzerland. In the present work, the model was calibrated on field experimental data, and satisfactorily reproduced soil respiration, organic matter stocks and inorganic nitrogen fluxes. Calibrated rates were in good agreement with the ranges reported in the literature. The main discrepancies between model and observations were for dissolved organic carbon. The sensitivity of the model to environmental factors was also analyzed. Soil temperature was the most influential factor at daily and seasonal scales while effects of soil moisture were weak overall. The ecosystem sensitivity to temperature changes was quantified using the Q10 index. The seasonal behavior observed was related to the influence of other forcing factors and to the different state (density and activity) of the microbial biomass pool during the year. Environmental factors influencing microbial decomposition, such as the C:N ratio and litter input rate, showed intermediate sensitivity. Since these parameters are tightly linked to the vegetation type, the analysis highlighted the effect of the aboveground ecosystem on soil functioning.

EEG dynamics of the frontoparietal network during reaching preparation in humans.

Visuomotor transformation processes are essential when accurate reaching movements towards a visual target have to be performed. In contrast, those transformations are not needed for similar, but non-visually guided, arm movements. According to previous studies, these transformations are carried out by neuronal populations located in the parietal and frontal cortical areas (the so-called "dorsal visual stream"). However, it is still debated whether these processes are mediated by the sequential and/or parallel activation of the frontoparietal areas. To investigate this issue, we designed a task where the same visual cue could represent either the target of a reaching/pointing movement or the go-signal for a similar but non-targeting arm movement. By subtracting the event-related potentials (ERPs) recorded from healthy subjects performing the two conditions, we identified the brain processes underlying the visuomotor transformations needed for accurate reaching/pointing movements. We then localized the generators by means of cortical current density (CCD) reconstruction and studied their dynamics from visual cue presentation to movement onset. The results showed simultaneous activation of the parietal and frontal areas from 140 to 260 ms. The results are interpreted as neural correlates of two critical phases of visuomotor integration, namely target selection and movement selection. Our findings suggest that the visuomotor transformation processes required for correct reaching/pointing movements do not rely on a purely sequential activation of the frontoparietal areas, but mainly on a parallel information processing system, where feedback circuits play an important role before movement onset.

Cell age-related monovalent cations content and density changes in stored human erythrocytes.

Conversion of erythrocyte membrane protein 4.1b to 4.1a occurs through a non-enzymatic deamidation reaction in most mammalian erythrocytes, with an in vivo half-life of approximately 41 days, making the 4.1a/4.1b ratio a useful index of red cell age [Inaba and Maede, Biochim. Biophys. Acta 944 (1988) 256-264]. Normal human erythrocytes distribute into subpopulations of increasing cell density and cell age when centrifuged in polyarabinogalactan density gradients. We have observed that, when erythrocytes were stored at 4 degrees C under standard blood bank conditions, the deamidation was virtually undetectable, as cells maintained the 4.1a/4.1b ratio they displayed at the onset of storage. By measuring the 4.1a/4.1b values in subpopulations of cells of different density at various time points during storage, a modification of the normal 'cell age/cell density' relationship was observed, as erythrocytes were affected by changes in cell volume in an age-dependent manner. This may stem from a different impact of storage on the imbalance of monovalent cations, Na(+) and K(+), in young and old erythrocytes, related to their different complement of cation transporters.

fMRI and EEG responses to periodic visual stimulation.

EEG/VEP and fMRI responses to periodic visual stimulation are reported. The purpose of these experiments was to look for similar patterns in the time series produced by each method to help understand the relationship between the two. The stimulation protocol was the same for both sets of experiments and consisted of five complete cycles of checkerboard pattern reversal at 1.87 Hz for 30 s followed by 30 s of a stationary checkerboard. The fMRI data was analyzed using standard methods, while the EEG was analyzed with a new measurement of activation-the VEPEG. Both VEPEG and fMRI time series contain the fundamental frequency of the stimulus and quasi harmonic components-an unexplained double frequency commonly found in fMRI data. These results have prompted a reappraisal of the methods for analyzing fMRI data and have suggested a connection between our findings and much older published invasive electrophysiological measurements of blood flow and the partial pressures of oxygen and carbon dioxide. Overall our new analysis suggests that fMRI signals are strongly dependant on hydraulic blood flow effects. We distinguish three categories of fMRI signal corresponding to: focal activated regions of brain tissue; diffuse nonspecific regions of steal; and major cerebral vessels of arterial supply or venous drainage. Each category of signal has its own finger print in frequency, amplitude, and phase. Finally, we put forward the hypothesis that modulations in blood flow are not only the consequence but are also the cause of modulations in functional activity.

Characterization of the hypertonically induced tyrosine phosphorylation of erythrocyte band 3.

Human erythrocyte band 3 becomes rapidly phosphorylated on tyrosine residues after exposure of erythrocytes to hypertonic conditions. The driving force for this phosphorylation reaction seems to be a decrease in cell volume, because (1) changes in band 3 phosphotyrosine content accurately track repeated changes in erythrocyte volume through several cycles of swelling and shrinking; (2) the level of band 3 phosphorylation is independent of the osmolyte employed but strongly sensitive to the magnitude of cell shrinkage; and (3) exposure of erythrocytes to hypertonic buffers under conditions in which intracellular osmolarity increases but volume does not change (nystatin-treated cells) does not promote an increase in tyrosine phosphorylation. We hypothesize that shrinkage-induced tyrosine phosphorylation results either from an excluded-volume effect, stemming from an increase in intracellular crowding, or from changes in membrane curvature that accompany the decrease in cell volume. Although the net phosphorylation state of band 3 is shown to be due to a delicate balance between a constitutively active tyrosine phosphatase and constitutively active tyrosine kinase, the increase in phosphorylation during cell shrinkage was demonstrated to derive specifically from an activation of the latter. Further, a peculiar inhibition pattern of the volume-sensitive erythrocyte tyrosine kinase that matched that of p72syk, a tyrosine kinase already known to associate with band 3 in vivo, suggested the involvement of this kinase in the volume-dependent response.

Tyrosine phosphorylation of band 3 protein in Ca2+/A23187-treated human erythrocytes.

Human erythrocytes were induced to release membrane vesicles by treatment with Ca2+ and ionophore A23187. In addition to the biochemical changes already known to accompany loading of human erythrocytes with Ca2+, the present study reveals that tyrosine phosphorylation of the anion exchanger band 3 protein also occurs. The relationship between tyrosine phosphorylation of band 3 and membrane vesiculation was analysed using quinine (a non-specific inhibitor of the Ca(2+)-activated K+ channel, and the only known inhibitor of Ca(2+)-induced vesiculation) and charybdotoxin, a specific inhibitor of the apamin-insensitive K(+)-channel. Both inhibitors suppressed tyrosine phosphorylation of band 3. In the presence of quinine, membrane vesiculation was also suppressed. In contrast, at the concentration of charybdotoxin required to suppress tyrosine phosphorylation of band 3, membrane vesiculation was only mildly inhibited (16-23% inhibition), suggesting that tyrosine phosphorylation of band 3 is not necessary for membrane vesiculation. Phosphorylation of band 3 was in fact observed when erythrocytes were induced to shrink in a Ca(2+)-independent manner, e.g. by treatment with the K+ ionophore valinomycin or with hypertonic solutions. These observations suggest that band 3 tyrosine phosphorylation occurs when cell volume regulation is required.

Oxidation state of glutathione and membrane proteins in human red cells of different age.

In this study the oxidation state of glutathione and membrane proteins was analyzed in red cells of different age in basal conditions. Red cells of different age were prepared by centrifugation and separated according to their density by two procedures: on self-forming gradients of autologous plasma (Murphy's procedure) and on discontinuous Stractan gradients. The efficiency of the two procedures in the isolation of senescent cells was compared. The results indicate that, despite the evidence that total cell GSH decreases with aging, its concentration, evaluated in the cell preparations of different ages, remains constant throughout the red cell life, when correlated with cell water content. Glutathione disulfide concentration increases with aging. The oxidation state of membrane proteins does not seem to change during the red cell life span.

Reduction of DABS-L-methionine-dl-sulfoxide by protein methionine sulfoxide reductase from polymorphonuclear leukocytes: stereospecificity towards the l-sulfoxide.

A new synthetic substrate for protein methionine sulfoxide reductase is proposed. We show that extracts from human polymorphonuclear leukocytes can reduce 4-dimethylaminoazobenzene-4'-sulfonyl-L-methionine-dl-sulfoxide [DABS-L-Met-dl-(O)] to the corresponding methionine derivative, in the presence of dithiothreitol or dithioerythritol. The product of the reaction (DABS-Met) was separated by reversed-phase HPLC and detected by reading the absorbance at 436 nm. Due to the chirality of the sulfur atom in the sulfoxide, two diastereomers of Met(O) exist, namely Met-l-sulfoxide and Met-d-sulfoxide. After separation of the two forms and preparation of the DABS-derivatives, we observed a preferential reduction of the l-sulfoxide by polymorphonuclear leukocytes extracts. We discuss the possibility that the observed stereospecificity might have physiological relevance in the field of the oxidative modifications of proteins.

Oxidation of membrane proteins and functional activity of band 3 in human red cell senescence.

The state of oxidation of membrane proteins was analyzed in red cell subpopulations of different age by quantifying the oxidation of methionine to its sulfoxide and by determining the amount of thiol groups in ghost membrane preparations and the reactivity of thiols of individual membrane proteins in intact cells. The results obtained show that oxidation of methionine occurs early during red cell life in the circulation, and can be detected in middle-aged and senescent cells. Thiol content of ghost membranes is kept constant in all the red cell subpopulations analyzed, but reactivity of thiol groups to the thiol reagent N-(7-dimethyl-amino-4-methyl-coumarinyl) maleimide (DACM) in intact cells decreased 30% in alpha-spectrin, band 3 (B3), 4.1 and 4.2 proteins, probably as a consequence of conformational changes of these molecules. Since the role played by band 3 in the exposure of senescence antigen has been described by many authors, the functional activity of the anion transporter has been analyzed by measuring the 4-4'-diisothiocyano-stilbene-2-2-'-disulfonate (DIDS) binding capacity in different red cell subpopulations. The results obtained are in agreement with the possibility that during senescence band 3 undergoes conformational changes involving the anion channel subsite being more exposed to the extracellular space and responsible for binding of DIDS.

Evidence for membrane protein oxidation during in vivo aging of human erythrocytes.

Oxidative lesions to membrane proteins were studied in human erythrocytes of different age and were evaluated on ghost membrane preparations by assaying thiol and methionine sulphoxide groups, and in situ on intact cells, after treating erythrocytes with the fluorochrome N-(7-dimethyl-amino-4-methyl-coumarinyl) maleimide (DACM). DACM reacts with thiol groups and the amount of this reagent bound by membrane proteins was quantified after SDS-PAGE separation. Results obtained show that during aging of normal cells the oxidative state of membrane proteins increases: this was better shown by the assay of methionine sulphoxide residues rather than by the thiol titration, when studies were carried out on ghost membranes. After separation of individual membrane proteins by SDS-PAGE, decreased accessibility of DACM to thiol groups of band 3 and of the main proteins of the membrane skeleton was evident in senescent erythrocytes. These results show that during aging, band 3 and membrane skeleton proteins undergo conformational changes and/or oxidation. Similar results were obtained when thiol distribution was studied in membrane proteins separated by SDS-PAGE in both reducing and non-reducing conditions.

Oxidative lesion to membrane proteins in senescent erythrocytes.

The oxidative lesion undergone by membrane proteins in senescent human erythrocytes was evaluated by assaying their MetSO and thiol group content in ghosts and the amount of a coumarinyl derivative of maleimide, the DACM, bound by individual membrane proteins after treatment of erythrocytes of different age with this reagent. Quantitation of MetSO content of ghost membranes indicates an increase of the oxidative state of membrane proteins from young to mature and senescent erythrocytes, while thiol group assay does not show significant differences among erythrocytes of different age. Quantitation of DACM bound in intact cells by individual membrane proteins shows a decreased accessibility of thiol groups of band 3 protein and of the main proteins of the membrane skeleton in senescent erythrocytes, and this could be partly due to oxidation. The decreased reactivity to DACM of senescent erythrocyte band 3 seems to concern thiols located on the cytoplasmic domain of this protein, since the anion channel binds the same amount of the anion transport inhibitor EM, in mature and senescent erythrocytes.

Re-evaluation of the structural integrity of red-cell glycoproteins during aging in vivo and nutrient deprivation.

Results presented in this paper show that removal of white-cell contaminations from human red blood cells by filtration through cellulose [Beutler, West & Blume (1976) J. Lab. Clin. Med. 88, 328-333] is a necessity whenever red cells are incubated at elevated temperatures or haemolysed after density separation. Omission of this precaution results in proteolysis of sialoglycoproteins in membranes from less-dense (young), but not dense (old), subpopulations. This proteolytic damage occurs during haemolysis of the cytoplasmic domain of glycophorin. A different type of proteolysis occurs if white-cell-contaminated red cells are incubated in the absence of glucose at elevated temperatures. Red cells release sialoglycopeptides. This process is stimulated by Ca2+ ions and is accompanied by the release of vesicles that differ from spectrin-free vesicles [Lutz, Liu & Palek (1977) J. Cell Biol. 73, 548-560]. This sialoglycopeptide release is dependent on white-cell contamination and is not required for the release of spectrin-free vesicles.

Organization of membrane constituents in human erythrocytes of different age.

The effect on the structural integrity of membrane glycoproteins of different methods for the isolation of human red cell subpopulations has been investigated. Moreover the extent of sialylation of membrane glycoconjugates in red cells of different age has been studied by a quantification of WGA binding sites at the single-cell level by cytofluorometric techniques, using the FITC-labeled lectin; the surface distribution of these sites has also been described by recording fluorescence intensity maps of cell surfaces.

Modification of membrane protein organization during in vitro aging of human erythrocytes.

In in vitro aged human erythrocytes, the presence of protein clusters can be found on the membrane; these clusters are made up of peptides held together by disulfide bridges, since they can be nearly completely dissociated by dithiothreitol treatment. SDS-polyacrylamide gel electrophoresis after dithiothreitol dissociation indicates that the aggregates are made of peptide fragments with a molecular weight ranging from 20 to approximately 110 kdalton; none of these fragments correspond to an intact protein component of the membrane. Their formation results from oxidation and proteolysis of membrane, and perhaps cytoplasmic proteins.

Electrophoretic and lectin-binding properties of glycopeptides released from the membrane during "in vitro" aging of human erythrocytes.

During in vitro aging of human erythrocytes sialopeptides are lost from the membrane in a process which appears to act on glycophorins. This glycopeptide material can be purified by affinity chromatography on Wheat germ agglutinin-Sepharose, as glycophorin does. The electrophoretic behaviour of the purified material suggests that the glycopeptide comes from the breakdown of the domain of glycophorin exposed on the surface of the membrane. The binding properties toward Phaseolus vulgaris E lectin indicate that the only N-linked sugar chain of glycophorin is present in the sialopeptide released from the membrane; therefore we can argue that the glycophorin breakdown during in vitro aging of red cell takes place beyond the 26th residue of the sequence, and probably quite near the lipid bilayer.